Dopamine D4 receptor, but not the ADHD-associated D4.7 variant, forms functional heteromers with the dopamine D2S receptor in the brain.

Polymorphic variants of the dopamine D(4) receptor have been consistently associated with attention-deficit hyperactivity disorder (ADHD). However, the functional significance of the risk polymorphism (variable number of tandem repeats in exon 3) is still unclear. Here, we show that whereas the most frequent 4-repeat (D(4.4)) and the 2-repeat (D(4.2)) variants form functional heteromers with the short isoform of the dopamine D(2) receptor (D(2S)), the 7-repeat risk allele (D(4.7)) does not. D(2) receptor activation in the D(2S)-D(4) receptor heteromer potentiates D(4) receptor-mediated MAPK signaling in transfected cells and in the striatum, which did not occur in cells expressing D(4.7) or in the striatum of knockin mutant mice carrying the 7 repeats of the human D(4.7) in the third intracellular loop of the D(4) receptor. In the striatum, D(4) receptors are localized in corticostriatal glutamatergic terminals, where they selectively modulate glutamatergic neurotransmission by interacting with D(2S) receptors. This interaction shows the same qualitative characteristics than the D(2S)-D(4) receptor heteromer-mediated mitogen-activated protein kinase (MAPK) signaling and D(2S) receptor activation potentiates D(4) receptor-mediated inhibition of striatal glutamate release. It is therefore postulated that dysfunctional D(2S)-D(4.7) heteromers may impair presynaptic dopaminergic control of corticostriatal glutamatergic neurotransmission and explain functional deficits associated with ADHD.

The dynamics of signaling at the histaminergic photoreceptor synapse of arthropods.

Histamine, a ubiquitous aminergic messenger throughout the body, also serves as a neurotransmitter in both vertebrates and invertebrates. In particular, the photoreceptors of adult arthropods use histamine, modulating its release to signal increases and decreases in light intensity. Strong evidence from various arthropod species indicates that histamine is synthesized and stored in photoreceptors, undergoes Ca-dependent release, inhibits postsynaptic interneurons by gating Cl channels, and is then recycled. In Drosophila, the synthetic enzyme, histidine decarboxylase, and the subunits of the histamine-gated chloride channel have been cloned. Possible histamine transporters at synaptic vesicles and for reuptake remain elusive. Indeed, the mechanisms that remove histamine from the synaptic cleft, and that help terminate histamine's action, are unexpectedly complex, their details remaining unresolved. A major pathway in Drosophila, and possibly other arthropod species, is by conjugation of histamine to beta-alanine to form carcinine in adjacent glia. This conjugate then returns to the photoreceptors where it is hydrolysed to liberate histamine, which is then loaded into synaptic vesicles. Evidence from other species suggests that direct reuptake of histamine into the photoreceptors may also occur. Light depolarizes the photoreceptors, causing histamine release and postsynaptic inhibition; dimming hyperpolarizes the photoreceptors, causing a decrease in histamine release and an "off" response in the postsynaptic cell. Further pursuit of histamine's action at these highly specialized synapses should lead to an understanding of how they signal minute changes in presynaptic membrane potential, how they reliably extract signals from noise, and how they adapt to a wide range of presynaptic membrane potentials.

Mitochondria are redistributed in Drosophila photoreceptors lacking milton, a kinesin-associated protein.

Photoreceptors are richly supplied with mitochondria, where they are required to meet the energetic demands, in the soma, of phototransduction and, in the terminal, of neurotransmitter release. Compromising the latter, we have made photoreceptors R1-R6 in Drosophila ommatidia homozygous for either of two alleles, milt(186) and milt(92), of milton in whole-eye mosaics. Such mutant photoreceptors fail to target mitochondria to their terminals. We show from quantitative electron microscopy (EM) that mitochondria are totally lacking at the terminal but nevertheless abundant and present throughout the soma, where their distribution differs from that of control ommatidia, however, being more heavily concentrated in the nuclear region. Mitochondria are sparse at the basalmost level of mutant ommatidia, and are lacking beneath the basement membrane, in the axons and terminals of these cells. The absence of mitochondria from R1-R6 terminals and concommitant reductions in synaptic vesicle packing density, previously reported, we show here are accompanied by reduced immunoreactivity to the photoreceptor transmitter histamine but not by any change in total head histamine content, as determined by high-performance liquid chromatography. Mutant terminals also contain vesicle profiles with a wider range of sizes. These two phenotypes suggest that the reduced availability of ATP when mutant terminals lack a mitochondrial supply compromises their ability to pump histamine into synaptic vesicles and perturbs membrane distribution within the terminal. In addition, a band of somata in the lamina cortex, at least some of which are postsynaptic neurons not homozygous for milton, also shows altered mitochondrial targeting, with abnormal clusters of mitochondria, as visualized by immunolabeling with anti-hsp and by serial EM. Within the lamina, terminals of mutant photoreceptors are penetrated by neighboring cells with invaginations that frequently contain mitochondria, suggesting that a mechanism exists for intercellular metabolic support. Our findings indicate the direct and compensatory responses in a population of neurons when mitochondria are not correctly targeted to their synaptic terminals.

On the bioactive conformation of NAN-190 (1) and MP3022 (2), 5-HT(1A) receptor antagonists.

Structural modifications of 1, a postsynaptic 5-HT(1A) receptor antagonist, provided its flexible (8, 12) and rigid (7, 9, 11, 13) analogues. Compounds 7, 8, 9, and 11 showed high 5-HT(1A) receptor affinity (K(i) = 4-72 nM). They acted as 5-HT(1A) postsynaptic receptor antagonists, since, like 1, they inhibited the behavioral syndrome, i.e., flat body posture (FBP) and forepaw treading (FT), in reserpine-pretreated rats as well as the lower lip retraction (LLR) in rats, both induced by 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), a 5-HT(1A) receptor agonist. Compound 12, which demonstrated high 5-HT(1A) receptor affinity (K(i) = 50 nM), revealed properties of a partial 5-HT(1A) receptor agonist: it induced LLR and, at the same time, inhibited FT in rats. Compound 13 (K(i) = 1600 nM) was not tested in a behavioral study. Restriction of the conformational freedom in 2, a full 5-HT(1A) receptor antagonist, yielded compound 14 with high 5-HT(1A) receptor affinity (K(i) = 47 nM) and partial agonist properties at postsynaptic 5-HT(1A) receptors in the above tests in vivo; i.e., it induced LLR and inhibited FBP and FT in rats. New constrained analogues of 1 and 2 (compounds 7 and 14, respectively) were also synthesized to recognize a bioactive conformation of those 5-HT(1A) receptor antagonists. On the basis of in vitro and in vivo investigations, binding and functional properties of compound 7 were found to reflect those of 1 at 5-HT(1A) receptors. On the other hand, compound 14, a rigid analogue of 2, showed a different activity in vivo in comparison with the parent compound. PM3 and MM calculations revealed the existence of three low-energy conformers of 7 and six of 14, all of them belonging to the extended family of conformations. The optimized structures of both analogues had a different angle between aromatic planes of terminal fragments; moreover, the heteroaromatic system of those molecules occupied various space regions. Our present study provides support to the hypothesis that the bioactive conformation of 1, responsible for its postsynaptic 5-HT(1A) receptor antagonism, is an extended linear structure represented by 7.

The determination of histamine in the Drosophila head.

Histamine is a neurotransmitter at arthropod photoreceptors. Even though the fruit fly, Drosophila melanogaster, is a widely used model in neuroscience research, the histamine content of its nervous system has not so far been reported. We have developed a high performance liquid chromatography (HPLC) method with pre-column o-phtaldialdehyde-mercaptoethanol (OPA-ME) derivatization and electrochemical detection, to determine this amine in Drosophila. The histamine content of the fly's head averages about 2.0 ng per head. In heads of the mutant hdc(JK910), a presumed null for the gene encoding the enzyme that synthesizes histamine, histamine was not detected in measurable amounts. In heads of the mutant sine oculis, which lacks compound eyes, only 28% of this amine was found compared with wild type flies, so histamine is mainly present in the compound eye photoreceptors. Also observed in histamine-deficient mutants was a decrease in the peak which contains a substance having the same retention time as carcinine (beta-alanyl-histamine). Our method was not able to detect compounds previously reported as histamine metabolites in insects. In spite of this, the method we have developed enables the fast and accurate measurement of histamine in the heads of Drosophila, suitable for screening mutants.

Social stress inhibits the nitric oxide effect on the corticotropin-releasing hormone- but not vasopressin-induced pituitary-adrenocortical responsiveness.

Putative involvement of endogenous nitric oxide (NO) in the corticotropin-releasing hormone (CRH, 1 microg/kg i.p.)- and vasopressin (AVP, 5 microg/kg i.p.)-induced ACTH and corticosterone secretion was investigated in both non-stressed and crowded rats. The NO synthase blocker Nomega-nitro-l-arginine (l-NNA, 2 mg/kg i.p. ) significantly augmented the AVP-induced ACTH and corticosterone secretion in control and stressed rats, but it increased the CRH-induced ACTH response only in control rats. Crowding stress did not affect the l-NNA evoked increase in AVP-induced hormone responses, but it abolished the CRH-induced ACTH response.

Crowding stress impairs the pituitary-adrenocortical responsiveness to the vasopressin but not corticotropin-releasing hormone stimulation.

To evaluate the effect of social crowding stress on the CRH and vasopressin-induced hypothalamic-pituitary-adrenocortical (HPA) response, both those neuropeptides were administered intracerebroventricularly and intraperitoneally to rats crowded for 3 days. Crowding stress did not affect the corticosterone response to CRH given by either route (1 micrograms i.c.v. or 2 micrograms/kg i.p.) but totally abolished or considerably diminished the response to vasopressin given i.p. (5 micrograms/kg) or i.c.v. (5 micrograms), respectively. Social crowding stress considerably impairs central vasopressin but does not change the CRH-system involved in the HPA stimulation.

Effect of indomethacin on the pituitary-adrenocortical response to adrenergic stimulation.

The role of prostaglandins (PGs) in stimulation of the hypothalamic-pituitary-adrenal (HPA) axis by adrenergic agonists and catecholamines was investigated in nonanesthetized rats. The cyclooxygenase and PGs synthesis inhibitor indomethacin was given systemically or intracerebroventricularly (icv) 15 min prior to phenylephrine (30 micrograms), clonidine (10 micrograms), and isoproterenol (20 micrograms), an alpha 1-, alpha 2-, and beta-adrenergic receptor agonists, respectively, or noradrenaline (10 micrograms) and adrenaline (10 micrograms). Indomethacin given ip (2 mg/kg) or icv (10 micrograms) almost abolished the increase in corticosterone secretion elicited by phenylephrine, considerably reduced the response to clonidine but did not markedly affect the response to isoproterenol. Pretreatment with indomethacin by either route strongly suppressed the corticosterone response to noradrenaline, but did not substantially affect the hormonal response to adrenaline. The above data indicate that prostaglandins considerably mediate the HPA axis response to central stimulation of alpha 1- and alpha 2-, but not beta-adrenergic receptors. They also point to significant involvement of prostaglandins in the noradrenaline-, but not adrenaline-induced HPA axis predominantly via alpha 1-and alpha 2-adrenergic receptors, whereas adrenaline exerts stimulation manly via beta-adrenergic receptors.

Effect of indomethacin on the CRH- and VP-induced pituitary-adrenocortical response during social stress.

The role of prostaglandins (PGs) on the corticotropin-releasing hormone (CRH)- and vasopressin (AVP)-induced pituitary-adrenocortical response under basal and social stress circumstances was investigated. Crowding stress applied for 3 days did not diminish the CRH-elicited corticosterone response, but it considerably reduced such a response to AVP. In control rats systemic or icv pretreatment with indomethacin, an inhibitor of PGs synthesis, did not affect the corticosterone response to ip or icv CRH administered 15 min later. By contrast, ip or icv pretreatment with indomethacin considerably reduced the corticosterone response to AVP given by either route in control rats. Similarly, ip pretreatment with indomethacin further reduced the corticosterone response to AVP already diminished by crowding stress. These results indicate that hypothalamic and anterior pituitary PGs are not involved in the CRH-elicited pituitary-adrenocortical response, but they significantly mediate this response to AVP under both basal and social stress circumstances.

Effect of gamma-aminobutyric acid and muscimol on corticosterone secretion in rats.

The effect of gamma-aminobutyric acid-receptor agonists, GABA and muscimol on the pituitary-adrenocortical activity, measured indirectly through corticosterone secretion, and the receptors involved were investigated in conscious rats. GABA given ip induced a dual effect, in lower dose (10 mg/kg) it significantly decreased the resting serum corticosterone levels while in higher doses (100-500 mg/kg) it considerably raised that level. Muscimol (0.5 mg/kg ip) also increased the corticosterone concentration. Both GABA and muscimol given intracerebroventricularly (icv) induced a significant, dose-related increase in serum corticosterone levels. Bicuculline, a GABAA-receptor antagonist, totally abolished the corticosterone response to GABA but did not influence the response to muscimol. Pretreatment with atropine did not affect the corticosterone response to GABA but significantly diminished the response to muscimol. These results suggest that GABA moderately inhibits the pituitary-adrenal axis at the pituitary level but significantly stimulates it at the hypothalamic level. The stimulatory effect of GABA, but not muscimol, is mediated by hypothalamic GABAA-receptors, and in the effect of muscimol hypothalamic cholinergic, muscarinic receptors are involved to a significant extent.

Social crowding stress diminishes the pituitary-adrenocortical and hypothalamic histamine response to adrenergic stimulation.

Social stress of crowding almost totally reduced the rise in serum corticosterone elicited by intracerebroventricular administration of isoprenaline, a beta-adrenergic receptor agonist, after 3 and 7 day of crowding and substantially diminished that response after 14 and 21 days. Crowding stress totally abolished the increase in hypothalamic histamine induced by isoprenaline in control rats. Crowding also significantly diminished the increase in serum corticosterone evoked by clonidine, an alpha 2-adrenergic agonist, and abolished the clonidine-induced elevation in hypothalamic histamine levels. The stimulatory effect of phenylephrine, an alpha 1-adrenergic agonist, on corticosterone secretion was only moderately diminished in crowded rats. Neither phenylephrine nor crowding stress changed significantly the hypothalamic histamine levels. These results indicate that social stress of crowding considerably impairs the hypothalamic-pituitary-adrenocortical responsiveness to central beta- and alpha 2-adrenergic receptor stimulation. Crowding also abolishes the rise in hypothalamic histamine induced by beta- and alpha 2-adrenergic agonist, suggesting a role of hypothalamic histamine in the HPA adaptation to the social stress of crowding.

Involvement of the central noradrenergic system in cholinergic stimulation of the pituitary-adrenal response.

Involvement of the central adrenergic system in stimulation of the hypothalamic-pituitary-adrenal (HPA) axis by carbachol, a cholinergic muscarinic agonist, was assessed indirectly through corticosterone secretion. Carbachol (2 micrograms) given intracerebroventricularly or intraperitoneally evoked a dose-related increase in serum corticosterone levels. On a molar basis, carbachol given i.c.v. was considerably more active than when injected i.p., indicating its central site of action. The corticosterone response to i.c.v. carbachol was significantly reduced by pretreatment of rats 15 min earlier with prazosin, an alpha 1-adrenergic receptor antagonist. Pretreatment with yohimbine, an alpha 2-adrenergic antagonists, did not significantly affect the carbachol-induced corticosterone response. Propranolol, a beta-adrenergic blocker, given i.c.v. or i.p. significantly impaired the carbachol-elicited corticosterone secretion. The selective noradrenergic neurotoxin DSP-4 (50 mg/kg) given i.p. 8 days before the experiment, also potently diminished the carbachol-induced rise in serum corticosterone levels. Carbachol markedly increased, while DSP-4 significantly diminished the hypothalamic noradrenaline levels. Likewise, DSP-4 significantly impaired the carbachol-induced rise in hypothalamic noradrenaline levels. Our present results indicate that the central adrenergic system is involved in the cholinergic muscarinic stimulation of the pituitary-adrenocortical response. Both hypothalamic noradrenaline and adrenergic alpha 1- and beta-receptors are significantly involved in the carbachol-induced HPA response.

Role of nitric oxide in the vasopressin-induced corticosterone secretion in rats.

The presence of nitric oxide synthase (NOS) in hypothalamic structures which control the activity of the hypothalamic-pituitary-adrenal (HPA) axis suggests a role for NO in regulation of ACTH and corticosterone secretion. We investigated the involvement of NO in the corticosterone secretion induced by vasopressin (AVP), a potent coregulator of the HPA activity. AVP injected i.p. was, on a molar basis, considerably more potent than administered intracerebroventricularly in inducing corticosterone secretion. This finding suggests a preferential action of AVP on pituitary corticotrop receptors, but not on central structures involved in stimulation of the HPA axis. Dexamethasone given before AVP totally abolished the AVP-elicited corticosterone response by a feedback mechanism and/or inhibition of the phospholipase A2 activity and prostaglandin synthesis. Pretreatment with the NOS inhibitors L-NAME and L-NNA augmented significantly and to a similar extent the corticosterone response to AVP administered both systemically and centrally and L-NNA was found to be more potent in this respect. Pretreatment with L-arginine markedly reduced the AVP-induced corticosterone response. These results suggest that endogenous nitric oxide is significantly involved in the AVP-elicited corticosterone secretion and NO-induced alterations in the prostaglandin synthesis may participate in this action.

Central histaminergic mechanisms in the corticosterone response to clonidine.

Involvement of central histaminergic mechanisms in stimulation of the hypothalamic-pituitary-adrenal (HPA) axis by clonidine was investigated in conscious rats. Clonidine as well as adrenergic and histamine receptor antagonists were administered intracerebroventricularly (icv), the antagonists always 15 min prior to clonidine, and 1 h later the trunk blood was collected for corticosterone determination. alpha-Fluoromethylhistidine (alpha-FMH), a neuronal histamine synthesis inhibitor, was given ip 2 h before clonidine. Immediately after decapitation, brains were exposed and hypothalami were isolated on ice and frozen for further spectrofluorimetric histamine determination. The clonidine-induced increase in the serum corticosterone level was considerably, but not totally, reduced by icv or ip pretreatment with yohimbine, an alpha 2-adrenergic receptor antagonist. The rise in the corticosterone level induced by clonidine was significantly diminished by mepyramine, a histamine H1-receptor antagonist, and moderately lowered by cimetidine, a histamine H2-receptor antagonist. Clonidine significantly augmented the histamine content in the hypothalamus and rest of the brain. The clonidine-induced increase in hypothalamic histamine might be the cause of an increased corticosterone secretion via stimulation of central H1-histamine receptors. On the other hand, alpha-FMH injected 2 h before clonidine considerably diminished both the histamine content in the hypothalamus and the corticosterone secretion induced by clonidine. These results indicate that clonidine given centrally stimulates the HPA activity via not only alpha-adrenergic but also histaminergic mechanisms. Clonidine augments the hypothalamic histamine which, in turn, stimulates the corticosterone secretion, predominantly via histamine H1-receptors. Neuronal histamine is considerably involved in the stimulatory action of clonidine since inhibition of the neuronal histamine synthesis by alpha-FMH significantly depresses the corticosterone response to clonidine.

Central histaminergic mechanisms mediate the vasopressin-induced pituitary adrenocortical stimulation.

Involvement of histamine receptors and hypothalamic and hippocampal histamine in stimulation of the hypothalamic-pituitary-adrenal (HPA) axis by vasopressin (AVP) was investigated in conscious rats. The HPA activity was assessed by measuring serum corticosterone levels. One hour after administration AVP, (5 micrograms/kg) given i.p. significantly raised the serum corticosterone and hippocampal histamine levels, while the hypothalamic histamine content was not affected. Pretreatment with the inhibitor of the brain histamine synthesis alpha-fluoromethylhistidine (alpha-FMH) (50 mg/kg i.p.) considerably reduced both the AVP-elicited serum corticosterone response and the hypothalamic and hippocampal histamine levels. The histamine H1- and H2-receptor-antagonists mepyramine (0.01 mg/kg) and ranitidine (0.1 mg/kg), given ip 15 min prior to AVP, significantly impaired the AVP-induced rise in the serum corticosterone level and totally abolished the AVP-elicited increase in the histamine content in the hippocampus; moreover mepyramine significantly lowered this content in hypothalamus. Pretreatment with the histamine H3-receptor antagonist thioperamide (5 mg/kg i.p.) also significantly decreased the AVP-elicited corticosterone response, but did not alter the histamine content in either brain structure examined. These results indicate that central histamine H1-, H20 and H3-receptors significantly mediate the stimulatory action of AVP on the pituitary-adrenocortical axis. Hippocampal histamine may be involved in mediation of the AVP-induced effect via H1- and H2-receptors. The inhibitory effect of thioperamide seems to be located directly at non H3-intracellular sites of the pituitary-adrenocortical axis.

Effect of indomethacin on nicotine-induced ACTH and corticosterone response.

The effect of nicotine on ACTH and corticosterone secretion and possible mediation of prostaglandins in this secretion was investigated in conscious rats. Nicotine (5 and 10 mg/kg i.p.) considerably increased the plasma ACTH and corticosterone levels, measured 1h after injection. Mecamylamine (10 and 50 micrograms i.c.v.), a nicotinic receptor antagonist, given 15 min prior to nicotine dose-dependently diminished the ACTH and corticosterone responses, by 59 and 30% respectively. Pretreatment with hexamethonium (2 mg/kg i.p.), a peripheral blocker of nicotinic receptors, diminished to a similar extent the nicotine-induced ACTH and corticosterone responses. On the other hand atropine, a muscarinic receptor antagonist, did not markedly alter those responses. Systemic or intracerebroventricular pretreatment with indomethacin (2 mg/kg i.p. or 0.1 and 1 microgram i.c.v.), a cyclooxygenase and endogenous prostaglandin synthesis blocker considerably reduced, by 58%, the nicotine-induced ACTH response, but did not alter the corticosterone response. These results show that nicotine given systemically stimulates ACTH and corticosterone secretion by selective activation of central and peripheral acetylcholine nicotinic receptors. Endogenous prostaglandins are significantly involved in the nicotine-induced central stimulation of ACTH secretion. Prostaglandins do not directly affect the nicotine-induced corticosterone secretion from the adrenal cortex.

Catecholaminergic regulation of the hypothalamic-pituitary-adrenocortical activity.

The significance and site of adrenergic receptors involved in the control of the hypothalamic-pituitary-adrenal axis (HPA) activity was assessed indirectly by estimation of serum corticosterone levels 1 h after drug administration to conscious rats. Adrenergic drugs were given intracerebroventricularly (icv) and intraperitoneally (ip), the antagonists 15 min prior to the agonists. Noradrenaline, adrenalin and isoproterenol given by either route increased dose-dependently the serum corticosterone levels. The corticosterone response to icv noradrenaline was almost abolished by icv pretreatment with propranolol, a beta-adrenergic antagonist, and yohimbine, and alpha 2-receptor blocker, and was also considerably reduced by prazosin, an alpha 1-adrenergic antagonist. When given ip, these antagonists did not significantly influence the noradrenaline induced corticosterone response, which suggests a suprapituitary site of action of noradrenaline in stimulation of the HPA. The corticosterone response to icv adrenalin was suppressed by prazosin given by either route. The corticosterone response to ip adrenalin was almost abolished by pretreatment with yohimbine, and also significantly diminished by propranolol given by the same route. The increase in corticosterone secretion, induced by isoproterenol given by either route, was abolished by ip injection of propranolol. These results indicate that noradrenaline stimulates the HPA via alpha- and beta-adrenergic receptors, mainly at the suprapituitary level. Adrenalin increases that activity both via central and pituitary alpha- and beta-adrenoceptors. Isoproterenol activates the HPA by stimulation of pituitary beta-receptors.

Effect of L-NAME, a specific nitric oxide synthase inhibitor, on corticotropin-releasing hormone-elicited ACTH and corticosterone secretion.

This study was designed to determine the role of endogenous nitric oxide (NO) in the corticotropin-releasing hormone (CRH)-induced ACTH and corticosterone secretion, as well as possible involvement of hypothalamic dopamine and noradrenaline in that secretion in conscious rats. CRH given i.p. stimulated dose-dependently the pituitary-adrenocortical activity measured 1 h later. Dexamethasone (0.2 mg/kg i.p.) injected 1 h before CRH (1 microg/kg i.p.) totally abolished the CRH-elicited ACTH and corticosterone secretion, indicating a predominantly pituitary site of CRH-evoked stimulation. L-arginine (120 mg/kg i.p.) and N(omega)-nitro-L-arginine methyl ester (L-NAME 5-10 mg/kg i.p.) did not markedly affect the basal plasma ACTH and corticosterone levels. L-NAME given 15 min before CRH markedly, but not significantly, augmented the CRH-induced ACTH response, and enhanced more potently and significantly the corticosterone response. Pretreatment with L-arginine, a substrate for NOS, slightly diminished the CRH-induced ACTH response and considerably reduced the corticosterone response. L-arginine also significantly reversed the L-NAME-evoked increase in the CRH-induced ACTH and corticosterone secretion. L-NAME did not markedly alter the CRH-induced hypothalamic dopamine and noradrenaline levels, while L-arginine significantly increased noradrenaline level. However, those alterations were not directly correlated with the observed changes in ACTH and corticosterone secretion. These results indicate that in conscious rats NO plays a marked inhibitory role in the CRH-induced ACTH secretion and inhibits more potently corticosterone secretion. Hypothalamic dopamine and noradrenaline do not seem to be directly involved in the observed alterations in ACTH and corticosterone secretion.

Influence of nitric oxide synthase inhibitors on the vasopressin-induced pituitary-adrenocortical activity and hypothalamic catecholamine levels.

In the present study the role of endogenous nitric oxide (NO) in the vasopressin-induced ACTH and corticosterone secretion was investigated in conscious rats. Vasopressin (AVP 5 microg/kg i.p.) considerably augmented ACTH and corticosterone secretion. L-arginine (120 and 300 mg/kg i.p.) did not significantly alter the AVP-induced secretion of those hormones. Nitric oxide synthase (NOS) blockers N(omega)-nitro-L-arginine (L-NNA) and its methyl ester (L-NAME) given i.p. 15 min before AVP markedly increased the AVP-induced ACTH secretion. L-NNA (2 mg/kg) more potently and significantly increased the AVP-induced ACTH secretion, whereas L-NAME elicited a weaker and not significant effect. Both those NOS antagonists intensified significantly and to a similar extent the AVP-induced corticosterone secretion. L-arginine (120 mg/kg i.p.) reversed the L-NNA-induced rise in the AVP-stimulated ACTH secretion and substantially diminished the accompanying corticosterone secretion. Neither vasopressin alone nor in combination with L-arginine and L-NAME evoked any significant alterations in the hypothalamic noradrenaline and dopamine levels. L-NNA (2 and 10 mg/kg i.p.) elicited a dose dependent and significant decrease in the hypothalamic noradrenaline level. The hypothalamic dopamine level was not significantly altered by any treatment. These results indicate that in conscious rats endogenous NO has an inhibitory influence on the AVP-induced increase in ACTH and corticosterone secretion. L-NNA is significantly more potent than L-NAME in increasing the AVP-induced ACTH secretion. This may be connected with a considerable increase by L-NNA of hypothalamic noradrenergic system activation which stimulates the pituitary-adrenal axis in addition to specific inhibition of NOS.

The role of prostaglandins and the hypothalamic and hippocampal histamine in the clonidine-induced pituitary-adrenocortical response.

Involvement of prostaglandins (PGs) and histamine in the hypothalamus and hippocampus in the clonidine-induced pituitary-adrenocortical response was investigated in conscious rats. The hypothalamic-pituitary adrenocortical (HPA) activity was assessed indirectly by measuring corticosterone secretion. Clonidine, an alpha 2-adrenergic agonist, given intracerebroventricularly (10 micrograms icv), considerably increased the serum corticosterone and hypothalamic histamine levels and markedly elevated the hippocampal histamine levels. Systemic or icv pretreatment with indomethacin (2 mg/kg or 10 micrograms), an inhibitor of prostaglandin synthesis, significantly reduced the clonidine-induced corticosterone response and abolished the increase in the hypothalamic and hippocampal histamine levels elicited by clonidine. Indomethacin in the doses used did not substantially change the resting serum corticosterone or hypothalamic and hippocampal histamine levels. These results indicate that prostaglandins and hypothalamic histamine are considerably involved in the HPA response to alpha 2-adrenergic receptor stimulation. They also suggest involvement of prostaglandins and histamine of the hippocampus in the clonidine-induced HPA response.