Intravascular pressure-evoked changes in intracellular calcium [Ca2+]i and tone in rat mesenteric and rabbit cerebral arteries in vitro.

The degree of myogenic response varies between different vascular beds and may contribute to differences in autoregulation. This study examined whether differences in pressure-induced changes in intracellular calcium ([Ca2+]i) account for this variability by comparing responses in rabbit cerebral arteries which possess a prominent myogenic response with rat mesenteric arteries where the response is much less marked. Rat mesenteric small arteries and small branches of the posterior cerebral artery were mounted on glass pipettes in a perfusion myograph containing physiological saline at 37 degrees C and aerated with 95% O2 and 5% CO2 under pressure and no flow conditions. Outer diameters of arteries were measured using a video dimension analyser. Vessel diameters were measured following exposure to 30, 60 and 90 mm Hg intralumenal distending pressure in the presence and absence of extracellular Ca2+ to determine the degree of active tone. In subsequent studies vessels were loaded with fura-2AM to allow measurement of changes in [Ca2+]i in response to the same conditions. Significant active tone was only seen at 60 mm Hg in rat mesenteric small arteries. In contrast, posterior cerebral arteries possessed active tone at all distending pressures. Increases in pressure were associated with rises in [Ca2+]i to similar extents in both vessel types. These data suggest that changes in [Ca2+]i in response to pressure changes in rabbit cerebral and rat mesenteric small arteries are similar despite differences in myogenic tone. The contrasting behaviour of these vessels may therefore reflect other factors such as differences sensitivity of the contractile machinery to [Ca2+]i.

Morphine tolerance and inhibition of oxytocin secretion by kappa-opioids acting on the rat neurohypophysis.

1. The present study investigated the mechanisms by which endogenous opioids regulate oxytocin secretion at the level of the posterior pituitary gland. Effects of the selective kappa-agonist U50,488 on oxytocin secretion were studied in urethane-anaesthetized lactating rats. Oxytocin secretion in response to electrical stimulation (0.5 mA, matched biphasic 1 ms pulses, 50 Hz, 60-180 pulses) of the neurohypophysial stalk was bioassayed on-line by measuring increases in intramammary pressure, calibrated with exogenous oxytocin. Intravenous (I.V.) U50,488 inhibited electrically stimulated oxytocin secretion, without affecting mammary gland sensitivity to oxytocin. The inhibition was dose related, with an ID50 of 441 (+194, -136) micrograms/kg and was naloxone reversible. Antagonism of endogenous beta-adrenoceptor activation by propranolol (1 mg/kg) reduced the potency of U50,488. The selective mu-agonist morphine (up to 5 mg/kg), had no effect on electrically stimulated oxytocin secretion, but depressed the mammary response to oxytocin. 2. In lactating rats given intracerebroventricular (I.C.V.) morphine infusion for 5 days to induce tolerance and dependence, I.V. U50,488 still inhibited electrically stimulated oxytocin secretion, but the ID50 was reduced to 170 (+78, -54) micrograms/kg; thus at the posterior pituitary the sensitivity of kappa-receptors is enhanced rather than reduced in morphine-tolerant rats, indicating the absence of cross-tolerance. In these rats, naloxone produced a large, sustained, fluctuating increase in intramammary pressure indicating morphine-withdrawal excitation of oxytocin secretion; I.V. U50,488 diminished this response, confirmed by radioimmunoassay, demonstrating the independence of mu- and kappa-receptors regulating oxytocin secretion. 3. In pregnant rats, I.C.V. infusion of morphine from day 17-18 of pregnancy delayed the start of parturition by 4 h, but did not significantly affect the progress of parturition once established, indicating tolerance to the inhibitory actions of morphine on oxytocin secretion in parturition, and lack of cross-tolerance to endogenous opioids restraining oxytocin in parturition. 4. Neurointermediate lobes from control and I.C.V. morphine-infused virgin rats were impaled on electrodes and perifused in vitro. Vasopressin and oxytocin release from the glands was measured by radioimmunoassay. Each gland was exposed to two periods of electrical stimulation (13 Hz, for 3 min). Naloxone (5 x 10(-6) M) was added before the second stimulation; half the lobes from each I.C.V. treatment were exposed to 5 x 10(-5) M morphine throughout.(ABSTRACT TRUNCATED AT 400 WORDS)

Pethidine (meperidine) inhibition of oxytocin secretion and action in parturient rats.

Pethidine (also known as meperidine and as Demerol) injected subcutaneously at 10 mg/kg into parturient rats on the birth of the second pup resulted in a marked slowing of the progress of parturition, associated with reduced plasma oxytocin concentrations. Injection of the opiate antagonist naloxone counteracted the inhibition of oxytocin secretion and largely prevented the slowing of parturition. In vitro, pethidine inhibited spontaneous, oxytocin-induced and acetylcholine-induced contractions of uteri from rats immediately post partum, and these effects were not reversed by naloxone. In anesthetized lactating rats, pethidine inhibited the suckling-induced milk-ejection reflex and attenuated oxytocin-induced contractions of mammary myoepithelium. Finally, pethidine depressed plasma oxytocin concentrations in rats given 2% saline to drink for 24 h to stimulate oxytocin secretion. Thus pethidine inhibits oxytocin secretion in all three conditions; this inhibition is probably mediated by central opioid receptors. In addition, however, pethidine depresses the oxytocin responsiveness both of mammary myoepithelium and of myometrium. The latter effect at least is not opioid mediated.

Release of oxytocin into blood and into cerebrospinal fluid induced by naloxone in anaesthetized morphine-dependent rats: the role of the paraventricular nucleus.

Abstract Opioid actions on oxytocin secretion into blood and cerebrospinal fluid (CSF) were investigated in urethane-anaesthetized female rats after intracerebroventricular (icv) infusion of morphine sulphate or vehicle for 5 days. Serial femoral arterial blood samples and cisterna magna CSF samples were collected for radioimmunoassay. Naloxone was given to assess endogenous opioid tone in icv vehicle-infused rats and to precipitate withdrawal in morphine-dependent animals. Initial plasma oxytocin concentration was not affected by icv morphine infusion. In control rats receiving icv vehicle, naloxone increased plasma oxytocin 11-fold within 5 min, and in icv morphine-infused rats, naloxone increased plasma oxytocin 80-fold within 5 min. In both groups, 90 min after naloxone plasma oxytocin was still 5 and 10 times, respectively, the initial concentration. Without naloxone, neither plasma nor CSF oxytocin concentration changed significantly with time (up to 90 min) in either icv treatment group. In the icv vehicle group, there was a 2-fold increase in CSF oxytocin 90 min after naloxone. In the icv morphine-infused group, CSF oxytocin was increased 5-fold 40 min after naloxone. In another group of icv morphine-infused rats, intravenous infusion of oxytocin to achieve plasma levels similar to those seen after naloxone, did not significantly increase CSF oxytocin. In a further group of icv morphine-infused rats, [(3)H]oxytocin was infused intravenously immediately after naloxone was given; in these rats oxytocin transfer from blood to CSF could account at most for only 20% of the increase in CSF oxytocin after naloxone. A further group of rats underwent bilateral microknife ablation of the paraventricular nuclei (PVN) 9 days before icv vehicle or morphine infusions were started; blood and CSF samples were collected under urethane anaesthesia. Initial concentrations of oxytocin in CSF and in plasma were similar in both groups with PVN ablation. In all PVN-lesioned rats initial plasma concentrations of oxytocin were undetectable (<5 pg/ml) and thus less than in intact rats. In contrast, initial levels of oxytocin in CSF were 8-fold greater in PVN-lesioned rats than in intact animals. Naloxone increased plasma oxytocin concentration in the icv vehicle group at least 10-fold within 30 min and in the icv morphine group at least 100-fold within 5 min. CSF oxytocin in the icv vehicle group was not altered by naloxone, but in the icv morphine group CSF oxytocin was increased 5-fold 40 min after naloxone. There were no consistent differences between the icv vehicle- and icv morphine-treated groups in the initial plasma levels of vasopressin, growth hormone and adrenocorticotrophin; PVN ablation did not affect adrenocorticotrophin levels. After naloxone growth hormone levels did not change, vasopressin concentration rose moderately only after 90 min and only in the icv vehicle-treated group, and adrenocorticotrophin concentrations decreased with time whether or not naloxone was given. The results imply an endogenous opioid tone on neurons releasing oxytocin into CSF, and morphine-dependence of these neurons. Furthermore, in PVN-lesioned rats, magnocellular supraoptic neurons could be a source of oxytocin release into CSF.

Release of oxytocin but not corticotrophin-releasing factor-41 into rat hypophysial portal vessel blood can be made opiate dependent.

The effects of morphine dependence and abrupt opiate withdrawal on the release of oxytocin and corticotrophin-releasing factor-41 (CRF-41) into hypophysial portal vessel blood in rats anaesthetized with urethane were investigated. Adult female Sprague-Dawley rats were made dependent upon morphine by intracerebroventricular infusion of morphine for 5 days; abrupt opiate withdrawal was induced by injection of the opiate antagonist naloxone. The basal concentrations of oxytocin in portal or peripheral plasma from morphine-dependent rats did not differ significantly from those in control, vehicle-infused rats. In rats in which the pituitary gland was not removed after stalk section, the i.v. injection of naloxone hydrochloride (5 mg/kg) resulted in a large and sustained increase in the concentration of oxytocin in both portal and peripheral plasma in control and morphine-dependent rats. The i.v. injection of naloxone resulted in a threefold increase in the secretion of oxytocin into portal blood in acutely hypophysectomized rats infused with morphine, but did not alter oxytocin secretion in vehicle-infused hypophysectomized rats. The concentration of oxytocin in peripheral plasma in both vehicle- and morphine-infused hypophysectomized rats was at the limit of detection of the assay and was unchanged by the administration of naloxone. There were no significant differences in the secretion of CRF-41 into portal blood in vehicle- or morphine-infused hypophysectomized rats either before or after the administration of naloxone. These data show that, as for oxytocin release from the neurohypophysis into the systemic circulation, the mechanisms which regulate oxytocin release into the portal vessel blood can also be made morphine dependent. The lack of effect of morphine or naloxone on the release of CRF-41 or other stress neuro-hormones suggests that the effect of opiate dependence and withdrawal is selective for oxytocin and is not simply a non-specific response to 'stress'.

Opioid receptor subtypes in the supraoptic nucleus and posterior pituitary gland of morphine-tolerant rats.

Morphine, given acutely, inhibits oxytocin secretion in adult female rats, but chronic intracerebroventricular infusion for five to six days induces tolerance and dependence in the mechanisms regulating oxytocin secretion. One explanation for tolerance could be that there is a loss of opioid receptors. To test this hypothesis cryostat sections of selected brain regions and the pituitary, from six control and six intracerebroventricular morphine-infused rats, were processed for quantitative in vitro receptor autoradiography. [3H]Etorphine or [3H](-)-bremazocine were used as ligands, and DAGO, DPDPE and U50,488H as selective displacers from mu-, delta-, and kappa-receptors, respectively. Control incubations had naloxone determined specificity. The supraoptic nucleus (site of oxytocin-secreting magnocellular perikarya) contained both mu- and kappa-receptors in control rats (mean +/- S.E.M. binding of mu-selective [3H]etorphine was 91.8 +/- 25.4 fmol/mg of tissue, and of kappa-selective [3H](-)-bremazocine was 130.4 +/- 25.6 fmol/mg). Chronic morphine treatment caused a 83.9% decrease in binding in mu-selective conditions (P less than 0.05), but no significant change in kappa-selective binding. In the median preoptic nucleus (which projects to the supraoptic nucleus) mean +/- S.E.M. binding of [3H]etorphine decreased by 77.0% (P less than 0.01) in chronic morphine-treated rats, from the control value of 76.2 +/- 9.8 fmol/mg of tissue. In the posterior pituitary gland (site of the terminals of the oxytocin-secreting magnocellular perikarya) binding with [3H](-)-bremazocine in controls was over 90% lower than in the supraoptic nucleus. No changes followed chronic morphine treatment. Thus chronic morphine exposure reduces the numbers of available mu-receptors in the supraoptic nucleus, and of opioid receptors in the median preoptic nucleus, perhaps accounting for morphine-tolerance in relation to oxytocin secretion.