The effect of PGE2 on the activation of quiescent lung fibroblasts

The effect of prostaglandin E₂ (PGE₂) on fibroblast proliferation was examined. The presence of PGE₂ for 24 h inhibited the growth of quiescent cells stimulated with serum, platelet-derived growth factor and macrophage-derived factors. Maximal inhibition of nuclear labeling with [³H]thymidine occurred at concentrations greater than 10⁻⁷ M. The inhibitory effect of PGE₂ was less potent in exponentially growing cells and was not the result of conversion of PGE₂ to PGA₂ during incubation in growth medium. The G₁ phase was determined to be 12–14 h in untreated cultures. The extent of growth inhibition by PGE₂ was similar with addition of PGE₂ at 0, 3, 6, or 9 h following restimulation of quiescent cell cultures. Approximately 25% of the cells that enter S phase are refractory to PGE₂-induced growth inhibition. Short-term exposure to PGE₂ (5 min and 30 min) caused substantial growth inhibition. The serum-induced proliferation was also inhibited by the cAMP analogue, dibutyrl cAMP. Our results suggest that PGE₂ affects a distinct subpopulation of cells. Restimulation of quiescent cells treated with PGE₂ for 24 h, indicated that release from PGE₂ exposure is associated with prolongation of the G₁ phase of the cell cycle.     

Role of the blood–cerebrospinal fluid barrier transporter as a cerebral clearance system for prostaglandin E2 produced in the brain

An increasing level of prostaglandin (PG) E₂ is involved in the progression of neuroinflammation induced by ischemia and bacterial infection. Although an imbalance in the rates of production and clearance of PGE₂ under these pathological conditions appears to affect the concentration of PGE₂ in the cerebrospinal fluid (CSF), the regulatory system remains incompletely understood. The purpose of this study was to investigate the cellular system of PGE₂ production via microsomal PGE synthetase‐1 (mPGES‐1), the inducible PGE₂‐generating enzyme, and PGE₂ elimination from the CSF via the blood–CSF barrier (BCSFB). Immunohistochemical analysis revealed that mPGES‐1 was expressed in the soma and perivascular sheets of astrocytes, pia mater, and brain blood vessel endothelial cells, suggesting that these cells are local production sites of PGE₂ in the CSF. The in vivo PGE₂ elimination clearance from the CSF was eightfold greater than that of d ‐mannitol, which is considered to reflect CSF bulk flow. This process was inhibited by the simultaneous injection of unlabeled PGE₂ and β‐lactam antibiotics, such as benzylpenicillin, cefazolin, and ceftriaxone, which are substrates and/or inhibitors of organic anion transporter 3 (OAT3). The characteristics of PGE₂ uptake by the isolated choroid plexus were at least partially consistent with those of OAT3. OAT3 was able to mediate PGE₂ transport with a Michaelis–Menten constant of 4.24 μM. These findings indicate that a system regulating the PGE₂ level in the CSF involves OAT3‐mediated PGE₂ uptake by choroid plexus epithelial cells, acting as a cerebral clearance pathway via the BCSFB of locally produced PGE₂.     

Effects of prostaglandins and other drugs on the cyclic AMP content of cultured bone cells

Prostaglandins of the E-series (PGE₁ and PGE₂) may be involved in disease-related, localized loss of bone. E-prostaglandins increase the cyclic AMP content of many cells; and, to determine if their effects on bone are mediated by cyclic AMP, we examined the effects of E-prostaglandins and of other agents on the cyclic AMP content of cultured bone cells. PGE₂ produced a rapid, marked and dose-related increase in the cyclic AMP content of confluent monolayers of bone cells isolated from newborn rat calvaria. At 2.8 × 10⁻⁶ M, PGE₁ and PGE₂ had approximately the same effect, while the effect of PGF_2α was much less pronounced. In the presence of theophylline, PGE₂ had a more marked effect than parathyroid hormone (PTH) and the combination of PGE₂ and PTH had a synergistic effect. The divalent, cationic, ionophore, A23187, produced an increase in cellular cyclic AMP and had an additive effect in combination with PGE₂. Synthetic salmon calcitonin (CT), which inhibits the bone resorptive effect of PGE₂, increased cellular cyclic AMP and had an additive effect in combination with PGE₂. A prostaglandin antagonist, SC-19220, partially inhibited the resorptive effect of PGE₂ and reduced its effect on cellular cyclic AMP. The calcium antagonist, D600, inhibited the bone resorptive effects of PGE₂ but had no effect on increased cellular cyclic AMP produced by PGE₂.The marked effect of PGE₂ on bone cell cyclic AMP suggests that this action is involved in the mechanism of PGE₂-related bone loss. The fact that agents with different effects on PGE₂-induced increases in cellular cyclic AMP can inhibit its resorptive actions, suggests that PGE₂-induced changes in cyclic AMP may be related less to its resorptive actions than to its inhibitory effect on bone formation.     

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 10⁻⁹g/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.     

Developmental changes in the effects of prostaglandin E<Subscript>2</Subscript> in the chicken ductus arteriosus

Prostaglandin E₂ (PGE₂) is the major vasodilator prostanoid of the mammalian ductus arteriosus (DA). In the present study we analyzed the response of isolated DA rings from 15-, 19- and 21-day-old chicken embryos to PGE₂ and other vascular smooth muscle relaxing agents acting through the cyclic AMP signaling pathway. PGE₂ exhibited a relaxant response in the 15-day DA, but not in the 19- and 21-day DA. Moreover, high concentrations of PGE₂ (≥3 μM in 15-day and ≥1 μM in 19-day and 21-day DA) induced contraction of the chicken DA. The presence of the TP receptor antagonist SQ29,548, unmasked a relaxant effect of PGE₂ in the 19- and 21-day DA and increased the relaxation induced by PGE₂ in the 15-day DA. The presence of the EP receptor antagonist AH6809 abolished PGE₂-mediated relaxation. The relaxant responses induced by PGE₂ and the β-adrenoceptor agonist isoproterenol, but not those elicited by the adenylate cyclase activator forskolin or the phosphodiesterase 3 inhibitor milrinone, decreased with maturation. High oxygen concentrations (95%) decreased the relaxation to PGE₂. The relaxing potency and efficacy of isoproterenol and milrinone were higher in the pulmonary than in the aortic side of the DA, whereas no regional differences were found in the response to PGE₂. We conclude that, in contrast to the mammalian situation, PGE₂ is a weak relaxant agent of the chicken DA and, with advancing incubation, it even stimulates TP vasoconstrictive receptors.     

On the multiplicity of platelet prostaglandin receptors I. Evaluation of competitive antagonism by aggregometry

Methods for the evaluation of competitive interactions at receptors associated with platelet activation and inhibition using aggregometry of human PRP have been developed. The evidence supports the suggestion that PGE₁ and PGI₂ share a common receptor for inhibition of platelet reactivity, but only a portion (if any) of the aggregation stimulation associated with PGE₂ is the result of PGE₂ binding (without efficacy) to this receptor. PGE₂ (@.3–20 μ ) is an effective antagonist of PGE₁, PGI₂, producing a shift of about one order of magnitude in the IC₅₀-values obtained from complete aggregation inhibition dose response curves. The antagonism of PGD₂ inhibition is particularly notable, 80 n PGE₂ levels are detectable. This and other actions of PGE₂ indicate another platelet receptor for PGE₂. PGE₁ acts at both the PGE₂ and PGI₂ receptor. Other substances showing PGI₂-like actions only at high doses (1–30 μ ), display additive responses with PGI₂ indicative of decreased affinity for the I₂/E₁ receptor and the absence of PGE₂-like aggregation stimulation activity.PGI₂ methyl ester has intrinsic inhibitory action not associated with in situ ester hydrolysis. The methyl ester is dissaggregatory showing particular specificity for inhibition of release and second wave aggregation.     

A dose-response comparison of PGA2, PGE1 and PGE2 using the phenylephrine-stimulated perfused oviduct of the rabbit

The phenylephrine-stimulated perfused oviduct of the rabbit was evaluated as a model for studying the activity of prostaglandins that produce inhibition of the oviducal smooth muscle. Elevation of the normal “tone” of the oviduct by perfusing phenylephrine through the lumen permitted quantitation of the responses to PGA₂, PGE₁ and PGE₂ by measuring the magnitude of the inhibitory response produced by the agents. PGE₂ was relatively more potent, efficacious and specific for the oviduct than PGA₂ or PGE₁. It was concluded that the model was suitable for comparative dose-response studies of PGA₂, PGE₁ and PGE₂ and their analogs.     

Selective antagonism of the systemic vasodepressor response to PGE1 by d,1–11, 15-bisdeoxy PGE1

Several bisdeoxy PGE₁ analogs are potent, competitive antagonists of PGE₁-induced colonic contractions in the gerbil. The efficacy of these analogs in antagonizing PGE₁-mediated systemic vasodepression has not been previously demonstrated. In this study, serial doses of PGs were administered before, during and after infusion of d,1–11, 15-bisdeoxy PGE₁. Bolus injections of PGE₁ (3.0 μk/kg), PGE₂ (3.0 μg/kg) and PGI₂ (0.3 μg/kg) were administered via the right external jugular vein to male Wistar rats. PGE₁, PGE₂ and PGI₂ decreased systemic arterial pressure 41%, 38% and 38%, respectively. The PGE₁ analog was infused (200 μg/kg/min) through the right common carotid artery. The analog itself had no effect on mean systemic arterial pressure, but maximum reversible inhibition (51%) of PGE₁-mediated vasodepression occurred following a 50 minute infusion. No significant effect of the PGE₁ analog was observed on PGE₂ or PGI₂-mediated vasodepression. These data demonstrate the ability to antagonize PGE₁-mediated vasodepression, and to differentiate the vascular responses to PGE₁ and PGE₂ or PGI₂.     

The simultaneous use of two prostaglandin E radioimmunoassays employing two antisera of differing specificity I. Determination of prostaglandin content in sera and culture medium of simian virus 40 transformed cells

Two antisera produced by employing a PGE₁ or PGE₂ protein conjugate have been titered and tested for crossreactivity with PGE₁ and PGE₂. These antibodies revealed marked differences in PGE₁ versus PGE₂ specificity. Anti-PGE₁ displayed only a low level of crossreactivity with PGE₂ (20%), while anti-PGE₂ was capable of detecting both PGE₁ and PGE₂ in known mixtures of PGs. These two antisera have been utilized to measure the same serum and culture medium samples. The differing values obtained with the two PGE antisera are a reflection of the known differences in PGE specificity. These results stress the fact that antisera with only partial crossreactivity with PGE₂ have only limited use in assessing experimental fluctuations in PGE level.     

Functional characterization of prostaglandin E2 inducible osteogenic colony forming units in cultures of cells isolated from the neonatal rat calvarium

Prostaglandin E₂ (PGE₂) increases the number of mineralized nodules that form in cultures of rat calvarial (RC) cells. The purpose of our study was to characterize PGE₂-inducible osteogenic colony forming units (CFU-Os) by determining their number, the cell populations from which they were released, their specific responsive period to PGE₂, and their proliferating and differentiating characteristics under the stimulation of PGE₂. Limiting dilution analysis was used to determine the number of PGE₂-inducible CFU-Os. Sequential digestion of intact rat parietal bones with collagenase isolated 5 subpopulations of RC cells that were used to estimate the cell populations where PGE₂-inducible CFU-Os resided. The responsive period of PGE₂-inducible CFU-Os to PGE₂ was evaluated by treating cultures of mixed RC cells for all possible combinations of days 1–10, 11–20, and 21–30. PGE₂ effects on proliferation and differentiation of CFU-Os were evaluated by comparing the DNA synthesis and AP activity in subpopulations I and IV on days 3, 6, and 9. Results showed: (1) PGE₂-inducible CFU-Os represent 0.27% of cells in the mixed RC population, (2) the majority of determined and PGE₂-inducible CFU-Os were found in the subpopulations released during the 60–100 min digestion periods, (3) the response of PGE₂-inducible CFU-Os is limited to the first 10 days of culture, and (4) PGE₂-stimulated nodule formation is associated with an early increase in DNA synthesis and a sustained increase in alkaline phosphatase activity. We conclude that, functionally, PGE₂-inducible CFU-Os are slowly proliferating AP negative cells primarily found in the subpopulations III-V. PGE₂ stimulates them to proliferate and become AP+, and function as determined CFU-Os to form mineralized nodules in vitro. © 1996 Wiley-Liss, Inc.     

Role of endogenous and exogenous prostaglandins on the contractile functioning of isolated sow (Sus scrofa) oviducts

The output prostaglandins (PHs) E₁, E₂ and F₂ α, from ampullary and isthmic portions of sow oviducts isolated during proestrus, estrus and metestrus, was explored. Moreover, $\text{in vitro}$ cumulative dose-response curves for the contractile effect of these three PGs, on identical oviductal segments, were constructed. Isthmic preparations form proestrous and metestrous animals released more PGE₁ and PGF₂ α than PGE₂ “like material”. During estrus, the outputs of PGE₁, PGE₂ and PGF₂ α were similar, whereas, oviducts from proestrous and metestrous sows released less PGE₁ and PGF₂ α than during estrus. Although the output of PGE₂ “like material” from isthmic and ampullary segments did not differ significantly during the three stages of the sex cycle, ampullary metestrous preparations released more PGE₁ and PGF₂ α, than estrous or proestrous ones. The addition of PGE₁, PGE₂ α, consistently stimulatedthe amplitude of contractions of isthmic oviductal segments isolated from proestrous and metestrous sows. Within the concentration-range explored, dose-response curves for PGE₂ and PGE₁ were to the left of those for PGF₂ α in the isthmus obtained before ovulation (proestrus) but not in segments isolated at later times (2–3 days) of the cycle (metestrus). The stimulatory dose-response curves for PGE₁, or PGE₂, in isthmic segments of metestrous preparations incubated with phentolamine (10^?6M) were shifted to the right of controls not exposed to the adrenoreceptor blocker, whereas, the curve for PGF₂ α without phentolamine, was identical to that obtained in its presence. PGE₁ and PGE₂ did not evoke significant contractile effects on oviductal ampullary protions from proestrous sows, wherea, PGF₂ α was clearly stimulatory at concentrations of 10^?9M and higher. In ampullary segments isolated after ovulation (metestrus) the threshold for contractile enhancement following PGF₂ α was greater than during proestrus, whereas, PGE₁ elicited a significant inhibition of contractions. The spontaneous contractile pattern exhibited by isthmic and ampullary oviductal regions, prior to and after ovulation, is discussed in terms of tissue PG generation and output and is compared with results regarding tubal motility following the exposure to exogenous PGs.     

Absorption of prostaglandin E2 and uterine sensitivity of the non-pregnant and pregnant monkey in vivo

Radioactive (11-³H) prostaglandin E₂(PGE₂) levels in plasma of non-pregnant Rhesus and Japanese monkeys were determined by radioimmunoassay. The amounts of PGE₂ in plasma increased gradually and reached a peak 90 minutes after oral administration. Comparatively low levels were detected 24 hours after oral administration. Plasma PGE₂ levels increased rapidly and disappeared within 5 minutes when 5 μg/kg of PGE₂ was administered intravenously.Uterine contractile sensitivity to PGE₂ and F_2α was measured by the threshold of a venous dosage required to evoke an elevation of uterine contractility in non-pregnant and pre- and post-labor Japanese monkeys. Uterine sensitivity to PGE₂ in the non-pregnant monkey appear to vary in accordance with the sexual life span. At term of pregnancy, PGE₂ was much more potent in causing uterine contraction than PGF_2α. During labor and at postpartum period with lactation, effectiveness of PGE₂ appear to be less than that of PGF_2α. The non-pregnant and pregnant uterus of the third trimester are more sensitive to PGE₂ than the laboring and postpartum uterus.The long latency of the elevation of uterine contractility induced by the intravenous administration of PG suggests that the PG compounds have potent actions on the central nervous system.     

Role of prostaglandins on the regulation of macrophage proliferation and cytotoxic functions

The data presented show different effects of prostaglandins on proliferation and cytotoxic effector functions of murine bone-marrow derived mononuclear cells. Colony stimulating factor (CSF)-dependent proliferation of colony forming unit-cells (CFU c) was inhibited by PGE₁, PGE₂ and PGB₂. Lymphokine induced cytotoxicity and antibody mediated cytotoxicity (ADCC) of monocytes and macrophages were also affected by PG. We conclude that PGE₂ may regulate macrophage mediated tumorcell-lysis mainly at the induction phase.If there processes function in vivo, one would therefore expect high affinity binding sites for PGE₂ on macrophages. The existence of a receptor for PGE₂ one murine bone marrow derived macrophages is described.     

Prostaglandin E2 inhibits the proliferation of human gingival fibroblasts via the EP2 receptor and Epac

Elevated levels of prostaglandins such as PGE₂ in inflamed gingiva play a significant role in the tissue destruction caused by periodontitis, partly by targeting local fibroblasts. Only very few studies have shown that PGE₂ inhibits the proliferation of a gingival fibroblast (GF) cell line, and we expanded this research by using primary human GFs (hGFs) and looking into the mechanisms of the PGE₂ effect. GFs derived from healthy human gingiva were treated with PGE₂ and proliferation was assessed by measuring cell number and DNA synthesis and potential signaling pathways were investigated using selective activators or inhibitors. PGE₂ inhibited the proliferation of hGFs dose‐dependently. The effect was mimicked by forskolin (adenylate cyclase stimulator) and augmented by IBMX (a cAMP‐breakdown inhibitor), pointing to involvement of cAMP. Indeed, PGE₂ and forskolin induced cAMP generation in these cells. Using selective EP receptor agonists we found that the anti‐proliferative effect of PGE₂ is mediated via the EP₂ receptor (which is coupled to adenylate cyclase activation). We also found that the effect of PGE₂ involved activation of Epac (exchange protein directly activated by cAMP), an intracellular cAMP sensor, and not PKA. While serum increased the amount of phospho‐ERK in hGFs by ～300%, PGE₂ decreased it by ～50%. Finally, the PGE₂ effect does not require endogenous production of prostaglandins since it was not abrogated by two COX‐inhibitors. In conclusion, in human gingival fibroblasts PGE₂ activates the EP₂—cAMP—Epac pathway, reducing ERK phosphorylation and inhibiting proliferation. This effect could hamper periodontal healing and provide further insights into the pathogenesis of inflammatory periodontal disease. J. Cell. Biochem. 108: 207–215, 2009. © 2009 Wiley‐Liss, Inc.     

Site of the in vivo stimulatory effect of prostaglandins on LH release

A possible direct effect of prostaglandins E₁ and E₂ (PGE₁ and PGE₂) on luteinizing hormone (LH) release at the pituitary level was studied using anterior pituitary cells in primary culture, a system approximately 10-fold more sensitive to stimulation of LH release than previously used hemipituitaries. No effect of PGE₁ or PGE₂ could be detected on the time course of basal or LH-RH-stimulated LH release or on the LH responsiveness to LH-RH. This absence of a direct effect of PGEs at the pituitary level on LH release was confirmed by experiments using female rats under Surital anesthesia in the afternoon of proestrus. After intravenous injection, under these conditions, 15(S)-15-methyl PGE₂ was 3–5 times more potent than PGE₂ to increase plasma LH levels while PGE₁ had about 50% the potency of PGE₂. Injection of sheep anti-LH-RH serum one hour before PGE₁ or PGE₂ injection not only lowered basal plasma LH levels but prevented the rise induced by PGEs. These data indicate clearly that the increased plasma LH levels observed after PGE injection are secondary to a stimulation of LH-RH release while PGEs do not appear to have a significant effect on LH release at the pituitary level.     

Interactions between prostaglandin E 1 and calcium at the level of the mitochondrial membrane

Low concentrations of PGE₁ facilitate the exit of actively accumulated Ca²⁺ from rat liver mitochondria. The effect is evident at pH 6,4 and disappears at neutral pH. Ca²⁺ bound to the mitochondrial membrane in the absence of energy is not discharged by PGE₁.Under conditions that lead to its active accumulation, Ca²⁺ stimulates the binding of PGE₁ to mitochondria. The effect is concentration dependent (maximal at 500 μM Ca²⁺), is evident only at slightly acid pH, and is transitory. The binding of PGE₁ reaches a maximum between 30 sec and 2 min and then declines very rapidly, returning to the baseline 2–5 min after the addition of Ca²⁺. The maximal amount of PGE₁ bound is 1.3 nmoles per mg of mitochondrial protein, i.e., about 1% of the Ca²⁺ taken up by mitochondria. No PGE₁ is bound when permeant anions are tranported into mitochondria together with Ca²⁺. Sr²⁺ and Mn²⁺ also stimulate the binding of PGE₁.Aspirin and indomethacin are powerful inhibitors of the binding of PGE₁ to mitochondria. This effect appears to be secondary to the inhibition of mitochondrial Ca²⁺ transport by the antiinflammatory drugs.     

Prostaglandin-induced enhancement of the anamnestic response

The ability of various prostaglandins (PGs) to affect the anamnestic immune response of keyhole limpet hemocyanin (KLH)-primed rabbit popliteal lymph node cells was investigated. Of the four PGs studied (PGA₁, PGE₂ and PGF_2α), PGE₁ was found to have a stimulatory effect, whereas PGA₁, PGE₂ and PGF_2α were ineffective in stimulating or inhibiting the anamnestic response. Under the conditions studied, a 3.5-fold increase in antibody production was obtained in PGE₁-treated, KLH-stimulated cultures. Maximum enhancement was obtained when 0.2 μg of PGE₁ were added at the time of culture initiation and were allowed to remain in contact with the lymph node cells for 24 hours.     

Metabolism of prostaglandins and xenobiotics by adrenal microsomal monooxygenase in the guinea pig

The possibility that prostaglandins could serve as substrates for the guinea pig adrenal microsomal monooxygenase was investigated. The binding of PGE₁ to adrenal microsomes was found to exhibit a reverse type I spectral change. Also PGE₁ diminished the magnitude of type I spectrum elicited by cortisol binding to adrenal microsomes. The incubation of [³H]PGE₁ or of [³H]PGE₂ with adrenal microsomes supplemented with NADPH yielded primarily the respective 19-hydroxy metabolite. The enzymatic activity catalyzing this hydroxylation appears to be a typical monooxygenase, requiring NADPH for activity and being strongly inhibited by metyrapone, SKF 525A, and cytochrome c. Carbon monoxide at a ratio of 9:1 to oxygen moderately inhibited the hydroxylation of PGE₁. Whereas the liver catalyzed the hydroxylation of PGE₁ and PGA₁ equally well, the adrenal microsomes preferentially catalyzed the hydroxylation of PGE₁. This finding and the observation that α-naphthoflavone is a weak inhibitor of the adrenal PGE₁ hydroxylation points to significant differences between the adrenal and liver prostaglandin hydroxylation activities. Cortisol, which is a substrate for adrenal monooxygenase, strongly inhibited PGE₁ and PGE₂ hydroxylation. By contrast, certain xenobiotics (ethylmorphine, hexobarbital, benzpyrene), which are also metabolized by adrenal microsomes, only slightly inhibited the hydroxylation of PGE₁. Similarly, PGE₁ only weakly inhibited ethylmorphine and benzphetamine demethylation and hexobarbital hydroxylation. These observations suggest that adrenal microsomes contain several monooxygenases with different affinities for prostaglandins and for the different xenobiotic substrates.     

Contractile effects of prostaglandin E2 in rat rectum: Sensitivity to the prostaglandin antagonists diphloretin phosphate and SC 19220

Prostaglandin E₂ (PGE₂) applied cumulatively (1 nM − 1 μM) induced concentration-dependent tonic contractions in the longitudinal muscle of isolated rat rectum. The PGE₂ effects were not altered by guanethidine (50 μM), whereas atropine (3 μM), guanethidine plus atropine or tetrodotoxin (0.1 μM) reduced them to an almost equal extent and increased the EC₅₀ values for PGE₂. The after-contractions following electrical stimulation were enhanced by PGE₂ (10 nM) and inhibited by atropine. Diphloretin phosphate (DPP, 100 μM) shifted the regression lines for PGE₂ to the right in both untreated and tetrodotoxin-treated preparations, and thereby increased the EC₅₀ values. Slopes of the concentration-effect lines for PGE₂ before and after DPP differed in the presence of tetrodotoxin. The regression line for PGE₂ with SC 19220 (100 μM) in tetrodotoxin-treated preparations was shifted to the right in a parallel fashion. It is concluded that PGE₂ exerted both a neural (cholinergic) and a smooth muscle effect. There may be a competitive antagonism between SC 19220 and PGE₂ but the block by DPP may be nonselective.     

Effect of exogenous phospholipase A2 and triacylglycerol lipase on the synthesis and release of monoenoic and bisenoic prostaglandins from isolated rat uterus

The possible existence of a selective and independent mechanism subserving the formation of prostaglandin E₁ (PGE₁) and of prostaglandin E₂ (PGE₂) has been reported in previous studies from our group. In the present experiments we have demonstrated that neutral lipid lipases play an important role yielding dihomo-gamma-linolenic acid for the formation of PGE₁. Indeed, exogenous triglyceride lipase added to the incubation bathing solution at a concentration of 150 U/ml increased several fold the production of PGE₁ by isolated uterine strips obtained from spayed rats. Nevertheless the presence of the enzyme did not modify significantly the synthesis and release of bisenoic PGs (PGE₂ and PGF_2α). When triarachidonin was added, as an artificial substrate into the incubating medium in order to detect the presence of endogenous triacylglycerol lipase, we observed a significant increment in the generation of PGE₂ (p < 0.005) and of PGF_2α (p < 0.001) without evident changes in the basal release of PGE₁. On the other hand, the addition of phospholipase A₂ (PLA₂) at 0.2 U/ml, increased significantly the production of PGE₂ (p < 0.001) but failed to alter the concentration of PGE₁ in the incubating solution. Surprisingly, PLA₂ did not enhance the synthesis of PGF_2α in the present experiments, a situation for which we do not have a clear explanation. Exogenous bradykinin (10⁻⁶ M), a well known stimulant of PLA₂ activity in several tissues, also increased significantly (p < 0.001) the production of PGE₂ without altering that of PGE₁. Results presented herein provide strong support to the notion that different enzyme mechanisms and different lipid stores are linked to the formation of PGE₁ and PGE₂ in isolated rat uteri.