Presynaptic alpha 2 -adrenergic stimulation leads to growth hormone release in the dog

In previous studies we have shown that the alpha 2 -adrenergic receptor agonist clonidine (CLON) releases growth hormone (GH) in conscious dogs, an effect abolished by the selective alpha 2-receptor antagonist yohimbine (YOH) and by reserpine, but not by the alpha 1-receptor antagonist prazosin (1). In the present work intravenous (iv) administration of CLON in conscious dogs evoked a dose-related rise in plasma GH at doses of 2-8 /micrograms/Kg, but not at 16 and 32 /micrograms/Kg. Acute pretreatment with the selective inhibitor of norepinephrine (NE) synthesis, DU-18288, or with a potent antagonist of presynaptic alpha 2-receptors, mianserin abolished the GH rise induced by CLON (4 /micrograms/Kg iv). In contrast, a 10-day-pretreatment with YOH greatly enhanced the GH-releasing effect of CLON (2 /micrograms/Kg iv). In all these data indicate that in the dog: 1) CLON induces GH release via activation of alpha 2-adrenergic receptors; 2) these receptors are likely located on presynaptic sites [experiments with reserpine (1), DU-18288, mianserin, dose-response curve with CLON 2-32/micrograms/kg iv]; 3) the adrenergic receptors involved in GH release exhibit supersensitivity upon (YOH-induced) chronic pharmacologic denervation. In view of the inhibitory action of presynaptic alpha 2-adrenergic receptors (autoreceptors) on NE function, it may be envisioned that in the dog noradrenergic activation is inhibitory and not stimulatory to GH release.     

The alpha2-adrenergic receptor system in the hypothalamus of the Pekin duck

Summary In the present study, we have employed the monoradioiodinated ₂-agonist clonidine ([¹²⁵I]-CLO) to characterize duck hypothalamic ₂-adrenoceptors and to localize ₂-specific binding sites in the duck brain. To validate the ₂-specificity of [¹²⁵I]-CLO using an enriched duck hypothalamic membrane fraction, a radioreceptor assay was established by altering the membrane protein concentration, time, temperature and ionic milieu of incubation, and in the presence or absence of protease inhibitors. Competitive displacement studies revealed the following sequence of potency to displace [¹²⁵I]-CLO: yohimbine>(-)-epinephrine>clonidine> (-)-norepinephrine>phentolamine>(-)-phenylephrine>(-)-isoproterenol>prazosin. The non-hydrolyzable guanosine 5-triphosphate analog guanylylimidodiphosphate markedly inhibited [¹²⁵I]-CLO binding suggestive of G-protein involvement. With regard to the histological distribution, diencephalic structures, such as the habenula and the nucleus reticularis of the thalamus, were densely labeled by [¹²⁵I]-CLO. In the hypothalamus, ₂-adrenoceptors were detected in the antidiuretic hormone-synthesizing nucleus paraventricularis, the nucleus praeopticus medialis, the nucleus anterior medialis hypothalami, the nucleus magnocellularis praeopticus, the nucleus commissurae pallii, the nucleus inferior hypothalami and the regio lateralis hypothalami. Circumventricular organs, such as the plexus choroidei, organum subfornicale, organum paraventriculare and the corpus pineale, were endowed with ₂-specific binding sites, as were the cell layers of the tectum opticum. In addition, telencephalic structures revealed high receptor densities. The presence of well characterized ₂-specific adrenoceptors in hypothalamic structures of the duck brain including associated telencephalic regions supports physiological investigations with regard to functional ₂-adrenergic modulation of antidiuretic hormone release in the duck.     

Sensory responses of neurons in the medial septal area to modulation of the theta-activity by alpha2-adrenergic receptor agonist clonidine

It was shown by us earlier that bilateral intracerebroventricular injection of alpha2-adrenoreceptor agonist clonidine produced a dose-dependent effect on theta oscillations in the septohippocampal system of awake rabbits. A relatively low dose of clonidine (0.5 microgram) attenuated and a high dose (5 micrograms) significantly enhanced the rhythmic activity. It was suggested that the effect of the low dose of clonidine is mediated by presynaptic alpha2-adrenoreceptors were as postsynaptic alpha2-adrenoreceptors. In this article sensory neuronal responses in the medial septal area (MS) were analyzed against the background of the theta activity modulation by different clonidine doses. Different effects of the low and high doses of the agonist were revealed. The low dose of clonidine (0.5 microgram in 5 microliters into each lateral ventricle) which produced a decrease in the theta activity resulted in attenuation of excitation and enhancement of inhibition, i.e., the number of activating effects significantly decreased and inhibitory responses were more frequent and distinct. The high dose of clonidine (5 micrograms in 5 microliters) which produced a sharp increase in the theta activity led to a significant decrease in the reactions of the MS cells to sensory stimuli (from 76.8% in the control to 45% under clonidine) independently on the initial reaction character. Persisted excitatory and inhibitory responses became less distinct than the initial ones except single excitatory reactions. The results suggest that alpha2-adrenoreceptors are involved in the control of the sensory reactivity of MS neurons. A sharp decrease in neuronal reactivity during stable rhythmical oscillations developing under the influence of high dose of clonidine confirm the role of the theta rhythm in the septohippocampal system as an active filter in information selection and registration.     

An affinity label for alpha 2-adrenergic receptors in rat brain

Clonidine, a potent and highly selective alpha 2-adrenergic agonist of the central nervous system, was modified. Insertion of the strong alkylating isothiocyanate group (NCS) group, at its aromatic residue, makes clonidine a potential affinity label of the alpha 2-adrenergic receptors. In displacement of [3H]clonidine and p-[3H]aminoclonidine from rat brain membrane preparations, clonidine-NCS demonstrates high affinity for the alpha 2-adrenergic receptors (Kd = 50 mM). The covalent labelling of the central alpha 2-receptors requires higher concentrations of the irreversible ligand (1-70 microM), thus indicating possible non-productive interactions at the environment of the receptor site. Only partial protection of the receptors is observed with a reversible alpha 2-agonist. The new clonidine analog appears to be a general ligand for the alpha 2-adrenergic receptors and might serve as a potential affinity probe for these receptors.     

Pretranslational regulation of alpha 2u-globulin in rat liver by growth hormone

Hypophysectomy is known to cause complete suppression of the hepatic synthesis alpha 2u-globulin. The effect of hypophysectomy on the synthesis of alpha 2u-globulin can be reversed by multiple hormone treatment. The role of pituitary growth hormone in the multihormonal regulation of alpha 2u-globulin in rat liver was examined in the hypophysectomized male rats with and without growth hormone supplementation. Daily treatment of hypophysectomized rats with 5 alpha-dihydrotestosterone, corticosterone, thyroxine, and growth hormone for 8 days caused about 80% recovery in the hepatic content of alpha 2u-globulin and its corresponding mRNA as determined by radioimmunoassay, in vitro translation, and liquid hybridization with a cloned cDNA probe. However, omission of growth hormone from the treatment regimen failed to raise hepatic alpha 2u-globulin and its mRNA to more than 5% of the normal control. The possible effect of growth hormone on the translation of the mRNA for alpha 2u-globulin was examined with cultured hepatocytes derived from growth hormone-deficient rats. Culture of these cells in the presence of growth hormone for 24 h did not turn on the synthesis of alpha 2u-globulin. These results indicate that growth hormone regulates the synthesis of alpha 2u-globulin by acting at a step antecedent to mRNA translation.     

Parallel inactivation of alpha 2-adrenergic agonist binding and Ni by alkaline treatment

The isolation of a cytochrome b₆-f complex from spinach, which is depleted of plastoquinone (and lipid), is reported. The depleted complex no longer functions as a plastoquinol-plastocyanin oxidoreductase but can be reconstituted with plastoquinone and exogenous lipids. The lipid classes digalactosyldiacylglycerol, phosphatidylglycerol and phosphatidylcholine were active in reconstitution while monogalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol were not. Neither plastoquinone nor lipid alone fully reconstitutes electron transport in the depleted complex. Saturation of plastoquinol-plastocyanin oxidoreductase activity in the depleted complex occurs at 1 plastoquinone per cytochrome f.     

Comparison of the inhibition of insulin release by activation of adenosine and alpha 2-adrenergic receptors in rat beta-cells.

Rat islets were used to compare the mechanisms whereby adenosine and adrenaline inhibit insulin release. Adenosine (1 microM-2.5 mM) and its analogue N6(-)-phenylisopropyladenosine (L-PIA) (1 nM-10 microM) caused a concentration-dependent but incomplete (45-60%) inhibition of glucose-stimulated release. L-PIA was more potent than D-PIA [the N6(+) analogue], but much less than adrenaline, which caused nearly complete inhibition (85% at 0.1 microM). 8-Phenyltheophylline prevented the inhibitory effect of L-PIA and 50 microM-adenosine, but not that of 500 microM-adenosine or of adrenaline. In contrast, yohimbine selectively prevented the inhibition by adrenaline. Adenosine and L-PIA thus appear to exert their effects by activating membrane A1 receptors, whereas adrenaline acts on alpha 2-adrenergic receptors. Adenosine, L-PIA and adrenaline slightly inhibited 45Ca2+ efflux, 86Rb+ efflux and 45Ca2+ influx in glucose-stimulated islets. The inhibition of insulin release by adenosine or L-PIA was totally prevented by dibutyryl cyclic AMP, but was only attenuated when adenylate cyclase was activated by forskolin or when protein kinase C was stimulated by a phorbol ester. Adrenaline, on the other hand, inhibited release under these conditions. It is concluded that inhibition of adenylate cyclase, rather than direct changes in membrane K+ and Ca2+ permeabilities, underlies the inhibition of insulin release induced by activation of A1-receptors. The more complete inhibition mediated by alpha 2-adrenergic receptors appears to result from a second mechanism not triggered by adenosine.     

Effects of the alpha 1-adrenergic agonist cirazoline on locomotion and brown adipose tissue thermogenesis in the rat.

Anorexia is induced by injection of alpha 1-adrenergic receptor agonists into the hypothalamic paraventricular nucleus (PVN) in rats. Of the agonists tested to date, cirazoline is the most potent when administered either into the PVN or systemically. The present experiments assess the effects of systemically administered cirazoline, at doses that suppress food intake, on dopamine and norepinephrine systems as evident in locomotion and stereotypy and in the induction of brown adipose tissue (BAT) thermogenesis. In Experiment 1, adult male rats were treated with either vehicle (0) or 0.05, 0.1, 0.2 or 0.4 mg/kg cirazoline (IP) prior to 30 minutes assessment of horizontal and vertical locomotion and stereotypy in Omnitech activity chambers. Horizontal activity and stereotypy were significantly suppressed at 0.05 mg/kg cirazoline but these effects waned at higher cirazoline doses. In Experiment 2, interscapular BAT temperature in adult male rats was monitored for 30 minutes after injection (IP) of either vehicle or 0.4 mg/kg cirazoline. Cirazoline, at 0.4 mg/kg did not influence BAT temperature whereas a positive control treatment (phenylpropanolamine: 40 mg/kg) rapidly increased BAT temperature during a 15 minute period after injection. These results suggest that cirazoline-induced anorexia is not the result of competing motor responses and that this drug, at a dose that produces maximal suppression of feeding, does not alter BAT thermogenesis.     

Interactions between an alpha2-adrenergic antagonist and a beta3-adrenergic agonist on the expression of UCP2 and UCP3 in rats

This experimental trial was devised to assess whether selective beta3-adrenergic receptor (AR) stimulation and simultaneous blockade of alpha2-AR would affect thermoregulation. With this purpose, the individual and combined administration of a beta-AR agonist, trecadrine, and an alpha2-AR antagonist, yohimbine, were evaluated. Yohimbine produced a marked decrease (p < 0.001) in body temperature one hour after administration (5 mg kg(-1), i.p.) and blocked the thermogenic effect of trecadrine (1 mg kg(-1), i.p.) when simultaneously administered. Uncoupling protein-2 expression in skeletal muscle was downregulated (p < 0.05) by trecadrine, while yohimbine had no effect. White adipose tissue UCP2 and muscle UCP3 were not modified by either trecadrine or yohimbine administration. Liver UCP2 mRNA expression was significantly decreased by yohimbine (p < 0.05). However, this downregulation does not seem to explain the reduction in temperature produced by yohimbine given the fact that trecadrine produced a similar downregulation of hepatic UCP2 (p < 0.05). The present work indicates that alpha2-AR antagonism blocks the thermogenic effects mediated by beta3-AR stimulation, contrary to our expectations, suggesting a possible interplay between both mechanisms. Moreover, these effects are not apparently explained by changes in UCP2 and UCP3.     

Evidence of functional alpha 2-adrenergic receptors in adult-rat adipocytes by using the agonist UK 14304.

The aim of this study was to re-assess whether alpha 2-adrenergic receptors were present in rat adipocytes, by using UK 14304, a new and very selective alpha 2-agonist. The following observations demonstrate the presence of functional alpha 2-adrenoceptors in rat adipocytes. (1) Adipocyte lipolysis was dose-dependently inhibited by UK 14304 (maximal effect 80% at 1 microM-UK 14304, 45% at 10 microM-UK 14304, under basal or theophylline-stimulated conditions respectively). (2) UK 14304 bound specifically to purified plasma membranes, with Bmax. = 744 fmol/mg of protein and KD = 9 nM. (3) The effect of UK 14304 was suppressed by alpha 2-antagonists. (4) Adrenaline inhibited lipolysis upon beta-adrenergic blockade (propranolol). (5) The anti-lipolytic effect of UK 14304 was modulated by the age of the rats.     

Processing of the luteinizing hormone-releasing hormone precursor in the preoptic area and hypothalamus of the rat

The LHRH precursor is known to contain the decapeptide and a 56 amino acid peptide termed gonadotropin-releasing hormone-associated peptide (GAP). The purpose of our study was to characterize the proLHRH and its processed products from the cell body and fiber region and from the nerve terminal region of LHRH neurons. The median eminence (ME) and a tissue block containing the preoptic area and hypothalamus (POH) were dissected separately. Tissues were homogenized and peptides were separated according to mol wt. Three different LHRH antisera bound to one immunoreactive (IR) substance which eluted at approximately 1200 mol wt. Subsequently, this material coeluted with synthetic LHRH on a reversed-phase column as a single peak. There was approximately 1.6-fold more LHRH-like IR in the ME than in the POH. The four different GAP antisera recognized multiple mol wt forms of GAP-like IR at approximately 16,000 to 14,000, 8,200, 6,500, 3,500, and 2,800 mol wt. There were more of the high mol wt materials and less of the 6500 and lower mol wt materials in the POH than in the ME. The most abundant species in both regions was the 6500 mol wt form. This IR substance coeluted with synthetic rat GAP1-56 on a reversed-phase column as a single peak. These experiments demonstrate 1) that multiple IR forms of the LHRH prohormone exist in the POH of the rat and 2) that nerve terminals of the LHRH neurons contain LHRH, GAP1-56, and some lower mol wt GAP-like substances. These results provide the first information concerning the processing scheme for the LHRH prohormone in the rat brain.     

Central alpha 2-adrenergic receptors in the rat: effect of sodium in vitro

Characteristics of the (3H) Rauwolscine binding were studied in homogenates of rat cerebral cortex, particularly in presence of NaCl. We had shown that (3H) Rauwolscine specifically bind the alpha 2-adrenoceptors. The Scatchard plot analysis had demonstrated 2 sites: a high affinity site (B 41,06 +/- 5,21 KD = 1,31 +/- 0,16) and a low affinity site (B = 203,41 +/- 13,37 KD = 11,62 +/- 0,55 nM). Incubation of cerebral cortex homogenates with NaCl 150 mM induced an increase (53,97 p less than 0.001) of the high affinity site density. These results indicate that (3H) Rauwolscine is a useful alpha 2-adrenoceptor ligand to study in animal models various central nervous system abnormalities or diseases induced by ion disorders.     

Localization of dopamine in the endocrine hypothalamus of the rat

Summary Microspectrofluorometry, fluorescence histochemistry and light and electron microscopic autoradiography have established the presence of sub-populations of neurons in the arcuate-periventricular region of the rat hypothalamus that sequester both radiolabeled dopamine and demonstrate formaldehyde-induced fluorescence. These characteristics are consistent with a catecholaminergic function. Selective sequestration of ³H-dopamine at the light and ultrastructural level is discussed in the context of an ultrashort loop autoregulatory mechanism for this neuronal population.Supported by USPHS Program Project Grants NS-11642, RR-05403USPHS Career Development Awardee K04 GM 70001.     

Localization of growth hormone receptors in rat and human thyroid cells

Growth hormone (GH) exerts its multiple actions by binding to a specific receptor (GHR) widely distributed in the organism. It is well established that, in acromegaly, the thyroid gland is larger than normal and that GH increases triiodothyronin concentrations and decreases those of tetraiodothyronin (thyroxine). The aim of the present study was to analyze the presence of GHR and its mRNA in rat and human thyroid gland by Western blot, in situ hybridization techniques, and immunohistochemistry. A band of the expected size for GHR was shown in rat and human thyroid by Western blot. GHR immunoreactivity was found in virtually all follicles. The signal was mainly localized in the cytoplasm, although a nuclear positivity was also found. In situ hybridization techniques demonstrated the presence of GHR messenger RNA in the thyroid gland (cytoplasm of the follicular cells). These results provide direct morphological evidence that GHR is localized in the thyroid gland of mammals and opens up the possibility that GH regulates thyroid cell function directly or via local autocrine or paracrine production of insulin-like growth factor I.     

Ethanol inhibits the naloxone-induced release of luteinizing hormone-releasing hormone from the hypothalamus of the male rat

It has been inferred that ethanol suppresses the secretion of luteinizing hormone (LH) in the male by depressing the release of LH-releasing hormone (LH-RH) from the hypothalamus. Direct support for this inference has been difficult to obtain, however, because of significant technical difficulties in measuring LH-RH release under in vivo conditions. To circumvent these problems, we made use of the opiate antagonist naloxone, as a neuroendocrine probe, to elicit the release of LH-RH under in vivo conditions. We found that ethanol was a potent suppressor of the increase in serum LH levels evoked by naloxone at extremely low blood ethanol concentrations ( less than 60 mg/dl). Furthermore, we observed that the antagonism between ethanol and naloxone appeared to be competitive in nature since a fixed dose of ethanol (1 g/kg, blood ethanol concentration 60 mg/dl) shifted the naloxone dose-response curve significantly to the right and high doses of the antagonist overcame ethanol's effects. Finally, we found that the interaction between ethanol and naloxone took place at the level of the hypothalamus. Our results, therefore, seem to provide the first in vivo evidence supporting the widely-held hypothesis that ethanol reduces serum LH levels by depressing the hypothalamically-medicated release of LH-RH. The mechanisms underlying ethanol's depression of naloxone-induced increases in the release of LH-RH are not fully understood at this time, but one prominent possibility is that ethanol enhances the synthesis or release of endogenous opioids which in turn override naloxone's effects.     

Modulation of lipoprotein lipase activity in the rat by the beta 2-adrenergic agonist clenbuterol.

This study evaluated the effects of beta 2-adrenoceptor stimulation on some determinants of triglyceride metabolism. Male Sprague-Dawley rats were injected twice daily with clenbuterol (30 micrograms.kg-1) for 7 days, or with an equivalent volume of vehicle. Serum triglycerides, hepatic triglyceride secretion rate, and lipoprotein lipase activity in white and brown adipose tissues as well as in red vastus lateralis muscle and heart were evaluated in the fasting state and following a fat-free, high-sucrose meal, 3 h after the last agonist injection. In rats killed in the fasting and postprandial states, clenbuterol reduced the mass of white adipose tissue (-25 and -12%, respectively; p < 0.02), whereas it increased the mass of vastus lateralis muscle (+11 and +7%; p < 0.002) and heart (+13 and %; p < 0.0001). In vehicle-injected animals, the fasting state was associated with lower lipoprotein lipase activity in white and brown adipose tissues, and higher enzyme activity in vastus lateralis and heart, compared with the postprandial state. Postprandially, treatment with clenbuterol reduced lipoprotein lipase activity in white adipose (-24%), whereas it increased enzyme activity in brown adipose (+107%) as well as in vastus lateralis (+35%). In fasted animals, no significant variation of enzyme activity in these tissues was observed following clenbuterol treatment, whereas in the heart, a decrease of lipoprotein lipase activity was observed (-22%). Clenbuterol lowered serum triglycerides significantly (-23%), but not their rate of secretion, whereas the agonist decreased the insulin to glucagon ratio only in the postprandial state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of growth hormone-release-inhibiting hormone (somatostatin) in the rat brain.

Utilizing an immunoperoxidase technique at the light microscope level, growth hormone-release-inhibiting hormone (somatostatin) was localized in the external zone of the median eminence, the subcommissural organ, the organum vasculosum of the lamina terminalis and the pineal gland. No positive reaction was detected in any other brain area.