A genetic determinant of the striatal dopamine response to alcohol in men.

Excessive alcohol use, a major cause of morbidity and mortality, is less well understood than other addictive disorders. Dopamine release in ventral striatum is a common element of drug reward, but alcohol has an unusually complex pharmacology, and humans vary greatly in their alcohol responses. This variation is related to genetic susceptibility for alcoholism, which contributes more than half of alcoholism risk. Here, we report that a functional OPRM1 A118G polymorphism is a major determinant of striatal dopamine responses to alcohol. Social drinkers recruited based on OPRM1 genotype were challenged in separate sessions with alcohol and placebo under pharmacokinetically controlled conditions, and examined for striatal dopamine release using positron emission tomography and [(11)C]-raclopride displacement. A striatal dopamine response to alcohol was restricted to carriers of the minor 118G allele. To directly establish the causal role of OPRM1 A118G variation, we generated two humanized mouse lines, carrying the respective human sequence variant. Brain microdialysis showed a fourfold greater peak dopamine response to an alcohol challenge in h/mOPRM1-118GG than in h/mOPRM1-118AA mice. OPRM1 A118G variation is a genetic determinant of dopamine responses to alcohol, a mechanism by which it likely modulates alcohol reward.

Substance P activity in the bullfrog retina: localization and identification in several vertebrate species.

Immunoreactive substance P is present in the bullfrog retina, possibly in two types of stratified amacrine cells, with their somas in the inner nuclear layer and their neuronal processes entering the inner plexiform layer and ramifying in sublayers 3 or 4 (or both). Occasionally, polygonal somas positive for substance P were found in the ganglion cell layer. Approximately 75 percent of the cell bodies positive for substance P and 65 percent of the radioimmunoassayable substance P were found in the superior half of the frog retina. On the basis of high-performance liquid chromatography, the immunoreactive substance P in the neural retina of the rat, monkey, or chick is similar to synthetic substance P, whereas this is not true of the immunoreactive substance P in the bullfrog or carp retina.

alpha-Melanocyte-stimulating hormone: reduction in adult rat brain after monosodium glutamate treatment of neonates.

Intraperitoneal injection of monosodium glutamate in neonatal rates resulted in a 90 percent loss of alpha-melanocyte-stimulating hormone in hypothalamic and extrahypothalamic areas of the brain, whereas the amount of hormone in the pituitary gland did not change. The dramatic reduction of alpha-melanocyte-stimulating hormone in the brain suggests that is primary source there is the neuronal perikarya of the arcuate nucleus.

Circulating atrial natriuretic peptides in conscious rats: regulation of release by multiple factors.

Cardiocytes in the atria contain a prohormone that gives rise to atrial natriuretic peptides (ANP's), which have intrinsic hemodynamic regulatory activity. The distribution of ANP's in the brain suggests the involvement of these peptides in central cardiovascular regulation. In conscious rats with chronic indwelling catheters, volume loading with isotonic saline or glucose increased the amount of circulating immunoreactive ANP's by a factor of 4 to 5, as determined by radioimmunoassay. Hyperosmotic challenge with a hypertonic NaCl solution or anesthesia with halothane caused similar increases in plasma ANP's. Results obtained with the denervated-heart preparation indicate that neuronal influences are important in the release of ANP's induced by volume loading. As judged from reversed-phase high-performance liquid chromatography of extracted plasma and radioimmunoassay of collected fractions, the circulating physiologically important ANP's in the conscious rodent appear to be alpha-rANP(5-28) (atriopeptin III) and either alpha-rANP(3-28) [ANF(8-33)] or alpha-rANP(1-28) (ANF).

Methionine and leucine enkephalin in rat neurohypophysis: different responses to osmotic stimuli and T2 toxin.

Specific radioimmunoassays were used to measure the effects of hypertonic saline (salt loading), water deprivation, and trichothecene mycotoxin (T2 toxin) on the content of methionine enkephalin (ME), leucine enkephalin (LE), alpha-neoendorphin, dynorphin A, dynorphin B, vasopressin, and oxytocin in the rat posterior pituitary. Concentrations of vasopressin and oxytocin decreased in response to both osmotic stimuli and treatment with T2 toxin, but the decrease was greater with osmotic stimulations. Similarly, concentrations of LE and dynorphin-related peptides declined after salt loading and water deprivation; LE concentrations also decreased after treatment with T2 toxin. The concentration of ME decreased after water deprivation, did not change after salt loading, and increased after T2 toxin treatment. The differentiating effects of these stimuli on the content of immunoreactive LE and ME are consistent with the hypothesis that LE and ME may be localized in separate populations of nerve endings with different roles in the posterior pituitary.

Hypothalamic-pituitary-adrenal axis functioning and cerebrospinal fluid corticotropin releasing hormone and corticotropin levels in alcoholics after recent and long-term abstinence.

We assessed the plasma corticotropin (adrenocorticotropic hormone) and cortisol responses to ovine corticotropin releasing hormone (oCRH) and the cerebrospinal fluid levels of CRH and corticotropin in alcoholics at various durations of abstinence and compared these variables with age-equivalent controls. Alcoholics who were tested at 1 week of abstinence (n = 11) demonstrated a significantly attenuated corticotropin response to oCRH compared with their response at 3 weeks of abstinence. Nine of these alcoholic patients demonstrated a significantly blunted corticotropin response at both 1 and 3 weeks of abstinence compared with controls (n = 15). A markedly exaggerated corticotropin response to oCRH, associated with tachycardia, was exhibited by 2 alcoholics at both 1 and 3 weeks of abstinence. Alcoholics who were abstinent greater than 3 weeks did not differ in their response to oCRH compared with controls. Controls demonstrated a significant inverse correlation between baseline cortisol levels and the cortisol response to oCRH. This correlation was not evident in any of the alcoholic groups, including those patients who were abstinent greater than 6 months. There was a positive correlation between cerebrospinal fluid concentrations of CRH and corticotropin in all patient groups. These findings indicated that alcoholics have significantly altered hypothalamic-pituitary-adrenal axis functioning up to 3 weeks following the cessation of drinking, with a more subtle impairment present for greater than 6 months following abstinence.

Interleukin-1 alpha and tumor necrosis factor-alpha differentially regulate enkephalin, vasoactive intestinal polypeptide, neurotensin, and substance P biosynthesis in chromaffin cells.

The pattern of expression of at least four neuropeptides contained in adrenomedullary chromaffin cells is altered by exposure to the cytokines interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF alpha), alone or in combination with stimulation of other second messenger pathways. Vasoactive intestinal polypeptide (VIP) was elevated 2- to 3-fold by 1 nM IL-1 alpha within 48 h of exposure, while neurotensin and substance P synthesis were unaffected, and met-enkephalin levels were decreased 25-35%. Stimulation of VIP and substance P biosynthesis by forskolin was markedly enhanced by IL-1 alpha, while forskolin stimulation of enkephalin and neurotensin biosynthesis was unaffected. IL-1 alpha amplified the effect of phorbol myristate acetate to increase the VIP content of chromaffin cells, but antagonized phorbol ester-induced elevation of neurotensin levels. TNF alpha also demonstrated a neuropeptide-specific pattern of modulation of second-messenger effects on chromaffin cell neuropeptide levels similar to those seen with IL-1 alpha. The neuroendocrine actions of IL-1 alpha described above, unlike IL-1 action in the immune system, do not appear to be mediated through IL-2 as this cytokine did not affect VIP or enkephalin expression in the presence or absence of protein kinase stimulation. Neither IL-1 alpha nor TNF alpha affected the calcium-coupled stimulation of neuropeptide secretion and biosynthesis that occurs in response to cell depolarization in these and other neuroendocrine cells in vitro and in vivo. These data provide a functional demonstration of IL-1 and TNF receptors in chromaffin cell cultures and suggest a physiological role for cytokine production in the adrenal medulla. Since both the magnitude and direction of neuropeptide synthesis modulation by IL-1 alpha and TNF alpha are highly peptide-specific, it appears that these cytokines do not merely augment second messenger pathways that affect neuropeptide synthesis, but potentially regulate the activity of factors controlling the pattern of neuropeptide gene expression in chromaffin cells.

Release of alpha-melanocyte-stimulating hormone from dispersed cells of the intermediate lobe of the rat pituitary gland: involvement of catecholamines and adenosine 3',5'-monophosphate.

Treatment of the neurointermediate lobe of the rat pituitary gland with mechanical agitation in the presence of trypsin and DNAse results in a preparation of cells which secrete alpha MSH and synthesize cAMP. The receptors for catecholamines present in the intact intermediate lobe remain functional on the dispersed cells. Thus, stimulation of a beta-adrenoceptor with (l)isoproterenol enhances the secretion of alpha MSH. This receptor is stereospecific and responds to low concentrations of isoproterenol (EC50 = 0.4 nM). Activation of a beta-adrenoceptor also increases the accumulation of cAMP. Furthermore, dopamine inhibits the basal release of alpha MSH but has no effect on basal levels of cAMP. In addition, dopamine inhibits the isoproterenol-enhanced release of alpha MSH as well as the isoproterenol-induced accumulation of cAMP.

Evidence that beta-endorphin binds to specific receptors in rat peripheral tissues and stimulates the adenylate cyclase-adenosine 3',5'-monophosphate system.

With the use of [125I]acetyl human beta-endorphin (Ac-hBE), specific binding sites for beta-endorphin (BE) were identified in the liver, kidney, adrenal, spleen, and testis of adult male rats, whereas specific BE-binding sites were not present in the ventral prostate or pancreas. In those tissues containing specific BE-binding sites, microsomal membranes (15,000-100,000 X g pellet) exhibited higher BE-binding capacity than the crude homogenate (125-100,000 X g pellet). The binding of BE was saturable, and maximal, specific binding was achieved with a 60-min incubation at 22 C. Furthermore, optimal BE binding was dependent on the presence of magnesium chloride. Scatchard analysis of BE binding to hepatic membranes revealed the existence of two classes of binding sites. One class had an apparent Ka of 0.019 X 10(9) M-1 and a lower number of binding sites (9.1 pmol BE/mg protein), whereas the other class had a lower affinity (apparent Ka of 0.0006 X 10(9) M-1) and a higher number of binding sites (159 pmol/mg protein). Specific BE binding to hepatic membranes was inhibited (80-100%) by rat AcBE-(1-27) and -(1-31), nonacetylated rat BE-(1-31), and human beta-lipotropin. At substantially higher peptide concentrations (greater than 10(-5) M), gamma-endorphin, met-enkephalin, or leu-enkephalin inhibited BE binding by 20-40%. In addition, opiate receptor-binding drugs, such as morphine and naloxone, at 10(-5) M did not alter BE binding to hepatic membranes. Incubation of hepatic membranes with BE induced a dose-related increase in membrane adenylate cyclase activity, and 0.5 X 10(-10) M BE resulted in a maximal enhancement of adenylate cyclase activity to 148% above control values. Water-deprived or salt-loaded male rats with chronically lowered immunoreactive plasma BE exhibited substantially increased BE binding to adrenal and kidney tissue. Specific binding sites for BE occur in a variety of peripheral tissues, and alterations of circulating BE result in changes in the capacity of certain peripheral tissues to bind BE. Finally, occupancy of specific BE-binding sites in peripheral tissue stimulates the adenylate cyclase-cAMP system, which suggests that the peripheral actions of circulating BE may be mediated via this system.

The course of thyrotropin-releasing hormone fibers to the median eminence in rats.

The topographical distribution of fibers containing TRH that run to the median eminence has been investigated by RIA of TRH after hypothalamic surgical transections in rats. Anterolateral or lateral cuts bilaterally transecting fibers at the caudal edge of the optic chiasm (lateral retrochiasmatic area) diminish TRH levels in the median eminence by 95% and 73%, respectively. Paramedian cuts are ineffective. These results suggest that TRH fibers reach the medial basal hypothalamus from an anterolateral direction just as several other classes of peptide containing fibers do.

Prostaglandin-induced release of anterior pituitary hormones: structure-activity relationships.

Prostaglandins (PG's) of the E, F, A, or B series were infused into a lateral ventricle of the brain of adult male rats, and the efficacy of each PG to stimulate the discharge of LH, prolactin, and TSH from the pituitary gland was determined. PGE2, PGF2alpha, and PGF2beta were found to be potent stimulators of LH release. The release was considerably less when PGE1, PGF1beta, PGA2, or PGB2 was infused. The basal release of LH was not altered by infusion of PGF1alpha, PGA1, PGB1, or the control solution. It is suggested that the cis double bond in the 5,6 position and the 11-hydroxyl group are essential for the LH releasing activity of PG's and that these functional groups may be of importance for activation of a receptor at the level of the brain. Prolactin secretion was stimulated 6 to 7-fold by the infusion of 5 mug of PGE2, but 20 mug had no stimulatory effect. PGF2beta caused a 3 to 4-fold increase in basal plasma prolactin concentration and was the only PG tested other than PGE2 that stimulated prolactin release. None of the PG's infused intraventricularly affected TSH secretion.

Biochemical identification of the beta-adrenoceptor and evidence for the involvement of an adenosine 3',5'-monophosphate system in the beta-adrenergically induced release of alph-melanocyte-stimulating hormone in the intermediate lobe of the rat pituitary gland.

The beta-adrenoceptor in the intermediate lobe (IL) of the hypophysis of the rat is characterized on the basis of the following: 1) the ability of beta-adrenergic agonists to increase adenylate cyclase activity in homogenates of the IL, and 2) the ability of drugs active upon the beta-adrenoceptor to compete with [125I]hydroxybenzylpindolol, a radiolabeled beta-adrenergic antagonist, for high affinity (Kd = 232 pM) binding sites. The values of the affinity of the beta-adrenoceptor for drugs obtained in either assay system are in good agreement. The relative potency among agonists, L-isoproterenol greater than L-epinephrine greater than L-norepinephrine, suggests that the receptor is of the beta-2 subcategory. cAMP, derivatives of cAMP, and a phosphodiesterase inhibitor, theophylline, mimic the ability of l-isoproterenol to enhance the release of alpha MSH from dispersed cells of the rat IL. The present results are in accord with the possibility that occupancy by agonists of the beta-adrenoceptor of the IL enhances adenylate cyclase activity, resulting in an accumulation of cAMP which initiates the intracellular events that are ultimately expressed as an enhanced release of alpha MSH. Pharmacological data suggest that stimulation of a dopamine receptor in the IL diminishes the response of the beta-adrenoceptor to agonists.

Opioid-mediated cardiovascular effects of clonidine in spontaneously hypertensive rats: elimination by neonatal treatment with monosodium glutamate.

The interaction between clonidine and opiate receptor antagonists on arterial blood pressure (BP) and heart rate were examined in normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). In conscious SHR, the hypotension and bradycardia caused by clonidine, 5 micrograms/kg iv, were significantly attenuated by naltrexone, 2 mg/kg ip. In urethane-anesthetized SHR, the reduction in mean BP and heart rate in response to 5 nmol clonidine microinjected into the nucleus of the solitary tract (NTS), were similarly inhibited after intra-NTS microinjection of 100 ng DL-naloxone but not after the same dose of D-naloxone. Neonatal treatment of SHR by monosodium glutamate (MSG) markedly reduced the beta-endorphin (BE) but not the leucin-enkephalin content of the arcuate nucleus and the NTS. MSG treatment did not affect the basal BP of these animals, but significantly reduced the hypotensive effect of clonidine and eliminated its susceptibility to opiate antagonists in both conscious and anesthetized SHR. In conscious and anesthetized WKY, the cardiovascular effects of clonidine were smaller than in SHR and were unaffected by naloxone or naltrexone. Neonatal treatment of WKY with MSG reduced the BE content of the arcuate nucleus but not of the NTS. MSG treatment of WKY did not influence either basal BP or the cardiovascular effects of clonidine, and the latter remained unaffected by opiate antagonists. These findings support the hypothesis that in SHR, but not in WKY, the centrally mediated cardiovascular effects of clonidine are partially mediated by the release of a BE-like opioid. They also strongly suggest that the site of both the release and the action of this opioid is in the NTS.

Prostaglandin E2-induced release of LHRH into hypophysial portal blood(1).

Prostaglandin E2 (PGE2) was infused into a lateral ventricle of adult male rats or infused into a hypophysial portal vessel. Intraventricularly administered PGE2 stimulated the release of LH, FSH, and prolactin but not TSH. Intraventricular infusion of the control solution did not alter the basal release of any of these hormones. PGE2 was found to be a potent stimulator of LH release, but larger quantities were needed to stimulate the release of FSH or prolactin. In addition, lateral ventricle infusion of 5 mug or 20 mug of PGE2 stimulated a 2- to 3-fold increase in the concentration of LHRH in hypophysial portal plasma but did not affect LHRH levels in arterial plasma. PGE2 infused into a hypophysial portal vessel at a rate of 0.167 mug/min for 30 min, or infusion of the control solution, resulted in similar changes in the concentration of FSH, LH, and prolactin in arterial plasma. The results of this investigation suggest that PGE2 stimulates the release of gonadotropins in vivo by enhancing the release of LHRH. The portal vessel infusion studies do not support a direct stimulatory effect of PGE2 on the pituitary gonadotrophs.

Corticotropin-releasing factor binding to peripheral tissue and activation of the adenylate cyclase-adenosine 3',5'-monophosphate system.

Specific binding sites for rat corticotropin-releasing factor (rCRF) are present in rat adrenal medulla, ventral prostate, spleen, liver, kidney, and testis and bovine chromaffin cells in culture. Maximal binding of [125I]rCRF occurred within 25 min at 4 C and was saturable. Scatchard analysis of rCRF binding to rat adrenal membranes and bovine chromaffin cells revealed the existence of two classes of binding sites. One class had a relatively higher apparent affinity and lower number of binding sites, whereas the other class had a relatively lower affinity and higher number of binding sites. CRF induced a dose-related increase in rat adrenal membrane adenylate cyclase activity and cAMP levels in bovine chromaffin cells. Nanomolar concentrations of rCRF maximally stimulated adenylate cyclase activity in rat adrenal membranes and maximally increased cAMP levels in bovine chromaffin cells to 86% and 130% above control values, respectively. The demonstration of specific CRF-binding sites in a variety of peripheral tissues and the finding that activation of specific CRF-binding sites in adrenal tissue stimulates the adenylate cyclase-cAMP system suggest that CRF may have an important regulatory role in various peripheral tissues.

alpha-Melanocyte-stimulating hormone-like peptides in the intermediate lobe of the rat pituitary gland: characterization of content and release in vitro.

Reverse phase high performance liquid chromatography (HPLC) followed by RIA of the chromatographic fractions was used to separate and quantify, respectively, the alpha MSH-like peptides stored in the intermediate lobe (IL) of the rat pituitary gland and released from IL cells in vitro. Immunoreactive material eluting with the same HPLC retention time as N,O-diacetyl alpha MSH accounted for approximately 80% of the total immunoreactive alpha MSH (IR-alpha MSH) in either the neurointermediate lobe or dispersed IL cells. The remainder of the IR-alpha MSH coeluted with either synthetic desacetyl alpha MSH or alpha MSH. Furthermore, the predominant alpha MSH-like compound released in vitro from dispersed IL cells eluted from the HPLC column with the same retention time as synthetic N,O-diacetyl alpha MSH. Treatment of dispersed IL cells with drugs known to enhance (l-isoproterenol or A 23187) or to inhibit (apomorphine or lisuride) the release of IR-alpha MSH revealed that N,O-diacetyl alpha MSH was the primary form released. Finally, an evaluation of the stability of the alpha MSH-like peptides indicated that N,O-diacetyl alpha MSH was readily converted to alpha MSH in the presence of 0.1 N hydrochloric acid.

Relationship between luteinizing hormone releasing hormone concentration in hypophysial portal blood and luteinizing hormone release in intact, castrated, and electrochemically-stimulated rats.

The concentration of luteinizing hormone releasing hormone (LHRH) in hypophysial portal plasma was determined in pentobarbital anesthetized,intact and castrated rats of both sexes, including proestrous rats following electrochemical stimulation of the medial preoptic area (MPOA). Mean LHRH levels in portal plasma obtained between 1400--1700 h from estrous and diestrous rats and from rats ovariectomized for 8 weeks were similar and ranged from 50--55 pg/ml, but the LHRH levels in proestrous rats were less than 12 pg/ml. In addition, hypophysial portal plasma collected during 1100 to 1400 h from animals orchidectomized for 8 weeks and from intact male rats contained mean LHRH concentrations that ranged from 50--65 pg/ml and 30--35 pg/ml, respectively. Electrochemical stimulation of the MPOA in the female rat on the afternoon of proestrus resulted in a marked increase in the concentration of LHRH in portal plasma. LHRH levels in portal plasma during the 0 to 30, 30 to 60, 60 to 90, 90 to 120, and 120 to 150-min periods after electrochemical stimulation of the MPOA were 105 +/- 24.2, 61 +/- 10.8, 51 +/- 8.2, 36 +/- 5.3, and 32 +/- 4.1 pg/ml, respectively. LHRH levels in portal plasma from the unstimulated rats were not detectable (less than 12 pg/ml) in most of the animals. In another group of proestrous rats, the effect of rabbit anti-LHRH serum or normal rabbit serum (NRS) on the release of LH after electrochemical stimulation of MPOA was examined. Pretreatment of proestrous rats with anti-LHRH serum blocked the release of LH due to MPOA stimulation, whereas pretreatment with NRS did not inhibit LH release. On the basis of these findings, it is concluded that electro-chemical stimulation of the MPOA in proestrous rats increases LHRH levels in portal blood and that the enhanced secretion of LHRH stimulates the release of LH from the pituitary gland.

Adrenergic regulation of beta-endorphin secretion from anterior pituitary in conscious rats: effects of thyroid state.

In conscious, chronically cannulated, unrestrained rats, systemic administration of catecholamines increases the plasma levels of beta-endorphin-like immunoreactivity (beta Ei). In euthyroid rats, this effect is mediated by both alpha 1 and beta-adrenergic receptors; the rise in plasma beta Ei caused by isoproterenol is blocked by 1 mg/kg propranolol, and the similar effects of norepinephrine and phenylephrine are blocked by 0.1 mg/kg prazosin. Both types of responses are completely suppressed by a 4-h pretreatment of rats with 0.1 mg/kg dexamethasone, indicating the anterior pituitary origin of the beta Ei released. Prior sectioning of the pituitary stalk does not significantly reduce the response to either phenylephrine or isoproterenol, suggesting that both agents act directly on the pituitary. Hypothyroidism induced by surgical thyroidectomy does not influence the beta Ei response to isoproterenol, which remains sensitive to block by propranolol or suppression by dexamethasone. However, neither norepinephrine nor phenylephrine is able to increase plasma beta Ei in the hypothyroid animals. Both isoproterenol and phenylephrine remain fully effective in rats made hyperthyroid by daily injections of 40 micrograms/kg T3 for 4 days. We propose that in unstressed rats catecholamines increase plasma beta Ei by a direct action on the anterior pituitary via either alpha 1- or beta-adrenergic receptors, and that expression of the alpha 1-, but not the beta-adrenergic response is regulated by thyroid hormones.

Onset of glucocorticoid responsiveness of anterior pituitary corticotrophs during development is scheduled by corticotropin-releasing factor.

Expression of the CRF gene in the hypothalamus and that of the POMC gene in the anterior pituitary are reduced during the first week of life in the rat. During this so-called stress nonresponsive period (SNRP), stimuli such as ether vapors, electroshocks, and hypoxia do not elicit ACTH secretion from the pituitary, as occurs later in development. The current hypothesis to explain the SNRP is an increased negative glucocorticoid feedback on POMC and CRF synthesis and/or release during this time. To test this hypothesis we studied the effects of adrenalectomy (ADX) on anterior pituitary POMC mRNA expression. In 7-day-old rats POMC mRNA levels were increased only 3-fold 48 h post-ADX, compared to a 7-fold increase in 14-day-old animals. This blunted effect of endogenous glucocorticoid removal on pituitary POMC mRNA could be due to decreased up-regulation of CRF after removal of glucocorticoids or normal up-regulation of CRF but decreased pituitary responsiveness to CRF relative to those in 14-day-old animals. Therefore, we studied in vitro beta-endorphin release from pituitaries obtained from 7- and 14-day-old rats. CRF stimulated basal beta-endorphin release to the same extent in pituitaries from both groups. The inhibition by corticosterone of CRF-stimulated beta-endorphin secretion was also indistinguishable in pituitaries obtained from 7- or 14-day-old rats. Since the responsiveness of the 7-day-old pituitary was normal, the blunted enhancement of POMC biosynthesis after ADX must be mediated at the level of the hypothalamus. Indeed, in situ hybridization showed that while in 14-day-old rats ADX induced a significant increase [190 +/- 10% (+/- SE) of control; n = 5; P less than 0.0005] in hypothalamic mRNA levels, ADX did not change the expression of the CRF gene in the paraventricular nucleus of 7-day-old rats, indicating a lack of glucocorticoid modulation of hypothalamic CRF synthesis. Finally, we studied the effects of 48 h CRF treatment on the post-ADX increase in POMC mRNA levels in 7-day-old rats. Daily injections of 200 ng CRF/rat induced an increase in anterior pituitary POMC mRNA concentrations [669 +/- 139% (+/- SE) of control; n = 6; P less than 0.02 vs. adrenalectomized vehicle-treated rats] comparable to that in adrenalectomized untreated 14-day-old rats. In conclusion, our data indicate that the glucocorticoid regulation of hypothalamic CRF gene expression is not mature during the first week of life, i.e. within the so-called SNRP.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of degradation and measurement of immunoreactive thyrotropin-releasing hormone in rat blood and plasma.

A radioimmunoassay (RIA) for thyrotropin-releasing hormone (TRH) is described. The cross-reactivity of the antiserum was tested with 26 analogs of TRH, 5 amino acids, and LH-releasing hormone. Use of this RIA revealed that inactivation of TRH by rat blood was prevented if the blood was frozen and thawed prior to its incubation with TRH. This procedure did not interfere with the binding of TRH to its antibody. The degradation of TRH by blood or plasma was inhibited by 2,3-dimercaptopropanol (BAL) or benzamidine, but these compounds nonspecifically inhibited the binding of [125I]TRH to anti-TRH. At 37 C, 50% of the synthetic TRH added to rat blood was degraded within seconds, whereas at 1C, 60-65% was recovered after 90 min. When blood was frozen and thawed prior to its incubation with TRH at 1C, essentially all of the hormone was recovered after a 90-min incubation period. In contrast, incubation of TRH with frozen and thawed blood at 37 C resulted in a rapid loss of TRH. BAL (10 mM) or bensamidine (100 mM) afforded complete protection for TRH for at least 30 min at 1 C. At 37 C, protection was incomplete. Exposure of rats to cold (2C) resulted in a significant increase in serum TSH levels, but TRH was undetectable (less than3 pg) in 100 mul of blood regardless of whether the blood contained BAL (10 mM) or benzamidine (100 mM), or was frozen quickly and thawed before RIA. However, when 5-8 ml of trunk blood was extracted with methanol, 8-11 pg/ml of TRH was found, and the TRH levels were slightly but significantly elevated after cold exposure.