Angiotensin II Inhibition of ATP-Sensitive K+ Currents in Rat Arterial Smooth Muscle Cells Through Protein Kinase C



  • 1The effects of the vasoconstrictor angiotensin II (Ang II) on whole-cell ATP-sensitive K+currents (Ik,atp) of smooth muscle cells isolated enzymatically from rat mesenteric arteries were investigated using the patch clamp technique.
  • 2Ang II, at a physiological concentration (100 nm), reduced Ik,atp activated by 0.1 mm internal ATP and 10 μm levcromakalim by 36.4 ± 2.3%.
  • 3The protein kinase C (PKC) activator 1-oleoyl-2-acetyl-sn-glycerol (OAG, 1 μm) reduced Ik,atp by 44.1 ± 2.7%. GDPβS (1 mm), included in the pipette solution, abolished the inhibition by Ang II, while that by OAG was unaffected.
  • 4Pretreatment with the PKC inhibitors staurosporine (100 nm) or calphostin C (500 nm) prevented the Ang II-induced inhibition of Ik,atp.
  • 5Ang II inhibition was unaffected by cell dialysis with PKA inhibitor peptide (5 μm), and the PKA inhibitor Rp-cAMPS (100 μ) did not reduce Ik,atp.
  • 6Our results suggest that Ang II modulates Katp channels through activation of PKC but not through inhibition of PKA.

Angiotensin II (Ang II) is a potent endogenous vasoconstrictor that acts to increase intracellular [Ca2+] and hence the contractile force of vascular smooth muscle cells. Vasoconstrictors may increase [Ca2+]i either by direct actions on Ca2+ entry through the plasma membrane or Ca2+ release from intracellular stores, or by causing membrane depolarization and so increasing the open probability of voltage-activated Ca2+ channels. This depolarization may be caused by activation of depolarizing channels such as Ca2+channels or non-selective cation channels, or by inhibition of potassium channels that maintain the resting potential at a negative level. Most vasoconstrictors appear to act via several mechanisms, and Ang II has been reported to activate a non-selective cation channel in rabbit ear artery cells (Hughes & Bolton, 1995), and to inhibit delayed rectifier K+ channels in rabbit portal vein (Clément-Chomienne, Walsh & Cole, 1996), and large conductance Ca2+-activated K+ (BKCa) channels in coronary artery (Toro, Amador & Stefani, 1990; Minami, Hirata, Tokumura, Nakaya & Fukuzawa, 1995).

Ang II receptors have been reported to couple to several cellular signalling pathways in vascular smooth muscle as in other tissues. Ang II receptor stimulation activates phospholipases to increase the concentration of inositol trisphosphate (IP3), which can cause Ca2+ release from intracellular stores, and diacylglycerol (DAG), which activates calcium/phospholipid-dependent protein kinase C (PKC) (Griendling, Rittenhouse, Brock, Ekstein, Gimbrone & Alexander, 1986; Nishizuka, 1992; Freeman & Tallant, 1994). Ang II has also been reported to inhibit adenylyl cyclase and so decrease intracellular cyclic AMP (Anand-Srivastava, 1983; Unger et al. 1996), and to increase protein tyrosine phosphorylation (Molloy, Taylor & Weber, 1993; Leduc & Meloche, 1995). The inhibitory action of Ang II on delayed rectifier K+ channels occurs through activation of PKC (Clément-Chomienne et al. 1996), though it has been suggested that inhibition by intracellular Ca2+ may also contribute to this action (Gelband & Hume, 1995). In contrast, inhibition of BKCa channels is independent of PKC (Minami et al. 1995).

Modulation of Katp channels can play an important role in controlling vascular tone by altering the resting membrane potential and so Ca2+ entry (Quayle, Nelson & Standen, 1997). Ang II has been reported to inhibit Katp channels in patches excised from cells cultured from pig coronary artery (Miyoshi & Nakaya, 1991), but the mechanism by which this effect occurs is unknown. We have therefore sought to establish whether Ang II can inhibit Katp channels in freshly isolated vascular smooth muscle cells, and to investigate the pathway involved in this action. Our results show that Ang II inhibits Ik,atp in cells from the rat mesenteric artery, and provide evidence that this inhibition occurs through activation of PKC. Further, the Ang II-mediated changes in Ik,atp occur through a cAMP-independent mechanism. The inhibitory action of Ang II on Katp channels may contribute to its physiological vasoconstrictor action.


Preparation of vascular smooth muscle cells

Vascular smooth muscle cells were isolated from rat mesenteric arteries. Male adult Wistar rats were killed by stunning followed by cervical dislocation. The care of the animals conformed to the requirements of the Animals (Scientific Procedures) Act 1986 (UK). Arteries were removed and cleaned of connective tissue in ice-cold solution containing (mm): 137 NaCl, 5.6 KCl, 0.42 Na2HPO4, 0.44 NaH2PO4, 1 MgCl2, 2 CaCl, 10 Hepes and 10 glucose; adjusted with NaOH to pH 7.4. The arteries were transferred to the same solution except that CaCl2 was reduced to 0.1 mm (low calcium solution) for 10 min, and warmed to 35 °C in a water bath. Arteries were digested for 30–35 min in low calcium solution containing (mg ml−1): 1.0 albumin, 1.5 papain and 1.0 dithioerythritol; and then for 16–20 min in low calcium solution containing (mg ml−1): 1.0 albumin, 1.5 collagenase type F (Sigma), and 1.0 hyaluronidase type I-S (Sigma). Arteries were then transferred to low calcium solution containing albumin (1.0 mg ml−1). Single smooth muscle cells were obtained by gentle trituration with a wide-bore pipette, stored at 4 °C, and used on the day of preparation.

Solutions and chemicals

For conventional whole-cell recordings, the intracellular solution contained (mm): 110 KCl, 30 KOH, 10 Hepes, 10 EGTA, 1 MgCl2, 1 CaCl2, 0.1 Na2ATP, 0.1 NaADP, 0.2 GTP; adjusted to pH 7.2. The calculated free [Ca2+] with 0.1 mm ATP was 20 nm. The ATP concentration in the pipette solution is enough to support PKC activity (Kishimoto, Takai, Mori, Kikkawa & Nishizuka, 1980). The 140 mmm K+ extracellular solution contained (mm): 140 KCl, 1 MgCl2, 0.1 CaCl2, 10 Hepes, 10 glucose; adjusted pH to 7.4. The 6 mm and 40 mm K+ solutions were made by substituting the appropriate amount of NaCl for KCl in the above solution. External Ca2+ was lowered to 0.1 mm to reduce Ca2+ influx. External solutions were changed by continuous perfusion of the experimental chamber (volume 0.4 ml); the dead time for the new solution to reach the bath was about 30 s and complete exchange took about 2 min. Calphostin C, guanosine-5′-(2-thiodiphosphate) (GDPβS), cAMP-dependent protein kinase inhibitor peptide (PKA inhibitor), and Rp-cyclic 3′,5′-hydrogen phosphothioate adenosine triethyl-ammonium salt (Rp-cAMPS) were obtained from Calbiochem (UK). Levcromakalim was a gift from SmithKline Beecham. Other chemicals were obtained from Sigma. Angiotensin II, PKA inhibitor, and Rp-cAMPS were dissolved in distilled water. Levcromakalim, staurosporine, calphostin C, 1-oleoyl-2-acetyl-sn-glycerol (OAG), and glibenclamide were dissolved in dimethylsulphoxide (DMSO). The final concentration of DMSO was less than 0.2 %.

Data recording and analysis

Whole-cell currents were recorded from single smooth muscle cells using the patch clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Membrane currents were recorded, and voltage controlled, using an Axolab interface and Axopatch 200 amplifier (Axon Instruments). Data were recorded on computer and analysed using pCLAMP6 (Axon Instruments). Currents were recorded in the conventional whole-cell configuration. Patch pipettes were made from thin-walled borosilicate glass (Clark Electromedical, Pangbourne, Berks, UK) and coated with sticky wax (Kemdent, Swindon, Wilts, UK) to reduce capacitance. Currents were filtered at 5kHz. Electrode resistance before sealing was 3–5 MΩ, and after sealing was > 10 GΩ. To minimize activity of voltage-dependent K+ (delayed rectifier) channels and BKCa channels, most experiments were performed at a negative holding potential of −60 mV and intracellular Ca2+ was buffered to a low level with EGTA. Experiments were done at 25–27 °C. Data are expressed as means ±s.e.m. Intergroup differences were analysed by analysis of variance followed by Duncan's multiple range test, and a value of P < 0.05 was considered statistically significant.


Angiotensin II inhibits Ik,atp

Smooth muscle cells isolated from rat mesenteric arteries had membrane capacitances of 15.9 ± 0.4 pF (n= 62). Since we wished to examine inhibition of Ik,atp, these currents were enhanced by using a low intracellular ATP (0.1 mm) solution, containing 0.1 mm ADP, and by the addition of the Katp channel opener levcromakalim. Wholecell current was recorded at a holding potential of −60 mV to minimize the activity of voltage-dependent K+ channels, and extracellular K+ was increased to 140 mm, so that K+currents were inward at this potential. Figure 1 shows a recording of whole-cell current under such conditions. Raising the extracellular K+ concentration from 6 to 140 mm caused a small increase in inward current, while levcromakalim (10 μm) activated a much more substantial inward current. The sulphonylurea glibenclamide (10 μm), which selectively inhibits Katp channels at this concentration (Quayle, Bonev, Brayden & Nelson, 1995; Quayle et al. 1997), inhibited both the levcromakalim-induced and the steady-state current. Thus, under our recording conditions, the levcromakalim-induced current and most of the background current appears to flow through Katp channels. In ten cells held at −60 mV in 140 mm [K+]o, the mean glibenclamide-sensitive current was −20 ± 5 pA, and was increased to −169 ± 24 pA by the addition of 10μm levcromakalim.

Figure 1.

Angiotensin II inhibits levcromakalim-induced Ik,atp

A, recording of whole-cell current from a cell held at −60 mV showing levcromakalim activation of glibenclamide-sensitive K+ currents. In all recordings shown the dashed hue indicates the zero current level. In this and the recordings shown in Figs 2, 3, 4 and 5, the cell was dialysed with a solution containing 140 mm K+, and extracellular K+ was changed from 6 to 140 mm as indicated by the vertical arrow. Levcromakalim (Lev) and glibenclamide (Glib) were added as indicated. B, current-voltage relations for glibenclamide-sensitive currents in the presence of levcromakalim, measured using 500 ms voltage ramps from −120 to 50 mV in a cell bathed first in 40 mm external K+ and then in 140 mm external K+ as indicated. Each relation was obtained by averaging the current from seven voltage ramps in 10 μm levcromakalim and subtracting the current averaged from seven ramps after the addition of 10μm glibenclamide. C, whole-cell current recording showing the response to 100 nm Ang II. Levcromakalim, Ang II and glibenclamide were applied in the extracellular solution as indicated.

The K+ selectivity of the glibenclamide-sensitive current induced by levcromakalim was confirmed by using voltage ramps from –120 to +50 mV to measure the current–voltage relation for this current in 140 and 40 mm extracellular K+. Figure 1B shows current-voltage relations measured in this way, and mean reversal potentials of the levcromakalim-induced currents with 140 and 40 mm external K+ were 4.1 ± 0.4 mV (n= 7 cells) and −24.9 + 0.5 mV (n= 3 cells), respectively. The calculated K+ equilibrium potentials (Eks) in 140 and 40 mm [K+]o are 0 and −31.3mV, respectively. The measured reversal potentials have not been corrected for the calculated 4 mV junction potential between the 6 mm K+ bath solution in which seals were made and the pipette solution. Allowing for this, the reversal potentials lie close to the EK values confirming that the glibenclamide-sensitive currents flowed through K+-selective channels.

In the absence of Ang II, levcromakalim-induced currents reached a steady level which was maintained in the presence of the channel opener (e.g. Figs 1A and 5B). Addition of 100 nm Ang II, however, caused a progressive reduction in the inward Ik,atp current. Figure 1C shows a recording from a cell in which levcromakalim increased the K+current to –360 pA, and addition of Ang II reduced this current by 130 pA, while the remaining current was inhibited by 10 μm glibenclamide. In similar experiments on ten cells, Ang II was effective on all the cells tested and inhibited glibenclamide-sensitive Ik,atp by 36.4 ± 2.3% (Fig. 2B).

Figure 5.

Inhibitors of protein kinase A do not alter the effects of Ang II

A, effects of PKA inhibitor peptide. The trace shows whole-cell current recorded from a cell dialysed with pipette solution containing 5 μm PKA inhibitor peptide. Levcromakalim, Ang II and glibenclamide were added as indicated. Holding potential, −60 mV. B, effect of Rp-cAMPS. The trace shows whole-cell current recorded from a cell exposed to levcromakalim, Rp-cAMPS and glibenclamide as indicated. Holding potential, −60 mV.

Figure 2.

I k,atp inhibition by a diacylglycerol analogue

A, whole-cell current recording showing the effect of the membrane-permeant DAG analogue OAG on levcromakalim-induced Ik,atp. OAG (1μm) inhibited Ik,atp by 44% in this cell, and glibenclamide inhibited the remaining current. Holding potential, −60 mV. B, inhibition of Ik,atp(induced by 0.1 mm intracellular ATP and levcromakalim) by Ang II and by OAG at 1 and 10 μm. The bars (left to right) show mean values ±s.e.m. from 10, 5 and 7 cells.

I k,atp is reduced by the diacylglycerol analogue OAG

Many of the physiological actions of Ang II have been reported to be mediated by activation of PKC by way of diacylglycerol (DAG). Such a pathway is involved in Ang II inhibition of Kv channels of vascular smooth muscle (Clément-Chomienne et al. 1996), and activation of PKC is involved in the inhibition of Katp channels in response to stimulation of several other vasoconstrictor receptors: neuropeptide Y, 5HT2, and histamine H1 receptors and α1-adrenoceptors (Bonev & Nelson, 1996). We therefore investigated the possibility that the Ang II inhibition of Ik,atp in our experiments occurred via activation of PKC. To establish that PKC activation could cause inhibition of Katp channels, we used the membrane-permeant analogue of diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol (OAG) to activate PKC. As shown in Fig. 2A, OAG (1 μm) markedly reduced levcromakalim-induced Ik,atp. In five cells 1 μm OAG decreased this current by 44.1 ± 2.7%, while 10 μm OAG caused a 63.7 ± 5.0% reduction (n= 7 cells, Fig. 2B).

GDPβS inhibits the action of Ang II

Ang II receptors are known to link to GTP-binding proteins (e.g. Unger et al. 1996). To provide evidence for G-protein involvement in the inhibition of Ik,atp by Ang II, we tested the effect of Ang II in cells dialysed with GDPβS (1 mm), a GDP analogue which competitively inhibits G-protein activation by GTP. Figure 3A shows that Ang II did not inhibit Ik,atp in a cell that was dialysed with GDPβS. Subsequent application of OAG (1 μm) caused a substantial reduction of Ik,atp, however, confirming that PKC activation downstream of the receptor and G-protein could still inhibit Katp channels in the presence of GDPβS.

Figure 3.

Ang II is ineffective in the presence of GDPβS

A, recording of whole-cell current made from a cell dialysed with pipette solution containing 1 mm GDPβS. Ang II did not inhibit Ik,atp under these conditions, while subsequent application of OAG was still effective. Holding potential, −60 mV. B, mean inhibition (±s.e.m.) of Ik,atp by Ang II (100 nm) and by OAG (1 μm) in cells dialysed with control intracellular solution (filled bars) and in cells dialysed with solution containing 1 mm GDPβS (open bars). The number of cells in each group was: Ang II, 10; Ang II + GDPβS, 5; OAG, 5; OAG + GDPβS, 7. * Results significantly different from control (P < 0.05).

Figure 3B summarizes the results from several cells, showing that the inhibition by Ang II was reduced 8-fold to 4.4 ± 3.1% (n= 5 cells) by GDPβS, while that to OAG (1μm) was unaffected (47.1 ±5.1 % inhibition, n= 6 cells).

PKC inhibitors block Ang II-mediated inhibition

The experiments of Fig. 3 are consistent with PKC activation (by OAG) causing inhibition of Ik,atp. To test whether stimulation of PKC is involved in the inhibition of Ik,atp by Ang II, we examined the effects of Ang II in the presence of PKC inhibitors. Staurosporine inhibits PKC by competing for the ATP-binding site of the enzyme with a reported half-inhibition constant (Ki) of 2.7 nm (Tamaoki, Nomoto, Takahashi, Kato, Morimoto & Tomita, 1986). Staurosporine is a potent inhibitor of PKC, but also has inhibitory effects on other protein kinases, including protein kinase A (PKA). Pretreatment (10 min) of rat mesenteric arterial cells with Staurosporine (100 nm) added to the bath solution prevented or greatly reduced Ang II inhibition of Ik,atp. Figure 4A shows an example of a recording made in staurosporine, and mean results from five cells under these conditions are shown in Fig. 6.

Figure 4.

Protein kinase C inhibitors prevent inhibition by Ang II

A, effects of staurosporine. The trace shows a recording of whole-cell current made from a cell that had been pretreated for 10 min with staurosporine (100 nm). The recording was made in the continued presence of staurosporine, and levcromakalim, Ang II and glibenclamide were added as indicated. Holding potential, −60 mV. B, effects of calphostin C. The trace shows a whole-cell current recording from a cell pretreated with (10 min), and in the continued presence of, calphostin C (500 nm). Levcromakalim, Ang II and glibenclamide were added as indicated. Holding potential, −60 mV.

Figure 6.

Effects of protein kinase inhibitors on Ik,atp inhibition by Ang II

The bars show mean (±s.e.m.) inhibition of Ik,atp by Ang II under control conditions (n= 10) and in cells pretreated with the PKC inhibitors staurosporine (100 nm, n= 5) or calphostin C (500 nm, n= 4), or in cells dialysed with PKA inhibitor peptide (5 μm, n= 4). *P < 0.05 compared with control.

We also tested calphostin C, which is a highly selective inhibitor of PKC, acting by competing at the binding site on PKC for diacylglycerol with a Ki of 50 nm (Gopalakrishna, Chen & Gundimeda, 1992). Pretreatment (10 min) with 500 nm calphostin C added to the bath solution also strongly inhibited the action of Ang II (Figs 4B and 6). Neither PKC inhibitor had any effect on Ik,atp currents activated by levcromakalim alone (data not shown, n= 3 in each case). These results suggest that activation of PKC provides the major pathway for inhibition of Katp channels by Ang II in our preparation.

PKA inhibitor peptide has no effect on Ang II inhibition

In addition to stimulating phosphoinositide hydrolysis Ang II receptors can cause inhibition of adenylyl cyclase in various tissues including arterial smooth muscle (Anand-Srivastava, 1983; Unger et al. 1996). Since activation of adenylyl cyclase and so PKA has been shown to activate Katp channels of arterial smooth muscle cells (Quayle, Bonev, Brayden & Nelson, 1994; Wellman, Quayle, Pveritt & Standen, 1997), it is possible that inhibition of adenylyl cyclase mediates part of the inhibitory action of Ang II on Ik,atp. To investigate this possibility, we studied the effects of a specific peptide inhibitor of PKA (Cheng et al. 1986) on Ang II-mediated inhibition. Since the Ki for this PKA inhibitor is 2.3 nm, intracellular dialysis with the peptide at 5 μm should effectively provide complete inhibition of PKA, and we would expect any effects of Ang II related to PKA inhibition to be abolished in its presence. Application of the inhibitor peptide in this way has been shown to abolish the PKA-mediated activation of Ik,atp seen when calcitonin gene-related peptide was applied to rabbit mesenteric arterial myocytes (Quayle et al. 1994). Figure 5A shows that Ang II, however, still inhibited Ik,atp in a cell dialysed with PKA inhibitor peptide, and in four cells we found that Ang II was just as effective in its presence, inhibiting current by 34.1 ± 3.2% (Fig. 6).

To provide further evidence that inhibition of the adenylyl cyclase-PKA system does not contribute to the inhibition of Ik,atp by Ang II we investigated whether it was possible to mimic any of the inhibitory effect of Ang II on levcromakalim-induced current by inhibiting PKA using the membrane-permeant analogue of cAMP, Rp-cAMPS. Rp-cAMPS inhibits PKA by binding to its regulatory subunit (Rothermel & Parker-Botelho, 1988), and has been shown to inhibit the effects of vasodilators that activate adenylyl cyclase (Quayle et al. 1994; Kleppisch & Nelson, 1995; Wellman et al. 1997). Figure 5B shows that 100 μm Rp-cAMPS did not inhibit Ik,atp a result that was confirmed in experiments on two further cells. Thus our results do not provide any evidence for the involvement of the adenylyl cyclase–PKA system in the inhibitory action of Ang II.


The results presented in this paper provide the first demonstration that Ang II can inhibit Katp channel activity in freshly isolated arterial smooth muscle cells, and provide evidence that this action occurs primarily through the activation of protein kinase C (PKC). First, the effects of Ang II were blocked by inhibition of PKC using either staurosporine or calphostin C. Second, application of a membrane-permeant analogue of diacylglycerol, OAG, which activates PKC, mimicked the action of Ang II in causing inhibition of Ik,atp. Evidence that the effects of Ang II involve G-protein activation comes from their block in the presence of intracellular GDPβS, which did not, however, affect Ik,atp inhibition by OAG, as expected since this should act downstream in the proposed signalling pathway from the G-protein. Ang II stimulation has been associated with activation of PKC in vascular smooth muscle: increases in phospholipase C activity and diacylglycerol have been demonstrated in response to Ang II in rat aortic cells, human subcutaneous and rat mesenteric arteries (Griendling et al. 1986; Ohanian, Izzard, Littlewood & Heagerty, 1993; Dixon, Sharma, Dickerson & Fortune, 1994). The mechanism by which PKC leads to inhibition of the Katp channel remains to be investigated. The cloned Kir6.0 family channel subunits and the sulphonylurea receptors (SUR) which form molecular components of Katp channels have been reported to have several consensus sequences for phosphorylation by PKC (Inagaki et al. 1995, 1996), but which, if any, of these lead to a reduction of channel activity remains unknown. It appears also that different subtypes of Katp channel can be modulated in different ways by PKC, since PKC can cause activation of Katp channels of cardiac muscle cells (Hu, Duan, Li & Nattel, 1996).

Our experiments have not directly addressed the question of the PKC isoform that is involved in Katp channel inhibition. The Ca2+-dependent isoforms α and β and the Ca2+-independent isoforms ɛ and ζ have all been shown to be expressed in vascular smooth muscle (Dixon et al. 1994; Lee & Severson, 1994; Clément-Chomienne et al. 1996). The low intracellular and extracellular [Ca2+] we used makes it unlikely that a Ca2+-dependent isoform is involved, while the ζ isoform is also an unlikely candidate as it is not activated by diacylglycerol or its analogues (Nishizuka, 1992; Lee & Severson, 1994). Further, PKCɛ has been shown to be translocated in response to Ang II stimulation in cultured pulmonary arterial smooth muscle cells (Nadim, Damron & Murray, 1995), and in response to stimulation of cells from ferret aorta with phenylephrine (Khalil, Lajoie, Resnick & Morgan, 1992). We think, therefore, that PKCɛ is the subtype most likely to be involved in the inhibition of Katp channels described here.

Four subtypes of Ang II receptor are currently known, designated AT1 to AT4 (Unger et al. 1996). The AT1 receptor is coupled to phospholipase C through Gq/G11 G-proteins, and is also negatively coupled to adenylyl cyclase. We have not investigated the type of receptor involved in Katpchannel inhibition in the present study, but it seems very likely to be AT1, which is responsible for most of the known effects of Ang II, including 80% of the vasoconstrictor response of portal vein (Pelet, Mironneau, Rakotoarisoa & Neuilly, 1995; Unger et al. 1996). In addition to stimulation of phospholipases and so PKC, Ang II acting at the AT1receptor can also decrease the activity of adenylyl cyclase, leading to inhibition of cAMP-dependent protein kinase (PKA) (e.g. Unger et al. 1996). Although activation of PKA can cause activation of Katp channels in response to vasodilators (Quayle et al. 1994; Kleppisch & Nelson, 1995; Wellman et al. 1997), our results suggest that inhibition of PKA does not play any role in the reduction in Ik,atp by Ang II. Intracellular dialysis with PKA inhibitor peptide at a concentration that should fully inhibit PKA did not alter the response to Ang II, and we also found that extracellular application of Rp-cAMPS, a membrane-permeant inhibitor of PKA, did not reduce Ik,atp.

Ang II has been shown to affect several different types of ion channel in vascular smooth muscle, and the signalling pathways involved appear to vary. The inhibition of delayed rectifier K+ current, like the inhibition of Ik,atp we report in the present paper, occurs through activation of PKC (Clément-Chomienne et al. 1996). However, Ang II also inhibits BKCa channels, and this action is independent of PKC (Toro et al. 1990; Minami et al. 1995). The mechanism by which Ang II activates a non-selective cation channel in rabbit ear artery cells remains to be elucidated, but appears to occur without a rise in IP3 (Hughes & Bolton, 1995). Interestingly, although the mechanism of the Ang II inhibition of Katp channels of cells cultured from porcine coronary artery is not known (Miyoshi & Nakaya, 1991), it also seems unlikely to involve PKC. Those experiments used cell-free excised membrane patches, where PKC is unlikely to be present, and translocation of PKC from cytoplasm to the cell membrane is necessary for its activation. It seems possible, therefore, that Ang II or its coupling G-protein may directly inhibit Katp channels in cultured myocytes. In our experiments on freshly isolated cells it seems that any such direct mechanism makes very little contribution, since Ang II had little effect in the presence of PKC inhibitors. It is possible that this may reflect a difference between freshly isolated cells and those maintained in culture.

Angiotensin II is a potent vasoconstrictor, known to activate channels that depolarize vascular smooth muscle and allow Ca2+ entry. Its effects in reducing the activity of K+ channels should enhance its action in causing membrane depolarization, and so contribute to its vasoconstrictor action, since voltage-activated Ca2+ channels, and so Ca2+ entry, are highly sensitive to small changes in membrane potential. Evidence that closure of Katp channels can contribute to depolarization of arterial smooth muscle comes from the 5–20 mV depolarizations that have been reported when they are blocked with glibenclamide in intact tissue (e.g. Murphy & Brayden, 1995; see Quayle et al. 1997 for review). Further, it is clear that activity of such channels contributes to resting blood flow in a number of vascular beds including those of the coronary, mesenteric, skeletal muscle and renal circulations, since glibenclamide increases resistance to blood flow in those tissues (e.g. Samaha, Heineman, Ince, Fleming & Balaban, 1992; see Quayle et al. 1997 for review). Since Katp channel activity is increased by a number of vasodilators and by metabolic stress such as hypoxia (Dart & Standen, 1995), the potential for inhibition by Ang II to reduce K+ conductance should also be increased under such conditions. Thus the action of Ang II in inhibiting Katp channels via activation of PKC described here may make an important contribution to the vasoconstrictor action of the peptide.


We thank Drs George Wellman and Herwig Köppel for discussions and comments on the manuscript. This work was supported by the MRC.