Cyclic AMP inhibits secretion from bovine adrenal chromaffin cells evoked by carbamylcholine but not by high K+

The role of cAMP in the control of secretion from bovine adrenal chromaffin cells was examined using the adenylate cyclase activator, forskolin. Treatment of chromaffin cells with forskolin resulted in a rise in cAMP levels. Forskolin inhibited catecholamine release elicited by carbamylcholine or nicotine but had no effect on secretion evoked by 55 mM K+. Inhibition of carbamylcholine-stimulated release by forskolin was half-maximal at 10 microM forskolin. The inhibition by forskolin of secretion evoked by carbamylcholine was at a step distal to the rise in intracellular free calcium concentration ([Ca2+]i), since this rise was not inhibited by forskolin, which itself produced a small rise in [Ca2+]i. The results suggest that secretion evoked by carbamylcholine is due to the activation of an additional second messenger pathway acting with the rise in [Ca2+]i. This additional pathway may be the target for cAMP action.     

Inhibition by forskolin of cytosolic calcium rise, shape change and aggregation in quin2-loaded human platelets

The adenylate cyclase stimulator forskolin was used to study the inhibitory effects of elevated cAMP on the activation of washed human platelets loaded with the fluorescent Ca2+ indicator quin2. In the presence of 10 microM isobutylmethylxanthine forskolin inhibited rises in [Ca2+]i evoked by thrombin and platelet-activating factor (PAF) due to both Ca2+ influx and release from internal stores with similar potency. Aggregation evoked by thrombin and PAF was suppressed whilst partial shape-change persisted, even in the absence of a measurable rise in [Ca2+]i. Forskolin did not affect the rise in [Ca2+]i evoked by Ca2+ ionophore; aggregation was suppressed but shape-change persisted.     

Cyclic AMP raises cytosolic Ca2+ and promotes Ca2+ influx in a clonal pancreatic beta-cell line (HIT T-15)

The effect on cytosolic Ca2+ concentration ([Ca2+]i) of cAMP analogues and the adenylate cyclase-stimulating agents forskolin, isoproterenol and glucagon has been examined in an insulin-secreting beta-cell line (HIT T-15) using fura 2. All these manipulations of the cAMP messenger system promoted a rise in [Ca2+]i which was blocked by the Ca2+ channel antagonists verapamil and nifedipine or by removal of extracellular Ca2+. The action of the adenylate cyclase activator forskolin was glucose-dependent. The results suggest that cAMP elevates [Ca2+]i in HIT cells by promoting Ca2+ entry through voltage-sensitive Ca2+ channels, not through mobilization of stored Ca2+. Activation of Ca2+ influx may be an important component of the mechanisms by which cAMP potentiates fuel-induced insulin release.     

Cytoplasmic Ca2+ in platelets is controlled by cyclic AMP: antagonism between stimulators and inhibitors of adenylate cyclase

Activation of platelets by thrombin rapidly increases cytoplasmic free calcium, [Ca2+]i, measured by Quin -2, and induces secretion. Stimulators of adenylate cyclase (i.e. PGI2, PGD2, forskolin) suppressed or reversed the increase of [Ca2+]i. Inhibitors of adenylate cyclase (i.e. epinephrine, ADP), added before or after thrombin, counteracted PGI2, PGD2 and forskolin and thereby increased [Ca2+]i and restored secretion. Responses to epinephrine (via alpha-2 adrenoreceptors) and ADP were independent of extracellular Ca2+, but required maintained occupancy of thrombin receptors and intact cAMP-phosphodiesterase activity. These results indicate that cAMP serves as an inhibitory second-messenger that antagonizes the mobilization of Ca2+, an activator second-messenger.     

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.     

Multiple signaling pathways of histamine H2 receptors. Identification of an H2 receptor-dependent Ca2+ mobilization pathway in human HL-60 promyelocytic leukemia cells

In order to analyze the complex activities of histamine H2 receptor activation on neutrophils, human HL-60 promyelocytic leukemia cells were differentiated into neutrophils by incubation with dimethyl sufoxide, loaded with the Ca2+-sensitive indicator dyes, indo-1 or fura-2, and the levels of intracellular Ca2+ ([Ca2+]i) measured in a fluorescent-activated cell sorter and fluorimeter, respectively. Histamine increased [Ca2+]i in a dose-dependent manner with a half-maximal concentration (EC50) of approximately 10(-6) to 10(-5) M, which exhibited H2 receptor specificity. Prostaglandin E2 and isoproterenol also induced [Ca2+]i mobilization in HL-60 cells, whereas the cell permeable form of cAMP and forskolin failed to increase [Ca2+]i. Since H2-receptor mediated [Ca2+]i mobilization was not inhibited by reducing the concentration of extracellular Ca2+ nor by the addition of Ca2+ channel antagonists, LaCl3 and nifedipine, [Ca2+]i mobilization is due to the release of Ca2+ from intracellular stores. Furthermore, both 10(-4) M histamine and 10(-6) M fMet-Leu-Phe increased the levels of 1,4,5-inositol trisphosphate. However, histamine-induced mobilization of [Ca2+]i was inhibited by cholera toxin but not by pertussis toxin, whereas the action of fMet-Leu-Phe was inhibited by pertussis toxin but not by cholera toxin. These data suggest that H2 receptors on HL-60 cells are coupled to two different cholera toxin-sensitive G-proteins and activate adenylate cyclase and phospholipase C simultaneously.     

Prostaglandin E2 stimulates the formation of mineralized bone nodules by a cAMP-independent mechanism in the culture of adult rat calvarial osteoblasts

The effects of prostaglandin E2 (PGE2) on the proliferation and differentiation of osteoblastic cells were studied in osteoblast-like cells isolated from adult rat calvaria. Treatment of the cells with PGE2 within the concentration range 10(-8)-10(-5) M resulted in a dose-dependent increase in alkaline phosphatase (ALP) activity, [3H]proline incorporation into collagenase-digestible protein, and mineralized bone nodule (BN) formation, as well as a dose-dependent decrease in [3H]thymidine incorporation into the cells. PGE2 also caused a dose-dependent increase in the intracellular cyclic adenosine monophosphate (cAMP) content, with a maximal effective concentration of 10(-5) M; this effect of PGE2 was mimicked by forskolin, an adenylate cyclase activator. The treatment of adult calvarial cells with forskolin decreased BN formation, ALP activity, and collagen synthesis. These results suggested that cAMP does not have a stimulatory, but rather a suppressive, effect on the differentiation of adult rat calvarial cells. A time-course study of cAMP accumulation showed that both PGE2- and forskolin-induced cAMP reached a maximum at 5 min after the treatment, but the former rapidly returned to the basal level by 40 min, while the latter declined slowly and was still at 70% of the maximal level at 60 min, suggesting that PGE2 activates phosphodiesterase as well as adenylate cyclase. The presence of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, reduced the rate of degradation of cAMP formed after PGE2 treatment, suggesting the involvement of calmodulin in the activation of phosphodiesterase. However, PGE2 also caused the production of inositol 1,4,5-triphosphate (IP3) and an elevation of the intracellular Ca2+ concentration ([Ca2+]i), both of which peaked at 15 s and returned to the basal level within 1 min. Submaximal responses of the IP3 production and the [Ca2+]i elevation to PGE2 were obtained at 10(-5) M. W-7 decreased both basal and PGE2-induced ALP activity, collagen synthesis and BN formation, indicating the involvement of Ca2+/calmodulin-dependent protein kinase in the PGE2-induced differentiation of calvarial cells. From these results, we concluded that PGE2 inhibits the proliferation and stimulates the differentiation of calvarial osteoblasts by elevating the [Ca2+]i through the activation of a phosphoinositide turnover, but not via an activation of adenylate cyclase. We also found that BN formation varies, depending on the time of PGE2 addition, suggesting that responsiveness of the cells to PGE2 may change during the culture period.     

Direct regulation of smooth muscle contractile elements by second messengers

The effects of adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP) and phorbol 12,13 dibutyrate (PDBu) on the Ca2+ sensitivity of the contractile elements in the rat mesenteric artery were investigated, using a method of permeabilizing smooth muscle with Staphylococcal alpha-toxin. Both cAMP and cGMP relaxed the permeabilized rat mesenteric artery at the intracellular Ca2+ concentrations [( Ca2+]i) held constant with Ca2+ EGTA buffer and Ca2+ ionophore, ionomycin. In addition, forskolin and sodium nitroprusside which activate adenylate and guanylate cyclases, respectively, also induced relaxation at a fixed [Ca2+]i. In contrast PDBu which stimulates protein kinase C caused an increase in force at a constant [Ca2+]i which could be partially reversed by cAMP or cGMP. These results indicate that second messengers exert direct control over smooth muscle Ca2+ sensitivity of the contractile elements, which is of physiologic and pharmacologic importance.     

Inhibition of Cyclic AMP Accumulation in Intact NCB-20 Cells as a Direct Result of Elevation of Cytosolic Ca2+

Earlier studies established that adenylyl cyclase in NCB-20 cell plasma membranes is inhibited by concentrations of Ca2+ that are achieved in intact cells. The present studies were undertaken to prove that agents such as bradykinin and ATP, which elevate the cytosolic Ca2+ concentration ([Ca2+]i) from internal stores in NCB-20 cells, could inhibit cyclic AMP (cAMP) accumulation as a result of their mobilization of [Ca2+]i and not by other mechanisms. Both bradykinin and ATP transiently inhibited [3H]cAMP accumulation in parallel with their transient mobilization of [Ca2+]i. The [Ca2+]i rise stimulated by bradykinin could be blocked by treatment with thapsigargin; this thapsigargin treatment precluded the inhibition of cAMP accumulation mediated by bradykinin (and ATP). A rapid rise in [Ca2+]i, as elicited by bradykinin, rather than the slow rise evoked by thapsigargin was required for inhibition of [3H]cAMP accumulation. Desensitization of protein kinase C did not modify the inhibitory action of bradykinin on [3H]cAMP. Effects of Ca2+ on phosphodiesterase were also excluded in the present studies. The accumulated data are consistent with the hypothesis that hormonal mobilization of [Ca2+]i leads directly to the inhibition of cAMP accumulation in these cells and presumably in other cells that express the Ca(2+)-inhibitable form of adenylyl cyclase.     

Cyclic AMP raises cytoplasmic calcium in pancreatic alpha 2-cells by mobilizing calcium incorporated in response to glucose

The cytoplasmic Ca2+ concentration ([Ca2+]i) was monitored in individual guinea-pig pancreatic alpha 2-cells exposed to modulators of glucagon release. Addition of the stimulatory amino acid arginine resulted in a sustained increase in [Ca2+]i, whereas the inhibitor glucose had the opposite effect. Epinephrine, the beta-adrenergic agonist isoproterenol, the adenylate cyclase activator forskolin and 8-bromo-cAMP transiently raised [Ca2+]i provided that the cells had been pretreated with glucose. However, simultaneous presence of glucose was not required and the effect occurred even in the absence of extracellular Ca2+. Carbachol, the alpha 2-adrenergic agonist clonidine and the sulfonylurea tolbutamide lacked effects on [Ca2+]i. In addition to providing support for the concept that glucagon release is positively modulated by [Ca2+]i, the results demonstrate that cAMP raises [Ca2+]i in the alpha 2-cells by mobilizing calcium incorporated in response to glucose.     

Activation of muscarinic receptors in PC12 cells. Correlation between cytosolic Ca2+ rise and phosphoinositide hydrolysis.

The intracellular signals generated by carbachol activation of the muscarinic receptor [release of inositol phosphates as a consequence of phosphoinositide hydrolysis and rise of the cytosolic Ca2+ concentration ([Ca2+]i, measured by quin2)] were studied in intact PC12 pheochromocytoma cells that had been differentiated by treatment with nerve growth factor. When measured in parallel samples of the same cell preparation 30 s after receptor activation, the release of inositol trisphosphate and of its possible metabolites, inositol bis- and mono-phosphate, and the [Ca2+]i rise were found to occur with almost superimposable carbachol concentration curves. At the same time carbachol caused a decrease in the radioactivity of preloaded phosphatidylinositol 4,5-bisphosphate, the precursor of inositol trisphosphate. Neither the inositol phosphate nor the [Ca2+]i signal was modified by preincubation of the cells with either purified Bordetella pertussis toxin or forskolin, the direct activator of adenylate cyclase. Both signals were partially inhibited by dibutyryl cyclic AMP, especially when the nucleotide analogue was applied in combination with the phosphodiesterase inhibitors RO 201724 and theophylline. The latter drug alone profoundly inhibited the carbachol-induced [Ca2+]i rise, with only minimal effect on phosphoinositide hydrolysis. Because of the diverging results obtained with forskolin on the one hand, dibutyryl cyclic AMP and phosphodiesterase inhibitors on the other, the effects of the latter drugs are considered to be pharmacological, independent of the intracellular cyclic AMP concentration. Two further drugs tested, mepacrine and MY5445, inhibited phosphoinositide hydrolysis at the same time as the 45Ca2+ influx stimulated by carbachol. Taken together, our results concur with previous evidence obtained with permeabilized cells and cell fractions to indicate phosphatidylinositol 4,5-bisphosphate hydrolysis and [Ca2+]i rise as two successive events in the intracellular transduction cascade initiated by receptor activation. The strict correlation between the carbachol concentration curves for inositol trisphosphate generation and [Ca2+]i rise, and the inhibition by theophylline of the Ca2$ signal without major effects on inositol phosphate generation, satisfy important requirements of the abovementioned interpretation.     

Melatonin inhibits pituitary adenylyl cyclase-activating polypeptide-induced increase of cyclic AMP accumulation and [Ca2+]i in cultured cells of neonatal rat pituitary

The effects of melatonin on pituitary adenylyl cyclase-activating polypeptide-induced increase of cyclic AMP and [Ca2+]i were studied in neonatal rat pituitary cells. The polypeptide increased cyclic AMP accumulation. In the presence of melatonin the increase of cyclic AMP was inhibited in a dose-dependent manner, the maximal inhibition was achieved with 1-10 nM melatonin. Pituitary adenylyl cyclase-activating polypeptide also increased [Ca2+]i in 30% of the pituitary cells and melatonin inhibited the effect. Most of the cells sensitive to adenylyl cyclase-activating polypeptide (77%) were also sensitive to GnRH, suggesting they are gonadotrophs. The remaining cells were not identified. The polypeptide-induced [Ca2+]i increase was inhibited in Ca2+-free medium in 2/3 of the cells indicating that Ca2+ influx was involved. To examine causal relationship between cyclic AMP and [Ca2+]i increase, we have studied the effect of adenylyl cyclase activation by forskolin on intracellular Ca2+ concentration. Forskolin had similar effects as adenylyl cyclase-activating polypeptide: it increased [Ca2+]i in the pituitary cells and the increase was dependent on presence of Ca2+ in the medium. Melatonin inhibited the forskolin induced [Ca2+]i increase. Our observations indicate that increase of cyclic AMP stimulates Ca2+ influx in the pituitary cells of neonatal rat and that this mechanism is involved in [Ca2+]i increase induced by the pituitary adenylyl cyclase-activating polypeptide. Because melatonin inhibits increase of cyclic AMP induced by pituitary adenylyl cyclase-activating polypeptide or forskolin, the inhibitory effect of melatonin on Ca2+-influx may be mediated by the decrease of cyclic AMP concentration. This mechanism of melatonin action has not been described previously. Because melatonin inhibits the polypeptide- or forskolin-induced [Ca2+]i also in the cells not sensitive to GnRH, melatonin receptors seem to be present on both gonadotrophs and non-gonadotrophic pituitary cells.     

Modulation of vincristine sensitivity of human kidney tumor cells by pharmacological agents interfering with intracellular signals. No apparent relationship to changes in cytoplasmic Ca2+ or pH

The effect of substances proposed to modulate intracellular signal systems on growth and sensitivity to vincristine in the human kidney tumor cell line ACHN was investigated and related to changes in cytoplasmic free Ca2+ concentration ([Ca2+]i) and cytoplasmic pH (pHi). Presence during culture of the protein kinase C (PKC) activator 12-O-tetradecanoyl phorbol 13-acetate (TPA) had no effect on cell growth but significantly increased the EC50 concentration for vincristine inhibited cell growth. There was no indication for endogenous PKC activity being responsible for basal vincristine insensitivity since it was not affected by the PKC inhibitor H-7. The Ca2+ ionophore ionomycin tended to increase cell growth and induced vincristine resistance, whereas the calmodulin inhibitor W-7 had opposite effects. Presence during culture of the adenylate cyclase activator forskolin did not affect basal cell growth but dose-dependently made the cells more sensitive to vincristine. The modulators of vincristine sensitivity had no immediate effect on pHi, whereas after 3 days of incubation ionomycin and forskolin tended to increase pHi. Ionomycin and forskolin induced an immediate increase in [Ca2+]i which remained after 3 days only for ionomycin, whereas TPA decreased [Ca2+]i, a change which tended to remain after 3 days of incubation. It is concluded that perturbation of the intracellular signal system may affect both cell growth and cytotoxic drug sensitivity. However, there is no apparent relationship between immediate or late changes in [Ca2+]i and pHi and vincristine sensitivity.     

Role of Ca2+ in H+ transport by rabbit gastric glands studied with A23187 and BAPTA, an incorporated Ca2+ chelator

The role of Ca2+ in stimulation of H+ gastric secretion by cAMP-dependent and -independent secretagogues was studied in isolated rabbit glands using Ca2+ ionophore, A23187, and an intracellular Ca2+ chelator (BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) incorporated as its acetoxymethyl ester (BAPTA-AM). Acetylcholine (ACh), tetragastrin (TG), histamine and forskolin induced a transitory increase of intracellular Ca2+ concentration, [Ca2+]i, measured in gastric glands loaded with Ca2+-sensitive dye fura-2, and provoked an acid secretory response evaluated with aminopyrine accumulation ratio (AP ratio). The Ca2+-ionophore A23187 also induced an increase in [Ca2+]i and in AP ratio. cAMP-dependent secretagogues were more potent stimulants of acid secretion than cAMP-independent secretagogues. cAMP analogue, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-BR-cAMP) induced an increase in AP ratio without modifying [Ca2+]i. BAPTA-AM (5-25 microM) induced a transient decrease of resting [Ca2+]i which returned to basal level due to extracellular Ca2+ entry. Increases in [Ca2+]i produced by ACh and TG were abolished by BAPTA and those produced by Ca2+ ionophore A23187 were partially buffered. BAPTA inhibited in a dose-dependent manner H+ secretion induced by cholinergic and gastrinergic stimulants in the presence of cimetidine. A23187 increased the AP ratio to values similar to those obtained with ACh or TG and was not inhibited by BAPTA. BAPTA partially inhibited (40%) the increase in AP ratio induced by forskolin and histamine inspite of the complete inhibition of the Ca2+ response. BAPTA did not inhibit the response to 8-BR-cAMP. BAPTA inhibition of forskolin stimulation was reversed by A23187 and the response was potentiated. These results indicate that ACh and TG response are completely dependent on an increase of [Ca2+]i. The response to cAMP-dependent agonists histamine and forskolin depend both on Ca2+ and cAMP. For forskolin stimulation the response may be the result of a potentiation between Ca2+ and cAMP.     

Multistep regulation of Leydig cell function

LH controls Leydig cell steroidogenesis by interaction with specific membrane receptors initiating membrane coupling events. Stimulation of the androgen pathways occurs mainly through cAMP mediated mechanism including LH induced guanyl nucleotide binding, membrane phosphorylation and adenylate cyclase activation. cAMP dependent kinase activation presumably causes phosphorylation of key proteins of the steroidogenic pathway and consequent increase in testosterone production. The hormone also appears to facilitate the androgen stimulus by a cyclic AMP independent mechanism located at the plasma membrane or intracellular sites. The stimulatory event can be negatively influenced by the action of certain peptide hormones (i.e. angiotensin II) through the guanyl nucleotide inhibitory subunit of adenylate cyclase (Gi). In recent studies we have presented evidence for a Ca2+ sensitive kinase system present in purified cell membranes. Gpp(NH)p, GTP, and phospholipid in presence of nanomolar Ca2+ induce phosphate incorporation into Mr 44,500 substrate with marked inhibition at microM Ca2+. Similarly a biphasic pattern of activation was observed with adenylate cyclase activity. Membrane phosphorylation may be a modifier of LH-stimulated adenylate cyclase activity and possibly other LH induced actions in the activated Leydig cell membrane. Furthermore we have defined the stimulatory effects of forskolin on all Leydig cell cyclic AMP pools and have provided additional evidence of functional compartmentalization and/or cAMP independent facilitory stimulus of steroidogenesis by the trophic hormone. The demonstration of a novel high affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation mediated by the Gi subunit of adenylate cyclase has provided a new approach for direct evaluation of functional inhibitory influence of Gi subunit in the Leydig cell. The cultured fetal Leydig cell system has provided a useful model to elucidate mechanisms involved in the development of gonadotropin induced estradiol mediated desensitization of steroidogenesis. We have isolated from the fetal testis a small population (2-5% of total) of transitional cells with morphological characteristics of cells found in 15 day postnatal testis but functional capabilities of the adult cell. We have also demonstrated after appropriate treatment (i.e. estrogen, and frequent or a high gonadotropin dose) the emergence of a functional adult-like cell type from the fetal Leydig cell population.     

Glucagon-like peptide-1 induces a cAMP-dependent increase of [Na+]i associated with insulin secretion in pancreatic beta-cells

Glucagon-like peptide-1 (GLP-1) elevates the intracellular free calcium concentration ([Ca2+]i) and insulin secretion in a Na+-dependent manner. To investigate a possible role of Na ion in the action of GLP-1 on pancreatic islet cells, we measured the glucose-and GLP-1-induced intracellular Na+ concentration ([Na+]i), [Ca2+]i, and insulin secretion in hamster islet cells in various concentrations of Na+. The [Na+]i and [Ca2+]i were monitored in islet cells loaded with sodium-binding benzofuran isophthalate and fura 2, respectively. In the presence of 135 mM Na+ and 8 mM glucose, GLP-1 (10 nM) strongly increased the [Na+]i, [Ca2+]i, and insulin secretion. In the presence of 13.5 mM Na+, both glucose and GLP-1 increased neither the [Na+]i nor the [Ca2+]i. In a Na+-free medium, GLP-1 and glucose did not increase the [Na+]i. SQ-22536, an inhibitor of adenylate cyclase, and H-89, an inhibitor of PKA, incompletely inhibited the response. In the presence of both 8 mM glucose and H-89, 8-pCPT-2'-O-Me-cAMP, a PKA-independent cAMP analog, increased the insulin secretion and the [Na+]i. Therefore, we conclude that GLP-1 increases the cAMP level via activation of adenylate cyclase, which augments the membrane Na+ permeability through PKA-dependent and PKA-independent mechanisms, thereby increasing the [Ca2+]i and promoting insulin secretion from hamster islet cells.     

Regulation of zymogen granule exocytosis by Ca2+, cAMP, and PKC in pancreatic acinar cells

The effect of cAMP and PKC on zymogen granule exocytosis was investigated by simultaneously measuring cytosolic Ca2+ concentration ([Ca2+]c) and individual zymogen granule exocytosis in isolated mouse pancreatic acini. When acinar cells were stimulated with acetylcholine (ACh, 10 microM), exocytic events were detected through granule-attached apical membranes with [Ca2+]c rise. Application of secretin, forskolin (an adenylate cyclase activator), or PMA (a PKC activator) alone did not elicit any [Ca2+]c rise or zymogen granule exocytosis, but co-stimulation with ACh led to exocytosis in that the total number of secreted granules increased markedly without a significant difference in [Ca2+]c rises. When we evoked exocytosis by [Ca2+]c ramps, pretreatment with forskolin or PMA elicited exocytosis at lower [Ca2+]c levels. These results indicate that PKC or cAMP alone could not directly elicit zymogen granule exocytosis, but that they increase the total releasable pool by rendering zymogen granules more sensitive to Ca2+.     

Forskolin refractoriness. Exposure to the diterpene alters guanine nucleotide-dependent adenylate cyclase and calcium-uptake activity of cells cultured from the rat aorta.

Cells with the morphological properties of endothelial cells were cultured from the rat aorta. The cultured cells accumulated 45Ca2+ from the medium in a manner which was stimulated by forskolin and by 8-bromo-cyclic AMP. Pretreating the cultures for 20 h with forskolin diminished forskolin-dependent Ca2+-uptake activity. Adenylate cyclase activity of cultured cell homogenates was stimulated by guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) and forskolin, and by isoprenaline in the presence, but not in the absence, of guanine nucleotide. p[NH]ppG increased forskolin sensitivity and caused a leftward shift in the forskolin dose-response curve. Pretreating the cultured cells with forskolin for 20 h, conditions that decreased forskolin-dependent Ca2+ uptake, increased basal and guanine nucleotide-dependent adenylate cyclase activity, but not forskolin-dependent activity determined in the absence of p[NH]ppG. Forskolin pretreatment diminished p[NH]ppG's capacity to increase forskolin sensitivity, but did not have a significant effect on either the sensitivity of adenylate cyclase to p[NH]ppG or its responsiveness to isoprenaline. These results suggest that the Ca2+-uptake mechanism is cyclic AMP-dependent and that guanine nucleotides mediated forskolin-dependent cyclic AMP production by the intact cells. In addition, there may be different guanine nucleotide requirements for hormone-receptor coupling and forskolin activation.     

Differential effects of parathyroid hormone and its analogues on cytosolic calcium ion and cAMP levels in cultured rat osteoblast-like cells

While the stimulatory effect of parathyroid hormone (PTH) on osteoblast-like cell adenylate cyclase is well known, the effect of PTH on cytosolic calcium ion ([Ca2+]i) mobilization is controversial, one group finding no effect but others reporting various increases. We investigated the effects on [Ca2+]i of synthetic rat PTH fragment 1-34 (rPTH(1-34)) and two bovine PTH analogues that inhibit PTH's stimulation of adenylate cyclase (bovine 8,18Nle, 34Tyr-PTH(3-34) and 34Tyr-PTH(7-34]. [Ca2+]i was measured before, during, and after exposure to PTH analogues in perifused, attached osteoblast-like rat osteosarcoma cells (ROS 17/2.8) that had been scrape-loaded with the luminescent photoprotein aequorin. Resting [Ca2+]i was 0.094 +/- 0.056 microM (mean +/- S.D., n = 103) and rose in a time- and dose-specific way after exposure to rPTH(1-34). At 10(-10) M rPTH(1-34), [Ca2+]i rose 100% within 30 s to a plateau; higher concentrations of PTH yielded increasing initial peaks of [Ca2+]i followed by lower plateaus. At 10(-6) M, the initial peak was 5-fold basal, or 0.64 +/- 0.07 microM. Both analogues of PTH were at least partial agonists for [Ca2+]i mobilization and did not reduce peak [Ca2+]i when co-perifused with rPTH(1-34). However, the analogues did reduce significantly rPTH(1-34)-induced cAMP accumulation and did not increase cAMP accumulation by themselves. Thus, rPTH(1-34) strongly mobilizes [Ca2+]i in ROS 17/2.8 cells, at near-physiologic concentrations. Failure of the PTH analogues to block the effect of PTH on [Ca2+]i while inhibiting the effect on cAMP accumulation suggests separate pathways for PTH activation of adenylate cyclase and mobilization of calcium.     

4 beta-Phorbol 12-myristate 13-acetate attenuates the glucagon-induced increase in cytoplasmic free Ca2+ concentration in isolated rat hepatocytes.

Hepatocytes were isolated from rats and then loaded with the fluorescent Ca2+ indicator quin2. Glucagon caused a sustained increase (at least 5 min) in the fluorescence of the quin2-loaded cells; the increase was much greater than that observed with control, non-quin2-loaded, cells. These observations indicate that glucagon caused an increase in cytoplasmic free Ca2+ concentration [( Ca2+]c). The effects of glucagon were mimicked if forskolin (to activate adenylate cyclase), dibutyryl cyclic AMP or bromo cyclic AMP were added directly to the cells. Thus an increase in cyclic AMP concentration may mediate the effect of glucagon on [Ca2+]c. If 4 beta-phorbol 12-myristate 13-acetate (PMA; an activator of protein kinase C) was added to the cells before glucagon, the magnitude of the increase in [Ca2+]c was greatly diminished. If PMA was added after glucagon it caused a lowering of [Ca2+]c. These effects of PMA on the glucagon-induced increase in [Ca2+]c could not be mimicked if [Ca2+]c was increased by the Ca2+-ionophore ionomycin. Thus an event involved in the mechanism by which glucagon increases [Ca2+]c appears to be required for the action of PMA. If [Ca2+]c was increased by forskolin, dibutyryl cyclic AMP or bromo cyclic AMP, the effect of PMA on [Ca2+]c was similar to that observed when glucagon was used to elevate [Ca2+]c. When [Ca2+]c was raised by dibutyryl cyclic AMP the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine did not prevent the subsequent addition of PMA from causing [Ca2+]c to decrease. These observations suggest that PMA can inhibit the cyclic AMP-induced increase in [Ca2+]c independently of any changes in cyclic AMP concentration. Glucagon appears to increase [Ca2+]c by releasing intracellular stores of Ca2+ and stimulating net influx of Ca2+ into the cell; PMA greatly diminishes both of these effects.