Effects of isolation and culture on prostaglandin synthesis by porcine aortic endothelial and smooth muscle cells

Freshly isolated neonatal porcine aortic tissue (smooth muscle with or without endothelium present) produced approximately 30 ng/mg wet tissue of 6-oxo-prostaglandin F1 alpha (the stable hydrolysis product from prostacyclin) and approximately 15 ng/mg of prostaglandin E2, as measured by radioimmunoassay after 24 h incubation in culture medium. Primary cultures of porcine endothelial and smooth muscle cells (isolated by enzymic digestion of aortic tissue) exhibited the same pattern of prostaglandin production, but absolute values were greater than for fresh tissue, particularly in the case of endothelium. Subcultures of endothelium produced smaller amounts of prostaglandins, although the pattern remained similar. In contrast, subcultures of smooth muscle cells produced a greater total amount of prostaglandins than did primary cultures, and the main product was prostaglandin E2. Experiments with [14C] prostaglandin H2 or [14C]arachidonic acid confirmed that aortic tissue, cultured endothelium, and primary cultures or aortic smooth muscle cells synthesized prostacyclin, and demonstrated that subcultured smooth muscle cells enzymically isomerised prostaglandin H2 to prostaglandin E2. Kinetic studies showed that prostaglandin production by cultured vascular cells was transiently increased by subculture or changing the growth medium, and that production per cell declined with increasing cell density. The change in pattern of prostaglandin production during culture was shown to be due to a rapid decline in the rate of prostacyclin production (which apparently began immediately after tissue isolation), together with a more gradual rise in prostaglandin E2 production. These results indicate that the amounts and ratios of prostaglandins produced by vascular endothelial and smooth muscle cells are greatly affected by the conditions used to isolate and culture the cells; vascular cells in vivo may similarly alter their pattern of prostaglandin production in response to local changes in their environment.     

Phorbol ester-induced phosphoinositide hydrolysis in rat aorta: role of cyclooxygenase products

This study investigates whether phorbol esters increase phosphoinositide hydrolysis in intact vascular smooth muscle, and the mechanism underlying the hydrolysis. Phorbol myristate acetate induced time- and concentration-dependent increases in phosphoinositide hydrolysis, as demonstrated by elevated inositol monophosphate levels, in deendothelialized rat aorta. The phorbol ester-elevated inositol monophosphate levels were abolished by indomethacin, a cyclooxygenase inhibitor, but were only partially decreased by SQ29548, a thromboxane A2/prostaglandin H2 receptor antagonist. SQ29548 also only partially decreased elevated inositol monophosphate levels due to prostaglandin E2, prostaglandin F2alpha, prostaglandin I2 and carbacyclin, a stable prostaglandin I2 analog. SQ29548 abolished elevated inositol monophosphate levels due to U46619, a stable thromboxane A2/prostaglandin H2 receptor agonist. These studies demonstrate that phorbol esters increase phosphoinositide hydrolysis in intact vascular smooth muscle, and that the increase is due, at lease in part, to endogenously released prostaglandins other than prostaglandin H2.     

Response of isolated coronary artery segments to serotonin in the presence of a flavinoid, indomethacin and acetylsalicylate

The aim of this work is to study the mechanism by which 4-metilesculetin (4-Me) cause the relaxation or inhibit the serotonin induced contraction in the smooth muscle. The effect of 4-Me, alone or associated with ascorbic-acid, on basal tone and serotonin induced contraction of isolated coronary strips have been studied. Experiments have been carried out in the presence of indomethacin (IN), specific inhibitor of prostaglandin synthetase. IN-decreased, but did not abolished, the 4-Me induced relaxation and reduced or suppressed the depressive effect of 4-Me on the serotonin dependent contraction. Therefore, it seems reasonable to conclude that the 4-Me influence could be mediated by prostaglandins release in the smooth muscle.     

Effect of a flavinoid (4-methylesculetin) on the response of isolated calf coronary arteries to histamine in the presence of indomethacin

The aim of this work is to study the mechanism by which 4-methylesculetin (4-Me) cause the relaxation or inhibit the histamine induced contraction in the smooth muscle. The effect of 4-Me, alone or associated with ascorbic-acid, on basal tone and histamine induced contraction of isolated coronary strips have been studied. Experiments have been carried out in the presence of indomethacin (IN), specific inhibitor of prostaglandin synthetase. IN-decreased, but did not abolished, the 4-Me induced relaxation and reduced or suppressed the depressive effect of 4-Me on the histamine dependent contraction. Therefore, it seems reasonable to conclude that the 4-Me influence could be mediated by prostaglandins release in the smooth muscle.     

Evidence for enhanced venous smooth muscle turnover of prostaglandin-like substance in portal veins from spontaneously hypertensive rats

The sensitivity of portal veins from 14 to 18 week-old Okamoto-Aoki spontaneously hypertensive rats to prostaglandins A₂, B₂, D₂ and F_2α were enhanced whereas the sensitivity to prostaglandin E₂ was diminished when compared with responses of veins from normotensive Wistar-Kyoto rats. Inhibition of prostaglandin synthesis with both eicosotetraynoic acid (ETYA) and indomethacin (INDO) abolished the observed differences in sensitivity to prostaglandins. Synthesis of prostaglandin-like substance (with arachidonic acid as precursor) was significantly enhanced in portal veins from spontaneously hypertensive rats. Metabolism of prostaglandins E₂ and F_2α, employing the oil-immersion technique of Kalsner and Nickerson, appeared to be similar in veins from normotensive and hypertensive rats. These findings suggest that prostaglandin synthesis is enhanced in venous smooth muscle from hypertensive rats. The increased concentration of endogenous prostaglandin at the venous smooth muscle cell may modify the responses to exogenously administered prostaglandins thus accounting, in part, for the altered sensitivity to these fatty acids.     

Glucocorticosteroid regulation of prostaglandin biosynthesis in human myometrial smooth muscle cells in monolayer culture

In the present investigation, we evaluated the production of prostaglandins by human myometrial smooth muscle cells maintained in monolayer culture in the absence or presence of glucocorticosteroids. In the presence of cortisol (10(-7) M) or dexamethasone (10(-8) M), the rate of production of prostacyclin (PGI2) by these cells was decreased significantly. The glucocorticosteroid-mediated inhibition of prostaglandin production was attenuated when cortisol-21-mesylate (10(-6) M), a glucocorticosteroid antagonist, was present in the culture medium. The rate of conversion of radiolabeled arachidonic acid to radiolabeled prostaglandins as determined by use of sonicates of myometrial cells and optimal assay conditions, however, was not affected significantly by treatment with cortisol or dexamethasone in concentrations sufficient to inhibit prostaglandin formation by more than 80%. These findings are suggestive that glucocorticosteroids act in human myometrial smooth muscle cells in culture to inhibit prostaglandin formation by way of a receptor-mediated process that does not involve inhibition of enzyme activities that are involved in the biosynthesis of prostaglandins, i.e. the conversion of arachidonic acid to prostaglandin.     

Non-specific prostaglandin antagonism by 8-ethoxycarbonyl-5-methyl-10, 11-dihydro-PGA1-ethyl ester (HR 546) on some smooth muscle preparations in vitro

The ability of 8-ethoxycarbonyl-10, 11 dihydro-A-prostaglandin(HR 546) to antagonise smooth muscle contracting effect of prostaglandins E2 and F2alpha on isolated preparations of rat and hamster stomach fundus, guinea pig ileum and gerbil colon has been studied. HR 546 was found to be a potent, non-specific, probably competitive, prostaglandin antagonist on these four smooth muscle preparations.     

Kam semen: Their role in the immune rejection of the nematode Nippostrongylus brasiliensis from the small intestine of rats

Recent work has established that chloroform extracts of ram semen and fractions of these extracts accelerate rejection of the nematode, Nippostrongylus brasiliensis, from the intestine of rats when injected intra-duodenally on day 6 of a primary infection (6). It was also shown that the administration of aspirin and d-propoxyphene hydrochloride (d-PP), potent inhibitors of prostaglandin action (7, 8), prevented the expulsion of worms which normally occurs between days 10 and 16 of a primary infection with N. brasiliensis. In the present study, we have established that there is a direct correlation between smooth muscle contracting activity and the capacity of individual semen fractions to accelerate worm expulsion. Methylation destroyed both smooth muscle contracting activity and the capacity of semen fractions to cause worm expulsion. Contraction of smooth muscle induced by the most active semen fraction (S.A.F. 1) was not inhibited by the amine antagonists mepyramine maleate and bromylsergic acid diethylamide. In addition, contractions induced in rabbit duodenum segments by 5-hydroxytryptamine were not inhibited by aspirin. These findings indicate that the semen fractions do not contain physiologically significant levels of the amines, histamine and 5-hydroxytryptamine, and this suggests that the capacity of semen fractions to cause worm expulsion is due to prostaglandins. This conclusion is supported by the observation that the most active fraction S.A.F. 1 contained bands with R_F values which corresponded with the R_F values of synthetic prostaglandins in thin layer chromatography. Furthermore, the intra-duodenal injection of synthetic prostaglandins also caused worm expulsion.     

16-Aryloxyprostaglandins: A new class of potent luteolytic agent

Recent work has established that chloroform extracts of ram semen and fractions of these extracts accelerate rejection of the nematode, Nippostrongylus brasiliensis, from the intestine of rats when injected intra-duodenally on day 6 of a primary infection (6). It was also shown that the administration of aspirin and d-propoxyphene hydrochloride (d-PP), potent inhibitors of prostaglandin action (7, 8), prevented the expulsion of worms which normally occurs between days 10 and 16 of a primary infection with N. brasiliensis. In the present study, we have established that there is a direct correlation between smooth muscle contracting activity and the capacity of individual semen fractions to accelerate worm expulsion. Methylation destroyed both smooth muscle contracting activity and the capacity of semen fractions to cause worm expulsion. Contraction of smooth muscle induced by the most active semen fraction (S.A.F. 1) was not inhibited by the amine antagonists mepyramine maleate and bromylsergic acid diethylamide. In addition, contractions induced in rabbit duodenum segments by 5-hydroxytryptamine were not inhibited by aspirin. These findings indicate that the semen fractions do not contain physiologically significant levels of the amines, histamine and 5-hydroxytryptamine, and this suggests that the capacity of semen fractions to cause worm expulsion is due to prostaglandins. This conclusion is supported by the observation that the most active fraction S.A.F. 1 contained bands with R_F values which corresponded with the R_F values of synthetic prostaglandins in thin layer chromatography. Furthermore, the intra-duodenal injection of synthetic prostaglandins also caused worm expulsion.     

Non-specific prostaglandin antagonism by 8-ethoxycarbonyl-5-methyl-10, 11-dihydro-PGA1-ethyl ester (HR 546) on some smooth muscle preparations in vitro

The ability of 8-ethoxycarbonyl-10, 11 dihydro-A-prostaglandin(HR 546) to antagonise smooth muscle contracting effect of prostaglandins E₂ and F_2α on isolated preparations of rat and hamster stomach fundus, guinea pig ileum and gerbil colon has been studied. HR 546 was found to be a potent, non-specific, probably competitive, prostaglandin antagonis on these four smooth muscle preparations.     

Non-specific prostaglandin antagonism by 8-ethoxycarbonyl-5-methyl-10, 11-dihydro-PGA1-ethyl ester (HR 546) on some smooth muscle preparations

The ability of 8-ethoxycarbonyl-10, 11 dihydro-A-prostaglandin(HR 546) to antagonise smooth muscle contracting effect of prostaglandins E₂ and F_2α on isolated preparations of rat and hamster stomach fundus, guinea pig ileum and gerbil colon has been studied. HR 546 was found to be a potent, non-specific, probably competitive, prostaglandin antagonis on these four smooth muscle preparations.     

Interaction of ouabain and diphenylhydantoin (DPH) with the cardiovascular actions of prostaglandin B2 and prostaglandin A2

Several reports have appeared indicating that ouabain may interact at sites on smooth muscle susceptible to activation by prostaglandins. This study reports on the interaction between ouabain (0) and diphenylhydantoin (DPH) with the cardiovascular actions of prostaglandin B₂ (PGB₂) and prostaglandin A₂ (PGA₂). Fifteen μg/kg i.v. of 0 enhanced the pressor response of the canine hindpaw to norepinephrine and tyramine but did not affect the pressor responses to sympathetic nerve stimulation (SNS), PGB₂ or PGA₂. PGB₂-induced bronchoconstriction (mediated solely by stimulation of smooth muscle) was reduced by ouabain. In a separate group of animals not receiving PG before 0, 0 reduced (p<0.05) the pressor responses to PGB₂. DPH enhanced the cutaneous pressor responses to SNS, NE, PGB₂ and PGA₂ but did not affect the bronchoconstrictor response to PGB₂. These data are consistent with the following conclusions: 1) Ouabain antagonizes the smooth muscle contractions produced by PGB₂. 2) The presence of PGB₂ antagonizes the prostaglandin inhibitory effects of ouabain suggesting that PGB₂ may compete for similar sites or allosterically interact with ouabain in smooth muscle. 3) DPH induced enhancement of PGB₂ and PGA₂ induced vasoconstriction may reflect DPH induced enhancement of adrenergic neurotransmitter release or inhibition of transmitter reuptake.     

Prostaglandin H synthase immunoreactivity localized by immunoperoxidase technique (PAP) in the small intestine and kidney of rabbit and guinea-pig.

Prostaglandins and inhibitors of prostaglandin synthesis have striking regulatory effects on intestinal muscularis externa. We suggested earlier that a population of macrophage-like cells, located between the external muscle layers might release prostaglandins with a local effect on enveloping interstitial cells of Cajal, postulated pacemaker cells of the gut. To determine cellular production site(s) of prostaglandin we applied monoclonal antibodies against prostaglandin H synthase combined with the PAP technique to sections of rabbit and guinea-pig small intestine and kidney. In rabbit small intestine muscle cells in the circular muscle layer and in the muscularis mucosae were positive, longitudinal muscle negative. Vascular endothelial cells and serosal mesothelial cells were stained. In guinea-pig all muscle layers were unstained but endothelial and mesothelial cells were stained together with unidentified cells in the outermost submucosa. In rabbit kidney, positive staining of collecting ducts, interstitial cells, the parietal layer of Bowman's capsule and arterial endothelial cells was present. Furthermore, we found prostaglandin synthase antigenicity in the epithelial cells lining the loop of Henle, not described before. In guinea-pig medullary collecting ducts were stained and the papilla was lined by stained epithelial cells. The results show a species variation in the distribution of recognizable levels of prostaglandin H synthase. The impressive reaction in the mesothelium must be considered, when enzyme distribution is examined biochemically with fractionated tissue. Our findings do not support our hypothesis that macrophage-like cells are more potent sources of prostaglandins than smooth muscle cells.     

Effects of lubiprostone on human uterine smooth muscle cells

Lubiprostone, a bicyclic fatty acid derivative and member of a new class of compounds called prostones, locally activates ClC-2 Cl(-) channels without activation of prostaglandin receptors. The present study was specifically designed to test and compare lubiprostone and prostaglandin effects at the cellular level using human uterine smooth muscle cells. Effects on [Ca(2+)](i), membrane potential and [cAMP](i) in human uterine smooth muscle cells were measured. 10 nM lubiprostone significantly decreased [Ca(2+)](i) from 188 to 27 nM, which was unaffected by 100 nM SC-51322, a prostaglandin EP receptor antagonist. In contrast 10nM PGE(2) and PGE(1) both increased [Ca(2+)](i) 3-5-fold which was blocked by SC-51322. Similarly, lubiprostone and prostaglandins had opposite/different effects on membrane potential and [cAMP](i). Lubiprostone caused SC-51322-insensitive membrane hyperpolarization and no effect on [cAMP](i). PGE(2) and PGE(1) both caused SC-51322-sensitive membrane depolarization and increased [cAMP](i). Lubiprostone has fundamentally different cellular effects from prostaglandins that are not mediated by EP receptors.     

Prostaglandin H synthase immunoreactivity localized by immunoperoxidase technique (PAP) in the small intestine and kidney of rabbit and guinea-pig

Summary Prostaglandins and inhibitors of prostaglandin synthesis have striking regulatory effects on intestinal muscularis externa. We suggested earlier that a population of macrophage-like cells, located between the external muscle layers might release prostaglandins with a local effect on enveloping interstitial cells of Cajal, postulated pacemaker cells of the gut.To determine cellular production site(s) of prostaglandin we applied monoclonal antibodies against prostaglandin H synthase combined with the PAP technique to sections of rabbit and guinea-pig small intestine and kidney. In rabbit small intestine muscle cells in the circular muscle layer and in the muscularis mucosae were positive, longitudinal muscle negative. Vascular endothelial cells and serosal mesothelial cells were stained. In guinea-pig all muscle layers were unstained but endothelial and mesothelial cells were stained together with unidentified cells in the outermost submucosa. In rabbit kidney, positive staining of collecting ducts, interstitial cells, the parietal layer of Bowman's capsule and arterial endothelial cells was present. Furthermore, we found prostaglandin synthase antigenicity in the epithelial cells lining the loop of Henle, not described before. In guinea-pig medullary collecting ducts were stained and the papilla was lined by stained epithelial cells.The results show a species variation in the distribution of recognizable levels of prostaglandin H synthase. The impressive reaction in the mesothelium must be considered, when enzyme distribution is examined biochemically with fractionated tissue. Our findings do not support our hypothesis that macrophage-like cells are more potent sources of prostaglandins than smooth muscle cells.     

The effect of leukotriene D4 on the isolated stomach and colon of the rat

The effect of synthetic leukotriene D4 (LTD4) was evaluated on isolated gastric longitudinal or circular smooth muscle and distal colon of the rat. The concentrations of LTD4, 2.5 X 10(-10)M to 5 X 10(-7)M, evoked minimal to maximal contractile responses. In addition, selected prostaglandins were used to identify the mediator of LTD4-induced contraction of gastric smooth muscle. FPL 55712 inhibited LTD4-induced contractions of gastric longitudinal or circular muscle. Indomethacin inhibited only LTD4-induced contractions of the longitudinal muscle. A combination of FPL 55712 and indomethacin produced greater inhibition of LTD4-induced contractions of longitudinal muscle than either agent alone. However, the same combination of inhibitors showed no greater effect than FPL 55712 alone on LTD4-induced contractions of circular smooth muscle. Unlike PGI2, PGF2, PGA2, or PGD2, PGE2 evoked contraction of the longitudinal muscle and relaxation of the circular muscle of the stomach. The dissimilar effect of PGE2 in the two smooth muscle layers of the rat stomach may signify that PGE2 is the prostaglandin released by LTD4 from the longitudinal and circular gastric muscle. However, the opposing pharmacologic effects following LTD4-induced release of prostaglandins in the circular muscle of the stomach would preclude the appearance of an inhibitory effect of indomethacin in this tissue. In contrast, PGE2 and other prostaglandins contract gastric longitudinal muscle in response to LTD4. Thus, these studies clearly suggest that LTD4 has both a direct and indirect effect on gastric smooth muscle of the rat. Unlike the stomach, LTD4-induced contraction of the distal colon was not inhibited by indomethacin while FPL 55712 antagonized contractions. Thus, these findings indicate a differential mechanism of stimulation of rat gastrointestinal tissue by LTD4.     

Effects of 7-oxa-13-prostynoic acid (7-OPyA) and prostaglandin E1 methyl ester on canine gastric secretion

The compound 7-OPyA has been reported to antagonize smooth muscle stimulatory effects of some prostaglandins (PG's) in vitro. The in vivo PG antagonist activity of 7-OPyA has not been adequately diarrhea in mice. We studied the effects of this compound on PGE1. Secretion was stimulated by continuous intravenous infusion of histamine. At the steady-state plateau of gastric secretion, PGE1 methyl ester (PGE1 ME) or PGE1 ME and 7-OPyA were simultaneously infused intravenously. The extent of gastric secretory inhibition afforded by PGE1 ME alone or in the presence of 7-OPyA was assessed. 7-OPyA did not modify PGE1 ME gastric antisecretory actions when administered at doses 20-50 times greater than the dose of PGE1 ME. These results suggest that the prostaglandin antagonist effects of 7-OPyA show organ specificity, which may be of clinical importance.     

Comparison of arachidonic acid metabolism between myofibroblasts and fibroblasts isolated from rat palatal mucoperiosteum

Myofibroblasts were cultured successfully from experimental wound tissue in rat palatal mucoperiosteum. Arachidonic acid metabolizing activity in cultured myofibroblasts was compared with that in fibroblasts cultured from normal mucoperiosteum. Prostaglandins biosynthesized from [14C]arachidonic acid in cell-free homogenates of both myofibroblasts and fibroblasts were prostaglandins D2, E2 and F2 alpha, and the activity producing each prostaglandin was not significantly different between the myofibroblasts and the fibroblasts, whereas smooth muscle cells, which are histologically similar to myofibroblasts, produced mainly 6-ketoprostaglandin F1 alpha, and relatively small amounts of prostaglandin E2. The release of arachidonic acid from cells prelabeled with [14C]arachidonic acid was compared among three types of cell. The calcium ionophore A23187 strongly enhanced arachidonic acid release in all three cell types. Bradykinin, 5-hydroxytryptamine and prostaglandin F2 alpha affected the stimulation of arachidonic acid release in the fibroblasts but were less or not effective in the myofibroblasts and smooth muscle cells. In addition, prostaglandin E2 biosynthesized in response to several stimuli was measured by radioimmunoassay. The content of prostaglandin E2 correlated closely with arachidonic acid release. In this study, we showed homogeneity between the myofibroblasts and fibroblasts in prostaglandin synthesizing activity and similarity in response to various stimuli between the myofibroblasts and smooth muscle cells, from the standpoint of arachidonic acid metabolism.     

Intramuscular accumulation of prostaglandins during static contraction of the cat triceps surae.

We previously demonstrated that muscle afferent endings are sensitized by exogenous prostaglandins during static contraction of skeletal muscle. The purpose of this study was to determine whether 30 s of static hindlimb contraction, induced by electrical stimulation of the cat sciatic nerve, increases the concentration of immunoreactive prostaglandin E2 (iPGE2) and 6-ketoprostaglandin F1 alpha (i6-keto-PGF1 alpha, the stable metabolite of prostaglandin I2) in muscle tissue. In addition, the role of ischemia in augmenting prostanoid production was examined. Gastrocnemius muscle was obtained by freeze-clamping tissue, and prostaglandins were extracted from muscle homogenates and measured by radioimmunoassay. Compared with precontraction values, high-intensity (68% of maximal tension) static contraction elevated gastrocnemius iPGE2 and i6-keto-PGF1 alpha by 45 and 53%, respectively (P less than 0.01). Likewise, when blood flow to the gastrocnemius was attenuated by arterial occlusion during and 2 min before low-intensity contraction (29% maximal tension), the intramuscular iPGE2 concentration was increased by 71% (P less than 0.01). Conversely, low-intensity contraction (30% of maximal tension) and arterial occlusion without contraction did not alter the concentration of either prostanoid. Our findings demonstrate that prostaglandins accumulate in muscle during static contraction. We believe that local muscle ischemia may provide a stimulus for this phenomenon. These prostaglandins therefore are available to sensitize afferent endings responsible for reflex adjustments during static muscle contraction.     

Prostaglandin synthesis along the gastrointestinal tract of the rabbit: Differences in total synthesis and profile

In the present study we systematically investigated the synthesis of prostaglandins in the mucosa and the muscle layer along the length of the rabbit gut. Homogenates of mucosa and muscle layer were incubated with (14C)-labelled arachidonic acid, and prostaglandin formation was determined using thin-layer chromatography.With respect to total prostaglandin synthesis the highest values in the mucosa were measured in fundus, antrum and colon, whereas the prostaglandin synthesis in the muscle layer was maximal in the small bowel, particularly the ileum.In the mucosa, the prostaglandins E2 and F2a predominated, and there were minor differences along the gastrointestinal tract. In the muscle layer of the stomach, high amounts of 6-keto prostaglandin Fla, the stable degradation product of prostacyclin were produced, while small and large bowel homogenates synthesized mostly F2a. Consistently the prostaglandins A2/B2 were a major product in most locations. In addition, PG E2 catabolism to 15-keto PG E2 and 13,14-dihydro-15-keto PG E2 in the absence of NAD was slow.No significant changes in total prostaglandin synthesis and prostaglandin profile were detected between 24 hrs fasted and normally fed rabbits at any part of the gastrointestinal tract.