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.     

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.     

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.     

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     

Substance P induces brief, localized increase in [Ca2+]i in dorsal horn neurons

We determined the spatial and temporal dynamics of the increase in intracellular Ca2+ levels [Ca2+]i produced by substance P (SP) in dorsal horn neurons. A microinjection technique was used to apply minute amounts of SP to small areas of cultured neurons loaded with the Ca2+ indicator fura-2. Five successive applications of SP to the soma produced short-lasting (< 50 s) increases in [Ca2+]i that became gradually smaller, indicating receptor desensitization. Focal application of SP to a distal locus in a neurite produced a brief (12 s) increase in [Ca2+]i that travelled down the dendrite but did not spread into cell soma. Prolonged application of SP to these neurons caused the appearance of varicosities in their dendrites.     

Ionotropic and metabotropic glutamate receptor agonist-induced [Ca2+]i increase in isolated rat supraoptic neurons

Although a number of studies have shown that various free fatty acids (FFAs) and monoacylglycerides (MGs) have bactericidal properties in vitro, the role of these compounds in vivo has not been determined. This study evaluated the antibacterial properties of medium-chain MGs and FFAs for different bacterial enteropathogens with an in-vitro bacterial killing assay and an in-vivo model of intestinal colonisation. Incubation of test bacteria with medium-chain MGs for 4 h led to 100-10,000-fold reductions in numbers of viable cells of Vibrio cholerae, Salmonella typhi, Shigella sonnei and enterotoxigenic Escherichia coli (ETEC). Lauric acid was the only medium-chain FFA to show comparable in-vitro bactericidal activity. The ability of dietary MGs to reduce or eliminate bacterial colonisation of the intestinal tract was evaluated in mice that were predisposed to bacterial colonisation by treatment with streptomycin (STR+). Mice were treated with streptomycin, challenged intragastrically with V. cholerae or ETEC, and given monocaprin (C10:0 MG) either concurrently or as part of the daily diet. Control mice given STR+ without MGs and challenged with V. cholerae or ETEC showed high numbers of challenged bacteria in gastrointestinal contents by 1 h after administration. Concurrent administration of V. cholerae and C10:0 MG (2.5 mg/ml) caused > 1000-fold reduction in numbers of V. cholerae recovered from the gastrointestinal tracts of STR+ mice. Concurrent administration of C10:0 MG with ETEC did not cause a reduction in the number of viable ETEC present in the intestinal tract of STR+ mice. Administration of C10:0 MG in the diet had no effect on the number of viable V. cholerae or ETEC associated with caecal or ileal tissue of STR+ mice when C10:0 MG in the diet was started 1 day before, the same day, or 2 days after bacterial challenge. Collectively, these results suggested that dietary MGs may prevent intestinal colonisation by bacterial enteropathogens if administered at the time of exposure, but have little effect on established intestinal infections.     

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.     

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.     

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)     

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.     

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.     

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.     

Relationships between dopamine-induced changes in cytosolic free calcium concentration ([Ca2+]i) and rate of prolactin secretion. Elevated [Ca2+]i does not indicate prolactin release

This study was undertaken to investigate the relationship between dopamine (DA) induced changes in the cytosolic calcium concentration ([Ca2+]i) and the rate of prolactin secretion using GH4ZR7, a rat pituitary cell line, which express only one subtype of D2 receptor. GH4ZR7 cells were loaded with Fluo-3, a fluorescent Ca2+ indicator, and then perifused with two different doses of DA (10(-7) mol/L and 5 x 10(-4) mol/L). We monitored changes in [Ca2+]i and rate of prolactin release simultaneously by attaching a spectrofluorometer to a dynamic perifusion system. DA has stimulatory and inhibitory effect on prolactin secretion in GH4ZR7 cells; 10(-7) mol/LDA slightly increased [Ca2+]i and stimulated prolactin release, whereas 5 x 10(-4) mol/LDA decreased [Ca2+]i and inhibited prolactin secretion. When the cells were pretreated with pertussis toxin (PTX), 10(-7) mol/L DA had no significant change in [Ca2+]i while stimulating prolactin release, and 5 x 10(-4) mol/L DA reduced [Ca2+]i without having any significant effect on the rate of prolactin secretion. The results of this study demonstrate that changes in [Ca2+]i do not always correlate with the rate of prolactin release from lactotrophs. The dissociation between [Ca2+]i and prolactin release is somewhat expected considering the diverse role of [Ca2+]i and post-[Ca2+]i events, which can change the rate of prolactin release.     

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.     

Endothelin-1-(1-31), a novel vasoactive peptide, increases [Ca2+]i in human coronary artery smooth muscle cells

We have previously found that human chymase cleaves big endothelins at the Tyr31-Gly32 bond and produces 31-amino acid long endothelins-(1-31), without any further degradation products. In this study, we investigated the effect of synthetic endothelin-1-(1-31) on the intracellular free Ca2+ concentration ([Ca2+]i) in cultured human coronary artery smooth muscle cells. Endothelin-1-(1-31) increased [Ca2+]i in a concentration-dependent manner (10(-14) to 10(-10) M). This endothelin-1-(1-31)-induced [Ca2+]i increase was not affected by phosphoramidon (N-(alpha-Rhamnopyranosyloxyhydroxyphosphinyl)-L-Leucyl-L-Tryptoph an), an inhibitor of endothelin-converting enzyme. It was, however, inhibited by 10(-10) M BQ123 (Cyclo-(-D-Trp-D-Asp(ONa)-Pro-D-Val-Leu-)), an endothelin ET(A) receptor antagonist, but not by 10(-10) M BQ788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-yMeLeu-D-Trp(COOM e)-D-Nle-ONa), an endothelin ET(B) receptor antagonist. These results suggest that endothelin-1-(1-31) by itself exhibits vasoactive properties probably through endothelin ET(A) receptors. Since human chymase has been reported to play a role in atherosclerosis, endothelin-1-(1-31) may be one of the candidate substances for its cause.     

Altered [Ca2+]i mobilization in diabetic cardiomyocytes: responses to caffeine, KCl, ouabain, and ATP

The available literature on 55Fe dosimetry has been devoted to environmental exposures and medical iron kinetic studies. For occupational dosimetry, ICRP (1979, 1988) published a non-recycling dosimetric model for iron. These ICRP publications do not provide information on iron excretion. Johnson and Dunford (1985) published dose conversion factors and urinary excretion curves based on the ICRP (1980) and MIRD (1983) iron metabolic models. A critical review of these models was undertaken to select a model for occupational dose assignment. The review indicated that the information and recommendations in ICRP (1988) and Johnson and Dunford (1985) are dependent on unrealistic assumptions that do not agree with known iron metabolism. Therefore, an alternative model is proposed for dosimetric application. Calculations of dose conversion factors and urinary excretion curve for class W55Fe inhalation exposure (1 micron AMAD) using the proposed model are compared with predictions based on ICRP (1980) and Johnson and Dunford (1985) models. The difference in the practical outcome (i.e., dose assignment) is examined by applying the proposed and reviewed models to a realistic bioassay case. The Johnson and Dunford (1985) model yields a dose estimate which is roughly a factor of ten higher than values predicted by ICRP (1980) and the proposed model. Some of the disagreement is due to uncertainty in the fraction of radio-iron excretion via urine. Further research on this subject is recommended. In the interim, the proposed model is recommended for occupational dose assignment.     

K(+)- and transmitter-induced rises in [Ca2+]i in auditory neurones of developing rats

Ca2+ ions are thought to play important roles in processes underlying neuronal plasticity such as synapse stabilization. We employed the Fura-2 technique on brainstem slices of neonatal rats to measure changes in intracellular Ca2+ ([Ca2+]i) in neurons of the lateral superior olive (LSO) in order to analyse whether these cells have functional Ca2+ channels when synaptic maturation takes place. Rises in intracellular Ca2+ could be induced by KCl-evoked depolarizations or by glutamate, but not by glycine or GABA. These results show that Ca2+ channels are present in developing LSO neurones and that many of them, if not all, belong to the voltage-sensitive type. We speculate that these channels play a role during ontogeny by mediating Ca(2+)-dependent mechanisms of synapse stabilization.     

Disrupted [Ca2+]i homeostasis contributes to the toxicity of nitric oxide in cultured hippocampal neurons

Nitric oxide (NO) has been shown to be an important mediator in several forms of neurotoxicity. We previously reported that NO alters intracellular Ca2+ concentration ([Ca2+]i) homeostasis in cultured hippocampal neurons during 20-min exposures. In this study, we examine the relationship between late alterations of [Ca2+]i homeostasis and the delayed toxicity produced by NO. The NO-releasing agent S-nitrosocysteine (SNOC; 300 microM) reduced survival by about one half 1 day after 20-min exposures, as did other NO-releasing agents. SNOC also was found to produce prolonged elevations of [Ca2+]i, persisting at 2 and 6 h. Hemoglobin, a scavenger of NO, blocked both the late [Ca2+]i elevation and the delayed toxicity of SNOC. Removal of extracellular Ca2+ during the 20-min SNOC treatment failed to prevent the late [Ca2+]i elevations and did not prevent the delayed toxicity, but removal of extracellular Ca2+ for the 6 h after exposure as well blocked most of the toxicity. Western blots showed that SNOC exposure resulted in an increased proteolytic breakdown of the structural protein spectrin, generating a fragment with immunoreactivity suggesting activity of the Ca2+-activated protease calpain. The spectrin breakdown and the toxicity of SNOC were inhibited by treatment with calpain antagonists. We conclude that exposures to toxic levels of NO cause prolonged disruption of [Ca2+]i homeostatic mechanisms, and that the resulting persistent [Ca2+]i elevations contribute to the delayed neurotoxicity of NO.