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PubMed2002

Prolactin signaling in porcine adrenocortical cells: involvement of protein kinases.

Prolactin (PRL) was found to have a stimulatory effect on adrenal steroidogenesis in vivo and in vitro in several species including pigs. PRL signal transduction pathways, however, in adrenocortical cells are poorly recognized. Therefore, the goal of this paper is to ascertain the involvement of protein kinase C (PKC) and tyrosine kinases in PRL signaling in porcine adrenal cortex. Adrenals were harvested from locally slaughtered mature gilts. Cortical cells were dispersed by sequential treatment with collagenase. The cells were seeded into 24-well culture plates at a density of 3 x 10(5)/mL. Cells were incubated with or without PRL (500 ng/mL), ACTH (5 nM--a positive control), tyrosine kinase inhibitor--genistein (1; 2.5 or 5 microM), PKC inhibitor--sphingosine (20-1000 nM) and PKC activators--diacylglycerol (DiC8; 10-100 microM) and phorbol ester (PMA; 1-1000 nM). All incubations were performed for 8 h (95% air and 5% CO(2), 37 degrees C). PRL and ACTH (P < 0.05) increased cortisol and androstenedione (A(4)) secretion. DiC8 and PMA mimicked the stimulatory effect of PRL. Sphingosine (P < 0.05) suppressed basal and PRL-stimulated steroid secretion. Genistein inhibited (P < 0.05) PRL-stimulated cortisol secretion and enhanced (P < 0.05) basal and PRL-stimulated A(4) secretion. Moreover, PKC activation was assessed by measuring the specific association of [3H]phorbol dibutyrate ([3H]PDBu) with adrenocortical cells after treatment with PRL or ionomycin (a positive control). PRL (within 2-3 min) and ionomycin (within 2-5 min) increased (P < 0.05) specific binding of [3H]PDBu to the porcine adrenocortical cells. In addition, PRL did not augment the cortisol and A(4) secretion by PKC-deficient adrenocortical cells. In conclusion, presented results support the hypothesis that PKC and tyrosine kinases are involved in PRL signaling in adrenocortical cells in pigs. Moreover, activation of PKC is associated with the increased secretion of cortisol and A(4).

Adrenal Cortex↗
PubMedDate not supplied

Phosphorylation of T-cell antigen receptor-associated proteins: correlation with activation for killing and/or for gamma-interferon production by a cytolytic T-cell clone.

The activation-induced phosphorylation of T-cell antigen receptor (Ti)-associated proteins was investigated in order to analyse possible signal-transduction mechanisms leading to two distinct effector functions of a mouse cytolytic T-cell clone (KB5.C20): target cell killing (independent of protein synthesis) and de novo production of gamma interferon (gIFN; dependent on gIFN gene expression). Ti-associated T3-like proteins were first identified by immunoprecipitation of 125I-labelled cell surface proteins from 1% digitonin lysates of clone KB5.C20 by 1- and 2-dimensional (non-reduced (NR)/reduced (R)) gel electrophoresis. In addition to the alpha and beta chains of the Ti (NR: 80-Kd; R: 43 and 40 Kd), two doublets of 35-37 Kd (NR) and 32-34 Kd (NR) leading to bands of 25, 16 and 14 Kd (R) were identified, as well as three bands (25, 23 and 22 Kd (NR)) leading to 27-, 25- and 21-Kd bands (R). Activation of clone KB5.C20 (prelabelled with 32P-orthophosphate) with either anti-Ti mAb or exposure to both ionomycin and phorbol myristic acetate (PMA) induced the phosphorylation of 21- and 25-27-Kd (R) Ti-associated proteins, whereas exposure to either ionomycin or PMA alone induced only weak phosphorylation of 21-Kd (R) components. A weak phosphorylation of 32- and 34-Kd Ti-associated proteins was sometimes observed after stimulation with anti-Ti mAb. Functional studies suggested that activation for gIFN production was observed only when both the 21- and 25-27-Kd proteins were phosphorylated, whereas activation for killing (when measured by PMA-induced non-specific killing) could occur in conditions where no phosphorylation of the 25-27-Kd protein was detected.

Antibodies, Monoclonal↗
PubMed1997

Ca2+ stores in the chick embryo retina cells.

The Ca2+ stores of digitonin permeabilized chick embryo retina cells in culture were characterized, by using the fluorescence of Fluo-3 potassium salt to follow continuously the free [Ca2+] in the medium. After ATP dependent Ca2+ accumulation, the Ca2+ release was induced by several agents; 10 microM cyclic-ADP-ribose (cADPR), 40 microM Ins (1,4,5)P3 10 microM thapsigargin (Th), 25 microM ionomycin (Ion), 15 microM CCCP together with 4.5 micrograms/ml oligomycin (CCCP/Olig), 50 microM arachidonic acid (AA). Neither Ins(1,4,5)P3 nor cADPR were able to mobilize Ca2+ from internal stores in these cells, but Th and AA were effective in releasing Ca2+. Four major Ca2+ stores in chick embryo retina cells were distinguished: i) the thapsigargin sensitive Ca2+ store, most likely the ER; ii) the Ca2+ store sensitive to oligomycin and CCCP, most likely the mitochondrial Ca2+ store, iii) an AA sensitive Ca2+ store, which is distinct from the previous two; and, iv) the Ca2+ store only sensitive to ionomycin. The capacities of these different Ca2+ stores of the chick embryo retina cells, relative to the total intracellular stores, are: 63.3%, 14.1%, 8.2%, for the ER, the mitochondrial and for the AA sensitive Ca2+ stores, respectively.

Adenosine Diphosphate Ribose↗
PubMed1995

Different calcium-dependent pathways control fertilisation-triggered glycoside release and the cortical contraction in ascidian eggs.

Fertilisation of ascidian eggs induces the rapid release of a cell surface N-acetylglycosaminidase that blocks sperm binding to vitelline coat sperm receptors resulting in a block to polyspermy. Fertilisation also triggers a large contraction of the egg (thus stimulating ooplasmic segregation) that is completed within 5 min of insemination. In eggs of the ascidian Phallusia mammillata, glycosidase release and cortical contractions are blocked by BAPTA-AM [bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester], a cell-permeant calcium chelator, indicating that both processes are probably dependent on a rise in intracellular calcium levels. Both glycosidase release and the cortical contraction are induced by treatment of the egg with the protein synthesis inhibitor emetine, while only the glycosidase release is induced by isoproterenol, carbachol or acetylcholine. Previous work with ryanodine demonstrated that ryanodine also caused glycosidase release but not the cortical contraction. Inversely, activation by ionomycin in calcium-free sea water causes cortical contractions but not glycosidase release. Thus the two processes can be activated independently. Dextran-coupled (10 kDa) calcium green-1 injected eggs show an increase in intracellular calcium 30-40 s before the cortical contraction is triggered by fertilisation or ionomycin-induced activation. This confirms previous findings that the cortical contraction is a consequence of the activation calcium wave triggered by the sperm. The extracellular calcium requirement for the glycosidase release suggests that calcium influx may be more important for this phase of egg activation. Thus activation of ascidian eggs appears to involve two independent pathways involving calcium.

Acetylcholine↗
PubMed1990

Bidirectional transbilayer lipid movement in human platelets as vizualized by the fluorescent membrane probe 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene.

Transbilayer movement of the fluorescent membrane probe TMA-DPH [1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene] in the plasma membrane of human platelets was investigated by measuring fluorescence intensity and fluorescence decay. Labeling of unstimulated platelets by TMA-DPH results in a rapid increase in fluorescence intensity, leveling off within 1 min. Dilution of platelets into buffer without TMA-DPH leads to an almost complete rapid efflux of TMA-DPH, indicating that TMA-DPH labels only the outer leaflet of the plasma membrane. Transbilayer movement of the fluorescent probe in unstimulated platelets could be observed upon prolonged incubation and occurs with a t1/2 of 60-90 min. Stimulation of platelets with thrombin directly after the initial rapid uptake of TMA-DPH results in a fast increase in membrane-bound TMA-DPH, fully explained by the increase in plasma membrane caused by secretion of intracellular storage organelles. No indications for increased transbilayer movement of the probe were found, since dilution of thrombin-stimulated TMA-DPH-labeled platelets into buffer without TMA-DPH indicated no uptake of TMA-DPH by intracellular membranes. In contrast to thrombin, stimulation of TMA-DPH-labeled platelets with the Ca2(+)-ionophore ionomycin results in a much larger increase in fluorescence intensity. This process is accompanied by labeling of intracellular membranes as indicated by incomplete efflux of TMA-DPH after dilution of the stimulated platelets. Thus, stimulation of platelets by ionomycin gives rise to rapid and massive inward movement of TMA-DPH (t1/2 approximately 10-12 s). Prolonged incubation of platelets in the absence of any stimulus allows labeling of the total lipid pool, including intracellular membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport↗
PubMed1999

Regulation of M1 muscarinic receptor-mediated signaling in intact cells by exogenous, but not endogenously produced, nitric oxide.

Regulation of nitric oxide (NO) formation is critical to ensure maintenance of appropriate cellular concentrations of this labile, signaling molecule. This study investigated the role exogenous and endogenously produced NO have in feeding back to regulate NO synthesis in intact cells. Two NO donors inhibited activation of neuronal NO synthase (nNOS) in response to the muscarinic receptor agonist carbachol in Chinese hamster ovary (CHO) cells stably transfected with the M1 muscarinic receptor and nNOS. The presence of the NO scavenger [2-(4-Carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide potassium salt] (C-PTIO) potentiated carbachol-induced activation of nNOS in transfected CHO cells. C-PTIO also potentiated nNOS activity in response to the Ca2+ ionophore ionomycin. In contrast, the NO scavenger oxyhemoglobin depressed carbachol- and ionomycin-induced NO formation. These discrepant results suggest that it is unlikely that endogenously produced NO induces feed back inhibition at the level of nNOS activation itself. Exogenous sources of NO inhibited carbachol-induced inositol phosphates formation. However, endogenously produced NO did not appear to feed back to regulate phosphoinositide hydrolysis as there was no difference in [3H]inositol phosphates formation between cells that do or do not express nNOS. There was also no change in carbachol-induced [3H]inositol phosphates formation in the presence or absence of a NOS inhibitor or the NO scavenger C-PTIO. A decrease in the carbachol-mediated transient Ca2+ peak was observed in cells that express nNOS as compared to cells lacking the enzyme, suggesting that endogenous NO might inhibit receptor mediated Ca2+ signaling. This conclusion, however, was not supported by the lack of ability of a NOS inhibitor to modulate carbachol-induced Ca2+ elevations. Taken together, these results highlight differences in the regulation of the nNOS activation cascade by endogenous vs. exogenous sources of NO.

Animals↗
PubMed1998

Regulatory mechanism of pepsinogen gene expression in monolayer cultured guinea pig gastric chief cells.

We have investigated the effects of dibutyryl cAMP, forskolin, carbamylcholine chloride (carbachol), ionomycin, and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of guinea pig pepsinogen mRNA in monolayer cultured gastric chief cells. After exposure of the cells to each of these compounds for 4 to 24 hr, and at 48 hr after primary culture, total cellular RNA was isolated using acid guanidium-phenol-chloroform and then was reverse transcribed to cDNA. Obtained cDNA was amplified by polymerase chain reaction (PCR) using primers detecting guinea pig pepsinogen mRNA and human beta-actin mRNA as an internal standard. The PCR products were separated and quantified using capillary electrophoresis. Dibutyryl cAMP and forskolin significantly increased pepsinogen mRNA, but carbachol, ionomycin, and TPA failed to increase that. These findings suggested that pepsinogen gene expression was up-regulated by intracellular cAMP, but not by intracellular calcium or protein kinase C in guinea pig chief cells.

Animals↗
PubMedDate not supplied

Early steps of lymphocyte activation bypassed by synergy between calcium ionophores and phorbol ester.

Although it has been proposed that the activation of T lymphocytes is mediated by an early rise in cytosolic calcium concentration, it has not been possible to mimic antigen- or mitogen-induced mouse lymphocyte activation by calcium ionophores that bypass receptor-mediated processes. There is now evidence from other systems that the rise in cytosolic calcium which follows receptor triggering is preceded by the breakdown of phosphatidylinositol bisphosphate into 1,2-diacylglycerol and inositol trisphosphate. The latter is known to cause release of calcium from intracellular stores. The cellular target for the former is now widely accepted to be protein kinase C. Therefore, ligand-induced cellular response follows a rise in cytosolic calcium concentration and protein kinase C activation. Here we confirm that the calcium ionophores A23187 and ionomycin do not activate mouse T lymphocytes. However, either one in combination with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which is structurally related to 1,2-diacylglycerol, induces in lymphoid cell populations the expression of receptors for interleukin-2 (IL-2), the secretion of IL-2 and cell proliferation as measured by 3H-thymidine uptake. The growth-promoting effect of IL-2 on an exogenous IL-2-dependent clone could not be substituted for by ionomycin either alone or with TPA. Thus, the combination of calcium ionophores and TPA bypasses the requirement for antigen- or lectin-induced signal at the onset of lymphocyte activation.

Animals↗
PubMed1994

Role of cytosolic Ca2+ and protein kinases in the induction of the hsp70 gene.

The role of cytosolic Ca2+ ([Ca2+]i) and protein kinases in the hsp70 induction following heat shock was investigated in cultured rat proximal tubular epithelial (PTE) cells. Changes in [Ca2+]i were measured by digital imaging fluorescence microscopy using fura 2. Steady state levels of hsp70 mRNA were examined by either Northern or dot blot analyses. [Ca2+]i increased within 10 minutes and continued to increase following heat shock. The increases in [Ca2+]i were reduced in nominally Ca(2+)-free media with or without EGTA. [Ca2+]i also increased within 0.5 minutes following ionomycin, but then declined to normal levels by 1.0 to 1.5 minutes. Heat shock induced hsp70 mRNA within 15 minutes, which continued to increase up to three hours. Ionomycin also induced hsp70 mRNA, which peaked at 30 minutes, and gradually decreased thereafter. The hsp70 induction following heat shock was attenuated when extracellular Ca2+ was reduced. Chelation of [Ca2+]i by quin-2 also reduced the hsp70 induction. Inhibitors of protein kinases, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), calphostin C, genistein, and 2-aminopurine, also had inhibitory effects on the hsp70 induction. In contrast, a calmodulin inhibitor, chlorpromazine, had little effect. These results suggest that heat shock increases [Ca2+]i in rat PTE cells and that [Ca2+]i and protein kinases are involved in the hsp70 induction following heat shock.

Animals↗
PubMed2004

A new simple whole blood flow cytometry-based method for simultaneous identification of activated cells and quantitative evaluation of cytokines released during activation.

The multiple cellular and soluble elements of the immune system respond in a coordinated way, orchestrated by cytokines, to preserve the integrity of the organism. In this study, we describe a new and unique whole blood method that, with minimal sample manipulation, allows an overall evaluation of immune responses by simultaneously measuring cell activation and cytokine secretion. The identification of cells actively secreting cytokines is based on the stabilization of tumor necrosis factor alpha (TNFalpha) at the cell surface through the use of a specific inhibitor of the TNFalpha-converting enzyme. This inhibitor does not affect the release of cytokines other than TNFalpha and makes it possible to assess, in the same measurement, the phenotype of TNFalpha(+)-secreting cells and quantify multiple secreted cytokines by using a specific and highly sensitive flow cytometry-based bead immunoassay. Upon stimulation of normal peripheral blood samples with either phorbol 12-myristate 13 acetate (PMA) plus ionomycin or lipopolysaccharide (LPS), both the number of TNFalpha+ cells and the amount of secreted cytokines progressively increased, the former becoming detectable first. After stimulation for 3 h with PMA plus ionomycin, cellular responses were associated with surface TNFalpha expression on the majority of CD3+ T cells and secretion of Th1-associated cytokines: interferon gamma, interleukin (IL)-2, and to a lesser extent IL4. In turn, stimulation with LPS induced a response mainly by inflammatory cells. After 4 h of LPS-stimulation, the majority of CD14+ monocytes showed surface TNFalpha expression; in parallel, high amounts of soluble IL1beta, IL6, and IL8 became detectable. Likewise, stimulation of blood samples with cytomegalovirus (CMV) lysates induced viral-specific immune responses detectable in seropositive but not seronegative volunteers; such responses were associated with the detection of increased numbers of TNFalpha+ monocytes, TNFalpha+/CD8+ T cells and TNFalpha+/CD8- T lymphocytes in association with an increased secretion of IFNgamma, IL6 and TNFalpha.

Adult↗
PubMed2000

Stimulus-specific alteration of the relationship between cytosolic Ca(2+) transients and nitric oxide production in endothelial cells ex vivo.

1. To investigate the quantitative relationship between elevation in the intracellular Ca(2+) concentration ([Ca(2+)](i)) and nitric oxide (NO) production, the changes in [Ca(2+)](i) and NO production were determined in parallel, using fluorimetry of fura-2 and 2, 3-diaminonaphthalene, respectively, in endothelial cells ex vivo of pig aortic valves. 2. The extent of [Ca(2+)](i) elevation was quantitatively assessed by two parameters: the level of peak [Ca(2+)](i) elevation and the area under the [Ca(2+)](i) curve during treatment (the integrated [Ca(2+)](i) elevation). The amount of NO production was expressed as a percentage of that obtained with 10 microM ATP for 3 min. 3. ATP, bradykinin, thrombin, and ionomycin were used as stimulation to induce NO production, and all these caused [Ca(2+)](i) increases and NO production in a concentration-dependent manner. 4. The relationships between the peak [Ca(2+)](i) and NO production or between the integrated [Ca(2+)](i) elevation and NO production were well described by a straight line. However, the slope value of the linear relationship in both cases varied with the type of stimulation, with thrombin giving the greatest value, followed by ATP, bradykinin and ionomycin. 5. These data suggest that in endothelial cells ex vivo: (1) [Ca(2+)](i) elevation regulates NO production, but (2) the peak [Ca(2+)](i) elevation- or the integrated [Ca(2+)](i) elevation-NO production relationships varies depending on the type of agonists. Our results thus demonstrate the presence of the agonists-dependent modulation of the relationship between [Ca(2+)](i) elevation and NO production in endothelial cells ex vivo.

Adenosine Triphosphate↗
PubMed2002

Capacitative Ca(2+) entry in vascular endothelial cells is mediated via pathways sensitive to 2 aminoethoxydiphenyl borate and xestospongin C.

1. Agonists increase endothelial cell intracellular Ca(2+), in part, by capacitative entry, which is triggered by the filling state of intracellular Ca(2+) stores. It has been suggested that depletion of endoplasmic reticulum (ER) Ca(2+) stores either leads to a physical coupling between the ER and a plasma membrane channel, or results in production of an intracellular messenger which affects the gating of membrane channels. As an axis involving the IP(3) receptor has been implicated in a physical coupling mechanism the aim of this study was to examine the effects of the putative IP(3) receptor antagonists/modulators, 2 aminoethoxydiphenyl borate (2APB) and xestospongin C, on endothelial cell Ca(2+) entry. 2. Studies were conducted in fura 2 loaded cultured bovine aortic endothelial cells and endothelial cells isolated from rat heart. 3. 2APB (30 - 300 microM) inhibited Ca(2+) entry induced by both agonists (ATP 1 microM, bradykinin 0.1 microM) and receptor-independent mechanisms (thapsigargin 1 microM, ionomycin 0.5 and 5 microM). 2APB did not diminish endothelial cell ATP-induced production of IP(3) nor effect in vitro binding of [(3)H]-IP(3) to an adrenal cortex binding protein. Capacitative Ca(2+) entry was also blocked by disruption of the actin cytoskeleton with cytochalasin (100 nM) while the initial Ca(2+) release phase was unaffected. 4. Similarly to 2APB, xestospongin C (3 - 10 microM) inhibited ATP-induced Ca(2+) release and capacitative Ca(2+) entry. Further, xestospongin C inhibited capacitative Ca(2+) entry induced by thapsigargin (1 microM) and ionomycin (0.5 microM). 5. The data are consistent with a mechanism of capacitative Ca(2+) entry in vascular endothelial cells which requires (a) IP(3) receptor binding and/or an event distal to the activation of the ER receptor and (b) a spatial relationship, dictated by the cytoskeleton, between Ca(2+) release and entry pathways.

Actins↗
PubMed2003

Cation channels trigger apoptotic death of erythrocytes.

Erythrocytes are devoid of mitochondria and nuclei and were considered unable to undergo apoptosis. As shown recently, however, the Ca(2+)-ionophore ionomycin triggers breakdown of phosphatidylserine asymmetry (leading to annexin binding), membrane blebbing and shrinkage of erythrocytes, features typical for apoptosis in nucleated cells. In the present study, the effects of osmotic shrinkage and oxidative stress, well-known triggers of apoptosis in nucleated cells, were studied. Exposure to 850 mOsm for 24 h, to tert-butyl-hydroperoxide (1 mM) for 15 min, or to glucose-free medium for 48 h, all elicit erythrocyte shrinkage and annexin binding, both sequelae being blunted by removal of extracellular Ca(2+) and mimicked by ionomycin (1 microM). Osmotic shrinkage and oxidative stress activate Ca(2+)-permeable cation channels and increase cytosolic Ca(2+) concentration. The channels are inhibited by amiloride (1 mM), which further blunts annexin binding following osmotic shock, oxidative stress and glucose depletion. In conclusion, osmotic and oxidative stress open Ca(2+)-permeable cation channels in erythrocytes, thus increasing cytosolic Ca(2+) activity and triggering erythrocyte apoptosis.

Amiloride↗
PubMed2001

Molecular events involved in up-regulating human Na+-independent neutral amino acid transporter LAT1 during T-cell activation.

We investigated the regulation of system-L amino acid transporter (LAT1) during T-cell activation. In quiescent T-cells, L-leucine transport is mediated mainly by the system-L amino acid transport system and is increased significantly during T-cell activation by PMA and ionomycin. In quiescent T-cells, the LAT1 protein was heterocomplexed with 4F2 heavy chain (4F2hc) in the plasma membrane. During T-cell activation, the amounts of 4F2hc and LAT1 heterocomplex were significantly elevated compared with those in quiescent T-cells. In addition, by Northern-blot analysis, these increments were found to be due to elevated levels of LAT1 and 4F2hc mRNA. Transient expression of constructs comprising various LAT1 gene promoter fragments, which contained all three of the GC boxes, was sufficient for promoting luciferase expression in Jurkat T-cells, but the promoter of the LAT1 gene did not respond to PMA and ionomycin. Similar observations were observed in the human 4F2hc gene promoter. In nuclear run-on assay, the LAT1 and 4F2hc genes were actively transcribed even in quiescent T-cells, but the low levels of both transcripts were shown to be the result of a block to transcription elongation within the exon 1 intron 1 regions. These findings indicated that a removal of the block to mRNA elongation stimulates the induction of system-L amino acid transporter gene transcripts (LAT1 and 4F2hc) in activated T-cells.

Amino Acid Sequence↗
PubMed1982

Phospholipid turnover in isolated rat pancreatic acini. Consideration of the relative roles of phospholipase A2 and phospholipase C.

The purpose of the present study was to explore the interaction of phosphatidylinositol breakdown and the turnover of arachidonic acid in isolated rat pancreatic acini by using receptor agonists and the calcium ionophore ionomycin. Acini prelabelled with myo-[(3)H]inositol in vivo responded to carbachol with a rapid breakdown of phosphatidylinositol. In the presence of [(32)P]P(i), carbachol increased labelling of phosphatidic acid and phosphatidylinositol within 1 and 5 min respectively. Carbachol also rapidly stimulated the incorporation of [(14)C]arachidonic acid into phosphatidylinositol within 2 min, and the peptidergic secretagogue caerulein caused the loss of radioactivity from phospholipids prelabelled with arachidonic acid. Ca(2+) deprivation partially impaired the stimulatory action of carbachol on arachidonic acid turnover. In contrast with its stimulatory effects on [(32)P]P(i) and [(14)C]arachidonate incorporation, carbachol inhibited the incorporation of the saturated fatty acid stearic acid into phosphatidylinositol. Whereas ionomycin stimulation of phosphatidylinositol breakdown and [(32)P]P(i) labelling of phospholipids was slower in onset and less effective than carbachol stimulation, the ionophore effectively promoted (arachidonyl) phosphatidylinositol turnover within 2 min. These results implicate two separate pathways for stimulated phosphatidylinositol degradation in the exocrine pancreas, involving phospholipases A(2) and C. Whereas mobilization of cellular Ca(2+) appears sufficient to cause activation of phospholipase A(2) and amylase secretion, additional events triggered by receptor activation may be required to act in concert with Ca(2+) to optimally stimulate phospholipase C. The nature of the interaction between phospholipases A(2) and C and their specific physiological roles in pancreatic secretion remain to be elucidated.

Animals↗
PubMed1986

Increase of adenylate cyclase catalytic-unit activity by dexamethasone in rat osteoblast-like cells.

Glucocorticoids are known to increase the cyclic AMP response to parathyroid hormone (PTH) in cultured bone organs or bone cells. Using the osteoblast-like cell line ROS 17/2.8, which possesses receptors for both PTH and glucocorticoids, we investigated which component of the complex hormone receptor-guanine nucleotide regulatory unit--adenylate cyclase was affected by dexamethasone treatment. In response to PTH, isoproterenol or forskolin, a compound that is supposed to act directly on the catalytic unit, cyclic AMP production by intact cells and adenylate cyclase activity in purified plasma membrane were markedly increased by dexamethasone. Whereas NaF, guanosine 5'-[beta gamma-imido]triphosphate and Mn/ stimulated adenylate cyclase activity were similarly enhanced in membranes isolated from glucocorticoid-treated cells, the activity of the stimulatory guanine nucleotide regulatory unit, as assessed by reconstitution into membranes from the CYC- clone, which is genetically devoid of this component, was not altered. Thus in osteoblast-like cells dexamethasone appears to increase cyclic AMP synthesis by influencing the catalytic unit. Moreover, since it has been reported that glucocorticoids may produce changes in cell calcium metabolism, we evaluated cytoplasmic free Ca2+ concentration ([Ca2+]i) and intracellular Ca2+ stores mobilizable by the bivalent-cationophore ionomycin, by using the intracellular fluorescent indicator Quin-2. The results indicated that dexamethasone treatment did not influence [Ca2+]i but markedly decreased ionomycin-releasable Ca2+ stores.

Adenylyl Cyclases↗
PubMed1988

Thrombin-stimulated elevation of human endothelial-cell cytoplasmic free calcium concentration causes prostacyclin production.

Endothelial cells are known to release prostacyclin (PGI2) in response to agonists, and this has generally been assumed to be caused, at least in part, by activation of a phospholipase A2 by elevated concentrations of cytoplasmic free calcium ([Ca2+]i). However, it has been shown in the blood platelet that agonists can cause arachidonate release without elevating [Ca2+]i. In the present study, rigorous analysis is made of the [Ca2+]i-dependence of PGI2 production in the human umbilical-vein endothelial cell. Thrombin caused a rapid increase in [Ca2+]i from the resting basal value of 0.1 microM to a peak, within 10-15 s, of approx. 2 microM. In the absence of extracellular Ca2+, [Ca2+]i then declined back to the resting value within 2-3 min. In the presence of extracellular Ca2+, [Ca2+]i partly decreased to a new steady-state value of approx. 1 microM. The elevated [Ca2+]i was maintained while the stimulus and the source of extracellular Ca2+ were present, suggesting that it was dependent on influx of Ca2+ across the plasma membrane. Thrombin stimulated the production of PGI2 in the presence or in the absence of extracellular Ca2+. However, the production of PGI2 was more prolonged in the presence of extracellular Ca2+. Total accumulated amounts of 6-oxo-prostaglandin F1 alpha on stimulation with thrombin without extracellular Ca2+ were only 65% of those accumulated with extracellular Ca2+ present. Cells depleted of extracellular and intracellular sources of Ca2+ by incubation with 1 mM extracellular EGTA and exposing them to ionomycin to discharge intracellular stores produced no elevation of [Ca2+]i on stimulation with thrombin or production of PGI2. The threshold [Ca2+]i required to support the production of PGI2 was measured to be 0.8-1.0 microM by using different doses of ionomycin selectively to increase [Ca2+]i. This relationship between [Ca2+]i and PGI2 production was similar to that produced by using different doses of thrombin. Our results show that the major and probably exclusive intracellular stimulus for the production of PGI2 by the vascular endothelial cell in response to thrombin is the elevation of [Ca2+]i.

6-Ketoprostaglandin F1 alpha↗
PubMed1989

Activation of protein kinase C in human neutrophils attenuates agonist-stimulated rises in cytosolic free Ca2+ concentration by inhibiting bivalent-cation influx and intracellular Ca2+ release in addition to stimulating Ca2+ efflux.

Stimulation of fura-2-loaded human neutrophils with formylmethionyl-leucyl-phenylalanine (FMLP) or ionomycin elevated the cytosolic free Ca2+ concentration, [Ca2+], to a maintained elevated level. Activation of protein kinase C (C-kinase) with phorbol 12-myristate 13-acetate, 4 beta-phorbol 12,13-didecanoate or dioctanoylglycerol caused decreases in [Ca2+]i from this level. 4 alpha-Phorbol didecanoate, which does not activate C-kinase, had no effect. These results confirm previous reports that C-kinase activation decreases neutrophil [Ca2+]i by stimulating removal of Ca2+ from the cytosol. Further experiments showed that activation of C-kinase attenuated the component of the FMLP-stimulated [Ca2+]i rise that was dependent on external Ca2+. C-kinase activation also inhibited FMLP-stimulated entry of the quenching cation, Mn2+, used as an indicator of bivalent-cation entry. In contrast, C-kinase activation caused only a partial inhibition of FMLP-stimulated release of Ca2+ from intracellular stores. 4 alpha-Phorbol didecanoate was ineffective in inhibiting Ca2+ entry, Mn2+ entry and intracellular Ca2+ release. Addition of FMLP also stimulated a decrease in the ionomycin-elevated [Ca2+]i, and this effect was blocked by staurosporine, a protein kinase inhibitor. These results show that, in addition to stimulating Ca2+ efflux, C-kinase activation in neutrophils inhibits FMLP-stimulated entry of bivalent cations, and partially inhibits intracellular release of Ca2+. Further, FMLP itself can modulate [Ca2+]i by activation of C-kinase.

Benzofurans↗