Almitrine mimics hypoxic vasoconstriction in isolated rat lungs E. BRIGITTE GOTTSCHALL, STEPHANIE FERNYAK, GEBHARD WUERTEMBERGER, AND NORBERT F. VOELKEL Cardiovascular Pulmonary Research Laboratory, University of Colorado Health Sciences Center, Denver, Colorado 80262; and Division of Pneumology, Department of Internal Medicine, University Hospital, Freiburg, Federal Republic of Germany Gottschall, E. Brigitte, Stephanie Fernyak, Gebhard Wuertemberger, and Norbert F. Voelkel. Almitrine mimics hypoxic vasoconstriction in isolated rat lungs. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H383-H391, 1992.-The effect of almitrine bimesylate or the solvent malic acid on pulmonary vascular perfusion pressure was assessed in isolated rat lungs and on the contractile behavior of rat aorta and main pulmonary artery rings. Addition of almitrine to the lung perfusate during normoxia caused a dose-dependent, transient increase in pulmonary artery pressure with no change of the lung microvascular pressure. In systemic or pulmonary conduit arteries, the contractile tension was unaffected by almitrine. This indicates a precapillary locus of drug action. We also examined almitrine’s effect on hypoxic pulmonary vasoconstriction (HPVC) in isolated lungs perfused with blood or with physiological salt solution (PSS). Low-dose almitrine potentiated hypoxic vasoconstriction in blood- but not in PSS-perfused lungs. However, a high dose of almitrine reduced hypoxic vasoconstriction dose dependently. When almitrine was added to the lung perfusate during hypoxiaor cyanide-induced (NaCN, 5 x 10e5 M) pulmonary vasoconstriction, almitrine caused no further vasoconstriction. However, when the pulmonary perfusion pressure was elevated by KC1 (20 mM) to the same magnitude as by alveolar hypoxia or cyanide, almitrine elicited a pressor response comparable to that observed during normoxia. Almitrine-induced pulmonary vasoconstriction resembled hypoxic vasoconstriction in that agents known to enhance hypoxic vasoconstriction (phorbol myristate acetate, vanadate, and 4aminopyridine) enhanced, and known inhibitors of HPVC (the Ca2+ entry blocker nifedipine and hypothermia) inhibited, the almitrine-induced vasoconstriction. These findings lead us to speculate that almitrine also affects the oxygen-sensing limb of the hypoxic pressor response, not simply the effector (contractile apparatus of the vascular muscle cell). hypoxic pulmonary vasoconstriction; oxygen sensing; vanadate; 4-aminopyridine
almitrine
bimesylate;
HYPOXIC pulmonary vasoconstriction occurs in all species so far investigated (10, 34,39), as well as in humans (25). However, the mechanism of this response, which primarily targets precapillary pulmonary vessels (14)) has been elusive. It is not known which cell type(s) provides the oxygen sensor, although it is clear that the contractile response occurs mainly in the smooth muscle cells (14, 16). Analogous to electrical circuits, a conceptual model of the hypoxic pressor response consists of a sensor or a receptor, a transducer, and an effector (43). Previously, a large number of investigations have been concerned with modulation of hypoxic vasoconstriction; a redundant system of amplifiers and dampers of this response has been described (34). Because it is possible that sensor and effector are linked, it is difficult to study the sensor or the effector separately. Thus an increase or decrease in the magnitude of the hypoxic pressure re0363-6135/92
$2.00 Copyright
sponse is likely to occur via a combined effect on sensor and effector (e.g., if both are calcium dependent), and, indeed, uncoupling of the sensor from the contractile effector has not been accomplished. In recent years, almitrine has been studied extensively in patients with chronic bronchitis (30, 46) because of its effect on the arterial PO, and has been used as an experimental tool to study chemoreceptor responses (13, 17). Ishii et al. (13) recorded increased oxygen-sensitive activity from the branchiocardiac vein of the crayfish after almitrine administration. In this study, both cyanide (NaCN) and almitrine mimicked hypoxia. Using various animal models and experimental procedures, several investigators reported that almitrine has an effect on the pulmonary circulation; however, controversy exists as to whether almitrine potentiates hypoxic vasoconstriction (1, 4, 5, 12, 15, 19, 27, 35). For example, in our own study, we found that almitrine caused pulmonary vasoconstriction in anesthetized, spontaneously breathing dogs but did not potentiate hypoxic vasoconstriction. However, in isolated rat lungs perfused with blood, Falus et al. (8) showed a potentiation of hypoxic vasoconstriction by a low dose of almitrine. Almitrine has been shown to affect energy metabolism and in this respect may be related to the hypoxia mimic cyanide, mentioned above. In liver mitochondria, almitrine inhibits electron transfer at the cytochrome bc complex of the ubiquinoneor proton-motive Q-cycle (26). In yeast cells, almitrine appeared to block the multisubunit mitochondrial FoFl ATP synthase (33). Thus almitrine joins the group of compounds that block mitochondrial electron transport and cause pulmonary vasoconstriction (36). Whereas cyanide blocks the cytochrome-c oxidase complex by binding to its heme Fe atom, almitrine may block ATP synthesis (like dinitrophenol) or impair electron transport downstream from the rotenone blocking site that is localized in the NADH-cytochrome-Q reductase complex. We wished to examine whether almitrine mimics hypoxia in its effects on the pulmonary circulation. If almitrine and hypoxia were indeed similar in their behavior in the lung circulation, then one would postulate that 1) almitrine causes pulmonary vasoconstriction, 2) almitrine potentiates or enhances hypoxic vasoconstriction (if hypoxia and almitrine use, at least in part, the same pathway), 3) agents that enhance hypoxic vasoconstriction also enhance almitrine-induced pulmonary vasoconstriction, and 4) agents that inhibit hypoxic pulmonary vasoconstriction (HPVC) preferentially also inhibit almitrine-induced vasoconstriction. Furthermore, if almitrine is a hypoxia mimic, one could postulate that
0 1992 the American
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the principal site of action for the drug was precapillary, i.e., the drug was not predominantly acting as a pulmonary venoconstrictor, and, finally, that almitrine had little or no effect on systemic vessels or the contractile machinery of large pulmonary conduit arteries. METHODS
We used male Sprague-Dawley rats (Harlan Industries, Indianapolis, IN) weighing 300-400 g, which were housed in the Animal Care Facility of the University of Colorado and had free access to food and water. Unless otherwise stated, all reagents were obtained from Sigma Chemical (St. Louis, MO). Isolated
Perfused
Rat Lung
Studies
Rat lungs were isolated as previously described (3). Briefly, a tracheal cannula was introduced, and the rats were ventilated with a Harvard small animal respirator at 55 breaths/min, initially using a gas mixture of 21% O,- 5% CO,-74% N2. A median sternotomy was performed, and after injection of 100 U of heparin into the right ventricle, the main pulmonary artery and the left ventricle were cannulated. The lungs and heart were removed from the chest and suspended in a warm, humid chamber. The lungs were perfused at a constant flow (0.03 ml/g body wt/min) with either heparinized homologous blood (taken from 3 ether-anesthetized rats) or with a physiological salt solution (PSS) containing (in mM) 119 NaCl, 4.7 KCl, 5.5 glucose, 1.17 MgS04, 22.6 NaHCO,, 1.18 KH2P04, and 3.2 CaCl,, as well as 4 vol/lO ml Ficoll (mol wt 70,000). When perfused with the PSS, the first 50 ml of perfusate containing residual blood were discarded so that the lungs were perfused with 30 ml recirculating, cell- and protein-free salt solution. In general, after an equili-
VASOCONSTRICTION
bration period of 20 min at 38”C, the vascular reactivity of each lung was tested with three pairs of a bolus injection of angiotensin II (ANG II, 0.1 pg/O.l ml) and 5 min of airway hypoxia [PSS-perfused lungs fractional concentration of 0, in inspired gas (FI,~), 0.00; blood-perfused lungs FI, , 0.031 separated by a 5-min period of normoxic ventilation (F1&21 0.21). Different inspired fractions of 0, were chosen because an FIN, of 0.03 in rat lungs perfused with blood produces a maximal pressor response, whereas an FI,~ of 0.00 is required to produce a maximal response in PSS-perfused lungs. The experimental period in each lung started after the third hypoxic challenge (Fig. I). ANG II may act through a different mechanism than hypoxia (23) and was therefore chosen as a nonhypoxic vasoconstricting stimulus to test vascular reactivity in each isolated lung. This also allowed us to use the ANG II pressor response as a reference response during the experimental period of each study. The pulmonary perfusion pressure was monitored continuously with a Statham P23 AA transducer and recorded on a Soltec recorder. In five blood-perfused lungs, the effect of IO pug almitrine on the microvascular pressure was measured using the well-established double-occlusion technique. Briefly, both inflow and outflow tubing were occluded simultaneously, causing arterial and venous pressures to equilibrate at the capillary pressure (7, 37, 40). Specific
Protocols
Effect of almitrine on pulmonary vascular pressure. In six lungs perfused with blood, vascular reactivity was tested with ANG II and hypoxia as described above, and then a single dose of almitrine (IO pug in perfusate reservoir) was added to observe its effects on pulmonary perfusion pressure. In all subsequent
4 -AMINO-PYRIDINE PMA VANADATE
COOLING NIFEDIPINE (or SOL.)
A
PAP = 7 00% PAP
[mmHC!Il 10
ANGII
HYPOXIA
02
4 &&/I
HYPOXIA
21% 02
ALMIhNE ‘Ogl
Fig. 1. Schematic time course of the experimental protocol. After a standard equilibration period and 3 pairs of ANG II (0.1 pg) and hypoxia challenges [fractional concentration of 0, in inspired gas (FI,J 0.00 physiological salt solution lungs], almitrine was added to perfusate reservoir. Schematic begins with (PSS) -perfused lungs; FI o2 0.03 blood-perfused second ANG II response. Large arrows, time points when compounds (as shown) were added to perfusate reservoir or perfusate temperature was changed. Third hypoxic pressor response was designated value 100%. APAP, change in mean pulmonary artery perfusion pressure. SOL, solvent; PMA, phorbol myristate acetate.
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experiments, lungs perfused with PSS were used. In isolated rat lungs perfused with PSS, a dose-response relationship between the amount of almitrine added to the reservoir and the rise in pulmonary perfusion pressure was established. Again, pulmonary vascular reactivity was tested with ANG II and hypoxia, as above, before almitrine was added to the perfusate reservoir and the rise in perfusion pressure was recorded. Four different doses of almitrine were tested in PSSperfused lungs: 5 pg (0.17 pg/ml, n = 5), 10 pug (0.33 pg/ml, n = 5), 20 pug(0.66 pg/ml, n = 5), and 40 pug(1.33 pg/ml, n = 5). However, only one particular dose was tested in any given lung. Five lungs were treated with the almitrine solvent, malic acid, at a final perfusate concentration of 0.01%. This concentration corresponds to the amount of malic acid added with the highest dose of almitrine given (40 pg) in this series of experiments. Effect of almitrine on hypoxic vasoconstriction. In six bloodperfused and six PSS-perfused lungs, we deviated from the procedure described above. These experiments were designed to examine whether low-dose almitrine enhanced hypoxic vasoconstriction. Therefore a submaximal hypoxic challenge was used. We wished to reproduce the data by Falus et al. (8), which demonstrated a potentiation of hypoxic vasoconstriction in rat lungs by a low dose of almitrine. To this end, in blood-perfused lungs we applied one ANG II challenge followed by a hypoxia challenge (FI,~ 0.03). The lungs were then challenged with two hypoxic challenges (FI, 0.05) separated by IO min of normoxia. Then either malic acid ‘
TIME (min)
Fig. 5. PAP tracings from representative experiments in isolated rat lungs perfused with PSS. A: almitrine (10 pg) added to reservoir during plateau phase of a hypoxic challenge had no vasoconstricting effect. B: at peak of a pressor response induced by a higher dose of almitrine (20 pg), a hypoxic challenge (FIN, 0.00) caused no further vasoconstriction. C: hypoxia-induced vasoconstriction was partially reduced when hypoxic (FIN, 0.00) challenge was started at peak of a pressor response due to 10 pg almitrine.
3otA
I PAP bmHg1
ALMITRINE/-
01 p ALMITRJNE
Pilot experiments indicated that hypoxia- and almitrine-induced vasoconstriction were both inhibited by nifedipine; subsequently, a dose of nifedipine (5 x 10e6 M) was found (43) that inhibited both hypoxia- and almitrine-induced vasoconstriction preferentially over ANG II-induced vasoconstriction (Fig. 8). A decrease in the perfusate temperature to 23°C led to similar findings, i.e., the hypoxia- and almitrineinduced pressure responses were slowed and blunted, whereas the ANG II response was nearly unchanged (Fig. 8). Thus the Ca2+ entry blocker and hypothermia had comparable effects on hypoxia- and almitrine-induced vasoconstriction. Tension
Development
in Isolated
Vessel Rings
Because we had established that almitrine caused precapillary vasoconstriction, we wished to examine whether almitrine contracted conduit arteries. Addition of almitrine to the bath fluid of isolated rat main pulmonary artery and aorta rings, in doses ranging from 0.5 to 30 pg, demonstrated that all of the rings from systemic (data not shown) and pulmonary conduit arteries were unresponsive to almitrine (Fig. 9). We concluded that these rings did not respond to almitrine after proper vascular reactivity had been established [A tension in grams: pulmonary artery 80 mM KCl-induced contraction = 0.88 t 0.06; phenylephrine (5 x lo-’ M)-induced contraction = 0.27 t 0.04; ACh (lo+ M) -in duced re 1axa t’ion = 0.2 t 0.02; almitrine 0.5-30 pg = 01. l
0
% ’
z
400 -
0
.
Fig. 6. A: almitrine added to reservoir at peak of a KCl-induced pressor response elicits additional vasoconstriction. B: almitrine (10 pg) added to perfusate reservoir at peak of a cyanide-induced pressor response leads to no additional vasoconstriction.
blocker 4-AP and vanadate, which are structurally unrelated and nonselective in enhancing the hypoxic pressor response, both increased pulmonary perfusion pressure by a small amount and enhanced both HPVC (APAP: 4-AP 363.56 t 16.85%; vanadate 327 t 45.9% of preceding hypoxic response) and almitrine-induced (10 pg) vasoconstriction (APAP: 4-AP 293.52 t 52.95%; vanadate 347.85 t 79.62% of preceding hypoxic response), whereas PMA enhanced both the almitrine pressor response
xw300 e ZO i&200
0
0%02
lopg group) group)
-
z:k! E IOO6 o\o
ALMITRINE (n=4,each (n=4,each
:
TIME (min)
n
r.l e cl II -----
0 .-CONTROL
PMA (1 O-8M)
4-AMINOPYRIDINE (5x1 O-3M)
VANADATE (3x1 O-5M)
Fig. 7. Effect of PMA, 4-aminopyridine, and vanadate on almitrineinduced vasoconstriction [expressed in % of (2nd) preceding hypoxic response in each lung]. All 3 compounds greatly enhanced almitrineinduced pressor response and hypoxic pressor response. Data are means t SE.
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ALMITRINE 0
100 ii 2 0 n 2 a (3 g 5 B
NIFEDIPINE SOLVENT
q 8o
I
60 40 20
0
I
hI ANG II
0%02
ALMITRINE TEMP
38°C
(n=4)
n
TEMP
23°C
(n=4)
I
ANG 11
(n=5)
compared to solvent
q
0%02
~~0.05
compared to 38°C
ALMITRINE
Fig. 8. Nifedipine (5 x 10e6 M) and cooling (23°C) of perfusate preferentially inhibit hypoxia- and almitrine-induced vasoconstriction over ANG II-induced vasoconstriction. ANG II is expressed in % of preceding ANG II response. Hypoxic pressor response and almitrine-induced pressor response are expressed in % of preceding hypoxic response. Time-matched pressor responses in lungs treated with nifedipine solvent (3% DMSO) or with nifedipine and lungs perfused at 38 or 23°C were compared. Data are means t SE. DISCUSSION
In this study, we show that almitrine induces pulmonary vasoconstriction in the isolated rat lung but has no effect on vascular tone in the isolated artery ring preparation. Almitrine, in a low dose, potentiates hypoxic vasoconstriction in blood-perfused rat lungs. Vasoconstriction caused by a large dose of almitrine reduces concomitant HPVC and vice versa, during HPVC, a high dose of almitrine is unable to elicit a pressor response. Considering this interaction between almitrine and hypoxia and also the finding that almitrine-induced vasoconstriction is enhanced by agents known to enhance HPVC and is inhibited by compounds known to inhibit preferentially the hypoxic pressor response (2, 23, 24, 28, 32,43), almitrine appears to be a hypoxia mimic, perhaps utilizing part of the same mechanism as hypoxia to induce vasoconstriction. In the isolated rat lung preparation, high doses of almitrine caused pulmonary vasoconstriction. We observed this vasoconstriction regardless of whether the lung was perfused with homologous blood or with a cell- and plasma-free salt solution. In the PSS-perfused lungs, almitrine caused a dose-dependent vasoconstriction; however, TENSION [91
VASOCONSTRICTION
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even at the highest dose used in our studies (40 pg or 1.33 hg/ml), almitrine caused less vasoconstriction than alveolar hypoxia. Insofar as we consider almitrine a hypoxia mimic, the fact that almitrine-induced vasoconstriction in the PSS-perfused lungs did not reach the magnitude of HPVC may indicate that almitrine is perhaps only a partial hypoxia mimic or a less potent agonist than hypoxia. Interestingly, almitrine caused a greater pressor response in blood-perfused lungs than in PSS-perfused lungs. This is particularly remarkable in view of the binding of almitrine by proteins (41). Having corrected for the difference in vascular reactivity between the two preparations (by expressing the almitrine response in percentage of the preceding hypoxic response), we question whether a blood component is responsible for this difference in the magnitude of almitrine-induced pulmonary vasoconstriction. Because there was no difference in the values of the microvascular pressure measured with or without almitrine in blood-perfused lungs, we conclude that almitrine acts on precapillary, nonconduit arteries. Pulmonary vasoconstriction during normoxia in response to almitrine has been described previously in in vivo dog studies (6, 12, 35) and in isolated rat and ferret lung studies by Bee et al. (l), Falus et al. (8), and Kato et al. (15). In Bee’s study, the vasoconstriction elicited by a bolus injection of 100 pg almitrine was immediately followed by vasodilation, whereas we observed no vasodilatory response with the doses used in our experiments. In the study by Falus et al. (8) in blood-perfused rat lungs, 0.5 and 2.0 pg/ml almitrine caused vasoconstriction. Thus our data are in agreement with these reports. The interaction between almitrine and hypoxia has been the concern of many previous studies. In in vivo experiments in dogs and in isolated lung studies, almitrine’s effect on HPVC has been described as enhancement (8, 27, 35), inhibition (5, 12), or no change (4). Based on the results of this study, we offer the explanation that these discrepancies may be due to the different effects of different doses of almitrine on the lung circulation. A review of the published experimental data and consideration of our own data lead us to conclude that almitrine in low doses potentiates hypoxic constriction, whereas in high doses (bolus injection of 20 or 40 pg) it causes pulmonary vasoconstriction and renders the pulmonary vasculature unresponsiveness to hypoxia. Addition of a high dose of almitrine to the PSS perfusate caused vasoconstriction but reduced the HPVC when the hypoxic challenge was started at the plateau of the almitrine-induced vasoconstriction. The higher the dose of almitrine added to the perfusate reservoir, the smaller the residual hypoxic pressor response. Because almitrine added to the perfusate in a high
(n=3)
NIFEDIPINE peO.05
HYPOXIC
*
T
8120
MIMICS
1
3
5 10 3opg
Fig. 9. Representative tracing of isolated main pulmonary artery ring from rat. Vessel reactivity was established by contracting rings with KCl. Almitrine (0.530 pg) caused no contractile response in reactive rings when added to bath fluid.
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dose during an ongoing hypoxic challenge caused no additional almitrine-induced pressor response, we questioned whether almitrine and hypoxia were acting through the same pathway, possibly by stimulating the same oxygen-sensing mechanism. Almitrine is clearly different from many other vasoconstricting agonists, which cause further vasoconstriction when added during established hypoxic vasoconstriction (44). In this context, it is of interest that cyanide, which causes pulmonary vasoconstriction (36) and may itself mimic hypoxia, also blocked almitrine-induced vasoconstriction, whereas KCl-induced vasoconstriction permitted a subsequent almitrine-induced pressor response. Thus elevation of vascular tone per se in the absence of hypoxia did not prevent almitrine-induced vasoconstriction. Three structurally distinct compounds, 4-AP, vanadate, and PMA (each known for its nonselective enhancing effect of HPV), all augmented almitrine-induced vasoconstriction. We believe that the small increase in vascular tone due to 4-AP and vanadate per se was not responsible for the enhancement of hypoxia- and almitrine-induced vasoconstriction because PMA did not affect baseline perfusion pressure but did substantially enhance hypoxia- and almitrine-induced vasoconstriction. If we compare the degree of enhancement of hypoxia- and almitrine-induced vasoconstriction elicited by 4-AP, vanadate, and PMA, it becomes apparent that almitrine and hypoxia were enhanced to a similar degree by the three enhancing compounds. Although the exact mechanism of HPVC enhancement by these agents remains uncertain, it is interesting that in carotid body type-1 cells both cyanide and almitrine suppress K+ currents (31) and also that hypoxia reduces K+ channel activity in these chemosensitive cells (20). If oxygen sensing or the transduction of a signal reflecting the reduction in PO, depends on cellular K+ kinetics, then a K+ channel blocker such as 4-AP could possibly affect HPVC and the constriction due to almitrine or cyanide. Indeed, the cyanide response also was enhanced by 4-AP (data not shown). However, 4-AP does not selectively enhance the vasoconstriction by hypoxia and the hypoxia mimics (cyanide and almitrine) but does enhance ANG II-induced vasoconstriction (23). Therefore our 4-AP experiments do not establish a relationship between oxygen sensing and cellular K+ kinetics, nor do they localize the site of action of almitrine. Ca2+ entry blockade with nifedipine and lowering of perfusate temperature, both of which have been shown to inhibit hypoxic vasoconstriction preferentially (2, 24, 28, 43)) inhibited hypoxia- and almitrine-induced vasoconstriction to a comparable degree, which supports our concept that hypoxia and almitrine bismesylate may act, in part, through a common mechanism. In the aggregate, our data, particularly the data showing that almitrine causes no further vasoconstriction concomitant to the action of hypoxia or cyanide, and the preferential inhibition of hypoxia and almitrine by Ca2+ channel blockade and hypothermia, lead us to hypothesize that the putative sensor for hypoxia in the lung may be altered or occupied by almitrine. Our experimental data support such a concept, especially if the oxygen sen-
VASOCONSTRICTION
sor has a limited capacity that can be exhausted. This concept is derived from a receptor model in which membrane receptors are occupied by ligands and in which the prior oxygen sensor or receptor occupancy by almitrine would find the system unresponsive to alveolar hypoxia. If hypoxia and almitrine compete in some fashion for this limited capacity, it follows that the combination of both stimuli may not necessarily yield an additive or potentiated response. However, the analogy between hypoxia and almitrine may not be perfect. Whereas severe hypoxia can inhibit pulmonary vasoconstriction (11, 39), high dose almitrine causes pulmonary vasoconstriction. In this respect, cyanide and almitrine may be more similar in their actions: both (in high doses) cause pulmonary vasoconstriction and impair subsequent hypoxia-induced vasoconstricion. Because almitrine has been shown to affect energy metabolism (26, 33) and because mitochondrial electron transport and/or ATP synthesis are possibly the locus of hypoxic sensing (35) where the signal may be generated by ATP reduction in a distinct cellular compartment (that is not involved in the contractile process), it is tempting to speculate that the “oxygen/almitrine receptor” is situated in the mitochondria. As new information is generated regarding the molecular mechanism of almitrine action, one may hope that, by analogy, this information may be pertinent for the understanding of oxygen sensing. The authors thank Dr. Ivan F. McMurtry for valuable discussion, Cheryl Pickett for preparation of the figures, and Rebecca Wolinsky for preparation of the manuscript. Almitrine bismesylate was kindly supplied by Servier Laboratories, France. This research was supported in part by the Deutsche Forschungsgemeinschaft Go 494/1-l (E. B. Gottschall) and National Heart, Lung, and Blood Institute Grant HL-14985 (N. F. Voelkel). Address for reprint requests: N. F. Voelkel, CVP Research Lab, B-133, Univ. of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262. Received
19 April
1991; accepted in final form 24 March
1992.
REFERENCES 1. Bee, D., G. W. Gill, C. J. Emery, G. L. Salmon, T. W. Evans, and G. R. Barer. Action of almitrine on the pulmonary vasculature in ferrets and rats. Bull. Eur. Physiopathol. Respir. 19: 539445, 1983. 2. Benumof, J. L., and E. A. Wahrenbrock. Dependency of hypoxic pulmonary vasoconstriction on temperature. J. Appl. Physiol. 42: 56-58, 1977. S. W., and N. F. Voelkel. Isolated perfused rat lung in 3. Chang, arachidonate studies. In: Methods in EnzymoLogy, edited by R. C. Murphy and F. A. Fitzpatrick. New York: Academic, vol. 187, p. 599-610. G. Malmkvist, F. X. Clergue, C. 4. Chen, L., F. L. Miller, Marshall, and B. E. Marshall. Effect of almitrine on hypoxic pulmonary vasoconstrition (HPV) (Abstract). Am. Rev. Respir. Dis. 133: A299, 1986. G. Malmkvist, F. X. Clergue, C. 5. Chen, L., F. L. Miller, Marshall, and B. E. Marshall. High-dose almitrine bismesylate inhibits hypoxic pulmonary vasoconstriction in closed-chest dogs. Anesthesiology 67: 534-542, 1987. 6. Chuma, R., 0. Tanaka, Y. Hoshino, H. Obara, and S. Iwai. Release of thromboxane A2 by low-dose almitrine in the hypoxic dog. Eur. Respir. J. 1: 706-710, 1988. C. A., J. H. Linehan, and D. A. Rickaby. Pulmo7. Dawson, nary microcirculatory hemodynamics. Ann. NY Acad. Sci. 384: 90-106, 1982.
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ALMITRINE
MIMICS
HYPOXIC
8. Falus, F., J. Herget, and V. Hampl. Almitrine in low dose potentiates vasosconstrictor responses of isolated rat lungs to moderate hypoxia. Eur. Respir. J. 4: 688-693, 1991. 9. Feddersen, C. O., S. Chang, J. Czartalomna, and N. F. Voelkel. Arachidonic acid causes cyclooxygenase-dependent and -independent pulmonary vasodilation. J. Appl. Physiol. 68: 1799-1808, 1990. 10. Grover, R. F., W. W. Wagner, Jr., I. F. McMurtry, and J. T. Reeves. Pulmonary circulation. In: Handbook of Physiology. The Cardiovascular System. Peripheral Circulation and Organ Blood now. Bethesda, MD: Am. Physiol. Sot., 1983, sect. 2, vol. III, chapt. 4, p. 103-136. 11. Harabin, A. L., M. D. Peake, and J. T. Sylvester. Effect of severe hypoxia on the pulmonary vascular response to vasoconstrictor agents. J. Appl. Physiol. 50: 561-565, 1981. 12. Hughes, J. M. B., D. J. Allison, A. Goatcher, and A. Tripath. Influence of alveolar hypoxia on pulmonary vasomotor responses in almitrine in the dog. Clin. Sci. Lond. 70: 555-564, 1986. 13. Ishii, K., K. Ishii, J.-C. Massabuau, and P. Dejours. Oxygen-sensitive chemoreceptors in the branchio-cardiac veins of the crayfish, Astacus leptodactylus. Respir. Physiol. 78: 73-81, 1989. 14. Kato, M., and N. C. Straub. Response of small pulmonary arteries to unilobar alveolar hypoxia and hypercapnia. Circ. Res. 19: 426-440, 1966. 15. Kato, S., S. Sato, and K. Takahashi. Almitrine bismesylate reduces hypoxic pulmonary vasoconstriction in isolated rat lungs. Tohoku J. Exp. Med. 157: 119-129, 1989. 16. Koyama, T., and M. Hormoto. Blood flow reduction in local pulmonary microvessels during acute hypoxia imposed on a small fraction of the lung. Respir. Physiol. 52: 181-189, 1983. 17. Laubie, M., and H. Schmitt. Long-lasting hyperventilation induced by almitrine: evidence for a specific effect on carotid and thoracic chemoreceptors. Eur. J. PharmacoZ. 61: 125-136, 1980. 18. Lloyd, T. C., Jr. Effect of alveolar hypoxia on pulmonary vascular resistance. J. Appl. Physiol. 19: 1086-1094, 1964. 19. Lockardt, A., and G. Mazmanian. Effects of almitrine bismesylate on pulmonary circulation and ventilation/perfusion ratios. Bull. Eur. Physiopathol. Respir. 18, Suppl. 4: 285-297, 1982. 20. Lopez-Lopez, J., C. Gonzalez, J. Urena, and J. LopezBarneo. Low PO, selectively inhibits K+ channel activity in chemoreceptor cell of the mammalian carotid body. J. Gen. Physiol. 93: 1001-1015, 1989. 21. Madden, J. A., C. A. Dawson, and D. R. Harder. Hypoxiainduced activation in small isolated pulmonary arteries from the cat. J. Appl. Physiol. 59: 113-118, 1985. 22. Maxwell, D. L., J. M. B. Hughes, and P. C. G. Nye. The effect of almitrine-bimesylate on the steady state responses of arterial chemoreceptor to CO2 and O2 in the cat. Respir. Physiol. 74: 275-284, 1988. 23. McMurtry, I. F. Angiotensin is not required for hypoxic constriction in salt solution-perfused rat lungs. J. Appl. Physiol. 56: 375-380, 1984. 24. McMurtry, I. F., A. B. Davidson, J. T. Reeves, and R. F. Grover. Inhibition of hypoxic pulmonary vasoconstriction by calcium antagonists in isolated rat lungs. Circ. Res. 38: 99-104, 1976. 25. Motley, H. L., A. Cournand, L. Werko, A. Himmelstein, and D. Dresdale. The influence of short periods of induced anoxia upon pulmonary artery pressures in man. Am. J. Physiol. 150: 315-320, 1947. 26. Mottershead, J. P., P. C. G. Nye, and P. G. Quirk. The effects of almitrine on electron transport in mitochondria isolated from rat liver. J. Physiol. Lond. 396: 91-99, 1987.
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27. Nakanishi, S., T. Hiramoto, N. Ahmed, and Y. Nishimoto. Almitrine enhances in low dose the reactivity of pulmonary vessels to hypoxia. Respir. Physiol. 74: 139-150, 1988. 28. Nilsen, K., and A. Hauge. Effects of temperature changes on pressor response to acute alveolar hypoxia in isolated rat lungs. Acta Physiol. Stand. 73: 111-120, 1968. 29. Orton, E. C., B. Raffestin, and I. F. McMurtry. Protein kinase C influences rat pulmonary vascular reactivity. Am. Rev. Respir. Dis. 141: 654-658, 1990. 30. Paramelle, B., P. Levy, and C. Pirotte. Long term follow-up of pulmonary arterial pressure evolution in COPD patients treated by almitrine bismesylate. Eur. J. Respir. Dis. 64, Suppl. 126: 333336, 1983. 31. Peers, C., and J. O’Donnell. Potassium currents in type I carotid body cells from the neonatal rat and their modulation by chemotactic agents. Brain Res. 522: 259-266, 1990. 32. Raffestin, B., and I. F. McMurtry. Effects of intracellular pH on hypoxic vasoconstriction in rat lungs. J. AppZ. Physiol. 63: 2524-2531, 1987. 33. Rigoulet, M., R. Ouhabi, X. Leverve, F. Putod-Paramelle, and B. Guerin. Almitrine, a new kind of energy-transduction inhibitor acting on mitochondrial ATP synthase. Biochim. Biophys. Acta 975: 325-329, 1989. 34. Rodman, D., and N. F. Voelkel. Hypoxic vasoconstriction, mediators and pharmacology. In: The Lung: Scientific Contribution, edited by R. Crystal, F. West, and P. Barnes. New York: Raven, 1990. 35. Romaldini, H., R. Rodriguez-Roisin, P. D. Wagner, and J. B. West. Enhancement of hypoxic pulmonary vasoconstriction by almitrine in the dog. Am. Rev. Respir. Dis. 128: 288-293, 1983. 36. Rounds, S., and I. F. McMurtry. Inhibitors of oxidative ATP production cause transient vasoconstriction and block subsequent pressor responses in rat lungs. Circ. Res. 48: 393-400, 1981. 37. Sakai, S., S. W. Chang, and N. F. Voelkel. Importance of vasoconstriction in lipid mediator-induced pulmonary edema. J. Appl. Physiol. 66: 2667-2674, 1989. 38. Shelton, G. The effects of lung ventilation on blood flow to the lungs and body of the amphibian, Xenopus laevis. Respir. Physiol. 9: 183-196, 1970. 39. Sylvester, J. T., A. L. Harabin, M. D. Peake, and R. S. Frank. Vasodilator and constrictor responses to hypoxia in isolated pig lungs. J. Appl. Physiol. 49: 820-825, 1980. 40. Townsley, M. I., R. J. Korthuis, B. Rippe, J. C. Parker, and A. E. Taylor. Validation of double vascular occlusion method for Pc,i in lung and skeletal muscle. J. Appl. Physiol. 61: 127-132, 1986. 41. Tweney, J. Almitrine bismesylate: current status. BUZZ. Eur. Physiopathol. Respir. 23, Suppl. 11: 153%163S, 1987. 42. Voelkel, N. F. Mechanisms of hypoxic pulmonary vasoconstriction. Am. Rev. Respir. Dis. 133: 1186-1195, 1986. 43. Voelkel, N. F., and J. Czartolomna. Vanadate potentiates hypoxic pulmonary vasoconstriction. J. PharmacoZ. Exp. Ther. 259: 666-672, 1991. 44. Voelkel, N. F., I. F. McMurtry, and J. T. Reeves. Hypoxia impairs vasodilation in the lung. J. CZin. Invest. 67: 238-246, 1981. 45. Voelkel, N. F., K. G. Morris, I. F. McMurtry, and J. T. Reeves. Calcium augments hypoxic vasoconstriction in lungs from high-altitude rats. J. Appl. Physiol. 49: 450-455, 1980. 46. Watanabe, S., R. E. Kanner, A. G. Cutillo, R. L. Menlove, R. T. Bachand, Jr., M. B. Szalkowski, and A. D. Renzetti, Jr. Long-term effect of almitrine bisymesylate in patients with hypoxemic chronic obstructive pulmonary disease. Am. Rev. Respir. Dis. 140: 1269-1273, 1989.
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almitrine
bimesylate;
HYPOXIC pulmonary vasoconstriction occurs in all species so far investigated (10, 34,39), as well as in humans (25). However, the mechanism of this response, which primarily targets precapillary pulmonary vessels (14)) has been elusive. It is not known which cell type(s) provides the oxygen sensor, although it is clear that the contractile response occurs mainly in the smooth muscle cells (14, 16). Analogous to electrical circuits, a conceptual model of the hypoxic pressor response consists of a sensor or a receptor, a transducer, and an effector (43). Previously, a large number of investigations have been concerned with modulation of hypoxic vasoconstriction; a redundant system of amplifiers and dampers of this response has been described (34). Because it is possible that sensor and effector are linked, it is difficult to study the sensor or the effector separately. Thus an increase or decrease in the magnitude of the hypoxic pressure re0363-6135/92
$2.00 Copyright
sponse is likely to occur via a combined effect on sensor and effector (e.g., if both are calcium dependent), and, indeed, uncoupling of the sensor from the contractile effector has not been accomplished. In recent years, almitrine has been studied extensively in patients with chronic bronchitis (30, 46) because of its effect on the arterial PO, and has been used as an experimental tool to study chemoreceptor responses (13, 17). Ishii et al. (13) recorded increased oxygen-sensitive activity from the branchiocardiac vein of the crayfish after almitrine administration. In this study, both cyanide (NaCN) and almitrine mimicked hypoxia. Using various animal models and experimental procedures, several investigators reported that almitrine has an effect on the pulmonary circulation; however, controversy exists as to whether almitrine potentiates hypoxic vasoconstriction (1, 4, 5, 12, 15, 19, 27, 35). For example, in our own study, we found that almitrine caused pulmonary vasoconstriction in anesthetized, spontaneously breathing dogs but did not potentiate hypoxic vasoconstriction. However, in isolated rat lungs perfused with blood, Falus et al. (8) showed a potentiation of hypoxic vasoconstriction by a low dose of almitrine. Almitrine has been shown to affect energy metabolism and in this respect may be related to the hypoxia mimic cyanide, mentioned above. In liver mitochondria, almitrine inhibits electron transfer at the cytochrome bc complex of the ubiquinoneor proton-motive Q-cycle (26). In yeast cells, almitrine appeared to block the multisubunit mitochondrial FoFl ATP synthase (33). Thus almitrine joins the group of compounds that block mitochondrial electron transport and cause pulmonary vasoconstriction (36). Whereas cyanide blocks the cytochrome-c oxidase complex by binding to its heme Fe atom, almitrine may block ATP synthesis (like dinitrophenol) or impair electron transport downstream from the rotenone blocking site that is localized in the NADH-cytochrome-Q reductase complex. We wished to examine whether almitrine mimics hypoxia in its effects on the pulmonary circulation. If almitrine and hypoxia were indeed similar in their behavior in the lung circulation, then one would postulate that 1) almitrine causes pulmonary vasoconstriction, 2) almitrine potentiates or enhances hypoxic vasoconstriction (if hypoxia and almitrine use, at least in part, the same pathway), 3) agents that enhance hypoxic vasoconstriction also enhance almitrine-induced pulmonary vasoconstriction, and 4) agents that inhibit hypoxic pulmonary vasoconstriction (HPVC) preferentially also inhibit almitrine-induced vasoconstriction. Furthermore, if almitrine is a hypoxia mimic, one could postulate that
0 1992 the American
Physiological
Society
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ALMITRINE
MIMICS
HYPOXIC
the principal site of action for the drug was precapillary, i.e., the drug was not predominantly acting as a pulmonary venoconstrictor, and, finally, that almitrine had little or no effect on systemic vessels or the contractile machinery of large pulmonary conduit arteries. METHODS
We used male Sprague-Dawley rats (Harlan Industries, Indianapolis, IN) weighing 300-400 g, which were housed in the Animal Care Facility of the University of Colorado and had free access to food and water. Unless otherwise stated, all reagents were obtained from Sigma Chemical (St. Louis, MO). Isolated
Perfused
Rat Lung
Studies
Rat lungs were isolated as previously described (3). Briefly, a tracheal cannula was introduced, and the rats were ventilated with a Harvard small animal respirator at 55 breaths/min, initially using a gas mixture of 21% O,- 5% CO,-74% N2. A median sternotomy was performed, and after injection of 100 U of heparin into the right ventricle, the main pulmonary artery and the left ventricle were cannulated. The lungs and heart were removed from the chest and suspended in a warm, humid chamber. The lungs were perfused at a constant flow (0.03 ml/g body wt/min) with either heparinized homologous blood (taken from 3 ether-anesthetized rats) or with a physiological salt solution (PSS) containing (in mM) 119 NaCl, 4.7 KCl, 5.5 glucose, 1.17 MgS04, 22.6 NaHCO,, 1.18 KH2P04, and 3.2 CaCl,, as well as 4 vol/lO ml Ficoll (mol wt 70,000). When perfused with the PSS, the first 50 ml of perfusate containing residual blood were discarded so that the lungs were perfused with 30 ml recirculating, cell- and protein-free salt solution. In general, after an equili-
VASOCONSTRICTION
bration period of 20 min at 38”C, the vascular reactivity of each lung was tested with three pairs of a bolus injection of angiotensin II (ANG II, 0.1 pg/O.l ml) and 5 min of airway hypoxia [PSS-perfused lungs fractional concentration of 0, in inspired gas (FI,~), 0.00; blood-perfused lungs FI, , 0.031 separated by a 5-min period of normoxic ventilation (F1&21 0.21). Different inspired fractions of 0, were chosen because an FIN, of 0.03 in rat lungs perfused with blood produces a maximal pressor response, whereas an FI,~ of 0.00 is required to produce a maximal response in PSS-perfused lungs. The experimental period in each lung started after the third hypoxic challenge (Fig. I). ANG II may act through a different mechanism than hypoxia (23) and was therefore chosen as a nonhypoxic vasoconstricting stimulus to test vascular reactivity in each isolated lung. This also allowed us to use the ANG II pressor response as a reference response during the experimental period of each study. The pulmonary perfusion pressure was monitored continuously with a Statham P23 AA transducer and recorded on a Soltec recorder. In five blood-perfused lungs, the effect of IO pug almitrine on the microvascular pressure was measured using the well-established double-occlusion technique. Briefly, both inflow and outflow tubing were occluded simultaneously, causing arterial and venous pressures to equilibrate at the capillary pressure (7, 37, 40). Specific
Protocols
Effect of almitrine on pulmonary vascular pressure. In six lungs perfused with blood, vascular reactivity was tested with ANG II and hypoxia as described above, and then a single dose of almitrine (IO pug in perfusate reservoir) was added to observe its effects on pulmonary perfusion pressure. In all subsequent
4 -AMINO-PYRIDINE PMA VANADATE
COOLING NIFEDIPINE (or SOL.)
A
PAP = 7 00% PAP
[mmHC!Il 10
ANGII
HYPOXIA
02
4 &&/I
HYPOXIA
21% 02
ALMIhNE ‘Ogl
Fig. 1. Schematic time course of the experimental protocol. After a standard equilibration period and 3 pairs of ANG II (0.1 pg) and hypoxia challenges [fractional concentration of 0, in inspired gas (FI,J 0.00 physiological salt solution lungs], almitrine was added to perfusate reservoir. Schematic begins with (PSS) -perfused lungs; FI o2 0.03 blood-perfused second ANG II response. Large arrows, time points when compounds (as shown) were added to perfusate reservoir or perfusate temperature was changed. Third hypoxic pressor response was designated value 100%. APAP, change in mean pulmonary artery perfusion pressure. SOL, solvent; PMA, phorbol myristate acetate.
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ALMITRINE
MIMICS
HYPOXIC
experiments, lungs perfused with PSS were used. In isolated rat lungs perfused with PSS, a dose-response relationship between the amount of almitrine added to the reservoir and the rise in pulmonary perfusion pressure was established. Again, pulmonary vascular reactivity was tested with ANG II and hypoxia, as above, before almitrine was added to the perfusate reservoir and the rise in perfusion pressure was recorded. Four different doses of almitrine were tested in PSSperfused lungs: 5 pg (0.17 pg/ml, n = 5), 10 pug (0.33 pg/ml, n = 5), 20 pug(0.66 pg/ml, n = 5), and 40 pug(1.33 pg/ml, n = 5). However, only one particular dose was tested in any given lung. Five lungs were treated with the almitrine solvent, malic acid, at a final perfusate concentration of 0.01%. This concentration corresponds to the amount of malic acid added with the highest dose of almitrine given (40 pg) in this series of experiments. Effect of almitrine on hypoxic vasoconstriction. In six bloodperfused and six PSS-perfused lungs, we deviated from the procedure described above. These experiments were designed to examine whether low-dose almitrine enhanced hypoxic vasoconstriction. Therefore a submaximal hypoxic challenge was used. We wished to reproduce the data by Falus et al. (8), which demonstrated a potentiation of hypoxic vasoconstriction in rat lungs by a low dose of almitrine. To this end, in blood-perfused lungs we applied one ANG II challenge followed by a hypoxia challenge (FI,~ 0.03). The lungs were then challenged with two hypoxic challenges (FI, 0.05) separated by IO min of normoxia. Then either malic acid ‘
TIME (min)
Fig. 5. PAP tracings from representative experiments in isolated rat lungs perfused with PSS. A: almitrine (10 pg) added to reservoir during plateau phase of a hypoxic challenge had no vasoconstricting effect. B: at peak of a pressor response induced by a higher dose of almitrine (20 pg), a hypoxic challenge (FIN, 0.00) caused no further vasoconstriction. C: hypoxia-induced vasoconstriction was partially reduced when hypoxic (FIN, 0.00) challenge was started at peak of a pressor response due to 10 pg almitrine.
3otA
I PAP bmHg1
ALMITRINE/-
01 p ALMITRJNE
Pilot experiments indicated that hypoxia- and almitrine-induced vasoconstriction were both inhibited by nifedipine; subsequently, a dose of nifedipine (5 x 10e6 M) was found (43) that inhibited both hypoxia- and almitrine-induced vasoconstriction preferentially over ANG II-induced vasoconstriction (Fig. 8). A decrease in the perfusate temperature to 23°C led to similar findings, i.e., the hypoxia- and almitrineinduced pressure responses were slowed and blunted, whereas the ANG II response was nearly unchanged (Fig. 8). Thus the Ca2+ entry blocker and hypothermia had comparable effects on hypoxia- and almitrine-induced vasoconstriction. Tension
Development
in Isolated
Vessel Rings
Because we had established that almitrine caused precapillary vasoconstriction, we wished to examine whether almitrine contracted conduit arteries. Addition of almitrine to the bath fluid of isolated rat main pulmonary artery and aorta rings, in doses ranging from 0.5 to 30 pg, demonstrated that all of the rings from systemic (data not shown) and pulmonary conduit arteries were unresponsive to almitrine (Fig. 9). We concluded that these rings did not respond to almitrine after proper vascular reactivity had been established [A tension in grams: pulmonary artery 80 mM KCl-induced contraction = 0.88 t 0.06; phenylephrine (5 x lo-’ M)-induced contraction = 0.27 t 0.04; ACh (lo+ M) -in duced re 1axa t’ion = 0.2 t 0.02; almitrine 0.5-30 pg = 01. l
0
% ’
z
400 -
0
.
Fig. 6. A: almitrine added to reservoir at peak of a KCl-induced pressor response elicits additional vasoconstriction. B: almitrine (10 pg) added to perfusate reservoir at peak of a cyanide-induced pressor response leads to no additional vasoconstriction.
blocker 4-AP and vanadate, which are structurally unrelated and nonselective in enhancing the hypoxic pressor response, both increased pulmonary perfusion pressure by a small amount and enhanced both HPVC (APAP: 4-AP 363.56 t 16.85%; vanadate 327 t 45.9% of preceding hypoxic response) and almitrine-induced (10 pg) vasoconstriction (APAP: 4-AP 293.52 t 52.95%; vanadate 347.85 t 79.62% of preceding hypoxic response), whereas PMA enhanced both the almitrine pressor response
xw300 e ZO i&200
0
0%02
lopg group) group)
-
z:k! E IOO6 o\o
ALMITRINE (n=4,each (n=4,each
:
TIME (min)
n
r.l e cl II -----
0 .-CONTROL
PMA (1 O-8M)
4-AMINOPYRIDINE (5x1 O-3M)
VANADATE (3x1 O-5M)
Fig. 7. Effect of PMA, 4-aminopyridine, and vanadate on almitrineinduced vasoconstriction [expressed in % of (2nd) preceding hypoxic response in each lung]. All 3 compounds greatly enhanced almitrineinduced pressor response and hypoxic pressor response. Data are means t SE.
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ALMITRINE 0
100 ii 2 0 n 2 a (3 g 5 B
NIFEDIPINE SOLVENT
q 8o
I
60 40 20
0
I
hI ANG II
0%02
ALMITRINE TEMP
38°C
(n=4)
n
TEMP
23°C
(n=4)
I
ANG 11
(n=5)
compared to solvent
q
0%02
~~0.05
compared to 38°C
ALMITRINE
Fig. 8. Nifedipine (5 x 10e6 M) and cooling (23°C) of perfusate preferentially inhibit hypoxia- and almitrine-induced vasoconstriction over ANG II-induced vasoconstriction. ANG II is expressed in % of preceding ANG II response. Hypoxic pressor response and almitrine-induced pressor response are expressed in % of preceding hypoxic response. Time-matched pressor responses in lungs treated with nifedipine solvent (3% DMSO) or with nifedipine and lungs perfused at 38 or 23°C were compared. Data are means t SE. DISCUSSION
In this study, we show that almitrine induces pulmonary vasoconstriction in the isolated rat lung but has no effect on vascular tone in the isolated artery ring preparation. Almitrine, in a low dose, potentiates hypoxic vasoconstriction in blood-perfused rat lungs. Vasoconstriction caused by a large dose of almitrine reduces concomitant HPVC and vice versa, during HPVC, a high dose of almitrine is unable to elicit a pressor response. Considering this interaction between almitrine and hypoxia and also the finding that almitrine-induced vasoconstriction is enhanced by agents known to enhance HPVC and is inhibited by compounds known to inhibit preferentially the hypoxic pressor response (2, 23, 24, 28, 32,43), almitrine appears to be a hypoxia mimic, perhaps utilizing part of the same mechanism as hypoxia to induce vasoconstriction. In the isolated rat lung preparation, high doses of almitrine caused pulmonary vasoconstriction. We observed this vasoconstriction regardless of whether the lung was perfused with homologous blood or with a cell- and plasma-free salt solution. In the PSS-perfused lungs, almitrine caused a dose-dependent vasoconstriction; however, TENSION [91
VASOCONSTRICTION
ALMITRINE 0.5
H389
even at the highest dose used in our studies (40 pg or 1.33 hg/ml), almitrine caused less vasoconstriction than alveolar hypoxia. Insofar as we consider almitrine a hypoxia mimic, the fact that almitrine-induced vasoconstriction in the PSS-perfused lungs did not reach the magnitude of HPVC may indicate that almitrine is perhaps only a partial hypoxia mimic or a less potent agonist than hypoxia. Interestingly, almitrine caused a greater pressor response in blood-perfused lungs than in PSS-perfused lungs. This is particularly remarkable in view of the binding of almitrine by proteins (41). Having corrected for the difference in vascular reactivity between the two preparations (by expressing the almitrine response in percentage of the preceding hypoxic response), we question whether a blood component is responsible for this difference in the magnitude of almitrine-induced pulmonary vasoconstriction. Because there was no difference in the values of the microvascular pressure measured with or without almitrine in blood-perfused lungs, we conclude that almitrine acts on precapillary, nonconduit arteries. Pulmonary vasoconstriction during normoxia in response to almitrine has been described previously in in vivo dog studies (6, 12, 35) and in isolated rat and ferret lung studies by Bee et al. (l), Falus et al. (8), and Kato et al. (15). In Bee’s study, the vasoconstriction elicited by a bolus injection of 100 pg almitrine was immediately followed by vasodilation, whereas we observed no vasodilatory response with the doses used in our experiments. In the study by Falus et al. (8) in blood-perfused rat lungs, 0.5 and 2.0 pg/ml almitrine caused vasoconstriction. Thus our data are in agreement with these reports. The interaction between almitrine and hypoxia has been the concern of many previous studies. In in vivo experiments in dogs and in isolated lung studies, almitrine’s effect on HPVC has been described as enhancement (8, 27, 35), inhibition (5, 12), or no change (4). Based on the results of this study, we offer the explanation that these discrepancies may be due to the different effects of different doses of almitrine on the lung circulation. A review of the published experimental data and consideration of our own data lead us to conclude that almitrine in low doses potentiates hypoxic constriction, whereas in high doses (bolus injection of 20 or 40 pg) it causes pulmonary vasoconstriction and renders the pulmonary vasculature unresponsiveness to hypoxia. Addition of a high dose of almitrine to the PSS perfusate caused vasoconstriction but reduced the HPVC when the hypoxic challenge was started at the plateau of the almitrine-induced vasoconstriction. The higher the dose of almitrine added to the perfusate reservoir, the smaller the residual hypoxic pressor response. Because almitrine added to the perfusate in a high
(n=3)
NIFEDIPINE peO.05
HYPOXIC
*
T
8120
MIMICS
1
3
5 10 3opg
Fig. 9. Representative tracing of isolated main pulmonary artery ring from rat. Vessel reactivity was established by contracting rings with KCl. Almitrine (0.530 pg) caused no contractile response in reactive rings when added to bath fluid.
TIME (min) Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on September 12, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
H390
ALMITRINE
MIMICS
HYPOXIC
dose during an ongoing hypoxic challenge caused no additional almitrine-induced pressor response, we questioned whether almitrine and hypoxia were acting through the same pathway, possibly by stimulating the same oxygen-sensing mechanism. Almitrine is clearly different from many other vasoconstricting agonists, which cause further vasoconstriction when added during established hypoxic vasoconstriction (44). In this context, it is of interest that cyanide, which causes pulmonary vasoconstriction (36) and may itself mimic hypoxia, also blocked almitrine-induced vasoconstriction, whereas KCl-induced vasoconstriction permitted a subsequent almitrine-induced pressor response. Thus elevation of vascular tone per se in the absence of hypoxia did not prevent almitrine-induced vasoconstriction. Three structurally distinct compounds, 4-AP, vanadate, and PMA (each known for its nonselective enhancing effect of HPV), all augmented almitrine-induced vasoconstriction. We believe that the small increase in vascular tone due to 4-AP and vanadate per se was not responsible for the enhancement of hypoxia- and almitrine-induced vasoconstriction because PMA did not affect baseline perfusion pressure but did substantially enhance hypoxia- and almitrine-induced vasoconstriction. If we compare the degree of enhancement of hypoxia- and almitrine-induced vasoconstriction elicited by 4-AP, vanadate, and PMA, it becomes apparent that almitrine and hypoxia were enhanced to a similar degree by the three enhancing compounds. Although the exact mechanism of HPVC enhancement by these agents remains uncertain, it is interesting that in carotid body type-1 cells both cyanide and almitrine suppress K+ currents (31) and also that hypoxia reduces K+ channel activity in these chemosensitive cells (20). If oxygen sensing or the transduction of a signal reflecting the reduction in PO, depends on cellular K+ kinetics, then a K+ channel blocker such as 4-AP could possibly affect HPVC and the constriction due to almitrine or cyanide. Indeed, the cyanide response also was enhanced by 4-AP (data not shown). However, 4-AP does not selectively enhance the vasoconstriction by hypoxia and the hypoxia mimics (cyanide and almitrine) but does enhance ANG II-induced vasoconstriction (23). Therefore our 4-AP experiments do not establish a relationship between oxygen sensing and cellular K+ kinetics, nor do they localize the site of action of almitrine. Ca2+ entry blockade with nifedipine and lowering of perfusate temperature, both of which have been shown to inhibit hypoxic vasoconstriction preferentially (2, 24, 28, 43)) inhibited hypoxia- and almitrine-induced vasoconstriction to a comparable degree, which supports our concept that hypoxia and almitrine bismesylate may act, in part, through a common mechanism. In the aggregate, our data, particularly the data showing that almitrine causes no further vasoconstriction concomitant to the action of hypoxia or cyanide, and the preferential inhibition of hypoxia and almitrine by Ca2+ channel blockade and hypothermia, lead us to hypothesize that the putative sensor for hypoxia in the lung may be altered or occupied by almitrine. Our experimental data support such a concept, especially if the oxygen sen-
VASOCONSTRICTION
sor has a limited capacity that can be exhausted. This concept is derived from a receptor model in which membrane receptors are occupied by ligands and in which the prior oxygen sensor or receptor occupancy by almitrine would find the system unresponsive to alveolar hypoxia. If hypoxia and almitrine compete in some fashion for this limited capacity, it follows that the combination of both stimuli may not necessarily yield an additive or potentiated response. However, the analogy between hypoxia and almitrine may not be perfect. Whereas severe hypoxia can inhibit pulmonary vasoconstriction (11, 39), high dose almitrine causes pulmonary vasoconstriction. In this respect, cyanide and almitrine may be more similar in their actions: both (in high doses) cause pulmonary vasoconstriction and impair subsequent hypoxia-induced vasoconstricion. Because almitrine has been shown to affect energy metabolism (26, 33) and because mitochondrial electron transport and/or ATP synthesis are possibly the locus of hypoxic sensing (35) where the signal may be generated by ATP reduction in a distinct cellular compartment (that is not involved in the contractile process), it is tempting to speculate that the “oxygen/almitrine receptor” is situated in the mitochondria. As new information is generated regarding the molecular mechanism of almitrine action, one may hope that, by analogy, this information may be pertinent for the understanding of oxygen sensing. The authors thank Dr. Ivan F. McMurtry for valuable discussion, Cheryl Pickett for preparation of the figures, and Rebecca Wolinsky for preparation of the manuscript. Almitrine bismesylate was kindly supplied by Servier Laboratories, France. This research was supported in part by the Deutsche Forschungsgemeinschaft Go 494/1-l (E. B. Gottschall) and National Heart, Lung, and Blood Institute Grant HL-14985 (N. F. Voelkel). Address for reprint requests: N. F. Voelkel, CVP Research Lab, B-133, Univ. of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262. Received
19 April
1991; accepted in final form 24 March
1992.
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