Nancy J. Rusch

Increased Expression of Ca2+-Sensitive K+ Channels in the Cerebral Microcirculation of Genetically Hypertensive Rats Evidence for Their Protection Against Cerebral Vasospasm

The Ca2+-sensitive K+ channel (K(Ca) channel) plays a key role in buffering pressure-induced constriction of small cerebral arteries. An amplified current through this channel has been reported in vascular smooth muscle cells obtained from hypertensive animals, implying that the expression or properties of K(Ca) channels may be regulated by in vivo blood pressure levels. In this study, we investigated this hypothesis and its functional relevance by comparing the properties, expression levels, and physiological role of K(Ca) channels in cerebral resistance arteries from normotensive and genetically hypertensive rats. Whole-cell patch-clamp experiments revealed a 4.7-fold higher density of iberiotoxin-sensitive K(Ca) channel current at physiological membrane potentials in spontaneously hypertensive rat (SHR) compared with Wistar-Kyoto (WKY) rat cerebrovascular smooth muscle cells (n = 18 and 21, respectively). However, additional single-channel analysis in detached patches showed similar levels of unitary conductance, voltage, and Ca2+ sensitivity in K(Ca) channels from WKY and from SHR membranes. In contrast, Western analysis using an antibody directed against the K(Ca) channel alpha-subunit revealed a 4.1-fold increase in the corresponding 125-kD immunoreactive signal in cerebrovascular membranes from SHR compared with WKY rats. The functional impact of this enhanced K(Ca) channel expression was assessed in SHR and WKY rat pial arterioles, which were monitored by intravital microscopy through in situ cranial windows. Progressive pharmacological block of K(Ca) channels by iberiotoxin (0.1 to 100 nmol/L) dose-dependently constricted pial arterioles from SHR and WKY rats (n = 6 to 8). The arterioles in SHR constricted 2- to 4-fold more intensely, and vasospasm occurred in some vessels. These data provide the first direct evidence that elevated levels of in situ blood pressure induce K(Ca) channel expression in cerebrovascular smooth muscle membranes. This homeostatic mechanism may critically regulate the resting tone of cerebral arterioles during chronic hypertension. Furthermore, the overexpression of distinct K+ channel types during specific cardiovascular pathologies may provide for the upregulation of novel disease-specific membrane targets for vasodilator therapies.

Increased Expression of Ca2+-Sensitive K+ Channels in Aorta of Hypertensive Rats

Potassium efflux through Ca2+-sensitive K+ channels (K[Ca] channels) is increased in arterial smooth muscle cells from hypertensive rats, but the molecular mechanism is unknown. The goal of this study was to compare the levels of K(Ca) channel current between aortic smooth muscle cells from adult Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) and then use Western blot methods and ribonuclease protection assays to examine the expression and mRNA levels for the K(Ca) channel in these same vascular tissues. Whole-cell patch-clamp methods indicated a larger component of K(Ca) channel current, sensitive to block by iberiotoxin (100 nmol/L), in single aortic smooth muscle cells from SHR compared with WKY. Subsequent Western blot analysis using a site-specific antibody (anti-alpha[913-926]) directed against the S9/S10 linker of the alpha-subunit of the K(Ca), channel revealed a 125-kD immunoreactive band in lanes loaded with either WKY or SHR aortic muscle membranes. The immunoreactive density of this band, which corresponded to the known molecular size of the alpha-subunit, was 2.2-fold greater in lanes loaded with aortic smooth muscle membranes from the hypertensive animals. However, despite this evidence for an increased expression and functional enhancement of K(Ca) channels in aortic smooth muscle membranes of SHR, ribonuclease protection assays with a 32P-labeled riboprobe targeted against the S9/S10 linker of the K(Ca) channel alpha-subunit revealed no difference in mRNA levels for the alpha-subunit between WKY and SHR aortic tissue. These findings provide initial evidence that (1) an increased expression of K(Ca) channels may be a mechanism for the enhanced K(Ca) current in aortic smooth muscle membranes of SHR, and (2) the upregulation of K(Ca) channels in arterial muscle membranes during hypertension, which is regarded as a homeostatic mechanism for buffering vascular excitability, may rely on posttranscriptional events.

High Blood Pressure Upregulates Arterial L-Type Ca2+ Channels Is Membrane Depolarization the Signal?

Long-lasting Ca2+ (CaL) channels of the Cav1.2 gene family contribute to the pathogenesis of abnormal arterial tone in hypertension. The physiological stimulus that enhances CaL channel current in the vascular smooth muscle cells (VSMCs) remains unknown. The present study investigated if high blood pressure triggers an upregulation of vascular CaL channel protein. Rat aortae were banded between the origins of the left renal (LR) and right renal (RR) arteries to selectively elevate blood pressure in the proximal RR arteries. After 2 days, the immunoreactivity on Western blots corresponding to the pore-forming &agr;1C subunit of the CaL channel was increased 3.25-fold in RR compared with LR arteries. This finding persisted at 28 days and was associated with abnormal Ca2+-dependent tone and higher CaL currents in the VSMCs exposed to high pressure. Based on microelectrode studies indicating that RR arteries were depolarized compared with LR arteries, further studies examined if membrane depolarization, an inherent response of VSMCs to high blood pressure, increased &agr;1C expression. Isolated rat renal arteries were cultured for 2 days in low K+ (4 mmol/L) or depolarizing high K+ (30 mmol/L) media. Arteries preconditioned in high K+ showed a 5.47-fold increase in &agr;1C expression, enhanced CaL channel current, and elevated Ca2+-dependent tone. These findings provide the first direct evidence that high blood pressure upregulates the CaL channel &agr;1C subunit in VSMCs in vivo and suggest that membrane depolarization is a potential signal involved in this interaction that may contribute to the development of abnormal vascular tone.

Voltage‐gated K+ channels in rat small cerebral arteries: molecular identity of the functional channels

Voltage‐gated potassium (KV) channels represent an important dilator influence in the cerebral circulation, but the composition of these tetrameric ion channels remains unclear. The goals of the present study were to evaluate the contribution of KV1 family channels to the resting membrane potential and diameter of small rat cerebral arteries, and to identify the α‐subunit composition of these channels using patch‐clamp, molecular and immunological techniques. Initial studies indicated that 1 μmol l−1 correolide (COR), a specific antagonist of KV1 channels, depolarized vascular smooth muscle cells (VSMCs) in pressurized (60 mmHg) cerebral arteries from ‐55 ± 1 mV to ‐34 ± 1 mV, and reduced the resting diameter from 152 ± 15 μm to 103 ± 20 μm. In patch clamped VSMCs from these arteries, COR‐sensitive KV1 current accounted for 65 % of total outward KV current and was observed at physiological membrane potentials. RT‐PCR identified mRNA encoding each of the six classical KV1 α‐subunits, KV1.1‐1.6, in rat cerebral arteries. However, only the KV1.2 and 1.5 proteins were detected by Western blot. The expression of these proteins in VSMCs was confirmed by immunocytochemistry and co‐immunoprecipitation of KV1.2 and 1.5 from VSMC membranes suggested KV1.2/1.5 channel assembly. Subsequently, the pharmacological and voltage‐sensitive properties of KV1 current in VSMCs were found to be consistent with a predominant expression of KV1.2/1.5 heterotetrameric channels. The findings of this study suggest that KV1.2/1.5 heterotetramers are preferentially expressed in rat cerebral VSMCs, and that these channels contribute to the resting membrane potential and diameter of rat small cerebral arteries.

Upregulation of L-Type Ca2+ Channels in Mesenteric and Skeletal Arteries of SHR

An increased Ca2+ influx attributed to dihydropyridine-sensitive L-type Ca2+ channels has been demonstrated in mesenteric vascular smooth muscle cells of spontaneously hypertensive rats (SHR). This study examined whether an upregulation of the pore-forming &agr;1C subunit of the L-type Ca2+ channel underlies this ionic defect. With the use of mesenteric arcade arteries from 12- to 16-week-old SHR and normotensive Wistar Kyoto (WKY) rats, reverse transcriptase–polymerase chain reaction demonstrated an increased level of amplified cDNA corresponding to the &agr;1C subunit mRNA in the SHR arteries. Western blots confirmed that the increased mRNA expression was associated with a 3.4-fold increase in the immunoreactive signal of the &agr;1C subunit protein in SHR compared with WKY mesenteric arteries, and immunocytochemistry confirmed this abnormality at the single-cell level. Finally, isolated mesenteric arteries from SHR were highly reactive to Bay K8644 and developed anomalous Ca2+-dependent tone, suggesting a functional role for &agr;1C subunit upregulation in vascular hyperreactivity. To determine if these Ca2+ channel abnormalities extended to the SHR skeletal muscle bed, we repeated a similar series of studies in WKY and SHR hind limb arteries. Skeletal muscle arteries from SHR also expressed higher levels of &agr;1C subunit mRNA and protein than WKY arteries and developed anomalous Ca2+-dependent tone attributed to L-type Ca2+ channels. Our data provide the first evidence that the &agr;1C subunit mRNA and protein are upregulated in SHR arteries and that the increased numbers of L-type Ca2+ channel pores are associated with the generation of abnormal vascular tone.

Enzymatic Isolation and Characterization of Single Vascular Smooth Muscle Cells from Cremasteric Arterioles

Objective: The goal of the present study was to develop a method to isolate viable arteriolar muscle cells from single cremasteric arterioles, which retain the contractile and electrophysiological phenotype of the donor microvessels.

Calcium currents are altered in the vascular muscle cell membrane of spontaneously hypertensive rats.

Calcium currents were recorded during whole-cell voltage damp in cultured azygos venous muscle cells from 1-3-day-old normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Different holding potentials were used to separate total cell current into its transient (T) and sustained or long-lasting (L) components. In recordings from 30 WKY and 30 SHR vascular cells, total cell calcium current was the same between cells from normotensive (167±20 pA) and hypertensive (139±15 pA) rats. However, the relative proportion of T and L calcium currents was different between WKY and SHR cells. In WKY cells, the peak amplitude of the L current was less than that of the T current (42±30% of total current), whereas in SHR cells, the L current was greater (62±3% of total current). Calcium currents in vascular muscle cells from SHR were activated and inactivated at more positive potentials than in cells from WKY. This study directly compares transmembrane calcium current in isolated cells from WKY and SHR blood vessels and shows that the proportions of T and L calcium channels activated by depolarization are altered in this genetic model of hypertension.

New expression profiles of voltage-gated ion channels in arteries exposed to high blood pressure.

The diameters of small arteries and arterioles are tightly regulated by the dynamic interaction between Ca2+ and K+ channels in the vascular smooth muscle cells. Calcium influx through voltage‐gated Ca2+ channels induces vasoconstriction, whereas the opening of K+ channels mediates hyperpolarization, inactivation of voltage‐gated Ca2+ channels, and vasodilation. Three types of voltage‐sensitive ion channels have been highly implicated in the regulation of resting vascular tone. These include the L‐type Ca2+ (CaL) channels, voltage‐gated K+ (KV) channels, and high‐conductance voltage‐ and Ca2+‐sensitive K+ (BKCa) channels. Recently, abnormal expression profiles of these ion channels have been identified as part of the pathogenesis of arterial hypertension and other vasospastic diseases. An increasing number of studies suggest that high blood pressure may trigger cellular signaling cascades that dynamically alter the expression profile of arterial ion channels to further modify vascular tone. This article will briefly review the properties of CaL, KV, and BKCa channels, present evidence that their expression profile is altered during systemic hypertension, and suggest potential mechanisms by which the signal of elevated blood pressure may result in altered ion channel expression. A final section will discuss emerging concepts and opportunities for the development of new vasoactive drugs, which may rely on targeting disease‐specific changes in ion channel expression as a mechanism to lower vascular tone during hypertensive diseases.

HYPOXIA INCREASES THE ACTIVITY OF CA2+-SENSITIVE K+ CHANNELS IN CAT CEREBRAL ARTERIAL MUSCLE CELL MEMBRANES

The cellular mechanisms mediating hypoxia-induced dilation of cerebral arteries have remained unknown, but may involve modulation of membrane ionic channels. The present study was designed to determine the effect of reduced partial pressure of O2, PO2, on the predominant K+ channel type recorded in cat cerebral arterial muscle cells, and on the diameter of pressurized cat cerebral arteries. A K+-selective single-channel current with a unitary slope conductance of 215 pS was recorded from excised inside-out patches of cat cerebral arterial muscle cells using symmetrical KCl (145 mM) solution. The open state probability (NPo) of this channel displayed a strong voltage dependence, was not affected by varying intracellular ATP concentration [(ATP]i) between 0 and 100 μM, but was significantly increased upon elevation of intracellular free Ca2+ concentration ([Ca2+]i). Low concentrations of external tetraethylammonium (0.1–3 mM) produced a concentration-dependent reduction of the unitary current amplitude of this channel. In cell-attached patches, where the resting membrane potential was set to zero with a high KCl solution, reduction of O2 from 21% to < 2% reversibly increased the NPo, mean open time, and event frequency of the Ca2+-sensitive, high-conductance single-channel K+ current recorded at a patch potential of + 20 mV. A similar reduction in PO2 also produced a transient increase in the activity of the 215-pS K+ channel measured in excised inside-out patches bathed in symmetrical 145 mM KCl, an effect which was diminished, or not seen, during a second application of hypoxic superfusion. Hypoxia had no effect on [Ca2+]i or intracellular pH (pHi) of cat cerebral arterial muscle cells, as measured using Ca2+- or pH-sensitive fluorescent probes. Reduced PO2 caused a significant dilation of pressurized cerebral arterial segments, which was attenuated by pre-treatment with 1 mM tetraethylammonium. These results suggest that reduced PO2 increases the activity of a high-conductance, Ca2+-sensitive K+ channel in cat cerebral arterial muscle cells, and that these effects are mediated by cytosolic events independent of changes in [Ca2+]i and pHi.

A Ca(2+)-dependent K+ current is enhanced in arterial membranes of hypertensive rats.

This study was designed to investigate the role and regulation of arterial membrane K+ channels in hypertension. Aortic segments from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were suspended for isometric tension recording. In other experiments, proximal aortic segments (PS) (exposed to high pressure) and distal aortic segments (DS) (exposed to lower pressure) were removed from surgically coarcted Sprague-Dawley rats and similarly prepared. Aortas from SHR and PS dose-dependently contracted to the K+ channel blocker tetraethylammonium (TEA) (0.1-10 mM), and this contraction was abolished by preincubation with 0.1 microM nifedipine. In contrast, the same concentrations of TEA did not contract either WKY or DS aortas. Since block of K+ channels by TEA had a different effect on aortic segments exposed to high versus low blood pressure, we compared whole-cell K+ currents in isolated vascular cells from the same aortas. The reversal potentials of depolarization-induced outward currents in WKY, SHR, DS, and PS aortic cells showed a Nernst relation to external K+ concentration indicative of selective K+ permeability. TEA (1 and 10 mM) was equipotent in blocking these K+ currents in patch-clamped cells from all aortic preparations, suggesting that the lack of TEA-induced contractions in WKY and DS aortas was not due to an absence of TEA-sensitive K+ channels in these arterial membranes. However, when the Ca2+ ionophore A23187 (10 microM) was used to increase the level of cytosolic Ca2+ in patch-clamped cells, the K+ current density in SHR and PS aortic cells was twofold or more higher than in WKY and DS cells.(ABSTRACT TRUNCATED AT 250 WORDS)