Prostaglandin receptors in rat kidney

The physiological effects of prostaglandins (PGs) are mediated through their interactions with specific binding sites (receptors) on effector cells. Since such receptors potentially regulate the action of PGs on the kidney, the distribution and properties of renal PG receptors in the rat were examined. The distribution of PGE2, PGE1, and PGF2 alpha receptors along the nephron was not uniform; the outer medulla had by far the greatest density of sites, followed by the inner medulla and cortex. Receptors were found exclusively in the particulate fractions, of which the 40,000g pellet had the highest specific activity. In the outer medulla, receptor density calculated from Scatchard plots was 2.12 pmol/mg for PGE2, 1.12 for PGE1, and 0.44 for PGF2 alpha; the KD's were similar for all prostaglandins. The conditions for optimal in vitro binding of PGE2 and PGF2 alpha by outer medullary membranes were investigated. In vivo administration of 16,16'-dimethyl-PGE2 resulted in a dose-dependent "down" regulation of PGE2 binding to outer medullary membranes due to changes in both the number and affinities of receptors. Changes in the numbers and/or properties of PG receptors may be an important mechanism for regulating the effects of PGs and renal function under normal and pathologic conditions.     

E and F alpha series prostaglandins in developing muscles

Prostaglandins are known to affect myoblast proliferation and fusion in vitro and are putative regulators of in vivo myogenesis. The levels of E and F alpha series prostaglandins in the thigh muscles of chicken embryos were measured by radioimmunoassays and correlated with indicators of muscle development. Just prior to the onset of secondary myogenesis, the amounts of PGE1, PGE2 and PGF1 alpha plus PGF2 alpha per mg of protein were high. In temporal association with myotube formation, the amount of PGE1 and PGE2 per mg of protein decreased. PGF alpha levels also fell, but at a slower rate than observed with the E series prostaglandins. The decreases in the amounts of prostaglandins per mg protein appeared to be due to a decline in the total amount of prostaglandin within each muscle. These observations are consistent with prostaglandins being one of the factors that controls in vivo muscle formation.     

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 role of renal metabolism of PGE2 in determining its activity as a renal vasodilator in the dog.

Since the mammalian renal cortex avidly metabolizes prostaglandin E2 (PGE2), we examined the importance of renal metabolism of PGE2 in determining its renal vascular activity in the dog. We used 13, 14 dihydro PGE2 (DHPGE2) as a model compound to study this because DHPGE2 retains similar activity to the parent prostaglandin, PGE2, but is a poorer substrate than PGE2 for both the metabolism and the cellular uptake of the prostaglandins. Using dog renal cortical slices, we found that under similar experimental conditions, PGE2 was metabolized several-fold faster than DHPGE2. Both prostaglandins were metabolized to the 15 keto 13, 14 dihydro PGE2, which was positively identified using GC-MS. In vivo, we infused increasing concentrations of DHPGE2 into the renal artery of dogs and measured renal hemodynamic changes using radioactive microspheres. DHPGE2 was a potent renal vasodilator beginning at an infusion rate of 10(-9)g/kg/min. When compared to PGE2, DHPGE2 was about 10 times more potent in affecting renal vasodilation. The intrarenal redistribution of blood flow towards the inner cortex seen with DHPGE2 was identical to that seen with PGE2. We conclude that renal catabolism of PGE2 is very important in limiting the in vivo biological activity of PGE2, but regional differences in metabolism of PGE2 within the cortex are an unlikely determinant of the pattern of redistribution of renal blood flow.     

Urinary excretion of prostaglandin E2 and prostaglandin F2alpha in potassium-deficient rats

Potassium-deficiency was induced in rats by dietary deprivation of potassium. The animals became polyuric and urine osmolality decreased more then three-fold compared to controls. Urinary excretion of prostaglandin E2 (PGE2) and prostaglandin F2alpha (PGF2alpha) did not increase during 2 weeks of potassium depletion. Partial inhibition of renal prostaglandin synthesis by meclofenamate did not increase the urine osmolality after water deprivation. These results make unlikely the hypothesis that the polyuria of potassium-deficiency, is the result of enhanced renal synthesis of prostaglandins with subsequent antagonism of the hydro-osmotic effect of vasopressin. Male animals consistently excreted less PGE2 than female animals.     

PPADS does not block contraction-induced prostaglandin E2 synthesis in cat skeletal muscle

Pyridoxal-phosphate-6-azophenyl-2'-4-disulfonate (PPADS), a purinergic 2 (P2) receptor antagonist, has been shown to attenuate the exercise pressor reflex in cats. In vitro, however, PPADS has been shown to block the production of prostaglandins, some of which play a role in evoking the exercise pressor reflex. Thus the possibility exists that PPADS blocks the exercise pressor reflex through a reduction in prostaglandin synthesis rather than through the blockade of P2 receptors. Using microdialysis, we collected interstitial fluid from skeletal muscle to determine prostaglandin E2 (PGE2) concentrations during the intermittent contraction of the triceps surae muscle before and after a popliteal arterial injection of PPADS (10 mg/kg). We found that the PGE2 concentration increased in response to the intermittent contraction before and after the injection of PPADS (both, P < 0.05). PPADS reduced the pressor response to exercise (P < 0.05) but had no effect on the magnitude of PGE2 production during contraction (P = 0.48). These experiments demonstrate that PPADS does not block the exercise pressor reflex through a reduction in PGE2 synthesis. We suggest that PGE2 and P2 receptors play independent roles in stimulating the exercise pressor reflex.     

Metabolism of prostaglandin E2 by mouse embryo palate mesenchyme cells in vitro

Mouse embryo palate mesenchyme cells synthesize a number of prostaglandins, particularly prostaglandin E2 (PGE2). However, the ability of such cells to metabolize prostaglandins was unknown. By use of radiolabeled PGE2 we determined that palate mesenchyme cells have little ability to degrade that prostaglandin in vitro but are able to metabolize products formed from its spontaneous degradation.     

Dissociation of hyperreninemia and renal prostaglandin synthesis in the adrenalectomized rat

The aim of this study was to determine whether hyperreninemia in the adrenalectomized (ADX) rat is dependent on renal prostaglandin synthesis, as has been suggested for two other hyperreninemic conditions, Bartter's syndrome and chronic liver disease. Plasma renin concentration (PRC) in anesthetized, ADX rats was significantly increased (delta +480%; p less than 0.001) compared to sham-operated controls. In vivo, indomethacin (10 mg/kg i.v.) significantly reduced PRC of anesthetized, ADX rats after both 45 min (delta -34%; p less than 0.05) and 90 min (delta -47%; p less than 0.05). In vitro renin release from renal cortical slices of ADX rats was also significantly greater (delta +130%; p less than 0.05) than from sham-operated control cortical slices. Renin release from cortical slices of ADX rats given dexamethasone (10 micrograms/kg/day) for 4 days prior to sacrifice did not differ from sham-operated control values. Prostaglandin E2 (PGE2) release from cortical slices of ADX rats did not differ significantly from controls. However, PGE2 synthesis in glomeruli microdissected from ADX rats was significantly increased (delta +110%; p less than 0.001) compared to controls. PGE2 synthesis in glomeruli of dexamethasone-treated ADX rats remained significantly elevated compared to controls. Ibuprofen (10(-6) M) decreased PGE2 synthesis in cortical slices by 80%. However, prostaglandin synthesis inhibition had no effect on renin release from either ADX or control renal cortical slices. These results suggest that despite increased glomerular synthesis, prostaglandins do not directly influence renin release in the ADX rat.     

Disruption of cyclooxygenase-1 gene results in an impaired response to radiation injury

Prostaglandins may play an important role in regulating normal renewal of gastrointestinal epithelium, epithelial injury repair, and initiation or progression of intestinal neoplasia. Synthesis of prostaglandins is catalyzed by either of two cyclooxygenase isoforms, Cox-1 and Cox-2. Cox-1 is the predominant cyclooxygenase isoform found in the normal intestine. In contrast, Cox-2 is present at low levels in normal intestine but is elevated at sites of inflammation and in adenomas and carcinomas. To determine directly whether prostaglandins synthesized by Cox-1 or Cox-2 regulate crypt epithelial cell fate after genotoxic or cytotoxic injury, we examined apoptosis, prostaglandin synthesis, and crypt stem cell survival after gamma-irradiation in Cox-1(-/-) and Cox-2(-/-) mice. Cox-1(-/-) mice had increased crypt epithelial cell apoptosis and decreased clonogenic stem cell survival compared with wild-type littermates. PGE(2) synthesis was also diminished in Cox-1(-/-) mice compared with wild-type controls in unstressed intestine and after radiation injury. In contrast, apoptosis, stem cell survival, and intestinal PGE(2) synthesis in Cox-2(-/-) mice after irradiation were the same as in wild-type littermates. Crypt stem cell survival after irradiation was inhibited by a highly specific neutralizing antibody to PGE(2), suggesting that this prostaglandin mediates stem cell fate in vivo. These data suggest that prostaglandins synthesized by Cox-1 regulate multiple steps that determine the fate of crypt epithelial cell after genotoxic or cytotoxic injury.     

Production of prostaglandins by porcine preovulatory follicular tissues and their roles in intrafollicular function

Prostaglandin production in vitro by theca and granulosa cells isolated from prepubertal pig ovaries was quantified in order to investigate the role of prostaglandins in intrafollicular function. Prepubertal gilts were slaughtered without treatment (O h, control) or treated with 1000 IU pregnant mare's serum gonadotropin (PMSG) and slaughtered at 36 or 72 h, or at 75 h following treatment with 500 IU of hCG at 72 h. Theca and granulosa cells were isolated from preovulatory follicles and cultured for 24 h alone or with follicle-stimulating hormone (FSH) or luteinizing hormone (LH). In vitro accumulation of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) was measured by radioimmunoassay. On a per follicle basis theca produced more of each prostaglandin (approx. 10-fold) than granulosa at each stage of follicular development; production by each tissue type increased with development of the follicle, responding to administration of gonadotropin (PMSG) in vivo. Neither tissue type was generally responsive to further gonadotropin stimulation in vitro. However, production of PGE2 by granulosa cells was increased by addition of gonadotropin, particularly LH, in vitro, with the greatest response observed in tissue obtained at 36 and 72 h after PMSG. There were no functional correlates between prostaglandin production and steroidogenesis by either tissue type and we conclude that prostaglandins do not have an obligatory role in follicular steroidogenesis. However, these data provide additional circumstantial evidence for a role of PGE2 in granulosa cell luteinization, and possibly in ovulation. The data also indicate that prostaglandins derived from thecal tissue in relatively large quantities may play an important role in ovulation.     

The unfolding and attempted refolding of mitochondrial aspartate aminotransferase from pig heart.

The role of prostaglandins in the regulation of muscle protein breakdown is controversial. We examined the influence of arachidonic acid (5 microM), prostaglandin E2 (PGE2) (2.8 microM) and the prostaglandin-synthesis inhibitor indomethacin (3 microM) on total and myofibrillar protein breakdown in rat extensor digitorum longus and soleus muscles incubated under different conditions in vitro. In other experiments, the effects of indomethacin, administered in vivo to septic rats (3 mg/kg, injected subcutaneously twice after induction of sepsis by caecal ligation and puncture) on plasma levels and muscle release of PGE2 and on total and myofibrillar protein breakdown rates were determined. Total and myofibrillar proteolysis was assessed by measuring production by incubated muscles of tyrosine and 3-methylhistidine respectively. Arachidonic acid or PGE2 added during incubation of muscles from normal rats did not affect total or myofibrillar protein degradation under a variety of different conditions in vitro. Indomethacin inhibited muscle PGE2 production by incubated muscles from septic rats, but did not lower proteolytic rates. Administration in vivo of indomethacin did not affect total or myofibrillar muscle protein breakdown, despite effective plasma levels of indomethacin with decreased plasma PGE2 levels and inhibition of muscle PGE2 release. The present results suggest that protein breakdown in skeletal muscle of normal or septic rats is not regulated by PGE2 or other prostaglandins.     

Competition for adenyl cyclase coupled (3H)-prostacyclin binding sites with prostaglandin E2 in rat peritoneal macrophages

Prostaglandins regulate macrophage function by their action on membrane-associated adenyl cyclase. In order to define more directly macrophage-prostaglandin interactions, a binding assay has been developed for macrophage receptors using (3H)-PGI2 as ligand. (3H)-PGI2 binding was specific, saturable and reversible. Moreover, specific binding showed to be enriched in a membrane-enriched fraction of the cells. The assay conditions ensured stability of (3H)-PGI2 during incubations and should exclude intracellular accumulation of the ligand in macrophages. Unlabelled PGE2 and PGI2 competed for (3H)-PGI2 specific binding in both macrophages and membrane preparations. PGE2 showed to be more potent in this respect than PGI2, a phenomena which was also observed for prostaglandin activation of cAMP production in macrophages. The data suggest an interaction at receptor level of endogenously released PGE2 and PGI2 by peritoneal macrophages in vivo and provide support for a previously proposed mechanism of action of low concentrations of PGE2, counteracting stimulation of cAMP production by PGI2 in macrophages.     

Cyclic AMP does not inhibit A23187-induced prostaglandin biosynthesis in cultured rabbit renal microvascular endothelial cells.

We have previously shown that cultured rabbit renal preglomerular microvascular endothelial cells have the ability to synthesize a number of common prostaglandins. In the present study we have examined whether endogenous cyclic AMP is involved in the regulation of PGI2 and PGE2 biosynthesis in these cultured cells. Isoproterenol and forskolin produced an increase in cyclic AMP accumulation in these cells but had no effect on PGI2 or PGE2 biosynthesis either in the presence or absence of A23187. Similar results were noted in the presence of 3-isobutyl-1-methylxanthine, a cyclic AMP-phosphodiesterase inhibitor. These studies suggested that endogenous cyclic AMP does not regulate the biosynthesis of PGI2 or PGE2 in cultured renal preglomerular microvascular endothelial cells either under basal or A23187-stimulated condition. They further suggested that the effect of 3-isobutyl-1-methylxanthine on prostaglandin biosynthesis in these cultured cells was not secondary to its effects on phosphodiesterase.     

Hypoxia enhances prostaglandin synthesis in renal mesangial cell cultures

In view of recent findings which suggest that renal prostaglandins mediate the effect of hypoxia on erythropoietin production, we have studied whether hypoxia is a stimulus for in vitro prostaglandin synthesis. Studies were carried out in rat renal mesangial cell cultures which produce erythropoietin in an oxygen-dependent manner. Production rates of PGE2 and in specified samples also of 6-keto-PGF1 alpha, as a measure of PGI2, and PGF2 alpha were determined by radioimmunoassay after incubation at either 20% O2 (normoxic) or 2% O2 (hypoxic) in gas permeable dishes for 24 hrs. Considerable variation in PGE2 production was noted among independent cell lines. PGE2 production appeared to be inversely correlated to the cellular density of the cultures. In addition, PGE2 production was enhanced in hypoxic cell cultures. The mean increase was 50 to 60%. PGF2 alpha and 6-keto-PGF1 alpha increased by about the same rate. These results indicate that hypoxia is a stimulus for in vitro prostaglandin production.     

Acute effects of prostaglandin E(1) and E(2) on vascular reactivity and blood flow in situ in the chick chorioallantoic membrane

The chick chorioallantoic membrane (CAM) subserves gas exchange in the developing embryo and shell-less culture affords a unique opportunity for direct observations over time of individual blood vessels to pharmacologic interventions. We tested a number of lipids including prostaglandins PGE(1&2) for vascular effects and signaling in the CAM. Application of PGE(1&2) induced a decrease in the diameter of large blood vessels and a concentration-dependent, localized, reversible loss of blood flow through small vessels. The loss of flow was also mimicked by misoprostol, an agonist for 3 of 4 known PGE receptors, EP(2-4), and by U46619, a thromboxane mimetic. Selective receptor antagonists for EP(3) and thromboxane each partially blocked the response. This is a first report of the effects of prostaglandins on vasoreactivity in the CAM. Our model allows the unique ability to examine simultaneous responses of large and small vessels in real time and in vivo.     

Some mechanisms involved in the prostaglandin E2 immunosuppressive effect in (CBA x C57BL)F1 mice in vivo

The cellular mechanisms involved in the immunosuppressive effect of prostaglandin E2 (PGE2) multiple injections into (CBA x C57Bl)F1 mice in vivo have been studied. PGE2 injection increases the induction of specific T-suppressors. In addition, there is a decrease in macrophage phagocytic activity and in the phagocytosis index, apparently mediated by Fc gamma receptors (Fc gamma R) and not by the macrophage complement receptor (C3R). The induction of antibody synthesis by using "immune" macrophages injected into a syngeneic recipient results in considerable decrease in the accumulation of antibody-forming cells if the macrophage donor has been pretreated with exogenous PGE2 in comparison with untreated controls. These cellular mechanisms are possibly one part of the diverse way in which PGE2 exerts an immunosuppressive effect in vivo and contributes to humoral immune response suppression.     

The interactions between indomethacin and cytotoxic drugs in mice bearing B-16 melanomas

We have compared the effects of indomethacin alone (100 microgram/mouse/day) with those of indomethacin plus adriamycin, 5-FU, nitrogen mustard, thioTEPA, and vincristine on B-16 tumor cell proliferation in vivo. As we have previously described, after four days of treatment with indomethacin, subcutaneous tumors were slightly smaller and lighter in weight, but contained more melanoma cells. Addition of indomethacin to cytotoxic regimens resulted in either no change or a decrease in the effectiveness of the chemotherapy. In previous studies we demonstrated that treatment of tumor-bearing mice with a long-acting synthetic analogue of PGE2 (di-M-PGE2) stimulated the synthesis of endogenous prostaglandins. In order to evaluate if these endogenously synthesized prostaglandins were responsible for the inhibition of B-16 growth in vivo, mice were treated with di-M-PGE2 or di-M-PGE2 plus indomethacin. Addition of indomethacin did not alter the tumor inhibitory effects of di-M-PGE2.     

Arginine vasotocin-induced prostaglandin synthesis in vitro by the reproductive tract of the viviparous lizard Sceloporus jarrovi

Two experiments were performed to determine whether arginine vasotocin (AVT) stimulates synthesis of prostaglandins (PGs) in reptilian oviducts. Homogenized oviducal tissue from female Sceloporus jarrovi in early and late pregnancy were cultured with radiolabeled (14C) prostaglandin precursor, arachidonic acid (AA). In late pregnancy, oviducts exposed to AVT exhibited a greater conversion of AA to PGF2 alpha than did controls, whereas in early pregnancy there was no difference. The conversion of AA to other prostaglandins (PGA2, PGD2, PGE2, PGI2) was not influenced by AVT. The second experiment examined whether endogenous in vitro synthesis of PGF and PGE2 from intact, pregnant oviducts was stimulated by AVT (50 ng/ml; 100 ng/ml). Both doses of AVT induced a similar, significant rise in PGF concentrations within 30 min whereas no significant increase was noted in PGE2 concentrations until 90 min after treatment. Indomethacin pretreatment blocked synthesis of both PGF and PGE2 for 30 min following AVT treatment. These data indicate that AVT induces a highly specific rise in the synthesis of PGF from the oviduct of female S. jarrovi in late pregnancy. Furthermore, the prostaglandin-stimulating effect of AVT in reptiles appears homologous with the effect of oxytocin in mammals and AVT in birds. We hypothesize that this interaction is an evolutionarily conserved relationship found in all amniote vertebrates.     

Prostaglandins can modify gamma-radiation and chemical induced cytotoxicity and genetic damage in vitro and in vivo

The effect of prostaglandin E1, E2, and F2 alpha on gamma-radiation, benzo(a)pyrene and diphenylhydantoin-induced cytotoxicity in vivo and genotoxicity in vitro was investigated. Prostaglandin E1 prevented both cytotoxic and genotoxic actions of all the three agents, where as both PGE2 and PGF2 alpha were ineffective. In fact, it was seen that both PGE2 and PGF2 alpha are genotoxic by themselves. Gamma-linolenic acid and dihomogamma-linolenic acid, the precursor of PGE1 were also as protective as that of PGE1, where as arachidonic acid, the precursor of 2 series PGs, has genotoxic actions to human lymphocytes in vitro. These results suggest that prostaglandins and their precursors can determine the susceptibility of cells to cytotoxic and genotoxic actions of chemicals and radiation. This study is particularly interesting since, it is known that some tumor cells contain excess of PGE2 and PGF2 alpha and many carcinogens can augment the synthesis of 2 series of PGs.