Activation of muscarinic acetylcholine receptors induces Ca(2+) mobilization in FRT cells

The effect of the muscarinic receptors agonist carbachol (Cch) on intracellular calcium concentration ([Ca(2+)](i)) and cAMP level was studied in polarized Fischer rat thyroid (FRT) epithelial cells. Cch provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Thapsigargin, a specific microsomal Ca(2+)-ATPase inhibitor, caused a rapid rise in [Ca(2+)](i) and subsequent addition of Cch was without effect. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Ryanodine, an agent that depletes intracellular Ca(2+) stores through stimulation of ryanodine receptors (RyRs), had no effect on [Ca(2+)](i). However, the transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with U73122, a specific inhibitor of phospholipase C (PLC). These data suggest that the Cch-stimulated increment of [Ca(2+)](i) required IP(3) formation and binding to its specific receptors in Ca(2+) stores. Further studies were performed to investigate whether the effect of Cch on Ca(2+) entry into FRT cells was via L-type voltage-dependent Ca(2+) channels (L-VDCCs). Nicardipine, a nonspecific L-type Ca(2+) channel blocker, decreased Cch-induced increase on [Ca(2+)](i), while Bay K-8644, an L-type Ca(2+) channel agonist, slightly increased [Ca(2+)](i) in FRT cells. These data indicate that Ca(2+) entry into these nondifferentiated thyroid cells occurs through an L-VDCC, and probably through another mechanism such as a capacitative pathway. Cch did not affect the intracellular cAMP levels, but its effects on [Ca(2+)](i) were significantly reduced when cells were pretreated with forskolin, suggesting the existence of an intracellular cross-talk between PLC and cAMP mechanisms in the regulation of intracellular Ca(2+) mobilization in neoplastic FRT cells.     

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.     

Modulation of Ca2+ signals by phosphatidylinositol-linked novel D1 dopamine receptor in hippocampal neurons

Recent evidence indicates the existence of a putative novel phosphatidylinositol-linked D1 dopamine receptor in brain that mediates phosphatidylinositol hydrolysis via activation of phospholipase Cbeta. The present work was designed to characterize the Ca(2+) signals regulated by this phosphatidylinositol-linked D(1) dopamine receptor in primary cultures of hippocampal neurons. The results indicated that stimulation of phosphatidylinositol-linked D1 dopamine receptor by its newly identified selective agonist SKF83959 induced a long-lasting increase in basal [Ca(2+)](i) in a time- and dose-dependent manner. Stimulation was observable at 0.1 microm and reached the maximal effect at 30 microm. The [Ca(2+)](i) increase induced by 1 microm SKF83959 reached a plateau in 5 +/- 2.13 min, an average 96 +/- 5.6% increase over control. The sustained elevation of [Ca(2+)](i) was due to both intracellular calcium release and calcium influx. The initial component of Ca(2+) increase through release from intracellular stores was necessary for triggering the late component of Ca(2+) rise through influx. We further demonstrated that activation of phospholipase Cbeta/inositol triphosphate was responsible for SKF83959-induced Ca(2+) release from intracellular stores. Moreover, inhibition of voltage-operated calcium channel or NMDA receptor-gated calcium channel strongly attenuated SKF83959-induced Ca(2+) influx, indicating that both voltage-operated calcium channel and NMDA receptor contribute to phosphatidylinositol-linked D(1) receptor regulation of [Ca(2+)](i).     

Effects of Staphylococcus aureus-hemolysin A on calcium signalling in immortalized human airway epithelial cells

Part of the innate defence of bronchial epithelia against bacterial colonization is secretion of salt and water which generally depends on coordinated actions of receptor-mediated cAMP- and calcium signalling. The hypothesis that Staphylococcus aureus-virulence factors interfere with endogenous signals in host cells was tested by measuring agonist-mediated changes in [Ca(2+)](i) in S9 cells upon pre-incubation with bacterial secretory products. S9 cells responded to mAChR-activation with calcium release from intracellular stores and capacitative calcium influx. Treatment of cells with culture supernatants of S. aureus (COL) or with recombinant alpha-hemolysin (Hla) resulted in time- and concentration-dependent changes in [Ca(2+)](i). High concentrations of Hla (2000 ng/ml) resulted in elevations in [Ca(2+)](i) elicited by accelerated calcium influx. A general Hla-mediated permeabilization of S9 cell membranes to small molecules, however, did not occur. Lower concentrations of Hla (200 ng/ml) induced a reduction in [Ca(2+)](i)-levels during the sustained plateau phase of receptor-mediated calcium signalling which was abolished by pre-incubation of cells with carboxyeosin, an inhibitor of the plasma membrane calcium-ATPase. This indicates that low concentrations of Hla change calcium signalling by accelerating pump-driven extrusion of Ca(2+) ions. In vivo, such a mechanism may result in attenuation of calcium-mediated cellular defence functions and facilitation of bacterial adherence to the bronchial epithelium.     

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.     

Intracellular angiotensin II activates rat myometrium

Angiotensin II is a modulator of myometrial activity; both AT(1) and AT(2) receptors are expressed in myometrium. Since in other tissues angiotensin II has been reported to activate intracellular receptors, we assessed the effects of intracellular administration of angiotensin II via microinjection on myometrium, using calcium imaging. Intracellular injection of angiotensin II increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in myometrial cells in a dose-dependent manner. The effect was abolished by the AT(1) receptor antagonist losartan but not by the AT(2) receptor antagonist PD-123319. Disruption of the endo-lysosomal system, but not that of Golgi apparatus, prevented the angiotensin II-induced increase in [Ca(2+)](i). Blockade of AT(1) receptor internalization had no effect, whereas blockade of microautophagy abolished the increase in [Ca(2+)](i) produced by intracellular injection of angiotensin II; this indicates that microautophagy is a critical step in transporting the peptide into the endo-lysosomes lumenum. The response to angiotensin II was slightly reduced in Ca(2+)-free saline, indicating a major involvement of Ca(2+) release from internal stores. Blockade of inositol 1,4,5-trisphosphate (IP(3)) receptors with heparin and xestospongin C or inhibition of phospholipase C (PLC) with U-73122 abolished the response to angiotensin II, supporting the involvement of PLC-IP(3) pathway. Angiotensin II-induced increase in [Ca(2+)](i) was slightly reduced by antagonism of ryanodine receptors. Taken together, our results indicate for the first time that in myometrial cells, intracellular angiotensin II activates AT(1)-like receptors on lysosomes and activates PLC-IP(3)-dependent Ca(2+) release from endoplasmic reticulum; the response is further augmented by a Ca(2+)-induced Ca(2+) release mechanism via ryanodine receptors activation.     

EGF enhances Ca(2+) mobilization and capacitative Ca(2+) entry in mouse mammary epithelial cells

Effects of epidermal growth factor (EGF) on the intracellular Ca(2+) ([Ca(2+)](i)) responses to nucleotides, Ca(2+) release from thapsigargin-sensitive stores and capacitative Ca(2+) entry were investigated in cultured mouse mammary epithelial cells. EGF treatment induced proliferation of mammary epithelial cells. We checked for mitotic activity by immunocytochemistry with an anti-PCNA (proliferating cell nuclear antigen) antibody, which stains nuclei of the cells in S-phase of cell cycle. EGF treatment apparently increased the number of PCNA-stained cells compared to those treated with differentiating hormones (insulin, prolactin and cortisol) or without any hormone. Application of EGF did not induce any acute [Ca(2+)](i) response. EGF treatment for 1-2 days in culture, however, enhanced [Ca(2+)](i) responses including [Ca(2+)](i) increase by ATP, UTP and other nucelotides, Ca(2+) release from thapsigargin-sensitive stores, as well as capacitative Ca(2+) entry. Genistein, a tyrosine kinase inhibitor, prevented EGF-induced cell proliferation and the [Ca(2+) ](i) responses in a dose-dependent manner. These results indicate that EGF treatment enhances Ca(2+) mobilization and capacitative Ca(2+) entry, well correlated with cellular proliferation in mammary epithelial cells.     

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.     

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.     

Effects of linoleic acid on intracellular calcium concentration in primarily cultured rat pancreatic β-cells and underlying mechanism

In this study, we investigated the mechanism of linoleic acid-stimulated increase in intracellular calcium concentration ([Ca(2+)](i)) in pancreatic islet β-cells. Pancreatic islet cells were primarily isolated from rats and cultured for the experiments. The cells were loaded with Fluo-3/AM, the indicator of [Ca(2+)](i), and the intensity of Fluo-3 was measured using confocal microscope. The islet β-cells were identified by immunocytochemical staining with insulin antibody after recording. The drugs were given by perfusion system. The results showed that linoleic acid (20 μmol/L) stimulated [Ca(2+)](i) increase with the first peak increase and the following plateau increase. Linoleic acid-stimulated [Ca(2+)](i) increase was partly inhibited by removal of extracellular calcium and by transient receptor potential (TRP) channel blocker, La(3+), and it was totally blocked by exhaustion of intracellular calcium stores and inhibition of phospholipase C. It is concluded that linoleic acid stimulates [Ca(2+)](i) increase in islet β-cells through both extracellular calcium influx via TRP channels and calcium release from intracellular calcium stores.     

Calcium dependence of proteinase-activated receptor 2 and cholecystokinin-mediated amylase secretion from pancreatic acini

Pancreatic acini secrete digestive enzymes in response to a variety of secretagogues including CCK and agonists acting via proteinase-activated receptor-2 (PAR2). We employed the CCK analog caerulein and the PAR2-activating peptide SLIGRL-NH(2) to compare and contrast Ca(2+) changes and amylase secretion triggered by CCK receptor and PAR2 stimulation. We found that secretion stimulated by both agonists is dependent on a rise in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and that this rise in [Ca(2+)](i) reflects both the release of Ca(2+) from intracellular stores and accelerated Ca(2+) influx. Both agonists, at low concentrations, elicit oscillatory [Ca(2+)](i) changes, and both trigger a peak plateau [Ca(2+)](i) change at high concentrations. Although the two agonists elicit similar rates of amylase secretion, the rise in [Ca(2+)](i) elicited by caerulein is greater than that elicited by SLIGRL-NH(2). In Ca(2+)-free medium, the rise in [Ca(2+)](i) elicited by SLIGRL-NH(2) is prevented by the prior addition of a supramaximally stimulating concentration of caerulein, but the reverse is not true; the rise elicited by caerulein is neither prevented nor reduced by prior addition of SLIGRL-NH(2). Both the oscillatory and the peak plateau [Ca(2+)](i) changes that follow PAR2 stimulation are prevented by the phospholipase C (PLC) inhibitor U73122, but U73122 prevents only the oscillatory [Ca(2+)](i) changes triggered by caerulein. We conclude that 1) both PAR2 and CCK stimulation trigger amylase secretion that is dependent on a rise in [Ca(2+)](i) and that [Ca(2+)](i) rise reflects release of calcium from intracellular stores as well as accelerated influx of extracellular calcium; 2) PLC mediates both the oscillatory and the peak plateau rise in [Ca(2+)](i) elicited by PAR2 but only the oscillatory rise in [Ca(2+)](i) elicited by CCK stimulation; and 3) the rate of amylase secretion elicited by agonists acting via different types of receptors may not correlate with the magnitude of the [Ca(2+)](i) rise triggered by those different types of secretagogue.     

P2Y-receptor regulates size of endothelial cells in an intracellular Ca2+ dependent manner

The effects of P2 receptor agonists on cell size and intracellular calcium levels, [Ca(2+)](i), was investigated using cultured endothelial cells isolated from the caudal artery of male Wistar rats. Cell size and [Ca(2+)](i) were measured using a phase-contrast and fluorescent confocal microscopic image analyzer and a Calcium Green fluorescence probe. P2Y receptor agonists, 2-methylthio ATP (2meS-ATP), ADP, UTP and ATP decreased the cell size and increased [Ca(2+)](i) in endothelial cells from rat caudal artery. However, alpha,beta-methylene ATP, a P2X receptor agonist, did not induce these responses. The decrease in size and the increase in [Ca(2+)](i), by 2meS-ATP were blocked by PPADS (P2-antagonist), suramin (P2-antagonist), thapsigargin (Ca(2+) pump inhibitor) and U-73122 (phospholipase C inhibitor). The present results show that activation of P2Y receptors, not P2X receptors, induces a decrease in cell size and an increase in [Ca(2+)](i), and the pharmacological properties of these two responses are the same. We concluded that the size of endothelial cells is regulated by P2Y receptors via intracelluar Ca(2+) derived from Ca(2+) stores.     

17beta-estradiol rapidly mobilizes intracellular calcium from ryanodine-receptor-gated stores via a PKC-PKA-Erk-dependent pathway in the human eccrine sweat gland cell line NCL-SG3

We describe a novel rapid non-genomic effect of 17beta-estradiol (E2) on intracellular Ca(2+) ([Ca(2+)](i)) signalling in the eccrine sweat gland epithelial cell line NCL-SG3. E2 had no observable effect on basal [Ca(2+)](i), however exposure of cells to E2 in the presence of the microsomal Ca(2+) ATPase pump inhibitor, thapsigargin, produced a secondary, sustained increase in [Ca(2+)](i) compared to thapsigargin treatment alone, where cells responded with a transient single spike-like increase in [Ca(2+)](i). The E2-induced increase in [Ca(2+)](i) was not dependent on the presence of extracellular calcium and was completely abolished by ryanodine (100muM). The estrogen receptor antagonist ICI 182,780 (1muM) prevented the E2-induced effects suggesting a role for the estrogen receptor in the release of [Ca(2+)](i) from ryanodine-receptor-gated stores. The E2-induced effect on [Ca(2+)](i) could also be prevented by the protein kinase C delta (PKCdelta)-specific inhibitor rottlerin (10muM), the protein kinase A (PKA) inhibitor Rp-adenosine 3',5'-cyclic monophosphorothioate (200muM) and the MEK inhibitor PD98059 (10muM). We established E2 rapidly activates the novel PKC isoform PKCvarepsilon, PKA and Erk 1/2 MAPK in a PKCdelta and estrogen-receptor-dependent manner. The E2-induced effect was specific to 17beta-estradiol, as other steroids had no effect on [Ca(2+)](i). We have demonstrated a novel mechanism by which E2 rapidly modulates [Ca(2+)](i) release from ryanodine-receptor-gated intracellular Ca(2+) stores. The signal transduction pathway involves the estrogen receptor coupled to a PKC-PKA-Erk 1/2 signalling pathway.     

Mechanisms of bradykinin-mediated Ca(2+) signalling in canine cultured corneal epithelial cells

Experiments were designed to differentiate the mechanisms of bradykinin receptors mediating the changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) in canine cultured corneal epithelial cells (CECs). Bradykinin and Lys-bradykinin caused an initial transient peak of [Ca(2+)](i) in a concentration-dependent manner, with half-maximal stimulation (pEC(50)) obtained at 6.9 and 7.1, respectively. Pretreatment of CECs with pertussis toxin (PTX) or cholera toxin (CTX) for 24 h did not affect the bradykinin-induced [Ca(2+)](i) changes. Application of Ca(2+) channel blockers, diltiazem and Ni(2+), inhibited the bradykinin-induced Ca(2+) mobilization, indicating that Ca(2+) influx was required for the bradykinin-induced responses. Addition of thapsigargin (TG), which is known to deplete intracellular Ca(2+) stores, transiently increased [Ca(2+)](i) in Ca(2+)-free buffer, and subsequently induced Ca(2+) influx when Ca(2+) was readded to this buffer. Pretreatment of CECs with TG completely abolished bradykinin-induced initial transient [Ca(2+)](i), but had slight effect on bradykinin-induced Ca(2+) influx. Pretreatment of CECs with 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF96365) and 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) inhibited the bradykinin-induced Ca(2+) release and Ca(2+) influx, consistent with the inhibition of receptor-gated Ca(2+) channels and phospholipase C (PLC) in CECs, respectively. These results demonstrate that bradykinin directly stimulates B(2) receptors and subsequently Ca(2+) mobilization via a PTX-insensitive G protein in canine CECs. These results suggest that bradykinin-induced Ca(2+) influx into the cells is not due to depletion of these Ca(2+) stores, as prior depletion of these pools by TG has no effect on the bradykinin-induced Ca(2+) influx that is dependent on extracellular Ca(2+) in CECs.     

Mechanism of endothelin-1-induced cytosolic Ca(2+) mobility in cultured H9c2 myocardiac ventricular cells

The effect of endothelin-1 (ET-1) on the intracellular free Ca(2+) ([Ca(2+)](i)) mobility in cultured H9c2 myocardiac ventricular cells was studied after loading with fura-2-AM. In Ca(2+)-containing buffer, ET-1 induced [Ca(2+)](i) rise from 10(-7) to 10(-9) M. ET-1 induced [Ca(2+)](i), which was composed of a first small peak and a secondary persistent plateau. In Ca(2+)-free buffer, pretreatment with 10(-7) M ET-1 inhibited the thapsigargin and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced [Ca(2+)](i) increase. Meanwhile, pretreatment with thapsigargin and CCCP also inhibited ET-1-induced [Ca(2+)](i) rise. In Ca(2+)-containing buffer, the ET(A) receptor antagonist (BQ123) completely abolished the secondary rising peak and plateau. Conversely, the ET(B) receptor antagonist (BQ788) completely inhibited the first small peak and secondary peak plateau. Nifedipine and La(3+) also abolished the 10(-7) M ET-1-induced [Ca(2+)](i) in the first rising peak. The internal Ca(2+) release induced by ET-1 was inhibited by U73122 (phospholipase C inhibitor), propranolol (phospholipase D inhibitor) and aristolochic acid (phospholipase A2 inhibitor). After incubation of 10(-7) M ET-1 in Ca(2+)-free buffer, the addition of 5 mM CaCl(2) increased Ca(2+) influx, implying that release of Ca(2+) from internal stores further induces capacitative Ca(2+) entry. Taken together, these results suggest that both ET(A) and ET(B) receptors are involved in ET-1-induced [Ca(2+)](i) rise in H9c2 myocardiac ventricular cells. Whereas ET(B) receptor seems to mediate the initial Ca(2+) influx via L-type Ca(2+) channel, ET(A) receptor appears to be involved in the subsequent Ca(2+) release from endoplasmic reticulum and mitochondria Ca(2+) stores.     

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.     

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.     

Sustained calcium entry through P2X nucleotide receptor channels in human airway epithelial cells

Purinergic receptor stimulation has potential therapeutic effects for cystic fibrosis (CF). Thus, we explored roles for P2Y and P2X receptors in stably increasing [Ca(2+)](i) in human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Cytosolic Ca(2+) was measured by fluorospectrometry using the fluorescent dye Fura-2/AM. Expression of P2X receptor (P2XR) subtypes was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP and other P2Y agonists caused only a transient increase in [Ca(2+)](i) derived from intracellular stores in a Na(+)-rich environment. In contrast, ATP induced an increase in [Ca(2+)](i) that had transient and sustained components in a Na(+)-free medium; the sustained plateau was potentiated by zinc or increasing extracellular pH. Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased [Ca(2+)](i) only in Na(+)-free medium, suggesting competition between Na(+) and Ca(2+) through P2XRs. Biochemical evidence showed that the P2X(4) receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca(2+) channels, voltage-dependent Ca(2+) channels, or Na(+)/Ca(2+) exchanger in the ATP-induced sustained Ca(2+) signal was ruled out. In conclusion, these data show that epithelial P2X(4) receptors serve as ATP-gated calcium entry channels that induce a sustained increase in [Ca(2+)](i). In airway epithelia, a P2XR-mediated Ca(2+) signal may have therapeutic benefit for CF.     

An oxidized metabolite of linoleic acid increases intracellular calcium in rat adrenal glomerulosa cells

EKODE, an epoxy-keto derivative of linoleic acid, was previously shown to stimulate aldosterone secretion in rat adrenal glomerulosa cells. In the present study, we investigated the effect of exogenous EKODE on cytosolic [Ca(2+)] increase and aimed to elucidate the mechanism involved in this process. Through the use of the fluorescent Ca(2+)-sensitive dye Fluo-4, EKODE was shown to rapidly increase intracellular [Ca(2+)] ([Ca(2+)](i)) along a bell-shaped dose-response relationship with a maximum peak at 5 microM. Experiments performed in the presence or absence of Ca(2+) revealed that this increase in [Ca(2+)](i) originated exclusively from intracellular pools. EKODE-induced [Ca(2+)](i) increase was blunted by prior application of angiotensin II, Xestospongin C, and cyclopiazonic acid, indicating that inositol trisphosphate (InsP(3))-sensitive Ca(2+) stores can be mobilized by EKODE despite the absence of InsP(3) production. Accordingly, EKODE response was not sensitive to the phospholipase C inhibitor U-73122. EKODE mobilized a Ca(2+) store included in the thapsigargin (TG)-sensitive stores, although the interaction between EKODE and TG appears complex, since EKODE added at the plateau response of TG induced a rapid drop in [Ca(2+)](i). 9-oxo-octadecadienoic acid, another oxidized derivative of linoleic acid, also increases [Ca(2+)](i), with a dose-response curve similar to EKODE. However, arachidonic and linoleic acids at 10 microM failed to increase [Ca(2+)](i) but did reduce the amplitude of the response to EKODE. It is concluded that EKODE mobilizes Ca(2+) from an InsP(3)-sensitive store and that this [Ca(2+)](i) increase is responsible for aldosterone secretion by glomerulosa cells. Similar bell-shaped dose-response curves for aldosterone and [Ca(2+)](i) increases reinforce this hypothesis.