Christopher Chapman

Effects of Opioid Agonists and Antagonists on Oxytocin and Vasopressin Release in vitro

The rat neurohypophysis contains both opioid receptors and substantial amounts of endogenous opioid peptides. Inhibitory influences of opioids on the secretion of both oxytocin and vasopressin have been described. We have examined the effects of a range of opioid agonists and antagonists with differing relative selectivities towards opioid receptor subclasses on the secretion of oxytocin and vasopressin from the isolated neurohypophysis. Oxytocin and vasopressin release evoked by brief periods of electrical stimulation in control experiments was compared to evoked release in the presence of test compounds. Oxytocin release was depressed approximately 25% by the delta-agonist (D-Ala2, D-Leu5)-enkephalin but not affected by putative kappa-agonists or by beta-endorphin. The use of opioid antagonists revealed a strong inhibition of oxytocin secretion by endogenous opioids released during electrical stimulation. Naloxone, relatively mu-selective, enhanced oxytocin secretion by up to 90% with a half-maximal effect at approximately 10(-6) M. MR2266, a relatively kappa-selective antagonist also enhanced oxytocin secretion but displayed agonist-like activity at high concentrations. ICI 154129, a delta-selective antagonist, was without effect on oxytocin secretion. Vasopressin release was unaffected by any of the agonists tested and not potentiated by antagonists at a range of stimulation frequencies. The data do not support the suggestion of an inhibitory endogenous opioid influence over vasopressin secretion within the neurohypophysis but indicate that an endogenous opioid peptide, possibly acting via mu- or kappa rather than delta-receptors, strongly suppresses the secretion of oxytocin.

Role of medial preoptic GABA neurones in regulating luteinising hormone secretion in the ovariectomised rat.

SummaryThe role of GABA neurones in the medial preoptic area (MPOA) in regulating the activity of the luteinising hormone-releasing hormone (LHRH) neurones projecting to the median eminence was investigated in the conscious ovariectomised rat. Plasma luteinising hormone (LH) concentrations were measured while (1) endogenous GABA release from the MPOA was monitored with the technique of microdialysis, or (2) activity at the GABA receptor was modulated by local infusions into the MPOA. Microdialysis studies revealed a fluctuating level of GABA release in the MPOA which did not correlate with pulsatile LH secretion. Infusion of 10 μM GABA (n=8) or bicuculline methiodide (BMI, n=6) into the MPOA, at a rate of l μ1/30 min, significantly inhibited mean LH concentrations (P< 0.05-0.001) and LH pulse frequency (P< 0.05-0.001) compared with controls (n = 8). LH pulse amplitude was not significantly altered by infusion of GABA (P> 0.05) while too few pulses were found after BMI treatment to enable statistical analysis. Infusions of GABA into the ventral half of the MPOA had a more significant inhibitory effect upon LH secretion compared with dorsal infusions (P=0.012). A similar relationship did not exist for BMI infusions. These results show that acute changes in preoptic GABA receptor occupancy result in disruption of pulsatile LH secretion in the ovariectomised rat. This suggests that GABA neurones provide a tonic input important for the functional integrity of the neural network controlling LH secretion. However, as changes in extracellular GABA concentrations in the MPOA do not correlate with pulsatile LH release, the preoptic GABA population, as a whole, is unlikely to be directly responsible for the pulsatile activity of the LHRH neurones in the ovariectomised rat.

Extracellular GABA concentrations in rat supraoptic nucleus during lactation and following haemodynamic changes: An in vivo microdialysis study

Morphological and pharmacological evidence suggest that the dense GABAergic innervation of the supraoptic nucleus is important for regulating the electrical activity of vasopressin and oxytocin neurons. We have employed the technique of intracranial microdialysis to examine extracellular GABA concentrations in the supraoptic nucleus of the anaesthetized rat and questioned whether differences exist in the dynamics of GABA release between virgin and lactating rats, and if events during lactation or following blood pressure manipulation alter endogenous GABA levels in this nucleus. No significant differences were detected between virgin and lactating animals in either basal or 100 mM potassium ion-evoked GABA release. The inclusion of the GABA uptake blocker nipecotic acid (0.5 mM) into the dialysate resulted in a six- to eight-fold increase (P < 0.01) in GABA outflow in both groups of animals. In lactating rats, GABA outflow measured at 4 min intervals was not altered during a 60 min period of suckling by a full litter of pups and no significant change in GABA outflow was detected in relation to individual milk ejections. In virgin rats, removal of 1.5-2 ml of blood resulted in a 30-60 mmHg fall in blood pressure and a non-significant decline in GABA outflow. Replacement of blood resulted in an abrupt 50 mmHg increase in blood pressure and a significant 22% increase in GABA outflow (P < 0.01), but no change in aspartate or methionine concentrations. Repeated intravenous injections of the alpha-adrenoceptor agonist, metaraminol, similarly evoked approximately 50 mmHg increments in blood pressure and a 26% increase in GABA outflow (P < 0.05). Electrical stimulation of the diagonal band of Broca for 10 min produced a two-fold increase in GABA outflow from the supraoptic nucleus (P < 0.05). These results show that the overall profile of basal and potassium-stimulated GABA concentrations in the supraoptic nucleus is not substantially different between lactating and virgin rats. In lactating animals we have found that GABA levels are not altered in response to suckling or at the time of high-frequency firing by oxytocin neurons to induce milk ejection. In contrast, our data further support the hypothesis that GABA inputs to supraoptic neurons are part of a baroreceptor reflex, relaying through the diagonal band of Broca, to signal periods of acute hypertension and inhibit the firing of vasopressin neurons. Such observations suggest the physiological importance of GABA inputs to the supraoptic nuclei and indicate that GABA may be used in a stimulus-specific manner to influence the activity of magnocellular neurons.

Bombesin Stimulates Growth Hormone Secretion from Cultured Bovine Pituitary Cells

Bombesin-like peptides are present in the mammalian hypothalamus. It has previously been reported that synthetic bombesin elevates plasma growth hormone (GH) and prolactin (PRL) concentrations in rats. To investigate whether bombesin has a direct action on the pituitary gland we measured GH and PRL secretion from 5-day cultures of bovine anterior pituitary cells. We compared the effects of bombesin with those of a previously described synthetic pentapeptide which releases rat GH in vitro (GHRP) and with the effects of acetylcholine which stimulates bovine GH secretion. Bombesin stimulated GH but not PRL secretion from pituitary cultures during 90-min incubations. Maximal stimulation to 157 +/- 16% of the control value was seen with 1.2 X 10(-7) M bombesin. Half-maximal stimulation occurred with 5 X 10(-9) M bombesin. The pentapeptide GHRP also stimulated GH but not PRL secretion. Maximal stimulation to 207 +/- 14% of the control value was seen with 1.3 X 10(-5) M GHRP. Half-maximal stimulation occurred with 10(-7) M GHRP. Acetylcholine stimulated secretion of both GH and PRL, both responses being abolished by the muscarinic antagonist atropine. Maximal stimulation with 10(-4) M acetylcholine was to 381 +/- 33% of the control value for GH and to 134 +/- 5% of the control value for PRL. The effects of bombesin and GHRP on GH secretion were not additive whilst the effects of both peptides were additive with those of acetylcholine. The data suggest that the synthetic GHRP peptide may possibly interact with native bombesin receptors on pituitary somatotrophs and that bombesin or a related peptide may act in vivo as a GH releasing factor.

Expression of pheromone receptor gene families during olfactory development in the mouse: expression of a V1 receptor in the main olfactory epithelium

In the mouse, two large gene families, V1R and V2R, encoding putative pheromone receptors have been described. Studies have suggested a homotypic recognition role for V1Rs and V2Rs during development in the targeting of vomeronasal axons to specific sets of glomeruli in the accessory olfactory bulb (AOB). Analysis of the onset of expression of the V1R and V2R gene families in developing vomeronasal neurons using polymerase chain reaction and in situ hybridization now suggests that a role for these receptors in the organization of axon projections is only likely at the final stages of targeting within the AOB. Surprisingly, our studies reveal expression of a V1Rd receptor in scattered cells within the main olfactory epithelium, suggesting that limited pheromone detection may also take place in this structure. The pheromone sensory neurons of the vomeronasal system and the neuroendocrine gonadotrophin‐releasing hormone (GnRH) neurons that regulate fertility both arise from progenitor cells of the nasal placode. The development of these two cell types is intimately linked, and the GnRH neuron population migrates into the forebrain during embryogenesis in close association with a subset of vomeronasal sensory axons; how GnRH neurons recognize this axon subset is unknown. We report selective expression of a V1Ra gene in the clonal NLT GnRH cell line, raising the possibility of a similar role for V1Rs or V2Rs in the directed migration of GnRH neurons. However, no expression of this gene or of other V1Rs and V2Rs is detectable at the cellular level in migrating GnRH neurons in the mouse.

Effects of central GABAB receptor modulation upon the milk ejection reflex in the rat.

In order to investigate the role of central GABAB receptors in the control of the milk ejection reflex, we have examined the effects of third ventricular and bilateral supraoptic nucleus (SON) injections of a GABAB receptor agonist (baclofen) and antagonist (hydroxy-saclofen) on the milk ejection reflex in the urethane-anaesthetised rat. In addition, microdialysis studies have evaluated whether the activation of GABAB receptors in the SON is able to modulate the release of GABA and glutamate, two major neurotransmitters involved in the regulation of the milk ejection reflex. Intracerebroventricular injections of baclofen (n = 9) in doses of 10, 50 and 100 pmol inhibited the milk ejection reflex in a dose-dependent manner, without affecting the electroencephalogram or attenuating the intramammary pressure response to intravenous injection of 0.5 mU exogenous oxytocin. Hydroxy-saclofen given into the third ventricle in doses of 100 pmol (n = 2) and 500 pmol (n = 4) did not modify the pattern of the milk ejection reflex. Bilateral SON microinfusions of baclofen in doses of 80 (n = 2) and 200 pmol (n = 4) did not modify the pattern of the milk ejection reflex. In microdialysis experiments (n = 8), inclusion of baclofen into the microdialysate at a concentration of 500 microM had no effect upon basal or potassium-stimulated GABA and glutamate outflow. These results show that the activation of GABAB receptors located outside, but not within, the SON are capable of inhibiting the milk ejection reflex. In contrast to our previous findings regarding the GABAA receptor, we found no evidence for a tonic role of GABAB receptors within the neural network inducing the periodic synchronous bursting of oxytocin neurons during suckling.

Stimulus-Induced Depletion of Pro-Enkephalins, Oxytocin and Vasopressin and Pro-Enkephalin Interaction with Posterior Pituitary Hormone Release in vitro

The secretion of oxytocin (OXT) from the neurohypophysis is modulated by the actions of opioids acting via kappa-receptors. The vasopressin (AVP)-containing nerve terminals in the neurohypophysis contain the kappa-opioid agonist dynorphin, but endogenous opioid restraint of OXT secretion is observed even when AVP release is not activated, suggesting that another source of opioids is responsible for modulating OXT secretion. We now report that acute stimulation of the rat neural lobe in vivo results in depletion of the neural lobe content of OXT, AVP, dynorphin A1-17, dynorphin A1-8 and metenkephalin (Met-Enk). The dynorphin content is depleted to a similar extent as that of OXT and AVP; a correlation analysis suggests that while most dynorphin is co-secreted with AVP, a significant portion is co-secreted with OXT, consistent with a co-localisation of dynorphin with OXT. Met-Enk was depleted to a lesser extent than either hormone, consistent with a partial localisation in non-releasable pools. However, depletion of Met-Enk was also observed following naloxone-precipitated opioid withdrawal accompanying selective hypersecretion of OXT, suggesting co-secretion of OXT and Met-Enk. Met-Enk is a mu-opioid receptor agonist, but extended forms of Met-Enk, as we now report, are active at neurohypophysial kappa-receptors.

Neurohypophysial opioids and oxytocin secretion: source of inhibitory opioids.

SummaryWhen electrical stimuli are applied to the neural stalk of the pituitary, oxytocin, vasopressin, and probably several opioid peptides also contained in nerve terminals in the gland are released: one action of the released opioids appears to be to inhibit oxytocin release by an action that has been likened to pre-synaptic inhibition. Thus, when Clarke et al. (1979) stimulated the neural stalk following intravenous injection of the opioid antagonist naloxone, they observed that the evoked oxytocin release was potentiated. In the present study we confirm this result and show that oxytocin release evoked by stimulation of the supraoptic nucleus is similarly potentiated by naloxone. This finding is consistent with the hypothesis that the opioid responsible for inhibition of oxytocin release coexists with either oxytocin or vasopressin. We further report that the specific δ-receptor antagonist ICI 174864 does not potentiate oxytocin release either in vivo or in vitro. Thus, it seems unlikely that the enkephalins, putative δ-receptor agonists present in neurohypophysial fibres, are the opioids responsible for the observed inhibition of oxytocin release.