Strong inward rectifying K + (K IR ) channels have been observed in vascular smooth muscle and can display negative slope conductance. In principle, this biophysical characteristic could enable K IR channels to 'amplify' responses initiated by other K + conductances. To test this, we have characterized the diversity of smooth muscle K IR properties in resistance arteries, confirmed the presence of negative slope conductance and then determined whether K IR inhibition alters the responsiveness of middle cerebral, coronary septal and third-order mesenteric arteries to K + channel activators. Our initial characterization revealed that smooth muscle K IR channels were highly expressed in cerebral and coronary, but not mesenteric arteries. These channels comprised K IR 2.1 and 2.2 subunits and electrophysiological recordings demonstrated that they display negative slope conductance. Computational modelling predicted that a K IR -like current could amplify the hyperpolarization and dilatation initiated by a vascular K + conductance. This prediction was consistent with experimental observations which showed that 30 μM Ba 2+ attenuated the ability of K + channel activators to dilate cerebral and coronary arteries. This attenuation was absent in mesenteric arteries where smooth muscle K IR channels were poorly expressed. In summary, smooth muscle K IR expression varies among resistance arteries and when channel are expressed, their negative slope conductance amplifies responses initiated by smooth muscle and endothelial K + conductances. These findings highlight the fact that the subtle biophysical properties of K IR have a substantive, albeit indirect, role in enabling agonists to alter the electrical state of a multilayered artery.

This study examined whether, and by what signaling and ionic mechanisms, pyrimidine nucleotides constrict rat cerebral arteries. Cannulated cerebral arteries stripped of endothelium and pressurized to 15 mmHg constricted in a dose-dependent manner to UTP. This constriction was partly dependent on the depolarization of smooth muscle cells and the activation of voltage-operated Ca(2+) channels. The depolarization and constriction induced by UTP were unaffected by bisindolylmaleimide I, a PKC inhibitor that abolished phorbol ester (PMA)-induced constriction in cerebral arteries. In contrast, the Rhokinase inhibitor Y-27632 attenuated the ability of UTP to both constrict and depolarize cerebral arteries. With patch-clamp electrophysiology, a voltage-dependent delayed rectifying K(+) (K(DR)) current was isolated and shown to consist of a slowly inactivating 4-aminopyridine (4-AP)-sensitive and an -insensitive component. The 4-AP-sensitive K(DR) current was potently suppressed by UTP through a mechanism that was not dependent on PKC. This reflects observations that demonstrated that 1) a PKC activator (PMA) had no effect on K(DR) and 2) PKC inhibitors (calphostin C or bisindolylmaleimide I) could not prevent the suppression of K(DR) by UTP. The Rho kinase inhibitor Y-27632 abolished the ability of UTP to inhibit the K(DR) current, as did inhibition of RhoA with C3 exoenzyme. Cumulatively, these observations indicate that Rho kinase signaling plays an important role in eliciting the cerebral constriction induced by pyrimidine nucleotides. Moreover, they demonstrate for the first time that Rhokinase partly mediates this constriction by altering ion channels that control membrane potential and Ca(2+) influx through voltage-operated Ca(2+) channels.

Abstract-Small arteries play an essential role in the regulation of blood pressure and organ-specific blood flow by contracting in response to increased intraluminal pressure, ie, the myogenic response. The molecular basis of the myogenic response remains to be defined. To achieve incremental changes in arterial diameter, as well as blood pressure or organ-specific blood flow, the depolarizing influence of intravascular pressure on vascular smooth muscle membrane potential that elicits myogenic contraction must be precisely controlled by an opposing hyperpolarizing influence. Here we use a dominant-negative molecular strategy and pressure myography to determine the role of voltage-dependent Kv1 potassium channels in vasoregulation, specifically, whether they act as a negative-feedback control mechanism of the myogenic response. Functional Kv1 channel expression was altered by transfection of endothelium-denuded rat middle cerebral arteries with cDNAs encoding c-myc epitope-tagged, dominant-negative mutant or wild-type rabbit Kv1.5 subunits. Expression of mutant Kv1.5 dramatically enhanced, whereas wild-type subunit expression markedly suppressed, the myogenic response over a wide range of intraluminal pressures. These effects on arterial diameter were associated with enhanced and reduced myogenic depolarization by mutant and wild-type Kv1.5 subunit expression, respectively. Expression of myc-tagged mutant and wild-type Kv1.5 subunit message and protein in transfected but not control arteries was confirmed, and isolated myocytes of transfected but not control arteries exhibited anti-c-myc immunofluorescence. No changes in message encoding other known, non-Kv1 elements of the myogenic response were apparent. These findings provide the first molecular evidence that Kv1-containing delayed rectifier K ϩ (K DR ) channels are of fundamental importance for control of arterial diameter and, thereby, peripheral vascular resistance, blood pressure, and organ-specific blood flow. Key Words: myogenic response Ⅲ delayed rectifier potassium channels Ⅲ vascular smooth muscle Ⅲ Kv1 channels T he intrinsic ability of resistance arteries to contract in response to elevations in intraluminal (or transmural) pressure, the myogenic response, was first described over 100 years ago by Bayliss. [1][2][3][4] This phenomenon is now well recognized to be an essential autoregulatory mechanism. [2][3][4] Myogenic tone development depends on L-type Ca 2ϩ channel (Cav1.2) activity within vascular myocytes. 5 The resulting rise in intracellular free Ca 2ϩ concentration via these Ca 2ϩ channels 6 activates cross-bridge cycling and contractile force development that may be enhanced and/or maintained by a Ca 2ϩ sensitization of the contractile machinery. 3,4 A current working hypothesis suggests that the activation of L-type Ca 2ϩ channels during the myogenic response is the result of low amplitude, steady-state depolarization of the vascular smooth muscle (VSM) cells attributable to increased intraluminal pressure. 2,3,6 However, very precise con...

This study sought to define whether inward rectifying K(+) (K(IR)) channels were modulated by vasoactive stimuli known to depolarize and constrict intact cerebral arteries. Using pressure myography and patch-clamp electrophysiology, initial experiments revealed a Ba(2+)-sensitive K(IR) current in cerebral arterial smooth muscle cells that was active over a physiological range of membrane potentials and whose inhibition led to arterial depolarization and constriction. Real-time PCR, Western blot, and immunohistochemical analyses established the expression of both K(IR)2.1 and K(IR)2.2 in cerebral arterial smooth muscle cells. Vasoconstrictor agonists known to depolarize and constrict rat cerebral arteries, including uridine triphosphate, U46619, and 5-HT, had no discernable effect on whole cell K(IR) activity. Control experiments confirmed that vasoconstrictor agonists could inhibit the voltage-dependent delayed rectifier K(+) (K(DR)) current. In contrast to these observations, a hyposmotic challenge that activates mechanosensitive ion channels elicited a rapid and sustained inhibition of the K(IR) but not the K(DR) current. The hyposmotic-induced inhibition of K(IR) was 1) mimicked by phorbol-12-myristate-13-acetate, a PKC agonist; and 2) inhibited by calphostin C, a PKC inhibitor. These findings suggest that, by modulating PKC, mechanical stimuli can regulate K(IR) activity and consequently the electrical and mechanical state of intact cerebral arteries. We propose that the mechanoregulation of K(IR) channels plays a role in the development of myogenic tone.

This study examined the role of the actin cytoskeleton in Rho-kinase-mediated suppression of the delayed-rectifier K(+) (K(DR)) current in cerebral arteries. Myocytes from rat cerebral arteries were enzymatically isolated, and whole cell K(DR) currents were monitored using conventional patch-clamp electrophysiology. At +40 mV, the K(DR) current averaged 19.8 +/- 1.6 pA/pF (mean +/- SE) and was potently inhibited by UTP (3 x 10(-5) M). This suppression was observed to depend on Rho signaling and was abolished by the Rho-kinase inhibitors H-1152 (3 x 10(-7) M) and Y-27632 (3 x 10(-5) M). Rho-kinase was also found to concomitantly facilitate actin polymerization in response to UTP. We therefore examined whether actin dynamics played a role in the ability of Rho-kinase to suppress K(DR) current and found that actin disruption using either cytochalasin D (1 x 10(-5) M) or latrunculin A (1 x 10(-8) M) prevented current modulation. Consistent with our electrophysiological observations, both Rho-kinase inhibition and actin disruption significantly attenuated UTP-induced depolarization and constriction of cerebral arteries. We propose that UTP initiates Rho-kinase-mediated remodeling of the actin cytoskeleton and consequently suppresses the K(DR) current, thereby facilitating the depolarization and constriction of cerebral arteries.

This study tested whether activation of protein kinase A (PKA) and G (PKG) pathways would attenuate the ability of RhoA to suppress the delayed rectifier K(+) (K(DR)) current and limit agonist-induced depolarization and constriction. Smooth muscle cells from rat cerebral arteries were enzymatically isolated, and whole cell K(DR) currents were monitored with conventional patch-clamp electrophysiology. The K(DR) current averaged 21.2 +/- 2.3 pA/pF (mean +/- SE) at +40 mV and was potently inhibited by UTP. Current suppression was eliminated in the presence of C3 exoenzyme, confirming that this modulation is dependent on RhoA. Activation of PKA (dibutyryl-cAMP, forskolin) or PKG (dibutyryl-cGMP, sodium nitroprusside, nitric oxide) similarly abolished the ability of UTP to suppress K(DR) and did so without effect on baseline current. Using pressure myography techniques, we stripped cerebral arteries of endothelium and preconstricted them with UTP; these were subsequently shown to hyperpolarize and dilate to both forskolin and sodium nitroprusside. An increase in K(V) channel activity was found to partly underlie these associated changes, as constriction to 4-aminopyridine (K(DR) channel blocker) was greater after PKA or PKG activation. We conclude from our electrophysiological and functional observations that the PKA and PKG pathways attenuate the ability of UTP to depolarize and constrict cerebral arteries in part by minimizing the RhoA-mediated suppression of the K(DR) current.

This study examined the role of the actin cytoskeleton in Rho‐kinase‐mediated suppression of the delayed rectifier K+ (KDR) current in cerebral arteries. Myocytes from rat cerebral arteries were enzymatically isolated and whole cell KDR currents monitored using conventional patch clamp electrophysiology. At +40 mV, the KDR current averaged 19.8 ± 1.6 pA/pF (mean ± SE) and was potently inhibited by uridine triphosphate (UTP; 50 μM). Consistent with past observations, KDR suppression was blocked by the Rho‐kinase inhibitor Y‐27632 (30 μM). Likewise, disruption of the actin cytoskeleton using cytochalasin D (10 μM) or latrunculin A (10 nM) abolished the ability of UTP to suppress KDR. Interestingly, these agents inhibited the current when used at higher concentrations, further suggesting a mechanistic link between the actin cytoskeleton and channels underlying the KDR current. These preliminary findings suggest that an intact cytoskeletal framework is a requisite for Rho‐kinase‐mediated suppression of KDR current and the subsequent depolarization and constriction of cerebral arteries. Supported by CIHR and HSFC.

This study sought to define whether inward rectifying K+ (KIR) channels were modulated by vasoactive stimuli known to depolarize and constrict intact cerebral arteries. Using pressure myography and patch clamp electrophysiology, initial experiments revealed a Ba2+‐sensitive KIR current in cerebral arterial smooth muscle cells that was active over a physiological range of membrane potentials and whose inhibition led to arterial depolarization and constriction. Subsequent real‐time PCR, western blot and immunohistochemical analyses established the expression of both KIR2.1 and KIR2.2 in cerebral arterial smooth muscle cells. Vasoconstrictor agonists known to depolarize and constrict rat cerebral arteries, including uridine triphosphate, U46619, and 5‐HT had no discernable effect on whole‐cell KIR activity. In contrast, a hyposmotic challenge that activates mechano‐sensitive ion channels, elicited a rapid and sustained inhibition of KIR. The hyposmotic induced‐inhibition of KIR was: mimicked by the PKC agonist, phorbol‐12‐myristate‐13‐acetate; and inhibited by Calphostin C, a PKC inhibitor. These findings suggest that by modulating PKC, mechanical stimuli can regulate KIR and consequently the electrical and mechanical state of intact cerebral arteries. We propose that the mechano‐regulation of KIR plays a role in the development of myogenic tone. Supported by CIHR, NIH, and AHFMR.