Non-additive potentiation of glutamate release by phorbol esters and metabotropic mGlu7 receptor in cerebrocortical nerve terminals

We recently showed that prolonged activation of metabotropic glutamate receptor 7 (mGlu7) potentiates glutamate release. This signalling involves phospholipase C activation via a pertussis toxin insensitive G protein and the subsequent hydrolysis of phosphatidylinositol (4,5)-bisphosphate. Release potentiation is independent of protein kinase C activation but it is dependent on the downstream release machinery, as reflected by the concomitant translocation of active zone Munc13-1 protein from the soluble to particulate fractions. Here we show that phorbol ester and mGlu7 receptor-dependent facilitation of neurotransmitter release is not additive, suggesting they share a common signalling mechanism. However, release potentiation is restricted to release sites that express N-type Ca(2+) channels, because phorbol ester and mGlu7 receptor-mediated release potentiation are absent in nerve terminals from mice lacking N-type Ca(2+) channels. In addition, phorbol esters but not mGlu7 receptors potentiate release at nerve terminals with P/Q-type Ca(2+) channels, although only under restricted conditions of Ca(2+) influx. The differential effect of phorbol esters at nerve terminals with either N- or P/Q-type Ca(2+) channels seems to be unrelated to the type Munc13 isoform expressed, and it is more likely dependent on other properties of the release machinery.     

The C terminus of the metabotropic glutamate receptor subtypes 2 and 7 specifies the receptor signaling pathways

There is accumulating evidence that the specificity of the transduction cascades activated by G protein-coupled receptors cannot solely depend on the nature of the coupled G protein. To identify additional structural determinants, we studied two metabotropic glutamate (mGlu) receptors, the mGlu2 and mGlu7 receptors, that are both coupled to G(o) proteins but are known to affect different effectors in neurons. Thus, the mGlu2 receptor selectively blocks N- and L-type Ca(2+) channels via a protein kinase C-independent pathway, whereas the mGlu7 receptor selectively blocks P/Q-type Ca(2+) channels via a protein kinase C-dependent pathway, and both effects are pertussis toxin-sensitive. We examined the role of the C-terminal domain of these receptors in this coupling. Chimeras were constructed by exchanging the C terminus of these receptors and transfected into neurons. Different chimeric receptors bearing the C terminus of mGlu7 receptor blocked selectively P/Q-type Ca(2+) channels, whereas chimeras bearing the C terminus of mGlu2 receptor selectively blocked N- and L-type Ca(2+) channels. These results show that the C terminus of mGlu2 and mGlu7 receptors is a key structural determinant that allows these receptors to select a specific signaling pathway in neurons.     

Reassessment of the Ca2+ sensing property of a type I metabotropic glutamate receptor by simultaneous measurement of inositol 1,4,5-trisphosphate and Ca2+ in single cells

Transient transfection of Chinese hamster ovary or baby hamster kidney cells expressing the Group I metabotropic glutamate receptor mGlu1alpha with green fluorescent protein-tagged pleckstrin homology domain of phospholipase Cdelta1 allows real-time detection of inositol 1,4,5-trisphosphate. Loading with Fura-2 enables simultaneous measurement of intracellular Ca(2+) within the same cell. Using this technique we have studied the extracellular calcium sensing property of the mGlu1alpha receptor. Quisqualate, in extracellular medium containing 1.3 mm Ca(2+), increased inositol 1,4,5-trisphosphate in all cells. This followed a typical peak and plateau pattern and was paralleled by concurrent increases in intracellular Ca(2+) concentration. Under nominally Ca(2+)-free conditions similar initial peaks in inositol 1,4,5-trisphosphate and Ca(2+) concentration occurred with little change in either agonist potency or efficacy. However, sustained inositol 1,4,5-trisphosphate production was substantially reduced and the plateau in Ca(2+) concentration absent. Depletion of intracellular Ca(2+) stores using thapsigargin abolished quisqualate-induced increases in intracellular Ca(2+) and markedly reduced inositol 1,4,5-trisphosphate production. These data suggest that the mGlu1alpha receptor is not a calcium-sensing receptor because the initial response to agonist is not sensitive to extracellular Ca(2+) concentration. However, prolonged activation of phospholipase C requires extracellular Ca(2+), while the initial burst of activity is highly dependent on Ca(2+) mobilization from intracellular stores.     

Spatial-temporal patterning of metabotropic glutamate receptor-mediated inositol 1,4,5-triphosphate, calcium, and protein kinase C oscillations: protein kinase C-dependent receptor phosphorylation is not required.

The metabotropic glutamate receptors (mGluR), mGluR1a and mGluR5a, are G protein-coupled receptors that couple via G(q) to the hydrolysis of phosphoinositides, the release of Ca(2+) from intracellular stores, and the activation of protein kinase C (PKC). We show here that mGluR1/5 activation results in oscillatory G protein coupling to phospholipase C thereby stimulating oscillations in both inositol 1,4,5-triphosphate formation and intracellular Ca(2+) concentrations. The mGluR1/5-stimulated Ca(2+) oscillations are translated into the synchronized repetitive redistribution of PKCbetaII between the cytosol and plasma membrane. The frequency at which mGluR1a and mGluR5a subtypes stimulate inositol 1,4,5-triphosphate, Ca(2+), and PKCbetaII oscillations is regulated by the charge of a single amino acid residue localized within their G protein-coupling domains. However, oscillatory mGluR signaling does not involve the repetitive feedback phosphorylation and desensitization of mGluR activity, since mutation of the putative PKC consensus sites within the first and second intracellular loops as well as the carboxyl-terminal tail does not prevent mGluR1a-stimulated PKCbetaII oscillations. Furthermore, oscillations in Ca(2+) continued in the presence of PKC inhibitors, which blocked PKCbetaII redistribution from the plasma membrane back into the cytosol. We conclude that oscillatory mGluR signaling represents an intrinsic receptor/G protein coupling property that does not involve PKC feedback phosphorylation.     

Ca2+ signaling and exocytosis in pituitary corticotropes

The secretion of adrenocorticotrophin (ACTH) from corticotropes is a key component in the endocrine response to stress. The resting potential of corticotropes is set by the basal activities of TWIK-related K(+) (TREK)-1 channel. Corticotrophin-releasing hormone (CRH), the major ACTH secretagogue, closes the background TREK-1 channels via the cAMP-dependent pathway, resulting in depolarization and a sustained rise in cytosolic [Ca(2+)] ([Ca(2+)](i)). By contrast, arginine vasopressin and norepinephrine evoke Ca(2+) release from the inositol trisphosphate (IP(3))-sensitive store, resulting in the activation of small conductance Ca(2+)-activated K(+) channels and hyperpolarization. Following [Ca(2+)](i) rise, cytosolic Ca(2+) is taken into the mitochondria via the uniporter. Mitochondrial inhibition slows the decay of the Ca(2+) signal and enhances the depolarization-triggered exocytotic response. Both voltage-gated Ca(2+) channel activation and intracellular Ca(2+) release generate spatial Ca(2+) gradients near the exocytic sites such that the local [Ca(2+)] is ~3-fold higher than the average [Ca(2+)](i). The stimulation of mitochondrial metabolism during the agonist-induced Ca(2+) signal and the robust endocytosis following stimulated exocytosis enable corticotropes to maintain sustained secretion during the diurnal ACTH surge. Arachidonic acid (AA) which is generated during CRH stimulation activates TREK-1 channels and causes hyperpolarization. Thus, corticotropes may regulate ACTH release via an autocrine feedback mechanism.     

Facilitatory effect of aspirin on glutamate release from rat hippocampal nerve terminals: involvement of protein kinase C pathway

The effect of aspirin on glutamate release from isolated nerve terminals (synaptosomes) from rat hippocampus was examined. The Ca(2+)-dependent release of glutamate evoked by 4-aminopyridine (4AP) was facilitated by aspirin in a concentration-dependent manner, but the 4AP-evoked Ca(2+)-independent release was not modified. Also, aspirin-mediated facilitation of glutamate release was completely inhibited by bafilomycin A1, which depletes vesicle content by inhibiting the synaptic vesicle H(+)-ATPase that drives glutamate uptake, not by l-trans-pyrrolidine-2,4-dicarboxylic acid (l-trans-PDC), a excitatory amino acid (EAA) transporter inhibitor, suggesting that the facilitation of glutamate release produced by aspirin originates from synaptic vesicle exocytosis rather than reversal of the plasma membrane glutamate transporter. In addition, aspirin did not alter either 4AP-evoked depolarization of the synaptosomal plasma membrane potential or Ca(2+) ionophore ionomycin-induced glutamate release, but significantly increased in 4AP-evoked Ca(2+) influx. A possible effect of aspirin on synaptosomal Ca(2+) channels was confirmed in experiments where synaptosomes pretreated with a combination of the N- and P/Q-type Ca(2+) channel blockers, which abolished the aspirin-mediated facilitation of glutamate release. The facilitatory action by aspirin observed in glutamate release was mimicked and occluded by arachidonic acid (AA) and eicosatetraynoic acid (ETYA), an analogue of AA that mimics the effect of AA but cannot be metabolized. Furthermore, this aspirin-mediated facilitation of glutamate release may depend on activation of protein kinase C (PKC), because PKC activator and PKC inhibitor, respectively, superseding or suppressing the facilitatory effect of aspirin. Together, these results suggest that aspirin exerts their presynaptic facilitatory effect, likely through AA directly to induce the activation of PKC, which subsequently enhances the Ca(2+) influx through voltage-dependent N- and P/Q-type Ca(2+) channels to cause an increase in evoked glutamate release from rat hippocampal nerve terminals.     

Urocortin 3 elevates cytosolic calcium in nucleus ambiguus neurons

J. Neurochem. (2012) 122, 1129-1136. ABSTRACT: Urocortin 3 (also known as stresscopin) is an endogenous ligand for the corticotropin-releasing factor receptor 2 (CRF(2) ). Despite predominant G(s) coupling of CRF(2) , promiscuous coupling with other G proteins has been also associated with the activation of this receptor. As urocortin 3 has been involved in central cardiovascular regulation at hypothalamic and medullary sites, we examined its cellular effects on cardiac vagal neurons of nucleus ambiguus, a key area for the autonomic control of heart rate. Urocortin 3 (1?nM-1000?nM) induced a concentration-dependent increase in cytosolic Ca(2+) concentration that was blocked by the CRF(2) antagonist K41498. In the case of two consecutive treatments with urocortin 3, the second urocortin 3-induced Ca(2+) response was reduced, indicating receptor desensitization. The effect of urocortin 3 was abolished by pre-treatment with pertussis toxin and by inhibition of phospolipase C with U-73122. Urocortin 3 activated Ca(2+) influx via voltage-gated P/Q-type channels as well as Ca(2+) release from endoplasmic reticulum. Urocortin 3 promoted Ca(2+) release via inositol 1,4,5 trisphosphate receptors, but not ryanodine receptors. Our results indicate a novel Ca(2+) -mobilizing effect of urocortin 3 in vagal pre-ganglionic neurons of nucleus ambiguus, providing a cellular mechanism for a previously reported role for this peptide in parasympathetic cardiac regulation.     

Cross-linking of surface immunoglobulin on B lymphocytes induces both intracellular Ca2+ release and Ca2+ influx: analysis with indo-1

The new Ca2+-probe indo-1 has a high fluorescence intensity, which allows low intracellular dye loadings. Stimulation of indo-1-loaded mouse B cells with anti-Ig antibodies provoked rapid rise of free cytoplasmic Ca2+ from 100 nM to greater than 1 microM, followed by a decline to a plateau at 300-400 nM. The initial rapid rise was not detected in quin2-loaded cells, presumably due to the Ca2+-buffering effects of the dye. The sustained Ca2+ increase was due to influx, whereas the initial rise was caused by release from intracellular stores. The magnitudes of Ca2+ release and inositol trisphosphate release were closely correlated. Concanavalin A does not provoke inositol trisphosphate release in mouse B cells. It did not induce a rapid initial Ca2+ rise in indo-1-loaded B cells either, but only a sustained increase to 200-300 nM. Finally, Ca2+ influx induced by both anti-Ig and concanavalin A were not affected by membrane depolarization.     

Regulation of neurotransmitter release by metabotropic glutamate receptors

The G protein-coupled metabotropic glutamate (mGlu) receptors are differentially localized at various synapses throughout the brain. Depending on the receptor subtype, they appear to be localized at presynaptic and/or postsynaptic sites, including glial as well as neuronal elements. The heterogeneous distribution of these receptors on glutamate and nonglutamate neurons/cells thus allows modulation of synaptic transmission by a number of different mechanisms. Electrophysiological studies have demonstrated that the activation of mGlu receptors can modulate the activity of Ca(2+) or K(+) channels, or interfere with release processes downstream of Ca(2+) entry, and consequently regulate neuronal synaptic activity. Such changes evoked by mGlu receptors can ultimately regulate transmitter release at both glutamatergic and nonglutamatergic synapses. Increasing neurochemical evidence has emerged, obtained from in vitro and in vivo studies, showing modulation of the release of a variety of transmitters by mGlu receptors. This review addresses the neurochemical evidence for mGlu receptor-mediated regulation of neurotransmitters, such as excitatory and inhibitory amino acids, monoamines, and neuropeptides.     

Receptor-activated cytoplasmic Ca2+ oscillations in pancreatic acinar cells: generation and spreading of Ca2+ signals.

Receptor-activated cytoplasmic Ca2+ oscillations have been investigated using both single cell microfluorometry and voltage-clamp recording of Ca(2+)-dependent Cl- current in single internally perfused acinar cells. In these cells there is direct experimental evidence showing that the ACh-evoked [Ca2+]i fluctuations are due to an inositol trisphosphate-induced small steady Ca2+ release which in turn evokes repetitive Ca2+ spikes via a caffeine-sensitive Ca(2+)-induced Ca2+ release process. There is indirect evidence suggesting that receptor-activation in addition to generating the Ca2+ releasing messenger, inositol trisphosphate, also produces another regulator involved in the control of Ca2+ signal spreading. Intracellular inositol trisphosphate or Ca2+ infusion produce short duration repetitive spikes confined to the cytoplasmic area close to the plasma membrane, but these signals can be made to progress throughout the cell by addition of caffeine or by receptor activation.     

Mobilization of intracellular Ca(2+) by endothelin-1 in rat intrapulmonary arterial smooth muscle cells

Endothelin-1 (ET-1) increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMCs); however, the mechanisms for Ca(2+) mobilization are not clear. We determined the contributions of extracellular influx and intracellular release to the ET-1-induced Ca(2+) response using Indo 1 fluorescence and electrophysiological techniques. Application of ET-1 (10(-10) to 10(-8) M) to transiently (24-48 h) cultured rat PASMCs caused concentration-dependent increases in [Ca(2+)](i). At 10(-8) M, ET-1 caused a large, transient increase in [Ca(2+)](i) (>1 microM) followed by a sustained elevation in [Ca(2+)](i) (<200 nM). The ET-1-induced increase in [Ca(2+)](i) was attenuated (<80%) by extracellular Ca(2+) removal; by verapamil, a voltage-gated Ca(2+)-channel antagonist; and by ryanodine, an inhibitor of Ca(2+) release from caffeine-sensitive stores. Depleting intracellular stores with thapsigargin abolished the peak in [Ca(2+)](i), but the sustained phase was unaffected. Simultaneously measuring membrane potential and [Ca(2+)](i) indicated that depolarization preceded the rise in [Ca(2+)](i). These results suggest that ET-1 initiates depolarization in PASMCs, leading to Ca(2+) influx through voltage-gated Ca(2+) channels and Ca(2+) release from ryanodine- and inositol 1,4,5-trisphosphate-sensitive stores.     

Glucose and endoplasmic reticulum calcium channels regulate HIF-1beta via presenilin in pancreatic beta-cells

Pancreatic beta-cell death is a critical event in type 1 diabetes, type 2 diabetes, and clinical islet transplantation. We have previously shown that prolonged block of ryanodine receptor (RyR)-gated release from intracellular Ca(2+) stores activates calpain-10-dependent apoptosis in beta-cells. In the present study, we further characterized intracellular Ca(2+) channel expression and function in human islets and the MIN6 beta-cell line. All three RyR isoforms were identified in human islets and MIN6 cells, and these endoplasmic reticulum channels were observed in close proximity to mitochondria. Blocking RyR channels, but not sarco/endoplasmic reticulum ATPase (SERCA) pumps, reduced the ATP/ADP ratio. Blocking Ca(2+) flux through RyR or inositol trisphosphate receptor channels, but not SERCA pumps, increased the expression of hypoxia-inducible factor (HIF-1beta). Moreover, inhibition of RyR or inositol trisphosphate receptor channels, but not SERCA pumps, increased the expression of presenilin-1. Both HIF-1beta and presenilin-1 expression were also induced by low glucose. Overexpression of presenilin-1 increased HIF-1beta, suggesting that HIF is downstream of presenilin. Our results provide the first evidence of a presenilin-HIF signaling network in beta-cells. We demonstrate that this pathway is controlled by Ca(2+) flux through intracellular channels, likely via changes in mitochondrial metabolism and ATP. These findings provide a mechanistic understanding of the signaling pathways activated when intracellular Ca(2+) homeostasis and metabolic activity are suppressed in diabetes and islet transplantation.     

Protein kinase C (PKC) phosphorylation of the Ca2+ o-sensing receptor (CaR) modulates functional interaction of G proteins with the CaR cytoplasmic tail

The extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) activates Ca(2+) influx independent of the release of intracellular Ca(2+) stores. The latter can be negatively regulated by protein kinase C (PKC) through phosphorylation of Thr-888 of the CaR. In this study, we substituted Thr-888 with various amino acid residues or a stop codon to understand how PKC phosphorylation of the CaR inhibits receptor-mediated release of intracellular Ca(2+) stores. Substitutions of Thr-888 with hydrophobic and hydrophilic amino acid residues had various effects on CaR-mediated release of intracellular Ca(2+) stores as well as activation of Ca(2+) influx. Several point mutations, such as T888D, had marked negative effects on CaR-mediated release of intracellular Ca(2+) stores but not on phorbol myristate acetate-insensitive activation of Ca(2+) influx. Presumably, the negatively charged aspartate mimics phospho-threonine. Interestingly, truncating the receptor at 888 had an even more pronounced negative effect on CaR-elicited release of intracellular Ca(2+) stores without significantly affecting CaR-mediated activation of Ca(2+) influx. Therefore, truncation at position 888 of the CaR affects the activity of the receptor in a manner that resembles PKC phosphorylation of the CaR. This in turn suggests that PKC phosphorylation of the CaR prevents G protein subtypes from interacting with the region of the receptor critical for releasing Ca(2+) stores, which is missing in the truncated receptor.     

Fundamental Ca2+ signaling mechanisms in mouse dendritic cells: CRAC is the major Ca2+ entry pathway

Although Ca(2+)-signaling processes are thought to underlie many dendritic cell (DC) functions, the Ca(2+) entry pathways are unknown. Therefore, we investigated Ca(2+)-signaling in mouse myeloid DC using Ca(2+) imaging and electrophysiological techniques. Neither Ca(2+) currents nor changes in intracellular Ca(2+) were detected following membrane depolarization, ruling out the presence of functional voltage-dependent Ca(2+) channels. ATP, a purinergic receptor ligand, and 1-4 dihydropyridines, previously suggested to activate a plasma membrane Ca(2+) channel in human myeloid DC, both elicited Ca(2+) rises in murine DC. However, in this study these responses were found to be due to mobilization from intracellular stores rather than by Ca(2+) entry. In contrast, Ca(2+) influx was activated by depletion of intracellular Ca(2+) stores with thapsigargin, or inositol trisphosphate. This Ca(2+) influx was enhanced by membrane hyperpolarization, inhibited by SKF 96365, and exhibited a cation permeability similar to the Ca(2+) release-activated Ca(2+) channel (CRAC) found in T lymphocytes. Furthermore, ATP, a putative DC chemotactic and maturation factor, induced a delayed Ca(2+) entry with a voltage dependence similar to CRAC. Moreover, the level of phenotypic DC maturation was correlated with the extracellular Ca(2+) concentration and enhanced by thapsigargin treatment. These results suggest that CRAC is a major pathway for Ca(2+) entry in mouse myeloid DC and support the proposal that CRAC participates in DC maturation and migration.     

Recent breakthroughs in the molecular mechanism of capacitative calcium entry (with thoughts on how we got here)

Activation of phospholipase C by G-protein-coupled receptors results in release of intracellular Ca(2+) and activation of Ca(2+) channels in the plasma membrane. The intracellular release of Ca(2+) is signaled by the second messenger, inositol 1,4,5-trisphosphate. Ca(2+) entry involves signaling from depleted intracellular stores to plasma membrane Ca(2+) channels, a process referred to as capacitative calcium entry or store-operated calcium entry. The electrophysiological current associated with capacitative calcium entry is the calcium-release-activated calcium current, or I(crac). In the 20 years since the inception of the concept of capacitative calcium entry, a variety of activation mechanisms have been proposed, and there has been considerable interest in the possibility of transient receptor potential channels functioning as store-operated channels. However, in the past 2 years, two major players in both the signaling and permeation mechanisms for store-operated channels have been discovered: Stim1 (and possibly Stim2) and the Orai proteins. Activation of store-operated channels involves an endoplasmic reticulum Ca(2+) sensor called Stim1. Stim1 acts by redistributing within a small component of the endoplasmic reticulum, approaching the plasma membrane, but does not appear to translocate into the plasma membrane. Stim1, either directly or indirectly, signals to plasma membrane Orai proteins which constitute pore-forming subunits of store-operated channels.     

Ion channels in smooth muscle: regulators of intracellular calcium and contractility

Smooth muscle (SM) is essential to all aspects of human physiology and, therefore, key to the maintenance of life. Ion channels expressed within SM cells regulate the membrane potential, intracellular Ca2+ concentration, and contractility of SM. Excitatory ion channels function to depolarize the membrane potential. These include nonselective cation channels that allow Na+ and Ca2+ to permeate into SM cells. The nonselective cation channel family includes tonically active channels (Icat), as well as channels activated by agonists, pressure-stretch, and intracellular Ca2+ store depletion. Cl--selective channels, activated by intracellular Ca2+ or stretch, also mediate SM depolarization. Plasma membrane depolarization in SM activates voltage-dependent Ca2+ channels that demonstrate a high Ca2+ selectivity and provide influx of contractile Ca2+. Ca2+ is also released from SM intracellular Ca2+ stores of the sarcoplasmic reticulum (SR) through ryanodine and inositol trisphosphate receptor Ca2+ channels. This is part of a negative feedback mechanism limiting contraction that occurs by the Ca2+-dependent activation of large-conductance K+ channels, which hyper polarize the plasma membrane. Unlike the well-defined contractile role of SR-released Ca2+ in skeletal and cardiac muscle, the literature suggests that in SM Ca2+ released from the SR functions to limit contractility. Depolarization-activated K+ chan nels, ATP-sensitive K+ channels, and inward rectifier K+ channels also hyperpolarize SM, favouring relaxation. The expression pattern, density, and biophysical properties of ion channels vary among SM types and are key determinants of electrical activity, contractility, and SM function.     

Role of intracellular Ca(2+) release in histamine-induced depolarization in rabbit middle cerebral artery

The role of Ca(2+) mobilization from intracellular stores and Ca(2+)-activated Cl(-) channels in caffeine- and histamine-induced depolarization and contraction of the rabbit middle cerebral artery has been studied by recording membrane potential and isometric force. Caffeine induced a transient contraction and a transient followed by sustained depolarization. The transient depolarization was abolished by ryanodine, DIDS, and niflumic acid, suggesting involvement of Ca(2+)-activated Cl(-) channels. Histamine-evoked transient contraction in Ca(2+)-free solution was abolished by ryanodine or by caffeine-induced depletion of Ca(2+) stores. Ryanodine slowed the development of depolarization induced by histamine in Ca(2+)-containing solution but did not affect its magnitude. In arteries treated with 1 mM Co(2+), histamine elicited a transient depolarization and contraction, which was abolished by ryanodine. DIDS and niflumic acid reduced histamine-evoked depolarization and contraction. Histamine caused a sustained depolarization and contraction in low-Cl(-) solution. These results suggest that Ca(2+) mobilization from ryanodine-sensitive stores is involved in histamine-induced initial, but not sustained, depolarization and contraction. Ca(2+)-activated Cl(-) channels contribute mainly to histamine-induced initial depolarization and less importantly to sustained depolarization, which is most likely dependent on activation of nonselective cation channels.     

A model of calcium waves in pancreatic and parotid acinar cells

We construct a mathematical model of Ca(2+) wave propagation in pancreatic and parotid acinar cells. Ca(2+) release is via inositol trisphosphate receptors and ryanodine receptors that are distributed heterogeneously through the cell. The apical and basal regions are separated by a region containing the mitochondria. In response to a whole-cell, homogeneous application of inositol trisphosphate (IP(3)), the model predicts that 1), at lower concentrations of IP(3), the intracellular waves in pancreatic cells begin in the apical region and are actively propagated across the basal region by Ca(2+) release through ryanodine receptors; 2), at higher [IP(3)], the waves in pancreatic and parotid cells are not true waves but rather apparent waves, formed as the result of sequential activation of inositol trisphosphate receptors in the apical and basal regions; 3), the differences in wave propagation in pancreatic and parotid cells can be explained in part by differences in inositol trisphosphate receptor density; 4), in pancreatic cells, increased Ca(2+) uptake by the mitochondria is capable of restricting Ca(2+) responses to the apical region, but that this happens only for a relatively narrow range of [IP(3)]; and 5), at higher [IP(3)], the apical and basal regions of the cell act as coupled Ca(2+) oscillators, with the basal region partially entrained to the apical region.     

Presynaptic mechanisms underlying the alpha-lipoic acid facilitation of glutamate exocytosis in rat cerebral cortex nerve terminals

The antioxidant alpha-lipoic acid has been reported to prevent and reverse age-related impairments in learning and memory. However, it is unclear how alpha-lipoic acid improves cognitive function. In this study, the effect of alpha-lipoic acid on the release of endogenous glutamate from rat cerebrocortical nerve terminals (synaptosomes) was examined. We found that alpha-lipoic acid potently facilitated 4-aminopyridine (4AP)-evoked glutamate release, and this release facilitation results from an enhancement of vesicular exocytosis and not from an increase of non-vesicular release. Examination of the effect of alpha-lipoic acid on cytosolic [Ca(2+)] revealed that the facilitation of glutamate release was associated with an increase in voltage-dependent Ca(2+) influx. Consistent with this, alpha-lipoic acid-mediated facilitation of glutamate release was completely prevented in synaptosomes pretreated with a wide spectrum blocker of the N- and P/Q-type Ca(2+) channels, omega-conotoxin MVIIC. The facilitatory effect of alpha-lipoic acid on Ca(2+) influx was not due to an increase of synaptosomal excitability because alpha-lipoic acid did not alter the 4AP-evoked depolarization of the synaptosomal plasma membrane potential. In addition, both ionomycin and hypertonic sucrose-induced glutamate release were enhanced by alpha-lipoic acid. Furthermore, disruption of cytoskeleton organization with cytochalasin D occluded the facilitatory effect of alpha-lipoic acid on 4AP or ionomycin-evoked glutamate release. These results suggest that the antioxidant alpha-lipoic acid enhances the Ca(2+) entry through presynaptic N- and P/Q-type Ca(2+) channels as well as the vesicular release machinery to cause an increase in evoked glutamate release from rat cerebrocortical synaptosomes. Also, activation of PKA and PKC may underlie, at least in part, the alpha-lipoic acid-mediated facilitation of glutamate release observed here as alpha-lipoic acid-enhanced 4AP and ionomycin-evoked glutamate release were significantly attenuated by PKA and PKC inhibitors. This finding may provide some information regarding the mechanism of action of alpha-lipoic acid in the central nervous system (CNS).     

Effects of Ca2+ channel antagonists on nerve stimulation-induced and ischemia-induced myocardial interstitial acetylcholine release in cats

Although an axoplasmic Ca(2+) increase is associated with an exocytotic acetylcholine (ACh) release from the parasympathetic postganglionic nerve endings, the role of voltage-dependent Ca(2+) channels in ACh release in the mammalian cardiac parasympathetic nerve is not clearly understood. Using a cardiac microdialysis technique, we examined the effects of Ca(2+) channel antagonists on vagal nerve stimulation- and ischemia-induced myocardial interstitial ACh releases in anesthetized cats. The vagal stimulation-induced ACh release [22.4 nM (SD 10.6), n = 7] was significantly attenuated by local administration of an N-type Ca(2+) channel antagonist omega-conotoxin GVIA [11.7 nM (SD 5.8), n = 7, P = 0.0054], or a P/Q-type Ca(2+) channel antagonist omega-conotoxin MVIIC [3.8 nM (SD 2.3), n = 6, P = 0.0002] but not by local administration of an L-type Ca(2+) channel antagonist verapamil [23.5 nM (SD 6.0), n = 5, P = 0.758]. The ischemia-induced myocardial interstitial ACh release [15.0 nM (SD 8.3), n = 8] was not attenuated by local administration of the L-, N-, or P/Q-type Ca(2+) channel antagonists, by inhibition of Na(+)/Ca(2+) exchange, or by blockade of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptor but was significantly suppressed by local administration of gadolinium [2.8 nM (SD 2.6), n = 6, P = 0.0283]. In conclusion, stimulation-induced ACh release from the cardiac postganglionic nerves depends on the N- and P/Q-type Ca(2+) channels (with a dominance of P/Q-type) but probably not on the L-type Ca(2+) channels in cats. In contrast, ischemia-induced ACh release depends on nonselective cation channels or cation-selective stretch activated channels but not on L-, N-, or P/Q type Ca(2+) channels, Na(+)/Ca(2+) exchange, or Ins(1,4,5)P(3) receptor-mediated pathway.