Otoneurologic and audiologic findings in fibromyalgia

In this study we developed a single cell analysis protocol with which protein tyrosine kinase (PTK)-dependent and independent Ca2+ fluxes occurring in human spermatozoa in response to progesterone were evaluated. By recording the fluorescence emitted by fluo-3-loaded spermatozoa using a confocal laser scanning microscopy system it was possible not only to monitor relative changes in the intracellular free Ca2+ concentration ([Ca2+]i) but also to determine the time at which the acrosomal exocytosis began. The addition of progesterone produced a rapid transient [Ca2+]i increase in 35% of spermatozoa. In approximately 10% of spermatozoa, this initial [Ca2+]i increase was followed by a secondary [Ca2+]i increase beginning 2-10 min after the progesterone addition and leading to the acrosomal exocytosis in most of these spermatozoa. On the other hand, a rapid triggering of exocytosis during the initial [Ca2+]i increase was a relatively infrequent observation. The inhibition of PTK with genistein or herbimycin A did not influence the initial progesterone-induced [Ca2+]i increase but inhibited the secondary [Ca2+]i increase and the ensuing acrosomal exocytosis. The initial PTK-independent Ca2+ response could be induced by progesterone in both non-capacitated and capacitated spermatozoa, whereas the ability to generate the secondary, PTK-dependent response developed during in-vitro capacitation.     

Diverse signal transduction pathways mediated by endogenous P2 receptors in cultured rat cerebral cortical neurons

The present study was conducted to assess the intracellular signaling pathways mediated by receptors for ATP, uridine triphosphate (UTP), and 2-methylthio ATP (2-MeSATP), by monitoring patch-clamp currents and intracellular calcium mobilization in cultured rat cortical cerebral neurons. All three agonists evoked potassium currents and increased the intracellular free Ca2+ concentration ([Ca2+]i), and these effects were inhibited by the broad G-protein inhibitor guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS) but not by the Gi/o-protein inhibitor pertussis toxin (PTX). UTP-evoked currents were inhibited by either the phospholipase C inhibitor neomycin or the selective protein kinase C (PKC) inhibitor GF109203X, and the rise in cytosolic Ca2+ was inhibited by either neomycin or the inositol 1,4,5-trisphosphate (IP3) receptor antagonist heparin, indicating that the UTP receptor involved phospholipase C-mediated phosphatidylinositol signaling. In contrast, 2-MeSATP-induced currents and rise in cytosolic Ca2+ were not inhibited by either neomycin, or GF109203X, or heparin. 2-MeSATP elicited single-channel currents in the cell-attached patch-clamp configuration and also in excised patches. The G-protein activator GTP gamma S induced single-channel currents in a fashion that mimicked the effect of 2-MeSATP. These data suggest that 2 MeSATP activated potassium channels by a direct action of G-protein beta gamma subunits and increased [Ca2+]i by a mechanism independent of phospholipase C stimulation and IP3 production. ATP-evoked currents were partially inhibited by either neomycin or GF109203X, although the rise in cytosolic Ca2+ was not affected by these inhibitors. ATP produced single-channel currents with two major classes of the slope conductance (86 and 95 pS) in cell-attached patches, each of which is consistent with that achieved by 2-MeSATP (85 pS) or UTP (96 pS); the currents with the lower conductance were observed in the outside-out patch-clamp configuration. These results indicate that P2 receptors for UTP and 2-MeSATP are linked to a PTX-insensitive G-protein involving different signal transduction pathways and that ATP responses are mediated by both of these P2 receptors.     

Cytoprotection and regulation of heat shock proteins induced by heat shock in human breast cancer T47-D cells: role of [Ca2+]i and protein kinases

Overexpression of heat shock protein 70 kDa alters the susceptibility of tumor cells to chemotherapeutic agents. We conducted experiments to study the regulation of expression of heat shock proteins (HSPs) in heat shock-treated T47-D cells, a human breast cancer cell line that expresses estrogen receptors. Cells exposed to heat shock at 44 degreesC displayed increased expression of heat shock protein 72 kDa (HSP-72), glucose-regulated protein 78 kDa (GRP-78), and GRP-94 in a time-dependent manner, as shown by [35S]methionine incorporation and Western blotting experiments. The maximal rate of synthesis occurred between 2 and 4 h after heat shock. Removal of external Ca2+ inhibited the synthesis of the heat shock-induced GRP-78 but not of HSP-72 and GRP-94, whereas treatment of cells with BAPTA (a Ca2+ chelator) inhibited HSP-72 and GRP-78. Treatment with H89 (a protein kinase A inhibitor) blocked the heat shock-induced GRP-78 synthesis, whereas GF-109203X (a protein kinase C inhibitor) attenuated the heat shock-induced HSP-72 synthesis and completely blocked synthesis of GRP-78 but not of GRP-94. These results indicate that protein kinase C is involved in regulation of the heat shock-induced synthesis of HSP-72, whereas PKA and PKC are involved in the regulation of GRP-78 synthesis. Cells overexpressing HSP-72 and GRPs after heat shock displayed resistance against lethal temperature (47 degreesC for 50 min) -induced death, which was diminished after removal of external Ca2+ and treatment with GF-109203X. Heat shock increased intracellular free Ca2+ concentration ([Ca2+]i) in a temperature- and heating duration-dependent fashion, and the increase was inhibited in the absence of external [Ca2+]i and significantly reduced by pretreatment with H89 and GF-109203X. The results suggest that different pathways are involved in the induction of synthesis of HSP-72, GRP-78, and GRP-94 by heat shock. It is highly likely that only HSP-72 and GRP-78 are involved in the process of cytoprotection from the thermal injury.     

Activation of the Ca2+ message system by parathyroid hormone is dependent on the cell cycle

To verify whether the heterogeneous intracellular calcium ([Ca2+]i) responses to PTH observed in the UMR 106-01 osteogenic sarcoma cells are secondary to cell cycle asynchrony or to genotypic differences within the population, we synchronized cell monolayers at the G1/S boundary using a sequential thymidine-aphidicolin block. Video image analysis of fura-2-loaded cells revealed that PTH (10(-7) M) induced transient increases of [Ca2+]i preferentially in cells in S phase (82% response frequency, n = 63; 286 +/- 33% of baseline, n = 29), whereas cells in G1 phase responded poorly to PTH (10% response frequency, n = 51; 140 +/- 8% of baseline, n = 5). In contrast, cell exposure to 2% fetal calf serum was followed by [Ca2+]i transients in 83% (n = 42) of cells in G1 phase, but in only 25% (n = 63) of cells in S phase, with similar response amplitude. Hormonal responsiveness was heterogeneous in small clones obtained from single UMR 106-01 cells, with response frequency similar to that observed in nonsynchronized cultures. Pretreatment with either La3+, nifedipine, or pertussis toxin reduced both frequency and amplitude of PTH response in S phase to levels close to G1 phase, whereas there was no significant difference in inositol trisphosphate generated by PTH stimulation in either phase. Therefore, the heterogeneous [Ca2+]i responses of UMR 106-01 cells to hormonal stimulation is dependent on the phase of the cell cycle, rather than on genotypic heterogeneity. The switch from the G1 to the S phase mode of response is driven by active coupling between the PTH receptor and a Ca2+ channel through a pertussis toxin-sensitive G protein.     

Effects of vasopressin and involvement of receptor subtypes in the rat central amygdaloid nucleus in vitro

Effects of arginine-vasopressin (AVP) on neurons in the central amygdaloid nucleus (ACe) were investigated with rat brain slice preparations using extracellular recording methods. Of 160 ACe neurons tested, 70 cells (44%) were excited and 9 cells (6%) were inhibited by bath application of AVP at 3 x 10(-7) M. The excitatory effects of AVP were dose-dependent and the threshold concentration was approximately 10(-10) to 10(-9) M. The excitatory effects of AVP persisted under blockade of synaptic transmission by perfusing with Ca2+-free and high-Mg2+ medium, whereas the inhibitory effects were abolished by synaptic blockade. AVP-induced effects were mimicked by a V1-receptor agonist and completely blocked by a selective V1-antagonist. V2-agonist produced no effects on ACe neurons and V2-antagonist had no effect on AVP-induced excitation. These results showed that the excitatory effect of AVP on ACe neurons was produced by a direct action through the V1-receptors, whereas the inhibitory response of ACe neurons to AVP seemed to be produced by an indirect action. The results of this study suggest that AVP is involved in the amygdala-relevant functions as a neurotransmitter or a neuromodulator.     

Involvement of extracellular and intracellular calcium sources in TRH-induced alpha-MSH secretion from frog melanotrope cells

The stimulatory effect of thyrotropin-releasing hormone (TRH) on alpha-melanocyte stimulating hormone (MSH) secretion from the frog pars intermedia is mediated through the phospholipase C (PLC) pathway but requires extracellular Ca2+. The aim of the present study was to investigate the respective contribution of extracellular and intracellular Ca2+ in the action of TRH on cytosolic calcium concentration ([Ca2+]i) and alpha-MSH release. In normal conditions, TRH (10(-7) M; 5 s) evoked two types of Ca2+ responses: in 63% of the cells, TRH caused a sustained and biphasic increase in [Ca2+]i while in 37% of the cells, TRH only induced a transient response. In the presence of EGTA or Ni2+, the stimulatory effect of TRH on [Ca2+]i and alpha-MSH secretion was totally suppressed. Nifedipine (10(-6) M) reduced by approximately 50% the amplitude of the two types of Ca2+ responses whereas omega-conotoxin GVIA (10(-7) M) suppressed the plateau-phase of the sustained response indicating that the activation of L-type Ca2+-channels (LCC) is required for initiation of the Ca2+ response while N-type Ca2+-channels (NCC) are involved in the second phase of the response. Paradoxically, neither nifedipine nor omega-conotoxin GVIA had any effect on TRH-induced alpha-MSH secretion. The PLC inhibitor U-73122 (10(-6) M) significantly reduced the transient increase in [Ca2+]i and totally suppressed the sustained phase of the Ca2+ response but had no effect on TRH-induced alpha-MSH secretion. The stimulatory effect of TRH on PLC activity was not effected by nifedipine and omega-conotoxin GVIA but was abolished in Ca2+-free medium. Ryanodine had no effect on the TRH-induced stimulation of [Ca2+]i and alpha-MSH secretion. Concomitant administration of nifedipine/omega-conotoxin GVIA or U-73122/omega-conotoxin GVIA markedly reduced the response to TRH but did not affect TRH-evoked alpha-MSH release. In contrast, concomitant administration of U-73122 and nifedipine significantly reduced the effect of TRH on both [Ca2+]i and alpha-MSH release. Taken together, these data indicate that, in melanotrope cells, activation of TRH receptors induces an initial Ca2+ influx through nifedipine- and omega-conotoxin-insensitive, Ni2+-sensitive Ca2+-channels which subsequently activates LCC and causes Ca2+ mobilization from intracellular pools by enhancing PLC activity. Activation of the PLC causes Ca2+ entry through NCC which is responsible for the plateau-phase of sustained Ca2+ response. Although nifedipine and U-73122, separately used, were devoid of effect on secretory response, Ca2+ entry through LCC and mobilization of intracellular Ca2+ are both involved in TRH-evoked alpha-MSH release because only one source of Ca2+ is sufficient for inducing maximal hormone release. In contrast, the Ca2+ influx through NCC does not contribute to TRH-induced alpha-MSH secretion.     

Role of PKC in the effects of alpha1-adrenergic stimulation on Ca2+ transients, contraction and Ca2+ current in guinea-pig ventricular myocytes

The effects of alpha1-adrenoceptor stimulation on intracellular Ca2+ transients, contractility and L-type Ca2+ current (ICa,L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanisms of the positive inotropic effect of alpha1-adrenergic stimulation by focussing on the role of protein kinase C (PKC). Phenylephrine, an alpha1-adrenergic agonist, at concentrations of 50-100 microM elicited a biphasic inotropic response: a transient negative inotropic response (22.9+/-6.0% of control) followed by a sustained positive inotropic response (61.0+/-8.4%, mean+/-SE, n=12). The Ca2+ transient decreased by 10.2+/-3.9% during the negative inotropic phase, while it increased by 67.7+/-10% (n=12) during the positive inotropic phase. These effects were inhibited by prazosin (1 microM), a alpha1-adrenergic antagonist. Phenylephrine increased the ICa,L by 60.8+/-21% (n=5) during the positive inotropic phase. To determine whether activation of PKC is responsible for the increases in Ca2+ transients, contractile amplitude and ICa,L during alpha1-adrenoceptor stimulation, we tested the effects of 4beta-phorbol 12-myristate 13-acetate (PMA), a PKC activator, and of bisindolylmaleimide I (GF109203X) and staurosporine, both of which are PKC inhibitors. PMA mimicked phenylephrine's effects on Ca2+ transients, contractile amplitude and ICa,L. PMA (100 nM) increased the Ca2+ transient, contractile amplitude and ICa,L by 131+/-17%, 137+/-25% (n=8), and 81.1+/-26% (n=5), respectively. Prior exposure to GF109203X (1 microM) or staurosporine (10 nM) prevented the phenylephrine-induced increases in Ca2+ transients, contractile amplitude and ICa,L. Our study suggests that during alpha1-adrenoceptor stimulation increase in ICa,L via PKC causes an increase in Ca2+ transients and thereby in the contractile force of the ventricular myocytes.     

ATP-regulated K+ channel and cytosolic Ca2+ mobilization in cultured rat spinal neurons

ATP activated the K+ channel responsible for outwardly rectifying currents via a P2Y purinoceptor linked to a pertussis toxin-insensitive G-protein in cultured rat spinal neurons. The evoked currents were inhibited by a selective protein kinase C inhibitor, GF109203X, whereas a phospholipase C inhibitor, neomycin had no effect. These indicate that the currents are regulated by phospholipase C-independent protein kinase C activation. In addition, ATP enhanced intracellular free Ca2+ concentration. The increase in intracellular free Ca2+ concentration was inhibited by a broad G-protein inhibitor, GDP beta S, but not affected by neomycin or an inositol 1,4,5-triphosphate receptor antagonist, heparin, suggesting that the cytosolic Ca2+ mobilization is regulated by a mechanism independent of a phospholipase C-mediated phosphatidylinositol signaling. These results, thus, demonstrate that ATP has dual actions on the coupled K+ channel and cytosolic Ca2+ release.     

Thrombin and phorbol esters potentiate Gs-mediated cAMP formation in intact human erythroid progenitors via two synergistic signaling pathways converging on adenylyl cyclase type VII

In intact, but not in permeabilized, human erythroid progenitor cells, thrombin and phorbol esters potentiate cellular cAMP formation in response to Gs-coupled receptor agonists such as prostaglandin E1 (PGE1). We show here that the two agonists achieve their phenotypically similar effects by using distinctly different signaling pathways, both of which require protein kinase C (PKC) activation. After short term exposure (11 min), phorbol esters caused an alkaline shift of cellular pH by approximately 0.1 unit, resulting in a 1.5-2-fold increase in PGE1-induced cAMP formation. The effect of phorbol esters was inhibited by 5-(N-ethyl-N-isopropyl)amiloride, a specific inhibitor of the Na+/H+ exchanger, and by the PKC inhibitors GF 109203X, G? 6976, and staurosporine. Thrombin increased cellular pH by only 0.02-0.05 unit but seemed to potentiate PGE1-stimulated cAMP formation by an effect on the Gs-activated adenylyl cyclase involving a Ca2+-independent (novel) PKC. This effect was inhibited by GF 109203X and staurosporine but was resistant to 5-(N-ethyl-N-isopropyl)amiloride or G? 6976. Inactivation of PKC by incubation of the cells in the presence of 10 nM phorbol-12-myristate-13-acetate for 18 hr completely abolished the potentiating effect of thrombin on cyclase activity, whereas the pH-dependent stimulation was fully retained. Northern blots with specific cDNA probes and a lack of Ca2+ sensitivity indicate that progenitor cells predominantly express adenylyl cyclase type VII. Our results suggest that in normal human erythroid progenitors, thrombin can activate pH-dependent and -independent, PKC-linked pathways converging on adenylyl cyclase type VII to potentiate cAMP formation in response to Gs-coupled receptor agonists.     

Pasteurella haemolytica A1-derived leukotoxin and endotoxin induce intracellular calcium elevation in bovine alveolar macrophages by different signaling pathways

Leukotoxin and endotoxin derived from Pasteurella haemolytica serotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381-388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237-252, 1995). Furthermore, extracellular Ca2+ is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca2+ in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca2+ by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca2+ regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca2+ in a concentration-dependent fashion by influx of extracellular Ca2+ through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca2+ even in the absence of extracellular Ca2+. Leukotoxin-induced intracellular Ca2+ elevation was inhibited by pertussis toxin, inhibitors of phospholipases A2 and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca2+ elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca2+ signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A2 and C and release of arachidonic acid in bovine alveolar macrophages. Ca2+ signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The differences in the Ca2+ signaling mechanisms may underlie the reported temporal differences in gene expression during leukotoxin and endotoxin activation.     

Pharmacological and biochemical evidence for the regulation of osteocalcin secretion by potassium channels in human osteoblast-like MG-63 cells

Previous reports have suggested the involvement of voltage-activated calcium (Ca2+) channels in bone metabolism and in particular on the secretion of osteocalcin by osteoblast-like cells. We now report that potassium (K+) channels can also modulate the secretion of osteocalcin by MG-63 cells, a human osteosarcoma cell line. When 1,25-dihydroxyvitamin D3(1,25(OH)2D3)-treated MG-63 cells were depolarized by step increases of the extracellular K+ concentration ([K+]out) from 5-30 mM, osteocalcin (OC) secretion increased from a control value of 218 +/- 13 to 369 +/- 18 ng/mg of protein/48 h (p < 0.005 by analysis of variance). In contrast, in the absence of 1,25(OH)2D3, there is no osteocalcin secretion nor any effect of cell depolarization on this activity. The depolarization-induced increase in 1,25(OH)2D3-dependent osteocalcin secretion was totally inhibited in the presence of 10 microM Nitrendipine (a Ca2+ channel blocker, p < 0.005) without affecting cellular alkaline phosphatase nor cell growth. Charybdotoxin, a selective blocker of Ca2+-dependent K+ channels (maxi-K) present in MG-63 cells, stimulated 1,25(OH)2D3-induced osteocalcin synthesis about 2-fold (p < 0.005) after either 30, 60, or 120 minutes of treatment. However, Charybdotoxin was without effect on basal release of osteocalcin in the absence of 1,25(OH)2D3 pretreatment. Using patch clamp technique, we occasionally observed the presence of a small conductance K+ channel, compatible with an ATP-dependent K+ channel (GK[ATP]) in nonstimulated cells, whereas multiple channel openings were observed when cells were treated with Diazoxide, a sulfonamide derivative which opens GK(ATP). Western blot analysis revealed the presence of the N-terminal peptide of GK(ATP) in MG-63 cells, and its expression was regulated with the proliferation rate of these cells, maximal detection by Western blots being observed during the logarithmic phase of the cycle. Glipizide and Glybenclamide, selective sulfonylureas which can block GK(ATP), dose-dependently enhanced 1,25(OH)2D3-induced OC secretion (p < 0.005). Reducing the extracellular calcium concentration with EGTA (microM range) totally inhibited the effect of Glipizide and Glybenclamide on osteocalcin secretion (p < 0.005), which remained at the same levels as controls. Diazoxide totally prevented the effect of these sulfonylureas. These results suggest that voltage-activated Ca2+ channels triggered via cell depolarization can enhance 1,25(OH)2D3-induced OC release by MG-63 cells. In addition, OC secretion is increased by blocking two types of K+ channels: maxi-K channels, which normally hyperpolarize cells and close Ca2+ channels, and GK(ATP) channels. The role of these channels is closely linked to the extracellular Ca2+ concentration.     

Contrasting effects of carbachol, McN-A-343 and AHR-602 on Ca(2+)-mobilization and Ca(2+)-influx pathways in taenia caeci

1. We compared the binding profiles and contractile mechanisms of putative muscarinic M1 agonists McN-A-343 and AHR-602 with those of carbachol in smooth muscle of guinea-pig taenia caeci. 2. McN-A-343 and AHR-602, as well as carbachol, completely displaced the atropine-sensitive binding of [3H]-quinuclidinyl benzilate to muscarinic receptors present in the membrane preparation. The potency order for the affinity of these agents for muscarinic receptors was carbachol > McN-A-343 > AHR-602. 3. In the presence of 2.2 mM extracellular Ca2+, McN-A-343 and AHR-602 induced contraction corresponding to 79 and 85%, respectively, of the maximal contraction to 0.1 mM carbachol. Contractions induced by these agents were mediated via activation of the muscarinic receptor subtype that had a high affinity for 4-DAMP (M3 selective) but a low affinity for pirenzepine (M1 selective) and AF-DX 116 (M2 selective). These contractions were inhibited by an L-type Ca2+ channel blocker, verapamil. 4. In Ca(2+)-free solution containing 2 mM EGTA, carbachol elicited a transient contraction whereas no contraction was observed in response to McN-A-343 and AHR-602. Application of McN-A-343 or AHR-602 inhibited the carbachol-induced contraction in Ca(2+)-free solution, and this inhibition was surmounted by a higher concentration of carbachol. 5. The EC50 value for carbachol-induced contraction in the presence of extracellular Ca2+ was approximately 175 times lower than that in the absence of Ca2+. After treatment with propylbenzilylcholine mustard, carbachol induced contraction only in the presence of extracellular Ca2+. 6. The results suggest that in the taenia caeci there is a greater receptor reserve for muscarinic M3 receptor-mediated Ca2+ influx than for M3 mediated Ca2+ release. The compounds McN-A-343 and AHR-602 are agonists of the Ca2+ influx pathway, but do not appear to stimulate the Ca2+ release pathway.     

Dual effect of the anti-allergic astemizole on Ca2+ fluxes in rat basophilic leukemia (RBL-2H3) cells: release of Ca2+ from intracellular stores and inhibition of Ca2+ release-activated Ca2+ influx

The antiallergic drugs astemizole and norastemizole inhibit exocytosis in mast cells, which might be relevant for their therapeutic action. From previous studies, it appeared that the drugs inhibited 45Ca2+ influx. Here, we present a more detailed study on the effects of astemizole and norastemizole on Ca2+ fluxes. Fura-2-loaded rat basophilic leukemia (RBL-2H3) cells were activated through the high-affinity receptor for IgE (FcepsilonRI) with antigen or by the endoplasmatic reticulum ATPase inhibitor thapsigargin, bypassing direct FcepsilonRI-related events. It appeared that astemizole (>15 microM), in contrast to norastemizole, showed a dual effect on intracellular calcium concentration ([Ca2+]i): a rise in intracellular calcium concentration was induced, which originated in the release of intracellular Ca2+ stores, whereas Ca2+ influx via store-operated Ca2+ (SOC) channels was inhibited. Ca2+ influx was further characterized using Ba2+ influx, whereas processes in the absence of Ca2+ influx were studied using Ni2+ or EGTA. It was concluded that the drugs most likely affect the store-operated Ca2+ channels in RBL cells directly. The two effects of astemizole on Ca2+ fluxes had opposing influences on exocytosis, thereby accounting for the biphasic effect of increasing astemizole concentration on mediator release in RBL cells.     

kappa-opioid agonist U50488 inhibits P-type Ca2+ channels by two mechanisms

The effects of U50488, kappa-opioid agonist on P-type Ca2+ channels, were studied. U50488 inhibited depolarization-induced Ca2+ uptake into rat brain synaptosomes, which was sensitive to omega-Agatoxin IVA (omega-AgaIVA; P-type Ca2+ channel blocker) and inhibited P-type Ca2+ channel currents recorded from rat cerebellar Purkinje neurons by the whole-cell patch clamp method. Dynorphin A also inhibited P-type Ca2+ channel currents. The inhibition by U50488 was biphasic; high affinity component (21%, IC50 = 8.9 x 10(-8) M) and low affinity component (79%, IC50 = 1.1 x 10(-5) M). At low concentrations of U50488 (10(-6) M), P-type Ca2+ channel current inhibition was attenuated by norbinartorphimine (nor-BNI), kappa-opioid antagonist, and by dialysis of cells with a pipette solution containing guanosine 5'-O-(2-thiodiphosphate) (GDP-beta S). At high concentrations of U50488 (10(-5) M), P-type Ca2+ channel current inhibition was frequency-dependent. Thus U50488-induced current inhibition is mediated by two mechanisms. Its high affinity component is produced by activation of kappa-opioid receptors, whereas the low affinity component is due to its direct action on the P-type Ca2+ channel.     

Purinergic receptor-induced changes in paracellular resistance across cultures of human cervical cells are mediated by two distinct cytosolic calcium-related mechanisms

In human cervical (CaSki) cells, extracellular adenosine triphosphate (ATP) induces an acute decrease in the resistance of the lateral intercellular space (RLIS), phase I response, followed by an increase in tight junctional resistance (RTJ), phase II response. ATP also stimulates release of calcium from intracellular stores, followed by augmented calcium influx, and both effects have similar sensitivities to ATP (EC50 of 6 microM). The objective of the study was to determine the degree to which the changes in [Ca2+]i mediate the responses to ATP. 1,2-bis (2-aminophenoxy) ethane-N,N,N1,N1-tetraacetic acid (BAPTA) abrogated calcium mobilization and phase I response; in contrast, nifedipine and verapamil inhibited calcium influx and attenuated phase II response. Barium, La3+, and Mn2+ attenuated phase I response and attenuated and shortened the ionomycin-induced phase I-like decrease in RLIS, suggesting that store depletion-activated calcium entry was inhibited. Barium and La3+ also inhibited the ATP-induced phase II response, but Mn2+ had no effect on phase II response, and in the presence of low extracellular calcium it partly restored the increase in RTJ. KCl-induced membrane depolarization stimulated an acute decrease in RLIS and a late increase in RTJ similar to ATP, but only the latter was inhibited by nifedipine. KCl also induced a nifedipine-sensitive calcium influx, suggesting that acute increases in [Ca2+]i, regardless of mobilization or influx, mediate phase I response. Phase II-like increases in RTJ could be induced by treatment with diC8, and were not affected by nifedipine. Biphasic, ATP-like changes in RTE could be induced by treating the cells with ionomycin plus diC8. We conclude that calcium mobilization mediates the early decrease in RLIS, and calcium influx via calcium channels activates protein kinase C and mediates the late increase in RTJ.     

Two separate mechanisms of T cell clonal anergy to Mls-1

T cell tolerance to superantigen can be mediated by clonal anergy in which Ag-specific mature T cells are physically present but are not able to mount an immune response. We induced T cell unresponsiveness to minor lymphocyte stimulations locus antigen (Mls)-1a in mice transgenic for TCR V beta 8.1 in three different systems: 1) injection of Mls-1a spleen cells, 2) mating with Mls-1a mice, and 3) bone marrow (BM) chimeras in which Mls-1a is present only on nonhematopoietic cells. CD4+8-V beta 8.1+ cells from all these groups did not proliferate in response to irradiated spleen cells from Mls-1a mice. We compared the response of these cells by T cell/stimulator cell conjugate formation, Ca2+ mobilization, and proliferation assays. The mechanisms underlying the unresponsiveness of these T cells appear to differ. CD4+8-V beta 8.1+ cells from Mls-1a spleen cell-injected mice mobilized cytoplasmic Ca2+ but proliferated at a reduced level in response to cross-linking with anti-TCR mAb. However, these cells formed conjugates, mobilized Ca2+, and proliferated in response to Mls-1a when activated B cells were used as stimulators, although they produced reduced levels of IL-2. In Mls-1a/b V beta 8.1 transgenic mice, a subset in CD4+8-V beta 8.1+ cells did not mobilize cytoplasmic Ca2+ after TCR cross-linking. Their conjugate formation, Ca2+ mobilization, or proliferation in response to Mls-1a on activated B cells was undetectable. Finally, CD4+8-V beta 8.1+ cells from the BM chimeras proliferated to TCR cross-linking at a partially reduced level and formed conjugates, mobilized Ca2+, and proliferated in response to Mls-1a on activated B cells. These features suggest that the mechanisms underlying the maintenance of anergy in Mls-1a spleen cell-injected mice are distinct from those in Mls-1a mice.     

Mechanisms involved in activation of human eosinophil exocytosis by substance P: an in vitro model of sensory neuroimmunomodulation

Substance P (SP), a tachykinin with a wide range of biological activities including a priming effect on human eosinophil chemotaxis, was investigated for its influence on eosinophil cytotoxic function measured as degranulation of eosinophil-derived neurotoxin (EDN). Peripheral blood was obtained from healthy volunteers and the degranulation assays were performed using radioimmunoassay (RIA). SP and its C-terminal elicited EDN release in a time-dependent mode at a narrow range of doses with optimal activity of 10(-6) M. FK888 (NK-1 receptor antagonist) inhibited EDN release stimulated by SP in dose dependency, also a complete inhibition was observed when eosinophils were preincubated with 1000 ng/ml pertussis toxin (PTX). Pre-exposure of eosinophils to staurosporine resulted in blockage of SP-induced EDN release in a dose-dependent mode. On the other hand, SP at 10(-7) M and 10(-8) M primed eosinophils to suboptimal dose (10(-8) M) of Platelet activating factor (PAF) resulting into significant enhancement of EDN release. SP(4-11) fragment showed a similar activity while SP(1-4) fragment was not active. SP priming of eosinophils was not affected by Ca2+ depletion, however, it caused a change in the pattern of the intracellular calcium influx against the suboptimal dose of PAF. These results suggest that SP i) may induced human eosinophil matrix protein degranulation through a receptor mediated mechanism coupled to PTX sensitive G protein(s) with the probability of linkage to phospholipase C activation, and, ii) primes human eosinophils for an exalted inflammatory response through a Ca2+ independent pathway.     

LTP induction dependent on activation of Ni2+-sensitive voltage-gated calcium channels, but not NMDA receptors, in the rat dentate gyrus in vitro

LTP induction dependent on activation of Ni2+-sensitive voltage-gated calcium channels, but not NMDA receptors, in the rat dentate gyrus in vitro. J. Neurophysiol. 78: 2574-2581, 1997. A N-methyl--aspartate receptor (NMDAR)-independent long-term potentiation (LTP) has been investigated in the dentate gyrus of the hippocampus in vitro in the presence of the NMDAR antagonist, -2-amino-phosphonopentanoate (50-100 mu M), at a concentration that completely blocked NMDAR-mediated excitatory postsynaptic currents (EPSCs). LTP of patch-clamped EPSCs was induced by pairing low-frequency evoked EPSCs (1 Hz) with depolarizing voltage pulses designed to predominately open low-voltage-activated (LVA) Ca2+ channels. Voltage pulses alone induced only a short-term potentiation. The LTP was blocked by intracellular application of the rapid Ca2+ chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, demonstrating that a rise in intracellular Ca2+ is required for the NMDAR-independent LTP induction. The NMDAR-independent LTP induction also was blocked by Ni2+ at a low extracellular concentration (50 mu M), which is known to strongly block LVA Ca2+ channels. However, Ni2+ did not inhibit the NMDAR-dependent LTP induced by high-frequency stimulation (HFS). The NMDAR-independent LTP induction was not blocked by high concentrations of the L-type Ca2+ channel blocker nifedipine (10 mu M). The NMDAR-independent LTP was inhibited by the metabotropic glutamate receptor ligand (+)-alpha-methyl-4-carboxyphenylglycine. These experiments demonstrate the presence of a NMDAR-independent LTP induced by Ca2+ influx via Ni2+-sensitive, nifedipine-insensitive voltage-gated Ca2+ channels, probably LVA Ca2+ channels. Induction of the NMDAR-independent LTP was inhibited by prior induction of HFS-induced NMDAR-dependent LTP, demonstrating that although the NMDAR-dependent and NMDAR-independent LTP use a different Ca2+ channel for Ca2+ influx, they share a common intracellular pathway.     

CD38 is functionally dependent on the TCR/CD3 complex in human T cells

One of the functions of surface CD38 is the induction of phosphorylation of discrete cytoplasmic substrates and mobilization of cytoplasmic calcium (Ca2+). The present work addresses the issue of whether the signaling mediated via CD38 operates through an independent pathway or, alternatively, is linked to the TCR/CD3 signaling machinery. We studied the signals elicited through CD38 by the specific agonistic IB4 monoclonal antibody (mAb) by monitoring the levels of cytoplasmic Ca2+ and the induced phenotypic and functional variations in T cell growth. IB4 mAb presented the unique ability to increase cytoplasmic Ca2+ levels, which correlated with the phosphorylation of the PLC-gamma1. These effects were blocked by phorbol 12-myristate 13-acetate (PMA) and were dependent on the presence of a functional TCR/CD3 surface complex, no effects being recorded on mutant Jurkat cells lacking part of the CD3 structures. CD38 signaling appeared to share with TCR/CD3 the ability to induce apoptotic cell death in Jurkat T cells, an event paralleled by specific up-regulation of the Fas molecule and inhibited by cyclosporin A. CD28, a costimulatory molecule, is synergized by increasing CD38-induced apoptotic cell death. The results indicate the existence of a strong functional interdependence between CD38 and TCR/CD3.     

Oscillatory Cl- current induced by angiotensin II in rat pulmonary arterial myocytes: Ca2+ dependence and physiological implication

We have previously reported that angiotensin II (ANG II) induces oscillations in the cytoplasmic calcium concentration ([Ca2+]i) of pulmonary vascular myocytes. The present work was undertaken to investigate the effect of ANG II in comparison with ATP and caffeine on membrane currents and to explore the relation between these membrane currents and [Ca2+]i. In cells clamped at -60 mV, ANG II (10 microM) or ATP (100 microM) induced an oscillatory inward current. Caffeine (5 mM) induced only one transient inward current. In control conditions, the reversal potential (Erev) of these currents was close to the equilibrium potential for Cl- ions (Ecl = -2.1 mV) and was shifted towards more positive values in low-Cl- solutions. Niflumic acid (10-50 microM) and DIDS (0.25-1 mM) inhibited this inward current. Combined recordings of membrane current and [Ca2+]i by indo-1 microspectrofluorimetry revealed that ANG II- and ATP-induced currents occurred simultaneously with oscillations in [Ca2+]i whereas the caffeine-induced current was accompanied by only one transient increase in [Ca2+]i. Niflumic acid (25 microM) had no effect on agonist-induced [Ca2+]i responses, whereas thapsigargin (1 microM) abolished both membrane current and the [Ca2+]i response. Heparin (5 mg/ml in the pipette solution) inhibited both [Ca2+]i responses and membrane currents induced by ANG II and ATP, but not by caffeine. In pulmonary arterial strips, ANG II-induced contraction was inhibited by niflumic acid (25 microM) or nifedipine (1 microM) to the same extent and the two substances did not have an additive effect. This study demonstrates that, in pulmonary vascular smooth muscle, ANG II, as well as ATP, activate an oscillatory calcium dependent chloride current which is triggered by cyclic increases in [Ca2+]i and that both oscillatory phenomena are primarily IP3-mediated. It is suggested that ANG II-induced oscillatory chloride current could depolarise the cell membrane leading to activation of voltage-operated Ca2+ channels. The resulting Ca2+ influx contributes to the component of ANG II-induced contraction that is equally sensitive to chloride or calcium channel blockade.