K(+)-evoked dopamine release depends on a cytosolic Ca2+ pool regulated by N-type Ca2+ channels.

Membrane depolarization evoked by 25-40 mM K+ elicited an immediate increase of somatic and neuritic [Ca2+]i in cultured dopaminergic neurons as measured by digital fluorescence microscope imaging. The rise of neuritic [Ca2+]i was inhibited by N-type but not L-type Ca2+ channel blockers, while the rise of somatic [Ca2+]i was prevented by both L- and N-type Ca2+ channel blockers. Similarly, depolarization-induced [3H]dopamine release was selectively attenuated by N-type Ca2+ channel blockers. The present results suggest that [3H]dopamine release from mesencephalic neuronal cell cultures relates to a Ca(2+)-dependent mechanism regulated by N-type channels located in the vicinity of the exocytotic sites within neuritic processes.     

Mechanisms of glutamate activation of axon-to-Schwann cell signaling in the squid.

Membrane potentials from Schwann cells associated with giant axons of the small squid (Alloteuthis and Loliguncula) and the large squid (Loligo) were monitored with glass microelectrodes following 100 Hz/15 s axonal stimulation, or the application of 10(-7) M glutamate and ion substitutions, in the presence or absence of 10(-7) M d-tubocurarine. Glutamate or stimulation caused the membrane of the Schwann cell to depolarize to approximately -32 mV. This was rapidly replaced by a transient hyperpolarization to approximately -55 mV; the potential returning to the resting level (-40 mV) in approximately 7 min. In the presence of d-tubocurarine only the initial depolarization was evident. Nominally zero [Na+]o or treatment with 10(-7) M tetrodotoxin (in normal [Na+]o) blocked the stimulation- and glutamate-induced depolarization while low Clo- hyperpolarized the Schwann cell without effect on the glutamate- or stimulation-induced depolarization. Nao+ depletion or pretreatment with tetrodotoxin in normal Nao+ did not affect the development of the Schwann cell hyperpolarization. These results do not support the hypothesis that the glutamate-induced depolarization is the trigger leading to the Schwann cell hyperpolarization. Preliminary experiments to test the possibility that inositol phosphate second messenger and an increase in [Ca2+]i are triggered by glutamate receptor activation showed that nominally 0 Cao2+/75 mM Mgo2+ only slightly reduced the hyperpolarizing response to stimulation or glutamate while intracellular Bapta (20-30 microM) blocked the hyperpolarization but not the depolarization. [3H]Myoinositol incorporation into axon-Schwann cell plasma membranes was high.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Reciprocal interactions between calcium and chloride in rod photoreceptors

This study used imaging and electrophysiological techniques in salamander retinal slices to correlate Ca2+ and Cl- levels in rods and thus test the hypothesis of a feedback interaction between Ca2+- and Ca2+-activated Cl- channels whereby Cl- efflux through Cl- channels can inhibit Ca2+ channels. Increasing [K+]o levels produced a concentration-dependent depolarization of rods accompanied by increases in [Ca2+]i measured with Fura-2. The voltage dependence of increases in [Ca2+]i was compared with the voltage dependence of the calcium current (ICa). [Cl-]i was measured with the dye, MEQ. Depolarization with high K+ to membrane potentials below -20 mV reduced [Cl-]i; larger depolarizations increased [Cl-]i. The Na/K/Cl cotransport inhibitor, bumetanide, shifted the apparent Cl- equilibrium potential (ECl) to more negative potentials, suggesting that this cotransporter helps establish a relatively depolarized ECl. MEQ fluorescence changes evoked by high K+ were inhibited by niflumic acid (0.1 mM), NPPB (2 microM), or replacement of Ca2+ with Ba2+, suggesting that depolarization-evoked Cl- changes result partly from stimulation of Ca2+-activated Cl- channels. Replacing >/=12 mM [Cl-]o with CH3SO4- produced a significant reduction in [Cl-]i. [Ca2+]i increases evoked by 20 or 50 mM K+ were also significantly inhibited by replacing >/=12 mM [Cl-]o with CH3SO4-. Thus modest depolarization can evoke increases in [Ca2+]i that lead to reductions in [Cl-]i, and conversely, reductions in [Cl-]i inhibit depolarization-evoked [Ca2+]i increases. These findings support the hypothesis that feedback interactions between Ca2+- and Ca2+-activated Cl- channels may contribute to the regulation of presynaptic Ca2+ currents involved in synaptic transmission from rod photoreceptors.     

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.     

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).     

Modulation of the Ca2+-activated large conductance K+ channel by intracellular pH in human renal proximal tubule cells

The Ca2+-activated and voltage-sensitive large conductance K+ channel (BK channel) with a slope conductance of about 300 pS is present in the surface membrane of cultured human renal proximal tubule epithelial cells (RPTECs). In this study we examined the effects of cytoplasmic pH (pH(i)) on activity and gating kinetics of the BK channel by using the inside-out configuration of the patch-clamp technique. At a constant cytoplasmic Ca(2+) concentration ([Ca2+]i), membrane depolarization raised channel open probability (P(o)), and lowering pH(i) shifted the P(o)-membrane potential (V(m)) relationship to the positive voltage direction. However, the value of the gating charge was not affected by changes in pH(i), suggesting that the effects of pH(i) on P(o) were not due to an alternation of the voltage sensitivity. At constant V(m), lowering pH(i) suppressed the [Ca2+]i-dependent channel activation and shifted the P(o)-[Ca2+]i relationship in the direction of higher [Ca2+]i with a reduction of maximal P(o). Furthermore, both the mean open and mean closed times of the BK channels at pH(i) 6.3 in the presence of 10(-4) M [Ca2+](i) were shorter than those at pH(i) 7.3 in the presence of 10(-5) M [Ca2+]i, even though these two different conditions gave a similar P(o). The data indicate that cytoplasmic H+ suppresses P(o) of the BK channel in RPTECs, which involves the mechanism independent of Ca2+ activation. Our preliminary kinetic analysis also supported this notion.     

The cytosolic free calcium in anti-mu-stimulated human B cells is derived partly from extracellular medium and partly from intracellular stores

The inositol phospholipid metabolism and the increase in cytosolic free Ca2+ concentration ([Ca2+]i) into the cell are recognized as two important events in the anti-mu-induced B cell activation. The anti-mu stimulation caused the [3H]inositol incorporation and also a rapid increase in [Ca2+]i from 85 nM to 285 nM. This signal returned to baseline a few minutes after stimulation. By using the fluorescent indicator quin-2 we demonstrated that this [Ca2+]i uptake was derived part from extracellular medium and part from intracellular stores. Both EGTA (a calcium chelator) and TMB.8 (a drug which interferes with Ca2+ sequestration by smooth endoplasmic reticulum) partially suppressed the intracellular Ca2+ uptake and were fully inhibitory when added together. The role of Ca2+ from intracellular stores may also be evidenced in calcium-free experiments, or in permeabilized experiments using exogenous inositol 1,4,5-trisphosphate (IP3, the putative mobilizer of intracellular Ca2+). Preventing the increase in [Ca2+]i also prevents the apparition of early activation makers. These results are consistent with the hypothesis that the Ca2+ increase in B cells stimulated by anti-mu is caused by the generation of IP3 during the phosphatidyl-inositol metabolism and also by the entry of extracellular Ca2+ through the plasma membrane.     

Cytoplasmic Ca2+ at low submicromolar concentration stimulates mitochondrial metabolism in rat luteal cells

The cytoplasmic Ca2+ signal is transferred to the mitochondrial matrix and activates mitochondrial dehydrogenases. The requirement for supramicromolar cytoplasmic [Ca2+] ([Ca2+]i) in perimitochondrial microdomains in this response has been suggested. We studied the correlation between [Ca2+]i, mitochondrial [Ca2+] ([Ca2+]m) and mitochondrial formation of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] in the presence of submicromolar [Ca2+]i in cultured rat "large" luteal cells. [Ca2+]i was monitored fluorimetrically with fura-PE3, [Ca2+]m with rhod-2 and NAD(P)H with autofluorescence. In intact cells, prostaglandin F2alpha, which induces both intracellular Ca2+ release and Ca2+ entry, stimulated mitochondrial NAD(P)H formation. Thapsigargin-induced Ca2+ release and subsequent capacitative Ca2+ entry, both resulting in Ca2+ responses not exceeding 150-200 nM, also enhanced the reduction of pyridine nucleotides. As shown in inhibitor studies, the increased steady-state NAD(P)H level was due to activation of Ca2+-dependent dehydrogenases. [Ca2+]m, measured in permeabilized cells, increased moderately, but significantly, following elevation of [Ca2+]i from 50 to 180 nM, showed a further gradual increase at higher submicromolar [Ca2+]i values and rose steeply at supramicromolar [Ca2+]i. In summary, our results demonstrate that, in a steroid-producing cell type, net mitochondrial Ca2+ uptake and mitochondrial dehydrogenation can be activated even by low submicromolar increases of [Ca2+]i.     

Tumor-promoting phorbol esters suppress receptor-stimulated inositol phospholipid degradation and Ca2+ mobilization in mouse lymphocytes

Anti-immunoglobulin antibodies (anti-Ig) provoke the rapid breakdown of phosphatidylinositol bisphosphate (PIP2), elevation of cytoplasmic Ca2+ levels ([Ca2+]i) and activation of protein kinase C (PKC) in B lymphocytes. Tumor-promoting phorbol esters, like phorbol myristate acetate, also activate PKC, but inhibit anti-Ig-induced B cell proliferation. To investigate the basis of the latter effect, we studied the influence of phorbol esters on PIP2 degradation and [Ca2+]i in murine B cells. The results show that PKC-activating phorbol esters cause marked inhibition of anti-Ig-stimulated PIP2 breakdown and Ca2+ mobilization. In addition, these agents inhibit concanavalin A-provoked Ca2+ influx, lower resting cytoplasmic Ca2+ levels and reduce ionophore-induced Ca2+ influx in B cells. Apparently, PKC stimulation causes feedback inhibition of receptor signalling, not only by suppressing PIP2 degradation, but also by exerting additional complex effects on the control of [Ca2+]i in B cells. It is, however, not clear how these findings relate to the anti-proliferative effects of phorbol esters on B cells.     

Regulation of intracellular Ca2+ concentration by interleukin-1beta in rat cortical synaptosomes: an age-related study

The pro-inflammatory cytokine interleukin-1beta (IL-1beta) is released by cells during injury and stress, and increased neuronal expression of IL-1beta is a feature of age-related neurodegeneration. We have recently reported that IL-1beta has a biphasic effect on the K+-induced rise in intracellular Ca2+ concentration ([Ca2+]i) in cortical synaptosomes, exerting an inhibitory effect on the K+-induced rise in [Ca2+]i at lower (3.5 ng/mL) concentrations and a stimulatory effect on the K+-induced rise in [Ca2+]i at higher (100 ng/mL) concentrations. In the present study, we observed that the K+-induced rise in [Ca2+]i was inhibited to a similar extent by the lower concentration of IL-1beta in cortical synaptosomes prepared from young (3-month-old), middle-aged (12-month-old) and aged (24-month-old) rats. In contrast, cortical synaptosomes prepared from the aged rats exhibited an increased susceptibility to the higher concentration of IL-1beta, resulting in a marked elevation in [Ca2+]i. We propose that the age-related increase in neuronal concentration of IL-1beta promotes a dramatic elevation in [Ca2+]i following membrane depolarization, thereby altering Ca2+ homeostasis and exacerbating neuronal vulnerability to excitotoxicity.     

A mouse with a point mutation in plasma membrane Ca2+-ATPase isoform 2 gene showed the reduced Ca2+ influx in cerebellar neurons

We analyzed mutant mice showing behavioral defects such as severe tremor, up-and-down and side-to-side wriggling of neck without coordination, and found that the gene causing the defects was located between 46 and 60.55 centimorgans (cM) on the mouse chromosome 6. In this region, nucleotide transition of the plasma membrane Ca2+-ATPase isoform 2 (PMCA2) gene was found, which caused a glutamic acid to change into lysine. Since PMCA2 is expressed in the cerebellum and plays an important role to maintain the homeostasis of the intracellular Ca2+ as a Ca2+ pump, the behavioral defect can be ascribed to the impairment of Ca2+ regulation in neurons of the cerebellum. To confirm the defect of Ca2+ homeostasis in the mutant mice, we measured high K+-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in the cerebellar neurons. Contrary to our expectation, the extent of the [Ca2+]i increase in all the regions tested in the cerebellar slice was far smaller than that of the wild type mice, while the resting [Ca2+]i remained almost unaltered. The rate of rise in [Ca2+]i during high K+-induced depolarization was significantly reduced, and the extrusion rate of increased [Ca2+]i was also reduced. These results suggested that voltage-gated Ca2+ channels were down-regulated in the mutant mice in order to regulate [Ca2+]i toward the normal homeostasis. The behavioral defects may be ascribed to the down-regulated Ca2+ homeostasis since dynamic changes in [Ca2+]i are important for various neuronal functions.     

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)     

Changes in mitochondrial function induced in isolated guinea-pig ventricular myocytes by calcium overload.

1. Changes in [Ca2+]i and pHi, mitochondrial membrane potential (psi m) and mitochondrial [NADH] have been measured independently using fluorescent techniques in single isolated guinea-pig ventricular myocytes subjected to Ca2+ overload. 2. The changes in NADH autofluorescence on the inhibition or uncoupling of respiration are consistent with the signal emanating from the mitochondrial NADH. 3. Removal of Ca2+ and Mg2+ from the bathing Tyrode solution induced a modest fall in both [Ca2+]i and pHi, a small slowly developing depolarization of psi m and an initial fall followed by a rise in mitochondrial [NADH]. 4. In myocytes that maintained an intact sarcolemma, return to Ca(2+)-containing fluid elicited a strong but brief intracellular acidification, a rise in [Ca2+]i which generally recovered more slowly to stabilize above the initial level in Tyrode solution, a steep fall in mitochondrial [NADH] and a brief transient recovery followed by a large sustained depolarization of psi m. NADH autofluorescence and mitochondrial depolarization often reached values that were not further increased by uncoupling respiration although recovery of NADH was elicited by inhibitors of respiration. 5. These changes were reduced when the Ca2+ overload was less severe as evidenced by a reduced hypercontracture upon Ca2+ repletion. A similar reduction could be routinely achieved by elevation of [Mg2+]o during the period of Ca2+ depletion. 6. These results suggest that the well-established depletion of energy-rich phosphates that occurs on Ca2+ overload is due to the combined effects of the failure of the citric acid cycle to provide sufficient mitochondrial NADH for the respiratory chain and an uncoupling of respiration from ATP production due to depolarization of psi m. The former effect could result from the depletion of sarcoplasmic amino acids and the latter from increased Ca2+ cycling across the mitochondrial wall provoked by the elevated [Na+]i and [Ca2+]i.     

Activation of human B lymphocytes by nanogram concentrations of anti-IgM-dextran conjugates.

Surface immunoglobulin (sIg) cross-linking on B lymphocytes by high concentrations of anti-Ig antibody has been used to mimic antigen-stimulated B cell activation. In order to develop a system to study sIg-mediated T cell-independent B cell activation using low concentrations of anti-Ig antibody that more closely resemble the concentrations of antigen that are achieved under in vivo conditions, we conjugated monoclonal anti-human IgM antibody (anti-mu) to dextran (molecular weight 2 X 10(6)) thereby increasing its valency. This dextran conjugate (anti-mu-Dex) stimulated comparable levels of thymidine incorporation and B cell size increases as were seen with unconjugated anti-mu but at 100- to 1000-fold lower concentrations. Anti-mu-Dex also stimulated increases in intracellular ionized calcium ([Ca2+]i) in a higher percentage of cells, of greater magnitude and of longer duration than that stimulated by unconjugated anti-mu. Interestingly, there was no direct correlation between the increases in [Ca2+]i that were stimulated by anti-mu-Dex and its ability to stimulate B cell proliferation. The concentrations of anti-mu-Dex (10 micrograms/ml) that led to the highest increase in [Ca2+]i resulted in thymidine incorporation that was no greater than that of medium control, whereas 0.01 to 0.1 microgram/ml stimulated significant thymidine incorporation with 50% lower levels of stimulation of [Ca2+]i. These data demonstrate that anti-mu-Dex is a potent activator of human B lymphocytes, is effective even at ng/ml concentrations which over a 2-h time period do not induce detectable modulation of sIg, and its stimulation of B cells into G1 and S may not be directly related to its ability to stimulate increases in levels of [Ca2+]i.     

Functional expression of a subtype of the Ca2+ channels in the embryonic rat hippocampus as detected by dual-fluorescence flow cytometric analysis

We have analyzed the expression of the Ca2+ channels in hippocampal cell suspensions from the 18- to 20-day-old rat embryo using dual-fluorescence flow cytometry. A high concentration of K+ induced elevation of the cytoplasmic free Ca2+ concentration ([Ca2+]i) as well as membrane depolarization. The high K+-evoked [Ca2+]i increase was inhibited by phenytoin, but not by either nifedipine or nicardipine. These agents had no effect on the high K+-induced membrane depolarization. These findings suggest that a subtype corresponding to the low voltage-activated Ca2+ channel is expressed in the embryonic rat hippocampal cells.     

Glutamate receptor agonists increase intracellular Ca2+ independently of voltage-gated Ca2+ channels in rat cerebellar granule cells

Changes in membrane potential and cytosolic free Ca2+ concentrations, [Ca2+]i, in response to L-glutamate and glutamate receptor agonists were measured in rat cerebellar granule cells grown on coverslips. The membrane was depolarized by the application of L-glutamate and kainate, and by elevating the extracellular K+ concentration, as determined by using the membrane potential probe bisoxonol (DiBA-C4-(3)). The [Ca2+]i as measured with fura-2 was 220 nM on average under resting conditions and increased by raising the extracellular K+ and by applying L-glutamate, kainate, quisqualate or N-methyl-D-aspartate (NMDA). Verapamil and nifedipine reduced the high-K+ induced rise in [Ca2+]i but did not significantly affect the responses produced by NMDA, quisqualate and kainate, suggesting that the increase in intracellular Ca2+ in response to glutamate receptor agonists is primarily due to Ca2+ influx through receptor-coupled ion channels.     

The relationship between acetylcholine-evoked Ca2+-dependent current and the Ca2+ concentrations in the cytosol and the lumen of the endoplasmic reticulum in pancreatic acinar cells

In a study of isolated mouse pancreatic acinar cells, we used the patch-clamp whole-cell recording configuration to monitor the Ca(2+)-dependent inward ionic current and simultaneously measured the Ca2+ concentration in either the cytosol ([Ca2+]i) or the lumen of the endoplasmic reticulum ([Ca2+]Lu), using appropriate Ca(2+)-sensitive fluorescent probes. A high concentration of acetylcholine (ACh, 10 microM) evoked an increase in [Ca2+]i, which resulted in the activation of Ca(2+)-dependent inward current. Continued ACh application for several minutes led to a marked reduction in both the current and the [Ca2+]i response and after about 4-10 min of sustained ACh stimulation, the inward current response had disappeared and [Ca2+]i was back to the pre-stimulation level. Repeated stimulation with shorter pulses of ACh (10 microM) resulted in responses of declining magnitude both in terms of inward current and [Ca2+]i rises. The ACh-activated inward current was entirely dependent on the elevation of [Ca2+]i, but at a relatively high [Ca2+]i the current was saturated. ACh caused a rapid release of Ca2+ from the lumen of the endoplasmic reticulum and after discontinuation of stimulation, [Ca2+]Lu was only very slowly (10-15 min) fully restored to the pre-stimulation level. Repeated applications of ACh did not change the relationships between the Ca(2+)-dependent current and [Ca2+]i or the current and [Ca2+]Lu. When [Ca2+]Lu was greater than 100 microM, the ACh-evoked Ca2+ release from the store was so large that the current response was initially saturated. We conclude that the ACh-evoked current response essentially depends on the release of stored Ca2+. Desensitization is mainly due to the relatively slow reloading of the intracellular stores with Ca2+.     

Inhibition of mitochondrial Ca2+ uptake affects phasic release from motor terminals differently depending on external [Ca2+

We investigated how inhibition of mitochondrial Ca2+ uptake affects stimulation-induced increases in cytosolic [Ca2+] and phasic and asynchronous transmitter release in lizard motor terminals in 2 and 0.5 mM bath [Ca2+]. Lowering bath [Ca2+] reduced the rate of rise, but not the final amplitude, of the increase in mitochondrial [Ca2+] during 50-Hz stimulation. The amplitude of the stimulation-induced increase in cytosolic [Ca2+] was reduced in low-bath [Ca2+] and increased when mitochondrial Ca2+ uptake was inhibited by depolarizing mitochondria. In 2 mM Ca2+, end-plate potentials (epps) depressed by 53% after 10 s of 50-Hz stimulation, and this depression increased to 80% after mitochondrial depolarization. In contrast, in 0.5 mM Ca2+ the same stimulation pattern increased epps by approximately 3.4-fold, and this increase was even greater (transiently) after mitochondrial depolarization. In both 2 and 0.5 mM [Ca2+], mitochondrial depolarization increased asynchronous release during the 50-Hz train and increased the total vesicular release (phasic and asynchronous) measured by destaining of the styryl dye FM2-10. These results suggest that by limiting the stimulation-induced increase in cytosolic [Ca2+], mitochondrial Ca2+ uptake maintains a high ratio of phasic to asynchronous release, thus helping to sustain neuromuscular transmission during repetitive stimulation. Interestingly, the quantal content of the epp reached during 50-Hz stimulation stabilized at a similar level ( approximately 20 quanta) in both 2 and 0.5 mM Ca2+. A similar convergence was measured in oligomycin, which inhibits mitochondrial ATP synthesis without depolarizing mitochondria, but quantal contents fell to <20 when mitochondria were depolarized in 2 mM Ca2+.     

Platelet-derived growth factor receptors expressed in response to injury of differentiated vascular smooth muscle in vitro: effects on Ca2+ and growth signals.

Vascular smooth muscle cells (VSMCs) in the intact vascular wall are differentiated for contraction, whereas the response to vascular injury involves transition towards a synthetic phenotype, with increased tendency for proliferation. Platelet-derived growth factor (PDGF) is thought to be important for this process. We investigated expression and functional coupling of PDGF receptors (PDGFRs) alpha and beta in rat tail arterial rings kept in organ culture, in order to capture early events in the phenotypic transition. In freshly dissected rings no PDGFR immunoreactivity was found in medial VSMCs, whereas PDGFR alpha was detected in nerve fibres. After organ culture for 1-4 days PDGFR alpha and beta as well as phospholipase Cgamma2 (PLCgamma2), known to couple to PDGFR, were expressed in VSMCs within 100 microm of the cut ends. Calponin, a marker for the contractile phenotype, was decreased near the injured area, suggesting that cells were in transition towards synthetic phenotype. In these cells, which showed functional Ca2+-release from the sarcoplasmic reticulum, PDGF-AB (100 ng x mL(-1)) had no effect on [Ca2+]i, whereas cultured VSMCs obtained from explants of rat tail arterial rings responded to PDGF-AB with an increase in [Ca2+]i. However, PDGFR within the cultured rings coupled to growth signalling pathways, as PDGF-AB caused a tyrphostin AG1295-sensitive activation of extracellular signal-regulated kinases 1 and 2 and of [3H]-thymidine incorporation. Thus, early expression of PDGFR in VSMC adjacent to sites of vascular injury coincides with signs of dedifferentiation. These receptors couple to growth signalling, but do not activate intracellular Ca2+ release.