Modulation of Ca²⁺ Channels by Cyclic Nucleotide Cross Activation of Opposing Protein Kinases in Rabbit Portal Vein

From the Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno.

Correspondence to Kathleen D. Keef, PhD, Anderson Medical Sciences Building, Reno, NV 89557. E-mail kathy{at}physio.unr.edu

	Abstract

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Abstract—Cyclic nucleotides are known to modify voltage-gated (L-type) Ca²⁺ channel activity in vascular smooth muscle cells, but the exact mechanism(s) underlying these effects is not well defined. The purpose of the present study was to investigate the modulatory role of the cAMP- and cGMP-dependent protein kinase (PKA and PKG, respectively) pathways in Ca²⁺ channel function by using both conventional and perforated-patch–clamp techniques in rabbit portal vein myocytes. The membrane-permeable cAMP derivative, 8-bromo cAMP (0.1 to 10 µmol/L), significantly increased (14% to 16%) peak Ba²⁺ currents, whereas higher concentrations (0.05 to 0.1 mmol/L) decreased Ba²⁺ currents (23% to 31%). In contrast, 8-bromo cGMP inhibited Ba²⁺ currents at all concentrations tested (0.01 to 1 mmol/L). Basal Ca²⁺ channel currents were significantly inhibited by the PKA blocker 8-Bromo-2'-O-monobutyryladenosine-3',5'-monophosphorothioate, Rp-isomer (Rp 8-Br-MP cAMPS, 30 µmol/L) and enhanced by the PKG inhibitor ß-Phenyl-1,N²-etheno-8-bromoguanosine-3',5'-monophosphorothioate, Rp-isomer (Rp-8-Br PET cGMPS, 10 nmol/L). In the presence of Rp 8-bromo PET cGMPS (10 to 100 nmol/L), both 8-bromo cAMP (0.1 mmol/L) and 8-bromo cGMP (0.1 mmol/L) enhanced Ba²⁺ currents (13% to 39%). The excitatory effect of 8-bromo cGMP was blocked by Rp 8-bromo MB-cAMPS. Both 8-bromo cAMP (0.05 mmol/L) and forskolin (10 µmol/L) elicited time-dependent effects, including an initial enhancement followed by suppression of Ba²⁺ currents. Ba²⁺ currents were also enhanced when cells were dialyzed with the catalytic subunit of PKA. This effect was reversed by the PKA blocker KT 5720 (200 nmol/L). Our results suggest that cAMP/PKA stimulation enhances and cGMP/PKG stimulation inhibits L-type Ca²⁺ channel activity in rabbit portal vein myocytes. Our results further suggest that both cAMP and cGMP have a primary action mediated by their own kinase as well as a secondary action mediated by the opposing kinase.

Key Words: L-type Ca²⁺ channel • vascular smooth muscle • 8-bromo cAMP • 8-bromo cGMP • cGMP-dependent protein kinase

	Introduction

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Voltage-dependent (L-type) Ca²⁺ channels play a major role in excitation-contraction coupling in vascular smooth muscle cells. L-type Ca²⁺ channels are known to be modulated by several intracellular second-messenger systems, including both the cAMP/cAMP-dependent protein kinase (PKA) and cGMP/cGMP-dependent protein kinase (PKG) pathways.¹ However, for vascular smooth muscle, little information is known regarding the exact mechanism(s) by which these processes take place. Patch-clamp studies in smooth muscle cells have shown that L-type Ca²⁺ channel activity can be enhanced by either low concentrations of 8-Br cAMP or the catalytic subunit of PKA.^{2 3 4} Stimulation of ß-adrenergic receptors with Iso has also been shown to increase Ca²⁺ channel currents.^{2 3 5 6 7} On the other hand, 8-Br cGMP or the NO-releasing agents sodium nitroprusside and SNAP have been reported to lead to a decrease of Ca²⁺ channel activity.^{2 8 9 10}

The precise mechanism underlying the effects of both PKA and PKG on L-type Ca²⁺ channels remains controversial. A previous study from this laboratory showed that a moderate increase in cAMP elicited with 1 µmol/L Iso, 1 µmol/L FSK, or 0.1 mmol/L 8-Br cAMP increased Ca²⁺ channel currents.² On the other hand, higher levels of cAMP elicited with 10 µmmol/L Iso, 10 µmol/L FSK, 1 mmol/L 8-Br cAMP, or 0.1 mmol/L 8-Br cGMP led to inhibition of Ca²⁺ channel currents. Experiments that measured the time course of responses to high concentrations of Iso or FSK revealed that Ca²⁺ channel currents were initially enhanced and subsequently inhibited. It has been suggested that moderate increases in cAMP enhance Ca²⁺ channel currents through PKA activation, whereas higher levels of cAMP lead to activation of PKG, which then predominates over the PKA effect (ie, cross activation of PKG by cAMP). Similar findings have been recently reported in colonic smooth muscle cells.³ In smooth muscle cells from the basilar artery, it has been shown that exposure of inside-out patches to the catalytic subunit of PKA increased L-type Ca²⁺ channel availability.⁴ In apparent conflict with these results, Sperelakis and coworkers^{11 12 13} have suggested that in rabbit portal vein cells the direct effects of the cAMP/PKA pathway are inhibitory and that only direct G-protein gating produces an increase in Ca²⁺ channel currents.

The exact role of both cyclic nucleotides may be complicated by the fact that they do not display absolute specificity for either PKA or PKG. For instance, when cAMP levels are raised sufficiently in smooth muscle cells, both PKA and PKG are activated.^{14 15 16 17} In the present study, the effects of PKG and PKA inhibitors were tested on Ca²⁺ channel activity under basal conditions and after maneuvers that elevate cyclic nucleotide levels in the cell. Specifically, we wanted to determine whether the inhibitory effects of high concentrations of cAMP are indeed mediated by "crossover" activation of PKG and whether the reverse situation occurs, ie, crossover activation of PKA by cGMP.

	Materials and Methods

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Smooth Muscle Cell Isolation
New Zealand male albino rabbits (1.5 to 2.0 kg) were killed with an overdose of sodium pentobarbital (50 mg/kg) administered intravenously into the ear vein. The portal vein was removed and cleaned of connective tissue in cold Krebs solution (mmol/L: NaCl 118.5, KCl 4.2, MgCl₂ 1.2, CaCl₂ 1.8, glucose 11, K₂HPO₄ 1.2, NaHCO₃ 23.8, and HEPES 11; titrated to pH 7.4 with Tris base; aerated with 95%O₂/5% CO₂) and cut into small segments (3x3 mm). The segments were then preincubated in a digestion solution¹⁸ (mmol/L: NaCl 90, MgCl₂ 1.2, K₂HPO₄ 1.2, glucose 20, taurine 50, and HEPES 5; titrated to pH 7.1 with NaOH) at 35°C for 60 minutes. After the preincubation period, the segments were then placed in the digestion solution containing 2 mg/mL collagenase (Sigma Chemical Co), 0.5 mg/mL protease (Sigma), and 2 mg/mL BSA and incubated for 7 to 14 minutes. After the digestion period, the segments were stored in a modified KB solution¹⁹ (mmol/L: KCl 55, K₂HPO₄ 30, MgSO₄ 5, ATP-Na 5, creatine 5, taurine 20, pyruvate 5, glutamate 10, oxalic acid 10, glucose 20, succinate 5, and EGTA 0.2, along with 1 mg/mL BSA; pH 7.4 with KOH) at 4°C for 4 to 5 hours before dispersion with gentle trituration.

Electrophysiology
To record whole-cell inward Ba²⁺ currents, the patch-clamp technique was used.²⁰ Ba²⁺ was used as the charge carrier to rule out the contamination of other currents activated by Ca²⁺. A cell suspension was placed on a recording chamber on the stage of an inverted microscope. The cells were superfused by gravity at a constant rate (1 to 2 mL/min), and the bath temperature was maintained at room temperature (20°C to 22°C). Micropipettes were made from borosilicate glass and had resistances of 2.0 to 3.5 M when filled with the pipette solution. The bath solution used to record inward Ba²⁺ currents was composed of the following (mmol/L): NaCl 117.5, TEACl 10, BaCl₂ 5, MgCl₂ 0.5, glucose 5.5, CsCl 5, and HEPES 10 (titrated to pH 7.4 with NaOH). The internal solution of the patch pipettes consisted of the following (mmol/L): glutamate 75, CsCl 55, BAPTA 10, K₂HPO₄ 1, ADP-Na 0.5, fructose-1,6-diphosphate 2, MgSO₄ 5.7, GTP 1, ATP-Na 5, NAD 1, and HEPES 10 (titrated to pH 7.2 with CsOH). When PKA was included in the patch pipette, glutamate was reduced from 75 to 60 mmol/L. Whole-cell Ba²⁺ currents were also recorded in some experiments using the perforated-patch technique with amphotericin B.²¹ For these experiments, the pipette solution consisted of the following (mmol/L): glutamate 120, CsCl 20, TEACl 10, and HEPES 10 (titrated to pH 7.2 with CsOH). Qualitatively, there was no discernible difference in current magnitude or responses to cyclic nucleotides in perforated-patch recordings versus the conventional whole-cell ruptured-patch technique, thus eliminating membrane current rundown as a potential artifact. For whole-cell recordings, 3 minutes was allowed after formation of a gigaseal and break-in before beginning experimental protocols. For perforated-patch recordings, 10 to 15 minutes was necessary to achieve a mean access resistance of 9.6±0.6 M (±SEM, n=26) before beginning experimental protocols.

Membrane currents were measured using an Axopatch 200A amplifier and digitized using a TL-1 A/D converter (Axon Instruments). Data were filtered at 10 kHz (-3 dB) and stored in a computer for later analysis. Voltage-clamp protocols and analysis were performed using the pCLAMP6.0 software package (Axon Instruments). Current amplitude was determined by averaging the currents attained between 4 and 8 milliseconds after initiation of the voltage step. Drug effects were determined by comparing the average of three such measurements both before and during drug application. Responses to cyclic nucleotides and kinase blockers were measured when the drug effect had reached steady state (ie, 2 to 5 minutes after application began) unless otherwise stated. Data are expressed as mean±SEM; statistical significance was tested with Student's t test for paired data, with P<.05 considered significant, using the Prism program (GraphPad).

Drugs
Rp 8-Br PET cGMPS (a generous gift from Dr Hans-G. Genieser, Biolog Life Science Institute, distributed by Ruth Langhorst International Marketing, La Jolla, Calif), Rp 8-Br cAMPS, Rp 8-Br MB-cAMPS, Rp 8-Br cGMPS (Biolog), 8-Br cAMP, and 8-Br cGMP (Sigma) were directly dissolved in the bath solution. FSK (Sigma) was prepared as stock a solution of 10 mmol/L in DMSO and diluted in the bath solution. Amphotericin B (Sigma) was first dissolved in DMSO (60 mg/mL) and diluted in the pipette solution to give a final concentration of 600 µg/mL. The catalytic subunit of PKA and KT 5720 were both obtained from Calbiochem. KT 5720 was dissolved in DMSO, and PKA was dissolved in pipette solution.

	Results

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Isolated portal vein myocytes were relaxed and spindle-shaped. Inward currents were recorded in 5 mmol/L Ba²⁺–containing solution in cells held at -80 mV. The membrane potential was stepped for 250 milliseconds to various test potentials (from -60 to +50 mV) at 20-second intervals. Fig 1A shows typical Ba²⁺ currents recorded using this method. Previous studies have shown that portal vein myocytes contain only one type of voltage-dependent Ca²⁺ channel (eg, L-type).^{2 22} Fig 1B shows the current-voltage relation with detectable inward current beginning near -20 mV. Peak inward current occurred at +10 mV and averaged 895±63 pA (n=30). The time courses of peak inward Ba²⁺ currents were subsequently monitored by applying repetitive test pulses to +10 mV from a holding potential of -60 mV every 20 to 25 seconds.

View larger version (22K): [in this window] [in a new window]   Figure 1. Current-voltage relationship of Ba2+ currents. A, Typical recordings for several voltage steps (250 milliseconds) between +10 and +50 mV from a holding potential of -80 mV. Pulses were applied every 20 seconds and recorded by the whole-cell ruptured-patch technique. B, Mean current-voltage relationship of Ba2+ currents in portal vein smooth muscle cells. Symbols represent mean±SE (n=30). Peak Ba2+ currents occurred, on average, between +10 and +20 mV, and activation of currents occurred at potentials positive to -20 mV.

Effect of 8-Br cAMP and 8-Br cGMP on Peak Ba²⁺ Currents
To investigate the actions of cyclic nucleotides on Ca²⁺ channel currents, the concentration-dependent effects of two cell-permeant analogues of cAMP and cGMP were examined (ie, 8-Br cAMP and 8-Br cGMP). Application of the lowest concentrations of 8-Br cAMP (ie, 0.1 to 10 µmol/L) significantly increased currents (Fig 2A), whereas higher concentrations (0.05 to 0.1 mmol/L) resulted in a significant reduction of currents (Fig 2B and 2C). The time to peak excitation with low concentrations of 8-Br cAMP averaged 3.7±0.5 minutes, whereas the time to peak inhibition with higher concentrations of 8-Br cAMP averaged 4.1±0.3 minutes. The effects of 8-Br cAMP on current amplitude are summarized in Fig 2E and support earlier findings of Ishikawa et al.² At the intermediate concentration of 0.05 mmol/L 8-Br cAMP, there was a brief (ie, 40- to 120-second) period of enhanced current amplitude in three of six cells before inhibition (see Fig 2B). Steady-state inhibition of Ba²⁺ currents was reached 5.3±1.1 minutes (n=6) after application of 0.05 mmol/L 8-Br cAMP. The highest concentration of 8-Br cAMP applied (ie, 0.1 mmol/L) produced inhibition of only Ba²⁺ currents.

View larger version (31K): [in this window] [in a new window]   Figure 2. Effect of cyclic nucleotides on inward Ba2+ currents. A through D, Time courses of peak currents before and after exposure to 0.01 (A), 0.05 (B), and 0.1 (C) mmol/L 8-Br cAMP and 0.1 mmol/L 8-Br cGMP (D). Washout period (WO) is also shown. Currents were elicited by voltage steps to +10 mV from a holding potential of -60 mV every 20 seconds. Panels A through D were recorded under the whole-cell ruptured-patch technique. E, Bar graph summarizing the effect of the cyclic nucleotides on inward currents. Graph shows mean±SE values determined as percent increase or decrease in currents from control (see "Materials and Methods"). *P<.05 compared with control.

In contrast to 8-Br cAMP, 8-Br cGMP decreased peak Ba²⁺ currents at all concentrations tested (0.01 to 1.0 mmol/L) and throughout the duration of exposure to 8-Br cGMP. The decrease in current amplitude was significant at higher concentrations (0.1 to 1.0 mmol/L, Fig 2D and 2E). Steady-state inhibition of current occurred after 5.0±0.6 and 4.1±0.4 minutes of exposure to 0.1 and 1 mmol/L 8-Br cGMP, respectively. The effects of 8-Br cGMP on current amplitudes are also summarized in Fig 2E.

Effect of PKG and PKA Inhibition on Basal Ca²⁺ Channel Currents
Our results indicate that the amplitude of Ca²⁺ channel currents can be modulated by raising cyclic nucleotide levels in the cell. Since there is also basal production of cyclic nucleotides in smooth muscle,^{23 24} it is possible that Ca²⁺ channel activity is modulated under basal conditions by PKA and PKG. To determine whether this is the case, we examined the effects of PKA and PKG inhibitors alone on voltage-dependent Ba²⁺ currents. Superfusion of myocytes with the PKG inhibitor Rp 8-Br PET cGMPS²⁵ (10 nmol/L) caused a significant reversible enhancement of Ba²⁺ currents elicited with depolarizing steps to +10 mV (Fig 3A). In contrast, when cells were treated with a PKA inhibitor, Rp 8-Br cAMPS (30 µmol/L) or Rp 8-Br MB-cAMPS (30 µmol/L), peak current was decreased. Fig 3B shows the time course of currents before and during treatment with Rp 8-Br MB-cAMPS, and the corresponding current recordings are shown to the right. The bar graph in Fig 3C summarizes the effect of the kinase blockers on peak currents. These results suggest that basal PKA activity tends to enhance and basal PKG activity tends to suppress Ca²⁺ channel activity. The net effect is likely to be dependent on both resting cyclic nucleotide and kinase levels.

View larger version (23K): [in this window] [in a new window]   Figure 3. Effect of the PKG and PKA inhibitors on basal Ca2+ channel currents. Time courses of peak Ba2+ currents were elicited by voltage steps to +10 mV from a holding potential of -60 mV, before and after exposure to the selective PKG inhibitor Rp 8-Br PET cGMPS (Rp PET, 10 nmol/L), shown in panel A. Washout period (WO) is also shown. This recording was obtained by the whole-cell ruptured-patch technique. Typical current tracings are presented for each condition to the right. Cells were also superfused with the selective PKA inhibitors Rp 8-Br MB-cAMPS (Rp MB-cAMPS, 30 µmol/L), shown in panel B (recorded by the perforated-patch technique), and Rp 8-Br cAMPS (30 µmol/L), shown in panel C. C, Bar graph summarizing the effect of both types of kinase inhibitors. Mean±SE values were determined as percent increase or decrease in Ba2+ currents from control (see "Materials and Methods"). *P<.05 compared with control.

Reversal of cAMP- and cGMP-Mediated Inhibitory Effects
Previous studies have suggested that the inhibitory effect of cAMP on Ba²⁺ currents is due to crossover activation of PKG.^{2 3} The PKG inhibitor Rp 8-Br PET cGMPS was therefore tested on a high concentration of 8-Br cAMP to provide direct evidence for this crossover effect. Under control conditions, 0.1 mmol/L 8-Br cAMP gave rise to significant inhibition of Ba²⁺ currents (eg, see Fig 2E). However, when cells were pretreated with Rp 8-Br PET cGMPS, the inhibitory effect of 8-Br cAMP was entirely abolished (compare Fig 2C with Fig 4A). In Fig 4B, the results with 10 and 100 nmol/L Rp 8-Br PET cGMPS are summarized.

View larger version (32K): [in this window] [in a new window]   Figure 4. Effect of high concentrations of 8-Br cAMP and 8-Br cGMP on peak inward Ba2+ currents during PKG inhibition. A and C, Time courses of peak Ba2+ currents elicited by voltage steps to +10 mV from a holding potential of -60 mV every 25 seconds and recorded by the perforated-patch technique. Each cell was first superfused with the PKG inhibitor Rp 8-Br PET cGMPS (Rp PET, 10 nmol/L), and this was followed by superfusion with both the inhibitor and 0.1 mmol/L 8-Br cAMP (A) or 0.1 mmol/L 8-Br cGMP (C). Note that both cyclic nucleotides caused an enhancement of Ba2+ currents. WO indicates washout period. B and D, Bar graphs summarizing the effects of two types of PKG inhibitors (Rp PET and Rp 8-Br cGMPS) used in the present study. Data (mean±SE) were determined as percent increase in Ba2+ current from control (see "Materials and Methods"). *P<.05 compared with control.

cGMP has also been reported to "cross over" and activate PKA.²⁶ Additional experiments were therefore undertaken to determine whether this pathway could be identified in portal vein myocytes. To examine this pathway, the PKG blocker Rp 8-Br PET cGMPS was tested on responses to 8-Br cGMP. Under control conditions, 0.1 mmol/L 8-Br cGMP inhibited Ba²⁺ currents in each cell tested (eg, see Fig 2E). However, when 8-Br cGMP was applied in the presence of Rp 8-Br PET cGMPS (10 nmol/L), Ba²⁺ current amplitude increased (Fig 4C), suggesting that cGMP also crosses over to activate PKA. To provide direct evidence for crossover activation of PKA, additional experiments were undertaken with the PKA blocker Rp 8-Br MB-cAMPS. The stimulatory response to 8-Br cGMP was entirely abolished in the presence of combined PKA and PKG blockade (Fig 4D). These results suggest that cGMP normally inhibits Ca²⁺ channel currents via PKG activation but may enhance Ca²⁺ channel currents via PKA activation when PKG is inhibited.

Time-Dependent Effects of 8-Br cAMP and Forskolin on Peak Ba²⁺ Currents
Our results suggest that an intermediate concentration of 8-Br cAMP (0.05 mmol/L) gives rise to time-dependent effects on Ba²⁺ currents, which include an initial brief excitation followed by sustained inhibition. Previously, we observed that direct activation of adenylyl cyclase with FSK also gave rise to an increase followed by a decrease in Ba²⁺ currents,² although the precise time course of these effects was not quantified. FSK was therefore reexamined in the present study so that its time dependent effects on Ba²⁺ currents could be directly compared with those of 8-Br cAMP (0.05 mmol/L) under the same conditions.

When cells were initially exposed to FSK, Ba²⁺ current amplitude increased by 40±10% (n=5). The time to reach peak current amplitude averaged 4.9±0.8 minutes (Fig 5A). This time was significantly longer than the time required to reach peak enhancement of current with 0.05 mmol/L 8-Br cAMP (ie, 1.4±0.3 minutes; Fig 5B). After the peak response to FSK, Ba²⁺ current amplitude declined and returned to the control level after 14.5 minutes. After 20 minutes, Ba²⁺ current amplitude had declined to a level that was 19% below the control amplitude. These data suggest that although 8-Br cAMP and FSK produce qualitatively similar effects on Ba²⁺ currents, their time courses of action differ significantly. The results also reveal that FSK enhances Ba²⁺ currents significantly more than does 8-Br cAMP (ie, 40% versus 16%). To determine whether greater enhancement of Ba²⁺ currents occurs when a lower concentration of FSK is applied, 1 µmol/L FSK was also tested. The enhancement observed with 1 µmol/L FSK (n=6) was not significantly different from that observed with 10 µmol/L FSK (ie, 34±10% increase versus 40±10% increase, respectively).

View larger version (19K): [in this window] [in a new window]   Figure 5. Effect of 8-Br cAMP and FSK on inward Ba2+ currents. Time course of peak currents is shown before and after exposure to 0.05 mmol/L 8-Br cAMP (A, n=3) and 10 µmol/L FSK (B, n=5). Currents were elicited by voltage steps to +10 mV from a holding potential of -60 mV every 20 seconds. Each point is expressed as the mean±SE value, determined as percent increase or decrease in currents from control (see "Materials and Methods").

Effect of Dialyzing Cells With the Catalytic Subunit of PKA
Our experiments with PKA and PKG blockers suggest that the increase in Ba²⁺ current elicited with cAMP is due to activation of PKA. This hypothesis was further investigated by dialyzing cells with the catalytic subunit of PKA (250 U/mL) over a 20-minute period of time. To ensure that the effect observed was due specifically to PKA, additional time-control experiments were performed using dialyzing pipettes that did not contain PKA. In addition, in some experiments, the PKA blocker KT 5720 (200 nmol/L) was added to the superfusate halfway through the protocol. KT 5720 is a relatively specific blocker of the catalytic subunit of PKA.²⁷ During the first few minutes after establishment of the whole-cell configuration, there was a variable degree of run-up of Ba²⁺ current. By 5 minutes, a steady state was reached. There was no significant difference between peak currents recorded from cells with and without PKA in the pipette at 5 minutes (839±63 pA [n=9, control] versus 719±35 pA [n=14, with PKA], P>.05). The currents plotted in Fig 6B were therefore normalized to the value obtained at 5 minutes. Dialysis of cells with PKA led to a 30% increase in Ba²⁺ current, which reached peak effect 13.5 minutes after cell break-in (n=8) and remained at this level for the next 6.5 minutes. When PKA was omitted from the dialyzing pipette, Ba²⁺ current amplitude reached steady state at 5 minutes and remained at this level for the next 15 minutes (n=9). Inclusion of KT 5720 in the superfusate after 13 minutes of dialysis with PKA fully reversed the PKA-induced increase in Ba²⁺ current amplitude (n=6). Sample traces are shown in Fig 6A, and a graph summarizing results is plotted in Fig 6B.

View larger version (25K): [in this window] [in a new window]   Figure 6. Effect of dialyzing cells with the catalytic subunit of PKA (250 U/mL). Ba2+ current was recorded every 30 seconds after cell break-in. A, Sample traces showing Ba2+ currents recorded at 5 minutes (1), 13.5 minutes (2), and 20 minutes (3) after break-in under various experimental conditions. Traces are as follows: trace a shows currents obtained in the absence of PKA; trace b, currents obtained when PKA was included in the pipette; and trace c, currents obtained with PKA in the pipette before and after application of KT 5720 (200 nmol/L). B, Summary graph of the time-dependent changes observed in Ba2+ currents using patch pipettes that contained PKA ( and ) and pipettes that did not (). Time after formation of whole-cell configuration is plotted on the x-axis. Cells dialyzed with PKA exhibited a significant increase in Ba2+ current compared with control currents. Application of KT 5720 at 13 minutes resulted in a significant decrease in current amplitude at later times (). Shown are mean±SE values. *P<.05 compared with control; +P<.05 compared with PKA alone.

	Discussion

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Agonists and maneuvers that raise cAMP levels in vascular smooth muscle have been reported to either enhance or suppress L-type Ca²⁺ channel activity. Presently, two hypotheses have been suggested to explain these conflicting results. The first hypothesis is that stimulation of the cAMP/PKA pathway causes inhibition of L-type Ca²⁺ channel activity and that enhancement of Ca²⁺ channels occurs via a direct membrane-delimited G-protein regulation of Ca²⁺ channels when agonists such as Iso are applied.^{11 12 13} The alternative hypothesis is that stimulation of the cAMP/PKA pathway leads to enhancement of L-type Ca²⁺ channel activity and that inhibition only occurs when cAMP levels are raised sufficiently to lead to crossover activation of PKG.^{2 3} In contrast to the controversy that surrounds the actions of PKA, there appears to be a general consensus that the cGMP/PKG pathway is coupled to inhibition of L-type Ca²⁺ channel currents.^{2 3 8 9 10 13} The present study provides additional evidence in support of the hypothesis that the inhibitory effects of cAMP are mediated by crossover activation of PKG. Our results further suggest that the actions of cGMP include crossover activation of PKA as well.

Addition of low concentrations of 8-Br cAMP led to a small but significant increase in Ba²⁺ currents, whereas higher concentrations of 8-Br cAMP reduced these currents. These results are in general agreement with previous studies suggesting that the actions of cAMP are concentration dependent; ie, low concentrations of cAMP enhance currents, whereas higher concentrations inhibit currents.^{2 3} The ability of cAMP to enhance L-type Ca²⁺ channel currents was also studied by Tewari and Simard,⁴ who found that 0.1 mmol/L 8-Br cAMP significantly increased single L-type Ca²⁺ channel activity in cell-attached patches of basilar artery smooth muscle cells. In addition, they found that exposure of inside-out patches to the PKA catalytic subunit increased channel availability. Other studies have shown that low concentrations of FSK can also lead to enhancement of L-type Ca channel currents.^{2 3 6 7 28} These observations are all difficult to reconcile with the hypothesis that stimulation of the cAMP/PKA pathway is exclusively inhibitory.^{11 12 13}

The precise concentration at which the inhibitory effect of 8-Br cAMP predominates over the excitatory effect appears to be both species and tissue dependent as well as being dependent on the recording conditions. Thus, in the present study performed on portal vein cells at 22°C with 5 mmol/L Ba²⁺ as charge carrier, 0.01 mmol/L 8-Br cAMP was excitatory, and 0.1 mmol/L 8-Br cAMP was inhibitory. In contrast, in a previous study of these cells performed at 35°C with 2.5 mmol/L Ba²⁺ as charge carrier, the concentration-response relationship was shifted to the right; ie, 0.1 mmol/L 8-Br cAMP was excitatory, whereas 1 mmol/L 8-Br cAMP was inhibitory. The reason for this difference may be related to a number of different factors, including isolation procedures as well as recording conditions. Nonetheless, in both the previous² and present portal vein studies as well as studies of canine colonic cells,³ the qualitative conclusion remains the same; ie, low concentrations of cAMP acting through PKA are excitatory, whereas higher concentrations of cAMP acting through PKG are inhibitory. The clear concentration dependence of effects and the differences in potency that are observed between preparations underscore the importance of testing a number of cyclic nucleotide concentrations. In a recent study of isolated rat portal vein myocytes by Liu et al,¹³ it was concluded that stimulation of the cAMP/PKA pathway produces only inhibition of Ca²⁺ channel currents. However, the lowest concentration of 8-Br cAMP tested was 0.1 mmol/L. Our results suggest that this concentration of 8-Br cAMP can produce inhibition, whereas lower concentrations are excitatory.

Direct stimulation of adenylyl cyclase with 1 to 10 µmol/L FSK enhanced Ba²⁺ currents to a greater extent than all concentrations of 8-Br cAMP tested (ie, 0.1 µmol/L to 0.1 mmol/L). This difference is unlikely to be related to access limitations for 8-Br cAMP, since higher concentrations of 8-Br cAMP inhibited Ba²⁺ currents, an action that we attribute to cyclic nucleotide levels that are sufficient to lead to crossover activation of PKG (eg, see Fig 2). We propose instead that the difference in efficacy of FSK versus 8-Br cAMP as activators of Ba²⁺ currents is related to differences in their relative potency as activators of PKG. Although cAMP and 8-Br cAMP activate PKA with similar potency,²⁹ 8-Br cAMP is a significantly more potent activator of PKG than is cAMP.^{29 30} For this reason, crossover activation of PKG by 8-Br cAMP may reach significant levels before maximum activation of PKA is attained, thereby limiting the enhancement of Ba²⁺ currents, which can occur with 8-Br cAMP. Thus, even differences in efficacy between FSK and 8-Br cAMP may be related to crossover activation of PKG.

FSK (10 µmol/L) led to time-dependent effects on Ba²⁺ currents, which began with excitation and progressed to inhibition. High concentrations of Iso also produce a similar time-dependent pattern.² This time dependence is likely to be related to time-dependent changes in PKA and PKG activity; ie, at early times (when cAMP levels are lower), the PKA effect may predominate, leading to excitation, whereas at later times (when cAMP levels are higher), the PKG effect may predominate, leading to inhibition. The present study revealed that an intermediate concentration of 8-Br cAMP (0.05 mmol/L) also produced similar time-dependent changes in Ba²⁺ current amplitude, although the period of excitation was much more limited. As discussed above, one reason for the difference between FSK and 8-Br cAMP may be related to the greater potency of 8-Br cAMP versus cAMP for PKG.^{29 30} However, it is also possible that the differences in time course reflect differences in the manner in which cyclic nucleotide levels rise in the cell when adenylyl cyclase is activated by FSK versus by diffusion of 8-Br cAMP across the sarcolemma.

The PKA blockers Rp 8-Br cAMPS (30 µmol/L) or Rp 8-Br MB-cAMPS (30 µmol/L) alone inhibited Ba²⁺ currents, whereas the PKG blocker Rp 8-Br PET cGMPS alone enhanced Ba²⁺ currents. These results suggest that Ca²⁺ channels are subject to PKA and PKG regulation under basal conditions. Kinase activation, in turn, is likely related to the basal production of both cAMP and cGMP that occurs in smooth muscle.^{23 24} The direction of change that we observed with each kinase blocker tested is commensurate with the proposed actions of PKA and PKG; ie, blockade of PKA (which we propose activates Ca²⁺ channels) inhibited Ba²⁺ currents, whereas blockade of PKG (which we propose inhibits Ca²⁺ channels) enhanced Ba²⁺ currents. It is difficult to predict which one of these effects predominates in the intact tissue. Indeed, it is likely that the balance continually shifts as changes in cyclic nucleotide levels occur in vivo. In addition, phosphorylation of either PKA or PKG may affect the modulatory role of these kinases. For instance, in one study that measured PKG activity purified from bovine lung, it was reported that autophosphorylation of PKG increased its affinity for cAMP 10-fold.³¹ Further experiments are needed to determine the relative contribution of phosphorylation and dephosphorylation to the activation of these kinases in portal vein myocytes.

Two observations made in the present study are of particular relevance in clarifying the direct role of the cAMP/PKA pathway in modulation of L-type Ca²⁺ channel activity. The first observation was that dialyzing cells with the catalytic subunit of PKA led to enhancement of Ca²⁺ channel currents, which was reversed by the PKA blocker KT 5720. This provides direct evidence that PKA can enhance L-type Ca²⁺ channel activity in portal vein cells. The second observation was that the inhibitory effect of 8-Br cAMP on Ca²⁺ channel currents was blocked by inhibition of PKG with Rp 8-Br PET cGMPS. This provides direct evidence that cAMP inhibits Ca²⁺ channel currents via cross activation of PKG. These data also strongly argue against the notion that the inhibitory action of cAMP on Ca²⁺ channel activity is mediated by PKA.^{11 12 13}

In the present study, we also obtained evidence suggesting that cGMP can activate PKA; ie, when PKG was blocked with Rp 8-Br PET cGMPS, addition of 8-Br cGMP enhanced Ba²⁺ currents. We propose that this effect is due to crossover activation of PKA by 8-Br cGMP. Recently, Cornwell et al²⁶ proposed a similar pathway in a study examining the proliferation of cultured rat aortic smooth muscle cells. In their study, both the NO generator (SNAP) and interleukin-1ß were reported to increase cGMP levels and PKA activity but not cAMP levels. These events, which are associated with proliferation of vascular smooth muscle cells, were inhibited by Rp 8-Br cAMPS but unaffected by Rp 8-Br cGMPS. An alternative mechanism for the stimulatory effects of 8-Br cGMP that cannot be ruled out is that it reduces cAMP breakdown by inhibiting PDE III. This would tend to raise cAMP levels and hence stimulate PKA.³² However, studies in other systems suggest that the affinity of 8-Br cGMP for PDE III is much less than that of authentic cGMP,³³ making this mechanism less likely than crossover activation of PKA. Furthermore, to adequately assess this pathway, it would be necessary to know the relative contribution of PDE III to cAMP breakdown in rabbit portal vein. Vascular smooth muscle cells are known to contain five different PDE isozymes, and the expression varies from tissue to tissue.^{32 34}

Our results indicate that 8-Br cAMP (and FSK) produce either an increase or decrease in Ca²⁺ channel current amplitude, depending on the time of exposure and the concentration of drug applied; ie, low concentrations enhance Ca²⁺ channel activity, whereas higher concentrations inhibit Ca²⁺ channel activity. In contrast, 8-Br cGMP inhibits Ca²⁺ channel currents at all concentrations tested, unless PKG activity is suppressed with a blocker. This suggests that when PKA and PKG are both fully activated, the PKG effect on Ca²⁺ channel currents predominates.

L-type Ca²⁺ channels are heteropentameric complexes composed of ₁, ₂, ß, , and subunits. The gene expressing the cardiac ₁ subunit has been reported to be alternatively spliced and expressed in smooth muscle.³⁵ In cardiac myocytes, activation of the cAMP/PKA pathway via ß-adrenoceptor activation leads to enhancement of L-type Ca²⁺ channel currents and a positive ionotrophic response.¹ However, in smooth muscle, activation of the cAMP/PKA pathway leads to muscle relaxation. The physiological role of PKA-induced enhancement of L-type Ca²⁺ channel currents is therefore likely to be quite different in smooth muscle than in cardiac muscle. Various membrane conductances can be modified by changes in [Ca]_i.^{36 37 38} It is possible that the activity of one or more of these conductances is modified when Ca²⁺ current is enhanced by cAMP-dependent mechanisms.

In summary, the present results support the hypothesis that L-type Ca²⁺ channel activity in the rabbit portal vein is enhanced by cAMP/PKA stimulation and inhibited by cGMP/PKG stimulation. Under basal conditions, both of these pathways appear to exert some modulatory influence over Ca²⁺ channel activity. Our results further suggest that cAMP and cGMP each has a primary action mediated by its own kinase as well as a secondary action mediated by the opposing kinase (an action that has been referred to as cross activation^{14 16} or "crossover"). These conclusions are in agreement with the findings of several other groups,^{2 3 4 7} all of which are difficult to reconcile with the hypothesis that cAMP activation of PKA is responsible for inhibition of L-type Ca²⁺ channel activity in smooth muscle cells.^{11 12 13}

	Selected Abbreviations and Acronyms

 

Br = bromo cAMPS = adenosine-3',5'-monophosphorothioate cGMPS = guanosine-3',5'-monophosphorothioate DMSO = dimethyl sulfoxide FSK = forskolin Iso = isoproterenol MB = monobutyryl PDE = phosphodiesterase PET = ß-phenyl-1, N2-etheno PKA, PKG = protein kinase A and G Rp = Rp isomer SNAP = S-nitroso-N-acetylpenicillamine TEACl = tetraethylammonium chloride

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-40399 to Dr Keef and HL-49254 to Dr Hume and a postdoctoral fellowship to Dr Ruiz-Velasco from the American Heart Association, Nevada Affiliate, Inc. We would also like to extend our sincere appreciation to Drs T. Lincoln and P. Komalavilas for supplying KT 5720 when none was available elsewhere.

Received July 21, 1997; accepted December 23, 1997.

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