Effect of platelet-activating factor on the electrophysiology of the human Fallopian tube: early mediation of embryo-maternal dialogue?

Platelet-activating factor (PAF) is produced by preimplantation embryos and may be involved in the earliest stages of embryo-maternal dialogue. This study explored the potential effects of PAF acting as a signalling agent on human Fallopian tubal epithelial cells grown as a polarized layer in primary culture. The response of the tubal epithelium was assessed in terms of the transepithelial potential difference and short-circuit current (I(scc)), which were recorded using a modified Ussing chamber. Resistance was calculated from the measurements of potential difference and I(scc). PAF (1.9 nmol to 1.9 micromol l(-1)) administered to the apical surface of the cells produced a marked, transient increase in both potential difference and I(scc) in a dose-dependent manner. The mode of action of PAF on the electrophysiological responses of human tubal epithelial cells was investigated. Blockers of Na(+), K(+) and voltage-operated Ca(2+) channels had little effect on PAF action. However, incubation of the epithelial cells in Cl(-)free medium or with a blocker of the Na(+)-K(+)-2Cl(-) cotransporter (Furosemide) reduced the effect of PAF. Blockade of chloride-bicarbonate channels with 4-acetamido-4'-iso-thiocyanostilbene-2.2'-disulphonic acid (SITS) reduced the effect of low doses of PAF only. These results indicate that PAF influences the movement of chloride ions across the tubal epithelial cell and is a candidate molecule for initial embryo-maternal dialogue.

Formation of Fallopian tubal fluid: role of a neglected epithelium.

Fluid produced and secreted by the Fallopian tube provides the environment in which gamete transport and maturation, fertilization and early embryo development occur. This review describes the composition of oviductal fluid in terms of ions and nutrients such as glucose, lactate, pyruvate and amino acids. The function of oestrogen-specific glycoprotein is discussed. The mechanisms of fluid secretion and agents known to influence fluid production and secretion are described. Clinical implications of abnormal oviductal fluid production and secretion in hydrosalpinx and pelvic inflammatory disease are also discussed.

Effect of inflammatory mediators on the electrophysiology of the human oviduct.

The effects of histamine and other inflammatory mediators on the electrophysiology and intracellular free calcium ([Ca(2+)](i)) of human oviductal epithelial cells, grown as a polarized layer in primary culture, were studied. Transepithelial potential difference (PD) and short-circuit current (I(scc)) were recorded using a modified Ussing chamber. Resistance (R) was calculated from the measurements of PD and I(scc). Basally applied histamine produced transient increases in PD and I(scc) with a small decrease in R. The histamine effect was reduced by triprolidine (H(1) receptor antagonist) but was unaffected by H(2) (ranitidine) or H(3) (thioperamide) receptor antagonists. Blockers of Na(+), K(+), or Na(+)/K(+)/2Cl(-) channels did not affect histamine action. Blockers of Cl(-)/HCO(3)(-) channels or Ca(2+) channels reduced the histamine effect. Platelet activating factor (PAF), applied apically, increased PD and I(scc). Histamine produced a transient increase in fluorescence of Fura 2-AM dye, indicating an increase in [Ca(2+)](i). Triprolidine pretreatment inhibited histamine-stimulated [Ca(2+)](i) increase. Cimetidine, (H(2) receptor antagonist), ranitidine, or thioperamide reduced the histamine effect. Histamine increased contractions of both circular and longitudinal smooth muscles in oviduct segments, an effect that was antagonized by triprolidine or thioperamide but not by ranitidine. Histamine's action on Ca(2+) and Cl(-) movements may adversely affect oviductal fluid production and decrease fertility. PAF's effects on Cl(-) movements may be important for normal embryo transport.

Electrophysiological basis of human fallopian tubal fluid formation.

A preparation for the maintenance of human Fallopian tubal epithelial cells as a polarized layer in primary culture was used to study the electrophysiological basis of tubal fluid formation in terms of the movement of Na+, K+ and Cl- ions. Transepithelial potential difference (PD) and short-circuit current (Iscc) were recorded by mounting the epithelial cells in a modified Ussing chamber. Resistance (R) was calculated from the measurements of PD and Iscc. The epithelia, although confluent, formed a 'leaky' electrical system and resistances greater than 300 omega cm-2 were rarely achieved. The sodium channel blocker, amiloride (100 mumol l-1), produced only small effects on PD and Iscc. The potassium channel blocker, tetraethylammonium chloride (TEA) (25 mmol l-1), also produced small, but significant changes in PD, Iscc and R while the chloride channel blocker, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) (1 mmol l-1), induced a marked increase in PD and Iscc, and a fall in R, when added to the basal surface of the cells. Bathing the apical surface of the cells with chloride-free medium also produced a marked increase in PD, Iscc and R: bathing the basal surface of the cells with chloride-free medium produced a marked decrease in PD and Iscc. Extracellular ATP (10 mumol l-1), added to either the apical or the basal surface of the cells, induced a transient increase in PD and Iscc and a decrease in R. Amiloride, TEA or furosemide had no effect on the response of the cells to ATP. SITS, applied to the apical surface, significantly reduced the response of the cells to ATP. We conclude that the major driving force for human tubal fluid formation is the transepithelial secretion of chloride ions into the oviduct lumen and that exogenous ATP is a potential modulator of secretion.

Uptake of relaxin in the uterus and cervix of rats in vivo: influence of ovarian steroids and tolerance.

The influence of oestradiol benzoate and progesterone treatment and of tolerance to relaxin on the uptake of 125I-labelled porcine relaxin by reproductive tissues was investigated in anaesthetized female rats. In ovary-intact rats, 125I-labelled relaxin uptake increased with time in reproductive tissues and in tissues concerned with metabolism and excretion. Administration of 50 micrograms unlabelled porcine relaxin before injection of 125I-labelled relaxin significantly reduced uptake of 125I-labelled relaxin into the uterus and cervix, but had no effect on uptake of 125I-labelled relaxin into other tissues, indicating that specific uptake of relaxin was occurring in the uterus and cervix. In ovariectomized rats, treatment with oestradiol benzoate or oestradiol benzoate plus progesterone for 1 day did not significantly increase uterine or cervical 125I-labelled relaxin uptake compared with corn oil-treated rats, but induced a significant increase in uterine uptake of 125I-labelled relaxin after treatment for 2 days. Induction of tolerance to relaxin by i.v. infusion of a high dose of relaxin significantly reduced uterine and cervical uptake of 125I-labelled relaxin at 3 h after termination of infusion compared with saline-infused rats. By 12 h after termination of infusion, 125I-labelled relaxin uptake in the uterus and cervix was similar in saline-infused rats and in rats given an infusion of relaxin. Infusion of glibenclamide (20 mg kg-1) did not influence uterine or cervical uptake of 125I-labelled relaxin; however, treatment with phentolamine (10 mg kg-1) significantly reduced 125I-labelled relaxin uptake in uterus, bladder and jejunum. This study demonstrates that steroid hormone treatment and tolerance modulate relaxin uptake in reproductive tissues.

Beta-adrenoceptor subtypes and the opening of plasmalemmal K(+)-channels in trachealis muscle: electrophysiological and mechanical studies in guinea-pig tissue.

1. Mechanical and electrophysiological studies of guinea-pig isolated trachealis have been made with the objectives of: (a) identifying which of the beta-adrenoceptor subtypes mediates the opening of plasmalemmal K(+)-channels, (b) gaining further insight into the properties of the novel, long-acting beta-adrenoceptor agonist, salmeterol and (c) clarifying the role of K(+)-channel opening in mediating the relaxant actions of agonists at beta-adrenoceptors. 2. Noradrenaline (10 nM-100 microM) caused a concentration-dependent increase in the rate of beating of guinea-pig isolated atria. The selective beta 1-adrenoceptor blocking drug, CGP 20712A (100 nM-10 microM) caused concentration-dependent antagonism of noradrenaline. The selective beta 2-adrenoceptor blocking drug, ICI 118551, also produced concentration-dependent antagonism of noradrenaline, but only when used in concentrations greater than 300 nM. 3. Cromakalim (100 nM-10 microM), isoprenaline (1-100 nM), procaterol (0.1-30 nM), salbutamol (1 nM-1 microM), salmeterol (1-100 nM) and theophylline (1 microM-1 mM) each caused concentration-dependent suppression of the spontaneous tone of guinea-pig isolated trachealis. 4. ICI 118551 (10 nM-1 microM) antagonized isoprenaline, procaterol and salmeterol in suppressing the spontaneous tone of the isolated trachea. The antagonism was concentration-dependent. In contrast, ICI 118551 (1 microM) antagonized neither cromakalim nor theophylline. CGP 20712A (up to 1 microM) failed to antagonize cromakalim, isoprenaline, procaterol, salmeterol or theophylline. In trachea treated with indomethacin (2.8 microM) and carbachol (10 microM), salmeterol (1 microM) antagonized the effects of isoprenaline but not aminophylline. 5. Intracellular electrophysiological recording from guinea-pig isolated trachealis showed that the relaxant effects of cromakalim (10 microM), isoprenaline (100 nM), procaterol (10 nM) and salbutamol(10 nM- 1 microM) were accompanied by the suppression of spontaneous electrical slow waves and by cellular hyperpolarization. In contrast, the relaxant effects of salmeterol (10 nM- 1 microM) were not accompanied by significant cellular hyperpolarization.6. CGP 20712A (1 microM) inhibited the hyperpolarization but not the relaxation induced by isoprenaline(100 nM). In contrast ICI 118551 (100 nM) inhibited both the hyperpolarization and the relaxation induced by isoprenaline (100 nM). Neither CGP 20712A (1 microM) nor ICI 118551 (100 nM) inhibited the hyperpolarization induced by cromakalim (10 microM). Salmeterol (1 microM) inhibited the hyperpolarization induced by isoprenaline (100 nM) but not that induced by cromakalim (10 microM).7. It is concluded that activation of either beta l- or beta 2-adrenoceptors can promote the opening of K+-channels in the trachealis plasmalemma. The poor ability of salmeterol to hyperpolarize trachealis muscle reflects neither its selectivity in activating beta 2-adrenoceptors as opposed to beta 1-adrenoceptors nor a non-specific action in stabilizing the cell membrane. Instead, it may reflect low intrinsic efficacy of the drug at beta 2-adrenoceptors. The opening of plasmalemmal K+-channels plays a supportive rather than a crucial role in mediating the tracheal relaxant actions of agonists at beta-adrenoceptors.

Beta-adrenoceptor subtypes and the opening of plasmalemmal K(+)-channels in bovine trachealis muscle: studies of mechanical activity and ion fluxes.

1. Studies of mechanical activity and 86Rb+ efflux have been made in bovine isolated trachealis with the objectives of: (a) identifying which of the beta-adrenoceptor subtypes mediates the opening of plasmalemmal K(+)-channels, (b) gaining further insight into the properties of the novel, long-acting beta 2-adrenoceptor agonist, salmeterol and (c) clarifying the role of K(+)-channel opening in mediating the mechano-inhibitory actions of agonists at beta-adrenoceptors. 2. In bovine trachealis muscle strips precontracted with histamine (460 microM), isoprenaline (0.1 nM-1 microM), procaterol (0.1-10 nM) and salmeterol (0.1-10 nM) each caused concentration-dependent relaxation. 3. ICI 118551 (10 nM-1 microM) antagonized isoprenaline, procaterol and salmeterol in suppressing histamine-induced tone of the isolated trachealis muscle. The antagonism was concentration-dependent. In contrast, CGP 20712A (10 nM-1 microM) failed to antagonize isoprenaline, procaterol or salmeterol. 4. Salmeterol (1-10 microM) antagonized isoprenaline in relaxing strips of bovine trachea which had been precontracted with carbachol (1 microM). 5. Cromakalim (10 microM), isoprenaline (100 nM-10 microM), procaterol (10 nM-1 microM) and salbutamol (100 nM-10 microM) each promoted the efflux of 86Rb+ from strips of bovine trachealis muscle preloaded with the radiotracer. In contrast, salmeterol (100 nM-10 microM) failed to promote 86Rb+ efflux. 6. CGP 201712A (1 microM), ICI 118551 (100 nM) and salmeterol (1 microM) did not themselves modify 86Rb+ efflux from trachealis muscle strips, nor did they affect the promotion of 86Rb+ efflux induced by cromakalim (10 microM). In contrast, CGP 20712A (1 microM) and ICI 118551 (100nM) were each able to inhibit the promotion of 86Rb+ efflux induced by isoprenaline (1 microM) or procaterol (100 nM). Furthermore,salmeterol (10 microM) inhibited isoprenaline (1 microM)-induced promotion of 86Rb+ efflux.7. It is concluded that, in bovine trachealis, activation of either beta l- or beta 2-adrenoceptors can promote the opening of 86Rb+-permeable K+-channels in the plasmalemma. The failure of salmeterol to promote plasmalemmal K+-channel opening may reflect, not its selectivity in activating beta 2- as opposed to beta 1-adrenoceptors, but rather its low intrinsic efficacy at beta 2-adrenoceptors. The opening of plasmalemmal K+-channels plays a supportive rather than a crucial role in mediating the mechano-inhibitory effects of agonists at beta-adrenoceptors acting on trachealis muscle.

The physiological effects of relaxin during pregnancy: studies in rats and pigs.

For 50 years after its discovery in 1926, there was a general lack of interest in relaxin among both reproductive biologists and clinicians. A key reason for this lack of interest was the lack of information concerning relaxin's physiological importance during pregnancy in any species. Research conducted since the early 1980s has established that the hormone relaxin is essential during pregnancy in at least two species--rats and pigs. Two vital roles for relaxin during pregnancy have been identified. Relaxin promotes growth and softening of the uterine cervix and thereby enables rapid and safe delivery in both rats and pigs. Relaxin also promotes growth and development of the mammary apparatus in both species. Interestingly, the major effects of relaxin on mammary growth and development are targeted on the nipple in the rat, whereas they are targeted on the glandular parenchyma in the pig. Relaxin-dependent growth of the nipple in rats is required for normal lactational performance. Although likely, it remains to be established that relaxin's profound effects upon mammary gland development in pigs are required for normal lactational performance. The fact that relaxin has effects upon cervical and mammary gland development during pregnancy in both rats and pigs encourages the view that relaxin may have similar effects during pregnancy in other species. Nevertheless, one must keep in mind that there is great diversity in the physiology of relaxin among species (reviewed by Sherwood 1988). This diversity includes not only relaxin's source, regulation of synthesis and secretion, and secretory profiles during pregnancy, but also its biological effects. It seems essentially certain that relaxin's effects during pregnancy differ among species. For example, transformation of the pubic joint cartilage to a flexible and elastic interpubic ligament occurs during pregnancy in several species, including guinea pigs, mice, and bats. This pelvic adaptation, which is nearly certainly relaxin dependent, does not occur in species such as rats and sheep. It is possible that relaxin may have little or no physiological significance during pregnancy in some species. Although considerable progress has been made toward an understanding of the physiological role(s) of relaxin in pregnant rats and pigs, many fundamental questions remain unanswered.(ABSTRACT TRUNCATED AT 400 WORDS)

Cyclic adenosine 3'5'-monophosphate and the relaxant action of relaxin in the rat uterus in vivo.

The relationship between relaxant actions of relaxin in the uterus and changes in uterine cAMP concentrations was assessed in anaesthetized bilaterally ovariectomized nonpregnant rats. Relaxin i.v. bolus (5 micrograms kg-1) did not change cAMP concentrations but inhibited uterine contractions with rapid onset. Uterine contractions were significantly reduced by 30-70% for 60 min. Relaxin (50 micrograms kg-1) produced a short-lived (up to 5 min) and small (up to 3.2-fold) increase in cAMP concentrations plus a marked (90%) and prolonged inhibition of uterine contractions (70-90% over 60 min). Salbutamol (an agonist at beta 2-adrenoceptors, 100 and 500 micrograms kg-1) produced a similar degree and time course of inhibition of uterine contractions to that of relaxin but a more marked (19-fold) increase in cAMP concentrations. Glibenclamide, a blocker of ATP-sensitive potassium channels, which has been shown to antagonize relaxin as a uterine relaxant, did not prevent the relaxin-induced rise in cAMP concentrations. It is suggested that the uterine relaxant action of relaxin may not result from an increase in uterine cAMP concentrations.

Influence of ovarian steroids on myometrial sensitivity and tolerance to relaxin in the rat in vivo: lack of cross-tolerance between relaxin, salbutamol and cromakalim.

The influence of oestradiol benzoate and progesterone on uterine sensitivity and development of tolerance to relaxin was investigated in bilaterally ovariectomized non-pregnant rats in vivo. Bolus doses of relaxin (2-20 micrograms/kg i.v.) produced rapid and reversible inhibition of uterine contractions in a dose-dependent manner. Treatment with oestradiol benzoate or oestradiol benzoate plus progesterone significantly increased uterine sensitivity to relaxin over 48 h by 2.4- to 8.5-fold. Tolerance to relaxin developed during continuous infusion of the hormone at 20 micrograms/kg per h for 40 h. A 7.8- to 17.4-fold reduction in sensitivity to relaxin was observed in relaxin-infused rats, whereas no change in sensitivity was observed in saline-infused rats. Infusion of relaxin at 50 micrograms/kg per h for 40 h produced a 131.8-fold reduction in uterine sensitivity to relaxin. The uterus remained tolerant to relaxin for up to 24 h after cessation of infusion. Treatment with oestradiol benzoate and/or progesterone did not influence the extent of tolerance development, but a more rapid recovery of uterine sensitivity to relaxin was observed in rats treated with oestradiol benzoate plus progesterone. Cross-tolerance with other uterine relaxant drugs was measured to investigate possible common mechanisms of action and sites of tolerance between relaxin and a beta-adrenoceptor agonist (salbutamol) and potassium channel openers (cromakalim and minoxidil sulphate). No cross-tolerance was observed between relaxin and salbutamol, or relaxin and cromakalim or minoxidil sulphate. Cross-tolerance between cromakalim and minoxidil sulphate was seen.

Interaction between myometrial relaxants and oxytocin: a comparison between relaxin, cromakalim and salbutamol.

The influence of treatment with oestradiol on the effects of the uterine relaxants, relaxin, salbutamol (an agonist at beta 2-adrenoceptors) and cromakalim (a potassium channel opener) and their interactions with the uterine stimulant oxytocin were investigated in vivo in the ovariectomized rat. Oestradiol benzoate (0.4 micrograms/kg per day) significantly increased sensitivity to cromakalim as an inhibitor of spontaneous uterine contractions compared with vehicle-treated rats by approximately threefold. The same dose of oestradiol benzoate had no effect on uterine sensitivity to salbutamol. Previous studies have shown that this dose of oestradiol benzoate produces a twofold increase in uterine sensitivity to relaxin as an inhibitor of spontaneous contractions. Oestradiol influenced the ability of relaxin to inhibit oxytocin-stimulated uterine contractions. In corn oil-treated rats, uterine responses to relaxin were markedly reduced during oxytocin infusion compared with responses to relaxin before oxytocin; the maximum obtainable response to relaxin was less than 50% inhibition. In oestradiol-treated rats, uterine sensitivity to relaxin during oxytocin infusion was similar to that observed against spontaneous contractions. Cromakalim was able to inhibit uterine contractions during oxytocin infusion in both corn oil- and oestradiol-treated rats, uterine sensitivity to cromakalim being similar in the absence and presence of oxytocin for both hormone treatment groups. Salbutamol was also able to inhibit uterine contractions during oxytocin infusion in both corn oil- and oestradiol-treated rats. Oestradiol treatment increased the potency of salbutamol as an inhibitor of oxytocin-stimulated uterine contractions compared with corn oil treatment by 3.5-fold. The interaction of oestradiol and relaxin during late pregnancy may be important for attenuation of the myometrial response to stimulants.

Cross tolerance between cromakalim and RP49356 in the uterus of the rat in vivo and in vitro.

We investigated tolerance to the potassium (K+) channel openers, cromakalim and RP49356 and cross tolerance between them in the uterus of the rat. Continuous infusion of RP49356 in vivo produced 5.2-fold (200 or 300 micrograms/kg per h) and 7.2-fold (600 micrograms/kg per h) desensitization of the uterus to RP49356. Uterine sensitivity to RP49356 was reduced 7.4-fold in cromakalim-tolerant rats and sensitivity to cromakalim was reduced 7.9-fold in RP49356-tolerant rats. Incubation of isolated uterus with 10 microM cromakalim for 4 h reduced uterine sensitivity to cromakalim by about 45-fold and to RP49356 by 2.1-fold but did not alter sensitivity to salbutamol. Incubation with 10 microM RP49356 for 4 h reduced sensitivity to RP49356 by 2.5-fold and to cromakalim by 3.8-fold. Occurrence of uterine tolerance to K+ channel openers in vitro suggests that tolerance in vivo has a pharmacodynamic basis. Cross tolerance between cromakalim and RP49356 provides evidence that these two K+ channel openers possess common binding sites and/or mechanism(s) of action. However, the greater extent of self-tolerance to cromakalim than to RP49356 suggests a difference in their actions.

One way cross tolerance between cromakalim and salbutamol in the uterus of the rat in vivo.

1. Cross tolerance between the potassium (K+) channel opener, cromakalim and the beta 2-adrenoceptor agonist, salbutamol, was investigated in the uterus of the non-pregnant rat in vivo. Uterine sensitivity to salbutamol was similar in both vehicle-treated and cromakalim-tolerant rats. In salbutamol-tolerant rats, uterine responses to cromakalim were markedly decreased compared with saline-infused rats, such that maximum inhibition of uterine contractions was less than 40%. 2. Propranolol treatment and salbutamol tolerance each produced similar reductions in sensitivity of the uterus to salbutamol of approximately 10 fold. The same dose of propranolol did not influence uterine sensitivity to cromakalim, which suggests that the relaxant action of cromakalim is not due to a direct or indirect activation of uterine beta 2-adrenoceptors. 3. Salbutamol produced a marked (11.7 fold) increase in uterine adenosine 3':5'-cyclic monophosphate (cyclic AMP) concentrations measured ex vivo, which was completely inhibited by propranolol pretreatment, but was unaffected by glibenclamide pretreatment. Cromakalim did not increase uterine cyclic AMP concentrations, suggesting that stimulation of adenylate cyclase is not significant in the uterine relaxant action of cromakalim. 4. The lack of propranolol antagonism of cromakalim and of cromakalim-induced changes in uterine cyclic AMP concentrations suggests that the cross tolerance observed between salbutamol and cromakalim may be at the level of K(+)-channels.

Antagonism of relaxin by glibenclamide in the uterus of the rat in vivo.

1. The effects of glibenclamide (a blocker of adenosine triphosphate [ATP]-dependent K(+)-channels) on the inhibition of uterine contractions by relaxin, salbutamol and cromakalim were compared in vivo. 2. Glibenclamide (20 mg kg-1) did not antagonize salbutamol. Glibenclamide produced a parallel rightward shift in the dose-response curve to cromakalim with a 5.5 fold decrease in uterine sensitivity (post-vehicle log ID50, -0.87 mg kg-1; post-glibenclamide log ID50, -0.07 mg kg-1). Glibenclamide produced a non-parallel rightward shift in the dose-response curve to relaxin (post-vehicle log ID50, 0.99 microgram kg-1; post-glibenclamide log ID50, 2.28 micrograms kg-1). 3. Glibenclamide reversed established inhibition of uterine contractions by cromakalim or relaxin but not that by salbutamol. 4. Insulin produced no antagonism of relaxin on isolated uterus of the rat, demonstrating that glibenclamide antagonism of relaxin in vivo is not by released insulin. Apamin did not antagonize relaxin in vivo, suggesting that small calcium-activated K(+)-channels are not involved in the action of relaxin. 5. Comparison of the lack of antagonism of salbutamol with the non-competitive-like antagonism of relaxin by glibenclamide suggests that relaxin does not relax uterine smooth muscle predominantly by increasing intracellular adenosine 3', 5'-cyclic monophosphate concentrations. Comparison of the non-competitive-like antagonism of relaxin and the competitive-like antagonism of cromakalim by glibenclamide suggests that the two relaxants may share, in part, a common mechanism of action and that additional mechanism(s) may also be involved in the inhibitory action of relaxin.

Effects of several potassium channel openers and glibenclamide on the uterus of the rat.

1. The ability of several potassium (K+) channel openers to inhibit spasm of the uterus of the nonpregnant rat and their susceptibility to antagonism by glibenclamide was assessed in vitro and in vivo. 2. In the isolated uterus exposed to oxytocin (0.2 nM), cromakalim, RP 49356 and pinacidil were of similar potency (mean pD2 = 6.4, 6.0 and 6.2 respectively) while minoxidil sulphate was of lower potency (pD2 = 4.7). Glibenclamide antagonized cromakalim and RP 49356 with the interactions consistent with competitive antagonism (mean pA2 of 6.57 and 7.00 respectively). Glibenclamide also antagonized pinacidil (pA2 = 6.22) but the slope of the Schild plot was significantly greater than -1. Neither salbutamol nor minoxidil sulphate was antagonized by glibenclamide (10 microM). 3. Cromakalim (1 and 10 microM), RP 49356 (1 and 10 microM), pinacidil (1 microM) and minoxidil sulphate (100 microM) suppressed spasm evoked by low (less than 40 mM) but not high (greater than or equal to 40 mM) KCl concentrations. Glibenclamide (10 microM) prevented cromakalim (10 microM)-, RP 49356 (10 microM)- and pinacidil (10 microM)-induced suppression of KCl (20 mM)-evoked spasm. Pinacidil (10 and 100 microM), cromakalim (100 microM) and salbutamol (0.01-1 microM) inhibited spasm evoked by all concentrations of KCl (10-80 mM). Suppression of spasm evoked by KCl (10-80 mM) by cromakalim (100 microM) and pinacidil (100 microM) was insensitive to glibenclamide (10 microM). 4. Cromakalim (0.1 mg kg-1) and RP 49356 (0.1 mg kg-1), given by i.v. bolus injection, inhibited uterine contractions, produced a fall in blood pressure and a slight tachycardia in the conscious ovariectomized rat. Glibenclamide (20mgkg-'), given by i.v. infusion, antagonized the vascular and uterine smooth muscle relaxant properties of cromakalim and RP 49356. 5. Several K+ channel openers are uterine relaxants. The antagonism of cromakalim, RP 49356 and pinacidil, at low concentrations, by glibenclamide suggests their actions may involve an ATP-sensitive K+channel. High concentrations of pinacidil (10 and 100 microM) and cromakalim (100 microM) may exert an additional action in the uterus. The low potency of minoxidil sulphate and its insensitivity to glibenclamide in the isolated uterus suggests that its mechanism of action may differ from that of the other K+ channel openers.

Tolerance to cromakalim in the rat uterus in vivo.

1. Cromakalim (0.1 and 1 mg kg-1) produced inhibition of uterine contractions and falls in mean blood pressure in ovariectomized, non-pregnant rats, the durations of which were dose-dependent. Frequency of contractions was inhibited selectively compared to amplitude. 2. The durations of the uterine effect of cromakalim (1 mg kg-1), when given at 12 h intervals, were less after the second, third and fourth doses compared to the first dose in non-pregnant rats. In ovariectomized rats treated from day 18 of pregnancy, using the same experimental design, no uterine relaxant effects were seen to the third and fourth doses. The timing of delivery and foetal viability were unaffected by cromakalim. 3. The sensitivity of the uterus to cromakalim was tested using small doses before and after three cromakalim doses (1 mg kg-1) given at 8 h intervals in non-pregnant rats. This regime resulted in a 25 fold decrease in the sensitivity of the uterus to cromakalim. 4. Cromakalim can produce long-lasting inhibition of uterine contractions in the rat after bolus i.v. dose but it exhibits little selectivity relative to its vasodepressor action and there appears to be tolerance to the uterine relaxant action of cromakalim in vivo.

Nifedipine kinetics in the rat and relationship between its serum concentrations and uterine and cardiovascular effects.

1. The kinetics of nifedipine and the relationship between its serum concentration and uterine and cardiovascular effects were investigated in 3 groups of animals. These were ovariectomized (ovx) anaesthetized non-pregnant rats following bolus i.v. injection (400 micrograms kg-1) and during 300 min infusion (10 micrograms kg-1 min-1) and ovx, progesterone-treated late pregnant rats during infusion. Also, the kinetics were determined in ovary-intact late pregnant rats following bolus i.v. injection (400 micrograms kg-1). 2. Measurement of serum nifedipine concentrations after bolus i.v. injection in ovx non-pregnant rats showed a biexponential decay with time from which the following parameters were calculated: V beta = 300 +/- 30 ml kg-1; rate constants k12 = 0.51 +/- 0.18 min-1; k21 = 0.07 +/- 0.02 min-1; ke1 = 0.10 +/- 0.05 min-1; elimination clearance = 2.4 +/- 0.2 (ml min-1) kg-1; t1/2 alpha = 2.5 +/- 1.0 min; t1/2 beta = 102 +/- 15 min. In intact pregnant rats, a biexponential decay of serum nifedipine concentrations with time was also observed after bolus i.v. administration with similar parameters to non-pregnant animals. These kinetic parameters, used to calculate serum nifedipine concentrations obtained during infusion, predicted values similar to experimental values for 180 min, but thereafter slightly underestimated experimental values. 3. Immediate reductions in uterine contractions, mean blood pressure and heart rate were observed following bolus i.v. injection of nifedipine to ovx non-pregnant rats, with returns towards control values as serum nifedipine concentrations declined. IC15 values (15% change from baseline), calculated from log10 serum concentration-response curves, of 0.3 +/- 0.05 micrograms ml-1 for inhibition of uterine contractions, 0.8 +/- 0.3 micrograms ml-1 for depression of blood pressure and 3.8 +/- 1.0 micrograms ml-1 for reduction in heart rate were obtained. 4. In ovx non-pregnant rats, nifedipine infusion produced a maximum reduction in integral of uterine contractions of 70% by 120 min and a maximum reduction of 15% in heart rate. Mean blood pressure was not significantly different from vehicle-treated rats. IC15 values were 0.7 +/- 0.1 micrograms ml-1 and 2.8 +/- 0.6 micrograms ml-1 for inhibition of uterine contractions and heart rate respectively. 5. In ovx, progesterone-treated late pregnant rats, nifedipine infusion produced similar serum concentrations to those of non-pregnant rats but completely abolished uterine contractions by 70 min. Maximum reductions of 30% in heart rate and blood pressure were observed. IC1S values were 0.5 + 0.1ygml-1 for uterine contractions, 0.9 + 0.3lpgmlP1 for blood pressure and 1.2 +/- 0.3 pg ml - 1 for heart rate. 6. The findings suggest that the kinetics of nifedipine are similar in pregnant and non-pregnant rats and support the idea that the drug exerts a slight selectivity for uterine inhibition relative to cardiovascular effects. The uterus of the late pregnant rat appears to be more sensitive to nifedipine than that of the non-pregnant animal.

The influence of oestrogen and progesterone on the actions of two calcium entry blockers in the rat uterus.

The potency and maximum effect of the calcium entry blocker nifedipine as an inhibitor of uterine contractions in vivo are increased in rats in late pregnant compared with non-pregnant rats. The influence of ovarian steroids produced during pregnancy (oestrogen and progesterone) on the potency and maximum effect of two calcium entry blockers (nifedipine and diltiazem) against uterine contractions during i.v. infusion was therefore investigated in anaesthetized non-pregnant rats. The influence of pregnancy on the relationship between serum concentrations of diltiazem during i.v. infusion and uterine and cardiovascular effects was also investigated. A twofold increase in the potency of nifedipine as an inhibitor of uterine contractions was observed in rats treated with oestrogen or oestrogen plus progesterone compared with rats treated with corn oil. There was no change in potency in rats receiving progesterone alone. Maximum inhibition of uterine contractions by nifedipine was significantly increased by all three hormone treatments. A twofold increase in the potency of diltiazem and a significant increase in maximum inhibition of uterine contractions was observed in rats in late pregnancy compared with non-pregnant rats. No increase in potency of diltiazem in reducing blood pressure or heart rate was observed in rats in late pregnancy. No significant difference in potency of diltiazem against uterine contractions was observed in rats treated with oestrogen, progesterone or oestrogen plus progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)