Presence Of External Ca2 Research Articles

N-Methyl- D-aspartate receptor activation inhibits protein synthesis in cortical neurons independently of its ionic permeability properties

Transient cerebral ischemia, which is accompanied by a sustained release of glutamate, strongly depresses protein synthesis. We have previously demonstrated in cortical neurons that a glutamate-induced increase in intracellular Ca 2+ is likely responsible for the blockade of the elongation step of protein synthesis. In this study, we provide evidence indicating that NMDA mobilizes a thapsigargin-sensitive pool of intracellular Ca 2+. Exposure of cortical neurons to NMDA, in the absence of external Ca 2+, produced a transient rise in intracellular Ca 2+ that was suppressed by pretreatment with thapsigargin. This rise in intracellular Ca 2+ did not result from an influx of Na + via reversal of the mitochondrial Na +/Ca 2+ exchanger since it persisted in a Na +-free medium or in the presence of CGP 37157, an inhibitor of the exchanger. Moreover, the NMDA-induced increase in intracellular Ca 2+ required the presence of D-serine, was blocked by D(−)-2-amino-5-phosphonopentanoic acid, but was not reduced in the presence of external Mg 2+. This unexpected non-ionotropic effect of NMDA was associated with an inhibition of protein synthesis that was also insensitive to the absence of external Ca 2+ or Na +, or presence of Mg 2+. NMDA treatment resulted in an increase in the phosphorylation of eEF-2 in the absence or presence of external Ca 2+. The initiation step of protein synthesis was not blocked by NMDA since the phosphorylation of initiation factor eIF-2α subunit was not altered by NMDA treatment. In conclusion, we provide evidence indicating that NMDA can inhibit protein synthesis in cortical neurons through a process that involves the mobilization of intracellular Ca 2+ stores via a mechanism that is not linked to the ionic properties of NMDA receptors.

Olfactory sensitivity to changes in environmental [Ca(2+)] in the freshwater teleost Carassius auratus: an olfactory role for the Ca(2+)-sensing receptor?

Olfactory sensitivity to changes in environmental Ca(2+) has been demonstrated in two teleost species; a salmonid (Oncorhynchus nerka) and a marine/estuarine perciform (Sparus aurata). To assess whether this phenomenon is restricted to species that normally experience large fluctuations in external ion concentrations (e.g. moving from sea water to fresh water) or is present in a much wider range of species, we investigated olfactory Ca(2+) sensitivity in the goldfish (Carassius auratus), which is a stenohaline, non-migratory freshwater cyprinid. Extracellular recording from the olfactory bulb in vivo by electroencephalogram (EEG) demonstrated that the olfactory system is acutely sensitive to changes in external Ca(2+) within the range that this species is likely to encounter in the wild (0.05-3 mmol l(-1)). The olfactory system responded to increases in external calcium with increasing bulbar activity in a manner that fitted a conventional Hill plot with an apparent EC(50) of 0.9+/-0.3 mmol l(-1) (close to both ambient and plasma free [Ca(2+)]) and an apparent Hill coefficient of 1.1+/-0.3 (means +/- S.E.M., N=6). Thresholds of detection were below 50 micro mol l(-1). Some olfactory sensitivity to changes in external [Na(+)] was also recorded, but with a much higher threshold of detection (3.7 mmol l(-1)). The olfactory system of goldfish was much less sensitive to changes in [Mg(2+)] and [K(+)]. Preliminary data suggest that Ca(2+) and Mg(2+) are detected by the same mechanism, although with a much higher affinity for Ca(2+). Olfactory sensitivity to Na(+) may warn freshwater fish that they are reaching the limit of their osmotic tolerance when in an estuarine environment. Olfaction of serine, a potent odorant in fish, was not dependent on the presence of external Ca(2+) or Na(+). Finally, the teleost Ca(2+)-sensing receptor (Ca-SR) was shown to be highly expressed in a subpopulation of olfactory receptor neurones by both immunocytochemistry and in situ hybridisation. The olfactory sensitivity to Ca(2+) (and Mg(2+)) is therefore likely to be mediated by the Ca-SR. We suggest that olfactory Ca(2+) sensitivity is a widespread phenomenon in teleosts and may have an input into the physiological mechanisms regulating internal calcium homeostasis.

The initial inositol 1,4,5-trisphosphate response induced by histamine is strongly amplified by Ca2+ release from internal stores in smooth muscle

We have studied the Ca2+-dependence and wortmannin-sensitivity of the initial inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) response induced by activation of either histamine or muscarinic receptors in smooth muscle from guinea pig urinary bladder. Activation of H1 receptors with histamine (100μM) produced a significant elevation in Ins(1,4,5)P3 levels with only 5s stimulation and in the presence of external Ca2+. However, this response was abolished fully by either the prolonged absence of external Ca2+ or the depletion of internal Ca2+ stores with thapsigargin (100nM) or ryanodine (10μM). In contrast, the same conditions only slightly reduced the initial Ins(1,4,5)P3 response induced by carbachol. The prolonged incubation of smooth muscle in 10μM wortmannin to inhibit type III PI 4-kinase abolished both the early histamine-evoked Ins(1,4,5)P3 and Ca2+ responses. Conversely, wortmannin did not alter Ca2+ release induced by carbachol, despite a partial reduction of its Ins(1,4,5)P3 response. Collectively, these data indicate that the detectable histamine-induced increase in Ins(1,4,5)P3 is more the consequence of Ca2+ release from internal stores than a direct activation of phospholipase C by H1 receptors. In addition, the effect of wortmannin implies the existence of a Ca2+-dependent amplification loop for the histamine-induced Ins(1,4,5)P3 response in smooth muscle.

External Bioenergy Increases Intracellular Free Calcium Concentration and Reduces Cellular Response to Heat Stress

BackgroundExternal bioenergy (energy emitted from the body) can influence a variety of biological activities. It has been shown to enhance immunity, promote normal cell proliferation, increase tumor cell death, and...

Lead induces endothelium- and Ca2+-independent contraction in rat aortic rings.

The contractile effect of lead on rat aortic rings was examined. Lead (0.1-3.1 mM) elicited concentration-dependent but endothelium-independent contractions, which were unaffected by prazosin (1 microM). The contractile effects of lead were similar when the aortic rings were bathed either in the absence or presence of external Ca2+. Lanthanum (1 mM) but not verapamil (I pM) inhibited the lead contractions; hence non-L-calcium channels are involved in such effect. In addition, lead induced contractions on aortic rings incubated in Ca2+-free EGTA-containing solution for 70 min., an experimental condition in which intracellular Ca2+-stores are depleted. Finally, the contractile effect of lead was not modified by calphostin C (an inhibitor of protein kinase C). In conclusion, the present results suggest that in rat aorta, the lead-induced contraction is independent of extra- and intracellular calcium stores. In addition, the effect of lead is independent of either catecholamines or protein kinase C. It is likely that in rat aorta, lead enters into the smooth muscle cells through non-L-calcium channels, and when acting like calcium on the contractile machinery it produces contraction. The differences observed between our results and those obtained by other authors may indicate that the mechanism of the contractile effect of lead varies among the different blood vessels.

Dipyridamole inhibits sickling-induced cation fluxes in sickle red blood cells

Sickling-induced cation fluxes contribute to cellular dehydration of sickle red blood cells (SS RBCs), which in turn potentiates sickling. This study examined the inhibition by dipyridamole of the sickling-induced fluxes of Na(+), K(+), and Ca(++) in vitro. At 2% hematocrit, 10 microM dipyridamole inhibited 65% of the increase in net fluxes of Na(+) and K(+) produced by deoxygenation of SS RBCs. Sickle-induced Ca(++) influx, assayed as (45)Ca(++) uptake in quin-2-loaded SS RBCs, was also partially blocked by dipyridamole, with a dose response similar to that of Na(+) and K(+) fluxes. In addition, dipyridamole inhibited the Ca(++)-activated K(+) flux (via the Gardos pathway) in SS RBCs, measured as net K(+) efflux in oxygenated cells exposed to ionophore A23187 in the presence of external Ca(++), but this effect resulted from reduced anion conductance, rather than from a direct effect on the K(+) channel. The degree of inhibition of sickling-induced fluxes was dependent on hematocrit, and up to 30% of dipyridamole was bound to RBC membranes at 2% hematocrit. RBC membrane content of dipyridamole was measured fluorometrically and correlated with sickling-induced flux inhibition at various concentrations of drug. Membrane drug content in patients taking dipyridamole for other clinical indications was similar to that producing inhibition of sickling-induced fluxes in vitro. These data suggest that dipyridamole might inhibit sickling-induced fluxes of Na(+), K(+), and Ca(++) in vivo and therefore have potential as a pharmacological agent to reduce SS RBC dehydration. (Blood. 2001;97:3976-3983)

P2X7 nucleotide receptor mediation of membrane pore formation and superoxide generation in human promyelocytes and neutrophils.

The P2X(7) receptor, which induces cation channel opening imparting significant permeability to Ca2+ and pore formation with changes in the plasma membrane potential, has been known to be rather restrictedly expressed in cells of the macrophage lineage including dendrites, mature macrophages, and microglial cells. However, we show here that the P2X(7) receptor is also expressed in cells of granulocytic lineage such as HL-60 promyelocytes, granulocytic differentiated cells, and neutrophils. Exposure of these cells to 2',3'-O-(4-benzoyl)benzoyl-ATP (BzATP) triggered intracellular Ca2+ rise through the mediation of phospholipase C-independent and suramin-sensitive pathways. BzATP also induced depolarization of the plasma membrane in the absence of extracellular Ca2+, whereas it hyperpolarized the cells in the presence of external Ca2+, probably in part through the activation of Ca2+-activated K(+) channels. However, the hyperpolarization phenomenon was markedly attenuated in differentiated HL-60 cells and neutrophils. RT-PCR and Northern blot analysis revealed the presence of P2X(7) receptors on both HL-60 and neutrophil-like cells. This was further confirmed by pore formation through which the uptake of Lucifer yellow and YO-PRO1 occurred on BzATP treatment. BzATP stimulated in a concentration-dependent manner the production of superoxide in differentiated HL-60 cells via a pathway partially dependent on extracellular Ca2+. Moreover, in human neutrophils, BzATP was a more effective inducer of superoxide generation than PMA. Taken together, this is a first demonstration of the expression of P2X(7) receptors on neutrophils, which shows that the receptor is functionally involved in the defense mechanism by activation of the respiratory burst pathway.

Fibrinogen binding to the integrin αIIbβ3 modulates store-mediated calcium entry in human platelets

AbstractEffects of the occupation of integrin αIIbβ3 by fibrinogen on Ca++signaling in fura-2–loaded human platelets were investigated. Adding fibrinogen to washed platelet suspensions inhibited increases in cytosolic [Ca++] concentrations ([Ca++]i) evoked by adenosine diphosphate (ADP) and thrombin in a concentration-dependent manner in the presence of external Ca++ but not in the absence of external Ca++ or in the presence of the nonselective cation channel blocker SKF96365, indicating selective inhibition of Ca++entry. Fibrinogen also inhibited store-mediated Ca++ entry (SMCE) activated after Ca++ store depletion using thapsigargin. The inhibitory effect of fibrinogen was reversed if fibrinogen binding to αIIbβ3 was blocked using RDGS or abciximab and was absent in platelets from patients homozygous for Glanzmann thrombasthenia. Fibrinogen was without effect on SMCE once activated. Activation of SMCE in platelets occurs through conformational coupling between the intracellular stores and the plasma membrane and requires remodeling of the actin cytoskeleton. Fibrinogen inhibited actin polymerization evoked by ADP or thapsigargin in control cells and in cells loaded with the Ca++ chelator dimethyl BAPTA. It also inhibited the translocation of the tyrosine kinase p60src to the cytoskeleton. These results indicate that the binding of fibrinogen to integrin αIIbβ3 inhibits the activation of SMCE in platelets by a mechanism that may involve modulation of the reorganization of the actin cytoskeleton and the cytoskeletal association of p60src. This action may be important in intrinsic negative feedback to prevent the further activation of platelets subjected to low-level stimuli in vivo.

Amperometric characterization of exocytotic events from single mast cells: dependence on external and internal Ca++sources

Mast cells exocytotically release histamine/serotonin in response to different secretagogues. We have used substance P and compound 48/80 to study the Ca++dependency of serotonin exocytosis from peritoneal mast cells using carbon fiber amperometric techniques. The exocytotic release pattern consists of a burst of events superimposed on a slow, transient, amperometric current baseline increase. Cellular re lease parameters (number, frequency and total charge of amperometric events) and individual event characteristics (charge integral, half width and peak amplitude) were similar for the two secretagogues used. Zero Ca++conditions greatly reduced, without completely abolishing,cellular release parameters. Cyclopiazonic acid, an inhibitor of the endoplasmatic Ca++ATPase, reduced the cellular exocytotic capacity and diminished the amplitude of individual exocytotic events more effectively than the 0 Ca++condition. The cyclopiazonic acid effects occurred in the presence of external Ca++, indicating that this condition is not sufficient for maintaining full exocytotic capacity. The results confirm the importance of intracellular Ca++for exocytotic activation. For the first time evidence is presented that the integrity of intracellular Ca++pools determines the amplitude and frequency of individual exocytotic events. Saponin, a non-specific detergent, also induced quantal release similar to that obtained with substance P and compound 48/80. This release was not dependent on extracellular C a++, but cyclopiazonic acid significantly reduced individual exocytotic release.

Comparison of intracellular calcium signals evoked by heat and capsaicin in cultured rat dorsal root ganglion neurons and in a cell line expressing the rat vanilloid receptor, VR1

The cloning of the receptor for capsaicin, vanilloid receptor 1, has shown it to be non-selective cation channel with a high calcium permeability which can be opened by noxious heat as well as capsaicin. Here we compare the calcium signals produced by native and recombinant capsaicin receptors when activated by either heat or capsaicin by imaging intracellular calcium levels ([Ca 2+] i) in rat dorsal root ganglion neurons and Chinese hamster ovary cells transfected with the rat vanilloid receptor, vanilloid receptor 1. Vanilloid receptor 1 transfected cells and a subset of dorsal root ganglion neurons responded to both capsaicin and to heating to 50°C with rapid, substantial and reversible rises in [Ca 2+] i. All except one of the dorsal root ganglion neurons responsive to capsaicin also showed sensitivity to heat, and most, but not all, heat-sensitive neurons also responded to capsaicin. Both capsaicin and heat responses were dependent on the presence of extracellular Ca 2+. Non-transfected Chinese hamster ovary cells and non-responsive dorsal root ganglion neurons showed only small rises in [Ca 2+] i in response to heat which did not depend on the presence of external Ca 2+. Responsive dorsal root ganglion neurons and vanilloid receptor 1 transfected cells showed a clear temperature threshold, above which [Ca 2+] i increased rapidly. This was estimated to be 42.6±0.3°C for vanilloid receptor 1 transfected cells and 42.0±0.6°C for dorsal root ganglion neurons. The competitive capsaicin antagonist capsazepine (10 μM) abolished [Ca 2+] i increases stimulated by capsaicin in both dorsal root ganglion neurons and vanilloid receptor 1 transfected cells. However, responses to heat of a similar magnitude in the same cells were inhibited by only 37% by capsazepine (10 μM). In vanilloid receptor 1 transfected cells, Ruthenium Red (10 μM) blocked responses to both capsaicin and heat. These results demonstrate that imaging of [Ca 2+] i can identify dorsal root ganglion neurons which are responsive to both heat and capsaicin. They show that heat and capsaicin responses mediated by native and recombinant capsaicin receptors are similar with respect to the characteristics and pharmacology examined, suggesting that expression of recombinant vanilloid receptor 1 in cell lines accurately reproduces the properties of the native receptor.

Two subtypes of G protein‐coupled nucleotide receptors, P2Y1 and P2Y2 are involved in calcium signalling in glioma C6 cells

1. In glioma C6 cells, the stimulation of P2Y receptors by ADP, ATP and UTP initiated an increase in the intracellular Ca2+ concentration, in a process that involved the release of Ca2+ from InsP(3)-sensitive store and the capacitative, extracellular Ca2+ entry. The presence of external Ca2+ was not necessary to elevate Ca(2+). 2. The rank order of potencies of nucleotide analogues in stimulating [Ca2+](i) was: 2MeSADP > ADP > 2MeSATP = 2ClATP > ATP > UTP. alpha,beta-Methylene ATP, adenosine and AMP were ineffective. 3. ADP and UTP effects were additive, while actions of ATP and UTP were not additive on [Ca2+](i) increase. Similarly, cross-desensitization between ATP and UTP but not between ADP and UTP occurred. 4. Suramin, a non-specific nucleotide receptors inhibitor, antagonized ATP-, UTP- and ADP-evoked Ca2+ responses. PPADS, a selective antagonist of the P2Y(1) receptor-generated InsP(3) accumulation, decreased ADP-initiated Ca2+ response with no effect on ATP and UTP. 5. Pertussis toxin (PTX) reduced ADP- and ATP-induced Ca2+ increases. Short-term treatment with TPA, inhibited both ATP and ADP stimulatory effects on [Ca2+](i). 6. ADP inhibited isoproterenol-induced cyclic AMP accumulation. PTX blocked this effect, but PPADS did not. 7. RT - PCR analysis revealed the molecular identity of P2Y receptors expressed by glioma C6 cells to be both P2Y(1) and P2Y(2). 8. It is concluded that both P2Y(1) and P2Y(2) receptors co-exist in glioma C6 cells. ADP acts as agonist of the first, and ATP and UTP of the second one. Both receptors are linked to phospholipase C (PLC).

Comparative Study on the Vasorelaxant Effects of Three Harmala Alkaloids In Vitro

Three psychological active principles from the seeds of Peganum harmala L., harmine, harmaline and harmalol, showed vasorelaxant activities in isolated rat thoracic aorta preparations precontracted by phenylephrine or KCl with rank order of relaxation potency of harmine > harmaline > harmalol. The vasorelaxant effects of harmine and harmaline (but not harmalol) were attenuated by endothelium removal or pretreatment with a nitric oxide (NO) synthase Nomega-nitro-L-arginine methyl ester. In cultured rat aortic endothelial cells, harmine and harmaline (but not harmalol) increased NO release, which was dependent on the presence of external Ca2+. In endothelium-denuded preparations, pretreatment of harmine, harmaline or harmalol (3-30 microM) inhibited phenylephrine-induced contractions in a non-competitive manner. Receptor binding assays indicated that all 3 compounds interacted with cardiac alpha1-adrenoceptors with comparable affinities (Ki value around 31 - 36 microM), but only harmine weakly interacted with the cardiac 1,4-dihydropyridine binding site of L-type Ca2+ channels (Ki value of 408 microM). Therefore, the present results suggested that the vasorelaxant effects of harmine and harmaline are attributed to their actions on the endothelial cells to release NO and on the vascular smooth muscles to inhibit the contractions induced by the activation of receptor-linked and voltage-dependent Ca2+ channels. The vasorelaxant effect of harmalol was not endothelium-dependent.

Two Independent Pathways Mediated by cAMP and Protein Kinase A Enhance Spontaneous Transmitter Release atDrosophilaNeuromuscular Junctions

cAMP is thought to be involved in learning process and known to enhance transmitter release in various systems. Previously we reported that cAMP enhances spontaneous transmitter release in the absence of extracellular Ca(2+) and that the synaptic vesicle protein neuronal-synaptobrevin (n-syb), is required in this enhancement (n-syb-dependent; Yoshihara et al., 1999). In the present study, we examined the cAMP-induced enhancement of transmitter release in the presence of external Ca(2+). We raised the intracellular concentration of cAMP by application of either forskolin, an activator of adenylyl cyclase, or by 4-chlorophenylthio-(CPT)-cAMP, a membrane-permeable analog of cAMP, in the presence of external Ca(2+), while recording miniature synaptic currents (mSCs) at the neuromuscular junction in n-syb null mutant embryos. The frequency of mSCs increased in response to elevation of cAMP, and this effect of cAMP was completely blocked by Co(2+) (n-syb-independent pathway). In contrast, in wild-type embryos the cAMP-induced mSC frequency increase was partially blocked by Co(2+). In a mutant, DC0, defective in protein kinase A (PKA), nerve-evoked synaptic currents were indistinguishable from the control, but mSCs were less frequent. In this mutant the enhancement by cAMP of both nerve-evoked and spontaneous transmitter release was completely absent, even in the presence of external Ca(2+). Taken together, these results suggest that cAMP enhances spontaneous transmitter release by increasing Ca(2+) influx (n-syb-independent) as well as by modulating the release mechanism without Ca(2+) influx (n-syb-dependent) in wild-type embryos, and these two effects are mediated by PKA encoded by the DC0 gene.

Pituitary adenylate cyclase-activating polypeptide may function as a neuromodulator in guinea-pig adrenal medulla.

The role of pituitary adenylate cyclase-activating polypeptide (PACAP) in catecholamine secretion from dissociated adrenal chromaffin cells of the guinea-pig was investigated using amperometry, the patch clamp technique and immunochemistry. Pretreatment of adrenal chromaffin cells with 0.3-10 nM PACAP for 2 min resulted in enhancement of nicotine- and muscarine-induced secretions in either the presence of external Ca2+ ions or nominally Ca2+-free solution, with no change in basal secretion or the holding current at -60 mV in most of the cells tested. Pretreatment with PACAP augmented the muscarine-induced non-selective cation current, but did not affect the muscarine-induced outward current or nicotine-induced current. PACAP-induced enhancement of nicotine- and muscarine-induced secretions was suppressed by the simultaneous application of PACAP and the protein kinase inhibitors 100 microM HA1004 or 2 microM H89. Application of forskolin enhanced both muscarine- and nicotine-induced secretions, whereas application of a phorbol ester augmented the nicotine-induced secretion, but suppressed the muscarine-induced secretion in a reversible manner. Immunohistochemical analysis of adrenal medullae revealed that PACAP-like immunoreactivity was present in nerve fibres surrounding putative chromaffin cells. PAC1R-like immunoreactivity was distributed diffusely in the plasma membrane, whereas nicotinic ACh receptor-like immunoreactivity was concentrated at the plasma membrane near the nucleus, where the synapses were mainly localized. These observations suggest that PACAP in the guinea-pig adrenal medulla functions as a neuromodulator to facilitate ACh-induced secretion through a cAMP-protein kinase A-dependent pathway.

Energy metabolism and its linkage to intracellular Ca 2+ and pH regulation in rat spermatogenic cells

Energy metabolism and intracellular adenine nucleotides of meiotic and postmeiotic spermatogenic cells are highly dependent on external substrates for oxidative phosphorylation and glycolysis. Using fluorescent probes to measure the changes in cytosolic [Ca 2+] ([Ca 2+] i) and pH (pH i), we were able to demonstrate that changes in energy metabolism of meiotic and postmeiotic spermatogenic cells were rapidly translated into changes of pH i and [Ca 2+] i in the absence or presence of external Ca 2+. Under these conditions, mitochondria were gaining cytosolic calcium in these cells. Our results indicate that Ca 2+ mobilised by changes in metabolic energy pathways originated in thapsigargin-sensitive intracellular Ca 2+ stores. Changes in intracellular adenine nucleotides, measured by HPLC, and a likely colocalization of ATP-producing and ATP-consuming processes in the cells seemed to provide the linkage between metabolic fluxes and the changes in pH i and [Ca 2+] i in pachytene spermatocytes and round spermatids. Glucose metabolism produced an increase of [Ca 2+] i in round spermatids but not in pachytene spermatocytes, and a decrease in pH i in both cell types. Hence, glucose emerges as a molecule that can differentially modulate [Ca 2+] i and pH i in pachytene spermatocytes and round spermatids in rats.

Tetracaine stimulates insulin secretion through the mobilization of Ca2+from thapsigargin- and IP3-insensitive Ca2+reservoir in pancreatic β-cells

The effect of tetracaine on45Ca efflux, cytoplasmic Ca2+concentration [Ca2+]i, and insulin secretion in isolated pancreatic islets and β-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the45Ca efflux from isolated islets in a dose-dependant manner. Tetracaine did not affect the increase in45Ca efflux caused by 50 mM K+or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+]iin isolated β-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 µM D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 µM thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in45Ca efflux caused by high concentrations of K+and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+pool in β-cells.

Extracellular pH changes and accompanying cation shifts during ouabain-induced spreading depression.

Interstitial ionic shifts that accompany ouabain-induced spreading depression (SD) were studied in rat hippocampal and cortical slices in the presence and absence of extracellular Ca(2+). A double-barreled ion-selective microelectrode specific for H(+), K(+), Na(+), or Ca(2+) was placed in the CA1 stratum radiatum or midcortical layer. Superfusion of 100 microM ouabain caused a rapid, negative, interstitial voltage shift (2-10 mV) after 3-5 min. The negativity was accompanied by a rapid alkaline transient followed by prolonged acidosis. In media containing 3 mM Ca(2+), the alkalosis induced by ouabain averaged 0.07 +/- 0.01 unit pH. In media with no added Ca(2+) and 2 mM EGTA, the alkaline shift was not significantly different (0.09 +/- 0.02 unit pH). The alkaline transient was unaffected by inhibiting Na(+)-H(+) exchange with ethylisopropylamiloride (EIPA) or by blocking endoplasmic reticulum Ca(2+) uptake with thapsigargin or cyclopiazonic acid. Alkaline transients were also observed in Ca(2+)-free media when SD was induced by microinjecting high K(+). The late acidification accompanying ouabain-induced SD was significantly reduced in Ca(2+)-free media and in solutions containing EIPA. The ouabain-induced SD was associated with a rapid but relatively modest increase in [K(+)](o). In the presence of 3 mM external Ca(2+), the mean peak elevation of [K(+)](o) was 12 +/- 0.62 mM. In Ca(2+)-free media, the elevation of [K(+)](o) had a more gradual onset and reached a significantly larger peak value, which averaged 22 +/- 1.1 mM. The decrease in [Na(+)](o) that accompanied ouabain-induced SD was somewhat greater. The [Na(+)](o) decreased by averages of 40 +/- 7 and 33 +/- 3 mM in Ca(2+) and Ca(2+)-free media, respectively. In media containing 1.2 mM Ca(2+), ouabain-induced SD was associated with a substantial decrease in [Ca(2+)](o) that averaged 0.73 +/- 0. 07 mM. These data demonstrate that in comparison with conventional SD, ouabain-induced SD exhibits ion shifts that are qualitatively similar but quantitatively diminished. The presence of external Ca(2+) can modulate the phenomenon but is irrelevant to the generation of the SD and its accompanying alkaline pH transient. Significance of these results is discussed in reference to the propagation of SD and the generation of interstitial pH changes.

Vasorelaxant effect of harman

The in vivo cardiovascular effect and in vitro vasorelaxant effect of harman, one of harmala alkaloids isolated from Peganum harmala, were examined in this study. Harman (1–10 mg/kg, i.v.) dose-dependently produced transient hypotension and long-lasting bradycardia in pentobarbital-anesthetized rats, which were attenuated by N G-nitro- l-arginine pretreatment. In isolated rat endothelium-intact thoracic aortic rings, harman concentration dependently relaxed phenylepherine-induced contractions with an IC 50 value around 9 μM. This vasorelaxant effect was attenuated by endothelium removal or N ω-nitro- l-arginine methyl ester pretreatment, but not by tetraethylammonium or indomethacin pretreatment. In cultured rat aortic endothelial cells, harman (1–100 μM) concentration dependently increased nitric oxide (NO) release, which was dependent on the presence of external Ca 2+. Harman pretreatment (3–30 μM) also concentration dependently inhibited the contractions induced by phenylephrine, 5-hydroxytryptamine (5-HT), and KCl in endothelium-denuded aortic rings in a non-competitive manner. In addition, harman pretreatment reduced both phasic and tonic phases of phenylephrine-induced contractions. Receptor binding assays further indicated that harman ( K i values around 5–141 μM) interacted with the cardiac α 1-adrenoceptors, brain 5-HT 2 receptors, and cardiac 1,4-dihydropyridine binding site of L-type Ca 2+ channels. Therefore, the present results suggested that the vasorelaxant effect of harman was attributed to its actions on the endothelial cells to release NO and on the vascular smooth muscles to inhibit the contractions induced by the activation of receptor-linked and voltage-dependent Ca 2+ channels. The vasorelaxant effect may be involved in the hypotensive effect of harman.

Surfactant modulates calcium response of neutrophils to physiologic stimulation via cell membrane depolarization.

Pulmonary surfactant (PS) reduces inflammation in the lung by poorly understood mechanisms. We have observed that surfactant-associated proteins (SAP) insert monovalent cation channels in artificial membranes. Neutrophils are primary mediators of acute pulmonary inflammation, and their functions are activated by increases in cytosolic ionized calcium concentration ([Ca2+]) and by changes in membrane potential. We hypothesize that PS inserts SAP-dependent cation channels in neutrophils, causing membrane depolarization, altered [Ca2+] response, and depressed activation. Human neutrophils were isolated, exposed to PS+SAP (1% Survanta), PS-SAP (1% Exosurf), or buffer, and washed before activating with selected stimulants. PS+SAP reduced phorbol ester- and formyl peptide-stimulated adherence and aggregation by 38% (p < 0.05) and 54% (p < 0.02), respectively. PS+SAP also inhibited the formyl peptide-induced [Ca2+] response of neutrophils (p < 0.01), but only in the presence of external Ca2+. Further characterization of this inhibition demonstrated that PS+SAP blocked formyl peptide-induced influx of both Ca2+ and Mn2+, and that this inhibition was present during activation by other neutrophil stimulants (IL-8, immune complexes). Prior depolarization of neutrophils with gramicidin-D similarly inhibited the [Ca2+] response of neutrophils to formyl peptide, and analysis of neutrophil membrane potential by 3,3'-dipentyloxaearbocyanine iodide (diOC5(3)) fluorescence revealed that PS+SAP induced rapid neutrophil depolarization. In contrast, PS-SAP exhibited little effect on neutrophil function, [Ca2+], or membrane potential. We conclude that PS+SAP decreases neutrophil adherence and aggregation responses, blocks Ca2+ influx after physiologic stimulation, and decreases membrane potential. We speculate that these effects are caused by membrane depolarization via SAP-dependent cation channel insertion, and that all of these effects contribute to the antiinflammatory properties of PS+SAP.

Vanadate induces calcium signaling, Ca2+ release-activated Ca2+ channel activation, and gene expression in T lymphocytes and RBL-2H3 mast cells via thiol oxidation.

Using ratiometric Ca2+ imaging and patch-clamp measurement of Ca2+ channel activity, we investigated Ca2+ signaling induced by vanadium compounds in Jurkat T lymphocytes and rat basophilic leukemia cells. In the presence of external Ca2+, vanadium compounds produced sustained or oscillatory Ca2+ elevations; in nominally Ca2+-free medium, a transient Ca2+ rise was generated. Vanadate-induced Ca2+ signaling was blocked by heparin, a competitive inhibitor of the 1,4, 5-inositol trisphosphate (IP3) receptor, suggesting that Ca2+ influx is secondary to depletion of IP3-sensitive Ca2+ stores. In Jurkat T cells, vanadate also activated the Ca2+-dependent transcription factor, NF-AT. Intracellular dialysis with vanadate activated Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels with kinetics comparable to those of dialysis with IP3. Neither phosphatase inhibitors nor nonhydrolyzable nucleotide analogues modified CRAC channel activation. The action of vanadate, but not IP3, was prevented by the thiol-reducing agent DTT. In addition, the activation of CRAC channels by vanadate was mimicked by the thiol-oxidizing agent chloramine T. These results suggest that vanadate enhances Ca2+ signaling via thiol oxidation of a proximal element in the signal transduction cascade.