Steroidal Antagonists of Progesterone- and Prostaglandin E1-Induced Activation of the Cation Channel of Sperm.

The cation channel of sperm (CatSper) is the principal entry point for calcium in human spermatozoa and its proper function is essential for successful fertilization. As CatSper is potently activated by progesterone, we evaluated a range of steroids to define the structure-activity relationships for channel activation and found that CatSper is activated by a broad range of steroids with diverse structural modifications. By testing steroids that failed to elicit calcium influx as inhibitors of channel activation, we discovered that medroxyprogesterone acetate, levonorgestrel, and aldosterone inhibited calcium influx produced by progesterone, prostaglandin E1, and the fungal natural product l-sirenin, but these steroidal inhibitors failed to prevent calcium influx in response to elevated K+ and pH. In contrast to these steroid antagonists, we demonstrated for the first time that the T-type calcium channel blocker ML218 acts similarly to mibefradil, blocking CatSper channels activated by both ligands and alkalinization/depolarization. These T-type calcium channel blockers produced an insurmountable blockade of CatSper, whereas the three steroids produced antagonism that was surmountable by increasing concentrations of each activator, indicating that the steroids selectively antagonize ligand-induced activation of CatSper rather than blocking channel function. Both the channel blockers and the steroid antagonists markedly reduced hyperactivated motility of human sperm assessed by computer-aided sperm analysis, consistent with inhibition of CatSper activation. Unlike the channel blockers mibefradil and ML218, which reduced total and progressive motility, medroxyprogesterone acetate, levonorgestrel, and aldosterone had little effect on these motility parameters, indicating that these steroids are selective inhibitors of hyperactivated sperm motility. SIGNIFICANCE STATEMENT: The steroids medroxyprogesterone acetate, levonorgestrel, and aldosterone selectively antagonize progesterone- and prostaglandin E1-induced calcium influx through the CatSper cation channel in human sperm. In contrast to T-type calcium channel blockers that prevent all modes of CatSper activation, these steroid CatSper antagonists preferentially reduce hyperactivated sperm motility, which is required for fertilization. The discovery of competitive antagonists of ligand-induced CatSper activation provides starting points for future discovery of male contraceptive agents acting by this unique mechanism.

Acute effects of prostaglandin E1 and E2 on vascular reactivity and blood flow in situ in the chick chorioallantoic membrane.

The chick chorioallantoic membrane (CAM) subserves gas exchange in the developing embryo and shell-less culture affords a unique opportunity for direct observations over time of individual blood vessels to pharmacologic interventions. We tested a number of lipids including prostaglandins PGE(1&2) for vascular effects and signaling in the CAM. Application of PGE(1&2) induced a decrease in the diameter of large blood vessels and a concentration-dependent, localized, reversible loss of blood flow through small vessels. The loss of flow was also mimicked by misoprostol, an agonist for 3 of 4 known PGE receptors, EP(2-4), and by U46619, a thromboxane mimetic. Selective receptor antagonists for EP(3) and thromboxane each partially blocked the response. This is a first report of the effects of prostaglandins on vasoreactivity in the CAM. Our model allows the unique ability to examine simultaneous responses of large and small vessels in real time and in vivo.

Transport of prostaglandin E1 across rat erythrocyte membrane.

In this study erythrocyte transport of prostaglandin E1 (PGE1) was investigated by employing inside-out membrane vesicles prepared from rat erythrocytes. The uptake of [3H]PGE1 in the presence of ATP was significantly higher than that of AMP, suggesting the involvement of an ATP-dependent efflux system in PGE1 transport across the erythrocyte membrane. Coincubation of glutathione with ATP further stimulated the uptake of [3H]PGE1. The uptake of [3H]PGE1 in the presence of ATP and glutathione was temperature-sensitive, and various eicosanoids including PGE2 and PGF2alpha decreased the uptake. Multidrug resistance-associated protein (MRP) 4 substrates/inhibitors including methotrexate, indomethacin, taurocholic acid and indocyanine green significantly inhibited [3H]PGE1 uptake. Western blot analysis revealed that Mrp4 is expressed in rat erythrocyte membrane. These results suggest that the release of PGE1 from the erythrocyte into the blood circulation may be mediated by ATP-dependent efflux pump(s) such as Mrp4.

Involvement of EP1 and EP2 receptors in the regulation of the Na,K-ATPase by prostaglandins in MDCK cells.

Prostaglandins are key regulators of ion transport in the kidney. In MDCK cells, which model distal tubule cells, the transcription of the Na,K-ATPase beta1 subunit is regulated by PGE1 and PGE2. To identify the EP receptors that mediate transcriptional regulation, transient transfection studies are conducted using the human beta1promoter/luciferase construct, pHbeta1-1141 Luc. The involvement of EP1 and EP2 receptors is indicated by studies with the EP1 selective agonist 17-phenyl trinor PGE2, and the EP2 selective agonist butaprost (which stimulate), as well as by studies with the antagonists SC-51089 (EP1 specific) and AH 6809 (EP1 and EP2 specific). Consistent with the involvement of Gs coupled EP2 receptors, is that the PGE1 stimulation is inhibited by the PKAI expression vector (encoding the protein kinase A (PKA) inhibitory protein), as well as by the myristolated PKA inhibitory peptide PKI. In addition to this evidence (for the involvement of EP2 receptors), evidence for the involvement of EP1 receptors in the PGE1 mediated stimulation of Na,K-ATPase beta subunit gene transcription includes the stimulatory effect of 17-phenyl trinor PGE2, as well as the inhibitory effects of SC-51089. Also consistent with the involvement of Gq coupled EP1 receptors, the PGE1 stimulation is inhibited by the PKCI vector (encoding the PKC inhibitory domain), the PKC inhibitor Go 6976, thapsigargin, as well as the calmodulin antagonists W7 and W13.

Platelet activation in cystic fibrosis.

Cystic fibrosis (CF) is caused by a mutation of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). We examined platelet function in CF patients because lung inflammation is part of this disease and platelets contribute to inflammation. CF patients had increased circulating leukocyte-platelet aggregates and increased platelet responsiveness to agonists compared with healthy controls. CF plasma caused activation of normal and CF platelets; however, activation was greater in CF platelets. Furthermore, washed CF platelets also showed increased reactivity to agonists. CF platelet hyperreactivity was incompletely inhibited by prostaglandin E(1) (PGE(1)). As demonstrated by Western blotting and reverse-transcriptase-polymerase chain reaction (RT-PCR), there was neither CFTR nor CFTR-specific mRNA in normal platelets. There were abnormalities in the fatty acid composition of membrane fractions of CF platelets. In summary, CF patients have an increase in circulating activated platelets and platelet reactivity, as determined by monocyte-platelet aggregation, neutrophil-platelet aggregation, and platelet surface P-selectin. This increased platelet activation in CF is the result of both a plasma factor(s) and an intrinsic platelet mechanism via cyclic adenosine monophosphate (cAMP)/adenylate cyclase, but not via platelet CFTR. Our findings may account, at least in part, for the beneficial effects of ibuprofen in CF.

Formation and antiproliferative effect of prostaglandin E(3) from eicosapentaenoic acid in human lung cancer cells.

We investigated the formation and pharmacology of prostaglandin E(3) (PGE(3)) derived from fish oil eicosapentaenoic acid (EPA) in human lung cancer A549 cells. Exposure of A549 cells to EPA resulted in the rapid formation and export of PGE(3.) The extracellular ratio of PGE(3) to PGE(2) increased from 0.08 in control cells to 0.8 in cells exposed to EPA within 48 h. Incubation of EPA with cloned ovine or human recombinant cyclooxygenase 2 (COX-2) resulted in 13- and 18-fold greater formation of PGE(3), respectively, than that produced by COX-1. Exposure of A549 cells to 1 microM PGE(3) inhibited cell proliferation by 37.1% (P < 0.05). Exposure of normal human bronchial epithelial (NHBE) cells to PGE(3), however, had no effect. When A549 cells were exposed to EPA (25 microM) or a combination of EPA and celecoxib (a selective COX-2 inhibitor), the inhibitory effect of EPA on the growth of A549 cells was reversed by the presence of celecoxib (at both 5 and 10 microM). This effect appears to be associated with a 50% reduction of PGE(3) formation in cells treated with a combination of EPA and celecoxib compared with cells exposed to EPA alone. These data indicate that exposure of lung cancer cells to EPA results in a decrease in the COX-2-mediated formation of PGE(2), an increase in the level of PGE(3), and PGE(3)-mediated inhibition of tumor cell proliferation.

Separation of Leucas aspera, a medicinal plant of Bangladesh, guided by prostaglandin inhibitory and antioxidant activities.

According to the traditional usage of the plant for antiinflammation and analgesia, Leucas aspera was tested for its prostaglandin (PG) inhibitory and antioxidant activities. The extract showed both activities, i.e., inhibition at 3 x 10(-4) g/ml against PGE(1)- and PGE(2)-induced contractions in guinea pig ileum and a 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging effect. The separation guided by the activities in these dual assay methods provided eight lignans and four flavonoids, LA-1- -12, among which LA-1- -7 and LA-10- -12 were identified as nectandrin B, meso-dihydroguaiaretic acid, macelignan, acacetin, apigenin 7-O-[6"-O-(p-coumaroyl)-beta-D-glucoside], chrysoeriol, apigenin, erythro-2-(4-allyl-2, 6-dimethoxyphenoxy)-1-(4-hydroxy-3-methoxyphenyl)propan-1-ol, myristargenol B, and machilin C, respectively. LA-8 was determined to be (-)-chicanine, the new antipode of the (+) compound, by spectroscopic methods including CD and ORD. Chiral-HPLC analysis of LA-9 showed that it was a mixture of two enantiomers, (7R, 8R)- and (7S, 8S)-licarin A. All of these components were first isolated from L. aspera. PG inhibition was observed in LA-1, LA-2, and LA-5, and antioxidant activity in LA-1- -3 and LA-8- -12.

Inhibition of protein kinase C decreases prostaglandin-induced breakdown of the blood-retinal barrier.

Breakdown of the blood-retinal barrier (BRB) occurs in several retinal diseases and is a major cause of visual loss. Vascular endothelial growth factor (VEGF) has been implicated as a cause of BRB breakdown in diabetic retinopathy and other ischemic retinopathies, and there is evidence to suggest that other vasopermeability factors may act indirectly through VEGF. In this study, we investigated the effect of several receptor kinase inhibitors on BRB breakdown resulting from VEGF, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), insulin-like growth factor-1 (IGF-1), prostaglandin E1 (PGE(1)), or PGE(2). Inhibitors of VEGF receptor kinase, including PKC412, PTK787, and SU1498, decreased VEGF-induced breakdown of the BRB. None of the inhibitors blocked leakage caused by TNF-alpha, IL-1beta, or IGF-1 and only PKC412, an inhibitor of protein kinase C (PKC) as well as VEGF and platelet-derived growth factor (PDGF) receptor kinases, decreased leakage caused by prostaglandins. Since the other inhibitors of VEGF and/or PDGF receptor kinases that do not also inhibit PKC had no effect on prostaglandin-induced breakdown of the BRB, these data implicate PKC in retinal vascular leakage caused by prostaglandins. PKC412 may be useful for treatment of post-operative and inflammatory macular edema, in which prostaglandins play a role, as well as macular edema associated with ischemic retinopathies.

Arg60 Leu mutation in the first cytoplasmic loop of the platelet thromboxane A2 receptor is not essential for mediating inhibitory coupling between the receptor and adenylyl cyclase.

Human platelet thromboxane A2 (TXA2) receptor (TXR) has been reported to functionally couple to the inhibitory GTP-binding protein for adenylyl cyclase (Gi). However, it still remains unclear which portions of the TXR structure are critical determinants in that coupling. We have previously reported several patients with platelet dysfunction, whose platelets showed impaired coupling between TXR and phospholipase C caused by an Arg60 to Leu mutation in the first cytoplasmic loop. To investigate whether this portion is essential for mediating inhibitory coupling between TXR and adenylyl cyclase, we analyzed the inhibition by the TXA2 analog of the PGE1 or forskolin-induced platelet cAMP increase in patients' platelets, and found that the inhibition occurred normally. This suggests that Arg60 in the first cytoplasmic loop of the TXR is not involved in TXR-Gi coupling.

Specific impairment of human platelet P2Y(AC) ADP receptor-mediated signaling by the antiplatelet drug clopidogrel.

Clopidogrel is an effective new antiplatelet agent useful for the treatment of ischemic cerebrovascular, cardiac, and peripheral arterial disease. However, the mechanism of clopidogrel action is not well understood, although it is known to inhibit ADP-evoked platelet aggregation. In the current study, the effect of clopidogrel on recently identified human platelet ADP receptors and their signaling pathways was investigated by using platelets from clopidogrel-treated subjects, 6 healthy volunteers (2 females and 4 males) who received 75 mg of clopidogrel daily for 7 days. Blood was taken and various platelet receptor signaling pathways were analyzed before treatment, after 7 days of medication, and 4 weeks after treatment had ceased. Platelet tests included the analysis of aggregation, rapid calcium influx, calcium mobilization from intracellular stores, adenylyl cyclase, and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The data indicate that clopidogrel does not affect those platelet ADP receptors coupled to cation influx (P2X1 ADP receptors) or calcium mobilization (P2Y1 ADP receptors). In contrast, clopidogrel treatment specifically impairs the ADP receptor coupled to G(i)/adenylyl cyclase (P2Y(AC) ADP receptors). Clopidogrel abolishes the inhibitory P2Y(AC) receptor-mediated ADP effects on prostaglandin E(1)-stimulated, cAMP-dependent phosphorylation of VASP without affecting epinephrine, thrombin, and thromboxane signaling. VASP phosphorylation is known to be closely correlated with the inhibition of platelet and fibrinogen receptor (glycoprotein IIb/IIIa) activation. Therefore, inhibition of the platelet P2Y(AC) ADP receptor and its intracellular signaling, including decreased VASP phosphorylation, is suggested as a molecular mechanism of clopidogrel action.

Effect of ganglionic blocking compounds on in-vivo fluid secretion in the rat small intestine.

It is well-known that enteric, secreto-motor nerves mediate cholera toxin-induced fluid secretion in the rat small intestine. This notion is, in part, derived from experiments on anaesthetized animals in which the response to cholera toxin was antagonized by the ganglionic nicotinic receptor antagonist, hexamethonium. In the current study, such anti-secretory action of ganglionic blocking compounds was analysed in an experiment designed to minimize any possible negative effect of general anaesthesia on intestinal secretion. Rats were anaesthetized with ether for 5-10 min, during which time a jejunal loop (10-12 cm) was constructed. The loop was challenged with one of the secretagogues, cholera toxin, prostaglandin E1 (PGE1) or okadaic acid. Saline (control) or either of the ganglionic blockers, hexamethonium and chlorisondamine, was administered intravenously. The rats were killed 5 h (cholera toxin) or 1.5 h (PGE1 and okadaic acid) after challenge, and the amount of fluid accumulated in the loops was determined. Cholera toxin-induced secretion was unchanged by hexamethonium but reduced by approximately 80% by chlorisondamine. The difference in effect between the two blockers might relate to the duration of ganglionic blockade. Chlorisondamine blocked secretion induced by either PGE1 or okadaic acid by approximately 60%. It is suggested that the anti-secretory effect of ganglionic blocking compounds might be a result of blockade of secreto-motor nerves but other mechanisms, for example interference with haemodynamic factors, cannot be ruled out.

Differential regulation of receptor-stimulated cyclic adenosine monophosphate production by polyvalent cations in MC3T3-E1 osteoblasts.

Extracellular cations have paradoxical trophic and toxic effects on osteoblast function. In an effort to explain these divergent actions, we investigated in MC3T3-E1 osteoblasts if polyvalent cations differentially modulate the agonist-stimulated cyclic adenosine monophosphate (cAMP) pathway, an important regulator of osteoblastic function. We found that a panel of cations, including gadolinium, aluminum, calcium, and neomycin, inhibited prostaglandin E1 (PGE)-stimulated cAMP accumulation but paradoxically potentiated parathyroid hormone (PTH)-stimulated cAMP production. In contrast, these cations had no effect on forskolin- or cholera toxin-induced increases in cAMP, suggesting actions proximal to adenylate cyclase and possible modulation of receptor interactions with G proteins. Phorbol 12-myristate 13-acetated (PMA) mimicked the effects of cations on PGE1- and PTH-stimulated cAMP accumulation in MC3T3-E1 cells, respectively, diminishing and augmenting the responses. Moreover, down-regulation of protein kinase C (PKC) by overnight treatment with PMA prevented gadolinium (Gd3+) from attenuating PGE1- and enhancing PTH-stimulated cAMP production, indicating involvement of PKC-dependent pathways. Cations, however, activated signal transduction pathways not coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), since there was no corresponding increase in inositol phosphate formation or intracellular calcium concentrations. In addition, pertussis toxin treatment failed to prevent Gd(3+)-mediated suppression of PGE1-stimulated cAMP, suggesting actions independent of Gm. Thus, polyvalent cations may either stimulate or inhibit hormone-mediated cAMP accumulation in osteoblasts. These differential actions provide a potential explanation for the paradoxical trophic and toxic effects of cations on osteoblast function that occur in vivo under different hormonal conditions.

A possible approach to the suppression of side effects induced by PGE1.

Prostaglandin E1 (PGE1) is known to possess various actions in vivo. Of these actions, the contraction of the ileum and inflammation are undesirable side effects. We previously proposed a hypothesis concerning the receptors for human blood platelet aggregation and its inhibition, and the contraction of the ileum and uterus based on a study of structure-activity relationships. If the same principle can be applied to contraction of the ileum and inflammation induced by PGE1, compounds that suppress the side effects of PGE1 can be developed. The antihistamine diphenylpyraline and the anticholinergic atropine antagonized PGE1-induced contraction of the ileum in guinea pigs. Papaverine, which is a smooth muscle relaxant, also acted as an antagonist. Gabexate mesylate (FOY), a non-peptide proteinase inhibitor, inhibits guinea pig ileum contraction induced by PGE1, but epsilon-guanidinocaproic acid (GCA), a metabolite of FOY, does not. Increased microvascular permeability of the abdominal skin in rats induced by the local injection of PGE1 and histamine was suppressed by atropine, papaverine and diphenylpyraline. FOY, not GCA, had a weak inhibitory action. We demonstrate the possibility of suppressing the side effects of PGE1 based on the results obtained in the present and previous studies.

Effects of extracellular divalent cations on responses of human blood platelets to adenosine 5'-diphosphate.

The effects of extracellular divalent cations on the responses of human platelets to adenosine 5'-diphosphate (ADP) and on its inhibition by the competitive antagonist adenosine 5'-triphosphate (ATP) were investigated. Two responses were studied, shape change and the inhibition of prostaglandin E1 (PGE1)-stimulated adenylate cyclase, and experiments were carried out in the presence of divalent cations (Ca2+ and Mg2+, 1 mM) or in their absence. For each response there was a small leftward shift of the concentration-response curve to ADP in the absence of divalent cations compared to that in their presence, and this leftward shift disappeared when the results were plotted in terms of ADP3- rather than total ADP concentration. The shape change results were, however, complicated by a reduction in the maximal response to ADP in the absence of divalent cations. For each response there was also a marked increase in the pA2 value of ATP in the absence of divalent cations compared to that in their presence, and this difference disappeared if the results were calculated in terms of ATP4- instead of total ATP. These results suggest that the human platelet ADP receptor, in common with other receptors for adenine nucleotides, recognises predominantly the uncomplexed forms of ADP and ATP as ligands.

Intracellular translocation of rap 1-B G-protein induced by prostaglandin E1 is blocked by phorbol ester in human platelets.

Prostaglandin E1 induced increase in cytosolic immunoreactive rap 1-B G-protein in a time and concentration dependent manner in human platelets. However, this increase was completely blocked by simultaneous treatment of platelets with phorbol 12-myristate 13-acetate (PMA).

Stimulatory (EP1 and EP3) and inhibitory (EP2) prostaglandin E2 receptors in isolated ileal smooth muscle cells.

Isolated smooth muscle cells from the circular layer of pig and guinea-pig ileum were used to study the effect of prostaglandin E2 (PGE2) and three PGE2 receptor (EP) agonists; iloprost (EP1), butaprost (EP2) and enprostil (EP3). In pig cells, PGE2 and enprostil induced cell contraction (22.1 and 21.5% shortening of cell length, obtained at 10 nM for PGE2 and 1 nM for enprostil, respectively). Iloprost and butaprost had no contractile effect. However, the cholecystokinin octapeptide (CCK-8; 10 nM)-induced contraction was inhibited when cells were preincubated with iloprost or butaprost. In guinea-pig cells, PGE2, butaprost and iloprost induced cell contraction, whereas enprostil had no effect (23.1% for 10 nM PGE2, 22.8% for 1 nM butaprost and 22.6% for 10 nM iloprost). Preincubation with SC19220 (EP1 antagonist) inhibited the PGE2-, butaprost- and iloprost-induced contractions. When the contractile effect of PGE2, butaprost and iloprost was inhibited by addition of SC19220, these agents inhibited the cell contraction induced by CCK-8 (1 nM). Smooth muscle cells from guinea-pig and pig ileum express two PGE2 receptor subtypes that induce opposite effect. EP1 and EP3 receptors mediate cell contraction in guinea-pig and pig, respectively, whereas EP2 receptors mediate cell relaxation in both species.

Cortical spreading depression blocks prostaglandin E1 and endotoxin fever in rats.

We have tested the hypothesis that the cortex may play a role in the development of fever. Male Sprague-Dawley rats equipped with AM transmitters for telemetric measurement of body temperature were given intracerebroventricular prostaglandin E1 (PGE1), corticotropin-releasing hormone (CRH), or intravenous E. coli endotoxin. Application of cotton pellets soaked with 3.3 M KCl to the frontal cortex (to induce spreading depression) significantly reduced fevers to PGE1 and endotoxin when compared with fever magnitude with 0.9% NaCl application to the cortex. Neither CRH-induced hyperthermia nor normal body temperatures were altered by the spreading depression. Our results reveal a novel action of spreading depression on thermoregulatory function and indicate cortical involvement in the development of fever.

Further studies on the potent positive chronotropic effect of (15S)-15-methyl-prostaglandin E1 on the guinea-pig isolated spontaneously beating right atrium.

1. 15-Me-PGE1 (10(-14)-10(-11) M) elicited a concentration-dependent and long-lasting increase in heart rate without an effect on contractility of the isolated spontaneously beating guinea-pig right atria. 2. Noradrenaline, histamine and PGE1 produced a concentration-dependent increase in both heart rate and contractility with relatively higher concentrations. 3. beta-Adrenoceptor blocker propranolol and histamine H2-receptor blocker cimetidine inhibited the effects of noradrenaline and histamine respectively without altering the positive chronotropic effect of 15-Me-PGE1. 4. Prazosin and lidocaine partially inhibited the effect of 15-Me-PGE1, while reducing Na+ concentration in the medium to 50 mM almost completely inhibited the positive chronotropic effect of the analog. 5. Calcium channel blocker, nicardipine, decreased the positive chronotropic effect of 15-Me-PGE1 in a concentration-dependent manner. 6. These results were taken as an evidence for the specific and unique effect of 15-Me-PGE1 in the guinea-pig sinoatrial node interacting with Na+ and Ca2+ fluxes probably through their specific exchangers.

Thromboxane receptor stimulation inhibits adenylate cyclase and reduces cyclic AMP-mediated inhibition of ADP-evoked responses in fura-2-loaded human platelets.

Stimulation of human platelets with the thromboxane A2 analogue, U46619, after treatment with prostaglandin E1 or forskolin, reduced the inhibition of ADP-evoked Mn2+ influx and the release of Ca2+ from intracellular stores. U46619 decreased the elevated concentration of 3',5'-cyclic AMP in platelets that were pretreated with prostaglandin E1. These results suggest that occupation of prostaglandin H2/thromboxane A2 receptors, like those for other agonists, inhibits adenylate cyclase activity, which can contribute to the promotion of platelet activation.