Developmental regulation of TCR-CD3-dependent [Ca2+]i responses of individual normal and pp59fyn-deficient T lymphocytes.

The aim of this study was to ascertain whether different types of T-cell receptor (TCR)-mediated [Ca2+]i signals could begin to explain the different cellular responses of mature and immature T cells to ligation of the TCR-CD3 complex. Using a digital fluorescence imaging system, we measured and compared [Ca2+]i of individual cells from immature and mature murine T-cell populations following application of CD3-epsilon monoclonal antibody (mAb). Our approach revealed distinctions among developmental subsets which were not seen by previous measurements of [Ca2+]i in bulk cell populations. The CD3-mediated [Ca2+]i responses of individual thymocytes were very complex. Latencies to peak [Ca2+]i varied greatly among thymocytes, but the responses of splenic T cells were synchronized, novel evidence that the timing of [Ca2+]i responses may be an important informative parameter for TCR-CD3 signalling. In addition, among cells responding to CD3 mAb, higher peak [Ca2+]i responses correlated with maturity (CD4+ CD8+ thymocytes < single-positive thymocytes < splenic T cells). Examination of cells from pp59fyn-deficient mice showed that pp59fyn deficiency affects the amplitude and probability, but not the latency or synchrony, of CD3-mediated [Ca2+]i responses of CD4+ CD8+ and CD4+ CD8- thymocytes. All subsets showed equivalent receptor-independent mobilization of [Ca2+]i. These developmentally distinct [Ca2+]i features most probably reflect meaningful developmental changes in how the TCR-CD3 complex couples to intracellular signalling machinery including pp59fyn. By clearly showing how [Ca2+]i responses change during development, these results support the hypothesis that distinctive types of [Ca2+]i responses drive thymocyte differentiation.     

FT-2 antigen for distinguishing lymphocyte subpopulations in the developing thymus

The surface phenotypes of lymphoid cells in the developing embryonic thymus were characterized by using monoclonal antibodies. FT-2 antigen thus defined was predominantly expressed on thymocytes in the earlier embryonic stages in all the inbred mouse strains tested. The immunofluorescence and immunoperoxidase tests indicated that, like FT-1 antigen, the proportion of FT-2+ fetal thymocytes rapidly decreased with increase in gestation time, and these cells disappeared by day 19 of gestation. The treatment of fetal thymocytes with anti-FT-1 plus complement eliminated not only FT-1+, but also FT-2+ cells, whereas the treatment with anti-FT-2 failed to eliminate approximately 40% of FT-1+ cells, suggesting that embryonic thymocytes can be provisionally divided into at least three subpopulations, FT-1+2+, FT-1+2- and FT-1-2-.     

Mitogenic response of human thymocytes: identification of functional Ca2+-dependent and independent signals.

In contrast to peripheral blood mononuclear cells (PBMC), human thymocytes do not exhibit a proliferative response to the T cell mitogens phytohaemagglutinin (PHA), concanavalin A (Con A), or Staphylococcal protein A (SPA). In thymocytes and PBMC, Con A and PHA induce increases in free cytosolic calcium concentrations [( Ca2+]i). Since both Con A and PHA induce similar increases in [Ca2+]i in thymocytes and PBMC, the absence of thymocyte proliferation was not due to an inability to induce an increase in [Ca2+]i. The lack of proliferative response was secondary to the failure of the mitogens to induce interleukin 2 (IL-2) production. Incubation of mitogen-treated thymocytes with phorbol esters reconstituted IL-2 production and the proliferative response indicating that the cells were indeed activated by the mitogens. Similarly, addition of exogenous recombinant IL-2 also induced mitogen-treated thymocytes to proliferate. This IL-2-dependent proliferation established that SPA, Con A, and PHA triggered the expression of biologically active IL-2 receptors. Since an increase in [Ca2+]i is a prerequisite, and possibly a trigger, for IL-2 production, the failure of PHA, Con A, or SPA to result in thymocyte proliferation may be due to an inability of thymocytes to respond to increases in [Ca2+]i with subsequent IL-2 production.     

Calcium homeostasis in dissociated embryonic neurons: a flow cytometric analysis.

1. Ca2+ homeostasis in freshly dissociated neurons from embryonic rat hypothalamus, cortex, and brain stem was investigated with flow cytometry. Cells were dissociated from embryonic brain by enzymatic and mechanical means and were incubated with the acetoxymethylester derivative of the Ca(2+)-sensitive dye indo-1. Neurons hydrolyzed and retained the dye as determined by the intensity of fluorescence emission, whereas similarly treated cultured astrocytes gave very low-level fluorescence. 2. The fluorescence of the indo-1 dye was measured at two wavelengths (405 and 485 nm) for each cell. Data were collected only from those cells (presumptive neurons) with high levels of fluorescence. Methods were developed to calibrate the level of intracellular free calcium ([Ca2+]i) as the ratio of fluorescence at 410 and 485 nm. The level of intracellular free Ca2+ was then calculated for each neuron. 3. A wide distribution of resting [Ca2+]i was found, with a median of approximately 90 nM. After addition of ionomycin to cells in Ca(2+)-free medium, there was a transient increase in [Ca2+]i, suggesting that all embryonic neurons had internal Ca2+ stores. The presence of active calcium extrusion mechanisms was demonstrated with the use of ionomycin in Ca(2+)-containing medium and with metabolic inhibitors. Furthermore, incubation in sodium-free medium resulted in a transient increase in [Ca2+]i and a reduced ability to eliminate elevated [Ca2+]i from the cytoplasm, suggesting that calcium homeostasis was dependent on the activity of the Na(+)-Ca2+ exchange mechanism. 4. Depolarization with K+ or veratrine increased [Ca2+]i in approximately 20% of the cells. This increase was blocked by eliminating extracellular free Ca2+ or adding Co2+, nifedipine, or verapamil, suggesting mediation by voltage-sensitive calcium channels. 5. Neurons were sorted on the basis of high [Ca2+]i and placed into dissociated culture. After 24 h, neurons in culture retained indo-1 fluorescence, suggesting that populations of neurons can be collected on the basis of their levels of [Ca2+]i. 6. These results demonstrate that flow cytometric analysis allows the characterization of a variety of Ca(2+)-regulatory mechanisms in populations of freshly dissociated embryonic neurons. Although only a proportion of embryonic day 17 neurons exhibit voltage-sensitive calcium channels, all neurons have developed the ability to sequester and extrude Ca2+.     

GABA-induced intracellular Ca2+ elevation in the embryonic chick brainstem slice

We examined the intracellular Ca2+ ([Ca2+]i) elevation evoked by GABA in an 8-day embryonic chick brainstem slice using a Ca imaging technique with Ca green-1 AM. When small quantities of GABA were pressure-ejected on the surface of the slice, the [Ca2+]i elevation was clearly detected. The GABA-induced [Ca2+]i elevation was eliminated in a Ca2+-free solution, whereas the previously reported GABA-induced light-scattering change was independent of extracellular Ca2+. Although, micro-application of glycine or glutamate also induced [Ca2+]i elevation, these changes were smaller than that by GABA. These results suggest that the GABA-induced [Ca2+]i elevation is due to Ca2+ entry resulting from membrane depolarization and may play an important role in the development of the central nervous system (CNS).     

Prevention of age-related dysregulation of calcium dynamics by estrogen in neurons

To determine the impact of aging and 17beta-estradiol on neuronal Ca2+ homeostasis, intracellular Fura-2 Ca2+-imaging was conducted during 20-pulses of glutamate in hippocampal neurons cultured from embryonic (E18), middle-age (10 months) and old (24 months) rat brain. Marked age-related differences in intracellular Ca2+ ([Ca2+]i) homeostasis and striking regulation by 17beta-estradiol were seen. Embryonic neurons exhibited the greatest capacity to regulate Ca2+ homeostasis followed by middle-age neurons. In old neurons, the first peak [Ca2+]i was substantially greater than at other ages and the return to baseline Ca2+ rapidly dysregulated with an inability to restore [Ca2+]i following the first glutamate pulse which persisted throughout the 20 pulses. 17beta-Estradiol pretreatment of old neurons profoundly attenuated the peak [Ca2+]i rise and delayed the age-associated dysregulation of baseline [Ca2+]i, normalizing responses to those of middle-age neurons treated with estradiol. The efficacy of 17beta-estradiol extended below 10 pg/ml with full protection against toxicity from glutamate and Abeta (1-40). These results demonstrate age-associated dysregulation of [Ca2+]i homeostasis which was largely prevented by 17beta-estradiol with implications for estrogen/hormone therapy.     

Elevations in cytosolic free Ca2+ are not required to trigger apoptosis in human leukaemia cells.

Previous studies have indicated that Ca2+ is a trigger for apoptosis (programmed cell death) in thymocytes and related cell lines. Recently we have shown that levels of apoptosis in leukaemic cells are diminished in Ca(2+)-deficient conditions, indicating that Ca2+ may be important in the mechanism of apoptosis in these cells. In the present study we investigated the possibility that Ca2+ serves as a trigger for apoptosis in the human leukaemic cell line, HL-60. Using fura-2 to measure cytosolic free Ca2+ concentrations, [Ca2+]i, in cell suspensions, and by using ratio imaging of fura-2 in single cells, we did not observe an early significant increase in [Ca2+]i in HL-60 cells undergoing apoptosis. The latter stages of apoptosis were, however, accompanied by increasing [Ca2+]i; these increases were apparently a result of, rather than a cause of, apoptosis. Furthermore, apoptosis could be induced in HL-60 cells under conditions of vastly reduced [Ca2+]i achieved by loading these cells with fura-2 in the presence of EGTA. These results indicate that elevation of [Ca2+]i is not a prerequisite for apoptosis in HL-60 cells and that apoptosis can occur in these cells in the presence of low [Ca2+]i.     

Ca2+]i following extrasystoles in guinea-pig trabeculae microinjected with fluo-3 - a comparison with frog skeletal muscle fibres

Force production of cardiac muscle is highly dependent on the interval between the excitations. The aim was to investigate relations between intracellular calcium ([Ca2+]i) and force when a stimulus protocol, with three extrasystoles (ESs) at various intervals, was used. The relation between [Ca2+]i and force was compared with that in frog skeletal muscle fibre. Fluo-3 was microinjected into thin cardiac trabeculae to monitor [Ca2+]i. During steady-state [Ca2+]i consisted of a rapid rise (phase 1) that lasted until peak dF/dt (rate of force development) and was followed by a slower rise (phase 2) that coincided with the action potential and had a peak after peak force. The decline in [Ca2+]i outlasted the duration of the contraction. As the ES intervals were prolonged, there was a gradual restitution of force and of the amplitude and rate of rise of phase 1 [Ca2+]i. Peak dF/dt was linearly related to the amplitude of phase 1 [Ca2+]i during restitution and potentiation of force. Skeletal muscle fibres were loaded with fluo-3-AM. From [Ca2+]i the amount of calcium bound to troponin ([Ca-T]) as a function of time was estimated. Force production of the skeletal muscle fibre could be predicted from [Ca-T] when the signal was delayed (time constant 36 ms). This finding indicates that the recorded [Ca2+]i in skeletal muscle represents activator calcium. In cardiac muscle probably only phase 1 [Ca2+]i represents activator calcium. Phase 2 [Ca2+]i probably represents calcium entry during the action potential and does not activate the contractile system to any significant extent.     

Simultaneous recordings of cytosolic Ca2+ level and membrane potential and current during the response to thyroliberin in clonal rat anterior pituitary cells

The response to thyroliberin in prolactin-producing rat GH4C1 clonal cells was studied using fura-2 to monitor the cytosolic Ca2+ level ([Ca2+]i) in single cells, combined with recordings of membrane potential and current. The average value of [Ca2+]i was 109 nM (mean +/- SD, n = 112), and evoked action potentials caused transient elevations of about 100 nM. At higher firing frequencies these transients merged to a sustained elevation. In 100% of the cells thyroliberin caused an instant rise in [Ca2+]i, peaking at 795 +/- 300 nM (n = 112). This first phase of the thyroliberin response was associated with hyperpolarization in current clamp and outward current in voltage clamp, caused by the opening of Ca2(+)-activated K+ channels. In 75% of the cells the initial peak in [Ca2+]i was followed by a prolonged plateau phase at 247 +/- 76 nM (n = 84). In current clamp the second-phase elevation of [Ca2+]i was linked to either a modest depolarization in combination with enhanced firing frequency or a more pronounced depolarization in silent cells. This elevation of [Ca2+]i was reversed by hyperpolarizing current injection. No second-phase elevation of [Ca2+]i was observed during voltage clamp at a holding potential of -50 mV. Short exposure to Ca2(+)-free conditions eliminated the second-phase elevation in [Ca2+]i, whereas the first phase remained intact. Our experiments show a direct relationship between electrical activity and [Ca2+]i in the GH4C1 cells. The second-phase elevation of [Ca2+]i caused by thyroliberin is the result of influx through voltage-sensitive Ca2+ channels, without involving agonist-gated channels.     

Dissociation between force and [Ca2+]i during extra systoles in guinea-pig ventricular muscle microinjected with fura-2

Thin trabeculae were dissected from the right ventricle of guinea-pig heart and stimulated to contract isometrically at 0.5 Hz (26 degrees C). Rapid and transient changes of force were obtained by inducing three extra systoles (ES1-3) at 450-ms intervals. The two regular contractions (P1-2) following (ES1-3) were potentiated. Fura-2 salt was microinjected into the preparation to monitor intracellular calcium ([Ca2+]i). Three distinct phases of [Ca2+]i were seen: (1) a rapid rising phase to about 200 nmol L(-1), (2) a slower rising phase to a peak at 400 nmol L(-1), and (3) a slow decline to about 50 nmol L(-1). During ES1, there was a discrepancy between force, which decreased, and peak [Ca2+]i, which increased to 600 nmol L(-1). It is likely that the increased [Ca2+]i during the extra systoles reflects increased sarcolemmal calcium inflow, causing post-extra-systolic potentiation. Ryanodine (1-2 microM) was added to inhibit the intracellular calcium release and thus reduce the intracellular [Ca2+]i gradients following excitation. Ryanodine inhibited phase 1 of [Ca2+]i and abolished post-extra-systolic potentiation. There was a close relationship between dF/dt and [Ca2+]i with ryanodine during control and ES1-3. It is likely that fura-2 reports a spatially averaged [Ca2+]i and that phase 1 of the signal therefore apparently underestimates activator calcium in the close vicinity of the contractile elements.     

Contribution of Na+/Ca2+ exchanger to glucose-induced [Ca2+]i increase in rat pancreatic islets

The present study was carried out to elucidate the role of the reverse mode of the Na+/Ca2+ exchanger in an increase in intracellular Ca2+ concentration ([Ca2+]i) induced by a stimulatory concentration of glucose in rat pancreatic islets. The effects of KB-R7943, a selective inhibitor of reverse Na+/Ca2+ exchanger, on Na+o removal-induced [Ca2+]i changes were examined by a microfluorimetric method using fura-2 in perifused preparations of isolated rat pancreatic islets. Na+o removal induced a rapid increase in [Ca2+]i under 100 or 5 mM K+ conditions, respectively. The increases in [Ca2+]i induced by Na+o removal were inhibited by KB-R7943. The net amount of the [Ca2+]i increases during Na+o removal (Delta[Ca2+]i), obtained by subtracting the KB-R7943-independent Delta[Ca2+]i in the presence of KB-R7943 from Delta[Ca2+]i in the absence of KB-R7943, was significantly increased when extracellular K+ was raised. Increasing the external glucose concentration from 3 to 20 mM caused a biphasic increase in [Ca2+]i, which exhibited a transient increase (first phase) followed by a sustained increase (second phase) in [Ca2+]i. KB-R7943 (10 microM) partially inhibited the second phase of the [Ca2+]i increase rather than the first phase. These results suggest that the increase in [Ca2+]i induced by Na+o removal may be enhanced when plasma membrane is depolarized, and consequently, Ca2+ influx through the reverse Na+/Ca2+ exchanger may partially contribute to the glucose-induced [Ca2+]i dynamics in rat pancreatic islet cells.     

Evidence for Na+/Ca2+ exchange in the rectal gland of Squalus acanthias

Previously we have shown that stimulation of in vitro perfused rectal gland tubules (RGT) of the dog-fish Squalus acanthias by adenosine 3',5'-cyclic monophosphate (cAMP), (as a cocktail comprising 0.1 mmol/l dibutyryl-cAMP, 10 micromol/l forskolin and 0.1 mmol/l adenosine, hereafter termed STIM) leads to an increase in cytosolic Ca2+ ([Ca2+]i) and that this assists Cl- secretion by enhancing basolateral K+ conductance. In the present study we examined the mechanism of the cAMP-induced increase in [Ca2+]i. [Ca2+]i was measured using the fura-2 technique in isolated in vitro perfused RGT. As before, STIM enhanced [Ca2+]i. This elevation of [Ca2+]i was prevented completely when STIM was added in the presence of the Na+2Cl-K+ cotransport inhibitor furosemide (0.5 mmol/l). This suggests that the increase in [Ca2+]i induced by STIM is caused by a concomitant increase in cytosolic Na+ ([Na+]i) and not by the activation of second messenger cascades. Furosemide prevents this increase in [Na+]i and hence the elevation of [Ca2+]i. Moreover, the plateau phase of the [Ca2+]i transient produced by carbachol (CCH, 0.1 mmol/l) was augmented strongly when bath Na+ was reduced to 5 mmol/l. These data suggest that the level of [Ca2+]i is determined by Na(+)-dependent Ca2+ export, most likely via a Na+/Ca2+ exchanger. The increase in [Na+]i accompanying stimulation of Cl- secretion reduces the rate of Ca2+ export leading to an elevation of [Ca2+]i, as does a reduction in bath Na+ which augments the [Ca2+]i plateau produced by CCH.     

Effects of hypoxia on membrane potential and intracellular calcium in rat neonatal carotid body type I cells.

1. We have studied the effects of hypoxia on membrane potential and [Ca2+]i in enzymically isolated type I cells of the neonatal rat carotid body (the principal respiratory O2 chemosensor). Isolated cells were maintained in short term culture (3-36 h) before use. [Ca2+]i was measured using the Ca(2+)-sensitive fluoroprobe indo-1. Indo-1 was loaded into cells using the esterified form indo-1 AM. Membrane potential was measured (and clamped) in single isolated type I cells using the perforated-patch (amphotericin B) whole-cell recording technique. 2. Graded reductions in PO2 from 160 Torr to 38, 19, 8, 5 and 0 Torr induced a graded rise of [Ca2+]i in both single and clumps of type I cells. 3. The rise of [Ca2+]i in response to anoxia was 98% inhibited by removal of external Ca2+ (+1 mM EGTA), indicating the probable involvement of Ca2+ influx from the external medium in mediating the anoxic [Ca2+]i response. 4. The L-type Ca2+ channel antagonist nicardipine (10 microM) inhibited the anoxic [Ca2+]i response by 67%, and the non-selective Ca2+ channel antagonist Ni2+ (2 mM) inhibited the response by 77%. 5. Under voltage recording conditions, anoxia induced a reversible membrane depolarization (or receptor potential) accompanied, in many cases, by trains of action potentials. These electrical events were coincident with a rapid rise of [Ca2+]i. When cells were voltage clamped close to their resting potential (-40 to -60 mV), the [Ca2+]i response to anoxia was greatly reduced and its onset was much slower.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP potentiates glucose-induced insulin release from mouse pancreatic islets without increasing cytosolic free Ca2+

The effects of various stimulants of insulin release on cytosolic free Ca2+, [Ca2+]i, in dispersed and cultured pancreatic beta-cells from ob/ob-mice were studied using the indicator quin-2, which in itself has only slight effects on the glucose-induced insulin release and the metabolism of the sugar. The resting [Ca2+]i was 158 +/- 7 nM. After increasing glucose to 20 mM there was a lag-period of 1-2 min before [Ca2+]i gradually rose, reaching a new plateau 60% higher after 5-6 min. Increasing intracellular cyclic AMP by adding forskolin did not further increase [Ca2+]i; on the contrary there was a slight temporary reduction despite a doubling of insulin secretion. The maintenance of the beta-cell function was evident from a marked increase of cytosolic [Ca2+]i after depolarization evoked by high extracellular K+. Also dibutyryl cyclic AMP and theophylline lacked the ability to raise [Ca2+]i beyond that obtained by glucose. The results suggest that cyclic AMP potentiates glucose-induced insulin release by sensitizing the secretory machinery to changes of [Ca2+]i rather than by increasing the cytosolic concentration of the ion.     

Measurement of calcium entry and exit in quiescent rat ventricular myocytes

The aim of this work was to obtain the first quantitative measurements of Ca2+ influx and efflux in quiescent cardiac cells. The relationship between free and total Ca2+ was obtained during a caffeine application. This buffering curve was then used to calculate changes of total Ca2+ from measurements of free cytosolic [Ca2+] ([Ca2+]i) made with Indo-1. The rate of Ca2+ removal from the cytoplasm was calculated by differentiating total Ca2+ with respect to time. The dependence of d(total Ca2+)/dt on [Ca2+]i was hyperbolic. Inhibition of either Na+-Ca2+ exchange (by addition of 10 mmol l(-1) NiCl2 or removal of external Na+) or the sarcolemmal Ca2+-activated adenosine triphosphatase (Ca2+-ATPase) (with carboxyeosin) decreased the calculated efflux. In both cases, the main effect was on the apparent maximum rate (Vmax) with little effect on the Michaelis-Menten constant (Km). These results suggest that the Na+-Ca2+ exchange and Ca2+-ATPase have very similar affinities for [Ca2+]i and that their fractional contributions do not change over the systolic range of [Ca2+]i. Ca2+ influx was quantified in two ways. The first method was to extrapolate the curve relating Ca2+ efflux to [Ca2+]i to zero [Ca2+]i. This gave a value of 4.49+/-0.54 micromol l(-1) s(-1) which was reduced to zero by either removal of external Ca2+ or addition of Ni2+. In other experiments external Ca2+ was removed and the maximum rate of fall of total Ca2+ calculated as 2.53+/-0.93 micromol l(-1) s(-1). This approach can be used to provide a quantitative analysis of the control of resting [Ca2+]i.     

Regulation of action potential size and excitability in substantia nigra compacta neurons: sensitivity to 4-aminopyridine

Application of the metabotropic glutamate receptor (mGluR) agonist (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) or the selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) depolarized both CA3 and CA1 pyramidal cells in guinea pig hippocampal slices. Simultaneous recordings of voltage and intracellular Ca2+ levels revealed that the depolarization was accompanied by a biphasic elevation of intracellular Ca2+ concentration ([Ca2+]i): a transient calcium rise followed by a delayed, sustained elevation. The transient [Ca2+]i rise was independent of the membrane potential and was blocked when caffeine was added to the perfusing solution. The sustained [Ca2+]i rise appeared when membrane depolarization reached threshold for voltage-gated Ca2+ influx and was suppressed by membrane hyperpolarization. The depolarization was associated with an increased input resistance and persisted when either the transient or sustained [Ca2+]i responses was blocked. mGluR-mediated voltage and [Ca2+]i responses were blocked by (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) or (S)-4-carboxy-3-hydroxyphenylglycine (4C3HPG). These data suggest that in both CA3 and CA1 hippocampal cells, activation of group I mGluRs produced a biphasic accumulation of [Ca2+]i via two paths: a transient release from intracellular stores, and subsequently, by influx through voltage-gated Ca2+ channels. The concurrent mGluR-induced membrane depolarization was not caused by the [Ca2+]i rise.     

Increase in cytoplasmic free Ca2+ elicited by noradrenalin and serotonin in cultured local interneurons of mouse olfactory bulb.

Effects of noradrenalin and serotonin on cytoplasmic free Ca2+ concentrations ([Ca2+]i) were studied by using the fluorescent indicator fura-2 in cultured local interneurons of mouse olfactory bulb. Application of noradrenalin (0.1-100 microM) caused a rapid and concentration-dependent rise in [Ca2+]i, while isoproterenol was ineffective at concentrations up to 100 microM. The noradrenalin (1 microM)-induced increase in [Ca2+]i was completely inhibited by pretreatment with alpha 1-antagonist, prazosin (100 nM), whereas the inhibitory effect of alpha 2-antagonist, yohimbine, was about 100-times less potent. Serotonin (0.1-100 microM) also caused the dose-dependent rise in [Ca2+]i, which was inhibited by serotonin2 antagonist, ketanserin. Even in the absence of the extracellular calcium, the noradrenalin- or serotonin-induced increase in [Ca2+]i was observed. These results indicate that both noradrenalin and serotonin elicit the rise in [Ca2+]i in local interneurons of the olfactory bulb. They also suggest that the rise in [Ca2+]i is mediated by alpha 1-adrenergic and serotonin2 receptors, and that the increased calcium is mainly derived from intracellular calcium storage sites. The above results provide evidence to suggest that in the olfactory bulb, noradrenergic and serotonergic centrifugal fibers exert modulatory influences on synaptic interactions between mitral/tufted cells and local interneurons by increasing cytoplasmic Ca2+ in local interneurons.     

Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts

Cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were measured with fluorescent indicators in CCL39 cells, a cell line established from Chinese hamster lung fibroblasts, transfected with complementary deoxyribonucleic acid (cDNA) of the Na+-Ca2+ exchanger isolated either from canine heart (NCX1) or from rat brain (NCX3). Raising extracellular [Mg2+] to 10 mM increased Mg2+ influx and the resultant change in [Mg2+]i (delta[Mg2+]i) was monitored with furaptra under Ca2+-free conditions. In control (vector-transfected) cells, delta[Mg2+]i at 45 min was similar with or without extracellular Na+ (130 mM or 0 mM) and when [Na+]i was raised by 1 mM ouabain treatment. delta[Mg2+]i in NCX1-transfected cells was attenuated significantly in the presence of 130 mM Na+, but became comparable to (or slightly larger than) that in control cells on either removal of extracellular Na+ or treatment with 1 mM ouabain. Cells expressing NCX3 showed an intermediate dependence of delta[Mg2+]i on Na+, probably reflecting a lower degree of expression of the exchanger protein. Extracellular Na+-dependent changes in [Ca2+]i (measured with fura-2 in the presence of extracellular Ca2+ and 10 microM ionomycin, a Ca2+ ionophore) were minimal in control cells, marked in the NCX1-transfected cells and intermediate in the NCX3-transfected cells. These results suggest that the Na+-Ca2+ exchanger (either NCX1 or NCX3) can transport Mg2+ and may play a role in the extrusion of magnesium from cells.     

Response to ATP is accompanied by a Ca2+ influx via P2X purinoceptors in the coronary arterioles of golden hamsters

In the vascular wall, adenosine-5'-triphosphate (ATP) released along with noradrenaline from sympathetic nerve terminals is considered to play an important role in controlling intracellular calcium ion ([Ca2+]i) levels in arteries. The present study examined how vascular smooth muscle cells in coronary arterioles respond to ATP in relation to [Ca2+]i dynamics. For this purpose, the dynamics of [Ca2+]i in the coronary arterioles of golden hamsters was examined by real-time laser scanning confocal microscopy. This technique enabled the visualization of [Ca2+]i changes in response to ATP in the intact coronary arterioles, the ultrastructure of which was well preserved. It was shown that an increase in [Ca2+]i in the arteriole smooth muscle cells was elicited by ATP. While P1 purinoceptor agonists have no effect on this process, P2 purinoceptor agonists were found to induce a [Ca2+]i increase in the smooth muscle cells. Suramin (an antagonist of P2X and P2Y receptors) completely inhibited ATP-induced [Ca2+]i dynamics, but reactive blue 2 (a P2Y receptor antagonist) did not. Uridine-5'-triphosphate (a P2Y receptor agonist) had no effect on [Ca2+]i, but alpha,beta-methylene ATP (a P2X receptor agonist) caused a strong increase in [Ca2+]i. We conclude that smooth muscle cells of the hamster coronary arterioles possess P2X, but not P1 or P2Y purinoceptors. The smooth muscle cells probably respond to extracellular ATP via P2X purinoceptors, resulting in the contraction of the coronary arterioles.