Stimulation of human spermatozoa with progesterone gradients to simulate approach to the oocyte. Induction of [Ca(2+)](i) oscillations and cyclical transitions in flagellar beating

Progesterone is present at micromolar concentrations in the cumulus matrix, which surrounds mammalian oocytes. Exposure of human spermatozoa to a concentration gradient of progesterone (0-3 microM) to simulate approach to the oocyte induced a slowly developing increase in [Ca(2+)](i) upon which, in many cells, slow oscillations were superimposed. [Ca(2+)](i) oscillations often started at very low progesterone (<10 nm), and their frequency did not change during the subsequent rise in concentration. Oscillations also occurred, but in a much smaller proportion of cells, in response to stepped application of progesterone (3 microM). When progesterone was removed, [Ca(2+)](i) oscillations often persisted or quickly resumed. Superfusion with low-Ca(2+) bathing medium (no added Ca(2+)) did not prevent [Ca(2+)](i) oscillations, but they could be abolished by addition of EGTA or La(3+). Inhibitors of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases or inositol trisphosphate signaling had no effect on [Ca(2+)](i) oscillations, but pharmacological manipulation of ryanodine receptors affected both their frequency and amplitude. Staining of live spermatozoa with BODIPY FL-X ryanodine showed localization of ryanodine binding primarily to the caudal part of the head and mid-piece. [Ca(2+)](i) oscillations did not induce acrosome reaction, but in cells generating oscillations, the flagellar beat mode alternated in synchrony with the oscillation cycle. Flagellar bending and lateral movement of the sperm head during [Ca(2+)](i) peaks were markedly increased compared with during [Ca(2+)](i) troughs. This alternating pattern of activity is likely to facilitate zona penetration. These observations show that progesterone initiates unusual and complex store-mediated [Ca(2+)](i) signaling in human spermatozoa and identify a previously unrecognized effect of progesterone in regulating sperm "behavior" during fertilization.     

A constitutively active nonselective cation conductance underlies resting Ca2+ influx and secretion in bovine adrenal chromaffin cells

We have combined fluorimetric measurements of the intracellular free Ca(2+) concentration ([Ca(2+)](i)) with the patch clamp technique, to investigate resting Ca(2+) entry in bovine adrenal chromaffin cells. Perfusion with nominally Ca(2+)-free medium resulted in a rapid, reversible decrease in [Ca(2+)](i), indicating a resting Ca(2+) permeability across the plasma membrane. Simultaneous whole-cell voltage-clamp showed a resting inward current that increased when extracellular Ca(2+) (Ca(2+)(o)) was lowered. This current had a reversal potential of around 0 mV and was carried by monovalent or divalent cations. In Na(+)-free extracellular medium there was a reduction in current amplitude upon removal of Ca(2+)(o), indicating the current can carry Ca(2+). The current was constitutively active and not enhanced by agents that promote Ca(2+)-store depletion such as thapsigargin. Extracellular La(3+) abolished the resting current, reduced resting [Ca(2+)](i) and inhibited basal secretion. Abolishment of resting Ca(2+) influx depleted the inositol 1,4,5-trisphosphate-sensitive Ca(2+) store without affecting the caffeine-sensitive Ca(2+) store. The results indicate the presence of a constitutively active nonselective cation conductance, permeable to both monovalent and divalent cations, that can regulate [Ca(2+)](i), the repletion state of the intracellular Ca(2+) store and the secretory response in resting cells.     

Dichlorodiphenylchloroethylene elevates cytosolic calcium concentrations and oscillations in primary cultures of human granulosa-lutein cells

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), a metabolite of DDT (1,1-dichlorodiphenyltrichloroethane), is a persistent hormonally active environmental toxicant that has been found in human serum and follicular fluid. The objective of this study was to determine whether DDE can alter free calcium ion concentrations in the cytosol ([Ca(2+)](cyt)) of human granulosa cells. Changes in [Ca(2+)](cyt) in single cells loaded with Fura-2 were studied using a dynamic digital Ca(2+) imaging system. At a concentration of 100 ng/ml, DDE stimulated small elevations of [Ca(2+)](cyt) accompanied by Ca(2+) oscillations. At 1 microg DDE/ml, there was a biphasic Ca(2+) response with marked elevations of [Ca(2+)](cyt) over time. In Ca(2+)-free medium, cells showed an initial small elevation of [Ca(2+)](cyt), which was magnified after addition of Ca(2+) to the medium. Washing the cells after DDE treatment failed to remove the elevated [Ca(2+)](cyt) and oscillations, both of which were eliminated by addition of EGTA. ATP also induced [Ca(2+)](cyt) elevations and oscillations, and these effects were potentiated when DDE was added. FSH induced transient [Ca(2+)](cyt) elevations, whereas hCG caused a prolonged elevation and marked oscillations in [Ca(2+)](cyt). These results suggest that DDE at concentrations normally found in human tissues induces elevations in [Ca(2+)](cyt) in granulosa-lutein cells. Our data therefore highlight a novel mechanism through which DDE can alter endocrine homeostasis and possibly act as an endocrine toxicant.     

Nifedipine reveals the existence of two discrete components of the progesterone-induced [Ca2+]i transient in human spermatozoa

The steroid progesterone, an agonist of acrosome reaction, induces a biphasic [Ca(2+)](i)-signal in human sperm comprising an initial transient [Ca(2+)](i) elevation, and a subsequent ramp or plateau. Nifedipine, an inhibitor of voltage-operated Ca(2+) channels, inhibits progesterone-induced acrosome reaction in human sperm, but fluorimetric studies have detected no effect of this compound on the progesterone-induced [Ca(2+)](i) signal. We have used single-cell imaging to study the effects of nifedipine on [Ca(2+)](i) signalling in human sperm. Analysis of mean responses from large numbers of cells showed that treatment with nifedipine reduced the duration but not the amplitude of the progesterone-induced [Ca(2+)](i) transient. In control cells, the latency of the transient peak (maximum fluorescence) fell within the range of 30-105 s. In the presence of nifedipine, very few cells peaked "late" (>60 s after application of progesterone). Analysis of transient responses in control cells revealed characteristic "early" and "late" responses, most cells showing both "early" and "late" transients, whereas "late" transients were rare and smaller in the presence of nifedipine. Sustained responses showed strong association with late transients, and occurrence and amplitude of sustained responses were significantly reduced in nifedipine pretreated cells.These findings are consistent with the occurrence of a discrete, nifedipine-sensitive component of the progesterone-induced [Ca(2+)](i) transient that peaks 1-2 min after exposure to the hormone and is crucial for the induction of the sustained [Ca(2+)](i) signal.     

Effect of clomiphene on Ca(2+) movement in human prostate cancer cells

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca(2+) indicator. Clomiphene at concentrations between 10-50 microM increased [Ca(2+)](i) in a concentration-dependent manner. The [Ca(2+)](i) signal was biphasic with an initial rise and a slow decay. Ca(2+) removal inhibited the Ca(2+) signal by 41%. Adding 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with clomiphene in Ca(2+)-free medium, confirming that clomiphene induced Ca(2+) entry. In Ca(2+)-free medium, pretreatment with 50 microM brefeldin A (to permeabilize the Golgi complex), 1 microM thapsigargin (to inhibit the endoplasmic reticulum Ca(2+) pump), and 2 microM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 microM clomiphene-induced store Ca(2+) release. Conversely, pretreatment with 50 microM clomiphene in Ca(2+)-free medium abolished the [Ca(2+)](i) increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 microM clomiphene-induced Ca(2+)release was unaltered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 microM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca(2+)](i) increases in PC3 cells by releasing store Ca(2+) from multiple stores in an phospholipase C-independent manner, and by activating Ca(2+) influx; and clomiphene was of mild cytotoxicity.     

Characterization of intracellular Ca(2+) increase in response to progesterone and cyclic nucleotides in mouse spermatozoa

Rises in intracellular Ca(2+) concentration ([Ca(2+)](i)) caused by progesterone, an inducer of the acrosome reaction, or by cyclic nucleotides, possible second messengers, were investigated by Ca(2+) imaging of the head of individual mouse sperm. Progesterone induced a [Ca(2+)](i) rise in a dose-dependent manner (4-40 microM), primarily in the postacrosomal region. For 20-microM progesterone, Ca(2+) responses occurred in 42% of sperm, separated into two types: transient type (60% of responding cells; duration, 1-1.5 min; mean amplitude, 335 nM) and prolonged type (40%; >3 min; 730 nM). Prolonged responses required higher doses of progesterone, and their occurrence was enhanced significantly by preincubation for 2-4 h as compared with transient responses. 8-Bromo-cGMP (0.3-3 mM) induced a [Ca(2+)](i) rise more effectively than did 8-bromo-cAMP. For 1-mM 8-bromo-cGMP, 90% of cells exhibited transient Ca(2+) responses (approximately 1 min; 220 nM), independently of the preincubation time. In Ca(2+)-free medium, most sperm showed no Ca(2+) response to progesterone and 8-bromo-cGMP. Pimozide, a Ca(2+) channel blocker, completely blocked prolonged responses and partially inhibited transient responses. These results suggest that progesterone activates at least two distinct Ca(2+) influx pathways, with fast or slow inactivation kinetics, and some sperm show both types of response. A cyclic nucleotide-mediated process could participate in the progesterone-induced [Ca(2+)](i) rise.     

Gonadotropin-releasing hormone-stimulated sperm binding to the human zona is mediated by a calcium influx

The mechanism by which GnRH increases sperm-zona pellucida binding in humans was investigated in this study. We tested whether GnRH increases sperm-zona binding in Ca(2+)-free medium and in the presence of Ca(2+) channel antagonists. We also examined the GnRH effect on the intracellular free Ca(2+) concentration ([Ca(2+)](i)). Sperm treatment with GnRH increased sperm-zona binding 300% but only when Ca(2+) was present in the medium. In Ca(2+)-free medium or in the presence of 400 nM nifedipine, 80 microM diltiazem, or 50 microM verapamil, GnRH did not influence sperm-zona binding. GnRH increased the [Ca(2+)](i) in the sperm in a dose-dependent manner. The maximum effect was reached with 75 nM GnRH. The GnRH-induced increase in [Ca(2+)](i) was fast and transient, from a basal [Ca(2+)](i) of 413 +/- 22 nM to a peak value of 797 +/- 24 nM. The GnRH-induced increase in [Ca(2+)](i) was entirely due to a Ca(2+) influx from the extracellular medium because the increase in [Ca(2+)](i) was blocked by the Ca(2+) chelator EGTA and by the Ca(2+) channel antagonists nifedipine and diltiazem. These antagonists, however, were not able to inhibit the progesterone-activated Ca(2+) influx. On the contrary, T-type calcium channel antagonists pimozide and mibefradil did not affect GnRH-activated Ca(2+) influx but inhibited the progesterone-activated Ca(2+) influx. Finally, the GnRH-induced Ca(2+) influx was blocked by two specific GnRH antagonists, Ac-D-Nal(1)-Cl-D-Phe(2)-3-Pyr-D-Ala(3)-Arg(5)-D-Glu(AA)(6)-GnRH and Ac-(3,4)-dehydro-Pro(1),-p-fluoro-D-Phe(2), D-Trp(3,6)-GnRH. These results suggest that GnRH increases sperm-zona binding via an elevation of [Ca(2+)](i) through T-type, voltage-operated calcium channels.     

Increase of [Ca(2+)](i) via activation of ATP receptors in PC-Cl3 rat thyroid cell line.

In PC-Cl3 rat thyroid cell line, ATP and UTP provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Thapsigargin (TG) caused a rapid rise in [Ca(2+)](i) and subsequent addition of ATP was without effect. The transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with the specific inhibitor of phospholipase C (PLC), U73122. These data suggest that the ATP-stimulated increment of [Ca(2+)](i) required InsP(3) formation and binding to its specific receptors in Ca(2+) stores. Desensitisation was demonstrated with respect to the calcium response to ATP and UTP in Fura 2-loaded cells. Further studies were performed to investigate whether the effect of ATP on Ca(2+) entry into PC-Cl3 cells was via L-type voltage-dependent Ca(2+) channels (L-VDCC) and/or by the capacitative pathway. Nifedipine decreased ATP-induced increase on [Ca(2+)](i). Addition of 2 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment of the cells with TG or with 100 microM ATP in Ca(2+)-free medium. These data indicate that Ca(2+) entry into PC-Cl3 stimulated with ATP occurs through both an L-VDCC and through a capacitative pathway. Using buffers with differing Na(+) concentrations, we found that the effects of ATP were dependent of extracellular Na(+), suggesting that a Na(+)/Ca(2+) exchange mechanism is also operative. These data suggest the existence, in PC-Cl3 cell line, of a P2Y purinergic receptor able to increase the [Ca(2+)](i) via PLC activation, Ca(2+) store depletion, capacitative Ca(2+) entry and L-VDCC activation.     

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.     

Effect of hydrogen peroxide on secretory response, calcium mobilisation and caspase-3 activity in the isolated rat parotid gland

The parotid glands are highly active secretory systems subjected to continuous stress, which in turn, can lead to several pathophysiological conditions. Damage of the parotid glands are caused by radical oxygen species (ROS) as by-products of oxygen metabolism. This study investigated the effect of hydrogen peroxide (H(2)O(2)) on Carbachol (CCh)-evoked secretory responses and caspase-3 activity in the isolated rat parotid gland to understand the role of oxidative stress on the function of the gland. Amylase secretion, cytosolic calcium concentration ([Ca(2+)](i)) and caspase-3 activity in parotid gland tissue were measured using fluorimetric methods. H(2)O(2) had little or no effect on amylase secretion compared to basal level. Combining H(2)O(2) with CCh resulted in an attenuation of the CCh-evoked amylase secretion compared to the effect of CCh alone. CCh can evoke a large increase in [Ca(2+)](i) comprising an initial peak followed by a plateau. In a Ca(2+)-free medium containing 1 mM EGTA, CCh evoked only the initial peak of [Ca(2+)](i). H(2)O(2) alone evoked a gradual and dose-dependent increase in [Ca(2+)](i). Combining H(2)O(2) with CCh resulted in a decrease in [Ca(2+)](i) compared to the effect of CCh alone. In a Ca(2+)-free medium, H(2)O(2) still evoked a small increase in [Ca(2+)](i), but this response was less compared to the results obtained with H(2)O(2) in normal [Ca(2+)](0). Combining H(2)O(2) with CCh resulted in only a small transient increase in [Ca(2+)](i). Following CCh stimulation, H(2)O(2) application resulted in a large increase in [Ca(2+)](i) in normal [Ca(2+)](0). This effect of H(2)O(2) was partially abolished in a nominally free Calcium medium containing EGTA. H(2)O(2) can stimulate caspase-3 activity in parotid gland tissue. Similar response was obtained with betulinic acid and thapsigargin (TPS) on caspase-3 activity compared to basal. The results have demonstrated that like CCh, H(2)O(2) can also mobilise Ca(2+) from intracellular stores and facilitate its influx into the cell from extracellular medium. This effect of H(2)O(2) may be due to its activity to induce apoptosis in the parotid gland, since H(2)O(2) can stimulate the activity of caspase-3, a marker of cellular apoptosis.     

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.     

Biphasic elevation of [Ca(2+)](i) in individual human spermatozoa exposed to progesterone

Fluorimetric studies on progesterone-induced [Ca(2+)](i) signalling in mammalian spermatozoa show both the well-characterised [Ca(2+)](i) transient and a subsequent sustained phase. However, the sustained phase is thought to reflect release of the fluorochrome during the acrosome reaction and has not been subject to critical investigation. We have used single-cell imaging of [Ca(2+)](i) to analyse the progesterone-induced [Ca(2+)](i) response in large numbers (>2000) of capacitated, human spermatozoa. In 70% of cells, treatment with progesterone induced a transient increase, which typically peaked within 1 min and decayed with a similar time course. Upon rapid application of progesterone this response peaked within 5-20 s. In 35% of progesterone-treated spermatozoa a sustained elevation of [Ca(2+)](i) occurred, which became discernible during the falling phase of the transient response and persisted for at least 20 min. Both [Ca(2+)](i) responses were localised to the postacrosomal region. Averaging of large numbers of single cell responses generated traces similar to those seen in fluorimetric studies. Although the sustained response was strongly associated with the initial, transient response, a few spermatozoa generated sustained responses that were not preceded by a significant transient response (5% of cells). It is concluded that a genuine biphasic [Ca(2+)](i) signal is activated by progesterone and that the sustained response is a discrete signalling event with biological significance.     

Thiol oxidation by 2,2'-dithiodipyridine induced calcium mobilization in MG63 human osteosarcoma cells

2,2'-dithiodipyridine (2,2'-DTDP), a reactive disulphide that mobilizes Ca(2+) in muscle, induced an increase in cytoplasmic free Ca(2+)concentrations ([Ca(2+)](i)) in MG63 human osteosarcoma cells loaded with the Ca(2+)-sensitive dye fura-2. 2,2'-DTDP acted in a concentration-independent manner with an EC(50) of 50 microM. The Ca(2+) signal comprised an initial spike and a prolonged increase. Removing extracellular Ca(2+) did not alter the Ca(2+) signal, suggesting that the Ca(2+) signal was due to store Ca(2+) release. In Ca(2+)-free medium, the 2,2'-DTDP-induced [Ca(2+)](i) increase was not changed by depleting store Ca(2+) with 50 microM bredfeldin A (a Golgi apparatus permeabilizer), 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler), 1 microM thapsigargin (an endoplasmic reticulum Ca(2+)pump inhibitor) or 5 microM ryanodine. Conversely, 2,2'-DTDP pretreatment abolished CCCP and thapsigargin-induced [Ca(2+)](i) increases. 2,2'-DTDP-induced Ca(2+) signals in Ca(2+)-containing medium were not affected by modulation of protein kinase C activity or suppression of phospholipase C activity. However, 2,2'-DTDP-induced Ca(2+) release was inhibited by a thiol-selective reducing reagent, dithiothreitol (5-25 microM) in a concentration-dependent manner. Collectively, this study shows that 2,2'-DTDP induced [Ca(2+)](i) increases in human osteosarcoma cells via releasing store Ca(2+)from multiple stores in a manner independent of protein kinase C or phospholipase C activity. The 2,2'-DTDP-induced store Ca(2+) release appeared to be dependent on oxidation of membranes.     

Characterization of intracellular Ca(2+) stores in gallbladder smooth muscle

The existence of functionally distinct intracellular Ca(2+) stores has been proposed in some types of smooth muscle. In this study, we sought to examine Ca(2+) stores in the gallbladder by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura 2-loaded isolated myocytes, membrane potential in intact smooth muscle, and isometric contractions in whole mount preparations. Exposure of isolated myocytes to 10 nM CCK caused a transient elevation in [Ca(2+)](i) that persisted in Ca(2+)-free medium and was inhibited by 2-aminoethoxydiphenylborane (2-APB). Application of caffeine induced a rapid spike-like elevation in [Ca(2+)](i) that was insensitive to 2-APB but was abolished by pretreatment with 10 muM ryanodine. These data support the idea that both inositol trisphosphate (IP(3)) receptors (IP(3)R) and ryanodine receptors (RyR) are present in this tissue. When caffeine was applied in Ca(2+)-free solution, the [Ca(2+)](i) transients decreased as the interval between Ca(2+) removal and caffeine application was increased, indicating a possible leakage of Ca(2+) in these stores. The refilling of caffeine-sensitive stores involved sarcoendoplasmic reticulum Ca(2+)-ATPase activation, similar to IP(3)-sensitive stores. The moderate Ca(2+) elevation caused by CCK was associated with a gallbladder contraction, but caffeine or ryanodine failed to induce gallbladder contraction. Nevertheless, caffeine caused a concentration-dependent relaxation in gallbladder strips either under resting tone conditions or precontracted with 1 muM CCK. Taken together, these results suggest that, in gallbladder smooth muscle, multiple pharmacologically distinct Ca(2+) pools do not exist, but IP(3)R and RyR must be spatially separated because Ca(2+) release via these pathways leads to opposite responses.     

Tamoxifen-induced [Ca2+]i rises and Ca2+-independent cell death in human oral cancer cells

The purpose of this study was to explore the effect of tamoxifen on cytosolic free Ca(2+) concentrations ([Ca(2+)](i)) and cell viability in OC2 human oral cancer cells. [Ca(2+)](i) and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Tamoxifen at concentrations above 2 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). The tamoxifen-induced Ca(2+) influx was sensitive to blockade of L-type Ca(2+) channel blockers but insensitive to the estrogen receptor antagonist ICI 182,780 and protein kinase C modulators. In Ca(2+)-free medium, after pretreatment with 1 muM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), tamoxifen-induced [Ca(2+)](i) rises were substantially inhibited; and conversely, tamoxifen pretreatment inhibited a part of thapsigargin-induced [Ca(2+)](i) rises. Inhibition of phospholipase C with 2 microM U73122 did not change tamoxifen-induced [Ca(2+)](i) rises. At concentrations between 10 and 50 microM tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 23 microM tamoxifen was not reversed by prechelating cytosolic Ca(2+) with BAPTA. Collectively, in OC2 cells, tamoxifen induced [Ca(2+)](i) rises, in a nongenomic manner, by causing Ca(2+) release from the endoplasmic reticulum, and Ca(2+) influx from L-type Ca(2+) channels. Furthermore, tamoxifen-caused cytotoxicity was not via a preceding [Ca(2+)](i) rise.     

Connexin 43 hemichannels contribute to cytoplasmic Ca2+ oscillations by providing a bimodal Ca2+-dependent Ca2+ entry pathway

Many cellular functions are driven by changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) that are highly organized in time and space. Ca(2+) oscillations are particularly important in this respect and are based on positive and negative [Ca(2+)](i) feedback on inositol 1,4,5-trisphosphate receptors (InsP(3)Rs). Connexin hemichannels are Ca(2+)-permeable plasma membrane channels that are also controlled by [Ca(2+)](i). We aimed to investigate how hemichannels may contribute to Ca(2+) oscillations. Madin-Darby canine kidney cells expressing connexin-32 (Cx32) and Cx43 were exposed to bradykinin (BK) or ATP to induce Ca(2+) oscillations. BK-induced oscillations were rapidly (minutes) and reversibly inhibited by the connexin-mimetic peptides (32)Gap27/(43)Gap26, whereas ATP-induced oscillations were unaffected. Furthermore, these peptides inhibited the BK-triggered release of calcein, a hemichannel-permeable dye. BK-induced oscillations, but not those induced by ATP, were dependent on extracellular Ca(2+). Alleviating the negative feedback of [Ca(2+)](i) on InsP(3)Rs using cytochrome c inhibited BK- and ATP-induced oscillations. Cx32 and Cx43 hemichannels are activated by <500 nm [Ca(2+)](i) but inhibited by higher concentrations and CT9 peptide (last 9 amino acids of the Cx43 C terminus) removes this high [Ca(2+)](i) inhibition. Unlike interfering with the bell-shaped dependence of InsP(3)Rs to [Ca(2+)](i), CT9 peptide prevented BK-induced oscillations but not those triggered by ATP. Collectively, these data indicate that connexin hemichannels contribute to BK-induced oscillations by allowing Ca(2+) entry during the rising phase of the Ca(2+) spikes and by providing an OFF mechanism during the falling phase of the spikes. Hemichannels were not sufficient to ignite oscillations by themselves; however, their contribution was crucial as hemichannel inhibition stopped the oscillations.     

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.     

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.     

cGMP stimulates endoplasmic reticulum Ca(2+)-ATPase in vascular endothelial cells

Calcium is a crucial regulator of many physiological processes such as cell growth, division, differentiation, cell death and apoptosis. In this study, we examined the effect of cGMP on agonist-induced [Ca(2+)](i) transient in isolated rat aortic endothelial cells. 100 microM ATP was applied to the cells bathed in a Ca(2+)-free physiological solution to induce a [Ca(2+)](i) transient that was caused by Ca(2+) release from intracellular stores. cGMP, which was applied after [Ca(2+)](i) reached its peak level, accelerated the falling phase of [Ca(2+)](i) transient. Pre-treatment of the cells with CPA abolished the accelerating effect of cGMP on the falling phase of [Ca(2+)](i) transient. The effect of cGMP was reversed by KT5823, a highly specific inhibitor of protein kinase G. Taken together, these data suggest that cGMP may reduce [Ca(2+)](i) level by promoting Ca(2+) uptake through sarcoplasmic/endoplasmic reticulum ATPase and that the effect of cGMP may be mediated by protein kinase G.