Desensitization of vasopressin sensitive adenylate cyclase by vasopressin and phorbol esters

Desensitization of vasopressin V2 receptor-mediated adenylate cyclase was studied in canine kidney cell line, MDCK cells. Overnight treatment of MDCK cells with arginine vasopressin (AVP) resulted in a loss of vasopressin receptors and an inhibition of cAMP accumulation in response to AVP. Both the loss of receptor and reduction in cAMP accumulation were time- and AVP concentration-dependent. Desensitization was selective for AVP because cAMP formation in response to isoproterenol, prostaglandin E1 (PGE1) and forskolin was not affected by AVP pre-treatment. Pre-treatment of MDCK cells with phorbol dibutyrate (PDBu) also caused a dose-dependent inhibition of AVP mediated cAMP accumulation, but not of isoproterenol-, PGE1- and forskolin-induced cAMP accumulation. PDBu pre-treatment did not cause loss of vasopressin receptors. Instead, the affinity for vasopressin was changed by PDBu treatment. Pre-treatment of the cells with pertussis toxin (PT) had no effect on the desensitization and downregulation of vasopressin (V2) receptors, suggesting that the desensitization may not be mediated by pertussis toxin sensitive G-protein. Our data suggest that pre-treatment of MDCK cells with AVP or PDBu caused desensitization of AVP-mediated cAMP accumulation and that downregulation of V2 receptors required agonist occupancy of the receptors, whereas the affinity of the receptors was changed by phorbol ester treatment.     

Autoregulation of prostaglandin E2-induced Ca2+ influx in osteoblast-like cells: inhibition by self-induced activation of protein kinase C.

In cloned osteoblast-like MC3T3-E1 cells, prostaglandin E2 (PGE2) stimulated 45Ca2+ influx even in the presence of nifedipine, a Ca2+ antagonist that inhibits voltage-dependent Ca2+ channel, in a dose-dependent manner, attaining a maximum at 0.5 microM. Dose of PGE2 above 0.5 microM caused less than maximal stimulation. While PGE2 stimulated the formation of inositol trisphosphate dose dependently in the range between 1 nM and 10 microM. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, which by itself had little effect on 45Ca2+ influx, significantly suppressed the 45Ca2+ influx induced by PGE2 in a dose-dependent manner between 1 nM and 1 microM. 4 alpha-Phorbol 12,13-didecanoate, a phorbol ester which is inactive for PKC, showed little effect in this capacity. Staurosporine, a PKC inhibitor, enhanced the PGE2-induced 45Ca2+ influx. On the other hand, dibutyryl cAMP had little effect on the 45Ca2+ influx induced by PGE2. Our data suggest that PGE2 regulates Ca2+ influx through self-induced activation of PKC. These results indicate that there is an autoregulatory mechanism in signal transduction by PGE2, and PGE2 modulates osteoblast functions through the interaction between Ca2+ influx and phosphoinositide hydrolysis in osteoblast-like cells.     

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.     

Activation of protein kinase C inhibits prostaglandin- and potentiates adenosine receptor-stimulated accumulation of cyclic AMP in a human T-cell leukemia line

Accumulation of cAMP in the human T-cell leukemia cell line Jurkat was stimulated by the adenosine analogue 5'-N-ethylcarboxamidoadenosine (NECA) and by prostaglandin E2 (PGE2). Addition of two phorbol esters, PDiBu and TPA, markedly enhanced the NECA-stimulated accumulation of cAMP whereas the PGE2-stimulated cAMP accumulation was substantially reduced. The non-tumor-promoting phorbol ester, 4 alpha-PDD, had no effect on either NECA- or PGE2-stimulated cAMP accumulation. The ability of PDiBu to inhibit the effect of PGE2 and to stimulate the effect of NECA remained in the presence a low concentration of forskolin (0.3 microM), which per se increased both NECA- and PGE2-stimulated cAMP accumulation. Our results suggest that the effect of PK-C-activating drugs on receptor-mediated cAMP accumulation is entirely dependent on which receptor is being stimulated.     

Protein kinase C activation reduces microglial cyclic AMP response to prostaglandin E2 by interfering with EP2 receptors

We studied the modulation by protein kinase C (PKC) of the cyclic AMP (cAMP) accumulation induced by prostaglandin (PG) E2 in rat neonatal microglial cultures. Short pretreatment of microglia with phorbol 12-myristate 13-acetate (PMA) or 4beta-phorbol 12,13-didecanoate, which activate PKC, but not with the inactive 4alpha-phorbol 12,13-didecanoate, substantially reduced cAMP accumulation induced by 1 microM PGE2. The action of PMA was dose and time dependent, and the maximal inhibition (approximately 85%) was obtained after 10-min preincubation with 100 nM PMA. The inhibitory effect of PMA was mimicked by diacylglycerol and was prevented by the PKC inhibitor calphostin C. As PMA did not affect isoproterenol- or forskolin-stimulated cAMP accumulation, we investigated whether activation of PKC decreased cAMP production by acting directly at PGE2 EP receptors. Neither sulprostone (10(-9)-10(-5) M), a potent agonist at EP3 receptors (coupled to adenylyl cyclase inhibition), nor 17-phenyl-PGE2 (10(-6)-10(-5) M), an agonist of EP1 receptors, modified cAMP accumulation induced by forskolin. On the contrary, 11-deoxy-16,16-dimethyl PGE2, which does not discriminate between EP2 and EP4 receptors, both coupled to the activation of adenylyl cyclase, and butaprost, a selective EP2 agonist, induced a dose-dependent elevation of cAMP that was largely reduced by PMA pretreatment, as in the case of PGE2. These results indicated EP2 receptors as a possible target of PKC and suggest that PKC-activating agents present in the pathological brain may prevent the cAMP-mediated microglia-deactivating function of PGE2.     

Modulation of responsiveness to cAMP stimulating agonists by phorbol ester in fetal rat osteoblasts.

We studied the effect of activation of protein kinase C (PKC) by a phorbol ester on cAMP accumulation in fetal rat osteoblasts. Activation of PKC by phorbol 12-myristate 13-acetate (PMA) caused a potentiation of cAMP accumulation induced by parathyroid hormone (PTH), forskolin, and cholera toxin. The results suggest that the potentiating effect of PMA on PTH-induced cAMP accumulation was not due to an effect on the PTH-receptor nor to an effect on cAMP degradation, as the effect of PMA persisted in the presence of a phosphodiesterase inhibitor. Pretreatment of the cells with pertussis toxin did not prevent the action of PMA, indicating that PMA does not act via the inhibitory G-protein. PMA had a biphasic effect on prostaglandin E2 (PGE2)-induced cAMP accumulation; i.e., at concentrations greater than or equal to 10(-6) M, PMA potentiated the PGE2-induced cAMP response but PMA attenuated cAMP accumulation induced by concentrations of PGE2 less than or equal to 5.10(77) M. From our data we conclude that PKC can interact with a stimulated cAMP pathway in a stimulatory and inhibitory manner. Potentiation of cAMP accumulation is probably due to modification of the adenylate cyclase complex, whereas attenuation of stimulated cAMP accumulation appears to be due to an effect on a different site of the cAMP generating pathway, which may be specific to PGE2-induced cAMP accumulation.     

Multifactorial regulation of prostaglandin synthesis in preovulatory goldfish ovarian follicles.

Goldfish preovulatory ovarian follicles (prior to germinal vesicle breakdown) were utilized for studies investigating the actions of activators of different signal transduction pathways on prostaglandin (PG) production. The protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA; 100-400 nM), 1-oleoyl-2-acetylglycerol (5 and 25 micrograms/ml), and 1,2-dioctanoylglycerol (10 and 50 micrograms/ml) stimulated PGE production; the inactive phorbol 4 alpha-phorbol didecanoate, which does not activate PKC, had no effect. Calcium ionophore A23187 (0.25-4.0 microM) stimulated PGE production and acted in a synergistic manner with activators of PKC. Although produced in lower amounts than PGE, PGF was stimulated by PMA and A23187. The direct activator of phospholipase A2, melittin (0.1-1.0 microM), stimulated a dose-related increase in PGE production, whereas chloroquine (100 microM), a putative inhibitor of phospholipase A2, blocked basal and PMA + A23187-stimulated PGE production. Several drugs known to elevate intracellular levels of cAMP including the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1-1.0 mM), forskolin (10 microM), and dibutyryl cAMP (dbcAMP; 5 mM) attenuate PMA + A23187-stimulated PGE production. Melittin-stimulated production of PGE was inhibited by dbcAMP, suggesting that the action of cAMP was distal to the activation of phospholipase A2. In summary, these studies demonstrate that activation of PKC and elevation of intracellular calcium levels stimulate PG production, in part, through activation of phospholipase A2. The adenylate cyclase/cAMP signalling pathway is inhibitory to PG production by goldfish ovarian follicles.     

Exogenous prostaglandin E2 inhibits TPA induced matrix metalloproteinase-9 production in MCF-7 cells

Elevated levels of prostaglandin E2 (PGE2) have been reported in many high metastatic human breast cancers, but no relationship between exogenous PGE2 activity, expression of matrix metalloproteinases (MMPs) and metastasis in human tumor cells has been reported. The poorly invasive human breast cancer cell line MCF-7 was cultured for 24h in the presence of both phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 nM) and PGE2 (1 microM) and the activity of MMP-9, one of the MMPs involved in metastasis, was measured, in growth medium by gelatin substrate zymography. TPA induced a strong production of MMP-9 while exogenous PGE2 had no effect on the basal MMP-9 level, but inhibited the TPA induced enzyme expression and matrigel invasiveness. We showed that MCF-7 cells expressed EP2, EP3 and EP4 receptors for PGE2 and that its action was probably mediated by EP4 receptor and adenylyl cyclase activation while cAMP dependent PKA was not involved in the process of inhibition of MMP-9 production. These findings suggest a possible inhibitory role for exogenous PGE2 in the metastatic process development.     

Phorbol esters and diacylglycerols amplify bradykinin-stimulated prostaglandin synthesis in Swiss 3T3 fibroblasts. Possible independence from protein kinase C

When Swiss 3T3 fibroblasts were incubated with bradykinin, prostaglandin E2 (PGE2) synthesis was stimulated. Phorbol esters or the diacylglycerol analog 1-oleoyl-2-acetylglycerol (OAG), by themselves, did not acutely stimulate PGE2 synthesis. However, when cells were preincubated with phorbol esters or OAG, bradykinin-stimulated PGE2 synthesis was potentiated markedly. When phorbol esters and OAG were added together, bradykinin-stimulated PGE2 synthesis was potentiated in an additive manner. When cells were preincubated for 48 h with phorbol esters, then bradykinin added, amplification of bradykinin-stimulated PGE2 synthesis by phorbol ester or OAG was still apparent, even though prolonged pretreatment with phorbol esters abolished protein kinase C (Ca2+/phospholipid-dependent enzyme) activity in cell-free preparations. Further, the protein kinase C antagonist, H-7, only slightly inhibited phorbol ester or OAG amplification of bradykinin-stimulated PGE2 synthesis. The possibility is raised that diacylglycerol, formed in response to many receptors, may serve as a transducer of receptor-receptor interactions. Since desensitization or inhibition of protein kinase C only partially reduced the amplification of bradykinin-stimulated PGE2 synthesis by phorbol esters or OAG, the possibility is raised that diacylglycerol mimetics may have actions in addition to activation of protein kinase C.     

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.     

Heterologous desensitization of adenylate cyclase with prostaglandin E1 alters sensitivity to inhibitory as well as stimulatory agonists

Adenylate cyclase in cultured human fibroblasts is activated by prostaglandin E1 (PGE1) or beta-adrenergic agonists, e.g., isoproterenol, and inhibited by muscarinic agonists. Incubation with PGE1 reduced adenylate cyclase responsiveness to both PGE1 and isoproterenol; this so-called heterologous desensitization is believed to result from impaired function of the stimulatory guanyl nucleotide-binding protein of the cyclase complex. The effect of heterologous desensitization by PGE1 on inhibition of adenylate cyclase by the muscarinic agonist oxotremorine was examined. Muscarinic inhibition of basal and isoproterenol-stimulated cAMP accumulation was attenuated following exposure to PGE1; the concentration of oxotremorine required for half-maximal inhibition of cAMP accumulation was increased. In both intact cells and membrane preparations the number of binding sites for [3H]scopolamine, a muscarinic antagonist, was unaltered by desensitization. Following exposure to PGE1, receptor affinity for oxotremorine, assessed by competition with [3H] scopolamine, and the guanyl nucleotide sensitivity of agonist binding were reduced. The amount of inhibitory guanyl nucleotide-binding regulatory protein available for [32P]ADP-ribosylation by pertussis toxin was unaltered by desensitization. Thus, heterologous desensitization of adenylate cyclase with the stimulatory agonist PGE1 alters sensitivity to inhibitory as well as stimulatory ligands.     

Keloid fibroblasts are refractory to inhibition of DNA synthesis by phorbol esters. Altered response is accompanied by reduced sensitivity to prostaglandin E2 and altered down-regulation of phorbol ester binding sites.

To investigate abnormal growth regulation in keloid fibroblasts, responses to phorbol esters were examined. Treatment of quiescent cultures with phorbol 12-myristate 13-acetate (PMA) blocked a normally occurring (20-24 h) peak of serum-stimulated thymidine incorporation in normal and keloid cells. In keloid fibroblasts PMA induced a delayed peak of DNA synthesis. When indomethacin was added with PMA the delayed peak appeared in normal fibroblasts. The ED50 for inhibition of the 20-24-h peak was 1 nM, whereas the delayed peak required a 50-fold-higher PMA concentration. In both cell types PMA induced prostaglandin E2 (PGE2) synthesis, and exogenous PGE2 caused 50% inhibition of the 20-24-h peak. When PMA and indomethacin were added with PGE2 the delayed peak was inhibited 90% in normal fibroblasts, whereas inhibition of keloid cells was the same as with PGE2 alone. Normal and keloid fibroblasts had the same number of phorbol ester binding sites. However, in normal cells, phorbol 12,13-dibutyrate bound with greater affinity, and down-regulation of phorbol ester binding occurred to a greater extent. These findings suggest that altered expression of protein kinase C isozymes or another molecule that binds phorbol esters may play a role in abnormal growth regulation of keloid cells.     

Molecular cloning and functional characterization of the canine prostaglandin E2 receptor EP4 subtype.

Prostaglandin E2 (PGE2) is an important mediator of diverse biologic functions in many tissues and binds with high affinity to four cell surface, seven-transmembrane domain, G protein-coupled receptors (EP1-EP4). The EP4 receptor subtype has a long intracellular carboxy-terminal region and is functionally coupled to adenylate cyclase, resulting in elevated intracellular cyclic adenosine 5' monophosphate (cAMP) levels upon activation. To further study EP4 receptor subtype function, a canine kidney cDNA library was screened and three clones were isolated and sequenced. The longest clone was 3,103 bp and contained a single open reading frame of 1,476 bp, potentially encoding a protein of 492 amino acids with a predicted molecular weight of 53.4 kDa. Sequence analysis of this open reading frame reveals 89% identity to the human EP4 protein coding region at the nucleotide level and 90% identity when the putative canine and human protein sequences are compared. Northern blot analysis showed relatively high levels of canine EP4 expression in heart, lung and kidney, while Southern blot analysis of canine genomic DNA suggests the presence of a single copy gene. Following transfection of canine EP4 into CHO-KI cells, Scatchard analysis revealed a dissociation constant of 24 nM for PGE, while competition binding studies using 3H-PGE2 as ligand demonstrated specific displacement by PGE2 prostaglandin E, (PGE1), and prostaglandin A3 (PGA3). Treatment with PGE2 also resulted in increased levels of cAMP in transfected, but not in parental, CHO-KI cells. In contrast, butaprost, an EP2 selective ligand, and sulprostone, an EP1/EP3 selective ligand, did not bind to this receptor at the maximal concentration used (320 nM). To further investigate secondary signaling, the canine EP4 cDNA was truncated to produce an 1,117 bp fragment encoding a 356 amino acid protein lacking the intracellular carboxy-terminus. When transfected, this truncated cDNA produced a protein with a dissociation constant of 11 nM for PGE2 and a binding and cAMP accumulation profile similar to that of the full-length protein. Both full-length and truncated canine EP4 underwent short term PGE2-induced desensitization as shown by a lack of continuing cAMP accumulation after the initial PGE2 stimulation, suggesting no involvement of the C-terminal intracellular tail. This result is in contrast to that reported for the human EP4 receptor, where residues within the C-terminal intracellular tail were shown to mediate short term, ligand induced desensitization.     

Modulation of cyclic AMP formation and progesterone secretion by human chorionic gonadotropin, epinephrine, buserelin and prostaglandins in normal or human chorionic gonadotropin desensitized rat immature luteal cells in monolayer culture

It is now well recognized that hCG-induced luteolysis is associated with hCG-induced desensitization, but the physiological significance of luteal cell GnRH, PGs and beta-receptors is still undefined. Therefore, we intend in this study to observe the effects of prostaglandin F2 alpha and prostaglandin E2 and the interactions between epinephrine, a potent LHRH agonist [(D-Ser-(TBu)6, des-Gly-NH10(2) LHRH ethylamide: Buserelin] and hCG in normal and in vitro hCG-desensitized rat immature luteal cells in monolayer culture, on basal, hCG or cholera toxin stimulated intracellular and extracellular cAMP and progesterone secretion. The present report shows that incubation of immature rat luteal cells in monolayer culture with Buserelin, led to 25-50% inhibition of the epinephrine-as well as PGE2-induced cAMP and progesterone responses. The LHRH agonist can also reverse the stimulatory effects of cholera toxin in the presence of hCG and led with PGF2 alpha, to additive inhibitory effects on extracellular cAMP accumulation induced by cholera toxin. Both Buserelin and PGF2 alpha can reverse the hCG-induced cAMP and progesterone release but no effect could be observed when the incubation was carried out with either substance in the absence of hCG. Prostaglandin E2, in acute conditions of incubation, seems to share agonist properties with hCG when both were incubated with luteal cells. Buserelin reversed the stimulatory effects of PGE2, hCG, epinephrine and cholera toxin on cAMP and progesterone responses to these substances. These results suggest that Buserelin and PGF2 alpha have luteolytic-like effects and that there may be a complementary action for the two substances. Preincubation of rat luteal cells in monolayer culture with 1 nM hCG for a 24 h period led to the inhibition of cAMP and progesterone responses after a subsequent exposure to hCG and epinephrine. Luteal cells were no longer responsive to hCG while the presence of epinephrine in hCG-desensitized cells led to a 40% stimulation of cAMP and progesterone production. These observations suggest that occurred a partial alteration of the N component activity of the adenylyl cyclase system.     

D1 Dopamine Receptors of NS20Y Neuroblastoma Cells Are Functionally Similar to Rat Striatal D1 Receptors

Dopamine or agonists with D1 receptor potency stimulated cyclic AMP (cAMP) accumulation in whole cell preparations of NS20Y neuroblastoma cells. The accumulation of cAMP after D1 stimulation was rapid and linear for 3 min. Both dopamine and the novel D1 receptor agonist dihydrexidine stimulated cAMP accumulation two- to three-fold over baseline. The pseudo-Km for dopamine was approximately 2 microM, whereas for dihydrexidine it was approximately 30 nM. The effects of both drugs were blocked by either the D1-selective antagonist SCH23390 (Ki, 0.3 nM) or the nonselective antagonist (+)-butaclamol (Ki, 5 nM). Both (-)-butaclamol and the D2-selective antagonist (-)-sulpiride were ineffective (Ki greater than 3 microM). Forskolin (10 microM), prostaglandin E1 (1 microM), and adenosine (10 microM) also stimulated cAMP accumulation, but none were antagonized by SCH23390 (1 microM). Finally, muscarinic receptor stimulation (100 microM carbachol) inhibited both D1- and forskolin-stimulated increases in cAMP accumulation by 80%. The present results indicate that NS20Y neuroblastoma cells have D1 receptors that are coupled to adenylate cyclase, and that these receptors have a pharmacological profile similar to that of the D1 receptor(s) found in rat striatum.     

Protein kinase C induces phosphorylation and desensitization of the human 5-HT1A receptor

The effects of short-term phorbol ester treatment of CHO cells that stably express 900 fmol of recombinant human serotonin 5-HT1A receptor/mg of protein on coupling to the inhibition of adenylyl cyclase and on phosphorylation of the receptor were studied. Pretreatment of cell monolayers with phorbol 12-myristate 13-acetate (PMA) caused a dose- and time-dependent shift of the half-maximal dose of serotonin (5-HT) required to inhibit membrane adenylyl cyclase (from IC50 approximately 100 nM to approximately 400 nM). This desensitization (shift in IC50) was rapid, occurring with 5 min of pretreatment and being maximal by 10-15 min; it was also dose-dependent, being half-maximal at approximately 300 nM PMA. Desensitization was also induced by sn-dioctanoylglycerol (DiC8) and blocked by the protein kinase C (PKC) inhibitors sphingosine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). In detached permeabilized cells, PMA pretreatment caused a rapid phosphorylation of immunoprecipitated 5-HT1A receptors, with an approximately 3-4-fold increase that was maximal after 15 min and persisted for 90 min. The phosphorylation occurred at a similar dose of PMA as that which induced desensitization (half-maximal at approximately 300 nM, maximal at 500 nM to 1 microM), could be reproduced by pretreatment with the PKC activators DiC8 or phorbol 12,13-dibutyrate (PDBu), and could be blocked by the PKC inhibitors sphingosine or H-7. The stoichiometry of the phosphorylation was approximately 2 mol of [32P]ATP/mol of receptor, suggesting the involvement at least two of three putative PKC sites within the 5-HT1A receptor. The close concordance between the PKC-induced desensitization and phosphorylation suggests a potential causative link between these two effects of PKC on the human 5-HT1A receptor.     

Effect of phorbol ester on prostaglandin regulation of proliferation in rabbit endometrial cells

We have proposed that two of the endogenously synthesized endometrial prostaglandins, prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E1 (PGE1), play a regulatory role in growth control of the endometrium. PGF2 alpha increases DNA synthesis and PGE1 inhibits that effect. Primary cultures of rabbit endometrial cells were used here to examine the effects of the tumor-promoting, diacylglycerol mimicking, phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on the prostaglandin control of cell proliferation. TPA treatment of these cultures results in: a decrease in control levels of proliferation and complete inhibition by TPA of PGF2 alpha stimulated DNA synthesis; a reduction in [3H]PGF2 alpha binding with short term treatment but an increase to above control binding level with long term treatment; an inhibition of the normal PGF2 alpha stimulated inositol polyphosphate synthesis; and a small increase in accumulation of PGF2 alpha in the culture media. Furthermore, in this culture system, TPA does not down regulate [3H]PGE1 binding; it does not alter the normal PGE1 stimulation of cAMP synthesis; and it has no effect on the normal endogenous PGE1 synthesis by these cultures. The above results are consistent with our previous observations that PGF2 alpha works through the intracellular messengers inositol polyphosphate/diacylglycerol whereas PGE1 works through cAMP.     

The time-course of cyclic AMP signaling is critical for leukemia U-937 cell differentiation

The regulation of the cAMP signaling is intimately involved in several cellular processes, including cell differentiation. Here, we provide strong evidence supporting that the time-course of cAMP signal is critical for leukemia U-937 cell differentiation. Three stimulating-cAMP agents were used to analyze the correlation between cAMP time-course and cell differentiation. All three agents denoted similar cAMP maximal responses in dose-response experiments. The kinetic of desensitization showed differential characteristics, while H2 receptor desensitized homologously without affecting PGE2 or forskolin effect, PGE2 response showed mixed desensitization characterized by a homologous initial phase followed by a heterologous phase. Regarding forskolin, long-term stimuli attenuated PGE2 and H2 agonist response without affecting adenylyl cyclase activity. In the absence of phosphodiesterase inhibitors, the three agents induced similar maximal cAMP levels after 5 min, but only that induced by the H2 agonist returned to basal levels. Consistent with this observation, H2 agonist was not able to induce U-937 cell maturation in contrast to PGE2 and forskolin, supporting the importance of time-course signaling in the determination of cell behavior.     

Effects of lubiprostone on human uterine smooth muscle cells

Lubiprostone, a bicyclic fatty acid derivative and member of a new class of compounds called prostones, locally activates ClC-2 Cl(-) channels without activation of prostaglandin receptors. The present study was specifically designed to test and compare lubiprostone and prostaglandin effects at the cellular level using human uterine smooth muscle cells. Effects on [Ca(2+)](i), membrane potential and [cAMP](i) in human uterine smooth muscle cells were measured. 10 nM lubiprostone significantly decreased [Ca(2+)](i) from 188 to 27 nM, which was unaffected by 100 nM SC-51322, a prostaglandin EP receptor antagonist. In contrast 10nM PGE(2) and PGE(1) both increased [Ca(2+)](i) 3-5-fold which was blocked by SC-51322. Similarly, lubiprostone and prostaglandins had opposite/different effects on membrane potential and [cAMP](i). Lubiprostone caused SC-51322-insensitive membrane hyperpolarization and no effect on [cAMP](i). PGE(2) and PGE(1) both caused SC-51322-sensitive membrane depolarization and increased [cAMP](i). Lubiprostone has fundamentally different cellular effects from prostaglandins that are not mediated by EP receptors.