Membrane Lipid-KIR2.x Channel Interactions Enable Hemodynamic Sensing in Cerebral Arteries.

Objective- Inward rectifying K+ (KIR) channels are present in cerebral arterial smooth muscle and endothelial cells, a tandem arrangement suggestive of a dynamic yet undiscovered role for this channel. This study defined whether distinct pools of cerebral arterial KIR channels were uniquely modulated by membrane lipids and hemodynamic stimuli. Approach and Results- A Ba2+-sensitive KIR current was isolated in smooth muscle and endothelial cells of rat cerebral arteries; molecular analyses subsequently confirmed KIR2.1/KIR2.2 mRNA and protein expression in both cells. Patch-clamp electrophysiology next demonstrated that each population of KIR channels was sensitive to key membrane lipids and hemodynamic stimuli. In this regard, endothelial KIR was sensitive to phosphatidylinositol 4,5-bisphosphate content, with depletion impairing the ability of laminar shear stress to activate this channel pool. In contrast, smooth muscle KIR was sensitive to membrane cholesterol content, with sequestration blocking the ability of pressure to inhibit channel activity. The idea that membrane lipids help confer shear stress and pressure sensitivity of KIR channels was confirmed in intact arteries using myography. Virtual models integrating structural/electrical observations reconceptualized KIR as a dynamic regulator of membrane potential working in concert with other currents to set basal tone across a range of shear stresses and intravascular pressures. Conclusions- The data show for the first time that specific membrane lipid-KIR interactions enable unique channel populations to sense hemodynamic stimuli and drive vasomotor responses to set basal perfusion in the cerebral circulation.

Caveolae Link CaV3.2 Channels to BKCa-Mediated Feedback in Vascular Smooth Muscle.

Objective- This study examined whether caveolae position CaV3.2 (T-type Ca2+ channel encoded by the α-3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca2+ influx to trigger Ca2+ sparks and large-conductance Ca2+-activated K+ channel feedback. Approach and Results- Using smooth muscle cells from mouse mesenteric arteries, the proximity ligation assay confirmed that CaV3.2 reside within 40 nm of caveolin 1, a key caveolae protein. Methyl-ß-cyclodextrin, a cholesterol depleting agent that disrupts caveolae, suppressed CaV3.2 activity along with large-conductance Ca2+-activated K+-mediated spontaneous transient outward currents in cells from C57BL/6 but not CaV3.2-/- mice. Genetic deletion of caveolin 1, a perturbation that prevents caveolae formation, also impaired spontaneous transient outward current production but did so without impairing Ca2+ channel activity, including CaV3.2. These observations indicate a mistargeting of CaV3.2 in caveolin 1-/- mice, a view supported by a loss of Ni2+-sensitive Ca2+ spark generation and colocalization signal (CaV3.2-RyR) from the proximity ligation assay. Vasomotor and membrane potential measurements confirmed that cellular disruption of the CaV3.2-RyR axis functionally impaired the ability of large-conductance Ca2+-activated K+ to set tone in pressurized caveolin 1-/- arteries. Conclusions- Caveolae play a critical role in protein targeting and preserving the close structural relationship between CaV3.2 and RyR needed to drive negative feedback control in resistance arteries.

Ca(V)3.2 channels and the induction of negative feedback in cerebral arteries.

RATIONALE: T-type (CaV3.1/CaV3.2) Ca(2+) channels are expressed in rat cerebral arterial smooth muscle. Although present, their functional significance remains uncertain with findings pointing to a variety of roles. OBJECTIVE: This study tested whether CaV3.2 channels mediate a negative feedback response by triggering Ca(2+) sparks, discrete events that initiate arterial hyperpolarization by activating large-conductance Ca(2+)-activated K(+) channels. METHODS AND RESULTS: Micromolar Ni(2+), an agent that selectively blocks CaV3.2 but not CaV1.2/CaV3.1, was first shown to depolarize/constrict pressurized rat cerebral arteries; no effect was observed in CaV3.2(-/-) arteries. Structural analysis using 3-dimensional tomography, immunolabeling, and a proximity ligation assay next revealed the existence of microdomains in cerebral arterial smooth muscle which comprised sarcoplasmic reticulum and caveolae. Within these discrete structures, CaV3.2 and ryanodine receptor resided in close apposition to one another. Computational modeling revealed that Ca(2+) influx through CaV3.2 could repetitively activate ryanodine receptor, inducing discrete Ca(2+)-induced Ca(2+) release events in a voltage-dependent manner. In keeping with theoretical observations, rapid Ca(2+) imaging and perforated patch clamp electrophysiology demonstrated that Ni(2+) suppressed Ca(2+) sparks and consequently spontaneous transient outward K(+) currents, large-conductance Ca(2+)-activated K(+) channel mediated events. Additional functional work on pressurized arteries noted that paxilline, a large-conductance Ca(2+)-activated K(+) channel inhibitor, elicited arterial constriction equivalent, and not additive, to Ni(2+). Key experiments on human cerebral arteries indicate that CaV3.2 is present and drives a comparable response to moderate constriction. CONCLUSIONS: These findings indicate for the first time that CaV3.2 channels localize to discrete microdomains and drive ryanodine receptor-mediated Ca(2+) sparks, enabling large-conductance Ca(2+)-activated K(+) channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.

Implications of αvß3 Integrin Signaling in the Regulation of Ca2+ Waves and Myogenic Tone in Cerebral Arteries.

OBJECTIVE: The myogenic response is central to blood flow regulation in the brain. Its induction is tied to elevated cytosolic [Ca(2+)], a response primarily driven by voltage-gated Ca(2+) channels and secondarily by Ca(2+) wave production. Although the signaling events leading to the former are well studied, those driving Ca(2+) waves remain uncertain. APPROACH AND RESULTS: We postulated that αvß3 integrin signaling is integral to the generation of pressure-induced Ca(2+) waves and cerebral arterial tone. This hypothesis was tested in rat cerebral arteries using the synergistic strengths of pressure myography, rapid Ca(2+) imaging, and Western blot analysis. GRGDSP, a peptide that preferentially blocks αvß3 integrin, attenuated myogenic tone, indicating the modest role for sarcoplasmic reticulum Ca(2+) release in myogenic tone generation. The RGD peptide was subsequently shown to impair Ca(2+) wave generation and myosin light chain 20 (MLC20) phosphorylation, the latter of which was attributed to the modulation of MLC kinase and MLC phosphatase via MYPT1-T855 phosphorylation. Subsequent experiments revealed that elevated pressure enhanced phospholipase Cγ1 phosphorylation in an RGD-dependent manner and that phospholipase C inhibition attenuated Ca(2+) wave generation. Direct inhibition of inositol 1, 4, 5-triphosphate receptors also impaired Ca(2+) wave generation, myogenic tone, and MLC20 phosphorylation, partly through the T-855 phosphorylation site of MYPT1. CONCLUSIONS: Our investigation reveals a hitherto unknown role for αvß3 integrin as a cerebral arterial pressure sensor. The membrane receptor facilitates Ca(2+) wave generation through a signaling cascade, involving phospholipase Cγ1, inositol 1,3,4 triphosphate production, and inositol 1, 4, 5-triphosphate receptor activation. These discrete asynchronous Ca(2+) events facilitate MLC20 phosphorylation and, in part, myogenic tone by influencing both MLC kinase and MLC phosphatase activity.

Genetic ablation of CaV3.2 channels enhances the arterial myogenic response by modulating the RyR-BKCa axis.

OBJECTIVE: In resistance arteries, there is an emerging view that smooth muscle CaV3.2 channels restrain arterial constriction through a feedback response involving the large-conductance Ca(2+)-activated K(+) channel (BKCa). Here, we used wild-type and CaV3.2 knockout (CaV3.2(-/-)) mice to definitively test whether CaV3.2 moderates myogenic tone in mesenteric arteries via the CaV3.2-ryanodine receptor-BKCa axis and whether this regulatory mechanism influences blood pressure regulation. APPROACH AND RESULTS: Using pressurized vessel myography, CaV3.2(-/-) mesenteric arteries displayed enhanced myogenic constriction to pressure but similar K(+)-induced vasoconstriction compared with wild-type C57BL/6 arteries. Electrophysiological and myography experiments subsequently confirmed the inability of micromolar Ni(2+), a CaV3.2 blocker, to either constrict arteries or suppress T-type currents in CaV3.2(-/-) smooth muscle cells. The frequency of BKCa-induced spontaneous transient outward K(+) currents dropped in wild-type but not in knockout arterial smooth muscle cells upon the pharmacological suppression of CaV3.2 channel. Line scan analysis performed on en face arteries loaded with Fluo-4 revealed the presence of Ca(2+) sparks in all arteries, with the subsequent application of Ni(2+) only affecting wild-type arteries. Although CaV3.2 channel moderated myogenic constriction of resistance arteries, the blood pressure measurements of CaV3.2(-/-) and wild-type animals were similar. CONCLUSIONS: Overall, our findings establish a negative feedback mechanism of the myogenic response in which CaV3.2 channel modulates downstream ryanodine receptor-BKCa to hyperpolarize and relax arteries.

Nitric oxide suppresses vascular voltage-gated T-type Ca2+ channels through cGMP/PKG signaling.

Recent reports have noted that T-type Ca2+ channels (CaV3.x) are expressed in vascular smooth muscle and are potential targets of regulation. In this study, we examined whether and by what mechanism nitric oxide (NO), a key vasodilator, influences this conductance. Using patch-clamp electrophysiology and rat cerebral arterial smooth muscle cells, we monitored an inward Ba2+ current that was divisible into a nifedipine-sensitive and -insensitive component. The latter was abolished by T-type channel blocker and displayed classic T-type properties including faster activation and steady-state inactivation at hyperpolarized potentials. NO donors (sodium nitroprusside, S-nitroso-N-acetyl-dl-penicillamine), along with activators of protein kinase G (PKG) signaling, suppressed T-type currents. Inhibitors of guanylyl cyclase/PKG {1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and KT5823, respectively}, had no effect on basal currents; KT5823 did, however, mask T-type Ca2+ channel current inhibition by NO/PKG. Functional experiments confirmed an inhibitory effect for NO on the T-type contribution to cerebral arterial myogenic tone. Cumulatively, our findings support the view that T-type Ca2+ channels are a regulatory target of vasodilatory signaling pathways. This targeting will influence Ca2+ dynamics and consequent tone development in the cerebral circulation.

Identification of L- and T-type Ca2+ channels in rat cerebral arteries: role in myogenic tone development.

L-type Ca(2+) channels are broadly expressed in arterial smooth muscle cells, and their voltage-dependent properties are important in tone development. Recent studies have noted that these Ca(2+) channels are not singularly expressed in vascular tissue and that other subtypes are likely present. In this study, we ascertained which voltage-gated Ca(2+) channels are expressed in rat cerebral arterial smooth muscle and determined their contribution to the myogenic response. mRNA analysis revealed that the α(1)-subunit of L-type (Ca(v)1.2) and T-type (Ca(v)3.1 and Ca(v)3.2) Ca(2+) channels are present in isolated smooth muscle cells. Western blot analysis subsequently confirmed protein expression in whole arteries. With the use of patch clamp electrophysiology, nifedipine-sensitive and -insensitive Ba(2+) currents were isolated and each were shown to retain electrical characteristics consistent with L- and T-type Ca(2+) channels. The nifedipine-insensitive Ba(2+) current was blocked by mibefradil, kurtoxin, and efonidpine, T-type Ca(2+) channel inhibitors. Pressure myography revealed that L-type Ca(2+) channel inhibition reduced tone at 20 and 80 mmHg, with the greatest effect at high pressure when the vessel is depolarized. In comparison, the effect of T-type Ca(2+) channel blockade on myogenic tone was more limited, with their greatest effect at low pressure where vessels are hyperpolarized. Blood flow modeling revealed that the vasomotor responses induced by T-type Ca(2+) blockade could alter arterial flow by ∼20-50%. Overall, our findings indicate that L- and T-type Ca(2+) channels are expressed in cerebral arterial smooth muscle and can be electrically isolated from one another. Both conductances contribute to myogenic tone, although their overall contribution is unequal.

Genetic ablation of smooth muscle KIR2.1 is inconsequential to the function of mouse cerebral arteries.

Cerebral blood flow is a finely tuned process dependent on coordinated changes in arterial tone. These changes are strongly tied to smooth muscle membrane potential and inwardly rectifying K+ (KIR) channels are thought to be a key determinant. To elucidate the role of KIR2.1 in cerebral arterial tone development, this study examined the electrical and functional properties of cells, vessels and living tissue from tamoxifen-induced smooth muscle cell (SMC)-specific KIR2.1 knockout mice. Patch-clamp electrophysiology revealed a robust Ba2+-sensitive inwardly rectifying K+ current in cerebral arterial myocytes irrespective of KIR2.1 knockout. Immunolabeling clarified that KIR2.1 expression was low in SMCs while KIR2.2 labeling was remarkably abundant at the membrane. In alignment with these observations, pressure myography revealed that the myogenic response and K+-induced dilation were intact in cerebral arteries post knockout. At the whole organ level, this translated to a maintenance of brain perfusion in SMC KIR2.1-/- mice, as assessed with arterial spin-labeling MRI. We confirmed these findings in superior epigastric arteries and implicated KIR2.2 as more functionally relevant in SMCs. Together, these results suggest that subunits other than KIR2.1 play a significant role in setting native current in SMCs and driving arterial tone.

Do TRPC-like currents and G protein-coupled receptors interact to facilitate myogenic tone development?

The objective of this study was to determine whether G(q/11)-coupled receptor activation can enhance the mechanosensitivity of a canonical transient receptor potential (TRPC)-like current and consequently the myogenic responsiveness of rat anterior cerebral arteries. Initial patch-clamp experiments revealed the presence of a basal cation current in isolated smooth muscle cells that displayed evidence of double rectification, which was blocked by trivalent cations (Gd(3+) and La(3+)). PCR analysis identified the expression of TRPC1, 3, 6 and 7 mRNA and, characteristic of TRPC-like current, the whole-cell conductance was insensitive to a Na(+)-dependent transport (amiloride), TRP vanilloid (ruthenium red), and chloride channel (DIDS, niflumic acid, and flufenamate) inhibitors. One notable exception was tamoxifen, which elicited a dual effect, blocking or activating the TRPC-like current at 1 and 10 µM, respectively. This TRPC-like current was augmented by constrictor agonists (uridine 5'-triphosphate and U46619) or hyposmotic challenge (303 to 223 mOsm/l), a mechanical stimulus. Although each stimulus was effective alone, smooth muscle cells pretreated with agonist did not augment the whole-cell response to hyposmotic challenge. Consistent with these electrophysiological recordings, functional experiments revealed that neither UTP nor U46619 enhanced the sensitivity of intact cerebral arteries to hyposmotic challenge or elevated intravascular pressure. In summary, this study found no evidence that G(q/11)-coupled receptor activation augments the mechanosensitivity of a TRPC-like current and consequently the myogenic responsiveness of anterior cerebral arteries.

Intravascular pressure augments cerebral arterial constriction by inducing voltage-insensitive Ca2+ waves.

This study examined whether elevated intravascular pressure stimulates asynchronous Ca(2+) waves in cerebral arterial smooth muscle cells and if their generation contributes to myogenic tone development. The endothelium was removed from rat cerebral arteries, which were then mounted in an arteriograph, pressurized (20-100 mmHg) and examined under a variety of experimental conditions. Diameter and membrane potential (V(M)) were monitored using conventional techniques; Ca(2+) wave generation and myosin light chain (MLC(20))/MYPT1 (myosin phosphatase targeting subunit) phosphorylation were assessed by confocal microscopy and Western blot analysis, respectively. Elevating intravascular pressure increased the proportion of smooth muscle cells firing asynchronous Ca(2+) waves as well as event frequency. Ca(2+) wave augmentation occurred primarily at lower intravascular pressures (<60 mmHg) and ryanodine, a plant alkaloid that depletes the sarcoplasmic reticulum (SR) of Ca(2+), eliminated these events. Ca(2+) wave generation was voltage insensitive as Ca(2+) channel blockade and perturbations in extracellular [K(+)] had little effect on measured parameters. Ryanodine-induced inhibition of Ca(2+) waves attenuated myogenic tone and MLC(20) phosphorylation without altering arterial V(M). Thapsigargin, an SR Ca(2+)-ATPase inhibitor also attenuated Ca(2+) waves, pressure-induced constriction and MLC(20) phosphorylation. The SR-driven component of the myogenic response was proportionally greater at lower intravascular pressures and subsequent MYPT1 phosphorylation measures revealed that SR Ca(2+) waves facilitated pressure-induced MLC(20) phosphorylation through mechanisms that include myosin light chain phosphatase inhibition. Cumulatively, our findings show that mechanical stimuli augment Ca(2+) wave generation in arterial smooth muscle and that these transient events facilitate tone development particularly at lower intravascular pressures by providing a proportion of the Ca(2+) required to directly control MLC(20) phosphorylation.

KIR channels function as electrical amplifiers in rat vascular smooth muscle.

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 mum 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.

Reactive Oxygen Species Mediate the Suppression of Arterial Smooth Muscle T-type Ca2+ Channels by Angiotensin II.

Vascular T-type Ca2+ channels (CaV3.1 and CaV3.2) play a key role in arterial tone development. This study investigated whether this conductance is a regulatory target of angiotensin II (Ang II), a vasoactive peptide that circulates and which is locally produced within the arterial wall. Patch clamp electrophysiology performed on rat cerebral arterial smooth muscle cells reveals that Ang II (100 nM) inhibited T-type currents through AT1 receptor activation. Blocking protein kinase C failed to eliminate channel suppression, a finding consistent with unique signaling proteins enabling this response. In this regard, inhibiting NADPH oxidase (Nox) with apocynin or ML171 (Nox1 selective) abolished channel suppression highlighting a role for reactive oxygen species (ROS). In the presence of Ni2+ (50 µM), Ang II failed to modulate the residual T-type current, an observation consistent with this peptide targeting CaV3.2. Selective channel suppression by Ang II impaired the ability of CaV3.2 to alter spontaneous transient outward currents or vessel diameter. Proximity ligation assay confirmed Nox1 colocalization with CaV3.2. In closing, Ang II targets CaV3.2 channels via a signaling pathway involving Nox1 and the generation of ROS. This unique regulatory mechanism alters BKCa mediated feedback giving rise to a "constrictive" phenotype often observed with cerebrovascular disease.

KIR channels tune electrical communication in cerebral arteries.

The conducted vasomotor response reflects electrical communication in the arterial wall and the distance signals spread is regulated by three factors including resident ion channels. This study defined the role of inward-rectifying K+ channels (KIR) in governing electrical communication along hamster cerebral arteries. Focal KCl application induced a vasoconstriction that conducted robustly, indicative of electrical communication among cells. Inhibiting dominant K+ conductances had no attenuating effect, the exception being Ba2+ blockade of KIR. Electrophysiology and Q-PCR analysis of smooth muscle cells revealed a Ba2+-sensitive KIR current comprised of KIR2.1/2.2 subunits. This current was surprisingly small and when incorporated into a model, failed to account for the observed changes in conduction. We theorized a second population of KIR channels exist and consistent with this idea, a robust Ba2+-sensitive KIR2.1/2.2 current was observed in endothelial cells. When both KIR currents were incorporated into, and then inhibited in our model, conduction decay was substantive, aligning with experiments. Enhanced decay was ascribed to the rightward shift in membrane potential and the increased feedback arising from voltage-dependent-K+ channels. In summary, this study shows that two KIR populations work collaboratively to govern electrical communication and the spread of vasomotor responses along cerebral arteries.

CaV1.2/CaV3.x channels mediate divergent vasomotor responses in human cerebral arteries.

The regulation of arterial tone is critical in the spatial and temporal control of cerebral blood flow. Voltage-gated Ca(2+) (CaV) channels are key regulators of excitation-contraction coupling in arterial smooth muscle, and thereby of arterial tone. Although L- and T-type CaV channels have been identified in rodent smooth muscle, little is known about the expression and function of specific CaV subtypes in human arteries. Here, we determined which CaV subtypes are present in human cerebral arteries and defined their roles in determining arterial tone. Quantitative polymerase chain reaction and Western blot analysis, respectively, identified mRNA and protein for L- and T-type channels in smooth muscle of cerebral arteries harvested from patients undergoing resection surgery. Analogous to rodents, CaV1.2 (L-type) and CaV3.2 (T-type) α1 subunits were expressed in human cerebral arterial smooth muscle; intriguingly, the CaV3.1 (T-type) subtype present in rodents was replaced with a different T-type isoform, CaV3.3, in humans. Using established pharmacological and electrophysiological tools, we separated and characterized the unique profiles of Ca(2+) channel subtypes. Pressurized vessel myography identified a key role for CaV1.2 and CaV3.3 channels in mediating cerebral arterial constriction, with the former and latter predominating at higher and lower intraluminal pressures, respectively. In contrast, CaV3.2 antagonized arterial tone through downstream regulation of the large-conductance Ca(2+)-activated K(+) channel. Computational analysis indicated that each Ca(2+) channel subtype will uniquely contribute to the dynamic regulation of cerebral blood flow. In conclusion, this study documents the expression of three distinct Ca(2+) channel subtypes in human cerebral arteries and further shows how they act together to orchestrate arterial tone.

The functional consequence of RhoA knockdown by RNA interference in rat cerebral arteries.

Uridine triphosphate (UTP) constricts cerebral arteries by activating transduction pathways that increase cytosolic [Ca(2+)] and myofilament Ca(2+) sensitivity. The signaling proteins that comprise these pathways remain uncertain with recent studies implicating a role for several G proteins. To start clarifying which G proteins enable UTP-induced vasoconstriction, a small interfering RNA (siRNA) approach was developed to knock down specified targets in rat cerebral arteries. siRNA directed against G(q) and RhoA was introduced into isolated cerebral arteries using reverse permeabilization. Following a defined period of organ culture, arteries were assayed for contractile function, mRNA levels, and protein expression. Targeted siRNA reduced RhoA or G(q) mRNA expression by 60-70%, which correlated with a reduction in RhoA but not G(q) protein expression. UTP-induced constriction was abolished in RhoA-depleted arteries, but this was not due to a reduction in myosin light chain phosphorylation. UTP-induced actin polymerization was attenuated in RhoA-depleted arteries, which would explain the loss of agonist-induced constriction. In summary, this study illustrates that siRNA approaches can be effectively used on intact arteries to induce targeted knockdown given that the protein turnover rate is sufficiently high. It also demonstrates that the principal role of RhoA in agonist-induced constriction is to facilitate the formation of F-actin, the physical structure to which phosphorylated myosin binds to elicit arterial constriction.

Inward rectifying potassium channels facilitate cell-to-cell communication in hamster retractor muscle feed arteries.

This study examined whether inward rectifying K+ (KIR) channels facilitate cell-to-cell communication along skeletal muscle resistance arteries. With the use of feed arteries from the hamster retractor muscle, experiments examined whether KIR channels were functionally expressed and whether channel blockade attenuated the conduction of acetylcholine-induced vasodilation, an index of cell-to-cell communication. Consistent with KIR channel expression, this study observed the following: 1) a sustained Ba2+-sensitive, K+-induced dilation in preconstricted arteries; 2) a Ba2+-sensitive inwardly rectifying K+ current in arterial smooth muscle cells; and 3) KIR2.1 and KIR2.2 expression in the smooth muscle layer of these arteries. It was subsequently shown that the discrete application of acetylcholine elicits a vasodilation that conducts with limited decay along the feed artery wall. In the presence of 100 microM Ba2+, the local and conducted response to acetylcholine was attenuated, a finding consistent with a role for KIR in facilitating cell-to-cell communication. A computational model of vascular communication accurately predicted these observations. Control experiments revealed that in contrast to Ba2+, ATP-sensitive- and large-conductance Ca2+ activated-K+ channel inhibitors had no effect on the local or conducted vasodilatory response to acetylcholine. We conclude that smooth muscle KIR channels play a key role in facilitating cell-to-cell communication along skeletal muscle resistance arteries. We attribute this facilitation to the intrinsic property of negative slope conductance, a biophysical feature common to KIR2.1- and 2.2-containing channels, which enables them to increase their activity as a cell hyperpolarizes.

Pyrimidine nucleotides suppress KDR currents and depolarize rat cerebral arteries by activating Rho kinase.

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.