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.     

The signals for starvation response are transduced through elevated [Ca2+]i in Dictyostelium cells

Selecting antiretroviral therapies for human immunodeficiency virus type 1-infected persons is complicated by the availability of a vast number of potentially useful drug combinations and by extensive variation among patients in their resistance to various drugs. AIDS clinical trials have used designs in which a handful of drug regimens in a few patient classes can be compared. Here is proposed implementation of innovative designs with factorial structure that permit assessment of many treatment arms and patient classes in a single trial; when and how they can be appropriately used are discussed. These designs are efficient, permit systematic investigation of correlations between genetic mutations and in vivo drug resistance, and provide insight into important drug interactions in people that conventional designs are unable to provide. Through creative application of these designs, identification of superior drug combinations and the science of understanding in vivo joint drug dynamics and genotypic resistance will progress at an optimum pace.     

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.     

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.     

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.     

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.     

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.     

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)     

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     

Characterization of action potential-triggered [Ca2+]i transients in single smooth muscle cells of guinea-pig ileum

Cytology can be a rewarding diagnostic technique in equine practice. The respiratory tract readily lends itself to sampling for cytologic evaluation from the upper to lower regions of the system. This article discusses preservation and staining techniques that will allow the practitioner to present satisfactory samples to the laboratory. General considerations for cytologic analysis are discussed as well as the specific findings for individual disorders of the respiratory tract. The proper use of cytologic findings in conjunction with other diagnostic techniques for the respiratory tract are also discussed.     

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.     

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.     

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.     

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.     

A putative tetrapeptide antagonist prevents beta-amyloid-induced long-term elevation of [Ca2+]i in rat astrocytes

Comparative fluorimetric studies on the long-term (8-hour) action of beta[1-42]amyloid and its shorter fragments beta[1-40], beta[25-35] and beta[31-35] on the steady-state intracellular Ca2+ concentration in primary cultures of rat astroglial cells using the Ca2+-sensitive fluorescent probe Fura-2 AM revealed higher 340/380 fluorescence excitation ratios in the treated cells as compared to the untreated controls. All the peptides were found to induce similar cellular effects, suggesting the [31-35] region as the putative active centre of the molecule. No significant alteration was detectable in Fura-2 fluorescence using the Ca2+-insensitive excitation wavelength of 367 nm, indicating that the observed changes reflect a real alteration in the Ca2+ concentration of the cells. Moreover, no considerable difference was observed in the total protein content of treated and untreated cells. Co-treatment of the cells with Pr-Ile-Ile-Gly-Leu-NH2 (Pr-IIGL) peptide, an analogue of the [31-34] region of beta[1-42]-amyloid, was found to effectively antagonize the beta[1-42]-amyloid-induced elevation of the fluorescence excitation ratio, leaving the 367-nm fluorescence unaffected. To the best of the authors' knowledge, this is the first report on an analogue of beta-amyloid peptide capable of blocking one of its physiological effects, thereby raising the possibility that this sequence could prove to be a lead compound for designing effective beta-amyloid antagonists.     

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.     

Mechanisms of stretch-induced changes in [Ca2+]i in rat atrial myocytes: role of increased troponin C affinity and stretch-activated ion channels

To study the effects of stretch on the function of rat left atrium, we recorded contraction force, calcium transients, and intracellular action potentials (APs) during stretch manipulations. The stretch of the atrium was controlled by intra-atrial pressure. The Frank-Starling behavior of the atrium was manifested as a biphasic increase of the contraction force after increasing the stretch level. The development of the contraction force after step increase of the stretch (intra-atrial pressure from 1 to 3 mm Hg) was accompanied by the increase in the amplitude of the calcium transients (P<0.05, n=4) and decrease in the time constant of the Ca2+ transient decay. The APs of the individual myocytes were also affected by stretch; the duration of the AP was decreased at positive voltages (AP duration at 15% repolarization level, P<0.001; n=13) and increased at negative voltages (AP duration at 90% repolarization level, P<0. 01; n=13). To study the mechanisms causing these changes we developed a mathematical model describing [Ca2+]i and electrical behavior of single rat atrial myocytes. Stretch was simulated in the model by increasing the troponin (TnC) sensitivity and/or applying a stretch-activated (SA) calcium influx. We mimicked the Ca2+ influx by introducing a nonselective cationic conductance, the SA channels, into the membrane. Neither of the 2 plausible mechanosensors (TnC or SA channels) alone could produce similar changes in the Ca2+ transients or APs as seen in the experiments. The model simulated the effects of stretch seen in experiments best when both the TnC affinity and the SA conductance activation were applied simultaneously. The SA channel activation led to gradual augmentation of Ca2+ transients, which modulated the APs through increased Na+/Ca2+-exchanger inward current. The role of TnC affinity change was to modulate the Ca2+ transients, stabilize the diastolic [Ca2+]i, and presumably to produce the immediate increase of the contraction force after stretch seen in experiments. Furthermore, we found that the same mechanism that caused the normal physiological responses to stretch could also generate arrhythmogenic afterpotentials at high stretch levels in the model.     

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.