Voltage-gated potassium channels (Kv) are important regulators of membrane potential in vascular smooth muscle cells, which is integral to controlling intracellular Ca2+ concentration and regulating vascular tone. Previous work indicates that Kv channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that Kv channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on Kv and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents. Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a β-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents. We also show that this modulation of Kv by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of Kv by KT5720 can be abrogated by pre-treatment with the PP2B inhibitor cyclosporin A, or inclusion of a PP2B auto-inhibitory peptide in the pipette solution. Finally, we demonstrate that tonic PKA-mediated modulation of Kv requires intact caveolae. Pre-treatment of the cells with methyl-β-cyclodextrin to deplete cellular cholesterol, or adding caveolin-scaffolding domain peptide to the pipette solution to disrupt caveolae-dependent signalling each attenuated PKA-mediated modulation of the Kv current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone.

Uridine triphosphate (UTP) can be released from damaged cells to cause vasoconstriction. Although UTP is known to act through P2Y receptors and PLC activation in vascular smooth muscle, the role of PKC in generating the response is somewhat unclear. Here we have used Tat-linked membrane permeable peptide inhibitors of PKC to assess the general role of PKC and also of specific isoforms of PKC in the UTP induced contraction of rat mesenteric artery. We examined the effect of PKC inhibition on UTP induced contraction, increased cytoplasmic Ca(2+) and reduction of K(+) currents and found that PKC inhibition caused a relatively small attenuation of contraction but had little effect on changes in cytoplasmic Ca(2+). UTP attenuation of both voltage-gated (Kv) and ATP-dependent (KATP) K(+) currents was abolished when intracellular Ca(2+) was decreased from 100 to 20 nM. PKC inhibition reduced slightly the ability of UTP to attenuate Kv currents but had no effect on KATP current inhibition. In conclusion, both UTP induced contraction of mesenteric artery and the inhibition of Kv and KATP currents of mesenteric artery smooth muscle cells by UTP are relatively independent of PKC activation; furthermore, the inhibition of both Kv and KATP currents requires intracellular Ca(2+).

We examined the regulatory role of tonic cAMP-dependent protein kinase (PKA) activity on Kv current of smooth muscle cells isolated from 3rd/4th-order rat mesenteric arteries using whole-cell recording. Applying KT5720 (1 µM), a PKA inhibitor, attenuated the Kv current by 40.2 ± 7.4% (n = 4) at +50 mV; a similar inhibition was seen with Rp-cAMPS (100 µM). Since PKA activity is dependent on adenylyl cyclase (AC)-mediated generation of cAMP, inhibiting AC ought to mimic PKA inhibition. Application of the AC inhibitor 2',5'-dideoxyadenosine inhibited the Kv current by 43.7 ± 7.1% (n=6).

Aims Membrane potential is a key determinant of vascular tone and many vasodilators act through the modulation of ion channel currents [e. g. the ATP-sensitive potassium channel (K-ATP)] involved in setting the membrane potential. Adenylyl cyclase (AC) isoenzymes are potentially important intermediaries in such vasodilator signalling pathways. Vascular smooth muscle cells (VSMCs) express multiple AC isoenzymes, but the reason for such redundancy is unknown. We investigated which of these isoenzymes are involved in vasodilator signalling and regulation of vascular ion channels important in modulating membrane potential.Methods and results AC isoenzymes were selectively depleted (by >75%) by transfection of cultured VSMCs with selective short interfering RNA sequences. AC6 was the predominant isoenzyme involved in vasodilator-mediated cAMP accumulation in VSMCs, accounting for similar to 60% of the total response to beta-adrenoceptor (beta-AR) stimulation. AC3 played a minor role in beta-AR signalling, whereas AC5 made no significant contribution. AC6 was also the principal isoenzyme involved in beta-AR-mediated protein kinase A (PKA) signalling (determined using the fluorescent biosensor for PKA activity, AKAR3) and the substantial beta-AR/PKA-dependent enhancement of K-ATP current. K-ATP current was shown to play a vital role in setting the resting membrane potential and in mediating the hyperpolarization observed upon beta-AR stimulation.Conclusion AC6, but not the closely related AC5, plays a principal role in vasodilator signalling and regulation of the membrane potential in VSMCs. These findings identify AC6 as a vital component in the vasodilatory apparatus central to the control of blood pressure.

BACKGROUND AND PURPOSEAnalogues of the endogenous diacylglycerols have been used extensively as pharmacological activators of protein kinase C (PKC). Several reports show that some of these compounds have additional effects that are independent of PKC activation, including direct block of K+ and Ca2+ channels. We investigated whether dioctanoyl‐sn‐glycerol (DiC8), a commonly used diacylglycerol analogue, blocks K+ currents of rat mesenteric arterial smooth muscle in a PKC‐independent manner.EXPERIMENTAL APPROACHConventional whole‐cell and inside‐out patch clamp was used to measure the inhibition of K+ currents of rat isolated mesenteric smooth muscle cells by DiC8 in the absence and presence of PKC inhibitor peptide.KEY RESULTSMesenteric artery smooth muscle Kv currents inactivated very slowly with a time constant of about 2 s following pulses from −65 to +40 mV. Application of 1 µM DiC8 produced an approximate 40‐fold increase in the apparent rate of inactivation. Pretreatment of the cells with PKC inhibitor peptide had a minimal effect on the action of DiC8, and substantial inactivation still occurred, indicating that this effect was mainly independent of PKC. We also found that DiC8 blocked BK and KATP currents, and again a significant proportion of these blocks occurred independently of PKC activation.CONCLUSIONS AND IMPLICATIONSThese results show that DiC8 has a direct effect on arterial smooth muscle K+ channels, and this precludes its use as a PKC activator when investigating PKC‐mediated effects on vascular K+ channels.

Arterial smooth muscle (ASM) contraction plays a critical role in regulating blood distribution and blood pressure. Vasoconstrictors activate cell surface receptors to initiate signaling cascades involving increased intracellular Ca(2+) concentration ([Ca(2+)](i)) and recruitment of protein kinase C (PKC), leading to ASM contraction, though the PKC isoenzymes involved vary between different vasoconstrictors and their actions. Here, we have used confocal microscopy of enhanced green fluorescence protein (eGFP)-labeled PKC isoenzymes to visualize PKC translocation in primary rat mesenteric ASM cells in response to physiological vasoconstrictors, with simultaneous imaging of Ca(2+) signaling. Endothelin-1, angiotensin II, and uridine triphosphate all caused translocation of each of the PKC isoenzymes alpha, delta, and epsilon; however, the kinetics of translocation varied between agonists and PKC isoenzymes. Translocation of eGFP-PKCalpha mirrored the rise in [Ca(2+)](i), while that of eGFP-PKCdelta or -epsilon occurred more slowly. Endothelin-induced translocation of eGFP-PKCepsilon was often sustained for several minutes, while responses to angiotensin II were always transient. In addition, preventing [Ca(2+)](i) increases using 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl) ester prevented eGFP-PKCalpha translocation, while eGFP-PKCdelta translocated more rapidly. Our results suggest that PKC isoenzyme specificity of vasoconstrictor actions occurs downstream of PKC recruitment and demonstrate the varied kinetics and complex interplay between Ca(2+) and PKC responses to different vasoconstrictors in ASM.

Since, in addition to its growth-promoting actions, insulin-like growth factor-I (IGF-I) has rapid vasoactive actions, we investigated the effects of IGF-I on whole-cell ATP-sensitive K+ (KATP) currents of rat mesenteric arterial smooth muscle cells. IGF-I (10 or 30 nM) reduced KATP currents activated by pinacidil or a membrane permeant cAMP analogue. Inhibition of phospholipase C, protein kinase C, protein kinase A, mitogen-activated protein kinase or mammalian target of rapamycin (mTOR) did not prevent the action of IGF-I. However, inhibition of KATP currents by IGF-I was abolished by the tyrosine kinase inhibitor genistein or the phosphoinositide 3-kinase inhibitors, LY 294002 and wortmannin. Intracellular application of either phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol 3,4,5-trisphosphate (PIP3) increased the KATP current activated by pinacidil and abolished the inhibitory effect of IGF-I. Thus, we show regulation of arterial KATP channels by polyphosphoinositides and report for the first time that IGF-I inhibits these channels via a phosphoinositide 3-kinase-dependent pathway.

AIMS:Voltage-gated K+ (Kv) channels of arterial smooth muscle (ASM) modulate arterial tone and are inhibited by vasoconstrictors through protein kinase C (PKC). We aimed to determine whether endothelin-1 (ET-1) and angiotensin II (AngII), which cause similar inhibition of Kv, use the same signalling pathway and PKC isoenzyme to exert their effects on Kv and to compare the involvement of PKC isoenzymes in contractile responses to these agents. METHODS AND RESULTS:Kv currents recorded using the patch clamp technique with freshly isolated rat mesenteric ASM cells were inhibited by ET-1 or AngII. Inclusion of a PKCepsilon inhibitor peptide in the intracellular solution substantially reduced inhibition by AngII, but did not affect that by ET-1. Kv inhibition by ET-1 was reduced by the conventional PKC inhibitor Gö 6976 but not by the PKCbeta inhibitor LY333531. Selective peptide inhibitors of PKCalpha and PKCepsilon were linked to a Tat carrier peptide to make them membrane permeable and used to show that inhibition of PKCalpha prevented ET-1 inhibition of Kv current, but did not affect that by AngII. In contrast, inhibition of PKCepsilon prevented Kv inhibition by AngII but not by ET-1. The Tat-linked inhibitor peptides were also used to investigate the involvement of PKCalpha and PKCepsilon in the contractile responses of mesenteric arterial rings, showing that ET-1 contractions were substantially reduced by inhibition of PKCalpha, but unaffected by inhibition of PKCepsilon. AngII contractions were unaffected by inhibition of PKCalpha but substantially reduced by inhibition of PKCepsilon. CONCLUSION:ET-1 inhibits Kv channels of mesenteric ASM through activation of PKCalpha, while AngII does so through PKCepsilon. This implies that ET-1 and AngII target Kv channels of ASM through different pathways of PKC-interacting proteins, so each vasoconstrictor enables its distinct PKC isoenzyme to interact functionally with the Kv channel.

Prolonged hyperglycaemia impairs vascular reactivity and inhibits voltage-activated K(+) (Kv) channels. We examined acute effects of altering glucose concentration on the activity and inhibition by endothelin-1 (ET-1) of Kv currents of freshly isolated rat arterial myocytes. Peak Kv currents recorded in glucose-free solution were reversibly reduced within 200 s by increasing extracellular glucose to 4 mm. This inhibitory effect of glucose was abolished by protein kinase C inhibitor peptide (PKC-IP), and Kv currents were further reduced in 10 mm glucose. In current-clamped cells, membrane potentials were more negative in 4 than in 10 mm glucose. In 4 mm d-glucose, 10 nm ET-1 decreased peak Kv current amplitude at +60 mV from 23.5 +/- 3.3 to 12.1 +/- 3.1 pA pF(-1) (n = 6, P < 0.001) and increased the rate of inactivation, decreasing the time constant around fourfold. Inhibition by ET-1 was prevented by PKC-IP. When d-glucose was increased to 10 mm, ET-1 no longer inhibited Kv current (n = 6). Glucose metabolism was required for prevention of ET-1 inhibition of Kv currents, since fructose mimicked the effects of d-glucose, while l-glucose, sucrose or mannitol were without effect. Endothelin receptors were still functional in 10 mm d-glucose, since pinacidil-activated ATP-dependent K(+) (K(ATP)) currents were reduced by 10 nm ET-1. This inhibition was nearly abolished by PKC-IP, indicating that endothelin receptors could still activate PKC in 10 mm d-glucose. These results indicate that changes in extracellular glucose concentration within the physiological range can reduce Kv current amplitude and can have major effects on Kv channel modulation by vasoconstrictors.

ATP-sensitive K+ (K-ATP) channels are involved in ischemic cardioprotection induced by preconditioning (IPC), though the relative role of sarcolemmal (sK(ATP)) and mitochondrial (mitoK(ATP)) channels remains controversial. The sK(ATP)-selective sulphonylthiourea HMR 1098 has often been reported to be without effect on ischemic cardioprotection, suggesting minimal involvement of sK(ATP). Since some sulphonylureas show reduced potency under conditions of metabolic stress, we used patch clamp to assess the ability of HMR 1098 to block sK(ATP) currents of adult rat ventricular myocytes activated by metabolic inhibition (MI, NaCN + iodoacetate). In contrast to the prototype sulphonylurea glibenclamide, HMR 1098 (10 mu M) was without effect on sK(ATP) currents, and also did not inhibit MI-induced action potential shortening However, HMR 1098 blocked sK(ATP) current induced by the K-ATP opener pinacidil (IC50 = 0.36 +/- 0.02 mu M), and reversed pinacidil-induced action potential shortening. In inside-out patches, block by HMR 1098 was relieved by increasing MgADP concentrations (1-100 mu M). HMR 1098 inhibited pinacidil-activated recombinant Kir6.2/SUR2A channels with a similar IC50 (0.30 +/- 0.04 mu M), but was less effective when channels were activated by low intracellular ATR HMR 1098 displaced binding of the pinacidil analogue [H-3]P1075 to native cardiac membranes with a biphasic inhibition curve. Our results show that HMR 1098 becomes a much less effective inhibitor of sK(ATP) during metabolic stress, and suggest that the lack of effect of HMR 1098 on ischemic cardioprotection reported in some studies may represent loss of block by the drug under these conditions rather than a lack of involvement of sK(ATP) channels. (c) 2005 Elsevier Ltd. All rights reserved.

OBJECTIVE:We have used isolated myocytes to investigate the effects of diazoxide on sarcolemmal KATP channel (sarcoKATP) activity and action potential failure during metabolic inhibition, and the role of these channels in protection of functional recovery on reperfusion. MATERIALS AND METHODS:Isolated adult rat ventricular myocytes were exposed to metabolic inhibition (NaCN and iodoacetate) and reperfusion. Functional recovery was assessed from the ability of cells to contract on electrical stimulation and to recover calcium homeostasis, measured with fura-2. Action potentials and KATP currents were measured using patch clamp. RESULTS:Pretreatment with diazoxide (100 microM, 5 min) increased the proportion of cells that recovered contractile function after MI and reperfusion from 16.8 +/- 2.4% to 65.0 +/- 2.2% (p<0.001) and the proportion of cells in which [Ca2+]i recovered to <250 nM. Pretreatment also accelerated action potential and contractile failure during MI. In cell-attached patches, MI activated sarcoKATP channels after 224 +/- 11 s, and diazoxide pretreatment decreased this to 145 +/- 24 s (p<0.01). However, diazoxide present in the patch pipette did not accelerate sarcoKATP channel activation. Intracellular Mg2+ rose earlier in diazoxide-pretreated cells. The sarcoKATP blocker HMR 1883 delayed action potential failure and reduced diazoxide protection. CONCLUSIONS:Diazoxide pretreatment increases recovery of function and [Ca2+]i following reperfusion. Protection is coupled with early action potential failure, due to early activation of sarcoKATP channels during metabolic inhibition (MI), which is likely to involve an indirect effect of diazoxide.

Functional KATP (ATP-sensitive potassium) channels are hetero-octamers of four Kir6 (inwardly rectifying potassium) channel subunits and four SUR (sulphonylurea receptor) subunits. Possible interactions between the C-terminal domain of SUR2A and Kir6.2 were investigated by co-immunoprecipitation of rat SUR2A C-terminal fragments with full-length Kir6.2 and by analysis of cloned KATP channel function and distribution in HEK-293 cells (human embryonic kidney 293 cells) in the presence of competing rSUR2A fragments. Three maltose-binding protein-SUR2A fusions, rSUR2A-CTA (rSUR2A residues 1254-1545), rSUR2A-CTB (residues 1254-1403) and rSUR2A-CTC (residues 1294-1403), were co-immunoprecipitated with full-length Kir6.2 using a polyclonal anti-Kir6.2 antiserum. A fourth C-terminal domain fragment, rSUR2A-CTD (residues 1358-1545) did not co-immunoprecipitate with Kir6.2 under the same conditions, indicating a direct interaction between Kir6.2 and a 65-amino-acid section of the cytoplasmic C-terminal region of rSUR2A between residues 1294 and 1358. ATP- and glibenclamide-sensitive K+ currents were decreased in HEK-293 cells expressing full-length Kir6 and SUR2 subunits that were transiently transfected with fragments rSUR2A-CTA, rSUR2A-CTC and rSUR2A-CTE (residues 1294-1359) compared with fragment rSUR2A-CTD or mock-transfected cells, suggesting either channel inhibition or a reduction in the number of functional KATP channels at the cell surface. Anti-KATP channel subunit-associated fluorescence in the cell membrane was substantially lower and intracellular fluorescence increased in rSUR2A-CTE expressing cells; thus, SUR2A fragments containing residues 1294-1358 reduce current by decreasing the number of channel subunits in the cell membrane. These results identify a site in the C-terminal domain of rSUR2A, between residues 1294 and 1358, whose direct interaction with full-length Kir6.2 is crucial for the assembly of functional KATP channels.

C‐terminal fragments of the sulphonylurea receptor SUR2A can alter the functional expression of cloned ATP‐sensitive K+ channels (KATP). To investigate the protective role of KATP channels during metabolic stress we transfected SUR2A fragments into adult rat cardiac myocytes. A fragment comprising residues 1294–1358, the A‐fragment, reduced sarcolemmal KATP currents by over 85% after 2 days (pinacidil‐activated current densities were: vector alone 7.04 ± 1.22; and A‐fragment 0.94 ± 0.07 pA pF−1, n= 6,6, P < 0.001). An inactive fragment (1358–1545, current density 6.30 ± 0.85 pA pF−1, n= 6) was used as a control. During metabolic inhibition (CN and iodoacetate) of isolated myocytes stimulated at 1 Hz, the A‐fragment delayed action potential shortening and contractile failure, but accelerated rigor contraction and increased Ca2+ loading. On reperfusion, A‐fragment‐transfected cells also showed increased intracellular Ca2+ and the proportion of cells recovering contractile function was reduced from 40.0 to 9.5% (P < 0.01). The protective effect of pretreatment with 2,4‐dinitrophenol, measured from increased functional recovery and reduced Ca2+ loading, was abolished by the A‐fragment. Our data are consistent with a role for KATP channels in causing action potential failure and reduced Ca2+ loading during metabolic stress, and with a major role in protection by preconditioning. The effects of the A‐fragment may arise entirely from reduced expression of the sarcolemmal KATP channel, but we also discuss the possibility of mitochondrial effects.

Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels are sensitive to both voltage and internal [Ca(2+)] and are found in many tissues. Their physiological roles range from causing relaxation of smooth muscle to regulating the frequency of action potential firing. There is considerable variation between different tissues in their Ca(2+)- and voltage-dependence. Much of this variation results from the association of the pore-forming alpha subunit (hSloalpha) with different beta subunits leading to altered channel properties. Since hSloalpha alone produces functional BK(Ca) channels, we have used a bicistronic expression method to ensure that both alpha and beta subunits are expressed, with the beta subunit being in excess. Using this method we have investigated the effect of four beta subunits (beta1 to beta4) on cloned BK(Ca) channels. The four beta subunits were individually cloned into a vector that had hSloalpha cDNA inserted downstream of an internal ribosome entry site. The constructs were transiently transfected into HEK293 cells together with a construct that expresses green fluorescent protein, as a marker for transfection. Fluorescent cells expressed BK(Ca) channels whose currents were recorded from inside-out or outside-out patches. The currents we measured using this expression system were similar to those expressed in Xenopus oocytes by Brenner et al. (Brenner, R., Jegla, T.J., Wickenden, A., Liu, Y., Aldrich, R.W. 2000. Cloning and functional expression of novel large-conductance calcium-activated potassium channel beta subunits, hKCNMB3 and hKCNMB4. J. Biol. Chem.275:6453-6461.)