Hormone-regulated Ca2+ channel in rat hepatocytes revealed by whole cell patch clamp.

An inward current responsible for hormone regulated Ca2+ entry has been identified in cultured rat hepatocytes using whole cell patch clamp. Addition of 20 nM vasopressin or of 100 microM ATP induced the inward current, which could be observed more clearly after blocking an outward K+ current. This large outward K+ current, which appeared after addition of vasopressin or ATP, could be blocked either by replacing K+ with Cs+ in the external medium and in the pipette solution, or by simply including 0.5 microM apamin in the K(+)-containing external medium. The outward current appears to be carried by a Ca2+ activated K+ channel. In the presence of apamin, hepatocytes pretreated with vasopressin in a Ca(2+)-free media reveal an inward current on addition of external Ca2+ (5 mM). The current could also be elicited by addition of vasopressin when cells are preincubated in the presence of 5 mM external Ca2+. No current is seen on addition of Ca2+ in the absence of vasopressin. Initially, the inward current was ca 200-300 pA at -60 mV, but it declined rapidly over 3 min to ca 20 pA. The current approached zero, as an asymptote at positive potential, and appeared to be somewhat inwardly rectifying. Additions of 5 mM Mn2+ or 5 mM Ba2+ in place of Ca2+ produced little or no current. An inhibitor of ER Ca(2+)-ATPase, thapsigargin, could also trigger the cascade of events leading to plasma membrane conductance of Ca2+. The data suggest that hormone-stimulated Ca2+ entry into hepatocytes is mediated by a Ca(2+)-release activated channel highly specific for Ca2+. This is the first demonstration of such a channel in hepatocytes, though similar ones have been described in mast cells, in vascular endothelial cells and T-lymphocytes.

Impaired cardiac function in rats with healed myocardial infarction: cellular vs. myocardial mechanisms.

The inotropic responsiveness of isolated perfused rat hearts and single left ventricular (LV) myocytes to extracellular Ca2+ ([Ca2+]o) was examined 3 wk after ligation of left main coronary artery. Myocytes isolated from myocardial infarcted (MI) hearts were 10% longer. At [Ca2+]o of 1.1 mM, cell shortening as well as intracellular Ca2+ concentration dynamics were similar between MI and sham LV myocytes. At [Ca2+]o of 4.9 mM, maximal extent of cell shortening was significantly less in MI myocytes (16 +/- 1 vs. 22 +/- 1%), and peak intracellular Ca2+ concentration was also substantially lower. Thus, under conditions of high [Ca2+]o, decreased sarcolemmal Ca2+ influx and Ca2+ release during excitation-contraction may contribute to systolic dysfunction in MI hearts. Perfused working hearts and isovolumic heart preparations with infarcted LV displayed depressed maximal systolic pressure and decreased sensitivity to the inotropic effects of [Ca2+]o. Our data also indicate that, in addition to possible abnormalities in the contractile response of single myocytes, global factors such as loss of functional myocardium, altered chamber geometry, tissue fibrosis, and/or subendocardial ischemia contributed to depressed LV function in post-MI hearts perfused at physiological [Ca2+]o.

Chronic exercise alters contractility and morphology of isolated rat cardiac myocytes.

Chronic exercise training elicits positive adaptations in cardiac contractile function and ventricular dimension. The potential contribution of single myocyte morphological and functional adaptations to these global responses to training was determined in this study. Left ventricular cardiac myocytes were isolated from the hearts of sedentary control (Sed) or exercise-trained (TR) rats. Training elicited an approximately 5% increase in resting myocyte length (Sed, 121.0 +/- 2.0 vs. TR, 126.7 +/- 2.0 microns; P < 0.05), whereas resting sarcomere length and midpoint cell width were unaffected. These data suggest that longitudinal myocyte growth contributes to the training-induced increase in end-diastolic dimension. Single myocytes (28 degrees C) were stimulated at 0.067 and 0.2 Hz and shortening dynamics assessed at extracellular Ca2+ concentrations ([Ca2+]o) of 0.6, 1.1, and 2.0 mM. In both groups, maximal extent of myocyte shortening (ESmax) increased as [Ca2+]o increased and decreased as contraction frequency increased. TR myocytes were more strongly influenced by the effects of [Ca2+]o and frequency. At 0.067 Hz and 2.0 mM, ESmax was greater in TR than in Sed myocytes. The magnitude of this difference decreased as [Ca2+]o was reduced. At 0.2 Hz, ESmax was similar in Sed and TR myocytes at 2.0 mM [Ca2+]o. As [Ca2+]o was reduced, ESmax decreased more rapidly in TR than in Sed myocytes; at 0.6 mM, ESmax was greater in Sed than in TR myocytes. Our data indicate that chronic exercise influences cardiac contractile function at the single myocyte level. This study also provides evidence in support of the hypothesis that chronic exercise influences myocyte Ca2+ influx and efflux pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Ion channels in human erythroblasts. Modulation by erythropoietin.

To investigate the mechanism of intracellular Ca2+ ([Cai]) increase in human burst-forming unit-erythroid-derived erythroblasts by erythropoietin, we measured [Cai] with digital video imaging, cellular phosphoinositides with high performance liquid chromatography, and plasma membrane potential and currents with whole cell patch clamp. Chelation of extracellular free Ca2+ abolished [Cai] increase induced by erythropoietin. In addition, the levels of inositol-1,4,5-trisphosphate did not increase in erythropoietin-treated erythroblasts. These results indicate that in erythropoietin-stimulated cells, Ca2+ influx rather than intracellular Ca2+ mobilization was responsible for [Cai] rise. Both Ni2+ and moderately high doses of nifedipine blocked [Cai] increase, suggesting involvement of ion channels. Resting membrane potential in human erythroblasts was -10.9 +/- 1.0 mV and was not affected by erythropoietin, suggesting erythropoietin modulated a voltage-independent ion channel permeable to Ca2+. No voltage-dependent ion channel but a Ca(2+)-activated K+ channel was detected in human erythroblasts. The magnitude of erythropoietin-induced [Cai] increase, however, was insufficient to open Ca(2+)-activated K+ channels. Our data suggest erythropoietin modulated a voltage-independent ion channel permeable to Ca2+, resulting in sustained increases in [Cai].

Relaxation abnormalities in single cardiac myocytes from renovascular hypertensive rats.

In myocardial hypertrophy secondary to renovascular hypertension, the rate of intracellular Ca2+ concentration decline during relaxation in paced left ventricular (LV) myocytes isolated from hypertensive (Hyp) rats is much slower compared with that from normotensive (Sham) rats. By use of a novel liquid-crystal television-based optical-digital processor capable of performing on-line real-time Fourier transformation and the striated pattern (similar to 1-dimensional diffraction grating) of cardiac muscle cells, sarcomere shortening and relaxation velocities were measured in single Hyp and Sham myocytes 18 h after isolation. There were no differences in resting sarcomere length, percent of maximal shortening, time to peak shortening, and average sarcomere shortening velocity between Sham and Hyp cardiac cells. In contrast, average sarcomere relaxation velocity and half-relaxation time were significantly prolonged in Hyp myocytes. Contractile differences between Sham and Hyp myocytes detected by the optical-digital processor are confirmed by an independent method of video tracking of whole cell length changes during excitation-contraction. Despite the fact that freshly isolated myocytes contract more rigorously than 18-h-old myocytes, the relaxation abnormality was still observed in freshly isolated Hyp myocytes, suggesting impaired relaxation is an intrinsic property of Hyp myocytes rather than changes brought about by short-term culture. We postulate that reduced sarcomere relaxation velocity is a direct consequence of impaired Ca2+ sequestration-extrusion during relaxation in Hyp myocytes and may be responsible for diastolic dysfunction in hypertensive hypertrophic myocardium at the cellular level.

Altered Ca2+ dynamics in single cardiac myocytes from renovascular hypertensive rats.

Several functional and biochemical characteristics of hypertrophied hearts isolated from rats with renovascular hypertension provide indirect evidence that cellular Ca2+ dynamics during myocardial contraction-relaxation are altered. In this study, intracellular Ca2+ concentration ([Ca2+]i) dynamics were examined in paced left ventricular (LV) myocytes isolated from rats with hypertension (HYP) induced by partial occlusion of the left renal artery and from normotensive rats (Sham). Characteristic myocardial changes produced by renovascular hypertension included a 40% increase in LV weight and a 3.6-fold increase in the fractional expression of the beta-heavy chain of myosin in isolated LV myocytes. In periods of mechanical quiescence between contractions, basal [Ca2+]i values were similar in Sham and HYP LV myocytes. During a contraction-relaxation cycle in HYP myocytes, peak [Ca2+]i, +d[Ca2+]i/dt, and -d[Ca2+]i/dt were reduced, whereas the time required for [Ca2+]i to rise from a basal value to a peak value (time-to-peak [Ca2+]i) was unaffected. In both Sham and HYP myocytes, the fall in [Ca2+]i from peak to basal values could be approximated by a monoexponential rate constant, kf. Values for kf were significantly smaller in HYP than in Sham myocytes. After treatment with 4 microM isoproterenol, peak [Ca2+]i, +[Ca2+]i/dt, -d[Ca2+]i/dt, and kf increased in both Sham and HYP myocytes. In contrast, basal [Ca2+]i and time-to-peak [Ca2+]i did not change. Thus, despite recent reports of inefficiencies of beta-adrenergic receptor coupling, there was no evidence of blunted beta-adrenergic responsiveness in HYP myocytes with respect to [Ca2+]i dynamics during contraction-relaxation. Finally, no Sham vs. HYP differences in the number of specific [3H]-PN200-110 binding sites per cell in quiescent, rod-shaped myocytes were detected, but a significant reduction in [3H]-PN200-110 binding sites in an enriched sarcolemmal membrane fraction isolated from HYP animals was observed. These observations are suggestive of a reduction in slow, Ca2+ channel surface density in HYP myocytes. The results of this study clearly indicate that [Ca2+]i dynamics during contraction-relaxation in single left ventricular myocytes are affected by residence in a chronic setting of renovascular hypertension. In addition, the prolonged [Ca2+]i removal phase observed in HYP myocytes can be restored toward normal by beta-adrenergic agonists.

Quantitation of proteins by dot-immunobinding assay. A comparison of visualization methods using eukaryotic initiation factor 2 and a monospecific antibody.

Various visualization methods were compared for quantitation of proteins by the dot-immunobinding assay. Comparisons were carried out using a multi-subunit protein, eukaryotic initiation factor 2, and monospecific antibodies directed against two of the factor's subunits. The protein was spotted onto nitrocellulose and the membranes were incubated with primary antibody. The antigen-antibody complex was visualized by one of six methods using either alkaline phosphatase-, horseradish peroxidase-, or glucose oxidase-conjugated IgG, or colloidal gold-labelled IgG, colloidal gold-labelled IgG with silver enhancement, or 125I-labelled protein A. The amount of secondary antibody bound was quantitated by densitometric scanning of the nitrocellulose membrane after staining or autoradiography. The sensitivity of each of the methods was similar; each of the visualization methods could detect less than 1 ng of protein by the dot-immunobinding assay. Curves of protein concentration vs. densitometric absorbance were found to fit a parabolic relationship (r2 = 0.99) over a wide range of concentrations.