Abstract-Arterial ATP-sensitive K ϩ (K ATP ) channels are critical regulators of vascular tone, forming a focal point for signaling by many vasoactive transmitters that alter smooth muscle contractility and so blood flow. Clinically, these channels form the target of antianginal and antihypertensive drugs, and their genetic disruption leads to hypertension and sudden cardiac death through coronary vasospasm. However, whereas the biochemical basis of K ATP channel modulation is well-studied, little is known about the structural or spatial organization of the signaling pathways that converge on these channels. In this study, we use discontinuous sucrose density gradients and Western blot analysis to show that K ATP channels localize with an upstream signaling partner, adenylyl cyclase, to smooth muscle membrane fractions containing caveolin, a protein found exclusively in cholesterol and sphingolipid-enriched membrane invaginations known as caveolae. Furthermore, we show that an antibody against the K ATP pore-forming subunit, Kir6.1 co-immunoprecipitates caveolin from arterial homogenates, suggesting that Kir6.1 and caveolin exist together in a complex. To assess whether the colocalization of K ATP channels and adenylyl cyclase to smooth muscle caveolae has functional significance, we disrupt caveolae with the cholesterol-depleting agent, methyl-␤-cyclodextrin. This reduces the cAMP-dependent protein kinase A-sensitive component of whole-cell K ATP current, indicating that the integrity of caveolae is important for adenylyl cyclase-mediated channel modulation. These results suggest that to be susceptible to protein kinase A-dependent activation, arterial K ATP channels need to be localized in the same lipid compartment as adenylyl cyclase; the results also provide the first indication of the spatial organization of signaling pathways that regulate K ATP channel activity.

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Objective: The vasoconstrictor angiotensin II (Ang II) acts at G q/11 -coupled receptors to suppress ATP-sensitive potassium (K ATP ) channel activity via activation of protein kinase C (PKC). The aim of this study was to determine the PKC isoforms involved in the Ang II-induced inhibition of aortic K ATP channel activity and to investigate potential mechanisms by which these isoforms specifically target these ion channels.Methods and results: We show that the inhibitory effect of Ang II on pinacidil-evoked whole-cell rat aortic K ATP currents persists in the presence of Gö6976, an inhibitor of the conventional PKC isoforms, but is abolished by intracellular dialysis of a selective PKCε translocation inhibitor peptide. This suggests that PKC-dependent inhibition of aortic K ATP channels by Ang II arises exclusively from the activation and translocation of PKCε. Using discontinuous sucrose density gradients and Western blot analysis, we show that Ang II induces the translocation of PKCε to cholesterol-enriched rat aortic smooth muscle membrane fractions containing both caveolin, a protein found exclusively in caveolae, and Kir6.1, the pore-forming subunit of the vascular K ATP channel. Immunogold electron microscopy of rat aortic smooth muscle plasma membrane sheets confirms both the presence of Kir6.1 in morphologically identifiable regions of the membrane rich in caveolin and Ang II-evoked migration of PKCε to these membrane compartments. Conclusions: Ang II induces the recruitment of the novel PKC isoform, PKCε, to arterial smooth muscle caveolae. This translocation allows PKCε access to K ATP channels compartmentalized within these specialized membrane microdomains and highlights a potential role for caveolae in targeting PKC isozymes to an ion channel effector.

The relative functional importance of potassium channels and cGMP-dependent pathways in the relaxation of vascular smooth muscle to the novel nitric oxide donor, diethylamine NONOate (DEA NONOate), was investigated in a resistance artery. The contribution from cGMP-dependent signalling pathways was examined by exposing arteries to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of soluble guanylyl cyclase, while the contribution through potassium channels was assessed with different sub-type-selective potassium channel blockers. DEA NONOate (3 nM-10 microM) evoked sustained relaxation in isolated segments of the rat small mesenteric artery contracted with phenylephrine (pEC50=6.7+/-0.2; n=11). The relaxation was attenuated significantly by either ODQ (10 microM; pEC50=5.8+/-0.4; n=7) or charybdotoxin (ChTX; 50 nM; pEC50=6.3+/-0.2; n=4), a peptide blocker of large conductance, calcium-activated potassium channels (BK(Ca)). The inhibitory effects of ODQ and ChTX were additive (pEC50=5.1+/-0.4; n=9). The selective inhibitor of BK(Ca) channels, iberiotoxin (IbTX; 30 nM), and 4-aminopyridine (4-AP; 1 mM), an inhibitor of voltage-gated potassium channels (Kv), failed to modify DEA NONOate-evoked relaxation. However, in the combined presence of both ODQ and either IbTX or 4-AP the relaxation was attenuated significantly (n=3). The blocker of ATP-modulated potassium channels (K(ATP)), glibenclamide (10 microM), and of small conductance calcium-activated potassium channels (SK(Ca)), apamin (30 nM), each failed to affect ODQ-sensitive or -resistant relaxations to DEA NONOate (n=3). In conclusion, relaxation to DEA NONOate in the rat isolated, small mesenteric artery can occur via both cGMP-dependent (ODQ-sensitive) and -independent (ODQ-resistant) mechanisms. However, the contribution made to relaxation by potassium channels appears to be unmasked following pharmacological attenuation of cGMP-dependent signalling pathways. The inhibitory action of ChTX suggests part of the cGMP-insensitive component involves the activation of potassium channels, a suggestion supported by the inhibitory actions of 4-AP and IbTX in the absence of cGMP.

1 Authentic nitric oxide (NO; 0.1 ± 10 mmoles) caused transient, dose-dependent relaxation of phenylephrine-induced tone without changing membrane potential in mesenteric arteries. Larger doses, above 10 mmoles, did not evoke more relaxation (maximal relaxation to 150 mmoles NO in denuded arteries, 69+7%, n=8) but stimulated muscle hyperpolarization (maximum 19+3 mV, n=5). 2 The soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 mM), abolished relaxation to low doses of NO (n=4), but did not modify hyperpolarization with higher doses of NO (n=4). The potassium channel blocker charybdotoxin (ChTX; 50 nM) abolished hyperpolarization to high doses of NO and signi®cantly reduced the maximal relaxation (to 43+6%, n=4; P50.01). ODQ and ChTX together abolished tension and membrane potential change to all doses of NO (n=4). 3 All relaxations to 3-morpholino-sydnonimine (SIN-1; 0.01 ± 10 mM) were associated with hyperpolarization. When the endothelium was intact, ChTX inhibited hyperpolarization and relaxation to SIN-1 (n=5), while iberiotoxin (IbTX; 50 nM) or 4-aminopyridine (4-AP; 500 mM) reduced relaxation by 40% and 20%, respectively and by 80% in combination (n=6 in each case). 4 In denuded arteries, relaxation to SIN-1 was una ected by either ChTX or ODQ alone, but abolished by the inhibitors together (n=6). Alone, 4-AP did not alter relaxation, but in the presence of ODQ it reduced the maximal response by around 45% (n=6; P50.01). 4-AP, ODQ and IbTX together inhibited relaxation to SIN-1 by 75% (n=6; P50.01). 5 Therefore, cyclic guanosine 3',5'-monophosphate (cyclic GMP)-independent smooth muscle hyperpolarization, possibly involving direct activation of calcium-activated and voltage-sensitive potassium channels, contributes to relaxation evoked by authentic NO and SIN-1. However, the importance of each pathway depends on the source of NO and with SIN-1 the relative contribution from each pathway is modi®ed by the endothelium.