Effect of forskolin on platelet deaggregation and cyclic AMP generation

Summary The diterpene, forskolin, induced a partial deaggregation of ADP-or collagen-aggregated human platelets in vitro. An increase in platelet cyclic AMP by forskolin was assumed to mediate the platelet deaggregation. PGE₁ also deaggregated these platelets, and a combination of forskolin and PGE₁ produced deaggregation greater than the maximum which could be obtained with each agent alone. A greater than additive effect was observed on the platelet cyclic AMP level in the presence of both forskolin and PGE₁. No additive effect was observed in the phosphorylation of molecular weight (Mr) 21K polypeptide using forskolin (0.1 mmol/l) and PGE₁ (5 mol/l) suggesting that although cyclic AMP is responsible for the deaggregation process a mechanism other than phosphorylation through cyclic AMP-dependent protein kinase may be responsible for the effect of forskolin on platelet deaggregation.     

Increased cyclic AMP response to forskolin in Epstein-Barr virus-transformed human B-lymphocytes derived from schizophrenics

Phorbol 12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator, elevated basal cyclic AMP levels and enhanced isoproterenol-, prostaglandin E₁- (PGE₁), forskolin- and cholera toxin-stimulated cyclic AMP accumulation in Epstein-Barr virus (EBV)-transformed human B-lymphocytes. Staurosporine, a PKC inhibitor, significantly antagonized the increase in cyclic AMP accumulation produced by PMA, whereas the inactive phorbol ester, 4α-phorbol 12,13-didecanoate (4αPDD), had no effect. Basal levels of cyclic AMP and the accumulation of cyclic AMP produced by PMA, isoproterenol, PGE₁, cholera toxin and the combination of these compounds with PMA were not significantly different between schizophrenics and controls. The cyclic AMP response to forskolin in the presence and absence of PMA was significantly greater in EBV-transformed human B-lymphocytes from schizophrenics. These results suggest that activation of adenylyl cyclase by forskolin is elevated in EBV-transformed B-lymphocytes derived from schizophrenics and that this elevation is further enhanced through a PKC-dependent phosphorylation mechanism. Received: 20 May 1996/Final version: 6 November 1996     

Effect of isoproterenol on the isolated pregnant rat myometrium

In pregnant rat myometrium, isoproterenol (10(-8) M) inhibited spontaneous contractions without causing hyperpolarization. Isoproterenol (10(-6) M) relaxed the depolarized muscle without affecting the membrane potential. The presence of 80 mM Na+ did not affect the degree of high-K+ depolarization. It was also without any influence on the effects of isoproterenol on the depolarized uterus. The results are consistent with the concept that hyperpolarization is not a prerequisite for beta-adrenoceptor induced relaxation of uterine smooth muscle.     

Effects of 8-S-benzyl cyclic AMP on mechanical characteristics and cyclic AMP levels of the canine ventricular myocardium

Effects of 8-S-benzyl cyclic AMP on the mechanical characteristics of cardiac contradiction and on teh intracellular cyclic AMP level were studied on the isolated canine right ventricular myocardium. For comparison, inotropic effects of dibutyryl and 8-bromo cyclic AMP were also investigated. 8-S-Benzyl cyclic AMP caused a concentration-dependent positive intropic action. The action of 8-S-benzyl cyclic AMP developed and reached a steady level much faster thant hat of dibutyryl cyclic AMP (dbcAMP), while the time courses of the disappearance of the actions following washout of the compounds did not differ. The dose-response curve for 8-S-benzyl cyclic AMP lay substantially in the same concentration range as did that for dbcAMP. The duration of a single contraction was decreased by 8-S-benzyl cyclic AMP in a concentration-dependent manner, chiefly because of shortening of the relaxation time. The cyclic AMP phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX) enhanced the positive inotropic action of 8-S-benzyl cyclic AMP; 8-bromo cyclic AMP caused a definite positive inotropic action only in the presence of IBMX. The intracellular cyclic AMP levels determined 5, 15 and 30 min after the administration of 1 mM 8-S-benzyl cyclic AMP were not significantly different from the corresponding control values determined in the paired muscle. The present results indicte that the changes in mechanical characteristics caused by 8-S-benzyl cyclic AMP were very similar to those induced by β-aderenoceptor stimulation. Since the intracellular cyclic AMP level was not changed after administration of 8-S-benzyl cyclic AMP, this compound is probably not metabolized intracellularly to cyclic AMP but acts directly as the original compound on the protein kinase. This compound provides an excellent pharmacological tool to mimic the effect of β-adrenoceptor stimulation on myocardial contractility.     

Negative functional effects of cyclic GMP are altered by cyclic AMP phosphodiesterases in rabbit cardiac myocytes

In this study, we tested the hypothesis that the negative functional effects of cyclic GMP on cardiac myocytes would be affected by the actions of cyclic GMP on cyclic AMP phosphodiesterases. Ventricular myocytes from eight rabbits were used to determine the functional and cyclic AMP changes caused by 10(-7), 10(-6), 10(-5) M 8-Bromo-cGMP alone and after the administration of 10(-6) M milrinone (cyclic GMP-inhibited cyclic AMP phosphodiesterase inhibitor) or 10(-6) M erythro-9-(2-Hydroxy-3-3-nonyl)adenine (EHNA, cyclic GMP-stimulated cyclic AMP phosphodiesterase inhibitor). 8-Br-cGMP dose-dependently reduced %shortening by 35+/-4% of baseline at 10(-5) M. This effect was significantly blunted by EHNA at all doses. The maximum rate of shortening was reduced by 31+/-3% by 10(-5) M 8-Br-cGMP. This effect of 8-Br-cGMP was significantly enhanced (42+/-4%) in the milrinone group. A similar pattern was observed in the maximum rate of relaxation data. Cyclic AMP levels were significantly increased from a baseline level of 4.0+/-0.8 pmol/10(5) myocytes by milrinone (+60%), EHNA (+61%) and 8-Br-cGMP (+47%). The combination of EHNA plus 8-Br-cGMP increased cyclic AMP levels significantly more that the combination of milrinone plus 8-Br-cGMP. Exogenous cyclic GMP reduces myocyte function, while raising cyclic AMP possibly through cyclic GMP-inhibited cyclic AMP phosphodiesterase effects. Blocking cyclic GMP-inhibited cyclic AMP phosphodiesterase enhances the functional effects cyclic GMP, while blocking cyclic GMP-stimulated cyclic AMP phosphodiesterase reduced these effects. The study demonstrated a functional interaction between cyclic GMP and cyclic AMP related to the cyclic GMP affected cyclic AMP phosphodiesterases.     

Relations between the effects of histamine,pheniramin and metiamide on spontaneous motility and the formation of cyclic AMP in the isolated rat uterus

Summary Histamine (5×10^–6 to 10^–3M) depressed the spontaneous motility of the isolated rat uterus in a dose-dependent manner. Under these conditions uterine cyclic AMP was raised up to 92%. Both effects, uterine relaxation and cyclic AMP accumulation after 2 min could be inhibited dose dependently by the H₂-antihistaminic compound metiamide (1.7×10^–6 M to 1.7×10^–4 M). By contrast, the H₁-antagonist pheniramin (4.4×10^–5 M) was ineffective. It was concluded that the histamine-induced inhibition of rat uterine motility is mediated by cyclic AMP which is formed in response to stimulation of H₂-histaminergic receptors.     

Effects of adenosine and related compounds on adenylate cyclase and cyclic AMP levels in smooth muscle.

The hypotheses were tested that the relaxant effect of adenosine and related compounds in the longitudinal muscle of the rabbit small intestine involves interaction with adenylate cyclase and/or the elevation of tissue cAMP levels. Adenylate cyclase was prepared by gentle homogenization of an isolated smooth muscle cell fraction obtained after collagenase digestion of longitudinal muscle strips. A number of analogs and derivatives of adenosine possessing a primary or secondary 6-amino group were found to inhibit the enzyme similarly to adenosine; however, there was no correlation between compounds known to relax the intact tissue and the existence, or the degree of, cyclase inhibition. Isolated muscle strips were exposed to adrenaline, isoprenaline, adenosine or ATP, at doses causing 30-60% relaxation, for 60 sec prior to sampling and analysis of cAMP content. While small increments in cAMP levels were found after administering adrenaline or isoprenaline, no change was found with adenosine in the absence or presence of theophylline of 1-methyl-3-isobutylxanthine. Neither adenylate cyclase inhibition nor changes in cAMP levels appear to be part of the mechanism of the smooth muscle relaxant action of adenosine or ATP.     

Fluoxetine affects cytosolic cAMP,ATP, Ca2+ responses to forskolin,and survival of human ovarian granulosa tumor COV434 cells

Fluoxetine (FLX), a selective serotonin reuptake inhibitor antidepressant, exhibits various other mechanisms of action in numerous cell types and has been shown to induce cell death in cancer cells, paving the way for its potential use in cancer therapy. The aim of this study was to determine the off-target effects of the anti-depressant drug FLX, on the human ovarian granulosa tumor COV434 cells stimulated by forskolin (FSK), by measuring the real-time kinetics of intracellular cyclic AMP (cAMP), ATP level, cytoplasmic calcium ([Ca²⁺]_cyt) and survival of COV434 cells. We show that incubating COV434 cells with FLX (between 0.6 and 10 µM) induces a decrease in intracellular cAMP response to FSK, a drop in ATP content and stimulates cytoplasmic Ca²⁺ accumulation in COV434 cells. Only the highest concentrations of FLX (5–10 µM) diminished cell viability. The present report is the first to identify an action mechanism of FLX in human tumor ovarian cells COV434 cells and thus opening the way to potential use of fluoxetine as a complementary tool, in granulosa tumor treatments.     

Effects of vasopressin on noradrenaline-induced cyclic AMP accumulation in rat brain slices

Addition of arginine vasopressin (AVP) or 1-desamino-8-D-arginine vasopressin (DDAVP) to rat cortical slices resulted in significant inhibition of the rise in cyclic AMP produced by incubation with 50 microM noradrenaline. A single injection of DDAVP (20 micrograms/rat) produced a reduced response to noradrenaline in derived cortex and caudate slices. In animals pretreated at day 5 of life with IP desipramine and intracisternal 6-hydroxydopamine (6-OHDA), both acute and chronic treatments with DDAVP resulted in a reduction in response in derived cortical, caudate and hippocampal slices. The 6-OHDA pretreated animals also showed reduced open-field behavioural activity after both acute and chronic DDAVP, while animals which were not pretreated responded to acute treatment only. The relationship between the effects of vasopressin on noradrenaline-induced cyclic AMP accumulation and its action on learning and memory is discussed.     

Response of rat small intestinal active aldohexose transport to elevation of mucosal cyclic AMP by forskolin and 3-isobutyl-1-methylxanthine in vitro

Summary The phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine (IBMX) was able to elevate rat small intestinal cyclic AMP levels to 300% of basal values. Active jejunal d-glucose transport was enhanced parallel to the rise of intracellular cyclic AMP levels to 140% of control values at 100 mol/l IBMX. Transport parameters, as determined in a three compartment model in vitro using a dual label method, indicate increased uphill glucose transport at the site of the brush border membrane, higher intracellular accumulation of the sugar, with unchanged passive permeabilities. Phlorizin-inhibited d-glucose transport and l-glucose transfer in the rat were not affected by the persisting cyclic AMP elevation produced by IBMX. Stimulating effects could also be demonstrated with d-galactose as a substrate. IBMX 100 mol/l also increased active d-glucose as well as 3-O-methylglucose transport in mouse jejunum.Stimulatory effects on intestinal hexose transport and mucosal cyclic AMP levels were also found with the adenylate-cyclase activator forskolin. In the present study, forskolin effects on jejunal mucosal cyclic AMP levels were enhanced in the presence of 100 mol/l IBMX, resulting in a 20-fold increase compared to controls at 20 mol/l forskolin. The concentration response for the effect of forskolin in the presence of 100 mol/l IBMX on d-glucose transport did not produce a significant increase compared to transport stimulation with IBMX alone. At higher concentrations of forskolin however, glucose transport decreased to levels well below the IBMX controls.The elevation of cellular cyclic AMP levels had no effects on passive permeability.Both IBMX 100 mol/l as well as forskolin 20 mol/l inhibited rat jejunal net fluid transport by 40%, combination of both agents resulted in a 55% reduction of net fluid absorption in everted sacs of rat jejunum.These results indicate a functional relationship between jejunal mucosal cyclic AMP levels and active hexose absorption different from the inhibitory role of cyclic AMP in intestinal fluid transport.A preliminary account of this work has been given at the 26th Spring Meeting (March 1985) of the Deutsche Pharmakologische Gesellschaft in Mainz, FRG     

Cyclic AMP and cyclic GMP response to stress in brain and pituitary: Stress elevates pituitary cyclic AMP

Male rats were exposed to six stressors (saline injection, cold, forced running, Formalin injection, immobilization, electric footshock) for 15, 30, or 60 min. Following sacrifice by microwave irradiation, cyclic AMP and cyclic GMP levels were measured in pituitary, pineal and 8 regions of rat brain. All stressors except saline increased plasma corticosterone, plasma prolactin and pituitary cyclic AMP levels compared to control animals. The magnitude of the pituitary cyclic AMP response was highly correlated with the intensity of the stress as determined by the levels of plasma prolactin. Electric footshock increased pituitary cyclic AMP levels over 10 fold and plasma prolactin over 60 fold. Cyclic AMP levels in other brain regions were not altered. Cerebellar cyclic GMP was increased only by stressors that involved increased motor activity.     

Effects of rolipram on scopolamine-induced impairment of working and reference memory in the radial-arm maze tests in rats

RATIONALE: Rolipram, a selective inhibitor of cyclic AMP-specific phosphodiesterase (PDE4), has been shown to enhance scopolamine-induced impairment of working memory. However, its effect on reference memory, which appears to be related to the level of cyclic AMP (cAMP), has not been investigated yet; in addition, the mechanism involved in its effects on memory remains to be elucidated. OBJECTIVES: To investigate the effects of rolipram on working and reference memories impaired by scopolamine and the involvement of cAMP. METHODS: By administration (IP) of rolipram and forskolin, an activator of adenylyl cyclase (AC), the effects of both drugs on the number of correct choices and errors in experiment 1 and, the frequency of both working memory errors and reference memory errors in experiment 2 were observed in two eight-arm radial maze tasks in rats. RESULTS: In experiment 1, rolipram (0.01-1.0 mg/kg) attenuated the scopolamine-induced (0.5 mg/kg) increase in the total number of errors in dose- and time-dependent manners. The minimum effective dose of rolipram was 0.05 mg/kg and the effects lasted nearly 60 min. By contrast, forskolin (1.0-10.0 mg/kg) failed significantly to affect any of the above indices altered by scopolamine. In experiment 2, rolipram (0.05 and 0.1 mg/kg) decreased the frequencies of both working and reference memory errors that were elevated by scopolamine. Forskolin did not alter either type of error at a dose that increased the exploration time. CONCLUSION: Rolipram may exert its effects of reversing both working and reference memory impairments via increased cyclic AMP concentrations in certain signal transduction pathways, rather than by a generalized increase in cAMP.     

The effects of theophylline and cyclic AMP on intestinal smooth muscle contractions

The effects of 10^?4-10^?3 M theopphylline, cyclic 3'5'-adenosine monophosphate (cyclic AMP), dibutyryl cyclic AMP (DBC) and theophylline plus DBC were observed on the spontaneous and potassium (K)-induced contractions of the guinea-pig taenia coli. All of the compounds inhibited spontaneous contractions as well as the phasic and tonic components of the K-contracture. The effects of cyclic AMP, a compound which will not readily penetrate the cell membrane, were similar to those of the other compounds in inhibiting the spontaneous and phasic contractions, but it was a less effective inhibitor of the tonic contracture. Inhibition of spontaneous and phasic activity may occur at the cell membrane while the inhibitory actions on the tonic contracture may depend on cellular penetration by the inhibitor. There was no evidence that theophylline and DBC have different sites of action.     

Effects of ethanol on the cyclic AMP system of the dog gastric mucosa.

The effect of ethanol on the cyclic AMP system of the dog fundic mucosa was studied in vitro. The gastric mucosal content of cyclic AMP was increased by 2.5% ethanol, whereas 10 and 20% ethanol decreased the mucosal content of cyclic AMP. The activity of adenylate cyclase was increased by 2.5 and 5% ethanol, whereas 10% ethanol did not significantly affect it. The activity of cyclic AMP phosphodiesterase was inhibited by ethanol in a competitive manner. The increase in the gastric mucosal content of cyclic AMP, induced by low concentrations of ethanol, is apparently due to the stimulation of adenylate cyclase and inhibition of phosphodiesterase. Changes in the phosphodiesterase or adenylate cyclase activites do not explain the decrease of the mucosal content of cyclic AMP by higher concentrations of ethanol. The mechanism of the decrease is discussed.     

Inducing coproporphyria in rat hepatocyte cultures using cyclic AMP and cyclic AMP-releasing agents

Cyclic AMP (c-AMP), added on its own to rat hepatocyte cultures, caused a marked accumulation of coproporphyrin III. The results obtained by comparing the effect of c-AMP to that of exogenous 5-aminolevulinate (ALA), and from adding c-AMP and ALA together, indicated that the coproporphyrinogen III metabolism was blocked, even though no inhibition of the relevant enzyme, coproporphyrinogen oxidase, could be demonstrated. Preferential accumulation of coproporphyrin could also be produced in cultures of rat hepatocytes by agents that raise the cellular levels of cyclic AMP, such as glucagon. The effect of supplementing the culture medium with triiodothyronine (T3) on the response of rat hepatocytes to c-AMP was also investigated. T3, which is known to stimulate mitochondrial respiration, uncoupling O₂ consumption from ATP synthesis, produced a c-AMP-like effect when given on its own and potentiated the effect of c-AMP, with an apparent increase in the severity of the metabolic block. It is suggested that an oxidative mechanism may be activated in c-AMP and T₃-induced coproporphyria, preferentially involving the mitochondrial compartment, leading to oxidation of porphyrinogen intermediates of haem biosynthesis, especially coproporphyrinogen. Coproporphyin, the fully oxidized aromatic derivative produced, cannot be metabolized and will therefore accumulate.     

Effect of serotonin on cyclic AMP level in rat hypothalamus slices during development.

The effect of serotonin (5-HT) on cyclic AMP levels in rat hypothalamic slices was age-dependent. The sensitivity of the cyclic AMP system to 5 X 10(-5) M 5-HT already existed in the foetus at 21 days of pregnancy. It reached a maximum on the 7th postnatal day and then decreased with age. In adult tissue the response was still present and was antagonized by two serotonergic antagonists (methiothepin and metergoline). When compared to the progressive increase of 5-HT level in the hypothalamus the data suggest a different evolution of serotonergic innervation and of 5-HT receptors.     

Cyclic AMP-dependent protein kinases and binding sites for cyclic AMP in rat erythrocytes

Summary In red cell preparations from reticulocyte-poor (untreated animals; 2% reticulocytes) and reticulocyte-rich blood (animals pretreated with acetylphenylhydrazide; 60% reticulocytes) of rats, cAMP binding sites and cAMP-dependent protein kinase activities were determined. High affinity binding sites for cAMP were present both in membrane and cytoplasmic preparations; while the apparent binding constants determined in both cell fractions (3×10^–9 M for membrane, 2×10^–8M for cytoplasmic fractions) were independent of the reticulocyte content of the preparations, the respective numbers of sites were about twice as high in the reticulocyte-rich as in the reticulocyte-poor preparations.In membrane preparations, significant cAMP-dependent protein kinase activity could be detected only in membrane fractions from reticulocyte-rich blood which were considerably contaminated by intracellular components (haemoglobin-containing membranes) while in washed (haemoglobin-free) membranes no cAMP-dependent protein kinase activity was found. In cytoplasmic preparations both from reticulocyte-poor and reticulocyte-rich blood, two different protein kinases, a low and a high K _aenzyme, were tentatively differentiated by kinetic data; the apparent activation constant for the high K _aenzyme (5×10^–8M) was in the concentration range of the binding constants determined on cytoplasmic preparations. The activity of the high K _aprotein kinase was several fold higher in reticulocyte-rich than in reticulocyte-poor cytoplasmic fractions, while the activity of the low K _aenzyme was obviously independent of the reticulocyte content.From the results obtained, it is concluded that in premature rat erythrocytes, membrane protein(s) may serve as protein substrates for cAMP-dependent protein kinase(s) located in the cytoplasm. This assumption was supported by experiments with intact erythrocytes (prelabelled with inorganic ³²P-phosphate) from reticulocyte-rich blood: isoprenaline, theophylline, and also dibutyryl-cAMP significantly increased phosphorylation of membrane protein of these cells.From the results presented (and others previously reported) it becomes evident that only premature rat erythrocytes, i. e., reticulocytes, are equipped with a -adrenergic receptor-effector system consisting of a -adrenergically stimulated adenyl cyclase and cAMP-dependent protein kinase(s). Obviously, the adrenergic receptor system and also part of the effector system is lost during the process of red cell maturation.This study was supported by a grant from the Deutsche Forschunsgemeinschaft.Preliminary accounts were presented at meetings of the Deutsche Pharmakologische Gesellschaft (Gauger et al., 1972; Gauger and Palm, 1973; Quiring et al., 1974).Deceased December 31, 1974.     

Effects of derivatives of cyclic amp and cyclic gmp on contraction force of cat papillary muscles.

Right ventricular kitten papillary muscles were incubated with dibutyryl adenosine 3',5'-monophosphate (dbcAMP) at varying concentrations as low as 1 X 10(-4)M. A positive inotropic effect was observed with all concentrations of dbcAMP. Concomitant administration of 5 X 10(-4)M monobutyryl guanosine 3',5'-monophosphate (mbcGMP) and 1-2 X 10(-4)M dbcAMP prevented the inotropic response observed when dbcAMP was used alone. When higher doses of dbcAMP were used (5 X 10(-4) M, 10 X 10(-4) M), there was no significant difference in the inotropic response seen between control tissues and papillary muscles pretreated with mbcGMP.     

Effect of forskolin on islet cyclic AMP,insulin secretion,blood glucose and intravenous glucose tolerance in rats

Summary The in vivo effect of forskolin on insulin release, blood glucose and intravenous glucose tolerance test has been studied in the rat. In addition in vitro experiments on the effect of forskolin on islet cAMP and insulin release have been performed for comparison purposes.In batch incubated islets forskolin increased cAMP levels concentration dependently, the EC₅₀ being approximately 25 M. The maximal effect occurred after 5 min. In the presence of 2.8 mM glucose 10M forskolin did not stimulate insulin release; however, it potentiated both phase of 11.1 mM glucose induced insulin secretion.I. v. administration of 1.5 mg/kg of forskolin increased blood glocose levels in rats, which was associated with significant elevation of serum insulin. During an i. v. glucose tolerance test forskolin potentiated the insulin releasing capacity of glucose but did not significantly affect blood glucose levels. It is conceivable that cAMP per se does not initiate but rather amplifies insulin release by glucose. Since the synergistic effect of forskolin and glucose on insulin release in vivo is not associated with increased elimination rate it is possible that forskolin exhibits additional effects which counteract the glucose lowering action of insulin.     

Glucocorticoids: Effects on prostaglandin release,cyclic AMP levels and glycosaminoglycan synthesis in fibroblast tissue cultures

Summary Glucocorticoids (GCs) reduced cyclic AMP levels and inhibited glycosaminoglycan (GAG) synthesis in secondary embryonic mouse fibroblast cultures, when cells were incubated for short periods (30 min). The order of potency was dexamethasone > prednisolone > hydrocortisone. The effect was more marked, when cyclic AMP levels and GAG synthesis were increased by addition of PGE₁.Glucocorticoids exerted no longer an inhibitory effect on cyclic AMP and GAG synthesis in cultures pretreated for 48 h with the steroids. Addition of PGE₁ caused a stronger rise in cyclic AMP and GAG synthesis than in controls without GC-preincubation. This enhancement was even more pronounced, when PGE₁ was added together with the GCs.The reversal of the inhibitory effect of the GCs into a potentiating effect following preincubation correlated to a reduction of endogenous PGE formation in the cultures. Short-term treatment with GCs did not reduce endogenous PGE levels, but prolonged incubation markedly decreased PGE levels. PGE formation recovered following addition of fresh medium after the 48 h incubation with the steroids, but the amount of PGE formed remained significantly lower than in untreated cultures. Non-glucocorticoid steroid hormones did not decrease PGE levels.The results indicate that the apparent loss of inhibitory activity of GCs on cyclic AMP and GAG synthesis observed after prolonged incubation may result from a reduction of endogenous PGE formation which renders the cells more sensitive to the stimulatory effect of exogenous PGE₁.with technical assistance of I. Klappstein