Differences in K+ current components in mesenteric artery myocytes from WKY and SHR

Previous studies have documented increased K⁺ permeability of arterial smooth muscle in hypertension and suggested a role in altered arterial contractile function. To characterize the mechanisms responsible for these alterations, we determined the contribution of K⁺ current (I_K) components to whole cell I_K in freshly dispersed myocytes and tetraethylammonium (TEA)-induced contractile responses in mesenteric arteries of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Tetraethylammonium produced a larger tonic contractile response in SHR with a lower threshold compared to WKY (ie, 0.1 v 1 mmol/L), which was due in part to the larger Ca²⁺ current in SHR. Whole cell I_K recorded by perforated patch methods was similar at a holding potential (HP) of −60 mV (I_K60), but were larger in SHR when recorded from a HP of −20 mV (I_K20). The selective blocker iberiotoxin (IbTX) was used to separate the contribution of voltage- (K_V) and calcium-dependent (K_Ca) components of I_K60. The I_K60 and I_K20 component inhibited by 100 nmol/L IbTX (ie, K_Ca) was larger in SHR than in WKY myocytes, whereas the IbTX-insensitive I_K60 component (ie, K_V) was larger in WKY. In the presence of IbTX, 1 and 10 mmol/L TEA inhibited a larger fraction of I_K60 in SHR myocytes compared with WKY. The activation properties of the TEA-sensitive and TEA-insensitive K_V components determined by fitting a Boltzmann activation function to the current-voltage data, exhibited both group and treatment differences in the half maximal activation voltage (V_0.5). The V_0.5 of the TEA-sensitive K_V component was more positive than that of the TEA-insensitive component in both groups, and values for the V_0.5 of both TEA-sensitive and TEA-insensitive components were more negative in SHR than WKY. These results show that SHR myocytes have larger K_Ca and smaller K_V current components compared with WKY. Furthermore, SHR myocytes have a larger TEA-sensitive K_V component. These differences may contribute to the differences in TEA contractions, resting membrane potential, Ca²⁺ influx, and K_Ca current reported in hypertensive arteries.     

Developmental changes in expression and biophysics of ion channels in the canine ventricle

Background: Developmental changes in the electrical characteristics of the ventricular myocardium are not well defined. This study examines the contribution of inwardly rectifying K⁺ current (I_K1), transient outward K⁺ current (I_to), delayed rectifier K⁺ currents (I_Kr and I_Ks) and sodium channel current (I_Na) to repolarization in the canine neonate myocardium. Methods: Single myocytes isolated from the left ventricle of 2–3 week old canine neonate hearts were studied using patch-clamp techniques. Results: Neonate cells were ~ 6-fold smaller than those of adults (28.8 ± 8.8 vs. 176 ± 6.7 pF). I_K1 was larger in neonate myocytes and displayed a substantial inward component and an outward component with negative slope conductance, peaking at − 60 mV (4.13 pA/pF). I_Kr tail currents (at − 40 mV), were small (< 20 pA). I_Ks could not be detected, even after exposure to isoproterenol (100 nM). I_to was also absent in the neonate, consistent with the absence of a phase 1 in the action potential. Peak I_Na, late I_Na and I_Ca were smaller in the neonate compared with adults. KCND3, KCNIP2 and KCNQ1 mRNA expression was half, while KCNH2 was equal and KCNJ2 was greater in the neonate when compared with adults. Conclusions: Two major repolarizing K⁺ currents (I_Ks and I_to) present in adult ventricular cells are absent in the 2 week old neonate. Peak and late I_Na are significantly smaller in the neonate. Our results suggest that the absence of these two currents in the neonate heart may increase the susceptibility to arrhythmias under certain long QT conditions.     

Altered Pharmacologic Responsiveness of Reduced L-Type Calcium Currents in Myocytes Surviving in the Infarcted Heart

Altered Pharmacology of I_ca,L in Myocytes From Infarcted Heart. The pharmacologic responses of macroscopic L-type calcium channel currents to the dihydropyridine agonist, Bay K 8644, and β-adrenergic receptor stimulation by isoproterenol were studied in myocytes enzymatically dissociated from the epicardial border zone of the arrhythmic 5-day infarcted canine heart (IZs). Calcium currents were recorded at 36° to 37° C using the whole cell, patch clamp method and elicited by applying step depolarizations from a holding potential of -40 mV to various test potentials for 250-msec duration at 8-second intervals. A Cs⁺ -rich and 10 mM EGTA-containing pipette solution and a Na⁺-and K⁺-free external solutions were used to isolate calcium currents from other contaminating currents. During control, peak I_ca,L, density was found to be significantly less in IZs (4.0 ± 1.1 pA/pF) than in myocytes dispersed from the epicardium of the normal noninfarcted heart (NZs; 6.5 ± 1.8 pA/pF). Bay K 8644 (I μM) significantly increased peak I_ca,L density 3.5-fold above control levels in both NZs (to 22.5 ± 6.2 pA/pF; n = 7) and IZs (to 12.8 ± 3.0 pA/pF; n = 5), yet peak I_ca,L density in the presence of drug was significantly less in IZs than NZs. The effects of Bay K 8644 on kinetics of current decay and steady-state inactivation relations of peak I_ca,L were similar in the two cell types. In contrast, the response of peak L-type current density to isoproterenol (1 μM) was significantly diminished in IZs compared to NZs regardless of whether Ba²⁺ or Ca²⁺ ions carried the current. Thus, these results indicate an altered responsiveness to β-adrenergic stimulation in cells that survive in the infarcted heart. Furthermore, application of forskolin (1 μM and 10 μM) or intracellular cAMP (200 μM), agents known to act downstream of the β-receptor, also produced a smaller increase in peak I_Ba density in IZs versus NZs, suggesting that multiple defects exist in the β-adrenergic signaling pathway of IZs. In conclusion, these studies illustrate that reduced macroscopic calcium currents of cells in the infracted heart exhibit an altered pharmacologic profile that has important implications in the development of drugs for the diseased heart.     

Regional Gradation of L-Type Calcium Currents in the Feline Heart with a Healed Myocardial Infarct

I_Ca in Healed Myocardial Infarction. Introduction: Abnormal action potentials in myocytes adjacent to > 2-month-uld feline LV myocardial infarcts (MI) may reflect alterations in Ca²⁺ currents (I_Ca). Methods and Results: We compared I_Ca, at 36°C, in subendocardial myocytes isolated from areas adjacent to MI and to I_Ca in cells from remote areas (> 4 mm away; REM) and control cells from similar regions in normal hearts. Control (CON) myocytes had membrane capacitance of 234 ± 10 pF (n = 81 cells) compared to 305 ± 14 pF in REM (71 cells; P < 0.05 from CON) and 237 ± 11 pF (n = 55 cells) in MI (not different from CON). From V_h=?40 mV, peak I_Ca elicited by test potentials (?35 to +70 mV) were significantly larger in CON (?1746 ± 123 pA) and REM (?1795 ± 142 pA) compared to Ml (?1352 ± 129 pA) (P < 0.05). Peak 1(11 density was significantly reduced in REM (?6.0 ± 0.4 pA/pF) or MI (?5.7 ± 0.4 pA/pF, P < 0.05) compared to CON (?7.5 ± 0.4 pA/pF). Double exponential I_Ca decay was similar among groups. Half-inactivation potential (V_0.5) was significantly shifted (hyperpolarizing direction) for MI (?29.1 ± 2.6 mV) and REM (?24.6 ± 1.2 mV) myocytes compared to ?20.3 ± 1.0 mV in CON. MI slope factor (k; 9.0 ± 0.5) was significantly different from CON (6.8 ± 0.3) and RKM (7.3 ± 0.4). No differences in time course of recovery from inactivation were noted. Five millimolar Ba²⁺₀ produced significant increases in I_Ca in CON and REM but an attenuated response in MI. Bay k8644 (1 μM) produced similar I_Ca increase in all groups. I_Ca increase due to isoproterenol (1 I_CaM) in MI and REM was half that in CON, but there were no differences in increased I_Ca responses among groups following phenylephrine (10 μM). Conclusion: Reduced I_Ca density in REM reflects cell hypertrophy, whereas altered I_Ca of MI may reflect altered channel structure and/or function.     

Augmented Contributions of Voltage-Gated Ca2 Channels to Contractile Responses in Spontaneously Hypertensive Rat Mesenteric Arteries

The observation that organic Ca²⁺ channel blockers are more effective in lowering blood pressure and peripheral resistance in hypertensive compared to normotensive subjects suggests that there is a greater contribution from voltage-gated Ca²⁺ channels (Ca_L) to vascular force maintenance in hypertensive arteries. This study tests this hypothesis by comparing the effects of Bay k 8644 and nisoldipine on basal force development, contractile responses to norepinephrine and serotonin, and Ca²⁺ currents (I_Ca) in mesenteric artery (MA) from Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). MA rings were used to record isometric contractions at L_max. Single cells were isolated by collagenase plus elastase for measurement of Ca_L properties by patch-clamp methods. Contractile responses to Bay k 8644 were larger and more sensitive in SHR than WKY, and were larger in endothelium-denuded compared to intact rings. In SHR, the addition of 10 nmol/L Bay k 8644 increased contractile sensitivity to norepinephrine (NE) and serotonin (5HT), and increased maximum response to 5HT. In WKY, 10 nmol/L Bay k 8644 produced a small increase in 5HT sensitivity with no effect on maximum response, and had no effect on NE responses. In the presence of 1 μmol/L nisoldipine, the maximum response and the sensitivity to both NE and 5HT were decreased in both WKY and SHR with the inhibitory effects of nisoldipine being larger in SHR than WKY. Peak I_Ca was larger in SHR, and current-voltage curves were shifted toward more negative voltages compared to WKY. Bay k 8644 increased I_Ca in both WKY and SHR myocytes with no apparent difference in the magnitude of its effect when expressed as a percent of control I_Ca. These results suggest that Ca_L contribute significantly to tonic force maintenance as well as to agonist responses in MA from both WKY and SHR, but with a much larger contribution in SHR. Differences in the sensitivity of Ca_L to Bay k 8644 were not responsible for the differences in contractile responses to this agonist.     

Electrophysiology of single cardiomyocytes isolated from rabbit pulmonary veins: implication in initiation of focal atrial fibrillation

Pulmonary veins (PVs) are important foci in initiation of paroxysmal atrial fibrillation. However, the mechanisms of the high arrhythmogenic activity of PVs are unclear. This study aimed to isolate single cardiomyocytes from PVs and evaluate their electrophysiological characteristics and arrhythmogenic potential. Cardiomyocytes of rabbit PVs were isolated by retrograde perfusion with digestive enzymes from aorta via left ventricle and left atrium. The action potentials and ionic currents were investigated in isolated single PV cardiomyocytes using the whole-cell clamp technique. Dissociation of PVs yielded single pacemaker cardiomyocytes (76 %) and non-pacemaker cardiomyocytes with a fast response action potential. Both the pacemaker and non-pacemaker cardiomyocytes had similar inward Ca²⁺ currents and transient outward K⁺ currents. However, the pacemaker cardiomyocytes had a smaller inward rectifier K⁺ current (1.50 ± 0.22 versus 4.21 ± 1.15 pA/pF, P < 0.005) and a larger delayed rectifier K⁺ current (0.60 ± 0.05 versus 0.24 ± 0.05 pA/pF, P < 0.005) than non-pacemaker cardiomyocytes. Acetylcholine induced hyperpolarization and inhibited the spontaneous action potential. Isoproterenol (10 nM) accelerated the spontaneous activity and induced early or delayed afterdepolarization, which could be suppressed by nifedipine. The PV cardiomyocytes with early afterdepolarization have a greater prolongation of action potential duration (ΔAPD, + 67 ± 17 versus −109 ± 20 ms, P < 0.0001) and a greater increase of inward Ca²⁺ current (0.90 ± 0.23 versus 0.38 ± 0.08 pA/pF, P < 0.05) after isoproterenol than those without early afterdepolarization. These findings suggest that PV cardiomyocytes have distinct action potentials and ionic current profiles, which may be responsible for the high arrhythmogenic activity of the PVs. Received: 17 April 2001, Returned for revision: 23 May 2001, Revision received: 11 July 2001, Accepted: 17 July 2001     

Reduction of Ventricular Sodium Current in a Mouse Model of HIV

Effect of HIV on Cardiac Sodium Current. Introduction: Cardiac arrhythmias have been reported in AIDS patients. Arrhythmias can arise from alterations in ventricular Na⁺ channel function. However, it is unknown whether HIV affects cardiac Na⁺ channel function. Therefore, the purpose of this study was to characterize the effect of HIV on ventricular Na⁺ current (I_Na) in a transgenic model of HIV (CD4C/HIV mice), which exhibit a severe AIDS‐like disease. Methods and Results: Patch‐clamp techniques were used to examine I_Na and action potentials (AP) in ventricular myocytes isolated from HIV and wild‐type (WT) mice. In HIV myocytes peak I_Na was reduced (at ?50 mV: HIV, ?55.3 ± 4.3 pA/pF, n = 15; WT, ?79.4 ± 5.2 pA/pF, n = 16, P < 0.05), whereas late I_Na was similar in both groups (HIV, ?4.3 ± 0.4 pA/pF; WT, ?4.4 ± 0.4 pA/pF, n = 22/group). AP amplitude (HIV 91.5 ± 4.7 mV, n = 12; WT 104.4 ± 3.1 mV, n = 15, P < 0.05) and the maximal velocity of the AP upstroke (V_max; HIV, 57.2 ± 9.3 mV/ms, n = 12; WT, 113.5 ± 8 mV/ms, n = 15, P < 0.05) were decreased in HIV myocytes. ECG recordings revealed that the QRS complex was prolonged in HIV mice (HIV, 15.7 ± 0.2 ms, n = 22; WT, 14.1 ± 0.5 ms, n = 10, P < 0.05). The serum levels of interleukin‐1β were elevated in HIV mice (HIV, 18.1 ± 3.1 pg/mL, n = 3; WT, 5.1 ± 1.1 pg/mL, n = 4, P < 0.05) in line with previous evidence that suggests that elevated levels of cytokines can affect cardiac ion currents. Conclusion: Overall, our observations suggest that elevated levels of proinflammatory cytokines in CD4C/HIV mice could alter Na⁺ channel function, thus altering cardiac depolarization and contribute to the generation of arrhythmias. (J Cardiovasc Electrophysiol, Vol. 21, pp. 916‐922, August 2010)     

Responses of Cremasteric Arterioles of Spontaneously Hypertensive Rats to Changes in Extracellular K+ Concentration

Objective : The goal of this study was to determine the effect of changes in extracellular K⁺ concentration ([K⁺]₀) on active tone in cremasteric arterioles of spontaneously hypertensive rats (SHR) and their normotensive Wistar-Kyoto (WKY) and Wistar controls. Methods : Diameters of third- and fourth-order arterioles were measured in the cremaster muscle of hypertensive and normotensive rats during abrupt changes in superfusate K⁺ concentration from 4.7 mM to 0 mM to 15 mM K⁺. Results : Arterioles constricted in response to superfusion with 0 mM K⁺ and exhibited a large, transient dilation in response to an abrupt change from 0 mM to 15 mM [K⁺]_o. Arteriolar dilation in response to 15 mM K⁺ was significantly larger in 12–15-week-old SHR than in WKY or Wistar controls. Arteriolar responses to 15 mM K⁺ were not significantly different in 4–6-week-old SHR and WKY. Dilator responses to 15 mM K⁺ were generally inhibited by 1 mM ouabain, although ouabain was less effective in inhibiting 15 mM K⁺-induced dilation in arterioles of SHR and WKY than in Wistar rats. Conclusions : Dilation of cremasteric arterioles in response to 15 mM [K⁺]₀, is mediated, at least in part, via stimulation of the electrogenic Na⁺/K⁺ pump, although Na⁺/K⁺-pump-independent components may also contribute to the response. Arterioles of SHR with established hypertension exhibit an altered response to elevated [K⁺]₀ which is not present in SHR in the early stage of hypertension.     

Cesium Abolishes the Barium-Induced Pacemaker Potential and Current in Guinea Pig Ventricular Myocytes

Cesium Abolishes Barium-Induced PM Current. Introduction: The ability of cesium to block barium-induced diastolic depolarization (“Ba-DD”) and pacemaker current was tested in isolated ventricular myocytes. Because Ba-DD is due to decreasing k conductance and there is no I_f at the resting potential, this approach permits verification of whether Cs⁺ is a specific blocker of I, or if it instead also blocks a K⁺ pacemaker current. Methods and Results: Guinea pig isolated ventricular myocytes were studied by a discontinuous, single electrode, voltage clamp method. During hyperpolarizing voltage clamp steps from -80 up to -140 mV in Tyrode's solution, the inward current increased as a function of voltage but did not change us a function of time (no I_f or K⁺ depletion). Cesium (4mM) reduced the current size during the hyperpolarizing steps hut did not induce or unmask time-dependent currents. Barium (0.05 to 0.1 mM) induced diastolic depolarization, and, in its presence, depolarizing voltage clamp steps were followed by an outward tail current that reversed at -92.0 ± 1.3 mV. Outward tail currents were larger at -50 mV than at the resting potential, and inward tail currents decayed more rapidly and to a larger extent during larger hyperpolarizing steps. In the presence of Ba²⁺, Cs⁺ (4 mM) had little effect on the steady-state current but markedly reduced or abolished undershoot, Ba-DD, and time-dependent tail currents at potentials both positive and negative to the resting potential. Cs⁺ had a smaller effect on the steady-state current-voltage (I-V) relation in the presence than in the absence of Ba²⁺, as part of the I_kl channels were already blocked by Ba²⁺ and the time-dependent changes induced by Ba²⁺ were not present. Both Ba²⁺ and Cs⁺ had little blocking effect on the steady-state current positive to the negative slope region of the I-V relation. Conclusion: In ventricular myocytes, Cs⁺ abolishes the Ba²⁺-induced pacemaker current by blocking the time-dependent change in K⁺ conductance, not by blocking I_f. Because Cs⁺ can also block a decaying K⁺ pacemaker current, the abolition of a pacemaker current by Cs⁺ in other cardiac tissues cannot be taken as unequivocal proof that the blocked current is I_f     

Altered Arachidonic Acid Metabolism Contributes to the Failure of Dopamine to Inhibit Na+, K+-ATPase in Kidney of Spontaneously Hypertensive Rats

Dopamine decreases tubular sodium reabsorption in part by inhibition of Na⁺, K⁺-ATPase activity in renal proximal tubules. The signaling mechanism involved in dopamine-mediated inhibition of Na⁺, K⁺-ATPase is known to be defective in spontaneously hypertensive animals. The present study was designed to evaluate the role of phospholipase A2 (PLA2) and its metabolic pathway in dopamine-induced inhibition of Na⁺, K⁺-ATPase in renal proximal tubules from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Renal proximal tubular suspensions were prepared and Na⁺, K⁺-ATPase activity was measured as ouabain-sensitive adenosine triphosphate hydrolysis. Dopamine inhibited Na⁺,K⁺-ATPase activity in a concentration (1 nM - 10 μM)-dependent manner in WKY rats while it failed to inhibit the enzyme activity in SHR. Dopamine (10μM)-induced inhibition of Na⁺,K⁺-ATPase activity in WKY rats was significantly blocked by mepacrine (10 μM), a PLA2 inhibitor, suggesting the involvement of PLA2 in dopamine-mediated inhibition of Na⁺,K⁺-ATPase. Arachidonic acid (a product released by PLA2 action) inhibited Na⁺,K⁺-ATPase in a concentration-dependent (1-100 μM) manner in WKY rats while the inhibition in SHR was significantly attenuated (IC₅₀: 7.5 and 80 μM in WKY rats and SHR, respectively). Furthermore, lower concentrations of arachidonic acid stimulated (30% at 1 μM) Na⁺,K⁺-ATPase activity in SHR. This suggests a defect in the metabolism of arachidonic acid in SHR. Proadifen (10 μM), an inhibitor of cytochrome P-450 monoxygenase (an arachidonic acid metabolizing enzyme) significantly blocked the inhibition produced by arachidonic acid in WKY rats and abolished the difference in arachidonic acid inhibition of Na⁺,K⁺-ATPase between WKY rats and SHR. These data suggest that PLA2 is involved in dopamine-induced inhibition of Na⁺,K⁺-ATPase and altered arachidonic acid metabolism may contribute to reduced dopaminergic inhibition of Na⁺,K⁺-ATPase activity in spontaneously hypertensive rats.     

Ventricular K currents are reduced in mice with elevated levels of serum TNFα

In the present study mice were treated with tumor necrosis factor alpha (TNFα) for 6 weeks to determine if chronic TNFα treatment could produce serum levels of TNFα similar to what has been observed in disease states (heart failure, HIV) and to determine if these levels of TNFα alter ventricular K⁺ currents. Mice chronically treated with TNFα and sham treated mice were utilized for experiments. Serum levels were measured with a Searchlight® protein array. Patch-clamp techniques, real-time PCR and Western blot analysis were used to study K⁺ current densities and K⁺ channel expression. Results showed that serum concentrations of TNFα were significantly higher in TNFα treated mice compared to controls (control: 9.5 ± 1.5 pg/ml, TNFα: 27.4 ± 5.0 pg/ml; p < 0.05) and comparable to serum TNFα levels observed in heart failure and HIV models. In ventricular myocytes from TNFα treated mice the outward K⁺ currents I_to and I_Kur were significantly reduced (at + 30 mV: I_to: control: 45.0 ± 2.9 pA/pF, TNFα: 34.5 ± 2.9 pA/pF; p < 0.05; I_Kur: control 34.1 ± 2.7 pA/pF, TNFα: 25.0 ± 2.2 pA/pF; p < 0.05). Expression studies revealed that ventricular mRNA and protein expression for the channels underlying I_to and I_Kur did not differ between the two groups. However, the recovery from inactivation for I_Kur was significantly longer in TNFα treated mice. Overall, this study shows that pathologically relevant levels of serum TNFα modulate K⁺ currents in mouse ventricle. These findings could help to explain the role of TNFα in the pathogenesis of cardiac arrhythmia.     

Altered insulin-like growth factor-1 and nitric oxide sensitivities in hypertension contribute to vascular hyperplasia

Vascular medial thickening, a hallmark of hypertension, is associated with vascular smooth muscle cell (VSMC) hypertrophy and hyperplasia. Although the precise mechanisms responsible are elusive, we have shown that strain induced regulation of autocrine insulin-like growth factor-1 (IGF-1) and nitric oxide (NO) reciprocally modulate VSMC proliferation. Therefore, we investigated potential IGF-1 and NO abnormalities in young (10-week-old) spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) and their respective VSMC ex vivo. The SHR had increased mean arterial pressure (173 ± 2 v 128 ± 3 mm Hg, n = 24, P < .05) but similar pulse pressures (31 ± 2 v 30 ± 3 mm Hg; P > .05) v WKY. The SHR exhibited increased aortic wall thickness in comparison with WKY (523 ± 16 v 355 ± 17μm; P < .05). No differences were seen in plasma combined NO₂ and NO₃ (NO_x) (0.48 ± 0.11 mmol/L for WKY v 0.58 ± 0.18 mmol/L for SHR) or plasma IGF-1 (1007 ± 28 ng/mL for WKY v 953 ± 26 ng/mL for SHR). Aortic VSMC from SHR displayed enhanced proliferation in comparison with WKY (P < .05). Underlying this enhanced proliferation was altered SHR VSMC sensitivity to the antiproliferative NO donor 2,2"[Hydroxynitrosohydrazono] bis-ethanimine (DETA-NO) (ID₅₀: 270 ± 20 mmol/L for SHR; 150 ± 11 mmol/L for WKY; P < .05). Basal cyclic guanosine monophosphate (cGMP) secretion from SHR VSMC was 65-fold greater than that seen from WKY (P < .001). In response to DETA-NO, cGMP secretion from SHR VSMC increased modestly (1.5-fold; P < .01), whereas treatment of WKY VSMC resulted in a 26-fold (P < .001) increase in cGMP. The SHR VSMC did not respond to exogenous IGF-1, whereas WKY VSMC exhibited a dose dependent increase in proliferation with IGF-1 (10⁻¹⁰ to 10⁻⁷ mol/L). These data suggest that VSMC hyperplasia in early hypertension is not reflected by imbalances in plasma IGF-1 or NO. Rather, altered SHR VSMC sensitivity to NO is likely responsible in part for the observed hyperproliferation seen in early stages of hypertension.     

Alterations in Membrane Fatty Acid Unsaturation and Chain Length in Hypertension as Observed by 1H NMR Spectroscopy

Alterations in fatty acids of membrane phospholipids in essential hypertension may account for altered membrane ion transport, elasticity, and contractility properties of hypertensive tissues. To investigate the abnormalities in membrane fatty acids in essential hypertension, the degree of fatty acid unsaturation ([–CHCH–]/[–CH₃]), the average carbon chain length, ratio of glycerol to fatty acyl chains, ratio of phosphatidylcholine to fatty acyl chains, and the ratio of free and acylated cholesterol to fatty acyl chains in fatty acid fractions of membrane phospholipids of aorta, kidney, and heart were determined in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats by ¹H nuclear magnetic resonance (NMR) spectroscopy. The degrees of fatty acid unsaturation in the aorta and the kidney membranes were significantly lower in SHR than in WKY rats (aorta, 0.53 ± 0.01 v 0.63 ± 0.01, n = 5, P = .01; kidney, 0.70 ± 0.01 v 0.84 ± 0.03, n = 10, P = .01). No significant difference could be detected in fatty acid unsaturation in heart membranes between these two strains. For aorta, kidney, and heart membranes, the average carbon chain lengths of fatty acid fractions of membrane phospholipids were significantly shorter for SHR than for WKY rats (aorta, 15.1 ± 0.2 v 18.3 ± 0.7, n = 5, P = .02; kidney, 14.5 ± 0.2 v 16.4 ± 0.4, n = 10, P = .01; heart, 17.3 ± 0.5 v 18.8 ± 0.6, n = 10, P = .05). The lower unsaturated fatty acid content in membrane phospholipids of the aorta and the kidney, with concomitant reduction in average chain length, may arise from increased oxidation of fatty acid double bonds in hypertensive tissues and may account, in part, for the increased aortic stiffness and abnormal kidney function associated with essential hypertension. Whether the lower unsaturated fatty acid content and decreased carbon chain length of phospholipid membranes in the aorta and the kidney are a cause or a consequence of the high blood pressure, however, remains unknown.     

ATP-dependent Ca2+ Transport and Na+/Ca2+ Exchange System in Renal Basolateral Plasmamembranes of Spontaneously Hypertensive Rats

A common abnormality of cellular Ca²⁺ handling in most tissues of spontaneously hypertensive rats (SHR) has been suggested. Therefore we investigated the ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange system in basolateral membrane vesicles (BLMV) of renal cortices from SHR and normotensive Wistar-Kyoto rats (WKY) at 12 and 20 weeks of age. In WKY the maximal transport rate of the ATP-dependent Ca²⁺ transport was 5.7 nmol/min/mg prot with an affinity for Ca²⁺ of 0.14 µM. These values were not significantly different in SHR at both ages studied. High concentrations of Na⁺ inhibited ATP-dependent Ca²⁺ uptake by 40% in BLMV of SHR and WKY. Low concentrations of Na⁺ stimulated ATP-dependent Ca²⁺ transport 20% in both rats. These findings suggest equal Na⁺/Ca²⁺ exchange activity in WKY and SHR. The present study failed to show a significant change in ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange activity in renal BLMV in SHR, suggesting that the Ca²⁺ homeostasis of the cortical cells is normal in SHR as far as the plasmamembrane is concerned.     

Role of Na+:Ca2+ Exchange Current in Cs+-Induced Early Afterdepolarizations in Purkinje Fibers

Na⁺:Ca²⁺ Exchanger and EADs. Introduction: The ionic mechanisms for early afterdepolarizations (EADs) have not been fully clarified. It has been suggested that L-type Ca²⁺ current (I_caL) is the primary current generating EADs that occur near the plateau level (E-EADs) of the membrane potential (Vm) when I_caL is enhanced. The purpose of these studies was to determine accurately the range of Vm at which EADs occur in Purkinje fibers with K⁺ currents blocked by Cs⁺ and to investigate the importance of Na⁺:Ca²⁺ exchange current (I_Na:ca) as opposed to l_CaL and other currents in the generation of EADs occurring later during repolarization (L-EADs). Methods and Results: Shortened Purkinje strands from dogs and guinea pigs were superfused with physiologic solution containing Cs⁺ (3.6 mM) and a low [K⁺]_o (3.0 or 2.0 mM) to induce EADs. The Vm of origin of EADs and their evolution were measured with the aid of phase plane plots of the rate of repolarization against Vm. L-EADs occurred over a wide range of Vm (?35 to ?90 mV), generally more negative in guinea pig than in dog. Elevation of [Ca²⁺]_o, from 1.8 to 3.6 mM suppressed L-EADs within a few cycles, and they returned with continued exposure. After repeated exposures to high [Ca^:2+]₀, L-EADs migrated toward less negative Vm when |Ca²⁺]₀, was reestablished to 1.8 mM in the presence of Cs⁺. Reduction of [Na⁺]₀ from 147.5 to 112.5 mM by substitution with Li⁺ or sucrose also rapidly depressed L-EADs. Conclusions: The observation of Cs⁺-induced L-EADs over a wide range of Vm indicates that there is not a single inward gated current as a common ionic mechanism for L-EADs but does not exclude an important role for I_Na:Ca, which can operate over a wide range of Vm. The rapid suppression of L-EADs with elevated [Ca²⁺]_o, and reduced [Na⁺]_o, and the migration of EADs to more positive Vm after exposures to high |Ca²⁺]_o, are compatible with I_Nc:Ca as the major charge carrier for L-EADs.     

Apparent Upregulation of Na+, K+ Pump Sites in SHR Skeletal Muscle with Reduced Transport Capacity

Slow-twitch, oxidative skeletal muscles in SHR exhibit several physiological defects, including a reduced ability to maintain force during high frequency repetitive stimulation (1). Muscle fatigue may be produced by one of a variety of factors acting at different levels of the neuromuscular system. Several lines of evidence, however, suggest that SHR soleus fatigues more rapidly than WKY soleus because SHR muscles allow more K⁺ to accumulate in the extracellular space during repetitive muscle activity. An increase in extracellular K⁺ can lead to a failure in the generation or conduction of muscle action potentials. Comparison of the compound action potentials recorded from SHR and WKY muscles during repetitive stimulation provided evidence for a decrease in excitability of SHR soleus. Since the K⁺ released from muscle fibers during exercise is returned to the fiber principally via the activity of the Na⁺, K⁺ pump, the increase in extracellular K⁺ in SHR muscle may reflect a decrease in pump capacity. Measurements including intracellular K⁺ and Na⁺ content at rest, the level of hyperpolarization produced by the addition of epinephrine and insulin to SHR soleus and the post-exercise recovery of resting membrane potentials all appear to indicate that Na⁺, K⁺ pump capacity is reduced in SHR soleus muscles. Nonetheless, ouabain binding studies show a significantly greater number of pump sites in SHR muscles. The data suggest that Na⁺ pump activity is decreased in SHR soleus muscles without an apparent reduction in either the number of pump sites or in pump binding affinity.     

Regional and frequency-dependent changes in action potentials and transient outward K<Superscript>+</Superscript> currents in ventricular myocytes from J-2-K cardiomyopathic hamsters

Abstract. Objectives Although lengthening of action potential duration (APD) and decreased transient outward K⁺ currents (I_to) have been observed in ventricular myocytes from cardiomyopathic hamsters, epi- and endo-cardial differences in I_to and their roles in frequency-dependent changes in APD have not been claried. Methods The patch-clamp technique of whole-cell conguration was used to record membrane potentials and currents in epicardial and endocardial myocytes of the J-2 hamster germline without (control) and with cardiomyopathy (CM). Results In control, APD in endocardial myocytes was longer than that in epicardial myocytes at 0.1 Hz. APD significantly lengthened with increased frequencies of stimulation from 0.1 to 6.0 Hz in both groups with the longer APD in endocardial myocytes. In CM, APD lengthened in epicardial myocytes exceeding the endocardial APD without a frequency-dependent prolongation. Pretreatment with 4 mM 4-aminopyridine completely abolished the frequency-dependent changes and abolished APD differences between epicardial and endocardial myocytes, and between control and CM hamsters. The transient outward K⁺ current (I_to) significantly decreased in epicardial myocytes from CM hamsters compared with that of control (17.5 ± 1.5 pA/pF in control vs. 9.5 ± 2.5 pA/pF in CM at +60 mV) with altered recovery from inactivation, without changes in the endocardial I_to. Moreover, the inward rectifier K⁺ current decreased in epicardial myocytes from CM hamsters and the L-type Ca2⁺ current reduced in both regions from CM compared to control. Conclusion Results indicate that differences in APD between epi- and endocardial myocytes in CM hamsters are mainly caused by a decreased current density and altered recovery from inactivation of I_to in epicardial myocytes.     

Effect of OPC-8490 on the membrane potentials and membrane currents of single guinea-pig myocytes

Summary The direct actions of OPC-8490 on mammalian myocardium were examined by determination of the drug's effects on the action potentials of isolated guinea-pig single ventricular cells and on the underlying ionic currents. OPC-8490 (10⁶ to 10⁴ M) did not alter the resting membrane potential, but rather produced a dose-dependent prolongation of the duration of the action potential. The amplitude of the action-potential plateau was also increased by OPC-8490. Whole-cell voltage clamp experiments revealed that OPC-8490 blocks myocardial delayed outward K⁺ current (I_K), which regulates repolarization of the action potentials. However I_KI, which regulates the resting membrane potential, was not changed by OPC-8490. Ca current (I_Ca) was increased by OPC-8490 in a dose-dependent and reversible manner. These results suggest that OPC-8490 augments the plateau amplitude and increases the duration of the action potentials by not only increasing I_Ca, but also by decreasing delayed out-ward K⁺ currents. Moreover, OPC-8490 did not affect the intracellular concentration of cyclic AMP in single cells. The OPC-8490 increase in I_Ca was thus unlikely to be mediated by a process involving cyclic AMP.     

Thyroid stimulating hormone directly modulates cardiac electrical activity

BackgroundThe electrocardiogram of hypothyroid patients shows a series of abnormalities of cardiac repolarization due to a reduction of some repolarizing K⁺ currents and an increase of the L-type calcium current. Experimental and clinical works call into question the unique role of T3 and T4 in these mechanisms and correlate increased serum TSH levels with the repolarization abnormalities in patients with both subclinical and overt hypothyroidism. In this context, the aim of the present study was to investigate the direct effects of TSH upon cardiac electrical properties.MethodsThe action potential recording and the ion channel subunits mRNA expression were obtained from left ventricle of adult rats. Additionally, the repolarizing K⁺ currents and the L-type Ca²⁺ current (ICa-L) were recorded in isolated rat adult ventricular myocytes by the patch-clamp technique.Results24 h exposure to TSH lengthened the action potential and slightly depolarized the resting membrane potential. TSH- receptor activation causes a reduction of the amplitude of I_to and I_K1 currents caused by a reduction in channels expression. However, TSH had no effect on I_Ca-L, I_K or I_Kur.ConclusionThese results support the idea that some of the electrical disturbances seen in hypothyroid hearts, such as the I_to and I_K1 current reduction, could be caused not by low T3 but by the elevation of circulating TSH.