Cardiotonic steroids differentially affect intracellular Na+ and [Na+]i/[K+]i-independent signaling in C7-MDCK cells

Recently, we reported that ouabain kills renal epithelial and vascular endothelial cells independently of elevation of the [Na(+)](i)/[K(+)](i) ratio. These observations raised the possibility of finding cardiotonic steroids (CTS) that inhibit the Na(+),K(+) pump without attenuating cell survival and vice versa. To test this hypothesis, we compared CTS action on Na(+),K(+) pump, [Na(+)](i) content, and survival of Madin-Darby canine kidney cells. At a concentration of 1 microM, ouabain and other tested cardenolides, as well as bufadienolides such as bufalin, cinobufagin, cinobufotalin, and telobufotoxin, led to approximately 10-fold inhibition of the Na(+),K(+) pump, a 2-3-fold decrease in staining with dimethylthiazol-diphenyltetrazolium (MTT), and massive death indicated by detachment of approximately 80% of cells and caspase-3 activation. In contrast, Na(+),K(+) pump inhibition and elevation of [Na(+)](i) seen in the presence of 3 microM marinobufagenin (MBG) and marinobufotoxin did not affect MTT staining and cell survival. Inhibition of the Na(+),Rb(+) pump in K(+)-free medium was not accompanied by a decline of MTT staining and cell detachment but increased sensitivity to CTS. In K(+)-free medium, half-maximal inhibition of (86)Rb influx was observed in the presence of 0.04 microM ouabain and 0.1 microM MBG, whereas half-maximal detachment and decline of MTT staining were detected at 0.03 and 0.004 microM of ouabain versus 10 and 3 microM of MBG, respectively. Both ouabain binding and ouabain-induced [Na(+)](i),[K(+)](i)-independent signaling were suppressed in the presence of MBG. Thus, our results show that CTS exhibit distinctly different potency in Na(+),K(+) pump inhibition and triggering of [Na(+)](i)/[K(+)](i)-independent signaling, including cell death.     

Intracellular [Ca2+] and K+ and Cl- efflux responses in submandibular cells of neonatal and adult rats.

The effects of graded doses (5 x 10(-8) to 10(-5)) acetylcholine on intracellular Ca2+ and on 86Rb and 36Cl efflux were compared in submandibular cell clusters of 1 and 7 day-old and adult rats. Initial Ca2+ peaks were similar at agonists concentrations lower than 10(-7) M but the release of Rb+ and Cl- were smaller in cells of young animals. At higher agonist concentrations, Ca2+ peaks were higher in immature cells; however, initial Cl- (but not Rb+) efflux was similar to that of mature cells. Plateau Ca2+ levels were independent of age and agonist concentrations but the content of Cl- and Rb+ varied greatly and differences between age groups were less evident. These data confirm a dissociation between intracellular Ca2+ levels and Ca(2+)-mediated ion transport in immature salivary cells.     

Intracellular free [Ca2+] in circulating lymphocytes of spontaneously hypertensive rats

In the light of previous reports suggesting a common abnormality of Ca handling in most tissues of hypertensive humans and rats, we applied a novel technique using the fluorescent probe Quin 2 for measurement of cytosolic free Ca2+ in lymphocytes of spontaneously hypertensive rats (SHR). (Ca2+)i is increased in SHR (122.1 +/- 7.4 nM) versus normotensive Wistar-Kyoto (WKY) control rats (81.1 +/- 6.3 nM) Membrane exchange, as challenged by varying the extracellular Ca concentration over a 10(5)-fold range proved to be relatively unimportant in regulating (Ca2+)i and did not significantly affect the difference between SHR and WKY. Catecholamines and ouabain had no appreciable effect on (Ca2+)i. The mechanisms of increased (Ca2+)i in SHR lymphocytes remain to be fully elucidated.     

Glycolysis and brain function: [K+]o stimulation of protein synthesis and K+ uptake require glycolysis

Glucose is essentially the sole energy substrate in the normally functioning brain. There are, however, situations in which other substrates can partially substitute for glucose and maintain an apparently normal brain function. However, in no case has it been possible to completely substitute other substrates for glucose and maintain normal brain function. Studies on insulin-induced hypoglycemia suggest that this glucose dependence does not result from its involvement in ATP generation. Two explantation that have been offered are that toxic catabolites arise if nonglucose substrates are oxidized or that glycolysis is necessary to maintain neurotransmitter metabolism. We consider a third basis for the glucose requirement: our past studies have shown that hippocampal slice protein synthesis is activated by small increases in extracellular [K+] ([K+]o), and that this results from activation of K+ uptake into brain cells. We find that this process specifically requires aerobic glycolysis. The basis for the requirement appears to be that [K+]o activation of the Na+-K+ pump is specifically dependent on glycolytically generated energy. Thus, it is possible that glucose is required to maintain normal K+ clearance from the extracellular space during neural activity. This could partially account for the dependence of brain function on glycolysis.     

Relationship between force and intracellular [Ca2+] in tetanized mammalian heart muscle

To determine features of the steady state [Ca2+]-tension relationship in intact heart, we measured steady force and intracellular [Ca2+] ([Ca2+]i) in tetanized ferret papillary muscles. [Ca2+]i was estimated from the luminescence emitted by muscles that had been microinjected with aequorin, a Ca2+-sensitive, bioluminescent protein. We found that by raising extracellular [Ca2+] and/or by exposing muscles to the Ca2+ channel agonist Bay K 8644, tension development could be varied from rest to an apparently saturating level, at which increases in [Ca2+]i produced no further rise in force. 95% of maximal Ca2+-activated force was reached at a [Ca2+]i of 0.85 +/- 0.06 microM (mean +/- SEM; n = 7), which suggests that the sensitivity of the myofilaments to [Ca2+]i is far greater than anticipated from studies of skinned heart preparations (or from previous studies using Ca2+-sensitive microelectrodes in intact heart). Our finding that maximal force was reached by approximately 1 microM also allowed us to calculate that the steady state [Ca2+]i-tension relationship, as it might be observed in intact muscle, should be steep (Hill coefficient of greater than 4), which is consistent with the Hill coefficient estimated from the entire [Ca2+]i-tension relationship derived from families of variably activated tetani (6.08 +/- 0.68; n = 7). Finally, with regard to whether steady state measurements can be applied directly toward understanding physiological contractions, we found that the relation between steady force and [Ca2+]i obtained during tetani was steeper than that between peak force and peak [Ca2+]i observed during physiological twitches.     

Graded response of K+ current,membrane potential,and [Ca2+]i to hypoxia in pulmonary arterial smooth muscle

Many studies indicate that hypoxic inhibition of some K+ channels in the membrane of the pulmonary arterial smooth muscle cells (PASMCs) plays a part in initiating hypoxic pulmonary vasoconstriction. The sensitivity of the K+ current (I(k)), resting membrane potential (E(m)), and intracellular Ca2+ concentration ([Ca2+]i) of PASMCs to different levels of hypoxia in these cells has not been explored fully. Reducing PO2 levels gradually inhibited steady-state I(k) of rat resistance PASMCs and depolarized the cell membrane. The block of I(k) by hypoxia was voltage dependent in that low O2 tensions (3 and 0% O2) inhibited I(k) more at 0 and -20 mV than at 50 mV. As expected, the hypoxia-sensitive I(k) was also 4-aminopyridine sensitive. Fura 2-loaded PASMCs showed a graded increase in [Ca2+]i as PO2 levels declined. This increase was reduced markedly by nifedipine and removal of extracellular Ca2+. We conclude that, as in the carotid body type I cells, PC-12 pheochromocytoma cells, and cortical neurons, increasing severity of hypoxia causes a proportional decrease in I(k) and E(m) and an increase of [Ca2+]i.     

Intracellular free [Ca2+] in human skeletal muscle with myopathic carnitine deficiency

Carnitine is required for the transport of activated long chain fatty acids through the mitochondrial inner membrane. We measured the intracellular free calcium concentration [( Ca2+]i) by means of a calcium selective microelectrode in skeletal muscle biopsies obtained from nine patients in which myopathic carnitine deficiency (MCD) was diagnosed, and from six subjects with no evidence of neuromuscular disease. Intact intercostal muscle bundles were dissected and then split for electron microscopic studies and electrophysiological measurements. The [Ca2+]i in muscle fibers from MCD patients was 0.46 +/- 0.02 mumol.l-1 (mean +/- SEM) and 0.10 +/- 0.01 mumol.l-1 in control subjects. At the electron microscopic level, the predominant abnormality was the presence of lipid vacuoles between the myofibrils. These results show that in patients with myopathic carnitine deficiency there is a significant increase in the resting myoplasmic calcium concentration which might be related to a malfunction of some mechanisms responsible for the homeostasis of intracellular calcium.     

Lymphocyte reaction in schizophrenia patients to the phytomitogens, concanavalin and phytohemagglutinin]

The capability of the schizophrenic patients' lymphocytes and lymphocytes of healthy persons to respond to the stimulating action of T-mutagens -- concanavalin A and PHA -- was studied. The T-cell count was determined by the method of rosette formation; the influence of adhesive cells on the lymphocyte response to mitogens was ascertained. The response to both the mitogens in the patients' lymphocyte cultures was reduced as compared to control, and the T-cell count failed to differ from the normal. The removal of adhesive lymphocytes results in the disappearance of differences between the response of the patients' lymphocytes and normal lymphocytes to both the mitogens.     

Transitional Changes in Membrane Potential and Intracellular [Ca2+] in Rat Basophilic Leukemia Cells

Using whole-cell current-clamp measurements we have found that thapsigargin-mediated activation of store-regulated Ca²⁺ entry in rat basophilic leukemia cells is accompanied by complex changes in membrane potential. These changes consisted of: (i) an initial slow, small depolarization, (ii) a transitional change in potential to a depolarized value and (iii) transitional changes between a hyperpolarized and a depolarized potential. These complex changes in potential can be explained by the interaction between the endogenous inwardly rectifying K⁺ conductance and the generation of a small inward current. To investigate the possible influence of these changes of potential on [Ca²⁺] _i , single cell measurements of fura2 fluorescence were undertaken alone or in combination with current-clamp measurements. Thapsigargin-mediated activation of the store-regulated Ca²⁺ entry pathway was accompanied by a marked increase of [Ca²⁺] _i . During this increase, transient, abrupt declines in [Ca²⁺] _i were detected in approximately 60% of the cells investigated. These changes of [Ca²⁺] _i are consistent with the observed changes of membrane potential recorded under current-clamp. Received: 1 December 1998/Revised: 30 March 1999     

How to find a prion: [URE3], [PSI+] and [beta

Infectious proteins (prions) in yeast or other microorganisms can be identified by genetic methods of rather general applicability. Infection in yeast means transfer by cytoplasmic mixing (cytoduction), a property of all non-chromosomal genetic elements whether plasmids, viruses, or prions. Prions can be diagnosed by reversible curability, increased occurrence when the corresponding protein is overproduced, a requirement for the gene for the corresponding protein for propagation, and, in some cases, similarity of phenotype of: (a) mutations in the gene for the protein and (b) the presence of the prion. This approach is illustrated with [URE3], an amyloid-based prion of the regulator of nitrogen catabolism, Ure2p and [PSI(+)] as a prion of the translation termination factor Sup35p. The prion concept is not limited to infectious amyloids, but includes proteins whose active form is necessary for the activation of the inactive precursor. We detail methods used in studies of [URE3] and [beta], a self-activating protease, some of which are of broad application.     

Extracellular [K+] fluctuations in voltage-clamped canine cardiac Purkinje fibers.

Membrane currents and extracellular [K+] were measured in canine Purkinje strands during voltage-clamp steps to plateau or diastolic potentials. Extracellular [K+] increased during step depolarizations and decreased during step hyperpolarizations. On hyperpolarization, the largest fraction of the K+ depletion occurred during the initial 500 ms of the voltage-clamp step and was correlated with a potassium depletion current, the id. A slower component of the depletion also occurred on hyperpolarization and had a time constant consistent with cylindrical diffusion of potassium within the Purkinje strands. On depolarization, there is an accumulation of K+ that is correlated with the plateau current ix. On termination of depolarizing test pulses, the K+ accumulation decays with a time course similar to the ix tail current. Surprisingly, no accumulation of K+ occurred during the arrhythmogenic transient inward current, TI, suggesting that the selectivity of this current should be reevaluated.