(circa -58 mV). TRAM-34 alone did not affect hyperpolarization to ACh but, in combination with apamin, ACh-evoked hyperpolarization was completely abolished. These data suggest that true endothelium-dependent hyperpolarization of smooth muscle cells in response to ACh is attributable to SK Ca channels, whereas IK Ca channels play an important role during the ACh-mediated repolarization phase only observed following depolarization.

The distribution of functionally active, inwardly rectifying K (K_IR) channels was investigated in the rat small mesenteric artery using both freshly isolated smooth muscle and endothelial cells and small arterial segments. In Ca²⁺-free solution, endothelial cells displayed a K_IR current with a maximum amplitude of 190 ± 16 pA at –150 mV and sensitivity to block with 30 µM Ba²⁺ (n = 7). In smooth muscle cells, outward K current was activated at around –47 ± 3 mV, but there was no evidence of K_IR current (n = 6). Furthermore, raising extracellular [K⁺] to either 60 or 140 mM, or applying the α₁-adrenoceptor agonist phenylephrine (PE; 30 µM), failed to reveal an inwardly rectifying current in the smooth muscle cells, although PE did stimulate an iberiotoxin-sensitive outward K current (n = 4). Exogenous K⁺ (10.8–16.8 mM) both relaxed and repolarized endothelium-denuded segments of the mesenteric artery contracted with PE. These effects were depressed by 100 µM ouabain but unaffected by either 30 µM BaCl₂ or 3 µM glibenclamide. These data suggest that functional, inwardly rectifying Ba²⁺-sensitive channels are restricted to the endothelial cell layer in the rat small mesenteric artery.

1 In rat small mesenteric arteries contracted with phenylephrine, 1-ethyl-2-benzimidazolinone (1-EBIO; 3 ± 300 mM) evoked concentration-dependent relaxation that, above 100 mM, was associated with smooth muscle hyperpolarization. 2 1-EBIO-evoked hyperpolarization (maximum 22.1+3.6 mV with 300 mM, n=4) was endothelium-dependent and inhibited by charybdotoxin (ChTX 100 nM; n=4) but not iberiotoxin (IbTX 100 nM; n=4). 3 In endothelium-intact arteries, smooth muscle relaxation to 1-EBIO was not altered by either of the potassium channel blockers ChTX (100 nM; n=7), or IbTX (100 nM; n=4), or raised extracellular K + (25 mM). Removal of the endothelium shifted the relaxation curve to the right but did not reduce the maximum relaxation. 4 In freshly isolated mesenteric endothelial cells, 1-EBIO (600 mM) evoked a ChTX-sensitive outward K-current. In contrast, 1-EBIO had no e ect on smooth muscle cell conductance whereas NS 1619 (33 mM) stimulated an outward current while having no e ect on the endothelial cells. 5 These data show that with concentrations greater than 100 mM, 1-EBIO selectively activates outward current in endothelial cells, which presumably underlies the smooth muscle hyperpolarization and a component of the relaxation. Sensitivity to block with charybdotoxin but not iberiotoxin indicates this current is due to activation of IK Ca . However, 1-EBIO can also relax the smooth muscle by an unde®ned mechanism, independent of any change in membrane potential.

We tested the hypothesis that cyclic changes in membrane potential (E_m) underlie spontaneous vasomotion in cheek pouch arterioles of anesthetized hamsters. Diameter oscillations (∼3 min^–1) were preceded (∼3 s) by oscillations in E_m of smooth muscle cells (SMC) and endothelial cells (EC). Oscillations in E_m were resolved into six phases: (1) a period (6 ± 2 s) at the most negative E_m observed during vasomotion (–46 ± 2 mV) correlating (r = 0.87, p < 0.01) with time (8 ± 2 s) at the largest diameter observed during vasomotion (41 ± 2 µm); (2) a slow depolarization (1.8 ± 0.2 mV s^–1) with no diameter change; (3) a fast (9.1 ± 0.8 mV s^–1) depolarization (to –28 ± 2 mV) and constriction; (4) a transient partial repolarization (3–4 mV); (5) a sustained (5 ± 1 s) depolarization (–28 ± 2 mV) correlating (r = 0.78, p < 0.01) with time (3 ± 1 s) at the smallest diameter (27 ± 2 µm) during vasomotion; (6) a slow repolarization (2.5 ± 0.2 mV s^–1) and relaxation. The absolute change in E_m correlated (r = 0.60, p < 0.01) with the most negative E_m. Sodium nitroprusside or nifedipine caused sustained hyperpolarization and dilation, whereas tetraethylammonium or elevated PO₂ caused sustained depolarization and constriction. We suggest that vasomotion in vivo reflects spontaneous, cyclic changes in E_m of SMC and EC corresponding with cation fluxes across plasma membranes.

1 The possibility that thromboxane (TXA 2 ) receptor stimulation causes differential block of the SK Ca and IK Ca channels which underlie EDHF-mediated vascular smooth muscle hyperpolarization and relaxation was investigated in the rat isolated mesenteric artery. 2 Acetylcholine (30 nM-3 mM ACh) or cyclopiazonic acid (10 mM CPA, SERCA inhibitor) were used to stimulate EDHF-evoked smooth muscle hyperpolarization. In each case, this led to maximal hyperpolarization of around 20 mV, which was sensitive to block with 50 nM apamin and abolished by repeated stimulation of mesenteric arteries with the thromboxane mimetic, U46619 (30 nM-0.1 mM), but not the a 1 -adrenoceptor agonist phenylephrine (PE). 3 The ability of U46619 to abolish EDHF-evoked smooth muscle hyperpolarization was prevented by prior exposure of mesenteric arteries to the TXA 2 receptor antagonist 1 mM SQ29548. 4 Similar-sized smooth muscle hyperpolarization evoked with the SK Ca activator 100 mM riluzole was also abolished by prior stimulation with U46619, while direct muscle hyperpolarization in response to either levcromakalim (1 mM, K ATP activator) or NS1619 (40 mM, BK Ca activator) was unaffected. 5 During smooth muscle contraction and depolarization to either PE or U46619, ACh evoked concentration-dependent hyperpolarization (to À67 mV) and complete relaxation. These responses were well maintained during repeated stimulation with PE, but with U46619 there was a progressive decline, so that during a third exposure to U46619 maximum hyperpolarization only reached -52 mV and relaxation was reduced by 20%. This relaxation could now be blocked with charybdotoxin alone. The latter responses could be mimicked with 300 mM 1-EBIO (IK Ca activator), an action not modified by exposure to U46619. 6 An early consequence of TXA 2 receptor stimulation is a reduction in the arterial hyperpolarization and relaxation attributed to EDHF. This effect appears to reflect a loss of SK Ca activity.