Effects of islet hormones on nerve-mediated and acetylcholine-evoked secretory responses in the isolated pancreas of normal and diabetic rats

This study employs the pancreas of normal and diabetic rats to investigate the relationship between the endocrine and exocrine pancreas in the control of exocrine secretion employing enzyme and immunohistochemical and physiological techniques. Acetylcholine esterase (ACh-E) positive nerves were distributed in the interacinar regions of the pancreas lying close to the exocrine cells. There was no difference between the cholinergic innervation of the pancreas in normal and diabetic rat. Insulin (INS) immunopositive cells were observed in the peripheral and central portions of the Islet of Langerhans in the pancreas of normal rat. In the diabetic animals the number of INS-positive cells were decreased. In contrast, glucagon (GLU) and somatostatin (SOM)-immunopositive cells were identified mainly in the peripheral parts of the Islets of Langerhans and their numbers increased markedly in the diabetic pancreas. Insulin alone had no significant effect on amylase secretion in the normal pancreas whereas GLU and SOM evoked small increases in amylase out compared to basal. In contrast, the islet hormones have no detectable secretory effect on the diabetic pancreas compared to control. Both electrical field stimulation (EFS) of intrinsic secretomotor nerves and exogenous application of acetylcholine (ACh) resulted in marked increases in amylase secretion. In pancreatic acini and acinar cells ACh evoked dose-dependent increases in amylase release. In normal pancreatic segments a combination of either INS or GLU with EFS or ACh resulted in marked potentiation of amylase output. In contrast, SOM inhibited the EFS-evoked amylase output but enhanced the secretory response to ACh. In pancreatic acini and acinar cells from normal rat and in pancreatic segments from diabetic rats, the islet hormones had no potentiating effect on the ACh-evoked secretory response. Similarly, in the diabetic rat the islet hormone had no effect on EFS-evoked amylase output. In fura-2 loaded pancreatic acinar cells ACh-induced a marked increase in intracellular free calcium concentration [Ca2+]i compared to basal. Either INS or GLU, but not SOM, elicited a small increase in [Ca2+]i. Combining either INS or GLU with ACh resulted in a potentiation of [Ca2+]i compared with ACh alone. In contrast, SOM had no significant effect on the ACh-induced [Ca2+]i compared to the response obtained with ACh alone. In pancreatic acinar cells of diabetic rat ACh-elicited similar magnitude of [Ca2+]i compared to acinar cells of normal rat. However, when the islet hormones were combined with ACh there was no enhancement of [Ca2+]i compared to ACh alone. The results indicate that the potentiation of either EFS or ACh-evoked secretory responses by the islet hormones seem to occur only in pancreatic segments which have intact viable Islets of Langerhans and not in either acini and acinar cells or from the pancreas of diabetic rat. Moreover, it is apparent that cellular Ca2+ is involved with the interaction of ACh with either INS or GLU.     

Absence of acetylcholine- and ionomycin-activated Cl- currents in submandibular cells of early postnatal rats

Using the whole-cell patch-clamp technique, we investigated developmental changes in the expression of an acetylcholine- (Ach-) activated Cl- conductance in rat submandibular acinar cells. ACh induced an oscillatory inward current in cells isolated from animals older than 5 weeks, but not in animals less than 2-3 weeks of age. The current/voltage (I/V) relationship of the ACh-induced current was that of an outward rectifier, and the current was inhibited by intracellular BAPTA, a Ca2+ buffer, indicating the current was Ca2+ activated. The ACh-induced current was also blocked in the presence of DPC and SITS, two Cl- current inhibitors in other tissues. Ionomycin mimicked the effect of ACh but in a nonoscillatory fashion. The appearance of the ionomycin-induced currents was also age related, as the current was not observed to occur in animals less than 2-3 weeks old. Since both ACh and ionomycin significantly increase cytosolic [Ca2+] in the acinar cells of young animals, the correlation between the age dependence of the ACh-activated Cl- current and the ionomycin-activated Cl- current responses suggests that the lack of responsiveness observed in the young animals is due to the absence of Ca2+-activated Cl- channels, rather than to a deficiency of a cellular mediator.     

Effects of 8-bromo-cyclic GMP on membrane potential of single swine tracheal smooth muscle cells

Cyclic GMP relaxes swine tracheal smooth muscle. Relaxation occurs because of decreases in intracellular calcium concentration ([Ca++]i) that are thought to occur through hyperpolarization which inhibits calcium influx. Activation of K+ channels has been suggested as the underlying mechanism for the hyperpolarization. In the present study, the effects of 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP, a membrane-permeable analog of cyclic GMP) on acetylcholine (ACh)-induced increases in [Ca++]i were examined by laser scanning confocal microscopy in fluo 3-loaded single cells. Membrane potential and currents were measured by the perforated-configuration of patch-clamp method, 8-Bromo-cGMP (1 microM-0.1 mM) inhibited 0.1 microM ACh-induced oscillations in [Ca++]i in a concentration-dependent manner. Spontaneous changes in membrane potential were observed by the patch-clamp method. Acetylcholine (0.03 microM) did not affect the time-averaged mean potential. The spontaneous changes in membrane potential were reduced and the cells were depolarized by 0.1 microM ACh and to a greater degree by 1 microM ACh. This result is consistent with previous observations of ACh-induced depolarization in intact tissue. The application of 0.1 mM 8-Br-cGMP had no significant effects on spontaneous changes in membrane potential and did not induce changes in membrane potential in cells treated with 0.1 microM ACh. In voltage-clamped cells, ACh (0.1 microM) induced oscillations in calcium-activated K+ currents. 8-Bromo-cGMP (0.1 mM) inhibited these ACh-induced oscillations in currents, but had no significant effects on spontaneous changes in membrane current in unstimulated cells. These data indicate that 8-Br-cGMP inhibits ACh-induced increases in [Ca++]i by mechanisms other than regulation of membrane potential.     

Activation of Ca(2+)-dependent K+ current by acetylcholine and histamine in a human gastric epithelial cell line

The effects of acetylcholine (ACh) and histamine (His) on the membrane potential and current were examined in JR-1 cells, a mucin-producing epithelial cell line derived from human gastric signet ring cell carcinoma. The tight-seal, whole cell clamp technique was used. The resting membrane potential, the input resistance, and the capacitance of the cells were approximately -12 mV, 1.4 G ohms, and 50 pF, respectively. Under the voltage-clamp condition, no voltage-dependent currents were evoked. ACh or His added to the bathing solution hyperpolarized the membrane by activating a time- and voltage-independent K+ current. The ACh-induced hyperpolarization and K+ current persisted, while the His response desensitized quickly (< 1 min). These effects of ACh and His were mediated predominantly by m3-muscarinic and H1-His receptors, respectively. The K+ current induced by ACh and His was inhibited by charybdotoxin, suggesting that it is a Ca(2+)-activated K+ channel current (IK.Ca). The measurement of intracellular Ca2+ ([Ca2+]i) using Indo-1 revealed that both agents increased [Ca2+]i with similar time courses as they increased IK.Ca. When EGTA in the pipette solution was increased from 0.15 to 10 mM, the induction of IK.Ca by ACh and His was abolished. Thus, both ACh and His activate IK.Ca by increasing [Ca2+]i in JR-1 cells. In the Ca(2+)-free bathing solution (0.15 mM EGTA in the pipette), ACh evoked IK.Ca transiently. Addition of Ca2+ (1.8 mM) to the bath immediately restored the sustained IK.Ca. These results suggest that the ACh response is due to at least two different mechanisms; i.e., the Ca2+ release-related initial transient activation and the Ca2+ influx-related sustained activation of IK.Ca. Probably because of desensitization, the Ca2+ influx-related component of the His response could not be identified. Intracellularly applied inositol 1,4,5-trisphosphate (IP3), with and without inositol 1,3,4,5-tetrakisphosphate (IP4), mimicked the ACh response. IP4 alone did not affect the membrane current. Under the steady effect of IP3 or IP3 plus IP4, neither ACh nor His further evoked IK.Ca. Intracellular application of heparin or of the monoclonal antibody against the IP3 receptor, mAb18A10, inhibited the ACh and His responses in a concentration-dependent fashion. Neomycin, a phospholipase C (PLC) inhibitor, also inhibited the agonist-induced response in a concentration-dependent fashion. Although neither pertussis toxin (PTX) nor N-ethylmaleimide affected the ACh or His activation of IK,Ca, GDP beta S attenuated and GTP gamma S enhanced the agonist response.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oscillatory Cl- current induced by angiotensin II in rat pulmonary arterial myocytes: Ca2+ dependence and physiological implication

We have previously reported that angiotensin II (ANG II) induces oscillations in the cytoplasmic calcium concentration ([Ca2+]i) of pulmonary vascular myocytes. The present work was undertaken to investigate the effect of ANG II in comparison with ATP and caffeine on membrane currents and to explore the relation between these membrane currents and [Ca2+]i. In cells clamped at -60 mV, ANG II (10 microM) or ATP (100 microM) induced an oscillatory inward current. Caffeine (5 mM) induced only one transient inward current. In control conditions, the reversal potential (Erev) of these currents was close to the equilibrium potential for Cl- ions (Ecl = -2.1 mV) and was shifted towards more positive values in low-Cl- solutions. Niflumic acid (10-50 microM) and DIDS (0.25-1 mM) inhibited this inward current. Combined recordings of membrane current and [Ca2+]i by indo-1 microspectrofluorimetry revealed that ANG II- and ATP-induced currents occurred simultaneously with oscillations in [Ca2+]i whereas the caffeine-induced current was accompanied by only one transient increase in [Ca2+]i. Niflumic acid (25 microM) had no effect on agonist-induced [Ca2+]i responses, whereas thapsigargin (1 microM) abolished both membrane current and the [Ca2+]i response. Heparin (5 mg/ml in the pipette solution) inhibited both [Ca2+]i responses and membrane currents induced by ANG II and ATP, but not by caffeine. In pulmonary arterial strips, ANG II-induced contraction was inhibited by niflumic acid (25 microM) or nifedipine (1 microM) to the same extent and the two substances did not have an additive effect. This study demonstrates that, in pulmonary vascular smooth muscle, ANG II, as well as ATP, activate an oscillatory calcium dependent chloride current which is triggered by cyclic increases in [Ca2+]i and that both oscillatory phenomena are primarily IP3-mediated. It is suggested that ANG II-induced oscillatory chloride current could depolarise the cell membrane leading to activation of voltage-operated Ca2+ channels. The resulting Ca2+ influx contributes to the component of ANG II-induced contraction that is equally sensitive to chloride or calcium channel blockade.     

Effect of caffeine on mucus secretion and agonist-dependent Ca2+ mobilization in human gastric mucus secreting cells

Caffeine is known to stimulate gastric acid secretion, but, the effects of caffeine on gastric mucus secretion have not been clarified. To elucidate the action of caffeine on gastric mucin-producing cells and its underlying mechanism, the effects of caffeine on mucus glycoprotein secretion and agonist-induced [Ca2+]i mobilization were examined in human gastric mucin secreting cells (JR-I cells). The measurement of [Ca2+]i using Indo-1 and the whole cell voltage clamp technique were applied. Mucus glycoprotein secretion was assessed by release of [3H]glucosamine. Caffeine by itself failed to increase [Ca2+]i and affect membrane currents, while it dose-dependently inhibited agonist (acetylcholine (ACh) or histamine)-induced [Ca2+]i rise, resulting in inhibiting activation of Ca2+-dependent K+ current (I(K.Ca)) evoked by agonists. The effect of caffeine was reversible, and the half maximal inhibitory concentration was about 0.5 mM. But, caffeine did not suppress [Ca2+]i rise and activation of I(K.Ca) induced by A23187 or inositol trisphosphate (IP3). Theophylline or 3-isobutyl-1-methyl-xanthine (IBMX) did not mimic the effect of caffeine. Caffeine failed to stimulate mucus secretion, while it significantly decreased ACh-induced mucus secretion. These results indicate that caffeine selectively inhibits agonist-mediated [Ca2+]i rise in human gastric epithelial cells, probably through the blockade of receptor-IP3 signaling pathway, which may affect the mucin secretion.     

Characterization and localization of P2 receptors in rat submandibular gland acinar and duct cells

[Ca2+]i and the Cl- current were measured in isolated submandibular gland acinar and duct cells to characterize and localize the purinergic receptors expressed in these cells. In both cell types 2'-3'-benzoylbenzoyl (Bz)-ATP and ATP increased [Ca2+]i mainly by activation of Ca2+ influx. UTP had only minimal effect on [Ca2+]i at concentrations between 0.1 and 1 mM. However, a whole cell current recording showed that all nucleotides effectively activated Cl- currents. Inhibition of signal transduction through G proteins by guanyl-5'-beta-thiophosphate revealed that the effect of ATP on Cl- current was mediated in part by activation of a G protein-coupled and in part by a G protein-independent receptor. BzATP activated exclusively the G protein-independent portion, whereas UTP activated only the G protein-dependent portion of the Cl- current. Measurement of [Ca2+]i in the microperfused duct showed that ATP stimulated a [Ca2+]i increase when applied to the luminal or the basolateral sides. BzATP increased [Ca2+]i only when applied to the luminal side, whereas UTP at 100 microM increased -Ca2+-i only when applied to the basolateral side. The combined results suggest that duct and possibly acinar cells express P2z receptors in the luminal and P2u receptors in the basolateral membrane.     

Imaging of intracellular calcium during desensitization of nicotinic acetylcholine receptors of rat chromaffin cells

1. The possible role of intracellular Ca2+ levels ([Ca2+]i) in desensitization of nicotinic acetylcholine receptors (AChRs) was investigated in rat cultured chromaffin cells by use of combined whole-cell patch clamping and confocal laser scanning microscopy with the fluorescent dye fluo-3. 2. On cells held at -70 mV, pressure-application of nicotine elicited inward currents with associated [Ca2+]i rises mainly due to influx through nicotinic AChRs. These responses were blocked by (+)-tubocurarine (10 microM) but were insensitive to alpha-bungarotoxin (1 microM) or Cd2+ (0.1 mM). 3. Pressure applications of 1 mM nicotine for 2 s (conditioning pulse) evoked inward currents which faded biexponentially to a steady state level due to receptor desensitization and were accompanied by a sustained increase in [Ca2+]i. Inward currents evoked by subsequent application of brief test pulses of nicotine were depressed but recovered with a time course reciprocal to the decay of the [Ca2+]i transient induced by the conditioning pulse. 4. Omission of intracellular Ca2+ chelators or use of high extracellular Ca2+ solution (10 mM) lengthened recovery of nicotinic AChRs from desensitization while adding BAPTA or EGTA intracellularly had the opposite effect. When the patch pipette contained fluo-3 or no chelators, after establishing whole cell conditions the rate of recovery became progressively longer presumably due to dialysis of endogenous Ca2+ buffers. None of these manipulations of external or internal Ca2+ had any effect on onset or steady state level of desensitization. 5. High spatial resolution imaging of [Ca2+]i in intact cells (in the presence of 0.1 mM Cd2+) showed that its level in the immediate submembrane area decayed at the same rate as in the rest of the cell, indicating that Ca2+ was in a strategic location to modulate (directly or indirectly) AChR desensitization. 6. The present data suggest that desensitized nicotinic AChRs are stabilized in their conformation by raised [Ca2+]i and that this phenomenon retards their recovery to full activity.     

Dendritic cell development: multiple pathways to nature''s adjuvants

The endothelin (ET) isoforms ET-1, ET-2 and ET-3 applied at 100 nM triggered a transient increase in [Ca2+]i in Bergmann glial cells in cerebellar slices acutely isolated from 20-25 day-old mice. The intracellular calcium concentration ([Ca2+]i) was monitored using Fura-2-based [Ca2+]i microfluorimetry. The ET-triggered [Ca2+]i transients were mimicked by ETB receptor agonist BQ-3020 and were inhibited by ETB receptor antagonist BQ-788. ET elevated [Ca2+]i in Ca(2+)-free extracellular solution and the ET-triggered [Ca2+]i elevation was blocked by 500 nM thapsigargin indicating that the [Ca2+]i was released from InsP3-sensitive intracellular pools. The ET-triggered [Ca2+]i increase in Ca(2+)-free solution was shorter in duration. Restoration of normal extracellular [Ca2+] briefly after the ET application induced a second [Ca2+]i increase indicating the presence of a secondary Ca2+ influx which prolongs the Ca2+ signal. Pre-application of 100 microM ATP or 10 microM noradrenaline blocked the ET response suggesting the involvement of a common Ca2+ depot. The expression of ETB receptor mRNAs in Bergmann glial cells was revealed by single-cell RT-PCR. The mRNA was also found in Purkinje neurones, but no Ca2+ signalling was triggered by ET. We conclude that Bergmann glial cells are endowed with functional ETB receptors which induce the generation of intracellular [Ca2+]i signals by activation of Ca2+ release from InsP3-sensitive intracellular stores followed by a secondary Ca2+ influx.     

Blockade by ifenprodil of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones: comparison with N-methyl-D-aspartate receptor antagonist actions

1. The block by ifenprodil of voltage-activated Ca2+ channels was investigated in intracellular free calcium concentration ([Ca2+]i) evoked by 50 mM K+ (high-[K+]o) in Fura-2-loaded rat hippocampal pyramidal neurones in culture and on currents carried by Ba2+ ions (IBa) through Ca2+ channels in mouse cultured hippocampal neurones under whole-cell voltage-clamp. The effects of ifenprodil on voltage-activated Ca2+ channels were compared with its antagonist actions on N-methyl-D-aspartate- (NMDA) evoked responses in the same neuronal preparations. 2. Rises in [Ca2+]i evoked by transient exposure to high-[K+]o in our preparation of rat cultured hippocampal pyramidal neurones are mediated predominantly by Ca2+ flux through nifedipine-sensitive Ca2+ channels, with smaller contributions from nifedipine-resistant, omega-conotoxin GVIA-sensitive Ca2+ channels and Ca2+ channels sensitive to crude funnel-web spider venom (Church et al., 1994). Ifenprodil (0.1-200 microM) reversibly attenuated high-[K+]o-evoked rises in [Ca2+]i with an IC50 value of 17 +/- 3 microM, compared with an IC50 value of 0.7 +/- 0.1 microM for the reduction of rises in [Ca2+]i evoked by 20 microM NMDA. Tested in the presence of nifedipine 10 microM, ifenprodil (1-50 microM) produced a concentration-dependent reduction of the dihydropyridine-resistant high-[K+]o-evoked rise in [Ca2+]i with an IC50 value of 13 +/- 4 microM. The results suggest that ifenprodil blocks Ca2+ flux through multiple subtypes of high voltage-activated Ca2+ channels. 3. Application of the polyamine, spermine (0.25-5 mM), produced a concentration-dependent reduction of rises in [Ca2+]i evoked by high-[K+]o. The antagonist effects of ifenprodil 20 micro M on high-[K+]0-evoked rises in [Ca2+]. were attenuated by spermine 0.25 mM but not by putrescine 1 or 5 mM. In contrast,spermine 0.1 mM increased rises in [Ca2+]i evoked by NMDA and enhanced the ifenprodil (5 micro M) block of NMDA-evoked rises in [Ca2+]i.4. Similar results were obtained in mouse cultured hippocampal pyramidal neurones under whole-cell voltage-clamp. Ifenprodil attenuated both the peak and delayed whole-cell IB. with an IC% value of 18 +/- 2 micro M, whilst it attenuated steady-state NMDA-evoked currents with an IC50 of 0.8 +/- 0.2 micro M. Block of IBa by ifenprodil 10 JaM was rapid in onset, fully reversible and occurred without change in thecurrent-voltage characteristics of Ba. The ifenprodil block of IBa was enhanced on membrane depolarization and was weakly dependent on the frequency of current activation. Spermine 0.1 mM potentiated control NMDA-evoked currents but attenuated IB,. In agreement with the microspectrofluorimetric studies, co-application of spermine produced a small enhancement of the inhibitory effect of ifenprodil 10 micro M on NMDA-evoked responses whereas the reduction of I4 by ifenprodil 10 micro M in the presence of spermine was less than expected if the inhibitory effects of ifenprodil and spermine on IBa were simply additive.5. The results indicate that ifenprodil blocks high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones. Although the Ca2+ channel blocking actions of ifenprodil are observed at higher concentrations than those associated with NMDA antagonist activity, Ca2+ channel blockade may contribute, at least in part, to the established neuroprotective and anticonvulsant properties of the compound.     

From angels to handmaidens: changing constructions of nursing''s public image in post-war Britain

The molecular and ionic mechanisms responsible for the regulation of mucus exocytosis in human airway gland cells remain poorly defined. To determine whether dynamic changes of intracellular free Ca2+ concentration [Ca2+]i can promote different exocytotic responses, we monitored dynamic changes in [Ca2+]i and secretory granule (SG) exocytosis in individual human tracheal submucosal serous gland (HTG) cells. These changes were in response to exposure of the cells to three different secretagogues associated with airway inflammation and disease: human neutrophil elastase (HNE), histamine, and ATP. Dynamic changes in [Ca2+]i from single cells were determined with Indo-1/AM using quantitative UV laser microspectrofluorometry. The rate of SG exocytosis was measured in single cells by fluorescence videomicroscopy of SG degranulation and by the ELISA method. Exposure of HTG cells to a low concentration of HNE (1.0 microM) caused a high rate of SG exocytosis (52% decrease in the initial quinacrine fluorescence) during the first 8-min stimulation period compared with that observed following exposure of the cells to 100 microM histamine (10% decrease) or 100 microM ATP (6% decrease). In contrast to a rapid and transient rise in [Ca2+]i induced by histamine (1.0-100 microM) and ATP (10-100 microM), HNE (0.01-1 microM) generated asynchronous oscillations in [Ca2+]i over the first 8-min period. Depletion of internal Ca2+ stores with thapsigargin (500 nM) induced a significant reduction (P < 0.01) in the observed increases in [Ca2+]i upon addition of each of the secretagogues, but did not greatly affect the SG exocytotic responses. Interestingly, the removal of extracellular Ca2+ (+5 mM EGTA) significantly reduced (P < 0.01) both the [Ca2+]i increases and the rate of SG exocytosis following exposure to the secretagogues. We also demonstrate that the influx of extracellular Ca2+ and [Ca2+]i oscillations rather than the absolute level of [Ca2+]i regulate the rapid onset and extent of exocytotic responses to HNE in airway gland cells. Taken together, these results provide strong evidence that [Ca2+]i is a critical intracellular messenger in the regulation of exocytosis process in human airway gland cells.     

Isoproterenol evokes extracellular Ca2+ spikes due to secretory events in salivary gland cells

Secretory cells should in principle export substantial amounts of calcium via exocytosis since Ca2+ is sequestered in secretory granules. Based on a new technique for measurements of the extracellular calcium concentration in the vicinity of the cell membrane and on the droplet technique, we have monitored the rate of calcium extrusion from salivary gland acinar cells. Isoproterenol (ISP), a beta-adrenergic agonist and powerful secretogogue, evoked no change in the cytosolic free Ca2+ concentration ([Ca2+]i) but induced vigorous extracellular Ca2+ concentration ([Ca2+]i) spiking. The absence of [Ca2+]i elevation and the pulsatile nature of the changes in [Ca2+]i indicate that these spikes are most likely due to calcium release from secretory granules. The cholinergic agonist acetylcholine (ACh), which induces moderate secretion, evoked a marked rise in [Ca2+]i and a smooth rise in [Ca2+]i, most likely induced by plasma membrane calcium pumps, on which shortlasting [Ca2+]i spikes were superimposed. The rate of ISP-induced calcium efflux was very substantial. The calculated calcium loss during the first 100 s of supramaximal stimulation corresponded to a reduction of the total cellular calcium concentration of approximately 0.4 mM. We conclude that in salivary glands, calcium release via exocytosis is one of the main mechanisms extruding calcium from cells to the extracellular milieu.     

Homologous and heterologous desensitisation of somatostatin-induced increases in intracellular Ca2+ and inositol 1,4,5-trisphosphate in CHO-K1 cells expressing human recombinant somatostatin sst5 receptors

The mechanisms responsible for somatostatin (SRIF)-induced increases in intracellular Ca2+ concentration ([Ca2+]i) and subsequent desensitisation were studied in CHO-K1 cells expressing human sst5 receptors (CHOsst5 cells). To study the nature of the desensitisation, interactions with uridine triphosphate (UTP) were examined. SRIF (pEC50 7.10) and UTP (pEC50) 5.14) caused concentration-dependent increases in [Ca2+]i but the SRIF maximum was about 40% of that to UTP. SRIF-, but not UTP-, induced increases in [Ca2+]i were transient and abolished by pertussis toxin. SRIF and UTP caused sustained increases in Ins(1,4,5)P3 but the SRIF maximum was about 30% of that to UTP. Removal of [Ca2+]e attenuated the SRIF-induced peak rise in [Ca2+]i but had no effect on the peak increases in Ins(1,4,5)P3. UTP-induced increases in [Ca2+]i and Ins(1,4,5)P3 were attenuated in the absence of [Ca2+]e. Following pre-exposure to SRIF (1 microM) or UTP (100 microM) for 5 min, subsequent SRIF responses were desensitised. Similar results were obtained in the absence of [Ca2+]e. Pre-exposure to SRIF had no effect on subsequent responses to UTP but in the absence of [Ca2+]e, responses to UTP were attenuated. The results suggest that SRIF but not UTP-induced increases in [Ca2+]i in CHOsst5 cells are mediated by pertussis toxin sensitive G proteins and are caused by an entry of extracellular Ca2+ and release from an Ins(1,4,5)P3 sensitive Ca2+ store. Homologous or heterologous desensitisation of agonist-induced increases in [Ca2+]i could be demonstrated in the presence or absence of extracellular Ca2+ respectively, and the latter appeared to involve depletion of a common intracellular Ca2+ store.     

Depletion of ryanodine-sensitive Ca2+ store activates Ca2+ entry in rat submandibular gland acinar cells

The existence of ryanodine-sensitive Ca2+ stores and their role in the Ca2+ entry mechanism were examined in the rat submandibular gland acinar cells, using the microfluorimetry of intracellular Ca2+ concentration ([Ca2+]i). In the presence of thapsigargin, a Ca(2+)-ATPase inhibitor of inositol (1, 4, 5) triphosphate (InsP3)-sensitive Ca2+ stores, caffeine caused an increase in [Ca2+]i, which was inhibited by treatment with ryanodine (a ligand to the Ca(2+)-induced Ca2+ release channels). In the cells treated with ryanodine, 1 mM Ca2+ addition to a Ca(2+)-free solution caused a marked increase in [Ca2+]i, which was eliminated by application of Ni2+ or SK & F 96365, suggesting a Ca2+ entry triggered by ryanodine. The maximal change in the net increase in [Ca2+]i caused by the ryanodine-coupled Ca2+ entry, was 104.0 +/- 16.0 nM, which intense was caused by 10 microM ryanodine. Emptying the InsP3-sensitive stores by treatment with thapsigargin also caused Ca2+ entry, which maximally changed [Ca2+]i by 349.6 +/- 15.1 nM. Ten mumol/liter ryanodine was confirmed to cause a release of 45Ca2+ from the parotidic microsomal fraction enriched in endopalsmic reticulum. We propose that ryanodine-sensitive Ca2+ stores are present in rat submandibular gland acinar cells. We further propose that release of Ca2+ from the ryanodine-sensitive stores, which means eventually depletion of the ryanodine-sensitive Ca2+ stores, can activate the Ca2+ entry. The ability for Ca2+ entry coupled with the ryanodine-sensitive Ca2+ stores seems to be about 30% of the ability for Ca2+ entry coupled with the thapsigargin-sensitive Ca2+ stores.     

Hydrogen peroxide induces intracellular calcium overload by activation of a non-selective cation channel in an insulin-secreting cell line

Fura-2 fluorescence was used to investigate the effects of H2O2 on [Ca2+]i in the insulin-secreting cell line CRI-G1. H2O2 (1-10 mM) caused a biphasic increase in free [Ca2+]i, an initial rise observed within 3 min and a second, much larger rise following a 30-min exposure. Extracellular calcium removal blocked the late, but not the initial, rise in [Ca2+]i. Thapsigargin did not affect either response to H2O2, but activated capacitive calcium entry, an action abolished by 10 microM La3+. Simultaneous recordings of membrane potential and [Ca2+]i demonstrated the same biphasic [Ca2+]i response to H2O2 and showed that the late increase in [Ca2+]i coincided temporally with cell membrane potential collapse. Buffering Ca2+i to low nanomolar levels prevented both phases of increased [Ca2+]i and the H2O2-induced depolarization. The H2O2-induced late rise in [Ca2+]i was prevented by extracellular application of 100 microM La3+. La3+ (100 microM) inhibited the H2O2-induced cation current and NAD-activated cation (NSNAD) channel activity in these cells. H2O2 increased the NAD/NADH ratio in intact CRI-G1 cells, consistent with increased cellular [NAD]. These data suggest that H2O2 increases [NAD], which, coupled with increased [Ca2+]i, activates NSNAD channels, causing unregulated Ca2+ entry and consequent cell death.     

Ca2+ mobilization in bovine corneal endothelial cells by P2 purinergic receptors

PURPOSE: To characterize Ca2+ mobilization by P2 receptors in the bovine corneal endothelial cells (BCEC). METHODS: Changes in intracellular Ca2+ ([Ca2+]i) were measured by fluorescence imaging of cultured and fresh BCEC cells loaded with the Ca2+-sensitive dye Fura-PE3. Relative rates of Ca2+ influx were measured employing Mn2+ as a surrogate for Ca2+. RESULTS: Exposure of cultured cells to uridine 5'-triphosphate (UTP), 2-methyl-thio ATP (msATP) and ATP caused biphasic changes in [Ca2+]i consisting of a peak followed by a plateau phase. Based on the peak responses to 100 microM agonist, the magnitude of UTP responses were similar to that of ATP but greater than that of msATP or ADP. UTP and msATP stimulated Mn2+ influx following [Ca2+]i peak similar to that observed in response to cyclopiazonic acid (CPA), an inhibitor of ER Ca2+-ATPase. Under Ca2+-free conditions, peak responses were similar to those in the presence of external Ca2+, but reduced when the cells were pre-exposed to CPA. Reactive Blue-2 (RB2), inhibited msATP responses by 60.4 +/- 18.8% but UTP responses by only 10.6 +/- 9.5%. Repeated exposures to UTP or msATP reduced [Ca2+]i mobilization indicating homologous desensitization. Response to UTP was not affected by a prior exposure to msATP. However, response to msATP was reduced by a prior exposure to UTP indicating mixed heterologous desensitization. Fresh cells responded to UTP (50 microM) with temporal characteristics of [Ca2+]i mobilization similar to that of cultured cells. CONCLUSION: BCEC express P2 receptors belonging to the P2Y subfamily. The emptying of the IP3-sensitive stores, leading to the initial peak in [Ca2+]i response, subsequently caused capacitative Ca2+ influx leading to the onset of the plateau phase. A significant homologous desensitization to UTP and msATP, selective heterologous desensitization between UTP and msATP, and selective inhibition by RB2 indicate the coexistence of multiple P2Y receptors.     

Calcium entry activated by store depletion in human umbilical vein endothelial cells

We have used the patch clamp technique combined with simultaneous measurement of intracellular Ca2+ to record ionic currents activated by depletion of intracellular Ca(2+)-stores in endothelial cells from human umbilical veins. Two protocols were used to release Ca2+ from intracellular stores, i.e. loading of the cells via the patch pipette with Ins(1,4,5)P3, and extracellular application of thapsigargin. Ins(1,4,5)P3 (10 microM) evoked a transient increase in [Ca2+]i in cells exposed to Ca(2+)-free extracellular solutions. A subsequent reapplication of extracellular Ca2+ induced an elevation of [Ca2+]i. These changes in [Ca2+]i were very reproducible. The concomitant membrane currents were neither correlated in time nor in size with the changes in [Ca2+]i. Similar changes in [Ca2+]i and membrane currents were observed if the Ca(2+)-stores were depleted with thapsigargin. Activation of these currents was prevented and holding currents at -40 mV were small if store depletion was induced in the presence of 50 microM NPPB. This identifies the large currents, which are activated as a consequence of store-depletion, as mechanically activated Cl- currents, which have been described previously [1,2]. Loading the cells with Ins(1,4,5)P3 together with 10 mM BAPTA induced only a very short lasting Ca2+ transient, which was not accompanied by activation of a detectable current, even in a 10 mM Ca(2+)-containing extracellular solution. Also thapsigargin does not activate any membrane current if the pipette solution contains 10 mM BAPTA (ruptured patches). The contribution of Ca(2+)-influx to the membrane current during reapplication of 10 mM extracellular calcium to thapsigargin-pretreated cells was estimated from the first time derivative of the corresponding Ca2+ transients at different holding potentials. These current values showed strong inward rectification, with a maximal amplitude of 1.0 +/- 0.3 pA at -80 mV (n = 8; membrane capacitance 59 +/- 9 pF).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cannabinoid receptor agonists protect cultured rat hippocampal neurons from excitotoxicity

Cannabinoid receptor agonists act presynaptically to inhibit the release of glutamate. Because other drugs with this action are known to reduce excitotoxicity, we tested several cannabimimetics in a model of synaptically mediated neuronal death. Reduction of the extracellular Mg2+ concentration to 0.1 mM evoked a repetitive pattern of intracellular Ca2+ concentration ([Ca2+]i) spiking that, when maintained for 24 hr, resulted in significant neuronal death. The [Ca2+]i spiking and cell death in this model result from excessive activation of N-methyl-D-aspartate receptors, as indicated by the inhibition of both [Ca2+]i spiking and neuronal death by the N-methyl-D-aspartate receptor antagonist CGS19755 (10 microM). The cannabimimetic drug Win55212-2 (100 nM) completely blocked [Ca2+]i spiking and prevented neuronal death induced by low extracellular Mg2+ concentrations. These effects on [Ca2+]i spiking and viability were stereoselective and were prevented by the CB1 receptor antagonist SR141716 (100 nM). The partial agonist CP55940 (100 nM) also afforded significant protection from excitotoxicity. Cannabimimetic drugs did not protect cells from the direct application of glutamate (30 microM). These data suggest that cannabimimetic drugs may slow the progression of neurodegenerative diseases.     

Sources of Ca2+ in relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery

1. The aim of the present study was to identify the sources of Ca2+ contributing to acetylcholine (ACh)-induced release of endothelium-derived hyperpolarizing factor (EDHF) from endothelial cells of rat mesenteric artery and to assess the pathway involved. The changes in membrane potentials of smooth muscles by ACh measured with the microelectrode technique were evaluated as a marker for EDHF release. 2. ACh elicited membrane hyperpolarization of smooth muscle cells in an endothelium-dependent manner. The hyperpolarizing response was not affected by treatment with 10 microM indomethacin, 300 microM NG-nitro-L-arginine or 10 microM oxyhaemoglobin, thereby indicating that the hyperpolarization is not mediated by prostanoids or nitric oxide but is presumably by EDHF. 3. In the presence of extracellular Ca2+, 1 microM ACh generated a hyperpolarization composed of the transient and sustained components. By contrast, in Ca(2+)-free medium, ACh produced only transient hyperpolarization. 4. Pretreatment with 100 nM thapsigargin and 3 microM cyclopiazonic acid, endoplasmic reticulum Ca(2+)-ATPase inhibitors, completely abolished ACh-induced hyperpolarization. Pretreatment with 20 mM caffeine also markedly attenuated ACh-induced hyperpolarization. However, the overall pattern and peak amplitude of hyperpolarization were unaffected by pretreatment with 1 microM ryanodine. 5. In the presence of 5 mM Ni2+ or 3 mM Mn2+, the hyperpolarizing response to ACh was transient, and the sustained component of hyperpolarization was not observed. On the other hand, 1 microM nifedipine had no effect on ACh-induced hyperpolarization. 6. ACh-induced hyperpolarization was nearly completely eliminated by 500 nM U-73122 or 200 microM 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate, inhibitors of phospholipase C, but was unchanged by 500 nM U-73343, an inactive form of U-73122. Pretreatment with 20 nM staurosporine, an inhibitor of protein kinase C, did not modify ACh-induced hyperpolarization. 7. These results indicate that the ACh-induced release of EDHF from endothelial cells of rat mesenteric artery is possibly initiated by Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pool as a consequence of stimulation of phospholipid hydrolysis due to phospholipase C activation, and maintained by Ca2+ influx via a Ni(2+)- and Mn(2+)-sensitive pathway distinct from L-type Ca2+ channels. The Ca(2+)-influx mechanism seems to be activated following IP3-induced depletion of the pool.