17-phenyl Trinor PGE2 Research Articles

Occupation of the prostaglandin E 2-type 1 receptor increases rat atrial contractility via a Y-27632-sensitive pathway

This study investigated whether rat left atria (LA) contain the prostaglandin E 2 type 1 receptor (EP1) and whether EP1 occupation induces positive inotropic responses in superfused LA. Western analysis demonstrated that LA contain EP1 and the EP1 splice variant. Exposing isolated, superfused LA to 17-phenyl trinor PGE 2, an EP1 agonist, increased isometric contractile force and its corresponding dF/dTs to ≈70% of the isoproterenol maximum with an EC 50 of ≈80 nM. In contrast, agonists for EP2, EP3, and EP4 caused little change in LA function. While the EP1 antagonists SC-51089 and SC-19220 blocked 17-phenyl trinor PGE 2-induced inotropy, neither prazosin, nadolol, atropine nor EI-283, a pan-specific protein kinase C inhibitor, affected 17-phenyl trinor PGE 2-induced inotropy. However, Y-27632 and HA-1077, inhibitors of rho A-activated protein kinases, prevented and reversed the increase in LA contractility that occurred in the presence of 17-phenyl trinor PGE 2. Thus, atria contain EP1 and EP1 occupation increases LA contractility via a pathway sensitive to inhibitors of rho A-activated protein kinases.

Effects of Tetramethylpyrazine on Prostaglandin E 2- and Prostaglandin E 2 Receptor Agonist-induced Disruption of Blood-Aqueous Barrier in Pigmented Rabbits

Purpose: To evaluate the effect of tetramethylpyrazine on the elevation of aqueous flare and intraocular pressure (IOP) induced by prostaglandin (PG) E 2 and PGE 2 receptor (EP) agonists. Methods: PGE 2 or EP agonists (11-deoxy PGE 1, EP 2 agonist; 17-phenyl trinor PGE 2, EP 1 and EP 3 agonist; or sulprostone, EP 1 and EP 3 agonist), 25 μg/mL, were transcorneally administered to pigmented rabbits. Animals were pretreated with tetramethylpyrazine intravenously (10 or 30 mg/kg) or topically (0.1% solution). Aqueous flare was measured using a laser flare-cell meter, and the intensity was expressed as the area under the curve (AUC). Intraocular pressure was measured using a noncontact tonometer. Results: After administration of PGE 2, aqueous flare and IOP increased and then gradually decreased. The AUC of eyes pretreated with tetramethylpyrazine, 10 or 30 mg/kg, intravenously, or topical 0.1% solution, was significantly smaller than that of the controls. The mean Δ IOP of eyes pretreated with tetramethylpyrazine, 30 mg/kg intravenously, was significantly lower than that of the controls. After administration of 11-deoxy PGE 1, aqueous flare increased and then gradually decreased. 17-phenyl trinor PGE 2 and sulprostone did not disrupt the blood-aqueous barrier. The AUC of eyes pretreated with tetramethylpyrazine, 10 or 30 mg/kg, intravenously, before 11-deoxy PGE 1 application was significantly smaller than that of the controls. Conclusion: The results indicated that tetramethylpyrazine inhibited PGE 2- or 11-deoxy PGE 1-induced elevation of aqueous flare and IOP.

Mechanisms involved in prostaglandin-induced increase in bone resorption in neonatal mouse calvaria

Prostaglandins (PG) E1, E2and F2 αinduce bone resorption in isolated neonatal parietal bone cultures, and an associated increase in interleukin-6 (IL-6) production. Indomethacin had little effect on the response to PGE2, or the relatively non-selective EP receptor agonists 11-deoxy PGE1and misoprostol, but blocked the effects of PGF2 αand the F receptor agonist fluprostenol, indicating an indirect action via release of other prostaglandins. It is more likely that there is positive autoregulation of prostaglandins production in this preparation mediated via stimulation of F receptors. The effects of selective EP receptor agonists sulprostone (EP1,3) and 17-phenyl trinor PGE2(EP1), indicated the involvement of EP2 and/or EP4 receptors, which signal via cAMP. The relatively weak increase in IL-6 production by misoprostol (with respect to resorption) suggests that these responses are controlled by different combination of EP2 and EP4 receptors. The PKA activator, forskolin, induced small increases in bone resorption at lower concentrations (50–500 ng/ml) but a reversal of this effect, and inhibition of resorption induced by other stimuli (PTH, PGE2), at higher concentrations (0.5–5 μg/ml). IL-6 production was markedly increased only at the higher concentrations. The inhibitory effect of forskolin may be a calcitonin-mimetic effect. PMA induced both resorption and IL-6 production which were both blocked by indomethacin, indicating a role for PKC in the control of prostaglandin production.

Prostaglandin E(2)-induced interleukin-6 release by a human airway epithelial cell line.

Human airway epithelial cell release of interleukin (IL)-6 in response to lipid mediators was studied in an airway cell line (BEAS-2B). Prostaglandin (PG) E(2) (10(-7) M) treatment caused an increase in IL-6 release at 2, 4, 8, and 24 h. IL-6 release into the culture medium at 24 h was 3,396 +/- 306 vs. 1,051 +/- 154 pg/ml (PGE(2)-treated cells vs. control cells). PGE(2) (10(-7) to 10(-10) M) induced a dose-related increase in IL-6 release at 24 h. PGF(2 alpha) (10(-6) M) treatment caused a similar effect to that of PGE(2) (10(-7) M). PGE(2) analogs with relative selectivity for PGE(2) receptor subtypes were studied. Sulprostone, a selective agonist for the EP-3 receptor subtype had no effect on IL-6 release. 11-Deoxy-16,16-dimethyl-PGE(2), an EP-2/4 agonist, and 17-phenyl trinor PGE(2), an agonist selective for the EP-1 > EP-3 receptor subtype (10(-6) to 10(-8) M), caused dose-dependent increases in IL-6 release. 8-Bromo-cAMP treatment resulted in dose-related increases in IL-6 release. RT-PCR of BEAS-2B cell mRNA demonstrated mRNA for EP-1, EP-2, and EP-4 receptors. After PGE(2) treatment, increases in IL-6 mRNA were noted at 4 and 18 h. Therefore, PGE(2) increases airway epithelial cell IL-6 production and release.

Prostaglandin E 2-stimulated secretion of protein in the salivary glands of the lone star tick via a phosphoinositide signaling pathway

Previous studies identified a prostaglandin E 2 (PGE 2) receptor in the salivary glands of partially fed female lone star ticks, Amblyomma americanum (L.). In the present studies, protein secretion from dispersed salivary gland acini was shown to be specific for PGE 2, as compared with PGF 2α or the thromboxane analog U-46619, in accordance with their respective binding affinities for the PGE 2 receptor. Furthermore, the selective PGE 2 EP1 receptor agonist, 17-phenyl trinor PGE 2, was as effective as PGE 2 in stimulating secretion of anticoagulant protein. Calcium ionophore A-23187 (1 to 100 μM) stimulated secretion of anticoagulant protein in a dose-dependent manner but the voltage-gated Ca 2+-channel blocker verapamil (1 to 1000 μM) and the receptor-mediated Ca 2+-entry antagonist, SK&F 96365 (1 and 10 μM), and 5 mM ethylene glycol bis(β-aminoethyl ether)-N,N N′, N′-tetraacetic acid (EGTA) had no appreciable effect on inhibiting PGE 2-stimulated secretion of anticoagulant protein. PGE 2 (0.1 μM) and the non-hydrolyzable analog of guanosine triphosphate (GTP), GTPγS (10 μM), directly activated phospholipase C (PLC) in a membrane-enriched fraction of the salivary glands after PLC was first incubated with the PGE 2 EP1 receptor antagonist AH-6809, which presumably antagonized endogenous PGE 2 (0.3 μM) in the broken-cell-membrane-enriched fraction. TMB-8, an antagonist of intracellular inositol trisphosphate (IP 3) receptors, inhibited PGE 2-stimulated secretion. The results support the hypothesis that PGE 2 stimulates secretion of tick salivary gland protein via a phosphoinositide signaling pathway and mobilization of intracellular Ca 2+.

Prostaglandin E receptor subtypes in cultured rat microglia and their role in reducing lipopolysaccharide-induced interleukin-1beta production.

Prostaglandins (PGs) are potent modulators of brain function under normal and pathological conditions. The diverse effects of PGs are due to the various actions of specific receptor subtypes for these prostanoids. Recent work has shown that PGE2, while generally considered a proinflammatory molecule, reduces microglial activation and thus has an antiinflammatory effect on these cells. To gain further insight to the mechanisms by which PGE2 influences the activation of microglia, we investigated PGE receptor subtype, i.e., EP1, EP2, EP3, and EP4, expression and function in cultured rat microglia. RT-PCR showed the presence of the EP1 and EP2 but not EP3 and EP4 receptor subtypes. Sequencing confirmed their identity with previously published receptor subtypes. PGE2 and the EP1 agonist 17-phenyl trinor PGE2 but not the EP3 agonist sulprostone elicited reversible intracellular [Ca2+] increases in microglia as measured by fura-2. PGE2 and the EP2/EP4-specific agonists 11-deoxy-PGE1 and 19-hydroxy-PGE2 but not the EP4-selective agonist 1-hydroxy-PGE1 induced dose-dependent production of cyclic AMP (cAMP). Interleukin (IL)-1beta production, a marker of activated microglia, was also measured following lipopolysaccharide exposure in the presence or absence of the receptor subtype agonists. PGE2 and the EP2 agonists reduced IL-1beta production. IL-1beta production was unchanged by EP1, EP3, and EP4 agonists. The adenylyl cyclase activator forskolin and the cAMP analogue dibutyryl cAMP also reduced IL-1beta production. Thus, the inhibitory effects of PGE2 on microglia are mediated by the EP2 receptor subtype, and the signaling mechanism of this effect is likely via cAMP. These results show that the effects of PGE2 on microglia are receptor subtype-specific. Furthermore, they suggest that specific and selective manipulation of the effects of PGs on microglia and, as a result, brain function may be possible.

PGE2, via EP3 receptors, regulates brain nitric oxide synthase in the perinatal period.

We tested the hypothesis that high prostaglandin levels during the perinatal period might regulate brain nitric oxide synthase (nNOS) expression. nNOS and cyclooxygenase (COX)-2 mRNAs were higher in brain cortex and the periventricular area of newborn rats and pigs compared with adult brain. Nitric oxide synthase activity was also 2. 5- to 4-fold higher in newborn than in adult brain. Administration of nonselective COX inhibitor ibuprofen or COX-2 inhibitor nimesulide every 8 h for 24 h to newborn rats and pigs reduced prostaglandin levels and caused comparable reductions in nNOS mRNA, protein, and activity to levels of adults; COX inhibitor-induced changes were prevented by cotreatment with PGE2 analog, 16, 16-dimethyl-PGE2, and agonist for the EP3 receptor of PGE2, sulprostone, but not by PGI2 analog carbaprostacyclin, PGD2, EP1 receptor agonist 17-phenyl trinor-PGE2, and EP2 agonist butaprost. Concordant observations were made in vitro and revealed that nNOS expression (detected by NADPH diaphorase reactivity) mostly present in neurons of the deeper cortical layers was reduced by COX inhibitor, and this effect was prevented by EP3 agonist. In conclusion, high levels of PGE2 in neonatal brain contribute to the increased expression of nNOS by acting on EP3 receptors; this positive interaction between PGE2 and nNOS might be required physiologically for normal brain development.

Growth regulation of primary human keratinocytes by prostaglandin E receptor EP 2 and EP 3 subtypes

We examined the contribution of specific EP receptors in regulating cell growth. By RT–PCR and northern hybridization, adult human keratinocytes express mRNA for three PGE 2 receptor subtypes associated with cAMP signaling (EP 2, EP 3, and small amounts of EP 4). In actively growing, non-confluent primary keratinocyte cultures, the EP 2 and EP 4 selective agonists, 11-deoxy PGE 1 and 1-OH PGE 1, caused complete reversal of indomethacin-induced growth inhibition. The EP 3/EP 2 agonist (misoprostol), and the EP 1/EP 2 agonist (17-phenyl trinor PGE 2), showed less activity. Similar results were obtained with agonist-induced cAMP formation. The ability of exogenous dibutyryl cAMP to completely reverse indomethacin-induced growth inhibition support the conclusion that growth stimulation occurs via an EP 2 and/or EP 4 receptor-adenylyl cyclase coupled response. In contrast, activation of EP 3 receptors by sulprostone, which is virtually devoid of agonist activity at EP 2 or EP 4 receptors, inhibited bromodeoxyuridine uptake in indomethacin-treated cells up to 30%. Although human EP 3 receptor variants have been shown in other cell types to markedly inhibit cAMP formation via a pertussis toxin sensitive mechanism, EP 3 receptor activation and presumably growth inhibition was independent of adenylyl cyclase, suggesting activation of other signaling pathways.

Generation second messengers by prostanoids in the iris-sphincter and ciliary muscles of cows, cats and humans

We have examined the generation of second messengers after stimulation of feline, bovine, human iris-sphincter and ciliary muscles by selected prostaglandins (PGs). The tissues, labeled or unlabeled with 3H-myo-inositol, were stimulated by a range of concentrations of 16, 16-dimethyl PGE 2, 11-deoxy PGE 1, 17-phenyl trinor PGE 2 and PGF 2α. In both tissues of all three species, 16, 16-dimethyl PGE 2 and 11-deoxy PGE 1 stimulated the formation of cyclic AMP. Butaprost, an EP 2 receptor agonist, which was tested only in feline ciliary muscle, generated cyclic AMP. In the feline iris-sphincter and in bovine and feline ciliary muscles, 17-phenyl trinor PGE 2, an EP 1 receptor agonist, significantly increased inositol phosphate turnover. The FP receptor agonist, PGF 2α stimulated inositol phosphate turnover in the bovine, feline, and human iris-sphincter muscles and in human ciliary muscles. Feline and bovine ciliary muscles did not respond to PGF 2α. These results suggest that EP 1 receptors are present in feline iris-sphincter muscle and in bovine and feline ciliary muscles. The EP 2 receptors exist in both tissue. These results also suggest the presence FP receptors in bovine, feline, and human iris-sphincter and in human ciliary muscles. Bovine and feline ciliary muscles do not appear to express FP receptors.

A specific prostaglandin E 2 receptor and its role in modulating salivary secretion in the female tick, Amblyomma americanum (L.)

Prostaglandins of the 2-series (e.g. PGE 2) are typically synthesized from arachidonic acid (AA) after AA is released from cellular phospholipids after activation of an intracellular phospholipase A 2 (PLA 2). Treatment of isolated salivary glands with PLA 2 inhibitor oleyloxyethyl phosphorylcholine (OPC) or prostaglandin synthetase inhibitors reduced dopamine-induced fluid secretion and cyclic AMP (cAMP) levels in isolated salivary glands. PGE 2 and its analog, 17-phenyl trinor PGE 2, partly reversed the inhibition of secretion and cAMP level by OPC, suggesting that prostaglandins may have an autocrine effect in modulating tick salivary gland function. A specific PGE 2 receptor was identified in the plasma membrane fraction of the salivary glands. The receptor exhibits a single, high affinity PGE 2 binding site with a K D≈ 29 nM, is saturable, reversible, and specific for PGE 2 and coupled to a cholera toxin-sensitive guanine nucleotide regulatory protein. Assay of adenylate cyclase activity in salivary gland membranes showed that PGE 2 neither stimulated nor inhibited adenylate cyclase activity, indicating that the PGE 2 effects on cAMP levels and possibly secretion are indirect, and that the PGE 2 receptor stimulates an alternate “second messenger” pathway.

PGE2, mainly through EP3 receptors, regulates expression of the constitutive neuronal and endothelial nitric oxide synthases (n- and e-NOS) in brain during the perinatal period. † 244

Increased NOS activity in brain during the perinatal period may be important for preservation of cellular and circulatory functions; but the mechanisms that regulate NOS activity are not known. Prostaglandins (PGs) may affect (inducible) NOS expression in cells. We postulated that high PG levels in the perinatal period control constitutive NOS expression in neural tissue. In rat brain n-NOS and cyclooxygenase (COX)-2 mRNA were highest on postnatal day 10 and thereafter declined by ≈4-fold to adult levels. Likewise in pig brain and microvessels, n-NOS, e-NOS and COX-2 mRNA were 3-4 fold higher in newborn (NB) than adult; (COX-1 was stable). Corresponding nitrite (stable product of NO) and PG synthesis were also 5-8 fold higher in NB than adult tissues. To assess if high PG levels control NOS expression in the perinatal period, NB rats were treated 24 h i.p. with COX-2 selective (nimesulide 1 mg/kg/day) or non-selective COX inhibitors (ibuprofen 40 mg/kg/day) to reduce brain PG levels to those of adult. Consistent with predominant COX-2 role in PG formation in NB brain, both COX inhibitors reduced comparably n-NOS mRNA. The in vivo effect of COX inhibitors in rat brain n-NOS was confirmed for e-NOS on isolated NB pig cerebral microvessels incubated 18 h with COX blockers: e-NOS mRNA and nitrite production in NB brain microvasculature decreased 4-fold to adult values after COX inhibition. In addition, the PGE2 analogue, 16,16-dimethyl PGE2, but not the PGI2 analogue, carbacyclin, or PGD2 prevented this downregulation of NOS by COX inhibition. Of the 3 PGE2 receptors in brain EP3 (sulprostone) and EP1 (17-phenyl trinor PGE2) stimulation respectively increased and slightly reduced NOS expression; EP2 stimulation was ineffective. Data indicate that high perinatal PGE2 levels control constitutive NOS expression in brain mainly through EP3 activation. Because PGE2 and NO exert important cellular functions including cytoprotection, we speculate that the positive interaction described between these agents could facilitate their concerted actions in perinatal neuroprotection.

SHORT COMMUNICATION Intracellular calcium mobilization following prostaglandin receptor activation in human ciliary muscle cells

PURPOSE. To investigate the presence of specific prostaglandin receptors in primary cultures of human ciliary muscle cells by measuring agonist-stimulated intracellular calcium mobilization. METHODS. The ciliary muscle cells, cultured from postmortem human ciliary muscle explants, were first characterized by anti-desmin and anti-smooth muscle a-actin antibodies. Increase in intracellular calcium concentrations, in fura 2-AM loaded human ciliary muscle cells stimulated by prostaglandin receptor agonists, were determined with a digital fluorescence imaging system. RESULTS. The resting intracellular calcium concentration in the fura 2-AM loaded cells was 60.0 ± 6.0 nM. The threshold concentration of PG receptor agonists needed to increase the concentration of intracellular calcium was 10 - 8 M. The stimulation of these cells by PGF 2a, 17-phenyl trinor PGE 2, and U46619, the FP, EP 1, and TP receptor agonists, resulted in the dose-dependent increase of intracellular calcium. CONCLUSION. The results of the present study suggest that EP 1, FP, and TP receptors are present in human ciliary muscle cells.

Regulation of cerebrovascular prostaglandin E 2 (PGE 2) and PGF 2α receptors and their functions during development

The density of PGF2 alpha (FP) and PGE2 (EP) receptors on the cerebral microvasculature of the newborn is less than on that of the adult animal. High levels of prostaglandins in the newborn brain could be responsible for down-regulation of FP and EP receptors and their functions in the cerebral microvasculature. Cerebrovascular FP and EP receptor density, receptor-coupled second messenger production and cerebral vasoconstrictor responses to PGF2 alpha and PGE2 were studied in newborn pigs (1 to 2 days old) treated intravenously with ibuprofen (40 mg/kg every 6 hours for 48 hours) or saline, and compared with adults. Ibuprofen treatment in the newborn increased brain microvascular FP and EP receptor densities to levels found in that of adult pigs. Likewise brain microvessel inositol 1,4,5-triphosphate production in response to PGF2 alpha, fenprostalene (PGF2 alpha analog), PGE2, 17-phenyl trinor PGE2 (EP1 receptor subtype agonist) and M&B 28,767 (EP3 agonist) was greater in ibuprofen-treated newborn pigs; the latter was associated with increased vasoconstriction to these agents to levels comparable with those of adults. Steady-state levels of FP receptor mRNA in cerebral microvas-culature did not differ between saline-treated newborn, and ibuprofen-treated newborn and adult pigs. It is concluded that the low FP and EP receptor densities and receptor-coupled functions on newborn brain microvessels seem to be secondary to high levels of brain prostaglandins; the changes in receptor levels do not seem to be related to steady-state levels of receptor mRNA in brain microvessels.

ONTOGENY AND PERINATAL REGULATION OF CHOROIDAL PGE2 AND PGF2α RECEPTORS IN THE PIG. • 411

PGE2 and PGF2α seem to be important in the regulation of choroidal blood flow (ChBF), the main source of O2 to the retina. In contrast to the adult (A), the newborn (NB) does not exhibit autoregulation of ChBF, which suggests a lack of vasoconstriction. We tested the hypothesis that the type and number of PGE2 (EP) and PGF2α (FP) receptors on the choroid (a vascular tissue) of the NB favored less vasoconstriction compared to that of the A, and that high levels of these prostaglandins (PG) in the NB (2-3 fold higher than A) down-regulate EP and FP receptors and their functions. Binding of PGE2 and PGF2α, as well as stimulation of inositol 1,4,5 triphosphate (IP3) and vasomotor reponses to these PG were tested in A, NB (2-3 days old) and NB treated with ibuprofen(Ibu, 30 mg/kg/12 h iv × 24 h [decreased PG to A levels]). Maximum binding (Bmax) of PGE2 and PGF2α was 3-4 times lower in NB than A choroid. EP receptors in NB were equally divided between EP1 and EP2 subtypes; EP3 and EP4 were not detected. In the A≈80% of EP receptors were EP1; the rest was EP2 and EP3 (≈10% each). PGE2 and butaprost (EP2 agonist) caused vasodilation of NB choroidal vessels but hardly none of A. PGF2α, 17-phenyl trinor PGE2 (EP1 agonist) and MB effects of EP1 and FP stimulation were associated with corresponding increases in IP3. Ibu increased Bmax of PGE2 and PGF2α in NB as well as the vasoconstrictor response to EP1, EP3 and FP agonists to levels of the A; Ibu virtually abolished dilation to PGE2 but did not affect response to butaprost. In summary, decreased constriction to EP and FP agonists in the NB is associated with fewer receptors and relatively higher proportion of EP2 in the NB compared to A. Reduction of PG levels in the NB induces up-regulation of FP, EP1 and EP3 (but not EP2) receptors and their functions to levels of the A. Data suggest that high levels of PG in the perinatal period down-regulate EP1, EP3 and FP receptors, which together with increased EP2 elicit decreased choroidal vasoconstriction to PGF2α and PGE2; the findings explain the absence of ChBF autoregulation in the NB leading to increased O2 delivery to the eye and a risk for retinopathy of prematurity.

Characterization and ontogeny of PGE 2 and PGF 2α receptors on the retinal vasculature of the pig

The vasoconstrictor effects of PGE 2 and PGF 2α are less pronounced on retinal vessels of the newborn than of the adult pig. We tested the hypothesis that the decreased vasomotor response to these prostaglandins might be due to relatively fewer receptors and/or different receptor subtypes (in the case of PGE 2) on retinal vessels of the newborn animal. Binding studies using [ 3H]PGE 2 and [ 3H]PGF 2α revealed that PGE 2 (EP) and PGF 2α (FP) receptor densities in retinal microvessel membrane preparations from newborn animals were approximately 25% of those found in vessels from the adult. The K d for PGF 2α did not differ; however, the K d for PGE 2 was less in newborn than in adult vessels. Competition binding studies using AH 6809 (EP 1 antagonist), butaprost (EP 2 agonist), M&B 28,767 (EP 3 agonist), and AH 23848B (EP 4 antagonist) suggested that the retinal vessels of the newborn contained approximately equal number of EP 1 and EP 2 receptor subtypes whereas the main receptor subtype in the adult vessels was EP 1. In addition, PGE 2 and butaprost produced comparable increases in adenosine 3′,5′-cyclic monophosphate synthesis in newborn and adult vessels. PGE 2, 17-phenyl trinor PGE 2 (EP 1agonist) and PGF 2α caused a 2.5 to 3-fold greater increase in inositol1,4,5-triphosphate (IP 3) formation in adult than in newborn preparations. It is concluded that fewer PGF 2α receptors and an associated decrease in receptor-coupled IP 3 formation in the retinal vessels of the newborn could lead to weaker vasoconstrictor effects of PGF 2α on retinal vessels of the newborn than of adult pigs; fewer EP 1 receptors (associated with vasoconstriction) and a relatively greater proportion of EP 2 receptors (associated with vasodilation) might be responsible for the reduced retinal vasoconstrictor effects of PGE 2 in the newborn.

Muscarinic Stimulation of Arachidonic Acid Release and Prostaglandin Synthesis in Bovine Ciliary Muscle: Prostaglandins Induce Cyclic AMP Formation and Muscle Relaxation

In the present study it is demonstrated that in bovine ciliary muscle, muscarinic stimulation results in; (a) release of 14C-arachidonic acid (14C-AA) and 14C-labeled prostaglandins (PGs) from muscle prelabeled with 14C-AA; (b) release of endogenous PGs, measured by means of radioimmunoassay; (c) enhanced IP3 production and (d) muscle contraction. In addition, PGs, such as PGE2 and PGD2, increased cAMP formation and induced muscle relaxation. The studies on the kinetics of 14C-AA metabolism revealed that incorporation of 14C-AA into glycerolipids and its conversion into PGs by the ciliary muscle were rapid and time-dependent. The amounts of 14 C-radioactivity recovered in the major PGs decreased in the following order: PGD2 > PGE2 < PGF2α > 6-keto-PGF1a. The rate of endogenous PGF2α synthesis by iris-ciliary body tissues from different mammalian species was found to be in the following order: ciliary muscle < ciliary processes < sphincter muscle. The EC50s for muscarinic-stimulated release of 14C-AA, 14C-labeled PGs, and endogenous PGF2α and PGE2, and for IP3 production and contraction of the ciliary muscle indicate that CCh is 2-16 times as potent as pilocarpine in eliciting these responses, with the greatest difference being for contraction. The maximal increase in ciliary muscle tension due to CCh was 48% greater than that evoked by pilocarpine. All PGs tested, including PGE2, 17-phenyl trinor PGE2, 11-deoxy PGE1, PGF2α and PGD2 had no effect on IP3 production and contraction in the ciliary muscle. However, PGE2 and PGD2 stimulated cAMP formation and inhibited CCh-induced IP3 production in a dose-dependent manner. In addition, PGE2 and PGD2 induced relaxation in ciliary muscle precontracted by CCh. In presence of indomethacin (1 μM), the CCh-induced contraction was greater than that observed in absence of the cyclo-oxygenase inhibitor. It is suggested that in the ciliary muscle certain PGs, such as PGE2 and PGD2, may function to modulate, via cAMP, the responses to muscarinic stimulation in this tissue.

Reduced responses of retinal vessels of the newborn pig to prostaglandins but not to thromboxane

The upper blood pressure limit of retinal blood flow autoregulation is lower in the newborn than in the adult; this suggests an insufficient vasoconstrictor response in the newborn when perfusion pressure is increased. Because prostaglandins (PGs) have an important role in autoregulation of retinal blood flow, we compared the effects of PGE2, PGF2 alpha, carbacyclin (PGI2 analogue), and U46619 (thromboxane analogue), as well as that of agonists for the three different PGE2 receptor subtypes, 17-phenyl trinor PGE2 (EP1), butaprost (EP2), and M&B 28,767 (EP3), on the retinal vasculature of newborn and adult pigs, using isolated eyecup preparations. PGF2 alpha and PGE2 caused a markedly greater constriction of retinal arteries and veins of the adult than of the newborn animals. Further analysis of the response to PGE2, using receptor subtype agonists, revealed that the EP1 receptor agonist, 17-phenyl trinor PGE2, and the EP3 receptor agonist, M&B 28,767, caused a significant constriction of adult arteries and veins but produced minimal effects on newborn vessels; the EP2 receptor agonist, butaprost, caused a small and comparable dilation of newborn and adult arteries and veins. The PGI2 analogue, carbacyclin, caused a greater dilation of the adult than of the newborn arteries, but produced comparable dilation of veins from both newborn and adult animals. In contrast to the effects of PGF2 alpha and PGE2, the thromboxane analogue, U46619, as well as the alpha 1-adrenoceptor agonist, phenylephrine, significantly constricted newborn arteries and veins, and this effect was comparable with that observed on retinal vessels of the adult.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and expression of cDNA for a mouse EP1 subtype of prostaglandin E receptor.

A functional cDNA clone encoding a mouse EP1 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse thromboxane A2 receptor cDNA. The clone isolated encodes a protein consisting of 405 amino acid residues with putative seven-transmembrane domains. [3H]PGE2 specifically bound to the membrane of Chinese hamster ovary cells expressing this clone. The binding to the membrane was displaced with unlabeled PGs in the order of PGE2 > iloprost (a prostacyclin analogue) > PGE1 > PGF2 alpha > U-46619 (a thromboxane A2 analogue) > PGD2. The binding was also inhibited by 17-phenyl trinor PGE2 (an EP1 agonist) and sulprostone (an EP1 and EP3 agonist) but not by 11-deoxy PGE1 (an EP2 and EP3 agonist) and butaprost (an EP2 agonist). PGE2 induced a rapid increase in intracellular Ca2+ concentration in Chinese hamster ovary cells expressing the receptor. These results suggest that this receptor belongs to EP1 subtype of PGE receptor. Northern blot analysis demonstrated that the mRNA of this receptor is expressed abundantly in kidney and in a lessor amount in lung.

Prostaglandin Receptors Coupled to Adenylyl Cyclase in the Iris-Ciliary Body of Rabbits, Cats and Cows

Stimulation of adenylyl cyclase by prostaglandin (PG) receptor agonists was examined in the iris-ciliary body of rabbits, cats and cows. Iris-ciliary bodies were incubated with or without agonists in Krebs pyruvate buffer containing protease and cyclooxygenase inhibitors. The formation of cAMP in a 15-min period in response to the agonists was measured. In the rabbit iris-ciliary body, all PG receptor agonists at concentrations of 0·062-10·0 μM with the exception of sulprostone stimulated adenylyl cyclase. In this species, 17-phenyl trinor PGE 2 and PGF 2α stimulated adenylyl cyclase but only at high concentrations (> 5 μM). The order of potency of the agonists was dimethyl PGE 2 > PGE 2 > 11-deoxy PGE 1 = PGD 2 > iloprost > PGF 2α. The DP receptor antagonist BWA868C had no effect on EP agonist responses, indicating that PGE 2, dimethyl PGE 2 and 11-deoxy PGE 1 were not stimulating DP receptors. In the cat, PGE 2, dimethyl PGE 2 and 11-deoxy PGE 1, were all equally potent in stimulating adenylyl cyclase. The order of potency in the cow was PGE 2 > dimethyl PGE 2 > 11-deoxy PGE 1. However, PGD 2, iloprost, 17-phenyl trinor PGE 2 and PGF 2α were inactive in this species. The results of this study indicate that EP receptor agonists stimulate adenylyl cyclase in the iris-ciliary body of the three species: stimulation by other agonists depends on the species. In the rabbit, the EP 2 receptor subtype appears to predominate; such a predominance of EP 2 receptors was not observed in the cat and cow.