Ionomycin-stimulated phasic myometrial contractions

1995 ◽  
Vol 269 (4) ◽  
pp. E779-E785
Author(s):  
M. Phillippe ◽  
E. M. Chien ◽  
M. Freij ◽  
T. Saunders
Keyword(s):  
Phospholipase C ◽  
Calcium Release ◽  
Calcium Influx ◽  
Inositol Phosphate ◽  
Calcium Ionophore ◽  
Cytosolic Calcium ◽  
Calcium Oscillations ◽  
Production Studies ◽  
Inositol Phosphate Production

Ionomycin, a calcium ionophore, facilitates the sustained entry of extracellular calcium; however, in myometrial tissue it stimulates phasic contractions. This study sought to define further this unanticipated effect of ionomycin and to begin to explore the possible mechanism(s) involved. Utilizing rat uterine strips, in vitro isometric contraction studies were performed to determine the effects of ionomycin with and without membrane-permeant inhibitors of cytosolic calcium oscillations. To determine the effects of ionomycin on phospholipase C, qualitative inositol phosphate production studies were performed. The in vitro contraction studies confirmed that ionomycin-stimulated phasic myometrial contractions were potentially dependent on stimulation of phospholipase C, calcium-induced calcium release, and additional calcium influx through dihydropyridine-sensitive membrane calcium channels. The inositol phosphate production studies confirmed that ionomycin stimulated phospholipase C in a dose-related fashion to levels comparable to oxytocin. In summary, these observations have confirmed the ability of ionomycin to generate dose-related phasic myometrial contractions through mechanisms potentially involving the phosphatidylinositol-signaling pathway.

Potassium chloride effects on the hormonal signal transduction mechanisms underlying phasic myometrial contractions

1995 ◽  
Vol 146 (3) ◽  
pp. 485-493 ◽  
Author(s):  
M Phillippe ◽  
E K Chien
Keyword(s):  
Signal Transduction ◽  
Potassium Chloride ◽  
Phospholipase C ◽  
Inositol Phosphate ◽  
Cytosolic Calcium ◽  
Calcium Oscillation ◽  
Uterine Tissue ◽  
Production Studies ◽  
Inositol Phosphate Production ◽  
Transduction Mechanisms

Abstract These studies sought to test the hypothesis that potassium-stimulated phasic myometrial contractions utilize cytosolic calcium oscillation-like mechanisms comparable to those activated in response to oxytocin. Uterine tissue was obtained from pro-oestrus/oestrus Sprague-Dawley rats. In vitro isometric contraction studies were performed using longitudinal myometrial strips; computer digitalized contraction data were analyzed for contraction area, and normalized for tissue cross-section area. Dose–response studies were performed using potassium chloride with and without inhibitors of cytosolic calcium oscillation mechanisms. Qualitative inositol-phosphate production studies were performed after preloading uterine tissue with [3H]inositol; subsequently, the individual inositol-phosphates produced in response to stimulation were isolated by anion exchange chromatography. Potassium chloride over a concentration of 10 to 30 mm produced a dose-related increase in phasic contractile activity. The potassium-stimulated phasic contractions were significantly suppressed in response to inhibition of phospholipase C, stimulation of protein kinase C, inhibition of calcium-induced calcium release, and prevention of extracellular calcium influx. The qualitative inositol-phosphate production studies confirmed activation of phospholipase C in response to 20 mm potassium. These studies have provided support for the hypothesis that potassium-stimulated phasic myometrial contractions activate intracellular signal transduction mechanisms comparable to those activated in response to hormonal uterotonic agonists. Journal of Endocrinology (1995) 146, 485–493

scholarly journals Cholecystokinin-stimulated tyrosine phosphorylation of p125FAK and paxillin is mediated by phospholipase C-dependent and -independent mechanisms and requires the integrity of the actin cytoskeleton and participation of p21rho

1997 ◽  
Vol 327 (2) ◽  
pp. 461-472 ◽  
Author(s):  
J. Luis GARCÍA ◽  
A. Juan ROSADO ◽  
Antonio GONZÁLEZ ◽  
T. Robert JENSEN
Keyword(s):  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Inositol Phosphate ◽  
Calcium Ionophore ◽  
Cytosolic Calcium ◽  
Significant Inhibition ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Pancreatic Acini ◽  
Pkc Activation

Recent studies show that the effects of some oncogenes, integrins, growth factors and neuropeptides are mediated by tyrosine phosphorylation of the cytosolic kinase p125 focal adhesion kinase (p125FAK) and the cytoskeletal protein paxillin. Recently we demonstrated that cholecystokinin (CCK) C-terminal octapeptide (CCK-8) causes tyrosine phosphorylation of p125FAK and paxillin in rat pancreatic acini. The present study was aimed at examining whether protein kinase C (PKC) activation, calcium mobilization, cytoskeletal organization and small G-protein p21rho activation play a role in mediating the stimulation of tyrosine phosphorylation by CCK-8 in acini. CCK-8-stimulated phosphorylation of p125FAK and paxillin reached a maximum within 2.5 min. The CCK-8 dose response for causing changes in the cytosolic calcium concentration ([Ca2+]i) was similar to that for p125FAK and paxillin phosphorylation, and both were to the left of that for receptor occupation and inositol phosphate production. PMA increased tyrosine phosphorylation of both proteins. The calcium ionophore A23187 caused only 25% of the maximal stimulation caused by CCK-8. GF109203X, a PKC inhibitor, completely inhibited phosphorylation with PMA but had no effect on the response to CCK-8. Depletion of [Ca2+]i by thapsigargin had no effect on CCK-8-stimulated phosphorylation. Pretreatment with both GF109203X and thapsigargin decreased CCK-8-stimulated phosphorylation of both proteins by 50%. Cytochalasin D, but not colchicine, completely inhibited CCK-8- and PMA-induced p125FAK and paxillin phosphorylation. Treatment with Clostridium botulinum C3 transferase, which inactivates p21rho, caused significant inhibition of CCK-8-stimulated p125FAK and paxillin phosphorylation. These results demonstrate that, in pancreatic acini, CCK-8 causes rapid p125FAK and paxillin phosphorylation that is mediated by both phospholipase C-dependent and -independent mechanisms. For this tyrosine phosphorylation to occur, the integrity of the actin, but not the microtubule, cytoskeleton is essential as well as the activation of p21rho.

scholarly journals Adenosine Triphosphate-Evoked Cytosolic Calcium Oscillations in Human Granulosa-Luteal Cells: Role of Protein Kinase C1

2001 ◽  
Vol 86 (2) ◽  
pp. 773-777 ◽  
Author(s):  
Chen-Jei Tai ◽  
Sung Keun Kang ◽  
Peter C. K. Leung
Keyword(s):  
Protein Kinase ◽  
Calcium Influx ◽  
Purinergic Receptor ◽  
Cytosolic Calcium ◽  
Inhibitory Effect ◽  
Calcium Oscillations ◽  
Dependent Manner ◽  
Luteal Cells

ATP has been shown to modulate progesterone production in human granulosa-luteal cells (hGLCs) in vitro. After binding to a G protein-coupled P2 purinergic receptor, ATP stimulates phospholipase C. The resultant production of diacylglycerol and inositol triphosphate activates protein kinase C (PKC) and intracellular calcium [Ca2+]i mobilization, respectively. In the present study, we examined the potential cross-talk between the PKC and Ca2+ pathway in ATP signal transduction. Specifically, the effect of PKC on regulating ATP-evoked[ Ca2+]i oscillations were examined in hGLCs. Using microspectrofluorimetry, [Ca2+]i oscillations were detected in Fura-2 loaded hGLCs in primary culture. The amplitudes of the ATP-triggered [Ca2+]i oscillations were reduced in a dose-dependent manner by pretreating the cells with various concentrations (1 nm to 10μ m) of the PKC activator, phorbol-12-myristate-13-acetate (PMA). A 10 μm concentration of PMA completely suppressed 10 μm ATP-induced oscillations. The inhibitory effect occurred even when PMA was given during the plateau phase of ATP evoked [Ca2+]i oscillations, suggesting that extracellular calcium influx was inhibited. The role of PKC was further substantiated by the observation that, in the presence of a PKC inhibitor, bisindolylmaleimide I, ATP-induced[ Ca2+]i oscillations were not completely suppressed by PMA. Furthermore, homologous desensitization of ATP-induced calcium oscillations was partially reversed by bisindolylmaleimide I, suggesting that activated PKC may be involved in the mechanism of desensitization. These results demonstrate that PKC negatively regulates the ATP-evoked [Ca2+]i mobilization from both intracellular stores and extracellular influx in hGLCs and further support a modulatory role of ATP and P2 purinoceptor in ovarian steroidogenesis.

scholarly journals Cdc42 and Rac Stimulate Exocytosis of Secretory Granules by Activating the Ip3/Calcium Pathway in Rbl-2h3 Mast Cells

2000 ◽  
Vol 148 (3) ◽  
pp. 481-494 ◽  
Author(s):  
Elizabeth Hong-Geller ◽  
Richard A. Cerione
Keyword(s):  
Calcium Release ◽  
Calcium Influx ◽  
Secretory Granules ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Dominant Negative ◽  
Ionophore A23187 ◽  
Ip3 Receptor ◽  
Extracellular Medium

We have expressed dominant-active and dominant-negative forms of the Rho GTPases, Cdc42 and Rac, using vaccinia virus to evaluate the effects of these mutants on the signaling pathway leading to the degranulation of secretory granules in RBL-2H3 cells. Dominant-active Cdc42 and Rac enhance antigen-stimulated secretion by about twofold, whereas the dominant-negative mutants significantly inhibit secretion. Interestingly, treatment with the calcium ionophore, A23187, and the PKC activator, PMA, rescues the inhibited levels of secretion in cells expressing the dominant-negative mutants, implying that Cdc42 and Rac act upstream of the calcium influx pathway. Furthermore, cells expressing the dominant-active mutants exhibit elevated levels of antigen-stimulated IP3 production, an amplified antigen-stimulated calcium response consisting of both calcium release from internal stores and influx from the extracellular medium, and an increase in aggregate formation of the IP3 receptor. In contrast, cells expressing the dominant-negative mutants display the opposite phenotypes. Finally, we are able to detect an in vitro interaction between Cdc42 and PLCγ1, the enzyme immediately upstream of IP3 formation. Taken together, these findings implicate Cdc42 and Rac in regulating the exocytosis of secretory granules by stimulation of IP3 formation and calcium mobilization upon antigen stimulation.

ATP induces contraction of cultured brain capillary pericytes, via activation of P2Y type purinergic receptors

2020 ◽  
Author(s):  
Sofie Hørlyck ◽  
Changsi Cai ◽  
Hans C Helms ◽  
Martin Lauritzen ◽  
Birger Brodin
Keyword(s):  
Intracellular Calcium ◽  
Purinergic Receptors ◽  
Calcium Release ◽  
Purinergic Receptor ◽  
Cytosolic Calcium ◽  
Receptor Activation ◽  
Confocal Imaging ◽  
Inositol Triphosphate ◽  
Brain Capillary

Brain capillary pericytes have been suggested to play a role in the regulation of cerebral blood-flow under physiological and pathophysiological conditions. ATP has been shown to cause constriction of capillaries under ischemic conditions and suggested to be involved in the "no-reflow" phenomenon. In order to investigate the effects of extracellular ATP on pericyte cell contraction, we studied purinergic receptor activation of cultured bovine brain capillary pericytes. We measured [Ca2+]i-responses to purinergic agonists with the fluorescent indicators fura-2 and Cal-520 and estimated contraction of pericytes as relative change in cell area, using real-time confocal imaging. Addition of ATP caused an increase in cytosolic calcium and contraction of the brain capillary pericytes, both reversible and inhibited by a purinergic receptor antagonist PPADS. Furthermore, we demonstrated that ATP-induced contraction could be eliminated by intracellular calcium-chelation with BAPTA, indicating that the contraction was mediated via purinergic P2 -type receptor-mediated [Ca2+]i-signaling. ATP stimulation induced inositol triphosphate signaling, consistent with the notion of P2Y receptor activation. Receptor profiling studies demonstrated presence of P2Y1 and P2Y2 receptors, using ATP, UTP, ADP and the subtype specific agonists MRS2365 (P2Y1) and 2-thio-UTP (P2Y2)). Addition of specific P2X agonists only caused a [Ca2+]i increase at high concentrations, attributed to activation of inositol triphosphate signaling. Our results suggest that contraction of brain capillary pericytes in vitro by activation of P2Y type purinergic receptors is caused by intracellular calcium release. This adds more mechanistic understanding to the role of pericytes in vessel constriction, and points towards P2Y receptors as potential therapeutic targets.

scholarly journals Sodium/Calcium Exchangers Selectively Regulate Calcium Signaling in Mouse Taste Receptor Cells

2010 ◽  
Vol 104 (1) ◽  
pp. 529-538 ◽  
Author(s):  
Steven A. Szebenyi ◽  
Agnieszka I. Laskowski ◽  
Kathryn F. Medler
Keyword(s):  
Calcium Release ◽  
Calcium Influx ◽  
Taste Receptor ◽  
Physiological Role ◽  
Cytosolic Calcium ◽  
Calcium Signals ◽  
Sodium Calcium ◽  
Signaling Mechanisms ◽  
Taste Cells ◽  
Mouse Taste

Taste cells use multiple signaling mechanisms to generate appropriate cellular responses to discrete taste stimuli. Some taste stimuli activate G protein coupled receptors (GPCRs) that cause calcium release from intracellular stores while other stimuli depolarize taste cells to cause calcium influx through voltage-gated calcium channels (VGCCs). While the signaling mechanisms that initiate calcium signals have been described in taste cells, the calcium clearance mechanisms (CCMs) that contribute to the termination of these signals have not been identified. In this study, we used calcium imaging to define the role of sodium-calcium exchangers (NCXs) in the termination of evoked calcium responses. We found that NCXs regulate the calcium signals that rely on calcium influx at the plasma membrane but do not significantly contribute to the calcium signals that depend on calcium release from internal stores. Our data indicate that this selective regulation of calcium signals by NCXs is due primarily to their location in the cell rather than to the differences in cytosolic calcium loads. This is the first report to define the physiological role for any of the CCMs utilized by taste cells to regulate their evoked calcium responses.

scholarly journals G-proteins are not directly involved in the CD3-antigen-mediated production of inositol phosphates in HPB-ALL T-leukaemia cells expressing phospholipase C isoforms γ1 and β3

1993 ◽  
Vol 289 (2) ◽  
pp. 387-394 ◽  
Author(s):  
M Biffen ◽  
M Shiroo ◽  
D R Alexander
Keyword(s):  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
G Protein ◽  
G Proteins ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Cross Linking ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Inositol Phosphate Production

The possible involvement of G-proteins in T cell antigen-receptor complex (TCR)-mediated inositol phosphate production was investigated in HPB-ALL T-cells, which were found to express the phospholipase C gamma 1 and beta 3 isoforms. Cross-linking the CD3 antigen on streptolysin-O-permeabilized cells stimulated a dose-dependent increase in inositol phosphate production, as did addition of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) or vanadate, a phosphotyrosine phosphatase inhibitor. It was possible, therefore, that the CD3-antigen-mediated production of inositol phosphates was either via a G-protein-dependent mechanism or by stimulation of protein tyrosine phosphorylation. The CD3-induced inositol phosphate production was potentiated by addition of vanadate, but not by addition of GTP[S]. Guanosine 5′-[beta-thio]diphosphate (GDP[S]) inhibited the rise in inositol phosphates induced by GTP[S], vanadate or cross-linking the CD3 antigen. The increase in protein tyrosine phosphorylation stimulated by vanadate or the OKT3 monoclonal antibody was not observed in the presence of GDP[S], showing that in permeabilized HPB-ALL cells, GDP[S] inhibits the actions of tyrosine kinases as well as G-protein function. Addition of either ADP[S] or phenylarsine oxide inhibited CD3- and vanadate-mediated increases in both tyrosine phosphorylation and inositol phosphate production, but did not inhibit GTP[S]-stimulated inositol phosphate production. On the other hand, pretreatment of cells with phorbol 12,13-dibutyrate inhibited subsequent GTP[S]-stimulated inositol phosphate production but did not inhibit significantly inositol phosphate production stimulated by either OKT3 F(ab')2 fragments or vanadate. Our results are consistent with the CD3 antigen stimulating inositol phosphate production by increasing the level of protein tyrosine phosphorylation, but not by activating a G-protein.

The acute hypocalcaemic effect of ethanol and its mechanism of action in the rat

1983 ◽  
Vol 61 (4) ◽  
pp. 388-394 ◽  
Author(s):  
Nateetip Krishnamra ◽  
Liangchai Limlomwongse
Keyword(s):  
Body Weight ◽  
Mechanism Of Action ◽  
Calcium Release ◽  
Calcium Influx ◽  
Plasma Calcium ◽  
In Vitro Studies ◽  
Calcium Efflux ◽  
Plasma Pool ◽  
Suppressive Action

The hypocalcaemic action of ethanol (3 g/kg body weight) was investigated in intact, thyroparathyroidectomized and antrectomized rats. It was found that ethanol administered either intraperitoneally or orally reduced plasma calcium concentrations within 30 min and that this response lasted for 8 h. Additional studies performed in antrectomized and thyroparathyroidectomized rats indicated that neither gastrin nor the hormones parathormone and calcitonin had any effect on the hypocalcaemic effect of ethanol. Investigation of the mechanism of action of ethanol-induced hypocalcaemia involved measurements of calcium efflux from and influx into the plasma pool. Ethanol did not have any effect on the disappearance from plasma of 45Ca administered intravenously at 0 min. In contrast, ethanol was found to enhance the disappearance of 45Ca administered intraperitoneally 17 h prior to the experiment. The interpretation of 45Ca studies was discussed and it was concluded that ethanol-induced hypocalcaemia resulted from a decrease in calcium influx into the plasma. Additional in vitro studies did not indicate the suppressive action of ethanol on the release of calcium from tibias. In conclusion, our results show that the mechanism of hypocalcaemia caused by ethanol is the suppression of calcium release from some tissue(s) into the plasma.

scholarly journals Activation of P2z purinoceptors diminishes the muscarinic cholinergic-induced release of inositol 1,4,5-trisphosphate and stored calcium in rat parotid acini. ATP as a co-transmitter in the stimulus-secretion coupling

1995 ◽  
Vol 312 (2) ◽  
pp. 457-464 ◽  
Author(s):  
T D Jørgensen ◽  
J Gromada ◽  
K Tritsaris ◽  
B Nauntofte ◽  
S Dissing
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphate ◽  
Acinar Cells ◽  
Inhibitory Effect ◽  
Coupled Processes ◽  
Parotid Acinar Cells ◽  
Inositol Phosphate Production ◽  
Dependent Inhibition ◽  
Muscarinic Cholinergic ◽  
Rat Parotid

The effect of extracellular ATP on the intracellular free Ca2+ concentration ([Ca2+]i) and inositol phosphate production following stimulation with the muscarinic cholinergic agonist acetylcholine (ACh) was investigated in isolated rat parotid acinar cells. Stimulation of rat parotid acinar cells with ATP4- results in a rise in [Ca2+]i that is due to influx of extracellular Ca2+ and mobilization of Ca2+ from intracellular stores. Stimulation with purinergic agonists revealed that both influx as well as Ca2+ release from intracellular stores was mediated through activation of P2z receptors. The Ca2+ mobilization from intracellular stores was due to production of Ins(1,4,5)P3 and was inhibited by U73122, an inhibitor of phospholipase C-coupled processes. Under Ca(2+)-free conditions ATP4- caused a dose-dependent inhibition (IC50 = 8 microM) of the ACh-evoked Ca2+ release. The inhibitory effect of ATP4- is due to activation of the P2z purinoceptors, which results in a strong reduction in the ACh-induced inositol phosphate production. Prestimulation with 100 microM ATP4- reduced the amount of Ins(1,4,5)P3 formed after maximal ACh stimulation by 91%. In conclusion, the inhibitory effect of ATP4- on the ACh-mediated response is due to interactions of the activated P2z receptor with the phospholipase C-coupled processes underlying the muscarinic cholinergic response.

scholarly journals The role of calcium in lymphocyte proliferation. (An interpretive review)

Blood ◽  
1983 ◽  
Vol 61 (3) ◽  
pp. 413-422
Author(s):  
AH Lichtman ◽  
GB Segel ◽  
MA Lichtman
Keyword(s):  
Plasma Membrane ◽  
Lymphocyte Proliferation ◽  
Calcium Influx ◽  
Lectin Binding ◽  
Calcium Ionophore ◽  
Total Cell ◽  
Ionized Calcium ◽  
Ionophore A23187 ◽  
Cell Calcium

A small quantity of extracellular calcium is required for the stimulation of lymphocytes by mitogens such as plant lectins. Lectin binding to the lymphocyte surface and early postbinding events that eventually lead to DNA synthesis are calcium dependent. Mitogenic lectins such as PHA and Con-A rapidly increase the size of an exchangeable pool of cell calcium and cause a smaller rise in intracellular ionized calcium. The increase in ionized calcium is so small (100–200 nM), however, that no increase in total cell calcium is measurable. When lymphocytes are stimulated by a lectin, the rate of calcium entry into the cell increases, but the plasma membrane calcium extrusion pump can prevent the total cell calcium from increasing measurably. The calcium ionophore A23187 is a lymphocyte mitogen and causes an increase in the exchangeable, ionized, and total cell calcium. The former two effects may be causal in mitogenesis; the latter effect is cytotoxic. With A23187 treatment, the rate of calcium influx exceeds the maximum rate of the plasma membrane extrusion pump and cell calcium increases in proportion to the concentration of A23187. The mitochondria, by virtue of their high membrane potential, provide a sink for the buffering of cytoplasmic calcium after A23187 treatment. Thus, the plasma membrane or mitochondria regulate the distribution of lymphocyte calcium when the cell is stimulated by mitogenic lectins or ionophores. The evidence strongly suggests that an alteration in calcium pools or an increase in cytoplasmic ionized calcium plays a role in the initiation of the biochemical reactions that lead to mitogen-induced lymphocyte proliferation in vitro and, perhaps, to the immune response.

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