Kay Marshall

In vitro characterization of prostanoid receptors on human myometrium at term pregnancy

1 Prostanoid receptors present on the pregnant human myometrium in vitro have been characterized according to the receptor classification proposed by Coleman et al. (1984) using natural prostanoids and synthetic, selective analogues and antagonists where available. 2 Prostaglandin E2 (PGE2) produced a biphasic effect consisting of an initial excitation followed by a dose‐related inhibition. The EP2/EP3‐receptor agonists, rioprostil and misoprostol, produced similar effects to PGE2, however, the excitatory event of the misoprostol response was related to dose. The EP1/EP3‐receptor agonist, sulprostone, evoked a purely excitatory response which was unaffected by AH6809. The selective EP2‐receptor agonist butaprost produced a long‐lasting dose‐dependent inhibition of activity. The results from these prostanoids indicated that inhibitory EP2‐ and excitatory EP3‐receptors are present on myometrium from pregnant donors at term. 3 PGF2α and the synthetic FP‐receptor agonist, fluprostenol, caused equipotent excitatory effects, indicating the presence of contractile FP‐receptors. 4 PGD2 produced a biphasic effect of which the inhibition appeared dose‐related and was antagonized by the selective DP‐receptor antagonist BW A868C. The selective DP‐receptor agonist, BW245C, produced a potent inhibitory effect that was competitively antagonized by BW A868C (pA2 = 8.6). 5 PGI2 produced a biphasic response qualitatively similar to PGE2. The EP1/IP‐receptor agonist, iloprost, produced an occasional unquantifiable excitation and dose‐related inhibition. The selective IP‐receptor prostanoid, cicaprost, evoked only an inhibitory response. 6 The stable thromboxane A2 (TXA2)‐mimetic, U46619, produced potent excitation which was competitively antagonized by the TP‐receptor antagonist, GR32191 (pA2 = 7.2). 7 The prostanoids tested indicate that a heterogeneous population of prostanoid receptors are present on human myometrium from pregnant donors. It may be concluded that excitation is EP3‐, FP‐ and TP‐receptor‐mediated and inhibition is EP2‐, DP‐ and IP‐receptor‐mediated. Comparison of data obtained from non‐pregnant specimens indicates that the lower segment tissue from pregnant donors demonstrated more pronounced responses to EP2 and IP‐receptor activation.

Influence of gender on working and spatial memory in the novel object recognition task in the rat.

Gender differences in many behavioural tasks have been observed in both humans and laboratory animals. The novel object recognition (NOR) task is increasingly used to investigate drug effects on working memory processes, although, the influence of sexually dimorphic behaviours have not yet been evaluated. In addition, the role of natural fluctuations in the sex steroids during the oestrous cycle has received little attention during object recognition tasks. Therefore, the aim of the current study was to investigate the influence of gender and oestrous cycle phase on working and spatial memory using the NOR task. Animals were tested in the NOR task and the spatial NOR task. Male and female rats completed an acquisition trial followed by an inter-trial interval of a specified length, then a final retention trial. Vaginal cytology enabled the influence of oestrous cycle phase to be determined in both the NOR and spatial NOR, each animal was tested during one phase of their regular oestrous cycle only. It was found that female rats performed significantly better than male rats in the standard NOR paradigm (p<0.05 compared to no significance (NS) at 3h, respectively), while male rats showed improved memory in the spatial NOR paradigm compared with female rats (p<0.05 compared to NS at 3h, respectively). There was no influence of phase of oestrous cycle on the NOR task, however, during the spatial NOR there was a significant improvement in ability when oestrogen and progesterone levels have been shown to be at their lowest (i.e. p<0.05 during oestrous compared to NS at other stages). In conclusion, it is clear that gonadal hormones can influence components of memory and gender is an important consideration in experimental design.

In vitro characterization of prostanoid FP‐, DP‐, IP‐ and TP‐receptors on the non‐pregnant human myometrium

1 Prostaglandin F (PGF), PGD, PGI and thromboxane A2 (TXA2) receptors have been pharmacologically characterized on the non‐pregnant human myometrium in vitro in accordance with the receptor classification proposed by Coleman et al. (1984). The tools for the classification include both natural prostanoids, synthetic, selective analogues and antagonists where available. 2 The potent excitatory actions of the natural FP‐receptor prostanoid, PGF2α, and the synthetic analogue, fluprostenol, indicate the presence of FP‐receptors mediating contraction on the human myometrium. 3 PGD2 produced a biphasic response consisting of excitation followed by relaxation of spontaneous activity of the myometrium. The selective DP‐receptor agonists, BW245C, produced purely inhibitory responses illustrating the presence of inhibitory DP‐receptors in this tissue. The inhibitory responses of both PGD2 and BW245C were antagonized by the competitive DP‐receptor antagonist, BWA 868C, providing conclusive evidence for the existence of DP‐receptors. 4 PGI2 produced a biphasic response similar to PGD2. Iloprost, the EP1/IP‐receptor agonist also produced a biphasic response, whilst the IP‐receptor selective agonist, cicaprost, caused inhibition only, suggesting that inhibitory IP‐receptors exist in the non‐pregnant human myometrium. 5 The TXA2‐mimetic, U46619, produced marked stimulation of the non‐pregnant human myometrium and was approximately equipotent to PGF2α and fluprostenol in this effect. The actions of U46619 were competitively antagonized by the TP‐receptor antagonist GR32191 showing that excitatory TP‐receptors exist in this tissue. 6 All prostanoids tested, both natural and synthetic, had activity on the non‐pregnant human myometrium in vitro, supporting the existence of a heterogeneous population of prostanoid receptors in this tissue. If the results from the present study are combined with those previously reported for EP‐receptor agonists (Senior et al., 1991), it may be concluded that excitation may occur through FP‐, TP‐, EP3‐ and few EP1‐receptors, whereas inhibition may occur through DP‐, IP‐ and EP2‐receptors.

In vitro characterization of prostanoid EP-receptors in the non-pregnant human myometrium

1 Prostaglandin receptors of the PGE type have been characterized in the non‐pregnant human myometrium in vitro according to the scheme of Coleman et al. (1984) by use of the agonists PGE2, sulprostone, rioprostil, AY23626, butaprost, misoprostol, 16,16‐dimethylprostaglandin E2, enprostil and iloprost, and, the antagonist AH6809. 2 All prostanoids tested were active in non‐pregnant human myometrium either as stimulators and/or inhibitors of spontaneous activity or both. Biphasic responses to PGE2 indicate that at least two receptor types of the EP‐receptor exist, one mediating relaxation and the other mediating contraction. 3 Further evidence for the EP‐receptor mediating excitation and relaxation was provided by the action of the EP2‐/EP3‐receptor selective prostanoids rioprostil, AY23626 and misoprostol, and the EP1‐/EP2‐receptor selective agonist 16,16‐dimethylprostaglandin E2. 4 Butaprost, an EP2‐receptor selective agonist, produced potent inhibition of spontaneous activity in the tissue which was generally longer‐lasting than that evoked by the natural prostanoid PGE2. 5 The EP1‐/EP3‐receptor selective agonist sulprostone and the EP3‐receptor agonist enprostil produced potent contractile responses supporting the presence of contractile EP3‐receptors in the non‐pregnant human myometrium in vitro. 6 The EP1‐/IP‐receptor selective agonist, iloprost, produced mixed responses in non‐pregnant human myometrium. The contractile response was inhibited by the EP1‐receptor antagonist AH6809. However, responses to the EP1‐/EP3‐receptor selective agonist sulprostone were unaffected by AH6809 which may indicate that only a small population of EP1‐receptors is present. 7 Therefore it would seem that a heterogeneous population of EP‐receptors is present in the non‐pregnant human myometrium.

Effects of the atypical antipsychotic olanzapine on reproductive function and weight gain in female rats

Sexual dysfunction is a major, although poorly understood, side-effect of treatment with antipsychotic drugs. We have recently show marked disruption of reproductive function and weight gain in female rats treated subchronically with risperidone and haloperidol. The aim of the present study was to examine further the potential relationship between reproductive dysfunction and weight gain in female rats treated with olanzapine. The effects of olanzapine on weight gain, food and water intake, intra-abdominal fat, the oestrous cycle and uterine weight were assessed in group-housed adult female hooded-Lister rats. Olanzapine (0.5-4.0 mg/kg i.p.) or vehicle was administered once daily for 21 days and body weight, food and water intake measured, with histological examination of vaginal lavage to determine the stage of the oestrous cycle. On day 22, animals were sacrificed and intra-abdominal fat, wet and dry uterine weights measured. Olanzapine induced significant weight gain with concomitant increases in food and water intake and intra-abdominal fat without an effect on the oestrous cycle, wet and dry uterine weights or plasma prolactin levels. These results confirm the ability of olanzapine to induce weight gain in female rats on unrestricted normal diet with a concomitant increase in food and water intake and increased intra-abdominal fat. These effects of olanzapine were produced in the absence of any apparent impairment in reproductive function, in contrast to the substantial disruption of oestrous and uterine atrophy previously shown in rats treated with risperidone and haloperidol.

Characterization of the prostanoid receptors mediating constriction and relaxation of human isolated uterine artery

1 This study was undertaken to characterize pharmacologically the prostanoid receptor subtypes mediating constriction and relaxation of human isolated uterine artery. 2 U‐46619 was a potent constrictor agonist on human uterine artery (EC50 [95% CL] = 3.5 [1.8‐6.7] μM). Prostaglandin E2 (PGE2α), PGF2, PGD2 and PGI2 only weakly constricted the uterine artery, being at least 100 times less potent than U‐46619. The PGE2 and PGI2 constrictor effects may be modified by the potent dilator effects of these compounds. A number of agonists which show selectivity for FP‐, DP‐ and EP‐receptors including ICI 81008, BW 245C, sulprostone, rioprostil and butaprost, failed to cause any constriction at concentrations up to 30 μM. 3 Constrictor responses induced by all agonists tested were reduced or abolished by the TP‐receptor blocking drugs, GR 32191 and EP 092. pA2 estimates for both antagonists versus U‐46619 were 8.50, values which are consistent with their affinities at TP‐receptors. 4 In preparations pre‐constricted with phenylephrine (1 μM) both PGI2 and PGE2 were potent relaxant agonists. The selective IP‐receptor agonists, cicaprost and iloprost, also dilated human uterine artery and were approximately 10 fold more potent than PGI2. The EP2‐receptor agonists, butaprost and rioprostil and the selective DP‐agonist, BW 245C, were at least 100 fold weaker than PGI2 and PGE2 suggesting that neither DP‐ nor EP2 receptors were involved. 5 We conclude that TP‐receptors mediate constriction, whereas IP‐ and possibly EP4‐receptors mediate relaxation of human uterine artery.

Studies using isolated uterine and other preparations show bimatoprost and prostanoid FP agonists have different activity profiles.

1 The pharmacology of bimatoprost, a synthetic prostaglandin‐amide, was examined in prostaglandin F2α (PGF2α)‐sensitive preparations. Bimatoprost potently contracted the rabbit isolated uterus (pEC50=7.92±0.16). In contrast, bimatoprost exhibited weak excitatory activity in human myometrium from pregnant and nonpregnant donors, mouse uterus, rat uterus, and endothelium‐intact rabbit jugular veins, and did not stimulate DNA synthesis in mouse fibroblasts. 2 The possibility that the effects of bimatoprost may reflect partial agonism at prostanoid FP receptors was examined and the contractile effects of full agonists, 17‐phenyl PGF2α (FP) and U‐46619 (TP, a control), were determined in the absence and presence of 1 μM bimatoprost on the mouse uterus. Analyses of the agonist–agonist functional studies showed no antagonism, indicating that bimatoprost is not a partial agonist. 3 Bioassay metabolism studies of bimatoprost and latanoprost (FP receptor agonist prodrug) in the rabbit uterus were conducted using recipient mouse uterus. Results indicated that the potent responses to bimatoprost in the rabbit uterus are produced by the intact molecule and not by its putative free acid metabolite, 17‐phenyl PGF2α. Some hydrolysis of latanoprost to latanoprost free acid appears to have occurred in the rabbit uterus, according to biological detection. 4 The pharmacology of bimatoprost could not be explained by its interaction with known prostanoid FP receptors and was independent of species‐, tissue‐, or preparation‐related factors. The potent contractile effects of bimatoprost in the rabbit uterus provide further pharmacological evidence for the presence of a novel receptor population that preferentially recognises bimatoprost.

Effects of the classical antipsychotic haloperidol and atypical antipsychotic risperidone on weight gain, the oestrous cycle and uterine weight in female rats

Antipsychotic drug-induced side effects of weight gain and sexual dysfunction have clinical significance adversely affecting both compliance and morbidity. This study evaluated the effects of haloperidol and the atypical antipsychotic risperidone (0.1-1.0 mg/kg) on weight gain, food and water intake, the oestrous cycle and uterine weight in female hooded Lister rats. Haloperidol and risperidone treated rats displayed marked weight gain, although only risperidone induced significant increases in food consumption over the 21-day period. Neither haloperidol nor risperidone influenced water consumption. Marked disruption of the oestrous cycle was observed in risperidone- and haloperidol-treated animals (0.5 and 1.0 mg/kg), which was supported by significantly reduced uterine weights. The findings presented here suggest that the weight gain and sexual dysfunction induced by antipsychotics may be modelled in rodents. This model may offer insight into the mechanisms involved in mediation of such side effects.

Evidence for human thromboxane receptor heterogeneity using a novel series of 9,11-cyclic carbonate derivatives of prostaglandin F2α

1 The pharmacological activity of a novel series of 9,11‐cyclic carbonate derivatives of prostaglandin F2α (PGF2α) was investigated in various isolated smooth muscle preparations possessing different prostanoid receptor subtypes as well as in human platelets. Since subdivision of thromboxane (TP‐) receptors into vascular/smooth muscle and platelet subtypes is a controversial subject, our studies included a human smooth muscle preparation (myometrium) in addition to the widely used rat aorta and human platelets as TP‐receptor preparations. 2 Two members of that series, AGN191976 and AGN192093 were found to be highly potent and selective thromboxane‐mimetics. AGN191976 and AGN192093 contracted isolated tissues of the rat thoracic aorta with EC50 values of 0.32±0.08 and 1.30±0.53 nM, respectively. Both agonists were at least 10 times more potent than the benchmark TP‐agonist, U‐46619, in this preparation, whilst being at least 500 times less potent at other prostanoid receptors (DP, EP1, EP3, FP, IP) in vitro. 3 In human myometrial strips from pregnant and non‐pregnant donors, both AGN191976 and AGN192093 were potent contractile agonists. The rank order of potency in myometrium of AGN191976>AGN192093>U‐46619 correlated well with that in the rat aorta. In human plateletrich plasma (PRP), however, AGN191976 had potent proaggregatory activity (EC50=16.3±1.4 nM), which is a TP‐receptor‐mediated event, whereas AGN192093 was a much weaker agonist (EC50= 37.9±2.0 μm). AGN192093 did not behave as an antagonist in the platelets, since it did not antagonize platelet aggregation induced by ADP, arachidonic acid, U‐46619 or AGN191976. In human washed platelets, the activity profile of AGN191976 (EC50=4.15±0.52 nM) and AGN192093 (no aggregation up to 10 μm) was similar to that obtained in PRP. 4 The involvement of TP‐receptors was verified with the potent TP‐antagonist, SQ29548. SQ29548 (0.1 μm in myometrium; 1 μm in aorta; 1 μm and 10 μm in platelets) antagonized responses to U‐46619, AGN191976 and AGN192093 as expected. 5 In conclusion, AGN191976 and AGN192093, both 9,11‐cyclic carbonate derivatives of PGF2α, were found to be highly potent and selective thromboxane‐mimetics in rat vascular and human myometrial smooth muscle. However, only AGN 191976 was a potent agonist at TP‐receptors in human platelets. The differential activity of AGN192093 on TP‐receptor‐mediated events in platelets and smooth muscle provides further evidence for a subdivision of TP‐receptors. AGN192093 appears to be a useful tool for the pharmacological distinction of TP‐receptor subtypes.

Investigation into the effects of the novel antipsychotic ziprasidone on weight gain and reproductive function in female rats

Weight gain and sexual dysfunction are serious side effects of certain antipsychotic drugs. Ziprasidone, a novel antipsychotic with a unique receptor binding profile, is reported to have a low propensity for such side effects. Previous results from this laboratory have demonstrated substantial weight gain following sub-chronic treatment with olanzapine and risperidone. Risperidone induced weight gain and markedly impaired reproductive function while olanzapine induced weight gain, without affecting reproductive function. The aim of this study was to investigate effects of ziprasidone on weight gain and reproductive function in female rats. Ziprasidone (1 and 2.5 mg/kg i.p.) or vehicle was administered once daily for 28 days and body weight, food and water intake measured, in addition to histological examination of vaginal lavage to determine the stage of the oestrous cycle. On day 28, the rats were sacrificed and the uterine weights recorded, intra-abdominal fat weight and plasma prolactin levels measured. Ziprasidone failed to induce significant weight gain during weeks 1-3, however, significant weight gain was observed on day 28 at 2.5 mg/kg (p < 0.05). Ziprasidone had no effect on food intake at any time point. A significant reduction in water intake (p < 0.05) was observed during the first week of treatment with 2.5 mg/kg ziprasidone. Ziprasidone had no effect on intra-abdominal fat weight, wet or dry uterine weight or plasma prolactin levels. All ziprasidone treated animals displayed a normal four-day oestrous cycle. This study is the first to report that ziprasidone is without effect on reproductive function or ingestive behaviour in the rat.