Phosphorylation down-regulates the store-operated Ca2+ entry pathway of human neutrophils

We have reported previously that the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) inhibits transiently Ca2+ entry through the plasma membrane Ca2+ pathway activated by emptying the intracellular Ca2+ stores (Montero, M., García-Sancho, J., and Alvarez, J. (1993) J. Biol. Chem. 268, 13055-13061). We show here that calyculin A and okadaic acid, inhibitors of protein phosphatases 1 and 2A, prevent the spontaneous reversion of the fMLP-induced inhibition of the entry of Ca2+ and Mn2+ (used as a Ca2+ surrogate), leading to a permanently inhibited Ca2+ entry pathway. At high concentrations or long incubation times the phosphatase inhibitors were even able to inhibit the store-operated Ca2+ entry pathway (SOCP) in the absence of fMLP. Inhibition of SOCP by phorbol dibutyrate, which is not reversible, was not modified by phosphatase inhibitors. These results provide additional support for the view that fMLP inhibits SOCP through phosphorylation of either the SOCP protein or a regulatory protein and indicate that dephosphorylation mediated by protein phosphatases 1 and/or 2A restores the activity of SOCP after inhibition by fMLP. The time course of the inhibition of SOCP by fMLP was similar to the one reported previously for the transient fMLP-induced phosphorylation of a 47-kDa protein involved in the generation of respiratory burst, which was similarly affected by the phosphatase inhibitors.     

BAPTA-AM blocks both voltage-gated and Ca2+-activated K+ currents in cultured bovine chromaffin cells

The effects of the membrane permeant Ca2+ chelator BAPTA-AM on voltage-gated Na+, Ca2+, K+ (I(Na), I(Ca) I(K), respectively) and Ca2+-activated K+ (I(KCa)) currents in cultured bovine chromaffin cells were investigated using the whole-cell patch-clamp technique. Superfusion with BAPTA-AM (50 microM) induced a rapid (< 60 s) and reversible block of both I(KCa) and I(K) (approximately 50%), without affecting either I(Ca) or I(Na). Preincubation with BAPTA-AM (50 microM, 30 min) or cell loading with the nonpermeable active form of BAPTA (10 mM in the pipette solution) permanently blocked I(KCa). BAPTA-AM superfusion (50 microM) also blocked I(K) (approximately 53%) after BAPTA-loading or BAPTA-AM preincubation. In conclusion, we show a fast and reversible block of I(KCa) and I(K) by BAPTA-AM, acting directly on K+ channels before it operates as a Ca2+ chelator, in cultured bovine chromaffin cells.     

Transient inhibition by chemotactic peptide of a store-operated Ca2+ entry pathway in human neutrophils

Emptying the intracellular calcium stores of fura-2-loaded human neutrophils by treatment with the endomembrane ATPase inhibitor thapsigargin leads to a maintained increase of [Ca2+]i by Ca2+ entry through a store-operated Ca2+ entry pathway. Under these conditions, [Ca2+]i was reduced transiently by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and permanently by phorbol 12,13-dibutyrate (PDB). Platelet-activating factor (PAF) had no effect. The fMLP- and PDB-induced [Ca2+]i decreases were not due to stimulated Ca2+ efflux but to inhibition of store-operated Ca2+ entry pathway. PDB and fMLP, but not PAF, inhibited the entry of Ca2+, Mn2+, and Ba2+ in thapsigargin-treated cells. This inhibition was dependent on [Ca2+]i, barely detectable at [Ca2+]i of 50 nM and increasingly strong and fast to appear at 170 and 630 nM. Inhibition of entry by fMLP was complete within 5-10s, disappeared within 2-3 min, and was partially prevented by staurosporin (100 nM). Inhibition by PDB was equally fast, but no recovery was detected within 5 min, and it was fully prevented by staurosporin. The inhibitory effect of fMLP had similar characteristics when PAF was used instead of thapsigargin to induce the entry of Ca2+ or Mn2+. We conclude that fMLP, but not PAF, is able to produce a transient inhibition of store-operated Ca2+ entry pathway, probably mediated by protein kinase C. This action could be part of a general homeostatic mechanism designed to moderate [Ca2+]i increases induced by some agonists.     

Pituitary adenylate cyclase-activating polypeptide causes rapid Ca2+ release from intracellular stores and long lasting Ca2+ influx mediated by Na+ influx-dependent membrane depolarization in bovine adrenal chromaffin cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to increase intracellular Ca2+ concentrations ([Ca2+]i) and catecholamine release in adrenal chromaffin cells. We measured [Ca2+]i with fura-2 and recorded ion currents and membrane potentials with the whole cell configuration of the patch-clamp technique to elucidate the mechanism of PACAP-induced [Ca2+]i increase in bovine adrenal chromaffin cells. PACAP caused [Ca2+]i to increase due to Ca2+ release and Ca2+ influx, and this was accompanied by membrane depolarization and inward currents. The Ca2+ release was suppressed by ryanodine, an inhibitor of caffeine-sensitive Ca2+ stores, but was unaffected by cinnarizine, an inhibitor of inositol trisphosphate-induced Ca2+ release. Ca2+ influx and inward currents were both inhibited by replacement of extracellular Na+, and Ca2+ influx was inhibited by nicardipine, an L-type Ca2+ channel blocker, or by staurosporine, a protein kinase C (PKC) inhibitor, but was unaffected by a combination of omega- conotoxin-GVIA, omega-agatoxin-IVA, and omega-conotoxin- MVIIC, blockers of N-, P-, and Q-type Ca2+ channels. Moreover, 1-oleoyl-2-acetyl-sn-glycerol, a PKC activator, induced inward currents and Ca2+ influx. These results indicate that PACAP causes both Ca2+ release, mainly from caffeine-sensitive Ca2+ stores, and Ca2+ influx via L-type Ca2+ channels activated by membrane depolarization that depends on PKC-mediated Na+ influx.     

Catecholamine secretion from bovine adrenal chromaffin cells: the role of the Na+/Ca2+ exchanger and the intracellular Ca2+ pool

The role of the Na+/Ca2+ exchanger and intracellular nonmitochondrial Ca2+ pool in the regulation of cytosolic free calcium concentration ([Ca2+]i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca2+]i were simultaneously monitored in a single chromaffin cell. After high-K+ stimulation, control cells and cells in which the Na+/Ca2+ exchange activity was inhibited showed similar rates of [Ca2+]i elevation. However, the recovery of [Ca2+]i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca2+ pump of the intracellular Ca2+ pool with thapsigargin caused a significant delay in the recovery of [Ca2+]i and greatly enhanced the secretory events. These data suggest that both the Na+/Ca2+ exchanger and the thapsigargin-sensitive Ca2+ pool are important in the regulation of [Ca2+]i and, by modulating the time course of secretion, are important in determining the extent of secretion.     

Pituitary adenylate cyclase-activating polypeptide causes Ca2+ release from ryanodine/caffeine stores through a novel pathway independent of both inositol trisphosphates and cyclic AMP in bovine adrenal medullary cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca2+ release and Ca2+ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca2+ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP3), cyclic AMP, and the intracellular Ca2+ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP3 and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP3, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP3 production, the Ca2+ release induced by PA-CAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP3 channels. The potencies of the peptides to cause Ca2+ release in the presence of cinnarizine were similar. The Ca2+ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca2+ release was unaffected by Rp-adenosine 3',5'-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca2+ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP3 production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca2+ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca2+ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP3 and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca2+ in adrenal chromaffin cells.     

Isolation of chromaffin cell thapsigargin-sensitive Ca2+ store in light microsomes from bovine adrenal medulla

1. A subcellular fractionation procedure for bovine adrenal glands was designed with the aim to study the biochemical properties of Ca2+ stores in chromaffin cells. 2. The thapsigargin-sensitive compartment of Ca2+ stores was found to be highly enriched in a light microsomal fraction (LMF) on a 15-30% linear sucrose gradient, and was found to be essentially devoid of contamination by plasma, mitochondrial or secretory granule membranes. 3. A Ca(2+)-pumping ATPase was identified in this LMF as a 97 kDa protein forming an acid-stable, Ca(2+)-dependent, thapsigargin-sensitive phosphorylated intermediate upon incubation with [gamma-32P]ATP, suggesting this protein to represent a SERCA-3 isoform of Ca2+ ATPases. 4. A major 162 kDa protein, previously demonstrated in the isolated chromaffin cells, was enriched in the LMF, distributing on sucrose gradients in parallel with the thapsigargin-sensitive Ca2+ uptake. 5. LMF appears to represent a part of the thapsigargin-sensitive Ca2+ store of chromaffin cells, and should be useful for further studies of the store properties at the subcellular and molecular level.     

Cd2+ and Co2+ at micromolar concentrations mobilize intracellular Ca2+ via the generation of inositol 1,4,5-triphosphate in bovine chromaffin cells

To understand the mechanisms underlying the Cd2+- and Co2+-induced intracellular Ca2+ mobilization, we measured the levels of inositol phosphates using bovine chromaffin cells. Studies using HPLC indicated that Cd2+, Co2+ and methacholine significantly increased the generation of 1,4,5-IP3. The results suggest that Cd2+ and Co2+ mobilize Ca2+ from IP3-sensitive Ca2+ stores, possibly through the presumptive Cd2+ receptor.     

Correlation between secretagogue-induced Ca2+ influx, intracellular Ca2+ levels and secretion of catecholamines in cultured adrenal chromaffin cells

BACKGROUND: Complex branched muscle fibers are frequently observed in the muscles of mdx mutant mice and/or in damaged muscles. To investigate whether the complex branched fibers were present in the compensatory hypertrophied muscles of rats, we examined the morphological changes in these muscles. METHODS: We examined the hypertrophied plantaris (PLA) muscle of the Wistar male rats, prepared by surgical ablation of synergistic muscles. The muscle was examined using three-dimensional analysis with scanning electron microscopy, immunohistochemical detection of proliferating cells using 5-bromo-2'-deoxyuridine (BrdU) and histological and histochemical characterization. Studies were performed at 48 hours, 2, 4, 6, 10, and 15 weeks after surgical preparation. RESULTS: The muscle hypertrophy ratio (muscle weight relative to the contralateral intact control side), gradually increased from 2 to 10 weeks, and the peak value (48.6%) occurred at the 10th week. The total number of fibers did not change significantly at any time interval. However, the number of branched muscle fibers increased significantly (P < 0.05) after 6 weeks, and accounted for about 2.5% of the total fibers at the 15th week. Most branched fibers showed complex features resembling the "anastomosing syncytial reticulum" described in myopathic animals. The fibers were observed mainly in the middle and distal portions of the PLA muscle. The proportion and distribution of proliferating cells in the entire PLA muscle corresponded with the distribution of the complex branched fibers. These results were also observed in muscle tissues prepared for histological and histochemical examination. CONCLUSIONS: The presence of a large proportion of complex branched fibers in a limited segment of the compensatory hypertrophied muscle suggests that this hypertrophy model represents a pathological and/or pathophysiological hypertrophy model rather than a normal physiological process.     

Possible involvement of intracellular Ca2+ in hyposmosis-evoked catecholamine release from adrenal chromaffin cells

The influence of hyposmotic conditions on catecholamine release was studied using cultured adrenal chromaffin cells. Incubation of the cells in hyposmotic solution led to the enhancement of catecholamine release in a manner dependent on the reduction of osmolarity. Hyposmosis-evoked catecholamine release was similarly observed in the presence or absence of extracellular Ca2+, and was not significantly affected by organic and inorganic Ca2+ entry blockers. These results indicated that the hyposmosis-evoked release might be associated with a rise in the intracellular Ca2+ concentration. Further studies showed that neither ryanodine nor thapsigargin caused any significant effect on hyposmosis-evoked catecholamine release, whereas pretreatment of chromaffin cells with carbonyl cyanide m-chlorophenyl hydrazone significantly enhanced the hyposmosis-evoked release. Catecholamine release evoked by exposure to hyposmotic medium is therefore thought to be mediated through intracellular Ca2+, which may be mainly sequestered by the mitochondrial pools. Neither caffeine- nor inositol 1,4,5-trisphosphate-sensitive Ca2+ pools seems likely to be involved in hyposmosis-evoked catecholamine release, although the Ca2+ pools that contribute to the elevation of intracellular Ca2+ observed under hyposmotic conditions are not yet completely identified.     

Ca2+-dependent capacitance increases in rat basophilic leukemia cells following activation of store-operated Ca2+ entry and dialysis with high-Ca2+-containing intracellular solution

Ca2+-dependent vesicular fusion was studied in single whole-cell patch-clamped rat basophilic leukemia (RBL) cells using the capacitance technique. Dialysis of the cells with 10 microM free Ca2+ and 300 microM guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) resulted in prominent capacitance increases. However, dialysis with either Ca2+ (225 nM to 10 microM) or GTP[gamma-S] alone failed to induce a capacitance change. Under conditions of weak Ca2+ buffering (0.1 mM EGTA), activation of Ca2+-release-activated Ca2+ (CRAC) channels by dialysis with inositol 1,4,5-trisphosphate (InsP3) failed to induce a capacitance increase even in the presence of GTP[gamma-S]. However, when Ca2+ATPases were inhibited by thapsigargin, InsP3 and GTP[gamma-S] led to a pronounced elevation in membrane capacitance. This increase was dependent on a rise in intracellular Ca2+ because it was abolished when cells were dialysed with a high level of EGTA (10 mM) in the recording pipette. The increase was also dependent on Ca2+ influx because it was effectively suppressed when external Ca2+ was removed. Our results demonstrate that ICRAC represents an important source of Ca2+ for triggering a secretory response.     

Ca2+-dependent K+ currents induced by muscarinic receptor activation in guinea pig adrenal chromaffin cells

The neuronal effects of the metabotropic glutamate receptor agonist (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid have been studied in cultured rat cerebellar granule cells, and compared with those of the endogenous excitotoxin glutamate, and the dietary excitotoxin beta-N-methylamino-L-alanine. Glutamate, beta-N-methylamino-L-alanine, and (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid all caused concentration-dependent cerebellar granule cell death over a 24-h exposure period. The metabotropic antagonist (RS)-alpha-methyl-4-carboxyphenylglycine reduced glutamate-, beta-N-methylamino-L-alanine-, and (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid-induced death by 50, 37, and 90%, respectively. (1S,3R)-Aminocyclopentane-1,3-dicarboxylic acid-induced death was unaffected by the group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid, increased by the group II antagonist ethylglutamic acid, and markedly decreased by the group III antagonist (RS)-alpha-methylserine-O-phosphate. Neither (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid nor the group I agonist (RS)-3,5-dihydroxyphenylglycine caused an increase in intracellular free calcium levels. The group III agonist L-(+)-2-amino-4-phosphonobutyric acid also induced concentration-dependent cerebellar granule cell death, and so it was suggested that the group III metabotropic glutamate receptors were responsible for (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid-induced death. Blocking these receptors with (RS)-alpha-methylserine-O-phosphate also prevented a proportion of glutamate- and beta-N-methylamino-L-alanine-induced death.     

Effects of a time-varying strong magnetic field on transient increase in cytosolic free Ca2+ induced by bradykinin in cultured bovine adrenal chromaffin cells

We tested the effects of exposure to a time-varying magnetic field changing between 0.07 and 1.7 T at an interval of 3 s on transient increase in intracellular Ca2+ stimulated by bradykinin in bovine adrenal chromaffin cells. Addition of bradykinin induced the increase in intracellular Ca2+ within a few minutes. The exposure to the magnetic field perfectly suppressed the increase in intracellular Ca2+ in Ca2+-free medium. The inhibition occurred for 15 min when the maximum magnetic flux density was more than 1.4 T. These results suggest that the exposure inhibits Ca2+ release from intracellular Ca2+ stores.     

Multiple forms of endocytosis in bovine adrenal chromaffin cells

BACKGROUND: Halothane is a potent dilator of cerebral arteries. The predominant site of cerebrovascular resistance is thought to be intracerebral arterioles, and the effects of halothane on these vessels were not previously examined. This study compared the effects of halothane with those of the vasodilator and nitric oxide donor, sodium nitroprusside, on intraparenchymal microvessel responsiveness in a brain slice preparation. METHODS: Anesthetized Sprague-Dawley rats underwent thoracotomy and intracardiac perfusion and then were decapitated. Hippocampal brain slices were prepared and placed in a perfusion/recording chamber and superfused with artificial cerebrospinal fluid. An arteriole was located within the brain parenchyma and its diameter was monitored with videomicroscopy before, during, and after various concentrations of halothane or sodium nitroprusside were equilibrated in the perfusate. All vessels were preconstricted with prostaglandin F2 alpha before halothane or sodium nitroprusside treatment. An observer blinded to treatment analyzed vessel diameter changes with a computerized videomicrometer. RESULTS: Baseline microvessel diameter was 18 +/- 2 microns in the halothane group (n = 14) and 15 +/- 1 microns in the sodium nitroprusside group (n = 15). Prostaglandin F2 alpha (0.5 micron) preconstricted vessels by approximately 15% from resting diameter in both groups. Halothane significantly and dose dependently dilated intracerebral microvessels by 54% +/- 6%, 74% +/- 8%, 108% +/- 13%, and 132% +/- 7% (normalized to the preconstricted diameter) at 0.5%, 1.0%, and 2.5% halothane, respectively. This dilatation corresponds to a decrease in a calculated index of cerebrovascular resistance index of up to 117% +/- 2% at 2.5% halothane. Sodium nitroprusside, in concentrations ranging from 10(-8) to 10(-3)M, also dose dependently dilated these intraparenchymal vessels by 129% +/- 7% at the highest concentration. These alterations in microvessel diameter corresponded to a decrease in the cerebrovascular resistance index of up to 116 +/- 4% for the largest dose. CONCLUSIONS: Halothane produces dose-dependent vasodilatation of intraparenchymal cerebral microvessels, thus predicting marked decreases in cerebrovascular resistance in this in vitro brain slice preparation. The effects of halothane on these cerebral microvessels are similar to those of the potent vasodilator sodium nitroprusside. These findings suggest that direct effects of halathane on cerebral microvessels diameter contribute substantially to alterations in cerebrovascular resistance and flow produced by this agent.     

Ca2+-channel-dependent and -independent inhibition of exocytosis by extracellular ATP in voltage-clamped rat adrenal chromaffin cells

Tooth development in urodele amphibians occurs from a restricted region of anterior cranial neural crest. An in vitro culture system was used to test the odontogenic potential of more caudal regions of neural crest, including an "intermediate region" of neural folds which has never previously been tested for either fate or potential. Explants of different axial levels of neural crest with stomodaeal ectoderm and endoderm demonstrated that odontogenic potential extends not only further caudally than the axial level fated to produce teeth, but also beyond that with potential to produce cartilage. Our results show that chondrogenic potential is found only within the most rostral portion of the intermediate region, but that odontogenic potential extends to its most caudal limit. This separation of skeletogenic cell lineages in the neural crest necessitates a reevaluation of the designations of "cranial" and "trunk" and a reconsideration of the evolutionary implications of developmentally distinct crest-derived mesenchyme populations. The proposal that odontogenic potential extends into the trunk neural crest may be explained as conserved from a phylogenetically older, more extensive skeletogenic ability which produced the exoskeleton of more basal vertebrates.     

Effect of dexamethasone on voltage-gated Ca2+ channels and cytosolic Ca2+ in rat chromaffin cells

The present study examined whether the synthetic glucocorticoid dexamethasone (DEX) can modulate voltage-gated Ca2+ channel (VGCC) activity, and as a consequence agonist-induced increases in cytosolic Ca2+, in cultured rat adrenal medullary chromaffin (RAMC) cells. Exposure to 1 microM DEX for 48 h significantly increased peak VGCC current (delta +140%). DEX treatment also significantly potentiated the increases in cytosolic Ca2+ in response to submaximal stimulatory concentrations of KCl (delta +64%) and nicotine (delta +32%). The Ca2+ channel agonist BAY K-8644 increased both VGCC current (delta +109%) and potentiated the KCl-stimulated increase in cytosolic Ca2+ (delta +35%) to a comparable extent to that seen with DEX. These data suggest that DEX treatment increases VGCC activity, and that this increased Ca2+ influx leads to potentiation of agonist-induced increases in cytosolic Ca2+ in RAMC cells.     

Localized secretion of ATP and opioids revealed through single Ca2+ channel modulation in bovine chromaffin cells

In bovine chromaffin cells, the Ca2+ channels involved in exocytosis are effectively inhibited by ATP and opioids that are coreleased with catecholamines during cell activity. This autocrine loop causes a delay in Ca2+ channel activation that is quickly removed by preceding depolarizations. Changes in Ca2+ channel gating by secreted products thus make it possible to correlate Ca2+ channel activity to secretory events. Here, using cell-attached patch recordings, we found a remarkable correlation between delayed Ca2+ channel openings and neurotransmitter secretion induced by either local or whole-cell Ba2+ stimulation. The action is specific for N- and P/Q-type channels and largely prevented by PTX and mixtures of purinergic and opioid receptor antagonists. Overall, our data provide evidence that exocytosis, viewed through the autocrine inhibition of non-L-type channels, is detectable in membrane patches of approximately 1 microm2 distributed over 30%-40% of the total cell surface, while Ca2+ channels and autoreceptors are uniformly distributed over most of the cell membrane.     

Ca2+-dependent inactivation of a store-operated Ca2+ current in human submandibular gland cells. Role of a staurosporine-sensitive protein kinase and the intracellular Ca2+ pump

Stimulation of human submandibular gland cells with carbachol, inositol trisphosphate (IP3), thapsigargin, or tert-butylhydroxyquinone induced an inward current that was sensitive to external Ca2+ concentration ([Ca2+]e) and was also carried by external Na+ or Ba2+ (in a Ca2+-free medium) with amplitudes in the order Ca2+ > Ba2+ > Na+. All cation currents were blocked by La3+ and Gd3+ but not by Zn2+. The IP3-stimulated current with 10 microM 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-triphosphate and 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette solution, showed 50% inactivation in <5 min and >5 min with 10 and 1 mM [Ca2+]e, respectively. The Na+ current was not inactivated, whereas the Ba2+ current inactivated at a slower rate. The protein kinase inhibitor, staurosporine, delayed the inactivation and increased the amplitude of the current, whereas the protein Ser/Thr phosphatase inhibitor, calyculin A, reduced the current. Thapsigargin- and tert-butylhydroxyquinone-stimulated Ca2+ currents inactivated faster. Importantly, these agents accelerated the inactivation of the IP3-stimulated current. The data demonstrate that internal Ca2+ store depletion-activated Ca2+ current (ISOC) in this salivary cell line is regulated by a Ca2+-dependent feedback mechanism involving a staurosporine-sensitive protein kinase and the intracellular Ca2+ pump. We suggest that the Ca2+ pump modulates ISOC by regulating [Ca2+]i in the region of Ca2+ influx.     

Does a decrease in subplasmalemmal Ca2+ explain how store-operated Ca2+ channels are opened?

The activity of each of 99 intraparietal neurons was studied in three awake-behaving rhesus monkeys (Macaca mulatta) while subjects performed 100-900 delayed saccade trials. On each trial, a saccadic target was presented at one location selected randomly from a grid of 441 locations spanning 40 degrees of horizontal and vertical visual space. Individual neurons in our population were sensitive to both the direction and amplitude of saccades. Response fields, which plotted firing rate as a function of the horizontal and vertical amplitude of movements for each neuron, were characterized by a Cartesian two-dimensional gaussian model. The goodness-of-fit of these gaussian models was tested by: (1) comparing observed responses with predicted responses for each movement; and (2) by computing the percentage of variance explained by each model. Cartesian Gaussian models provided a good fit to the response fields of most neurons. Across our population, the Gaussian fit to the response field of each neuron accounted for more of the variance in neuronal activity when the data were plotted with regard to the horizontal and vertical amplitude of the saccade than when the same data were plotted with regard to the position of the saccadic target. The Gaussian functions were used to estimate the eccentricity and spatial tuning breadth of each neuronal response field. Modal response field radius was less than 5 degrees, whereas mean response field radius was about 10 degrees. Linear regression analysis demonstrated that response field eccentricity accounted for less than 30% of the variance in response field radius. Analysis of the horizontal distribution of response field centers showed an approximately normal distribution around central fixation. Most histologically recovered neurons were located on the lateral bank of the intraparietal sulcus, although a small number of saccade-related neurons were recorded from Brodmann's area 5 on the medial bank of the intraparietal sulcus.     

Ca2+-induced Ca2+ release in chromaffin cells seen from inside the ER with targeted aequorin

The presence and physiological role of Ca2+-induced Ca2+ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca2+] ([Ca2+]c). Using targeted aequorin, we have directly monitored [Ca2+] changes inside the ER ([Ca2+]ER) in bovine adrenal chromaffin cells. Ca2+ entry induced by cell depolarization triggered a transient Ca2+ release from the ER that was highly dependent on [Ca2+]ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca2+ release was quantal in nature due to modulation by [Ca2+]ER. Whereas caffeine released essentially all the Ca2+ from the ER, inositol 1,4, 5-trisphosphate (InsP3)- producing agonists released only 60-80%. Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca2+ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR.