ATP-sensitive K⁺ channel activation by calcitonin gene-related peptide and protein kinase A in pig coronary arterial smooth muscle

Abstract

1. We used patch clamp to study whole-cell K⁺ currents activated by calcitonin gene-related peptide (CGRP) in smooth muscle cells freshly dissociated from pig coronary arteries.

2. CGRP (50 nm) activated an inward current at −60 mV in symmetrical 140 mm K⁺ that was blocked by glibenclamide (10 μm), an inhibitor of ATP-sensitive potassium (K_ATP) channels. CGRP-induced currents were larger in cells dialysed with 0.1 mm ATP than with 3.0 mm ATP.

3. Forskolin (10 μm) activated a glibenclamide-sensitive current, as did intracellular dialysis with cAMP (100 μm). The catalytic subunit of cAMP-dependent protein kinase (protein kinase A, PKA), added to the pipette solution, activated equivalent currents in five out of twelve cells.

4. CGRP-induced currents were reduced by the PKA inhibitors adenosine 3′,5′-cyclic monophosphorothioate, R_P-isomer, triethylammonium salt (Rp-cAMPS; 100 μm) and N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulphonamide dihydrochloride (H-89; 1 μm), and abolished by inclusion of a PKA inhibitor peptide in the pipette solution.

5. The β-adrenergic agonist isoprenaline (10 μm) also activated a glibenclamide-sensitive K⁺ current.

6. CGRP-induced currents were unaffected by the inhibitor of cGMP-dependent protein kinase (PKG) KT5823 (1 μm). Sodium nitroprusside (10 μm) did not activate a glibenclamide-sensitive current in cells held at −60 mV, but did activate an outward current at +60 mV that was abolished by KT5823, or by 100 nm iberiotoxin (an inhibitor of BK_Ca channels).

7. Our findings suggest that CGRP activates coronary K_ATP channels through a pathway that involves adenylyl cyclase and PKA, but not PKG.

Calcitonin gene-related peptide (CGRP) is an extremely potent vasodilator in the coronary circulation of several species, including humans, pigs and rats (for review see Bell & McDermott, 1996). CGRP-immunoreactive nerve fibres are present throughout the non-adrenergic non-cholinergic innervation of the vasculature, and in the coronary circulation local release of CGRP from these fibres may contribute substantially to basal coronary blood flow (Yaoita, Sato, Kawaguchi, Saito, Maehara & Maruyama, 1994). The release of CGRP is greatly enhanced in cardiac ischaemia, where its effects may contribute both to subsequent vasodilatation and to protection of the myocardium (Franco-Cerceda, Saria & Lundberg, 1989; Li, Xiao, Peng & Deng, 1996).

One possible mechanism that might link CGRP to coronary artery vasodilatation is the activation of ATP-sensitive K⁺ channels (K_ATP channels), which have been implicated in the regulation of coronary blood flow under both normoxic and hypoxic conditions (Daut, Maier-Rudolph, von Beckerath, Mehrke, Günther & Goedel-Meinen, 1990; also see Quayle, Nelson & Standen, 1997, for review). K⁺ channel activation will lead to membrane hyperpolarization, so decreasing intracellular [Ca²⁺] and contractile force (Nelson, Patlak, Worley & Standen, 1990b; Quayle et al. 1997). Indeed CGRP-induced vasorelaxation and hyperpolarization have been shown to be inhibited by the K_ATP channel blocker glibenclamide in both mesenteric and cerebral arteries (Nelson, Huang, Brayden, Hescheler & Standen, 1990a; Kitazono, Faraci & Heistad, 1993). It has also recently been demonstrated that CGRP and adenosine A₂ receptor activation can increase K_ATP currents in rabbit mesenteric arteries through a pathway involving activation of adenylyl cyclase and cAMP-dependent protein kinase (protein kinase A, PKA) (Quayle, Bonev, Brayden & Nelson, 1994; Kleppisch & Nelson, 1995). It is not clear, however, that CGRP can activate K_ATP channels in coronary arteries, since CGRP-induced relaxations of rat and porcine coronary arterial rings in vitro have been reported to be insensitive to glibenclamide (Prieto, Benedito & Nyborg, 1991; Kageyama, Yanagisawa & Taira, 1993).

CGRP has, however, been reported to activate ATP-sensitive K⁺ channels in cells that migrate out of coronary artery explants maintained in culture (Miyoshi & Nakaya, 1995). But the properties of these channels differ substantially from those reported for K_ATP channels of native smooth muscle cells freshly isolated from vascular tissue. For example, the channels of cultured cells are expressed at a much higher density than are native vascular K_ATP channels, and are extremely sensitive to activation by extracellular Ca²⁺ (Miyoshi et al. 1992). At external [Ca²⁺] greater than 100 μM, the open probability of the channels in cultured cells is near unity, so that these channels do not exhibit ATP dependence when exposed to physiological [Ca²⁺]_o. In contrast, K_ATP channels of native vascular smooth muscle cells, including those of coronary arteries, show very low open probabilities in the absence of exogenous or endogenous activators, and high sensitivity to [Ca²⁺]_o has not been reported (Dart & Standen, 1993; Quayle et al. 1997).

Thus, despite the likely importance of CGRP in the coronary circulation, it remains unclear whether CGRP can activate K_ATP channels in coronary arterial smooth muscle cells and, if so, what signalling pathway(s) are involved. In the present study, we have measured whole-cell K_ATP currents in smooth muscle cells freshly isolated from pig coronary arteries. We find that CGRP is an effective activator of K_ATP channels in these cells, and provide evidence that this action is mediated by production of cAMP and activation of PKA. While it has been shown that cAMP can also cause cross-activation of cGMP-dependent protein kinase (PKG) in pig coronary arteries (Jiang, Colbran, Francis & Corbin, 1992), our experiments suggest that activation of PKG is not involved in K_ATP activation by CGRP, and indeed that activation of PKG with sodium nitroprusside does not activate K_ATP channels. Further, we provide the first direct evidence that the β-receptor agonist isoprenaline can also activate K_ATP currents in native vascular smooth muscle. A brief report of some of these findings has been published (Wellman, Quayle, Everitt & Standen, 1997).

METHODS

Tissue preparation and cell isolation

Pig hearts were obtained from a local abattoir, and first order branches (approximately 1-2 mm outer diameter) of the left anterior descending coronary artery were dissected and cut into 2 mm ring segments while in cold saline solution containing (mM): 137 NaCl, 5.4 KCl, 0.44 NaH₂PO₄, 0.42 Na₂HPO₄, 1 MgCl₂, 2 CaCl₂, 10 Hepes, 10 glucose; pH adjusted to 7.4 with NaOH. Half of the segments were used immediately while the others were stored for up to 24 h in either cold saline (4°C) or tissue culture medium (Dulbecco's modified Eagle's medium F-12, Ham's nutrient mix) supplemented with penicillin-streptomycin (10 i.u. ml⁻¹ and 10 μg ml⁻¹, respectively) and bovine albumin fraction V at 37°C. Similar results were observed using either storage condition, and therefore have been pooled.

Single vascular smooth muscle cells were isolated from coronary arteries using an enzymatic dissociation procedure similar to that which has been described previously (Quayle, Dart & Standen, 1996). Arteries were first incubated at 35°C for 45 min in saline solution containing 2 mM Ca²⁺, and then transferred to saline containing 0.1 mM Ca²⁺ for 5 min before being placed into a similar 0.1 mM Ca²⁺ solution with 1-1.5 mg ml⁻¹ papain and 1 mg ml⁻¹ dithioerythritol for about 30 min at 35°C. Arteries were then transferred into a 0.1 mM Ca²⁺ solution containing 1-1.5 mg ml⁻¹ collagenase F and 1 mg ml⁻¹ hyaluronidase for 15-20 min, rinsed in 0.1 mM Ca²⁺ solution, and single cells dispersed by trituration using a polished pasteur pipette. Cells were stored on ice and used for up to 8 h after isolation.

Data recording

The conventional whole-cell configuration of the patch clamp technique was used to measure membrane currents in single arterial smooth muscle cells. Patch electrodes were fabricated from thin-walled borosilicate glass (1.5 mm outer diameter; Clarke Electromedical, Pangbourne, Berks, UK), coated with sticky wax (Kemdent, Swindon, Wilts, UK) to reduce capacitance, and fire polished. Electrode resistances were around 5 MΩ before sealing to the cell, and seal resistances were 5-10 GΩ. Following the establishment of whole-cell recordings, the mean series resistance was 8.7 ± 0.4 MΩ (n= 112). Since the currents recorded in these experiments were less than 250 pA, corresponding to a maximum voltage error of 3 mV, we did not compensate for series resistance. Data were recorded and cell voltage controlled using a personal computer and either pCLAMP 6 or Axotape 2 software and an Axopatch 200A amplifier and Digidata 1200 interface (Axon Instruments). Data were also stored on FM tape (Racal Store 4DS) at a bandwidth of 625 Hz. For display, whole-cell currents were filtered at 2 kHz using an 8-pole Bessel filter and sampled at a frequency of 6.7 Hz using Axotape 2. Data were analysed using pCLAMP 6 software.

Solutions

The pipette (intracellular) solution had the following composition (mM): 107 KCl, 33 KOH, 10 Hepes, 10 EGTA, 1 MgCl₂, 0.1 Na₂ATP, 0.1 NaADP, 0.3 GTP (Li⁺ salt); pH adjusted to 7.2 with NaOH. Seals were made in a 6 mM K⁺ extracellular solution containing (mM): 6 KCl, 134 NaCl, 1 MgCl₂, 0.1 CaCl₂, 10 Hepes, 10 glucose; pH adjusted to 7.4 with NaOH, but most recordings were made under conditions of symmetrical 140 mM K⁺ using an extracellular solution of the same composition as that above, except that KCl was 140 mM and NaCl was omitted. The experimental bath (volume, 0.5 ml) was perfused continuously with extracellular solution at a rate of about 2 ml min⁻¹.

All chemicals and reagents were obtained from Sigma except for pinacidil (Research Biochemicals) and Rp-cAMPS (the R_P isomer of adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt), H-89 (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulphonamide dihydrochloride), KT5823 and the purified catalytic subunit of PKA (which were from Calbiochem). Synthetic rat CGRP (Sigma) was frozen in aliquots of 100 μM stock solution in distilled water, which were added to the extracellular solution to give a final concentration of 50 nM.

Experiments were conducted at a temperature of 20-26°C. Results are given as means ±s.e.m. Where control and test measurements were made on the same cell, we have expressed mean results directly as whole-cell current (in pA). Where, however, different cells were used, as they were when applying different ATP concentrations or cAMP in the pipette solution used to dialyse the cell interior, mean results are expressed relative to membrane capacitance (in pA pF⁻¹) to allow for differences in cell size. Statistical significance was assessed using Student's paired or unpaired t test as appropriate.

RESULTS

In this study we used 112 isolated coronary arterial myocytes. These cells had a membrane capacitance of 21.1 ± 0.5 pF, similar to values we have reported previously in the same preparation (Dart & Standen, 1995; Quayle et al. 1996).

CGRP activates K_ATP current in pig coronary arterial myocytes

Our initial experiments were designed to establish whether CGRP could activate K_ATP currents in smooth muscle cells freshly isolated from pig coronary arteries. The recording conditions (cell dialysis with 10 mM EGTA and a holding potential of -60 mV) were therefore chosen to minimize activity of Ca²⁺-activated and delayed rectifier potassium channels. Unless otherwise stated, cells were also dialysed with 0.1 mM ATP and 0.1 mM ADP to enhance K_ATP currents. Figure 1A shows a typical recording of whole-cell current made under these conditions. Increasing the extracellular potassium concentration from 6 to 140 mM, so that the driving force on K⁺ became inward, caused a modest increase in inward membrane current. Application of CGRP (50 nM) produced a substantial additional inward membrane current. In thirty-two cells, 50 nM CGRP activated a mean membrane current of -67.4 ± 11.4 pA. Figure 1A also shows that the CGRP-induced current was abolished by the sulphonylurea K_ATP channel inhibitor glibenclamide (10 μM), which shows good selectivity for K_ATP channels at this concentration (Wellman, Quayle & Standen, 1996; Quayle et al. 1997). The voltage dependence of the CGRP-induced current was also consistent with the involvement of K_ATP channels. Figure 1B shows that the current-voltage relationship measured in 140 mM [K⁺]_o in response to a voltage ramp from -100 to +40 mV (at a rate of 0.3 mV ms⁻¹) is linear, as expected for a channel such as K_ATP whose gating shows little or no voltage dependence. This CGRP-activated current reversed direction at +1.0 mV, very close to the calculated reversal potential for K⁺ of 0 mV. This finding is consistent with our previous work showing that the reversal potentials of glibenclamide-sensitive currents activated in these cells by adenosine and by hypoxia change with changes in [K⁺]_o, as expected for currents flowing through K⁺ channels (Dart & Standen, 1993, 1995).

Figure 1. K_ATP current activation by CGRP in pig coronary arterial smooth muscle cells.

A, whole-cell current recorded in a cell dialysed with 0.1 mM ATP at a holding potential of -60 mV. Intracellular K⁺ was 140 mM, and extracellular K⁺ was changed from 6 to 140 mM (indicated by arrow) prior to addition of CGRP. The dotted line represents zero current level in this and subsequent figures. Cell capacitance was 23.5 pF. B, current-voltage relationship of the glibenclamide-sensitive current obtained in the presence of 50 nM CGRP. The cell was dialysed with 0.1 mM ATP and exposed to symmetrical 140 mM K⁺. Voltage was ramped from -100 to +40 mV at a rate of 0.3 mV ms⁻¹. The displayed trace represents averaged ramp currents obtained in the presence of CGRP from which averaged ramp currents obtained in the presence of both CGRP and 10 μM glibenclamide have been subtracted. Ramp currents averaged (n= 20) in each condition were obtained from a single cell. Cell capacitance was 21 pF. C, K_ATP current density in cells measured under basal conditions (140 mM K⁺) and in the presence of 50 nM CGRP. Cells were dialysed with either 0.1 mM ATP (▪, n= 6) or 3.0 mM ATP (, n= 3). The bars show glibenclamide-sensitive current density (mean +s.e.m.). *P < 0.05 compared with 0.1 mM ATP and extracellular CGRP. Mean currents before normalization to capacitance under each condition were as follows: 0.1 mM ATP (basal current), -17.8 ± 2.9 pA; 0.1 mM ATP + 50 nM CGRP, -80.4 ± 12.8 pA; 3.0 mM ATP (basal current), -10.5 ± 6.0 pA; 3.0 mM ATP + 50 nM CGRP, -34.6 ± 6.0 pA.

Further evidence that the CGRP-activated current flows through K_ATP channels comes from experiments in which we used an intracellular solution containing 3 mM ATP, rather than 0.1 mM ATP used in most of this study. Figure 1C shows that CGRP-induced glibenclamide-sensitive currents were significantly smaller in cells dialysed with 3.0 mM rather than 0.1 mM ATP; -1.37 ± 0.22 pA pF⁻¹ (n= 3) as opposed to -3.86 ± 0.64 pA pF⁻¹ (n= 6). In most cells we found that part of the basal inward current recorded in 140 mM external K⁺ could also be blocked by glibenclamide, consistent with this component flowing through K_ATP channels, while a further part is likely to represent current through inward rectifier K⁺ channels (Quayle et al. 1996).

Activation of adenylyl cyclase or PKA increases K_ATP current

Forskolin

Many of the actions of CGRP have been ascribed to an intracellular signal transduction pathway involving activation of adenylyl cyclase, leading to intracellular accumulation of cAMP and so activation of PKA (Bell & McDermott, 1996). To investigate whether such a pathway might underlie K_ATP channel activation by CGRP in coronary arterial myocytes, we first sought to establish whether activation of adenylyl cyclase using forskolin could itself lead to activation of K_ATP channels. Forskolin is known to activate adenylyl cyclase in many cellular systems, and Fig. 2A shows that in coronary arterial myocytes, 10 μM forskolin mimicked the effect of CGRP in activating a membrane current that was completely inhibited by the addition of 10 μM glibenclamide. In four cells the glibenclamide-sensitive current was increased from -15.8 ± 1.4 pA under control conditions to -51.0 ± 5.3 pA following the addition of forskolin (Fig. 2B).

Figure 2. Forskolin activation of K_ATP current.

A, whole-cell current recorded in a cell held at -60 mV. Arrows indicate that the K⁺ concentration in the bath superfusate was changed to 140 mM prior to addition of forskolin, and returned to 6 mM K⁺ at the end of the experiment. Forskolin and glibenclamide were added as indicated. Intracellular K⁺ was 140 mM and the cell was dialysed with 0.1 mM ATP. Following the inhibition of the forskolin-induced current by glibenclamide, the K⁺ concentration in the bath solution was changed from 140 to 6 mM demonstrating that the leak current in the cell was not altered from the start of the experiment. Cell capacitance was 20 pF. B, magnitude of glibenclamide-sensitive currents obtained in 140 mM K⁺ in the absence and presence of 10 μM forskolin. The bars show means +s.e.m. from 4 cells. **P < 0.01 compared with control.

An alternative way to increase intracellular cAMP levels is to inhibit the degradation of the cyclic nucleotide by use of the non-specific phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). If CGRP was enhancing K_ATP current through stimulation of adenylyl cyclase, phospho-diesterase inhibition leading to an increase in cAMP accumulation in response to CGRP should result in a potentiation of the resulting K_ATP current. Consistent with the involvement of cAMP, we found that CGRP activated a glibenclamide-sensitive current of -46.7 ± 15.4 pA, and that when IBMX (100 μM) was added in the continued presence of CGRP the current became -82.5 ± 19.6 pA (n= 4).

cAMP and the catalytic subunit of PKA

To investigate the ability of increased levels of cAMP to enhance K_ATP currents more directly, we also did experiments in which the cyclic nucleotide was included in the intracellular (pipette) solution. As described above, a small basal K_ATP current was normally seen in cells dialysed with our standard pipette solution, and an example is shown in Fig. 3A. Dialysis with pipette solution containing 100 μM cAMP led to development of a much greater K_ATP current (Fig. 3B). Glibenclamide-sensitive currents averaged 6.6 ± 1.0 pA pF⁻¹ (n= 8) in the presence of cAMP compared with 1.1 ± 0.2 pA pF⁻¹ in five control cells studied in the same series of experiments (Fig. 3D).

Figure 3. K_ATP current activation by cAMP and the catalytic subunit of PKA.

A, whole-cell current record demonstrating the background glibenclamide-sensitive current in a cell dialysed with control (140 mM K⁺, 0.1 mM ATP) pipette solution and held at -60 mV. In this and in B and C, whole-cell recording was established at the beginning of the record shown, and the potassium concentration of the external superfusate was changed from 6 to 140 mM and back to 6 mM as indicated by the arrows. Glibenclamide was applied as shown by the bar. Cell capacitance was 16 pF. B, whole-cell current record made using a pipette solution containing 100 μM cAMP. Cell capacitance was 15.6 pF. C, whole-cell current record from a cell made using a pipette solution containing 100 units ml⁻¹ of the catalytic subunit of PKA. Cell capacitance was 20.5 pF. D, mean (+s.e.m.) K_ATP current densities. The left-hand bars show current density in cells dialysed with pipette solution containing 100 μM cAMP (n= 8,) and in cells dialysed with control pipette solution (n= 5, ▪) recorded in the same series of experiments. The right-hand bars show the current density in 5 cells dialysed with pipette solution that contained 100 units ml⁻¹ PKA catalytic subunit () and in 5 control cells recorded in the same series of experiments (▪). **P < 0.01 compared with control. Mean currents before normalization to capacitance under each condition were as follows: left-hand control, -18.7 ± 4.4 pA; cAMP, -99.6 ± 35.2 pA; right-hand control, -15.5 ± 3.8 pA; PKA catalytic subunit, -109.7 ± 18.8 pA.

The above experiments are all consistent with an increase in cAMP causing activation of cAMP-dependent protein kinase to increase K_ATP current. We therefore also performed experiments to examine whether application of the purified catalytic subunit of PKA could itself enhance K_ATP channel activity. We anticipated the possibility that the catalytic subunit might not be effective in all experiments, since the large size of the molecule (40 kDa) might restrict its diffusion from the pipette solution into the cell if the recording geometry was not ideal. We found that K_ATP currents similar in size to those seen in response to cAMP were obtained in five out of twelve cells dialysed with the PKA catalytic subunit (100 units ml⁻¹). One such cell is illustrated in Fig. 3C. Glibenclamide-sensitive currents in these five cells averaged 5.0 ± 0.9 pA pF⁻¹ compared with 0.9 ± 0.3 pA pF⁻¹ (n= 5) in cells dialysed with control pipette solution (Fig. 3D).

Inhibition of CGRP-induced currents by inhibitors of cAMP-dependent protein kinase

Increased adenylyl cyclase activity and the consequent increase in intracellular cAMP have been shown to activate PKA in pig coronary arteries (Jiang et al. 1992). If CGRP causes K_ATP channel activation by such a pathway, it should be possible to inhibit the effect of CGRP using agents that inhibit PKA. We used the cAMP analogue Rp-cAMPS, which inhibits PKA by binding to its regulatory subunit (Rothermel & Parker-Botelho, 1988). Figure 4A shows that Rp-cAMPS, applied in the continued presence of CGRP, caused a reduction in the K_ATP current activated by CGRP. In four cells, 100 μM Rp-cAMPS reduced the CGRP-activated current by 61.5 ± 2.2 % (Fig. 4C). We considered the possibility that Rp-cAMPS might have a direct blocking action on the K_ATP channel in addition to causing inhibition of PKA, and that such a direct block might explain part of the observed reduction in the CGRP-induced K_ATP current. We therefore investigated the effect of Rp-cAMPS on currents activated by the K_ATP channel opener pinacidil, thought to act directly on K_ATP channels. Rp-cAMPS (100 μM) had no significant effect on the glibenclamide-sensitive current activated by 10 μM pinacidil (Fig. 4B); mean results for four cells are shown in Fig. 4C. This suggests that the reduction of CGRP-activated current by Rp-cAMPS shown in Fig. 4 was primarily caused by inhibition of PKA.

Figure 4. Inhibition of CGRP-induced current by the PKA inhibitor Rp-cAMPS.

A, whole-cell current record demonstrating the inhibition by Rp-cAMPS of a CGRP-induced current obtained in a cell dialysed with 0.1 mM ATP and 140 mM K⁺ and held at -60 mV. The potassium concentration of the superfusate bathing the cell was changed from 6 to 140 mM prior to addition of CGRP. Upon establishing a stable current level in the presence of CGRP, Rp-cAMPS was added to the superfusion solution. The cell was also exposed to glibenclamide at the end of the recording. Cell capacitance was 21 pF. B, whole-cell current record demonstrating that Rp-cAMPS did not reduce the current produced in response to the direct K_ATP channel opener pinacidil in a myocyte dialysed with 0.1 mM ATP and 140 mM K⁺ and held at -60 mV. The K⁺ concentration of the superfusate bathing the cell was changed from 6 to 140 mM prior to addition of pinacidil. Cell capacitance was 24.5 pF. C, histogram showing the mean +s.e.m. percentage inhibition by Rp-cAMPS of CGRP-induced (▪) and pinacidil-induced current (), respectively (n= 4 in each case). *P < 0.05 compared with control (CGRP current before application of Rp-cAMPS). Rp-cAMPS did not affect the current in response to pinacidil (P= 0.39).

The staurosporine analogue H-89 is believed to inhibit PKA in a different way from Rp-cAMPS, binding to the ATP-binding site of the enzyme and so preventing it from causing phosphorylation of its target protein (Hidaka, Watanabe & Kobayashi, 1991). H-89 was also an effective inhibitor of CGRP-induced K_ATP current, as 1 μM of this compound caused a 68.3 ± 11.8 % inhibition of the CGRP-activated current (n= 4 cells). H-89 (1 μM), though, also reduced pinacidil-induced current by 28.7 ± 2.6 % (n= 7 cells, P < 0.01), suggesting that H-89, unlike Rp-cAMPs, may cause some direct inhibition of K_ATP channels. However, H-89 was considerably less effective on pinacidil-induced current than on that activated by CGRP, consistent with PKA inhibition contributing substantially to its action in reducing CGRP-activated current.

As external application of either Rp-cAMPS or H-89 only partially reversed the effects of CGRP, and since both compounds show only about 10-fold selectivity for PKA over cGMP-dependent protein kinase, it is unclear whether CGRP was activating K_ATP currents primarily through a mechanism involving PKA. We therefore sought to achieve complete inhibition of PKA by including a highly selective twenty amino acid synthetic PKA inhibitor peptide (Cheng et al. 1986) in the intracellular solution. In cells dialysed with 5 μM of the PKA inhibitor peptide, CGRP failed to activate a membrane current, as illustrated in Fig. 5A. However, subsequent addition of 10 μM pinacidil always activated a glibenclamide-sensitive membrane current in these cells, demonstrating the presence of functional K_ATP channels. In seven cells studied under these conditions, 10 μM pinacidil increased the glibenclamide-sensitive current from a basal value in 140 mM K⁺ current of -35.0 ± 11.0 to -175.3 ± 26.6 pA, while CGRP was without effect (Fig. 5B). Further, in the same series of experiments, in control cells where the PKA inhibitor peptide was not included in the intracellular solution, CGRP activated a glibenclamide-sensitive current of -60.7 ± 28.8 pA (n= 3), demonstrating the presence of functional CGRP receptors in comparable cells. These results strongly support the concept that activation of K_ATP current by CGRP is primarily through a pathway involving PKA.

Figure 5. PKA inhibitor peptide blocks CGRP activation of K_ATP current.

A, whole-cell current record showing the absence of current activation by CGRP in a cell held at -60 mV and dialysed with pipette solution containing 5 μM PKA inhibitor peptide. The potassium concentration of the superfusate bathing the cell was changed from 6 to 140 mM prior to addition of 50 nM CGRP. After testing CGRP, 10 μM pinacidil and then 10 μM glibenclamide were added as indicated. Cell capacitance was 11 pF. B, mean (+s.e.m.) basal glibenclamide-sensitive current (▪) and glibenclamide-sensitive current in the presence of CGRP (50 nM,) and pinacidil (10 μM,□) in 7 cells dialysed with PKA inhibitor peptide. **P < 0.01 compared with basal current.

Activation of K_ATP currents by isoprenaline

In addition to CGRP, certain other vasodilators that are effective in the coronary circulation are known to activate adenylyl cyclase and PKA. These include β-adrenoceptor agonists and prostacyclins (Quayle et al. 1997). We therefore examined whether activation of the adenylyl cyclase-PKA pathway with a different receptor, the β-adrenoceptor, was also able to activate K_ATP currents in isolated coronary arterial smooth muscle cells. Figure 6 shows that, like CGRP, the β-adrenoceptor agonist isoprenaline (10 μM) produced an increase in the membrane current (n= 5 cells) that was completely reversed by the addition of glibenclamide.

Figure 6. Activation of K_ATP current by isoprenaline.

A, whole-cell current record in a cell held at -60 mV. The arrow indicates that the K⁺ concentration in the bath superfusate was changed to 140 mM prior to addition of isoprenaline. Intracellular K⁺ was 140 mM and the cell was dialysed with 0.1 mM ATP. Cell capacitance was 31 pF. B, magnitude of glibenclamide-sensitive currents obtained in 140 mM K⁺ in the absence and presence of 10 μM isoprenaline. The bars show the means +s.e.m. from 5 cells. **P < 0.01 compared with control.

The role of PKG in the response to CGRP and sodium nitroprusside

In tissue extracts of pig coronary arteries, isoprenaline and forskolin have been shown to elevate cAMP to levels which increase the activity of cGMP-dependent protein kinase (PKG) (Jiang et al. 1992). The effectiveness of the PKA inhibitor peptide in abolishing K_ATP current activation by CGRP (Fig. 5) argues against such cross-activation of PKG being involved in the response to CGRP in our experiments. To confirm that PKG was not involved in K_ATP current activation by CGRP, we used the membrane-permeant inhibitor of PKG, KT5823 (Kase et al. 1987). Figure 7A shows that KT5823 (1 μM) did not reduce CGRP-induced glibenclamide-sensitive membrane current; mean results for five cells are shown in Fig. 7B.

Figure 7. Inhibition of PKG does not block activation of K_ATP current by CGRP.

A, whole-cell current recording from a myocyte held at -60 mV. Intracellular K⁺ was 140 mM and the cell was dialysed with 0.1 mM ATP. CGRP, the PKG inhibitor KT5823 and glibenclamide were added as indicated. Cell capacitance was 19.5 pF. B, the magnitude of the glibenclamide-sensitive current in the presence of 50 nM CGRP (Control), and of 50 nM CGRP + 1 μM KT5823. The bars show the means +s.e.m. from experiments like those of Fig. 7A on 5 cells. KT5823 had no effect on CGRP-induced currents (P= 0.47).

Although activation of PKG is not involved in the response to CGRP, it is possible that PKG, like PKA, could cause activation of K_ATP channels. To investigate this, we tested whether the nitrovasodilator sodium nitroprusside (SNP), which is known to increase guanylyl cyclase and so PKG activity (Lincoln, Komalavilas & Cornwell, 1994), could increase K_ATP currents in coronary vascular smooth muscle cells. Figure 8A and B show that SNP (10 μM) did not increase glibenclamide-sensitive currents (n= 6) under the recording conditions (symmetrical 140 mM K⁺; holding potential, -60 mV) we used to examine K_ATP currents activated by other agents. In the same cells, however, subsequent application of 10 μM pinacidil increased K_ATP currents by -104.3 ± 31.6 pA.

Figure 8. Sodium nitroprusside does not activate a K_ATP current.

A, whole-cell current recording from a myocyte held at -60 mV. Intracellular K⁺ was 140 mM and the cell was dialysed with 0.1 mM ATP. Arrows indicate when the K⁺ concentration in the bath superfusate was changed to 140 mM prior to addition of SNP, and a return to 6 mM K⁺ at the end of the experiment. SNP and pinacidil were added as indicated. Cell capacitance was 18 pF. B, glibenclamide-sensitive current in 140 mM K⁺ solution, 10 μM SNP and 10 μM pinacidil. The bars show the means +s.e.m. from experiments like those of Fig. 8A on 6 cells. **P < 0.01 compared with 140 mm K⁺. C, whole-cell current recording from a myocyte held at +60 mV. Extracellular K⁺ was 6 mM, intracellular K⁺ was 140 mM and the cell was dialysed with 0.1 mM ATP, and extracellular SNP, KT5823 and iberiotoxin were added as indicated. Cell capacitance was 15 pF. D, SNP-induced current recorded at +60 mV in the absence (▪) and presence () of 1 μM KT5823. The bars show means +s.e.m. from 4 cells. *P < 0.05 compared with control.

PKG has been shown to activate large conductance Ca²⁺-activated potassium (BK_Ca) channels in cerebral arterial smooth muscle cells (Robertson, Schubert, Hescheler & Nelson, 1993). In our experiments, however, we did not see activation of any K⁺ current by SNP at -60 mV. This is not surprising in view of the strong buffering of intracellular [Ca²⁺] by our internal solution and the low open probability of BK_Ca channels at -60 mV. To provide evidence that SNP was increasing PKG activity, therefore, we also measured membrane currents in cells held at +60 mV, where BK_Ca channels are much more likely to be open. Under these conditions SNP activated an outward potassium current of 49.5 ± 10.3 pA (n= 9) that was abolished by 100 nM iberiotoxin, a selective inhibitor of BK_Ca channels (n= 5). Inhibition of PKG by KT5823 (1 μM) also abolished BK_Ca currents activated by SNP (n= 4), returning outward currents in these cells to near baseline levels (Fig. 8C and D). These results suggest that SNP was able to increase PKG activity in our coronary arterial myocytes, and that this could cause activation of BK_Ca channels but not K_ATP channels.

DISCUSSION

The results presented in this paper provide strong evidence that CGRP can activate K_ATP currents in freshly isolated coronary arterial myocytes. CGRP-activated currents were inhibited by the sulphonylurea glibenclamide, had current-voltage relations which suggested that channel gating was essentially voltage independent, and were reduced in amplitude when intracellular [ATP] was increased. These properties are all consistent with the CGRP-induced current flowing through K_ATP channels similar to those activated by adenosine or hypoxia in this tissue (Dart & Standen, 1993, 1995).

Modulation of K_ATP channels by cAMP-dependent protein kinase

Our results also provide evidence that activation of K_ATP channels in coronary smooth muscle can occur through a signal transduction pathway involving stimulation of adenylyl cyclase, increased production and accumulation of cAMP, and activation of PKA. Firstly, the direct activator of adenylyl cyclase, forskolin, activated a glibenclamide-sensitive current, and the phosphodiesterase inhibitor IBMX potentiated the currents induced by CGRP. Secondly, dialysis of cells with either cAMP or the purified catalytic subunit of PKA also activated K_ATP currents. Finally, CGRP-induced K_ATP currents were reduced by two structurally dissimilar drugs that inhibit PKA (Rp-cAMPS and H-89), and abolished by a highly selective PKA inhibitor peptide. We also found that activation of a different receptor known to stimulate adenylyl cyclase, the β-adrenoceptor, with isoprenaline activated a glibenclamide- sensitive current.

These findings obtained from the coronary vasculature are consistent with previous observations of K_ATP channel activation by PKA in smooth muscle cells isolated from rabbit mesenteric artery (Quayle et al. 1994; Kleppisch & Nelson, 1995). It has also been suggested that activation of K_ATP channels can occur through GTP-binding proteins (G-proteins) independent of the adenylyl cyclase-PKA pathway. For example, exposure of preactivated G_iα-subunits to the cytosolic surface of cardiac myocyte membrane patches resulted in increased K_ATP channel activity (Terzic, Tung, Inanobe, Katada & Kurachi, 1994). Receptor-mediated activation of K_ATP channels independent of cAMP or PKA has also been suggested from studies using rat mesenteric (Randall & McCulloch, 1995) and hamster cheek pouch (Jackson, 1993) vascular beds; this evidence is based on the ability of glibenclamide to reduce dilatations to isoprenaline and vasoactive intestinal polypeptide (VIP) but not to forskolin or membrane-permeant analogues of cAMP. However, our results using the conventional whole-cell patch clamp technique demonstrate that exposure to forskolin as well as cell dialysis with cAMP can activate glibenclamide-sensitive K_ATP currents in coronary vascular smooth muscle. Furthermore, our observation that cell dialysis with the PKA inhibitor peptide completely abolished CGRP-induced glibenclamide-sensitive currents argues against the existence of additional receptor-mediated mechanisms of K_ATP channel activation under our experimental conditions.

Evidence against the activation of K_ATP currents by cGMP-dependent protein kinase

It has been proposed that cAMP produced in response to activators of adenylyl cyclase can cause cross-activation of cGMP-dependent protein kinase (PKG; Lincoln, Cornwell & Taylor, 1990). Consistent with this hypothesis, Jiang et al. (1992) have found that forskolin and isoprenaline increased cAMP concentrations to levels which could activate PKG in pig coronary vascular smooth muscle. More recently, it has been suggested that PKA activation by low concentrations of cAMP, and PKG activation at higher concentrations of cAMP could account for the biphasic action of forskolin and membrane-permeant analogues of cAMP on Ca²⁺ currents in canine colonic myocytes (Koh & Sanders, 1996). However, involvement of PKG in the activation of K_ATP currents by CGRP in our study appears unlikely. This conclusion is supported by the effectiveness of inhibitors of PKA but not PKG to diminish CGRP-induced K_ATP currents.

In addition to the question of cross-activation of PKG by activators of adenylyl cyclase, we were also interested in whether PKG activation by nitric oxide could increase K_ATP currents in coronary vascular smooth muscle. The nitrovasodilator sodium nitroprusside (SNP) is known to activate PKG (Lincoln et al. 1994), and has been found to produce glibenclamide-sensitive membrane hyperpolarizations in rabbit mesenteric arteries (Murphy & Brayden, 1995). SNP did not increase K_ATP currents under the same recording conditions (symmetrical 140 mM K⁺; holding potential, -60 mV) in which we observed activation of K_ATP currents by activators of adenylyl cyclase. We did, however, observe increased iberiotoxin-sensitive outward potassium currents at positive membrane potentials in response to SNP, which were greatly reduced by inhibition of PKG. This outward current is most probably due to increased activity of large conductance Ca²⁺-activated K⁺ channels (BK_Ca channels). This effect of SNP is consistent with previous reports demonstrating activation of BK_Ca channels through a pathway involving stimulation of guanylyl cyclase and activation of PKG (e.g. Robertson et al. 1993), and demonstrates the presence of functional PKG under our recording conditions. These findings are consistent with previous observations that BK_Ca, but not K_ATP, channel activity is increased by nitrovasodilators in the coronary vasculature (Wellman, Bonev, Nelson & Brayden, 1996; see Quayle et al. 1997 for review).

In conclusion, therefore, activation of PKG with SNP did not lead to activation of K_ATP channels under our experimental conditions, and we found no evidence that cross-activation of PKG by cAMP contributed to CGRP activation of K_ATP channels. It remains possible, of course, that such cross activation could occur under conditions in which cAMP levels are higher than those achieved with CGRP in our experiments, and that it could play a role in the coronary circulation in vivo, or in other vascular beds.

Does CGRP activate coronary K_ATP channels under physiological conditions?

CGRP may play an important role in the physiological regulation of the coronary circulation. Basal release of CGRP from afferent nerve terminals within the heart can contribute substantially to resting blood flow, and it has been suggested that CGRP might also play a part in the autoregulation of coronary blood flow in response to changes in pressure (Yaoita et al. 1994; Bell & McDermott, 1996). Further, CGRP release is increased during ischaemia (Franco-Cerceda et al. 1989), so that CGRP release and the consequent vasodilatation may be a mechanism that helps to match coronary blood flow to the metabolic needs of the myocardium (Bell & McDermott, 1996). CGRP can cause vasorelaxation by several mechanisms, including direct inhibition of Ca²⁺ influx, inhibition of intracellular Ca²⁺ release and a decrease in Ca²⁺ sensitivity of the contractile apparatus (Bell & McDermott, 1996; Fukuizumi, Kobayashi, Nishimura & Kanaide, 1996). It is interesting, therefore, to consider whether K_ATP channel activation by CGRP might be important in the physiological regulation described above.

While our results show that CGRP can activate K_ATP channels in coronary smooth muscle, we used high extracellular [K⁺] and low intracellular [ATP] in our experiments to make it easier to record the K_ATP currents activated by CGRP. It is clear from our previous work with adenosine, however, that K_ATP currents can be activated in the same preparation using physiological [K⁺]_o and permeabilized-patch recording, where intracellular nucleotide levels are likely to be maintained at more physiological levels (Dart & Standen, 1993). This suggests that CGRP should also be able to cause activation of outward K_ATP current and so hyperpolarization under physiological conditions. While hyperpolarization by CGRP remains to be demonstrated in coronary arteries, CGRP has been shown to hyperpolarize both mesenteric and ophthalmic arteries by activating K_ATP channels (Nelson et al. 1990a; Zschauer, Uusitalo & Brayden, 1992), and K_ATP channel activation by CGRP contributes to vasodilatation in cerebral blood vessels in vivo (Kitazono et al. 1993).

Studies of CGRP-induced relaxations of coronary arteries have given variable evidence as to the role of K⁺ channel activation. Kageyama et al. (1993) found that CGRP relaxations of pig coronary arterial rings in vitro were insensitive to glibenclamide and to high extracellular K⁺, suggesting that K⁺ channel activation was not involved under their experimental conditions. In porcine coronary arterial strips, however, Fukuizumi et al. (1996) have reported that the CGRP-induced reduction in intracellular [Ca²⁺] was abolished in arteries contracted with 90 mM K⁺, implying the involvement of potassium channels, though they did not examine the effect of glibenclamide. One factor that may be important in the responsiveness of coronary K_ATP channels to activation by CGRP, and other agents, is the oxygen tension experienced by the coronary smooth muscle cells. Moderate hypoxia can activate K_ATP channels of coronary arteries (Daut et al. 1990; Nakhostine & Lamontagne, 1993; Dart & Standen, 1995), and P_O2 levels in vivo may fall from around 70 mmHg in distributing arterioles to as low as 20 mmHg in terminal arterioles (Duling & Berne, 1980). Thus a background activation of K_ATP channels by physiological oxygen tensions may increase their responsiveness to activation by other agents, including CGRP. Consistent with this, relaxations of sheep coronary artery segments in response to CGRP in vitro were enhanced when the P_O2 of the extracellular solution was decreased from 630 to 20 mmHg (Kwan, Wadsworth & Kane, 1990), while metabolic inhibition with 2-deoxyglucose also enhanced glibenclamide-sensitive relaxations to CGRP in the guinea-pig ureter (Maggi, Santicioli & Giuliani, 1996). It is possible, therefore, that the lack of effect of glibenclamide in the study by Kageyama et al. (1993) results from a high P_O2, since their solutions were aerated with 100 % oxygen, and that K_ATP channel activation might make an important contribution to the dilator action of CGRP at more physiological O₂ levels. Clearly, experimental studies examining coronary dilatations to CGRP in the intact heart will be needed to assess the extent to which K_ATP channel activation is involved, both under normoxic conditions and in ischaemia.