Beta-endorphin cells in the arcuate nucleus: projections to the supraoptic nucleus and changes in expression during pregnancy and parturition.

Supraoptic nucleus oxytocin neurone activity and secretion are inhibited in late pregnancy and parturition by endogenous opioids. Here, we investigated alterations in the projections and gene expression of beta-endorphin/pro-opiomelanocortin neurones in the arcuate nucleus in the pregnant rat. All regions of the arcuate nucleus were found to contain cells immunoreactive for beta-endorphin fluorescent microbeads retrogradely transported from the supraoptic nucleus, and double-labelled neurones (beta-endorphin plus microbeads), showing that beta-endorphin neurones throughout the arcuate nucleus project to the supraoptic nucleus. There was an increase in the number of beta-endorphin-immunoreactive cells in the arcuate nucleus and an increase in the density of beta-endorphin fibres within the supraoptic nucleus and peri-supraoptic region in late pregnancy and parturition, suggesting enhanced expression of beta-endorphin and increased beta-endorphin innervation of the supraoptic nucleus. Pro-opiomelanocortin mRNA expression in the arcuate nucleus increased in late compared to early pregnancy: the number of positive neurones significantly increased in the caudal region. Fos expression (an indicator of neuronal activation) in the arcuate nucleus was colocalized in beta-endorphin neurones in both proestrus and parturient rats, but the number of positive cells did not increase during parturition, suggesting lack of activation of beta-endorphin neurones at birth. Thus, beta-endorphin cells in the arcuate nucleus project to the supraoptic nucleus and increased innervation during pregnancy may explain the enhanced endogenous opioid inhibition of oxytocin neurones.

Direct pathways to the supraoptic nucleus from the brainstem and the main olfactory bulb are activated at parturition in the rat.

Sensory input from female reproductive structures is paramount for the co-ordination of neuroendocrine changes at parturition. Using a retrograde tracer (fluorescent latex microspheres) in combination with Fos (as an indicator of neuronal activation) and tyrosine hydroxylase (to identify catecholaminergic neurons) immunocytochemistry we identified cells within the brainstem and main olfactory bulb that project to the supraoptic nucleus, and which become significantly activated at parturition (compared to virgin rats and rats on the day of expected parturition). Within the A2/C2 region in the nucleus tractus solitarii, 60% of the projecting activated cells were catecholaminergic, as were 59% of such cells in the A1/C1 region of the ventrolateral medulla. This suggests that oxytocin and vasopressin neurons within the supraoptic nucleus are stimulated at parturition via afferent inputs from the brainstem, but the input is not exclusively noradrenergic. Within the mitral layer of the main olfactory bulb, cells that projected to the supraoptic nucleus were significantly activated, suggesting that the olfactory system may regulate supraoptic nucleus cell firing at parturition. The preoptic area, organum vasculosum of the lamina terminalis and medial amygdala contained cells that projected to the supraoptic nucleus but these projections were not significantly activated at parturition, although non-projecting cells in these regions were. On the expected day of parturition, but before birth, projections from the organum vasculosum of the lamina terminalis to the supraoptic nucleus became significantly activated. These findings provide evidence of direct afferent pathways to the supraoptic nucleus from the brain stem and olfactory bulbs that are activated at parturition.

Effect of progesterone on the activation of neurones of the supraoptic nucleus during parturition.

Parturition is driven by a pulsatile pattern of oxytocin secretion, resulting from burst firing activity of supraoptic oxytocin neurones and reflected by induction of Fos expression. Rats were injected with progesterone on day 20 of pregnancy to investigate the role of the decreasing progesterone:ratio oestrogen ratio, which precedes delivery, in the activation of supraoptic neurones. Progesterone delayed the onset of birth by 28 h compared with vehicle (control) and prolonged the duration of delivery, which was overcome by pulsatile injections of oxytocin, indicating that the slow delivery may reflect impaired oxytocin secretion. Parturient rats pretreated with progesterone had fewer Fos immunoreactive nuclei in the supraoptic nucleus than did parturient rats pretreated with vehicle. The number of Fos immunoreactive nuclei was not restored after oxytocin injection, indicating that appropriate activation of oxytocin neurones is impaired by progesterone and also that there is a lack of stimulatory afferent drive. Fos expression increased in the nucleus of the tractus solitarius during parturition in rats pretreated with either vehicle or progesterone, but not in rats that had been pretreated with progesterone and induced with oxytocin, indicating that this input was inhibited. Endogenous opioids inhibit oxytocin neurones in late pregnancy and the opioid antagonist, naloxone, increases Fos expression in supraoptic nuclei by preventing inhibition. However, progesterone attenuated naloxone-induced Fos expression in the supraoptic nucleus in late pregnancy and naloxone administered during parturition did not accelerate the duration of births delayed by progesterone administration, indicating that progesterone does not act by hyperactivation of endogenous opioid tone. RU486, a progesterone receptor antagonist, enhanced supraoptic neurone Fos expression in late pregnancy, indicating progesterone receptor-mediated actions. Thus, progesterone withdrawal is necessary for appropriate activation of supraoptic and tractus solitarius neurones during parturition.

Oestrogen modulation of noradrenaline neurotransmission.

Noradrenaline (NA) exerts an important neuromodulatory role within diverse neuronal networks and is also likely to be a target for oestrogen in the brain. Distinct, highly organized sub-populations of brainstem NA neurons express oestrogen receptors (ERs) and some of these display species differences. A number of genes expressed by NA neurons, ranging from transcription factors to co-released neuropeptides, are influenced by oestrogen and may have roles in the predominant enhancement in NA activity in response to oestrogen. The effects of oestrogen on genes involved directly in NA biosynthesis are less clear, although promoter transgenic work suggests oestrogen to have a powerful influence upon tyrosine hydroxylase gene transcription. In addition to direct actions on NA neurons, evidence suggests that oestrogen also regulates adrenergic receptor expression and function within the ER-rich hypothalamus as well as the cerebral cortex. Together, these investigations point to a multifaceted pre- and postsynaptic regulation of NA transmission by oestrogen. While the hypothalamic neuronal networks controlling reproduction remain the principal site of investigation of oestrogen regulated NA transmission, the role of oestrogen and NA and their potential interactions in cortical functioning are becoming of equal interest.

Fluctuating estrogen and progesterone receptor expression in brainstem norepinephrine neurons through the rat estrous cycle.

Norepinephrine (NE) neurons within the nucleus tractus solitarii (NTS; A2 neurons) and ventrolateral medulla (A1 neurons) represent gonadal steroid-dependent components of several neural networks regulating reproduction. Previous studies have shown that both A1 and A2 neurons express estrogen receptors (ERs). Using double labeling immunocytochemistry we report here that substantial numbers of NE neurons located within the NTS express progesterone receptor (PR) immunoreactivity, whereas few PRs are found in ventrolateral medulla. The evaluation of ERa and PR immunoreactivity in NE neurons through the estrous cycle revealed a fluctuating pattern of expression for both receptors within the NTS. The percentage of A2 neurons expressing PR immunoreactivity was low on metestrus and diestrus (3-7%), but increased significantly to approximately 24% on proestrous morning and remained at intermediate levels until estrus. The pattern of ERalpha immunoreactivity in A2 neurons was more variable, but a similar increment from 11% to 40% of NE neurons expressing ERa was found from diestrus to proestrus. Experiments in ovariectomized, estrogen-treated and estrogen-plus progesterone-treated rats revealed that PR immunoreactivity in A2 neurons was induced strongly by estrogen treatment, whereas progesterone had no significant effect. The numbers of ERalpha-positive NE neurons were not influenced by steroid treatment. These observations provide direct evidence for PRs in NE neurons of the brainstem and show that cyclical patterns of gonadal steroid receptor expression exist in A2, but not A1, neurons through the rat estrous cycle. The expression of PR in A2 neurons appears to be driven principally by circulating estrogen concentrations. The fluctuating levels of ERalpha and PR expression in these brainstem NE neurons may help generate cyclical patterns of biosynthetic and electrical activity within reproductive neural networks.

Sex-steroid actions on neurotransmission.

Although it is well recognized that sex-steroids exert both developmental and activational influences on the brain, the cellular and molecular mechanisms underlying their actions are less well understood. Progress is rapid, however, and this paper reviews recent insights gained through manipulation of sex-steroid receptor genes, identification of phenotypes expressing these receptors and how these proteins may also be activated to regulate transcription by ligand-independent pathways. Advances in our understanding of more rapid actions of sex-steroids and the molecular targets involved are also reviewed, as are new studies describing effects on synaptic plasticity and the recent excitement regarding the neuroprotective effects of oestrogen replacement therapy in Alzheimer's disease.

Estrogen uncouples noradrenergic activation of Fos expression in the female rat preoptic area.

The preoptic area of the rat brain is a site at which gonadal steroids act to regulate sexual behaviour and gonadotrophin secretion. The expression of the immediate-early gene product, Fos, in the preoptic area was investigated in conscious ovariectomised, vehicle and estrogen-treated animals which had received an intracerebroventricular (i.c.v.) infusion of noradrenaline, and also in anaesthetised proestrous and ovariectomised rats following electrical stimulation of the brainstem A1 or A2 noradrenergic cell groups. In ovariectomised oil-treated rats, a third ventricular infusion of noradrenaline (45 micrograms) resulted in a significant (P < 0.05) increase in the numbers of Fos-immunoreactive cell nuclei throughout the preoptic area, compared to vehicle controls. In contrast, Fos expression in animals which had received estrogen replacement showed no change in response to i.c.v. noradrenaline compared with saline-treated controls. In anaesthetised, ovariectomised animals electrical stimulation of the A1 cell group resulted in a significant increase (P < 0.05) in Fos-like immunoreactivity compared with sham controls, specifically within the ventral preoptic area whilst stimulation of the A2 cell group had no significant effect. In anaesthetised, proestrous rats receiving electrical stimulation no significant changes in Fos-like immunoreactivity were detected within the preoptic area after either A1 or A2 stimulation compared with paired controls. These results show that noradrenaline-induced Fos expression in the preoptic area is dependent on estrogen status and suggest that the estrogenic regulation of reproductive functions may thus involve altered responses to noradrenaline in sub-populations of preoptic neurones.

Differential cellular localization of oestrogen receptor immunoreactivity and oxytocin mRNA and immunoreactivity in the rat preoptic area.

Oestrogen influences both oxytocin mRNA and peptide immunoreactivity in the preoptic area and the rat oxytocin gene contains functional oestrogen response elements. However, using combinations of immunocytochemistry and in situ hybridization for oxytocin and oestrogen receptor, we found that preoptic oxytocin neurons do not possess oestrogen receptors. This finding implies oestrogen actions on oxytocin synthesis in preoptic neurones are unlikely to be mediated directly.

Oestrogenic activity of an environmentally persistent alkylphenol in the reproductive tract but not the brain of rodents.

Compounds with oestrogenic actions present in the environment as a result of human activity may represent a threat to health and reproductive efficiency in human and wildlife populations. We show here that parenteral administration of octylphenol, a recently described environmental oestrogen derived from one group of non-ionic surfactants, is active in stimulating oestrogen-dependent uterine growth in prepubertal rats, but has no influence on perinatal sexual differentiation of the rat brain. These results extend previous in vitro findings to show that alkylphenols exert weak oestrogenic activity in vivo in mammals.

Changing patterns of Fos expression in brainstem catecholaminergic neurons during the rat oestrous cycle.

Brainstem catecholaminergic neurons are believed to play an important role in the activation of luteinising hormone-releasing hormone (LHRH) neurons on the afternoon of proestrus which results in the luteinising hormone (LH) surge. To examine the respective roles of brainstem A1 and A2 neurons and the adjoining C1 and C2 adrenergic cells at this time, we have examined the patterns of Fos-immunoreactivity within tyrosine hydroxylase (TH) and phenylethanolamine-N-methyl transferase (PNMT) neurons during diestrus and proestrus. Initial studies demonstrated that the LH surge commenced at approximately 15:00 h in proestrous animals and that peak plasma levels of LH were observed between 16:00 and 17:00 h. Groups of cycling female rats (n = 6) were then perfused between 09:00 and 11:00 (diestrus early) and 18:00 to 19:30 h (diestrus late) on diestrus and at the same times on proestrus (proestrus early and proestrus late). Double-labelling immunocytochemistry revealed little Fos expression by adrenergic neurons of the C1 or C2 cell groups and this did not change significantly between any of the experimental groups. Analysis of the A2 region was divided into rostral, middle and caudal divisions and all regions showed a significant (P < 0.01) increase in the number of Fos-expressing TH neurons (up to 35% of TH cells) in proestrus early animals compared with diestrus and proestrus late rats. In the A1 region, a significant increase in the number of TH neurons expressing Fos (approximately 33%) was detected in both proestrus early (P < 0.05) and diestrus early (P < 0.01) rats compared with animals perfused in the late afternoon (approximately 12%).(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of uterine activity with oxytocin in late pregnant rats replicates the expression of c-fos in neuroendocrine and brain stem neurons as seen during parturition.

We investigated whether delivery could be induced with pulsatile oxytocin and whether such treatment activated neurons in the supraoptic nucleus (SON) and putative afferent neurons in the nucleus tractus solitarii and ventrolateral medulla, as seen during spontaneous parturition. Rats were implanted with a jugular venous cannula 1 day before the expected term and on the next morning were given a pulse of oxytocin or saline every 10 min for 4 h. Pulses of 10 and 20 mU oxytocin induced delivery in 77% (14 of 18) of rats, whereas none of the control animals (0 of 9) gave birth during the treatment. Lower doses of oxytocin were not effective at this time in inducing delivery. Animals were killed either before (prepartum groups) or during (parturient groups) delivery, and the brains were processed for immunocytochemistry. Oxytocin treatment induced Fos expression in SON and brain stem neurons in both parturient rats and rats in which parturition was not induced. Fos expression in all sites was significantly higher than that in control prepartum rats, but was similar in extent and distribution to that in spontaneous parturient rats. In the brain stem, a substantial proportion of Fos-immunoreactive cells contained tyrosine hydroxylase-like immunoreactivity, and the number of these cells was increased in response to oxytocin treatment. As only very few Fos-immunoreactive nuclei in either the SON or the nucleus tractus solitarii were observed in virgin rats injected with oxytocin, we suggest that intermittent oxytocin injections in late pregnant rats induce strong uterine activity, which can stimulate magnocellular and putative afferent neurons even before the expulsion of pups.

Endogenous opioid regulation of oxytocin release during parturition is reduced in ovariectomized rats.

Pregnant rats were ovariectomized (or sham-ovariectomized) on days 17, 18 or 21 of pregnancy and oestradiol-17 beta and progesterone were replaced. Prepartum oxytocin concentrations were significantly lower in ovariectomized steroid-treated rats than in intact controls, and on day 21 of pregnancy injection of relaxin into acutely ovariectomized rats significantly increased plasma oxytocin concentrations. During parturition, injection of the opioid antagonist naloxone induced significant increases in plasma oxytocin concentration compared with saline-injected rats. The naloxone-induced increase was significantly less in ovariectomized steroid-treated rats than in rats with intact ovaries, indicating that endogenous opioid activity is less in ovariectomized rats than in intact rats. The progress of parturition in the ovariectomized steroid-treated rats was severely disrupted compared with sham-ovariectomized rats despite similar plasma oxytocin levels at the birth of pup number 2; this disruption was not overcome by injection of naloxone or by the consequent increase in oxytocin secretion, indicating deficient preparation of the uterus and birth canal in the absence of relaxin. We conclude that the decreased oxytocin concentrations prepartum, the prolongation of parturition and the decrease in opioid tone in ovariectomized steroid-treated rats may be partly due to a lack of relaxin produced by the ovary.

The maintenance of normal parturition in the rat requires neurohypophysial oxytocin.

The neuropeptide oxytocin has long been known as a potent contractor of the uterus. However, it has remained difficult to attribute a definite role for neurohypophysial oxytocin in either the initiation or continuation of labour. Most recently, Lefebvre and colleagues have suggested that oxytocin produced in the uterus, rather than in the hypothalamus, may be more important in parturition since at term the uterus of the rat contains 70-fold more mRNA for oxytocin than the hypothalamus, and this disappears at about the time of parturition. Despite the high levels of mRNA the uterus contains only nanogram quantities of immunoreactive oxytocin per gram wet weight at term, compared to microgram quantities present in the pituitary. Here we show that activation of the neurohypophysial oxytocin system occurs, as reflected by expression of immunoreactivity for Fos in the hypothalamic supraoptic nucleus, and that this activation is indeed critical for normal parturition, since its inhibition results in a significant prolongation of parturition. In addition, we present evidence that pulsatile delivery of oxytocin into the circulation is important for the efficient progress of parturition, indicating that a major role of the neuronal circuits regulating oxytocin secretion for parturition, as is already known for suckling, is to produce an appropriately patterned hormonal output for efficient biological action.

Intravenous hypertonic saline induces Fos immunoreactivity in neurons throughout the lamina terminalis.

Expression of Fos, the protein product of c-fos, was studied immunohistochemically in the forebrain of rats infused intravenously with hypertonic solutions. Intravenous 1.5 or 0.75 mol/l NaCl or 1.2 mol/l sucrose in 0.15 mol/l NaCl, but not isotonic 0.15 mol/l NaCl, caused increased Fos expression in the hypothalamic paraventricular and supraoptic nuclei and throughout the lamina terminalis (organum vasculosum laminae terminalis, median preoptic nucleus and subfornical organ). These results show that neurons in the lamina terminalis are activated by physiological increases in plasma tonicity and support an involvement of the lamina terminalis in osmoregulation.

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.

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.

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)

Intracellular recordings from bovine anterior pituitary cells: modulation of spontaneous activity by regulators of prolactin secretion.

The electrophysiological properties of cultured bovine anterior pituitary cells were examined using intracellular recordings. Although all cells had high input resistances (mean 332 M omega), membrane potentials (Vm) showed a wide distribution (-32 to -80 mV). In 9 cells with low Vm (mean -38 mV) spontaneous Ca2+-dependent action potentials were observed (mean frequency 3.4 hertz). In the majority of silent cells (mean Vm -58 mV), application of TRH caused a reduction in membrane resistance and a transient hyperpolarization of Vm. This initial response was followed by a small, sustained depolarization. In 4/18 cells this depolarization led to the appearance of action potentials in previously silent cells. In 15/18 cells application of dopamine caused an increase in the membrane conductance and led to a sustained hyperpolarization of 30-35 mV. The response reversibly blocked the propagation of action potentials in spontaneously active cells. This effect could also be reversed by the dopamine receptor antagonists, sulpiride and haloperidol. In all cells, elevating the extracellular potassium concentration caused a concentration-dependent depolarization and decrease in the membrane input resistance. In some cells this effect was associated with an increased frequency of action potentials. Electrophysiological responses to TRH, dopamine, and elevated potassium were correlated with changes in the release of PRL. It is concluded that by their effects on Vm these factors modulate spontaneous electrical properties and may regulate the entry of calcium necessary for hormone secretion from lactotrophs.

Synergistic interaction in bovine pituitary cultures between growth hormone-releasing factor and other hypophysiotrophic factors.

Synthetic human pancreatic GH-releasing factor (1-44)NH2 (GRF) and acetylcholine (ACh) were shown to evoke a dose-related release of GH from cultured bovine pituitary cells with half-maximal effective doses of 0.3 and 500 nmol/l respectively. Concentrations of ACh (10 mumol/l) and GRF (25 nmol/l) which were shown to give near maximal responses when presented alone, produced highly synergistic responses when tested in combination. This synergism was related to the ACh concentration employed, and both the ACh-induced release and ACh-induced synergism were abolished by the muscarinic antagonist, atropine. A synergistic interaction was also demonstrated between GRF and concentrations of thyrotrophin-releasing hormone (TRH) and bombesin which, in the absence of GRF, failed to elicit significant GH release. Acetylcholine stimulated a similar dose-dependent release of prolactin, but GRF was ineffectual in either directly stimulating prolactin release or affecting the response to ACh or TRH. No synergistic interaction could be detected between combinations of ACh and TRH or between ACh and bombesin. The data suggest that, in the somatotroph, GRF acts through a different second messenger pathway to ACh, TRH and bombesin and that these two pathways can be activated to produce a potentiated response. Growth hormone-releasing factor is, therefore, not only a specific GH secretagogue, but may act in concert with other hypophysiotrophic factors to regulate GH secretion from the bovine anterior pituitary.