Electrical field stimulation causes endothelium-dependent and nitric oxide-mediated relaxation of pulmonary artery GEORGETTE Department
M. BUGA of Pharmacology,
AND
LOUIS
University
J. IGNARRO of California
Buga, Georgette M., and Louis J. Ignarro. Electrical field stimulation causes endothelium-dependent and nitric oxide-mediated relaxation of pulmonary artery. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H973-H979,1992.-The objective of this study was to ascertain the mechanism by which electrical field stimulation (EFS) of bovine pulmonary arterial rings causes endothelium-dependent smooth muscle relaxation. Like acetylcholine-elicited relaxation, EFS-elicited relaxation was endothelium-dependent and accompanied by accumulation of guanosine 3’,5’-cyclic monophosphate (cGMP) in the vascular smooth muscle. Relaxation in response to EFS was unaltered by tetrodotoxin, guanethidine, atropine, propranolol, chlorpheniramine, cimetidine, indomethacin, aminophylline, cy, pmethylene ATP, nifedipine, capsaicin, and certain antioxidants and free radical scavengers. Thus the relaxation was not neurogenically mediated and was not attributed to free radical formation during EFS. Like nitric oxide-elicited relaxation, EFS-elicited relaxation was antagonized by oxyhemoglobin and methylene blue. Relaxation was also antagonized by the three NC-substituted L-arginine analogues: NG-methyl-L-arginine, NG-nitro+arginine, and NG-amino-L-arginine. NG-amino-Larginine also inhibited the tissue cGMP accumulation in response to EFS. The inhibitory effect of the NG-substituted Larginine analogues was reversed by addition of excess L-arginine but not D-arginine. Relaxation in response to EFS was dependent on the presence of extracellular calcium and intracellular calmodulin, as removal of extracellular calcium or addition of trifluoperazine nearly abolished relaxation. EFSelicited relaxation was inhibited also by tetraethylammonium chloride and elevated extracellular potassium concentration. These observations indicate that EFS-elicited relaxation of bovine pulmonary artery is mediated by neuronally independent, but endothelium- and calcium-dependent, stimulation of nitric oxide and cGMP formation. bovine pulmonary artery; guanosine 3’,5’-cyclic monophosphate; L-arginine; electrical stimulation THE MAMMALIAN PULMONARY CIRCULATION, in contrast to most systemic vascular beds, is characterized by a relatively low resistance to blood flow and a high compliance. Although this is attributed primarily to the low level of pulmonary arterial smooth muscle tone, the precise mechanisms involved are unknown. Decreased sensitivity of pulmonary arterial smooth muscle to endogenous vasoconstrictor agents or increased sensitivity to endogenous vasodilator agents could explain the unique characteristics of the pulmonary circulation. Evidence exists, for example, that pulmonary arteries are more sensitive than are systemic arteries to the vasodilator action of oxygen (39). The pulmonary circulation is very sensitive to nitrovasodilators both in vivo (25) and in vitro (5, 14, 17, 19, 20, 23). Isolated preparations of the first through fourth intralobar branches of bovine pulmonary artery and vein are highly sensitive to the direct relaxant actions of nitrovasodilators and to the indirect relaxant effects of endothelium-dependent, endothelium-derived relaxing
School of Medicine, Los Angeles, California
90024
factor (EDRF)-mediated agents. Pulmonary veins are more sensitive than are arteries to nitrovasodilators and to certain endothelium-dependent relaxants (5, 14, 19, 21). The mechanism by which acetylcholine (ACh) relaxes bovine pulmonary artery and bradykinin relaxes pulmonary vein involves the endothelium-dependent formation of NO and guanosine 3’,5’-cyclic monophosphate (cGMP) (16,19,20). Some endothelium-dependent vasodilators of bovine pulmonary artery such as vasoactive intestinal polypeptide and arachidonic acid involve multiple mechanisms, including the NO-cGMP pathway and the prostacyclin-adenosine 3’,5’-cyclic monophosphate (CAMP) pathway (23). Perfusion of isolated segments of bovine pulmonary artery and vein results in basally released and agonist-stimulated release of endotheliumderived NO, as assessed by bioassay cascade (15). Thus the pulmonary vascular bed is very sensitive to exogenous NO and to chemical agents and procedures that provoke endothelium-dependent NO formation. The existence of neuronal mechanisms for pulmonary vasodilation involving both sympathetic and vagal pathways was first described by Hyman and co-workers (13) in 1981. A variety of isolated systemic arterial preparations relax in response to electrical field stimulation (EFS) by endothelium-independent mechanisms that appear to involve adrenergic, cholinergic, and nonadrenergic-noncholinergic neurotransmission (1, 2, 4, 18, 31, 32,35,38). Vascular smooth muscle relaxation caused by EFS of mammalian blood vessels entails multiple mechanisms that vary with the vascular bed and species (see Ref. 12 for review). Isolated canine and bovine coronary arterial preparations relaxed in response to EFS by endothelium-independent mechanisms that did not involve classic neurotransmitters (6, 26, 36). EFS of pulmonary arterial rings from rabbit, cat, and monkey, however, was reported to cause endothelium-dependent relaxation that did not involve classical neurotransmitter release or arachidonic acid metabolism (8). The objective of the present study was to ascertain the mechanism by which EFS of bovine pulmonary arterial rings causes endotheliumdependent smooth muscle relaxation. MATERIALS AND METHODS Chemicals and solutions. Phenylephrine hydrochloride, acetylcholine chloride, indomethacin, hemoglobin (human), methylene blue, L-arginine, D-arginine, N”-nitro-L-arginine, tetrodotoxin (TTX), atropine sulfate, trifluoperazine dihydrochloride, guanethidine sulfate, propranolol hydrochloride, chlorpheniramine maleate, cimetidine, aminophylline, cy, pmethylene ATP, nifedipine, capsaicin, tetraethylammonium chloride, sodium ascorbate, reduced glutathione, superoxide dismutase (bovine erythrocytes), and catalase (bovine liver) were purchased from Sigma Chemical. U-46619 ([ 15S]-hydroxy-llar,9a,[epoxymethano] prosta-5Z, 13E-dienoic acid) was
0363-6135/92 $2.00 Copyright 0 1992 the American Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 13, 2019.
H973
H974
ELECTRICAL
STIMULATION
CAUSES
+Endothelium -8
tPE
-5
- Endothelium
t
’
1
1Omin
W/E m
-I t PE -5
i
J t PE -5
Fig. 1. Influence of endothelium on responses of arterial rings to electrical field stimulation (EFS) and acetylcholine (ACh). Rings were submaximally precontracted with phenylephrine (PE). Indomethacin (10 PM) was present in the bathing medium. EFS was conducted at 4 Hz as indicated. ACh was added cumulatively at indicated final bath concentrations. W/E signifies washing 3 times followed by 30 min of equilibration. Tracings are representative of 18 rings from 4 to 5 animals. In each experiment responses varied by no more than 20% from those illustrated.
provided* by Upjohn and was dissolved and stored in absolute ethanol (10 mg/ml). Solutions of hygroscopic acetylcholine chloride were prepared in distilled water, divided into aliquots, and stored at -20°C. Amino acids were soluble and prepared fresh daily in distilled water. Oxyhemoglobin was prepared from hemoglobin as described previously (19). S-nitroso-Nacetylpenicillamine (SNAP) was prepared as described previously (24), and fresh aqueous solutions (1 mM) were prepared in ice-cold distilled water and discarded after 4 h because of chemical instability. NG-methyl+arginine acetate and iVGamino-L-arginine acetate were synthesized and purified as described previously (9, 11). The highly water-soluble, crystalline hydrochloride salt of NC-nitro-L-arginine was prepared by dissolving the free base in 3 N HCl, followed by rotary evaporation of the solvent. The residue was then subjected to a high vacuum until completely dry and stored in a desiccated container under nitrogen at -2OOC. Krebs bicarbonate solution consisted of (in mM) 118 NaCl, 4.7 KCl, 1.5 CaCIZ, 25 NaHC03, 1.2 MgSO,, 1.2 KH2POr, 11 glucose, 0.023 disodium EDTA. Preparation of arterial rings. Bovine lungs were obtained from an abattoir located nearby and transported to the laboratory. The second intrapulmonary arterial branch (4-5 mm outside diameter) extending into the larger lobe was rapidly isolated; cleaned of parenchyma, fat, and adhering connective tissue; and placed in cold, preoxygenated Krebs bicarbonate solution. Arterial segments were sliced into rings (4 mm wide) as described previously (17,21). Rings prepared in this manner possessedan intact endothelium as assessedpharmacologically by 80-100% relaxation in response to 0.1-l PM ACh and histologically after silver staining. In some arterial rings the endothelial layer was completely removed without appreciable damage to the underlying smooth muscle by gently inverting the rings inside out, rubbing the intimal surface lightly with moistened filter paper for 3 min, and returning the rings to
NITRIC
OXIDE
FORMATION
their original configuration. Endothelium-denuded arterial rings contracted in response to ACh. Recording of muscle tension. Arterial rings were mounted by means of fine nichrome wires in jacketed, 25ml-capacity, dropaway chambers (Metro Scientific) containing Krebs bicarbonate solution (37°C) gassed with 95% 02-5% CO* (17). The upper nichrome wire of each ring was attached to a force-displacement transducer (Grass FT03C), and changes in isometric force were recorded on a Grass polygraph (model 79D). Length-tension relationships were determined initially for unrubbed and rubbed arterial rings. Tension was adjusted to the optimal length for maximal isometric contractions to potassium by progressively stretching the rings and repeatedly obtaining contractile responses to 80 mM KC1 with 15 min of equilibration between each contractile response (21). The optimal tensions determined in these initial experiments were employed in all subsequent experiments and did not vary significantly as a function of intimal rubbing. Optimal tensions and maximal contractile tensions developed in response to KC1 at optimal lengths, respectively, were 6 and 20-24 g for arterial rings. Rings were routinely depolarized by addition of 120 mM KC1 following 2 h of equilibration at optimal tension, then washed and allowed to equilibrate for 45 min before initiating any given protocol (17, 21). This procedure increases and stabilizes any subsequent submaximal precontractile responses to phenylephrine, presumably by loading the smooth muscle cells with calcium. This procedure has been employed routinely for bovine pulmonary vessels in this laboratory. Submaximal precontractile responses to l-10 PM phenylephrine ranged from 60 to 80% of maximal contractions produced by 120 mM KCl. In the experiments conducted in the absence of extracellular calcium (see Fig. 6), precontractile tensions generated by a given concentration of phenylephrine was reduced by 20-30%. Therefore, the concentration of phenylephrine was increased (generally 3-. fold) to maintain precontractile contractions at 60-80% of maximum. EFS. EFS was conducted with the aid of two parallel platinum electrodes placed 4-5 mm apart, on either side of the rings, and connected to a current amplifier and stimulator (SD9 Grass) as described previously (18). EFS was conducted at 25 V at a frequency of 4 Hz in the form of square wave pulses of 0.2 ms duration and delivered as 20-s trains. Pulse durations in excess of 0.2 ms and trains in excess of 10 s were not used in this study to avoid the possibility of generating free radicals in solution. Rings were washed three times and allowed to equilibrate for 30 min after EFS to permit the rings to recover completely from the relaxant response to EFS. Less than 10% variability in magnitudes of relaxation elicited by EFS (4 Hz, 25 V) was observed for a given ring during a typical experiment. Determination of CGA@ levels. Determinations were made in arterial rings that had been equilibrated under tension and depolarized with KCl, as was done routinely for all ring experiments. Tone was monitored until the time of quick-freezing. The use of rapid drop-away bath chambers, quick-freezing, tissue extraction, and radioimmunoassay procedures were described previously (17). None of the test agents added to bath chambers interfered with the radioimmunoassay procedure. Recoveries of standard quantities of added cGMP, determined periodically, were 92-104%. Calculations and statistical analysis. Relaxation or contraction of arterial rings was measured as the decrease or increase, respectively, in tension below or above the elevated tension elicited by submaximal precontraction with phenylephrine. Values in Figs. 3-7 are expressed as means & SE. Comparisons were made using Duncan’s multiple range test, where comparisons with a common control were made (see Figs. 3-7). The level of statistically significant difference was P < 0.05.
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 13, 2019.
ELECTRICAL
STIMULATION
CAUSES
NITRIC
OXIDE
FORMATION
H975
25 VOLTS 4 Hz
2 Hz
-It
J
t PE -5
PE %I
-5
t
4 Hz 15 v
c
f
I
10 min
t
-5
1
2ov
25V
I
c
PE
Fig. 2. Voltage-dependent and frequency-dependent relaxation of arterial rings in response to EFS. Rings were submaximally precontracted with PE. Indomethacin (10 PM) was present in the bathing medium. EFS was conducted at various frequencies (2-16 Hz) at 25 V (top) or at various voltages (15-30 V) at 4 Hz (bottom). Strips were washed 3 times and allowed to equilibrate for 30 min between each EFS applied to precontracted rings. Tracings are representative of 12 rings from 3 animals. In each experiment responses varied by no more than 25% from those illustrated.
r
J t PE
-J t
-5
t PE
t PE -5
PE
-5
-5
RESULTS
Characteristics of EFS-elicited pulmonary laxation. EFS caused endothelium-dependent
arterial
re-
relaxation of precontracted rings of pulmonary artery (Fig. 1). The endothelium-dependent relaxant responses to EFS were compared with those caused by ACh. Although Fig. 1 illustrates the responses to 4 Hz at 25 V, EFS conducted at 2, 4,8, and 16 Hz (25 V) or at 15, 20, 25, and 30 V (4 Hz) caused relaxant responses that were completely endothelium dependent. EFS-elicited relaxation was both frequency dependent and voltage dependent (Fig. 2).
80
q
O.lpM
ACh
Arterial relaxation in response to EFS was relatively long lasting, and repeated EFS caused a gradual but reversible decline in contractile tone (Fig. 1). Washing the arterial ring preparations after each EFS was necessary to obtain highly reproducible magnitudes of relaxant responses to EFS (Fig. 2). EFS-elicited relaxation was completely resistant to blockade by either atropine or TTX, whereas atropine but not TTX blocked the relaxant response to ACh (Fig. 3). Moreover, EFS-elicited relaxation was unaffected by 5 PM guanethidine, 10 PM propranolol, 10 PM indomethacin, 10 PM chlorpheniramine, 10 PM cimetidine, 100 PM aminophylline, 10 PM cy, ,&methylene ATP, 1 PM nifedipine, 1 PM capsaicin, 1 mM sodium ascorbate, 50 PM reduced glutathione, 100 units/ml of superoxide dismutase, or 300 units/ml of catalase (data not shown; 3-4 experiments, each using 4-6 arterial rings for each test condition, were conducted). Inhibition of EFS-elicited pulmonary arterial relaxation by chemical agents that interfere with the actions or synthesis of NO. Relaxant responses to EFS were mark-
Control
+At ropine hJM)
+Tet rodotox (1PW
Fig. 3. Effects of atropine and tetrodotoxin (TTX) on responses of arterial rings to EFS and ACh. Rings were submaximally precontracted with PE. Responses to EFS (4 Hz) and ACh were obtained at ,peak contractile responses to PE. Atropine and TTX were added to bathing media 30 min before precontraction. Values are means t SE using 24 rings (for each test condition) from 4 animals (6 rings/animal). Values for ACh in the presence of atropine are significantly different (P < 0.001) from values for ACh alone (control
edly inhibited by hemoglobin and methylene blue (Fig. 4). Similarly, by using the same arterial ring preparations, the relaxant responses to ACh and SNAP were markedly inhibited by hemoglobin and methylene blue (Fig. 4). Three NG-substituted analogues of L-arginine were tested for their effects on EFS-elicited relaxation. NG-methyl-L-arginine (100 PM), NG-amino+arginine (100PM), and NG-nitro-L-arginine (10PM) caused comparably marked inhibitory effects on EFS-elicited relaxation (Fig. 5). NG-nitro-L-arginine was the most potent of the three L-arginine analogues tested. Addition of 300 PM L-arginine, but not its enantiomer D-arginine, to the bathing medium containing one of the three inhibitory
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 13, 2019.
H976
ELECTRICAL
STIMULATION
CAUSES
NITRIC
.
100
- Controls
80
80 .-: zn60 Q 8
60
, T
+~DM
/
Hemoglobin
OXIDE
.
FORMATION
cl
No Arginine
El
0.3mM
Ea
0.3mM +NMA,
L- Arginine D-Arginine NAA
or NNA
E40 3 at 20
0 +NAA +NNA +NMA (0.1 mM) (0.1 mM) (0.01 mM) Fig. 5. Effects of NO synthase inhibitors in the absence and presence of L-arginine or D-arginine on responses of arterial rings to EFS. Rings were submaximally precontracted with PE. EFS was conducted at 4 Hz. NG-methyl-L-arginine (NMA), NC-amino-L-arginine (NAA), and NC-nitro-L-arginine (NNA) were added to bathing media at peak contractile responses to PE, and EFS was conducted 10 min later. Rings were washed 3 times, allowed to equilibrate for 30 min, precontracted with PE, and L-arginine or D-arginine was added, followed within 1 min by addition of NMA, NAA, or NNA. EFS was conducted 10 min later. Values are means t SE using 16-24 rings (for each test condition) for 4-6 animals (4 rings/animal). Values obtained in the presence of NMA, NAA, or NNA alone or with D-arginine are significantly different (P < 0.001) from values for control or NO synthase inhibitors with L-arginine. Control
0 +lflM
Mmthylene
Blue
40 20 0 -7 -6 -8 -7 -6 ACh SNAP Fig. 4. Influence of hemoglobin and methylene blue on responses of arterial rings to EFS, ACh, and S-nitroso-N-acetylpenicillamine (SNAP). Rings were submaximally precontracted with PE. Responses to EFS (4 Hz), ACh, and SNAP were obtained at peak contractile responses to PE. Concentrations of ACh and SNAP represent final bath concentrations. Hemoglobin and methylene blue were added to bathing media 5 and 30 min, respectively, prior to precontraction. Values are means k SE using 18-24 rings (for each test condition) from 3 to 4 animals (6 rings/animal). All values obtained in the presence of hemoglobin or methylene blue are significantly different (P < 0.01) from values for corresponding controls. 4Hz
-8
L-arginine analogues nearly completely restored arterial relaxation in response to EFS (Fig. 5). Dependence of EFS-elicited relaxation of pulmonary artery on extraceZZuZar calcium. Replacement of the nor-
mal Krebs-bicarbonate bathing solution with calciumfree solution resulted in near abolition of relaxation in response to either EFS or ACh (Fig. 6). Replenishment of the bathing solution with calcium and further tissue incubation for 45 min restored by -75% the capacity of EFS and ACh to cause relaxation. Trifluoperazine, a well-known calmodulin antagonist, nearly abolished arterial relaxation in response to EFS. Tetraethylammonium chloride (10 PM-3 mM) and 30 mM KC1 caused 70-80% (range) inhibition of EFS-elicited relaxation (data not shown; 3 experiments each using 4-6 arterial rings for each test condition were conducted). cGMP formation in response to EFS of pulmonary artery and inhibition by NG-amino-L-arginine. EFS (4
Hz; 25 V) caused a five- to sixfold increase in arterial levels of cGMP that accompanied the smooth muscle relaxation (Fig. 7). Both relaxation and cGMP accumulation were inhibited by 10 PM NG-amino-L-arginine.
DISCUSSION
Frank and Bevan (8) first reported that isolated rings of pulmonary artery from rabbits, cats, and monkeys relaxed in response to EFS in an endothelium-dependent manner. The precise mechanism, however, was not elucidated because the study was conducted soon after the discovery of EDRF (10).The data from the present study indicate that EFS causes relaxation of isolated rings of bovine pulmonary artery by mechanisms involving the L-arginine-NO-cGMP pathway. The evidence for this conclusion is 1) the relaxation is endothelium dependent and is accompanied by tissue accumulation of cGMP, 2) relaxation is antagonized by oxyhemoglobin and methylene blue, chemical agents that inhibit the actions of NO on smooth muscle, and 3) relaxation is antagonized by NG-substituted analogues of L-arginine by mechanisms that are reversed in an enantiomerically specific manner by excess L-arginine. The observations that EFS- and ACh-elicited relaxation are dependent on extracellular calcium and intracellular calmodulin are consistent with the above hypothesis that endotheliumderived NO is largely responsible for EFS-elicited relaxation of bovine pulmonary artery. As reported previously for pulmonary artery from rabbits, cats, and monkeys (8), EFS-elicited relaxation of bovine pulmonary artery was completely resistant to alteration by a wide range of pharmacological agents that are well known to interfere with chemical neurotransmission and prostaglandin formation. Although guanethidine and atropine failed to influence EFS-elicited relax-
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 13, 2019.
ELECTRICAL
STIMULATION
CAUSES
NITRIC
OXIDE
FORMATION
H977
endothelium dependent and there is no evidence for the direct innervation of vascular endothelial cells. Thus the present observations strengthen the view that EFS-elic80 ited relaxation of pulmonary artery does not involve E neuronal transmitter release (8). Free radical generation in oxygenated salt solution in response to EFS does not appear to be involved in the mechanism by which EFS causes relaxation of pulmonary artery in the present study. The evidence for this conclusion is that neither antioxidants nor oxygen-derived radical scavengers added to tissue baths altered the relaxation responses. Moreover, no evolution of gas bubbles, such as chlorine gas, was ever observed at the 0 +Ca2+ Control +TFP -Ca2+ electrode surface at any time during or after EFS, as has (3uM) been reported in certain studies by others (12, 30, 37). Fig. 6. Influence of extracellular calcium and trifluoperazine on re- Although relaxant responses to EFS were relatively well sponses of arterial rings to EFS and ACh. Rings were submaximally maintained in pulmonary artery compared with blood precontracted with PE. Responses to EFS (4 Hz) and ACh were vessel preparations where relaxation is neurogenically obtained at peak contractile responses to PE. -Ca2’ signifies replacemediated (1, 31, 32), phenylephrine-induced precontracment of normal Krebs bicarbonate solution with calcium-free Krebs bicarbonate solution, followed by 30 min of equilibration and precontile tone consistently returned after washing the prepatraction with PE. +Ca2’ signifies replacement of calcium-free Krebs rations. Under these conditions, the magnitudes of relaxbicarbonate solution with normal Krebs bicarbonate solution, followed ation elicited by EFS at 4 Hz were highly reproducible by 30 min of equilibration and precontraction with PE. Trifluoperazine for a given arterial ring for the duration of the experi(TFP) was added to bathing media 30 min before precontraction. Values are means & SE using 18 rings (for each test condition) from 3 ment. These observations argue against involvement of animals (6 rings/animal). Values obtained for -Ca2’ and in the presfree radicals in the relaxant response, as no functional ence of TFP are significantly different (P < 0.001) from corresponding vascular smooth muscle damage was evident during the control values. Values for +Ca2’ in the presence of ACh are significourse of any experiment. Finally, the electrical stimucantly different (P < 0.05) from corresponding ACh control values. lation parameters employed, such as square wave pulses of very short duration (0.2 ms) and short stimulation 100 200 q Rdaxation trains of 20 s, are not conducive to the generation of free Cyclic GMP fz radicals in aqueous solution (6). Prolonging the electrical t 160 80 stimulation to 2-ms pulses delivered as 20-min trains, yo c) however, can produce secondary relaxant responses that E are attributed to free radical generation (6). 120 G) z Although the present observations indicate clearly that Q EFS stimulates the formation and/or release of endothe80 $ 0 hum-derived NO in pulmonary artery, studies were not ‘( focused on the precise mechanism by which the electric to 40 current is coupled or gated to the L-arginine-NO pathway. Nevertheless, several points are noteworthy. At least one of the constitutive, cytosolic isoforms of mam0 malian NO synthase in vascular endothelial cells requires Control +NAA (0.01 mM) both calcium and calmodulin for catalytic activity (7). Fig. 7. Alteration of guanosine 3’,5’-cyclic monophosphate (cGMP) The dependence of EFS-elicited relaxation on extracellevels and tone of arterial rings by EFS and the influence of NC-aminolular calcium, together with the inhibitory action of L-arginine (NAA). Rings were submaximally precontracted with PE. trifluoperazine, tetraethylammonium chloride, and eleEFS was conducted at 4 Hz. When tested, NAA was added to bathing suggest that media at peak contractile responses to PE, and EFS was conducted 10 vated extracellular potassium concentration, min later. Rings were quick-frozen 60 s after EFS or at peak contractile some type of potential-dependent calcium channels on responses to PE (basal). Values are means * SE using 12 rings (for the endothelial cells may be involved, and that calcium each test condition) from 3 animals (4 rings/animal). Control values influx triggers NO formation via activation of calciumare significantly different (P c 0.001) from basal values. Values obcalmodulin-dependent NO synthase. For example, as tained in the presence of NAA are significantly different (P < 0.001) reported previously by Luckhoff and Busse (33, 34), for from corresponding control values. endothelial cells in response to certain agonists, EFS may initially mobilize intracellular calcium, thereby ation, nonadrenergic-noncholinergic neurotransmission opening calcium-activated potassium channels, which was not likely involved because relaxation was TTX insensitive. TTX-resistant responses do not necessarily leads to hyperpolarization. Endothelial cell hyperpolarirule out the possibility of a neurogenic mechanism be- zation would augment the driving force for potentialdependent transmembrane calcium influx, and thereby cause EFS can promote neurotransmitter release from provide the signal for NO synthesis. In this manner, the nerve terminal varicosities in the absence of propagated action potentials (27, 28). The involvement of nerve relationship between transmembrane calcium influx and terminals in EFS-elicited relaxation of pulmonary artery the membrane potential in endothelial cells may be opposite to that characteristic of excitable vascular smooth is unlikely, however, because relaxation is completely
1
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 13, 2019.
H978
ELECTRICAL
STIMULATION
CAUSES
muscle cells (3, 33). Similarly, EFS caused relaxation of rat tail artery by mechanisms that appear to involve potassium-mediated membrane hyperpolarization and calcium influx, as this was inhibited by elevating the extracellular potassium concentration to 20-30 mM or by addition of 1 mM tetraethylammonium chloride (29). Regardless of the precise mechanisms involved, the physiological or pathophysiological significance of electrically induced, nonneurogenically mediated, endothelium- and calcium-dependent relaxation of pulmonary artery still remains to be determined. We thank Dr. Jon M. Fukuto for synthesizing the NG-substituted analogues of L-arginine and Diane Rome Peebles for preparing the illustrations. This investigation was supported in part by National Heart, Lung, and Blood Institute Grants HL-35014 and HL-40922, a grant from the Laubisch Fund for Cardiovascular Research, and the Tobacco-Related Disease Research Program. Address for reprint requests: L. J. Ignarro, Dept. of Pharmacology, UCLA School of Medicine, CHS, Los Angeles, CA 90024. Received 22 July 1991; accepted in final form 26 November 1991. REFERENCES Bevan, J. A., G. M. Buga, A. Snowden, and S. I. Said. Is the neural vasodilator mechanism to cerebral and extracerebral arteries the same? In: Cerebral Blood Flow: Effect of Nerves and Neurotransmitters, edited by M. L. Marcus and D. D. Heistad. New York: Elsevier, 1982, p. 421-439. 2. Bowman, A., and J. S. Gillespie. Block of some non-adrenergic inhibitory responses of smooth muscle by a substance from haemolyzed erythrocytes. J. Physiol. Lond. 328: 11-25, 1982. 3. Busse, R., H. Fichtner, A. Luckhoff, and M. Kohlhardt. Hyperpolarization and increased free calcium in acetylcholinestimulated endothelial cells. Am. J. Physiol. 255 (Heart Circ. Physiol. 24): H965-H969, 1988. 4. Cohen, R. A., J. T. Shepherd, and P. M. Vanhoutte. Prejunctional and postjunctional actions of endogenous norepinephrine at the sympathetic neuroeffector junction in canine coronary arteries. 1.
Circ. 5.
Res. 52: 16-25,
1983.
Edwards, J. C., L. J. Ignarro, K. S. Wood, A. L. Hyman, and P. J. Kadowitz. Relaxation of intrapulmonary artery and vein by nitrogen oxide-containing vasodilators and cyclic GMP. J. Pharmacol.
Exp.
Ther.
228: 33-42,1984.
Feletou, M., and P. M. Vanhoutte. Relaxation of canine coronary artery to electrical stimulation: limited role of free radicals. Am. J. Physiol. 253 (Heart Circ. Physiol. 22): H884-H889, 1987. 7. Forstermann, U., J. S. Pollock, H. H. H. W. Schmidt, M. Heller, and F. Murad. Calmodulin-dependent endothelium-derived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells. 6.
Proc. 8.
9.
10.
11.
12.
Natl.
Acad.
Sci.
USA
88: 1788-1792,
1991.
Frank, G. W., and J. A. Bevan. Electrical stimulation causes endothelium-dependent relaxation in lung vessels. Am. J. Physiol. 244 (Heart Circ. Physiol. 13): H793-H798, 1983. Fukuto, J. M., K. S. Wood, R. E. Byrns, and L. J. Ignarro. NG-amino-L-arginine: a new potent antagonist of L-arginine-mediated endothelium-dependent relaxation. Biochem. Biophys. Res. Commun. 168: 458-465,199O. Furchgott, R. F., and J. V. Zawadzki. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature Lond. 288: 373-376, 1980. Gold, M. E., K. S. Wood, R. E. Byrns, J. M. Fukuto, and L. J. Ignarro. NG-methyl+arginine causes endothelium-dependent contraction and inhibition of cyclic GMP formation in artery and vein. Proc. Natl. Acad. Sci. USA 87: 4430-4434, 1990. Hardebo, J. E., J. Kahrstrom, and C. Owman. Characterization of dilatation induced by electrical field stimulation in mammalian cerebral and peripheral vessels. Q. J. Exp. Physiol. 74: 475491,1989.
13.
Hyman,
D. S. Knight,
and P. J.
OXIDE
FORMATION
Kadowitz. Pulmonary vasodilator responses to catecholamines and sympathetic nerve stimulation in the cat. Circ. Res. 48: 407415,1981.
14 Ignarro,
L. J., G. M. Buga, R. E. Byrns, K. S. Wood, and G. Chaudhuri. Endothelium-derived relaxing factor and nitric oxide possess identical pharmacologic properties as relaxants of bovine arterial and venous smooth muscle. J. Pharmacol. Exp. Ther. 246: 218-226,1988. 15 Ignarro, L. J., G. M. Buga, and G. Chaudhuri. EDRF gener’ ation and release from perfused bovine pulmonary artery and vein. l
Eur.
J. Pharmacol.
149: 79-88,1988.
16. Ignarro,
L. J., G. M. Buga, K. S. Wood, R. E. Byrns, and G. Chaudhuri. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc. Natl. Acad. Sci. USA
84: 9265-9269,1987.
Exp.
Ther.
17 Ignarro, L. J., T. Burke, K. S. Wood, and P. J. Kadowitz. ’ Association between cyclic GMP accumulation and acetylcholineelicited relaxation of bovine intrapulmonary artery. J. Pharmacol. 228: 682-690,1984.
18 Ignarro, L. J., P. A. Bush, G. M. Buga, K. S. Wood, J. M. ’ Fukuto, and J. Rajfer. Nitric oxide and cyclic GMP formation upon electrical stimulation cause relaxation of corpus cavernosum smooth muscle. B&hem. Biophys. Res. Commun. 170: 843-850, 1990.
L. J., R. E. Byrns, G. M. Buga, and K. S. Wood. 19. Ignarro, Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacological and chemical properties that are identical to those for nitric oxide radical. Circ. Res. 61: 866-879, 1987. 2. Ignarro, L. J., R. E. Byrns, G. M. Buga, K. S. Wood, and G. Chaudhuri. Pharmacological evidence that endothelium-derived relaxing factor is nitric oxide: use of pyrogallol and superoxide dismutase to study endothelium-dependent and nitric oxide-elicited vascular smooth muscle relaxation. J. Pharmacol. Exp. Ther. l
244: 181-190,1988. 21.
Ignarro, L. J., R. E. Byrns, and K. S. Wood. Endotheliumdependent modulation of cyclic GMP levels and intrinsic smooth muscle tone in isolated bovine intrapulmonary artery and vein. Circ.
Res. 60: 82-92,
1987.
Ignarro, L. J., R. G. Harbison, K. S. Wood, and P. J. Kadowitz. Activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor from intrapulmonary artery and vein: stimulation by acetylcholine, bradykinin and arachidonic acid. J. Pharmacol. Exp. Ther. 237: 893-900, 1986. 23* Ignarro, L. J., R. G. Harbison, K. S. Wood, M. S. Wolin, D. B. McNamara, A. L. Hyman, and P. J. Kadowitz. Differences in responsiveness of intrapulmonary artery and vein to arachidonic acid: mechanism of arterial relaxation involves cyclic GMP and cyclic AMP. J. Pharmacol. Exp. Ther. 233: 560-569,1985. L. J., H. Lippton, J. C. Edwards, W. H. Baricos, 24* Ignarro, A. L. Hyman, P. J. Kadowitz, and C. A. Gruetter. Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement of SExp. Ther. 218: nitrosothiols as active intermediates. J. Pharmacol.
22.
739-749,198l. 25.
Kadowitz, P. J., P. Nandiwada, C. A. Gruetter, L. J. Ignarro, and A. L. Hyman. Pulmonary vasodilator responses to nitroprusside and nitroglycerin in the dog. J. Clin. Invest. 67: 893902,198l.
26. Kalsner, S., and M. Quillan. Nonneurogenic relaxation to field stimulation in coronary arteries. J. Pharmacol. Exp. Ther. 250: 461-469,1989. 27. Katz, B., and R. Miledi. Spontaneous and evoked activity of motor nerve endings in calcium ringer. J. Physiol. Lond. 203: 689706,1969.
Katz, B., and R. Miledi. Further study of the role of calcium in synaptic transmission. J. Physiol. Lond. 207: 789-801, 1970. 29. Kotecha, N., and T. 0. Neild. Relaxation and hyperpolarization of the smooth muscle of the rat tail artery following electrical stimulation. J. Physiol. Land. 397: 489-501, 1988. 30. Lamb, E. S., and R. C. Webb. Vascular effects of free radicals generated by electrical stimulation. Am. J. Physiol. 257 (Heart Circ.
28.
Physiol. 31.
A. L., P. Nandiwada,
NITRIC
26): H709-H714,
1984.
Lee, T. J. F., L. R. Kinkeas, and S. Sarwinski. Norepinephrine and acetvlcholine transmitter mechanisms in large cerebral
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 13, 2019.
ELECTRICAL
STIMULATION
CAUSES
arteries of the pig. J. Cereb. Blood Flow Metab. 2: 439-450, 1982. 32. Lee, T. J. F., C. Su, and J. A. Bevan. Nonsympathetic dilator innervation of cat cerebral arteries. Experientia Base1 31: 14241425, 1975. 33. Liickhoff, A., and R. Busse. Activators of potassium channels enhance calcium influx into endothelial cells as a consequence of potassium currents. Naunyn-Schmiedeberg’s Arch. Pharmacol. 342: 94-99,199o. 34. Liickhoff, A., and R. Busse. Calcium influx into endothelial cells and formation of endothelium-derived relaxing factor is controlled by the membrane potential. Pjluegers Arch. 416: 305-311, 1990. 35. Mackenzie, I., and G. Burnstock. Evidence against vasoactive intestinal polypeptide being the non-adrenergic non-cholinergic inhibitory transmitter released from nerves supplying the smooth
NITRIC
36.
37.
38.
39.
OXIDE
FORMATION
H979
muscle of the guinea-pig taenia coli. Eur. J. PharmacoZ. 67: 255264, 1980. Rooke, T., R. A. Cohen, T. J. Verbeuren, and P. M. Vanhoutte. Nonneurogenic inhibitory effect of electrical impulses in isolated canine coronary arteries. Eur. J. PharmacoZ. 80: 251-254, 1982. Rubanyi, G. M., and P. M. Vanhoutte. Oxygen-derived free radicals, endothelium and responsiveness of vascular smooth muscle. Am. J. Physiol. 250 (Heart Circ. Physiol. 19): H815-H821,1986. Toda, N. Response of isolated monkey coronary arteries to catecholamines and to transmural electrical stimulation. Circ. Res. 49: 1228-1236, 1981. Weir, E. K. Does normoxic pulmonary vasodilation rather than hypoxic vasoconstriction account for the pulmonary pressor response to hypoxia? Lancet 1: 476-477, 1978.
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 13, 2019.
M. BUGA of Pharmacology,
AND
LOUIS
University
J. IGNARRO of California
Buga, Georgette M., and Louis J. Ignarro. Electrical field stimulation causes endothelium-dependent and nitric oxide-mediated relaxation of pulmonary artery. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H973-H979,1992.-The objective of this study was to ascertain the mechanism by which electrical field stimulation (EFS) of bovine pulmonary arterial rings causes endothelium-dependent smooth muscle relaxation. Like acetylcholine-elicited relaxation, EFS-elicited relaxation was endothelium-dependent and accompanied by accumulation of guanosine 3’,5’-cyclic monophosphate (cGMP) in the vascular smooth muscle. Relaxation in response to EFS was unaltered by tetrodotoxin, guanethidine, atropine, propranolol, chlorpheniramine, cimetidine, indomethacin, aminophylline, cy, pmethylene ATP, nifedipine, capsaicin, and certain antioxidants and free radical scavengers. Thus the relaxation was not neurogenically mediated and was not attributed to free radical formation during EFS. Like nitric oxide-elicited relaxation, EFS-elicited relaxation was antagonized by oxyhemoglobin and methylene blue. Relaxation was also antagonized by the three NC-substituted L-arginine analogues: NG-methyl-L-arginine, NG-nitro+arginine, and NG-amino-L-arginine. NG-amino-Larginine also inhibited the tissue cGMP accumulation in response to EFS. The inhibitory effect of the NG-substituted Larginine analogues was reversed by addition of excess L-arginine but not D-arginine. Relaxation in response to EFS was dependent on the presence of extracellular calcium and intracellular calmodulin, as removal of extracellular calcium or addition of trifluoperazine nearly abolished relaxation. EFSelicited relaxation was inhibited also by tetraethylammonium chloride and elevated extracellular potassium concentration. These observations indicate that EFS-elicited relaxation of bovine pulmonary artery is mediated by neuronally independent, but endothelium- and calcium-dependent, stimulation of nitric oxide and cGMP formation. bovine pulmonary artery; guanosine 3’,5’-cyclic monophosphate; L-arginine; electrical stimulation THE MAMMALIAN PULMONARY CIRCULATION, in contrast to most systemic vascular beds, is characterized by a relatively low resistance to blood flow and a high compliance. Although this is attributed primarily to the low level of pulmonary arterial smooth muscle tone, the precise mechanisms involved are unknown. Decreased sensitivity of pulmonary arterial smooth muscle to endogenous vasoconstrictor agents or increased sensitivity to endogenous vasodilator agents could explain the unique characteristics of the pulmonary circulation. Evidence exists, for example, that pulmonary arteries are more sensitive than are systemic arteries to the vasodilator action of oxygen (39). The pulmonary circulation is very sensitive to nitrovasodilators both in vivo (25) and in vitro (5, 14, 17, 19, 20, 23). Isolated preparations of the first through fourth intralobar branches of bovine pulmonary artery and vein are highly sensitive to the direct relaxant actions of nitrovasodilators and to the indirect relaxant effects of endothelium-dependent, endothelium-derived relaxing
School of Medicine, Los Angeles, California
90024
factor (EDRF)-mediated agents. Pulmonary veins are more sensitive than are arteries to nitrovasodilators and to certain endothelium-dependent relaxants (5, 14, 19, 21). The mechanism by which acetylcholine (ACh) relaxes bovine pulmonary artery and bradykinin relaxes pulmonary vein involves the endothelium-dependent formation of NO and guanosine 3’,5’-cyclic monophosphate (cGMP) (16,19,20). Some endothelium-dependent vasodilators of bovine pulmonary artery such as vasoactive intestinal polypeptide and arachidonic acid involve multiple mechanisms, including the NO-cGMP pathway and the prostacyclin-adenosine 3’,5’-cyclic monophosphate (CAMP) pathway (23). Perfusion of isolated segments of bovine pulmonary artery and vein results in basally released and agonist-stimulated release of endotheliumderived NO, as assessed by bioassay cascade (15). Thus the pulmonary vascular bed is very sensitive to exogenous NO and to chemical agents and procedures that provoke endothelium-dependent NO formation. The existence of neuronal mechanisms for pulmonary vasodilation involving both sympathetic and vagal pathways was first described by Hyman and co-workers (13) in 1981. A variety of isolated systemic arterial preparations relax in response to electrical field stimulation (EFS) by endothelium-independent mechanisms that appear to involve adrenergic, cholinergic, and nonadrenergic-noncholinergic neurotransmission (1, 2, 4, 18, 31, 32,35,38). Vascular smooth muscle relaxation caused by EFS of mammalian blood vessels entails multiple mechanisms that vary with the vascular bed and species (see Ref. 12 for review). Isolated canine and bovine coronary arterial preparations relaxed in response to EFS by endothelium-independent mechanisms that did not involve classic neurotransmitters (6, 26, 36). EFS of pulmonary arterial rings from rabbit, cat, and monkey, however, was reported to cause endothelium-dependent relaxation that did not involve classical neurotransmitter release or arachidonic acid metabolism (8). The objective of the present study was to ascertain the mechanism by which EFS of bovine pulmonary arterial rings causes endotheliumdependent smooth muscle relaxation. MATERIALS AND METHODS Chemicals and solutions. Phenylephrine hydrochloride, acetylcholine chloride, indomethacin, hemoglobin (human), methylene blue, L-arginine, D-arginine, N”-nitro-L-arginine, tetrodotoxin (TTX), atropine sulfate, trifluoperazine dihydrochloride, guanethidine sulfate, propranolol hydrochloride, chlorpheniramine maleate, cimetidine, aminophylline, cy, pmethylene ATP, nifedipine, capsaicin, tetraethylammonium chloride, sodium ascorbate, reduced glutathione, superoxide dismutase (bovine erythrocytes), and catalase (bovine liver) were purchased from Sigma Chemical. U-46619 ([ 15S]-hydroxy-llar,9a,[epoxymethano] prosta-5Z, 13E-dienoic acid) was
0363-6135/92 $2.00 Copyright 0 1992 the American Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 13, 2019.
H973
H974
ELECTRICAL
STIMULATION
CAUSES
+Endothelium -8
tPE
-5
- Endothelium
t
’
1
1Omin
W/E m
-I t PE -5
i
J t PE -5
Fig. 1. Influence of endothelium on responses of arterial rings to electrical field stimulation (EFS) and acetylcholine (ACh). Rings were submaximally precontracted with phenylephrine (PE). Indomethacin (10 PM) was present in the bathing medium. EFS was conducted at 4 Hz as indicated. ACh was added cumulatively at indicated final bath concentrations. W/E signifies washing 3 times followed by 30 min of equilibration. Tracings are representative of 18 rings from 4 to 5 animals. In each experiment responses varied by no more than 20% from those illustrated.
provided* by Upjohn and was dissolved and stored in absolute ethanol (10 mg/ml). Solutions of hygroscopic acetylcholine chloride were prepared in distilled water, divided into aliquots, and stored at -20°C. Amino acids were soluble and prepared fresh daily in distilled water. Oxyhemoglobin was prepared from hemoglobin as described previously (19). S-nitroso-Nacetylpenicillamine (SNAP) was prepared as described previously (24), and fresh aqueous solutions (1 mM) were prepared in ice-cold distilled water and discarded after 4 h because of chemical instability. NG-methyl+arginine acetate and iVGamino-L-arginine acetate were synthesized and purified as described previously (9, 11). The highly water-soluble, crystalline hydrochloride salt of NC-nitro-L-arginine was prepared by dissolving the free base in 3 N HCl, followed by rotary evaporation of the solvent. The residue was then subjected to a high vacuum until completely dry and stored in a desiccated container under nitrogen at -2OOC. Krebs bicarbonate solution consisted of (in mM) 118 NaCl, 4.7 KCl, 1.5 CaCIZ, 25 NaHC03, 1.2 MgSO,, 1.2 KH2POr, 11 glucose, 0.023 disodium EDTA. Preparation of arterial rings. Bovine lungs were obtained from an abattoir located nearby and transported to the laboratory. The second intrapulmonary arterial branch (4-5 mm outside diameter) extending into the larger lobe was rapidly isolated; cleaned of parenchyma, fat, and adhering connective tissue; and placed in cold, preoxygenated Krebs bicarbonate solution. Arterial segments were sliced into rings (4 mm wide) as described previously (17,21). Rings prepared in this manner possessedan intact endothelium as assessedpharmacologically by 80-100% relaxation in response to 0.1-l PM ACh and histologically after silver staining. In some arterial rings the endothelial layer was completely removed without appreciable damage to the underlying smooth muscle by gently inverting the rings inside out, rubbing the intimal surface lightly with moistened filter paper for 3 min, and returning the rings to
NITRIC
OXIDE
FORMATION
their original configuration. Endothelium-denuded arterial rings contracted in response to ACh. Recording of muscle tension. Arterial rings were mounted by means of fine nichrome wires in jacketed, 25ml-capacity, dropaway chambers (Metro Scientific) containing Krebs bicarbonate solution (37°C) gassed with 95% 02-5% CO* (17). The upper nichrome wire of each ring was attached to a force-displacement transducer (Grass FT03C), and changes in isometric force were recorded on a Grass polygraph (model 79D). Length-tension relationships were determined initially for unrubbed and rubbed arterial rings. Tension was adjusted to the optimal length for maximal isometric contractions to potassium by progressively stretching the rings and repeatedly obtaining contractile responses to 80 mM KC1 with 15 min of equilibration between each contractile response (21). The optimal tensions determined in these initial experiments were employed in all subsequent experiments and did not vary significantly as a function of intimal rubbing. Optimal tensions and maximal contractile tensions developed in response to KC1 at optimal lengths, respectively, were 6 and 20-24 g for arterial rings. Rings were routinely depolarized by addition of 120 mM KC1 following 2 h of equilibration at optimal tension, then washed and allowed to equilibrate for 45 min before initiating any given protocol (17, 21). This procedure increases and stabilizes any subsequent submaximal precontractile responses to phenylephrine, presumably by loading the smooth muscle cells with calcium. This procedure has been employed routinely for bovine pulmonary vessels in this laboratory. Submaximal precontractile responses to l-10 PM phenylephrine ranged from 60 to 80% of maximal contractions produced by 120 mM KCl. In the experiments conducted in the absence of extracellular calcium (see Fig. 6), precontractile tensions generated by a given concentration of phenylephrine was reduced by 20-30%. Therefore, the concentration of phenylephrine was increased (generally 3-. fold) to maintain precontractile contractions at 60-80% of maximum. EFS. EFS was conducted with the aid of two parallel platinum electrodes placed 4-5 mm apart, on either side of the rings, and connected to a current amplifier and stimulator (SD9 Grass) as described previously (18). EFS was conducted at 25 V at a frequency of 4 Hz in the form of square wave pulses of 0.2 ms duration and delivered as 20-s trains. Pulse durations in excess of 0.2 ms and trains in excess of 10 s were not used in this study to avoid the possibility of generating free radicals in solution. Rings were washed three times and allowed to equilibrate for 30 min after EFS to permit the rings to recover completely from the relaxant response to EFS. Less than 10% variability in magnitudes of relaxation elicited by EFS (4 Hz, 25 V) was observed for a given ring during a typical experiment. Determination of CGA@ levels. Determinations were made in arterial rings that had been equilibrated under tension and depolarized with KCl, as was done routinely for all ring experiments. Tone was monitored until the time of quick-freezing. The use of rapid drop-away bath chambers, quick-freezing, tissue extraction, and radioimmunoassay procedures were described previously (17). None of the test agents added to bath chambers interfered with the radioimmunoassay procedure. Recoveries of standard quantities of added cGMP, determined periodically, were 92-104%. Calculations and statistical analysis. Relaxation or contraction of arterial rings was measured as the decrease or increase, respectively, in tension below or above the elevated tension elicited by submaximal precontraction with phenylephrine. Values in Figs. 3-7 are expressed as means & SE. Comparisons were made using Duncan’s multiple range test, where comparisons with a common control were made (see Figs. 3-7). The level of statistically significant difference was P < 0.05.
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 13, 2019.
ELECTRICAL
STIMULATION
CAUSES
NITRIC
OXIDE
FORMATION
H975
25 VOLTS 4 Hz
2 Hz
-It
J
t PE -5
PE %I
-5
t
4 Hz 15 v
c
f
I
10 min
t
-5
1
2ov
25V
I
c
PE
Fig. 2. Voltage-dependent and frequency-dependent relaxation of arterial rings in response to EFS. Rings were submaximally precontracted with PE. Indomethacin (10 PM) was present in the bathing medium. EFS was conducted at various frequencies (2-16 Hz) at 25 V (top) or at various voltages (15-30 V) at 4 Hz (bottom). Strips were washed 3 times and allowed to equilibrate for 30 min between each EFS applied to precontracted rings. Tracings are representative of 12 rings from 3 animals. In each experiment responses varied by no more than 25% from those illustrated.
r
J t PE
-J t
-5
t PE
t PE -5
PE
-5
-5
RESULTS
Characteristics of EFS-elicited pulmonary laxation. EFS caused endothelium-dependent
arterial
re-
relaxation of precontracted rings of pulmonary artery (Fig. 1). The endothelium-dependent relaxant responses to EFS were compared with those caused by ACh. Although Fig. 1 illustrates the responses to 4 Hz at 25 V, EFS conducted at 2, 4,8, and 16 Hz (25 V) or at 15, 20, 25, and 30 V (4 Hz) caused relaxant responses that were completely endothelium dependent. EFS-elicited relaxation was both frequency dependent and voltage dependent (Fig. 2).
80
q
O.lpM
ACh
Arterial relaxation in response to EFS was relatively long lasting, and repeated EFS caused a gradual but reversible decline in contractile tone (Fig. 1). Washing the arterial ring preparations after each EFS was necessary to obtain highly reproducible magnitudes of relaxant responses to EFS (Fig. 2). EFS-elicited relaxation was completely resistant to blockade by either atropine or TTX, whereas atropine but not TTX blocked the relaxant response to ACh (Fig. 3). Moreover, EFS-elicited relaxation was unaffected by 5 PM guanethidine, 10 PM propranolol, 10 PM indomethacin, 10 PM chlorpheniramine, 10 PM cimetidine, 100 PM aminophylline, 10 PM cy, ,&methylene ATP, 1 PM nifedipine, 1 PM capsaicin, 1 mM sodium ascorbate, 50 PM reduced glutathione, 100 units/ml of superoxide dismutase, or 300 units/ml of catalase (data not shown; 3-4 experiments, each using 4-6 arterial rings for each test condition, were conducted). Inhibition of EFS-elicited pulmonary arterial relaxation by chemical agents that interfere with the actions or synthesis of NO. Relaxant responses to EFS were mark-
Control
+At ropine hJM)
+Tet rodotox (1PW
Fig. 3. Effects of atropine and tetrodotoxin (TTX) on responses of arterial rings to EFS and ACh. Rings were submaximally precontracted with PE. Responses to EFS (4 Hz) and ACh were obtained at ,peak contractile responses to PE. Atropine and TTX were added to bathing media 30 min before precontraction. Values are means t SE using 24 rings (for each test condition) from 4 animals (6 rings/animal). Values for ACh in the presence of atropine are significantly different (P < 0.001) from values for ACh alone (control
edly inhibited by hemoglobin and methylene blue (Fig. 4). Similarly, by using the same arterial ring preparations, the relaxant responses to ACh and SNAP were markedly inhibited by hemoglobin and methylene blue (Fig. 4). Three NG-substituted analogues of L-arginine were tested for their effects on EFS-elicited relaxation. NG-methyl-L-arginine (100 PM), NG-amino+arginine (100PM), and NG-nitro-L-arginine (10PM) caused comparably marked inhibitory effects on EFS-elicited relaxation (Fig. 5). NG-nitro-L-arginine was the most potent of the three L-arginine analogues tested. Addition of 300 PM L-arginine, but not its enantiomer D-arginine, to the bathing medium containing one of the three inhibitory
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 13, 2019.
H976
ELECTRICAL
STIMULATION
CAUSES
NITRIC
.
100
- Controls
80
80 .-: zn60 Q 8
60
, T
+~DM
/
Hemoglobin
OXIDE
.
FORMATION
cl
No Arginine
El
0.3mM
Ea
0.3mM +NMA,
L- Arginine D-Arginine NAA
or NNA
E40 3 at 20
0 +NAA +NNA +NMA (0.1 mM) (0.1 mM) (0.01 mM) Fig. 5. Effects of NO synthase inhibitors in the absence and presence of L-arginine or D-arginine on responses of arterial rings to EFS. Rings were submaximally precontracted with PE. EFS was conducted at 4 Hz. NG-methyl-L-arginine (NMA), NC-amino-L-arginine (NAA), and NC-nitro-L-arginine (NNA) were added to bathing media at peak contractile responses to PE, and EFS was conducted 10 min later. Rings were washed 3 times, allowed to equilibrate for 30 min, precontracted with PE, and L-arginine or D-arginine was added, followed within 1 min by addition of NMA, NAA, or NNA. EFS was conducted 10 min later. Values are means t SE using 16-24 rings (for each test condition) for 4-6 animals (4 rings/animal). Values obtained in the presence of NMA, NAA, or NNA alone or with D-arginine are significantly different (P < 0.001) from values for control or NO synthase inhibitors with L-arginine. Control
0 +lflM
Mmthylene
Blue
40 20 0 -7 -6 -8 -7 -6 ACh SNAP Fig. 4. Influence of hemoglobin and methylene blue on responses of arterial rings to EFS, ACh, and S-nitroso-N-acetylpenicillamine (SNAP). Rings were submaximally precontracted with PE. Responses to EFS (4 Hz), ACh, and SNAP were obtained at peak contractile responses to PE. Concentrations of ACh and SNAP represent final bath concentrations. Hemoglobin and methylene blue were added to bathing media 5 and 30 min, respectively, prior to precontraction. Values are means k SE using 18-24 rings (for each test condition) from 3 to 4 animals (6 rings/animal). All values obtained in the presence of hemoglobin or methylene blue are significantly different (P < 0.01) from values for corresponding controls. 4Hz
-8
L-arginine analogues nearly completely restored arterial relaxation in response to EFS (Fig. 5). Dependence of EFS-elicited relaxation of pulmonary artery on extraceZZuZar calcium. Replacement of the nor-
mal Krebs-bicarbonate bathing solution with calciumfree solution resulted in near abolition of relaxation in response to either EFS or ACh (Fig. 6). Replenishment of the bathing solution with calcium and further tissue incubation for 45 min restored by -75% the capacity of EFS and ACh to cause relaxation. Trifluoperazine, a well-known calmodulin antagonist, nearly abolished arterial relaxation in response to EFS. Tetraethylammonium chloride (10 PM-3 mM) and 30 mM KC1 caused 70-80% (range) inhibition of EFS-elicited relaxation (data not shown; 3 experiments each using 4-6 arterial rings for each test condition were conducted). cGMP formation in response to EFS of pulmonary artery and inhibition by NG-amino-L-arginine. EFS (4
Hz; 25 V) caused a five- to sixfold increase in arterial levels of cGMP that accompanied the smooth muscle relaxation (Fig. 7). Both relaxation and cGMP accumulation were inhibited by 10 PM NG-amino-L-arginine.
DISCUSSION
Frank and Bevan (8) first reported that isolated rings of pulmonary artery from rabbits, cats, and monkeys relaxed in response to EFS in an endothelium-dependent manner. The precise mechanism, however, was not elucidated because the study was conducted soon after the discovery of EDRF (10).The data from the present study indicate that EFS causes relaxation of isolated rings of bovine pulmonary artery by mechanisms involving the L-arginine-NO-cGMP pathway. The evidence for this conclusion is 1) the relaxation is endothelium dependent and is accompanied by tissue accumulation of cGMP, 2) relaxation is antagonized by oxyhemoglobin and methylene blue, chemical agents that inhibit the actions of NO on smooth muscle, and 3) relaxation is antagonized by NG-substituted analogues of L-arginine by mechanisms that are reversed in an enantiomerically specific manner by excess L-arginine. The observations that EFS- and ACh-elicited relaxation are dependent on extracellular calcium and intracellular calmodulin are consistent with the above hypothesis that endotheliumderived NO is largely responsible for EFS-elicited relaxation of bovine pulmonary artery. As reported previously for pulmonary artery from rabbits, cats, and monkeys (8), EFS-elicited relaxation of bovine pulmonary artery was completely resistant to alteration by a wide range of pharmacological agents that are well known to interfere with chemical neurotransmission and prostaglandin formation. Although guanethidine and atropine failed to influence EFS-elicited relax-
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 13, 2019.
ELECTRICAL
STIMULATION
CAUSES
NITRIC
OXIDE
FORMATION
H977
endothelium dependent and there is no evidence for the direct innervation of vascular endothelial cells. Thus the present observations strengthen the view that EFS-elic80 ited relaxation of pulmonary artery does not involve E neuronal transmitter release (8). Free radical generation in oxygenated salt solution in response to EFS does not appear to be involved in the mechanism by which EFS causes relaxation of pulmonary artery in the present study. The evidence for this conclusion is that neither antioxidants nor oxygen-derived radical scavengers added to tissue baths altered the relaxation responses. Moreover, no evolution of gas bubbles, such as chlorine gas, was ever observed at the 0 +Ca2+ Control +TFP -Ca2+ electrode surface at any time during or after EFS, as has (3uM) been reported in certain studies by others (12, 30, 37). Fig. 6. Influence of extracellular calcium and trifluoperazine on re- Although relaxant responses to EFS were relatively well sponses of arterial rings to EFS and ACh. Rings were submaximally maintained in pulmonary artery compared with blood precontracted with PE. Responses to EFS (4 Hz) and ACh were vessel preparations where relaxation is neurogenically obtained at peak contractile responses to PE. -Ca2’ signifies replacemediated (1, 31, 32), phenylephrine-induced precontracment of normal Krebs bicarbonate solution with calcium-free Krebs bicarbonate solution, followed by 30 min of equilibration and precontile tone consistently returned after washing the prepatraction with PE. +Ca2’ signifies replacement of calcium-free Krebs rations. Under these conditions, the magnitudes of relaxbicarbonate solution with normal Krebs bicarbonate solution, followed ation elicited by EFS at 4 Hz were highly reproducible by 30 min of equilibration and precontraction with PE. Trifluoperazine for a given arterial ring for the duration of the experi(TFP) was added to bathing media 30 min before precontraction. Values are means & SE using 18 rings (for each test condition) from 3 ment. These observations argue against involvement of animals (6 rings/animal). Values obtained for -Ca2’ and in the presfree radicals in the relaxant response, as no functional ence of TFP are significantly different (P < 0.001) from corresponding vascular smooth muscle damage was evident during the control values. Values for +Ca2’ in the presence of ACh are significourse of any experiment. Finally, the electrical stimucantly different (P < 0.05) from corresponding ACh control values. lation parameters employed, such as square wave pulses of very short duration (0.2 ms) and short stimulation 100 200 q Rdaxation trains of 20 s, are not conducive to the generation of free Cyclic GMP fz radicals in aqueous solution (6). Prolonging the electrical t 160 80 stimulation to 2-ms pulses delivered as 20-min trains, yo c) however, can produce secondary relaxant responses that E are attributed to free radical generation (6). 120 G) z Although the present observations indicate clearly that Q EFS stimulates the formation and/or release of endothe80 $ 0 hum-derived NO in pulmonary artery, studies were not ‘( focused on the precise mechanism by which the electric to 40 current is coupled or gated to the L-arginine-NO pathway. Nevertheless, several points are noteworthy. At least one of the constitutive, cytosolic isoforms of mam0 malian NO synthase in vascular endothelial cells requires Control +NAA (0.01 mM) both calcium and calmodulin for catalytic activity (7). Fig. 7. Alteration of guanosine 3’,5’-cyclic monophosphate (cGMP) The dependence of EFS-elicited relaxation on extracellevels and tone of arterial rings by EFS and the influence of NC-aminolular calcium, together with the inhibitory action of L-arginine (NAA). Rings were submaximally precontracted with PE. trifluoperazine, tetraethylammonium chloride, and eleEFS was conducted at 4 Hz. When tested, NAA was added to bathing suggest that media at peak contractile responses to PE, and EFS was conducted 10 vated extracellular potassium concentration, min later. Rings were quick-frozen 60 s after EFS or at peak contractile some type of potential-dependent calcium channels on responses to PE (basal). Values are means * SE using 12 rings (for the endothelial cells may be involved, and that calcium each test condition) from 3 animals (4 rings/animal). Control values influx triggers NO formation via activation of calciumare significantly different (P c 0.001) from basal values. Values obcalmodulin-dependent NO synthase. For example, as tained in the presence of NAA are significantly different (P < 0.001) reported previously by Luckhoff and Busse (33, 34), for from corresponding control values. endothelial cells in response to certain agonists, EFS may initially mobilize intracellular calcium, thereby ation, nonadrenergic-noncholinergic neurotransmission opening calcium-activated potassium channels, which was not likely involved because relaxation was TTX insensitive. TTX-resistant responses do not necessarily leads to hyperpolarization. Endothelial cell hyperpolarirule out the possibility of a neurogenic mechanism be- zation would augment the driving force for potentialdependent transmembrane calcium influx, and thereby cause EFS can promote neurotransmitter release from provide the signal for NO synthesis. In this manner, the nerve terminal varicosities in the absence of propagated action potentials (27, 28). The involvement of nerve relationship between transmembrane calcium influx and terminals in EFS-elicited relaxation of pulmonary artery the membrane potential in endothelial cells may be opposite to that characteristic of excitable vascular smooth is unlikely, however, because relaxation is completely
1
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 13, 2019.
H978
ELECTRICAL
STIMULATION
CAUSES
muscle cells (3, 33). Similarly, EFS caused relaxation of rat tail artery by mechanisms that appear to involve potassium-mediated membrane hyperpolarization and calcium influx, as this was inhibited by elevating the extracellular potassium concentration to 20-30 mM or by addition of 1 mM tetraethylammonium chloride (29). Regardless of the precise mechanisms involved, the physiological or pathophysiological significance of electrically induced, nonneurogenically mediated, endothelium- and calcium-dependent relaxation of pulmonary artery still remains to be determined. We thank Dr. Jon M. Fukuto for synthesizing the NG-substituted analogues of L-arginine and Diane Rome Peebles for preparing the illustrations. This investigation was supported in part by National Heart, Lung, and Blood Institute Grants HL-35014 and HL-40922, a grant from the Laubisch Fund for Cardiovascular Research, and the Tobacco-Related Disease Research Program. Address for reprint requests: L. J. Ignarro, Dept. of Pharmacology, UCLA School of Medicine, CHS, Los Angeles, CA 90024. Received 22 July 1991; accepted in final form 26 November 1991. REFERENCES Bevan, J. A., G. M. Buga, A. Snowden, and S. I. Said. Is the neural vasodilator mechanism to cerebral and extracerebral arteries the same? In: Cerebral Blood Flow: Effect of Nerves and Neurotransmitters, edited by M. L. Marcus and D. D. Heistad. New York: Elsevier, 1982, p. 421-439. 2. Bowman, A., and J. S. Gillespie. Block of some non-adrenergic inhibitory responses of smooth muscle by a substance from haemolyzed erythrocytes. J. Physiol. Lond. 328: 11-25, 1982. 3. Busse, R., H. Fichtner, A. Luckhoff, and M. Kohlhardt. Hyperpolarization and increased free calcium in acetylcholinestimulated endothelial cells. Am. J. Physiol. 255 (Heart Circ. Physiol. 24): H965-H969, 1988. 4. Cohen, R. A., J. T. Shepherd, and P. M. Vanhoutte. Prejunctional and postjunctional actions of endogenous norepinephrine at the sympathetic neuroeffector junction in canine coronary arteries. 1.
Circ. 5.
Res. 52: 16-25,
1983.
Edwards, J. C., L. J. Ignarro, K. S. Wood, A. L. Hyman, and P. J. Kadowitz. Relaxation of intrapulmonary artery and vein by nitrogen oxide-containing vasodilators and cyclic GMP. J. Pharmacol.
Exp.
Ther.
228: 33-42,1984.
Feletou, M., and P. M. Vanhoutte. Relaxation of canine coronary artery to electrical stimulation: limited role of free radicals. Am. J. Physiol. 253 (Heart Circ. Physiol. 22): H884-H889, 1987. 7. Forstermann, U., J. S. Pollock, H. H. H. W. Schmidt, M. Heller, and F. Murad. Calmodulin-dependent endothelium-derived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells. 6.
Proc. 8.
9.
10.
11.
12.
Natl.
Acad.
Sci.
USA
88: 1788-1792,
1991.
Frank, G. W., and J. A. Bevan. Electrical stimulation causes endothelium-dependent relaxation in lung vessels. Am. J. Physiol. 244 (Heart Circ. Physiol. 13): H793-H798, 1983. Fukuto, J. M., K. S. Wood, R. E. Byrns, and L. J. Ignarro. NG-amino-L-arginine: a new potent antagonist of L-arginine-mediated endothelium-dependent relaxation. Biochem. Biophys. Res. Commun. 168: 458-465,199O. Furchgott, R. F., and J. V. Zawadzki. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature Lond. 288: 373-376, 1980. Gold, M. E., K. S. Wood, R. E. Byrns, J. M. Fukuto, and L. J. Ignarro. NG-methyl+arginine causes endothelium-dependent contraction and inhibition of cyclic GMP formation in artery and vein. Proc. Natl. Acad. Sci. USA 87: 4430-4434, 1990. Hardebo, J. E., J. Kahrstrom, and C. Owman. Characterization of dilatation induced by electrical field stimulation in mammalian cerebral and peripheral vessels. Q. J. Exp. Physiol. 74: 475491,1989.
13.
Hyman,
D. S. Knight,
and P. J.
OXIDE
FORMATION
Kadowitz. Pulmonary vasodilator responses to catecholamines and sympathetic nerve stimulation in the cat. Circ. Res. 48: 407415,1981.
14 Ignarro,
L. J., G. M. Buga, R. E. Byrns, K. S. Wood, and G. Chaudhuri. Endothelium-derived relaxing factor and nitric oxide possess identical pharmacologic properties as relaxants of bovine arterial and venous smooth muscle. J. Pharmacol. Exp. Ther. 246: 218-226,1988. 15 Ignarro, L. J., G. M. Buga, and G. Chaudhuri. EDRF gener’ ation and release from perfused bovine pulmonary artery and vein. l
Eur.
J. Pharmacol.
149: 79-88,1988.
16. Ignarro,
L. J., G. M. Buga, K. S. Wood, R. E. Byrns, and G. Chaudhuri. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc. Natl. Acad. Sci. USA
84: 9265-9269,1987.
Exp.
Ther.
17 Ignarro, L. J., T. Burke, K. S. Wood, and P. J. Kadowitz. ’ Association between cyclic GMP accumulation and acetylcholineelicited relaxation of bovine intrapulmonary artery. J. Pharmacol. 228: 682-690,1984.
18 Ignarro, L. J., P. A. Bush, G. M. Buga, K. S. Wood, J. M. ’ Fukuto, and J. Rajfer. Nitric oxide and cyclic GMP formation upon electrical stimulation cause relaxation of corpus cavernosum smooth muscle. B&hem. Biophys. Res. Commun. 170: 843-850, 1990.
L. J., R. E. Byrns, G. M. Buga, and K. S. Wood. 19. Ignarro, Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacological and chemical properties that are identical to those for nitric oxide radical. Circ. Res. 61: 866-879, 1987. 2. Ignarro, L. J., R. E. Byrns, G. M. Buga, K. S. Wood, and G. Chaudhuri. Pharmacological evidence that endothelium-derived relaxing factor is nitric oxide: use of pyrogallol and superoxide dismutase to study endothelium-dependent and nitric oxide-elicited vascular smooth muscle relaxation. J. Pharmacol. Exp. Ther. l
244: 181-190,1988. 21.
Ignarro, L. J., R. E. Byrns, and K. S. Wood. Endotheliumdependent modulation of cyclic GMP levels and intrinsic smooth muscle tone in isolated bovine intrapulmonary artery and vein. Circ.
Res. 60: 82-92,
1987.
Ignarro, L. J., R. G. Harbison, K. S. Wood, and P. J. Kadowitz. Activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor from intrapulmonary artery and vein: stimulation by acetylcholine, bradykinin and arachidonic acid. J. Pharmacol. Exp. Ther. 237: 893-900, 1986. 23* Ignarro, L. J., R. G. Harbison, K. S. Wood, M. S. Wolin, D. B. McNamara, A. L. Hyman, and P. J. Kadowitz. Differences in responsiveness of intrapulmonary artery and vein to arachidonic acid: mechanism of arterial relaxation involves cyclic GMP and cyclic AMP. J. Pharmacol. Exp. Ther. 233: 560-569,1985. L. J., H. Lippton, J. C. Edwards, W. H. Baricos, 24* Ignarro, A. L. Hyman, P. J. Kadowitz, and C. A. Gruetter. Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement of SExp. Ther. 218: nitrosothiols as active intermediates. J. Pharmacol.
22.
739-749,198l. 25.
Kadowitz, P. J., P. Nandiwada, C. A. Gruetter, L. J. Ignarro, and A. L. Hyman. Pulmonary vasodilator responses to nitroprusside and nitroglycerin in the dog. J. Clin. Invest. 67: 893902,198l.
26. Kalsner, S., and M. Quillan. Nonneurogenic relaxation to field stimulation in coronary arteries. J. Pharmacol. Exp. Ther. 250: 461-469,1989. 27. Katz, B., and R. Miledi. Spontaneous and evoked activity of motor nerve endings in calcium ringer. J. Physiol. Lond. 203: 689706,1969.
Katz, B., and R. Miledi. Further study of the role of calcium in synaptic transmission. J. Physiol. Lond. 207: 789-801, 1970. 29. Kotecha, N., and T. 0. Neild. Relaxation and hyperpolarization of the smooth muscle of the rat tail artery following electrical stimulation. J. Physiol. Land. 397: 489-501, 1988. 30. Lamb, E. S., and R. C. Webb. Vascular effects of free radicals generated by electrical stimulation. Am. J. Physiol. 257 (Heart Circ.
28.
Physiol. 31.
A. L., P. Nandiwada,
NITRIC
26): H709-H714,
1984.
Lee, T. J. F., L. R. Kinkeas, and S. Sarwinski. Norepinephrine and acetvlcholine transmitter mechanisms in large cerebral
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 13, 2019.
ELECTRICAL
STIMULATION
CAUSES
arteries of the pig. J. Cereb. Blood Flow Metab. 2: 439-450, 1982. 32. Lee, T. J. F., C. Su, and J. A. Bevan. Nonsympathetic dilator innervation of cat cerebral arteries. Experientia Base1 31: 14241425, 1975. 33. Liickhoff, A., and R. Busse. Activators of potassium channels enhance calcium influx into endothelial cells as a consequence of potassium currents. Naunyn-Schmiedeberg’s Arch. Pharmacol. 342: 94-99,199o. 34. Liickhoff, A., and R. Busse. Calcium influx into endothelial cells and formation of endothelium-derived relaxing factor is controlled by the membrane potential. Pjluegers Arch. 416: 305-311, 1990. 35. Mackenzie, I., and G. Burnstock. Evidence against vasoactive intestinal polypeptide being the non-adrenergic non-cholinergic inhibitory transmitter released from nerves supplying the smooth
NITRIC
36.
37.
38.
39.
OXIDE
FORMATION
H979
muscle of the guinea-pig taenia coli. Eur. J. PharmacoZ. 67: 255264, 1980. Rooke, T., R. A. Cohen, T. J. Verbeuren, and P. M. Vanhoutte. Nonneurogenic inhibitory effect of electrical impulses in isolated canine coronary arteries. Eur. J. PharmacoZ. 80: 251-254, 1982. Rubanyi, G. M., and P. M. Vanhoutte. Oxygen-derived free radicals, endothelium and responsiveness of vascular smooth muscle. Am. J. Physiol. 250 (Heart Circ. Physiol. 19): H815-H821,1986. Toda, N. Response of isolated monkey coronary arteries to catecholamines and to transmural electrical stimulation. Circ. Res. 49: 1228-1236, 1981. Weir, E. K. Does normoxic pulmonary vasodilation rather than hypoxic vasoconstriction account for the pulmonary pressor response to hypoxia? Lancet 1: 476-477, 1978.
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 13, 2019.
