Differential effects of histamine, vasoactive intestinal polypeptide, prostaglandin E2 and somatostatin on cyclic AMP-dependent protein kinase activation in gastric glands isolated from the guinea pig fundus and antrum

Histamine, vasoactive intestinal polypeptide (VIP), secretin and prostaglandin E2 (PGE2) stimulate cyclic AMP-dependent protein kinase activity in gastric glands isolated from the guinea pig fundus and antrum. The effects are observed in the absence of any cyclic AMP phosphodiesterase inhibitor and maximal stimulation of the protein kinases occurs within 0.5 min of incubation at 20 degrees C. As shown by dose-response studies, VIP is equally potent in the antrum as in the fundus (identical values of the activation constant are found in both types of gland, Ka = 2.5 . 10(-9) M); a similar situation occurs for PGE2 action (but with Ka = 2.0 . 10(-8) M), whereas the potency of histamine is higher in the fundus (ka = 8.0 . 10(-6)M) than in the antrum (Ka = 5.0 . 10(-5) M). Secretin also increases the protein kinase activity ratio but with a 1000 times lower potency than VIP. In fundic glands, histamine (10(-3) M) is the activator of by far the greatest efficacy (increasing protein kinase activity at 4 times of the basal value) as compared with the effect obtained with 10(-6) M PGE2 (2.7 times) and 10(-7) M VIP (1.4 times). In contrast, VIP has greater efficacy (2.3 times) than histamine (2.1 times) in antral glands, whereas PGE2 is equally active in the two parts of the gastric mucosa. In addition, somatostatin (10(-6) M) inhibits partially (30%) and specifically the protein kinase activation stimulated by histamine, whereas it has no effect on VIP- and PGE2-induced activation. The results are consistent with increased cyclic AMP levels in response to these effectors in this system. A physiological role of histamine on acid-secreting parietal cells, of VIP on nonparietal cells and of PGE2 on both cell types, mediated by the cyclic AMP/protein kinase system is proposed.     

Differential effects of prostaglandin E2 on contractions of airway smooth muscle

In an in vitro muscle bath, the active tension generated by strips of canine tracheal smooth muscle responding to cumulative additions of either histamine (10(-8) to 10(-3) M) or acetylcholine (10(-9) to 10(-3) M) was measured in the absence and presence of prostaglandin E2 (PGE2) (10(-6) to 10(-5) M). When contractile responses of equal magnitude were compared, the contractions elicited by acetylcholine were resistant to the inhibitory effects of PGE2, relative to comparable contractions elicited by histamine. To assess the role of adenylate cyclase in determining the different responses to histamine and acetylcholine in the presence of PGE2, we assayed adenylate cyclase activity in membranes prepared from canine tracheal smooth muscle and found that acetylcholine, but not histamine, decreased PGE2-stimulated adenylate cyclase activity by 48 +/- 2% (mean +/- SE; n = 5). However, in other experiments, we found that even large pharmacological increases in tissue adenosine 3',5'-cyclic monophosphate (cAMP) content only partially inhibited muscarinic tone. Also, exogenously applied analogues of cyclic AMP inhibited contractions induced by histamine more effectively than comparable contractions induced by acetylcholine. We concluded that acetylcholine decreased adenylate cyclase activity in membranes prepared from canine tracheal smooth muscle and that this effect may have contributed to, but did not completely account for, the relative resistance of muscarinic contractions to the inhibitory effects of PGE2.     

Factors regulating the production of prostaglandin E2 and prostacyclin (prostaglandin I2) in rat and human adipocytes.

The regulation of PGE2 (prostaglandin E2) and PGI2 (prostaglandin I2; prostacyclin) formation was investigated in isolated adipocytes. The formation of both PGs was stimulated by various lipolytic agents such as isoproterenol, adrenaline and dibutyryl cyclic AMP. During maximal stimulation the production of PGE2 and PGI2 (measured as 6-oxo-PGF1 alpha) was 0.51 +/- 0.04 and 1.21 +/- 0.09 ng/2 h per 10(6) cells respectively. Thus PGI2 was produced in excess of PGE2 in rat adipocytes. The production of the PGs was inhibited by indomethacin and acetylsalicylic acid in a concentration-dependent manner. The half-maximal effective concentration of indomethacin was 328 +/- 38 nM and that of acetylsalicylic acid was 38.5 +/- 5.3 microM. The PGs were maximally inhibited by 70-75% after incubation for 2 h. In contrast with their effect on PG production, the two agents had a small potentiating effect on the stimulated lipolysis (P less than 0.05). The phospholipase inhibitors mepacrine and chloroquine inhibited both PG production and triacylglycerol lipolysis and were therefore unable to indicate whether the PG precursor, arachidonic acid, originates from phospholipids or triacylglycerols in adipocytes. Angiotensin II significantly (P less than 0.05) stimulated both PGE2 and PGI2 production in rat adipocytes without affecting triacylglycerol lipolysis. Finally, it was shown that PGE2 and PGI2 were also produced in human adipocytes, although in smaller quantities than in rat adipocytes. It is concluded that the production of PGs in isolated adipocytes is regulated by various hormones. Moreover, at least two separate mechanisms for PG production may exist in adipocytes: (1) a mechanism that is activated concomitantly with triacylglycerol lipolysis (and cyclic AMP) and (2) an angiotensin II-sensitive, but lipolysis (and cyclic AMP)-independent mechanism.     

Endothelin-1 and endothelin-3 stimulate prostaglandin E2 secretion from the rat median eminence.

The in vitro effects of endothelin-1 (ET-1) and endothelin-3 (ET-3) on the release of prostaglandin (PG)E2 from the rat median eminence were investigated. The addition of ET-1 from 10(-9) M to 10(-6) M stimulated PGE2 release in a dose-dependent manner (from 10.5 +/- 2.1 to 54.4 +/- 5.6 pg/ME fragment/30 min; mean +/- SEM, p less than 0.001). ET-3 also stimulated the release of PGE2 from 10(-7) M to 10(-5) M dose dependently (from 18.1 +/- 0.7 to 60.9 +/- 17.4 pg/ME fragment/30 min p less than 0.05). The time course effect of ET-3 (10(-6) M) showed that PGE2 release was stimulated within five minutes (control, 1.5 +/- 0.5; ET-3, 15.8 +/- 3.0 pg/ME fragment/5 min, p less than 0.01). These results suggest that ET-1 and ET-3 have some physiological effects on the rat median eminence.     

Inhibition of prostaglandin E2-induced cyclic amp accumulation in the rat anterior pituitary by 11-deoxyprostaglandin E analogs (9-ketoprostynoic acids).

The effects of various 11-deoxyprostaglandin E analogs on the basal and prostaglandin E2 (PGE2)-induced cyclic AMP accumulation in the rat anterior pituitary were studied in vitro. 13-Hydroxy-9-oxoprost-14-ynoic acid at 5 X 10(-4)M but not 5 X 10(-5)M, decreased (45%) the induced accumulation and did not alter the basal accmulation; 15-hydroxy-9-oxoprost-13-ynoic acid at 5 X 10(-4)M caused less of a decrease (29%) in the induced and also did not alter the basal accumulation. (14Z)-13-Hydroxy-9-oxoprost-14-enoic acid at 5 X 10(-4)M did not alter the induced and caused a slight increase (5 fold) in the basal accumulation. 7-Oxa-13-prostynoic acid increased slightly the basal accumulation at 5 X 10(-5)M (2 fold) and 2.33 X 10(-4)M (6-fold) and did not antagonize the induced accumulation. Thus, the 9-ketoprostynoic acids are effective PGE2 antagonists in this system.     

Insulin-dependent contractility of glomerular mesangial cells in response to angiotensin II, platelet-activating factor and endothelin is attenuated by prostaglandin E2.

Culture of glomerular mesangial cells in the absence of insulin decreased the degree of contraction of individual cells in response to vasoconstrictive agonists, angiotensin II, platelet-activating factor and endothelin 1, as compared with cells cultured in the presence of insulin (0.7 nM). This change was associated with a decreased sensitivity of the intracellular Ca2+ response to vasoactive agents in fura-2-loaded cells and with an increase in the basal level of prostanoid [prostaglandins (PG) E1 and E2] production estimated by radioimmunoassay. Addition of exogenous PGE2 to insulin-exposed cells decreased the contractile response to that observed in insulin-deficient cells. Inclusion of 8-bromo cyclic AMP had a similar effect. In 45Ca2(+)-release studies it was shown that, in saponin-permeabilized insulin-exposed cells, preincubation with exogenous PGE2 or 8-bromo cyclic AMP decreased the sensitivity of 45Ca2+ release in response to Ins(1,4,5)P3, as demonstrated by an increase in the EC50 (concn. giving half-maximal effect) to 0.182 +/- 0.024 microM and 0.457 +/- 0.031 microM respectively, as compared with untreated permeabilized cells (EC50 0.091 +/- 0.021 microM). A similar decrease in Ins(1,4,5)P3-sensitive 45Ca2+ release was seen in permeabilized cells from insulin-free conditions of culture (EC50 0.20 +/- 0.061 microM). As altered glomerular haemodynamics are found in insulinopaenic diabetic conditions, it is proposed that a decrease in intracellular Ca2+ availability in response to vasoactive agonists and consequent decrease in mesangial-cell contractility contributes to the hyperfiltration seen in this condition.     

Phorbol esters modulate cyclic AMP accumulation in porcine thyroid cells

In cultured porcine thyroid cells, during 60 min incubation phorbol 12-myristate 13-acetate (PMA) had no effect on basal cyclic AMP accumulation and slightly stimulated cyclic AMP accumulation evoked by thyroid stimulating hormone (TSH) or forskolin. Cholera toxin-induced cyclic AMP accumulation was significantly stimulated by PMA. On the other hand, cyclic AMP accumulation evoked by prostaglandin E1 or E2 (PGE1 or PGE2) was markedly depressed by simultaneous addition of PMA. These opposing effects of PMA on cyclic AMP accumulation evoked by PGE and cholera toxin were observed in a dose-related fashion, with half-maximal effect of around 10(-9) M in either case. The almost same effects of PMA on cyclic AMP accumulation in basal and stimulated conditions were also observed in freshly prepared thyroid cells. The present study was performed in the presence of phosphodiesterase inhibitor, 3-iso-butyl-1-methylxanthine (IBMX), indicating that PMA affected adenylate cyclase activity. Therefore, it is suggested that PMA may modulate the production of cyclic AMP in response to different stimuli, possibly by affecting several sites in the adenylate cyclase complex in thyroid cells.     

Platelet aggregation. II. Adenyl cyclase, prostaglandin E1, and calcium

In exploration of the proposal that prostaglandin E1 (PGE1) inhibits platelet aggregation via stimulation of adenyl cyclase, the temporal relationship of adenosine cyclic 3',5' monophosphate (cyclic AMP) synthesis and inhibition of ADP-induced aggregation in response to PGE1 was studied. The requirement for calcium in aggregation led to the investigation of the effects of calcium ions on platelet adenyl cyclase activity. PGE1 stimulated the synthesis of cyclic AMP from adenosine-5'-triphosphate-8-14-C by platelet membrane fractions and also increased cyclic AMP synthesis in intact platelets previously incubated for 2 hours with adenosine-14-C. The accumulation of cyclic AMP increased signficiantly at low concentrations of PGE1 and reached a maximum at about 1 mug. Regardless of the inducing agent, calcium ions are an absolute requirement for the aggregation of platelets.     

The effect of turpentine-induced inflammation on rat liver glycosyltransferases and Golgi complex ultrastructure

The effect of vasopressin on the toad urinary bladder has been shown to be mediated by cyclic AMP. It has been assumed that, as demonstrated for other systems, this involves activation of cyclic AMP-dependent protein kinase. In order to test this hypothesis we investigated the effect of vasopressin on cyclic AMP-dependent protein kinases in epithelial cells of toad bladders. About 80% of protein kinase activity and cyclic AMP-binding capacity was found to be in the cytosol. DEAE-cellulose chromatography showed a pattern of 15--20% type I and 80--85% type II cyclic AMP-dependent protein kinase. Cytosolic kinase was activated 3--4-fold by cyclic AMP with half-maximal activation at 5 . 10(-8) M. Similarly, half-maximal binding of cyclic AMP occurred at 7 . 10(-8) M. Incubation of toad bladders in Ringer's solution containing 0.1 mM 3-isobutyl-1-methylxanthine, prior to homogenization and assay, showed stable cyclic AMP-binding capacity and protein kinase ratio --cyclic AMP/+cyclic AMP. Exposure of bladders to 10 mU/ml of vasopressin for 10 min caused intracellular activation of protein kinase and decrease in cyclic AMP-binding capacity that were maintained for at least 30 min. Incubation of bladders with increasing concentrations of vasopressin (0.5--100 mU/ml) resulted in a discrepancy between a progressive increase in cyclic AMP levels and a levelling off at 10 mU/ml of vasopressin for the changes in protein kinase ratio and cyclic AMP-binding capacity. The increase in kinase ratio was due to higher activity in the absence of exogenous cyclic AMP and was fully inhibitable by a specific protein kinase inhibitor. Using Sephadex G-25-CM50 column chromatography for separation of holoenzyme and free catalytic subunit we demonstrated that the activation of protein kinase in the vasopressin-treated bladders is due to intracellular dissociation of the kinase. These results show that the effect of vasopressin on the toad bladder involves activation of a cytosolic cyclic AMP-dependent protein kinase. The time course and the dose-response curve of the kinase activation closely parallel vasopressin's effect on osmotic water flow.     

Stimulation of bone formation by prostaglandin E2

We examined the effect of prostaglandin E2 (PGE2), in the presence or absence of cortisol, on bone formation in 21-day fetal rat calvaria maintained in organ culture for 24 to 96 h. [3H]Thymidine and [3H] proline incorporation were used to assess DNA and collagen synthesis, respectively. Changes in dry weight and DNA content were assessed after 96 h. PGE2 (10(-7) M) stimulated both DNA and collagen synthesis in calvaria. The effect on DNA synthesis was early (24 h), transient and limited to the periosteum. Collagen synthesis was stimulated at a later time (96 h), predominantly in the central bone. Cortisol (10(-7) M) inhibited DNA and collagen synthesis. The addition of PGE2 reversed the inhibitory effects of cortisol on DNA synthesis and content and increased collagen synthesis in central bone to levels above control untreated cultures. We conclude that PGE2 has stimulatory effects on bone formation and can reverse the inhibitory effects of cortisol. Hence the effects of cortisol may be mediated in part by their ability to reduce the endogenous production of prostaglandins.     

Various effects of prostaglandin E2 on reabsorption of water and urea in the amphibia osmosis-regulating epithelium

Principal similarities between molecular pathways providing the enhancement of water and urea reabsorption under the action of argininvasotocin (AVT) in amphibian urinary bladder suggest that prostaglandin E2 (PGE2) could be a negative regulator of urea transport. To analyse this hypothesis, the role of PGE2 in regulation of urea transport was studied in isolated frog (Rana temporaria L.) urinary bladder. The urea permeability (Pu) was determined from the rate of efflux of (14) Curea from mucosal to serosal solution in isoosmotic conditions. The water permeability was measured in separate experiments in presence of an osmotic gradient. In contrast to water permeability, we were unable to demonstrate any inhibitory effect of 10-1000 nM PGE2 on AVT-stimulated urea transport using a variety of protocols. It was found that basolateral PGE2 exposure (10 nM-1 microM) caused an increase in Pu with no effect on osmotic water flow. The PGE2 effect was markedly inhibited by phloretin, a specific inhibitor of urea transporter. Sulprostone, an EP1/EP3 prostaglandin E2 receptor agonist, had no effect on Pu suggesting the contribution of EP2/EP4 receptor subtypes. In presence of osmotic water flow, the AVT-induced urea transport was significantly higher. This water flow-dependent urea permeability was inhibited by PGE2 although the inhibitory effect was less pronounced in comparison to the action of PGE2 on osmotic water flow. On the basis of these results we can make a conclusion that PGE2 has different role in regulation of water and urea transport in the frog urinary bladder. PGE2 could be considered as a stimulator of urea transport and an inhibitor of osmotic water flow activated by the AVT. The ability of PGE2 to regulate various types of cAMP-dependent transport by different mechanisms seems to be based on the presence of multiple basolateral PGE2 receptor subtypes in amphibian osmosis-regulatory epithelium.     

A prostaglandin E receptor coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rabbit cortical collecting tubule cells

At different concentrations, prostaglandin E2 (PGE2) can either stimulate or inhibit cAMP formation in freshly isolated rabbit cortical collecting tubule (RCCT) cells, but in cultured RCCT cells PGE2 can only stimulate cAMP synthesis (Sonnenburg, W. K., and Smith W. L. (1989) J. Biol. Chem. 263, 6155-6160). Here, we report characteristics of [3H]PGE2 binding to membrane receptor preparations from both freshly isolated and cultured RCCT cells. [3H]PGE2 binding to membranes from freshly isolated RCCT cells was saturable and partially reversible. Equilibrium binding analyses indicated that in the absence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) there is a single class of PGE2 binding sites (KD = 4.2 +/- 0.4 nM; Bmax = 583 +/- 28 fmol/mg); in the presence of 100 microM GTP gamma S, there is also only one class of binding sites but with a somewhat lower KD = 1.2 +/- 0.5 nM (Bmax = 370 +/- 40 fmol/mg). This stimulatory effect of GTP gamma S was blocked by pretreatment of the freshly isolated RCCT cells with pertussis toxin. The relative affinities of prostanoids for the [3H]PGE2-binding site were determined to be 17,18,19,20-tetranor-16-phenoxy-PGE2-methylsulfonylamide (sulprostone) approximately PGE2 approximately PGE1 approximately 16,16-dimethyl-PGE2 greater than carbacyclin approximately PGF2 alpha greater than PGD2. This is the order of potency with which prostaglandins inhibit arginine vasopressin-induced cAMP formation in fresh RCCT cells. Interestingly, [3H]PGE2 binding to membranes from cultured cells, which, unlike fresh cells, fail to show an inhibitory response to PGE2, was only 10-20% of that observed with membranes from fresh cells; moreover, binding of [3H]PGE2 to membranes from cultured cells was neither stimulated by GTP gamma S nor inhibited by sulprostone. The prostanoid binding specificities and the unusual pertussis toxin-sensitive, stimulatory effect of GTP gamma S on binding of [3H]PGE2 to membranes from freshly isolated RCCT cells are characteristics shared by a Gi-linked PGE receptor from renal medulla (Watanabe, T., Umegaki, K., and Smith, W. L. (1986) J. Biol. Chem. 261, 14340-14349). Our results suggest that the [3H]PGE2 binding site of freshly isolated RCCT cells is the PGE receptor which is coupled to a Gi to attenuate arginine vasopressin-induced cAMP synthesis in the renal collecting tubule.     

Cell-specific localization of prostaglandin E2-sensitive adenylate cyclase in rabbit endometrium

Epithelial and stromal cells were isolated from endometrium of Day 1 pseudopregnant rabbits by enzymatic digestion with trypsin or trypsin:collagenase:deoxyribonuclease. Dispersed cells were grown in RPMI 1640 supplemented with 10% whole or steroid-depleted fetal bovine serum (FBS). Epithelial and stromal cells reached confluency after 6 to 7 days in culture and showed specific characteristics. Cells could be differentiated according to morphology, growth patterns, electrophoretic patterns, and response to estrogen or progesterone. Hormonal stimulation of adenylate cyclase activity was measured in broken cell preparations by catalytic transformation of alpha-32P-adenosine triphosphate into 32P-adenosine 3'-5' cyclic monophosphate (cAMP). Adenylate cyclase activity was present in fresh endometrial tissue and in dispersed cells after 7 days in culture. The enzyme activity was significantly higher in stromal than in epithelial cells at all stimulation levels: basal (9.2 +/- 1.0 vs. 2.3 +/- 0.6, p less than 0.001) and guanosine triphosphate (GTP, 300 microM) (25.4 +/- 2.9 vs. 7.0 +/- 1.6, p less than 0.001). Net response to prostaglandin E2 (PGE2, 10 microM) was three times higher (p less than 0.001) in stromal (17 +/- 2) than in epithelial (5.0 +/- 1) cells. These results suggest that PGE2 can stimulate adenylate cyclase in rabbit endometrium and that the enzyme is preferentially localized in the stroma. Our results are in agreement with the hypothesis that cAMP formed in endometrium in response to PGE2 might be involved in the decidual reaction.     

Effect of prostaglandin E1 on the cyclic adenosine-3',5'--monophosphate system and radiosensitivity of B-82 cells cultured in vitro

The cyclic AMP (cAMP) system of mice fibroblasts L, clone B-82 has been studied. B-82 cells possess receptors to prostaglandin E1 (PGE1) and are deficient in beta-adrenoreceptors. It has been shown that 10 mM NaF, 1.10(-4) M GTP and 1.10(-5) M PGE1 stimulate adenylate cyclase of B-82 cells. PGE1 causes increase in the intracellular content of cAMP and protects B-82 cells against irradiation. Specifict beta-agonist isoproterenol fails to modulate adenylate cyclase activity and does not change cAMP content in B-82 cells. Isoproterenol has no effect on the B-82 cells radiosensitivity, although it is known to be a potent radioprotector. It is presumed that the stimulation of cAMP system by radioprotector is necessary for the performance of radioprotective effect.     

Interaction between prostaglandin E2 and leukotriene D4 on the excretion of electrolytes by the dog kidney in vivo

Recent experiments indicate that prostaglandin E2 potentiates the vasodilatory properties of leukotrienes in the skin microcirculation. The present experiments were undertaken to study the effect of leukotriene D4 and prostaglandin E2 on renal hemodynamics and urinary electrolytes in the dog. Experiments were performed in three groups of anesthetized Mongrel dogs: the first group was studied under hydropenia, whereas the two remaining groups were studied during water diuresis with (Group 3) or without indomethacin (Group 2). LTD4 (100 ng/min) and PGE2 (3 ug/min) were infused in the left renal artery to minimize systemic effects of these compounds. LTD4 alone failed to influence urinary sodium excretion in all 3 groups. In Group 1, urinary sodium increased from 77 +/- 6 to 393 +/- 74 uEq/min during PGE2, and further increased to 511 +/- 52 uEq/min during LTD4 + PGE2. No change occurred in the contralateral right kidney. In this group, glomerular filtration as well as renal plasma flow were not statistically influenced. In Group 2, the same phenomenon was observed for urinary sodium. The combined infusion of LTD4 + PGE2 increased urinary sodium without significant changes in glomerular filtration and renal plasma flow. Finally, in Group 3, indomethacin was shown to reduce the natriuretic effects of LTD4 and PGE2: during PGE2 alone, urinary sodium increased from 90 +/- 14 to 260 +/- 66 uEq/min, and only rose from 80 +/- 10 to 175 +/- 19 uEq/min during the combined infusion of LTD4 and PGE2. In groups 2 and 3, free water clearance was utilized as an index of sodium chloride reabsorption in the thick ascending limb: this parameter increased from 2.35 +/- 0.25 to 4.70 +/- 0.30 ml/min, while urinary volume was increasing from 3.55 +/- 0.25 to 10.05 +/- 0.65 ml/min, during LTD4 + PGE2. Indomethacin, administered in Group 3, (3 mg/kg/hr) again abolished the effect of combined PGE2 + LTD4. These results indicate a potentiating effect of leukotriene D4 on the PGE2-induced natriuresis in the anesthetized dog. These phenomena occurred in the absence of significant changes in renal hemodynamics, therefore suggesting a direct tubular effect of these arachidonic acid metabolites. Finally, the water diuresis experiments suggest a proximal site of action of PGE2 and LTD4.     

cAMP and human neutrophil chemotaxis. Elevation of cAMP differentially affects chemotactic responsiveness.

Neutrophils (PMN) treated with cAMP elevating agents were evaluated for their chemotactic responsiveness to FMLP and leukotriene B4 (LTB4). PGE1 and isoproterenol, increased PMN cyclic AMP production and inhibited chemotaxis to both FMLP and LTB4. In contrast, forskolin, which activates adenylate cyclase directly, inhibited chemotaxis to FMLP but not to LTB4. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), was required for inhibition of PMN chemotaxis to FMLP by forskolin, PGE1, and isoproterenol. Isoproterenol and PGE1 inhibited PMN chemotaxis to LTB4 in the absence of IBMX and chemotaxis was further inhibited in the presence of IBMX. PMN cAMP levels were stimulated 2- to 3-fold with isoproterenol, 6- to 10-fold with PGE1, and 5- to 7-fold with forskolin over basal levels in the presence of IBMX. These observations demonstrate that total cellular cAMP concentration is not correlated with inhibition of PMN chemotaxis to all stimuli; forskolin, which increased cyclic AMP 5- to 7-fold over basal levels, did not inhibit chemotaxis to LTB4, whereas isoproterenol, which increased cyclic AMP only 2- to 3-fold over basal levels, inhibited chemotaxis to LTB4. PMN cAMP extrusion was determined under basal conditions and in the presence of PGE1, isoproterenol, or forskolin. PMN extruded cAMP under all conditions examined.     

Changes in responsiveness of rat granulosa cells to prostaglandin E2 and follicle-stimulating hormone during culture

The changes in responsiveness of granulosa cells to either FSH or prostaglandin E2 (PGE2), during culture of the cells, have been examined. In freshly isolated cells, FSH and PGE2 stimulated both cyclic AMP and progesterone production in a dose-dependent manner. In these cells, FSH stimulated cyclic AMP production to a greater extent than did PGE2. After the cells had been cultured for 2 days, neither FSH nor PGE2 stimulated progesterone production to any detectable extent. In these cells the ability of FSH to stimulate cyclic AMP was decreased, and that of PGE2 was increased markedly, such that PGE2 was far more effective than FSH in stimulating cyclic AMP. After culture of the cells for a further 2 days (4 days total), the FSH stimulation of cyclic AMP returned to that seen in freshly isolated cells, whereas the stimulation by PGE2 remained elevated. The acute stimulation of progesterone production could be restored by chronic exposure of the cells to either FSH or PGE2. These results demonstrate that dramatic changes in responsiveness of granulosa cells take place during culture. The results also suggest that some stimulating factor must be present to maintain the steroidogenic capabilities of the cells. Without this, although the cells are able to produce cyclic AMP in response to FSH, they cannot produce progesterone.     

Cyclic AMP and platelet prostaglandin synthesis.

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3'5'-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of 14C-arachidonic acid by washed platelets. Prostaglandin E1 (PGE1), PGE1 with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B2. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE1 with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of 14C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.     

Cyclic AMP response to prostaglandin E1 in mononuclear cells from peripheral blood and synovial fluid of patients with rheumatoid arthritis.

The cyclic AMP response to prostaglandin E1 (PGE1) was studied in peripheral blood (PB) and synovial fluid (SF) mononuclear cells from patients with rheumatoid arthritis (RA). The PGE1 induced accumulation of cyclic AMP was consistently (7 of 8 patients) less in cell suspensions derived from SF than in suspensions of equivalent numbers of mononuclear cells obtained simultaneously from PB. The high PB/SF cyclic AMP ratio was seen most clearly at the lowest concentration (10(-6)M) of PGE1 tested. There was no correlation between the patients' therapy and cyclic AMP response to PGE1. The high PB/SF cyclic AMP ratio was not accounted for by the presence of platelets in PB cell suspensions.     

Impaired bone resorption to prostaglandin E2 in prostaglandin E receptor EP4-knockout mice

Prostaglandin E(2) (PGE(2)) acts as a potent stimulator of bone resorption. In this study, we first clarified in normal ddy mice the involvement of protein kinase A and induction of matrix metalloproteinases (MMPs) in PGE(2)-induced bone resorption, and then identified PGE receptor subtype(s) mediating this PGE(2) action using mice lacking each subtype (EP1, EP2, EP3, and EP4) of PGE receptor. In calvarial culture obtained from normal ddy mice, both PGE(2) and dibutyryl cyclic AMP (Bt(2)cAMP) stimulated bone resorption and induced MMPs including MMP-2 and MMP-13. Addition of an inhibitor of protein kinase A, H89, or an inhibitor of MMPs, BB94, significantly suppressed bone-resorbing activity induced by PGE(2.) In calvarial culture from EP1-, EP2-, and EP3-knockout mice, PGE(2) stimulated bone resorption to an extent similar to that found in calvaria from the wild-type mice. On the other hand, a marked reduction in bone resorption to PGE(2) was found in the calvarial culture from EP4-knockout mice. The impaired bone resorption to PGE(2) was also detected in long bone cultures from EP4-knockout mice. Bt(2)cAMP greatly stimulated bone resorption similarly in both wild-type and EP4-knockout mice. Induction of MMP-2 and MMP-13 by PGE(2) was greatly impaired in calvarial culture from EP4-knockout mice, but Bt(2)cAMP stimulated MMPs induction similarly in the wild-type and EP4-knockout mice. These findings suggest that PGE(2) stimulates bone resorption by a cAMP-dependent mechanism via the EP4 receptor.