Thyrotropin-releasing hormone activates Na+/H+ exchange in rat pituitary cells.

The effects of thyrotropin-releasing hormone (TRH) and 12-O-tetradecanoylphorbol 13-acetate (TPA) on cytosolic pH (pHi) were studied on GH4C1 pituitary cells loaded with the fluorescent pH indicator bis(carboxyethyl)carboxyfluorescein (BCECF) and the fluorescent Ca2+ indicator quin2. TRH, which was minimally effective at around 10(-9) M, and TPA, 100 nM, produced very small elevations in pHi of about 0.05 pH units from the normal basal resting pHi of GH4C1 cells of around 7.05. The effects were more marked after acid-loading the cells using 1 micrograms of nigericin/ml. Preincubation with amiloride or replacing the extracellular Na+ with choline+ completely blocked the elevations stimulated by TRH or TPA, consistent with an activation of the Na+/H+ antiport mechanism. The effects were completely independent of the cytoplasmic free calcium concentration ([Ca2+]i). The calcium ionophore ionomycin produced an elevation in [Ca2+]i with no concomitant effect on pHi, and amiloride, although completely inhibiting the pH change stimulated by TRH, failed to affect the initial stimulated [Ca2+]i transient. Although the data are consistent with an elevation in pHi by TRH which is caused by stimulation of a protein kinase C and subsequent activation of the antiporter, the rapidity of the onset of the pHi response to TRH could not be mimicked by a combination of TPA and ionomycin. These results, together with previous findings which show that secretion can be mimicked by TPA and ionomycin, suggest that TRH-stimulated Na+/H+ exchange plays no part in the acute stimulation of secretion, but that TRH increases the pH-sensitivity of the antiport system during increased synthesis of prolactin and growth hormone.     

Relationship between cytosolic Ca2+ concentration and amylase release in rat parotid acinar cells following muscarinic stimulation.

Carbachol (CCh), a muscarinic-cholinergic agonist, increased both cytosolic free calcium concentration ([Ca2+]i) and amylase release in rat parotid acinar cells or acini in a dose-dependent manner. Treatment of acinar cells with the intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), or the intracellular Ca2+ chelator, 1,2-bis(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA), strongly attenuated the increases in [Ca2+]i evoked by CCh, but amylase release from acini was not significantly suppressed by the treatment with TMB-8 or BAPTA. Low concentrations (0.02-0.5 microM) of ionomycin, a Ca2+ ionophore, caused increases in [Ca2+]i comparable to those induced by CCh, but the same concentrations had only a little effect on amylase release. The protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated amylase release in quantities similar to those induced by CCh, although TPA alone did not cause any change in [Ca2+]i. Combined addition of TPA and ionomycin potentiated only modestly amylase release stimulated by TPA alone. Staurosporine, a protein kinase C-inhibitor, similarly inhibited both the CCh- and TPA-induced amylase release. These results suggest that an increase in [Ca2+]i elicited by CCh does not play an essential role for inducing amylase release in rat parotid acini. Amylase release by muscarinic stimulation may be mediated mainly by activation of protein kinase C.     

Relationship of cytosolic ion fluxes and protein kinase C activation to platelet-derived growth factor induced competence and growth in BALB/c-3T3 cells

Platelet-derived growth factor (PDGF) and other agents that activate protein kinase C (PKC) rapidly alter cytosolic pH (pHi) and intracellular free calcium ([Ca++]i) in BALB/c-3T3 fibroblasts. To define whether changes in pHi or [Ca++]i are linked to PDGF-stimulated mitogenesis, these parameters were assessed in control and PKC depleted fibroblasts. PDGF addition to BALB/c-3T3 fibroblasts resulted in transient acidification of the cytoplasm followed by prolonged cytosolic alkalinization. Exposure of cells to 12-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester that activates PKC, resulted in cytosolic alkalinization without prior acidification. Overnight incubation with 600 nM TPA decreased the total cell PKC histone phosphorylating activity in BALB/c-3T3 fibroblasts by greater than 90%. In PKC-deficient fibroblasts, TPA, and PDGF-induced alkalinization was abolished. In addition, the transient drop in pHi seen initially in control cells treated with PDGF is sustained to the point where pHi is fully 0.6-0.7 pH units below control cell values for up to 30 minutes. PDGF increased [Ca++]i threefold; this transient rise in [Ca++]i was only minimally affected (less than 15%) by lowering of the extracellular calcium level with ethylene glycol bis(b-aminoethyl ether)0 N,N,N' tetraacetic acid (EGTA) or blocking calcium influx with CoCl2. In contrast, 8-(diethylamine)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an agent thought to inhibit calcium release from intracellular stores, substantially inhibited the rise in [Ca++]i caused by PDGF. TPA and 1-oleoyl-2-acetylglycerol (OAG) increased [Ca++]i but in contrast to PDGF this effect was blocked by pretreatment of cells with EGTA or CoCl2. In PKC-deficient fibroblasts, PDGF still increased [Ca++]i and stimulated DNA synthesis as effectively as in controls. TPA and OAG however, no longer increased [Ca++]i. The continued ability of PDGF to stimulate DNA synthesis in the face of sustained acidification and the absence of PKC activity suggests that cytosolic alkalinization and PKC activation are not essential for PDGF-induced competence in BALB/c-3T3 fibroblasts.     

Role of Ca2+ in activation of reactive oxygen species production in polymorphonuclear leukocytes during tumour growth in rats.

The role of Ca(2+) ions in PMA-induced generation of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNL) was studied during Zajdela hepatoma growth in the peritoneal cavity of rats. In PMNL from control healthy animals, a manifold Ca(2+)-induced enhancement of ROS generation and its significant reduction in the presence of Ca(2+) binding agent (BAPTA-AM) were observed. In contrast, ROS generation by PMNL from tumour-carrying animals dramatically increased in Ca(2+)-free medium, being practically insensitive to the agents, which can increase or decrease intracellular Ca(2+) levels. Free cytosolic Ca(2+) ([Ca(2+)](i)) in control PMNL was found to be relatively low ( approximately 250 nmol/L), rising slowly after Ca(2+) addition and further to two-fold in the presence of Ca(2+) and ionomycin in the incubating medium. Tumour growth in animals was accompanied with a significant [Ca(2+)](i) elevation. In Ca(2+)-free medium, [Ca(2+)](i) elevation was up to 480 nmol/L in tPMNL with the additions of Ca(+) and ionomycin as well as EGTA and ionomycin being able to increase [Ca(2+)](i) to 700-900 nmol/L onward. It was concluded that a higher Ca(2+) permeability of the plasma membrane and higher Ca(2+) accumulation in intracellular pools of PMNL was developed at the advanced stages of malignant disease. These results indicate the primed state of circulating PMNL and the independence of PMA-induced ROS generation at intra- and extracellular Ca(2+) levels at the advanced stages of tumour growth in animals.     

Activation of protein kinase C inhibits sodium fluoride-induced elevation of human platelet cytosolic free calcium and thromboxane B2 generation

Addition of NaF to washed platelets produces a dose-dependent and transient elevation of the intracellular free calcium concentration ([Ca++]i), thromboxane B2 (TxB2) generation and dense granule release, all of which are significantly inhibited when the extracellular calcium concentration ([Ca++]e) is reduced with EGTA. Inhibition of platelet cyclo-oxygenase by acetylsalicylic acid (ASA) does not affect NaF-induced elevation of [Ca++]i and dense granule release in the presence of 1 mM [Ca++]e. Pre-incubation of the platelets with the phorbol ester TPA produces a marked inhibition of NaF-induced elevation of [Ca++]i and TxB2 generation without affecting dense granule release. Thus, NaF may have more than one site of action. Pretreatment of the platelets with the selective protein kinase C inhibitor H7 prevents TPA induced inhibition of NaF mediated rise in [Ca++]i and TxB2 generation. Thus we propose that NaF induced calcium mobilisation is analogous to receptor-operated calcium mobilisation in platelets, as it is readily inhibited by protein kinase C activation or by the reduction of [Ca++]e and is independent of platelet cyclo-oxygenase activity.     

Proliferative effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophores on human large granular lymphocytes (LGL)

The proliferative responses of natural killer (NK) cells to 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly activates protein kinase c(PKC), and to the Ca2+ ionophores A23817 and ionomycin, known to enhance the intracellular calcium, have been investigated. Highly purified large granular lymphocytes (LGL) were cultured for 12-30 hr in the presence of TPA, ionomycin, or A23817. TPA alone (1-20 ng/ml) triggered rapid LGL proliferation, whereas the calcium ionophores were ineffective. The addition of either calcium ionophore to suboptimal doses or TPA (0.1-0.5 ng/ml) resulted in a synergistic effect on LGL proliferation. Under these conditions high levels of IL-2 activity were released by the LGL. Phenotypic analysis revealed the rapid loss of the Fc gamma receptors (CD16) on LGL and the induction of the expression of IL-2 (CD25) and transferrin receptors and of HLA-DR, but not of CD3. Removal of extracellular Ca2+ by addition of EGTA at the beginning of the culture greatly depressed LGL proliferation and IL-2 production, and blocked phenotypic changes, such as the expression of Tac antigen. Finally, progression to the proliferative phase of LGL, activated by TPA alone or with ionomycin, was completely abrogated by a hyperimmune anti-IL-2 antiserum.     

Intracellular Ca2+ stores are essential for injury induced Ca2+ signaling and re-endothelialization

Endothelialization repairs the lining of damaged vasculature and is a key process in preventing thrombosis and restenosis. It has been demonstrated that extracellular calcium ([Ca2+](o)) influx is important for subsequent endothelialization. The role of intracellular Ca2+ stores in mechanical denudation induced intracellular calcium ([Ca2+](i)) rise and endothelialization remains to be demonstrated. Using monolayer culture of a human endothelial cell line (human umbilical vein endothelial cell, HUVEC), we investigated [Ca2+](i) wave propagation and re-endothelialization following mechanical denudation. Consistent with previous reports for other types of cells, mechanical denudation induces calcium influx, which is essential for [Ca2+](i) rise and endothelialization. Moreover, we found that intracellular Ca(2+) stores are also essential for denudation induced [Ca2+](i) wave initiation and propagation, and the subsequent endothelialization. Thapsigargin which depletes intracellular Ca2+ stores completely abolished [Ca2+](i) wave generation and endothelialization. Xestospongin C (XeC), which prevents Ca2+ release from intracellular Ca2+ stores by inhibition of inositol 1,4,5-trisphosphate (IP(3)) receptor, inhibited intercellular Ca2+ wave generation and endothelialization following denudation. Purinergic signaling through a suramin sensitive mechanism and gap junction communication also contribute to in intercellular Ca(2+) wave propagation and re-endothelialization. We conclude that intracellular Ca2+ stores, in addition to extracellular Ca2+, are essential for intracellular Ca2+ signaling and subsequent endothelialization following mechanical denudation.     

Phorbol esters modulate thrombin-operated calcium mobilisation and dense granule release in human platelets

Human alpha-thrombin-induced elevation of cytosolic free calcium ([Ca2+]i) and dense granule release was examined in platelets preincubated with either activators or an inhibitor of protein kinase C. 12-O-Tetradecanoylphorbol 13-acetate (TPA) or two 12-deoxy analogues of TPA, when added alone to platelets, did not elevate [Ca2+]i, as monitored by quin2 fluorescence, though small amounts of dense granule release were detected. Preincubation of the platelets with either TPA or 12-deoxyphorbol 13-phenylacetate, but not the parent, 4-beta-phorbol, produced a dose-dependent inhibition of the elevation of [Ca2+]i and 5-hydroxytryptamine release induced by human alpha-thrombin. Furthermore, this phorbol ester-mediated inhibition of human alpha-thrombin-induced activation could be prevented by H7 (1-[5-isoquinolinesulphonyl]-2-methylpiperazine), the recently described inhibitor of protein kinase C. These results suggest a role for protein kinase C as a modulator of receptor-operated calcium fluxes in human platelets.     

Intercellular communication of transformed and non-transformed rat liver epithelial cells : Modulation by TPA

Gap-junctional intercellular communication of transformed and non-transformed rat liver epithelial cell lines was compared using a dye transfer method in the presence and absence of 12-O-tetradecanoylphorbol 13-acetate (TPA). Whereas non-transformed cells (IAR 20, non-tumorigenic in newborn rats and in nude mice) showed very high communication capacity throughout a culture period of 3 weeks, transformed cells (IAR 6-1, tumorigenic in newborn rats and in nude mice) were less able to communicate. Similar correlation between intercellular communication and expression of transformed phenotypes were also found in newly cloned epithelial cell lines, IAR 27 E and IAR 27F. When TPA was added to culture medium at 100 ng/ml, intercellular communication in all lines tested was reduced within 60 min. However, communication recovered completely from the effect within 10 h after addition of TPA. Further addition of TPA to the cultures every 24 h for 3 weeks had no effect on intercellular communication (measured 30 min after each TPA addition), suggesting that a single application of TPA made these cells refractory to further doses. A known stimulator of gap-junctional communication, db-cAMP, also increased dye transfer in IAR 20 and IAR 6-1 cells. TPA added to db-cAMP-treated cultures of IAR 20 and IAR 6-1 cells inhibited intercellular communication, suggesting that cAMP is not an antagonist of the effect of TPA on intercellular communication in these cell lines. These results are in sharp contrast to those obtained with the fibroblast cell line BALB/c 3T3, in which db-cAMP antagonized TPA effect [1] and inhibition by TPA of intercellular communication was transient only when administered during their growth phase, and was stable and continuous when TPA was applied at confluence [2], and suggest that TPA may not be an effective tumour promoter in rat liver.     

A role for protein kinase C during rat egg activation

Upon sperm-egg interaction, an increase in intracellular calcium concentration ([Ca(2+)](i)) is observed. Several studies reported that cortical reaction (CR) can be triggered not only by a [Ca(2+)](i) rise but also by protein kinase C (PKC) activation. Because the CR is regarded as a Ca(2+)-dependent exocytotic process and because the calcium-dependent conventional PKCs (cPKC) alpha and beta II are considered as exocytosis mediators in various cell systems, we chose to study activation of the cPKC in the rat egg during in vivo fertilization and parthenogenetic activation. By using immunohistochemistry and confocal microscopy techniques, we demonstrated, for the first time, the activation of the cPKC alpha, beta I, and beta II during in vivo fertilization. All three isozymes examined presented translocation to the egg's plasma membrane as early as the sperm-binding stage. However, the kinetics of their translocation was not identical. Activation of cPKC alpha was obtained by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) or by 1-oleoyl-2-acetylglycerol (OAG) but not by the calcium ionophore ionomycin. PKC alpha translocation was first detected 5-10 min after exposure to TPA and reached a maximum at 20 min, whereas in eggs activated by OAG, translocation of PKC alpha was observed almost immediately and reached a maximum within 5 min. These results suggest that, although [Ca(2+)](i) elevation on its own does not activate PKC alpha, it may accelerate OAG-induced PKC alpha activation. We also demonstrate a successful inhibition of the CR by a myristoylated PKC pseudosubstrate (myrPKCPsi), a specific PKC inhibitor. Our study suggests that exocytosis can be triggered independently either by a [Ca(2+)](i) rise or by PKC.     

Prolactin induces calcium influx and release from intracellular pools in human T lymphocytes by activation of tyrosine kinases

The early events related to intracellular signals after prolactin (PRL) activation in T lymphocytes are not clearly established. The aim of this work was to study the effect of PRL in cytosolic calcium levels in human T lymphocytes. By using the dye FURA-2 AM, the variations in cytosolic Ca(2+) were studied in peripheral human T lymphocytes isolated from extracted blood from healthy donors. Fifty nanograms per milliliter PRL induces a small increase in cytosolic calcium. When the cells are preincubated overnight (16-20 h) in the presence of PRL, the increase in calcium is higher. This high increase is due to the release from intracellular pools and to the influx from the extracellular media. That is, after overnight incubation with PRL, calcium influx in T cells follows the capacitative model. Since PRL receptor (PRL-R) activation involves the tyrosine kinase pathway, we check calcium effect in the presence of genistein, a known inhibitor of tyrosine kinases. When cells are preincubated in the presence of 10 microM genistein, and PRL is immediately added, no increase in cytosolic calcium is observed. The presence of genistein also completely blocks the increase in cytosolic calcium stimulated by PRL after overnight incubation with PRL. In the presence of PRL and N,N-dimethyl-D-erythro-sphingosine (DMS), a stimulus that increases cytosolic calcium in T cells by tyrosine kinase stimulation, a high, even insignificant, calcium influx is induced. However, when the cells are incubated overnight in the presence of PRL, and then DMS is added, a significant increase in cytosolic calcium levels takes place. This increase is associated with an increase in calcium release from intracellular pools and an increase in calcium uptake. Genistein reduces the influx of external calcium induced by DMS after short incubation with PRL and significantly inhibits both, calcium pools empty, and calcium influx is induced by DMS after overnight incubation with PRL. In summary, PRL induces calcium influx in normal T lymphocytes. The influx is magnified after long PRL exposures, intracellular Ca(2+) pool-dependent, and activated through tyrosine kinases.     

Importance of bicarbonate transport for protection of cardiomyocytes against reoxygenation injury

Isolated cardiomyocytes from adult rats were incubated in anoxic bicarbonate-buffered media at extracellular pH (pH(o)) 6.4 until a cytosolic Ca(2+) overload and intracellular pH (pH(i)) of 6.4 were reached. On reoxygenation, the pH of the medium was changed to 7.4 to activate the Na(+)/H(+)exchanger (NHE) and the Na(+)-HCO(-)(3) symporter (NBS). The reoxygenation was performed in the absence or presence of the NHE inhibitor HOE-642 (3 micromol/l) and/or the NBS inhibitor DIDS (0.5 mmol/l), as in bicarbonate-free media. In reoxygenated control cells pH(i) rapidly recovered to the preanoxic level, and a burst of spontaneous oscillations of cytosolic Ca(2+) occurred, accompanied by the development of hypercontracture. When NBS and NHE were simultaneously inhibited during reoxygenation, pH(i) recovery was prevented, Ca(2+) oscillations were attenuated, and hypercontracture was abolished. Sole inhibition of NBS or NHE showed no protection against hypercontracture. In the absence of cytosolic acidosis, HOE-642 or DIDS did not prevent hypercontracture induced by Ca(2+) overload. The results demonstrate that simultaneous inhibition of NHE and NBS is needed to protect myocardial cells against reoxygenation-induced hypercontracture.     

Ouabain-induced enhancement of rat mast cells response. Modulation by protein phosphorylation and intracellular pH

The digitalic glicoside ouabain induces potentiation of rat mast cell histamine release in response to several stimuli, which is mediated by Na+/Ca2+ exchanger. In this work, we studied the effect of ouabain on cytosolic calcium, intracellular pH and histamine release with Ca2+ ionophore A23187 in conditions designed to maximize ouabain-induced potentiation of rat mast cells response. The effect of protein kinase C (PKC), cAMP and phosphatase inhibition was also tested. Ouabain induced an enhancement in histamine release, cytosolic calcium and intracellular pH. The adenylate cyclase activator forskolin reduced the effect of ouabain on histamine release and intracellular pH, but enhanced the effect on cytosolic calcium. PKC activator PMA enhanced the effect of ouabain on histamine release and cytosolic calcium, without affecting intracellular pH. A PKC inhibitor, GF-109203X, reduced ouabain-induced enhancement of histamine release and intracellular pH, but increased the enhancement on cytosolic calcium. Finally, inhibition of protein phosphatases 1 and 2A with okadaic acid, increased the effect of ouabain on histamine release and intracellular pH, but reduced cytosolic calcium in presence of ouabain. This result suggest that ouabain-induced potentiation of rat mast cell histamine release with A23187 is modulated by kinases, and this modulation may be carried out by changes in intracellular alkalinization. However, the mechanism underlying cellular alkalinization remains to be elucidated.     

Intracellular alkalinization induces Ca2+ influx via non-voltage-operated Ca2+ channels in rat aortic smooth muscle cells

In smooth muscle, the cytosolic Ca2+ concentration ([Ca2+](i)) is the primary determinant of contraction, and the intracellular pH (pH(i)) modulates contractility. Using fura-2 and 2',7'-biscarboxyethyl-5(6) carboxyfluorescein (BCECF) fluorometry and rat aortic smooth muscle cells in primary culture, we investigated the effect of the increase in pH(i) on [Ca2+](i). The application of the NH(4)Cl induced concentration-dependent increases in both pH(i) and [Ca2+](i). The extent of [Ca2+](i) elevation induced by 20mM NH(4)Cl was approximately 50% of that obtained with 100mM K(+)-depolarization. The NH(4)Cl-induced elevation of [Ca2+](i) was completely abolished by the removal of extracellular Ca2+ or the addition of extracellular Ni2+. The 100mM K(+)-induced [Ca2+](i) elevation was markedly inhibited by a voltage-operated Ca2+ channel blocker, diltiazem, and partly inhibited by a non-voltage-operated Ca2+ channel blocker, SKF96365. On the other hand, the NH(4)Cl-induced [Ca2+](i) elevation was resistant to diltiazem, but was markedly inhibited by SKF96365. It is thus concluded that intracellular alkalinization activates the Ca2+ influx via non-voltage-operated Ca2+ channels and thereby increases [Ca2+](i) in the vascular smooth muscle cells. The alkalinization-induced Ca2+ influx may therefore contribute to the enhancement of contraction.     

Intracellular calcium regulation of connexin43

The mechanism by which intracellular Ca(2+) concentration ([Ca(2+)](i)) regulates the permeability of gap junctions composed of connexin43 (Cx43) was investigated in HeLa cells stably transfected with this connexin. Extracellular addition of Ca(2+) in the presence of the Ca(2+) ionophore ionomycin produced a sustained elevation in [Ca(2+)](i) that resulted in an inhibition of the cell-to-cell transfer of the fluorescent dye Alexa fluor 594 (IC(50) of 360 nM Ca(2+)). The Ca(2+) dependency of this inhibition of Cx43 gap junctional permeability is very similar to that described in sheep lens epithelial cell cultures that express the three sheep lens connexins (Cx43, Cx44, and Cx49). The intracellular Ca(2+)-mediated decrease in cell-to-cell dye transfer was prevented by an inhibitor of calmodulin action but not by inhibitors of Ca(2+)/calmodulin-dependent protein kinase II or protein kinase C. In experiments that used HeLa cells transfected with a Cx43 COOH-terminus truncation mutant (Cx43(Delta257)), cell-to-cell coupling was similarly decreased by an elevation of [Ca(2+)](i) (IC(50) of 310 nM Ca(2+)) and similarly prevented by the addition of an inhibitor of calmodulin. These data indicate that physiological concentrations of [Ca(2+)](i) regulate the permeability of Cx43 in a calmodulin-dependent manner that does not require the major portion of the COOH terminus of Cx43.     

Cytosolic zinc release and clearance in hippocampal neurons exposed to glutamate--the role of pH and sodium

Although Zn(2+) homeostasis in neurons is tightly regulated and its destabilization has been linked to a number of pathologies including Alzheimer's disease and ischemic neuronal death, the primary mechanisms affecting intracellular Zn(2+) concentration ([Zn(2+) ](i)) in neurons exposed to excitotoxic stimuli remain poorly understood. The present work addressed these mechanisms in cultured hippocampal neurons exposed to glutamate and glycine (Glu/Gly). [Zn(2+)](i) and intracellular Ca(2+) concentration were monitored simultaneously using FluoZin-3 and Fura-2FF, and intracellular pH (pH(i)) was studied in parallel experiments using 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Glu/Gly applications under Na(+)-free conditions (Na(+) substituted with N-methyl-D-glucamine(+)) caused Ca(2+) influx, pH(i) drop, and Zn(2+) release from intracellular stores. Experimental maneuvers resulting in a pH(i) increase during Glu/Gly applications, such as stimulation of Na(+) -dependent pathways of H(+) efflux, forcing H(+) efflux via gramicidin-formed channels, or increasing extracellular pH counteracted [Zn(2+)](i) elevations. In the absence of Na(+), the rate of [Zn(2+)](i) decrease could be correlated with the rate of pH(i) increase. In the presence of Na(+), the rate of [Zn(2+) ](i) decrease was about twice as fast as expected from the rate of pH(i) elevation. The data suggest that Glu/Gly-induced cytosolic acidification promotes [Zn(2+) ](i) elevations and that Na(+) counteracts the latter by promoting pH(i)-dependent and pH(i)-independent mechanisms of cytosolic Zn(2+) clearance.     

Analysis of calcium homeostasis in activated human polymorphonuclear leukocytes. Evidence for two distinct mechanisms for lowering cytosolic calcium

The stimulation of polymorphonuclear leukocytes (PMNs) by chemoattractants triggers a rapid rise in cytosolic free calcium concentration(s) ([Ca2+]i), which quickly returns to base line, suggesting a role for calcium removal in the homeostasis of activated PMNs. To investigate cytosolic calcium homeostasis, PMNs were treated with a fluoroprobe and ionomycin to induce a sustained elevation of [Ca2+]i. The cells were then stimulated, and attenuation of the fluorescence signal was measured as an indication of calcium loss from the cytosol. The formyl peptide chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), and 1,2-dioctanoyl-sn-glycerol, but not the inactive phorbol ester 4 alpha-phorbol didecanoate, induced a dose-dependent decrease in [Ca2+]i in ionomycin-pretreated cells. However, the decline in [Ca2+]i caused by PMA was sustained and occurred following a lag time, whereas the response to fMLP was immediate, lasted approximately 2 min, and then was followed by a return of [Ca2+]i to its initial level. The restoration of [Ca2+]i required extracellular calcium. Varying the ionomycin concentration allowed studies at different initial [Ca2+]i, which in untreated PMNs was approximately 135 nM. In contrast to fMLP, PMA did not lower calcium at concentrations below 200 nM. The decline in [Ca2+]i induced by fMLP, but not PMA, was blocked by pertussis toxin. In contrast, the decrease in [Ca2+]i caused by PMA and 1,2-dioctanoyl-sn-glycerol, but not fMLP, was inhibited by the protein kinase C antagonists staurosporine, H-7, and sphingosine. These results suggest that formyl peptide chemoattractants transiently stimulate an activity which lowers [Ca2+]i to normal intracellular levels. Activation of this process appears to be independent of protein kinase C. An additional cytosolic calcium lowering activity, dependent on protein kinase C, operates at [Ca2+]i above 200 nM. Thus, activated PMNs can use at least two processes for attentuation of elevated cytosolic calcium levels.     

Local Ca2+ rise near store operated Ca2+ channels inhibits cell coupling during capacitative Ca2+ influx

Using a new fluorescence imaging technique, LAMP, we recently reported that Ca(2+) influx through store operated Ca(2+) channels (SOCs) strongly inhibits cell coupling in primary human fibroblasts (HF) expressing Cx43. To understand the mechanism of inhibition, we studied the involvement of cytosolic pH (pH(i)) and Ca(2+)([Ca(2+)](i)) in the process by using fluorescence imaging and ion clamping techniques. During the capacitative Ca(2+) influx, there was a modest decline of pH(i) measured by BCECF. Decreasing pH(i) below neutral using thioacetate had little effect by itself on cell coupling, and concomitant pH(i) drop with thioacetate and bulk [Ca(2+)(i) rise with ionomycin was much less effective in inhibiting cell coupling than Ca(2+) influx. Moreover, clamping pH(i) with a weak acid and a weak base during Ca(2+) influx largely suppressed bulk pH(i) drop, yet the inhibition of cell coupling was not affected. In contrast, buffering [Ca(2+)(i) with BAPTA, but not EGTA, efficiently prevented cell uncoupling by Ca(2+) influx. We concluded that local Ca(2+) elevation subjacent to the plasma membrane is the primary cause for closing Cx43 channels during capacitative Ca(2+) influx. To assess how Ca(2+) influx affects junctional coupling mediated by other types of connexins, we applied the LAMP assay to Hela cells expressing Cx26. Capacitative Ca(2+) influx also caused a strong reduction of cell coupling, suggesting that the inhibitory effect by Ca(2+) influx may be a more general phenomenon.     

Functional characterization of thapsigargin and agonist-insensitive acidic Ca2+ stores in Drosophila melanogaster S2 cell lines

The role of acidic intracellular calcium stores in calcium homeostasis was investigated in the Drosophila Schneider cell line 2 (S2) by means of free cytosolic calcium ([Ca2+]i) and intracellular pH (pHi) imaging together with measurements of total calcium concentrations within intracellular compartments. Both a weak base (NH4Cl, 15 mM) and a Na+/H+ ionophore (monensin, 10 microM) evoked cytosolic alkalinization followed by Ca2+ release from acidic intracellular Ca2+ stores. Pretreatment of S2 cells with either thapsigargin (1 microM), an inhibitor of endoplasmic reticulum Ca(2+)-ATPases, or with the Ca2+ ionophore ionomycin (10 microM) was without effect on the amplitude of Ca2+ release evoked by alkalinization. Application of the cholinergic agonist carbamylcholine (100 microM) to transfected S2-DM1 cells expressing a Drosophila muscarinic acetylcholine receptor (DM1) emptied the InsP3-sensitive Ca2+ store but failed to affect the amplitude of alkalinization-evoked Ca2+ release. Glycyl-L-phenylalanine-beta-naphthylamide (200 microM), a weak hydrophobic base known to permeabilize lysosomes by osmotic swelling, triggered Ca2+ release from internal stores, while application of brefeldin A (10 microM), an antibiotic which disperses the Golgi complex, resulted in a smaller increase in [Ca2+]i. These results suggest that the alkali-evoked calcium release is largely attributable to lysosomes, a conclusion that was confirmed by direct measurements of total calcium content of S2 organelles. Lysosomes and endoplasmic reticulum were the only organelles found to have concentrations of total calcium significantly higher than the cytosol. However, NH4Cl (15 mM) reduced the level of total calcium only in lysosomes. Depletion of acidic Ca2+ stores did not elicit depletion-operated Ca2+ entry. They were refilled upon re-exposure of cells to normal saline ([Ca2+]o = 2 mM), but not by thapsigargin-induced [Ca2+]i elevation in Ca(2+)-free saline.