Myocardial infarction in rat eliminates regional heterogeneity of AP profiles,I(to) K(+) currents,and [Ca(2+)](i) transients

Transient outward K(+) current density (I(to)) has been shown to vary between different regions of the normal myocardium and to be reduced in heart disease. In this study, we measured regional changes in action potential duration (APD), I(to), and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients of ventricular myocytes derived from the right ventricular free wall (RVW) and interventricular septum (SEP) 8 wk after myocardial infarction (MI). At +40 mV, I(to) density in sham-operated hearts was significantly higher (P < 0.01) in the RVW (15.0 +/- 0.8 pA/pF, n = 47) compared with the SEP (7.0 +/- 1.1 pA/pF, n = 18). After MI, I(to) density was not reduced in SEP myocytes but was reduced (P < 0.01) in RVW myocytes (8.7 +/- 1.0 pA/pF, n = 26) to levels indistinguishable from post-MI SEP myocytes. These changes in I(to) density correlated with Kv4.2 (but not Kv4.3) protein expression. By contrast, Kv1.4 expression was significantly higher in the RVW compared with the SEP and increased significantly after MI in RVW. APD measured at 50% or 90% repolarization was prolonged, whereas peak [Ca(2+)](i) transients amplitude was higher in the SEP compared with the RVW in sham myocytes. These regional differences in APD and [Ca(2+)](i) transients were eliminated by MI. Our results demonstrate that the significant regional differences in I(to) density, APD, and [Ca(2+)](i) between RVW and SEP are linked to a variation in Kv4.2 expression, which largely disappears after MI.     

Deranged Kv channel regulation in fibroblasts from mice lacking the serum and glucocorticoid inducible kinase SGK1

Coexpression of the serum and glucocorticoid inducible kinase 1 (SGK1) up-regulates Kv channel activity in Xenopus oocytes and human embryonic kidney cells. To investigate the physiological impact of SGK1 dependent Kv channel regulation, we recorded whole-cell currents in lung fibroblasts from SGK1 knockout mice (sgk1-/-) and wild-type littermates (sgk1+/+). Serum-grown mouse lung fibroblasts (MLF) from both genotypes exhibited voltage-gated outwardly rectifying K(+)-currents with time-dependent activation (tau(act) approximately 3 msec), slow inactivation (tau(inact) approximately 700 msec), use-dependent inactivation, and (partial) inhibition by K(+) channel blockers TEA, 4-AP, and margatoxin. In serum grown MLF peak Kv current density at +100 mV was significantly lower in sgk1-/- (14 +/- 2 pA/pF, n = 13) than in sgk1+/+ (31 +/- 4 pA/pF, n = 16). PCR amplification of different Kv1 and Kv3 subunits from mouse fibroblasts demonstrated the expression of Kv1.1-1.7, Kv3.1, and Kv3.3 mRNA in both sgk1+/+ and sgk1-/- cells. Upon serum deprivation Kv currents almost disappeared in sgk1+/+ (4 +/- 1 pA/pF, n = 11) but not in sgk1-/- (10 +/- 1 pA/pF, n = 6) MLF. Accordingly, following serum deprivation Kv current density was significantly lower in sgk1+/+ than in sgk1-/-. Stimulation of serum-depleted cells with dexamethasone (dex) (1 microM, 1 day), IGF-1 (6.7 microM, 4-6 h) or both, significantly activated Kv currents in sgk1+/+ but not in sgk1-/- MLF. In the presence of both, dex and IGF-1, the Kv current density was significantly larger in sgk1+/+ (27 +/- 3 pA/pF, n = 12) than in sgk1-/- (13 +/- 3 pA/pF, n = 10) cells. Similar to MLF, Kv currents were significantly higher in sgk1+/+ mouse tail fibroblasts (MTF). In sgk1+/+ but not sgk1-/- MTF the Kv currents were inhibited upon serum deprivation and reincreased after stimulation of serum deprived MTF with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h). According to Fura-2-fluorescence capacitative Ca(2+) entry was lower in sgk1-/- MTF compared to sgk1+/+ MTF. Upon serum deprivation capacitative Ca(2+) entry decreased significantly in sgk1+/+ but not in sgk1-/- MTF. Stimulation of depleted cells with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h) reincreased capacitative Ca(2+) entry in sgk1+/+ MTF, whereas in sgk1-/- cells it remained unchanged. In conclusion, lack of SGK1 does not abrogate Kv channel activity but abolishes regulation of those channels by serum, glucocorticoids and IGF-1, an effect influencing capacitative Ca(2+) entry.     

Shared requirement for dynein function and intact microtubule cytoskeleton for normal surface expression of cardiac potassium channels

Potassium channels at the cardiomyocyte surface must eventually be internalized and degraded, and changes in cardiac potassium channel expression are known to occur during myocardial disease. It is not known which trafficking pathways are involved in the control of cardiac potassium channel surface expression, and it is not clear whether all cardiac potassium channels follow a common pathway or many pathways. In the present study we have surveyed the role of retrograde microtubule-dependent transport in modulating the surface expression of several cardiac potassium channels in ventricular myocytes and heterologous cells. The disruption of microtubule transport in rat ventricular myocytes with nocodazole resulted in significant changes in potassium currents. A-type currents were enhanced 1.6-fold at +90 mV, rising from control densities of 20.9 +/- 2.8 to 34.0 +/- 5.4 pA/pF in the nocodazole-treated cells, whereas inward rectifier currents were reduced by one-third, perhaps due to a higher nocodazole sensitivity of Kir channel forward trafficking. These changes in potassium currents were associated with a significant decrease in action potential duration. When expressed in heterologous human embryonic kidney (HEK-293) cells, surface expression of Kv4.2, known to substantially underlie A-type currents in rat myocytes, was increased by nocodazole, by the dynein inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, and by p50 overexpression, which specifically interferes with dynein motor function. Peak current density was 360 +/- 61.0 pA/pF in control cells and 658 +/- 94.5 pA/pF in cells overexpressing p50. The expression levels of Kv2.1, Kv3.1, human ether-a-go-go-related gene, and Kir2.1 were similarly increased by p50 overexpression in this system. Thus the regulation of potassium channel expression involves a common dynein-dependent process operating similarly on the various channels.     

Effects of carvedilol on transient outward and ultra-rapid delayed rectifier potassium currents in human atrial myocytes

Carvedilol is a beta- and alpha(1)-adrenoceptor antagonist. It is widely used in the treatment of cardiovascular diseases including atrial arrhythmias. However, it is unclear whether carvedilol may affect the repolarization currents, transient outward K(+) current (I(to)) and ultra-rapid delayed rectifier K(+) current (I(Kur)) in the human atrium. The present study evaluated effects of carvedilol on I(to) and I(Kur) in isolated human atrial myocytes by whole-cell patch-clamp recording technique. We found that carvedilol reversibly inhibited I(to) and I(Kur) in a concentration-dependent manner. Carvedilol (0.3 microM) suppressed I(to) from 9.2+/-0.5 pA/pF to 4.8+/-0.5 pA/pF (P<0.01) and I(Kur) from 3.6+/-0.5 pA/pF to 1.9+/-0.3 pA/pF (P<0.01) at +50 mV. I(to) was inhibited in a voltage-dependent manner, being significantly attenuated at test potentials from +10 to +50 mV, whereas the inhibition of I(Kur) was independent. The concentration giving a 50% inhibition was 0.50 microM for I(to) and 0.39 microM for I(Kur). Voltage-dependence of activation, inactivation and time-dependent recovery from inactivation of I(to) were not altered by carvedilol. However, time to peak and time-dependent inactivation of I(to) were significantly accelerated, indicating an open channel blocking action. The findings indicate that carvedilol significantly inhibits the major repolarization K(+) currents I(to) and I(Kur) in human atrial myocytes.     

Sex-based transmural differences in cardiac repolarization and ionic-current properties in canine left ventricles

The female sex is associated with longer electrocardiographic QT intervals and increased proarrhythmic risks of QT-prolonging drugs. This study examined the hypothesis that sex differences in repolarization may be associated with differential transmural ion-current distribution. Whole cell patch-clamp and current-clamp were used to study ionic currents and action potentials (APs) in isolated canine left ventricular cells from epicardium, midmyocardium, and endocardium. No sex differences in AP duration (APD) were found in cells from epicardium versus endocardium. In midmyocardium, APD was significantly longer in female dogs (e.g., at 1 Hz, female vs. male: 288 +/- 21 vs. 237 +/- 8 ms; P < 0.05), resulting in greater transmural APD heterogeneity in females. No sex differences in inward rectifier K+ current (I(K1)) were observed. Transient outward K+ current (I(to)) densities in epicardium and midmyocardium also showed no sex differences. In endocardium, female dogs had significantly smaller I(to) (e.g., at +30 mV, female vs. male: 2.5 +/- 0.2 vs. 3.5 +/- 0.3 pA/pF; P < 0.05). Rapid delayed-rectifier K+ current (I(Kr)) density and activation voltage-dependence showed no sex differences. Female dogs had significantly larger slow delayed-rectifier K+ current (I(Ks)) in epicardium and endocardium (e.g., at +40 mV; tail densities, female vs. male; epicardium: 1.3 +/- 0.1 vs. 0.8 +/- 0.1 pA/pF; P < 0.001; endocardium: 1.2 +/- 0.1 vs. 0.7 +/- 0.1 pA/pF; P < 0.05), but there were no sex differences in midmyocardial I(Ks). Female dogs had larger L-type Ca2+ current (I(Ca,L)) densities in all layers than male dogs (e.g., at -20 mV, female vs. male, epicardium: -4.2 +/- 0.4 vs. -3.2 +/- 0.2 pA/pF; midmyocardium: -4.5 +/- 0.5 vs. -3.3 +/- 0.3 pA/pF; endocarium: -4.5 +/- 0.4 vs. -3.2 +/- 0.3 pA/pF; P < 0.05 for each). We conclude that there are sex-based transmural differences in ionic currents that may underlie sex differences in transmural cardiac repolarization.     

Increased focal Kv4.2 channel expression at the plasma membrane is the result of actin depolymerization

Voltage-dependent potassium channel trafficking and localization are regulated by proteins of the cytoskeleton, but the mechanisms by which these occur are still unclear. Using human embryonic kidney (HEK) cells as a heterologous expression system, we tested the role of the actin cytoskeleton in modulating the function of Kv4.2 channels. Pretreatment (>or=1 h) of HEK cells with 5 microM cytochalasin D to disrupt the actin microfilaments greatly augmented whole cell Kv4.2 currents at potentials positive to -20 mV. However, no changes in the voltage dependence of activation and inactivation of macroscopic currents were observed to account for this increase. Similarly, single channel recordings failed to reveal any significant changes in the single channel conductance, open probability, and kinetics. However, the mean patch current was increased from 0.9 +/- 0.2 pA in control to 6.7 +/- 3.0 pA in the presence of cytochalasin D. Imaging experiments revealed a clear increase in the surface expression of the channels and the appearance of "bright spot" features, suggesting that large numbers of channels were being grouped at specific sites. Our data provide clear evidence that increased numbers and altered distribution of Kv4.2 channels at the cell surface are primarily the result of reorganization of the actin cytoskeleton.     

Functional alterations in cerebrovascular K(+) and Ca(2+) channels are comparable between simulated microgravity rat and SHR

Exposure to microgravity leads to a sustained elevation in transmural pressure across the cerebral vasculature due to removal of hydrostatic pressure gradients. We hypothesized that ion channel remodeling in cerebral vascular smooth muscle cells (VSMCs) similar to that associated with hypertension may occur and play a role in upward autoregulation of cerebral vessels during microgravity. Sprague-Dawley rats were subjected to 4-wk tail suspension (Sus) to simulate the cardiovascular effect of microgravity. Large-conductance Ca(2+)-activated K(+) (BK(Ca)), voltage-gated K(+) (K(V)), and L-type voltage-dependent Ca(2+) (Ca(L)) currents of Sus and control (Con) rat cerebral VSMCs were investigated with a whole cell voltage-clamp technique. Under the same experimental conditions, K(V), BK(Ca), and Ca(L) currents of cerebral VSMCs from adult spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were also investigated. K(V) current density decreased in Sus rats vs. Con rats [1.07 +/- 0.14 (n = 22) vs. 1.31 +/- 0.28 (n = 16) pA/pF at +20 mV (P < 0.05)] and BK(Ca) and Ca(L) current densities increased [BK(Ca): 1.70 +/- 0.37 (n = 23) vs. 0.88 +/- 0.22 (n = 19) pA/pF at +20 mV (P < 0.05); Ca(L): -2.17 +/- 0.21 (n = 35) vs. -1.31 +/- 0.10 (n = 26) pA/pF at +10 mV (P < 0.05)]. Similar changes were also observed in SHR vs. WKY cerebral VSMCs: K(V) current density decreased [1.03 +/- 0.33 (n = 9) vs. 1.62 +/- 0.64 (n = 9) pA/pF at +20 mV (P < 0.05)] and BK(Ca) and Ca(L) current densities increased [BK(Ca): 2.54 +/- 0.47 (n = 11) vs. 1.12 +/- 0.33 (n = 12) pA/pF at +20 mV (P < 0.05); Ca(L): -3.99 +/- 0.53 (n = 12) vs. -2.28 +/- 0.20 (n = 10) pA/pF at +20 mV (P < 0.05)]. These findings support our hypothesis, and their impact on space cardiovascular research is discussed.     

Characterization of slowly inactivating KV{alpha} current in rabbit pulmonary neuroepithelial bodies: effects of hypoxia and nicotine

Pulmonary neuroepithelial bodies (NEB) form innervated cell clusters that express voltage-activated currents and function as airway O(2) sensors. We investigated A-type K(+) currents in NEB cells using neonatal rabbit lung slice preparation. The whole cell K(+) current was slowly inactivating with activation threshold of approximately -30 mV. This current was blocked approximately 27% by blood-depressing substance I (BDS-I; 3 microM), a selective blocker of Kv3.4 subunit, and reduced approximately 20% by tetraethylammonium (TEA; 100 microM). The BDS-I-sensitive component had an average peak value of 189 +/- 14 pA and showed fast inactivation kinetics that could be fitted by one-component exponential function with a time constant of (tau1) 77 +/- 10 ms. This Kv slowly inactivating current was also blocked by heteropodatoxin-2 (HpTx-2; 0.2 microM), a blocker of Kv4 subunit. The HpTx-2-sensitive current had an average peak value of 234 +/- 23 pA with a time constant (tau) 82 +/- 11 ms. Hypoxia (Po(2) = 15-20 mmHg) inhibited the slowly inactivating K(+) current by approximately 47%, during voltage steps from -30 to +30 mV, and no further inhibition occurred when TEA was combined with hypoxia. Nicotine at concentrations of 50 and 100 microM suppressed the slowly inactivating K(+) current by approximately 24 and approximately 40%, respectively. This suppression was not reversed by mecamylamine suggesting a direct effect of nicotine on these K(+) channels. In situ hybridization experiments detected expression of mRNAs for Kv3.4 and Kv4.3 subunits, while double-label immunofluorescence confirmed membrane localization of respective channel proteins in NEB cells. These studies suggest that the hypoxia-sensitive current in NEB cells is carried by slowly inactivating A-type K(+) channels, which underlie their oxygen-sensitive potassium currents, and that exposure to nicotine may directly affect their function, contributing to smoking-related lung disease.     

Quantitative analysis of voltage-gated potassium currents from primary equine (Equus caballus) and elephant (Loxodonta africana) articular chondrocytes

In this comparative study, we have established in vitro models of equine and elephant articular chondrocytes, examined their basic morphology, and characterized the biophysical properties of their primary voltage-gated potassium channel (Kv) currents. Using whole cell patch-clamp electrophysiological recording from first-expansion and first-passage cells, we measured a maximum Kv conductance of 0.15 +/- 0.04 pS/pF (n = 10) in equine chondrocytes, whereas that in elephant chondrocytes was significantly larger (0.8 +/- 0.4 pS/pF, n = 4, P 相似文献   

A comparison of transient outward currents in canine cardiac Purkinje cells and ventricular myocytes

Although abnormalities in Purkinje cell (PC) repolarization are important causes of cardiac arrhythmias, the detailed properties of repolarizing currents in PCs are incompletely understood. We compared transient outward K(+) current (I(to)) in single PCs from canine false tendons with midmyocardial ventricular myocytes (VMs). I(to) reactivation was biexponential, with a similar rapid-phase time constant (30 +/- 5 and 35 +/- 4 ms for VM and PC, respectively) but a large, slow component in PCs with a much greater time constant than VM (1,427 +/- 70 vs. 181 +/- 24 ms, P < 0.001). Tetraethylammonium had no effect on VM I(to) but reversibly inhibited PC I(to) (IC(50) = 2.4 +/- 0.4 mM). PC I(to) was also more sensitive to 4-aminopyridine (IC(50) = 50 +/- 7 vs. 526 +/- 49 microM in VM, P < 0.0001). H(2)O(2) slowed I(to) inactivation in PCs but did not affect VM I(to). We conclude that PC I(to) shows significant differences from VM I(to), with some features, such as tetraethylammonium sensitivity, that have been reported in neither cardiac I(to) of atrial or ventricular myocytes nor cloned K(+) channel subunits (Kv1.4, Kv4.2, or Kv4.3) known to participate in cardiac I(to).     

Kir2.3 knock-down decreases IK1 current in neonatal rat cardiomyocytes

Inward rectifier potassium Kir2.x channels mediate cardiac inward rectifier potassium currents (I(K1)). As a subunit of Kir2.x, the physiological role of Kir2.3 in native cardiomyocytes has not been reported. This study shows that Kir2.3 knock-down remarkably down-regulates Kir2.3 expression (Kir2.3 protein was reduced to 19.91+/-3.24% on the 2nd or 3rd day) and I(K1) current densities (at -120 mV, control vs. knock-down: -5.03+/-0.24 pA/pF, n=5 vs. -1.16+/-0.19 pA/pF, n=7, P<0.001) in neonatal rat cardiomyocytes. The data suggest that Kir2.3 plays a potentially important role in I(K1) currents in neonatal rat cardiomyocytes.     

L-type Ca2+ currents in ventricular myocytes from neonatal and adult rats

Postnatal changes in the slow Ca2+ current (I(Ca)(L)) were investigated in freshly isolated ventricular myocytes from neonatal (1-7 days old) and adult (2-4 months old) rats, using whole-cell voltage clamp and single-channel recordings. The membrane capacitance (mean+/-SEM) averaged 23.2+/-0.5 pF in neonates (n = 163) and 140+/-4.1 pF in adults (n = 143). I(Ca)(L) was measured as the peak inward current at a test potential of +10 mV (or +20 mV) by applying a 300-ms pulse from a holding potential of -40 mV; 1.8 mM Ca2+ was used as charge carrier. The basal ICa(L) density was 6.7+/-0.2 pA/pF in neonatal and 7.8+/-0.2 pA/pF in adult cells (p < 0.05). The time course of inactivation of the fast component (at +10 ms) was significantly longer in the neonatal (10.7+/-1.4 ms) than in the adult (6.6+/-0.4 ms) cells (p < 0.05). Ryanodine (10+/-M) significantly increased this value to 18.0+/-1.9 in neonate (n = 8) and to 17.7+/-2.0 in adult (n = 9). For steady-state inactivation, the half-inactivation potential (Vh) was not changed in either group. For steady-state activation, Vh was 5.1 mV in the neonatal (n = 6) and -7.9 mV in the adult cells (n = 7). Single-channel recordings revealed that long openings (mode-2 behavior) were occasionally observed in the neonatal cells (11 events from 1080 traces/11 cells), but not in the adult cells (400 traces/4 cells). Slope conductance was 24 pS in both the neonatal and adult cells. Results in rat ventricular myocytes suggest the following: (i) the peak Ca2+ current density is already well developed in the neonatal period (being about 85% of the adult value); (ii) the fast component of inactivation is slower in neonates than in adults; and (iii) naturally occurring long openings are occasionally observed in the neonatal stage but not in the adult. Thus, the L-type Ca2+ channels of the neonate were slightly lower in density, were inactivated more slowly, and occasionally exhibited mode-2 behavior as compared with those of the adult.     

Molecular correlates of altered expression of potassium currents in failing rabbit myocardium

Action potential (AP) prolongation is a hallmark of failing myocardium. Functional downregulation of K currents is a prominent feature of cells isolated from failing ventricles. The detailed changes in K current expression differ depending on the species, the region of the heart, and the mechanism of induction of heart failure. We used complementary approaches to study K current downregulation in pacing tachycardia-induced heart failure in the rabbit. The AP duration (APD) at 90% repolarization was significantly longer in cells isolated from failing hearts compared with controls (539 +/- 162 failing vs. 394 +/- 114 control, P < 0.05). The major K currents in the rabbit heart, inward rectifier potassium current (I(K1)), transient outward (I(to)), and delayed rectifier current (I(K)) were functionally downregulated in cells isolated from failing ventricles. The mRNA levels of Kv4.2, Kv1.4, KChIP2, and Kir2.1 were significantly downregulated, whereas the Kv4.3, Erg, KvLQT1, and minK were unaltered in the failing ventricles compared with the control left ventricles. Significant downregulation in the long splice variant of Kv4.3, but not in the total Kv4.3, Kv4.2, and KChIP2 immunoreactive protein, was observed in cells isolated from the failing ventricle with no change in Kv1.4, KvLQT1, and in Kir2.1 immunoreactive protein levels. Multiple cellular and molecular mechanisms underlie the downregulation of K currents in the failing rabbit ventricle.     

Gastric ulcers reduce A-type potassium currents in rat gastric sensory ganglion neurons

Voltage-dependent potassium currents are important contributors to neuron excitability and thus also to hypersensitivity after tissue insult. We hypothesized that gastric ulcers would alter K(+) current properties in primary sensory neurons. The rat stomach was surgically exposed, and a retrograde tracer (1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine methanesulfonate) was injected into multiple sites in the stomach wall. Inflammation and ulcers were produced by 10 injections of 20% acetic acid (HAc) in the gastric wall. Saline (Sal) injections served as control. Nodose or T9-10 dorsal root ganglia (DRG) cells were harvested and cultured 7 days later to record whole cell K(+) currents. Gastric sensory neurons expressed transient and sustained outward currents. Gastric inflammation significantly decreased the A-type K(+) current density in DRG and nodose neurons (Sal vs. HAc-DRG: 82.9 +/- 7.9 vs. 46.5 +/- 6.1 pA/pF; nodose: 149.2 +/- 10.9 vs. 71.4 +/- 11.8 pA/pF), whereas the sustained current was not altered. In addition, there was a significant shift in the steady-state inactivation to more hyperpolarized potentials in nodose neurons (Sal vs. HAc: -76.3 +/- 1.0 vs. -83.6 +/- 2.2 mV) associated with an acceleration of inactivation kinetics. These data suggest that a reduction in K(+) currents contributes, in part, to increased neuron excitability that may lead to development of dyspeptic symptoms.     

DITPA restores the repolarizing potassium currents Itof and Iss in cardiac ventricular myocytes of diabetic rats

Diabetes Mellitus (DM) can produce an increase in the cardiac action potential duration and QT interval that can be associated with sudden death. These cardiac effects are due to a region-specific decrease in repolarizing outward K(+) currents. Some authors have suggested that the proarrhythmic effects of diabetes can be due to diabetes-induced hypothyroidism. Thus, we have examined the effect of the thyroid hormone analog diiodothyropropionic acid (DITPA) on calcium-independent outward potassium currents in ventricular myocytes from diabetic rats. Sustained (I(ss)) and fast transient outward (I(tof)) K(+) currents were recorded using the whole-cell configuration of the patch-clamp technique. Myocytes were enzymatically isolated from the free wall of the right ventricle, and the epicardial and endocardial layers of the left ventricle of healthy, diabetic and DITPA-treated diabetic rats. Circulating thyroid hormones were measured by electrochemiluminescence. DITPA-treatment of diabetic rats restored I(tof) and I(ss) current densities in cardiac myocytes from the three regions studied, but did not alter current densities in myocytes of control rats. T(3) and T(4) levels were reduced by diabetes, and DITPA-treatment increased circulating T(3) levels. T(3)-treatment of diabetic rats also restored current densities to control values. However, direct incubation of diabetic myocytes with DITPA did not restore current densities. In summary, DITPA-treatment of diabetic rats restored the potassium current (I(tof) and I(ss)) densities in myocytes from all ventricular regions.     

Reduced functional expression of K(+) channels in vascular smooth muscle cells from rats made hypertensive with N{omega}-nitro-L-arginine

Smooth muscle membrane potential is determined, in part, by K(+) channels. In the companion paper to this article, we demonstrated that superior mesenteric arteries from rats made hypertensive with N(omega)-nitro-l-arginine (l-NNA) are depolarized and express less K(+) channel protein compared with those from normotensive rats. In the present study, we used patch-clamp techniques to test the hypothesis that l-NNA-induced hypertension reduces the functional expression of K(+) channels in smooth muscle. In whole cell experiments using a Ca(2+)-free pipette solution, current at 0 mV, largely due to voltage-dependent K(+) (K(V)) channels, was reduced approximately 60% by hypertension (2.7 +/- 0.4 vs. 1.1 +/- 0.2 pA/pF). Current at +100 mV with 300 nM free Ca(2+), largely due to large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels, was reduced approximately 40% by hypertension (181 +/- 24 vs. 101 +/- 28 pA/pF). Current blocked by 3 mM 4-aminopyridine, an inhibitor of many K(V) channel types, was reduced approximately 50% by hypertension (1.0 +/- 0.4 vs. 0.5 +/- 0.2 pA/pF). Current blocked by 1 mM tetraethylammonium, an inhibitor of BK(Ca) channels, was reduced approximately 40% by hypertension (86 +/- 14 vs. 53 +/- 19 pA/pF). Differences in BK(Ca) current magnitude are not attributable to changes in single-channel conductance or Ca(2+)/voltage sensitivity. The data support the hypothesis that l-NNA-induced hypertension reduces K(+) current in vascular smooth muscle. Reduced molecular and functional expression of K(+) channels may partly explain the depolarization and augmented contractile sensitivity of smooth muscle from l-NNA-treated rats.     

神经肽Y对心室肌细胞离子通道的影响

采用全细胞膜片钳技术观察神经肽Y(neuropeptide Y,NPY)对心室肌细胞离子通道的影响。结果如下：(1)NPY浓度在1．0～100nmol/L范围内剂量依赖性抑制大鼠心室肌细胞I_(Ca-L),IC_(50)值为1．86nmol/L。NPY对I_(Ca-L)的I-V曲线的最大峰值电位、激活和失活电位均无显著影响。NPY对去甲肾上腺素(norepinephrine,NE)增加的I_(Ca-L)有显著抑制作用。(2)NPY对人鼠心室肌细胞I_(Na/Ca)有显著抑制作用。10nmol/L NPY使前向I__(Na/Ca)由(0．27±0．11)pA/pF减小为(0．06±0．01)pA/pF;反向I__(Na/Ca)由(0．45±0．12)pA/pF降为(0．27±0．09)pA/pF(P＜0．05,n=4)。(3)NPY对大鼠心室肌细胞I_(to)有显著增强作用。10 nmol/L NPY使I_(to)由(12．5±0．70)pA/pF增加至(14．7±0．59)pA/pF(P＜0．05,n=4)。(4)10nmol/L NPY对大鼠心室肌细胞I_(Na)没有显著影响。(5)10nmol/L NPY对豚鼠心室肌细胞I_K无明显影响。研究结果证实,NPY抑制大鼠心室肌细胞I_(Ca-L)和I_(Na/Ca),增强I_(to)对I_Na和豚鼠心审肌细胞I_K没有显著作用,表明NPY对上述主要离子通道的效应与NE的效应相拮抗。     

Effects of endothelin-1 on K(+) currents from rat ventricular myocytes.

It has been suggested that the positive inotropic effect of the vasoactive peptide hormone, endothelin-1 (ET-1), involves inhibition of cardiac K(+) currents. In order to identify the K(+) currents modulated by ET-1, the outward K(+) currents of isolated rat ventricular myocytes were investigated using whole-cell patch-clamp recording techniques. Outward currents were elicited by depolarisation to +40 mV for 200 ms from the holding potential of -60 mV. Currents activated rapidly, reaching a peak (I(pk)) of 1310 +/- 115 pA and subsequently inactivating to an outward current level of 1063 +/- 122 pA at the end of the voltage-pulse (I(late)) (n = 11). ET-1 (20 nM) reduced I(pk) by 247.6 +/- 60.7 pA (n = 11, P < 0.01) and reduced I(late) by 323.2 +/- 43.9 pA (P < 0.001). The effects of ET-1 were abolished in the presence of the nonselective ET receptor antagonist, PD 142893 (10 microM, n = 5). Outward currents were considerably reduced and the effects of ET-1 were not observed when K(+) was replaced with Cs(+) in the experimental solutions; this indicates that ET-1 modulated K(+)-selective currents. A double-pulse protocol was used to investigate the inactivation of the currents. The voltage-dependent inactivation of the currents from potentials positive to -80 mV was fitted by a Boltzmann equation revealing the existence of an inactivating transient outward component (I(to)) and a noninactivating steady-state component (I(ss)). ET-1 markedly inhibited I(ss) by 43.0 +/- 3.8% (P < 0.001, n = 7) and shifted the voltage-dependent inactivation of I(to) by +3.3 +/- 1.2 mV (P < 0.05). Although ET-1 had little effect on the onset of inactivation of the currents elicited from a conditioning potential of -70 mV, the time-independent noninactivating component of the currents was markedly inhibited. In conclusion, the predominant effect of ET-1 was to inhibit a noninactivating steady-state background K(+) current (I(ss)). These results are consistent with the hypothesis that I(ss) inhibition contributes to the inotropic effects of ET-1.