Decreasing extracellular Na+ concentration triggers inositol polyphosphate production and Ca2+ mobilization

Removing extracellular Na+ (Na+o) evoked a large increase in cytosolic free Ca2+ concentration ([Ca2+]i in human skin fibroblasts. Decreasing [Na+]o from 120 to 14 mM caused the half-maximal peak increase in [Ca2+]i. Removing Na+o strongly stimulated 45Ca2+ efflux and decreased total cell Ca2+ by about 40%. Bradykinin caused changes in [Ca2+]i, total Ca2+, and 45Ca2+ fluxes similar to those evoked by removing Na+o. Prior stimulation of the cells with bradykinin prevented Na+o removal from increasing [Ca2+]i and vice versa. Na+o removal rapidly increased [3H]inositol polyphosphate production. Loading the cells with Na+ had no effect on the increase in 45Ca2+ efflux produced by Na+o removal. Therefore, decreasing [Na+]o probably stimulates a "receptor(s)" which is sensitive to extracellular, not intracellular, Na+. Removing Na+o also mobilized intracellular Ca2+ in smooth muscle and endothelial cells cultured from human umbilical and dog coronary arteries, respectively.     

Turgor regulation and cytoplasmic free Ca2+ in the algaLamprothamnium

Summary In internodal cells ofLamprothamnium succinctum, turgor regulation in response to hypotonie treatment is inhibited by lowering external Ca²⁺ concentration ([Ca²⁺]_e) from 3.9 (normal) to 0.01 (low) mM. In order to clarify whether a change in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_c) is involved in turgor regulation, the Ca²⁺ sensitive protein aequorin was injected into the cytoplasm of internodal cells. A large transient light emission was observed upon hypotonic treatment under normal [Ca²⁺]_e but not under low [Ca²⁺]_e. Thus hypotonic treatment induces a transient increase in [Ca²⁺]_c under normal [Ca²⁺]_e but not under low [Ca²⁺]_e.Abbreviations ASW artificial sea water - _i cellular osmotic pressure - [Ca²⁺]_c cytoplasmic free Ca²⁺ concentration - EDTA ethylenediamine-tetraacetic acid - EGTA ethylenglycol-bis(-aminoethyl ether(N,N-tetraacetic acid - [Ca²⁺]_e external Ca²⁺ concentration - _e external osmotic pressure - GM glass micropipette - GP glass plate - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid - MS microscope stage - OL objective lens - PIPES piperazine-N-N-bis(2-ethanesulfonic acid) - W Weight     

Cytosolic free Ca2+ concentration and intracellular calcium distribution of Ca2+-tolerant isolated heart cells

The cytosolic free Ca2+ concentration of calcium-tolerant rat myocytes has been measured by the null point titration technique using arsenazo III as a Ca2+ indicator and digitonin to permeabilize the plasma membrane. The mean value obtained for 8 separate preparations was 270 +/- 35 nM. The distribution of releasable calcium between the mitochondrial and sarcoplasmic reticular compartments was measured by the successive additions of uncoupler and A23187 to cells pretreated with ruthenium red. The relative distribution of calcium in each pool was independent of the cell calcium content up to the maximum value of releasable calcium investigated (4.5 nmol/mg of cell dry weight) and was distributed in the approximate ratio of 2:1 in favor of the sarcoplasmic reticulum. The cells contained 1 nmol of calcium/mg of cell dry weight in a form nonreleasable by A23187, which was independent of the total cell calcium content as measured by atomic absorption spectroscopy. It is calculated that the calcium content of mitochondria in heart under physiological conditions is about 5 nmol/mg of mitochondrial protein. At this level, the mitochondria are likely to provide effective buffering of the cytosolic free Ca2+ concentration of quiescent heart cells. The corresponding intramitochondrial free Ca2+ is in a range above values needed to regulate the activity of Ca2+-dependent enzymes of the citric acid cycle in heart. The physiological calcium content of the sarcoplasmic reticulum in heart cells is estimated to be about 2.5 nmol/mg of cell dry weight, which is at least 5-fold greater than the amount of calcium release calculated to cause maximum tension development of cardiac muscle.     

Cytosolic Ca2+ concentration during Ca2+ depletion of isolated rat hearts

Reperfusion of isolated mammalian hearts with a Ca²⁺-containing solution after a short Ca²⁺-free period at 37°:C results in massive influx of Ca²⁺ into the cells and irreversible cell damage: the Ca²⁺paradox. Information about the free intracellular, cytosolic [Ca²⁺] ([Ca²⁺]_i) during Ca²⁺ depletion is essential to assess the possibility of Ca²⁺ influx through reversed Na⁺/Ca²⁺ exchange upon Ca²⁺ repletion. Furthermore, the increase in end-diastolic pressure often seen during Ca²⁺-free perfusion of intact hearts may be similar to that seen during ischemia and caused by liberation of Ca²⁺ from intracellular stores. Therefore, in this study, we measured [Ca²⁺]i during Ca²⁺- free perfusion of isolated rat hearts. To this end, the fluorescent indicator Indo-1 was loaded into isolated Langendorff-perfused hearts and Ca²⁺-transients were recorded. Ca²⁺-transients disappeared within 1 min of Ca²⁺ depletion. Systolic [Ca²⁺]i during control perfusion was 268±54 nM. Diastolic [Ca²⁺]i during control perfusion was 114±34 nM and decreased to 53±19 nM after 10 min of Ca²⁺ depletion. Left ventricular end-diastolic pressure (LVEDP) significantly increased from 13±4 mmHg during control perfusion after Indo-1 AM loading to 31±5 mmHg after 10 min Ca²⁺ depletion. Left ventricular developed pressure did not recover during Ca²⁺ repletion, indicating a full Ca²⁺ paradox. These results show that LVEDP increased during Ca²⁺ depletion despite a decrease in [Ca²⁺]i, and is therefore not comparable to the contracture seen during ischemia. Furthermore, calculation of the driving force for the Na⁺/Ca²⁺ exchanger showed that reversed Na⁺/Ca²⁺ exchange during Ca²⁺ repletion is not able to increase [Ca²⁺]i to cytotoxic levels.     

Cytosolic free Ca2+ concentration in canine aortic endothelial cells lining the polyester arterial prosthesis

A rise in baseline cytosolic free Ca2+ in canine vascular endothelial-like cells (VEC) lining the luminal surface of the polyester arterial prosthesis is described. In one, three and six month implantation experiments we employed six adult mongrel dogs, polyester arterial prostheses Arteknit Ra K, fluorescent Ca2+ indicator Fura-2 and digital imaging microscopy to study cytosolic free Ca2+ in cultured VEC. The electron microscopy scanning of the luminal surface in different regions of the graft were also performed. A rise in cytosolic free Ca2+ in the VEC lining the luminal surface of the prosthesis is probably the result of the immunologic reaction and mechanical stress which stimulate the proliferation activity of the endothelial cells. It seems that the baseline cytosolic free Ca2+ reflects the course of the endothelization process on the polyester arterial prosthesis.     

Relationship between inositol polyphosphate production and the increase of cytosolic free Ca2+ induced by vasopressin in isolated hepatocytes

Addition of vasopressin to rat hepatocytes prelabeled with myo-[2-3H]inositol resulted in a very rapid decrease [3H]phosphatidylinositol 4,5-bisphosphate (Ptd-Ins-4,5-P2) which was paralleled by increases of up to 3-fold in the levels of [3H]inositol trisphosphate (Ins-P3) and [3H]inositol bisphosphate (Ins-P2). Increases of [3H]inositol phosphate (Ins-P) were not detected until about 5 min after hormone addition. These data indicate that the major pathway for hormone-induced lipid breakdown in liver is through a phosphodiesterase for PtdIns-4,5-P2 and that decreases of phosphatidylinositol are a secondary result of increased PtdIns-4,5-P2 resynthesis. Using the fluorescent Ca2+ indicator Quin 2, cytosolic free Ca2+ increased from 160 nM to about 400 nM after vasopressin addition to hepatocytes and preceded the conversion of phosphorylase b to a. Half-maximal and maximal increases of cytosolic free Ca2+ and phosphorylase a activity were observed at 0.2 and 1 nM vasopressin, respectively. The dose-response curve for the initial rate of cytosolic free Ca2+ increase was very similar to those obtained for the initial rates of Ins-P3 production and PtdIns-4,5-P2 breakdown. Pretreatment of hepatocytes with Li+ caused a 3--4-fold potentiation of vasopressin-induced elevations of Ins-P, Ins-P2, and Ins-P3, with half-maximal effects at 0.5, 1, and 5 mM, respectively. The calculated maximal concentrations of Ins-P3 in cells treated with 20 nM vasopressin were 10 and 30 microM, respectively, without and with Li+. Lithium did not affect the initial rate of inositol polyphosphate production or Ca2+ mobilization. The increase of Ins-P3 which correlated with peak cytosolic free Ca2+ elevation was about 0.6 microM. In a saponin-permeabilized hepatocyte preparation, Ins-P3 (1 microM) caused Ca2+ release from a vesicular, ATP-dependent Ca2+ pool. The data presented here suggest that Ins-P3 may be a second messenger for the mobilization of intracellular Ca2+ by hormones in liver.     

Measurements of cytoplasmic free Ca2+ concentration in human pancreatic islets and insulinoma cells

In human pancreatic islets an increase in the glucose concentration from 3 to 20 mM raised the free cytoplasmic Ca2+ concentration [( Ca2+]i), an effect being reversible upon withdrawal of the sugar. Depolarization with a high concentration of K+ or the sulphonylurea tolbutamide also raised [Ca2+]i. Addition of extracellular ATP produced a transient rapid rise in [Ca2+]i. Oscillations in [Ca2+]i were observed in the presence of 10 mM glucose. Insulinoma cells responded to glucose and tolbutamide with increases in [Ca2+]i, whereas the sulphonamide diazoxide caused a decrease in [Ca2+]i. These findings confirm previous results obtained in rodent beta-cells.     

Cytosolic Ca2+ buffers

"Ca(2+) buffers," a class of cytosolic Ca(2+)-binding proteins, act as modulators of short-lived intracellular Ca(2+) signals; they affect both the temporal and spatial aspects of these transient increases in [Ca(2+)](i). Examples of Ca(2+) buffers include parvalbumins (α and β isoforms), calbindin-D9k, calbindin-D28k, and calretinin. Besides their proven Ca(2+) buffer function, some might additionally have Ca(2+) sensor functions. Ca(2+) buffers have to be viewed as one of the components implicated in the precise regulation of Ca(2+) signaling and Ca(2+) homeostasis. Each cell is equipped with proteins, including Ca(2+) channels, transporters, and pumps that, together with the Ca(2+) buffers, shape the intracellular Ca(2+) signals. All of these molecules are not only functionally coupled, but their expression is likely to be regulated in a Ca(2+)-dependent manner to maintain normal Ca(2+) signaling, even in the absence or malfunctioning of one of the components.     

Inhibition of ATP-regulated K+ channels precedes depolarization-induced increase in cytoplasmic free Ca2+ concentration in pancreatic beta-cells

The effects of glucose, diazoxide, K+, and tolbutamide on the activity of K+ channels, membrane potential, and cytoplasmic free Ca2+ concentration were investigated in beta-cells from the Uppsala colony of obese hyperglycemic mice. With [K+]e = [K+]i = 146 mM, it was demonstrated that the dominating channel at the resting potential is a K+ channel with a single-channel conductance of about 65 picosiemens and a reversal potential of about +70 mV (pipette potential). This channel is characterized by complex kinetics with openings grouped in bursts. The channel was completely inhibited by 20 mM glucose in intact cells or by intracellularly applied Mg-ATP (1 mM). The number of active channels was markedly reduced already by 5 mM glucose. However, the single channel current of the channels remaining active was unaffected, indicating no major depolarization. To evoke a substantial depolarization of the membrane and thereby action potentials, a total block in channel activity was necessary. This could be achieved either by increasing the concentration of glucose to 20 mM or by combining 5 mM glucose with 100 microM tolbutamide. In both cases, the effect was counteracted by the hyperglycemic sulfonamide diazoxide. The effects on single channel activity were paralleled by changes in membrane potential and cytoplasmic free Ca2+ concentration, also when the latter measurements were performed at room temperature. The transient increase in the number of active channels and the resulting hyperpolarization observed after raising the glucose concentration to 20 mM probably reflected a drop in cytoplasmic ATP concentration. It is suggested that ATP works as a key regulator of the beta-cell membrane potential and thereby the opening of voltage-activated Ca2+ channels.     

The importance to chondrocyte differentiation of changes in expression of the multiple inositol polyphosphate phosphatase

It is important to both physiological and pathological osteogenesis to understand the significance of changes in gene expression in growth-plate chondrocytes that transit between the proliferative and hypertrophic states. MINPP is one such gene of interest. The Minpp protein dephosphorylates highly phosphorylated inositol signaling molecules InsP(5) and InsP(6). We show here that the ATDC5 chondrocyte progenitor cell line can recapitulate developmentally specific changes in MINPP expression previously only seen in longitudinal bone growth plates-both an initial 2-3-fold increase and a subsequent decrease back to initial levels during transition to hypertrophy. The increase in MINPP expression was accompanied by a 40% decrease in InsP(6) levels in ATDC5 cells. However, InsP(5) levels were not modified. Furthermore, throughout the hypertrophic phase, during which MINPP expression decreased, there were no alterations in InsP(5) and InsP(6) levels. We also created an ATDC5 line that stably overexpressed Minpp at 2-fold higher levels than in wild-type cells. This had no significant effect upon cellular levels of InsP(5) and InsP(6). Thus, substantial changes in MINPP expression can occur without a net effect upon InsP(5) and InsP(6) turnover in vivo. On the other hand, Minpp-overexpressing cells showed impaired chondrogenesis. We noted that the expression of alkaline phosphatase activity was inversely correlated with the expression of MINPP. The ATDC5 cells that overexpress Minpp failed to show an insulin-dependent increase in alkaline phosphatase levels, which presumably affects phosphate balance [J. Biol. Chem. 276 (2001) 33995], and may be the reason cellular differentiation was impaired. In any case, we conclude that Minpp is important to chondrocyte differentiation, but in a manner that is, surprisingly, independent of inositol polyphosphate turnover.     

The relationship between mitogen-induced changes in the cytoplasmic pH and free Ca2+ concentration in rat thymocytes

The relationship between pHi and [Ca]i signals generated in rat thymocytes by the mitogen Con A has been investigated. It is shown that the mitogen-induced [Ca]i rise is dependent on Na+/H+ exchange or some other Na(+)-sensitive process. This conclusion is based on the following findings: (i) [Ca]i response to Con A weakens upon decreasing the concentration of extracellular Na+, or inhibiting Na+/H+ exchange; (ii) agents that alkalinize the cytoplasm (the phorbol ester TPA, the Na+/H+ ionophore monensin and NH4Cl) cause an increase in [Ca]i (Klip, A., Rothstein, A. and Mack, E. (1984) Biochem. Biophys. Res. Commun. 124, 14-22; Grinstein, S. and Goetz, J.D. (1985) Biochim. Biophys. Acta 819, 267-270); (iii) The effects of Con A, TPA and monensin on [Ca]i are not additive. The last observation suggests that all these agents activate the same Na+/H+ (Na+ and/or H+)-dependent system of Ca2+ transport. It is found that the pH i and [Ca]i responses in rat thymocytes are sensitive to changes in the intracellular levels of cyclic nucleotides, ATP and in temperature. These regulatory effects on the ionic signals are different for Con A, TPA and monensin. In particular, both the stimulation of Na+/H+ antiport and the [Ca]i rise brought about by Con A or TPA are inhibited upon elevating the cellular cAMP. In contrast, the monensin-induced [Ca]i signal is almost independent of cAMP but is highly sensitive to changes in cGMP and temperature. Reducing the ATP level eliminates both the pHi and [Ca]i responses to Con A but not to monensin. These different characteristics of [Ca]i signals elicited by the mitogen and the Na+/H+ ionophore indicate that these agents use different mechanisms to activate the Na+/H(+)-dependent Ca2+ transporting system. A [Ca]i response to monensin has been obtained in some other cell types, namely, in lymphoblastoid Raji cells, Ehrlich ascites tumor cells and also in platelets.     

Cytosolic free Ca2+ level in isolated hepatocytes: the effect of insulin

Cytosolic free Ca2+ level was estimated in rat hepatocytes using the method described by Murphy et al. (1980). For control hepatocytes, a value of 0.20 +/- 0.06 mumol/l was obtained. Insulin increased cytosolic free Ca2+ level to 0.63 +2- 0.24 mumol/l. No net fluxes of Ca2+ across the plasma membrane were observed during incubation of hepatocytes with insulin. Mitochondria were shown to be the main Ca2+ buffering system. FCCP released 77-88% of releasable calcium from the cell. Dibucaine increased cytosolic free Ca2+ level to 1.16 mumol/l.     

Novel role for K+-dependent Na+/Ca2+ exchangers in regulation of cytoplasmic free Ca2+ and contractility in arterial smooth muscle

Cytoplasmic free Ca2+ ([Ca2+]cyt) is essential for the contraction and relaxation of blood vessels. The role of plasma membrane Na+/Ca2+ exchange (NCX) activity in the regulation of vascular Ca2+ homeostasis was previously ascribed to the NCX1 protein. However, recent studies suggest that a relatively newly discovered K+-dependent Na+/Ca2+ exchanger, NCKX (gene family SLC24), is also present in vascular smooth muscle. The purpose of the present study was to identify the expression and function of NCKX in arteries. mRNA encoding NCKX3 and NCKX4 was demonstrated by RT-PCR and Northern blot in both rat mesenteric and aortic smooth muscle. NCXK3 and NCKX4 proteins were also demonstrated by immunoblot and immunofluorescence. After voltage-gated Ca2+ channels, store-operated Ca2+ channels, and Na+ pump were pharmacologically blocked, when the extracellular Na+ was replaced with Li+ (0 Na+) to induce reverse mode (Ca2+ entry) activity of Na+/Ca2+ exchangers, a large increase in [Ca2+]cyt signal was observed in primary cultured aortic smooth muscle cells. About one-half of this [Ca2+]cyt signal depended on the extracellular K+. In addition, after the activity of NCX was inhibited by KB-R7943, Na+ replacement-induced Ca2+ entry was absolutely dependent on extracellular K+. In arterial rings denuded of endothelium, a significant fraction of the phenylephrine-induced and nifedipine-resistant aortic or mesenteric contraction could be prevented by removal of extracellular K+. Taken together, these data provide strong evidence for the expression of NCKX proteins in the vascular smooth muscle and their novel role in mediating agonist-stimulated [Ca2+]cyt and thereby vascular tone.     

Mitochondrial free Ca2+ concentration in living cells

Evidence has accrued during the past two decades that mitochondrial Ca²⁺ plays an important role in the regulation of numerous cell functions such as energy metabolism. This implies that mitochondrial Ca²⁺ transport systems might be able to relay the changes of cytosolic Ca²⁺ concentration ([Ca²⁺]_c) into mitochondrial matrix for regulating biochemical activities. To substantiate this idea, measurements of intramitochondrial free Ca²⁺ concentration ([Ca²⁺]_m) become essential. In this article, we review the results from recent studies attempting to measure [Ca²⁺]_m in living cells. In addition, the significance of each study is discussed.     

Effect of extracellular Ca2+ on plasma membrane Ca2+ inflow and cytoplasmic free Ca2+ in isolated hepatocytes

An initial rapid phase and a subsequent slow phase of 45Ca2+ uptake were observed following the addition of 45Ca2+ to Ca2+-deprived hepatocytes. The magnitude of the rapid phase increased 15-fold over the range 0.1-11 mM extracellular Ca2+ (Ca2+o) and was a linear function of [Ca2+]o. The increases in the rate of 45Ca2+ uptake were accompanied by only small increases in the intracellular free Ca2+ concentration. In cells made permeable to Ca2+ by treatment with saponin, the rate of 45Ca2+ uptake (measured at free Ca2+ concentrations equal to those in the cytoplasm of intact cells) increased as the concentration of saponin increased from 1.4 to 2.5 micrograms per mg wet weight cells. Rates of 45Ca2+ uptake by cells permeabilized with an optimal concentration of saponin were comparable with those of intact cells incubated at physiological [Ca2+o], but were substantially lower than those for intact cells incubated at high [Ca2+o]. It is concluded that Ca2+ which enters the hepatocyte across the plasma membrane is rapidly removed by binding and transport to intracellular sites and by the plasma membrane (Ca2+ + Mg2+)-ATPase and the plasma membrane Ca2+ inflow transporter is not readily saturated with Ca2+o.     

Receptor-activated cytoplasmic Ca2+ spiking mediated by inositol trisphosphate is due to Ca2(+)-induced Ca2+ release.

Receptor-mediated inositol 1,4,5-trisphosphate (Ins-(1,4,5)P3) generation evokes fluctuations in the cytoplasmic Ca2+ concentration ([Ca2+]i). Intracellular Ca2+ infusion into single mouse pancreatic acinar cells mimicks the effect of external acetylcholine (ACh) or internal Ins(1,4,5)P3 application by evoking repetitive Ca2+ release monitored by Ca2(+)-activated Cl- current. Intracellular infusion of the Ins(1,4,5)P3 receptor antagonist heparin fails to inhibit Ca2+ spiking caused by Ca2+ infusion, but blocks ACh- and Ins(1,4,5)P3-evoked Ca2+ oscillations. Caffeine (1 mM), a potentiator of Ca2(+)-induced Ca2+ release, evokes Ca2+ spiking during subthreshold intracellular Ca2+ infusion. These results indicate that ACh-evoked Ca2+ oscillations are due to pulses of Ca2+ release through a caffeine-sensitive channel triggered by a small steady Ins(1,4,5)P3-evoked Ca2+ flow.     

Biphasic regulation of mitochondrial Ca2+ uptake by cytosolic Ca2+ concentration

A rise in cytosolic Ca(2+) concentration is used as a key activation signal in virtually all animal cells, where it triggers a range of responses including neurotransmitter release, muscle contraction, and cell growth and proliferation [1]. During intracellular Ca(2+) signaling, mitochondria rapidly take up significant amounts of Ca(2+) from the cytosol, and this stimulates energy production, alters the spatial and temporal profile of the intracellular Ca(2+) signal, and triggers cell death [2-10]. Mitochondrial Ca(2+) uptake occurs via a ruthenium-red-sensitive uniporter channel found in the inner membrane [11]. In spite of its critical importance, little is known about how the uniporter is regulated. Here, we report that the mitochondrial Ca(2+) uniporter is gated by cytosolic Ca(2+). Ca(2+) uptake into mitochondria is a Ca(2+)-activated process with a requirement for functional calmodulin. However, cytosolic Ca(2+) subsequently inactivates the uniporter, preventing further Ca(2+) uptake. The uptake pathway and the inactivation process have relatively low Ca(2+) affinities of approximately 10-20 microM. However, numerous mitochondria are within 20-100 nm of the endoplasmic reticulum, thereby enabling rapid and efficient transmission of Ca(2+) release into adjacent mitochondria by InsP(3) receptors on the endoplasmic reticulum. Hence, biphasic control of mitochondrial Ca(2+) uptake by Ca(2+) provides a novel basis for complex physiological patterns of intracellular Ca(2+) signaling.