An embryonic Ca++ mobilizing muscarinic system in the chick embryo heart

In the embryonic and in the adult heart muscarinic stimulation reduces the heart rate. Here we demonstrate that in the embryonic heart an additional muscarinic system is present, which is characterized by Ca++ mobilization and corresponds to the embryonic muscarinic system in other organ anlagen. In suspensions of embryonic chick heart cells we measured release of Ca++ from intracellular stores and influx of extracellular Ca++ with fura-2 and chlorotetracycline after muscarinic stimulation and determined the [3H]quinuclidinylbenzilate binding sites. We observed intense Ca++ mobilization at day 4, weaker reactions between day 4.5 and 11, and no Ca++ response at day 13. The pharmacological profile was identical to the profile of Ca++ mobilization in cells from other embryonic tissues in which the general embryonic muscarinic system is expressed. In parallel, we studied the effect of muscarinic agonists and antagonists on the heart rate of isolated embryonic hearts at day 4 and 5 in a perfusion chamber. Oxotremorine and bethanechol being antagonists or weak partial agonists in Ca++ mobilization, behaved as full agonists in frequency regulation. Thus, the pharmacological profile of the transient embryonic muscarinic system was different from that of the definitive adult form.     

Interrelations of cyclic adenosine monophosphate and calcium in regulating glycogen synthetase in developing skeletal and cardiac muscles in chickens]

The decrease of glycogen synthetase activity under the effect of Ca++ and cAMP is observed in the skeletal muscles of chick embryos only at the later stages of embryogenesis (15-18 days). In the cardiac muscle the activity of glycogen synthetase is inhibited by Ca++ apparently prior to the 12th day of development, long before the effect of cAMP which appears, as in the skeletal muscles, on the 18th day. Under the combined effect of cAMP and Ca++ in vitro and that of Ca++ and epinephrine in vivo, no additivity was observed. The possibility of Ca++ participation in the realization of catecholamine effect on the glycogen synthetase system of muscle and their interrelations with cAMP are discussed.     

Transduction persists in rod photoreceptors after depletion of intracellular calcium

We have examined the role of Ca++ in phototransduction by manipulating the intracellular Ca++ concentration in physiologically active suspensions of isolated and purified rod photoreceptors (OS-IS). The results are summarized by the following. Measurement of Ca++ content using arsenazo III spectroscopy demonstrates that incubation of OS-IS in 10 nM Ca++-Ringer's solution containing the Ca++ ionophore A23187 reduces their Ca++ content by 93%, from 1.3 to 0.1 mol Ca++/mol rhodopsin. Virtually the same reduction can be accomplished in 10 nM Ca++-Ringer's without ionophore, presumably via the plasma membrane Na/Ca exchange mechanism. Hundreds of photoresponses can be obtained from the Ca++-depleted OS-IS for at least 1 h in 10 nM Ca++-Ringer's with ionophore. The kinetics and light sensitivity of the photoresponse are essentially the same in the presence or absence of the ionophore in 10 nM Ca++. The addition of A23187 in 1 mM Ca++-Ringer's results in a Ca++ influx that rapidly suppresses the dark current and the photoresponse. This indicates that there is an intracellular site at which Ca++ can modulate the light-regulated conductance. Both the current and photoresponse can be restored if intracellular Ca++ is reduced by lowering the external Ca++ to 10 nM. During the transition from high to low Ca++, the response duration becomes shorter, which suggests that it can be regulated by a Ca++-dependent mechanism. If the dark current and the photoresponse are suppressed by adding A23187 in 1 mM Ca++-Ringer's, the subsequent addition of the cyclic GMP phosphodiesterase inhibitor isobutylmethylxanthine can restore the current and photoresponse. This implies that under conditions where the rod can no longer control its intracellular Ca++, the elevation of cyclic GMP levels can restore light regulation of the channels. The persistence of normal flash responses under conditions where intracellular Ca++ levels are reduced and perturbed suggests that changes in the intracellular Ca++ concentration do not cause the closure of the light-regulated channel.     

A possible regulatory role of Ca++-calmodulin system in cellular cholesterol ester hydrolysis in the steroidogenic response to ACTH in bovine adrenocortical cells

In order to corroborate the regulatory role of Ca++-calmodulin system in the steroidogenic response to adrenocorticotropic hormone (ACTH), the effects of calmodulin inhibitors (chlorpromazine, trifluoperazine, and W-7) on cortisol production and cellular cholesterol ester hydrolysis induced by ACTH in bovine adrenocortical cells were examined. Three calmodulin inhibitors diminished not only the cholesterol ester hydrolysis and cortisol production induced by ACTH in the presence of Ca++, but also inhibited the Ca++-induced hydrolysis and cortisol production in the absence of ACTH. Neither cortisol production in crude mitochondrial fraction nor the ACTH-induced Ca++-influx was affected by chlorpromazine. These results indicate that Ca++f-calmodulin system plays a significant regulatory role in the supply of free cholesterol to the adrenal mitochondria in the steroidogenic response to ACTH.     

Comparison of Ca++-regulated events in the intestinal brush border

The intestinal epithelial cell and specifically the cytoskeleton of the brush border are thought to be controlled by micromolar levels of free calcium. Calcium-binding proteins of this system include intestinal calcium binding protein (CaBP), calmodulin (CaM), villin, and a 36,000-mol-wt protein substrate of tyrosine kinases. To assess the sequence of events as the intracellular Ca++ level rises, we determined the amount of CaM and CaBP in the intestinal epithelium by western blotting and tested the Ca++ binding of CaM and CaBP by equilibrium dialysis. The Ca++-dependent actin severing activity of villin was analyzed in the presence of physiological CaM levels and increasing calcium concentrations. In addition, we analyzed the Ca++ levels required for interaction between CaM and the microvillus 110,000-mol-wt protein as well as fodrin and the interaction between a polypeptide of 36,000 mol wt (P-36) and actin. The results suggest that CaBP serves as the predominant Ca++ buffer in the cell, but CaM can effectively buffer ionic calcium in the microvillus and thus protect actin from the severing activity of villin. CaM binds to its cytoskeletal receptors, 110,000-mol-wt protein and fodrin differently, governed by the free Ca++ and pH. The interaction between P-36 and actin, however, appears to require an unphysiologically high calcium concentration (10(-4) to 10(-3) M) to be meaningful. The results provide a coherent picture of the different Ca++ regulated events occurring when the free calcium rises into the micromolar level in this unique system. This study would suggest that as the Ca++ rises in the intestinal epithelial cell an ordered sequence of Ca++ saturation of intracellular receptors occurs with the order from the lowest to highest Ca++ requirements being CaBP less than CaM less than villin less than P-36.     

Intracellular calcium pools and their metabolic dependence in normal versus simian virus 40-transformed human fibroblasts

Previous studies indicate that although normal and Simian virus (SV40)-transformed WI38 human fibroblasts have similar levels of intracellular Ca++ on a per mg protein basis, their ability to maintain this intracellular Ca++ against a low concentration of extracellular Ca++ differs markedly. The transformed but not the normal cells rapidly lose Ca++ when exposed to low extracellular Ca++, suggesting Ca++ transport and/or sequestration differ in the two cell types. In this study we have extended our investigations of Ca++ metabolism in the two cell types. We observe that normal WI38 cells, when exposed to metabolic inhibitors to deplete intracellular ATP, undergo a twofold increase in intracellular Ca++ levels. Under similar conditions and over the same time course, no comparable change in Ca++ level is observed in the SV40-transformed cell, despite the extensive depletion of ATP. 45Ca++ desaturation curves indicate that the bulk of the net increase in cell Ca++ following ATP depletion of the normal WI38 cell comes to reside in a slowly exchanging Ca++ pool. The data also indicate that glycolysis, and not oxidative phosphorylation, drives the active extrusion of Ca++ from these cells, an observation consistent with previous studies on the Na+-K+ pump in other cell types. Finally, the data indicate that in these cells mitochondria do not appear to be the major subcellular organelle responsible for regulation of at least the two cellular Ca++ pools measurable using isotope desaturation analysis. This is based on the inability of the respiratory inhibitor rotenone to alter significantly the size of either of these Ca++ pools. These pools compose 80-90% of total cell Ca++ in both cell types.     

Lanthanum: inhibition of ACTH-stimulated cyclic AMP and corticosterone synthesis in isolated rat adrenocortical cells

Lanthanum (La+++) is a well-known Ca++ antagonist in a number of biological systems. It was used in the present study to examine the role of Ca++ in the regulation of adenyl cyclase of the adrenal cortex by ACTH. In micromolar concentrations, .La+++ inhibited both cyclic AMP and corticosterone response of isolated adrenal cortex cells to ACTH. However, a number of intracellular processes were not affected by La+++. These include the stimulation of steroidogenesis by dibutyryl cyclic AMP, conversion of several steroid precursors into corticosterone, and stimulation of the latter by glucose. Thus, inhibition of steroidogenesis by La+++ appears to be solely due to an inhibition of ACTH-stimulated cyclic AMP formation. Electron microscope examination showed that La+++ was localized on plasma membrane of the cells and did not appear to penetrate beyond this region. Since La+++ is believed to replace Ca++ at superficial binding sites on the cell membrane, it is proposed that Ca++ at these sites plays an important role in the regulation of adenyl cyclase by ACTH. Similarities in the role of Ca++ in "excitation-contraction" coupling and in the ACTH-adenyl cyclase system raise the possibility that a contractile protein may be involved in the regulation of adenyl cyclase by those hormones which are known to require Ca++ in the process.     

Microwave enhancement of membrane conductance: calmodulin hypothesis

It has been found that 2450 MHz microwave radiation increases membrane conductance in molluscan neurons. Analysis of this effect points to the important role of Ca++ in the mechanism of neuron microwave response. However, regulation of many intracellular processes is not a direct Ca++ effect, but is mediated through calmodulin, a Ca++-binding multifunctional protein. Furthermore, there is some evidence showing that Ca++ regulation of a Ca pump, endoplasmic reticulum Ca++ buffering, and Ca++-activated K+ conductance are mediated via calmodulin. Based on that, calmodulin is hypothesized to be a microwave susceptible protein, and a qualitative model of microwave enhancement of membrane conductance is suggested.     

Induction of cytotoxic lymphocyte responses by antigen-specific helper factors

Antigen-specific helper factors (ASHF) were purified by antigen-affinity chromatography from supernatants of long-term helper T lymphocyte (TH) lines. We have modified an established helper-dependent assay system to demonstrate the antigen specificity and H-2 restriction properties of ASHF in the induction of cytotoxic T lymphocyte precursors (CTLp). Antigen specificity is demonstrated by the binding of ASHF molecules only to nominal antigen, both during purification and in tests of functional activity. Our ASHF preparations do not contain any interleukin 2 (IL 2) activity. The ASHF, purified by antigen-affinity chromatography in the presence of Ca++, is defined as Ca++-sufficient ASHF, whereas ASHF purified on antigen-affinity columns in the absence of Ca++ is defined to be Ca++ deficient. Ca++-sufficient ASHF is not H-2 restricted (as defined by the phenotype of the ASHF-producing cells) in the recognition of nominal antigen or in its interactions with CTLp or adherent stimulator cells. In contrast, when the "complete" (Ca++-sufficient) ASHF is functionally dissociated into subunits by removal of Ca++, the "incomplete" antigen-specific subunit of ASHF (Ca++-deficient ASHF) is still H-2-unrestricted in its ability to bind nominal antigen, but requires products from syngeneic adherent cells to trigger CTLp. When adherent cells that are H-2 identical to the ASHF are provided in culture, the "incomplete" ASHF is able to trigger either syngeneic or allogeneic CTLp in an antigen-specific manner. We interpret the results of our experiments to suggest that an H-2-restricted molecular interaction occurs in CTLp induction by ASHF. An antigen-specific, TH-derived receptor appears to require association with Ca++ and self major histocompatibility complex (MHC)-encoded molecules to form a "complete" ASHF that is able to trigger CTLp in an apparently H-2-unrestricted manner.     

"Calcium antagonists": a class of drugs with a bright future. Part I. Cellular calcium homeostasis and calcium as a coupling messenger

The aim of this series of minireviews is to present material from multidisciplinary sources to facilitate the understanding of the pharmacology and the ample clinical potential of a class of drugs that were originally designated as "calcium antagonists" and more recently have been referred to as "calcium entry blockers", "calcium slow channel blockers" or "calcium modulators". In this first report our attention will be focussed on the pivotal role of Ca++ as a messenger linking stimuli of extracellular origin to the intracellular environment. Eucaryotic cells have a number of powerful means to control their cytosolic Ca++ concentration. Firstly, in a cell at rest the cellular membrane is relatively impermeable to passive Ca++ movements. This property of the plasmalemma prevents the high free Ca++ concentration (approximately 1 mM) of the extracellular compartment from invading the cytosol (approximately 0.1 microM). However, extracellular Ca++ can reach the cytosol through the Na+/Ca++ exchange mechanism and the plasmalemma possesses special Ca++ channels the conductance of which is controlled by gates that are opened by critical changes in cellular polarization (voltage-operated channels: VOC) or by receptor activation (receptor-operated channel: ROC). The Ca++ entering via VOC or ROC can subsequently trigger the liberation of Ca++ from the sarcoplasmic reticulum or from calcium stores located in the inner side of the plasmalemma. The intracellular message generated by external stimuli is transferred to the response mechanism by several cytosolic proteins that require Ca++ as activator. Finally, the termination of the response is the result of a reduction in the cytosolic Ca++ concentration that is accomplished by the Na+/Ca++ exchange mechanism or by energy-dependent pumps which extrude Ca++ from the cell or store it in subcellular organelles. Therefore, any of the numerous steps of the excitation-response coupling which employ Ca++ as a messenger or as a protein activator can be the site of action of a pharmacological agent. In the follow-up minireview, some methods to determine the basic pharmacological profile of compounds interfering with cellular Ca++-dependent functions will be described.     

Influence of sodium-calcium exchange on calcium current rundown and the duration of calcium-dependent chloride currents in pituitary cells, studied with whole cell and perforated patch recording

The whole cell patch-clamp technique, in both standard and perforated patch configurations, was used to study the influence of Na+-Ca++ exchange on rundown of voltage-gated Ca++ currents and on the duration of tail currents mediated by Ca++-dependent Cl- channels. Ca++ currents were studied in GH3 pituitary cells; Ca++-dependent Cl- currents were studied in AtT-20 pituitary cells. Na+-Ca++ exchange was inhibited by substitution of tetraethylammonium (TEA+) or tetramethylammonium (TMA+) for extracellular Na+. Control experiments demonstrated that substitution of TEA+ for Na+ did not produce its effects via a direct interaction with Ca++-dependent Cl- channels or via blockade of Na+-H+ exchange. When studied with standard whole cell methods, Ca++ and Ca++-dependent Cl- currents ran down within 5-20 min. Rundown was accelerated by inhibition of Na+-Ca++ exchange. In contrast, the amplitude of both Ca++ and Ca++-dependent Cl- currents remained stable for 30-150 min when the perforated patch method was used. Inhibition of Na+-Ca++ exchange within the first 30 min of perforated patch recording did not cause rundown. The rate of Ca++-dependent Cl- current deactivation also remained stable for up to 70 min in perforated patch experiments, which suggests that endogenous Ca++ buffering mechanisms remained stable. The duration of Ca++-dependent Cl- currents was positively correlated with the amount of Ca++ influx through voltage-gated Ca++ channels, and was prolonged by inhibition of Na+-Ca++ exchange. The influence of Na+-Ca++ exchange on Cl- currents was greater for larger currents, which were produced by greater influx of Ca++. Regardless of Ca++ influx, however, the prolongation of Cl- tail currents that resulted from inhibition of Na+-Ca++ exchange was modest. Tail currents were prolonged within tens to hundreds of milliseconds of switching from Na+- to TEA+-containing bath solutions. After inhibition of Na+-Ca++ exchange, tail current decay kinetics remained complex. These data strongly suggest that in the intact cell, Na+-Ca++ exchange plays a direct but nonexclusive role in limiting the duration of Ca++-dependent membrane currents. In addition, these studies suggest that the perforated patch technique is a useful method for studying the regulation of functionally relevant Ca++ transients near the cytoplasmic surface of the plasma membrane.     

Strongylocentrotus purpuratus spindle tubulin. II. Characteristics of its sensitivity to Ca++ and the effects of calmodulin isolated from bovine brain and S. purpuratus eggs

Tubulin was extracted from spindles isolated from embryos of the sea urchin Strongylocentrotus purpuratus and purified through cycles of temperature-dependent assembly and disassembly. At 37 degrees C, the majority of the cycle-purified spindle tubulin polymer is insensitive to free Ca++ at concentrations below 0.4 mM, requiring free Ca++ concentrations greater than 1 mM for complete depolymerization. However, free Ca++ at concentrations above 1 microM inhibits initiation of polymer formation without significantly inhibiting the rate of elongation onto existing polymer. At 15 degrees C and 18 degrees C, temperatures that are physiological for S. purpuratus embryos, spindle tubulin polymer is sensitive to free Ca++ at micromolar concentrations such that 3-20 microM free Ca++ causes complete depolymerization. Calmodulin purified from either bovine brain or S. purpuratus eggs does not affect the Ca++ sensitivity of the spindle tubulin at 37 degrees C, although both increase the Ca++ sensitivity of cycle-purified bovine brain tubulin. These results indicate that cycle-purified spindle tubulin and cycle-purified bovine brain tubulin differ significantly in their responses to calmodulin and in their Ca++ sensitivities at their physiological temperatures. They also suggest that, in vivo, spindle tubulin may be regulated by physiological levels of intracellular Ca++ in the absence of Ca++-sensitizing factors.     

cAMP-dependent protein kinase substrates in platelets. Evidence that thrombolamban, a 22,000 dalton substrate, and the Ca++-ATPase are not associated proteins

Inhibition of platelet function by cAMP is due at least in part to a reduction in the agonist stimulated increase in cytoplasmic calcium during cell activation. This inhibition is also associated with cAMP-dependent phosphorylation of thrombolamban, a 22 kDa phosphoprotein which is present in the same membrane fraction as the calcium-dependent ATPase. Phosphorylation of this protein has been correlated with increased uptake of calcium by microsomal membranes. The present study was undertaken to examine the interaction of thrombolamban with the Ca++-ATPase in order to assess the possibility that the increased calcium uptake was by a direct effect of thrombolamban on Ca++-ATPase activity or that thrombolamban was a component of the Ca++-ATPase. Several approaches were utilized to assess the interaction of thrombolamban with the microsomal Ca++-ATPase. Gel filtration of labeled microsomes solubilized under non-denaturing conditions showed a major peak of radioactivity (Kav 0.64) corresponding to thrombolamban which was well separated from the Ca++-ATPase activity (Kav 0.09). Chemical cross-linking studies using partially purified thrombolamban and intact microsomes showed incorporation of the phosphoprotein into a 147,000 dalton complex. Indirect immunostaining with an anti-Ca++-ATPase antibody failed to demonstrate the Ca++-ATPase in the 147,000 dalton complex. Recombination of the phosphorylated thrombolamban with the Ca++-ATPase had no effect on Ca++-ATPase activity. These results indicate that, under the conditions used in these experiments, there was no apparent interaction between thrombolamban and the microsomal Ca++-ATPase. We conclude that thrombolamban is covalently bound to the Ca++-ATPase.     

Separate effects of mercurial compounds on the ionophoric and hydrolytic functions of the (Ca++ +Mg++)-ATPase of sarcoplasmic reticulum.

We have shown that a Ca++-ionophore activity is present in the (Ca++ +Mg++)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (A. E. Shamoo & D. H. MacLennan, 1974. Proc. Nat. Acad. Sci. USA 71:3522). Methylmercuric chloride inhibited the (Ca++ +Mg++)-ATPase and Ca++ transport, but had no effect on the activity of the Ca++ ionophore. Mercuric chloride inhibited ATPase, transport and ionophore activity. The ATPase and transport functions were more sensitive to methylmercuric chloride than to mercuric chloride. The two functions were inhibited concomitantly by methylmercuric chloride but slightly lower concentrations of mercuric chloride were required to inhibit Ca++ transport than were required to inhibit ATPase. Methylmercuric chloride and mercuric chloride probably inhibited ATPase and Ca++ transport by blocking essential -SH groups. However, it appears that there are no essential -SH groups in the Ca++ ionophore and that mercuric chloride inhibited the Ca++ ionophore activity by competition with Ca++ for the ionophoric site. Blockage of Ca++ transport by mercuric chloride probably occurs both at sites of essential -SH groups and at sites of ionophoric activity. These data suggest the separate identity of the sites of ATP hydrolysis and of Ca++ ionophoric activity.     

Hepatic insulin effects independent of changes in Ca++ metabolism

The possibility of Ca++ acting as second messenger for insulin in rat liver was investigated using the net stimulation of 14C-glucose incorporation into glycogen by isolated hepatocytes as an index of insulin action. An insulin effect could be partially sustained in the virtual absence of Ca++ and Mg++ and a maximal insulin effect could be observed in the presence of either Ca++ or Mg++, suggesting that extracellular Ca++ is not required for insulin action. Inhibiting the activity of calmodulin, an intracellular mediator of Ca++ action, with trifluoperazine had little effect on insulin action. The efflux of 45Ca from prelabeled hepatocytes was not altered by the presence of insulin arguing against insulin-induced changes in Ca++ fluxes. Collectively, these results do not support the role of Ca++ as second messenger for insulin action in liver.     

Inositol phosphates turnover, cytosolic Ca++ and pH: putative signals for the control of cell growth

The signals that induce a cell to divide are usually external and in the form of a binding of growth factors. We focussed our attention in defining the sequence of events which occurs after the binding of the mitogens to their surface receptors. One of the early membrane events stimulated by growth factors is a Na+ flux coupled to a H+ efflux that is typically inhibited by amiloride. The importance of this event and of the consequent cytoplasmic alkalinization for the cell proliferation is discussed. Recent data indicate that mitogens increase intracellular Ca++ levels and activate protein kinase C by inducing the hydrolysis of membrane phosphoinositides. A role for Ca++ and protein kinase C in activating Na+/H+ A role for Ca++ and protein kinase C in activating Na+/H+ exchange system is discussed. Finally a model is presented that illustrates the first membrane events stimulated by the growth factors. The model reveals an intimate interconnections between phosphoinositide metabolism, cytosolic Ca++ rise, protein kinase C and cytoplasmic alkalinization.     

Calcium-mediated decrease of a voltage-dependent potassium current.

Elevated intracellular Ca++ concentration reduces the amplitude of an early, voltage-dependent K+ current (IA) in the Type B photoreceptor of Hermissenda crassicornis. Internal Ca++ is increased by activating a voltage and light-dependent Ca++ current present in these cells or by direct iontophoresis of Ca++ ions. Substitution of Ba++ for Ca++ or elimination of Ca++ from the sea water bathing the cells abolishes the reduction in IA during paired light and depolarizing voltage steps. The delayed K+ current (IB) in these cells is also reduced during paired light and voltage steps, but this decrease of IB is not affected by removal of extracellular Ca++. IB (but not IA), apparently much less dependent on intracellular Ca++ levels, is reduced by light alone. Ca++ iontophoresis also abolishes the light-dependent Na+ current, which recovers with a time course of minutes.     

Depolarizing response of rat parathyroid cells to divalent cations

Membrane potentials were recorded from rat parathyroid glands continuously perfused in vitro. At 1.5 mM external Ca++, the resting potential averages -73 +/- 5 mV (mean +/- SD, n = 66). On exposure to 2.5 mM Ca++, the cells depolarize reversibly to a potential of -34 +/- 8 mV (mean +/- SD). Depolarization to this value is complete in approximately 2-4 min, and repolarization on return to 1.5 mM Ca++ takes about the same time. The depolarizing action of high Ca++ is mimicked by all divalent cations tested, with the following order of effectiveness: Ca++ greater than Sr++ greater than Mg++ greater than Ba++ for alkali-earth metals, and Ca++ greater than Cd++ greater than Mn++ greater than Co++ greater than Zn++ for transition metals. Input resistance in 1.5 mM Ca++ was 24.35 +/- 14 M omega (mean +/- SD) and increased by an average factor of 2.43 +/- 0.8 after switching to 2.5 mM Ca++. The low value of input resistance suggests that cells are coupled by low-resistance junctions. The resting potential in low Ca++ is quite insensitive to removal of external Na+ or Cl-, but very sensitive to changes in external K+. Cells depolarize by 61 mV for a 10- fold increase in external K+. In high Ca++, membrane potential is less sensitive to an increase in external K+ and is unchanged by increasing K+ from 5 to 25 mM. Depolarization evoked by high Ca++ may be slowed, but is unchanged in amplitude by removal of external Na+ or Cl-. Organic (D600) and inorganic (Co++, Cd++, and Mn++) blockers of the Ca++ channels do not interfere with the electrical response to Ca++ changes. Our results show remarkable parallels to previous observations on the control of parathormone (PTH) release by Ca++. They suggest an association between membrane voltage and secretion that is very unusual: parathyroid cells secrete when fully polarized, and secrete less when depolarized. The extraordinary sensitivity of parathyroid cells to divalent cations leads us to hypothesize the existence in their membranes of a divalent cation receptor that controls membrane permeability (possibly to K+) and PTH secretion.     

细胞膜毒素对线粒体上钙通道的抑制

本文探讨膜毒素对鼠肝线粒体Ca~(++)传递和Ca~(++)结合亲和力的影响。当膜毒素的浓度为7.14毫微克分子/毫克线粒体蛋白时,处理过的线粒体传递Ca~(++)能力下降至原来一半左右。本实验做Ca~(++)结合膜毒素处理线粒体的Scatchard图呈直线(K_d=48.2μM,结合Ca~(++)数目N=341毫微克分子/毫克线粒体蛋白)。就是说,膜毒素抑制线粒体高亲和力Ca~(++)结合部位,而不影响低亲和力Ca~(++)结合部位。我们认为膜毒素作用位点在于线粒体高亲和力Ca~(++)结合部位。     

Thrombin-induced calcium oscillation in human platelets and MEG-01, a megakaryoblastic leukemia cell line.

Digital imaging microscopy revealed that human platelets show periodic intracellular Ca++ elevation in response to 0.01 U/ml thrombin. MEG-01, a megakaryoblastic leukemia cell line, also responded with oscillatory intracellular Ca++ elevation (0.7-1 times/min) to thrombin (0.001-0.003U/ml). Ca++ transients appears to be fused with higher thrombin doses. With extracellular Ca++ concentrations of 0.1 mM or less, Ca++ oscillation could not be elicited, or even when present, it disappeared after a few spikings of [Ca++]i. Extracellular Ca++ concentrations of 0.3 mM or more were required to facilitate ongoing Ca++ oscillation, suggesting an important role of Ca++ influx for Ca++ oscillation.