Characteristics of a store-operated calcium-permeable channel: sarcoendoplasmic reticulum calcium pump function controls channel gating

We examined the single channel properties and regulation of store-operated calcium channels (SOCC). In human submandibular gland cells, carbachol (CCh) induced flickery channel activity while thapsigargin (Tg) induced burst-like activity, with relatively lower open probability (NP(o)) and longer mean open time. Tg- and CCh-activated channels were permeable to Na(+) and Ba(2+), but not to NMDG, in the absence of Ca(2+). The channels exhibited similar Ca(2+), Na(+), and Ba(2+) conductances and were inhibited by 2-aminoethoxydiphenylborate, xestospongin C, Gd(3+), and La(3+). CCh stimulated flickery activity changed to burst-like activity by (i) addition of Tg, (ii) using Na(+) instead of Ca(2+), (iii) using Ca(2+)-free bath solution, or (iv) buffering [Ca(2+)](i) with BAPTA-AM. Buffering [Ca(2+)](i) induced a 2-fold increase in NP(o) of Tg-stimulated SOCC. Reducing free [Ca(2+)] in the endoplasmic reticulum with the divalent cation chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), induced burst-like channel activity similar to that seen with CCh + Tg. Thus, SOCC is activated by stimulation of muscarinic receptors, inhibition of the sarcoendoplasmic Ca(2+) pump, and lowering [Ca(2+)] in the internal store. Importantly, SOCC activity depends on [Ca(2+)](i) and the free [Ca(2+)] in the internal store. These novel findings reveal that SERCA plays a major role in the gating of SOCC by (i) refilling the internal Ca(2+) store(s) and (ii) decreasing the [Ca(2+)](i)-dependent inhibition.     

Effect of hydrogen peroxide and superoxide anions on cytosolic Ca2+: comparison of endothelial cells from large-sized and small-sized arteries

We compared the Ca(2+) responses to reactive oxygen species (ROS) between mouse endothelial cells derived from large-sized arteries, aortas (aortic ECs), and small-sized arteries, mesenteric arteries (MAECs). Application of hydrogen peroxide (H(2)O(2)) caused an increase in cytosolic Ca(2+) levels ([Ca(2+)](i)) in both cell types. The [Ca(2+)](i) rises diminished in the presence of U73122, a phospholipase C inhibitor, or Xestospongin C (XeC), an inhibitor for inositol-1,4,5-trisphosphate (IP(3)) receptors. Removal of Ca(2+) from the bath also decreased the [Ca(2+)](i) rises in response to H(2)O(2). In addition, treatment of endothelial cells with H(2)O(2) reduced the [Ca(2+)](i) responses to subsequent challenge of ATP. The decreased [Ca(2+)](i) responses to ATP were resulted from a pre-depletion of intracellular Ca(2+) stores by H(2)O(2). Interestingly, we also found that Ca(2+) store depletion was more sensitive to H(2)O(2) treatment in endothelial cells of mesenteric arteries than those of aortas. Hypoxanthine-xanthine oxidase (HX-XO) was also found to induce [Ca(2+)](i) rises in both types of endothelial cells, the effect of which was mediated by superoxide anions and H(2)O(2) but not by hydroxyl radical. H(2)O(2) contribution in HX-XO-induced [Ca(2+)](i) rises were more significant in endothelial cells from mesenteric arteries than those from aortas. In summary, H(2)O(2) could induce store Ca(2+) release via phospholipase C-IP(3) pathway in endothelial cells. Resultant emptying of intracellular Ca(2+) stores contributed to the reduced [Ca(2+)](i) responses to subsequent ATP challenge. The [Ca(2+)](i) responses were more sensitive to H(2)O(2) in endothelial cells of small-sized arteries than those of large-sized arteries.     

Role of TRPM2 in H(2)O(2)-Induced Cell Apoptosis in Endothelial Cells

Melastatin-like transient receptor potential channel 2 (TRPM2) is an oxidant-sensitive and cationic non-selective channel that is expressed in mammalian vascular endothelium. Here we investigated the functional role of TRPM2 channels in hydrogen peroxide (H(2)O(2))-induced cytosolic Ca(2+) ([Ca(2+)](i)) elavation, whole-cell current increase, and apoptotic cell death in murine heart microvessel endothelial cell line H5V. A TRPM2 blocking antibody (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the [Ca(2+)](i) rise and whole-cell current change in response to H(2)O(2). Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect. H(2)O(2)-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect against H(2)O(2)-induced apoptotic cell death. TM2E3 and TRPM2-specific shRNA also protect the cells from tumor necrosis factor (TNF)-α-induced cell death in MTT assay. In contrast, overexpression of TRPM2 in H5V cells resulted in an increased response in [Ca(2+)](i) and whole-cell currents to H(2)O(2). TRPM2 overexpression also aggravated the H(2)O(2)-induced apoptotic cell death. Downstream pathways following TRPM2 activation was examined. Results showed that TRPM2 activity stimulated caspase-8, caspase-9 and caspase-3. These findings strongly suggest that TRPM2 channel mediates cellular Ca(2+) overload in response to H(2)O(2) and contribute to oxidant-induced apoptotic cell death in vascular endothelial cells. Down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death.     

The effects of hydrogen peroxide on intracellular calcium handling and contractility in the rat ventricular myocyte

Elevations in reactive oxygen species are implicated in many disease states and cause systolic and diastolic myocardial dysfunction. To understand the underlying cellular dysfunction, we characterised the effects of H?O? on [Ca(2+)](i) handling and contractility in the rat ventricular myocyte. This was achieved using patch clamping, [Ca(2+)](i) measurement using Fluo-3, video edge detection and confocal microscopy. All experiments were performed at 37°C. 200 μM H?O? resulted in a 44% decrease in the [Ca(2+)](i) transient amplitude, a 30% increase in diastolic [Ca(2+)](i) and an 18% decrease in the rate of systolic Ca(2+) removal. This was associated with a 61% reduction in systolic shortening, a contracture of 3 μm and a 42% increase in relaxation time respectively. The decrease in the [Ca(2+)](i) transient amplitude could be explained by a 27% decrease in SR Ca(2+) content. This, in turn results from a 22% decrease of SERCA activity. The decreased SR Ca(2+) content also provides a mechanism for a reduction in [Ca(2+)](i) spark frequency with no evidence for a Ca(2+) independent modification of ryanodine receptor open probability. We conclude that decreased SERCA activity is the major factor responsible for the changes of the systolic [Ca(2+)](i) transient.     

Tyrosine phosphorylation is involved in Ca(2+)entry in human gingival fibroblasts

Bradykinin (1 microM) and histamine (100 microM) evoked an initial transient increase and a subsequent sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded human gingival fibroblasts, which may be attributed to Ca(2+) release from intracellular stores and Ca(2+) entry from extracellular sites, respectively. In fibroblasts pretreated with tyrosine kinase inhibitors such as herbimycin A (1 microM) and tyrphostin 47 (20 microM), the sustained level of [Ca(2+)](i) induced by bradykinin and histamine increased, but not the initial peak level. In the absence of external Ca(2+), bradykinin and histamine induced only the transient increase in [Ca(2+)](i), but a subsequent addition of Ca(2+) to the medium resulted in a sustained increase in [Ca(2+)](i) caused by Ca(2+)entry. Thapsigargin, an inhibitor of Ca(2+)-ATPase in inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores, mimicked the effect of bradykinin and histamine. In the fibroblasts pretreated with tyrosine kinase inhibitors, the bradykinin-, histamine- and thapsigargin-induced Ca(2+) entry was clearly enhanced, but not the transient [Ca(2+)](i) increase. Tyrosine phosphatase inhibitor benzylphosphonic acid (200 microM) had no effect on Ca(2+)entry or transient [Ca(2+)](i) increase. These results suggest that tyrosine phosphorylation is involved in Ca(2+) entry in human gingival fibroblasts.     

Effect of diethylstilbestrol (DES) on intracellular Ca(2+) levels in renal tubular cells

The effect of the estrogen diethylstilbestrol (DES) on intracellular Ca(2+) concentrations ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) cells was investigated, using the fluorescent dye fura-2 as a Ca(2+) indicator. DES (10-50 microM) evoked [Ca(2+)](i) increases in a concentration-dependent manner. Extracellular Ca(2+) removal inhibited 45 +/- 5% of the Ca(2+) response. In Ca(2+)-free medium, pretreatment with 50 microM DES abolished the [Ca(2+)](i) increases induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) and 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor); and pretreatment with CCCP and thapsigargin partly inhibited DES-induced [Ca(2+)](i) signals. Adding 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 50 microM DES in Ca(2+)-free medium, suggesting that DES may induce capacitative Ca(2+) entry. 17beta-Estradiol (2-20 microM) increased [Ca(2+)](i), but 100 microM diethylstilbestrol dipropionate had no effect. Pretreatment with the phospholipase C inhibitor U73122 (1 microM) to abolish inositol 1,4,5-trisphosphate formation inhibited 30% of DES-induced Ca(2+) release. DES (20 microM) also increased [Ca(2+)](i) in human normal hepatocytes and osteosarcoma cells. Cumulatively, this study shows that DES induced rapid and sustained [Ca(2+)](i) increases by releasing intracellular Ca(2+) and triggering extracellular Ca(2+) entry in renal tubular cells.     

Phosphorylation of inositol 1,4,5-trisphosphate receptors in parotid acinar cells. A mechanism for the synergistic effects of cAMP on Ca2+ signaling.

Acetylcholine-evoked secretion from the parotid gland is substantially potentiated by cAMP-raising agonists. A potential locus for the action of cAMP is the intracellular signaling pathway resulting in elevated cytosolic calcium levels ([Ca(2+)](i)). This hypothesis was tested in mouse parotid acinar cells. Forskolin dramatically potentiated the carbachol-evoked increase in [Ca(2+)](i), converted oscillatory [Ca(2+)](i) changes into a sustained [Ca(2+)](i) increase, and caused subthreshold concentrations of carbachol to increase [Ca(2+)](i) measurably. This potentiation was found to be independent of Ca(2+) entry and inositol 1,4,5-trisphosphate (InsP(3)) production, suggesting that cAMP-mediated effects on Ca(2+) release was the major underlying mechanism. Consistent with this hypothesis, dibutyryl cAMP dramatically potentiated InsP(3)-evoked Ca(2+) release from streptolysin-O-permeabilized cells. Furthermore, type II InsP(3) receptors (InsP(3)R) were shown to be directly phosphorylated by a protein kinase A (PKA)-mediated mechanism after treatment with forskolin. In contrast, no evidence was obtained to support direct PKA-mediated activation of ryanodine receptors (RyRs). However, inhibition of RyRs in intact cells, demonstrated a role for RyRs in propagating Ca(2+) oscillations and amplifying potentiated Ca(2+) release from InsP(3)Rs. These data indicate that potentiation of Ca(2+) release is primarily the result of PKA-mediated phosphorylation of InsP(3)Rs, and may largely explain the synergistic relationship between cAMP-raising agonists and acetylcholine-evoked secretion in the parotid. In addition, this report supports the emerging consensus that phosphorylation at the level of the Ca(2+) release machinery is a broadly important mechanism by which cells can regulate Ca(2+)-mediated processes.     

The essential role of H2O2 in the regulation of intracellular Ca2+ by epidermal growth factor in rat-2 fibroblasts

We have investigated a new mechanism by which epidermal growth factor (EGF) increases intracellular Ca(2+) ([Ca(2+)](i)) in Rat-2 fibroblasts. EGF induced a transient increase of [Ca(2+)](i), and sustained Ca(2+) increase disappeared in the absence of extracellular Ca(2+). However, EGF had no effect on the formation of inositol phosphates. Expression of N17Rac or scrape-loading of C3 transferase blocked the elevation of [Ca(2+)](i) by EGF, but not by lysophosphatidic acid (LPA). EGF increased intracellular H(2)O(2), with a maximal increase at 5 min, which was blocked by catalase, scrape-loading of C3 transferase, or expression of N17Rac. H(2)O(2) scavengers, catalase and N-acetyl-L-cysteine, also blocked the Ca(2+) response to EGF, but not to LPA. In the presence of EGTA, preincubation with EGF completely inhibited subsequent Ca(2+) response to extracellular H(2)O(2) and vice versa. Incubation with EGF or phosphatidic acid abolished subsequent elevation of [Ca(2+)](i) by phosphatidic acid or EGF, respectively. Furthermore, preincubation with LPA inhibited the subsequent Ca(2+) response to EGF, but not vice versa. These results suggested that intracellular H(2)O(2) regulated by Rac and RhoA, but not inositol phosphates, was responsible for the EGF-stimulated elevation of [Ca(2+)](i). It was also suggested that EGF cross talked with LPA in the regulation of [Ca(2+)](i) by producing intracellular H(2)O(2).     

Effect of hydrogen peroxide on reoxygenation-induced Ca2+ accumulation in rat cardiomyocytes

Reactive oxygen species (ROS) contribute to cell damage during reperfusion of the heart. ROS may exert their effects partly by interfering with Ca(2+) homeostasis of the myocardium. The purpose of this study was to investigate the effects of hydrogen peroxide (H(2)O(2)) on Ca(2+) accumulation during reoxygenation of isolated adult rat cardiomyocytes exposed to 1 h of hypoxia and to relate the effects to possible changes in release of lactate dehydrogenase (LDH), free intracellular Ca(2+) ([Ca(2+)](i)) and Mg(2+)([Mg(2+)](i)), and mitochondrial membrane potential (Deltapsim). Cell Ca(2+) was determined by (45)Ca(2+) uptake. Free [Mg(2+)](i) and [Ca(2+)](i) and Deltapsim were measured by flow cytometry. Reoxygenation-induced Ca(2+) accumulation was attenuated by 23 and 34% by 10 and 25 microM H(2)O(2), respectively, added at reoxygenation. H(2)O(2) at 100 and 250 microM increased cell Ca(2+) by 50 and 83%, respectively, whereas 500 microM H(2)O(2) decreased cell Ca(2+) by 20%. H(2)O(2) at (25 microM) reduced LDH release and [Mg(2+)](i) and increased Deltapsim, indicating cell protection, whereas 250 microM H(2)O(2) increased LDH release and [Mg(2+)](i) and decreased Deltapsim, indicating cell damage. Clonazepam (100 microM) attenuated the increase in Ca(2+) accumulation, the elevation of [Ca(2+)](i), and the decrease in Deltapsim induced by 100 and 250 microM H(2)O(2) during reoxygenation. We report for the first time that 25 microM H(2)O(2) attenuates Ca(2+) accumulation, LDH release, and dissipation of Deltapsim during reoxygenation of hypoxic cardiomyocytes, indicating cell protection.     

Effects of chronic hypoxia on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells.

Microfluorimetric measurements of intracellular calcium ion concentration [Ca(2+)](i) were employed to examine the effects of chronic hypoxia (2.5% O(2), 24 h) on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells. Activation of muscarinic receptors evoked rises in [Ca(2+)](i) which were enhanced in chronically hypoxic cells. Transient rises of [Ca(2+)](i) evoked in Ca(2+)-free solutions were greater and decayed more slowly following exposure to chronic hypoxia. In control cells, these transient rises of [Ca(2+)](i) were also enhanced and slowed by removal of external Na(+), whereas the same manoeuvre did not affect responses in chronically hypoxic cells. Capacitative Ca(2+) entry, observed when re-applying Ca(2+) following depletion of intracellular stores, was suppressed in chronically hypoxic cells. Western blots revealed that presenilin-1 levels were unaffected by chronic hypoxia. Exposure of cells to amyloid beta peptide (1-40) also increased transient [Ca(2+)](i) rises, but did not mimic any other effects of chronic hypoxia. Our results indicate that chronic hypoxia causes increased filling of intracellular Ca(2+) stores, suppressed expression or activity of Na(+)/Ca(2+) exchange and reduced capacitative Ca(2+) entry. These effects are not attributable to increased amyloid beta peptide or presenilin-1 levels, but are likely to be important in adaptive cellular remodelling in response to prolonged hypoxic or ischemic episodes.     

Ca2+ release dynamics in parotid and pancreatic exocrine acinar cells evoked by spatially limited flash photolysis

Intracellular calcium concentration ([Ca(2+)](i)) signals are central to the mechanisms underlying fluid and protein secretion in pancreatic and parotid acinar cells. Calcium release was studied in natively buffered cells following focal laser photolysis of caged molecules. Focal photolysis of caged-inositol 1,4,5 trisphosphate (InsP(3)) in the apical region resulted in Ca(2+) release from the apical trigger zone and, after a latent period, the initiation of an apical-to-basal Ca(2+) wave. The latency was longer and the wave speed significantly slower in pancreatic compared with parotid cells. Focal photolysis in basal regions evoked only limited Ca(2+) release at the photolysis site and never resulted in a propagating wave. Instead, an apical-to-basal wave was initiated following a latent period. Again, the latent period was significantly longer under all conditions in pancreas than parotid. Although slower in pancreas than parotid, once initiated, the apical-to-basal wave speed was constant in a particular cell type. Photo release of caged-Ca(2+) failed to evoke a propagating Ca(2+) wave in either cell type. However, the kinetics of the Ca(2+) signal evoked following photolysis of caged-InsP(3) were significantly dampened by ryanodine in parotid but not pancreas, indicating a more prominent functional role for ryanodine receptor (RyR) following InsP(3) receptor (InsP(3)R) activation. These data suggest that differing expression levels of InsP(3)R, RyR, and possibly cellular buffering capacity may contribute to the fast kinetics of Ca(2+) signals in parotid compared with pancreas. These properties may represent a specialization of the cell type to effectively stimulate Ca(2+)-dependent effectors important for the differing primary physiological role of each gland.     

Nifedipine-activated Ca(2+) permeability in newborn rat cortical collecting duct cells in primary culture

To characterize Ca(2+) transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca(2+) concentration ([Ca(2+)](i)) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 microM) produced an increase in [Ca(2+)](i) from 87.6 +/- 3.3 nM to 389.9 +/- 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca(2+)](i) in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca(2+)](i). Experiments in the presence of EGTA showed that external Ca(2+) was required for the nifedipine effect, while lanthanum (20 microM), gadolinium (100 microM), and diltiazem (20 microM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K(+) channels were not involved in the nifedipine-induced [Ca(2+)](i) rise. H(2)O(2) also triggered [Ca(2+)](i) rise. However, nifedipine-induced [Ca(2+)](i) increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca(2+) transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca(2+) channel of capacitive type (either transient receptor potential or leak channel).     

Novel effects of gossypol, a chemical contraceptive in man: mobilization of internal Ca(2+) and activation of external Ca(2+) entry in intact cells

The effect of gossypol on Ca(2+) signaling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Gossypol evoked a rise in cytosolic free Ca(2+) levels ([Ca(2+)](i)) concentration-dependently between 2 and 20 microM. The response was decreased by external Ca(2+) removal. In Ca(2+)-free medium pretreatment with gossypol nearly abolished the [Ca(2+)](i) increase induced by carbonylcyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, and thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) pump; but pretreatment with CCCP and thapsigargin only partly inhibited gossypol-induced Ca(2+) release. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) increase after pretreatment with 5 microM gossypol in Ca(2+)-free medium. This Ca(2+) entry was decreased by 25 microM econazole, 50 microM SKF96365 and 40 microM aristolochic acid (a phospholipase A(2) inhibitor). Pretreatment with aristolochic acid inhibited 5 microM gossypol-induced internal Ca(2+) release by 55%, but suppression of phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) had no effect. Gossypol (5 microM) also increased [Ca(2+)](i) in human bladder cancer cells and neutrophils. Collectively, we have found that gossypol increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from multiple Ca(2+) stores in a manner independent of the production of inositol-1,4,5-trisphosphate, followed by Ca(2+) influx from external space.     

Effects of capsaicin on Ca(2+) release from the intracellular Ca(2+) stores in the dorsal root ganglion cells of adult rats

We have investigated the effect of capsaicin on Ca(2+) release from the intracellular calcium stores. Intracellular calcium concentration ([Ca(2+)](i)) was measured in rat dorsal root ganglion (DRG) neurons using microfluorimetry with fura-2 indicator. Brief application of capsaicin (1 microM) elevated [Ca(2+)](i) in Ca(2+)-free solution. Capsaicin-induced [Ca(2+)](i) transient in Ca(2+)-free solution was evoked in a dose-dependent manner. Resiniferatoxin, an analogue of capsaicin, also raised [Ca(2+)](i) in Ca(2+)-free solution. Capsazepine, an antagonist of capsaicin receptor, completely blocked the capsaicin-induced [Ca(2+)](i) transient. Caffeine completely abolished capsaicin-induced [Ca(2+)](i) transient. Dantrolene sodium and ruthenium red, antagonists of the ryanodine receptor, blocked the effect of capsaicin on [Ca(2+)](i). However, capsaicin-induced [Ca(2+)](i) transient was not affected by 2-APB, a membrane-permeable IP(3) receptor antagonist. Furthermore, depletion of IP(3)-sensitive Ca(2+) stores by bradykinin and phospholipase C inhibitors, neomycin, and U-73122, did not block capsaicin-induced [Ca(2+)](i) transient. In conclusion, capsaicin increases [Ca(2+)](i) through Ca(2+) release from ryanodine-sensitive Ca(2+) stores, but not from IP(3)-sensitive Ca(2+) stores in addition to Ca(2+) entry through capsaicin-activated nonselective cation channel in rat DRG neurons.     

Effect of H2O2 on CCK-8-evoked changes in mitochondrial activity in isolated mouse pancreatic acinar cells

BACKGROUND INFORMATION: This paper studies the effect of H(2)O(2) on mitochondrial responses evoked by CCK-8 (cholecystokinin 8) in mouse pancreatic acinar cells. Cytosolic ([Ca(2+)](c)) and mitochondrial ([Ca(2+)](m)) free-calcium concentrations, mitochondrial inner membrane potential (psi(m)) and FAD autofluorescence were monitored using confocal laser scanning microscopy. RESULTS: CCK-8 induced an increase in [Ca(2+)](m) that slowly declined towards the prestimulation level. Depolarization of psi(m) that partially recovered, as well as increases in FAD autofluorescence, could also be observed in response to the hormone. Pretreatment of cells with 1 mM H(2)O(2) alone resulted in marked changes in mitochondrial parameters and, moreover, H(2)O(2) inhibited the CCK-8-evoked changes in [Ca(2+)](m), psi(m) and FAD autofluorescence. The results of the present study have demonstrated that CCK-8 can evoke marked changes in pancreatic acinar cell mitochondrial activity and that CCK-8-evoked responses are blocked by H(2)O(2). Additionally, H(2)O(2) releases Ca(2+) from intracellular stores and inhibits pancreatic acinar cell responses to CCK-8. CONCLUSION: The effects observed reflect an impairment of mitochondrial activity in the presence of H(2)O(2) that could represent some of its mechanisms of action to induce cellular damage leading to cell dysfunction and generation of pathologies.     

Effects of pilocarpine on the secretory acinar cells in human submandibular glands

Pilocarpine has been used as a choice of drugs for treatment of impaired salivary flow. Although considerable data are available as to the stimulatory effect of pilocarpine on the salivary secretion in human, its underlying mechanism, at the cellular level, has not been rigorously studied. In this experiment, we studied the effect of pilocarpine on the ion channel activity, cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) and aquaporin (AQP)-5 expression, which play key roles in the secretary process and determine the capacity of fluid secretion. In human submandibular gland (SMG) acinar cells, 10(-5) M pilocarpine activated the outward rectifying-current, which was predominantly K(+) selective in the whole cell patch clamp study. The pilocarpine increased [Ca(2+)](i) in a concentration-dependent manner in the range of 10(-6) M to 10(-4) M. We found that both increases of [Ca(2+)](i) and outward rectifying- K(+) current were inhibited by 10(-5) M U-73122, a specific phospholipase C inhibitor. The magnitudes of pilocarpine-induced [Ca(2+)](i) transients were approximately 55% lower than those with the same concentration of carbachol (CCh). Pilocarpine also increased the amount of AQP-5 protein in the apical membrane (APM) in human SMG acinar cells. Our results suggest that pilocarpine induce salivary secretions in human by activating K(+) channels, increasing [Ca(2+)](i) via phospholipase C dependent pathway, and increasing AQP-5 protein expression in the APM of SMG acinar cells.     

Different Ca2+ signalling cascades manifested by mastoparan in the prothoracic glands of the tobacco hornworm, Manduca sexta, and the silkworm, Bombyx mori

Application of the tetradecapeptide mastoparan to the prothoracic glands (PGs) of the tobacco hornworm, Manduca sexta, and the silkworm, Bombyx mori, resulted in increases in intracellular Ca(2+) ([Ca(2+)](i)). In M. sexta, Gi proteins are involved in the mastoparan-stimulated increase in [Ca(2+)](i). However, there is no involvement of Gi proteins in the mastoparan-stimulated increase in [Ca(2+)](i) in prothoracic gland cells from B. mori. Unlike in M. sexta prothoracic glands, in B. mori prothoracic glands mastoparan increases [Ca(2+)](i) even in the absence of extracellular Ca(2+). Pharmacological manipulation of the Ca(2+) signalling cascades in the prothoracic glands of both insect species suggests that in M. sexta prothoracic glands, mastoparan's first site of action is influx of Ca(2+) through plasma membrane Ca(2+) channels while in B. mori prothoracic glands, mastoparan's first site of action is mobilization of Ca(2+) from intracellular stores. In M. sexta, the combined results indicate the presence of mastoparan-sensitive plasma membrane Ca(2+) channels, distinct from those activated by prothoracicotropic hormone or the IP(3) signalling cascade, that coordinate spatial increases in [Ca(2+)](i) in prothoracic gland cells. We propose that in B. mori, mastoparan stimulates Ca(2+) mobilization from ryanodine-sensitive intracellular Ca(2+) stores in prothoracic gland cells.     

Swelling of mitochondria in cultured rat hippocampal astrocytes is induced by high cytosolic Ca(2+) load,but not by mitochondrial depolarization

The influence of cytosolic Ca(2+) load and of mitochondrial membrane potential change on mitochondrial morphology was investigated in cultured rat hippocampal astrocytes. The uncoupler FCCP, applied together with oligomycin, depolarized mitochondria rapidly but did not change their morphology. Depolarization was associated with a moderate cytosolic [Ca(2+)](i) rise of up to 0.3 microM. Only high cytosolic Ca(2+) load (above a threshold of 50 microM), which was evoked by application of the ionophore 4-Br-A23187 in Ca(2+)-containing medium, caused drastic change of mitochondrial morphology. The shape change from the typical rod-like to a spherical shape, indicating mitochondrial swelling, was associated with depolarization. Cyclosporin A sensitivity suggests involvement of permeability transition. Thus, a dramatic cytosolic [Ca(2+)](i) rise is required to induce mitochondrial swelling and depolarization. A large but still moderate [Ca(2+)](i) rise evoked by physiological stimulation, however, has no comparable effect.