Bovine cyclic endometrium contains high-affinity luteinizing hormone/human chorionic gonadotropin binding sites

High-affinity LH/hCG binding sites have been characterized in porcine, lepine, and murine uteri. In the present study, LH/hCG binding sites were characterized in bovine endometrium. Radioreceptor assays were performed with membrane homogenates of endometrial tissues and analyzed for binding site specificity and capacity. There was little competition for receptor occupancy between hCG and ovine FSH (5%) or ovine prolactin (< 0.1%), but there was a 20% cross-reaction with eCG. There was no affinity for LH/hCG in crude membrane preparations of kidney, skeletal muscle, or vascular tissues. Concentrations of endometrial LH/hCG binding sites were determined during the bovine estrous cycle. LH/hCG receptors were found in cell preparations from Days 2-4 and 15-17 of the cycle, but not in preparations from the other stages of the cycle tested (Days 8-12, pre- and post-estrus, and ovulation). The concentration of uterine LH/hCG receptor varied during the estrous cycle, with higher values at Days 15-17 (3.1 fmol/mg protein) and lower values at Days 2-4 (1.2 fmol/mg protein). However, the binding capacity of hCG by luteal cells (9.7 fmol/mg protein) was 3-fold higher (p < 0.01) than that by endometrial tissue on any day studied. No differences in affinity constant (Ka) were seen between endometrial LH/hCG receptors (either) from Days 2-4 or 15-17) and mid-cycle luteal cells (0.60 x 10(11) M-1). Using Western blot analysis, we determined the expression of cyclooxygenase (COX) during the estrous cycle of the cow. It was found that the signal for COX was strongest at 15-17 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

2-aminotetralin-derived substituted benzamides with mixed dopamine D2, D3, and serotonin 5-HT1A receptor binding properties: a novel class of potential atypical antipsychotic agents

A new chemical class of potential atypical antipsychotic agents, based on the pharmacological concept of mixed dopamine D2 receptor antagonism and serotonin 5-HT1A receptor agonism, was designed by combining the structural features of the 2-(N,N-di-n-propylamino)tetralins (DPATs) and the 2-pyrrolidinylmethyl-derived substituted benzamides in a structural hybrid. Thus, a series of 35 differently substituted 2-aminotetralin-derived substituted benzamides was synthesized and the compounds were evaluated for their ability to compete for [3H]-raclopride binding to cloned human dopamine D2A and D3 receptors, and for [3H]-8-OH-DPAT binding to rat serotonin 5-HT1A receptors in vitro. The lead compound of the series, 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (12a), displayed high affinities for the dopamine D2A receptor (Ki = 3.2 nM), the dopamine D3 receptor (Ki = 0.58 nM) as well as the serotonin 5-HT1A receptor (Ki = 0.82 nM). The structure-affinity relationships of the series suggest that the 2-aminotetralin moieties of the compounds occupy the same binding sites as the DPATs in all three receptor subtypes. The benzamidoethyl side chain enhances the affinities of the compounds for all three receptor subtypes, presumably by occupying an accessory binding site. For the dopamine D2 and D3 receptors, this accessory binding site may be identical to the binding site of the 2-pyrrolidinylmethyl-derived substituted benzamides.     

Demonstration of a novel circulating anti-prostacyclin receptor antibody

Although coronary artery disease (CAD) is appreciated to be accelerated in patients with chronic spinal cord injury (SCI), the underlying mechanism of CAD in SCI remains obscure. We have recently shown that platelets from subjects with SCI develop resistance to the inhibitory effect of prostacyclin (PGI2) on the platelet stimulation of thrombin generation. The loss of the inhibitory effect was due to the loss of high-affinity prostanoid receptors, which may contribute to atherogenesis in SCI. Incubation of normal, non-SCI platelets in SCI plasma (n = 12) also resulted in the loss of high-affinity binding of PGI2 (Kd1 = 9.1 +/- 2.0 nM; n1 = 170 +/- 32 sites per cell vs. Kd1 = 7.2 +/- 1.1 nM; n1 = 23 +/- 8 sites per cell), with no significant change in the low-affinity receptors (Kd2 = 1.9 +/- 0.1 microM; n2 = 1,832 +/- 232 sites per cell vs. Kd2 = 1. 6 +/- 0.1 microM; n2 = 1,740 +/- 161 sites per cell) as determined by Scatchard analysis of the binding of [3H]PGE1. The loss of high-affinity PGI2 binding led to the failure of PGI2 to inhibit the platelet-stimulated thrombin generation. The increase of cellular cyclic AMP level, mediated through the binding of PGI2 to low-affinity receptors in platelets, was unaffected in SCI platelets. PAGE and immunoblot of SCI plasma showed the presence of an IgG band, which specifically blocked the binding of [3H]PGE1 to the high-affinity PGI2 receptors of normal platelets. PAGE of the reduced IgG band, the amino acid sequence of the novel band as a heavy chain of IgG that inhibits the binding of [3H]PGE1 to the high-affinity platelet PGI2 receptor, demonstrates that the specific recognition and inhibition of high-affinity PGI2 binding to platelets was due to an anti-prostacyclin receptor antibody present in SCI plasma.     

Identification and characterization of cytosolic sulfotransferases in normal human endometrium

Understanding the factors which alter estrogen metabolism and activity in endometrial tissue is important because unopposed estrogen stimulation is an important risk factor in the development of endometrial carcinoma. The cyclic progression of the endometrium through proliferative and secretory phases is normally under the control of the ovarian hormones beta-estradiol (E2) and progesterone. One mechanism by which progesterone inhibits the activity of E2 in secretory endometrium is by elevating the degree of E2 sulfation, thereby reducing its ability to bind to the estrogen receptor and elicit a cellular response. Our laboratories have investigated the cytosolic sulfotransferases (STs) found in biopsies of both proliferative and secretory endometrium obtained from five normal pre-menopausal women who were not taking any drugs or steroids. Two of the human cytosolic STs were detected in human endometrial tissues. The phenol-sulfating form of phenol ST (P-PST) was found at varying levels in cytosol from both proliferative and secretory endometrium in all of the women studied but with no consistent correlation to the phase of the menstrual cycle. In contrast, estrogen ST (EST) was not detected in the proliferative endometrial cytosol of any of the women studied but was consistently found in all of the secretory endometrial cytosols. The presence and levels of these STs was confirmed by ST activity studies, immunoblot analysis and Northern blot analysis. These results indicate that the expression of EST in human endometrial tissues varies with the phase of the menstrual cycle and is most likely regulated by progesterone secreted from the ovaries.     

Cytologic study of ascites and the endometrium in ovarian carcinoma. Clinical significance

OBJECTIVE: To ascertain the usefulness of endometrial cytology with ovarian cancers when examining extension of the disease and to analyze significant factors associated with migration of ovarian cancer cells into the uterine cavity. STUDY DESIGN: Cytologic results on ascites and the endometrium were analyzed in 87 patients with primary ovarian cancer in the absence of metastasis to the endometrium or cervicovagina. RESULTS: Positive results for cytology were found in 62/87 of ascites cases (71.3%) and in 20/87 endometrium cases (23.0%). The 15 cases (15/62 or 24.2%) positive for ascitic and endometrial cytology, divided clinically into stage III (6 cases) and stage IV (9 cases), were classified histologically as serous, 7 cases; mucinous, 2 cases; clear cell, 4 cases; endometrioid, 1 case; and unclassified, 1 case. Half the clear cell carcinomas (4/8 or 50.0%) were positive in the ascites and endometrium. The ascitic volume at surgery exceeded 500 mL in 9/15 cases (60.0%). CONCLUSION: Papillae with basement membrane material in the cores may be structurally associated with migration of ovarian cancer cells into the uterine cavity, especially in clear cell carcinomas. Cytologic positivity of the endometrium and ascites significantly correlated with ascitic volume.     

Iron deposition at mineralization fronts and osteoid formation following chronic cadmium exposure in ovariectomized rats

Integrins are heterodimeric glycoproteins that have been found to undergo dynamic temporal and spatial changes in distribution in the endometrium during the menstrual cycle in women. Likewise the extracellular matrix (ECM) ligands for these receptors are likely to play a role in the establishment of a receptive endometrium. To develop primate models to study the role of these molecules in the cascade of molecular events leading to implantation, integrin expression and associated changes in ECM were investigated during the menstrual cycle and in early pregnancy in the baboon. Antibodies specific for the integrins (alpha(1-6) and alpha(v); beta1, beta3, and beta4) and ECM (laminin, collagen IV, fibronectin) were utilized. In addition, cytokeratin and alpha-smooth muscle actin were used as epithelial, stromal, and smooth muscle cell markers, respectively. Endometrium was obtained in duplicate or triplicate during the menstrual cycle and early pregnancy. Changes observed during the natural menstrual cycle were confirmed using ovariectomized, steroid-treated animals. Constitutively expressed integrins on the endometrial epithelium included the collagen/laminin receptors: alpha2, alpha3, alpha6, and beta4. The pattern of expression correlated well with the distribution of ECM in this tissue. Collagen IV was confined to the basement membrane of glandular epithelium and blood vessels. Laminin immunostaining was found in the basement membrane, mostly in the stroma of the basal region, in the glandular endometrium and vasculature. Fibronectin was present throughout the stroma but not in the basement membrane. The collagen receptor alpha1 beta1 and fibronectin receptor alpha4 beta1 appeared in the glandular epithelium in the luteal phase. As in the human, alpha1 and alpha4 disappeared from the glandular epithelium with the establishment of pregnancy. In contrast, the alpha4 beta3 vitronectin receptor appeared in the glandular epithelium only in pregnancy or following long-term steroid treatment with estrogen and progesterone but not during the time of uterine receptivity associated with the initial period of embryo attachment. Osteopontin, an ECM ligand for alpha(v) beta3, was coexpressed with this integrin in invading cytotrophoblasts, glandular epithelium, and decidualizing stromal cells. Decidualization in the baboon was associated with changes in integrin expression similar to those found in humans: there was an increase in alpha1, alpha3, alpha6, beta1, and alpha(v) beta3 in the decidualized stromal cells. Laminin and collagen IV expression also increased at the implantation site and throughout the endometrium. In contrast, fibronectin expression was most evident at the implantation site and corresponded to alpha5 expression on the invading cytotrophoblasts. In summary, marked similarities were found in the expression of ECM and the integrin receptors between the baboon and the human endometrium throughout the menstrual cycle and in pregnancy. Cycle-specific integrins, alpha1, and alpha4, were present on epithelial cells during the secretory phase. Delayed expression of alpha(v) beta3 in baboon endometrial glands correlated closely with the time of enhanced glandular secretory activity in this primate. The baboon appears to be an excellent model for the investigation of the role of integrins and ECM leading to successful implantation.     

Characterization of desamino-5-[125I]iodo-3-methoxy-zacopride ([125I]MIZAC) binding to 5-HT3 receptors in the rat brain

1. Antagonists at 5-HT3 receptors have shown activity in animal models of mental illness, however, few radiolabeled 5-HT3 ligands are available for preclinical studies. MIZAC, an analogue of the selective 5-HT3 antagonist, zacopride, binds with high affinity (1.3-1.5 nM) to CNS 5-HT3 sites. The authors report here the selectivity of MIZAC for these sites in rat brain homogenates. 2. Ninety-seven percent of total specific binding of [125I]MIZAC (0.1 nM) of was displaced by bemesetron (3 microM), a selective 5-HT3 antagonist. Competition studies using ligands with known affinities for 5-HT3 sites give a high correlation with reported pKi values (r2 0.98). Bemesetron displaceable binding has a regional distribution consistent with that of the 5-HT3 receptor, i.e. highest in cortex and hippocampus, and lowest in striatum and cerebellum. 3. Potent antagonists present at concentrations sufficient to occupy 95% of other 5-HT receptor populations (1A, 1B, 1D, 2A, 2B, 2C, 5A, 5B, 6, and 7) showed minimal ability to displace [125I]MIZAC binding (3 nM). Specificity studies using radioligand binding assays selective for 5-HT4, 5-HT6, and 5-HT7 receptors, and for binding sites of other neurotransmitters indicate a high degree of selectivity of [125I]MIZAC for the 5-HT3 receptor. 4. [125I]MIZAC binds to an apparent low affinity (benzac) site having a unique pharmacology. Low affinity binding was displaceable by benztropine, but not by other muscarinic agents nor inhibitors of dopamine uptake. The regional distribution of the low affinity site differed markedly from that of the high affinity site. The apparent affinity of [125I]MIZAC for the benzac site is two orders of magnitude lower than for the 5-HT3 receptor. Given its high selectivity for 5-HT3 binding sites, [125I]MIZAC appears to be a promising ligand for labeling 5-HT3 receptors in vitro and in vivo.     

Technical features of a CCD video camera system to record cardiac fluorescence data

Prostaglandin E2 (PGE2) is a potent local mediator of cell growth and differentiation in various tissues. The receptors for PGE2 have been classified into four pharmacological subtypes, EP1, EP2, EP3, and EP4, based on the responses to selective agonists and antagonists. We have cloned a functional cDNA for the rat EP2 receptor subtype from a rat lung cDNA library. The rat EP2 receptor cDNA encodes 357 amino acid residues having high homology with the human and mouse EP2 receptors and containing seven putative transmembrane domains. In COS-7 cells transfected with rat EP2 cDNA, specific [3H]PGE2 binding was found with a dissociation constant of 14.9 nM, and this binding was inhibited by unlabeled PGE2 and PGE2 alpha. PGE2 and butaprost, an EP2 selective agonist, were effective in increasing the cAMP level in the COS-7 cell transfectants. Northern blot and RT-PCR analysis showed widespread distribution of the EP2 receptor in various tissues. Higher EP2 expression was found in fetal long bones and calvariae than in adult by RT-PCR and in situ hybridization, suggesting a role for this receptor in rapidly growing skeletal tissue.     

Endothelin-1 and endothelin receptors are present in the sheep uterus and conceptus at implantation

Previous studies have demonstrated that endothelin is present in the ovine endometrium and increases at around the expected time of implantation. To characterize further uterine endothelin at the time of establishment of pregnancy in sheep, endothelin was measured by radioimmunoassay in uterine flushings obtained during the oestrous cycle and in pregnant ewes up to the time of implantation (day 16). During the oestrous cycle, the highest amounts of endothelin were present in uterine flushings on day 14 (1.1 +/- 0.2 ng endothelin/uterus). During early pregnancy, basal levels of endothelin (0.5-0.6 ng endothelin/uterus) were present in uterine flushings for the first 10 days and then increased on day 14 to levels similar to those found at the equivalent stage of the oestrous cycle. On days 15 and 16 of pregnancy, endothelin content in the uterine lumen increased to significantly (P < 0.05) higher concentrations (2.9 +/- 0.4 ng endothelin/uterus) when compared with the non-fertile cycle. The principal isoform present in flushings at the time of implantation was endothelin-1, as determined by reverse-phase HPLC. Endothelin was released principally by purified endometrial epithelial cells in culture, with barely detectable amounts released by endometrial stromal cells or conceptus tissue, which is consistent with the epithelium being the principal source of endothelin in the uterine lumen. Endothelin binding sites were present in endometrium and myometrium, as demonstrated by specific binding of 125I-labelled endothelin-1, which was saturable and displaced by endothelin-1. Both endothelinA and B sub-types of receptors were present as demonstrated by the biphasic displacement of 125I-labelled endothelin-1 binding by the specific endothelinB agonist BQ3020. These were localised principally on luminal and glandular epithelium and in the vasculature of the endometrium and myometrium as shown by autoradiography. Endothelin receptors were also present on the conceptus obtained at the time of implantation. In the day 20 conceptus, endothelin immunostaining was localised principally in the heart, in trophoblast in uninucleate but not in binucleate cells, and in fetal membranes. This immunostaining of the conceptus may represent binding to receptor sites. It is concluded that endothelin-1 is present in the uterine lumen and may play an important role in the paracrine regulation of the conceptus and endometrium at the time of rapid embryo development, implantation and early placentation.     

Survey on the pharmacodynamics of the new antipsychotic risperidone

This review reports on the pharmacodynamics of the new antipsychotic risperidone. The primary action of risperidone is serotonin 5-HT2 receptor blockade as shown by displacement of radioligand binding (Ki: 0.16 nM), activity on isolated tissues (EC50: 0.5 nM), and antagonism of peripherally (ED50: 0.0011 mg/kg) and centrally (ED50: 0.014 mg/kg) acting 5-HT2 receptor agonists in rats. Risperidone is at least as potent as the specific 5-HT2 receptor antagonist ritanserin in these tests. Risperidone is also a potent dopamine D2 receptor antagonist as indicated by displacement of radioligand binding (Ki: 1.4 nM), activity in isolated striatal slices (IC50: 0.89 nM), and antagonism of peripherally (ED50: 0.0057 mg/kg in dogs) and centrally acting D2 receptor agonists (ED50: 0.056-0.15 mg/kg in rats). Risperidone shows all effects common to D2 antagonists, including enhancement of prolactin release. However, some central effects such as catalepsy and blockade of motor activity occur at high doses only. Risperidone is 4-10 times less potent than haloperidol as a central D2 antagonist in rats and it differs from haloperidol by the following characteristics: predominant 5-HT2 antagonism; LSD antagonism; effects on sleep; smooth dose-response curves for D2 antagonism; synergism of combined 5-HT2/D2 antagonism; pronounced effects on amphetamine-induced oxygen consumption; increased social interaction; and pronounced effects on dopamine (DA) turnover. Risperidone displays similar activity at pre- and postsynaptic D2 receptors and at D2 receptors from various rat brain regions. The binding affinity for D4 and D3 receptors is 5 and 9 times weaker, respectively, than for D2 receptors; interaction with D1 receptors occurs only at very high concentrations. The pharmacological profile of risperidone includes interaction with histamine H1 and alpha-adrenergic receptors but the compound is devoid of significant interaction with cholinergic and a variety of other types of receptors. Risperidone has excellent oral activity, a rapid onset, and a 24-h duration of action. Its major metabolite, 9-hydroxyrisperidone, closely mimics risperidone in pharmacodynamics. Risperidone can be characterized as a potent D2 antagonist with predominant 5HT2 antagonistic activity and optimal pharmacokinetic properties.     

Effect of post-coital contraceptive methods on the endometrium and the menstrual cycle

STUDY OBJECTIVE: To evaluate the effect of treatment with ethinylesteradiol-levonorgestrel or danazol on ovarian function, gonadotrophin release and endometrial development during the time when a pregnancy may occur following unprotected intercourse. METHODS: Women with regular menstrual cycles were followed during one control, one treatment and one follow-up month. The women obtained either a combination of 0.5 mg levonorgestrel and 0.1 mg ethinylestradiol (Yuzpe regimen: n = 16) or 600 mg danazol orally and repeated after 12 hours (n = 16). The treatment was administered on either cycle day (cd) 12 or day LH +2. An endometrial biopsy was obtained once on cd LH +6 to +8 in the subjects treated on cd LH +2 both in control and treatment cycles, and morphometric analysis was performed. The concentrations of LH, pregnandiol (P2G), and estrone (EIG) glucuronide were followed daily in morning urine during control and treatment cycles. RESULTS: Following treatment with the Yuzpe regimen on cd 12 the LH surge was either undetectable (three subjects), postponed to cd 16 to 22 (three subjects) or cd 38 to 39 (two subjects) with lower P2G and LH levels than in the control cycle. Following preovulatory treatment with danazol, no LH peak could be detected in four subjects and in the remaining four subjects the LH peak varied between cd 13 and cd 24. The mean area under the curve for LH was significantly lower, the levels of EIG were slightly higher and the P2G levels were unaffected in comparison with the control cycle. Neither of the two treatments administered on cd LH +2 affected the hormonal pattern and only a discreet effect on the development of the endometrium was seen after the EE/LNG treatment. CONCLUSION: The findings indicate that the contraceptive effect of postcoital treatment with EE/LNG and danazol is mainly due to an inhibition or delay of ovulation and insufficient corpus luteum function. The direct effect on the endometrium is limited, if any.     

Augmentation of a humanized anti-HER2 mAb 4D5 induced growth inhibition by a human-mouse chimeric anti-EGF receptor mAb C225

Overexpression of epidermal growth factor (EGF) receptor and HER2 (p185neu) may both contribute to the growth of human cancers. A humanized anti-HER2 monoclonal antibody (mAb) 4D5 and a human-mouse chimeric anti-EGF receptor mAb C225 are currently being investigated in clinical trials for their anti-tumor activities. In the present study, we have examined the effect of concurrent treatment of OVCA 420 human ovarian cancer cells with mAb C225 and mAb 4D5. Exposure of OVCA420 cells to saturating concentrations of C225 (20 nM) for 7 days resulted in 40-50% growth inhibition, and exposure to 20 nM mAb 4D5 also resulted in 30-40% growth inhibition. The growth inhibition of OVCA420 cells by mAb C225 or 4D5 was associated with an increased G1 cell population; an increased level of a cyclin-dependent kinase (CDK) inhibitor p27Kip1 with increased association of p27kip1 with CDK2, CDK4 and CDK6; and decreased activities of these CDKs. Combination treatment with concurrent exposure to mAbs C225 and 4D5 resulted in additive anti-proliferative effects on these cells, which was accompanied by enhanced G1 cell distribution, a greater increase in the levels of p27Kip1 and a greater decrease in the activities of CDK kinases. The anti-proliferative effects and related changes in cell cycle regulators induced by mAb 4D5, mAb C225 or the combination of the two mAbs could be reversed by concurrent exposure to exogenous EGF. Our data suggest the potential fruitful cooperation of anti-EGF receptor mAb and anti-HER2 mAb in the treatment of human cancers stimulated by EGF receptor and HER2 signals.     

N-[2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide: a potent and selective dopamine D4 ligand

A series of new 1-aryl-4-alkylpiperazines containing a terminal benzamide fragment or a tetralin-1-yl nucleus on the alkyl chain were synthesized and tested for binding at cloned human dopamine D4 and D2 receptor subtypes. A SAFIR (structure-affinity relationship) study on this series is herein discussed. The most relevant D4 receptor affinities were displayed by N-[omega-[4-arylpiperazin-1-yl]alkyl]-methoxybenzamides (compounds 5, 16-20), their IC50 values ranging between 0.057 and 7.8 nM. Among these, N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide (17) emerged since it exhibited very high affinity for dopamine D4 receptor (IC50 = 0.057 nM) with selectivity of >10 000 for the D4 versus the D2 receptor; compound 17 was also selective versus serotonin 5-HT1A and adrenergic alpha1 receptors.     

Erythromycin modulates IL-8 expression in normal and inflamed human bronchial epithelial cells

Previous studies have shown that blood plasma levels of 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP) and 17alpha, 20beta, 21-trihydroxy-4-pregnen-3-one (20beta-S) increase in striped bass (Morone saxatilis) undergoing final oocyte maturation (FOM). Both hormones are produced by ovarian fragments undergoing hCG-induced germinal vesicle breakdown (GVBD) in vitro. In the present study, we investigated binding of DHP and 20beta-S to ovarian membranes from striped bass undergoing FOM. Saturable binding sites for DHP were not detected. Saturation of 20beta-S binding sites with 5 nM [3H]20beta-S occurred within 40 min at 0 degrees C (at 3 min, half of the maximum specific binding of steroid was calculated to have occurred), and the binding was pH-dependent. Scatchard analyses revealed the presence of a single class of high-affinity (dissociation constant [Kd] = 1.4 +/- 0.2 nM), limited-capacity (estimated concentration [Bmax] = 2.7 +/- 0.3 pmol/g ovary) 20beta-S binding sites on membranes from striped bass ovaries undergoing FOM. In contrast, only low levels of specific binding (Bmax < 0.04 pmol/g tissue) were detected on membranes from testes, liver, brain, and muscle. Ovarian membranes prepared from vitellogenic females also had low levels (Bmax < 0.1 pmol/g ovary) of specific 20beta-S binding, less than 5% of that found during FOM. Results of competition assays showed that DHP was approximately 250 times less effective than 20beta-S for displacing 20beta-S from ovarian membranes. In contrast, 20beta, 21-dihydroxy-4-pregnen-3-one was a very effective competitor, although it is only a weak inducer of oocyte GVBD in vitro. Of several other steroids tested, only progesterone showed affinity for the 20beta-S binding site within a physiological range of concentrations. Taken together with previous studies of striped bass FOM, these findings indicate that 20beta-S is the oocyte maturation-inducing steroid hormone in striped bass.     

Inhibition of purinergic transmission by prostaglandin E1 and E2 in the guinea-pig vas deferens: an electrophysiological study

1. The effects of prostaglandin E1 (PGE1) and E2 (PGE2) on postjunctional electrical activity in the guinea-pig vas deferens evoked by sympathetic nerve stimulation were investigated using both intracellular and focal extracellular recording techniques in vitro. 2. Bath application of PGE1 (1-100 nM) or PGE2 (0.1-100 nM) concentration-dependently inhibited the amplitudes of all excitatory junction potentials (e.j.ps) evoked during short trains of stimuli (10 stimuli at 1 Hz). Increasing the duration of nerve stimulation (100 stimuli at 1 Hz) did not overcome this inhibitory effect. At these concentrations PGE1 and PGE2 were without any apparent inhibitory effect on the amplitudes of spontaneous e.j.ps. 3. Local application of PGE1 (10-100 nM) or PGE2 (10-30 nM) markedly reduced the frequency of occurrence of excitatory junction currents (e.j.cs) evoked by trains of 20-100 stimuli at 1 to 4 Hz without changing the amplitudes of spontaneous e.j.cs or the configuration of the nerve terminal impulse. 4. In the presence of PGE1 or PGE2, raising the frequency of stimulation (from 1 to 4 Hz), increased the likelihood of e.j.c. occurrence. 5. The postjunctional electrical activity recorded in the guinea-pig vas deferens is believed to be due to ATP released from the sympathetic nerve endings. Thus the present study demonstrates that both PGE1 and PGE2 powerfully inhibit quantal ATP release in the guinea-pig vas deferens.     

Genetic resistance to parasitic infection

Intraventricular administration of carbachol can induce phase shifts in wheel-running activity in rodents, which depend on circadian phase and are mediated via muscarinic cholinergic receptors in Syrian hamsters. We studied the circadian variation in binding of [3H]-N-methylscopolamine ([3H]NMS), a hydrophilic muscarinic receptor antagonist, in micropunches obtained from the anterior hypothalamus and occipital cortex of Syrian hamsters housed in a 14:10 light:dark cycle. Binding sites were characterized on cells contained within 1 mm punches (obtained from slices 300 microm thick), using a method to selectively detect cell surface (functional) receptors. Atropine sulphate was used to determine nonspecific binding. Cortex showed a significant daily rhythm in [3H]NMS binding with a peak occurring late in the light phase and a trough at lights on, while the hypothalamus showed no detectable rhythm. Following suprachiasmatic nucleus (SCN) ablation or maintenance in constant darkness, the rhythm in the cortex was abolished. These findings suggest that photic information conveyed via the SCN is responsible for the receptor binding rhythm in the cortex. Autoradiographic studies ([3H]NMS; 2 nM, 3 weeks exposure) clearly revealed both M1 and M2 subtypes of muscarinic receptors in the region of the SCN and the visual cortex.     

Effect of lung volume reduction surgery on gas exchange and pulmonary hemodynamics at rest and during exercise

The present study was an investigation of the regulation of anion secretion across cultured mouse endometrial epithelium by prostaglandin E2 (PGE2) using the short-circuit current (ISC) technique. The cultured endometrial monolayers responded to both apical and basolateral application of PGE2 with a sustained rise in ISC in a concentration-dependent manner. However, the potencies of apical and basolateral addition of PGE2 were different, with apparent EC50 of 200 and 4 nM, respectively. Replacement of Cl- or HCO3- in the bathing solution significantly reduced the ISC responses to both apical and basolateral addition of PGE2; however, the apical response exhibited greater dependence on HCO3- . Pretreatment with diphenylamine 2,2'-dicarboxylic acid, a Cl- channel blocker, significantly reduced both PGE2-induced ISC responses, while pretreatment with amiloride, a Na+ channel blocker, did not exert any effect. Forskolin, an adenylate cyclase activator, and 3-isobutyl-dihydro-testosterone-1-methyl-xanthine, a cAMP phosphodiesterase inhibitor, mimicked the ISC response to PGE2 while MDL12330A, an adenylate cyclase inhibitor, completely abolished the PGE2-induced ISC. The results of the present study indicate that the anion secretion across the mouse endometrial epithelium may be regulated by PGE2 involving a cAMP-dependent mechanism predominantly. The differential responses to apical and basolateral challenge with PGE2 also suggest that PGE2 of different origins may play different roles in uterine function.     

Isolation of rat chromosome-specific paint probes by bivariate flow sorting followed by degenerate oligonucleotide primed-PCR

Angiogenesis is an essential component of endometrial regeneration after menses in preparation for implantation. Vascular endothelial growth factor (VEGF) is a secreted angiogenic peptide with mitogenic activity specific for endothelial and trophoblast cells. VEGF-immunoreactivity was detected in glandular epithelium throughout the menstrual cycle by immunohistochemistry, but, showed cyclic variation in the stroma and the blood vessels. During the early proliferative phase, strong staining was seen in the glandular epithelial cells while staining in the stroma was confined to a subpopulation of stromal cells and endometrial blood vessels appeared negative. In contrast, very intense staining of the endometrial stromal cells was seen in the mid proliferative endometrium possibly due to increased synthesis of VEGF by oestrogen. In the late proliferative endometrium, staining was seen in the endothelial cells and the perivascular stromal cells around the endometrial blood vessels. The greatest degree of immunostaining of stromal cells was observed in the mid to late proliferative endometrium. Throughout the secretory phase no staining was seen around the endometrial blood vessels and staining of endometrial stromal cells was confined to early secretory endometrium. In the late secretory endometrium only the glands were positive to VEGF antibody. The observed increase in the immunostaining of stroma suggests increased production of VEGF from early to mid and late proliferative endometrium which parallels the increase in the oestradiol levels in the proliferative phase of the menstrual cycle. It is proposed that VEGF may serve as a paracrine mediator of the effects of ovarian steroids on endometrial vascular development.     

Regulation of cyclooxygenase-2 and prostaglandin F synthase gene expression by steroid hormones and interferon-tau in bovine endometrial cells

Estradiol (E2) and progesterone are responsible for regulating PG synthesis in the endometrium during the estrous cycle and interferon-tau (IFN-tau) alters PG synthesis during early pregnancy in ruminants. In this study, we examined the effects of these steroid hormones and recombinant bovine IFN-tau (rbIFN-tau) on PG production and on cyclooxygenase-2 (COX-2) and PG F (PGF) synthase (PGFS) gene expression in isolated endometrial cells. E2 decreased both PGF2alpha and PG E2 (PGE2) whereas progesterone increased PGF2alpha secretion in epithelial cells. Steroid hormones had no effect on PG production in stromal cells. rbIFN-tau attenuated both PGF2alpha and PGE2 production in epithelial cells and enhanced their production, and the ratio of PGE2 to PGF2alpha, in stromal cells. Northern blot analysis showed that E2 and rbIFN-tau decreased COX-2 messenger RNA (mRNA) levels in epithelial cells. Conversely, rbIFN-tau increased COX-2 mRNA in stromal cells. Furthermore, rbIFN-tau decreased PGFS mRNA in both cell types and this was associated with the increase in PGE2/PGF2alpha ratio. These results show that the regulation of PG synthesis by steroid hormones is different in endometrial epithelial and stromal cells in vitro. The attenuation of PGF2alpha secretion from epithelial cells and increased PGE2 production in stromal cells by rbIFN-tau are modulated by steroid hormones.     

Ethanol administration delays recovery from acute pancreatitis induced by exocrine hyperstimulation

A cholera toxin-sensitive, prostaglandin E2 (PGE2) specific receptor has been identified in the plasma membrane fraction of tick salivary glands. In the present study, we report that stimulation of dispersed salivary glands of the lone star tick Amblyomma americanum (L.) with 1 nM to 10 microM PGE2 increased the intracellular concentration of inositol trisphosphate (IP3) in a dose-dependent manner. Incubation of dispersed tissue with 1 nM to 10 microM PGE2 also stimulated release of 45Ca2+ from preloaded tissue. PGE2 (10 microM) did not stimulate an influx of 45Ca2+. Therefore, the PGE2 receptor in the salivary glands appears to activate a phosphoinositide phospholipase C signalling pathway to increase formation of intracellular IP3 and, thus, mobilize Ca2+ from intracellular stores. Incubation of dispersed salivary glands with 1 nM to 1 microM PGE2 stimulated secretion of anticoagulant protein, but not at < 1 nM or > 1 microM PGE2. In addition, the mammalian PGE2 EP1 receptor antagonist AH-6809 affected secretion of anticoagulant by dispersed salivary gland tissue at a low concentration supporting the hypothesis that the PGE2 receptor in tick salivary glands is EP1-like. We propose that a major function for PGE2 in tick salivary glands is to mobilize Ca2+ and stimulate secretion (exocytosis) of bioactive proteins into the tick's saliva during feeding.