CP55,940 increases intracellular Ca2+ levels in Madin-Darby canine kidney cells

The effect of CP55,940, a presumed CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in Madin-Darby canine kidney cells was examined by using the fluorescent dye fura-2 as a Ca2+ indicator. CP55,940 (2-50 microM) increased [Ca2+]i concentration-dependently with an EC50 of 8 microM. The [Ca2+]i signal comprised an initial rise and a sustained phase. Extracellular Ca2+ removal decreased the maximum [Ca2+]i signals by 32+/-12%. CP55,940 (20 microM)-induced [Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists, AM-251 and AM-281. CP55,940 (20 microM)-induced [Ca2+]i increase in Ca2+-free medium was inhibited by 86+/-3% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 20 microM CP55,940 in Ca2+-free medium for 6 min abolished thapsigargin-induced [Ca2+]i increases. CP55,940 (20 microM)-induced intracellular Ca2+ release was not inhibited when inositol 1,4,5-trisphosphate formation was abolished by suppressing phospholipase C with 2 microM U73122. Collectively, this study shows that CP,55940 induced significant [Ca2+]i increases in canine renal tubular cells by releasing stored Ca2+ from the thapsigargin-sensitive pools in an inositol 1,4,5-trisphosphate-independent manner, and also by causing extracellular Ca2+ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors.     

Selective inhibition of collagen synthesis by the Ca2+ ionophore A23187 in cultured human fibroblasts

The question of whether the Ca2+ ionophore A23187 affects collagen production relative to total protein synthesis or has possible effects on collagen degradation was investigated. Cultured normal human fibroblasts were incubated with radioactive proline, and the radioactivity of collagenase-sensitive and -resistant proteins was used to calculate the rates of protein production. The net production of collagen relative to total proteins was inhibited by A23187 in a dose-related manner, and 50% inhibition of basal collagen production was achieved with 0.6 microM A23187. There was a 70% decrease in the absolute rate of collagen production in the presence of 0.6 microM A23187 which represented a 4-fold greater inhibition of collagen production than of noncollagen protein production. The major mechanism for the decreased net production of collagen was decreased synthesis, rather than increased degradation. Ca2+ mobilization induced by cholecystokinin octapeptide was also associated with selective inhibition of collagen production in normal human fibroblasts. These studies establish that the Ca2+ ionophore A23187 induces a selective decrease in collagen polypeptide synthesis by normal human fibroblasts and suggest a modulatory role of Ca2+ on collagen metabolism.     

Synergistic stimulation of S-adenosylmethionine decarboxylase activity by Ca2+ ionophore A23187, cholera toxin and 1-oleoyl-2-acetylglycerol

The Ca2+ ionophore A23187 induced S-adenosylmethionine decarboxylase in guinea-pig lymphocytes, and cholera toxin stimulated the induction synergistically. The activator of protein kinase C, 1-oleoyl-2-acetylglycerol, did not induce S-adenosylmethionine decarboxylase activity but potentiated the enzyme activity induced by A23187 or by A23187 and cholera toxin. The addition of both A23187 and cholera toxin induced S-adenosylmethionine decarboxylase, but the further addition of 1-oleoyl-2-acetylglycerol or 12-O-tetradecanoylphorbol 13-acetate did not potentiate the enzyme induction in protein kinase-C-down-regulated cells that had been treated with 12-O-tetradecanoylphorbol 13-acetate for 18 h. These results suggest that a Ca2+-dependent pathway, other than that for protein kinase C, is essential for the induction of S-adenosylmethionine decarboxylase and that a cAMP-dependent pathway and also protein kinase C are involved in the potentiation of the induction.     

On the mechanism of stimulation of H+ transport in gastric mucosa by Ca++ ionophore A23187. II. Ca++-cyclic AMP interactions

The possibility of interactions between calcium and cyclic AMP (cAMP) in the mechanism of stimulation of H+ transport by A23187 was studied in the isolated gastric mucosa of the toad Bufo marinus. A23187 stimulated H+ secretion and histamine release. The amount of histamine released by A23187 did not explain the degree of stimulation. Metiamide partially inhibited the response to A23187. Ca++ ionophore produced an overstimulation of secretion after H+ transport had been induced by supramaximal effective concentrations of histamine (10-4 M). In the presence of metiamide, IMX potentiated the response to A23187. Also, in the same condition (metiamide treated) the effects of db-cAMP and A23187 were additive. The results are consistent with an interaction between Ca++ and ionophore-released histamine at the oxyntic cell in the stimulation by A23187. The stimulatory response may be the result of a potentiation between calcium and cAMP at the intracellular level.     

Effects of Ca2+ and ionophore A23187 on protein synthesis in intact rabbit reticulocytes

1. Amino acid incorporation in intact rabbit reticulocytes was unaffected by depletion of Ca2+ with EGTA. 2. The Ca2+ ionophore A23187 strongly inhibited incorporation in reticulocytes incubated in 1 mM Ca2+ but not in EGTA. Polysomal profiles and average ribosomal transit times of cells treated with Ca2+ ionophore at 1 mM Ca2+ were characteristic of translational elongation block. 3. The behavior of reticulocytes with respect to Ca2+ and A23187 contrasts with that of nucleated cells possessing endoplasmic reticulum in which protein synthesis is inhibited at translational initiation by either Ca2+ depletion or by exposure to Ca2+ ionophore.     

Regulation of arachidonic acid metabolism in Madin-Darby canine kidney cells. Comparison of A23187 and 12-O-tetradecanoyl-phorbol-13-acetate

Challenge of Madin-Darby canine kidney (MDCK) cells with the divalent cation ionophore A23187 caused a marked increase in the deacylation of [3H]arachidonic acid but not of [14C]palmitic acid. When the cells were treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and A23187, there was an additional increase in the deacylation of [3H]arachidonic acid compared to that observed with either agent alone. In contrast to deacylation, the stimulation of prostaglandin production by A23187 was small compared to the stimulation by TPA. Cycloheximide inhibited synthesis of prostaglandins in TPA-treated cells, but did not block the stimulated deacylation caused by either TPA or A23187. These data indicate that, while both TPA and A23187 stimulated the deacylation of [3H]arachidonic acid, TPA had an additional, cycloheximide-sensitive effect that was required for efficient conversion of the release fatty acids to prostaglandins. Thus, although required, deacylation appeared to be independent of and insufficient to stimulate maximum prostaglandin synthesis in these cells.     

Arachidonate mobilization in diacyl, alkylacyl and alkenylacyl phospholipids on stimulation of rat platelets by thrombin and the Ca2+ ionophore A23187.

Platelet stimulation by thrombin or Ca2+ ionophore induces mobilization of arachidonate from lipid stores. We have previously shown that, in [14C]arachidonic acid-prelabelled resting platelets, [14C]arachidonate was transferred from diacyl-sn-glycerophosphocholine to ethanolamine and choline-containing ether phospholipids. This transfer reached an equilibrium after 5 h incubation [Colard, Breton & Bereziat (1984a) Biochem. J. 222, 657-662]. [14C]Arachidonate-prelabelled platelets having reached this transfer equilibrium were used to study the mobilization of arachidonate in etheracyl and diacyl phospholipids. Upon thrombin stimulation, arachidonate decreased in diacyl-sn-glycero-3-phosphoinositol, in alkylacyl- and diacyl-sn-glycero-3-phosphocholine and increased in alkenylacyl- and diacyl-sn-glycero-3-phosphoethanolamine. Upon challenge with Ca2+ ionophore A23187, arachidonate decreased in diacyl-sn-glycero-3-phosphoethanolamine, in diacyl- and alkylacyl-sn-glycero-3-phosphocholine and increased in alkenylacyl-sn-glycero-3-phosphoethanolamine. We also compared arachidonate mobilization in platelets stimulated immediately after [14C]arachidonic acid chase with platelets stimulated after 5 h reincubation. We observed that the arachidonate newly incorporated into diacyl-sn-glycero-3-phosphocholine and triacylglycerols was rapidly released upon stimulation. This suggests the presence in these two lipids of a rapidly-turning-over arachidonate pool.     

Calcium ionophore A23187 elevates angiotensin-converting enzyme in cultured bovine endothelial cells

Calcium ionophore A23187 (0.3-0.4 microM) elevated cellular angiotensin-converting enzyme activity (ACE) 2-7-fold after 48 h incubation with bovine pulmonary artery endothelial cells in culture. Cycloheximide (0.1 micrograms/ml) blocked the elevation in ACE produced by A23187. The increase in ACE was inhibited by 0.2 mM EGTA, 50 microM verapamil and 50 microM nifedipine, and was not associated with changes in cellular cAMP. Melittin, a phospholipase A2 activator, or addition of exogenous arachidonic acid failed to reproduce the elevation, and indomethacin only partially blocked the A23187 effect. The elevation of ACE was also inhibited by the calcium-calmodulin inhibitor, calmidazolium. Thus, we postulate that the ionophore A23187 elevates ACE in endothelial cells through a calcium-dependent mechanism other than phospholipase A2 activation. The elevation depends on new protein synthesis and involves calcium-calmodulin-dependent cellular mechanisms.     

The Ca2+-sensing receptor couples to Galpha12/13 to activate phospholipase D in Madin-Darby canine kidney cells

The Ca²⁺-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: G_i to inhibit the activity of adenylyl cyclase and activate ERK, G_q to stimulate phospholipase C and phospholipase A₂, and G to stimulate phosphatidylinositol 3-kinase. To determine whether the receptor also couples to G_12/13, we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known G_12/13 target. We established Madin-Darby canine kidney (MDCK) cell lines that stably overexpress the wild-type CaR (CaR^WT) or the nonfunctional mutant CaR^R796W as a negative control, prelabeled these cells with [³H]palmitic acid, and measured CaR-stimulated PLD activity as the formation of [³H]phosphatidylethanol (PEt). The formation of [³H]PEt increased in a time-dependent manner in the cells that overexpress the CaR^WT but not the CaR^R796W. Treatment of the cells with C₃ exoenzyme inhibited PLD activity, which indicates that the CaR activates the Rho family of small G proteins, targets of G_12/13. To determine which G protein(s) the CaR couples to in order to activate Rho and PLD, we pretreated the cells with pertussis toxin to inactivate G_i or coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for G_i and G_q, and a p115RhoGEF construct containing the RGS domain for G_12/13). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress CaR^WT inhibited extracellular Ca²⁺-stimulated PLD activity, but pretreatment of cells with pertussis toxin and overexpression of RGS4 were without effect. The involvement of other signaling components such as protein kinase C, ADP-ribosylation factor, and phosphatidylinositol biphosphate was excluded. These findings demonstrate that the CaR couples to G_12/13 to regulate PLD via a Rho-dependent mechanism and does so independently of G_i and G_q. This suggests that the CaR may regulate cytoskeleton via G_12/13, Rho, and PLD. calcium-sensing receptor; G proteins; RGS proteins     

On the mechanism of stimulation of H+ transport in gastric mucosa by Ca++ ionophore A23187: I. Pathways for cytosolic calcium increase

The pathways for cytosolic Ca⁺⁺ increase under A23187 stimulation of H⁺ secretion were studied in the isolated gastric mucosa of the toad $\text{Bufo marinus}$. A23187 produced a more potent stimulation of secretion when added to the mucosal side which did not contain calcium. Measurements of ionophore incorporation by fluorometric methods indicated that A23187 incorporates into oxyntic cells intracellularly. The presence of divalent cations inhibited incorporation. This may be the reason for a more potent action when A23187 was added from the mucosal side. With-drawal of calcium from serosal solution largely inhibited the secretory response to A23187 added to the mucosal side. Reintroduction of calcium into the serosal side in the presence of ionophore elicited H⁺ secretion. The results are consistent with an uptake of A23187 from the mucosal side into cellular organelles and basolateral membranes. Calcium entry through the serosal side may be responsible for triggering secretion. Although A23187 likely releases calcium from intracellular stores, its rate of release may not be sufficient to bring about a full stimulation of secretion in serosal-Ca⁺⁺-free conditions.     

Extracellular pH determines the rate of Ca2+ entry into Madin-Darby canine kidney-focus cells

We investigated the relationship between intracellular Ca²⁺ and pH homeostasis in Madin-Darby canine kidney-focus (MDCK-F) cells, a cell line exhibiting spontaneous oscillations of intracellular Ca²⁺ concentration (Ca _i ²⁺ ). Ca _i ²⁺ and intracellular pH (pH _i ) were measured with the fluorescent dyes Fura-2 and BCECF by means of video imaging techniques. Ca²⁺ influx from the extracellular space into the cell was determined with the Mn²⁺ quenching technique. Cells were superfused with HEPES-buffered solutions. Under control conditions (pH 7.2), spontaneous Ca _i ²⁺ oscillations were observed in virtually all cells investigated. Successive alkalinization and acidification of the cytoplasm induced by an ammonia ion prepulse had no apparent effect on Ca _i ²⁺ oscillations. On the contrary, changes of extracellular pH value strongly affected Ca _i ²⁺ oscillations. Extracellular alkalinization to pH 7.6 completely suppressed oscillations, whereas extracellular acidification to pH 6.8 decreased their frequency by 40%. Under the same conditions, the respective pH _i changes were less than 0. 1 pH units. However, experiments with the Mn²⁺ quenching technique revealed that extracellular alkalinization significantly reduced Ca²⁺ entry from the extracellular space. Large increases of Ca _i ²⁺ triggered by the blocker of the cytoplasmic Ca²⁺-ATPase, thapsigargin, had no effect on pH _i We conclude: intracellular Ca²⁺ homeostasis in MDCK-F cells is pH dependent. pH controls Ca²⁺ homeostasis mainly by effects on the level of Ca²⁺ entry across the plasma membrane. On the contrary, the intracellular pH value seems to be insensitive to rapid changes of Ca _i ²⁺ .The project was supported by the Deutsche Forschungsgemeinschaft, SFB-176 (A6) and by the Jubilämusstiftung of the University of Würzburg.The authors gratefully acknowledge the valuable discussions with Drs. M.J. Berridge, M. Carew, I. Davidson, G. Law and B. Somasundraman. We are grateful to Applied Imaging for financial and technical support and to the Medical Research Council for financial support.     

Effect of the antidepressant sertraline on Ca2+fluxes in Madin-Darby canine renal tubular cells

The effect of the antidepressant sertraline on cytosolic-free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether sertraline changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Sertraline at concentrations between 1and 100 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+ implicating Ca2+ entry and release both contributed to the [Ca2+]i rise. Sertraline induced Mn2+ influx, leading to quench of fura-2 fluorescence, suggesting Ca2+ influx. This Ca2+ influx was inhibited by suppression of phospholiapase A2 but not by store-operated Ca2+ channel blockers and protein kinase C/A modulators. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitors nearly abolished sertraline-induced Ca2+ release. Conversely, pretreatment with sertraline partly reduced inhibitor-induced [Ca2+]i rise, suggesting that sertraline released Ca2+ from endoplasmic reticulum. Inhibition of phospholipase C did not much alter sertraline-induced [Ca2+]i rise. Collectively, in MDCK cells, sertraline induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive Ca2+ channels.     

Phospholipase activation in the IgE-mediated and Ca2+ ionophore A23187-induced release of histamine from rat basophilic leukemia cells

The importance of phospholipase(s) activation in the IgE-mediated and ionophoreinduced histamine release from the rat basophilic leukemia cell line has been examined. The activation of phospholipase(s) as measured by [¹⁴C]arachidonic acid release and the release of histamine both required Ca²⁺ and were temporally parallel. Inhibition of phospholipase(s) activity by the inhibitors mepacrine and α-parabromoacetophenone also correlated with the inhibition of histamine release. [¹⁴C]Arachidonic acid released by the phospholipase(s) was mainly metabolized to prostaglandin D₂. The inhibition of the cyclooxygenase pathway by indomethacin did not affect histamine release. 5,8,11,14-Eicosatetraynoic acid inhibited both histamine and [¹⁴C]arachidonic acid release suggesting an effect not only on the cyclooxygenase and lipoxygenase pathways but also on the phospholipase(s). These results suggest that activation of phospholipase appears to be necessary for histamine release in the rat bosophilic leukemia cells.     

A phospholipase D-like mechanism in pancreatic islet cells: stimulation by calcium ionophore, phorbol ester and sodium fluoride

In neonatal rat islet cells prelabelled with [14C-methyl] choline, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate rapidly activated a phospholipase D-like mechanism as suggested by the accumulation in cells and medium of choline (but not of phosphorylcholine or glycerophosphorylcholine, markers for phospholipase C and phospholipase A2 action on phosphatidylcholine). This finding was confirmed by a rise in phosphatidic acid (but not diglyceride or arachidonic acid) in fatty acid-labelled cells. Phospholipase D was also activated by ionomycin or sodium fluoride; however, this was accompanied by parallel increases in diglyceride, monoacylglycerol and arachidonic acid in the absence of phosphorylcholine generation, suggesting that these agents also activated a phospholipase C-diglyceride lipase pathway acting on non-choline-containing phosphoglycerides (presumably phosphoinositides). In conjunction with our recent demonstration of insulinotropic effects of phosphatidic acid (M. Dunlop and R. Larkins, Diabetes, in press), our findings suggest for the first time a possible role for phospholipase D activation in the stimulation of insulin release and may imply a novel site of action for phorbol esters in the regulation of exocytosis.     

Granulosa cells from pig follicles of different sizes demonstrate maturational differences in their steroidogenic responses to FSH, calcium ionophore A23187, and phorbol diester

Cultures of granulosa cells from small (less than 3 mm), medium (3-6 mm), or large (8-10 mm) pig follicles were treated as follows: (1) basal controls, (2) cyclic adenosine 3',5'-monophosphate (cAMP) pathway agonists (pig FSH: 100 ng/ml; forskolin: 10 microM; dibutyryl cAMP; 1 mM), (3) calcium ionophore A23187 (0.005-1 micrograms), or (4) phorbol 12-myristate 13-acetate (TPA; 0.05-4 ng/ml). The combination of A23187 or TPA together with cAMP agonists was also examined in cultures of granulosa cells from follicles of different sizes. All substances were added at the time of culture, and oestradiol and progesterone were measured in the culture media after 48 h. All cAMP agonists were most potent in their stimulation of steroidogenesis (as a % of control) in cells from small follicles (P less than 0.05) with the exception of forskolin, which increased oestradiol in cells from large follicles to a greater extent than in cells of small follicles (P less than 0.05) (cells from medium follicles demonstrated less stimulation than those from small follicles except in progesterone production, for which FSH was equipotent). With the exception of forskolin, however, granulosa from large follicles showed little (oestradiol) or no stimulation (progesterone) with cAMP agonists. Under basal conditions, A23187 inhibited progesterone in all groups (P less than 0.05), and oestradiol production was reduced in granulosa cells from small follicles (P less than 0.05), unchanged in cells from medium follicles, and significantly stimulated in cells from large follicles. A23187 inhibited the enhanced production of both hormones after administration of cAMP agonists from cells of small and medium follicles (P less than 0.05), with inhibition significantly greater in cells of small follicles compared with medium. In cells from large follicles challenged with cAMP agonists, A23187 inhibited progesterone but stimulated oestradiol production; substitution of TPA (a protein kinase C stimulator) for A23187 gave identical results under basal or FSH-treated cultures of granulosa cells from small-, medium- or large-sized follicles. Our results suggest that TPA, A23187 and cAMP agonists modulate steroidogenesis differently in pig granulosa cells, depending on the stage of maturation of the follicle. Oestradiol production in granulosa cells from large preovulatory follicles may come under the stimulatory control of regulators of protein kinase C as in follicles near ovulation.     

Effects of ionophore A23187 on calcium fluxes from cultured adrenal cells

The effect of the calcium ionophore A23128 on calcium fluxes from Y-1 adrenal cortical cells was investigated. Conditions were chosen which are known to result in an inhibition of steroidogenesis (6 . 10(-6) M ionophore and 3 . 10(-4) M extracellular calcium). Calcium efflux from Y-1 cells exhibited two distinct phases. A fast phase which was insensitive to the mitochondrial poison sodium azide and a slow, azide-sensitive phase. The ionophore brought about a rapid increase in the rate of calcium efflux and an 84% reduction in the size of the calcium pool which was associated with the slow efflux phase as well as a reduction in its rate constant. A decrease in the size of the rapidly exchanging calcium pool was also detected. Ethanol, the solvent which was used for the ionophore, slightly increased the rate constant of the rapidly exchanging pool. Conditions which resulted in diminished steroidogenic capacity also brought about a reduction in the size of an energy dependent, intracellular pool. The data is interpreted as being consistent with a hypothesis that the ionophore-induced inhibition of steroidogenesis may be causatively related to the loss of intracellular calcium or to the mechanism which brings about the loss.     

Modulation of the epithelial barrier by dexamethasone and prolactin in cultured Madin-Darby canine kidney (MDCK) cells

Glucocorticoids and prolactin (PRL) have a direct effect on the formation and maintenance of tight junctions (TJs) in cultured endothelial and mammary gland epithelial cells. In this work, we investigated the effect of a synthetic glucocorticoid dexamethasone (DEX) and PRL on the paracellular barrier function in MDCK renal epithelial cells. DEX (4 microM)+PRL (2 microg/ml) and DEX alone increased significantly the transepithelial electrical resistance after chronic treatment (4 days) of confluent MDCK monolayers or after 24 h treatment of subconfluent monolayers. Immunoblotting and immunocytochemistry revealed no changes in the expression and distribution of TJ-associated proteins occludin, ZO-1 and claudin-1 in confluent monolayers after hormone addition. However, a marked increase in junctional content for occludin and ZO-1 with no changes in their total expression was observed in subconfluent MDCK monolayers 24 h exposed to DEX or DEX+PRL. No change in cell proliferation/growth was detected at subconfluent conditions following hormone treatment. An increase in the total number of viable cells was observed only in confluent MDCK monolayers after exposure to DEX+PRL suggesting that the main effect of these hormones on already established barrier may be associated with the inhibition of cell death. In conclusion, our data suggest that these hormones (specially dexamethasone) have an effect on TJ structure and function only during the formation of MDCK epithelial barrier by probably modulating the localization, stability or assembly of TJ proteins to membrane sites of intercellular contact.