Interrelationships among prostaglandins, vasopressin and cAMP in renal papillary collecting tubile cells in culture

To determine the influence of prostaglandins on cAMP metabolism in renal papillary collecting tubule (RPCT) cells, intracellular cAMP levels were measured after incubating cells with prostaglandins (PGs) alone or in combination with arginine vasopressin (AVP). PGE₁, PGE₂ and PGI₂, but not PGD₂ or PGF_2α, increased intracellular cAMP concentrations. At maximal concentrations (10⁻⁵ tthe effects of PGE₂ plus PGI₂ (or PGE₁), but not of PGI₂ plus PGE₁, were additive suggesting that at least two different PG receptors may be present in RPCT cell populations. Bradykinin treatment of RPCT cells caused an accumulation of intracellular cAMP which was blocked by aspirin and was quantitatively similar to that observed with 10⁻⁵ PGE₂. PGs, when tested at concentrations (e.g. 10⁻⁹ ) which had no independent effect on intracellular cAMP levels, did not inhibit the AVP-induced accumulation of intracellular cAMP in RPCT cells. These results indicate that PGs do not block AVP-induced accumulation of intracellular cAMP in RPCT cells at concentrations of PGs which have been shown to inhibit the hydroosmatic effect of AVP on perfused collecting tubule segments. However, at higher concentrations of PGs (e.g. 10⁻⁵ ), the effects of AVP plus PGE₁, PGE₂, PGI₂ or bradykinin on intracellular cAMP levels were not additive. Thus, under certain conditions, there is an interaction between PGs and AVP at the level of cAMP metabolism in RPCT cells.     

Regulation of cyclic AMP metabolism in rabbit cortical collecting tubule cells by prostaglandins

Prostaglandin E1 (PGE1) at 1 nM inhibits arginine-vasopressin (AVP)-induced water reabsorption in the rabbit cortical collecting tubule (RCCT), while 100 nM PGE1, by itself, stimulates water reabsorption (Grantham, J. J., and Orloff, J. (1968) J. Clin. Invest. 47, 1154-1161). To investigate the basis for these two responses, we measured the effects of prostaglandins on cAMP metabolism in purified RCCT cells. In freshly isolated cells, PGE2, PGE1, and 16,16-dimethyl-PGE2 acting at high concentrations (0.1-10 microM) stimulated cAMP accumulation; however, one PGE2 analog, sulprostone (16-phenoxy-17,18,19,20-tetranor-PGE2 methylsulfonilamide), failed to stimulate cAMP accumulation or to antagonize PGE2-induced cAMP formation; PGD2, PGF2 alpha, and a PGI2 analog, carbacyclin (6-carbaprostaglandin I2), also failed to stimulate cAMP synthesis. These results suggest that there is a PGE-specific stimulatory receptor in RCCT cells which mediates activation of adenylate cyclase. Occupancy of this receptor would be anticipated to cause water reabsorption by the collecting tubule. At lower concentrations (0.1-100 nM) PGE2, PGE1, 16,16-dimethyl-PGE2, and, in addition, sulprostone inhibited AVP-induced cAMP accumulation by fresh RCCT cells in the presence of cAMP phosphodiesterase inhibitors. Pertussis toxin pretreatment of RCCT cells blocked the ability of both PGE2 and sulprostone to inhibit AVP-induced cAMP accumulation. In membranes prepared from RCCT cells, sulprostone prevented stimulation of adenylate cyclase by AVP. These results suggest that E-series prostaglandins (including sulprostone) can act through an inhibitory PGE receptor(s) coupled to the inhibitory guanine nucleotide regulatory protein, Gi, to block AVP-induced cAMP synthesis by RCCT cells. Occupancy of this receptor would be expected to cause inhibition of AVP-induced water reabsorption in the intact tubule. Curiously, after RCCT cells were cultured for 5-7 days, PGE2 no longer inhibited AVP-induced cAMP accumulation, but PGE2 by itself could still stimulate cAMP accumulation. In contrast to PGE2, epinephrine acting via an alpha 2-adrenergic, Gi-linked mechanism did block AVP-induced cAMP formation by cultured RCCT cells. This implies that some component of the inhibitory PGE response other than Gi is lost when RCCT cells are cultured.     

Interactions between parathyroid hormone and prostaglandins on renal cortical cyclic AMP

Effects of parathyroid hormone (PTH) and several prostaglandins (PGs) on cyclic AMP (cAMP) metabolism were studied and compared in isolated renal cortical tubules from male hamsters. Both production and intracellular degradation of cAMP were increased by PTH and each of the PGs tested (PGE2, PGE1, PGI2). Production of cAMP was increased to similar levels by maximal concentrations of PTH and each PG, however, degradation of cAMP was significantly higher in response to PTH than with any of the PGs. This difference in intracellular degradation of cAMP was responsible for the much higher concentrations of cAMP in renal cortical tubules exposed to PGs (PGE1, PGE2, PGI2) than to PTH. Submaximal amounts of each PG produced additive increases in cAMP concentrations in the presence of maximal amounts of PTH. Additivity of the combined responses was lost, however, as the PGs concentrations reached their maxima. The results suggest that renal PGs (PGE2 and PGI2) may modulate the effects of PTH on cAMP concentrations in renal cortical tubules.     

Stimulatory and inhibitory effects of prostaglandins on the gonadotropin-sensitive adenylate cyclase in the monkey corpus luteum

Detailed analysis of the action of prostaglandins (PGs) on the corpus luteum in primate species is very limited. In this study we examined the response of the adenylate cyclase system to PGs in homogenates prepared from the corpus luteum of rhesus monkeys at midluteal phase of the menstrual cycle. The conversion of [alpha 32p] ATP to [32p] cyclic AMP (cAMP) was assessed in the absence (control activity; 50 microM GTP) and presence of various concentrations of seven PGs and arachidonic acid, either alone or in combination with 250 nM hCG. Cyclic AMP production increased up to three-fold in the presence of PGD2, PGE2, PGI2 or PGF2 alpha; however PGA2, PGB2, 13, 14-dihydro-15-keto PGE2 and arachidonic acid alone did not alter cAMP levels. In dose-response studies, adenylate cyclase was 10 and 100-fold more sensitive to PGD2 (Vmax at 1 X 10(-5) M) than to PGE2 or to PGI2 and PGF2 alpha, respectively. Activity in the presence of hCG plus either PGD2, PGE2, PGI2 or PGF2 alpha did not differ from that for hCG (or the PG) alone. In contrast, addition of PGA2 or arachidonate inhibited (p less than 0.05) hCG-stimulated cAMP production by 50 and 100 percent. We conclude that the gonadotropin-sensitive adenylate cyclase of the macaque corpus luteum is also modulated by several PGs. These factors may either mimic (e.g., PGD2, PGE2, PGI2) or suppress (PGA2) gonadotropin-stimulated cAMP production and possibly cAMP-mediated events in luteal cells.     

Effect of vasopressin on Na+ kinetics in cultured rat vascular smooth muscle cells

The effect of arginine vasopressin (AVP) on Na+ kinetics was examined in cultured rat vascular smooth muscle cells (VSMC) and rat renal papillary collecting tubule cells (RPCT) by the direct measurement of intracellular sodium concentration [(Na+]i) using fluorescence dye; SBFI. AVP increased [Na+]i in a dose-dependent manner at a concentration of 10(-9) M or higher in rat VSMC but did not affect [Na+]i in rat RPCT. The calcium (Ca2+)-free solution completely blocked the increasing effect of AVP on [Na+]i in rat VSMC. A Ca2+ ionophore, ionomycin (1-2 x 10(-6) M) increased [Na+]i both in rat VSMC and RPCT. The Ca2(+)-free solution abolished the ionomycin-increased [Na+]i both in rat VSMC and RPCT. These results therefore indicate that after binding the V1 receptor AVP increases [Na+]i mediated through an increase in cellular Ca2+ uptake in VSMC.     

Vasopressin-induced increases in cellular free calcium concentration measured in single cells of rat renal papillary collecting tubule

We determined the cellular free calcium concentration [Ca2+]i in response to arginine vasopressin (AVP) using single cells of cultured rat renal papillary collecting tubule cells. AVP at a concentration of 1 x 10(-10) M or higher significantly increased [Ca2+]i in a dose-dependent manner. The prompt increase in [Ca2+]i induced by AVP was completely blocked by the V1V2 antagonist, but not by the V1 antagonist. Also, an antidiuretic agonist of 1-deamino-8-D-arginine vasopressin (dDAVP) increased [Ca2+]i, which was blocked by the pretreatment with the V1 V2 antagonist. An AVP-induced increase in [Ca2+]i was still demonstrable in cells pretreated with Ca2(+)-free medium containing 1 x 10(-3) M EGTA, or a blocker of cellular Ca2+ uptake, 5 x 10(-5) M verapamil. These results indicate that AVP increases [Ca2+]i through the V2 receptor in renal papillary collecting tubule cells where cAMP is a well-known second messenger for AVP, and that cellular free Ca2+ mobilization depends on both the intracellular and extracellular Ca2+.     

Functional compartmentalization of arginine-vasopressin-activated cyclic AMP in anterior pituitary gland: the presence of a compartment activated by prostaglandin E2

AVP(10(-8)-10(-6)M) increased ACTH as well as PGE2 release from rat anterior pituitary quarters in vitro in a concentration dependent manner. IBMX (0.1 mM), a phosphodiesterase inhibitor, increased the ACTH response to AVP. The cAMP content in pituitary tissue was increased by AVP. Cyclooxygenase inhibition by indomethacin(1.4 X 10(-5) M) or diclofenac (1.8 X 10(-5)M) led to a potentiation of AVP-evoked ACTH secretion and to a decrease in AVP-stimulated cAMP formation. PGE2(10(-6)M) significantly increased pituitary cAMP content and indomethacin did not affect cAMP levels activated by PGE2. PGE2 attenuated the AVP-induced ACTH release. These results indicate that at least two functional compartments of AVP-activated cAMP responses are involved in the AVP-induced ACTH release. One compartment is directly activated by AVP and participates in the propagation of AVP-induced ACTH release. The second compartment is activated by PGE2. The contribution of the second compartment to the regulation of ACTH secretion is not well understood since PGE2 shows an inhibitory effect on AVP-induced ACTH secretion.     

Augmentation of vasopressin-induced cAMP production by high potassium in rat renal papillary collecting tubule cells in culture.

The effect of high potassium, 60 mM KCl, on the cellular action of arginine vasopressin (AVP) was studied in rat renal papillary collecting tubule cells in culture. In the presence of 0.5 mM 3-isobutyl-1-methylxanthine AVP-induced cAMP production was enhanced by pretreatment of the cells with 60 mM KCl. Such an enhancement was not found in cells pretreated with Ca(2+)-free medium containing 1 mM EGTA or in Na(+)-free medium, which rather reduced AVP-induced cAMP production. Similar results were obtained with the blockers of cellular Ca2+ uptake, 1 x 10(-4) M verapamil and 1 x 10(-5) M nifedipine. The 60 mM KCl elevated the cellular sodium concentration ([Na+]i) from 15.1 to 18.8 mM, cellular pH (pHi) from 7.18 to 7.32, and basal cellular free calcium concentration ([Ca2+]i). These results indicate that high potassium promptly augments AVP-induced cAMP production in renal papillary collecting tubule cells. This effect is based on the alkalinized pHi and the increased [Ca2+]i.     

Prostaglandin inhibition of testosterone production induced by luteinizing hormone, dibutyryl cyclic AMP or 3-isobutyl-1-methyl xanthine in dispersed rat testicular interstitial cells.

Testicular interstitial cells were utilized to study the effects of prostaglandins (PG) on in vitro testosterone production and to examine the role of cyclic adenosine-3',5'-monophosphate (cAMP) in this process. Testosterone production was assessed after 3 hour incubations while cAMP accumulation was examined after a 0.5 hour incubation period. Testosterone and cAMP were measured by radioimmunoassay. None of the PGs tested (PGA, PGA2, PGB1, PGE1, PGE2, PGF1alpha PGF2alpha) altered basal testosterone production when present in incubates at concentrations of 1.3 X 10(-8) M to 1.3 X 10(-4). However, at concentrations of 1.3 X 10(-4) M all of these PGs were capable of decreasing Luteinizing Hormone (LH; 100ng)-induced testosterone production. The inhibition of LH-induced testosterone production by the B, E and F series PGs was less pronounced than that for the A series. PGA1 and PGA2 exhibited 80% and 95% inhibition, respectively, at 1.3 X 10(4) M. The inhibitory action of 4 X 10(5) M PGA1 or PGA2 was evident within 30 minutes. Preincubation of interstitial cells with indomethacin (10(-5) or 10(-6) M) for 30 minutes did not alter subsequent basal or LH (100ng)-induced testosterone production. Accumulation of cAMP was stimulated by LH (10 microgram) or by PGs (1.3 X 10(-4) M PGA1, PGA2, PGB1, PGE1 or PGF2alpha). The PG-induced cAMP accumulation thus occurred at concentrations where LH-stimulated testosterone production was inhibited. Furthermore, PGA1 and PGA2 (1.3 X 10(-4) M) inhibited testosterone production induced by either 3-isobutyl-1-methyl xanthine (MIX; 10(-4) M or 10(-3) M) or dibutyryl cAMP (dbcAMP; 10(-4) M or 10(-3) M). These results indicate that PGs can block testosterone production by a direct effect on testicular interstitial cells and suggest that PGs exert their inhibitory action distal to stimulation of cAMP formation. PGs do not appear to play a role in the mechanism of LH action.     

Effects of prostaglandins on cyclic AMP levels in isolated cells from developing chick limbs

Effects of prostaglandins (PGs) on accumulation of cyclic AMP (cAMP) in the presence of a phosphodiesterase inhibitor were investigated in cells isolated from avian limb buds at various stages of development. Cells were responsive to PGE2 at the earliest stage investigated (stage 20-21) which was well in advance of specific cytodifferentiation of limb tissues. At three later stages (24-25; 26-28; 30-32), the responsiveness of cells isolated from the developing skeletal anlagen of the limb progressively increased coincident with the differentiation and maturation of the cartilage phenotype. Cells isolated from stage 26-28 cartilage rods were responsive also to prostacyclin (PGI2); however, the response produced was only about 50% of the response to an equivalent concentration of PGE2. Cells were not responsive to either PGF2 alpha or 6-keto PGF1 alpha, at concentrations of 30-33 micrograms/ml demonstrating a degree of specificity for PGE2 and PGI2. In the absence of the phosphodiesterase inhibitor, PGE2 increased cAMP accumulation two-fold over the controls and produced a concentration-dependent response between 0.3-30 micrograms/ml. The results demonstrate that PGs are capable of modulating cAMP levels of undifferentiated limb mesenchymal cells as well as embryonic cartilage cells and suggest a role for these compounds in limb chondrogenesis.     

Effects of vasopressin and prostaglandin synthesis inhibition on cyclic AMP accumulation in the dog renal inner medulla

The interaction of arginine vasopressin (AVP) and endogenous prostaglandins on cAMP production was investigated in the dog. Cyclic AMP content of dog inner medullary tissue slices exposed to different concentrations of AVP in the presence and absence of various prostaglandin synthesis inhibitors was determined. If the slices were incubated in isotonic media with 95% O2 and 5% CO2 gas phase, inhibition of prostaglandin synthesis decreased cAMP accumulation. A significant correlation was found between the decrements in cAMP content and basal cAMP levels. AVP-induced increments in cAMP accumulation was, however, unaffected by prostaglandin synthesis inhibitors. If incubation was performed in a hypertonic medium and at low O2 concentration, basal cAMP content was significantly reduced and it was not altered by inhibition of prostaglandin synthesis. The cAMP response to AVP was practically identical in the presence and absence of prostaglandin synthesis inhibitors. In conscious dogs AVP and indomethacin in itself had no effect on urinary cAMP excretion, but there was a significant decrease if the two compounds were combined. These results fail to lend support the hypothesis that endogenous prostaglandins modulate AVP-induced cAMP accumulation in the inner medulla.     

Tolvaptan inhibits ERK-dependent cell proliferation, Cl⁻ secretion, and in vitro cyst growth of human ADPKD cells stimulated by vasopressin

In autosomal dominant polycystic kidney disease (ADPKD), arginine vasopressin (AVP) accelerates cyst growth by stimulating cAMP-dependent ERK activity and epithelial cell proliferation and by promoting Cl(-)-dependent fluid secretion. Tolvaptan, a V2 receptor antagonist, inhibits the renal effects of AVP and slows cyst growth in PKD animals. Here, we determined the effect of graded concentrations of tolvaptan on intracellular cAMP, ERK activity, cell proliferation, and transcellular Cl(-) secretion using human ADPKD cyst epithelial cells. Incubation of ADPKD cells with 10(-9) M AVP increased intracellular cAMP and stimulated ERK and cell proliferation. Tolvaptan caused a concentration-dependent inhibition of AVP-induced cAMP production with an apparent IC(50) of ～10(-10) M. Correspondingly, tolvaptan inhibited AVP-induced ERK signaling and cell proliferation. Basolateral application of AVP to ADPKD cell monolayers grown on permeable supports caused a sustained increase in short-circuit current that was completely blocked by the Cl(-) channel blocker CFTR(inh-172), consistent with AVP-induced transepithelial Cl(-) secretion. Tolvaptan inhibited AVP-induced Cl(-) secretion and decreased in vitro cyst growth of ADPKD cells cultured within a three-dimensional collagen matrix. These data demonstrate that relatively low concentrations of tolvaptan inhibit AVP-stimulated cell proliferation and Cl(-)-dependent fluid secretion by human ADPKD cystic cells.     

PGE2 enhances cytokine-elicited nitric oxide production in mouse cortical collecting duct cells.

It has been documented that arginine vasopressin (AVP) and prostaglandin E(2) (PGE(2)) regulate water reabsorption in renal tubular cells. The present study was attempted to delineate the downstream signaling of AVP and PGE(2) in a cortical collecting duct cell line (M-1 cell). Using RT-PCR, we detected mRNA for V2 and VACM-1 but not for V1a and AII/AVP receptors of AVP. Furthermore, neither AVP nor V2 receptor agonist and antagonist alter cellular cAMP. These together with unchanged cellular Ca(2+) by AVP suggested that AVP pathway was not operating in M-1 cells. All four classical PGE(2) receptors with EP3 and EP4 as the most prominent were detected in M-1 cells. PGE(2), 11-deoxy-PGE(1) (EP2 and EP4 agonist), and 17-phenyl-trinor-PGE(2) (EP1 agonist) increased cellular concentration of cAMP. There was no effect of PGE(2) or EP1 agonist on cellular Ca(2+). These findings provide evidence of the involvement of PGE(2) cascade in M-1 cells. M-1 cells were capable of synthesizing nitric oxide (NO). Although individual cytokines did not affect NO production, a mixture of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma elevated NO concentration to 4.5-fold of the control. Addition of PGE(2) and db-cAMP to the cytokine mixture further increased NO production to 7.0- and 9.8-fold, respectively, of that seen in non-treated cells. PGE(2) or db-cAMP alone, however, had no effect on NO production. The results of the study led us to speculate that enhanced production of cAMP via PGE(2) signaling pathway in M-1 cells could either stimulate or attenuate water reabsorption in renal tubule. While an increase in cAMP alone may enhance water reabsorption, a concomitant increase in cAMP and cytokines may inhibit water reabsorption in renal tubule.     

Evidence for 6-keto-PGF1 alpha in adrenal cortex of the rat and effects of 6-keto-PGF1 alpha and PGI2 on adrenal cAMP levels and steroidogenesis.

Both intact cortical tissue and isolated cortical cells from the adrenal gland of the rat were analyzed for 6-keto-PGF1 alpha, the hydrolysis metabolite of PGI2, using high-performance liquid chromatography and gas chromatography-mass spectrometry. 6-Keto-PGF1 alpha was present in both incubations of intact tissue and isolated cells of the adrenal cortex, at higher concentrations than either PGF2 alpha or PGE2. Thus, the cortex does not depend upon vascular components for the synthesis of the PGI2 metabolite. Studies in vitro, using isolated cortical cells exposed to 6-keto-PGF1 alpha (10(-6)-10(-4)M), show that this PG does not alter cAMP levels or steroidogenesis. Cells exposed to PGI2 (10(-6)-10(-4)M), however, show a concentration-dependent increase of up to 4-fold in the levels of cAMP without altering cortico-sterone production, ACTH (5-200 microU/ml) increased cAMP levels up to 14-fold, and corticosterone levels up to 6-fold, in isolated cells. ACTH plus PGI2 produced an additive increase in levels of cAMP, however, the steroidogenic response was equal to that elicited by ACTH alone. Adrenal glands of the rat perfused in situ with PGI2 showed a small decrease in corticosterone production, whereas ACTH greatly stimulated steroid release. Thus, while 6-keto-PGF1 alpha is present in the rat adrenal cortex, its precursor, PGI2, is not a steroidogenic agent in this tissue although it does stimulate the accumulation of cAMP.     

Prostaglandins: specific inhibition of platelet adhesion to collagen and relationship with cAMP level

The effect of 3 prostaglandins (PG's) (I2, D2 and E1) on the adhesion of platelets to purified type III collagen has been investigated. A quantitative method for a specific evaluation of the adhesion has been applied and has revealed an inhibition of adhesion by low concentrations (10(-10)M) of PGs added before collagen; the effect varied as a function of the dose of PGs (maximum at 10(-6)M) which also induced an increase in the level of platelet cAMP. The inhibition of adhesion and the elevation of platelet cAMP followed the same time course and were either of short duration (rapid decrease in the induced effects after 15 and 45 seconds in the case of PGE1) or longer lasting (maximum effect maintained for 5 minutes in the case of PGI2 and D2). These effects were potentiated by a phosphodiesterase inhibitor such as theophylline (10(-3)M). The addition of PGs after collagen resulted in a reduction of the enhancement of cAMP, associated with a decrease in the inhibition of adhesion. Moreover, the addition of exogenous cAMP (dibutyryl N6-02' cAMP) induced a comparable inhibition. A correlation between the adhesion of platelets to collagen and the level of either endogenous or exogenous cAMP has been established. The PGs also inhibited the platelet release reaction from the alpha granules (beta TG) and the dense bodies. (5-HT and ADP). A greater inhibition of release than of adhesion was observed for the same doses of PGs added.     

Effects of prostaglandins and arachidonic acid on baboon cerebral and mesenteric arteries

Effects of prostaglandins (PGs) E1, E2, F2 alpha and I2 in a wide range of concentration were examined in mesenteric and cerebral arteries isolated from mature baboons. PGs E1, E2 and F2 alpha at low concentrations (10(-10) to 10(-7) M) elicited relaxation in helically cut strips of cerebral arteries precontracted with phenylephrine. In contrast, the PGs did not cause relaxation in the mesenteric artery. PGI2 (10(-9) to 10(-6) M) produced marked relaxation in both arteries. The EC25 for PGI2 in the mesenteric artery was significantly lower than that in the cerebral artery. During baseline conditions, cerebral arteries contracted in response to high concentrations (greater than 10(-7) M) of PGs E1, E2 and F2 alpha. In mesenteric arteries, a large contraction was induced by PGs F2 alpha and E2 but not by PGE1. Arachidonic acid (10(-6) M) produced an aspirin-inhibitable relaxation in both arteries to a similar extent, so that the vasodilator PG(s) formed in the two different arterial walls appear to exert a similar relaxant action. Thus, the baboon mesenteric artery was more sensitive to PGI2 for the relaxant effect than was the cerebral artery, while PGs F2 alpha, E1 and E2 caused only a contraction in the mesenteric artery but both relaxation and contraction in the cerebral artery.     

Inductive effects of prostaglandins on alkaline phosphatase in osteoblastic cells, clone MC3T3-E1

The effects of prostaglandins (PGs) on the induction of alkaline phosphatase (ALP) were investigated in osteoblastic clone MC3T3-E1 cells cultured in serum-free medium. Prostaglandin E2 (PGE2) stimulated ALP activity in the cells in a dose-dependent fashion with a maximal effect which was about twice that in the control cells at concentrations of 100-500 ng/ml. Actinomycin D and cycloheximide inhibited the stimulative effect of PGE2 on ALP activity in the cells. PGE2-induced and native ALPs in the cells were of the same type as that in adult mouse calvaria, being heat-labile, L-homoarginine- and levamisole-sensitive, and L-phenylalanine-insensitive. Isobutyl methylxanthine (IBMX), a cAMP phosphodiesterase inhibitor, stimulated the inductive effect of PGE2 on ALP activity at 0.1 mM, at which concentration IBMX alone had little effect on the activity. PGE2 also increased the intracellular cAMP content in a dose-dependent fashion with a maximal effect at 100 ng/ml. PGE1, PGF1 alpha, and PGF2 alpha (primary PGs like PGE2) increased the activity. Our present results suggest that PGs stimulate the differentiation of osteoblasts and are involved in bone formation in vivo, as well as in bone resorption.     

Blood pressure and cardiac tissue responses to prostacyclin (PGI2) in various species

The effect of prostacyclin (PGI2) on blood pressure and heart rate (in vivo) and on isolated heart tissue has been investigated in different species. Isolated cardiac tissue had limited responses to PGI2 tested at 10(-13) to 10(-5) M. Cultured neonatal rat heart cells did not respond to PGI2, neither did intact rat hearts or rabbit cardiac tissue. Guinea pig and rat atria showed limited dose-dependent responses to PGI2 at concentrations greater than 10(7) M. In rat atria, 10(-5) M PGI2 produced a limited elevation of tissue cAMP content. When given by intravenous injection or infusion, PGI2 produced hypotension in anaesthetized primates (three species), rat, rabbit, pig, and dog. As a vasodepressor in all species, PGI2 (on a weight basis) was more active than prostaglandins of the B or E type and, in most species tested, it was approximately five times more active than PGE2. Heart responses in intact animals were often paradoxical in that decreases in heart rate often accompanied blood pressure falls.     

Pertussis toxin abolishes the inhibitory effects of prostaglandins E1, E2, I2 and F2 alpha on hormone-induced cAMP accumulation in cultured hepatocytes

Several prostaglandins inhibit the cAMP response to glucagon and beta-adrenergic stimulation in hepatocytes. To probe the mechanism of this inhibition, we have examined in primary hepatocyte cultures how pretreatment with pertussis toxin (islet-activating protein) influences the ability of the cells to respond to hormones and prostaglandins. Pertussis toxin augmented the effects of glucagon, epinephrine and isoproterenol, and also markedly enhanced the cAMP response to prostaglandin E1 (PGE1). Furthermore, whereas PGE1, PGE2, PGI2 and PGF2 alpha attenuated the cAMP responses to glucagon in control cultures, this inhibition was abolished in cells pretreated with pertussis toxin. A more detailed comparison was made of the effects of PGE1 and PGF2 alpha. In cells not treated with pertussis toxin, both these prostaglandins at high concentrations reduced the cAMP response to glucagon and isoproterenol by approximately 50%, but dose-effect curves showed that PGE1 was about 100-fold more potent as an inhibitor than PGF2 alpha. Pertussis toxin abolished the inhibitory effects of PGE1 and PGF2 alpha with almost identical time and dose requirements. The results obtained with PGE1, PGE2, PGI2 and PGF2 alpha suggest that prostaglandins of different series attenuate hormone-activable adenylate cyclase in hepatocytes through a common mechanism, dependent on the inhibitory GTP-binding protein.     

Differential effects of prostaglandin F2 alpha and of prostaglandins E1 and E2 on cyclic 3',5'-monophosphate production and intracellular calcium mobilization in avian uterine smooth muscle cells

The effects of prostaglandins (PGs) E1 (PGE1), E2 (PGE2) and F2 alpha (PGF2 alpha) on cyclic 3',5'-adenosine monophosphate (cAMP) production and intracellular Ca mobilization were examined in smooth muscle cells of chicken uterus grown in primary culture. At subnanomolar concentrations, both PGE1 and PGE2 significantly suppressed cAMP levels. However, at higher concentrations (0.1-100 microM), both agonists caused a dose-related increase in cAMP production. PGF2 alpha, on the other hand, had no effect on cAMP production. Forskolin (1-100 microM), which also stimulated cAMP production in a dose-dependent fashion, potentiated the effects of both PGE1 and PGE2. In digitonin-permeabilized uterine cells preloaded with 45Ca2+, the addition of PGF2 alpha caused a biphasic 45Ca2+ efflux. There was a small but significant 45Ca2+ release (10.0 +/- 1.5%) within 30 s (rapid phase), followed by a larger one (32.0 +/- 2.0%) within 5 min (slow phase). PGE2, at doses above 1 nM (which significantly increased cAMP accumulation), promoted 45Ca2+ sequestration. This action of PGE2 was observed as early as 1 min and was complete by 5 min. In addition, 0.001 nM PGE2 (a dose that was ineffective on 45Ca2+ mobilization) enhanced PGF2 alpha-induced 45Ca2+ mobilization from 22.5 +/- 5% to 57.0 +/- 3.5%. These results show that PGs of the E series have distinctly different effects on cAMP production and intracellular Ca mobilization. PGF2 alpha action may be linked directly to intracellular Ca mobilization, whereas the effects of PGE may be exerted at multiple sites depending on its local concentration. At low concentrations, its action may be mediated by the suppression of cAMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)