Rise In Cytosolic Ca2 Research Articles

Rise of cytosolic Ca2+ and activation of membrane-bound guanylyl cyclase activity in rat enterocytes by heat-stable enterotoxin of Vibrio cholerae non-01.

The cytosolic calcium level ([Ca2+]i) and the membrane-bound guanylyl cyclase activity in the isolated rat intestinal epithelial cells were investigated. Heat-stable enterotoxin of Vibrio cholerae non-01 (NAG-ST) was found to increase both the [Ca2+]i and the enzyme activity. These changes occur similarly until 5 min of incubation with NAG-ST, indicating that these changes might be involved in NAG-ST induced signal transduction in rat enterocytes.

Rise of cytosolic Ca 2+ and activation of membrane-bound guanylyl cyclase activity in rat enterocytes by heat-stable enterotoxin of Vibrio cholerae non-01

The cytosolic calcium level ([Ca 2+] i) and the membrane-bound guanylyl cyclase activity in the isolated rat intestinal epithelial cells were investigated. Heat-stable enterotoxin of Vibrio cholerae non-01 (NAG-ST) was found to increase both the [Ca 2+] i and the enzyme activity. These changes occur similarly until 5 min of incubation with NAG-ST, indicating that these changes might be involved in NAG-ST induced signal transduction in rat enterocytes.

Characterization of an intracellular alkaline shift in rat astrocytes triggered by metabotropic glutamate receptors.

The modulation of intracellular pH by activation of metabotropic glutamate receptors was investigated in cultured and acutely dissociated rat astrocytes. One minute superfusion of 100 microM (1S,3R)-1-aminocyclopentane-1, 3-dicarboxcylic acid (ACPD) evoked an alkaline shift of 0.13 +/- 0. 013 (mean +/- SE) and 0.16 +/- 0.03 pH units in cultured (cortical or cerebellar) and acutely dissociated cortical astrocytes, respectively. Alkalinizations were elicited by concentrations of ACPD as low as 1 muM. The ACPD response was mimicked by S-3-hydroxyphenylglycine (3-HPG) and by (s)-4-carboxy-3-hydroxyphenylglycine (4C-3HPG) but was not blocked by alpha-methyl-4-carboxyphenylglycine (MCPG) or (RS)-1-aminoindan-1, 5-dicarboxcylic acid (AIDA), features consistent with an mGluR5 receptor-mediated mechanism. The ACPD-evoked alkaline shift was insensitive to amiloride, 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS), and the v-type ATPase inhibitors 7-chloro-4-nitrobenz-2-oxa-1,3-diazol (NBD-Cl), bafilomycin, and concanamycin. The alkaline response persisted in Na+- or Cl--free saline, but was reversibly blocked in bicarbonate-free, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions. A bicarbonate-dependent and Na+-independent alkaline shift could also be elicited by either 3 mM caffeine or 1 muM ionomycin. These data suggest that a rise in cytosolic Ca2+ activity is instrumental in triggering the alkalinizing mechanism and that this response is independent of the classic depolarization-induced alkalinization mediated by electrogenic sodium-bicarbonate cotransport.

Measurement of Ca2+ Fluxes during Elicitation of the Oxidative Burst in Aequorin-transformed Tobacco Cells

We have employed suspension cultured aequorin-transformed tobacco cells to examine the involvement of Ca2+ in signal transduction of the oxidative burst. Use of cultured cells for this purpose was validated by demonstrating that the cells responded to cold shock quantitatively and qualitatively similarly to the intact transgenic plants from which they were derived. Stimulation of the oxidative burst in the cell suspension was achieved by administration of oligogalacturonic acid, Mas-7 (a peptide known to activate G proteins and Ca2+ fluxes), hypo-osmotic stress, or harpin (a protein from the pathogenic bacterium Erwinia amylovora). The latter failed to promote any detectable increase in cytoplasmic Ca2+ concentration, whereas each of the former three triggered a rapid rise in cytosolic Ca2+ followed by a return within seconds to basal Ca2+ levels. Peak Ca2+ concentrations induced by the former three elicitors were approximately 0.7, 1.4, and 1.3 microM, respectively. Three lines of evidence suggest that the observed Ca2+ pulses are essential to transduction of the oxidative burst signals by their respective elicitors: (i) inhibition of the Ca2+ transients with Ca2+ chelators or Ca2+ channel blockers prevented expression of the oxidative burst, (ii) introduction of exogenous Ca2+ into the same cells initiated the burst even in the absence of other inducers of the response, and (iii) the observed Ca2+ transients often returned to near basal levels well before any H2O2 synthesis could be detected, suggesting that the Ca2+ influx is required to communicate the burst signal but not maintain the defense response. These data suggest that Ca2+ pulses serve frequently, but not invariably, to transduce an oxidative burst signal.

Hormone-induced rise in cytosolic Ca2+ in axolotl hepatocytes: properties of the Ca2+ influx channel

Calcium entry in nonexcitable cells occurs through Ca(2+)-selective channels activated secondarily to store depletion and/or through receptor- or second messenger-operated channels. In amphibian liver, hormones that stimulate the production of adenosine 3',5'-cyclic monophosphate (cAMP) also regulate the opening of an ion gate in the plasma membrane, which allows a noncapacitative inflow of Ca2+. To characterize this Ca2+ channel, we studied the effects of inhibitors of voltage-dependent Ca2+ channels and of nonselective cation channels on 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP)-dependent Ca2+ entry in single axolotl hepatocytes. Ca2+ entry provoked by 8-BrcAMP in the presence of physiological Ca2+ followed first-order kinetics (apparent Michaelis constant = 43 microM at the cell surface). Maximal values of cytosolic Ca2+ (increment approximately 300%) were reached within 15 s, and the effect was transient (half time of 56 s). We report a strong inhibition of cAMP-dependent Ca2+ entry by nifedipine [half-maximal inhibitory concentration (IC50) = 0.8 microM], by verapamil (IC50 = 22 microM), and by SK&F-96365 (IC50 = 1.8 microM). Depolarizing concentrations of K+ were without effect. Gadolinium and the anti-inflammatory compound niflumate, both inhibitors of nonselective cation channels, suppressed Ca2+ influx. This "profile" indicates a novel mechanism of Ca2+ entry in nonexcitable cells.

Large plasma membrane disruptions are rapidly resealed by Ca2+-dependent vesicle-vesicle fusion events.

A microneedle puncture of the fibroblast or sea urchin egg surface rapidly evokes a localized exocytotic reaction that may be required for the rapid resealing that follows this breach in plasma membrane integrity (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC). 263:390-393). How this exocytotic reaction facilitates the resealing process is unknown. We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle. When an approximately 40 by 10 microm surface patch was torn off, entry of fluorescein stachyose (FS; 1, 000 mol wt) or fluorescein dextran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not detected by confocal microscopy. Moreover, only a brief (approximately 5-10 s) rise in cytosolic Ca2+ was detected at the wound site. Several lines of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. When we injected FS-containing SW deep into the cells, a vesicle formed immediately, entrapping within its confines most of the FS. DiI staining and EM confirmed that the barrier delimiting injected SW was a membrane bilayer. The threshold for vesicle formation was approximately 3 mM Ca2+ (SW is approximately 10 mM Ca2+). The capacity of intracellular membranes for sealing off SW was further demonstrated by extruding egg cytoplasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca2+ -free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet-rich domain formed a membrane, suggesting that the yolk platelet is a critical element in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca2+ regulated vesicle-vesicle (including endocytic compartments but possibly excluding ER) fusion reactions. The function in resealing of this cytoplasmic fusion reaction is to form a replacement bilayer patch. This patch is added to the discontinuous surface bilayer by exocytotic fusion events.

Effects of PKC alpha activation on Ca2+ pump and K(Ca) channel in deoxygenated sickle cells.

We have previously shown that a pretreatment with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), reduced deoxygenation-induced K+ loss and Ca2+ uptake and prevented cell dehydration in sickle anemia red blood cells (SS cells) (H. Fathallah, E. Coezy, R.-S. De Neef, M.-D. Hardy-Dessources, and F. Giraud. Blood 86: 1999-2007, 1995). The present study explores the detailed mechanism of this PMA-induced inhibition. The main findings are, first, the detection of PKC alpha and PKC zeta in normal red blood cells and the demonstration that both isoforms are expressed at higher levels in SS cells. The alpha-isoform only is translocated to the membrane and activated by PMA and by elevation of cytosolic Ca2+. Second, PMA is demonstrated to activate Ca2+ efflux in deoxygenated SS cells by a direct stimulation of the Ca2+ pump. PMA, moreover, inhibits deoxygenation-induced, charybdotoxin-sensitive K+ efflux in SS cells. This inhibition is partly indirect and explained by the reduced deoxygenation-induced rise in cytosolic Ca2+ resulting from Ca2+ pump stimulation. However, a significant inhibition of the Ca2+-activated K+ channels (K(Ca) channels) by PMA can also be demonstrated when the channels are activated by Ca2+ plus ionophore, under conditions in which the Ca2+ pump is operating near its maximal extrusion rate, but swamped by Ca2+ plus ionophore. The data thus suggest a PKC alpha-mediated phosphorylation both of the Ca2+ pump and of the K(Ca) channel or an auxiliary protein.

Ryanodine receptor expression is associated with intracellular Ca2+ release in rat parotid acinar cells.

The ryanodine receptor mediates intracellular Ca2+ mobilization in muscle and nerve, but its physiological role in nonexcitable cells is less well defined. Like adenosine 3',5'-cyclic monophosphate and inositol 1,4,5-trisphosphate, cyclic ADP-ribose (0.3-5 microM) and ADP (1-25 microM) produced a concentration-dependent rise in cytosolic Ca2+ in permeabilized rat parotid acinar cells. Adenosine and AMP were less effective. Ryanodine markedly depressed the Ca2+-mobilizing action of the adenine nucleotides and forskolin in permeabilized cells and was likewise effective in depressing the action of forskolin in intact cells. Cyclic ADP-ribose-evoked Ca2+ release was enhanced by calmodulin and depressed by W-7, a calmodulin inhibitor. A fluorescently labeled ligand, 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3,4-diaza-s-indac ene-3-propionic acid-glycyl ryanodine, was synthesized to detect the expression and distribution of ryanodine receptors. In addition, ryanodine receptor expression was detected in rat parotid cells with a sequence highly homologous to a rat skeletal muscle type 1 and a novel brain type 1 ryanodine receptor. These findings demonstrate the presence of a ryanodine-sensitive intracellular Ca2+ store in rat parotid cells that shares many of the characteristics of stores in muscle and nerve and may mediate Ca2+-induced Ca2+ release or a modified form of this process.

Simultaneous recording of cytosolic Ca2+ levels inDidinium andParamecium during aDidinium attack onParamecium

The carnivorous ciliateDidinium nasutum captures prey such asParamecium by discharging extrusomes, known as toxicysts, while the attackedParamecium defensively discharges trichocysts. Several authors have suggested that both discharges, the toxicysts ofDidinium and the trichocysts ofParamecium, are evoked by the rise in cytosolic Ca2+ level in each cell. However, these putative increases in cytosolic Ca2+ levels have not as yet been recorded simultaneously in these cells during aDidinium attack onParamecium. We injected the fluorescent Ca2+ indicator Ca-Green 1 dextran into bothDidinium andParamecium, and simultaneously observed the cytosolic Ca2+ levels in these cells asDidinium attackedParamecium. When aParamecium came into contact with theDidinium proboscis, theDidinium showed a significant rise in cytosolic Ca2+ in the basal portion of the proboscis. One video frame (33 ms) after the onset of the Ca2+ rise inDidinium, theParamecium also showed an increase in cytosolic Ca2+. This is the first simultaneous recording of changes in the Ca2+ level during a predator-prey interaction in ciliates. The possible roles of these Ca2+ increases are discussed in relation to the discharge of toxicysts during theDidinium attack and of trichocysts as a defensive behavior ofParamecium.

Endoplasmic Reticulum Chaperones GRP78 and Calreticulin Prevent Oxidative Stress, Ca2+ Disturbances, and Cell Death in Renal Epithelial Cells

Activation of stress response genes can impart cellular tolerance to environmental stress. Iodoacetamide (IDAM) is an alkylating toxicant that up-regulates expression of hsp70 (Liu, H., Lightfoot, D. L., and Stevens, J. L. (1996) J. Biol. Chem. 271, 4805-4812) and grp78 in LLC-PK1 renal epithelial cells. Therefore, we used IDAM to determine the role of these genes in tolerance to toxic chemicals. Prior heat shock did not protect cells from IDAM but pretreatment with trans-4,5-dihydroxy-1,2-dithiane (DTTox), thapsigargin, or tunicamycin enhanced expression of the endoplasmic reticulum (ER) chaperones GRP78 and GRP94 and rendered cells tolerant to IDAM. Cells expressing a 524-base pair antisense grp78 fragment (pkASgrp78) had a diminished capacity to up-regulate grp78 and grp94 expression after ER stress. Protection against IDAM due to prior ER stress was also attenuated in pkASgrp78 cells suggesting that ER chaperones of the GRP family are critical for tolerance. Covalent binding of IDAM to cellular macromolecules and depletion of cellular thiols was similar in tolerant and naïve cells. However, DTTox pretreatment blocked the increases in cellular Ca2+ and lipid peroxidation observed after IDAM treatment. Overexpressing the ER Ca2+-binding protein calreticulin prevented IDAM-induced cell death, the rise in cytosolic Ca2+, and oxidative stress. Although activation of the ER stress response did not prevent toxicity due to Ca2+ influx, EGTA-AM and ruthenium red both blocked cell death suggesting that redistribution of intracellular Ca2+ to the mitochondria may be important in toxicity. The data support a model in which induction of ER stress proteins prevents disturbances of intracellular Ca2+ homeostasis, thus uncoupling toxicant exposure from oxidative stress and cell death. Multiple ER stress proteins are likely to be involved in this tolerance response.

Vinculin phosphorylation and barrier failure of coronary endothelial monolayers under energy depletion.

We studied the hypothesis that, in energy-depleted endothelial cells, Ca(2+)-dependent activation of protein kinase C (PKC) causes phosphorylation of vinculin and that this effect is involved in the early loss of endothelial barrier function. Vinculin localization and phosphorylation, PKC activity, and albumin permeability were studied in cultured coronary endothelial monolayers from rats. Ten minutes after the onset of metabolic inhibition by 5 mM potassium cyanide and 5 mM 2-deoxy-D-glucose, immunofluorescence of vinculin at cell-to-cell and cell-to-matrix contacts faded, whereas total cellular vinculin content remained unchanged. During the same time period, vinculin phosphorylation at tyrosine and serine sites increased by 3.9- and 3.5-fold, respectively. Vinculin phosphorylation was related to activation of PKC and an unidentified tyrosine kinase and was elicited by a rise in cytosolic Ca2+ within energy-depleted endothelial cells. Conditions inhibiting vinculin phosphorylation also reduced monolayer permeability induced by energy depletion. These data indicate that vinculin phosphorylation is involved in the progression of hyperpermeability during energy depletion in coronary endothelial monolayers.

Interaction between adrenaline and epidermal growth factor in the control of liver glycogenolysis in mouse.

Epidermal growth factor (EGF) stimulates glycogenolysis in mouse liver, but the effect requires concentrations that are only achieved in plasma upon adrenergic stimulation of EGF release from submandibular salivary glands. Thus, we studied the interaction between adrenaline and EGF in liver glycogen metabolism, both in whole animals and in isolated hepatocytes. Adrenaline administered to anesthetized mice stimulated both the endocrine secretion of EGF from submandibular salivary glands and the degradation of glycogen in the liver. In sialoadenalectomized mice, adrenaline administration did not increase plasma EGF concentration. In these animals, the glycogenolytic response to adrenaline was enhanced. The sensitivity of hepatocytes to adrenaline was similar in cells from sialoadenalectomized and sham-operated mice. EGF, added to isolated hepatocytes, reduced the glycogenolytic effect of adrenaline (the maximal effect but not the ED50). Adrenaline stimulated glycogen degradation through both an alpha1-adrenergic mediated Ca2+ increase and a beta-adrenergic-mediated cAMP increase. EGF did not interfere with the rise of cytosolic Ca2+ but decreased the cAMP signal. EGF did not decrease the glycogenolytic effect of phenylephrine or VP (which increased cytosolic Ca2+ but not cAMP), but EGF decreased both the glycogenolytic effect and the cAMP signal generated by glucagon or forskolin. EGF did not interfere with the glycogenolytic effect of CPT-cAMP or bt2-cAMP. The effect of EGF on cAMP was blocked by 3-isobutyl-1-methylxanthine. These results demonstrate that the effect of EGF on the glycogenolytic action of adrenaline involves interference with the generation of the cAMP signal. We suggest that EGF induces such an effect through the activation of a phosphodiesterase.

Prostaglandin E1 potentiation of the spontaneous phasic contraction of rat isolated portal vein by a cyclopiazonic acid-sensitive mechanism.

1. The effect of prostaglandin E1 (PGE1) on the spontaneous phasic contraction of the rat isolated portal vein was studied. 2. The isolated portal vein exhibited spontaneous phasic contractions. Removal of Ca2+ from Krebs-Ringer solution or application of nifedipine abolished the spontaneous contraction, indicating that the contraction depends exclusively on Ca2+ influx through L-type Ca2+ channels. On the other hand, cyclopiazonic acid (CPA), a specific inhibitor of Ca(2+)-ATPase of sarcoplasmic reticulum (SR) increased the amplitude of the contractions, suggesting that the SR regulates the spontaneous contractions negatively by sequestration of Ca2+ entering through L-type Ca2+ channels and buffering the rise in cytosolic Ca2+. 3. PGE1 increased the amplitude of the spontaneous contraction in a concentration-dependent manner without affecting the resting tension. The effect was completely abolished by nifedipine. Bay K 8644 and phenylephrine (PE) also increased the amplitude of the contraction in a concentration-dependent manner. PGE1 at a concentration of 1 microM. Bay K 8644 at 100 nM and PE at 30 nM doubled the amplitude, respectively. 4. Pretreatment with 1 microM CPA abolished the effect of PGE1, but the effects of Bay K 8644 and PE were not inhibited by pretreatment with CPA. In contrast, 10 microM ryanodine attenuated the effect of PE without affecting the contractile effect of PGE1. 5. When the SR was depleted of Ca2+ by repeated applications of caffeine in a nominally Ca(2+)-free Krebs-Ringer solution, it took about 120 s to restore the spontaneous contraction after addition of Ca2+ into the solution. In CPA-treated veins, the time taken to restore the contraction was shortened significantly. Pretreatment with 1 microM PGE1 shortened the time to the same extent as pretreatment with CPA did. 6. These results suggest that PGE1 increases the amplitude of the spontaneous phasic contraction by a different mechanism from those by which PE and Bay K 8644 increase it. Inhibition of Ca(2+)-ATPase of the SR might be involved in the vasoactive effect of PGE1.

An essential role of the yeast pheromone-induced Ca2+ signal is to activate calcineurin.

Previous studies showed that, in wild-type (MATa) cells, alpha-factor causes an essential rise in cytosolic Ca2+. We show that calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, is one target of this Ca2+ signal. Calcineurin mutants lose viability when incubated with mating pheromone, and overproduction of constitutively active (Ca(2+)-independent) calcineurin improves the viability of wild-type cells exposed to pheromone in Ca(2+)-deficient medium. Thus, one essential consequence of the pheromone-induced rise in cytosolic Ca2+ is activation of calcineurin. Although calcineurin inhibits intracellular Ca2+ sequestration in yeast cells, neither increased extracellular Ca2+ nor defects in vacuolar Ca2+ transport bypasses the requirement for calcineurin during the pheromone response. These observations suggest that the essential function of calcineurin in the pheromone response may be distinct from its modulation of intracellular Ca2+ levels. Mutants that do not undergo pheromone-induced cell cycle arrest (fus3, far1) show decreased dependence on calcineurin during treatment with pheromone. Thus, calcineurin is essential in yeast cells during prolonged exposure to pheromone and especially under conditions of pheromone-induced growth arrest. Ultrastructural examination of pheromone-treated cells indicates that vacuolar morphology is abnormal in calcineurin-deficient cells, suggesting that calcineurin may be required for maintenance of proper vacuolar structure or function during the pheromone response.

Frequency detection and temporally dispersed synaptic signal association through a metabotropic glutamate receptor pathway.

In the classical view, a central neuron integrates incoming synaptic information by simple algebraic summation of the resultant bioelectrical signals that coincide in time. The voltage dependence of the NMDA (N-methyl-D-aspartate) type of ionotropic glutamate receptor endows neurons with an additional tool that allows one synaptic input to influence another, providing, again, that the two are active simultaneously. Here we identify a new mechanism by which non-coincident signals generated by different synaptic inputs are integrated. The device serves to regulate neuronal excitation through G-protein-coupled, metabotropic glutamate receptors (mGluRs) in a powerful and specific manner. We show that, in cerebellar Purkinje cells, a single activation of the climbing-fibre input markedly potentiates mGluR-mediated excitation at parallel-fibre synapses. The potentiation results from a transient rise in cytosolic Ca2+ which is 'memorized' in such a way that it promotes excitation through mGluRs for about two minutes. A Ca2+-transient is also effective if imposed up to two seconds after parallel-fibre stimulation. By allowing temporally and spatially dispersed synaptic signals to be assimilated, this mechanism adds a new element to the computational power of central neurons.

1303 Type 1 InsP3 receptor required in LTD in cerebellar purkinje neuron

Acute hydrolysis of phosphoinositides has been demonstrated in bovine aortic endothelial cells (BAEC) treated with bradykinin (BK) (10−7M). The first phosphoinositide to decrease was phosphatidylinositol-4,5-bisphosphate (PIP2) indicating this to be the initial substrate of phospholipase action.Other lipid changes associated with the stimulation of BAEC were an increase in diacylglycerol (DAG) and arachidonic (AA) with a sustained production of phosphatidic acid (PA). The changes in cell phospholipids were accompanied by the release of inositol phosphates. Inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) was produced within 10 s of stimulation with BK. There was no evidence for the production of inositol-1,3,4-trisphosphate.The release of ionic calcium (Ca2+) intracellularly was demonstrated. The timecourse of the rise in intracellular Ca2+ was consistent with the timecourse of production of IP3. Intracellular Ca2+ rose from 127 ± 21 nm to 462 ± 27 nM. The Ca2+ peak was at 7.0 ± 0.4 s and took 3 min to reach a steady state which remained above the basal level. When extracellular Ca2+ was depleted in the extracellular medium a spike of intracellular Ca2+ release was measured with an immediate return to basal. Entry of extracellular Ca2+ into the cell after ionophore A23187 treatment does not induce inositol phosphate release, indicating that phosphoinositide hydrolysis is likely to be the cause rather than consequence of the elevation in cytosolic Ca2+.These data indicate action of phospholipase C (PLC) on PIP2 after BK stimulation of BAEC with the subsequent production of InsP3 causing the resulting intracellular Ca2+ release. Extracellular Ca2+ is not required for the initial rise in cytosolic Ca2+ concentration but is required for the maintenance of a sustained response.

Characteristics of palytoxin-induced cation currents and Ca2+ mobilization in smooth muscle cells of rabbit portal vein.

We examined the nature of the palytoxin (PTX)-induced channel and its relevance to the Ca2+ mobilizing effect of the toxin on smooth muscle cells isolated from rabbit portal vein using whole-cell voltage-clamp and microfluorimetric techniques. PTX (1 nM) induced a sustained, irreversible inward current at a holding potential of -40 mV. The PTX-induced current reversed at 0.5 +/- 0.6 mV, and the PTX-induced channel permitted the passage of Na+, K+, Cs+ and, to a lesser extent, Li+, but not choline+ or Ca2+. During the sustained phase of the current, superfusion of Ni2+ (5 mM), La3+ (0.5 mM) or 2,4-dichlorobenzamil (2,4-DCB, 25 microM) reduced the current amplitude and decreased the slope conductance without changing the reversal potential. In 5 of 7 experiments, ouabain transiently increased the PTX-induced inward current and shifted the reversal potential in a positive direction. Subsequently, ouabain inhibited the current in every cell. PTX (10 nM) induced a sustained rise in cytosolic Ca2+ ([Ca2+]i), which was resistant to verapamil but suppressed by omission of extracellular Ca2+. When external Na+ was replaced by choline+, PTX did not increase [Ca2+]i. Pretreatment with 2,4-DCB prevented the elevation of [Ca2+]i due to PTX. These results suggest that PTX does not directly stimulate Ca2+ entry but induces entry through Na(+)-Ca2+ exchange as a consequence of increased cytosolic Na+. Ni2+, La3+, 2,4-DCB and ouabain were shown to act as blockers of the PTX-induced channel. Ouabain may also inhibit Na+ pump current activated by cytosolic Na+.

Calcium signaling and secretion in pituitary cells

An important trigger of hormone secretion from pituitary cells is a rise in cytosolic Ca 2+ ([Ca 2+] i). Pituitary cells may modulate [Ca 2+] i by an increased membrane flux from the extracellular space and/or by a release from intracellular stores. Both mechanisms can support exocytosis, although in different pituitary cell types one or the other mechanism may predominate. Molecular events transducing a rise in [Ca 2+] i into hormone secretion are still poorly understood. Here, the exocytotic machinery in pituitary cells is briefly reviewed in terms of the spatial organization of [Ca 2+] i elevation relative to the Ca 2+ sensor(s).

Rise in cytosolic Ca2+ and collapse of mitochondrial potential in anoxic, but not hypoxic, rat proximal tubules.

It has been suggested that ischemic renal proximal tubular cell injury is mediated by an increase in cytosolic calcium concentrations ((Ca2+)i). However, measurements of (Ca2+)i in rat or rabbit proximal tubules exposed to hypoxia or anoxia have yielded ambiguous results. This study explored the possibility that the severity of oxygen deprivation and the energy state of the mitochondria are important determinants of (Ca2+)i. To this end, (Ca2+)i (measured with fura-2) and the mitochondrial membrane potential (measured with rhodamine 123) were studied simultaneously in individual rat proximal tubules in hypoxic and anoxic conditions. (Ca2+)i did not change during hypoxia, but increased rapidly during anoxia. Increases in (Ca2+)i were only observed in parallel with a decrease of rhodamine 123 fluorescence, which indicates a collapse of the mitochondrial membrane potential. The increase in (Ca2+)i during anoxia was prevented by incubating the tubules in a low Ca2+ medium, which did not interfere with the collapse of the mitochondrial membrane potential. Both hypoxic and anoxic incubation led to cell death, as assessed by the fluorescent dye propidium iodide. These results clearly demonstrate that the level of oxygen deprivation is critical in determining changes in (Ca2+)i. Because cell damage occurred in both hypoxic and anoxic conditions. It was concluded that an increase in (Ca2+)i is not a necessary prerequisite for the development of ischemic cell injury.

Identification and function of type-2 and type-3 ryanodine receptors in gut epithelial cells.

Reverse transcription-PCR (RT-PCR) techniques were used to identify the expression of ryanodine receptor (RyR) isoforms in gut epithelial cells. Restriction digest and sequence analysis of the PCR product showed the presence of RyR 2 and RyR 3. [3H]Ry binding studies on a microsome preparation, in a high-salt buffer, showed specific binding with an EC50 of 15 microM. In order to determine a potential functional role for these RyRs, we first characterized the response of the cells to acetylcholine. At all concentrations used acetylcholine induced sinusoidal cytosolic Ca2+ concentration ([Ca2+]i) oscillations. In response to 10(-4) M acetylcholine, levels of inositol 1,4,5-trisphosphate (InsP3) showed a peak of six times the basal level, at 30 s after stimulation. Application of caffeine alone failed to elicit a rise in cytosolic Ca2+. However, caffeine (5-50 mM) did rapidly and reversibly inhibit the acetylcholine-induced [Ca2+]i oscillations. The effects of Ry were more complex. Applied alone, Ry had no effect on the [Ca2+]i signal. When applied during agonist-evoked [Ca2+]i oscillations, Ry (10 microM) slowly blocked the response. In the continuous presence of Ry (10 microM) a short application of acetylcholine elicited a [Ca2+]i response that continued as oscillations even when the agonist was removed. The oscillations, in the presence of Ry (10 microM) but absence of agonist, were blocked either by removal of extracellular Ca2+ or by an application of a higher concentration of Ry (100 microM). These effects are consistent with the known use-dependence and dose-dependence for Ry action at the RyR. We conclude that the RyR 2 and RyR 3, identified by RT-PCR, play a central role in [Ca2+]i oscillations in gut epithelial cells.