Relaxin affects the central control of oxytocin release

In several species the myometrium is quiescent shortly before parturition. At this time high titres of relaxin are present in the plasma and there is evidence that the hormone has a direct inhibitory action on the uterine muscle. Relaxin could also contribute to uterine quiescence by inhibiting oxytocin release. To determine whether relaxin has a central action on the release of oxytocin, we have studied the effect of intravenous injections of porcine relaxin on milk ejection in the anaesthetized lactating rat. We report that reflex milk ejection was suppressed by relaxin in a dose-dependent manner, the onset of inhibition being rapid and lasting from 10 to 60 min. After the period of inhibition the normal temporal pattern of reflex milk ejection was resumed. Mammary sensitivity to exogenous or endogenous oxytocin was reduced by relaxin but not sufficiently to explain the effects observed. Furthermore, relaxin (1 microgram per rat) injected into the cerebral ventricles profoundly disturbed the pattern of reflex milk ejection without affecting the response of the mammary gland to oxytocin. These results suggest a novel role for relaxin within the central nervous system. The site in the brain at which the effects of relaxin are exerted remains unknown.     

Oxytocin-immunoreactive terminals synapse on oxytocin neurones in the supraoptic nucleus

The neuropeptide oxytocin, synthesized by magnocellular neurones in the hypothalamus, is well known for its peripheral action during lactation and parturition after its release into the bloodstream from axons in the neurohypophysis. However, it may also be released centrally to control the activity of oxytocinergic neurones themselves. Oxytocin release has been measured from isolated magnocellular nuclei in vitro and in the cerebrospinal fluid. When injected into the third ventricle, the peptide increases the basal firing rate of oxytocinergic neurones as well as the frequency and amplitude of the bursts of action potentials they normally show before each reflex milk ejection. Oxytocin also excites magnocellular neurones when applied microiontophoretically. I now report that immunocytochemical staining reveals synapses in the supraoptic nucleus of the hypothalamus where both the pre- and postsynaptic elements contain oxytocin. These oxytocinergic synapses, impinging on their own neurones, may represent the anatomical basis for the hypothalamic release of this peptide and for the facilitatory action on its own secretion.     

Oxytocin induces morphological plasticity in the adult hypothalamo-neurohypophysial system

The hypothalamo-neurohypophysial system offers a unique example in the adult mammalian central nervous system (CNS) of a functional and structural plasticity related to a physiological state. During lactation, oxytocin neurones evolve a synchronized electrical activation which permits pulsatile hormone release at milk ejection. At the same time, in the supraoptic (SON) and paraventricular nuclei, glial coverage of neurones diminishes, so that large portions of their surface membrane become directly juxtaposed; synaptic remodelling also associates pairs of neurones through the formation of common presynaptic terminals. These structural changes, reversible after weaning, affect exclusively oxytocinergic neurones and could facilitate their synchronized electrical activity. As several observations suggest that oxytocin itself is released centrally, we have examined the effect of prolonged intracerebroventricular infusions of oxytocin on the structure of the SON of non-lactating animals. We report here that the peptide indeed engenders the structural reorganization characteristic of the oxytocin system when it is physiologically activated. Similar infusion of vasopressin has no effect. Our observations thus demonstrate that a central neuropeptide can induce anatomical changes in the adult CNS, and suggest that oxytocin can regulate its own release by contributing to the dramatic restructuring of the nuclei containing the neurones responsible for its secretion.     

Magnocellular axons in passage through the median eminence release vasopressin

Vasopressin (arginine vasopressin, AVP) is present in two types of nerve fibres in the median eminence (ME). First, it is found in nerve terminals that originate in the parvicellular neurones of the hypothalamic paraventricular nucleus (PVN) and abut on the pericapillary space surrounding the fenestrated capillaries of the primary pituitary portal plexus in the external zone (EZ) of the ME. These neurones also synthesize corticotropin-releasing factor (CRF), which acts synergetically with vasopressin to stimulate release of adrenocorticotropin (ACTH) from the pituitary gland (see ref. 7). Second, vasopressinergic axons of the magnocellular neurosecretory system pass through the internal zone (IZ) of the ME to terminate in the neurohaemal contact zone of the neurohypophysis. The involvement of vasopressinergic magnocellular neurones in the control of ACTH secretion is much debated. Of particular interest in this context is the origin of the vasopressin found in pituitary portal blood. Although it has been demonstrated that vasopressin and CRF are present in the same neurosecretory granules of EZ fibres, parallel determinations of vasopressin and CRF in pituitary portal blood have shown alterations of the concentration of vasopressin without a concomitant change in that of CRF. Such a dissociation suggests that either differential release of vasopressin and CRF can occur from a single population of nerve endings, or there are fibres in the pituitary-stalk ME which release vasopressin but not CRF. Here we present evidence for the latter. Our results indicate that stimuli causing depolarization of the axonal membrane in vitro elicit release of vasopressin from nerve fibres in the external and internal zones of the ME.     

Intracellular calcium stores regulate activity-dependent neuropeptide release from dendrites

Information in neurons flows from synapses, through the dendrites and cell body (soma), and, finally, along the axon as spikes of electrical activity that will ultimately release neurotransmitters from the nerve terminals. However, the dendrites of many neurons also have a secretory role, transmitting information back to afferent nerve terminals. In some central nervous system neurons, spikes that originate at the soma can travel along dendrites as well as axons, and may thus elicit secretion from both compartments. Here, we show that in hypothalamic oxytocin neurons, agents that mobilize intracellular Ca(2+) induce oxytocin release from dendrites without increasing the electrical activity of the cell body, and without inducing secretion from the nerve terminals. Conversely, electrical activity in the cell bodies can cause the secretion of oxytocin from nerve terminals with little or no release from the dendrites. Finally, mobilization of intracellular Ca(2+) can also prime the releasable pool of oxytocin in the dendrites. This priming action makes dendritic oxytocin available for release in response to subsequent spike activity. Priming persists for a prolonged period, changing the nature of interactions between oxytocin neurons and their neighbours.     

Direct measurement of exocytosis and calcium currents in single vertebrate nerve terminals

The release of neurohormone is widely thought to be exocytotic, involving Ca2(+)-dependent fusion of secretory vesicles with the plasma membrane. The inaccessibility of most nerve ending has so far hampered direct time-resolved measurements of neuronal exocytosis in response to brief depolarization. By using 'whole-terminal' patch-clamp and circuit-analysis techniques to measure membrane capacitance, we have now monitored changes in the surface membrane area of individual nerve terminals isolated from the mammalian neurohypophysis. A single depolarizing pulse leading to Ca2+ entry through voltage-gated calcium channels, rapidly and reproducibly increases the membrane area by an amount corresponding to the fusion of 1-100 secretory vesicles. The magnitude of the capacitance increase depends not only on Ca2+ entry and buffering, but also on the pattern of stimulation revealing facilitation, fatigue and recovery of the release process.     

Immunoreactive beta-endorphin in sheep ovary

Although the ovarian production of sex steroids is of obvious physiological importance, recent studies suggest that peptides such as oxytocin, relaxin and inhibin are also synthesized in the ovary. We report here the presence of immunoreactive (ir) beta-endorphin, ir-adrenocorticotropic hormone (ACTH) and presumptive high molecular weight forms of both in extracts of sheep ovary, consistent with ovarian production from a common precursor. Our findings suggest that beta-endorphin and ACTH are produced and secreted by the follicular cells, and that their production may be related to the oestrous cycle.     

CD38 is critical for social behaviour by regulating oxytocin secretion

CD38, a transmembrane glycoprotein with ADP-ribosyl cyclase activity, catalyses the formation of Ca2+ signalling molecules, but its role in the neuroendocrine system is unknown. Here we show that adult CD38 knockout (CD38-/-) female and male mice show marked defects in maternal nurturing and social behaviour, respectively, with higher locomotor activity. Consistently, the plasma level of oxytocin (OT), but not vasopressin, was strongly decreased in CD38-/- mice. Replacement of OT by subcutaneous injection or lentiviral-vector-mediated delivery of human CD38 in the hypothalamus rescued social memory and maternal care in CD38-/- mice. Depolarization-induced OT secretion and Ca2+ elevation in oxytocinergic neurohypophysial axon terminals were disrupted in CD38-/- mice; this was mimicked by CD38 metabolite antagonists in CD38+/+ mice. These results reveal that CD38 has a key role in neuropeptide release, thereby critically regulating maternal and social behaviours, and may be an element in neurodevelopmental disorders.     

Corticotropin releasing activity of the new CRF is potentiated several times by vasopressin

Initially the hypothalamic factor responsible for the release of corticotropin (CRF), was thought to be a simple peptide. More recent work has led to the conclusion that CRF is a multifactorial complex. In 1979 we proposed that vasopressin, much disputed as a CRF candidate, was a major constituent of the complex, interacting with a potentiating the CRF activity of the other component(s). The recent characterization of a 41 residue ovine hypothalamic peptide capable of releasing adrenocorticotropic hormone (ACTH) in a dose-related manner has allowed us to compare its CRF bioactivity with that of vasopressin and simple extracts of the hypothalamus, and to investigate any interaction it may have with vasopressin and other hypothalamic factors in the release of ACTH. We report here that the new CRF is more potent than vasopressin in releasing ACTH. When given simultaneously with vasopressin a fourfold potentiation of CRF activity with steep dose-response characteristics were observed. It also potentiated vasopressin-free hypothalamic extracts, suggesting that a new CRF does not account for all the nonvasopressin portion of the CRF complex.     

Modulation of stress-induced ACTH release by corticotropin-releasing factor, catecholamines and vasopressin

The stress-induced release of ACTH is believed to involve the activation of several humoral and neural pathways, including corticotropin-releasing factor (CRF), catecholamines and vasopressin. The essential role of CRF was supported by our observation that immunoneutralization of this releasing factor significantly lowers plasma ACTH levels of ether-stressed rats. However, the presence of a small but measurable residual ACTH secretion suggested the possible involvement of factors other than CRF in the stress response. We report here that pretreatment with a vasopressin antagonist decreases the plasma ACTH levels of ether-stressed rats in later (10-20 min), but not earlier (0-10 min), phases of ether stress. The ganglionic blocker chlorisondamine, inhibits ACTH release during both phases of the response to ether by 40-60% when used alone, and by 100% when administered with anti-CRF antibody. These results support a role of CRF, catecholamines and vasopressin in mediating ACTH release by ether stress.     

Inhibition of the firing of vasopressin neurons by atriopeptin

Atriopeptin, the atrial natriuretic peptide, is a circulating hormone that is released from the atria of mammalian hearts in response to volume expansion and acts upon the kidneys, adrenal glands and vasculature to regulate fluid and electrolyte homeostasis. Atriopeptin is also present in the brain of the rat. Atriopeptin immunoreactive cell bodies and fibres are found in many areas known to be involved in the central regulation of the cardiovascular system, suggesting that it may be a neuromediator in the central control of fluid and electrolyte balance. The paraventricular nucleus of the hypothalamus, which contains the cell bodies of neurons that secrete vasopressin from the posterior pituitary gland, receives a dense innervation from atriopeptin-like immunoreactive fibres. We have studied the effect of atriopeptin on the electrical activity of single neurons in the paraventricular nucleus of anaesthetized rats and found that atriopeptin is a potent inhibitor of putative vasopressin neurons. Atriopeptin, which has systemic actions that oppose those of vasopressin, may act as a neuromodulator in the brain to prevent vasopressin secretion.     

Somatostatin selectively inhibits noradrenaline release from hypothalamic neurones

Somatostatin in a hypothalamic peptide hormone which inhibits growth hormone release from the anterior pituitary. However, biochemical and morphological investigations have revealed that somatostatin is located not only in the hypothalamus but also in other brain areas (for example the cerebral cortex) where it occurs and in nerve cell bodies and fibres from which it can be released in a Ca2+-dependent manner. It has therefore been suggested that the neuropeptide may have functions in the central nervous system other than its effect on growth hormone release; one possible action is that of a neuromodulator. Therefore, hypothalamic and cerebral cortical slices of the rat were used to examine whether somatostatin modifies the electrically or CaCl2-evoked release of tritiated monoamines from monoaminergic neurones. it is reported here that somatostatin inhibits 3H-noradrenaline release from the hypothalamus (but not from the cerebral cortex) but does not affect the release of 3H-dopamine and 3H-serotonin.     

Calcium/calmodulin-dependent protein kinase II increases glutamate and noradrenaline release from synaptosomes

A variety of evidence indicates that calcium-dependent protein phosphorylation modulates the release of neurotransmitter from nerve terminals. For instance, the injection of rat calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-dependent PK II) into the preterminal digit of the squid giant synapse leads to an increase in the release of a so-far unidentified neurotransmitter induced by presynaptic depolarization. But until now, it has not been demonstrated that Ca2+/CaM-dependent PK II can also regulate neurotransmitter release in the vertebrate nervous system. Here we report that the introduction of Ca2+/CaM-dependent PK II, autoactivated by thiophosphorylation, into rat brain synaptosomes (isolated nerve terminals) increases the initial rate of induced release of two neurotransmitters, glutamate and noradrenaline. We also show that introduction of a selective peptidergic inhibitor of Ca2+/CaM-dependent PK II inhibits the initial rate of induced glutamate release. These results support the hypothesis that activation of Ca2+/CaM-dependent PK II in the nerve terminal removes a constraint on neurotransmitter release.     

Insulin-like growth factor II precursor gene organization in relation to insulin gene family

The insulin gene family, comprised of insulin, relaxin, insulin-like growth factors I and II (IGF-I and IGF-II) and possibly the beta-subunit of 7S nerve growth factor, represents a group of structurally related polypeptides whose biological functions have diverged. The IGFs, or somatomedins, constitute a class of polypeptides that have a key role in pre-adolescent mammalian growth (see ref. 4 for review). IGF-I expression is regulated by growth hormone and mediates postnatal growth, while IGF-II appears to be induced by placental lactogen during prenatal development. The primary structures of both human IGFs have been determined and are closely related. A polypeptide highly homologous to human IGF-II is secreted by the rat liver cell line, BRL-3A. As this polypeptide, termed multiplication stimulating activity (MSA), differs from human IGF-II by only five amino acid residues, MSA probably represents the rat IGF-II protein. Using molecular cloning techniques, we have isolated cDNA and chromosomal genes coding for the MSA and human IGF-II precursors, respectively. Our data, presented here, indicate that both MSA and human IGF-II are synthesized initially as larger precursor molecules. The deduced preprohormones both have molecular weights (MWs) of 20,100 and contain C-terminal propeptides of 89 amino acid residues, which we have named E-peptides. The organization of the IGF-II precursor gene is discussed in relation to that of other insulin gene family members.     

Structure of a genomic clone encoding biologically active human relaxin

Relaxin is a peptide hormone synthesized in the corpora lutea of ovaries during pregnancy and is released into the blood stream prior to parturition. Its major biological effect is to remodel the mammalian reproductive tract to facilitate the birth process. Determination of the structure of human relaxin is thus a first step in opening up the possibility of clinical intervention in cases of difficult labour. However, the limited availability of human ovaries during pregnancy has prevented both direct amino acid sequence determination and isolation of cDNA clones obtained from relaxin producing tissue. Our approach has therefore been to screen directly for a human relaxin gene using an homologous porcine relaxin cDNA probe. We report here the successful identification of a genomic clone from which the structure of the entire coding region of a human preprorelaxin gene has been determined. Synthesis of biologically active relaxin has shown that the novel gene structure described herein codes for an authentic human relaxin. We believe this is the first successful synthesis of a biologically active hormone whose structure was predicted solely from the structure of a genomic clone.     

Evidence for opiate receptors on pituicytes

A hypothalamo-neurohypophyseal enkephalinergic pathway has been described and the pars nervosa of the rat pituitary contains enkephalin-like material which may coexist in vasopressin and oxytocin terminals. At the level of the pars nervosa itself, stereospecific opiate receptors with properties very similar to those of brain receptors have been described, and opiates have been shown to inhibit the release of both vasopressin and oxytocin. The location of the opiate receptors involved has been presumed to be pre-terminal on the neurosecretory fibres. Using an autoradiographic technique to visualize opiate receptors, however, we now report that destruction of the neurosecretory fibres following pituitary stalk section does not result in a significant change in the neural lobe opiate receptor population. This suggests that the opiate receptors within the neural lobe may be present on pituicytes rather than on neurosecretory fibres.     

Activation by adrenaline of a low-conductance G protein-dependent K+ channel in mouse pancreatic B cells

Insulin is produced and secreted by the B cells in the endocrine pancreas. In vivo, insulin secretion is under the control of a number of metabolic, neural and hormonal substances. It is now clear that stimulation of insulin release by fuel secretagogues, such as glucose, involves the closure of K+ channels that are sensitive to the intracellular ATP concentration (KATP channels). This leads to membrane depolarization and the generation of Ca2(+)-dependent action potentials. The mechanisms whereby hormones and neurotransmitters such as adrenaline, galanin and somatostatin, which are released by intraislet nerve endings and the pancreatic D cells, produce inhibition of insulin secretion are not clear. Here we show that adrenaline suppresses B-cell electrical activity (and thus insulin secretion) by a G protein-dependent mechanism, which culminates in the activation of a sulphonylurea-insensitive low-conductance K+ channel distinct from the KATP channel.     

Glucose modulates Mg2+ fluxes in pancreatic islet cells

Magnesium, the most abundant intracellular divalent cation, is an essential cofactor for many enzyme systems, but it remains unknown as to whether variations in the cytoplasmic concentration of ionized Mg2+ directly control cellular processes. Experiments with adrenal medullary cells made 'leaky' by exposure to high electric fields provided evidence that Mg2+ could influence hormone release not only by competing with Ca2+ for entry into the cell, but also at intracellular sites controlling exocytosis. A similar conclusion was reached for insulin release in a study using isolated rat islets also subjected to high voltage discharges. There is no experimental evidence, however, that physiological stimuli influence Mg2+ movements in intact secretory cells. We report here that 28Mg2+ fluxes in pancreatic islet cells are markedly modified by glucose, the physiological stimulus of insulin release, but not by its non-insulinotropic analogue, 3-O-methylglucose.     

Axonal transport of vasoactive intestinal peptide in sciatic nerve.

Dense plexuses of neurones containing immunoreactive vasoactive intestinal peptide (VIP) have been found in discrete areas of the central nervous system and in peripheral organs, including the gastrointestinal tract, pancreas and urogenital system. In many of these locations VIP is concentrated in nerve endings, where it can be released by high K+ concentrations in a Ca2+-dependent manner. VIP release may also be provoked by electrical stimulation of nerves, for example the vagus. VIP thus shows some of the features of neurotransmitter or neuromodulator substances. The presence of immunoreactive VIP in the fine terminal varicosities as well as in the cell bodies of neurones suggests that it might be transported from the perikaryon, where it is presumably formed, to the nerve endings, through the axonal transport system. Such transport would be in keeping with a role for the peptide as a neurohumor or neurohormone. We report here that VIP accumulates in constricted rat sciatic nerves in a manner suggesting fast, anterograde axonal flow.     

Molecular cloning and expression of a rat V1a arginine vasopressin receptor.

The neurohypophyseal hormone arginine vasopressin has diverse actions, including the inhibition of diuresis, contraction of smooth muscle, stimulation of liver glycogenolysis and modulation of adrenocorticotropic hormone release from the pituitary. Arginine vasopressin receptors are G protein-coupled and have been divided into at least three types; the V1a (vascular/hepatic) and V1b (anterior pituitary) receptors which act through phosphatidylinositol hydrolysis to mobilize intracellular Ca2+, and the V2 (kidney) receptor which is coupled to adenylate cyclase. We report here the cloning of a complementary DNA encoding the hepatic V1a arginine vasopressin receptor. The liver cDNA encodes a protein with seven putative transmembrane domains, which binds arginine vasopressin and related compounds with affinities similar to the native rat V1a receptor. The messenger RNA corresponding to the cDNA is distributed in rat tissues known to contain V1a receptors.