Mechanisms underlying changes of tetanic [Ca2+]i and force in skeletal muscle

Force development in skeletal muscle is driven by an increase in myoplasmic free [Ca2+]i ([Ca2+]i) due to Ca2+ release from the sarcoplasmic reticulum (SR). The magnitude of [Ca2+]i elevation during stimulation depends on: (a) the rate of Ca2+ release from the SR; (b) the rate of Ca2+ uptake by the SR; and (c) the myoplasmic Ca2+ buffering. We have used fluorescent Ca2+ indicators to measure [Ca2+]i in intact, single fibres from mouse and Xenopus muscles under conditions where one or more of the above factors are changed. The following interventions resulted in increased tetanic [Ca2+]i: beta-adrenergic stimulation, which potentiates the SR Ca2+ release; application of 2.5-di(tert-butyl)-1,4-benzohydroquinone, which inhibits SR Ca2+ pumps; application of caffeine, which facilitates SR Ca2+ release and inhibits SR Ca2+ uptake; early fatigue, where the rate of SR Ca2+ uptake is reduced; acidosis, which reduces both the myoplasmic Ca2+ buffering and the rate of SR Ca2+ uptake. Reduced tetanic [Ca2+]i was observed in late fatigue, due to reduced SR Ca2+ release, and in alkalosis, due to increased myoplasmic Ca2+ buffering. Force is monotonically related to [Ca2+]i but depends also on the myofibrillar Ca2+ sensitivity and the maximum force cross-bridges can produce. This is clearly illustrated by changes of intracellular pH where, despite a lower tetanic [Ca2+]i, tetanic force is higher in alkalosis than acidosis due to increases of myofibrillar Ca2+ sensitivity and maximum cross-bridge force.     

Different mechanisms for [Ca2+]i oscillations induced by carbachol and high concentrations of [Ca2+]o in the rat ventricular myocyte

1. The purpose of the present study was to explore the different mechanisms of [Ca2+]i oscillations induced by high concentrations of either carbachol (CCh) or extracellular Ca2+ ([Ca2+]o). First, we compared the oscillations induced by CCh at concentrations of 100-300 micromol/L and [Ca2+]o (5 mmol/L) in the single rat ventricular myocyte. Second, we studied CCh- and [Ca2+]o-induced [Ca2+]i oscillations following either interference with the production of inositol trisphosphate (IP3), reductions in cytosolic Ca2+ ([Ca2+]i), inhibition of Ca2+ influx and Na+-Ca2+ exchange or depletion of Ca2+ from its intracellular store. 2. The [Ca2+]i oscillations induced by CCh were frequent and were superimposed on [Ca2+]i transients in electrically stimulated cells, whereas those induced by high [Ca2+]o were occasional and occurred in quiescent cells and between [Ca2+]i transients in electrically stimulated cells. In both cases, [Ca2+]i oscillations were preceded by an increase in resting levels of [Ca2+]i. 3. Carbachol-induced [Ca2+]i oscillations were accompanied by an increase in amplitude and prolongation of the time of decline to 80% of the peak of the [Ca2+]i transient, while high [Ca2+]o-induced [Ca2+]i oscillations were the opposite. 4. A reduction of [Ca2+]o to 0.1 mmol/L and treatment with Ni2+ or ryanodine or 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid AM (BAPTA-AM) abolished the [Ca2+]i oscillations induced by both CCh and high [Ca2+]o. 5. The calcium channel blockers verapamil and nifedipine and inhibitors of phospholipase C (neomycin and U-73122) abolished the [Ca2+]i oscillations induced by CCh; Li+ accelerated the onset of the [Ca2+]i oscillations induced by CCh. 6. These observations suggest that the mechanisms responsible for the [Ca2+]i oscillations induced by CCh and high [Ca2+]o are different from each other. Other than an increase in extracellular Ca2+ influx as a mechanism common for both CCh- and high [Ca2+]o-induced [Ca2+]i oscillations, the CCh-induced [Ca2+]i oscillations involve influx of Ca2+ via L-type Ca2+ channels, Na+-Ca2+ exchange, mobilization of intracellular Ca2+ and IP3 production.     

Fluorescence and laser photon counting: measurements of epithelial [Ca2+]i or [Na+]i with ciliary beat frequency

We describe a system we developed that enabled simultaneous measurements of either epithelial calcium ion concentration ([Ca2+]i) or sodium ion concentration ([Na+]i) with the ciliary beat frequency (CBF) in native ciliated epithelia using either Fura-2 (AM) or SBFI (AM) ratiometric fluorescence photon counting along with nonstationary laser light scattering. Studies were performed using native epithelial tissues obtained from ovine tracheae. The dynamic range of the laser light-scattering system was determined by a simulated light "beating" experiment. The nonstationary CBF was demonstrated by the time-frequency analysis of the raw photon count sequences of backscattered heterodyne photons from cultured and native epithelia. Calibrations of calcium and sodium ion concentrations were performed using the respective Fura-2 and SBFI impermanent salts as well as in native epithelia. The cumulative responses of 10(-6), 10(-5), and 10(-4) M nifedipine on [Ca2+]i together with the CBF as well as the cumulative responses of 10(-5), 10(-4), and 10(-3) M amiloride on [Na+]i together with the CBF were also determined. Nifedipine decreased [Ca2+]i but had no effect on CBF. Amiloride decreased [Na+]i and CBF. Stimulation of CBF corresponded with either an increase of [Na+]i or an increase of [Ca2+]i. Decreases of [Na+]i or substantial decreases of [Ca2+]i were associated with decreases in the CBF. These data demonstrate the utility of this system for investigating the regulatory mechanisms of intracellular ions dynamics and the CBF in native epithelia.     

Agonist-induced intracellular Ca2+ transients in HT29 cells

In the present study we have investigated the mechanism of intracellular Ca2+ activity ([Ca2+]i) changes in HT29 cells induced by adenosine triphosphate (ATP), carbachol (CCH), and neurotensin (NT). [Ca2+]i was measured with the fluorescent Ca2+ indicator fura-2 at the single-cell level or in small cell plaques with high time resolution (1-40Hz). ATP and CCH induced not only a dose-dependent [Ca2+]i peak response, but also changes of the plateau phase. The [Ca2+]i plateau was inversely dependent on the ATP concentration, whereas the CCH-induced [Ca2+]i plateau increased at higher CCH concentrations. NT showed (from 10(-10) to 10(-7) mol/l) in most cases only a [Ca2+]i spike lasting 2-3 min. The [Ca2+]i plateau induced by ATP (10(-6) mol/l) and CCH (10(-5) mol/l) was abolished by reducing the Ca2+ activity in the bath from 10(-3) to 10(-4) mol/l (n = 7). In Ca(2+)-free bathing solution the [Ca2+]i peak value for all three agonists was not altered. Using fura-2 quenching by Mn2+ as an indicator of Ca2+ influx the [Ca2+]i peak was always reached before Mn2+ influx started. Every agonist showed this delayed stimulation of the Ca2+ influx with a lag time of 23 +/- 1.5 s (n = 15) indicating a similar mechanism in each case. Verapamil (10(-6)-10(-4) mol/l) blocked dose dependently both phases (peak and plateau) of the CCH-induced [Ca2+]i increase. Short pre-incubation with verapamil augmented the effect on the [Ca2+]i peak, whereas no further influence on the plateau was observed. Ni2+ (10(-3) mol/l) reduced the plateau value by 70%.     

The thapsigargin-evoked increase in [Ca2+]i involves an InsP3-dependent Ca2+ release process in pancreatic acinar cells

In pancreatic acinar cells, as in many other cell types, the tumour promoter thapsigargin (TG) evokes a significant increase of intracellular free Ca2+ ([Ca2+]i). The increases of [Ca2+]i evoked by TG was associated with significant changes of plasma membrane Ca2+ permeability, with [Ca2+]i values following changes in extracellular [Ca2+]. Plasma membrane Ca2+ extrusion is activated rapidly as a consequence of the rise in [Ca2+]i evoked by TG and the rate of extrusion is linearly dependent on [Ca2+]i up to 1 microM Ca2+. In contrast, the activation of the Ca2+ entry pathway is delayed and the apparent rate of Ca2+ entry is independent of [Ca2+]i. In the presence of 20 mM caffeine, which reduces the resting levels of inositol trisphosphate (InsP3), the increase of [Ca2+]i evoked by TG was significantly reduced. The reduction was manifest both as a decrease of the amplitude of the [Ca2+]i peak (30% reduction) and, more importantly, as a reduction of the apparent maximal rate of [Ca2+]i increase (from 12.3 +/- 1.0 to 6.1 +/- 0.6 nM Ca2+/s). The inhibition evoked by caffeine was reversible and the removal of caffeine in the continuous presence of TG evoked a further increase of [Ca2+]i. The amplitude of the [Ca2+]i increase upon caffeine removal was reduced as a function of the time of TG exposure. Addition of TG in the presence of 1 mM La3+, which is known to inhibit the plasma membrane Ca(2+)-activated adenosine triphosphatase, induced a much higher peak of [Ca2+]i. This increase was associated with an augmentation of the apparent rate of [Ca2+]i increase (from 12.3 +/- 1.2 to 16.1 +/- 1.9 nM Ca2+/s).(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelin-induced calcium signaling and secretion in chief cells and fibroblasts from pathological human parathyroid glands

Endothelins (ETs) are 21 amino acid peptides with vasoactive and mitogenic properties. The three isopeptides (ET-1, -2, and -3) and their receptors (E1A and ETB subtypes) display expression in numerous tissues and possibly mediate autocrine/paracrine actions. The present investigation shows that ET-1 triggers biphasic increases of the concentration of cytoplasmic Ca2+ ([Ca2+]i) in pathological human parathyroid cells. Both the peak and sustained [Ca2+]i increase, as well as the proportion of responding cells, are dose-dependent in the 10(-10)-10(-7) mol/L range of ET-1. In absence of external Ca2+, the ET-1-induced [Ca2+]i peak is attenuated. ET-3 has no effect on [Ca2+]i indicating functional dominance of the ETA receptor subtype. ET-1 (10 nmol/L) lowers parathyroid hormone secretion in 0.5 mmol/L but not in higher external Ca2+ concentrations, and parathyroid cell ET release is inhibited by increases of external Ca2+. Fibroblasts overgrowing the parathyroid chief cells during monolayer culture respond to ET-1 with biphasic [Ca2+]i increases or repetitive [Ca2+]i spikes, but show no response to elevation of external Ca2+. These findings imply that ET secretion and ET receptor expression may constitute an autocrine/paracrine mechanism in the regulation of human PTH secretion.     

Effects of pHi on isometric force and [Ca2+]i in porcine coronary artery smooth muscle

During ischemia or hypoxia, alterations in pHi may play a significant role in alteration of vessel wall function. We studied the effects of altering pHi on isometric force and [Ca2+]i in porcine coronary artery. pHi was altered at constant pHo by use of NH4Cl and measured with the fluorescent dye BCECF. [Ca2+]i was monitored with fura 2 and ratiometric fluorescence measurements. Addition of NH4Cl elicited a concentration-dependent (2 to 30 mmol/L) sustained increase in isometric force in unstimulated tissues. In tissues stimulated with KCl (29 mmol/L) or U46619 (1 mumol/L), addition of NH4Cl elicited a rapid but transient decrease followed by a sustained increase in force above the initial stimulated levels. Removal of NH4Cl was associated with a transient decrease and increase followed by a prolonged depression of force and slow recovery to initial levels. Addition of NH4Cl elicited a rapid monotonic increase in pHi and then a slow recovery toward initial levels; washout of NH4Cl led to a rapid acidification followed by recovery. In contrast to the steady state effects of NH4Cl on force, its effects on [Ca2+i were in the opposite direction. During the sustained increase in force elicited by NH4Cl alkalinization, [Ca2+]i was substantially decreased, whereas when force was depressed during the acidification elicited by NH4Cl washout, [Ca2+i was increased to values observed before addition of NH4Cl. The initial transients in force elicited by NH4Cl addition or washout were also associated with opposite changes in [Ca2+]i. Thus, the effects on force of the NH4Cl-induced changes in pHi are associated with changes in the Ca2+ sensitivity of the contractile apparatus rather than mediated through changes in [Ca2+]i.     

Delta9-tetrahydrocannabinol activates [Ca2+]i increases partly sensitive to capacitative store refilling

Delta9-tetrahydrocannabinol induces [Ca2+]i increases in DDT1MF-2 smooth muscle cells. Both Ca2+ entry and release from intracellular Ca2+ stores were concentration dependently activated. The Ca2+ entry component contributed most to the increases in [Ca2+]i. Stimulation with delta9-tetrahydrocannabinol after functional downregulation of intracellular Ca2+ stores by longterm thapsigargin treatment, still induced a major Ca2+ entry and a minor Ca2+ release component. Thapsigargin sensitive influx and release were selectively inhibited by the cannabinoid CB1 receptor antagonist SR141716A. No effects on [Ca2+]i were obtained after stimulation with the CB2 receptor agonist palmitoylethanolamide. This study is the first demonstration of (1) Ca2+ release from thapsigargin sensitive intracellular stores and capacitative Ca2+ entry via CB1 receptor stimulation and of (2) an additional delta9-tetrahydrocannabinol induced thapsigargin insensitive component, mainly representing Ca2+ influx which is neither mediated by CB1 nor CB2 receptor stimulation.     

Glutamatergic and GABAergic agonists increase [Ca2+]i in avian cochlear nucleus neurons

Neurons of the avian cochlear nucleus, nucleus magnocellularis (NM), are stimulated by glutamate, released from the auditory nerve, and GABA, released from both interneurons surrounding NM and from cells located in the superior olivary nucleus. In this study, the Ca2+ indicator dye Fura-2 was used to measure Ca2+ responses in NM stimulated by glutamate- and GABA-receptor agonists using a chicken brainstem slice preparation. Glutamatergically stimulated Ca2+ responses were evoked by kainic acid (KA), alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA), and N-methyl-D-aspartate (NMDA). KA- and AMPA-stimulated changes in [Ca2+]i were also produced in NM neurons stimulated in the presence of nifedipine, an L-type Ca2+ channel blocker, suggesting that KA- and AMPA-stimulated changes in [Ca2+]i were carried by Ca2(+)-permeable receptor channels. Significantly smaller changes in [Ca2+]i were produced by NMDA. When neurons were stimulated in an alkaline (pH 7.8) superfusate, NMDA responses were potentiated. KA- and AMPA-stimulated responses were not affected by pH. Several agents known to stimulate metabotropic receptors in other systems were tested on NM neurons bathed in a Ca2+ free-EGTA--buffered media, including L-cysteine sulfinic acid (L-CSA), trans-azetidine dicarboxylic acid (t-ADA), trans-aminocyclo-pentanedicarboxylic acid (t-ACPD), and homobromoibotenic acid (HBI). The only agent to reliably and dose-dependently increase [Ca2+]i was HBI, an analog of ibotenate. GABA also stimulated increases in [Ca2+]i in NM neurons. GABA-stimulated responses were reduced by agents that block voltage-operated channels and by agents that inhibit Ca2+ release from intracellular stores. Whereas GABA-A receptor agonist produced increases in [Ca2+]i GABA-B and GABA-C receptor agonists had no effect. There appear to be several ways for [Ca2+]i to increase in NM neurons. Presumably, each route represents a means by which Ca2+ can alter cellular processes.     

CD38 disruption impairs glucose-induced increases in cyclic ADP-ribose, [Ca2+]i, and insulin secretion

Increases in [Ca2+]i in pancreatic beta cells, resulting from Ca2+ mobilization from intracellular stores as well as Ca2+ influx from extracellular sources, are important in insulin secretion by glucose. Cyclic ADP-ribose (cADPR), accumulated in beta cells by glucose stimulation, has been postulated to serve as a second messenger for intracellular Ca2+ mobilization for insulin secretion, and CD38 is thought to be involved in the cADPR accumulation (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). Here we created "knockout" (CD38(-/-)) mice by homologous recombination. CD38(-/-) mice developed normally but showed no increase in their glucose-induced production of cADPR in pancreatic islets. The glucose-induced [Ca2+]i rise and insulin secretion were both severely impaired in CD38(-/-) islets, whereas CD38(-/-) islets responded normally to the extracellular Ca2+ influx stimulants tolbutamide and KCl. CD38(-/-) mice showed impaired glucose tolerance, and the serum insulin level was lower than control, and these impaired phenotypes were rescued by beta cell-specific expression of CD38 cDNA. These results indicate that CD38 plays an essential role in intracellular Ca2+ mobilization by cADPR for insulin secretion.     

Effect of cilostazol, a phosphodiesterase type III inhibitor, on histamine-induced increase in [Ca2+]i and force in middle cerebral artery of the rabbit

1. The effect of cilostazol, an inhibitor of phosphodiesterase type III (PDE III), on the contraction induced by histamine was studied by making simultaneous measurements of isometric force and the intracellular concentration of Ca2+ ([Ca2+]i) in endothelium-denuded muscle strips from the peripheral part of the middle cerebral artery of the rabbit. 2. High K+ (80 mM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Cilostazol (10 microM) did not modify the resting [Ca2+]i, but it did significantly decrease the tonic contraction induced by high K+ without a corresponding change in the [Ca2+]i response. 3. Histamine (3 microM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Cilostazol (3 and 10 microM) significantly reduced both the phasic and tonic increases in [Ca2+]i and force induced by histamine, in a concentration-dependent manner. 4. Rp-adenosine-3':5'-cyclic monophosphorothioate (Rp-cAMPS, 0.1 mM), a PDE-resistant inhibitor of protein kinase A (and as such a cyclic AMP antagonist), did not modify the increases in [Ca2+]i and force induced by histamine alone, but it did significantly decrease the cilostazol-induced inhibition of the histamine-induced responses. 5. In Ca2+-free solution containing 2 mM EGTA, both histamine (3 microM) and caffeine (10 mM) transiently increased [Ca2+]i and force. Cilostazol (1-10 microM) (i) significantly reduced the increases in [Ca2+]i and force induced by histamine, and (ii) significantly reduced the increase in force but not the increase in [Ca2+]i induced by caffeine. 6. In ryanodine-treated strips, which had functionally lost the histamine-sensitive Ca2+ storage sites, histamine (3 microM) slowly increased [Ca2+]i and force. Cilostazol (3 and 10 microM) lowered the resting [Ca2+]i, but did not modify the histamine-induced increase in [Ca2+]i, suggesting that functional Ca2+ storage sites are required for the cilostazol-induced inhibition of histamine-induced Ca2+ mobilization. 7. The [Ca2+]i-force relationship was obtained in ryanodine-treated strips by applying ascending concentrations of Ca2+ (0.16-2.6 mM) in Ca2+-free solution containing 100 mM K+. Histamine (3 microM) shifted the [Ca2+]i-force relationship to the left and increased the maximum Ca2+-induced force. Under the same conditions, whether in the presence or absence of 3 microM histamine, cilostazol (3-10 microM) shifted the [Ca2+]i-force relationship to the right without producing a change in the maximum Ca2+-induced force. 8. It is concluded that, in smooth muscle of the peripheral part of the rabbit middle cerebral artery, cilostazol attenuates the histamine-induced contraction both by inhibiting histamine-induced Ca2+ mobilization and by reducing the myofilament Ca2+ sensitivity. It is suggested that the increase in the cellular concentration of cyclic AMP that will follow the inhibition of PDE III may play an important role in the cilostazol-induced inhibition of the histamine-contraction.     

ATP stimulation of Ca2+ -dependent plasminogen release from cultured microglia

1. ATP (10-100 microM), but not glutamate (100 microM), stimulated the release of plasminogen from microglia in a concentration-dependent manner during a 10 min stimulation. However, neither ATP (100 microM) nor glutamate (100 microM) stimulated the release of NO. A one hour pretreatment with BAPTA-AM (200 microM), which is metabolized in the cytosol to BAPTA (an intracellular Ca2+ chelator), completely inhibited the plasminogen release evoked by ATP (100 microM). The Ca2+ ionophore A23187 induced plasminogen release in a concentration-dependent manner (0.3 microM to 10 microM). 2. ATP induced a transient increase in the intracellular calcium concentration ([Ca2+]i) in a concentration-dependent manner which was very similar to the ATP-evoked plasminogen release, whereas glutamate (100 microM) had no effect on [Ca2+]i (70 out of 70 cells) in microglial cells. A second application of ATP (100 microM) stimulated an increase in [Ca2+]i similar to that of the first application (21 out of 21 cells). 3. The ATP-evoked increase in [Ca2+]i was totally dependent on extracellular Ca2+, 2-Methylthio ATP was active (7 out of 7 cells), but alpha,beta-methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca2+]i. Suramin (100 microM) was shown not to inhibit the ATP-evoked increase in [Ca2+]i (20 out of 20 cells). 2'- and 3'-O-(4-Benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), a selective agonist of P2X7 receptors, evoked a long-lasting increase in [Ca2+]i even at 1 microM, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5'-triphosphate-2', 3'-dialdehyde (oxidized ATP, 100 microM), a selective antagonist of P2X7 receptors, blocked the increase in [Ca2+]i induced by ATP (10 and 100 microM). 4. These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca2+]i via the ionotropic P2X7 receptor which stimulates the release of plasminogen from the microglia.     

Effects of urinary ouabain-like factors and vanadium diascorbate on calcium mobilization in porcine inner medullary collecting duct cells: comparison with the effects of ouabain and vasopressin

It is speculated that ouabain-like factors (OLF) play a role in the pathogenesis of volume-dependent hypertension. In previous studies we isolated a more polar OLF-1 and a more apolar OLF-2 from the urine of healthy subjects after 5 days on a high sodium intake (>400 mmol/day) by gel chromatography (Sephadex G-25 and G-10) and reverse-phase HPLC. We subsequently identified the chemical structure of OLF-2 as vanadium (V(IV)) diascorbate. OLF-1, OLF-2, and vanadium diascorbate inhibited dose-dependently porcine Na-K-ATPase in vitro. Because the inner medullary collecting duct (IMCD) plays a crucial role in the long-term regulation of body fluid volume, in the present study we investigated the effects of urinary OLF-1 and OLF-2, and of vanadium diascorbate in comparison to ouabain and vasopressin (AVP) on calcium mobilization, ie, on free calcium concentration [Ca2+]i, in cultured porcine IMCD cells. [Ca2+]i was determined by the fura-2 method in IMCD cells isolated by hypotonic treatment and density gradient centrifugation from fresh porcine kidneys. Assuming an approximate molecular weight (MW) of 400 for OLF-1 and OLF-2, OLF-1 (10(-4) mol/L) produced a slow increase in [Ca2+]i from 39 +/- 10 to 169 +/- 21 nmol/L (n = 7 ) after 4 min. Similarly, OLF-2 (10(-4) mol/L) resulted in an increase in [Ca2+]i from 74 +/- 20 to 216 +/- 52 nmol/L (n = 7) after 4 min. Vanadium diascorbate (MW 403) dose-dependently increased [Ca2+]i . At a concentration of 10(-6) mol/L it increased [Ca2+]i from 46 +/- 5 to 149 +/- 9 nmol/L (n = 5) after 4 min. A similar slow increase in [Ca2+]i was found with ouabain (10(-6) mol/L), which increased [Ca2+]i from 61 +/- 22 to 180 +/- 29 nmol/L (n = 5) after 4 min in contrast to AVP (10(-7) mol/L), which rapidly increased [Ca2+]i from 48 +/- 10 to 299 +/- 32 nmol/L (n = 4) within 30 sec. Thus, OLF-1, OLF-2, and Vanadium diascorbate, the active component of OLF-2, reveal similar effects as ouabain on IMCD cells, ie, they produce a slow increase in [Ca2+]i as expected from inhibition of Na-K-ATPase. The physiologic or pathologic roles of these and additional OLF in body fluid and blood pressure regulation and in hypertension have yet to be evaluated.     

HIV-1 envelope proteins gp120 and gp160 potentiate NMDA-induced [Ca2+]i increase, alter [Ca2+]i homeostasis and induce neurotoxicity in human embryonic neurons

The envelope glycoprotein gp120 of the human immunodeficiency virus HIV-1 has been proposed to cause neuron death in developing murine hippocampal cultures and rat retinal ganglion cells. In the present study, cultured human embryonic cerebral and spinal neurons from 8- to 10-week-old embryos were used to study the neurotoxic effect of gp120 and gp160. Electrophysiological properties as well as N-methyl-D-aspartate (NMDA)-induced current were recorded from neurons maintained in culture for 10-30 days. Neither voltage-activated sodium or calcium currents nor NMDA-induced currents were affected by exposure of neurons to 250 pM gp120 or gp160. In contrast, when neurons were subjected to photometric measurements using the calcium dye indo-1 to monitor the intracellular free Ca2+ concentration ([Ca2+])i, gp120 and gp160 (20-250 pM) potentiated the large rises in [Ca2+]i induced by 50 microM NMDA. The potentiation of NMDA-induced Ca2+ responses required the presence of Ca2+ in the medium, and was abolished by the NMDA antagonist D-2-amino-5-phosphonovalerate (AP5) and the voltage-gated Ca2+ channel inhibitor nifedipine. Moreover, exposure of a subpopulation of spinal neurons (25% of the cells tested) to 20-250 pM gp120 or gp160 resulted in an increase in [Ca2+]i that followed three patterns: fluctuations not affected by AP5, a single peak, and the progressive and irreversible rise of [Ca2+]i. The neurotoxicity of picomolar doses of gp120 and gp160 cultures was estimated by immunofluorescence and colorimetric assay. Treatment of cultures with AP5 or nifedipine reduced gp120-induced toxicity by 70 and     

High PCO does not alter pHi, but raises [Ca2+]i in cultured rat carotid body glomus cells in the absence and presence of CdC12

We measured the effect of high PCO (500-550 Torr) on the pHi and [Ca2+]i in cultured glomus cells of adult rat carotid body (CB) as a test of the two models currently proposed for the mechanism of CB chemoreception. The metabolic model postulates that the rise in glomus cell [Ca2+]i, the initiating reaction in the signalling pathway leading to chemosensory neural discharge, is due to [Ca2+] release from intracellular Ca2+ stores. The membrane potential model postulates that the rise in [Ca2+]i comes from influx of extracellular Ca2+ through voltage-dependent Ca2+ channels (VDCC) of the L-type. High PCO did not change pHi at PO2 of 120-135 Torr, showing that CO-induced changes in [Ca2+]i are not due to changes in pHi. High PCO caused a highly significant rise in [Ca2+]i from 90+/-12 nM to 675+/-65 nM, both in the absence and in the presence of 200 microM CdCl2, a potent blocker of L-type VDCCs. This result is fully consistent with release of Ca2+ from glomus cell intracellular stores according to metabolic model, but inconsistent with influx of extracellular Ca2+ through VDCCs according to the membrane potential model.     

Effects of SCN substitution for Cl- on tension, [Ca2+]i, and ionic currents in vascular smooth muscle

Substitution of thiocyanate ions (SCN-) for chloride ions (Cl-) in the extracellular medium of aortic rings and strips causes a biphasic contractile response; initial relaxation followed by sustained contraction. Alterations in these responses are sex-specific, and may elucidate fundamental differences in vascular function between males and females. In order to investigate the role of changes in intracellular Ca2+ ([Ca2+]i) in these changes in tension, we investigated effects of SCN- on [Ca2+]i and ionic currents in vascular smooth muscle cells (VSMC). Extracellular substitution of SCN- for Cl- caused a biphasic change in [Ca2+]i. Initially, [Ca2+]i decreased, reaching a minimum within 1-2 min, subsequently returned to original levels within 4-5 min, and then increased to a higher plateau over the next 10 minutes. This pattern of change in [Ca2+]i is identical to the pattern of tension changes in aortic rings, but it occurs somewhat faster. Partial substitution of SCN- for Cl- elicited increased, but no preceding decrease in [Ca2+]i. In the absence of external Ca2+, anion substitution elicited the decrease in [Ca2+]i but not the subsequent increase. Verapamil (1 microM) blocked the increased [Ca2+]i phase but not the decreased [Ca2+]i phase; whereas, R+ verapamil (up to 5 microM for 20 min), an inactive enantiomer, caused no change. Ionic current measurements obtained using whole cell patch and current clamp techniques revealed two responses to anion substitution: (a) a rapid, transient outward shift in holding current, and (b) a sustained increase in peak current and a hyperpolarizing shift in voltage sensitivity of Ca2+ channels. The calcium channel blocker PN200-110 blocked SCN(-)-enhanced current but had no effect on the changes in holding current. S- verapamil, but not R+ verapamil, reduced SCN(-)-enhanced current. In current clamp mode, SCN- caused an initial hyperpolarization followed by a slow depolarization punctuated by spikes. Thus, SCN- causes changes in vascular smooth muscle [Ca2+]i that could underlie both phases of its effects on tension in isolated aortas and may be explained by the following model: an initial outward shift in current causes hyperpolarization with a consequent decrease in cell excitability, and the somewhat slower increase in Ca2+ channel excitability eventually leads to enhanced calcium influx and tension. These data shed light on possible mechanisms underlying gender-related differences in VSMC physiology.     

Time course of the initial [Ca2+]i response to extracellular ATP in smooth muscle depends on [Ca2+]e and ATP concentration

In response to extracellular application of 50 microM ATP, all individual porcine aortic smooth muscle cells respond with rapid rises from basal [Ca2+]i to peak [Ca2+]i within 5 s. The time from stimulus to the peak of the [Ca2+]i response increases with decreasing concentration of ATP. At ATP concentrations of 0.5 microM and below, the time to the [Ca2+]i peak varies more significantly from cell to cell than at higher concentrations, and each cell shows complicated initiation and decay kinetics. For any individual cell, the lag phase before a response decreases with increasing concentration of ATP. An increase in lag time with decreasing ATP concentration is also observed in the absence of extracellular Ca2+, but the lag phase is more pronounced, especially at concentrations of ATP below 0.5 microM. Whole-cell patch-clamp electrophysiology shows that in porcine aortic smooth muscle cells, ATP stimulates an inward current carried mainly by Cl- ion efflux with a time course similar to the [Ca2+]i changes and no detectable current from an ATP-gated cation channel. A simple signal cascade initiation kinetics model, starting with nucleotide receptor activation leading to IP3-mediated Ca2+ release from IP3-sensitive internal stores, fits the data and suggests that the kinetics of the Ca2+ response are dominated by upstream signal cascade components.     

Pathways through which glucose induces a rise in [Ca2+]i of polymorphonuclear leukocytes of rats

Basal levels of [Ca2+]i are elevated in diabetes mellitus. Such an abnormality is most likely due to both increased calcium influx into cells and decreased efflux of this ion out of the cells. The present study examined the cellular pathways that are responsible for hyperglycemia-induced acute rise in polymorphonuclear leukocytes (PMNL), and explored whether such a rise is due to increased calcium entry into PMNL and/or to calcium release from their intracellular stores. There were dose dependent and time dependent rises in the [Ca2+]i of PMNL exposed to high concentrations of glucose. Similar effects were observed when the PMNL were exposed to high concentrations of choline chloride or mannitol. A substantial part of the rise in [Ca2+]i was inhibited when the media contained verapamil or nifedipine or when the PMNL were placed in calcium free media, and the rise in [Ca2+]i was completely abolished when the PMNL were placed in calcium free media containing ryanodine. GDP beta S or pertussis toxin almost completely prevented the glucose-induced rise in [Ca2+]i of PMNL. Rp-cAMP, H-89 or staurosporine produced significant inhibition of the rise in [Ca2+]i. High concentrations of glucose produced a dose dependent shrinkage of PMNL volume over a period of two hours. The volume of PMNL, however, was normal after 24 hours in vitro incubation studies as well as after 1, 2 and 12 days of streptozotocin-induced hyperglycemia in rats. The results are consistent with the formulation that the osmotic activity (cell shrinkage) of the high glucose concentrations activates G protein(s) which then stimulates the adenylate-cAMP-protein kinase A pathway, phospholipase C system and calcium channels. The stimulation of these cellular pathways permits both calcium influx into the PMNL as well as mobilization of calcium from their intracellular stores. Both of these events contribute to the acute rise in their [Ca2+]i. It is possible that the rise in [Ca2+]i is critical for the stimulation of the events that lead to the generation and accumulation of inorganic osmolytes to restore cell volume to normal.     

Effects of MK-447 on thrombin-induced aggregation, secretion of ATP, and [Ca2+]i mobilization in rabbit platelets

AIM: To study the effects of MK-447 on aggregation release reaction and intracellular calcium mobilization by thrombin. METHODS: Aggregation and release reaction were assessed by light transmission and ATP content in rabbit citrate platelet-rich plasma (PRP), and cytosolic-free calcium was measured by fluorescence and imaging. RESULTS: MK-447 (2-aminomethyl-4-t-butyl-6-iodophenol hydrochloride) induced a decrease in light transmission (DLT), so called platelet shape change, without detectable aggregation and secretion of ATP, and increased intracellular calcium concentration ([Ca2+]i) slightly in washed single platelet loaded with Fura 2, the peak value being about 160 nmol.L-1. These effects were not inhibited by egtazic acid 3 mmol.L-1 or indometacin 3 mumol.L-1. The pretreatment of PRP with MK-447 700 mumol.L-1 reduced the DLT by thrombin, potentiated and enhanced thrombin-induced aggregation and secretion of ATP in a concentration-dependent manner. Thrombin-induced [Ca2+]i mobilization (peak value: 369 +/- 45 nmol.L-1) was further enhanced by the administration of MK-447 at 2 min before the addition of thrombin, and the peak value reached 623 +/- 121 nmol.L-1 (P < 0.01). CONCLUSION: MK-447-induced platelet shape change was involved in intracellular calcium release in this preparation. MK-447 enhanced thrombin-induced aggregation and release reaction and these effects of MK-447 on aggregation and release reaction by thrombin might result from the synergistic effect of intracellular calcium mobilization.