Possible involvement of inositol phosphates and calmodulin in calcitonin-induced stimulation of phosphate transport in LLC-PK1 cells

The present study investigated the possible involvement of phosphatidylinositol breakdown and Ca2+-calmodulin complex in the calcitonin-induced stimulation of phosphate transport in LLC-PK1 cells. This cell line with calcitonin receptors possesses Na+-dependent phosphate transport and has been employed as a model for studying the mechanism of renal tubular phosphate transport. (Asu1,7) eel calcitonin stimulated the phosphate transport in LLC-PK1 cells in a dose-dependent fashion with accompanying increase of inositol triphosphate (IP3) production. When the cells were preincubated with the potent calmodulin antagonist, w-7 or w-13, the stimulatory effect of calcitonin on phosphate transport was significantly inhibited. These findings indicate that Ca2+-calmodulin complex formed by increased cytosolic Ca2+, which is mobilized from intracellular pools by IP3, may be involved in the signal transduction of calcitonin in these cells.     

Phorbol esters stimulate phosphate accumulation synergistically with A23187 in cultured renal tubular cells

The effects of phorbol esters and diacylglycerol on phosphate accumulation in the cultured mouse kidney cells were investigated to assess the possible role of Ca2+-activated, phospholipid dependent protein kinase (protein kinase C) on the renal phosphate handling. 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated phosphate accumulation dose-dependently. TPA-induced phosphate accumulation was synergistically enhanced with A23187. 4 alpha-phorbol 12,13-didecanoate did not stimulate the phosphate accumulation, while 4 beta-phorbol 12,13-didecanoate stimulated it. Additionally, 1-oleoyl-2-acetyl-glycerol exhibited a stimulatory effect on phosphate accumulation. These data indicated that protein kinase C is one of possible regulators of phosphate transport at the renal tubules.     

Activators of Protein Kinase C Act at a Postreceptor Site to Amplify Cyclic AMP Production in Rat Pinealocytes

Activation of alpha 1-adrenoceptors appears to amplify beta-adrenergic stimulation of cyclic AMP (cAMP) accumulation in rat pinealocytes severalfold by a mechanism involving activation of a Ca2+-, phospholipid-dependent protein kinase (protein kinase C). The mechanism of action of protein kinase C was investigated in this report using intact cells. Activation of protein kinase C with 4 beta-phorbol 12-myristate 13-acetate (PMA; 10(-7) M) or the alpha 1-adrenergic agonist phenylephrine (PE; 10(-6) M) did not inhibit cAMP efflux in beta-adrenergically stimulated cells. The amplification of the beta-adrenergic cAMP response by these agents also occurred in the presence of isobutylmethylxanthine (10(-3) M) and Ro 20-1724 (10(-4) M), an observation suggesting that inhibition of cAMP phosphodiesterase activity is not the mechanism of action. Furthermore, although PMA (10(-7) M) caused a sixfold increase in the magnitude of the cAMP response to isoproterenol, it did not alter the EC50 of the response (1.7 X 10(-8) M), a result indicating that protein kinase C activation does not alter beta-adrenoceptor sensitivity. The cAMP response following cholera toxin pretreatment (60-120 min) was rapidly and markedly enhanced by alpha 1-adrenergic agonists (cirazoline greater than PE greater than methoxamine), by phorbol esters (PMA greater than 4 beta-phorbol 12,13,-dibutyrate much greater than 4 alpha-phorbol 12,13-didecanoate), and by synthetic diacylglycerols (1,2-dioctanoylglycerol greater than 1-oleoyl 2-acetylglycerol much greater than diolein). The cAMP response to forskolin (10(-5)-10(-3) M) was also increased by PE (3 X 10(-6) M) and PMA (10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-1 stimulation of arachidonic acid release from human synovial fibroblasts; blockade by inhibitors of protein kinases and protein synthesis

Addition of IL-1 (interleukin-1) to human synovial fibroblasts radiolabelled with [3H]arachidonic acid caused a linear dose-dependent increase in arachidonic acid release and a transient rise in labelled diacylglycerol. Protein kinase C activators PMA 4-phorbol 12-myristate 13-acetate and DiC8 (1,2-dioctanoyl-sn-glycerol) also increased arachidonic acid release, but the time course observed with PMA was different from that of IL-1. When cultures were treated with PMA for 16-24 h to down regulate protein kinase C, the ability of IL-1 to increase arachidonic acid release persisted to the same extent as in nontreated cultures. In contrast, PMA pretreatment prevented the eight-fold stimulation of arachidonic acid release in response to PMA observed in cultures not previously exposed to PMA. To examine the role of other kinases in IL-1 stimulated arachidonic acid release, cultures were treated with H-7 (1-(5-isoquinolinesulphonyl)-2-methylpiperazine dichloride), H-8 (N-[2-(methylamino) ethyl]-5-isoquinolinesulphonamide dichloride), HA1004 (N-(2-guanidoinoethyl)-5-isoquinolinesulphonamide hydrochloride), and staurosporine. IL-1 stimulation of arachidonic acid release was blocked by H-7, H-8 and staurosporine. H-7 was a more potent inhibitor than H-8, suggesting that cAMP dependent kinase did not mediate IL-1 action. Addition of H-7 at various times following IL-1 decreased IL-1 stimulated arachidonic acid release, suggesting that continued protein kinase activity was necessary for IL-1 action. Cycloheximide and actinomycin D inhibited the stimulation of arachidonic acid release by IL-1, PMA or DiC8. The addition of cycloheximide or actinomycin D 15-45 min after IL-1 also inhibited IL-1 stimulated arachidonic acid release, indicating that continued protein synthesis was required for IL-1 action. These results suggest that IL-1 stimulation of acylhydrolyase activity in human synovial cells occurs by a mechanism requiring continued protein synthesis and protein kinase activity and that neither protein kinase C nor cAMP dependent protein kinase is involved.     

Cross-talk regulation between cyclic AMP production and phosphoinositide hydrolysis induced by prostaglandin E2 in osteoblast-like cells.

In cloned osteoblast-like MC3T3-E1 cells, PGE2 stimulated both cAMP accumulation and the formation of inositol trisphosphate (IP3) dose dependently. The cAMP accumulation showed the peak value at 5 min and decreased thereafter, whereas the IP3 formation reached a plateau almost within 10 min and sustained it up to 30 min. The effect of PGE2 on cAMP accumulation (EC50 was 80 nM) was more potent than that on IP3 formation (EC50 was 0.8 microM). 12-O-Tetradecanoyl-phorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, reduced the PGE2-induced cAMP accumulation, whereas 4 alpha-phorbol 12,13-didecanoate, a PKC-nonactivating phorbol ester, had little effect on the cAMP accumulation. 1-Oleoyl-2-acetyl-glycerol, a specific activator for PKC, inhibited PGE2-induced cAMP accumulation. TPA had little effect on cAMP accumulation induced by forskolin or NaF, a GTP-binding protein activator. So, the effect of TPA is presumed to be exerted at the point between the PGE2 receptor and Gs. On the other hand, forskolin and dibutyryl cAMP had little effect on the IP3 formation stimulated by PGE2. H-7, a PKC inhibitor, enhanced the PGE2-induced cAMP accumulation in comparison with HA1004, a control for H-7. Our data suggest that PGE2 regulates cAMP production through self-induced activation of PKC. These results strongly suggest that there is an autoregulatory mechanism in PGE2 signaling, and PGE2 modulates osteoblast functions through a cross-talk interaction between cAMP production and phosphoinositide hydrolysis in osteoblast-like cells.     

Protein kinase A and/or C inhibitors potentiate isoproterenol-induced cyclic AMP accumulation in intact human lymphocytes

The objective of the present study was to investigate the roles of protein kinase A and/or C in agonist-induced beta adrenoceptor activation in intact human lymphocytes. LYmphocytes from healthy subjects were incubated with isoproterenol and phosphodiesterase inhibitor (IBMX, 1.0 mM) after 20 minutes of preincubation with (or without) various compounds possessing protein kinase A and/or C inhibitory activities. These compounds included the relatively selective protein kinase C (PK-C) inhibitors (W-7, calmidazolium, polymyxin B, neomycin, tamoxifen and clomiphene), purified protein inhibitors of protein kinase A (PK-A) (obtained synthetically, or purified from bovine hearts and porcine hearts) and the two compounds (H-7, H-9), which have been found to inhibit both PK-A and PK-C. The results showed that all PK-C inhibitors alone decreased cellular basal cAMP levels while inhibitors of PK-A as well as both H-7 and H-9 increased basal cAMP levels in a dose dependent manner at certain concentrations. All inhibitors studied potentiated isoproterenol-induced cAMP accumulation. The protein kinase A and C inhibitor, H-7, also potentiated PGE1 (but not forskolin)-induced cAMP accumulation. In contrast, the protein kinase C activator, PMA, inhibited isoproterenol- and PGE1- (but not forskolin) induced cAMP accumulation. These data suggest that the potentiating effects of PK-A and/or C inhibitors may be related to the inhibition of PK-A and/or PK-C, both of which have been shown to be involved in beta 2 adrenoceptor desensitization and phosphorylation.     

Role for protein kinase C in the modulation of glomerular PGE2 production by angiotensin II

Angiotensin II increased PGE2 release from superfused glomeruli, and stimulated labeled inositol phosphate production. 12-O-Tetradecanoyl phorbol -13-acetate (TPA, 10(-7) M), which stimulates protein kinase C activity in soluble fractions of glomerular homogenates, suppressed angiotensin II actions on inositol phosphate production and PGE2. By contrast, 4a phorbol 12,13 di-decanoate and phorbol had no effect on protein kinase C activity or angiotensin II induced increases in inositol phosphate or PGE2. 1-(5-Isoquinolinyl)-2-methylpiperazine (H-7), which inhibits protein kinase C activity in soluble fractions of glomerular homogenates, prevented TPA induced suppression of angiotensin II actions on inositol phosphate production and PGE2. Moreover H-7 prolonged the time course of angiotensin II induced inositol phosphate production and enhanced angiotensin II actions on glomerular PGE2 production. The results support a role for inositol phospholipid hydrolysis through the phospholipase C pathway in the mediation of angiotensin II actions on PGE2 in glomeruli and are consistent with negative modulation of these actions by protein kinase C.     

Stimulation of Na+/phosphate cotransport in LLC-PK1 cells by 12-O-tetradecanoylphorbol 13-acetate (TPA)

We have tested for the effect of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on Na+/phosphate cotransport in an established epithelial cell line of renal origin (LLC-PK1). Incubation of LLC-PK1 cells with TPA produced an increase in Na+/phosphate (Pi) cotransport. The maximal response was reached at a TPA concentration of 10 ng/ml. Other phorbol esters which have no potency or a smaller one to activate protein kinase C had no effect on Na+/Pi cotransport. Incubation of LLC-PK1 cells with 10 ng/ml TPA for 8 h led to a 300% increase in Na+/Pi cotransport; in the presence of cycloheximide the increase amounted only to a 100% and was reached within 2 h. Kinetic analysis of Na+/Pi cotransport indicated an increase in the apparent Vmax without an effect on the apparent Km. The increased Pi transport was retained in isolated apical vesicles. Na+-dependent alanine transport into LLC-PK1 monolayers was affected by TPA administration in a similar manner. TPA had under the chosen experimental conditions no effect on [3H]thymidine incorporation into DNA excluding a general proliferative effect. We conclude that TPA via activation of protein kinase C regulates the number of operating transport systems. As also other Na+-coupled transport systems are influenced, the TPA effect appears to be related to the expression of a general 'adaptive' alteration of membrane transport in LLC-PK1 cells.     

Evidence that protein kinase C may not be involved in the insulin action on cAMP phosphodiesterase: studies with electroporated rat adipocytes that were highly responsive to insulin.

Partially permeabilized rat adipocytes with a high responsiveness to insulin were prepared by electroporation and used to study the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on insulin actions in adipocytes. H-7 is a well-documented inhibitor of several protein kinases, including protein kinase C; however, it does not rapidly enter adipocytes protected with the intact plasma membrane. The cells were suspended in Buffer X [4.74 mM NaCl, 118.0 mM KCl, 0.38 mM CaCl2, 1.00 mM EGTA, 1.19 mM Mg2SO4, 1.19 mM KH2PO4, 25.0 mM Hepes/K, 20 mg/ml bovine serum albumin, and 3 mM pyruvate/Na, pH 7.4] and electroporated six times with a Gene-Pulser (from Bio-Rad) set at 25 microF and 2 kV/cm. In cells electroporated as above, insulin stimulated (a) membrane-bound, cAMP phosphodiesterase approximately 2.6-fold when the hormone concentration was 10 nM and (b) glucose transport activity approximately 4.5-fold when the hormone concentration was raised to 100 nM. H-7 strongly inhibited the actions of insulin on both glucose transport (apparent Ki = 0.3 mM) and cAMP phosphodiesterase (apparent Ki = 1.2 mM) in electroporated adipocytes. H-7 also inhibited lipolysis in adipocytes; the apparent Ki value for the reaction in intact cells was 0.45 mM, and that in electroporated cells was 0.075 mM. It is suggested that a certain protein kinase or kinases that are significantly sensitive to H-7 may be involved in the insulin-dependent stimulation of glucose transport and that of phosphodiesterase. However, protein kinase C (or Ca2+/phospholipid-dependent protein kinase) may not be involved, at least, in the hormonal action on phosphodiesterase since the apparent Ki value of H-7 for the reaction is too high.     

Second messenger systems and progesterone secretion in the small cells of the bovine corpus luteum: effects of gonadotropins and prostaglandin F2a

The present studies were conducted to determine the effects of gonadotropins (LH and hCG) and prostaglandin F2a (PGF2a) on the production of "second messengers" and progesterone synthesis in purified preparations of bovine small luteal cells. Corpora lutea were removed from heifers during the luteal phase of the normal estrous cycle. Small luteal cells were isolated by unit-gravity sedimentation and were 95-99% pure. LH provoked rapid and sustained increases in the levels of [3H]inositol mono-, bis-, and trisphosphates (IP, IP2, IP3, respectively), cAMP and progesterone in small luteal cells. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH but had no effect on LH-stimulated cAMP or progesterone accumulation. Time course studies revealed that LH-induced increases in IP3 and cAMP occurred simultaneously and preceded the increases in progesterone secretion. Similar dose-response relationships were observed for inositol phosphate and cAMP accumulation with maximal increases observed with 1-10 micrograms/ml of LH. Progesterone accumulation was maximal at 1-10 ng/ml of LH. LH (1 microgram/ml) and hCG (20 IU/ml) provoked similar increases in inositol phosphate, cAMP and progesterone accumulation in small luteal cells. 8-Bromo-cAMP (2.5 mM) and forskolin (1 microM) increased progesterone synthesis but did not increase inositol phosphate accumulation in 30 min incubations. PGF2a (1 microM) was more effective than LH (1 microgram/ml) at stimulating increases in inositol phosphate accumulation (4.4-fold vs 2.2-fold increase for PGF2a and LH, respectively). The combined effects of LH and PGF2a on accumulation of inositol phosphates were slightly greater than the effects of PGF2a alone. In 30 min incubations, PGF2a had no effect on cAMP accumulation and provoked small increases in progesterone secretion. Additionally, PGF2a treatment had no significant effect on LH-induced cAMP or progesterone accumulation in 30 min incubations of small luteal cells. These findings provide the first evidence that gonadotropins stimulate the cAMP and IP3-diacylglycerol transmembrane signalling systems in bovine small luteal cells. PGF2a stimulated phospholipase C activity in small cells but did not reduce LH-stimulated cAMP or progesterone accumulation. These results also demonstrate that induction of functional luteolysis in vitro requires more than the activation of the phospholipase C-IP3/calcium and -diacylglycerol/protein kinase C transmembrane signalling system.     

A transfected m1 muscarinic acetylcholine receptor stimulates adenylate cyclase via phosphatidylinositol hydrolysis

The m1 muscarinic acetylcholine receptor gene was transfected into and stably expressed in A9 L cells. The muscarinic receptor agonist, carbachol, stimulated inositol phosphate generation, arachidonic acid release, and cAMP accumulation in these cells. Carbachol stimulated arachidonic acid and inositol phosphate release with similar potencies, while cAMP generation required a higher concentration. Studies were performed to determine if the carbachol-stimulated cAMP accumulation was due to direct coupling of the m1 muscarinic receptor to adenylate cyclase via a GTP binding protein or mediated by other second messengers. Carbachol failed to stimulate adenylate cyclase activity in A9 L cell membranes, whereas prostaglandin E2 did, suggesting indirect stimulation. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated arachidonic acid release yet inhibited cAMP accumulation in response to carbachol. PMA also inhibited inositol phosphate release in response to carbachol, suggesting that activation of phospholipase C might be involved in cAMP accumulation. PMA did not inhibit prostaglandin E2-, cholera toxin-, or forskolin-stimulated cAMP accumulation. The phospholipase A2 inhibitor eicosatetraenoic acid and the cyclooxygenase inhibitors indomethacin and naproxen had no effect on carbachol-stimulated cAMP accumulation. Carbachol-stimulated cAMP accumulation was inhibited with TMB-8, an inhibitor of intracellular calcium release, and W7, a calmodulin antagonist. These observations suggest that carbachol-stimulated cAMP accumulation does not occur through direct m1 muscarinic receptor coupling or through the release of arachidonic acid and its metabolites, but is mediated through the activation of phospholipase C. The generation of cytosolic calcium via inositol 1,4,5-trisphosphate and subsequent activation of calmodulin by m1 muscarinic receptor stimulation of phospholipase C appears to generate the accumulation of cAMP.     

Prostaglandin I(2) production and cAMP accumulation in response to acidic extracellular pH through OGR1 in human aortic smooth muscle cells

Ovarian cancer G-protein-coupled receptor 1 (OGR1) and GPR4 have recently been identified as proton-sensing or extracellular pH-responsive G-protein-coupled receptors stimulating inositol phosphate production and cAMP accumulation, respectively. In the present study, we found that OGR1 and GPR4 mRNAs were expressed in human aortic smooth muscle cells (AoSMCs). Acidic extracellular pH induced inositol phosphate production, a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), and cAMP accumulation in these cells. When small interfering RNAs (siRNAs) targeted for OGR1 and GPR4 were transfected to the cells, the acid-induced inositol phosphate production and [Ca(2+)](i) increase were markedly inhibited by the OGR1 siRNA but not by the GPR4 siRNA. Unexpectedly, the acid-induced cAMP accumulation was also largely inhibited by OGR1 siRNA but only slightly by GPR4 siRNA. Acidic extracellular pH also stimulated prostaglandin I2 (PGI(2)) production, which was again inhibited by OGR1 siRNA. The specific inhibitors for extracellular signal-regulated kinase kinase and cyclooxygenase attenuated the acid-induced PGI(2) production and cAMP accumulation without changes in the inositol phosphate production. A specific inhibitor of phospholipase C also inhibited the acid-induced cAMP accumulation. In conclusion, OGR1 is a major receptor involved in the extracellular acid-induced stimulation of PGI(2) production and cAMP accumulation in AoSMCs. The cAMP accumulation may occur through OGR1-mediated stimulation of the phospholipase C/cyclooxygenase/PGI(2) pathway.     

A novel LLC-PK1 renal epithelial cell mutant impaired in in vivo down-regulation of cAMP-mediated hormonal response.

A novel "cAMP-resistant" variant of LLC-PK1 renal epithelial cells which is impaired in in vivo down-regulation of response following hormonal stimulation of adenylate cyclase (AC) is described. Compared to parental cells, the BIB27 mutant exhibited markedly higher in vivo activation of cAMP-dependent protein kinase (cAMP-PK) in response to the hormones salmon calcitonin (SCT) or [Arg8]-vasopressin (AVP) or the AC activator forskolin. The activation of cAMP-PK subsequent to agonist stimulation also persisted much longer in the mutant than in LLC-PK1 cells, although the cAMP-PK of BIB27 cells was normal in terms of both absolute levels and regulation by cAMP in vitro. Intracellular cAMP accumulation was also much higher in BIB27 than in LLC-PK1 cells following agonist stimulation. Production of cAMP could be detected in BIB27 cells even 12 h after treatment with AVP or SCT, whereas cAMP production in LLC-PK1 had returned to basal within 1 and 8 h, respectively. High levels of free cAMP-PK catalytic (C) subunit in BIB27 persisted even 12 h after hormone addition, meaning that the higher cAMP production in BIB27 did not result in the normal down-regulation of cAMP-PK C subunit levels. In vitro AC activity in BIB27 cell homogenates could be stimulated by hormones or receptor-independent agonists, but to a lesser extent than in LLC-PK1 cell homogenates. The SCT and AVP concentrations promoting half-maximal AC activation in BIB27 cells were about 10- and 3-fold higher than parental, respectively. BIB27 accordingly appeared to possess a mutation in AC responsible for the impairment of both in vitro response to agonists and the normal in vivo down-regulation processes following hormonal stimulation.     

Cytoskeleton-dependent tyrosine phosphorylation of the p130(Cas) family member HEF1 downstream of the G protein-coupled calcitonin receptor. Calcitonin induces the association of HEF1, paxillin, and focal adhesion kinase.

HEF1 is a recently described p130(Cas)-like docking protein that contains one SH3 domain and multiple SH2 binding motifs. In B cells, HEF1 is phosphorylated by a cytoskeleton-dependent mechanism that is triggered by integrin ligation. However, the induction of HEF1 phosphorylation by G protein-coupled receptors has not been reported. We found that HEF1, but not p130(Cas), is tyrosine-phosphorylated following stimulation of the rabbit C1a calcitonin receptor stably expressed in HEK-293 cells. The calcitonin-induced tyrosine phosphorylation of HEF1 increased in a time- and dose-dependent manner. Dibutyryl cAMP and forskolin had little or no effect on HEF1 phosphorylation, and the protein kinase A inhibitor H89 failed to detectably inhibit the response to calcitonin, indicating that the G(s)/cAMP/protein kinase A pathway does not mediate the calcitonin effect. Pertussis toxin, which selectively blocks G(i/o) signaling, also had no effect. Increasing cytosolic Ca(2+) with ionomycin stimulated HEF1 phosphorylation and preventing any calcitonin-induced change in cytosolic calcium by a combination of BAPTA and extracellular EGTA completely blocked the calcitonin-induced tyrosine phosphorylation of HEF1. Phorbol 12-myristate 13-acetate also induced HEF1 tyrosine phosphorylation, and the protein kinase C inhibitor calphostin C completely inhibited both calcitonin- and phorbol 12-myristate 13-acetate-stimulated HEF1 phosphorylation. Calcitonin also induced the tyrosine phosphorylation of paxillin and focal adhesion kinase, and the association of these two proteins with HEF1. Pretreatment with cytochalasin D, which disrupts actin microfilaments, prevented the calcitonin-induced HEF1 and paxillin phosphorylation. In conclusion, the calcitonin-stimulated tyrosine phosphorylation of HEF1 is mediated by calcium- and protein kinase C-dependent mechanisms and requires the integrity of the actin cytoskeleton.     

Accumulation of Cyclic AMP Elicited by Vasoactive Intestinal Peptide Is Potentiated by Noradrenaline, Histamine, Adenosine, Baclofen, Phorbol Esters, and Ouabain in Mouse Cerebral Cortical Slices: Studies on the Role of Arachidonic Acid Metabolites and Protein Kinase C

In mouse cerebral cortical slices, noradrenaline (NA) potentiates cyclic AMP (cAMP) accumulation elicited by vasoactive intestinal peptide (VIP) through alpha 1-adrenergic receptors. This synergism is inhibited by indomethacin, and the prostaglandins E2 and F2 alpha mimic the effect of NA. In the present study, we observed that the synergism between VIP and NA is not inhibited by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) or the diacylglycerol-lipase inhibitor RHC 80267, thus further stressing the role of phospholipase A2 activation. Various neuroactive agents that potentiate the stimulatory effect of VIP on cAMP formation were also examined. As with NA, the potentiation by histamine and adenosine is inhibited by indomethacin. In contrast to NA, histamine, and adenosine, the synergistic interaction between phorbol esters and VIP on cAMP formation is abolished by H-7 but not by indomethacin. The potentiation by baclofen, a gamma-aminobutyric acidB receptor agonist, is partially inhibited by the 5-lipoxygenase inhibitor nafazatrom. The synergism between ouabain and VIP is reduced by H-7 but not by indomethacin and nafazatrom. These data indicate that the stimulation of cAMP formation elicited by VIP is under the modulation of various neuroactive agents that trigger diverse intracellular mechanisms to potentiate the effect of the peptide.     

Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca2+ and protein kinase C.

The mechanisms of muscarinic receptor-linked increase in cAMP accumulation in SH-SY5Y human neuroblastoma cells has been investigated. The dose-response relations of carbachol-induced cAMP synthesis and carbachol-induced rise in intracellular free Ca2+ were similar. The stimulated cAMP synthesis was inhibited by about 50% when cells were entrapped with the Ca2+ chelator BAPTA or in the presence of the protein kinase C (PKC) inhibitor staurosporine. Production of cAMP could be induced also by the Ca2+ ionophore, ionomycin and by TPA, an activator of PKC. When added together TPA and ionomycin had a synergistic effect. When cAMP synthesis was activated with cholera toxin, PGE1 or PGE1 + pertussis toxin carbachol stimulated cAMP production to the same extent as in control cells. Ca2+ and protein kinase C thus seem to be the mediators of muscarinic-receptor linked cAMP synthesis by a direct action on adenylate cyclase.     

Parathyroid hormone receptor internalization is independent of protein kinase A and phospholipase C activation

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) binding to their common receptor stimulates second messenger accumulation, receptor phosphorylation, and internalization. LLC-PK(1) cells expressing a green fluorescent protein-tagged PTH/PTHrP receptor show time- and dose-dependent receptor internalization. The internalized receptors colocalize with clathrin-coated pits. Internalization is stimulated by PTH analogs that bind to and activate the PTH/PTHrP receptor. Cell lines expressing a mutant protein kinase A regulatory subunit that is resistant to cAMP and/or a mutant receptor (DSEL mutant) that does not activate phospholipase C internalize their receptors normally. In addition, internalization of the wild-type receptor and the DSEL mutant is stimulated by the PTH analog [Gly(1),Arg(19)]hPTH-(1-28), which does not stimulate phospholipase C. Forskolin, IBMX, and the active phorbol ester, phorbol-12-myristate-13-acetate, did not promote receptor internalization or increase PTH-induced internalization. These data indicate that ligand-induced internalization of the PTH/PTHrP receptor requires both ligand binding and receptor activation but does not involve stimulation of adenylate cyclase/protein kinase A or phospholipase C/protein kinase C.     

Protein kinase C activators and bradykinin selectively inhibit vasopressin-stimulated cAMP synthesis in MDCK cells

To evaluate a possible modulation by protein kinase C of hormonal, cAMP-mediated effects on renal epithelial cells, we studied the effect of protein kinase C activators and of bradykinin on intracellular cAMP accumulation in MDCK cells. A 15-min pretreatment of cells with phorbol 12-myristate 13-acetate or 1-oleoyl-2-acetylglycerol induced a dose-dependent inhibition of vasopressin-stimulated cAMP synthesis, but not of basal or glucagon-, prostaglandin E2-, and forskolin-stimulated cAMP generation. 4 alpha-Phorbol 12,13-didecanoate, inactive on protein kinase C, did not affect cAMP accumulation. Bradykinin (0.1-10 microM) also inhibited the stimulatory effect of vasopressin on cAMP synthesis in a concentration-dependent manner, but affected neither basal cAMP content, nor its stimulation by glucagon, prostaglandin E2 and forskolin. The effect of activators of protein kinase C and of bradykinin occurred while renal prostaglandin synthesis was blocked with indomethacin. The inhibitory effect of protein kinase C activators and bradykinin on cAMP generation was reversed by the protein kinase C inhibitor H7, was enhanced by monensin, one effect of which is to block the recycling of membrane receptors, and persisted when the GTP-binding protein N1 was blocked with 1 mM Mn2+. Our data suggest that: protein kinase C can modulate the tubular effects of vasopressin by inhibiting cAMP generation; this effect is not mediated by renal prostaglandins, and might result from a direct action on the vasopressin receptor, or on its coupling with Ns; the modulation by bradykinin of vasopressin effects are likely to be exerted, at least partly, through activation of protein kinase C.     

Involvement of protein kinase A in the regulation of intracellular free calcium and phosphoinositide turnover in rat myometrium

Preincubation of Fura 2-loaded rat myometrial cells with H-8, an inhibitor of protein kinase A, for 1 h reversed the inhibitory effects of 8-(4-chlorophenylthio)-cAMP (CPTcAMP) on the oxytocin-stimulated increase in (Ca2+)i (intracellular free calcium), with an EC50 of 47 microM. H-8 also prevented the inhibition by relaxin and isoproterenol of the oxytocin-induced increase in (Ca2+)i. The EC50 of H-8 in reversing the relaxin effect was 42 microM. H-8 reversal of the effect of relaxin on (Ca2+)i was evident both in the absence of extracellular calcium and in cells pretreated with pertussis toxin. H-8 also reversed the inhibitory effects of relaxin and CPTcAMP on the oxytocin-induced increase in [3H]inositol phosphate formation and [3H]phosphoinositide hydrolysis. Preincubation of myometrial cells for 1 h with H-7, another protein kinase inhibitor, only partially attenuated the inhibition by relaxin and CPTcAMP of the oxytocin-induced increase in (Ca2+)i and [3H]inositol phosphate formation at concentrations 4-5 times greater than those of H-8. Acute (15-min) exposure to phorbol myristate acetate (1.0 microM) did not affect basal (Ca2+)i or the oxytocin-stimulated increases in (Ca2+)i or inositol phosphate formation. These results imply a regulatory role for protein kinase A in the inhibition of the oxytocin-induced increase in (Ca2+)i and inositol phosphate formation by relaxants.