Effects of an inhibitor of myosin light chain kinase on amylase secretion from rat pancreatic acini

Ca(2+)/calmodulin-dependent protein (CaM) kinases play an important role in Ca(2+)-mediated secretory mechanisms. Previously, we demonstrated that a CaM kinase II inhibitor KN-62 had a small inhibitory effect on amylase secretion stimulated by CCK. In the present study, we investigated the effects of a myosin light chain kinase (MLCK) inhibitor on amylase secretion and Ca(2+) signaling in rat pancreatic acini. A specific inhibitor of MLCK, wortmannin, inhibited amylase secretion stimulated by CCK-8 (30 pM) in a concentration-dependent manner. Wortmannin (10 microM) had no effects on basal secretion but reduced amylase secretion stimulated by CCK-8 (30 pM) by 67 +/- 3%. Wortmannin inhibited amylase secretion stimulated by calcium ionophore (A23187) and phorbol ester (TPA). Wortmannin also inhibited amylase response to thapsigargin by 76 +/- 8% and to both thapsigargin and TPA by 52 +/- 10%. Ca(2+) oscillations evoked by CCK-8 (10 pM) were inhibited by wortmannin (10 microM). Wortmannin had a little inhibitory effect on an initial rise in [Ca(2+)](i), and abolished a subsequent sustained elevation of [Ca(2+)](i) evoked by 1 nM CCK-8. In conclusion, MLCK plays a crucial role in amylase secretion from pancreatic acini and regulates Ca(2+) entry from the extracellular space.     

Intracellular Ca(2+)-Mg(2+)-ATPase regulates calcium influx and acrosomal exocytosis in bull and ram spermatozoa.

Calcium influx is required for the mammalian sperm acrosome reaction (AR), an exocytotic event occurring in the sperm head prior to fertilization. We show here that thapsigargin, a highly specific inhibitor of the microsomal Ca(2+)-Mg(2+)-ATPase (Ca(2+) pump), can initiate acrosomal exocytosis in capacitated bovine and ram spermatozoa. Initiation of acrosomal exocytosis by thapsigargin requires an influx of Ca(2+), since incubation of cells in the absence of added Ca(2+) or in the presence of the calcium channel blocker, La(3+), completely inhibited thapsigargin-induced acrosomal exocytosis. ATP-Dependent calcium accumulation into nonmitochondrial stores was detected in permeabilized sperm in the presence of ATP and mitochondrial uncoupler. This activity was inhibited by thapsigargin. Thapsigargin elevated the intracellular Ca(2+) concentration ([Ca(2+)](i)), and this increase was inhibited when extracellular Ca(2+) was chelated by EGTA, indicating that this rise in Ca(2+) is derived from the external medium. This rise of [Ca(2+)](i) took place first in the head and later in the midpiece of the spermatozoon. However, immunostaining using a polyclonal antibody directed against the purified inositol 1,4,5-tris-phosphate receptor (IP(3)-R) identified specific staining in the acrosome region, in the postacrosome, and along the tail, but not in the midpiece region. No staining in the acrosome region was observed in sperm without acrosome, indicating that the acrosome cap was stained in intact sperm. The presence of IP(3)-R in the anterior acrosomal region as well as the induction, by thapsigargin, of intracellular Ca(2+) elevation in the acrosomal region and acrosomal exocytosis, implicates the acrosome as a potential cellular Ca(2+) store. We suggest here that the cytosolic Ca(2+) is actively transported into the acrosome by an ATP-dependent, thapsigargin-sensitive Ca(2+) pump and that the accumulated Ca(2+) is released from the acrosome via an IP(3)-gated calcium channel. The ability of thapsigargin to increase [Ca(2+)](i) could be due to depletion of Ca(2+) in the acrosome, resulting in the opening of a capacitative calcium entry channel in the plasma membrane. The effect of thapsigargin on elevated [Ca(2+)](i) in capacitated cells was 2-fold higher than that in noncapacitated sperm, suggesting that the intracellular Ca pump is active during capacitation and that this pump may have a role in regulating [Ca(2+)](i) during capacitation and the AR.     

Effect of diindolylmethane on Ca(2+) homeostasis and viability in PC3 human prostate cancer cells

The effect of the natural product diindolylmethane on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in PC3 human prostate cancer cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Diindolylmethane at concentrations of 20-50 μM induced [Ca(2+)](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). Diindolylmethane-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca(2+)](i) rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca(2+)](i) rise. At concentrations of 50-100 μM, diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/PI staining data implicate that diindolylmethane (50 and 100 μM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca(2+)](i) rise in PC3 cells by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via phospholipase A(2)-sensitive store-operated Ca(2+) channels. Diindolylmethane caused cell death in which apoptosis may participate.     

CaV1.3 channels and intracellular calcium mediate osmotic stress-induced N-terminal c-Jun kinase activation and disruption of tight junctions in Caco-2 CELL MONOLAYERS

We investigated the role of a Ca(2+) channel and intracellular calcium concentration ([Ca(2+)](i)) in osmotic stress-induced JNK activation and tight junction disruption in Caco-2 cell monolayers. Osmotic stress-induced tight junction disruption was attenuated by 1,2-bis(2-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-mediated intracellular Ca(2+) depletion. Depletion of extracellular Ca(2+) at the apical surface, but not basolateral surface, also prevented tight junction disruption. Similarly, thapsigargin-mediated endoplasmic reticulum (ER) Ca(2+) depletion attenuated tight junction disruption. Thapsigargin or extracellular Ca(2+) depletion partially reduced osmotic stress-induced rise in [Ca(2+)](i), whereas thapsigargin and extracellular Ca(2+) depletion together resulted in almost complete loss of rise in [Ca(2+)](i). L-type Ca(2+) channel blockers (isradipine and diltiazem) or knockdown of the Ca(V)1.3 channel abrogated [Ca(2+)](i) rise and disruption of tight junction. Osmotic stress-induced JNK2 activation was abolished by BAPTA and isradipine, and partially reduced by extracellular Ca(2+) depletion, thapsigargin, or Ca(V)1.3 knockdown. Osmotic stress rapidly induced c-Src activation, which was significantly attenuated by BAPTA, isradipine, or extracellular Ca(2+) depletion. Tight junction disruption by osmotic stress was blocked by tyrosine kinase inhibitors (genistein and PP2) or siRNA-mediated knockdown of c-Src. Osmotic stress induced a robust increase in tyrosine phosphorylation of occludin, which was attenuated by BAPTA, SP600125 (JNK inhibitor), or PP2. These results demonstrate that Ca(V)1.3 and rise in [Ca(2+)](i) play a role in the mechanism of osmotic stress-induced tight junction disruption in an intestinal epithelial monolayer. [Ca(2+)](i) mediate osmotic stress-induced JNK activation and subsequent c-Src activation and tyrosine phosphorylation of tight junction proteins. Additionally, inositol 1,4,5-trisphosphate receptor-mediated release of ER Ca(2+) also contributes to osmotic stress-induced tight junction disruption.     

A sustained increase in intracellular Ca(2+) is required for the acrosome reaction in sea urchin sperm.

The acrosome reaction (AR), necessary for fertilization in many species, requires an increase in intracellular Ca(2+) ([Ca(2+)](i)). In sea urchin sperm, the AR is triggered by an egg-jelly factor: the associated [Ca(2+)](i) elevation lasts minutes and involves two Ca(2+) permeable channels. Both the opening of the second channel and the onset of the AR occur approximately 5 s after treatment with egg factor, suggesting that these events are linked. In agreement, removal of Ca(2+) from sea water or addition of Ca(2+) channel blockers at the time when opening of the second channel is first detected inhibits AR and causes a "rapid" (t(1/2) = 3--15 s) decrease in [Ca(2+)](i) and partial inhibition of the intracellular pH change associated with the AR. Simultaneous addition of NH(4)Cl and either EGTA, Co(2+), or Ni(2+) 5 s after egg factor prevents the partial inhibition of the evoked pH(i) change observed but does not reverse AR inhibition. Therefore, the sustained increase in [Ca(2+)](i) caused by the second Ca(2+) channel is needed for the sperm AR. Experiments with agents that induce capacitative Ca(2+) uptake (thapsigargin and cyclopiazonic acid) suggest that the second channel opened during the AR could be a store-operated Ca(2+) channel.     

VEGF and ATP act by different mechanisms to increase microvascular permeability and endothelial [Ca(2+)](i)

Vascular endothelial growth factor (VEGF) increases hydraulic conductivity (L(p)) by stimulating Ca(2+) influx into endothelial cells. To determine whether VEGF-mediated Ca(2+) influx is stimulated by release of Ca(2+) from intracellular stores, we measured the effect of Ca(2+) store depletion on VEGF-mediated increased L(p) and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) of frog mesenteric microvessels. Inhibition of Ca(2+) influx by perfusion with NiCl(2) significantly attenuated VEGF-mediated increased [Ca(2+)](i). Depletion of Ca(2+) stores by perfusion of vessels with thapsigargin did not affect the VEGF-mediated increased [Ca(2+)](i) or the increase in L(p). In contrast, ATP-mediated increases in both [Ca(2+)](i) and L(p) were inhibited by thapsigargin perfusion, demonstrating that ATP stimulated store-mediated Ca(2+) influx. VEGF also increased Mn(2+) influx after perfusion with thapsigargin, whereas ATP did not. These data showed that VEGF increased [Ca(2+)](i) and L(p) even when Ca(2+) stores were depleted and under conditions that prevented ATP-mediated increases in [Ca(2+)](i) and L(p). This suggests that VEGF acts through a Ca(2+) store-independent mechanism, whereas ATP acts through Ca(2+) store-mediated Ca(2+) influx.     

Capsazepine elevates intracellular Ca2+ in human osteosarcoma cells, questioning its selectivity as a vanilloid receptor antagonist

Capsazepine is thought to be a selective antagonist of vanilloid type 1 receptors; however, its other in vitro effect on different cell types is unclear. In human MG63 osteosarcoma cells, the effect of capsazepine on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and cytotoxicity was explored by using fura-2 and tetrazolium, respectively. Capsazepine caused a rapid rise in [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 100 microM. Capsazepine-induced [Ca(2+)](i) rise was partly reduced by removal of extracellular Ca(2+), suggesting that the capsazepine-induced [Ca(2+)](i) rise was composed of extracellular Ca(2+) influx and intracellular Ca(2+). In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of capsazepine on [Ca(2+)](i) was inhibited by 75%. Conversely, pretreatment with capsazepine to deplete intracellular Ca(2+) stores totally prevented thapsigargin from releasing more Ca(2+). U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca(2+) mobilizer)-induced, but not capsazepine-induced, [Ca(2+)](i) rise. Overnight treatment with 1-100 microM capsazepine inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human MG63 osteosarcoma cells, capsazepine increases [Ca(2+)](i) by stimulating extracellular Ca(2+) influx and also by causing intracellular Ca(2+) release from the endoplasmic reticulum via a phospholiase C-independent manner. Capsazepine may be mildly cytotoxic.     

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

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

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.     

CA(2+) mobilization in the human submandibular duct cell line A253

Ca(2+)mobilization induced by ATP, isoproterenol and the Ca(2+)-ATPase inhibitor thapsigargin in the human submandibular duct cell line A253 was investigated using the Ca(2+)-sensitive fluorescent indicator fura-2. ATP and isoproterenol increased cytosolic free Ca(2+)([Ca(2+)](i)) and subsequent exposure to thapsigargin after ATP or isoproterenol stimulation caused a further increase in [Ca(2+)](i). However, ATP and isoproterenol were not able to elicit a further increase in [Ca(2+)](i)after exposure of the cells to thapsigargin. Relatively few cells reacted to isoproterenol stimulation, but nearly all cells reacted to isoproterenol if ATP was added together with, or prior to isoproterenol stimulation. Moreover, the effect of ATP was potentiated by prior or simultaneous addition of isoproterenol. Furthermore, ATP decreased [Ca(2+)](i)in the presence of thapsigargin probably due to agonist-induced export of intracellular calcium. The results may suggest the existence of three thapsigargin sensitive pools; one opened by ATP acting through P(2)-purinergic receptors and IP(3), one opened by isoproterenol acting through beta2-adrenergic receptors, and a third pool not sensitive to ATP or isoproterenol.     

Developmental changes in capacitative Ca(2+) entry in mouse mammary epithelial cells

Developmental changes in capacitative Ca(2+) entry and Ca(2+) release from intracellular stores were measured using fura-2 fluorescence method during the pregnancy period (day 3-;18) in mouse mammary epithelial cells. Ca(2+) release was identified with the transient intracellular Ca(2+) ([Ca(2+)](i)) increase induced by thapsigargin addition in a Ca(2+)-free solution. Capacitative Ca(2+) entry was measured by the transient [Ca(2+)](i) increase induced by re-addition of extracellular Ca(2+) after depletion of Ca(2+) stores by thapsigargin. The capacitative Ca(2+) entry was greatest at the early stage of pregnancy (i.e. day 3 of pregnancy) and decreased as pregnancy progressed, while Ca(2+) release remained unchanged throughout the developmental stages. These findings indicate that in contrast to Ca(2+) release, a close correlation exists between capacitative Ca(2+) entry and pregnancy-induced development in mammary epithelial cells.     

Mechanism of bradykinin-induced Ca(2+) mobilization in MG63 human osteosarcoma cells.

BACKGROUND: The effect of bradykinin on intracellular free Ca(2+) levels ([Ca(2+)](i)) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca(2+) dye. METHODS/RESULTS: Bradykinin (0.1 nM-1 microM) increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 0.5 nM. The [Ca(2+)](i) signal comprised an initial peak and a fast decay which returned to baseline in 2 min. Extracellular Ca(2+) removal inhibited the peak [Ca(2+)](i )signals by 35 +/- 3%. Bradykinin (1 nM) failed to increase [Ca(2+)](i) in the absence of extracellular Ca(2+ )after cells were pretreated with thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor; 1 microM). Bradykinin (1 nM)-induced intracellular Ca(2+) release was nearly abolished by inhibiting phospholipase C with 2 microM 1-(6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). The [Ca(2+)](i )increase induced by 1 nM bradykinin in Ca(2+)- free medium was abolished by 1 nM HOE 140 (a B2 bradykinin receptor antagonist) but was not altered by 100 nM Des-Arg-HOE 140 (a B1 bradykinin receptor antagonist). Pretreatment with 1 pM pertussis toxin for 5 h in Ca(2+) medium inhibited 30 +/- 3% of 1 nM bradykinin-induced peak [Ca(2+)](i) increase. CONCLUSIONS: Together, this study shows that bradykinin induced [Ca(2+)](i) increases in a concentration-dependent manner, by stimulating B2 bradykinin receptors leading to mobilization of Ca(2+) from the thapsigargin-sensitive stores in a manner dependent on inositol-1,4,5-trisphosphate, and also by inducing extracellular Ca(2+) influx. The bradykinin response was partly coupled to a pertussis toxin-sensitive G protein pathway.     

Calcium elevation elicited by reverse mode Na+/Ca2+ exchange activity is facilitated by intracellular calcium stores in bovine chromaffin cells

The Na(+)/Ca(2+) exchanger (NCX) in plasma membranes either moves Ca(2+) out of (forward mode) or into (reverse mode) cells depending on the electrochemical gradient of these ions across the membrane. In this report, we characterize the sources responsible for the elevation in [Ca(2+)](i) elicited by reverse mode NCX activity. The elevation in [Ca(2+)](i) elicited by reverse mode NCX activity was significantly diminished by thapsigargin. KB-R7943 could only partially suppress the [Ca(2+)](i) change. Measurement of the [Ca(2+)](i) concurrent with reverse mode NCX current by perforated whole-cell patch showed that elevation in [Ca(2+)](i), but not the current, was inhibited by thapsigargin. The change in [Ca(2+)](i) response elicited by nicotinic acetylcholine receptor agonist was inhibited by thapsigargin. These suggest the importance of intracellular Ca(2+) stores in facilitating the [Ca(2+)](i) elevation elicited by reverse mode NCX activity under physiological condition.     

Role of sarcoplasmic reticulum in regulation of tonic contraction of rabbit basilar artery

Superficial sarcoplasmic reticulum (SR) regulates smooth muscle force development directly by Ca(2+) release and removal to and from the cytoplasm (Somlyo and Somlyo. J Cardiovasc Pharmacol 8, Suppl 8: S42-S47, 1986) by buffering Ca(2+) influx and contributing to Ca(2+) extrusion (Mueller and van Breemen. Nature 281: 682-683, 1979) and indirectly by releasing Ca(2+) near Ca(2+)-activated K(+) channels (K(Ca)) to hyperpolarize the plasma membrane (Bolton and Imaizumi. Cell Calcium 20: 141-152, 1996 and Nelson et al. Science 270: 633-637, 1995). In the rabbit basilar artery, relative contributions of direct effects and those mediated through activation of K(Ca) were evaluated by measuring force and intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to the SR-depleting agents thapsigargin and ryanodine and the large conductance K(Ca) (BK(Ca)) blockers iberiotoxin (IbTX) and tetraethylammonium ion (TEA). A large contraction was observed in response to K(Ca) blockade with either 3 mM TEA or 100 nM IbTX and also after addition of 10 microM ryanodine or 2 microM thapsigargin. When K(Ca) was blocked first with TEA or IbTX, subsequent addition of thapsigargin or ryanodine also increased force. Measurements of fura 2 fluorescence showed parallel increases in [Ca(2+)](i) in response to sequential blockade of sarco(endo)plasmic reticulum Ca(2+)-ATPase and K(Ca) regardless of the order of application. It appears that a significant fraction of K(Ca) remains activated in the absence of SR function and that SR contributes to relaxation after blockade of K(Ca). We found that depletion of SR before stimulating Ca(2+) influx through voltage-gated Ca(2+) channels markedly reduced force development rate and that thapsigargin abolished this effect. We conclude that the SR of rabbit cerebral arteries modulates constriction by direct and indirect mechanisms.     

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.     

Rapid turnover of calcium in the endoplasmic reticulum during signaling. Studies with cameleon calcium indicators

HEK293 cells expressing the thyrotropin-releasing hormone (TRH) receptor were transfected with cameleon Ca(2+) indicators designed to measure the free Ca(2+) concentration in the cytoplasm, [Ca(2+)](cyt), and the endoplasmic reticulum (ER), [Ca(2+)](er). Basal [Ca(2+)](cyt) was about 50 nm; thyrotropin-releasing hormone (TRH) or other agonists increased [Ca(2+)](cyt) to 1 micrometer or higher. Basal [Ca(2+)](er) averaged 500 micrometer and fell to 50-100 micrometer over 10 min in the presence of thapsigargin. TRH consistently decreased [Ca(2+)](er) to 100 micrometer, independent of extracellular Ca(2+), whereas agonists for endogenous receptors generally caused a smaller decline. When added with thapsigargin, all agonists rapidly decreased [Ca(2+)](er) to 5-10 micrometer, indicating that there is substantial store refilling during signaling. TRH increased [Ca(2+)](cyt) and decreased [Ca(2+)](er) if applied after other agonists, whereas other agonists did not alter [Ca(2+)](cyt) or [Ca(2+)](er) if added after TRH. When Ca(2+) was added back to cells that had been incubated with TRH in Ca(2+)-free medium, [Ca(2+)](cyt) and [Ca(2+)](er) increased rapidly. The increase in [Ca(2+)](er) was only partially blocked by thapsigargin but was completely blocked if cells were loaded with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In conclusion, these new Ca(2+) indicators showed that basal [Ca(2+)](er) is approximately 500 micrometer, that [Ca(2+)](er) has to be >100 micrometer to support an increase in [Ca(2+)](cyt) by agonists, and that during signaling, intracellular Ca(2+) stores are continuously refilled with cytoplasmic Ca(2+) by the sarcoendoplasmic reticulum Ca(2+)-ATPase pump.     

Evidence that IP3 and ryanodine-sensitive intra-cellular Ca2+ stores are not involved in acute hyposmotically-induced prolactin release in Tilapia.

Prolactin (PRL) cells from the euryhaline tilapia, Oreochromis mossambicus, behave like osmoreceptors by responding directly to reductions in medium osmolality with increased secretion of the osmoregulatory hormone PRL. Extracellular Ca(2+) is essential for the transduction of a hyposmotic stimulus into PRL release. In the current study, the presence and possible role of intracellular Ca(2+) stores during hyposmotic stimulation was investigated using pharmacological approaches. Changes in intracellular Ca(2+) concentration were measured with fura-2 in isolated PRL cells. Intracellular Ca(2+) stores were depleted in dispersed PRL cells with thapsigargin (1 microM) or cyclopiazonic acid (CPA, 10 microM). Pre-incubation with thapsigargin prevented the rise in [Ca(2+)](i) induced by lysophosphatidic acid (LPA, 1 microM), an activator of the IP(3) signalling cascade, but did not prevent the hyposmotically-induced rise in [Ca(2+)](i) in medium with normal [Ca(2+)] (2mM). Pre-treatment with CPA produced similar results. Prolactin release from dispersed cells followed a pattern that paralleled observed changes in [Ca(2+)](i). CPA inhibited LPA-induced prolactin release but not hyposmotically-induced release. Xestospongin C (1microM), an inhibitor of IP(3) receptors, had no effect on hyposmotically-induced PRL release. Pre-exposure to caffeine (10mM) or ryanodine (1microM) did not prevent a hyposmotically-induced rise in [Ca(2+)](i). Taken together these results indicate the presence of IP(3) and ryanodine-sensitive Ca(2+) stores in tilapia PRL cells. However, the rapid rise in intracellular [Ca(2+)] needed for acute PRL release in response to hyposmotic medium can occur independently of these intracellular Ca(2+) stores.     

Activation of Rac1 increases c-Jun NH(2)-terminal kinase activity and DNA fragmentation in a calcium-dependent manner in rat myoblast cell line H9c2.

We examined the role of intracellular Ca(2+) in c-Jun NH(2)-terminal kinase (JNK) activation and DNA fragmentation in the rat myoblast cell line H9c2 using small GTP-binding protein Rac1. A constitutively active mutant of Rac1 (V12-Rac1) increased JNK-responsive gene expression 6-fold, although this increase was attenuated by the intracellular Ca(2+) chelator BAPTA-AM. V12-Rac1 also increased the number of DNA fragmentated cells. However, V12-Rac1-mediated JNK activation was not affected by BAPTA-AM as determined by direct measurement of active forms, and V12-Rac1 did not affect intracellular Ca(2+) concentration. These results suggest that Rac1 can activate JNK and induces cell injury, but [Ca(2+)](i) is necessary for V12-Rac1 to induce DNA fragmentation downstream of JNK activation.     

Effects of a time varying strong magnetic field on release of cytosolic free Ca2+ from intracellular stores in cultured bovine adrenal chromaffin cells

This study was made to explain the mechanisms for the effects of exposure to a time varying 1.51 T magnetic field on the intracellular Ca(2+) signaling pathway. The exposure inhibited an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine chromaffin cells induced by addition of bradykinin (BK) to a Ca(2+) free medium. The exposure did not change BK induced production of inositol 1,4,5-trisphosphate (IP(3)). [Ca(2+)](i) was markedly increased in IP(3) loaded cells, and this increase was inhibited by the magnetic field exposure. A similar increase in [Ca(2+)](i) by other drugs, which stimulated Ca(2+) release from intracellular Ca(2+) stores, was again inhibited by the same exposure. However, transmembrane Ca(2+) fluxes caused in the presence of thapsigargin were not inhibited by the magnetic field exposure in a Ca(2+) containing medium. Inhibition of the BK induced increase in [Ca(2+)](i) by the exposure for 30 min was mostly recovered 1 h after exposure ended. Our results reveal that the magnetic field exposure inhibits Ca(2+) release from intracellular Ca(2+) stores, but that BK bindings to BK receptors of the cell membrane and intracellular inositol IP(3) production are not influenced.     

Immediate cell signal induced by laminin in rat sertoli cells.

Rat Sertoli cells in primary culture have been studied for their ability to respond to extracellular matrix macromolecules by increases of [Ca(2+)](i). We observed that cells seeded on glass coverslips, loaded with the intracellular Ca(2+) indicator fura-2, responded to laminin, but not to fibronectin, with an immediate [Ca(2+)](i) raise, with a peak followed by a prolonged plateau. [Ca(2+)](i) increases were dependent upon Ca(2+) influx across the plasma membrane and Ca(2+) release from intracellular Ca(2+) pools. Ca(2+) influx was inhibited by extracellular Ca(2+) removal by EGTA, and by treatment with La(3+), or with the L-type voltage operated Ca(2+) channel blocker, nifedipine. Ca(2+) release from intracellular Ca(2+) storing organelles, was inhibited by the microsomal Ca(2+)-ATPase blocker thapsigargin. Responses were mimicked by synthetic peptides carrying the Arg-Gly-Asp adhesion sequence, but not by the control Arg-Gly-Glu-containing peptide, in which aspartic acid was replaced by glutamic acid. Laminin-dependent [Ca(2+)](i) increases were down-regulated by the follicle-stimulating hormone. However, this occurred only when cells were not subjected to homotypic cell-cell contact, and responded to the hormone with a significant [Ca(2+)](i) elevation. These results indicate that laminin may regulate Sertoli cells by intracellular signals that perturb Ca(2+) homeostasis. This role may be related to an effect exerted by the seminiferous epithelium basement membrane on the regulation of spermatogenesis.