CRF-related Peptides Research Articles

The role of CRF receptor subtypes in stress-induced behavioural responses

The actions of corticotropin-releasing factor (CRF) and CRF-related peptides in the brain and periphery are mediated through multiple receptors. Two CRF receptor subtypes that differ markedly in their pharmacological profiles and anatomical distribution have been identified and characterized. Important advances have been made in understanding CRF and its actions through the development of specific CRF receptor antagonists, application of antisense oligonucleotides, and the production of transgenic mice lacking functional CRF 1 receptors. This chapter describes recent findings with respect to CRF-related peptides and CRF receptors and their role in stress-induced behaviours.

Molecular integration of hypothalamo-pituitary-adrenal axis-related neurohormones on the GnRH neuron

Gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus is pivotal to the regulation of reproductive physiology in vertebrates. GnRH and the reproductive axis, in general, can be inhibited during periods of stress or injury. Stress, in the form of mechanical, psychological or immunological insult to an organism results in the activation of the hypothalamo-pituitary-adrenal (HPA) axis initiated by the hypothalamic release of corticotropin-releasing factor (CRF). Recent studies indicate that CRF may act either directly on the GnRH neuron to down-regulate GnRH synthesis, or indirectly via a beta-endorphin-mediated pathway. Moreover, in vitro studies suggest that CRF-related peptides can increase the sensitivity of the GnRH neuron to prolactin by increasing the synthesis of the prolactin receptor.

Do Centrally Administered Neuropeptides Access Cognate Receptors?: An Analysis in the Central Corticotropin-Releasing Factor System

To determine the extent to which centrally administered corticotropin-releasing factor (CRF) activates neurons that express CRF receptors (CRF-Rs), we followed the kinetics and distribution (relative to those of CRF-Rs) of Fos induction seen in response to intracerebroventricular (icv) injection of the peptide (1-10 microg). CRF provoked widespread Fos expression: its strength was dose-related, it peaked at 2 hr after injection, and it was antagonized in a dose-dependent manner by coinjection of CRF-R antagonists. The activation pattern closely mimicked the distribution of CRF-R1 mRNA, in including widespread Fos induction throughout the cortical mantle, in cell groups involved in sensory information processing, and in the cerebellum and several of its major afferents and targets. Dual labeling revealed extensive correspondence of CRF-stimulated Fos-immunoreactivity (Fos-ir) and CRF-R1 mRNA at these and other loci. Unique sites of CRF-R2 expression were relatively unresponsive to CRF but were more so after icv administration of urocortin (UCN), a new mammalian CRF-related peptide. Both CRF and UCN elicited activational responses in cell groups that are involved in central autonomic control but that express neither CRF-R, including the central amygdaloid and paraventricular hypothalamic nuclei, and brainstem catecholaminergic cell groups. The results support an ability of CRF-related peptides in the ventricular system to access receptor-expressing cells directly but leave open questions as to the basis for the recruitment of central autonomic structures, many of which have been identified as stress-related sites of CRF action.

Molecular integration of hypothalamo-pituitary-adrenal axis-related neurohormones on the GnRH neuron

Gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus is pivotal to the regulation of reproductive physiology in vertebrates. GnRH and the reproductive axis, in general, can be inhibited during periods of stress or injury. Stress, in the form of mechanical, psychological or immunological insult to an organism results in the activation of the hypothalamo-pituitary-adrenal (HPA) axis initiated by the hypothalamic release of corticotropin-releasing factor (CRF). Recent studies indicate that CRF may act either directly on the GnRH neuron to down-regulate GnRH synthesis, or indirectly via a β-endorphin-mediated pathway. Moreover, in vitro studies suggest that CRF-related peptides can increase the sensitivity of the GnRH neuron to prolactin by increasing the synthesis of the prolactin receptor.

Distribution of mRNAs encoding CRF receptors in brain and pituitary of rat and mouse.

Two G protein-coupled receptors have been identified that bind corticotropin-releasing factor (CRF) and urocortin (UCN) with high affinity. Hybridization histochemical methods were used to shed light on controversies concerning their localization in rat brain, and to provide normative distributional data in mouse, the standard model for genetic manipulation in mammals. The distribution of CRF-R1 mRNA in mouse was found to be fundamentally similar to that in rat, with expression predominating in the cerebral cortex, sensory relay nuclei, and in the cerebellum and its major afferents. Pronounced species differences in distribution were few, although more subtle variations in the relative strength of R1 expression were seen in several forebrain regions. CRF-R2 mRNA displayed comparable expression in rat and mouse brain, distinct from, and more restricted than that of CRF-R1. Major neuronal sites of CRF-R2 expression included aspects of the olfactory bulb, lateral septal nucleus, bed nucleus of the stria terminalis, ventromedial hypothalamic nucleus, medial and posterior cortical nuclei of the amygdala, ventral hippocampus, mesencephalic raphe nuclei, and novel localizations in the nucleus of the solitary tract and area postrema. Several sites of expression in the limbic forebrain were found to overlap partially with ones of androgen receptor expression. In pituitary, rat and mouse displayed CRF-R1 mRNA signal continuously over the intermediate lobe and over a subset of cells in the anterior lobe, whereas CRF-R2 transcripts were expressed mainly in the posterior lobe. The distinctive expression pattern of CRF-R2 mRNA identifies additional putative central sites of action for CRF and/or UCN. Constitutive expression of CRF-R2 mRNA in the nucleus of the solitary tract, and stress-inducible expression of CRF-R1 transcripts in the paraventricular nucleus may provide a basis for understanding documented effects of CRF-related peptides at a loci shown previously to lack a capacity for CRF-R expression or CRF binding. Other such "mismatches" remain to be reconciled.

Urocortin Expression in Rat Brain: Evidence Against a Pervasive Relationship of Urocortin-Containing Projections With Targets Bearing Type 2 CRF Receptors

Histochemical and axonal transport methods were used to clarify the central organization of cells and fibers that express urocortin (UCN), a recently discovered corticotropin-releasing factor (CRF)-related neuropeptide, which has been proposed as an endogenous ligand for type 2 CRF receptors (CRF-R2). Neurons that display both UCN mRNA and peptide expression were found to be centered in the Edinger-Westphal (EW), lateral superior olivary (LSO), and supraoptic nuclei; lower levels of expression are seen in certain cranial nerve and spinal motoneurons and in small populations of neurons in the forebrain. Additional sites of UCN mRNA and peptide expression detected only in colchicine-treated rats are considered to be minor ones. UCN-immunoreactive projections in brain are predominantly descending and largely consistent with central projections attributed to the EW and LSO, targeting principally accessory optic, precerebellar, and auditory structures, as well as the spinal intermediate gray. Although neither the EW nor LSO are known to project to the forebrain, UCN-ir neurons in the EW were identified that project to the lateral septal nucleus, which houses a prominent UCN-ir terminal field. Although substantial UCN-ir projections were observed to several brainstem cell groups that express CRF-R2, including the dorsal raphe and interpeduncular nuclei and the nucleus of the solitary tract (NTS), most prominent seats of CRF-R2 expression were found to contain inputs immunopositive for piscine urotensin I, but not rat UCN. The results define a central UCN system whose organization suggests a principal involvement in motor control and sensorimotor integration; its participation in stress-related mechanisms would appear to derive principally by virtue of projections to the spinal intermediolateral column, the NTS, and the paraventricular nucleus. Several observations, including the lack of a pervasive relationship of UCN-ir projections with CRF-R2-expressing targets, support the existence of still additional CRF-related peptides in mammalian brain.

Modes of control of insect Malpighian tubules: synergism, antagonism, cooperation and autonomous regulation

Rates of fluid and ion secretion by insect Malpighian tubules are controlled by peptides, including CRF-related peptides and kinins, and in some species by serotonin. It now appears to be a general rule that tubule secretion rate is controlled through the interaction of two or more haemolymph-borne factors. In this review we suggest that these interactions may be classified as synergistic, cooperative, or antagonistic. When presented together, two diuretic factors may act in synergism, so that fluid secretion is stimulated to a greater extent than the sum of their individual effects. Synergism may involve one or more second messenger systems. Alternatively, diuretic factors may act in cooperation, so that although their overall effects are additive, cation and anion transport pathways are controlled separately by distinct second messenger systems. There is also one example of antagonism between factors controlling tubule secretion and between their respective second messengers; one factor is stimulatory, the other is inhibitory. In addition to the complex control of fluid and ion transport by haemolymph-borne factors, sophisticated autonomous regulatory mechanisms have been identified in Malpighian tubules. When triggered by appropriate stimuli, these mechanisms play homeostatic roles, preserving haemolymph osmolality or ionic composition.

Characterization of the relaxant action of urocortin, a new peptide related to corticotropin-releasing factor in the rat isolated basilar artery.

In addition to its well established neuroendocrine and neurotransmitter effects, corticotropin releasing factor (CRF) exerts a potent vasorelaxant action. Recently, a CRF-related peptide, urocortin, has been identified in the mammalian brain. In the present study, the cerebral vasomotor action of this peptide and the mechanism underlying its relaxant effect are characterized. Ring segments obtained from the rat basilar artery were used for measurement of isometric force. The relaxant action of urocortin, CRF and sauvagine was studied in segments with a functionally intact endothelium. In segments precontracted with prostaglandin F2alpha, urocortin, CRF and sauvagine induced concentration-related relaxation. The order of potency was as follows (pD2+/-s.e.m. given in brackets): urocortin (9.32+/-0.07) > sauvagine (9.08+/-0.08) > CRF (7.50+/-0.07). Complete relaxation was achieved with each agonist. Relaxation was not affected by removal of the endothelium but was markedly attenuated in segments precontracted with 50 mM K+ Krebs solution. The relaxant effect of urocortin was inhibited by astressin in an apparently competitive manner. A pA2 value of 7.52 was estimated for astressin. Inhibition of urocortin-induced relaxation was also observed in the presence of the adenylate cyclase inhibitor SQ22536 (pD2 in the presence of 300 microM SQ22536, 9.36+/-0.05) and the K+ channel blockers tetraethylammonium (10 mM; pD2, 8.65+/-0.07), iberiotoxin (100 nM; pD2, 8.88+/-0.08) and apamin (10 nM; pD2, 8.94+/-0.07). However, the inhibitory actions of SQ22536 and apamin or iberiotoxin were not additive. The results suggest that urocortin induces relaxation of cerebral arteries by activating CRF-R2 receptors present in the vascular wall. Relaxation appears to be mediated by adenylate cyclase stimulation and activation of Ca2+-dependent K+ channels.

Central CRF inhibits gastric emptying of a nutrient solid meal in rats: the role of CRF2 receptors.

Corticotropin-releasing factor (CRF)-related peptides exhibit different affinity for the receptor subtypes 1 and 2 cloned in the rat brain. We investigated, in conscious rats, the effects of intracisternal (i.c.) injection of CRF (rat/human) on the 5-h rate of gastric emptying of a solid nutrient meal (Purina chow and water ad libitum for 3 h) and the CRF receptor subtype involved. CRF, urotensin I (suckerfish), and sauvagine (frog) injected i.c. inhibited gastric emptying in a dose-dependent manner, with ED50 values of 0.31, 0.13, and 0.08 microgram/rat, respectively. Rat CRF-(6-33) (0.1-10 micrograms i.c.) had no effect. The nonselective CRF1 and CRF2 receptor antagonist, astressin, injected i.c. completely blocked the inhibitory effect of i.c. CRF, urotensin I, and sauvagine with antagonist-to-agonist ratios of 3:1, 10:1, and 16:1, respectively. The CRF1-selective receptor antagonist NBI-27914 injected i.c. at a ratio of 170:1 had no effect. These data show that central CRF and CRF-related peptides are potent inhibitors of gastric emptying of a solid meal with a rank order of potency characteristic of the CRF2 receptor subtype affinity (sauvagine > urotensin I > CRF). In addition, the reversal by astressin but not by the CRF1-selective receptor antagonist further supports the view that the CRF2 receptor subtype is primarily involved in central CRF-induced delayed gastric emptying.

Pharmacological characterisation of the recombinant human CRF binding protein using a simple assay

We present the pharmacological characterisation of the recombinant human corticotropin releasing factor binding protein (hCRF-BP) using a simple assay. In this assay we employed [ 3H]urocortin as the radioligand and, as a means to separate bound and free ligand, adsorption to activated charcoal. Using this method, approximately 60–70% of total binding was specific. Kinetic analysis revealed that association of specific [ 3H]urocortin binding was monophasic and slow and that the binding was irreversible. Saturation analysis showed a single saturable site of relatively high density (94 fmol per 10 μl of medium from cells transfected with the recombinant CRF binding protein). The apparent K d for [ 3H]urocortin binding of 0.25 nM is similar to previously reported affinities of rat urocortin for hCRF-BP. A range of CRF-related peptides potently competed for specific [ 3H]urocortin binding. The rank order of potency of these agents was human/rat CRF=urotensin 1>human urocortin>CRF 6–33>sauvagine>ovine CRF. The non-peptide CRF 1 receptor antagonists CP 154,526 ( N-butyl- N-[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3- d]pyrimidin-4-yl]- N-ethylamine) and SC 241 ([3-(2-bromo-4-isopropyl-phenyl)-5-methyl-3H-[1,2,3]triazo lo[4,5- d]pyrimidin-7-yl]-bis-(2-methoxy-ethyl)-amine) were not active at the highest concentration tested (10 −6 M). We conclude that this is a simple and accurate assay for characterisation of the pharmacology of the recombinant CRF-BP. This assay should assist with further study of the pharmacology and function of the CRF-BP.

Corticotropin-releasing factor (CRF) and endocrine responses to stress: CRF receptors, binding protein, and related peptides.

Corticotropin-releasing factor (CRF) is a 41-amino acid neuropeptide, which is recognized as a critical mediator of complimentary, stress-related endocrine, autonomic, and behavioral responses in mammalian species. CRF belongs to a family of structurally related peptides including frogskin sauvagine and fish urotensin I. The effects of CRF and related peptides are mediated by two distinct receptors, which differ in their anatomical distribution, as well as in their pharmacological characteristics. In addition, CRF is bound with high affinity by a CRF binding protein (CRF-BP), which is a putative inhibitor of CRF action. CRF is probably not the sole endogenous ligand for CRF receptors or the CRF-BP, since a second mammalian member of the CRF family, urocortin, has recently been identified. This article describes recent findings with respect to CRF, its receptors, binding protein, and CRF-related peptides, which provide further insights into the role and mechanisms of CRF action in stress responses.

Quantification of Locusta diuretic hormone in the central nervous system and corpora cardiaca: influence of age and feeding status, and mechanism of release

Locusta-DH is known to have a hormonal function in the control of post-feeding diuresis in the migratory locust. This study has quantified Locusta-DH in tissues from V th instar nymphs and adults, and investigated the K +-induced release of the peptide from corpora cardiaca. Locusta-DH is present in thoracic and abdominal ganglia, but the amounts are small (25–200 fmol) compared with brain (∼1 pmol) and corpora cardiaca (>5 pmol) from 14-day old locusts. About 50% of the immunoreactive material in corpora cardiaca coelutes with Locusta-DH on reversed-phase HPLC. An earlier eluting fraction is also biologically active, suggesting locusts have a second, previously undetected, CRF-related peptide. The amount of peptide stored in corpora cardiaca varies with age and physiological status. Reductions on day 1 of the adult instar and immediately after feeding suggest Locusta-DH controls post-eclosion as well as post-feeding diureses. Locusta-DH is released by a Ca 2+-dependent mechanism from corpora cardiaca held in salines containing ≥40 mM K +. This is blocked by verapamil, implicating L-type Ca 2+ channels. Release is most rapid shortly after transfer to a high K + saline, and more peptide is released from glands allowed to recover in normal saline between successive K + depolarisations.

Circulating Levels of Locusta Diuretic Hormone: The Effect of Feeding

Audsley, N., G. J. Goldsworthy and G. M. Coast. Circulating levels of Locusta diuretic hormone: The effect of feeding. Peptides 18(1) 59–65, 1997.—A radioimmunoassay was developed using 125I-labeled-[Tyr 0] Locusta-DH and polyclonal antibodies raised against Locusta-DH(29–46). The assay had a detection limit of 50 p M, and displayed limited crossreactivity for other CRF-related peptides, but not for unrelated peptides. About 60% of the total immunoreactive material in locust hemolymph was attributable to Locusta-DH. The circulating level of diuretic hormone increases fivefold in fed insects, sufficient to stimulate primary urine production, and is correlated with the duration of the meal. This is consistent with the role of Locusta-DH in the control of postfeeding diuresis. DH in a pooled sample of hemolymph from locusts that were starved overnight.

Urocortin interaction with corticotropin-releasing factor (CRF) binding protein (CRF-BP) : a novel mechanism for elevating ‘free’ CRF levels in human brain

Here we demonstrate that urocortin, a new mammalian member of the corticotropin-releasing factor (CRF) neuropeptide family has high affinity for both the recombinant human CRF binding protein (CRF-BP) and for a membrane-associated form of the protein solubilized from postmortem human cerebrocortical brain tissue. The rank order of binding potency for both the human recombinant CRF-BP and for the solubilized human brain CRF-BP is: urotensin > hCRF > urocortin > sauvagine. The bound hCRF/hCRF-BP complex was detected in the postmortem human brain tissue using an ELISA assay specific for the hCRF/hCRF-BP complex. A large proportion (65%) of the endogenous hCRF was found to be complexed to the CRF-BP and thus unavailable for CRF receptor activation. Incubation of human brain postmortem tissue extracts with urocortin and urotensin resulted in a dramatic decrease in hCRF/hCRF-BP levels and a concomitant increase in ‘free’ hCRF levels. Thus, urocortin and other putative CRF-related peptides may elevate endogenous levels of ‘free’ hCRF in brain by displacing hCRF from the binding protein. These data define an indirect endogenous mechanism for activation of CRF receptors by new mammalian members of the CRF family of neuropeptides.

Neuropeptides implicated in the control of diuresis in insects.

Primary urine production in insect Malpighian tubules is stimulated by two classes of neuropeptides, CRF-related diuretic peptides and insect kinins. The CRF-related peptide of the locust, Locusta migratoria, has a hormonal role in the control of postfeeding diuresis, but the functional role of the kinins has yet to be defined. The two classes of peptide act synergistically to stimulate tubule secretion, and the kinins may therefore have a modulatory action in the control of diuresis. The peptides differ in their effects on Malpighian tubule ion transport, and this could be important for the regulation of hemolymph volume and composition.

Synergism between diuretic peptides controlling ion and fluid transport in insect malpighian tubules

This study provides clear evidence for synergism between diuretic peptides from Locusta migratoria, Locusta-DP (a CRF-related peptide) and locustakinin (an insect myokinin). At low concentrations, the peptides act co-operatively to stimulate Malpighian tubule fluid secretion by more than the sum of their separate responses. They work via different second messengers, and their synergistic actions can be mimicked with exogenous 8-bromo-cyclic AMP replacing Locusta-DP, and/or with thapsigargin substituting for locustakinin. Additionally, 5-HT, which had been shown previously to stimulate tubule secretion via a cyclic AMP-independent mechanism, potentiated the response to Locusta-DP. Potassium is invariably the dominant cation secreted by locust tubules, but Locusta-DP, whether in the absence or presence of locustakinin, increases J Na at the expense of J K. In contrast, J Na and J K increase in parallel with J V after stimulation by locustakinin. These findings are consistent with the known actions of cyclic AMP and leucokinin-VIII on mosquito tubules. The ability to vary the relative amounts of Na + and K + delivered to the hindgut may be important for the maintenance of haemolymph volume and composition in recently fed locusts.

Cloning and characterization of a functionally distinct corticotropin-releasing factor receptor subtype from rat brain.

The present study reports the isolation of a cDNA clone that encodes a second member of the corticotropin-releasing factor (CRF) receptor family, designated as the CRF2 receptor. The cDNA was identified using oligonucleotides of degenerate sequence in a PCR paradigm. A PCR fragment obtained from rat brain was utilized to isolate a full-length cDNA from a rat hypothalamus cDNA library that encoded a 411-amino acid protein with approximately 70% identity to the known CRF1 receptor over the entire coding region. When expressed in mouse Ltk- cells, this receptor stimulates cAMP production in response to CRF and known CRF-like agonists. CRF and the nonmammalian CRF-related peptides sauvagine and urotensin I stimulate adenylate cyclase activity in a dose-dependent manner with a rank order of potency different from that of the CRF1 receptor: sauvagine > urotensin > or = rat/human CRF > ovine CRF. Tissue distribution analysis of the mRNAs by reverse transcriptase-PCR shows CRF2 receptor mRNA is present in rat brain and detectable in lung and heart. In situ hybridization studies indicate specific expression within the brain in the ventromedial nuclei of the hypothalamus, the lateral septum, the amygdala, and entorhinal cortex, but there is unremarkable expression in the pituitary. An additional splice variant of the CRF2 receptor with a different N-terminal domain has been identified by PCR, encoding a putative protein of 431 amino acids. Thus, the data demonstrate the presence of another functional CRF receptor, with significant differences in the pharmacological profile and tissue distribution from the CRF1 receptor, which would predict important functional differences between the two receptors.

Cross reactivity studies of CRF-related peptides on insect Malpighian tubules

Manduca sexta diuretic peptide II (Mas-DPII) stimulates fluid secretion by adult Malpighian tubules and cyclic AMP production by larval proximal and adult tubules of M. sexta in a dose-dependent manner. Mas-DPII has no effect on fluid transport across the larval cryptonephric complex. M. sexta diuretic hormone (Mas-DH) and CRF-related insect diuretic peptides from Acheta domesticus, Locusta migratoria, and Periplaneta americana also cause similar increases in the production of cyclic AMP by the Malpighian tubules of both larval and adult M. sexta. Insect CRF-related diuretic peptides exhibit varying degrees of potency when assayed on Malpighian tubules from L. migratoria and A. domesticus. Sauvagine, bovine-CRF, and human-CRF have only a small, but significant, effect on cyclic AMP production by M. sexta Malpighian tubules. However, sauvagine, bovine-CRF, and sucker fish urotensin-I have no effect on L. migratoria tubules. Stimulation of cyclic AMP production by M. sexta Malpighian tubules could potentially be used as a screening assay to identify other insect CRF-related diuretic peptides.

Effect of Manduca Sexta Diuretic Hormone and Related Peptides on Isolated Malpighian Tubules of the House Cricket Acheta Domesticus (L.)

ABSTRACT Previously, a corticotropin releasing factor (CRF)-like diuretic peptide (Man-duca-DH) has been isolated from Manduca sexta and shown to stimulate fluid excretion in vivo in post-eclosion Pieris rapae adults and in pre-wandering postfeeding Manduca sexta larvae. However, Manduca-DH was reported to have no effect on Malpighian tubules in vitro. Manduca-DH and [Nle2,11]-Manduca-DH were synthesized in Texas and assayed in London on isolated Malpighian tubules of Acheta domesticus. Man-duca-DH stimulated fluid secretion by about 60% of the maximum response achievable with extracts of corpora cardiaca and increased the production of cyclic AMP. In combination with 10−4moll−1 3-isobutyl-l-methyl xanthine (IBMX), Manduca-DH stimulated maximal secretion. A number of CRF-related peptides also stimulated fluid secretion and cyclic AMP production in cricket tubules, and the CRF antagonist α-helical-CRF[9–41] blocked the stimulation of fluid secretion by Manduca-DH. [Nle2,11]-Manduca-DH was more active than Manduca-DH in both assays, suggesting that methionine residues in the natural peptide may become oxidized. Taken in conjunction with previous in vivo studies, the present findings suggest that a Manduca-DH-like diuretic peptide is the hormone controlling post-eclosion diuresis in butterflies, and Manduca-DH was shown to stimulate both fluid secretion and cyclic AMP production in Malpighian tubules from 1–12 h posteclosion Pieris rapae adults. The function of the peptide in Manduca sexta is discussed.

The CRF-related peptide sauvagine stimulates and the GABA B receptor agonist baclofen inhibits cyclic-AMP production in melanotrope cells of Xenopus laevis

Release of α-MSH from the pars intermedia melanotrope cells of Xenopus laevis is regulated by various classical neurotransmitters and neuropeptides. We have examined the effect of two of these regulatory substances, the neurotransmitter GABA and the CRF-related peptide sauvagine, on the adenylate cyclase system of the melanotrope cells. Sauvagine treatment, which stimulates α-MSH release, lead to an elevation in the level of cyclic-AMP, an effect which was potentiated by cholera toxin. Treatment with baclofen, a GABA B receptor agonist, gave a pertussis toxin-sensitive decrease in the cyclic-AMP level and an inhibition of α-MSH release. We conclude that sauvagine stimulates α-MSH secretion through activation of adenylate cyclase and that GABA B receptor activation inhibits secretion through inhibition of cyclic-AMP production. Baclofen treatment sensitized melanotrope cells to the stimulatory action of 8-bromo-cyclic-AMP on the secretion of α-MSH. This observation supports the conclusion that GABA B receptor activation inhibits cyclic-AMP production.