Mechanisms Responsible for Forskolin-Induced Relaxation of Rat Tail Artery
Abstract—The goal of the present study was to determine the physiologically relevant mechanisms for forskolin-induced relaxation of intact rat tail artery. We stimulated deendothelialized rat tail artery with phenylephrine and then relaxed the tissue with the addition of forskolin, a specific activator of adenylyl cyclase. We measured membrane potential with the use of microelectrodes, estimated intracellular Ca2+ concentration ([Ca2+]i) with the use of fura 2, and measured isometric force with a strain-gauge transducer. We found that 0.3 to 1.0 μmol/L forskolin relaxed 0.3 to 1.0 μmol/L phenylephrine-stimulated rat tail artery by decreasing the [Ca2+]i sensitivity of force as well as through repolarization. There was no evidence for forskolin-induced inhibition of Ca2+ influx beyond that associated with repolarization. There also was no evidence for forskolin-induced enhancement of Ca2+ efflux or sequestration. Inhibition of ATP-activated K+ channels with 10 μmol/L glibenclamide, Ca2+-activated K+ channels with 50 nmol/L iberiotoxin, Ca2+-activated K+ channels with 3 or 10 mmol/L tetraethylammonium ion, inwardly rectified K+ channels with 20 μmol/L Ba2+, and voltage-activated K+ channels with 0.5 mmol/L 4-aminopyridine did not significantly attenuate forskolin-induced reductions in [Ca2+]i or force. Forskolin-induced repolarization was not altered by 10 μmol/L glibenclamide or 0.5 mmol/L 4-aminopyridine. These data suggest that these K+ channels were not individually involved in forskolin-induced relaxation and that other channels and/or multiple channels are involved in forskolin-induced repolarization of intact rat tail artery. Our data also suggest that forskolin-induced relaxation of intact rat tail artery occurred primarily through repolarization and reductions in the [Ca2+]i sensitivity of force.
There are five mechanisms responsible for agonist-induced arterial smooth muscle contraction (for a review, see Reference 11 ): (1) depolarization, (2) release of Ca2+ from the sarcoplasmic reticulum, (3) direct activation of L-type Ca2+ channels to increase Ca2+ influx beyond that expected for the level of depolarization, (4) activation of other Ca2+-permeable channels, and (5) increasing the [Ca2+]i sensitivity of myosin light chain phosphorylation.
Increases in cAMP levels are hypothesized to induce smooth muscle relaxation by decreasing [Ca2+]i and/or the sensitivity of the contractile apparatus to [Ca2+]i. The action of cAMP may not only be through activation of cAMP-dependent protein kinase because large increases in [cAMP] can also activate cGMP-dependent protein kinase.2 3 Most4 5 6 7 but not all8 9 studies in smooth muscle found that elevations in [cAMP] decreased [Ca2+]i. Adenosine decreased 45Ca2+ influx in coronary artery,10 and forskolin reduced Mn2+ influx (a surrogate for Ca2+ influx) in swine carotid artery.11 These results suggest that increases in cAMP concentration may decrease [Ca2+]i, at least partially by decreasing Ca2+ influx.
Increases in cAMP levels are hypothesized to induce relaxation through at least six specific mechanisms. Five of these six mechanisms are the reverse of the above agonist-dependent contractile mechanisms: (1) increases in [cAMP] can repolarize some smooth muscles, potentially by activating KCa channels.12 13 (2) Increases in [cAMP], via activation of cGMP-dependent protein kinase, could inhibit release of Ca2+ from the intracellular Ca2+ store.14 15 (3) cAMP, under certain conditions, may inactivate voltage-dependent L-type Ca2+ channels.16 (4) It is possible, but not reported, that cAMP could inhibit Ca2+ influx through Ca2+-permeable nonselective Ca2+ channels. (5) cAMP can decrease the [Ca2+]i sensitivity of phosphorylation.7 17 This effect was not caused by cAMP-dependent protein kinase phosphorylation of myosin light chain kinase.18 19 Finally, (6) cAMP may enhance Ca2+ efflux or sequestration. This is the only mechanism that is not the reverse of a mechanism responsible for contraction. There are few data on cAMP, but there are several proposed mechanisms for cGMP-dependent increases in Ca2+ extrusion and sequestration, such as cGMP-dependent protein kinase can phosphorylate phospholamban, a process that activates the sarcoplasmic reticulum Ca2+-ATPase by removing the inhibition caused by unphosphorylated phospholamban.4 20 21 Second, cGMP-dependent protein kinase also may activate a plasma membrane Ca2+-ATPase.22 23 Third, 8-bromo-cGMP increased Na+/Ca2+ exchange in isolated rat aortic smooth muscle cells.24
The first goal of this study was to determine which of these mechanisms were physiologically relevant during cAMP-dependent rat tail artery relaxation. The second goal was to characterize pharmacologically which membrane ion channels were involved in cAMP-induced repolarization. Unlike prior studies, we performed these experiments in sections of intact arteries rather than isolated smooth muscle cells. This approach allowed determination of changes in Em and isometric force in one set of intact tissues and of [Ca2+]i and isometric force in a second set of intact tissues. We stimulated deendothelialized rat tail artery with 0.3 to 1.0 μmol/L phenylephrine and then relaxed the tissue by the addition of low concentrations of forskolin (0.3 to 1.0 μmol/L), a specific activator of adenylyl cyclase.
Rat tail arteries were obtained from 180- to 250-g male Sprague-Dawley rats that were humanely killed with 0.15 mg/g pentobarbital according to a protocol approved by the Animal Use Committee of the University of Virginia. The adventitia was dissected, and the arterial tissue was immobilized on a dual-wire myograph (Mulvany-Halperin type; University of Vermont [Burlington]) in a temperature-controlled tissue bath at 35°C (the physiological temperature for this vessel). The physiological salt solution contained (in mmol/L) 140 NaCl, 4.7 KCl, 5 MOPS, 1.2 Na2HPO4, 1.6 CaCl2, 1.2 MgSO4, and 5.6 d-glucose, pH adjusted to 7.4 at 35°C. The endothelium was removed through gently rubbing with a wire, a procedure that eliminated acetylcholine-induced relaxation. Removal of the endothelium was necessary to prevent contamination of fura 2 signals by changes in endothelial [Ca2+]i. At the end of each experiment, the tissue was depolarized with 90 mmol/L [K+]o, and the fura 2 and force responses were normalized to the 90 mmol/L [K+]o response. When tissues were depolarized with high [K+]o, we added 3 μmol/L phentolamine to block the effect of endogenously released norepinephrine.
The experimental design consisted of (1) the addition of the K+ channel blockers (if included) for 5 minutes, (2) stimulation with various concentrations of phenylephrine for 5 minutes, and (3) the addition of increasing concentrations of forskolin at 5-minute intervals. The only exception to this was the iberiotoxin experiments, in which the high cost of iberiotoxin required that iberiotoxin be added in only the forskolin-containing solutions.
Measurement of Em
Em was measured in one set of tissues. High-impedance microelectrodes (50 to 80 MΩ filled with 2 mol/L KCl) were pulled with a Flaming Brown P87 puller (Sutter). Cells were impaled with microelectrodes held in a Leitz micromanipulator, and Em was measured with a Dagan model 8700 electrometer. Criteria for acceptance of recordings were (1) an abrupt decrease in voltage on impalement, (2) stable baseline for 3 minutes, and (3) unchanged electrode resistance and potential after removal of the microelectrode from the tissue. Isometric force was measured with a strain-gauge transducer (Kulite BG-10; University of Vermont [Burlington]), and solutions were perfused through the 6-mL chamber at 6 to 10 mL/min with a roller pump (perfusion was necessary to prevent artifacts from changing solution level). Measurements were made in tissues from four to six arteries and presented as mean±1 SEM. The perfusion setup required large solution volumes (120 mL) to maintain constant solution depth; therefore, measurement of Em was not performed when reagents were expensive (ie, iberiotoxin). In these cases, only experiments with fura 2 were performed.
Measurement of Myoplasmic [Ca2+]i
[Ca2+]i was estimated with fura 2 in a second set of tissue. Fura 2 was loaded intracellularly via a 60-minute incubation in the AM ester of fura 2 (20 μmol/L). The fura 2 was washed out for 30 minutes before recording to allow adequate hydrolysis and washout of fura 2-AM (see Refs 11 and 25 for details of fura 2 loading and fluorescence measurement equipment [University of Pennsylvania, Philadelphia]). Isometric force was measured with a capacitative force transducer (Harvard Apparatus). Measurements were made in four to six tissue samples and presented as mean±1 SEM. Fura 2 signals were calibrated through incubation of the tissue sequentially in solutions containing ionomycin with low Ca2+ (10 min) and high Ca2+ (5 min); then, the fura 2 signal was quenched with Mn2+ (5 min). The low-Ca2+ calibration solution contained (in mmol/L) 0.04 ionomycin, 2 EGTA, 145 KCl, 5 MOPS, 1.2 MgSO4, and 5.6 d-glucose, pH adjusted to 7.4 at 35°C. The high-Ca2+ calibration solution contained (in mmol/L) 0.04 ionomycin, 5 CaCl2, 145 KCl, 5 MOPS, 1.2 MgSO4, and 5.6 d-glucose, pH adjusted to 7.4 at 35°C. The Mn2+ quench solution contained (in mmol/L) 5 MnCl2, 145 KCl, 5 MOPS, 1.2 MgSO4, and 5.6 d-glucose. There are many uncertainties in fura 2 calibration26 27 ; for example, the presence of various proteins (0.3 to 1 mmol/L) in a calibration solution increased the measured Kd value of fura 2 by as much as 200%.27 For these reasons, we primarily present data as background-subtracted 340 nm/380 nm ratios normalized to the response observed 5 minutes after stimulation with 90 mmol/L [K+]o. The force reported in the figures was that measured in the experiments with fura 2. The force measured in the Em experiments was similar to that observed in the experiments with fura 2.
Measurements were compared with the use of Student’s t test if there were two groups or with an ANOVA and the Student-Newman-Keuls test if there were more than two groups. Regression analysis was performed with the least-squares method, and slopes were compared by calculating residual variances and a t test.28 When possible (see Fig 2A⇓ and 2B⇓), regression was performed on measured values from individual experiments. However, this was not possible in the unpaired comparison of Em and fura 2–estimated [Ca2+]i (see Fig 2C⇓). A value of P<.05 was considered significant.
Stimulation with phenylephrine induced a dose-dependent depolarization, increase in fura 2–estimated [Ca2+]i, and contraction (Fig 1⇓). The addition of 0.3 and 1.0 μmol/L forskolin to 0.3 and 1.0 μmol/L phenylephrine–stimulated tissue induced repolarization, reductions in fura 2–estimated [Ca2+]i, and reduction in force. These data show that forskolin induced repolarization and suggest that the repolarization may be at least partially responsible for relaxation.
Further evaluation of the data in Fig 1⇑ revealed that repolarization was not responsible for the entire relaxation. First, we compared the combination of 1 μmol/L phenylephrine and 0.3 μmol/L forskolin with 0.3 μmol/L phenylephrine alone (Fig 1⇑, dashed line 1). The measured fura 2 signals did not statistically differ (t test, P=.71), but the contraction was statistically larger than that with phenylephrine alone (t test, P=.002). Second, we compared the combination of 1 μmol/L phenylephrine and 1.0 μmol/L forskolin, the combination of 0.3 μmol/L phenylephrine and 0.3 μmol/L forskolin, and 0.1 μmol/L phenylephrine alone (Fig 1⇑, dashed line 2). The measured fura 2 signals did not statistically differ (ANOVA, P=.39), but the contraction was statistically larger with phenylephrine alone than the contraction observed in the presence of 0.3 or 1.0 μmol/L forskolin (ANOVA, P<.001). These data suggest that some of the forskolin-induced relaxation was caused by reductions in the [Ca2+]i sensitivity of force.
These conclusions were supported with a comparison of the relationships among Em, fura 2 signals, and contraction. First, we compared Em and force. The addition of 0.3 or 1.0 μmol/L forskolin to 0.3 or 1.0 μmol/L phenylephrine–stimulated rat tail artery decreased contractile force at intermediate and high Em levels compared with phenylephrine stimulation alone (Fig 2A⇓, ○ and •, respectively; the regression lines are based on a log10 transform of force data, and they differed with a value of P<.001). This suggests that factors beyond repolarization were responsible for at least part of the relaxation.
Second, we compared fura 2–estimated [Ca2+]i and force. The addition of 0.3 or 1.0 μmol/L forskolin to 0.3 or 1.0 μmol/L phenylephrine–stimulated rat tail artery decreased contractile force at intermediate and high levels of fura 2–estimated [Ca2+]i compared with phenylephrine stimulation alone (Fig 2B⇑, ○ and •, respectively; the linear regression lines differed with a value of P<.001). These data suggest that forskolin decreased the [Ca2+]i sensitivity of force.
Third, we compared the relation between Em and fura 2–estimated [Ca2+]i. The plot in Fig 2C⇑ suggests that the addition of forskolin was associated with a modestly higher fura 2–estimated [Ca2+]i at lower Em values compared with the use of phenylephrine alone. Because the Em and fura 2 data are not paired, statistical comparison regarding individual data points is not possible. Evaluation of the regression line through the mean values revealed no significant difference. These data suggest that the combination of phenylephrine and forskolin either had no effect or, possibly, increased [Ca2+]i above that expected on the basis of the Em. More importantly, this finding demonstrates that forskolin is not inhibiting potential independent Ca2+ influx, inhibiting nonselective cation channels, or activating Ca2+ efflux/sequestration (mechanisms 3, 4, and 6 listed in the introduction, respectively). These three mechanisms should reduce [Ca2+]i below that expected on the basis of measured Em.
We examined the relative importance of forskolin-induced repolarization compared with other mechanisms by reversing the repolarization with increasing [K+]o (Fig 3⇓). Stimulation with 0.3 μmol/L phenylephrine depolarized and contracted the tissues. The addition of 1 μmol/L forskolin repolarized and relaxed the tissues. The further addition of 30, 35, and 40 mmol/L [K+]o depolarized the tissues and increased force. The addition of 35 mmol/L [K+]o depolarized the tissues to a level that did not statistically differ from that observed with 0.3 μmol/L phenylephrine alone. The force observed with 0.3 μmol/L phenylephrine plus 1 μmol/L forskolin plus 35 mmol/L [K+]o was 49% of the value observed with 0.3 μmol/L phenylephrine alone. These data suggest that approximately half of the forskolin-induced relaxation was caused by the repolarization and the other half by decreases in the [Ca2+]i sensitivity of force.
We next evaluated the role of K+ channels in forskolin-induced relaxation. We inhibited KATP channels with 10 μmol/L glibenclamide, KCa channels with 50 nmol/L iberiotoxin, both KATP and KCa channels with 10 μmol/L glibenclamide and 50 nmol/L iberiotoxin, KCa channels with 3 or 10 mmol/L TEA+, KIR channels with 20 μmol/L Ba2+, and KV channels with 0.5 mmol/L 4-aminopyridine. Preliminary studies found that 10 μmol/L glibenclamide, a specific inhibitor of KATP channels, completely inhibited 10 μmol/L pinacidil–induced relaxation of rat tail artery precontracted with 1 μmol/L phenylephrine (data not shown). There is no good agonist for KCa channels; therefore, we used a standard dose (50 nmol/L29 ) of iberiotoxin. TEA+ is a less-specific K+ channel antagonist, acting at low concentrations on KCa channels (EC50 ≈0.2 μmol/L) and at higher concentrations on KATP channels (EC50 ≈7 mmol/L) and KV channels (EC50 >5 mmol/L) channels.29 The KIR channel is inhibited by Ba2+ with an IC50 value of 2 μmol/L.29 The KV channel is inhibited by 4-aminopyridine with an IC50 value of 0.3 to 1 mmol/L.29
We incubated rat tail artery tissue with the above K+ channel blockers, stimulated the tissue with 0.3 μmol/L phenylephrine, and then evaluated the response to 0.3 μmol/L forskolin. None of tested K+ channel blockers significantly attenuated the forskolin-induced reduction in fura 2–estimated [Ca2+]i or force (Fig 4⇓). Em was measured with only some of the K+ inhibitors. In the presence of TEA+, 0.3 μmol/L forskolin induced APs and transient contractions in five of seven preparations. The reported mean Em value is from the two preparations that did not exhibit APs (Fig 4⇓, AP). The forskolin-induced repolarization was not significantly altered by 10 μmol/L glibenclamide or 0.5 mmol/L 4-aminopyridine. Similar results were observed with 1.0 μmol/L forskolin–induced relaxation of 0.3 μmol/L phenylephrine–stimulated rat tail artery (data not shown). This higher forskolin concentration abolished TEA+-induced APs, and the mean repolarization did not differ from that observed with 1.0 μmol/L forskolin without TEA+. These data suggest that these K+ channels were not individually responsible for forskolin-induced relaxation of phenylephrine-induced contraction in the deendothelialized rat tail artery.
The first goal of the present study was to determine the physiologically relevant mechanism or mechanisms responsible for forskolin-induced rat tail artery relaxation. Our approach was to measure Em, fura 2–estimated [Ca2+]i, and force in intact phenylephrine-stimulated deendothelialized rat tail artery. This approach allows delineation of some but not all of the known mechanisms. Specifically, we can determine whether relaxation is caused by repolarization (mechanism 1, as given), by decreases in the [Ca2+]i sensitivity of force (mechanism 5), or by an alteration in the relation between Em and fura 2–estimated [Ca2+]i that would indicate mechanism 3 (direct inactivation of Ca2+ channels), mechanism 4 (inactivation of nonselective cation channels), or mechanism 6 (enhancement of Ca2+ efflux or sequestration) was involved in the relaxation. In this study, we did not investigate the role of Ca2+ release inhibition (mechanism 2).
We found that forskolin-induced both a repolarization (Figs 1⇑ and 2A⇑) and a decrease in the [Ca2+]i sensitivity of force (Fig 2B⇑). Both were observed in maximally as well as submaximally phenylephrine-stimulated tissues (Fig 1⇑). Approximately half of the overall relaxation of 0.3 μmol/L phenylephrine–stimulated rat tail artery was associated with repolarization (Fig 3⇑). When the forskolin-induced repolarization was reversed by the addition of 35 mmol/L [K+]o, force increased to 49% of the value observed with phenylephrine alone. The other half of the overall relaxation was associated with a decrease in the [Ca2+]i sensitivity of force.
Forskolin was associated with the similar or slightly higher [Ca2+]i for a certain Em compared with that observed with phenylephrine alone (Fig 2C⇑). This suggests that there may be a mechanism keeping [Ca2+]i higher than that expected given the measured Em. It is possible that (1) forskolin is activating L channels and enhancing Ca2+ influx,16 (2) forskolin is inhibiting Ca2+ efflux, or 3) forskolin-induced repolarization is uncovering phenylephrine-dependent activation of L channels (contractile mechanism 330 31 ). However, statistical analysis of our data did not clearly demonstrate this effect. More importantly, our data suggest that forskolin did not relax phenylephrine-stimulated rat tail artery by relaxing mechanism 3 (voltage-independent inactivation of L channels), relaxing mechanism 4 (inactivation of nonselective cation channels), or mechanism 6 (enhanced Ca2+ efflux or sequestration). These three mechanisms would reduce [Ca2+]i below that expected given the relative depolarization; this was not observed (Fig 2C⇑).
In the histamine-stimulated swine carotid artery, we found that forskolin reduced Mn2+ influx (a surrogate for Ca2+ influx), fura 2–estimated [Ca2+]i, and force.11 We suggested that the reduction in Mn2+ influx resulted from repolarization or voltage-independent inactivation of Ca2+ channels. The results of the present study suggest that the former is more important in the rat tail artery. It appears that forskolin-induced repolarization and the resulting decrease in Ca2+ influx occur in both the swine carotid and rat tail artery (although we cannot measure Em in the former for technical reasons). It should be noted that high concentrations of forskolin (50 μmol/L) have been reported to alter Na+ and K+ currents independent of cAMP concentration.32 33 Our experiments were performed with low forskolin concentrations (≤1 μmol/L), at which forskolin is a specific activator of adenylyl cyclase rather than a channel blocker.
The second goal of this study was to determine whether regulation of K+ channels was physiologically relevant in forskolin-induced repolarization. The results of several studies suggested that increases in cAMP concentration activate KCa channels in bilayer12 and whole-cell studies.13 Calcitonin gene–related peptide, a stimulus that increases cAMP concentration, also activates KATP channels.34 The activation of these or other K+ channels could induce repolarization, thereby decreasing Ca2+ influx and [Ca2+]i. We found that inhibition of KATP channels with glibenclamide, KCa channels with TEA+, and KV channels with 4-aminopyridine did not alter forskolin-induced repolarization, reduction in fura 2–estimated [Ca2+]i, or the relaxation (Fig 4⇑). In addition, the blockage of KCa channels with iberiotoxin and KIR channels with Ba2+ did not alter forskolin-induced reductions in fura 2–estimated [Ca2+]i or relaxation. These data suggest that other mechanisms, perhaps inactivation of Na+ channel(s), activation of other K+ channel(s), and/or inactivation of multiple channels, are responsible for forskolin-induced repolarization in intact rat tail artery. It is possible that blockage of one K+ channel may not significantly alter repolarization if the other channel or channels compensate for the block. These data do not rule out a role for individual K+ channels in forskolin-induced repolarization of other arteries.
In conclusion, we demonstrated that repolarization and decreases in the [Ca2+]i sensitivity of force appear to equally participate in forskolin-induced relaxation of phenylephrine-stimulated rat tail artery. The activation of glibenclamide-, iberiotoxin-, TEA+-, Ba2+-, or 4-aminopyridine–sensitive K+ channels did not appear to be individually responsible for forskolin-induced repolarization. Potentially, other channels or multiple channels are involved in forskolin-induced repolarization in deendothelialized rat tail artery.
Selected Abbreviations and Acronyms
|[Ca2+]i||=||intracellular Ca2+ concentration|
|[K+]o||=||extracellular K+ concentration|
|KATP||=||ATP-activated K+ channel|
|KCa||=||Ca2+-activated K+ channel|
|KIR||=||inwardly rectified K+ channel|
|KV||=||voltage-activated K+ channel|
This work was supported by grant J-360 from the Jeffress Trust. The authors thank Barbara Weaver for technical support and Dr Terrence Smith for helpful discussions.
Reprint requests to Christopher M. Rembold, MD, Box 146, Cardiovascular Division, University of Virginia Health Sciences Center, Charlottesville, VA 22908.
- Received September 10, 1997.
- Revision received October 10, 1997.
- Accepted October 29, 1997.
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