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Previous research demonstrated that rotenone (RT) induces neuronal injury partially by increasing intracellular Ca(2+) concentrations ([Ca(2+)]i), and inducing oxidative stress, leading to a neurodegenerative disorder. However, the mechanism of RT-induced injury remains elusive. Recent work revealed that Ca(2+) signaling is important for RT-induced senescence in human neuroblastoma SH-SY5Y cells. In the present study, we found that in SH-SY5Y cells, RT increased [Ca(2+)]i, senescence associated β-galactosidase activity, aggregation of lipofuscin, production of reactive oxygen species, G1/G0 cell cycle arrest, and activation of p53/p21 signaling proteins. In addition, RT decreased the expression of the signaling proteins for cell proliferation and survival, Cyclin-dependent kinase 2 (CDK2), cyclin D1, and Akt. Pretreatment of SH-SY5Y cells with isradipine, an L-type Ca(2+) channel blocker, or EGTA antagonized these effects of RT. These results suggested that application of isradipine might be a novel approach to prevent RT-induced neurodegenerative disorder such as Parkinson's disease., (Copyright © 2013 IBRO. All rights reserved.)

1. The mechanisms underlying AVP-induced increase in [Ca(2+)](i) and glucagon release in clonal alpha-cells In-R1-G9 were investigated. 2. AVP increased [Ca(2+)](i) and glucagon release in a concentration-dependent manner. After the administration of AVP, glucagon was released within 30 s, quickly reached the maximum within 2 min, and maintained a steady-state concentration for at least 15 min. 3. In Ca(2+)-containing medium, AVP increased [Ca(2+)](i) in a biphasic pattern; a peak followed by a sustained plateau. In Ca(2+)-free medium, the Ca(2+) response to AVP became monophasic with lower amplitude and no plateau. Both the basal and AVP-induced glucagon releases were lower in the absence than in the presence of extracellular Ca(2+). When [Ca(2+)](i) was stringently deprived by BAPTA, a Ca(2+) chelator, AVP still significantly increased glucagon release. 4. Pretreatment with thapsigargin, a microsomal Ca(2+) ATPase inhibitor, abolished both the Ca(2+) peak and sustained plateau. 5.AVP increased intracellular concentration of IP(3). 6. U-73122 (8 microM), a phospholipase C inhibitor, abolished AVP-induced increases in [Ca(2+)](i), but only reduced AVP-induced glucagon release by 39%. 7. Pretreatment with nimodipine, an L-type Ca(2+) channel blocker failed to alter AVP-induced glucagon release or increase in [Ca(2+)](i). 8. The results suggest that AVP causes glucagon release through both Ca(2+)-dependent and -independent pathways. For the Ca(2+)-dependent pathway, the G(q) protein activates phospholipase C, which catalyzes the formation of IP(3). IP(3) induces Ca(2+) release from the endoplasmic reticulum, which, in turn, triggers Ca(2+) influx. Both Ca(2+) release and Ca(2+) influx may contribute to AVP-induced glucagon release.

Aim: To study the effect of U-73122, putative phospholipase C (PLC) inhibitor, on cytosolic Ca2+ concentration ([Ca2+]i) and voltage-dependent calcium channels (VDCC)., Methods: To record whole cell calcium current (ICa) by perforated patch-clamp and to measure cytosolic free calcium concentration ([Ca2+]i) with Fura-2., Results: U-73122 inhibited KCl-induced and Bay-K-8644-induced increase in [Ca2+]i dose-dependently. U-73122 decreased markedly depolarization-induced ICa both in RINm5F and porcine myometrial cells in a concentration-dependent manner. The effect of U-73122 was more potent in porcine myometrial cells than that in RINm5F cells. U-73343 (an analog of U-73122), that does not inhibit PLC, also reduced the ICa by 50%., Conclusion: U-73122 inhibited VDCC and [Ca2+]i in both RINm5F and porcine myometrial cells.

We studied the mechanisms underlying alpha2-adrenergic receptor (AR)-mediated increase in intracellular free calcium ([Ca2+]i) in freshly dispersed myometrial cells from sows in the luteal phase of the estrous cycle. After the blockade of beta-ARs with propranolol, epinephrine increased [Ca2+]i dose-dependently in both the presence and absence of extracellular Ca2+. The rank order of alpha antagonists in inhibiting [Ca2+]i response to epinephrine was yohimbine > WB4101 >> prazosin in both the presence and absence of extracellular Ca2+, suggesting that epinephrine acts on alpha(2A)-ARs to increase Ca2+ influx as well as Ca2+ release from intracellular stores. Thapsigargin, the blocker of the Ca2+ pump in the sarcoplasmic reticulum, abolished the release but did not affect the influx. Pertussis toxin (PTX) inhibited the influx but failed to change the release. Nimodipine, an L-type Ca2+ channel blocker, nearly abolished the influx. The peak increase in [Ca2+]i caused by epinephrine was reached within 20 sec of administration. Intracellular cAMP concentrations were also decreased at 20 sec post-epinephrine. Epinephrine enhanced the L-type Ca2+ channel current, whereas forskolin suppressed it. Maximization of intracellular cAMP content by applying 8-bromo-cAMP (100 microM) blocked the effect of epinephrine on the current. U-73122, a phospholipase C inhibitor, reduced the Ca2+ release by epinephrine and oxytocin. Our results suggested that 1) activation of alpha2-ARs induces Ca2+ influx through opening L-type Ca2+ channels as well as inducing Ca2+ release from intracellular stores, and 2) a PTX-sensitive G protein couples negatively to adenylyl cyclase, leading to a decrease in cAMP formation which may be involved in the activation of Ca2+ channels. In addition, our results are consistent with the coupling of alpha2-ARs to a PTX-insensitive G protein (G(q)) to release Ca2+ from intracellular stores.

We studied the effects of caffeine on intracellular calcium in a clonal insulin-secreting cell line RINm5F. Caffeine (1-30 mM) induced a dose-dependent intracellular Ca2+ release and Ca2+ influx. Pretreatment with combination of ryanodine (10 microM) and caffeine (10 mM), but not ryanodine alone, abolished subsequent caffeine-induced release of intracellular Ca2+. Pretreatment with another ryanodine receptor blocker procaine (0.1, 0.3, and 1mM) antagonized the caffeine-induced release of intracellular Ca2+. Pretreatment with thapsigargin (2 microM), the endoplasmic reticular Ca2+-ATPase inhibitor, abolished both the caffeine- and arginine vasopressin (AVP)-induced Ca2+ release. AVP induces Ca2+ release by increasing the formation of IP3. Pretreatment with AVP greatly reduced caffeine-induced Ca2+ release whereas pretreatment with caffeine also reduced AVP-induced Ca2+ release. The L-type Ca2+ channel blocker nimodipine (1 microM) inhibited caffeine-induced Ca2+ influx. In addition, depletion of intracellular Ca2+ stores with thapsigargin did not affect caffeine-induced Ca2+ influx. These results suggested that: 1) the ryanodine- and caffeine-sensitive Ca2+ store exists in RINm5F cells, 2) most of these Ca2+ release channels in RINm5F cells are closed in the resting state, since pretreatment with ryanodine alone failed to block the caffeine-induced Ca2+ release, 3) there is a "cross-talk" between the caffeine- and IP3-sensitive pools in RINm5F cells, 4) caffeine increases Ca2+ entry in RINm5F cells through L-type voltage-dependent Ca2+ channels, and 5) the caffeine-induced Ca2+ influx is independent of the Ca2+ release from the intracellular Ca2+ stores.

The mechanisms for Ca++ release from caffeine-sensitive stores were investigated in freshly dispersed porcine myometrial cells utilizing the fura-2 method. Because the caffeine-sensitive Ca++ store has not been detected in myometrium of mammals, we first determined the existence of this type of store in porcine myometrial cells. The evidence includes: 1) caffeine (1-33 mM)-induced concentration-dependent increase in the intracellular Ca++ concentration ([Ca++]i) in both the presence and absence of extracellular Ca++ and 2) although ryanodine alone (< or = 10 microM) failed to change [Ca++]i, it inhibited the response to caffeine in a use-, concentration- and time-dependent manner. In the cell suspension study, the amount of Ca++ released by 10 mM caffeine was found to be inversely proportional to the amount released by preadministration of caffeine (1-33 mM). In the single cell study, about 30% of cells responded to only a certain concentration of caffeine and the others responded to caffeine gradually. Thapsigargin, an inhibitor of Ca(++)-adenosine triphosphatase in sarcoplasmic reticulum, failed to increase [Ca++]i. Pretreatment with thapsigargin inhibited the response to caffeine in a time- and concentration-dependent manner. These results suggest that in porcine myometrial cells: 1) the Ca++ released from the caffeine- and ryanodine-sensitive store is in an all-or-none manner through compartments of stores or the entire store of a cell and 2) the release process is regulated by luminal Ca++ content of the stores.

This study investigated the underlying mechanisms of oxytocin (OT)-induced increases in intracellular Ca2+ concentrations ([Ca2+]i) in acutely dispersed myometrial cells from prepartum sows. A dose-dependent increase in [Ca2+]i was induced by OT (0.1 nM to 1 microM) in the presence and absence of extracellular Ca2+ ([Ca2+]e). [Ca2+]i was elevated by OT in a biphasic pattern, with a spike followed by a sustained plateau in the presence of [Ca2+]e. However, in the absence of [Ca2+]e, the [Ca2+]i response to OT became monophasic with a lower amplitude and no plateau, and this monophasic increase was abolished by pretreatment with ionomycin, a Ca2+ ionophore. Administration of OT (1 microM) for 15 sec increased inositol 1,4,5-trisphosphate (IP3) formation by 61%. Pretreatment with pertussis toxin (PTX, 1 microgram/ml) for 2 hr failed to alter the OT-induced increase in [Ca2+]i and IP3 formation. U-73122 (30 nM to 3 microM), a phospholipase C (PLC) inhibitor, depressed the rise in [Ca2+]i by OT dose dependently. U-73122 (3 microM) also abolished the OT-induced IP3 formation. Thapsigargin (2 microM), an inhibitor of Ca(2+)-ATPase in the endoplasmic reticulum, did not increase [Ca2+]i. However, it did time-dependently inhibit the OT-induced increase in [Ca2+]i. Nimodipine (1 microM), a voltage-dependent Ca2+ channel (VDCC) blocker, inhibited the OT-induced plateau by 26%. La3+ (1 mM), a nonspecific Ca2+ channel blocker, abrogated the OT-induced plateau. In whole-cell patch-clamp studies used to evaluate VDCC activities, OT (0.1 microM) increased Ca2+ current (ICa) by 40% with no apparent changes in the current-voltage relationship.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study was undertaken to investigate whether lysine vasopressin (LVP) induces porcine myometrial contractions by activating vasopressin or oxytocin (OT) receptors. Both LVP (3 x 10(-9)-10(-6) M) and OT (3 x 10(-11)-10(-8) M) increased contractility dose-dependently in myometrium from both the luteal phase and prepartum period. Comparison of EC50s showed that OT was 75 and 57 times more potent than LVP in increasing myometrial contractility in pregnant and nonpregnant sows, respectively. L-366,948 (10(-8), 3 x 10(-8), 10(-7) M), a highly selective OT receptor antagonist, inhibited LVP- and OT-induced increases in myometrial contractility dose-dependently in both pregnant and nonpregnant tissues with similar antagonist affinity values (pA2). The V1 antagonist d(CH2)5[D-[Tyr(Me)2]AVP also antagonized both LVP- and OT-induced increases in myometrial contractility, but higher concentrations (10(-7) and 10(-6) M) were required to achieve the antagonism. The V2 antagonist Aaa-D-Tyr(Et)-Phe-Val-Asn-Abu-Pro-Arg-Arg-NH2 (10(-6) M) did not alter this effect of LVP and OT. LVP (10(-9)-10(-6) M) and OT (10(-10)-10(-7) M) also increased intracellular Ca2+ ([Ca2+]i) concentrations in porcine myometrial cells from sows during the prepartum period in a dose-dependent manner, with OT being 14 times more potent than LVP. L-366,948 (10(-9)-3 x 10(-8) M) antagonized the effect of OT (10(-7) M) and LVP (10(-6) M) in a similar dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied the effects of the aminosteroid U-73122, a putative phospholipase C (PLC) inhibitor, on carbachol-induced increases in insulin release, [Ca2+]i, and IP3 in beta-TC3 cells. Carbachol (0.1-100 microM) increased [Ca2+]i and carbachol (0.1-1000 microM) increased insulin release dose-dependently. Carbachol (100 microM) also increased inositol 1,4,5-trisphosphate (IP3) production. U-73122 (2-12 microM) inhibited the effects of carbachol on [Ca2+]i and insulin release in a dose-dependent manner, and at the highest dose studied (12 microM) it abolished or greatly attenuated all three effects of carbachol. In contrast, U-73343 (12 microM), the analog of U-73122 that does not inhibit PLC, only inhibited the effect of carbachol on [Ca2+]i by 20%, and did not inhibit the effect of carbachol on insulin release. Since carbachol increases IP3, [Ca2+]i, and insulin release by activating PLC, these results suggested that U-73122 inhibits phospholipase C-dependent processes in beta-TC3 cells.

The present study investigated the mechanism by which arginine vasopressin (AVP) increases insulin secretion in rat insulinoma (RINm5F) cells by using a specific phospholipase C (PLC) inhibitor, 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17- yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), and a phospholipase A2 (PLA2) inhibitor, N-(p-amylcinnamoyl)anthranilic acid (ACA). AVP (0.1-100 nM) increased insulin secretion and cytosolic free Ca++ concentration ([Ca++]i) dose-dependently. AVP-induced increases in the intracellular concentration of inositol 1,4,5-trisphosphate (IP3) and [Ca++]i were dose-dependently inhibited by U-73122 (2-8 microM). At 8 microM, U-73122 abolished AVP's effect on IP3 and [Ca++]i, but AVP-induced increases in insulin secretion were only reduced by 35%. In contrast, 8 microM U-73122 did not reduce the ionomycin (a Ca++ ionophore, 100 nM)-induced increase in [Ca++]i. The discrepancy between the results of [Ca++]i and insulin secretion in U-73122 experiments is indicative of the multiple signal transduction pathways associated with the activation of AVP receptors, specifically the Ca(++)-independent pathway. The phospholipase A2 inhibitor ACA (100 microM) did not antagonize AVP (10 nM)-induced increases in insulin secretion. These results suggested: 1) U-73122 blocks PLC activities but fails to block other signal transduction pathways that trigger insulin secretion in these cells and 2) AVP increases insulin release from RINm5F cells through both the PLC-mediated Ca(++)-dependent and Ca(++)-independent pathways.

Activation of the sympathetic nervous system inhibits insulin secretion. We tested the hypothesis that activation of alpha 2-adrenergic receptors on the beta-cell by epinephrine or clonidine attenuates insulin release by an effect on the voltage-dependent Ca2+ channel (VDCC) and examined the role of G-proteins in this signal transduction pathway. Using a cultured SV40-transformed hamster beta-cell line (HIT cells) as a model system, we determined the effect of alpha 2-adrenergic agonists on insulin secretion, 86Rb+ efflux (a marker for K+ channel flux), and the free cytosolic Ca2+ level [( Ca2+]i) monitored in fura-2-loaded cells. In a dose-dependent manner, epinephrine and clonidine (10(-8)-10(-5)M) attenuated the increase in [Ca2+]i and insulin secretion induced by either K+ depolarization or stimulation of the VDCC with the agonist Bay K 8644. Epinephrine failed to affect the rise in [Ca2+]i induced by carbamylcholine, an agent that mobilizes intracellular Ca2+. Epinephrine also did not changes 86Rb+ efflux from HIT cells. The inhibitory effects of epinephrine were prevented by the alpha 2-adrenergic antagonist idazoxan, but were unaffected by the alpha 1-adrenergic antagonist phenoxybenzamine. Pretreatment of HIT cells with pertussis toxin (0.1 micrograms/ml) overnight abolished the inhibitory effects of epinephrine and clonidine on both [Ca2+]i and insulin secretion. These data suggest that one mechanism by which alpha 2-adrenergic agonists inhibit insulin secretion is by inhibiting Ca2+ influx through VDCC, an action that is mediated through a pertussis toxin-sensitive G-protein.

We tested the hypothesis that somatostatin (SRIF) inhibits insulin secretion from an SV40 transformed hamster beta cell line (HIT cells) by an effect on the voltage-dependent Ca2+ channels and examined whether G-proteins were involved in the process. Ca2+ currents were recorded by the whole cell patch-clamp method, the free cytosolic calcium, [Ca2+]i, was monitored in HIT cells by fura-2, and cAMP and insulin secretion were measured by radioimmunoassay. SRIF decreased Ca2+ currents, [Ca2+]i, and basal insulin secretion in a dose-dependent manner over the range of 10(-12)-10(-7)M. The increase in [Ca2+]i and insulin secretion induced by either depolarization with K+ (15 mM) or by the Ca2+ channel agonist, Bay K 8644 (1 microM) was attenuated by SRIF in a dose-dependent manner over the same range of 10(-12)-10(-7) M. the half-maximal inhibitory concentrations (IC50) for SRIF inhibition of insulin secretion were 8.6 X 10(-12) M and 8.3 X 10(-11) M for K+ and Bay K 8644-stimulated secretion and 1 X 10(-10) M and 2.9 X 10(-10) M for the SRIF inhibition of the K+ and Bay K 8644-induced rise in [Ca2+]i, respectively. SRIF also attenuated the rise in [Ca2+]i induced by the cAMP-elevating agent, isobutylmethylxanthine (1 mM) in the presence of glucose. Bay K 8644, K+ and SRIF had no significant effects on cAMP levels and SRIF had no effects on adenylyl cyclase activity at concentrations lower than 1 microM. SRIF (100 nM) did not change K+ efflux (measured by 86Rb+) through ATP-sensitive K+ channels in HIT cells. SRIF (up to 1 microM) had no significant effect on membrane potential measured by bisoxonol fluorescence. Pretreatment of the HIT cells with pertussis toxin (0.1 microgram/ml) overnight abolished the effects of SRIF on Ca2+ currents, [Ca2+]i and insulin secretion implying a G-protein dependence in SRIF's actions. Thus, one mechanism by which SRIF decreases insulin secretion is by inhibiting Ca2+ influx through voltage-dependent Ca2+ channels, an action mediated through a pertussis toxin-sensitive G-protein.