The effects of prostaglandins E2 and F2α on synaptosomal accumulation and release of 3H-norepinephrine

Prostaglandins (PG) of both the E and F series may serve as modulators of norepinephrine (NE) release from peripheral sympathetic neurons. We have studied the effects of PGE₂ and PGF_2α on the accumulation and release of ³H-NE in the CNS using synaptosomes isolated from rat hypothalami.The release of ³H-NE from synaptosomes superfused with Krebs-Ringer bicarbonate buffer was multiphasic with an initial fast release phase followed by a slower release. Raising KC1 concentration of the superfusion medium to 56mM during the slow release phase is known to stimulate ³H-NE release. PGE₂ (1 × 10^?6M) attenuated ³H-NE release during the fast phase and reduced the amount of ³H-NE released due to KC1 stimulation. At lower concentrations of PGE₂ there was no change in the release profile. PGF_2α was without effect on ³H-NE release at all concentrations tested.The accumulation of ³H-NE was significantly diminished by PGE₂ at a concentration of 1 × 10^?6M, while a lower concentration (1 × 10^?7M) was ineffective. PGF_2α had no effect on ³H-NE accumulation at all concentrations investigated.     

Interaction between phenylephrine and prostaglandins E1 and F1α on the contractile responses of vascular muscle

Prostaglandins (PGs) E₁ or F_1α (1.4−8.4 × 10⁻⁸ M) contracted strips of rabbit aorta and increased the contractions produced by 1−6 × 10⁻⁷ M phenylephrine (PE). The addition of the PGs simultaneously with PE or after a low concentration of PE (2 × 10⁻⁷ M) significantly increased the PE-induced contractions. However, when the PGs were added after a higher concentration of PE (6 × 10⁻⁷ M) an additional increase in the PE-induced contraction was produced with PGF_1α but not with PGE₁. Isobolic plots of the data obtained from the simultaneous addition of PE and the PGs indicate that both PGs interact with PE in a synergistic or potentiative manner, suggesting that their effects are mediated through different receptor mechanisms. Addition of the PGs after a high dose of PE indicates that there may also be either qualitative or quantitative differences between PGE₁ and PGF_1α.     

Prostaglandin E1 and F2α specific binding in bovine corpora lutea: Comparison with luteolytic effects

Preliminary characterization indicated the presence of separate prostaglandin (PG)E₁ and (PG)F_2α binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 × 10⁻⁹M and 2.1 × 10⁻⁸M for PGE₁ and PGF_2α, respectively. Competition of several natural prostaglandins for the PGE₁ and PGF_2α bovine luteal specific binding sites indicates specificity for the 9-keto or 9α-hydroxyl moiety, respectively. Differences in relative ability to inhibit ³H-PG binding were found due to sensitivity to the absence or presence of the 5,6-cis-double bond as well.Bovine luteal function was affected following treatment of heifers with 25 mg PGF_2α as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contrast, treatment with 25 mg PGE₁ resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE₁ treatment compared to pretreatment values. In so far as data obtained on PGF_2α relative binding affinity to the bovine CL can be compared to data obtained independently on PGF_2α induced luteolysis in the bovine, PGF_2α relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE₁ relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.     

The effects of prostaglandins PGE1, PGE2, PGF1a, and PGF2a on canine synovial perfusion

In these experiments we have examined the effects of PGE₁, PGE₂, PGF_1α and PGF_2α on synovial perfusion in the normal canine synovial microcirculation. The effects of the drugs on synovial perfusion were determined indirectly from the changes produced in the rate of clearance of ¹³³Xenon from the joint by their intra-articular injection. Prostaglandins PGE₁ and PGE₂ were found to be strongly vasodilator with PGE₁ being the more active. PGF_1α appeared to have little or no vasoactive properties in doses up to 1 ugm. (2.8 × 10^−5M) in our I preparation while PGF_2α was vasodilator at this high dosage only. Neither SC19920 nor diphloretin phosphate antagonised the effects of PGE₁ in these experiments.     

Differential effects of prazosin and rauwolsin on the release of prostaglandins I2 and E2 from the perfused mesenteric arterial bed of the rabbit following nerve stimulation

Sympathetic nerve stimulation of the perfused mesenteric arterial bed of the rabbit, , increase the secretion of prostaglandin (PG)I₂ and PGE₂. Prazosin (4.8 × 10⁻⁶), and α₁ adrenergic receptor antagonist, inhibited this inrease in release of PGI₂ but not of PGE₂ whereas rauwolsin (10⁻⁷ M), an α₂ adrenergic receptor antagonist, inhibited the increase in release of PGE₂ but not of PGI₂. Prazosin (10⁻⁶ M) completely blocked the vasoconstrictor response to nerve stimulation, and to norepinephrine and phenylephrine administration, suggesting there to be little of an α₂ adrenergic receptor component in this response. It is concluded that the increase in PGI₂ release follows the activation of α₁ adrenergic receptors and is therefore post-junctional in origin, whereas the increase in PGE₂ release follows the activation of α₂ adrenergic receptors and may be pre- and/or post-junctional in origin.Indomethacin (2.8 × 10⁻⁷, 5.6 × 10⁻⁷ and 1.12 × 10^{−6 M} did not affect the vasoconstrictor responses to nerve stimulation at 10 Hz, whereas rauwolsin (10⁻⁷ M) in the presence of indomethacin substantially increased them. These results indicate that PGE₂ does not regulate norepinephrine release following nerve stimulation at 10 Hz to rabbit mesenteric arteries, and that the inhibition of norepinephrine release following stimulation of α₂ pre-junctional receptors is independent of PG involvement.     

Prostaglandin E1 and F2α specific binding in bovine corpora lutea: Comparison with luteolytic effects

Preliminary characterization indicated the presence of separate prostaglandin (PG)E₁ and (PG)F_2α binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 × 10⁻⁹M and 2.1 × 10⁻⁸M for PGE₁ and PGF_2α, respectively. Competition of several natural prostaglandins for the PGE₁ and PGF_2α bovine luteal specific binding sites indicates specificity for the 9-keto or 9α-hydroxyl moiety, respectively. Differences in relative ability to inhibit ³H-PG binding were found due to sensitivity to the absence or presence of the 5,6-cis-double bond as well.Bovine luteal function was affected following treatment of heifers with 25 mg PGF_2α as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contrast, treatment with 25 mg PGE₁ resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE₁ treatment compared to pretreatment values. In so far as data obtained in vitro on PGF_2α relative binding affinity to the bovine CL can be compared to data obtained independently in vitro on PGF_2α induced luteolysis in the bovine, PGF_2α relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE₁ relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.     

Effect of prostaglandin F2α on propulsive activity of the isolated segmental colon of the guinea-pig

The effects of prostaglandin F_2α (PGF_2α) on propulsive activity in segments of isolated colon and on isolated strips of guinea-pig colon were investigated.Using experimental conditions under which spontaneous propulsive activity was negligible, PGF_2α (5×10⁻⁸×1×10⁻⁶M), added to the bathing medium, increased propulsive activity in a concentration dependent manner. This increase of propulsive activity was abolished in the presence of atropine or tetrodotoxin (1×10⁻⁷g/ml).The contractions produced by PGF_2α(5×10⁻⁷ − 1×10⁻⁵M) in isolated longitudinal and circular smooth muscle strips of guinea-pig colon were unaffected in the presence of atropine or tetrodotoxin (1×10⁻⁷ g/ml).From these results it is concluded that under the conditions employed in this study propulsive activity stimulated by PGF_2α may depend on the contractions of both muscle layers and stimulation of the peristalic reflex.     

Stimulation by prostaglandin E2 of glucagon and insulin release from isolated rat pancreas

To ascertain whether prostaglandins (PG) may play a role in the secretion of glucagon and in an attempt to elucidate the conflicting observations on the effects of PG on insulin release, the isolated intact rat pancreas was perfused with solutions containing 1.1 × 10⁻⁹ to 1.8 × 10⁻⁵M PGE₂. In the presence of 5.6 mM glucose significant increments in portal venous effluent levels of glucagon and insulin were observed in response to minimal concentrations of 2.8 × 10⁻⁸ and 1.4 × 10⁻⁷M PGE₂, respectively; a dose-response relationship was evident for both hormones at higher concentrations of PGE₂. When administered over 60 seconds, 1.4 × 10⁻⁶M PGE₂ resulted in a significant increase in glucagon levels within 24 seconds and in insulin within 48 seconds. Ten-minute perfusions of 1.4 × 10⁻⁶M PGE₂ elicited biphasic release of both islet hormones; Phase I glucagon release preceded that of insulin. Both phases of the biphasic glucagon and insulin release which occurred in response to 15-minute perfusions of 10 mM arginine were augmented by PGE₂. These observations indicate that PGE₂ can evoke glucagon and insulin release at concentrations close to those observed by others in the extracts of rat pancreas. We conclude that PG may be involved in the regulation of secretion of glucagon and insulin and may mediate and/or modify the pancreatic islet hormone response to other secretagogues.     

Regulations of macrophage-mediated tumor cell killing by prostaglandins: Comparison of the effects of PGE2 and PGI2

Mouse resident peritoneal macrophages stimulated by purified bacterial lipopolysaccharide (LPS) produced both prostaglandin E₂ (PGE₂) and prostaglandin I₂ (PGI₂), the latter detected as its stable metabolite, 6-keto PGF_1α. Maximum production, induced in each case by 1 ng/ml purified LPS, was in the range of 10⁻⁷M for PGI₂ and 3 × 10⁻⁸M for PGE₂. A quantitatively similar increase in intracellular levels of macrophage cyclic AMP (measured on a whole cell basis), with a similar duration of effect, was stimulated by PGE₂ and PGI₂; however, only PGE₂ had a negative regulatory effect on macrophage activation for tumor cell killing. These data confirm that more than a whole cell increase in the concentration of cyclic AMP is needed to shut off nonspecific tumor cell killing mediated by LPS-activated resident peritoneal macrophages.     

Stimulation by prostaglandin E2 of glucagon and insulin release from isolated rat pancreas

To ascertain whether prostaglandins (PG) may play a role in the secretion of glucagon and in an attempt to elucidate the conflicting observations on the effects of PG on insulin release, the isolated intact rat pancreas was perfused with solutions containing 1.1 × 10⁻⁹ to 1.8 × 10⁻⁵M PGE₂. In the presence of 5.6 mM glucose significant increments in portal venous effluent levels of glucagon and insulin were observed in response to minimal concentrations of 2.8 × 10⁻⁸ and 1.4 × 10⁻⁷M PGE₂, respectively; a dose-response relationship was evident for both hormones at higher concentrations of PGE₂. When administered over 60 seconds, 1.4^−10−6M PGE₂ resulted in a significant increase in glucagon levels within 24 seconds and in insulin within 48 seconds. Ten-minute perfusions of 1.4 × 10⁻⁶M PGE₂ elicited biphasic release of both islet hormones; Phase I glucagon release preceded that of insulin. Both phases of the biphasic glucagon and insulin release which occurred in response to 15-minute perfusions of 10 mM arginine were augmented by PGE₂. These observations indicate that PGE₂ can evoke glucagon and insulin release at concentrations close to those observed by others in the extracts of rat pancreas. We conclude that PG may be involved in the regulation of secretion of glucagon and insulin and may mediate and/or modify the pancreatic islet hormone response to other secretagogues.     

Effect of PGI2, PGE2 and 6-keto PGF1α on canine gastric blood flow and acid secretion

Prostaglandins (PG)I₂, PGE₂ and 6-keto PGF₁α were infused directly into the gastric arterial supply at 10⁻⁹, 10⁻⁸ and 10⁻⁷ g/kg/min during an intra-gastric artery pentagastrin infusion in anesthetized dogs. 6-keto PGF₁α was also infused at 10⁻⁶ g/kg/min. Gastric arterial blood flow was measured continuously with a non-cannulating electromagnetic flow probe and gastric acid collected directly from the stomach. PGI₂ and PGE₂ produced similar dose-dependent increases in blood flow with an increase of more than four-fold at the highest dose. Both PGs inhibited acid output over this dose range with PGE₂ having 10 times the potency of PGI₂. 6-keto PGF₁α was at least 1000 times less active than PGI₂ or PGE₂ at increasing blood flow and failed to inhibit acid output even at 10⁻⁶ g/kg/min.     

Histamine alters prostaglandin output from diestrous rat uteri. Involvement of H2-receptors and 9-keto-reductase

The effects of exogenous histamine (H) on prostaglandin (PG) generation and release in uteri isolated from diestrous rats and the influences of H₂-receptors blockers (cimetidine and mitiamide) on the output of uterine PGs, were explored. Moreover, the action of H on the uterine 9-keto-reductase, was also studied. Histamine (10⁻⁴M) failed to alter the basal output of PGE₁ but reduced significantly the generation and release of PGE₂ and augmented the output of PGF_2α. On the other hand, cimetidine (10⁻⁵M) enhanced the basal release of PGE₂ but had no action on the outputs of PGs E₁ or F_2α. The enhancing effect of H on the production and release of PGF_2α was abolished in the presence of cimetidine. Also, the antagonist reversed the influence of H on the output of PGE₂. Metiamide, another H₂-receptor antagonist, did not alter the basal control generation and release of uterine PGs, but antagonized the augmenting influence of H on PGF_2α uterine output, as much as cimetidine did, and prevented the depressive action of H on the release of PGE₂ from uteri. Histamine (10⁻⁴M) significantly stimulated uterine formation of cyclic-adenosine monophosphate, an action which was antagonized by the presence of cimetidine (10⁻⁵M), a blocker of H₂ receptors. Also, histamine (10⁻⁵M) and dibutyril-cyclic-adenosine monophosphate (DB-cAMP) at 10⁻³M, enhanced significantly the formation ³H-PGF_2α from ³H-PGE₂. Results presented herein demonstrate that H is able to diminish the generation of PGE₂ in uteri from rats at diestrus augmenting the synthesis of PGF_2α, apparently via the activation of H₂-receptors, enhancing adenylate-cyclase. These effects appear to increase uterine 9-keto-reductase activity which transforms PGE₂ into PGF_2α. Relationships between the foregoing results and those evoked by estradiol, are also discussed.     

The in vitro effect of indomethacin on basal bone resorption, on prostaglandin production and on the response to added prostaglandins

Mouse calvaria were maintained in organ culture without serum additives. Basal active resorption, as measured by ⁴⁵Ca and hydroxyproline release, was significantly inhibited to 74% control levels by indomethacin (1.4 × 10⁻⁷ M). Prostaglandin F and prostaglandin E₂ production, determined by radioimmunoassay, were both significantly lowered by this concentration of indomethacin. DNA, protein and hydroxyproline synthesis, as indices of cell toxicity, were unaffected by low concentrations of indomethacin, while concentrations of 1.4 × 10⁻⁶M inhibited protein synthesis (p<0.005). In the presence of indomethacin (1.4 × 10⁻⁷M) both PGE₂ and PGF_2α stimulated resorption in a dose-dependent manner, with PGE₂ being the more potent. Neither prostaglandin affected hydroxyproline synthesis at low concentrations, but PGE₂ had a marked inhibitory action at a higher concentration (10⁻⁶M). In combination, the effects of PGE₂ and PGF_2α showed no evidence of synergism or any antagonistic action. The study shows that in vitro calcium and hydroxyproline resorption in the unstimulated mouse calvaria are inhibited by indomethacin at concentrations measured in serum during human therapy. The decreased PGF and PGE₂ production associated with this decreased bone resorption in the presence of non-toxic concentrations of indomethacin would suggest a role for these prostaglandins in maintaining the basal resorption of cultured bone.     

Dose and time dependent luteotropic and luteolytic action of prostaglandin F2α in the luteinized rabbit ovary

The mechanism of stimulatory and inhibitory action of PGF_2α on ovarian steroidogenesis both under and conditions has been studied in the pseudo-pregnant rabbits. Short term incubation of the ovaries with PGF_2α (2.82 × 10⁻⁵M) resulted in an increased synthesis of progesterone and 20α-OH P. The addition of PGF_2α in the medium and further incubation of the ovaries obtained from rabbits that had been constantly infused with PGF_2α (0.5 μg/min.) for two hours resulted in increased synthesis of these progestins. The ratio of progesterone to 20α -OH P was also enhanced under these conditions and thus supported the luteotropic action of small doses of PGF_2α under short term incubations. However, as the amount of PGF_2α infused was increased to 5 μg/min., the addition of PGF_2α under conditions strikingly decreased the production of these progestins. The ratio of progesterone to 20α -OH P was also decreased and thus was indicative of luteolytic action of higher doses of PGF_2α. High doses of PGF_2α (5.64 × 10⁻⁴M) failed to I cause any significant change in the progestin synthesis under short term incubation. These results thus suggest that the luteotropic and luteolytic action of PGF_2α in the luteinized rabbit ovary is dose and time dependent.     

Action of 7-oxa-13-prostynoic acid on rat uterine contractility and sensitivity to PGF2α and angiotensin II

The action of 7-oxa-13-prostynoïc acid on spontaneous contractions and on PGF_2α and angiotensin II elicited contractions has been studied on rat uteri excised during pro and metaoestrus At the concentration of 5 × 10⁻⁶M, the compound does not alter the spontaneous contractions of proestrus uteri and suppresses the phasic component of PGF_2α-elicited contractions. At the concentration of 5 × 10⁻⁵M, 7-oxa-13-prostynoïc acid inhibits spontaneous contractions and inhibits PGF_2α-elicited contractions. In metaoestrus uteri, the inhibitory effect is not competitive. 7-oxa-13-prostynoïc acid is also able to inhibit non competitively the contractor effect of angiotensin II in proestrus uteri. This result, together with those of previous investigations performed using other prostaglandin inhibitors, suggest that the contractor effect of angiotensin II on the uterus is partially mediated by endogenous prostaglandins.     

PGE2 is a more potent vasodilator of the lamb ductus arteriosus than is either PGI2 or 6 keto PGFα

It has been shown in vitro that the lamb ductus arteriosus forms prostaglandins PGE₂, PGF_2α, 6 keto PGF_1α (and its unstable precursor PGI₂). In this study the relative potencies of these endogenous prostaglandins were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO₂ and indomethacin. All the prostaglandins (except PGF_2α) relaxed the vessel. This is consistent with the hypothesis that endogenous prostaglandins inhibit the tendency of the vessel to contract in response to oxygen. Only PGE₂, however, relaxed the vessel at concentrations below 10⁻⁸M. PGI₂ and 6 keto PGF_1α had approximately 0.001 and 0.0001 times the activity of PGE₂. Although PGE₂ has been observed to be a minor product of prostaglandin production in the lamb ductus arteriosus, the tissue's marked sensitivity to PGE₂ might make it the most significant prostaglandin in regulating the patency of the vessel.     

Prostaglandin I2 as a potentiator of acute inflammation in rats

Prostaglandin I₂ potentiated the paw swelling induced by carrageenin in rats. Prostaglandin I₂ (0.1 μg) showed similar activity to PGE₁ (0.01 μg). This potentiating property disappeared in 60 minutes and was completely abolished by diphenhydramine (25 mg kg⁻¹, i.p.). In vascular permeability tests, PGI₂ itself (2.5 × 10⁻¹⁰ mol, 88 ng) caused no dye leakage reaction, but PGE₁ (2.5 × 10⁻¹⁰ mol, 88.5 ng) caused a significant dye leakage. This effect of PGE₁ was statistically significant compared with vehicle- or PGI₂-treated group (p<0.05). Prostaglandin I₂ potentiated the increased vascular permeability induced by 5-hydroxytriptamine (2.5 × 10⁻¹⁰ mol), bradykinin (5 × 10⁻¹⁰ mol) and histamine (2 × 10⁻¹⁰ to 2 × 10⁻⁸ mol). The potentiation was the most evidence in the case of histamine.     

Prostaglandin-E2-9-ketoreductase in rabbit kidney

The 100,000 xg supernatant of rabbit kidney contains a prostaglandin-E₂-9-ketoreductase which has an obligatory requirement for NADPH. This enzyme is localised in the renal cortex and is able to quantitatively convert PGE₂ to PGF_2α. A broad pH profile was evident with an optimum at pH 7·5. Kinetic studies indicated a K_m of 3·2 × 10⁻⁴M PGE₂. The isoelectric point was at pH 5·65 and the molecular weight, as estimated by gel filtration, was 21,800. These values differ from those obtained with enzyme from monkey brain tissue and suggest a tissue specificity of PGE₂-9-ketoreductase. By combining isoelectric focussing techniques with sephadex filtration considerable purification of the renal enzyme was achieved.     

Effects of prostacyclin on coronary circulation, heart rate and myocardial contractile force in isolated hearts of guinea PIG and rabbit — Comparison with prostaglandin E2

Infusions of prostacyclin (PGI₂) (3 × 10⁻¹⁰ − 3 × 10⁻⁷M) into the coronary circulation of isolated hearts from guinea pigs or rabbits resulted in a concentration-dependent decrease in the coronary perfusion pressure (CPP). There was a slight decrease in left ventricular systolic pressure in the heart of the rabbit, whereas the heart rate remained unchanged. PGE₂ was without effect on the heart of the rabbit but was as potent as PGI₂ in decreasing the CPP in the guinea pig heart. 6-oxo-PGF_1α (up to 3 × 10⁻⁶ M) did not affect any of the parameters measured.     

Pre-junctional inhibitory action of prostaglandin E2 on excitatory neuro-effector transmission in the human bronchus

The effects of prostaglandin E₂ (PGE₂) and indomethacin on excitatory neuro-effector transmission in the human bronchus were investigated by tension recording and microelectrode methods. PGE₂ (10⁻¹⁰–10⁻⁹M) suppressed the amplitude of twitch contractions and excitatory junction potentials (e.j.ps) evoked by field stimulation at a steady level of basal tension obtained by the combined application of indomethacin (10⁻⁵M) and FPL55712 (10⁻⁶M). In doses over 10⁻⁸M, PGE₂ reduced the muscle tone and dose-dependently suppressed the amplitude of twitch contractions. Indomethacin (10⁻⁵ or 5 × 10⁻⁵M) reduced the muscle tone and enhanced the amplitude of twitch contractions and e.j.ps evoked by field stimulation in the presence of FPL55712. PGE₂ (10⁻⁹M) had no effect on the post-junctional response of smooth muscle cells to exogenously applied acetylcholine (ACh) (4 × 10⁻⁷M). However, indomethacin (10⁻⁵M) significantly enhanced the ACh-induced contraction of the human bronchus. These results indicate that PGE₂ in low concentrations has a pre-junctional action to inhibit excitatory neuro-effector transmission in addition to a post-junctional action, presumably by suppressing transmitter release from the vagus nerve terminals in the human bronchial tissues.