Threshold conditions for synaptically evoking Ca(2+) waves in hippocampal pyramidal neurons

Regenerative Ca(2+) release from inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular stores in the form of Ca(2+) waves leads to large-amplitude [Ca(2+)](i) increases in the apical dendrites of hippocampal CA1 pyramidal neurons. Release is generated following synaptic activation of group I metabotropic glutamate (mGlu) receptors. We systematically examined the conditions for evoking these waves in transverse slices from 2- to 3-wk-old rats. Using a sharpened asymmetrical bipolar tungsten stimulating electrode placed in the stratum radiatum, we varied the lateral position of the electrode, the number of stimulating pulses, the train frequency, and stimulus current. Several trends were clear. Increasing the frequency of stimulation from 20 to 100 Hz, keeping the total number of pulses constant, lowered the required stimulus current. Stimulation at frequencies below 20 Hz made it difficult to evoke release. Increasing the number of stimulation pulses, keeping the frequency constant, lowered the threshold current. A minimum of five pulses at 100 Hz was required to evoke release reliably, but several examples of success with three pulses were recorded. Theta-burst stimulation was as effective as tetanic stimulation. Placing the point of the stimulation electrode closer to the pyramidal neuron made it easier to evoke release, although stimulation at a lateral distance of 500 microm with unsharpened electrodes was sometimes successful. The simplest explanation for these results is that a bolus of IP(3) must be produced quickly in a restricted region of the dendrites to generate Ca(2+) waves. The conditions necessary for evoking regenerative Ca(2+) release have many parallels (and some differences) with the conditions required to evoke long-term potentiation in these cells following tetanic stimulation.     

Dopamine modulates exocytosis independent of Ca(2+) entry in melanotropic cells

Dopamine is a known inhibitor of pituitary melanotropic cells. It reduces Ca(2+) influx by hyperpolarizing the cell membrane and by modulating high- and low-voltage-activated (HVA and LVA) Ca(2+) channels. As a result, dopamine reduces the hormonal output of the cell. However, it is unknown how dopamine affects each of the four different HVA Ca(2+) channel types individually. Moreover, it is unknown whether dopamine interacts with exocytosis independent of Ca(2+) channels. Here we show that dopamine differentially modulates the HVA Ca(2+) channels and that it affects the stimulus-secretion coupling through a direct effect on the exocytotic machinery. Sustained L- and P-type Ba(2+) currents are reduced in amplitude and inactivating N- and Q-type currents acquire different activation and inactivation kinetics in the presence of dopamine. The Q-type current shows slow activation, which is a hallmark for direct G-protein modulation. We used membrane capacitance measurements to monitor exocytosis. Surprisingly, we find that the amount of exocytosis per step depolarization is not diminished by dopamine despite the reduction in Ca(2+) current. To test whether dopamine affects the release machinery downstream of Ca(2+) entry, we stimulated exocytosis by dialyzing cells with buffered high-Ca(2+) solutions. Dopamine increased the amount and the rate of exocytosis. In the first 90 s, the rate of secretion was increased two- to threefold, but it was normalized again at 180 s, suggesting that predominantly vesicles that fuse early in the exocytotic phase are modulated by dopamine. Thus while Ca(2+) channels are inhibited by dopamine, the exocytotic machinery downstream of Ca(2+) influx is sensitized. As a result, release is more effectively stimulated by Ca(2+) influx during dopamine inhibition.     

Initiation site of Ca(2+) entry evoked by endoplasmic reticulum Ca(2+) depletion in mouse parotid and pancreatic acinar cells

Purpose

In non-excitable cells, which include parotid and pancreatic acinar cells, Ca²⁺ entry is triggered via a mechanism known as capacitative Ca²⁺ entry, or store-operated Ca²⁺ entry. This process is initiated by the perception of the filling state of endoplasmic reticulum (ER) and the depletion of internal Ca²⁺ stores, which acts as an important factor triggering Ca²⁺ entry. However, both the mechanism of store-mediated Ca²⁺ entry and the molecular identity of store-operated Ca²⁺ channel (SOCC) remain uncertain.

Materials and Methods

In the present study we investigated the Ca²⁺ entry initiation site evoked by depletion of ER to identify the localization of SOCC in mouse parotid and pancreatic acinar cells with microfluorometeric imaging system.

Results

Treatment with thapsigargin (Tg), an inhibitor of sarco/ endoplasmic reticulum Ca²⁺-ATPase, in an extracellular Ca²⁺ free state, and subsequent exposure to a high external calcium state evoked Ca²⁺ entry, while treatment with lanthanum, a non-specific blocker of plasma Ca²⁺ channel, completely blocked Tg-induced Ca²⁺ entry. Microfluorometric imaging showed that Tg-induced Ca²⁺ entry started at a basal membrane, not a apical membrane.

Conclusion

These results suggest that Ca²⁺ entry by depletion of the ER initiates at the basal pole in polarized exocrine cells and may help to characterize the nature of SOCC.     

Cyclic AMP inhibits chemotactic-peptide-induced but not Ca2+-ionophore- or tetradecanoylphorbol-acetate-induced enzyme secretion in guinea pig neutrophils

cAMP-increasing agents such as prostaglandin El, dibutyryl cAMP and 8-bromo-cAMP inhibited N-acetyl-beta-D-glucosaminidase secretion induced by the chemotactic peptide formylmethionyl-leucyl-phenylalanine in guinea pig neutrophils. However, they could not inhibit but rather potentiated the secretion significantly when secretion was induced by the Ca2+ ionophore A23187. Similarly, these agents did not inhibit tetradecanoylphorbol acetate (TPA)-induced secretion. Moreover, the secretion induced by combined treatment with Ca2+ ionophore A23187 and TPA was hardly blocked by cAMP-increasing agents. These results strongly suggest that cAMP inhibits the secretion through suppression of a signal transduction from receptor activation to events such as Ca2+ mobilization and protein kinase C activation, probably of the inositol phospholipid metabolism.     

Biphasic actions of topiramate on monoamine exocytosis associated with both soluble N-ethylmaleimide-sensitive factor attachment protein receptors and Ca(2+)-induced Ca(2+)-releasing systems

To explore the pharmacological mechanisms of topiramate (TPM), we determined the effects of TPM on monoamine (dopamine and serotonin) exocytosis associated with N-ethylmaleimide-sensitive factor attachment protein receptors and Ca(2+)-induced Ca(2+)-releasing systems, including inositol-triphosphate receptor and ryanodine receptor in freely moving rat pre-frontal cortex using in vivo microdialysis. During resting stage, Ca(2+) output from endoplasmic reticulum Ca(2+) store via inositol-triphosphate receptor regulates syntaxin-associated monoamine exocytosis mechanism, whereas during neuronal hyperexcitable stage, Ca(2+) output via ryanodine receptor regulates synaptobrevin-associated monoamine exocytosis mechanism. Basal monoamine releases were increased and decreased by therapeutically relevant and supratherapeutic concentration of TPM, respectively. The therapeutic-relevant concentration of TPM increased Ca(2+)-evoked release concentration-dependently; however, its stimulatory effect was attenuated in the supratherapeutic range. The K(+)-evoked releases were reduced by TPM concentration-dependently (from therapeutic to supratherapeutic ranges). The therapeutic-relevant concentration of TPM-induced elevation of basal release was reduced by cleavage with syntaxin and inhibition of inositol-triphosphate receptor predominantly, by cleavage with SNAP-25 and synaptobrevin weakly, but not by ryanodine receptor inhibitor. The therapeutic-relevant concentration of TPM-induced elevation of Ca(2+)-evoked release was reduced by cleavage with syntaxin and inositol-triphosphate receptor inhibitor selectively. The therapeutic-relevant concentration of TPM-induced reduction of K(+)-evoked monoamine release was abolished by cleavage with synaptobrevin, but was not affected by cleavage with SNAP-25 or synaptobrevin. The stimulatory effect of ryanodine receptor agonist on K(+)-evoked monoamine release was reduced by TPM, whereas that of inositol-triphosphate receptor agonist was not affected by TPM. Therefore, these results indicate that the combination of the effects of TPM on exocytosis mechanisms associated with SNARE and Ca(2+)-induced Ca(2+)-releasing systems, enhancement of inositol-triphosphate receptor/syntaxin and inhibition of ryanodine receptor/synaptobrevin in pre-frontal cortex, may be involved in clinical actions of TPM.     

Role of Ca(2+) in the rapid cooling-induced Ca(2+) release from sarcoplasmic reticulum in ferret cardiac muscles

Rapid lowering of the solution temperature (rapid cooling, RC) from 24 to 3°C within 3 s releases considerable amounts of Ca²⁺ from the sarcoplasmic reticulum (SR) in mammalian cardiac muscles. In this study, we investigated the intracellular mechanism of RC-induced Ca²⁺ release, especially the role of Ca²⁺, in ferret ventricular muscle. Saponin-treated skinned trabeculae were placed in a glass capillary, and the amount of Ca²⁺ released from the SR by RC and caffeine (50 mM) was measured with fluo-3. It was estimated that in the presence of ATP about 45% of the Ca²⁺ content in the SR was released by RC. The amount of SR Ca²⁺ released by RC was unchanged by the replacement of ATP by AMP-PCP (a non-hydrolysable ATP analogue and agonist for the ryanodine receptor but not for the Ca²⁺ pump of SR), suggesting that the suppression of the Ca²⁺ pump of SR at low temperature might not be a major mechanism in RC-induced Ca²⁺ release. The free Ca²⁺ concentration of the solution used for triggering RC-induced Ca²⁺ release was estimated to be only about 20 nM with fluo-3 or aequorin. When this solution was applied to the preparation at 3°C, only a small amount of Ca²⁺ was released from SR presumably by the Ca²⁺-induced Ca²⁺ release (CICR) mechanism. Thus, in mammalian cardiac muscles, RC releases a part of the (<50%) stored Ca²⁺ contained in the SR, and the mechanism of RC-induced Ca²⁺ release may differ from that of CICR, which is thought to play a role in frog skeletal muscle fibres that express ryanodine receptors of different types.     

Ca(2+)-independent but voltage-dependent secretion in mammalian dorsal root ganglion neurons

We have investigated the Ca(2+) dependence of vesicular secretion from the soma of dorsal root ganglion (DRG) neurons, which secrete neuropeptides by exocytosis of dense-core vesicles. In patch-clamped somata of rat DRG neurons, we found a depolarization-induced membrane capacitance increase (DeltaC(m)) in the absence of extracellular Ca(2+) and in the presence of a Ca(2+) chelator (BAPTA) in the intracellular solution. Depletion of internal Ca(2+) stores by thapsigargin in the Ca(2+)-free bath also did not block the DeltaC(m), indicating that Ca(2+) release from internal Ca(2+) stores may not have been involved. Furthermore, the Ca(2+)-independent DeltaC(m) was blocked by whole-cell dialysis with tetanus toxin and was accompanied by pulsatile secretion of false transmitters, as detected by amperometric measurements. These results indicate the existence of Ca(2+)-independent but voltage-dependent vesicular secretion (CIVDS) in a mammalian sensory neuron.     

Contribution of Ca(2+)-permeable AMPA/KA receptors to glutamate-induced Ca(2+) rise in embryonic lumbar motoneurons in situ

Intracellular Ca(2+) ([Ca(2+)](i)) was fluorometrically measured with fura-2 in lumbar motoneurons of acutely isolated spinal cord slices from embryonic rats. In ester-loaded cells, bath-applied glutamate (3 microM to 1 mM) evoked a [Ca(2+)](i) increase by up to 250 nM that was abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) plus 2-amino-5-phosphonovalerate (APV). CNQX or APV alone reduced the response by 82 and 25%, respectively. The glutamatergic agonists kainate (KA), quisqualate (QUI), and S-alpha-amino-3-hydroxy-5-methyl-4-isoxalone (S-AMPA) evoked a similar [Ca(2+)](i) transient as glutamate. N-methyl-D-aspartate (NMDA) was only effective to increase [Ca(2+)](i) in Mg(2+)-free saline, whereas [1S,3R]-1-aminocyclopentane-1,3-dicarboxylic acid ([1S,3R]-ACPD) had no effect. The glutamate-induced [Ca(2+)](i) rise was suppressed in Ca(2+)-free superfusate. Depletion of Ca(2+) stores with cyclopiazonic acid (CPA) did not affect the response. Thirty-six percent of the [Ca(2+)](i) increase in response to membrane depolarization induced by a 50 mM K(+) solution persisted on combined application of the voltage-gated Ca(2+) channel blockers nifedipine, omega-conotoxin-GVIA and omega-agatoxin-IVA. In fura-2 dialyzed motoneurons, the glutamate-induced [Ca(2+)](i) increase was attenuated by approximately 70% after changing from current to voltage clamp. Forty percent of the remaining [Ca(2+)](i) transient and 20% of the concomitant inward current of 0.3 nA were blocked by Joro spider toxin-3 (JSTX). The results show that voltage-gated Ca(2+) channels, including a major portion of R-type channels, constitute the predominant component of glutamate-induced [Ca(2+)](i) rises. NMDA and Ca(2+)-permeable KA/AMPA receptors contribute about equally to the remaining component of the Ca(2+) rise. The results substantiate previous assumptions that Ca(2+) influx through JSTX-sensitive KA/AMPA receptors is involved in (trophic) signaling in developing motoneurons.     

Activation of BK channels in rat chromaffin cells requires summation of Ca(2+) influx from multiple Ca(2+) channels

Large-conductance Ca(2+) and voltage-dependent K(+) channels (BK channels) in many tissues require high Ca(2+) concentrations for activation and therefore might be expected to be tightly coupled to Ca(2+) channels. However, in most cases, little is known about the relative organization of the BK channels and the Ca(2+) channels involved in their activation. We probed the nature of the organization of BK and Ca(2+) channels in rat chromaffin cells by manipulating Ca(2+) influx through Ca(2+) channels and by altering cellular Ca(2+) buffering using EGTA and bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid (BAPTA). The results were analyzed to determine the distance between Ca(2+) and BK channels that would be most consistent with the experimental data. Most BK channels are close enough to Ca(2+) channels to be resistant to the buffering action of millimolar of EGTA, but are far enough to be inhibited by BAPTA. Analysis of the EGTA/BAPTA results suggests that BK channels are at a distance of 50 to 160 nm from Ca(2+) channels. A model that assumes random distribution of Ca(2+) and BK channels fails to account for the observed [Ca(2+)](i) detected by BK channels, suggesting that a specific mechanism may exist to mediate the functional coupling between these channels. Importantly, the effects of EGTA and BAPTA cannot be explained by assuming a one-to-one coupling between Ca(2+) and BK channels. Rather, Ca(2+) influx through a number of Ca(2+) channels appears to act in concert to regulate the behavior of any individual BK channel. Thus differences in BK channel open probabilities may be explained by differences in the extent of Ca(2+) domain overlap at the sites of individual BK channels.     

Effect of uncoupling NO/cGMP pathways on carbachol- and CCK-stimulated Ca2+ entry and amylase secretion from the rat pancreas

 Nitric oxide (NO) production reportedly regulates guanosine 3′,5′-cyclic monophosphate (cGMP) formation and Ca²⁺ influx in pancreatic acini. We have investigated the functional roles of the NO/cGMP messenger system in rat pancreatic acini. In dispersed acini, the levels of amylase secretion, cytosolic [Ca²⁺]([Ca²⁺]_i), NO synthase, and cGMP were measured. The NO synthase inhibitor N ^G-nitro-L-arginine methyl ester (L-NAME, 0.01–100 μM) had no effect on amylase secretion induced by various concentrations of carbachol, cholecystokinin octapeptide (CCK-8) or the high affinity CCK agonist, JMV-180. Similarly, L-NAME up to 100 μM did not affect the changes in Ca²⁺ spiking evoked by these secretagogues; nor was Ca²⁺ entry, refilling or oscillation altered by L-NAME. Sub- and supramaximal concentrations of these secretagogues did not change NO synthase activities compared with basal levels. While sodium nitroprusside (SNP), a NO donor, caused a 9.4-fold increase in cGMP levels compared with basal levels, carbachol, CCK-8 and JMV-180 had no effect. In addition, the guanylate cyclase inhibitor LY 83583 (10 nM to 10 μM) altered neither amylase secretion nor Ca²⁺ signaling induced by these secretagogues. These findings indicate that the stimulatory action of carbachol or CCK-8 is not mediated by NO or cGMP. To investigate whether cGMP stimulates pancreatic secretion we showed that both SNP and a cell-permeant cGMP analog at 0.1–1 mM stimulated amylase secretion and Ca²⁺ transients to a level equal to 10–15% and 13–24%, respectively, of those observed with maximal concentrations of secretagogues. The guanylate cyclase activator guanylin (1–10 μM), which increased cGMP levels 2.4-fold compared with basal levels, elicited a small amount of amylase secretion and a small Ca²⁺ transient. In conclusion, exogenous NO is capable of increasing endogenous cGMP, which results in a modest increase in the [Ca²⁺]_i transient and pancreatic amylase secretion. However, the NO/cGMP system does not appear to be involved significantly in the mediation of Ca²⁺ signaling and amylase secretion stimulated by carbachol and CCK-8. Received: 30 October 1996 / Received after revision and accepted: 13 January 1997     

Ca(2+)-independent and Ca(2+)-dependent stimulation of quantal neurosecretion in avian ciliary ganglion neurons.

1. Although it is generally agreed that Ca2+ couples depolarization to the release of neurotransmitters, hypertonic saline and ethanol (ETOH) evoke neurosecretion independent of extracellular Ca2+. One possible explanation is that these agents release Ca2+ from an intracellular store that then stimulates Ca(2+)-dependent neurosecretion. An alternative explanation is that these agents act independently of Ca2+. 2. This work extends previous observations on the action of ETOH and hypertonic solutions (HOSM) on neurons to include effects on [Ca2+]i. We have looked for Ca(2+)-independent or -dependent neurosecretion evoked by these agents in parasympathetic postganglionic neurons dissociated from chick ciliary ganglia and maintained in tissue culture. The change in concentration of free Ca2+ in the micromolar range inside neurons ([Ca2+]i) was measured with indo-1 with the use of a Meridian ACAS 470 laser scanning microspectrophotometer. 3. Elevated concentration of extracellular KCl increased [Ca2+]i and the frequency of quantal events. Also, a twofold increase in osmotic pressure (HOSM) produced a similar increase in quantal release and a significant rise in [Ca2+]i; however, the Ca2+ appeared to come from intracellular stores. 4. In contrast, ETOH stimulated quantal neurosecretion without a measurable change in [Ca2+]i. It appears the alcohol exerts its influence on some stage in the process of exocytosis that is distal to or independent of the site of Ca2+ action. 5. The effects of high [KCl]o and osmotic pressure were occlusive. This is explained in part by the observation that hypertonicity reduced Ca2+ current, but an action on Ca2+ stores is also likely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca(2+)-dependent Ca(2+) clearance via mitochondrial uptake and plasmalemmal extrusion in frog motor nerve terminals

Ca(2+) clearance in frog motor nerve terminals was studied by fluorometry of Ca(2+) indicators. Rises in intracellular Ca(2+) ([Ca(2+)](i)) in nerve terminals induced by tetanic nerve stimulation (100 Hz, 100 or 200 stimuli: Ca(2+) transient) reached a peak or plateau within 6-20 stimuli and decayed at least in three phases with the time constants of 82-87 ms (81-85%), a few seconds (11-12%), and several tens of seconds (less than a few percentage). Blocking both Na/Ca exchangers and Ca(2+) pumps at the cell membrane by external Li(+) and high external pH (9.0), respectively, increased the time constants of the initial and second decay components with no change in their magnitudes. By contrast, similar effects by Li(+) alone, but not by high alkaline alone, were seen only on 200 stimuli-induced Ca(2+) transients. Blocking Ca(2+) pumps at Ca(2+) stores by thapsigargin did not affect 100 stimuli-induced Ca(2+) transients but increased the initial decay time constant of 200 stimuli-induced Ca(2+) transients with no change in other parameters. Inhibiting mitochondrial Ca(2+) uptake by carbonyl cyanide m-chlorophenylhydrazone markedly increased the initial and second decay time constants of 100 stimuli-induced Ca(2+) transients and the amplitudes of the second and the slowest components. Plotting the slopes of the decay of 100 stimuli-induced Ca(2+) transients against [Ca(2+)](i) yielded the supralinear [Ca(2+)](i) dependence of Ca(2+) efflux out of the cytosol. Blocking Ca(2+) extrusion or mitochondrial Ca(2+) uptake significantly reduced this [Ca(2+)](i)-dependent Ca(2+) efflux. Thus Ca(2+)-dependent mitochondrial Ca(2+) uptake and plasmalemmal Ca(2+) extrusion clear out a small Ca(2+) load in frog motor nerve terminals, while thapsigargin-sensitive Ca(2+) pump boosts the clearance of a heavy Ca(2+) load. Furthermore, the activity of plasmalemmal Ca(2+) pump and Na/Ca exchanger is complementary to each other with the slight predominance of the latter.     

Involvement of cytoskeletal integrity in the regulation of Cl- and amylase secretion from rat parotid acinar cells

The cytoskeleton serves as a signal modulator for Ca2+ and cAMP-regulated cell functions including the secretion of ions and granule contents. The interaction between Ca2+ and cAMP signaling systems potentiates amylase secretion and suppresses Cl- secretion in the parotid glands. In this study, we investigated the role of the cytoskeleton in the modulation of Cl- and amylase secretion from rat parotid acinar cells upon activation of each intracellular signaling system and their interaction. Cytochalasin D markedly inhibited the Ca2+-activated outwardly rectifying Cl- current at positive membrane potentials and carbachol (CCh)-induced Cl- currents in the whole-cell configuration at -80 mV, whereas colchicine enhanced Cl- currents. Cytochalasin D, but not colchicine, markedly inhibited CCh-induced Cl- secretion. Synergistic actions of CCh and forskolin on Cl- and amylase secretion were observed even in the presence of cytochalasin D. These results suggest that the synergistic effects of Ca2+ and cAMP signaling systems on amylase and Cl- secretion do not require actin filament integrity but that secretion by the two signals themselves does require actin filament integrity.     

Acceleration of Ca(2+) repletion in the junctional sarcoplasmic reticulum and alternation of the Ca(2+)-induced Ca(2+)-release mechanism in hypertensive rat (SHR) cardiac muscle

We estimated the time taken for a repletion of the junctional sarcoplasmic reticulum (JSR) Ca(2+) stores from a family of mechanical restitution curves after twitches of various magnitudes in the cardiac muscle of hypertensive rats (SHR), using a method described previously (Tameyasu et al. Jpn J Physiol. 2004;54:209-19), to evaluate abnormality in Ca(2+) handling by cardiac JSR in hypertension. We found no differences in contractility or in the time course of mechanical restitution between SHR and the controls (WKY) at 3 weeks of age. In comparison to WKY, 7- and 20-week-old SHR showed a greater rested state contraction (RST) and similar or smaller rapid cooling contracture, suggesting that their JSR contains a similar amount of Ca(2+) at saturation, but releases more Ca(2+) upon stimulation. The adult SHR and WKY showed similar mechanical restitution time courses, but the adults had longer pretwitch latencies. The function G(t) representing the time course of JSR Ca(2+) store repletion in adult SHR exceeded the WKY value at t < or = 0.5 s, but the function H(t) representing JSR [Ca(2+)] change corresponding to the mechanical restitution after RST was smaller in the adult SHR at t < or = 0.5 s, resulting in smaller H(t)/G(t) in adult SHR at t < or = 0.5 s. Deviations of G(t), H(t), and H(t)/G(t) from WKY were greater at 20 weeks than at 7. The results suggest an acceleration of JSR Ca(2+) store repletion and an alternation of the Ca(2+)-induced release of Ca(2+ )from the JSR in young adult SHR.     

Stimulation of Ca(2+)-dependent exocytosis and release of arachidonic acid in cultured mast cells (RBL-2H3) by quercetin

Basic secretagogues, such as compound 48/80, stimulate secretion in rat peritoneal mast cells by directly activating the heterotrimeric G-protein Gi(3) (Aridor M, et al. Science 1993;262:1569-72). Cultured RBL-2H3 mast cells do not normally respond to basic secretagogues, but acquire such responsiveness upon prolonged exposure to the kinase inhibitor, quercetin, which also increases the cellular level of Gi(3) (Senyshyn J, Baumgartner RA, Beaven MA. J Immunol 1998;160:5136-44). Expression of a GTPase-deficient mutant of Galphai(3) in RBL-2H3 cells results in the stimulation of Ca(2+)-triggered exocytosis and release of arachidonic acid (AA) (Zussman A, Hermuet S, Sagi-Eisenberg R. Eur J Biochem 1998;258:144-6). Here we show that long-term incubation with quercetin markedly stimulates Ca(2+)-triggered exocytosis and release of AA from the RBL-2H3 cells. We further show that membranes derived from such quercetin-treated cells display a reduced GTPase, but not ATPase, activity. Taken together with our previous observations, these results further implicate Gi(3) as one of the cellular targets through which quercetin confers responsiveness towards the family of basic secretagogues.     

Amplification of odor-induced Ca(2+) transients by store-operated Ca(2+) release and its role in olfactory signal transduction

A critical role of Ca(2+) in vertebrate olfactory receptor neurons (ORNs) is to couple odor-induced excitation to intracellular feedback pathways that are responsible for the regulation of the sensitivity of the sense of smell, but the role of intracellular Ca(2+) stores in this process remains unclear. Using confocal Ca(2+) imaging and perforated patch recording, we show that salamander ORNs contain a releasable pool of Ca(2+) that can be discharged at rest by the SERCA inhibitor thapsigargin and the ryanodine receptor agonist caffeine. The Ca(2+) stores are spatially restricted; emptying produces compartmentalized Ca(2+) release and capacitative-like Ca(2+) entry in the dendrite and soma but not in the cilia, the site of odor transduction. We deplete the stores to show that odor stimulation causes store-dependent Ca(2+) mobilization. This odor-induced Ca(2+) release does not seem to be necessary for generation of an immediate electrophysiological response, nor does it contribute significantly to the Ca(2+) transients in the olfactory cilia. Rather, it is important for amplifying the magnitude and duration of Ca(2+) transients in the dendrite and soma and is thus necessary for the spread of an odor-induced Ca(2+) wave from the cilia to the soma. We show that this amplification process depends on Ca(2+)-induced Ca(2+) release. The results indicate that stimulation of ORNs with odorants can produce Ca(2+) mobilization from intracellular stores without an immediate effect on the receptor potential. Odor-induced, store-dependent Ca(2+) mobilization may be part of a feedback pathway by which information is transferred from the distal dendrite of an ORN to its soma.     

Neuroprotective effects of increased extracellular Ca(2+) during aglycemia in white matter

We investigated the effects of extracellular [Ca(2+)] ([Ca(2+)](o)) on aglycemia-induced dysfunction and injury in adult rat optic nerves. Compound action potentials (CAPs) from adult rat optic nerve were recorded in vitro, and the area under the CAP was used to monitor nerve function before and after 1 h periods of aglycemia. In control artificial cerebrospinal fluid (ACSF) containing 2 mM Ca(2+), CAP function fell after 29.9 +/- 1.5 (SE) min and recovered to 48.8 +/- 3.9% following aglycemia. Reducing bath [Ca(2+)] during aglycemia progressively improved recovery. For example, in Ca(2+)-free ACSF, the CAP recovered to 99.1 +/- 3.8%. Paradoxically, increasing bath [Ca(2+)] also improved recovery from aglycemia. In 5 or 10 mM bath [Ca(2+)], CAP recovered to 78.8 +/- 9.2 or 91.6 +/- 5.2%, respectively. The latency to CAP failure during aglycemia increased as a function of bath [Ca(2+)] from 0 to 10 mM. Increasing bath [Mg(2+)] from 2 to 5 or 10 mM, with bath [Ca(2+)] held at 2 mM, increased latency to CAP failure with aglycemia and improved recovery from this insult. [Ca(2+)](o) recorded with calcium-sensitive microelectrodes in control ACSF, dropped reversibly during aglycemia from 1.54 +/- 0.03 to 0.45 +/- 0.04 mM. In the presence of higher ambient levels of bath [Ca(2+)] (i.e., 5 or 10 mM), the aglycemia-induced decrease in [Ca(2+)](o) declined, indicating that less Ca(2+) left the extracellular space to enter an intracellular compartment. These results indicate that the role of [Ca(2+)], and divalent cations in general, during aglycemia is complex. While extracellular Ca(2+) was required for irreversible aglycemic injury to occur, higher levels of [Ca(2+)] or [Mg(2+)] increased the latency to CAP failure and improved the extent of recovery, apparently by limiting Ca(2+) influx. These effects are theorized to be mediated by divalent cation screening.     

Early effects of denervation on Ca(2+)-handling proteins in skeletal muscle

The adaptive response of skeletal muscle fibres depends on a variety of biological factors including loading conditions and neuromuscular activity. An extreme type of atrophy-inducing change in contractile activity is represented by the physical disconnection between the motor nerve and its respective fibre unit. Since fibre type alterations have a striking effect on the Ca(2+)-regulatory apparatus, we have investigated the fate of a key Ca(2+)-pump and essential Ca(2+)-binding proteins in extensor digitorum longus specimens two weeks after nerve crush or complete denervation. In contrast to increased levels of sarcalumenin, immunoblotting revealed that the expression of the fast SERCA1 Ca(2+)-ATPase isoform is drastically decreased and fast calsequestrin is slightly reduced. Analysis of myosin heavy chain isoforms agreed with this result and showed a fast-to-slow fibre type shifting process following denervation. Hence, changes in muscle activity appear to have a profound effect on the abundance and isoform expression pattern of Ca(2+)-handling elements.