Role of CAMP-dependent protein in CAMP-mediated vasodilation
kinase
JOHNSON HAYNES, JR., JULIET ROBINSON, LETETIA SAUNDERS, AUBREY E. TAYLOR, AND SAMUEL J. STRADA Pulmonary and Critical Care Division, Departments of Medicine, Physiology and Pharmacology, University of South Alabama, College of Medicine, Mobile, Alabama 36688 Haynes, Johnson, Jr., Juliet Robinson, Letetia Saunders, Aubrey E. Taylor, and Samuel J. Strada. Role of CAMP-dependent protein kinase in CAMP-mediated vasodilation. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H511-H516, 1992.-In this study, the role of adenosine 3’,5’-cyclic monophosphate (CAMP)-dependent protein kinase A (PKA) in CAMP-dependent relaxation was assessed in the isolated-perfused rat lung using a PKA inhibitor, &-CAMPS, &bromoCAMP (&BrcAMP), and the diterpene activator of adenylate cyclase (AC), forskolin (FSK). A role for K’ channels was also assessed with the nonselective K+ channel blocker, tetraethylammonium (TEA, 10 mM), and an ATP-sensitive K+ channel inhibitor, glibenclamide (GLI, 100 PM). Both 8-BrcAMP (O.l1.0 mM) and FSK (0.1-10 PM) dose-dependently attenuated the peak pressor response to alveolar hypoxia (HPR). RpCAMPS potentiated the HPR and attenuated B-BrcAMP-mediated vasodilation but had no effect on FSK-mediated vasodilation. FSK-mediated vasodilation was not mimicked by 1,9dideoxy-FSK, which is biologically inactive on AC but alters K+ channels identically to FSK, nor was it attenuated by the platelet-activating factor antagonist SRI 63-441 or the cyclooxygenase inhibitor indomethacin. TEA, but not GLI, attenuated FSK-mediated vasodilation. Similarly, TEA attenuated 8BrcAMP-mediated vasodilation. These results support roles for PKA and indirect gating of a non-ATP-sensitive K+ channel in mediating CAMP-dependent pulmonary vasodilation. pulmonary circulation; adenosine 3’,5’-cyclic monophosphate; adenosine 3’,5’-cyclic monophosphate-dependent protein kinase; hypoxic vasoconstriction; pulmonary vasodilation; potassium channels RELAXATION OFCONTRACTEDVASCULAR smoothmuscle by ,&adrenergic agents and other agents that activate adenylate cyclase, such as forskolin (FSK), are thought to produce their biological effect by increasing the intracellular concentration of adenosine 3’,5’-cyclic monophosphate (cAMP)(4, 22-28). We have previously demonstrated that N6, 02-dibutyryl-CAMP (DBcAMP) and the selective phosphodiesterase inhibitor indolidan will relax pulmonary vascular beds precontracted with alveolar hypoxia (12). Although the precise mechanism by which CAMP causes relaxation in smooth muscle is not known, it is generally accepted that the activation of CAMP-dependent protein kinase (PKA) is involved (4, 13, 27). Several recent studies have challenged the concept that PKA is not the sole mediator of CAMP-dependent relaxation of vascular smooth muscle (13, 16, 27). Some studies have concluded that CAMP-dependent effects on intracellular Ca2+ may be more important in mediating relaxation (18, 27), whereas others have suggested that relaxation involves the activation of cGMPdependent protein kinase (PKG) (13, 16). Hei et al. (13) compared the effects of CAMP analogues on tension and PKA activities in the rat vas deferens and demonstrated 0363-6135/92
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that 8bromo-CAMP (8BrcAMP), which relatively selectively activates PKA, does not inhibit phenylephrineinduced contractions of vas deferens. In contrast, DBcAMP, a less potent activator of PKA than 8BrcAMP, does relax the phenylephrine-contracted vas deferens. They concluded that PKA is not responsible for the relaxant effect of CAMP analogues, at least in vas deferens smooth muscle. The objective of this study was to test whether the CAMP attenuation of the hypoxic pressor response (HPR) is mediated via PKA. Our findings suggest that PKA does play a role in CAMP-dependent relaxation in pulmonary vascular beds precontracted with alveolar hypoxia but that other mechanisms involving a non-ATP-sensitive K+ channel and possibly PKG are also involved. MATERIALS
AND
METHODS
Materials All materials were obtained from Sigma Chemical (St. Louis, MO) unless otherwise specified. Angiotensin II (ANG II), 8BrcAMP, the nonselective K’ channel antagonist tetraethylammonium chloride (TEA) (5,10,19), and the platelet-activating factor (PAF) antagonist SRI 63-441 (a generous gift from Dr. W. S. Houlihan, Sandoz Research Institute, East Hanover, NJ) (11) were dissolved in normal saline. The R, diastereoisomer of adenosine 3’,5’-cyclic phosphorothioate (&-CAMPS; also a generous gift from Dr. W. S. Houlihan), which is an intracellular antagonist of PKA, (1) and the cyclooxygenase inhibitor sodium indomethacin trihydrate (Merck Sharpe and Dohme Research Laboratories, West Point, PA) were dissolved in distilled water. The ATP-sensitive K+ channel antagonist glibenclamide (19) was dissolved in 0.25% dimethyl sulfoxide with 0.0002 N NaOH. The diterpene activator of adenylate cyclase FSK (7, 24) and 1,9-dideoxy-FSK (7,14) were dissolved in 0.001% ethanol. Isolated Perfused
Lung
Male Sprague-Dawley rats (260-320 g body wt), were anesthetized with pentobarbital sodium (20-25 mg ip), and the lungs were removed for extracorporeal perfusion. After placement of a tracheal cannula, the lungs were ventilated with a Harvard rodent ventilator (model 683) at 55 breaths/min, a tidal volume of 2.5 ml, and positive-end expiratory pressure of 2 cmHtLO using a gas mixture of 95% air-5% COa (normoxic gas). During the hypoxic stimuli, the lungs were ventilated with a 3% 02-5% C02-balance NP (hypoxic) gas mixture. A median sternotomy was then performed; then heparin (100 IU) was injected into the right ventricle, and cannulas were placed into the pulmonary artery and left ventricle. The heart, lungs, and mediastinal structures were removed en bloc and placed into a humidified chamber. The lungs were perfused by a Gilson Minipuls 2 peristaltic pump at a constant flow of 0.03 ml-g body wt-l. min. Lungs were perfused with homologous blood
0 1992 the American
Physiological
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(27 ml) previously collected in a heparinized syringe by cardiac puncture from two adult male Sprague-Dawley rats anesthetized with ether. The perfusate pH was maintained between 7.30 and 7.42, and the temperature of the recirculating perfusate and lungs was maintained at 38°C. Pulmonary arterial (P& and venous pressures were continuously monitored with Cope pressure transducers (model 040-500-503) and recorded on a Grass polygraph recorder (model 7E). The peak increase in P,, in response to a given stimulus was measured. Specific Protocols Effects of FSK and 8-BrcAMP on HPR. Lungs were isolated from 36 rats and perfused with whole blood. After a 30-min period of equilibration the lungs were challenged with 0.1 ,ug ANG II injected as a bolus into the pulmonary artery to assess the ANG II pressor response during normoxia. Ten minutes after the ANG II challenge, the inspired gas mixture (95% air5% COa) was changed to a 3% 02-5% C02-balance N2 mixture and ventilated for IO min to assess the HPR. The peak increase in P,, in response to ANG II and hypoxia was recorded. In each study there were three sets of ANG II and hypoxic challenges. Each hypoxic and ANG II challenge was separated by a IO-min period of equilibration. The lungs were subsequently divided into groups that were administered varying concentrations of FSK or &BrcAMP, and dose-response data were obtained. In the FSK treatment groups, a loo-p1 bolus of FSK was injected into the pulmonary artery during the plateau of the second HPR, which resulted in final perfusate concentrations of 0.1 (n = 9), 0.5 (n = 5), 1.0 (n = 5), or 10 PM (n = 4). In the 8 BrcAMP treatment groups, final perfusate concentrations of 0.1 (n = 5), 0.5 (n = 5), and 1 mM (n = 4) were assessed using the protocol as described above. Any decrease in the P,, from the plateau of the HPR observed at 6 min following FSK and 8-BrcAMP was recorded and compared. The 6-min time point was chosen because of the different kinetics of relaxation seen with FSK and 8BrcAMP. With FSK, there was an immediate decrease in P,, that plateaued, whereas with 8-BrcAMP the decrease in P,, was gradual and plateaued at -6 min. Based on the data obtained from these studies, all subsequent studies were performed using 0.5 PM FSK and 0.5 mM 8-BrcAMP unless otherwise indicated. Effects of R,-CAMPS on HPR. To test the hypothesis that CAMP decreases pulmonary vasoreactivity through the activation of PKA, the effect of PKA inhibition by &,-CAMPS (1), was assessed. In control (no drug intervention, n = 8), 0.5 PM FSK (n = 5), 0.5 mM &-CAMPS + 0.5 PM FSK (n = 4), 0.5 mM 8-BrcAMP (n = 5), and 0.5 mM &-CAMPS + 0.5 mM 8BrcAMP (n = 5) treatment groups, the peak P,, of the second HPR was compared with the peak P,, of the third HPR using the same protocol as above. In the &-CAMPS groups, R,CAMPS was added to the perfusate reservoir 15 min before the second hypoxic challenge. In addition, the second HPR in lungs pretreated with &-CAMPS was compared with the second HPR in lungs not treated with &-CAMPS. Other potential mechanisms involved in FSK effect on HPR. Although FSK may increase intracellular CAMP levels by directly activating adenylate cyclase (24), it may also exert effects through G proteins, which can result in direct or indirect gating of K+ channels (2, 7, 14), and/or by increasing the production of prostacyclin (PGI& and PAF through the activation of phospholipase A2 (PLAJ (3, 8, 11, 20). By use of the same protocol (see above: Effects of FSK and 8-BrcAMP on HPR), 1,9-dideoxy-FSK (0.5 PM, n = 4), which is biologically inactive on adenylate cyclase but does alter K+ channel activity (7, 14), was studied. Its effect on the ANG II responses and HPRs were compared with the ANG II responses and HPRs of control and FSK (0.5 PM)-treated groups. Subsequent studies were per-
VASODILATION
formed that assessed FSK-mediated attenuation of the HPR at 0.1 PM. By use of the same protocol (see above: Effects of FSK and 8-BrcAMP on HPR), the groups assessed were as follows: FSK alone (n = 9), FSK + R,-CAMPS (0.5 mM, n = 5), FSK + glibenclamide (0.1 mM, n = 4), FSK + TEA (10 mM,n= 5), FSK + indomethacin (0.1 mM, n = 6), and FSK + SRI 63-441 (0.26 mM, n = 4). TEA and indomethacin were added to the perfusate reservoir during the equilibration period prior to the initial ANG II challenge. R,-CAMPS, glibenclamide, and SRI 63-441 were added to the perfusate reservoir 5 min before the second ANG II challenge. An additional group was assessed that compared the decrease in the P,, from the plateau of the second HPR observed with 8-BrcAMP (0.1 mM) to the 0.1 mM 8-BrcAMP effect in lungs pretreated with TEA (10 mM, n = 5). Statistics The results are presented as means t SE. Statistical analyses were performed using the paired and unpaired Student’s t test and one-way analysis of variance (ANOVA). Tukey’s test (30) was used for multiple comparisons when ANOVA indicated statistically significant differences between groups. Differences were considered to be significant when P c 0.05. RESULTS
Effect of FSK and B-BrcAMP Blood-Perfused Rat Lungs
on HPR in Isolated
Figures 1 and 2 demonstrate that FSK (0.1-10 PM) and 8-BrcAMP (0.1-l mM) dose-dependently attenuate the HPR. In the 0.1 PM and IO PM FSK-treated groups, the magnitudes of the decrease in P,, were signi ficantly different from each other (P < 0.05) and from tl le 0.5-l
FSKI .lpM
.5pM
WM
1 OpM
* Fig. 1. Immediate pulmonary arterial pressure (P,,) response to forskolin (FSK) at 0.1, 0.5, 1.0, or 10 PM during acute hypoxic [fractional concentration of O2 in inspired gas (FIO~,) = 0.031 pulmonary vasoconstriction in isolated blood-perfused rat lung. *p < 0.05 compared with all groups.
[8Br-CAMP] .lmM
E 0 w
.SmM
1mM
-8
n-
a -18
Fig. 2. Immediate P,, response to 8-bromoadenosine 3’,5’-cyclic monophosphate (8BrcAMP) at 0.1, 0.5, or 1 mM during acute hypoxic = 0.03) pulmonary vasoconstriction in isolated blood-perfused Woo, rat lung. *p < 0.01 compared with all groups.
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MECHANISM
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PM groups. The decreases in P,, seen in lungs treated with FSK (0.5 and 1 PM) were not significantly different. In the 8-BrcAMP group, the decrease in P,, in lungs treated with 1 mM 8-BrcAMP was significantly (P C 0.01) greater than the decrease observed in the 0.1 and 0.5 mM treated groups. The difference between the decrease in P,, seen with 0.1 mM and 0.5 mM 8-BrcAMP was not significant. Effects of R,-CAMPS on FSK- and 8-BrcAMP-Mediated Decrease in Pulmonary Vasoreactivity in Response to Alveolar Hypoxia Using an intracellular inhibitor of PKA, R,-CAMPS, the role of PKA in mediating the relaxant effect of FSK and 8-BrcAMP was assessed.Figure 3 demonstrates that the first HPR in each group was not significantly different. Figure 3 also compares the second HPR to the third HPR (see Specific Protocols) in control, FSK (0.5 PM)-, and 8-BrcAMP (0.5 mM)-treated groups, and in groups where lungs were pretreated with R,-CAMPS (0.5 mM) before receiving FSK or 8-BrcAMP. In control lungs, the second and third HPR were 39.4 t 3 and 41.4 t 2 cmH20, respectively, and were not significantly different. In contrast, there was a significant decrease (P c 0.01) in the third HPR compared with the second HPR in lungs treated with FSK and 8-BrcAMP. In lungs that received &-CAMPS before FSK administration, the decrease in the third HPR remained significant (P < 0.01) compared with the second HPR. In contrast, R,-CAMPS blocked the previously observed decrease in the third HPR compared with the second HPR in the 8-BrcAMP group. The decrease in pulmonary vasoreactivity with FSK and B-BrcAMP was not specific for the HPR but was also observed with the ANG II pressor response (data not shown). Because the first HPRs in each group were not significantly different, the second HPRs in lungs that did not receive R,-CAMPS were grouped and compared with those that did receive R,-CAMPS. There was a
H513
VASODILATION
significant (P < 0.01) increase (40.8 t 2 to 51.5 t 3 cmHzO) in the HPR of lungs pretreated with R,-CAMPS. Other Potential Mechanisms Involved in FSK-Mediated Decrease in Pulmonary Vasoreactivity Because the R,-CAMPS concentration (0.5 mM) may not have been adequate to block the FSK (0.5 PM)induced decrease in the HPR and because FSK may also gate K+ channels (7, 14) and/or increase phospholipase A, (PLA,) activity, (20) additional studies to assessthis effect(s) at a lower effective dose of FSK (0.1 PM) were performed. In pulmonary vascular beds precontracted with a hypoxic gas mixture, 0.1 PM FSK resulted in a decrease in P,, of 6 t 0.8 cmH20. Under the same hypoxic conditions, the decrease in P,, observed with 0.1 PM FSK alone was compared with the decrease in P,, seen with 0.1 PM FSK in lungs that had been pretreated with R,-CAMPS, indomethacin, SRI 63-441, glibenclamide, and TEA. Of the groups studied, only lungs pretreated with TEA resulted in a significant (P < 0.05) attenuation of the FSK-induced decrease in P,, (Fig. 4). In the SRI 63-441 pretreatment group, there was a significantly greater (P < 0.05) decrease in the P,, seen with FSK compared with FSK alone (Table 1). In addition, the effect(s) of 1,9-dideoxy-FSK on the ANG II response and HPR was compared with the effect of FSK and controls. FSK, but not the 1,9dideoxy derivative, caused a significant decrease (P < 0.05) in the P,, in lungs contracted with the hypoxic gas mixture and ANG II (Fig. 5, A and B). To assess whether cAMP/PKA played a role in the observed TEA attenuation of FSKmediated decrease in pulmonary vasoreactivity, 8BrcAMP-mediated relaxation was assessedin the presence of TEA. In this group TEA almost completely FSK + TEA -0.5 s
- -
-1.5..
I"
-2.3.. E 0
-J.S*-
E
-5.5 -4.5..
* -
a -6.5..
L
-7.51
Fig. 4. Tetraethylammonium chloride (TEA; 10 yM final perfusate concentration) attenuates FSK (0.1. PM final perfusate concentration) mediated relaxation in pulmonary vascular bed precontracted with a with FSK. hypoxic gas mixture (FIOO, = 0.03). *P < 0.01 compared
Table 1. Agents not effective in attenuating forskolin-mediated pulmonary vasodilation Group Fig. 3. Three sequential hypoxic pressor responses (HPR) observed in control lungs (no drug intervention; n = 8) and in lungs treated with either FSK (0.5 PM; n = 5), the R, diastereoisomer of adenosine 3’,5’cyclic phosphorothioate (R,-CAMPS, 0.5 mM) + 0.5 PM FSK (n = 4), 8-BrcAMP (0.5 mM; n = 5), or R,-CAMPS + 8-BrcAMP (n = 5). Closed bar, first HPR; open bar, second HPR; and hatched bar, third HPR. FSK and 8-BrcAMP were administered as bolus injections into pulmonary artery during plateau of second HPR. In lung receiving R,CAMPS, R,-CAMPS was added to perfusate reservoir 15 min before second HPR. *P < 0.01 for HPR 3 comnared with HPR 2.
FSK FSK FSK FSK FSK
+ 500 ,uM R,-CAMPS + 100 ELM INDO + 260 PM SRI + 100 pM GLI
n
Ppa, cm&O
9
-6t0.8 -6t0.9 -9t0.8 -11*0.2* -6kl
5 6 6 4
For each group, Values are means t SE; n = no. of experiments. dose of forskolin (FSK) was 0.1 PM. Ppa, pulmonary arterial pressure; R,-CAMPS, R, diastereomer of adenosine 3’,5’-cyclic phosphorothioate; INDO, indomethacin; SRI, SRI 63-441; and GLI, glibenclamide. * P < 0.05 compared with FSK alone.
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H514
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OF
CAMP-MEDIATED
VASODILATION FSK, CAMP,
P-agonist
ddFSK z
125
E 100 E CL
75
#2
#l
PGI, I v Adenylate Cyclase
#3
\
HYPOXIC PRESSOR RESPONSE
CONTROL
4 t CAMP
\
ddFSK
(Rp)-CAMPS II II PG r
FSK
$
+
tCa+ +
+Ca+ + TEA
ANGIOTENSIN
II PRESSOR
RESPONSE
Fig. 5. FSK (0.5 PM), but not 1,9-dideoxy-FSK (ddFSK, 0.5 PM), attenuates the hypoxic (A) and ANG II (B) pressor responses in isolated blood-perfused rat lung. Arrows show point of FSK or ddFSK administration. Data are as percent of initial response. *p < 0.05 compared with control and ddFSK. Note that no immediate decrease in P,, was observed with ddFSK as reported with FSK in Fig. 1. BBr-CAMP
BBr-CAMP
71
K’ channel Fig. 7. Potential mechanisms of CAMP-mediated relaxation of smooth muscle. LTs, leukotrienes; PG12, prostacyclin; PAF, platelet-activating factor; PKA, CAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; &-CAMPS, a PKA inhibitor; and TEA, a nonselective K+ channel blocker.
+
TEA
a t -6.5
Fig. 6. TEA (10 PM) attenuates 8-BrcAMP (0.1 mM)-mediated relaxation in pulmonary vascular beds precontracted with a hypoxic gas mixture (FIN, = 0.03). *P < 0.01 compared with 8-BrcAMP.
blocked 8-BrcAMP-mediated (Fig. 6).
$ Ca++-activated
attenuation
of the HPR
DISCUSSION
A number of potential mechanisms can mediate CAMP relaxation of smooth muscle as outlined in Fig. 7. For example, agents that increase intracellular CAMP levels may lead to changes in PLA2 activity (20) and/or protein kinase(s) with subsequent effects on the concentration of cytosolic Ca2+ (16). During this cascade of events, other known potential vasodilators are produced such as prostacyclin (PG12) (8, 29) and PAF (11, 29), with the former initiating its effect at least in part by increasing adenylate cyclase activity (26). In addition, the gating of K+ channels may be involved (10, 17). In the present study, we assessedthe role of PKA in CAMP-dependent relaxation of pulmonary vascular beds precontracted with alveolar hypoxia using FSK, which
activates adenylate cyclase and increases intracellular CAMP (23, 24), 8-BrcAMP, which is relatively selective in activating PKA (6, 13), and &-CAMPS, a PKA inhibitor (1). The major findings in this study are 1) FSK and 8-BrcAMP dose dependently attenuate the HPR, 2) R,CAMPS blocks the relaxation effect of 8-BrcAMP but not that of FSK in pulmonary vascular beds precontracted with alveolar hypoxia, 3) &-CAMPS potentiates the HPR, and 4) the nonspecific K+ channel blocker TEA almost completely blocked the 8-BrcAMP-mediated attenuation of the HPR. A criterion used in assigning a role for intracellular CAMP as a mediator of a particular response is the ability of CAMP and its analogues, such as 8-BrcAMP, to mimic the response when administered extracellularly (21). 8BrcAMP is a CAMP analogue that effectively activates PKA but is much less effective in activating PKG (6, 13). Thus use of this relatively selective CAMP analogue allowed us to assessthe role of PKA in CAMP-dependent relaxation of smooth muscle. In this study, the findings of increased pulmonary vasoreactivity in response to alveolar hypoxia and the attenuation of the decrease in P,, during the HPR by 8-BrcAMP in lungs pretreated with &-CAMPS support a role for PKA in CAMP-mediated relaxation of vascular smooth muscle. In contrast to 8-BrcAMP, &CAMPS did not block FSK (0.5 PM)-mediated relaxation of the HPR. Why this difference occurred is not clear, but possible explanations include the following: 1) the intracellular CAMP level obtained with FSK was greater than that obtained with 8-BrcAMP, and thus the concentration of R,-
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MECHANISM
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CAMP-MEDIATED
CAMPS used was insufficient to antagonize the FSKmediated increase in intracellular CAMP and activation of PKA; 2) FSK permanently activates adenylate cyclase, and consequently the CAMP turnover is maintained in contrast to 8BrcAMP hydrolysis by CAMP phosphodiesterase(s); 3) the affinities of endogenous CAMP and 8BrcAMP for PKA could be different; 4) there was poor membrane permeability of &,-CAMPS; or 5) the mechanism(s) involved was independent of PKA. Complete dose-response data could not be obtained with this intracellular antagonist of CAMP because of a lack of its availability, but even at a lower concentration (0.1 PM) of FSK, &-CAMPS did not block FSK-mediated relaxation (Table 1). Because &-CAMPS treatment did attenuate %BrcAMP-mediated relaxation, neither inadequate concentration nor poor membrane permeability of the antagonist adequately explains the lack of effect of &-CAMPS in FSK-mediated relaxation, but the involvement of mechanisms independent of PKA is suggested. CAMP and its analogues can activate both PKA and PKG; however, relaxation of smooth muscle correlates best with PKG activation (6). The lack of effect with R,CAMPS in FSK-treated lungs may be due in part to the activation of PKG by increased intracellular CAMP in response to FSK. Several recent studies support this concept and suggest that agents such as FSK can activate PKG by increasing intracellular CAMP levels (6, 13, 16). A second potential PKA-independent mechanism for FSK-mediated relaxation is the gating of ionic channels. FSK can activate adenylate cyclase by postreceptor activation of a G stimulatory protein or by directly activating its catalytic unit (23,24). G proteins can indirectly gate ionic channels [i.e., Ca2+-activated K+ channels (K&J] using effecters such as adenylate cyclase (2) or by directly altering K+ channels independent of adenylate cyclase activation (7,14). Because activation of K+ channels has been found to mediate a pulmonary vasodilator response (17), we could not exclude this as a possibility for the FSK effect observed in our study. To assess a possible direct FSK effect, 1,9-dideoxy-FSK, which is unable to activate adenylate cyclase but does alter K+ channels indistinguishably from FSK (7, 14), was studied. This FSK derivative did not decrease pulmonary vasoreactivity, suggesting that a direct effect of FSK on K+ channels was not involved. The ATP-sensitive K+ channel antagonist glibenclamide attenuates pulmonary vasodilation to endothelin isopeptides in the cat (17). In our study, glibenclamide did not attenuate FSK-mediated vasodilation in pulmonary vascular beds precontracted with an hypoxic gas mixture. However, the nonselective K+ channel blocker TEA did attenuate FSKmediated pulmonary vasodilation, as has been observed for histamine-mediated pulmonary vasodilation in the rat lung (10). In this study, TEA also decreased 8 BrcAMP-mediated vasodilation. This suggests that the activation of adenylate cyclase by FSK indirectly gates a non-ATP-sensitive K+ channel through a PKA-dependent phosphorylation. Such an effect has not been previously described in vascular smooth muscle, but it has been demonstrated that FSK can activate a K& channel in cultured kidney cells (9) and that TEA-
Hi515
VASODILATION
sensitive K+ channels are important in maintaining the normal quiescent state of airway smooth muscle (15). Although speculative, phosphorylation of Ca2+ channels by PKA could lead to the activation of K& channels, hyperpolarization of the vascular smooth muscle membrane, and vasorelaxation (Fig. 7). These findings, along with the lack of effects of the FSK derivative and glibenclamide, suggest that the FSK effect is in part mediated indirectly by a non-ATP-sensitive K+ channel. A third potential PKA-independent mechanism for FSK-mediated relaxation involves the vasodilator products of membrane phospholipid hydrolysis, PAF and PGI2 (3, 8, 11, 20, 29) (Fig. 7). However, this mechanism is not supported by this study, because neither the cyclooxygenase blocker indomethacin nor the PAF receptor antagonist SRI 63-441 blocked FSK-mediated pulmonary vasodilation. In conclusion, our results support important roles for PKA and indirect gating of a K& channel in the mediation of smooth muscle relaxation induced by CAMP. We thank Sandy Mead for preparation of this manuscript and Bonnie White and Sandy Worley for graphic support. We also thank Dr. Thomas M. Lincoln for constructive comments regarding the manuscript. This work was supported by a grant from the American Heart Association, Alabama Affiliate, by National Heart, Lung, and Blood Institute Clinical Investigator Award HL-02352, and by a Florence Foundation Research Career Development Grant to J. Haynes, Jr. Received
18 June
1991; accepted
in final
form
26 September
1991.
REFERENCES 1. Botelho, L. H. P., J. D. Rothermel, R. V. Coombs, and B. Jastorff. CAMP analog antagonists of CAMP action. lMethods EnzymoZ. 159: 159-172, 1988. 2. Brown, A. M., and L. Birnbaumer. Ion channels and G proteins. Hosp. Pratt. 24: 189-204, 1989. 3. Chang, J., J. H. Musser, and H. McGregor. Phospholipase AZ: function and pharmacological regulation. Biochem. PharmacoZ. 36: 2429-2436,1987. 4. Conti, M. A., and R. S. Adelstein. The relationship between calmodulin binding and phosphorylation of smooth muscle myosin kinase by the catalytic subunit of 3’:5’ CAMP-dependent protein kinase. J. Biol. Chem. 256: 3178-3181, 1981. 5. Cook, N. S. The pharmacology of potassium channels and their therapeutic potential. Trends Pharmacol. Sci. 9: 21-28, 1988. 6. Francis, S. H., B. D. Noblett, B. W. Todd, J. N. Wells, and J. D. Corbin. Relaxation of vascular and tracheal smooth muscle by cyclic nucleotide analogs that preferentially activate purified cGMP-dependent protein kinase. MOL. Pharmacol. 34: 506-517, 1988. 7. Garber, S. S., T. Hoshi, and R. W. Aldrich. Interaction of forskolin with voltage-gated K+ channels in PC12 cells. J. Neurosci. 10: 3361-3368,199O. J. G., N. Voelkel, A. S. Nies, I. F. McMurtry, and 8. Gerber, J. T. Reeves. Moderation of hypoxic vasoconstriction by infused arachidonic acid: role of PG12. J. AppZ. Physiol. 49: 107-112, 1980. 9. Guggino, S. E., B. A. Suarez-Isla, W. B. Guggino, and B. Sacktor. Forskolin and antidiuretic hormone stimulate a Ca2+activated K+ channel in cultured kidney cells. Am. J. Physiol. 249 (Renal Fluid Electrolyte Physiol. 18): F448-F455, 1985. 10. Hasunuma, K., T. Yamaguchi, D. M. Rodman, R. F. O’Brien, and I. F. McMurtry. Effects of inhibitors of EDRF and EDHF on vasoreactivity of perfused rat lungs. Am. J. Physiol. 260 (Lung CeZZ MOL. Physiol. 4): L97-L104, 1991. 11. Haynes, J., S. W. Chang, K. G. Morris, and N. F. Voelkel. Platelet-activating factor antagonists increase vascular reactivity in perfused rat lungs. J. AppZ. Physiol. 65: 1921-1928, 1988. 12. Haynes, J., P. A. Kithas, A. E. Taylor, and S. J. Strada.
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Selective inhibition of cGMP-inhibitable CAMP phosphodiesterase decreases pulmonary vasoreactivity. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H487-H492, 1991. Hei, Y., K. L. MacDonell, J. H. McNeil, and J. Diamond. Lack of correlation between activation of cyclic AMP-dependent protein kinase and inhibition of contraction of rat vas deferens by cyclic AMP analogs. Mol. PharmacoZ. 39: 233-238, 1991. Hoshi, T., S. S. Garber, and R. W. Aldrich. Effect of forskolin on voltage-gated K+ channels is independent of adenylate cyclase activation. Science Wash. DC 240: 1652-1655, 1988. Kotlikoff, M. I. Potassium currents in canine airway smooth muscle cells. Am. J. Physiol. 259 (Lung Cell Mol. Physiol. 3): L384L395,1990. Lincoln, T. M., T. L. Cornwell, and A. E. Taylor. cGMPdependent protein kinase mediates the reduction of Ca2+ by CAMP in vascular smooth muscle cells. Am. J. Physiol. 258 (Cell Physiol. 27): C399-C407,1990. Lippton, H. L., G. A. Cohen, I. F. McMurtry, and A. L. Hyman. Pulmonary vasodilation to endothelin isopeptides in vivo is mediated by potassium channel activation. J. AppZ. Physiol. 70: 947-952,199l. Miller, J. R., P. J. Silver, and J. T. Stull. The role of myosin light chain kinase phosphorylation in beta-adrenergic relaxation of tracheal smooth muscle. MOL. PharmacoZ. 24: 235-242,1983. Nelson, M. T., J. B. Patlak, J. F. Worley, and N. B. Standen. Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone. Am. J. Physiol. 259 (Cell Physiol. 28): C3-Cl8,1990. Pfeilschifter, J., W. Pignat, F. Mark, and I. Wiesenberg. Release of phospholipase A2 activity from rat vascular smooth
21. 22.
23.
24.
25.
26.
27.
28. 29.
30.
VASODILATION muscle cells mediated by CAMP. Eur. J. Biochem. 181: 237-242, 1989. Robinson, G. A., R. W. Butcher, and E. W. Sutherland. Cyclic AMP. New York: Academic, 1971, p. 72-90. Sakai, A., and N. F. Voelkel. Dibutyryl cyclic adenosine monophosphate inhibits pulmonary vasoconstriction. Lung 166: 22l231,1988. Scarpace, P. J. Forskolin activation of adenylate cyclase in rat myocardium with age: effects of guanine nucleotide analogs. Mech. Ageing Deu. 52: 169-178,199O. Seamon, K., and J. W. Daly. Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulatory protein. J. BioZ. Chem. 256: 9799-9801, 1981. Seamon, K. B., J. W. Daly, H. Metzger, N. J. desouza, and J. Reden. Structure-activity relationships for activation of adenylate cyclase by the diterpene forskolin and its derivatives. J. Med. Chem. 26: 436-439,1983. Shaul, P., R. Magness, M. Moore, and L. M. Buja. Regulation of adenylate cyclase activity by prostacyclin in pulmonary and systemic arteries of the rat (Abstract). FASEB J. 3: A569, 1989. Takuwa, Y., N. Takuwa, and H. Rasmussen. The effects of isoproterenol on intracellular calcium concentration. J. BioZ. Chem. 263: 762-768,1988. Voelkel, N. F. Drug effects on the pulmonary circulation: an overview. Semin. Respir. Med. 7: 141-146, 1985. Voelkel, N. F., S. W. Chang, T. J. McDonnell, J. Y. Westcott, and J. Haynes. Role of membrane lipids in the control of normal vascular tone. Am. Rev. Respir. Dis. 136: 214-217, 1987. Zar, J. H. StatisticaL Analysis. Englewood Cliffs, NJ: PrenticeHall, 1974, p. 153.
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kinase
JOHNSON HAYNES, JR., JULIET ROBINSON, LETETIA SAUNDERS, AUBREY E. TAYLOR, AND SAMUEL J. STRADA Pulmonary and Critical Care Division, Departments of Medicine, Physiology and Pharmacology, University of South Alabama, College of Medicine, Mobile, Alabama 36688 Haynes, Johnson, Jr., Juliet Robinson, Letetia Saunders, Aubrey E. Taylor, and Samuel J. Strada. Role of CAMP-dependent protein kinase in CAMP-mediated vasodilation. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H511-H516, 1992.-In this study, the role of adenosine 3’,5’-cyclic monophosphate (CAMP)-dependent protein kinase A (PKA) in CAMP-dependent relaxation was assessed in the isolated-perfused rat lung using a PKA inhibitor, &-CAMPS, &bromoCAMP (&BrcAMP), and the diterpene activator of adenylate cyclase (AC), forskolin (FSK). A role for K’ channels was also assessed with the nonselective K+ channel blocker, tetraethylammonium (TEA, 10 mM), and an ATP-sensitive K+ channel inhibitor, glibenclamide (GLI, 100 PM). Both 8-BrcAMP (O.l1.0 mM) and FSK (0.1-10 PM) dose-dependently attenuated the peak pressor response to alveolar hypoxia (HPR). RpCAMPS potentiated the HPR and attenuated B-BrcAMP-mediated vasodilation but had no effect on FSK-mediated vasodilation. FSK-mediated vasodilation was not mimicked by 1,9dideoxy-FSK, which is biologically inactive on AC but alters K+ channels identically to FSK, nor was it attenuated by the platelet-activating factor antagonist SRI 63-441 or the cyclooxygenase inhibitor indomethacin. TEA, but not GLI, attenuated FSK-mediated vasodilation. Similarly, TEA attenuated 8BrcAMP-mediated vasodilation. These results support roles for PKA and indirect gating of a non-ATP-sensitive K+ channel in mediating CAMP-dependent pulmonary vasodilation. pulmonary circulation; adenosine 3’,5’-cyclic monophosphate; adenosine 3’,5’-cyclic monophosphate-dependent protein kinase; hypoxic vasoconstriction; pulmonary vasodilation; potassium channels RELAXATION OFCONTRACTEDVASCULAR smoothmuscle by ,&adrenergic agents and other agents that activate adenylate cyclase, such as forskolin (FSK), are thought to produce their biological effect by increasing the intracellular concentration of adenosine 3’,5’-cyclic monophosphate (cAMP)(4, 22-28). We have previously demonstrated that N6, 02-dibutyryl-CAMP (DBcAMP) and the selective phosphodiesterase inhibitor indolidan will relax pulmonary vascular beds precontracted with alveolar hypoxia (12). Although the precise mechanism by which CAMP causes relaxation in smooth muscle is not known, it is generally accepted that the activation of CAMP-dependent protein kinase (PKA) is involved (4, 13, 27). Several recent studies have challenged the concept that PKA is not the sole mediator of CAMP-dependent relaxation of vascular smooth muscle (13, 16, 27). Some studies have concluded that CAMP-dependent effects on intracellular Ca2+ may be more important in mediating relaxation (18, 27), whereas others have suggested that relaxation involves the activation of cGMPdependent protein kinase (PKG) (13, 16). Hei et al. (13) compared the effects of CAMP analogues on tension and PKA activities in the rat vas deferens and demonstrated 0363-6135/92
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that 8bromo-CAMP (8BrcAMP), which relatively selectively activates PKA, does not inhibit phenylephrineinduced contractions of vas deferens. In contrast, DBcAMP, a less potent activator of PKA than 8BrcAMP, does relax the phenylephrine-contracted vas deferens. They concluded that PKA is not responsible for the relaxant effect of CAMP analogues, at least in vas deferens smooth muscle. The objective of this study was to test whether the CAMP attenuation of the hypoxic pressor response (HPR) is mediated via PKA. Our findings suggest that PKA does play a role in CAMP-dependent relaxation in pulmonary vascular beds precontracted with alveolar hypoxia but that other mechanisms involving a non-ATP-sensitive K+ channel and possibly PKG are also involved. MATERIALS
AND
METHODS
Materials All materials were obtained from Sigma Chemical (St. Louis, MO) unless otherwise specified. Angiotensin II (ANG II), 8BrcAMP, the nonselective K’ channel antagonist tetraethylammonium chloride (TEA) (5,10,19), and the platelet-activating factor (PAF) antagonist SRI 63-441 (a generous gift from Dr. W. S. Houlihan, Sandoz Research Institute, East Hanover, NJ) (11) were dissolved in normal saline. The R, diastereoisomer of adenosine 3’,5’-cyclic phosphorothioate (&-CAMPS; also a generous gift from Dr. W. S. Houlihan), which is an intracellular antagonist of PKA, (1) and the cyclooxygenase inhibitor sodium indomethacin trihydrate (Merck Sharpe and Dohme Research Laboratories, West Point, PA) were dissolved in distilled water. The ATP-sensitive K+ channel antagonist glibenclamide (19) was dissolved in 0.25% dimethyl sulfoxide with 0.0002 N NaOH. The diterpene activator of adenylate cyclase FSK (7, 24) and 1,9-dideoxy-FSK (7,14) were dissolved in 0.001% ethanol. Isolated Perfused
Lung
Male Sprague-Dawley rats (260-320 g body wt), were anesthetized with pentobarbital sodium (20-25 mg ip), and the lungs were removed for extracorporeal perfusion. After placement of a tracheal cannula, the lungs were ventilated with a Harvard rodent ventilator (model 683) at 55 breaths/min, a tidal volume of 2.5 ml, and positive-end expiratory pressure of 2 cmHtLO using a gas mixture of 95% air-5% COa (normoxic gas). During the hypoxic stimuli, the lungs were ventilated with a 3% 02-5% C02-balance NP (hypoxic) gas mixture. A median sternotomy was then performed; then heparin (100 IU) was injected into the right ventricle, and cannulas were placed into the pulmonary artery and left ventricle. The heart, lungs, and mediastinal structures were removed en bloc and placed into a humidified chamber. The lungs were perfused by a Gilson Minipuls 2 peristaltic pump at a constant flow of 0.03 ml-g body wt-l. min. Lungs were perfused with homologous blood
0 1992 the American
Physiological
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H511
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(27 ml) previously collected in a heparinized syringe by cardiac puncture from two adult male Sprague-Dawley rats anesthetized with ether. The perfusate pH was maintained between 7.30 and 7.42, and the temperature of the recirculating perfusate and lungs was maintained at 38°C. Pulmonary arterial (P& and venous pressures were continuously monitored with Cope pressure transducers (model 040-500-503) and recorded on a Grass polygraph recorder (model 7E). The peak increase in P,, in response to a given stimulus was measured. Specific Protocols Effects of FSK and 8-BrcAMP on HPR. Lungs were isolated from 36 rats and perfused with whole blood. After a 30-min period of equilibration the lungs were challenged with 0.1 ,ug ANG II injected as a bolus into the pulmonary artery to assess the ANG II pressor response during normoxia. Ten minutes after the ANG II challenge, the inspired gas mixture (95% air5% COa) was changed to a 3% 02-5% C02-balance N2 mixture and ventilated for IO min to assess the HPR. The peak increase in P,, in response to ANG II and hypoxia was recorded. In each study there were three sets of ANG II and hypoxic challenges. Each hypoxic and ANG II challenge was separated by a IO-min period of equilibration. The lungs were subsequently divided into groups that were administered varying concentrations of FSK or &BrcAMP, and dose-response data were obtained. In the FSK treatment groups, a loo-p1 bolus of FSK was injected into the pulmonary artery during the plateau of the second HPR, which resulted in final perfusate concentrations of 0.1 (n = 9), 0.5 (n = 5), 1.0 (n = 5), or 10 PM (n = 4). In the 8 BrcAMP treatment groups, final perfusate concentrations of 0.1 (n = 5), 0.5 (n = 5), and 1 mM (n = 4) were assessed using the protocol as described above. Any decrease in the P,, from the plateau of the HPR observed at 6 min following FSK and 8-BrcAMP was recorded and compared. The 6-min time point was chosen because of the different kinetics of relaxation seen with FSK and 8BrcAMP. With FSK, there was an immediate decrease in P,, that plateaued, whereas with 8-BrcAMP the decrease in P,, was gradual and plateaued at -6 min. Based on the data obtained from these studies, all subsequent studies were performed using 0.5 PM FSK and 0.5 mM 8-BrcAMP unless otherwise indicated. Effects of R,-CAMPS on HPR. To test the hypothesis that CAMP decreases pulmonary vasoreactivity through the activation of PKA, the effect of PKA inhibition by &,-CAMPS (1), was assessed. In control (no drug intervention, n = 8), 0.5 PM FSK (n = 5), 0.5 mM &-CAMPS + 0.5 PM FSK (n = 4), 0.5 mM 8-BrcAMP (n = 5), and 0.5 mM &-CAMPS + 0.5 mM 8BrcAMP (n = 5) treatment groups, the peak P,, of the second HPR was compared with the peak P,, of the third HPR using the same protocol as above. In the &-CAMPS groups, R,CAMPS was added to the perfusate reservoir 15 min before the second hypoxic challenge. In addition, the second HPR in lungs pretreated with &-CAMPS was compared with the second HPR in lungs not treated with &-CAMPS. Other potential mechanisms involved in FSK effect on HPR. Although FSK may increase intracellular CAMP levels by directly activating adenylate cyclase (24), it may also exert effects through G proteins, which can result in direct or indirect gating of K+ channels (2, 7, 14), and/or by increasing the production of prostacyclin (PGI& and PAF through the activation of phospholipase A2 (PLAJ (3, 8, 11, 20). By use of the same protocol (see above: Effects of FSK and 8-BrcAMP on HPR), 1,9-dideoxy-FSK (0.5 PM, n = 4), which is biologically inactive on adenylate cyclase but does alter K+ channel activity (7, 14), was studied. Its effect on the ANG II responses and HPRs were compared with the ANG II responses and HPRs of control and FSK (0.5 PM)-treated groups. Subsequent studies were per-
VASODILATION
formed that assessed FSK-mediated attenuation of the HPR at 0.1 PM. By use of the same protocol (see above: Effects of FSK and 8-BrcAMP on HPR), the groups assessed were as follows: FSK alone (n = 9), FSK + R,-CAMPS (0.5 mM, n = 5), FSK + glibenclamide (0.1 mM, n = 4), FSK + TEA (10 mM,n= 5), FSK + indomethacin (0.1 mM, n = 6), and FSK + SRI 63-441 (0.26 mM, n = 4). TEA and indomethacin were added to the perfusate reservoir during the equilibration period prior to the initial ANG II challenge. R,-CAMPS, glibenclamide, and SRI 63-441 were added to the perfusate reservoir 5 min before the second ANG II challenge. An additional group was assessed that compared the decrease in the P,, from the plateau of the second HPR observed with 8-BrcAMP (0.1 mM) to the 0.1 mM 8-BrcAMP effect in lungs pretreated with TEA (10 mM, n = 5). Statistics The results are presented as means t SE. Statistical analyses were performed using the paired and unpaired Student’s t test and one-way analysis of variance (ANOVA). Tukey’s test (30) was used for multiple comparisons when ANOVA indicated statistically significant differences between groups. Differences were considered to be significant when P c 0.05. RESULTS
Effect of FSK and B-BrcAMP Blood-Perfused Rat Lungs
on HPR in Isolated
Figures 1 and 2 demonstrate that FSK (0.1-10 PM) and 8-BrcAMP (0.1-l mM) dose-dependently attenuate the HPR. In the 0.1 PM and IO PM FSK-treated groups, the magnitudes of the decrease in P,, were signi ficantly different from each other (P < 0.05) and from tl le 0.5-l
FSKI .lpM
.5pM
WM
1 OpM
* Fig. 1. Immediate pulmonary arterial pressure (P,,) response to forskolin (FSK) at 0.1, 0.5, 1.0, or 10 PM during acute hypoxic [fractional concentration of O2 in inspired gas (FIO~,) = 0.031 pulmonary vasoconstriction in isolated blood-perfused rat lung. *p < 0.05 compared with all groups.
[8Br-CAMP] .lmM
E 0 w
.SmM
1mM
-8
n-
a -18
Fig. 2. Immediate P,, response to 8-bromoadenosine 3’,5’-cyclic monophosphate (8BrcAMP) at 0.1, 0.5, or 1 mM during acute hypoxic = 0.03) pulmonary vasoconstriction in isolated blood-perfused Woo, rat lung. *p < 0.01 compared with all groups.
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MECHANISM
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PM groups. The decreases in P,, seen in lungs treated with FSK (0.5 and 1 PM) were not significantly different. In the 8-BrcAMP group, the decrease in P,, in lungs treated with 1 mM 8-BrcAMP was significantly (P C 0.01) greater than the decrease observed in the 0.1 and 0.5 mM treated groups. The difference between the decrease in P,, seen with 0.1 mM and 0.5 mM 8-BrcAMP was not significant. Effects of R,-CAMPS on FSK- and 8-BrcAMP-Mediated Decrease in Pulmonary Vasoreactivity in Response to Alveolar Hypoxia Using an intracellular inhibitor of PKA, R,-CAMPS, the role of PKA in mediating the relaxant effect of FSK and 8-BrcAMP was assessed.Figure 3 demonstrates that the first HPR in each group was not significantly different. Figure 3 also compares the second HPR to the third HPR (see Specific Protocols) in control, FSK (0.5 PM)-, and 8-BrcAMP (0.5 mM)-treated groups, and in groups where lungs were pretreated with R,-CAMPS (0.5 mM) before receiving FSK or 8-BrcAMP. In control lungs, the second and third HPR were 39.4 t 3 and 41.4 t 2 cmH20, respectively, and were not significantly different. In contrast, there was a significant decrease (P c 0.01) in the third HPR compared with the second HPR in lungs treated with FSK and 8-BrcAMP. In lungs that received &-CAMPS before FSK administration, the decrease in the third HPR remained significant (P < 0.01) compared with the second HPR. In contrast, R,-CAMPS blocked the previously observed decrease in the third HPR compared with the second HPR in the 8-BrcAMP group. The decrease in pulmonary vasoreactivity with FSK and B-BrcAMP was not specific for the HPR but was also observed with the ANG II pressor response (data not shown). Because the first HPRs in each group were not significantly different, the second HPRs in lungs that did not receive R,-CAMPS were grouped and compared with those that did receive R,-CAMPS. There was a
H513
VASODILATION
significant (P < 0.01) increase (40.8 t 2 to 51.5 t 3 cmHzO) in the HPR of lungs pretreated with R,-CAMPS. Other Potential Mechanisms Involved in FSK-Mediated Decrease in Pulmonary Vasoreactivity Because the R,-CAMPS concentration (0.5 mM) may not have been adequate to block the FSK (0.5 PM)induced decrease in the HPR and because FSK may also gate K+ channels (7, 14) and/or increase phospholipase A, (PLA,) activity, (20) additional studies to assessthis effect(s) at a lower effective dose of FSK (0.1 PM) were performed. In pulmonary vascular beds precontracted with a hypoxic gas mixture, 0.1 PM FSK resulted in a decrease in P,, of 6 t 0.8 cmH20. Under the same hypoxic conditions, the decrease in P,, observed with 0.1 PM FSK alone was compared with the decrease in P,, seen with 0.1 PM FSK in lungs that had been pretreated with R,-CAMPS, indomethacin, SRI 63-441, glibenclamide, and TEA. Of the groups studied, only lungs pretreated with TEA resulted in a significant (P < 0.05) attenuation of the FSK-induced decrease in P,, (Fig. 4). In the SRI 63-441 pretreatment group, there was a significantly greater (P < 0.05) decrease in the P,, seen with FSK compared with FSK alone (Table 1). In addition, the effect(s) of 1,9-dideoxy-FSK on the ANG II response and HPR was compared with the effect of FSK and controls. FSK, but not the 1,9dideoxy derivative, caused a significant decrease (P < 0.05) in the P,, in lungs contracted with the hypoxic gas mixture and ANG II (Fig. 5, A and B). To assess whether cAMP/PKA played a role in the observed TEA attenuation of FSKmediated decrease in pulmonary vasoreactivity, 8BrcAMP-mediated relaxation was assessedin the presence of TEA. In this group TEA almost completely FSK + TEA -0.5 s
- -
-1.5..
I"
-2.3.. E 0
-J.S*-
E
-5.5 -4.5..
* -
a -6.5..
L
-7.51
Fig. 4. Tetraethylammonium chloride (TEA; 10 yM final perfusate concentration) attenuates FSK (0.1. PM final perfusate concentration) mediated relaxation in pulmonary vascular bed precontracted with a with FSK. hypoxic gas mixture (FIOO, = 0.03). *P < 0.01 compared
Table 1. Agents not effective in attenuating forskolin-mediated pulmonary vasodilation Group Fig. 3. Three sequential hypoxic pressor responses (HPR) observed in control lungs (no drug intervention; n = 8) and in lungs treated with either FSK (0.5 PM; n = 5), the R, diastereoisomer of adenosine 3’,5’cyclic phosphorothioate (R,-CAMPS, 0.5 mM) + 0.5 PM FSK (n = 4), 8-BrcAMP (0.5 mM; n = 5), or R,-CAMPS + 8-BrcAMP (n = 5). Closed bar, first HPR; open bar, second HPR; and hatched bar, third HPR. FSK and 8-BrcAMP were administered as bolus injections into pulmonary artery during plateau of second HPR. In lung receiving R,CAMPS, R,-CAMPS was added to perfusate reservoir 15 min before second HPR. *P < 0.01 for HPR 3 comnared with HPR 2.
FSK FSK FSK FSK FSK
+ 500 ,uM R,-CAMPS + 100 ELM INDO + 260 PM SRI + 100 pM GLI
n
Ppa, cm&O
9
-6t0.8 -6t0.9 -9t0.8 -11*0.2* -6kl
5 6 6 4
For each group, Values are means t SE; n = no. of experiments. dose of forskolin (FSK) was 0.1 PM. Ppa, pulmonary arterial pressure; R,-CAMPS, R, diastereomer of adenosine 3’,5’-cyclic phosphorothioate; INDO, indomethacin; SRI, SRI 63-441; and GLI, glibenclamide. * P < 0.05 compared with FSK alone.
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H514
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VASODILATION FSK, CAMP,
P-agonist
ddFSK z
125
E 100 E CL
75
#2
#l
PGI, I v Adenylate Cyclase
#3
\
HYPOXIC PRESSOR RESPONSE
CONTROL
4 t CAMP
\
ddFSK
(Rp)-CAMPS II II PG r
FSK
$
+
tCa+ +
+Ca+ + TEA
ANGIOTENSIN
II PRESSOR
RESPONSE
Fig. 5. FSK (0.5 PM), but not 1,9-dideoxy-FSK (ddFSK, 0.5 PM), attenuates the hypoxic (A) and ANG II (B) pressor responses in isolated blood-perfused rat lung. Arrows show point of FSK or ddFSK administration. Data are as percent of initial response. *p < 0.05 compared with control and ddFSK. Note that no immediate decrease in P,, was observed with ddFSK as reported with FSK in Fig. 1. BBr-CAMP
BBr-CAMP
71
K’ channel Fig. 7. Potential mechanisms of CAMP-mediated relaxation of smooth muscle. LTs, leukotrienes; PG12, prostacyclin; PAF, platelet-activating factor; PKA, CAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; &-CAMPS, a PKA inhibitor; and TEA, a nonselective K+ channel blocker.
+
TEA
a t -6.5
Fig. 6. TEA (10 PM) attenuates 8-BrcAMP (0.1 mM)-mediated relaxation in pulmonary vascular beds precontracted with a hypoxic gas mixture (FIN, = 0.03). *P < 0.01 compared with 8-BrcAMP.
blocked 8-BrcAMP-mediated (Fig. 6).
$ Ca++-activated
attenuation
of the HPR
DISCUSSION
A number of potential mechanisms can mediate CAMP relaxation of smooth muscle as outlined in Fig. 7. For example, agents that increase intracellular CAMP levels may lead to changes in PLA2 activity (20) and/or protein kinase(s) with subsequent effects on the concentration of cytosolic Ca2+ (16). During this cascade of events, other known potential vasodilators are produced such as prostacyclin (PG12) (8, 29) and PAF (11, 29), with the former initiating its effect at least in part by increasing adenylate cyclase activity (26). In addition, the gating of K+ channels may be involved (10, 17). In the present study, we assessedthe role of PKA in CAMP-dependent relaxation of pulmonary vascular beds precontracted with alveolar hypoxia using FSK, which
activates adenylate cyclase and increases intracellular CAMP (23, 24), 8-BrcAMP, which is relatively selective in activating PKA (6, 13), and &-CAMPS, a PKA inhibitor (1). The major findings in this study are 1) FSK and 8-BrcAMP dose dependently attenuate the HPR, 2) R,CAMPS blocks the relaxation effect of 8-BrcAMP but not that of FSK in pulmonary vascular beds precontracted with alveolar hypoxia, 3) &-CAMPS potentiates the HPR, and 4) the nonspecific K+ channel blocker TEA almost completely blocked the 8-BrcAMP-mediated attenuation of the HPR. A criterion used in assigning a role for intracellular CAMP as a mediator of a particular response is the ability of CAMP and its analogues, such as 8-BrcAMP, to mimic the response when administered extracellularly (21). 8BrcAMP is a CAMP analogue that effectively activates PKA but is much less effective in activating PKG (6, 13). Thus use of this relatively selective CAMP analogue allowed us to assessthe role of PKA in CAMP-dependent relaxation of smooth muscle. In this study, the findings of increased pulmonary vasoreactivity in response to alveolar hypoxia and the attenuation of the decrease in P,, during the HPR by 8-BrcAMP in lungs pretreated with &-CAMPS support a role for PKA in CAMP-mediated relaxation of vascular smooth muscle. In contrast to 8-BrcAMP, &CAMPS did not block FSK (0.5 PM)-mediated relaxation of the HPR. Why this difference occurred is not clear, but possible explanations include the following: 1) the intracellular CAMP level obtained with FSK was greater than that obtained with 8-BrcAMP, and thus the concentration of R,-
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MECHANISM
OF
CAMP-MEDIATED
CAMPS used was insufficient to antagonize the FSKmediated increase in intracellular CAMP and activation of PKA; 2) FSK permanently activates adenylate cyclase, and consequently the CAMP turnover is maintained in contrast to 8BrcAMP hydrolysis by CAMP phosphodiesterase(s); 3) the affinities of endogenous CAMP and 8BrcAMP for PKA could be different; 4) there was poor membrane permeability of &,-CAMPS; or 5) the mechanism(s) involved was independent of PKA. Complete dose-response data could not be obtained with this intracellular antagonist of CAMP because of a lack of its availability, but even at a lower concentration (0.1 PM) of FSK, &-CAMPS did not block FSK-mediated relaxation (Table 1). Because &-CAMPS treatment did attenuate %BrcAMP-mediated relaxation, neither inadequate concentration nor poor membrane permeability of the antagonist adequately explains the lack of effect of &-CAMPS in FSK-mediated relaxation, but the involvement of mechanisms independent of PKA is suggested. CAMP and its analogues can activate both PKA and PKG; however, relaxation of smooth muscle correlates best with PKG activation (6). The lack of effect with R,CAMPS in FSK-treated lungs may be due in part to the activation of PKG by increased intracellular CAMP in response to FSK. Several recent studies support this concept and suggest that agents such as FSK can activate PKG by increasing intracellular CAMP levels (6, 13, 16). A second potential PKA-independent mechanism for FSK-mediated relaxation is the gating of ionic channels. FSK can activate adenylate cyclase by postreceptor activation of a G stimulatory protein or by directly activating its catalytic unit (23,24). G proteins can indirectly gate ionic channels [i.e., Ca2+-activated K+ channels (K&J] using effecters such as adenylate cyclase (2) or by directly altering K+ channels independent of adenylate cyclase activation (7,14). Because activation of K+ channels has been found to mediate a pulmonary vasodilator response (17), we could not exclude this as a possibility for the FSK effect observed in our study. To assess a possible direct FSK effect, 1,9-dideoxy-FSK, which is unable to activate adenylate cyclase but does alter K+ channels indistinguishably from FSK (7, 14), was studied. This FSK derivative did not decrease pulmonary vasoreactivity, suggesting that a direct effect of FSK on K+ channels was not involved. The ATP-sensitive K+ channel antagonist glibenclamide attenuates pulmonary vasodilation to endothelin isopeptides in the cat (17). In our study, glibenclamide did not attenuate FSK-mediated vasodilation in pulmonary vascular beds precontracted with an hypoxic gas mixture. However, the nonselective K+ channel blocker TEA did attenuate FSKmediated pulmonary vasodilation, as has been observed for histamine-mediated pulmonary vasodilation in the rat lung (10). In this study, TEA also decreased 8 BrcAMP-mediated vasodilation. This suggests that the activation of adenylate cyclase by FSK indirectly gates a non-ATP-sensitive K+ channel through a PKA-dependent phosphorylation. Such an effect has not been previously described in vascular smooth muscle, but it has been demonstrated that FSK can activate a K& channel in cultured kidney cells (9) and that TEA-
Hi515
VASODILATION
sensitive K+ channels are important in maintaining the normal quiescent state of airway smooth muscle (15). Although speculative, phosphorylation of Ca2+ channels by PKA could lead to the activation of K& channels, hyperpolarization of the vascular smooth muscle membrane, and vasorelaxation (Fig. 7). These findings, along with the lack of effects of the FSK derivative and glibenclamide, suggest that the FSK effect is in part mediated indirectly by a non-ATP-sensitive K+ channel. A third potential PKA-independent mechanism for FSK-mediated relaxation involves the vasodilator products of membrane phospholipid hydrolysis, PAF and PGI2 (3, 8, 11, 20, 29) (Fig. 7). However, this mechanism is not supported by this study, because neither the cyclooxygenase blocker indomethacin nor the PAF receptor antagonist SRI 63-441 blocked FSK-mediated pulmonary vasodilation. In conclusion, our results support important roles for PKA and indirect gating of a K& channel in the mediation of smooth muscle relaxation induced by CAMP. We thank Sandy Mead for preparation of this manuscript and Bonnie White and Sandy Worley for graphic support. We also thank Dr. Thomas M. Lincoln for constructive comments regarding the manuscript. This work was supported by a grant from the American Heart Association, Alabama Affiliate, by National Heart, Lung, and Blood Institute Clinical Investigator Award HL-02352, and by a Florence Foundation Research Career Development Grant to J. Haynes, Jr. Received
18 June
1991; accepted
in final
form
26 September
1991.
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