Biomedical subjects

R J Bicknell

Publications and source records attributed to R J Bicknell.

At least 55 records · Page 3Linked to original sources

PubMed1991

Binding sites for atrial natriuretic peptide (ANP) on cultured pituicytes: lack of effect of ANP on release of neurohypophysial hormones in vitro.

Receptors for atrial natriuretic peptide (ANP) are known to be present in the posterior pituitary gland and this is a possible site of action of ANP to modulate neurohypophysial hormone release. Pituicytes cultured from adult rat neurohypophyses are shown to possess high affinity binding sites for ANP, suggesting that in vivo a population of neurohypophysial ANP receptors are present on these astrocytic glial cells. alpha-rANP (1-100 nM) did not modulate the basal or electrically stimulated release of oxytocin or vasopressin from the isolated neurohypophysis in vitro. The physiological significance of the glial ANP binding sites thus remains unknown.

Animals↗

PubMed1991

Opioid binding in the rostral hypothalamus is reduced following lesion of the ventral noradrenergic tract in female rats.

Experiments were undertaken to establish whether opioid receptors exert a direct presynaptic influence on noradrenergic (NA) terminals in the preoptic/anterior hypothalamus (PO/AH) of the female rat. Thus, opioid binding studies were performed in rats with lesions of the ventral NA tract (VNAT; the main NA projection to the hypothalamus) to assess whether a loss of NA terminals may also result in a decrease in opioid binding in the PO/AH. In the first experiment, unilateral electrolytic lesions of the VNAT caused a significant reduction in both the NA content and specific [3H]-diprenorphine binding to membrane homogenates in the ipsilateral PO/AH. In the second experiment bilateral 6-hydroxydopamine (6-OHDA)-induced lesions of the VNAT caused a significant reduction in NA levels in the PO/AH as well as significant decreases in the density of [3H]-diprenorphine binding to tissue sections of the PO/AH when compared to control animals. These results strongly suggest that the NA input to the PO/AH is regulated by endogenous opioid peptides, and provide an anatomical substrate to explain opioid-NA interactions in the control of gonadotrophin releasing hormone (GnRH) and gonadotrophin secretion.

Animals↗

PubMed1991

Adrenalin activation of beta 2-adrenoceptors stimulates morphological changes in astrocytes (pituicytes) cultured from adult rat neurohypophyses.

Neurohypophysial astrocytes, the pituicytes, are known to undergo morphological changes in vivo in response to stimuli that increase the demand for hormone secretion. Similar changes have been induced by beta-adrenergic stimulation both in the isolated, but otherwise intact, neural lobe and in pituicytes cultured from adult rats. Since the predominant beta-receptor subtype in the neural lobe is beta 2, we investigated the possibility that beta 2-receptor activation is mainly responsible for the observed pituicyte responses. In one experiment, cultured pituicytes were induced by noradrenalin to change from flattened amorphous to stellate morphology. Addition of the beta 2-antagonist IPS 339, but not the beta 1-antagonist practolol, significantly reduced (by 30-60%) the number of cells transformed by noradrenalin. In a second experiment, adrenalin, by definition a more potent beta 2-agonist, transformed significantly more pituicytes into stellate shapes than did noradrenalin at the same concentrations (100% vs. 60% increase, respectively). These results support the idea that beta 2-adrenergic receptors are involved in neurohypophysial plasticity. Also, since the neural lobe is outside of the bloodbrain barrier, these findings suggest that adrenal catecholamines participate in altering pituicyte morphology.

Adrenergic beta-Agonists↗

PubMed1990

Morphological plasticity that occurs in the neurohypophysis following activation of the magnocellular neurosecretory system can be mimicked in vitro by beta-adrenergic stimulation.

The osmotic stress to rats of replacing drinking water with 2% NaCl for four days (salt loading) led to dramatic ultrastructural morphological changes in the neural lobe of the pituitary, including a decrease in the ratio of glial to neurosecretory terminal coverage of the pericapillary basal lamina. The decrease in the proportion of the basal lamina covered by pituicytes, the specialized astrocytic glial cells of the neural lobe, was due to a decrease in the number of pituicyte processes reaching the pericapillary spaces. The concomitant increase in the proportion of neuronal coverage was due to the combination of an increase in the length of individual nerve terminals and a change in the number of terminals. Similar structural changes to those seen in vivo were produced by incubation of the isolated neural lobe with the beta-adrenergic agonist isoprenaline. A decrease in the ratio of glial to neuronal coverage of the basal lamina was achieved within 1 h and could be blocked by inclusion of the beta-adrenergic antagonist propranolol. It is proposed that the morphological plasticity is explained by the active movement of pituicyte cytoplasm.

Animals↗

PubMed1990

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'.

Animals↗

PubMed1989

Radiometric assay for carboxypeptidase H (EC 3.4.17.10) and other carboxypeptidase B-like enzymes.

Carboxypeptidase H, EC 3.4.17.10, also known as enkephalin convertase, carboxypeptidase E, and crino carboxypeptidase B, is an important enzyme involved in the biosynthesis of bioactive peptides. To assay the enzyme, tissues are homogenized in at least 20 vol (ml/g) of 0.025 M Tris-HCl buffer, pH 8, with 5 mg/ml of bovine serum albumin. After centrifugation, the supernatant is brought to pH 5.6 and centrifuged again. Following a 20-min preincubation in 2 mM CoCl2, the supernatant is incubated with 0.1 mM (final concentration) of the radioactive substrate [3H]benzoyl-Phe-Ala-Arg. The 100-microliters assay is stopped by the addition of 680 microliters of acetonitrile/0.25 M HCl (0.7/1). The 1.5-ml tube is transferred into a scintillation vial and is flushed with 4 ml of Econofluor, a water-immiscible scintillation fluid. The product, [3H]benzoyl-Phe-Ala, recovered in the organic phase, is counted directly with no interference from the substrate remaining in the aqueous phase. The blank is below 1%. Expressed in nanomoles per minute per milligram of tissue, the activity of the soluble enzyme in rat is 0.34 for striatum, 21.0 for pancreatic islet, 16.6 for anterior pituitary, 46.0 for intermediate pituitary, and 10.9 for neural pituitary. In every case 25 microM guanidinoethylmercaptosuccinic acid, an active site-directed inhibitor of carboxypeptidase H, completely inhibits the activity.

Animals↗

PubMed1989

Beta-adrenergic and opioid receptors on pituicytes cultured from adult rat neurohypophysis: regulation of cell morphology.

Explants of adult rat neurohypophysis were maintained in culture for 14 days. The majority of cells present in the outgrowth of such cultures were identified as pituicytes on the basis of immunostaining for glial fibrillary acidic protein. Pituicytes were also stained by antisera to the membrane glycoprotein antigen Thy-1 and the extracellular matrix glycoprotein fibronectin. The cultures contained naloxone sensitive binding sites for the opioid receptor ligand [3H] dynorphin A 1-8 and peripheral-type benzodiazepine binding sites. Dynorphin binding was visualised over pituicytes following autoradiography. The morphology of cultured pituicytes was regulated by beta-adrenergic receptors present on the cells which, when activated, stimulated rapid transformation from a flattened irregular morphology to a stellate, process-bearing morphology. Dynorphin was without effect on the morphology of cultured pituicytes. These findings are discussed in the context of the known morphological plasticity of pituicytes in vivo.

Animals↗

PubMed1988

Opioid inhibition of secretion from oxytocin and vasopressin nerve terminals following selective depletion of neurohypophysial catecholamines.

Opioids intrinsic to the neurohypophysis inhibit secretion from magnocellular neurosecretory terminals. This study examined whether the actions of opioids are mediated via interactions with neurohypophysial catecholamine systems. Blocking the action of intrinsic opioids in the isolated neurohypophysis with naloxone enhanced evoked secretion of oxytocin (OXT) by 150% and of vasopressin (AVP) by 30%. The enhancement of OXT secretion was not significantly altered in neurohypophyses depleted of greater than 90% of noradrenaline content by prior lesion of the ventral noradrenergic tract, or depleted of greater than 90% of both noradrenaline and dopamine content by prior reserpine treatment. Significant enhancement of AVP secretion by naloxone did not occur following depletion of catecholamines. The data suggest: (1) the majority of the influence of intrinsic opioids on secretion of OXT is not mediated via interaction with noradrenaline or dopamine systems, (2) the weaker influence of intrinsic opioids over AVP secretion may be mediated via catecholamines, (3) the majority of neurohypophysial noradrenaline is derived from projections of ascending medullary cell groups.

Animals↗

PubMed1988

Functional kappa-opioid receptors on oxytocin and vasopressin nerve terminals isolated from the rat neurohypophysis.

Opioids intrinsic to the rat neurohypophysial system act to inhibit secretion from the terminals of magnocellular neurones. Opioid receptors in the neurohypophysis are predominantly of the kappa-subtype and selective kappa-agonists suppress electrically evoked release of oxytocin (OXT) and vasopressin (AVP). We have looked for the presence of functional kappa-receptors on neurohypophysial nerve terminals by examining effects of kappa-agonists on secretion from suspensions of isolated neurohypophysial nerve terminals (neurosecretosomes) retained on filters in a perifusion system. Release of both OXT and AVP evoked by K+-depolarisation was inhibited by the kappa-agonists U-50,488H (34% and 45% respectively) and dynorphin A1-13 (68% and 51% respectively). Inhibition by dynorphin A was only observed in the presence of peptidase inhibitors. The actions of both kappa-agonists were prevented by the opioid receptor antagonist naloxone. The experiments indicate the presence of kappa-receptors on terminals of OXT and AVP neurones. This receptor population is in addition to those previously described on pituicytes and those influencing release of neurohypophysial noradrenaline.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1988

Morphological characterisation of lactotrophs separated from the bovine pituitary by a rapid enrichment technique.

The morphological characteristics of bovine pituitary cells separated by a rapid enrichment procedure are described. Single-cell suspensions were prepared from pituitary glands of steers by use of a collagenase technique and separated by discontinuous gradient centrifugation. The separation of prolactin- and growth hormone-containing cells was assessed by radioimmunoassay of hormone content and immunocytochemistry, and the distribution of fibroblasts assessed after establishing cell cultures. Morphometric analysis of the fine structure of two fractions respectively enriched and depleted in the proportion of immuno-cytochemically-identified lactotrophs was performed after labelling with anti-prolactin antiserum coupled to immunogold complex. Cells recovered from the higher-density fraction were more highly granulated, suggesting that this was a major characteristic determining separation. Cells labelled for prolactin could not be distinguished from unlabelled cells on the basis of their granule size range, but unlabelled cells had a significantly greater coefficient of variation. These data suggest that granule density and distribution, but not granule size per se, are useful characteristics for the identification of bovine lactotrophs.

Animals↗

PubMed1988

Opioid peptides inhibit noradrenergic transmission in the preoptic area to block LH secretion: evidence from neonatally androgenised rats.

A series of experiments are described which show (a) that the release of noradrenaline in the preoptic area of rats is regulated by a presynaptic opioid receptor and, (b) that changes in the opioid control of preoptic noradrenaline release are associated with the anovulatory condition found in adult female rats which have been exposed to androgen neonatally. The experiments support the hypothesis that opioid-noradrenergic interaction in the preoptic area is an important component in the neural mechanism regulating gonadotrophin secretion.

Action Potentials↗

PubMed1988

Hypothalamic opioid mechanisms controlling oxytocin neurones during parturition.

The influences of opioids on oxytocin secretion and parturition were investigated in the rat. Morphine, administered centrally or peripherally, severely delays the course of established parturition. This delay is accompanied by reduced plasma oxytocin levels and is overcome by treatment either with the opioid antagonist naloxone, or by infusion of oxytocin. An endogenous opioid regulatory mechanism inhibiting oxytocin secretion becomes activated immediately prior to and during parturition. This mechanism does not operate earlier in pregnancy or during normal lactation and is not seen in nonpregnant animals. Naloxone acutely speeds up the course of established parturition, an effect accompanied by greatly elevated plasma oxytocin levels. The mechanisms underlying opioid regulation of oxytocin neurones were investigated at two sites. Precipitated withdrawal from chronic morphine treatment causes hypersecretion of oxytocin. This response is mediated by greatly enhanced electrical activity in the perikarya of oxytocin neurones indicating the presence of opioid receptors on oxytocin neurones and/or on their afferent input. Opioid receptors are also present in the neurohypophysis where they exert direct and noradrenaline mediated effects on secretion from oxytocin terminals in vitro.

Animals↗

PubMed1988

Naloxone excites oxytocin neurones in the supraoptic nucleus of lactating rats after chronic morphine treatment.

1. Lactating rats were implanted with a cannula in a lateral cerebral ventricle to deliver morphine (up to 50 micrograms/h) chronically from a subcutaneous osmotically driven mini-pump. After infusion of morphine for 5 days the rats were anaesthetized with urethane and prepared with ventral surgery for recording the electrical activity of single, antidromically identified neurones in the supraoptic nucleus. 2. A single I.V. injection of naloxone (5 mg/kg) in these rats provoked a long-lasting, large increase in intramammary pressure, but in control rats had negligible effects. Concentrations in plasma of oxytocin, measured by radioimmunoassay in samples of femoral arterial blood, rose from 44.7 +/- 2.5 to 1072.1 +/- 89.5 pg/ml (means +/- S.E.M.) 6 min after naloxone in the morphine-treated rats. In control rats, the concentration of oxytocin in plasma rose only from 42.1 +/- 2.9 to 125.1 +/- 28.2 pg/ml after naloxone. 3. Naloxone produced a transient increase in arterial blood pressure in morphine-treated but not control rats. Concentrations in plasma of vasopressin, measured by radioimmunoassay in samples of femoral arterial blood, rose in morphine-treated rats from 7.4 +/- 2.4 to 29.2 +/- 3.7 pg/ml after naloxone, but did not rise significantly in control rats. 4. Naloxone (1-5 mg/kg) produced a prompt and prolonged increase in the discharge rate of each of ten continuously active (putative oxytocin) cells recorded from ten morphine-treated rats. The discharge rate of the six cells tested at the highest dose (5 mg/kg) increased by an average of 6.3 Hz (360%) within 5 min, and the firing rate remained elevated for at least 30 min; the discharge rate of six continuously active supraoptic neurones recorded in control rats was not affected by naloxone. 5. The firing activity of five phasic (putative vasopressin) supraoptic neurones in morphine-treated rats was increased for at least 30 min by the injection of naloxone; these increases were the result of a raised intraburst firing rate with no change in burst duration or frequency. One phasic neurone was inhibited for 15 min, and one phasic neurone was unaffected. 6. The excitatory effects of naloxone on neurones in the supraoptic nucleus of morphine-treated rats were not explained by changes in blood pressure or osmolarity and did not depend on suckling or a cholinergic pathway. 7. The concentrations of oxytocin in plasma and the operation of the milk-ejection reflex were similar in the controls and morphine-treated rats, prior to naloxone. These findings indicate tolerance to initial inhibitory effects of morphine on oxytocin secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1988

Opioid-noradrenergic interactions in the neurohypophysis. I. Differential opioid receptor regulation of oxytocin, vasopressin, and noradrenaline release.

The actions of opioids on electrically evoked release of oxytocin, vasopressin, and noradrenaline-using the [3H]-noradrenaline technique-from the rat neurohypophysis were examined in vitro. Antagonism of the action of endogenous neurohypophysial opioids with naloxone enhanced release of peptides and [3H]-noradrenaline differentially. Naloxone enhanced oxytocin release by 100 and 173% in two series of experiments (ED50 7 x 10(-7) M), whilst vasopressin release was enhanced by only 30 and 20%, respectively. [3H]-noradrenaline release was maximally enhanced by 41% (ED50 2 x 10(-7) M). We examined the opioid receptor subtypes mediating these effects using selective receptor agonists. The kappa-agonist U-50,488H inhibited oxytocin and vasopressin release to a similar extent, but did not modify [3H]-noradrenaline release. The effects of U-50,488H were completely prevented by a tenfold molar excess of naloxone. The mu-agonist (D-Ala2, MePhe5 Gly-ol)-enkephalin also failed to inhibit [3H]-noradrenaline release and caused only a minor inhibition of oxytocin and vasopressin secretion. The delta-agonist (D-Pen2, D-Pen5)-enkephalin was without effect. We conclude that (1) kappa-receptors sensitive to U-50,488H mediate opioid inhibition of secretion from oxytocin and vasopressin nerve terminals; (2) when opioid actions are blocked by naloxone, opioid peptides within the neurohypophysis are shown to exert a much greater influence over oxytocin compared to vasopressin terminals; (3) neurohypophysial opioids also regulate release from noradrenergic terminals, although the nature of the receptors involved remains unclear, and (4) kappa-receptors can mediate inhibition of neurohormone secretion by an action independent of the neurohypophysial noradrenergic innervation.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1988

Opioid-noradrenergic interactions in the neurohypophysis. II. Does noradrenaline mediate the actions of endogenous opioids on oxytocin and vasopressin release?

The function of noradrenaline in the rat neurohypophysis was investigated by examining the effects of selective adrenergic receptor agents on electrically evoked release of oxytocin, arginine vasopressin, and noradrenaline using the [3H]-noradrenaline technique. Since endogenous opioids in the neurohypophysis suppress release of both neurohormones and of noradrenaline, we assessed the role of noradrenaline in mediating opioid actions on neurohormone secretion by examining modification of the action of the opioid antagonist naloxone by adrenergic receptor agents. The data suggest (1) that in addition to opioid receptors, 'presynaptic' alpha 2-receptors regulate release from neurohypophysial noradrenaline terminals; (2) noradrenaline released from neurohypophysial terminals acts on beta- and alpha 1-receptors to facilitate both oxytocin and arginine vasopressin release: this action only becoming evident at elevated levels of endogenous noradrenaline release attained following removal of presynaptic opioid or alpha 2-regulation, and (3) opioid peptides within the neurohypophysis act to inhibit oxytocin and, to a lesser extent, arginine vasopressin secretion, partly through inhibiting release of facilitatory noradrenaline. We propose a model in which opioids act in the neurohypophysis both independently of noradrenaline via kappa-receptors on neurosecretory terminals or pituicytes and also via interaction with the noradrenaline system.

Animals↗

PubMed1988

Optimizing release from peptide hormone secretory nerve terminals.

Secretion of the peptide neurohormones oxytocin and vasopressin from terminals of magnocellular neurones in the mammalian neurohypophysis is elicited by conduction of depolarizing action potentials into terminal membranes, inducing opening of voltage-sensitive Ca2+ channels, entry of Ca2+ from the extracellular space and a rise in cytoplasmic Ca2+ concentration. The amount of peptide released per action potential is not immutable. In particular, the patterns in which action potentials are generated at the cell somata of the two types of neurone each appear exquisitely suited to optimize the release process at the terminal by utilizing a frequency-facilitation mechanism and by minimizing a mechanism of fatigue in the release process. The different properties of oxytocin and vasopressin neurones are of important physiological significance. The secretory terminals are also a site of receptor-mediated influences of neuromodulators which can greatly alter release efficiency. The mechanisms underlying facilitation and fatigue are not clearly understood. The evidence suggests that processes both prior to depolarization of the terminals (propagation and form of the action potentials) and directly at the terminals (frequency/pattern-dependent Ca2+ entry and channel openings) are involved. Transient activity-related increases in extracellular K+ concentration may be involved at both sites. Two types of neuromodulation have been partly characterized. Kappa-Opioid receptors in secretory terminal membranes directly modulate depolarization-evoked peptide release probably via interactions with Ca2+ channels. beta-Adrenergic receptors localized on neurohypophyseal astroglial cells mediate more subtle effects of noradrenaline. In the more chronic situation the neurohypophyseal astroglia alter their morphological relationships with neurosecretory elements and the basal lamina at release sites, changes which may also serve to optimize the secretory process.

Animals↗

PubMed1988

Endogenous opioid actions and effects of environmental disturbance on parturition and oxytocin secretion in rats.

Blood samples were taken from conscious, chronically-catheterized rats during parturition for measurement of oxytocin by specific radioimmunoassay. After the birth of the 3rd pup, rats were allowed to remain in their nesting cage (undisturbed rats) or were transferred for 45 min to a glass bowl (disturbed rats); at the time of transfer, rats were given an i.v. injection of the opioid antagonist naloxone or saline vehicle. Subsequent parturition was prolonged in saline-treated disturbed rats, but not in naloxone-treated disturbed rats. Parturition was significantly hastened in naloxone-treated undisturbed rats. Naloxone injections were followed by a large rise in plasma oxytocin concentrations in disturbed and undisturbed rats. We conclude, from a statistical analysis of the relationship within experimental groups between plasma oxytocin concentration and speed of parturition, that the effects of disturbance and of naloxone upon parturition may be accounted for, at least in part, by their effects upon oxytocin release. However, the effects of disturbance on parturition may not be mediated entirely by activation of opioid pathways. Naloxone did not potentiate oxytocin release in non-pregnant rats, or on Day 1 post partum, but did potentiate oxytocin release on Day 22 of pregnancy even in rats before the onset of parturition. Endogenous opioid pathways regulating oxytocin release therefore appear to be active during late pregnancy and during parturition itself.

Animals↗

PubMed1987

Effect of neonatal testosterone upon opioid receptors and the content of beta-endorphin, neuropeptide Y and neurotensin in the medial preoptic and the mediobasal hypothalamic areas of the rat brain.

The content of beta-endorphin, neuropeptide Y and neurotensin-like immunoreactivity (beta-End, NPY and NT), and the total number of opioid binding sites, were measured in the medial preoptic area (MPOA) and mediobasal hypothalamus (MBH) of ovariectomized adult rats which were oestrogen-primed. The rats had been injected neonatally with either testosterone propionate (TP) or vehicle (oil). NPY content was found to be higher in the MPOA of animals which received TP, whereas no significant difference was observed in the MBH NPY content. However, the NT concentration in the MBH of TP-treated rats was almost twice the amount detected in oil-treated rats and with this peptide no significant changes were detected in the MPOA. Finally, beta-End and the total number of opioid binding sites were reduced in both the MPOA and MBH of the rats which were exposed to TP neonatally. Since exposure to testosterone neonatally masculinises the rat hypothalamus, to the extent that female rats cannot generate oestrogen-stimulated prolactin and luteinizing hormone surges, we suggest that the neurochemical changes reported in this paper reflect some aspects of this sexual differentiation of the rat hypothalamus.

Animals↗