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1.
To define sites of prostaglandin action of renal tubules, the distribution of adenylate cyclase sensitive to prostaglandin E2 (PGE2) was examined in single nephron segments dissected from rat kidney. Further, the interaction between PGE2 and vasopressin on adenylate cyclase activity in nephron segments sensitive to vasopressin was evaluated. Procedures involved in isolating nephron segments were without effects on adenylate cyclase stimulation by PGE2. PGE2 stimulated adenylate cyclase activity of the thin descending limb of Henle (tDL), cortical collecting tubules (CCT), and medullary collecting tubules (MCT) at concentrations of 1.4 × 10?5 to 2.8 × 10?5 M. PGE2 was without effects in other nephron segments tested including proximal convoluated tubules, proximal pars recta, the thin and thick ascending limb of Henle's loop, and distal and connecting tubules. PGE2, at both high (2.8 × 10?5 M) and low (2.8 × 10?8 M) concentrations, did not inhibit adenylate cyclase activity stimulated by submaximal doses of vasopressin in medullary thick ascending limb of Henle (MTAL), CCT, and MCT. These data define the distribution of PGE2-sensitive adenylate cyclase in the rat nephron, i.e., tDL, CCT, and MCT, and show the lack of direct inhibitory actions of PGE2 on vasopressin sensitive adenylate cyclase in MTAL, CCT, and MCT.  相似文献   

2.
Prostacyclin, (PGI2) is a potent but unstable inhibitor of platelet aggregation, probably acting through stimulation of adenylate cyclase.A stable analogue of prostacyclin with antiaggregatory properties, 5,6-dihydro-PGI2 (6β-PGI), and PGE1 can compete for the binding sites labelled by 3H-PGI2 in human platelet membranes (the affinity being PGI2 > PGE1 > 6β -PGI1). Both 6β-PGI1 and PGE1, as well as PGI2, bind to two classes of binding sites. 6β -PGI1 and PGE1 activate adenylate cyclase to the same extent as PGI2,with a rank order of potency which parallels that observed in binding experiments. The stimulation of this enzyme is brought about by interaction of each these prostanoids with two different classes of components. The comparison of binding and adenylate cyclase data suggests that the sites to which PGI2, 6β -PGI1 and PGE1 bind might be coupled to the activation of adenylate cyclase. Since 6β-PGI1 seems to act through the same molecular mechanisms as PGI2, because of its stability it is an useful tool to investigate the mode of action of prostacyclin in platelets.  相似文献   

3.
Abstract

Prolonged (16 h) preexposure to prostaglandin E1 (PGE1) of cells from a murine virus-induced T lymphoma cell line BL/VL3 provoked, in their membranes, a dose-dependent reduction of PGE1-mediated adenylate cyclase stimulation. Smaller (but significant) decreases of helodermin- and isoproterenol-mediated stimulations were also observed. After a 16 h incubation of these cells with 1 µM PGE1, that reduced by 85%, the PGE1-mediated adenylate cyclase stimulation in membranes, 50% of the PGE1 response recovered after 2 h of PGE1 withdrawal from the incubation medium. Over the following 2 - 24 h time interval, further recovery was limited. Protein synthesis was required for this resensitization mechanism of functional PGE1 receptors coupled to adenylate cyclase, as judged by the inhibitory effects of cycloheximide.  相似文献   

4.
Metabolism and action of the prostaglandin endoperoxide PGH2 in rat kidney   总被引:3,自引:0,他引:3  
Kidney membrane fractions metabolized [1-14C]PGH2 to TXB2, PGE2, PGF, PGD2, 6-keto PGF, and HHT. TXA2, as measured by TXB2, was enzymatically formed in cortex microsomes and was identified by thin layer chromatography and gas chromatography - mass spectrometry. PGH2 caused a labile inhibition of cortical PGE2-stimulated adenylate cyclase. PGE2, PGF, and PGD2 are stimulators of cortical adenylate cyclase. The inability of two thromboxane synthetase inhibitors, imidazole and 9,11-azoprosta-5,13 dienoic acid, to block PGH2 inhibition suggested that TXA2 was not an obligatory intermediate in this process. Therefore, a potential function of cortical PGH2 is inhibition of adenylate cyclase.  相似文献   

5.
The adenosine derivative, N6-phenylisopropyladenosine (PIA), which inhibits adenylate cyclase in adipocyte membranes by a GTP-dependent and sodium-amplified process, was studied on GTPase activity in hamster adipocyte ghosts. PIA stimulated a high affinity GTPase without apparent lag phase. Both unstimulated and PIA-stimulated GTPases exhibited very similar Km values of about 0.2 μM GTP. PIA-induced low Km GTPase stimulation was amplified by sodium ions and was half-maximal and maximal at about 0.02 and 0.1 μM PIA, respectively. Stimulations of the low Km GTPase by PIA and PGE1, both inhibiting adipocyte adenylate cyclase, were not additive. Similar to PIA-induced adenylate cyclase inhibition, stimulation of the GTPase by PIA but not by PGE1 was prevented by the adenosine receptor antagonist, 3-isobutyl-1-methylxanthine. The data suggest that PIA-induced stimulation of a high affinity GTPase is an essential mechanism of adenosine receptor-mediated adipocyte adenylate cyclase inhibition.  相似文献   

6.
Prostaglandins E1 or E2 (PGE1, PGE2)1 stimulated adenylate cyclase(s) from particulate fractions of whole liver homogenates 5- to 6-fold, but caused only slight (1.5- to 2-fold) stimulation of the enzyme from homogeneous hepatocytes. In contrast, glucagon stimulated enzyme from hepatocytes 12- to 15-fold and enzyme from whole liver 8- to 10-fold. Accordingly, most of the total prostaglandin-sensitive adenylate cyclase in cell suspensions was recovered in fractions containing non-parenchymal cells, and most of the total glucagon-sensitive activity was recovered with hepatocytes. PGE1 did not change adenosine-3′,5′-monophosphate (cyclic AMP) concentrations, or alter cyclic AMP increases caused by glucagon in hepatocytes. Glucagon consistently increased hepatocyte cyclic AMP concentrations and stimulated glycogenolysis by 35 to 40%. PGE1 did not affect basal or glucagon-stimulated glycogenolysis in the intact cells.  相似文献   

7.
Preparations of small and large steroidogenic cells from enzymatically dispersed ovine corpora lutea were utilized to study the invitro effects of luteinizing hormone (LH) and prostaglandins (PG) E1, E2 and I2. Cells were allowed to attach to culture dishes overnight and were incubated with either LH (100 ng/ml), PGE2, PGE2, or PGI2 (250 ng/ml each). The secretion of progesterone by large cells was stimulated by all prostaglandins tested (P < 0.05) while the moderate stimulation observed after LH treatment was attributable to contamination of the large cell population with small cells. Prostaglandins E1 and E2 had no effect on progesterone secretion by small cells, while LH was stimulatory at all times (0.5 to 4 hr) and PGI2 was stimulatory by 4 hr. Additional studies were conducted to determine if the effects of PGE2 upon steroidogenesis in large cells were correlated with stimulated activity of adenylate cyclase. In both plated and suspended cells PGE2 caused an increase (P < 0.05) in the rate of progesterone secretion but had no effect upon the activity of adenylate cyclase or cAMP concentrations within cells or in the incubation media. Exposure of luteal cells to forskolin, a nonhormonal stimulator of adenylate cyclase, resulted in marked increases in all parameters of cyclase activity but had no effect on progesterone secretion. These data suggest that the actions of prostaglandins E1, E2 and I2 are directed primarily toward the large cells of the ovine corpus luteum and cast doubt upon the role of adenylate cyclase as the sole intermediary in regulation of progesterone secretion in this cell type.  相似文献   

8.
Prostaglandin D2 (PGD2) is one of several prostaglandins that can inhibit platelet aggregation and activate adenylate cyclase. Platelets were exposed to varying concentrations of PGD2, washed, and the adenylate cyclase response to prostaglandins, epinephrine, and sodium fluoride determined. Incubating platelets with 5 × 10?5 M PGD2 for 2 hr resulted in a 45% decrease in PGD2 activation of adenylate cyclase and a 25% decrease in stimulation by PGE1. Fluoride activation (7-fold) epinephrine inhibition (30%) and basal enzyme activity were unchanged by exposure of the platelets to PGD2. Desensitization was concentration dependent, with loss of enzyme activity first noted when platelets were incubated with 10?7 M PGD2. Enzyme sensitivity could be partially restored when desensitized platelets were washed free of PGD2 and incubated in buffer for 2 hr; complete resensitization required incubation for 24 hr in plasma. Regulation of prostaglandin sensitive platelet adenylate cyclase could be of importance in mediating the response of platelets to aggregating agents.  相似文献   

9.
Agonist-specific desensitization of prostaglandin I2-stimulated (PGI2)1 adenosine 3′:5′-monophosphate (cyclic AMP) accumulation can be demonstrated in intact human foreskin fibroblasts (HFF) following a single exposure to PGE1 or a stable PGI2 analog (nitrilo-PGI2). A single PGI2-stimulation of HFF cells does not result in desensitization. Continuous re-addition of PGI2 over a 4 hr period does induce desensitization to subsequent PGI2-stimulation. HFF cells that are desensitized to PGI2 are also desensitized to PGE1 or nitrilo-PGI2 stimulation indicating that these agonists share a common adenylate cyclase complex. Desensitization to PGI2 can be measured after a 60 min, but not after a 30 min, exposure to PGE1 or nitrilo-PGI2. Once HFF cells are desensitized, a 12–24 hr period is required for the recovery of PGI2 sensitivity.The adenylate cyclase in membranes prepared from intact cells that were preincubated with PGE1 is also desensitized to subsequent PGI2-stimulation. Preincubation of cells with PGI2 does not induce desensitization of PGI2-stimulated adenylate cyclase. These data suggest that HFF cells must be constantly exposed to a biologically active prostaglandin for desensitization to occur. The intrinsic chemical lability of PGI2 may be a biochemical protection mechanism against desensitization in cells that normally respond to PGI2.  相似文献   

10.
Preparations of small and large steroidogenic cells from enzymatically dispersed ovine corpora lutea were utilized to study the effects of luteinizing hormone (LH) and prostaglandins (PG) E1, E2 and I2. Cells were allowed to attach to culture dishes overnight and were incubated with either LH (100 ng/ml), PGE2, PGE2, or PGI2 (250 ng/ml each). The secretion of progesterone by large cells was stimulated by all prostaglandins tested (P < 0.05) while the moderate stimulation observed after LH treatment was attributable to contamination of the large cell population with small cells. Prostaglandins E1 and E2 had no effect on progesterone secretion by small cells, while LH was stimulatory at all times (0.5 to 4 hr) and PGI2 was stimulatory by 4 hr. Additional studies were conducted to determine if the effects of PGE2 upon steroidogenesis in large cells were correlated with stimulated activity of adenylate cyclase. In both plated and suspended cells PGE2 caused an increase (P < 0.05) in the rate of progesterone secretion but had no effect upon the activity of adenylate cyclase or cAMP concentrations within cells or in the incubation media. Exposure of luteal cells to forskolin, a nonhormonal stimulator of adenylate cyclase, resulted in marked increases in all parameters of cyclase activity but had no effect on progesterone secretion. These data suggest that the actions of prostaglandins E1, E2 and I2 are directed primarily toward the large cells of the ovine corpus luteum and cast doubt upon the role of adenylate cyclase as the sole intermediary in regulation of progesterone secretion in this cell type.  相似文献   

11.
12.
《Journal of Physiology》1997,91(3-5):229-234
The effects of pituitary adenylate cyclase activating polypeptides (PACAPs) on gastroduodenal HCO3 secretion were investigated in anesthetized rats and compared with those of vasoactive intestinal polypeptide (VIP). Under urethane anesthesia, a rat stomach mounted in an ex vivo chamber (in the absence of acid secretion) or a rat proximal duodenal loop was perfused with saline, and the HCO3 secretion was measured at pH 7.0 using a pH-stat method and by adding 10 mM HCl. Intravenous injection of PACAP-27 stimulated HCO3 secretion in a dose-dependent manner in the duodenum but not in the stomach; at 8 nmol/kg PACAP-27 increased the HCO3 secretion to maximal values of four times greater than basal levels, although this peptide had no effect on duodenal HCO3 secretion after intracisternal administration (1 nmol/rat). PGE2 (300 μg/kg, iv) significantly increased HCO3 secretion in both the stomach and the duodenum. The potency of duodenal HCO3 secretory action was in the following order; PACAP-27 > PACAP-38 = VIP, and that of PACAP-27 was about 100-fold greater than that of PGE2. The duodenal HCO3 secretory action of PACAP-27 as well as PGE2 was markedly potentiated by prior administration of isobutylmethyl xanthine (10 mg/kg, sc), the inhibitor of phosphodiesterase. Folskolin (250 μg/kg, iv), the stimulator of adenylate cyclase, also increased HCO3 secretion in the duodenum but not in the stomach. These results suggest that: 1) PACAPs are potent stimulators of HCO3 secretion in the duodenum but not in the stomach; 2) this action is mediated by cAMP through stimulation of adenylate cyclase; 3) cAMP is a mediator in duodenal but not gastric HCO3 secretion; and 4) PACAPs may be involved in the peripheral regulation of duodenal HCO3 secretion.  相似文献   

13.
The effects of prostaglandin (PG) E1, E2, A1, F, F or D2 on the rat renal cortical, outer medullary and inner medullary adenylate cyclase-cyclic AM systems were examined. While high concentrations (8X10−4M) of each prostaglandin stimulated adenylate cyclase activity in each area of the kidney, PGE1 was the only prostaglandin to stimulate at 10−7M. PGA's were the only prostaglandins tested besides PGE's which stimulated adenylate cyclase at less than 10−4M. This effect of PGA's was limited to the outer medulla. PGD2 was the least stimulatory. Observations with renal slices yielded qualitatively results. The PGE's were the most potent in each area with PGA's only stimulatory in the outer medulla. O2 deprivation (5% O2) lowered the slice cyclic AMP content in each area of the kidney. In the cortex and outer medulla, prostaglandin mediated increases in cyclic AMP content were either lower or absent at 5% O2 compared to 95% O2. However, in the inner medulla PGE stimulation was observed only at 5% O2 and not 95% O2. No other prostaglandins were found to increase inner medullary cyclic AMP content at 95% or 5% O2. These results illustrate that the adenylate cyclase-cyclic AMP system responds uniquely to prostaglandins in each area of the kidney. Consideration of these results along with correlative observations suggests that inner medullary produced PGE's may act as local modulators of inner medullary adenylate cyclase.  相似文献   

14.
Ovine luteal slices were used to study the effects of prostaglandins (PG) F2α on luteinizing hormone (LH)-stimulated secretion of progesterone and adenylate cyclase activity. The accumulation of progesterone in incubation medium and adenylate cyclase activity was similar after incubation of luteal slices with Medium 199 alone or Medium 199 containing PGF2α (250 ng/ml) for 3 hr. Addition of luteinizing hormone (LH; 100 ng/ml) resulted in a 2–3 fold increase in both the rate of progesterone accumulation and adenylate eyclase activity by 3 hr. When luteal slices were incubated in the presence of both LH and PGF2α the rates of progesterone accumulation and adenylate cyclase activity were identical to those in flasks containing LH alone after 1 hr; however, after 3 hr both LH stimulated progesterone accumulation and adenylate cyclase activity were inhibited to levels similar to those observed in control slices.In a second experiment, after 60–120 min of exposure to PGF2α the rate of progesterone accumulation in the medium was not different from that in untreated control slices. In addition, after this experiment the luteal slices were homogenized and the basal, sodium fluoride, LH, isoproterenol (ISO) and PGE2 sensitive adenylate cyclase activities were determined to evaluate the hormonal specificity of the negative effect of the pretreatment with PGF2α. Both LH and ISO stimulated adenylate cyclase activities were reduced after PGF2α pretreatment. However, fluoride ion stimulated adenylate cyclase activity was not significantly effected by PGF2α pretreatment and PGE2 sensitive adenylate cyclase was effected only slightly.  相似文献   

15.
Human decidua contains an active adenylate cyclase, and a number of studies indicate that adenylate cyclase is functionally linked to increased in vitro prostaglandin synthesis. Increased decidual prostaglandin synthesis is associated with parturition, and therefore activation of adenylate cyclase may be involved in the control of human parturition. In this study, third trimester human decidual cells were preincubated for no more than 24 h prior to stimulation with a number of reagents which increase cellular cyclic AMP levels. Forskolin rapidly increased intracellular and extracellular cyclic AMP levels, but there was no increase in prostaglandin E2 biosynthesis during incubations ranging from 5 min up to 24 h. Dibutyryl cyclic AMP or 8-bromo-cyclic AMP were also without effect on PGE2 production, which suggests that the adenylate cyclase was not linked to the mechanisms regulating prostaglandin production. Cholera toxin increased basal cyclic AMP and PGE2 synthesis, and was without effect on IL-1β-stimulated PGE2 levels. PGE2 synthesis was increased by 24 h culture with IL-1β in all the cell preparations, indicating that the cells were biologically active, and that the lack of effect of changes in cyclic AMP synthesis on PGE2 levels could not be attributed to a defect in the prostaglandin synthetic pathway. Our findings did not agree with earlier work which showed that changes in cyclic AMP were correlated with changes in PGE2 production by human decidual cells. It is clear that in the previous studies the decidual cells were preincubated for 4–7 days prior to stimulation, in contrast with 24 h in our investigation. We suggest that the functional link between cyclic AMP and PGE2 synthesis reported previously may develop during culture, and not be a part of normal decidual cell function, but further studies are needed to test this hypothesis.  相似文献   

16.
—Adenylate cyclase activity of permeabilized neuroblastoma cells was measured by the conversion of [α32P]ATP into labelled cyclic AMP. Adenosine (10?6 - 10?4m ) induced a dose-dependent increase in cyclic AMP formation. This effect could not be accounted for either by an adenosine-induced inhibition of the phosphodiesterase activity present in the enzyme preparation, or by a direct conversion of adenosine into cyclic AMP. This indicates that the observed increase in cyclic AMP accumulation reflected an activation of adenylate cyclase. Adenosine is partially metabolized during the course of incubation with the enzyme preparation. However, none of the identified non-phosphorylated adenosine metabolites were able to induce an adenylate cyclase activation. This suggests that adenosine itself is the stimulatory agent. The apparent Km of the adenylate cyclase for adenosine was 5 ± 10?6-10?5m . Maximal activation represented 3-4 times the basal value (10-100 pmol cyclic AMP formed/10 min/mg protein). The adenosine effect was stereospecific, since structural analogues of adenosine were inactive. Adenosine increased the maximal velocity of the adenylate cyclase reaction. The stimulatory effect of adenosine was inhibited by theophylline. Prostaglandin PGE1 had a stimulatory effect much more pronounced than that of adenosine (6-10-fold the basal value at 10?6m ). Dopamine and norepinephrine induced a slight adenylate cyclase activation which was not potentiated by adenosine. It is concluded that adenosine is able to activate directly neuroblastoma cell adenylate cyclase. It seems very likely that such a direct activation is also present in intact nervous tissue and account, at least partly, for the observed cyclic AMP accumulation in response to adenosine.  相似文献   

17.
The response of endothelial adenylate cyclase (AC) to prostaglandins (PGE1, PGE2, PGF, PGF, PGD2 and PGI2) and the relationship of PGE2 to adrenergic systems were investigated in cerebrovascular endothelial cultures. E-type prostaglandins and PGI2 were more effective in stimulating endothelial AC (EC50 = 3 × 10?7M, and 3 × 10?7M, respectively) than prostaglandins of the F-series and PGD2 which activated AC at high doses only. A modulation of endothelial AC response to either PGE2 or norepinephrine (NE) was observed in the presence of both agents in the system. It was manifested by a dose-dependent NE inhibition of the PGE2-stimulated formation of cAMP, which was partially restored by phentolamine. Alpha and β-adrenergic agonists (α, clonidine and 6-fluoronorepinephrine; β, isoproterenol) also partly blocked while forskolin and PGE2 synergistically stimulated the production of cAMP in the endothelial cultures. These findings strongly suggest that the interaction of prostaglandins and α- and β-adrenergic agonists with the AC system in cerebrovascular endothelium may play a role in the regulation of the cerebral microcirculation and/or blood pressure.  相似文献   

18.
The ionophore A23187 stimulated adenylate cyclase activity in intact macrophages within 1 min. This action was blocked by pretreatment with indomethacin (25 μmol/l) suggesting the involvement of a prostaglandin (PG). PGE2 (500 nmol/l) also stimulated adenylate cyclase activity in intact cells, but this was not prevented by indomethacin pretreatment. Colchicine (100 μmol/l) potentiated the increases in macrophage cyclic AMP production seen after addition of PGE2 or A23187. The high affinity form of cyclic AMP phosphodiesterase (PDE) was activated within 1 min of the addition of A23187 to intact macrophages. The data suggest that the increase in macrophage cyclic AMP production after A23187 is a consequence of adenylate cyclase activation and not PDE inhibition. The endogenous production of a prostaglandin probably mediates this effect of A23187, emphasizing the importance of arachidonic acid metabolites in the regulation of macrophage functions.  相似文献   

19.
Prostaglandins and activation of AC/cAMP prevents anoikis in IEC-18   总被引:2,自引:0,他引:2  
Recent data indicates that chronic inflammation of the intestine such as Crohn's or ulcerative colitis puts those individuals at heightened risk for colorectal adenocarcinoma. In this study, we examine the effect of the inflammatory mediator PGE2 and associated signalling on detachment-induced cell death (anoikis) in intestinal epithelial cells. Treatment of detached IEC-18 with 0.01–0.05 μM PGE2 increased cell viability as well as induced aggregation. As EP4 prostaglandin receptors on IEC are coupled to adenylate cyclase, we next treated cells with agents that promote cAMP signalling (Forskolin, dbcAMP, and etazolate), all of which promoted IEC aggregation as well as survival. We next treated detached IECs with specific inhibitors of adenylate cyclase or PKA, which accelerated anoikis. To explore the mechanism of cell-cell adhesion, we next treated detached IECs with an anti-E-cadherin blocking antibody which dispersed aggregates induced by dbcAMP, and an adenovirus expressing a dominant negative E-cadherin (EcadΔEC) prevented aggregate formation. Interestingly EcadΔEC prevented aggregation of IEC induced by dbcAMP but did not significantly reduce viability. This suggests that cAMP signalling is important in both aggregate formation and promoting viability but these are distinct events. Taken together, these data support a mechanism whereby elevated PGE2 levels characteristic of colitis prevent anoikis by activating an AC-, cAMP-, and PKA-dependent signalling pathway. The delay of apoptosis by PGE2 may be one mechanism by which inflammation may contribute to carcinogenesis.  相似文献   

20.
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