Increased intracellular Ca2+ signaling caused by the antitumor agent helenalin and its analogues

The antitumor sesquiterpene lactone helenalin, which is found in species of the plant genus Helenium, caused a marked potentiation of the increases in intracellular free Ca2+ concentration ([Ca2+]i) produced by mitogens such as vasopressin, bradykinin, and platelet-derived growth factor in Swiss mouse 3T3 fibroblasts. Removing external Ca2+ partly attenuated the increased [Ca2+]i responses caused by helenalin. The increased [Ca2+]i responses occurred at concentrations of helenalin that inhibited cell proliferation. At higher concentrations, helenalin inhibited the [Ca2+]i responses. No change in resting [Ca2+]i was caused by helenalin even at high concentrations. Other helenalin analogues also increased the [Ca2+]i response. Helenalin did not inhibit protein kinase C (PKC) and PKC appeared to play a minor role in the effects of helenalin on [Ca2+]i responses in intact cells. Studies with saponin-permeabilized HT-29 human colon carcinosarcoma cells indicated that helenalin caused an increased accumulation of Ca2+ into nonmitochondrial stores and that the potentiating effect of helenalin on mitogen-stimulated [Ca2+]i responses was due in part to an increase in the inositol-(1,4,5)-trisphosphate-mediated release of Ca2+ from these stores.     

Studies on cytosolic 5SrRNA-L5 protein particles (5SRNP) of rat liver

Effects of antiallergic drugs on bradykinin-induced histamine release and intracellular Ca2+ release from peritoneal mast cells were studied in rats. Bradykinin caused a concentration-dependent histamine release as well as Ca2+ release from the intracellular Ca store of peritoneal mast cells. Antiallergic drugs used in this study showed an inhibition of not only histamine release but also Ca2+ release. The Ca2+ release from the intracellular Ca store induced by bradykinin was more sensitive to antiallergic drugs than histamine release from mast cells. Mequitazine and terfenadine caused potent inhibitory effects on both responses, whereas effects of ketotifen and cromolyn sodium were relatively weak. In conclusion, histamine release from mast cells and intracellular C2+ release induced by bradykinin were inhibited by antiallergic drugs similar to those induced by substance P and compound 48/80.     

An immunosuppressive agent, FTY720, increases intracellular concentration of calcium ion and induces apoptosis in HL-60

We previously reported that FTY720 is an efficient inducer of apoptosis in lymphocytes and cultured cell lines. In the present study, HL-60 human promyerocytoma cells also induced apoptosis through in vitro treatment with the drug, demonstrating extensive DNA fragmentation 6 hr after incubation. The major target of FTY720 was the common signalling pathway of apoptosis, since a rapid (< 1 min) increase in the intracellular Ca2+ concentration ([Ca2+]i) was found in the cells treated with the drug. Calcium chelation in the culture medium with EGTA did not affect the [Ca2+]i mobilization. A phospholipase C inhibitor, U73122, inhibited the increase in [Ca2+]i as well as the fragmentation of the nuclear DNA, whereas U73343, a non-effective analogue of U73122, had little effect. These results suggest that FTY720-induced apoptosis is mediated through an activation of phospholipase C and the subsequent release of Ca2+ from intracellular calcium pools. In addition, the treatment of HL-60 with pertussis toxin (PTX) did not inhibit Ca2+ mobilization or apoptosis, suggesting that the activation of phospholipase C is independent of PTX-sensitive G-proteins.     

Cranial asymmetries. Reflexions on plagiocephalies. Premature sutural synostosis or extracranial origin?

The effects of various Ca2+ channel agonists and antagonists on tumor cell growth were investigated using U-373 MG human astrocytoma and SK-N-MC human neuroblastoma cell lines. Classical Ca2+ channel antagonists, verapamil, nifedipine, and diltiazem, and inorganic Ca2+ channel antagonists, Ni2+ and Co2+, inhibited growth of these tumor cells in a dose-dependent manner. Except Ni2+, these Ca2+ channel antagonists did not induce a significant cytotoxicity, suggesting that the growth-inhibitory effects of these drugs may be the result of the influence on the proliferative signaling mechanisms of these tumor cells. In contrast, Bay K-8644, a Ca2+ channel agonist, neither enhanced the growth of tumor cells nor increased intracellular Ca2+ concentration, indicating that voltage-sensitive Ca2+ channels may not be involved in tumor cell proliferation. Moreover, growth-inhibitory concentrations of Ca2+ channel antagonists significantly blocked agonist (carbachol or serum)-induced intracellular Ca2+ mobilization, which was monitored using Fura-2 fluorescence technique. These results suggest that the inhibition of the growth of human brain tumor cells induced by Ca2+ channel antagonists may not be the result of interaction with Ca2+ channels, but may be the result of the interference with agonist-induced intracellular Ca2+ mobilization, which is an important proliferative signaling mechanism.     

Expression of functional P2-purinergic receptors in primary cultures of human colorectal carcinoma cells

Primary cell cultures of human colorectal carcinomas were established and characterized immunocytochemically. In the isolated cancer cells intracellular Ca2+ concentrations ([Ca2+]i) were measured by the fura-2 method. Stimulation with either extracellular ATP or UTP caused a biphasic rise of [Ca2+]i in a dose-dependent manner and cross-desensitization between both nucleotides was observed. The rank order of potency was ATP >== UTP > ATP-gamma-S > ADP > adenosine which is characteristic for a P2U-receptor subtype. Selective agonists of P1-, or P2X- purinoceptors had no effect on [Ca2+]i. The initial rise in [Ca2+]i was independent of extracellular calcium [Ca2+]e, whereas the second phase was not observed under [Ca2+]e-free conditions suggesting a capacitative Ca2+-entry-mechanism. Intracellular Ca2+ mobilization was proven by use of the Ca2+-ATPase inhibitor thapsigargin. P2U-specific mRNA could be detected by RT-PCR in both colorectal tumor tissues and in the human colorectal cancer cell line HT 29. In HT 29 cells, the hydrolysis-resistant ATP analog ATP-gamma-S inhibited cell proliferation and, also, induced apoptosis in a dose-dependent manner. Thus, human colorectal cancer cells express functional P2U-receptors which may play a role in the regulation of cell proliferation and apoptosis.     

LTP induction dependent on activation of Ni2+-sensitive voltage-gated calcium channels, but not NMDA receptors, in the rat dentate gyrus in vitro

LTP induction dependent on activation of Ni2+-sensitive voltage-gated calcium channels, but not NMDA receptors, in the rat dentate gyrus in vitro. J. Neurophysiol. 78: 2574-2581, 1997. A N-methyl--aspartate receptor (NMDAR)-independent long-term potentiation (LTP) has been investigated in the dentate gyrus of the hippocampus in vitro in the presence of the NMDAR antagonist, -2-amino-phosphonopentanoate (50-100 mu M), at a concentration that completely blocked NMDAR-mediated excitatory postsynaptic currents (EPSCs). LTP of patch-clamped EPSCs was induced by pairing low-frequency evoked EPSCs (1 Hz) with depolarizing voltage pulses designed to predominately open low-voltage-activated (LVA) Ca2+ channels. Voltage pulses alone induced only a short-term potentiation. The LTP was blocked by intracellular application of the rapid Ca2+ chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, demonstrating that a rise in intracellular Ca2+ is required for the NMDAR-independent LTP induction. The NMDAR-independent LTP induction also was blocked by Ni2+ at a low extracellular concentration (50 mu M), which is known to strongly block LVA Ca2+ channels. However, Ni2+ did not inhibit the NMDAR-dependent LTP induced by high-frequency stimulation (HFS). The NMDAR-independent LTP induction was not blocked by high concentrations of the L-type Ca2+ channel blocker nifedipine (10 mu M). The NMDAR-independent LTP was inhibited by the metabotropic glutamate receptor ligand (+)-alpha-methyl-4-carboxyphenylglycine. These experiments demonstrate the presence of a NMDAR-independent LTP induced by Ca2+ influx via Ni2+-sensitive, nifedipine-insensitive voltage-gated Ca2+ channels, probably LVA Ca2+ channels. Induction of the NMDAR-independent LTP was inhibited by prior induction of HFS-induced NMDAR-dependent LTP, demonstrating that although the NMDAR-dependent and NMDAR-independent LTP use a different Ca2+ channel for Ca2+ influx, they share a common intracellular pathway.     

Dependence of hepatocytic autophagy on intracellularly sequestered calcium

Autophagic sequestration of endogenous lactate dehydrogenase or electroinjected [3H]raffinose in isolated rat hepatocytes was strongly suppressed by the Ca2+ chelator EGTA, unless the cells had previously been electroloaded in the presence of high concentrations of Ca2+ (1.2 mM). The extracellular Ca2+ chelator bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) and the intracellular Ca2+ chelator BAPTA/tetra(acetoxymethyl)-ester (BAPTA/AM) both inhibited autophagy to the same extent as did EGTA. Inhibitors of Ca(2+)-activated protein kinases (KN-62, H-7, W-7) had little or no effect on autophagy, indicating that the Ca2+ requirement of autophagy was not mediated by such kinases. Agents that elevate cytosolic Ca2+ by releasing Ca2+ from intracellular stores, like thapsigargin, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and the ionophores A23187 and ionomycin, inhibited autophagy strongly, implicating depletion of sequestered rather than of cytosolic intracellular Ca2+ as a common mechanism of inhibition. Lysosomal (propylamine-sensitive) protein degradation, known to be largely autophagy-dependent, was inhibited by thapsigargin and tBuBHQ. Thapsigargin had no effect on cellular ATP levels, but all agents tested (thapsigargin, tBuBHQ, ionophores) inhibited protein synthesis. Our results suggest that autophagy, like protein synthesis, is dependent on the presence of Ca2+ in some intracellular storage compartment.     

Testosterone acts directly at the pituitary to regulate gonadotropin-releasing hormone-induced calcium signals in male rat gonadotropes

We have recently shown that castration alters GnRH-induced calcium (Ca2+) signaling in the gonadotropes of male rats. Instead of generating spike-plateau Ca2+ responses to high concentrations of GnRH (100 nM), the majority of gonadotropes from castrated rats have oscillatory Ca2+ responses, which are generally only seen with low concentrations of GnRH in the gonadotropes of intact rats. This change in the nature of GnRH-induced Ca2+ responses is prevented by in vivo testosterone treatment. The aims of the present study were, therefore, to determine if testosterone acts directly at the pituitary or via the regulation of hypothalamic GnRH secretion. Accordingly, castrated male rats were treated with a GnRH antagonist to ablate the effects of increased GnRH secretion at the pituitary gland. GnRH antagonist treatment (10 microg/100 g BW, twice daily for 7 days from the time of castration) decreased the concentration of LH in the serum of castrated rats (0.4 +/- 0.1 ng/ml vs. 11.2 +/- 0.4 ng/ml in untreated castrated rats, mean +/- SEM) but had no effect on the proportion of gonadotropes having oscillatory Ca2+ responses to 100 nM GnRH when compared with untreated castrated rats (63% in antagonist-treated castrated rats vs. 70% in untreated castrated rats). The GnRH antagonist treatment did not, however, interfere with the ability of in vivo testosterone treatment (100 microg/100 g body weight/day) to decrease the proportion of gonadotropes having oscillatory Ca2+ responses to 100 nM GnRH (26% in testosterone-treated rats vs. 25% in testosterone and antagonist-treated rats). These findings indicate that testosterone acts directly at the pituitary, and not by altered GnRH secretion, to modulate GnRH-induced Ca2+ signals. To confirm this suggestion, cultured gonadotropes of castrated male rats were treated in vitro with 10 nM testosterone. Testosterone treatment for twelve, but not 4 h, restored the proportion of gonadotropes having oscillatory Ca2+ responses to that seen in gonadotropes from intact rats. The in vitro effects of testosterone over 12 h were prevented by concomitant treatment with the protein synthesis inhibitor cycloheximide (10 microM), which, when given alone, had no effect on GnRH-induced Ca2+ signals in cells from castrate male rats. Taken together, these findings suggest that testosterone has a direct genomic action at the pituitary to regulate GnRH-induced Ca2+ signals, via a process that involves new protein synthesis.     

Inhibition by mibefradil, a novel calcium channel antagonist, of Ca(2+)- and volume-activated Cl- channels in macrovascular endothelial cells

1. We have studied the effects of mibefradil, a novel calcium antagonist, on the resting potential and ion channel activity of macrovascular endothelial cells (calf pulmonary artery endothelial cells, CPAE). The patch clamp technique was used to measure ionic currents and the Fura-II microfluorescence technique to monitor changes in the intracellular Ca2+ concentration, [Ca2+]i. 2. Mibefradil (10 microM) hyperpolarized the membrane potential of CPAE cells from its mean control value of -26.6 +/- 0.6 mV (n = 7) to -59.8 +/- 1.7 mV (n = 6). A depolarizing effect was observed at higher concentrations (-13.7 +/- 0.6 mV, n = 4, 30 microM mibefradil). 3. Mibefradil inhibited Ca(2+)-activated Cl- currents, ICl,Ca, activated by loading CPAE cells via the patch pipette with 500 nM free Ca2+ (Ki = 4.7 +/- 0.18 microM, n = 8). 4. Mibefradil also inhibited volume-sensitive Cl- currents, ICl,vol, activated by challenging CPAE cells with a 27% hypotonic solution (Ki = 5.4 +/- 0.22 microM, n = 6). 5. The inwardly rectifying K+ channel, IRK, was not affected by mibefradil at concentrations up to 30 microM. 6. Ca2+ entry activated by store depletion, as assessed by the rate of [Ca2+]i-increase upon reapplication of 10 mM extracellular Ca2+ to store-depleted cells, was inhibited by 17.6 +/- 6.5% (n = 8) in the presence of 10 microM mibefradil. 7. Mibefradil inhibited proliferation of CPAE cells. Half-maximal inhibition was found at 1.7 +/- 0.12 microM (n = 3), which is similar to the concentration for half-maximal block of Cl- channels. 8. These actions of mibefradil on Cl- channels and the concomitant changes in resting potential might, in addition to its effect on T-type Ca2+ channels, be an important target for modulation of cardiovascular function under normal and pathological conditions.     

Replacement of troponin-I in slow-twitch skeletal muscle alters the effects of the calcium sensitizer EMD 53998

A component of fungus Thielavia minor, OPC-15161, has been shown to inhibit the proliferation and extracellular matrix production of extracellular matrix-producing mesangial cells in the kidney in vivo. In this study, we examined the effects of OPC-15161 on the proliferation and extracellular matrix production of rat cultured hepatic stellate cells (HSCs). To determine the effect of OPC-15161 on proliferation of HSCs, the cell number and the uptake of [3H]thymidine were investigated in the presence and absence of interleukin-1beta (IL-1beta). IL-1beta significantly increased the uptake of [3H]thymidine in the HSCs, and the addition of OPC-15161 inhibited the uptake in a dose-dependent manner. The cell number of HSCs was also increased by IL-1beta, which was inhibited by OPC-15161. Production of extracellular matrix by OPC-15161 was studied by the production of [3H]-hydroxyproline in the presence and absence of transforming growth factor-beta1 (TGF-beta1). TGF-beta1 significantly increased the production of [3H]-hydroxyproline in the cells, whereas the addition of OPC-15161 inhibited this effect dose dependently. We also investigated the effects of OPC-15161 on Ca2+ mobilization and measured D-myo-inositol 1,4,5-triphosphate (IP3) in the HSCs. IL-1beta induced the increase of intracellular Ca2+ and IP3 concentrations in the HSCs, which were decreased by OPC-15161. Based on these results, we conclude that OPC-1 5161 inhibited the proliferation and production of hydroxyproline in cultured rat HSCs, and thus, it may have a role in prevention of liver fibrosis in vivo.     

Effects of morphine on monosodium glutamate neurotoxicity and its mechanism

The enhancing effects of morphine on monosodium glutamate (MSG) neurotoxicity and its blocking by naloxone were studied through morphological observation, together with detection of concentrations of intracellular free Ca2+ ([Ca2+]i) by Ca2+ indicator Fura-2/AM and lactate dehydrogenase (LDH) efflux in the bathing medium in primary cultures from 14-17 d old mouse fetal cortex. It was found that 10 min pre-incubation of young cortical neurons (7 day in vitro) with morphine 10(-7) or 10(-6) mol.L-1 substantially increased LDH release from 105.7% +/- 19.0% (treated with MSG alone) to 194.5% +/- 17.7% and 214.0% +/- 9.5% respectively after exposure to MSG 0.1 mmol.L-1, but pre-incubation with morphine (10(-7) or 10(-6) mol.L-1) plus naloxone (0.1 mmol.L-1) reversed the LDH release after treatment with the same concentration of MSG. Morphine (10(-7) or 10(-6) mol.L-1) produced little elevation of [Ca2+]i. However, when combined with MSG (0.1 mmol.L-1) morphine elevated the [Ca2+]i level much more than MSG alone. These results suggest that morphine markedly enhances excitotoxic neuron damage, which can be reversed by naloxone. Overloading of intracellular Ca2+ may be a simultaneous pathological mechanism underlying the neuronal damage and death that occur in excitatory toxicity.     

Regulated binding of the protein kinase C substrate GAP-43 to the V0/C2 region of protein kinase C-delta

The interaction between protein kinase C-delta and its neuronal substrate, GAP-43, was studied. Two forms of protein kinase C-delta were isolated from COS cells and characterized by differences in gel mobility, GAP-43 binding, and specific GAP-43 and histone kinase activities. A slow migrating, low specific activity form of protein kinase C-delta bound directly to immobilized GAP-43. Binding was abolished in the presence of EGTA, suggesting Ca2+ dependence of the interaction. The free catalytic domain of protein kinase C-delta did not bind GAP-43, suggesting the existence of a binding site in the regulatory domain. Glutathione S-transferase-protein kinase C-delta regulatory domain fusion proteins were generated and tested for binding to GAP-43. The V0/C2-like amino-terminal domain was defined as the GAP-43-binding site. GAP-43 binding to this region is inhibited by EGTA and regulated at Ca2+ levels between 10(-7) and 10(-6) M. The interaction between protein kinase C-delta and GAP-43 was studied in intact cells by coexpression of the two proteins in human embryonic kidney cells followed by immunoprecipitation. Complex formation occurred only after treatment of the cells with the Ca2+ ionophore ionomycin, indicating that elevation of intracellular Ca2+ is required for interaction in vivo. It is concluded that protein kinase C-delta interacts with GAP-43 through the V0/C2-like domain, outside the catalytic site, and that this interaction is modulated by intracellular Ca2+.     

Calmodulin is involved in membrane depolarization-mediated survival of motoneurons by phosphatidylinositol-3 kinase- and MAPK-independent pathways

In the present work, we find that the elevation of extracellular K+ concentration promotes the survival of chick spinal cord motoneurons in vitro deprived of any neurotrophic support. This treatment induces chronic depolarization of the neuronal plasma membrane, which activates L-type voltage-dependent Ca2+ channels, resulting in Ca2+ influx and elevation of the cytosolic free Ca2+ concentration. Pharmacological reduction of intracellular free Ca2+ or withdrawal of extracellular Ca2+ reversed the effects of depolarization on survival. The intracellular Ca2+ response to membrane depolarization developed as an initial peak followed by a sustained increase in intracellular Ca2+ concentration. The depolarizing treatment caused tyrosine phosphorylation of mitogen-activated protein kinase (MAPK) without involving tyrosine kinase receptor activation. The calmodulin antagonist W13 inhibited the survival-promoting effect induced by membrane depolarization but not the tyrosine phosphorylation of MAPK. Moreover, depolarization did not induce phosphatidylinositol-3 kinase (PI-3K) phosphorylation in our cells, and the PI-3K inhibitor wortmannin did not suppress the survival-promoting effect of K+ treatment. These results suggest that calmodulin is involved in calcium-mediated survival of motoneurons through the activation of PI-3K- and MAPK-independent pathways.     

Calcium-dependent potassium exchange in human red cell ghosts

1. Ca buffers may be introduced into human red cells by reversible haemolysis. The resealed ghosts retain Ca and chelating anions in the same ratio as in the haemolysing solution, enabling the intracellular Ca2+ concentration to be calculated simply. 2. The passive permeability of the ghosts to Na and Cl is unaffected by intracellular Ca2+ concentrations in the 10(-8)-10(-4) M range, whereas the K permeability is greatly increased at concentrations above 10(-7) M. 3. These preparations enable Ca-dependent K movements to be studied under stable conditions. When the ghosts contain about 5 X 10(-6) M-Ca2+, over 96% of K transport occurs via the Ca-sensitive route.     

Expression in animal cells and characterization of the hepatitis E virus structural proteins

Capacitation of spermatozoa, a complex process occurring after sperm ejaculation, is required to obtain fertilization of the oocyte in vivo and in vitro. Although most of the biochemical/ biophysical events that occur during capacitation in vitro have been characterized, the molecular mechanisms underlying these complex events are still obscure. Increases of intracellular free Ca2+ concentrations ([Ca2+]i) and protein tyrosine phosphorylation have previously been demonstrated during in vitro capacitation of human spermatozoa. In the present study we investigated the relationship between extracellular/intracellular Ca2+, protein tyrosine phosphorylation, and tyrosine kinase and phosphatase activities during sperm capacitation. We report that the increase in tyrosine phosphorylation of several protein bands that occurs during sperm capacitation is independent of the presence of Ca2+ in the external medium and, at least partially, of the increase in [Ca2+]i occurring during the process. Indeed, the spontaneous increase in phosphorylation was still present in Ca(2+)-free/EGTA-containing-medium and in the presence of the intracellular Ca2+ chelator BAPTA/AM. Moreover, phosphorylation of proteins and protein tyrosine kinase (PTK) activity was enhanced if spermatozoa were incubated in Ca(2+)-free medium, suggesting the presence of Ca(2+)-inhibited tyrosine kinase(s) in human sperm. This hypothesis is further substantiated by the lower tyrosine phosphorylation observed after incubation with the ionophore A23187 and the endoplasmic Ca(2+)-ATPase inhibitor thapsigargin, which promote Ca2+ influx in human sperm. The ability of the cells to undergo acrosome reaction in response to progesterone, which can be considered a functional endpoint of capacitation, was highly compromised when spermatozoa were incubated in Ca(2+)-free medium or in the presence of EGTA, confirming that Ca2+ is required for sperm capacitation. Conversely, in the presence of erbstatin, a inhibitor of tyrosine kinase activity, which blunts tyrosine phosphorylation during capacitation, response to progesterone was maintained, suggesting that tyrosine phosphorylation must be kept at a low level (physiologically by the presence of Ca2+ in the external medium, or pharmacologically by the presence of erbstatin) in order to obtain response to progesterone. This mechanism may be important in vivo during sperm transit in the female genital tract to ensure appropriate timing of full capacitation in the proximity of the oocyte.     

Involvement of calcium in ACC-oxidase activity from Cicer arietinum seed embryonic axes

Both in vivo and in vitro ACC-oxidase activities as well as ethylene production from embryonic axes of chickpea seeds were strongly inhibited by EGTA, a selective extracellular Ca2+ ion chelator, indicating that the influx of Ca2+ is important for enzymatic activity. EGTA inhibition was restored by exogenous Ca2+. Treatments of embryonic axes with either Verapamil and LaCl3 (both Ca2+ channel blockers) or TMB-8 (an intracellular Ca2+ antagonist) provoked an inhibition of both ACC-oxidase activity and ethylene production. These results suggest an involvement of calcium fluxes and intracellular calcium levels in the activity of the last step of the ethylene biosynthetic pathway, which is, in turn, intimately correlated with germination of Cicer arietinum seeds.     

Effect of transforming growth factor beta-1 on the intracellular calcium in cardiac fibroblasts

We attempted to determine the effects of transforming growth factor beta-1 on intracellular Ca2+ concentration changes in the presence of isoproterenol in cardiac fibroblasts. Transforming growth factor beta-1 inhibited the increase of intracellular Ca2+ concentration in the presence of isoproterenol in fibroblasts. It also inhibited the production of cyclic-AMP in fibroblasts in the presence of isoproterenol. Islet-activating protein did not block these reactions of transforming growth factor beta-1. Forskolin did not affect the intracellular calcium concentration change resulting from treatment with transforming growth factor beta-1. Binding of [3H]CGP-12177 was decreased to 47% of control preincubated for 24 hours with transforming growth factor beta-1 in fibroblasts. Scatchard plots suggested a decrease in beta-adrenergic receptor number without specific change in receptor affinity. These results suggested that transforming growth factor beta-1 modulates the signal transduction through beta-adrenergic receptor and intracellular Ca2+ concentration by regulating the number of receptors in fibroblasts.     

Complete activation of porcine oocytes induced by the sulfhydryl reagent, thimerosal

Thimerosal (200 microM) triggered Ca2+ oscillations in 56 of 56 mature porcine oocytes. The Ca2+ oscillations were blocked by the sulfhydryl-reducing agent dithiothreitol (DTT), thus supporting the hypothesis that thimerosal acts by oxidizing critical sulfhydryl groups on intracellular Ca2+-release proteins. Thimerosal treatment alone arrested the oocytes in metaphase, probably by oxidizing tubulin sulfhydryl groups and thus destroying the spindle. However, a 10-min exposure to 200 microM thimerosal followed by a 30-min incubation in 8 mM DTT induced complete activation, as 73.8% of the oocytes formed pronuclei. The second polar body was visible in 73.3% (55 of 75) of the activated oocytes. Combined thimerosal/DTT treatment of the oocytes also induced cortical granule exocytosis, as revealed by confocal microscopy, and the subsequent hardening of the zona pellucida. After activation, some oocytes were incubated in vitro, or in vivo in a ligated porcine oviduct, for 6 days. When cultured in vitro, 42.0% (37 of 88) of the oocytes developed to the compact morula or blastocyst stage; the average number of inner cell mass (ICM) and trophectoderm (TE) nuclei in the blastocysts was 8.6 +/- 0.7 and 20.1 +/- 1.3, respectively. Culture in a ligated oviduct resulted in 42.9% development to the compact morula or blastocyst stage, with the blastocysts having a mean number of 12.5 +/- 1.0 ICM and 63.6 +/- 9.2 TE nuclei.     

Vasopressin stimulates Ca2+ spiking activity in A7r5 vascular smooth muscle cells via activation of phospholipase A2

[Arg8]-vasopressin (AVP) is both a potent vasoconstrictor and a mitogen for vascular smooth muscle cells. AVP binds to a single class of receptors (V1a) in the A7r5 rat aortic smooth muscle cell line (Kd approximately 2 nmol/L). Stimulation of these cells with AVP results in an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) by releasing intracellular Ca2+ stores and increasing Ca2+ influx; the EC50 for these effects is approximately 5 nmol/L. AVP has recently been reported to stimulate arachidonic acid release in primary cultures of rat aortic smooth muscle over a much lower concentration range (EC50 approximately 0.05 nmol/L). The present study examined the effects of varying concentrations of AVP on spontaneous Ca2+ spiking activity in fura 2-loaded A7r5 cells. Frequency of CA2+ spiking increased with increasing [AVP] in the range of 10 to 500 pmol/L. Higher concentrations of AVP inhibited spiking but elicited the characteristic [Ca2+]i changes ascribed to the release of Ca2+ stores and increased Ca2+ entry. The effects of both low and high concentrations of AVP were inhibited by [1-(beta-mercapto-beta,beta,-pentamethylenepropionic acid),2-0-methyltyrosine]arginine vasopressin, a selective V1a vasopressin antagonist. Nimodipine (50 nmol/L), a blocker of L-type voltage-sensitive Ca2+ channels, abolished the Ca(2+)-spiking activity without inhibiting a maximal [Ca2+]i response to AVP (1 mumol/L). AVP-stimulated Ca2+ spiking, but not release of intracellular Ca2+ stores, was also abolished by ONO-RS-082 (1 mumol/L), an inhibitor of phospholipase A2. These results suggest that occupation of a small fraction of V1a vasopressin receptors by AVP results in stimulation of phospholipase A2 and leads to increased Ca(2+)-spiking activity. This effect may be important for fine tuning of vascular tone, whereas maximal stimulation by AVP (full receptor occupancy) may be required for more vigorous or sustained vasoconstriction or mitogenesis.