Agonist-promoted down-regulation and functional desensitization in two naturally occurring variants of the human serotonin1A receptor.

We recently reported two naturally occurring polymorphisms of the human serotonin1A (5-HT1A) receptor: glycine22-->serine (Ser22) and isoleucine28-->valine (Val28) in the putative aminoterminal domain of the receptor. To investigate the regulatory properties of these variants, the wild type (WT) and variant 5-HT1A receptors were stably expressed in CHO-K1 cells. WT, Ser22, and Val28 displayed similar high-affinity binding to [3H]-8-OH-DPAT. Competition experiments with 5-HT1A agonists and antagonists demonstrated similar pharmacological profiles. Receptor agonist-promoted down-regulation was tested by exposure to 100 mumol/L 8-OH-DPAT. After 24-h exposure, WT and Val28 underwent 59.3 +/- 3.9% and 59.5 +/- 1.4% reduction in receptor density respectively, whereas the degree of down-regulation was significantly lower for Ser22 (21.4 +/- 4.2%). Cell treatment for 24 h with 100 mumol/L 8-OH-DPAT reduced the 5-HT-induced inhibition of cAMP accumulation by 24.9 +/- 5.1% for WT and 16.4 +/- 0.8% for Val28, but only by 4.8 +/- 3% for Ser22. We conclude that the Ser22 variant is capable of attenuating agonist-mediated receptor down-regulation and desensitization.

Lack of cross-tolerance for hypophagia induced by DOI versus m-CPP suggests separate mediation by 5-HT2A and 5-HT2C receptors, respectively.

Intraperitoneal administration of 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) produced significant decreases in the first-hour food intake on day 1 and on day 2 relative to saline-treated animals. Complete tolerance developed to DOI-induced hypophagia by day 3. However, there was no cross-tolerance to m-chlorophenyl-piperazine (m-CPP)-induced hypophagia. Similarly, complete tolerance developed to m-CPP-induced hypophagia by day 3, but again there was no cross-tolerance to DOI-induced hypophagia. These findings suggest that DOI and m-CPP-induced hypophagia are mediated by different mechanisms, most likely by selective stimulation of 5-HT2A receptors by DOI and 5-HT2C receptors by m-CPP. The functional sensitivity changes did not parallel changes in hypothalamic [3H]-mesulergine-labeled 5-HT2C receptors or [3H]-ketanserin-labeled 5-HT2A receptors following chronic m-CPP or DOI treatment, although both treatments significantly reduced 5-HT2A and 5-HT2C receptors in cortex. Thus, future studies investigating the effects of daily m-CPP and DOI administration on phosphoinositide hydrolysis or mRNA for 5-HT2C and 5-HT2A receptors in the hypothalamus might help explain the functional sensitivity changes observed in the present study.

Differential effects of chronic antidepressant treatment on 5-HT1C receptor binding sites in Wistar rat brain.

The effects of chronic clomipramine, imipramine and clorgyline on 5-HT1C receptors were studied in discrete brain regions, in male Wistar rats, using [3H]mesulergine to label the receptor binding sites. Clorgyline treatment significantly reduced [3H]mesulergine binding (Bmax values) in both the hypothalamus and striatum compared to saline-treated animals. There were no differences in the maximum number of [3H]mesulergine binding sites following clorgyline in the hippocampus, frontal cortex or brainstem. Neither clomipramine or imipramine treatment resulted in any significant changes in 5-HT1C receptor number in the brain regions examined here. Furthermore, the Kd values (receptor affinity) for [3H]mesulergine binding were not significantly different comparing treatment groups to control animals. The significant changes in discrete brain regions following chlorgyline treatment suggest that 5-HT1C receptors may be involved in the clinical efficacy for the treatment of depression and other neuropsychiatric disorders.

Analysis of the 5-HT1C receptor and the serotonin uptake site in fawn-hooded rat brain.

Both the 5-HT1C receptor and the 5-HT uptake binding sites were measured in Fawn-Hooded, Sprague-Dawley and Wistar rats. Five brain regions were examined: frontal cortex, hippocampus, striatum, hypothalamus, and brainstem. We found significant differences in the Bmax and Kd values in various brain regions comparing Fawn-Hooded rats, with Sprague-Dawley and Wistar animals. The regional differences in receptor number and affinity in both the 5-HT1C receptor and the 5-HT uptake site in the Fawn-Hooded strain, relative to Wistar and Sprague-Dawley animals, provide support for the use of the Fawn-Hooded rat in serotonin dysfunction studies.

Diurnal rhythms of 5-HT1A and 5-HT2 receptor binding in euthermic and torpor prone deermice, Peromyscus maniculatus.

Deermice display both spontaneous and induced daily torpor bouts, attaining minimum body temperatures of 15-20 degrees C. There is evidence that brain serotonin may be involved in the initiation and/or maintenance of torpor. Inhibition of serotonin [5-hydroxytryptamine (5-HT)] synthesis markedly reduces the duration and depth of torpor. Because a certain percentage of deermice will not enter torpor under any circumstances, we were able to compare 5-HT receptor subtypes in deermice that readily enter into torpor (TP) and in non-torpor prone (NTP) animals. Deermice were trapped in the wild and subjected to food rationing and low ambient temperature and then sacrificed either in a normothermic or torpid state at 11:00 p.m. or 11:00 a.m. Whole brain was assayed for 5-HT1A and 5-HT2 receptor differences using [3H]8-OH-DPAT and [3H]ketanserin, respectively. The Bmax values for 5-HT1A receptors were significantly greater in both TP and NTP animals sacrificed at 11:00 p.m. compared to animals sacrificed at 11:00 a.m. In contrast, the density of 5-HT2 receptors was significantly greater in animals sacrificed at 11:00 a.m. compared to animals sacrificed at 11:00 p.m. This is consistent with the opposing functions of these receptors in the regulation of temperature and sleep. The affinity (Kd) of each receptor was unchanged. A comparison of TP and NTP animals sacrificed at the same time of day revealed no significant differences in either Bmax or in Kd values, indicating that differences in 5-HT1A and 5-HT2 receptors may not explain the heterogeneity of deermice in their ability to enter torpor.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional analysis of 5-HT1A receptors in two species of Peromyscus.

Two species of deer-mice, Peromyscus maniculatus (P. man) and Peromyscus leucopus (P. leu), were compared for differences in 5-hydroxytryptamine1A (5-HT1A) receptor number and affinity. Both species enter into torpor; however, P. man enters spontaneous torpor with a higher frequency and for a longer duration than P. leu. Further, compared to P. leu a higher percentage of P. man exhibit daily torpor. Deer mice can be induced to enter torpor by a reduction in food supply, shortened photoperiods, and decreasing ambient temperature. Under these conditions, P. man enters into torpor more frequently, for longer durations, and with a higher percentage of individuals as compared to P. leu. [3H]8-OH-DPAT was used to label 5-HT1A brain receptors in three brain regions: the frontal cortex, brainstem, and striatum. In addition, the hypothalamus and hippocampus were examined for 5-HT1A receptor differences; however, no measurable specific binding could be determined in these regions. In the frontal cortex, the Bmax values were significantly lower in P. man compared to P. leu. There were no significant differences in the Bmax values in the striatum and brainstem between P. man and P. leu. Further, there were no significant differences in the Kd values between the two species in any of the brain regions examined. The absence of any difference in receptor number or affinity in any of the brain regions examined, except the cortex, suggests that the 5-HT1A receptor is most likely not involved in a more efficient pathway to torpor.

Identification of tryptophan 2,3-dioxygenase RNA in rodent brain.

The gene for tryptophan 2,3-dioxygenase (TDO) heretofore was believed to be expressed only in liver. The data presented here demonstrate that RNA encoding TDO is present in rodent brain. Oligonucleotide primers based on the rat liver TDO cDNA sequence were synthesized and used to amplify RNA derived from mouse whole brain and liver and rat brain regions by the RNA-PCR. Reaction products were purified and subjected to DNA sequencing. Identical sequences were obtained when mouse whole brain and liver RNAs were amplified, and these sequences were shown to be 96% identical to the published rat liver tryptophan TDO cDNA sequence. In addition, TDO sequences were found in RNA derived from rat brainstem, cerebellum, cortex, hypothalamus, and the remainder of the brain.

Regional analysis of 5-HT1A and 5-HT2 receptors in the fawn-hooded rat.

The Fawn-Hooded strain of rats exhibits a hemorrhagic disorder, known as platelet storage pool deficiency. In addition to the platelet dysfunction, there is an altered response to certain serotonin drugs. To assess the characteristics of the binding to 5-HT1A and 5-HT2 receptors in this strain, regions of the brain from Fawn-Hooded, Sprague-Dawley and Wistar male rats were examined. The drug [3H]8-OH-DPAT was used to label 5-HT1A receptors and the Kd values for frontal cortex, hippocampus, striatum, hypothalamus and brainstem were similar in all three strains of rat. As with the 5-HT1A receptors, no differences were observed in the Kd values for 5-HT2 receptors, in any of the regions examined, among the three strains. However, the Bmax for the binding of [3H]8-OH-DPAT in the striatum and brainstem of Fawn-Hooded rats was less than in the Sprague-Dawley and Wistar animals. Furthermore, 5-HT2 receptors displayed a greater Bmax value in the striatum and in the frontal cortex of Fawn-Hooded animals, compared to Sprague-Dawley and Wistar rats. These differences in receptors are consistent with previous studies in which Fawn-Hooded rats were found to have altered serotonergic function, relative to Wistar and Sprague-Dawley animals.

Stimulation of serotonin1A receptors increases release of prolactin in the rat.

The effect of serotonin1A receptor agonists on release of prolactin was examined in awake, freely-moving male rats in which a catheter in the jugular vein allowed samples of blood to be collected periodically after intravenous injection of the agonist. The serotonin1A receptor agonist, 8-hydroxy-2(di-n-propylamino) tetralin (8-OHDPAT) increased concentrations of prolactin in plasma rapidly and in a dose-related manner. Concentrations of prolactin peaked within 9 min after intravenous injection of 8-OHDPAT and returned to baseline values within 30 min. Another serotonin1A receptor agonist, 5-methylurapidil (5-MeU), produced a similar response of prolactin. The effects of these agonists on release of prolactin were completely blocked by pretreatment with the serotonin receptor antagonists, methysergide and metergoline, administered 1 or 2 hr before the agonist. These results demonstrated that serotonin1A receptors can mediate the effects of serotonin on release of prolactin in the male rat.

Partial characterization of a neurotransmitter pathway regulating the in vivo release of prolactin.

Nicotinic cholinergic, opiate and serotonergic agonists as well as dopaminergic antagonists induce the release of pituitary prolactin. The purposes of the present studies were to determine if nicotine, morphine and the serotonin1A (5-HT1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) utilize a common synaptic pathway to release prolactin and, if so, to establish the serial order of the receptors involved. We also sought to determine whether the pathway under investigation leads to the secretion of prolactin via a mechanism involving dopamine, the prolactin inhibitory factor. Male rats with indwelling jugular catheters were pretreated with saline, mecamylamine, naltrexone, methysergide or bromocriptine. In the saline-treated animals, administration of nicotine, morphine, 8-OH-DPAT and haloperidol resulted in significant increases in plasma prolactin levels. Mecamylamine pretreatment prevented the prolactin response to nicotine only. Naltrexone blocked the stimulation of prolactin release by morphine and by nicotine. Methysergide inhibited the effects of 8-OH-DPAT, morphine and nicotine but not haloperidol. Bromocriptine blocked the prolactin secretion induced by haloperidol as well as by each of the above agonists. Also, in dual-immunocytochemically stained sections, tyrosine hydroxylase-immunoreactive cells and serotonin-immunoreactive processes were detected in close anatomical proximity in the dorsomedial arcuate nucleus. These data indicate that nicotine, morphine and 8-OH-DPAT act to release prolactin via a common synaptic pathway expressing nicotinic cholinergic, opiate, and 5-HT1A receptors at synapses arranged serially in that functional order. Furthermore, the data indicate that the in vivo secretion of prolactin via this pathway may ultimately occur through the inhibition of dopamine release.

Regulation of the anorectic drug recognition site during glucoprivic feeding.

The acute effects of 2-deoxy-D-glucose (2-DG)-induced glucoprivic feeding on the anorectic drug recognition site and Na+K(+)-ATPase in the brain were examined in adult rats and in lean and genetically obese mice. The marked hyperglycemia and the induction of feeding caused by the administration of 2-DG to satiated rats and lean mice were associated with significant increases in Na+K(+)-ATPase activity, and in [3H]ouabain binding and [3H]mazindol binding to the anorectic drug recognition site in hypothalamic membranes. Basal and 2-DG-stimulated levels of blood glucose were significantly correlated to the levels of hypothalamic [3H]ouabain (r = + .91, p less than 0.01) and [3H]mazindol (r = + .87, p less than 0.01) binding. A significant correlation (r = .74, p less than 0.05) was also observed between [3H]mazindol binding and [3H]ouabain binding supporting the hypothesis that these hypothalamic binding sites are functionally coupled in their response to circulating glucose. Following the intracerebroventricular (ICV) administration of the diabetogenic drug alloxan, 2-DG did not stimulate feeding or increase [3H]mazindol and [3H]ouabain binding sites in the hypothalamic paraventricular area. Since 2-DG still caused hyperglycemia in alloxan-treated rats, alloxan-induced inactivation of glucoreceptor mechanisms led to an uncoupling of the anorectic drug recognition site from a hypothalamic glucostat. In genetically obese mice (ob/ob), 2-DG also could not induce feeding or increase hypothalamic [3H]ouabain or [3H]mazindol binding, despite a significant hyperglycemic response. In contrast, 2-DG did increase feeding and the binding of [3H]ouabain and [3H]mazindol to the hypothalamus of lean littermates.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute effects of nicotine on prolactin release in the rat: agonist and antagonist effects of a single injection of nicotine.

The effects of nicotine on prolactin release were studied in conscious, unrestrained rats in which an indwelling jugular cannula allowed multiple samplings of blood after i.v. administration of nicotine. Intravenous administration of nicotine bitartrate dihydrate increases plasma prolactin concentrations in a dose-dependent manner with an ED50 of approximately 100 micrograms/kg (200 nmol/kg) and this effect is blocked completely by pretreatment with mecamylamine, indicating that it is mediated by a nicotinic cholinergic receptor. Intracerebral ventricular injection of 1 microgram of nicotine also increases plasma prolactin levels, but i.v. injection of this same amount of nicotine has no effect, indicating that nicotine acts within the brain to release prolactin. A single i.v. injection of nicotine resulted in desensitization of the prolactin response to a subsequent injection of nicotine given 1 to 2 hr later, thus confirming a previous report by Sharp and Beyer (J. Pharmacol. Exp. Ther. 238: 486-491, 1986). The prolactin response to nicotine was restored within 24 hr after a single injection. The acute desensitization after a single injection of nicotine appears to be specific to release of prolactin by nicotine because the prolactin response to morphine was unaffected 1 hr after injection of nicotine. A single injection of nicotine appears to desensitize the prolactin response to a subsequent injection of nicotine with an ED50 of approximately 20 micrograms/kg (40 nmol/kg), indicating that nicotine is even more potent in stimulating desensitization of nicotinic cholinergic receptors than in stimulating prolactin release. These results support the concept that nicotine acts as a time-averaged antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chronic administration of nicotine on prolactin release in the rat: inactivation of prolactin response by repeated injections of nicotine.

The effects of chronic injections of nicotine on nicotine-induced prolactin release in the rat were measured and compared to the effects of this treatment on [3H]acetylcholine binding to nicotinic cholinergic sites in the hypothalamus. Treatment with nicotine for 10 days (s.c. injections twice daily) abolished prolactin release in response to an acute i.v. injection of nicotine given 2, 6 or 8 days after the last of the chronic injections of nicotine. At each of these time points, the binding of [3H]acetylcholine in the hypothalamus from rats treated chronically with nicotine was significantly higher than in the hypothalamus from control rats. By 14 days after the last chronic injection of nicotine, the prolactin response to an acute injection of nicotine was restored. Coinciding with the return of the nicotine-induced prolactin response, the binding of [3H]acetylcholine had returned to control values. These results are consistent with the hypothesis that nicotine inactivates nicotinic cholinergic receptors in brain by an allosteric mechanism, and that prolonged inactivation of nicotinic cholinergic receptors leads to their increased number.

Naltrexone blocks nicotine-induced prolactin release.

Two receptor populations involved in the release of prolactin were examined in conscious, freely moving, male rats bearing indwelling jugular cannulae. The intravenous administration of either nicotine or morphine increased plasma prolactin levels. Pretreatment with the nicotinic antagonist mecamylamine blocked the prolactin response to nicotine only. In contrast, the opiate antagonist naltrexone blocked the prolactin response to both nicotine and morphine. These findings indicate that the nicotine stimulated release of prolactin is dependent not only on functional nicotinic cholinergic receptors but on opiate receptors as well. This suggests that nicotine and morphine release prolactin via a common pathway containing nicotinic cholinergic and opiate synapses in series.

Glucostatic regulation of hypothalamic and brainstem [3H](+)-amphetamine binding during food deprivation and refeeding.

Saturable and stereospecific binding sites for [3H](+)-amphetamine have been identified in rat hypothalamus and structure-activity studies suggest that these sites may mediate the anorectic actions of amphetamine and related phenylethylamines. In order to determine whether these binding sites may be involved in the physiological control of appetite, [3H](+)-amphetamine binding was measured in crude synaptosomal membranes prepared from various brain regions following food deprivation and refeeding. Rats deprived of food for 24 to 72 h exhibited time-dependent reductions in body weight, blood glucose concentration, and specific [3H](+)-amphetamine binding in hypothalamus and brainstem. No change in specific [3H](+)-amphetamine binding was found in the frontal cortex, striatum, cerebellum, or liver prepared from these same animals. The decrease in specific [3H](+)-amphetamine binding in both the hypothalamus and brain stem following food deprivation was rapidly and completely reversed by allowing animals brief access to food or a 10% glucose solution. The changes in [3H](+)-amphetamine binding in hypothalami and brainstem of food-deprived and refed rats were also highly correlated with blood glucose concentration. Further, parenteral administration of D-glucose, but not L-glucose, to either fed or food-deprived rats significantly increased the number of [3H](+)-amphetamine binding sites in hypothalamus. These data suggest that the [3H](+)-amphetamine binding site in the hypothalamus and (or) brainstem may function in the 'glucostatic' regulation of appetite.

Increased number of hypothalamic [3H] (+)-amphetamine binding sites in genetically obese (ob/ob) mice.

In genetically obese (ob/ob) mice the development of obesity was correlated with the binding of [3H] (+)-amphetamine to the hypothalamus. In 39-day-old ob/ob mice, which had obtained a body weight greater than 150% of their lean littermates, hypothalamic [3H] (+)-amphetamine binding had increased by approximately 60% above levels of their lean littermates. This increased number of hypothalamic [3H] (+)-amphetamine binding sites appeared during the onset of obesity and then remained elevated following the most prominent weight gain. Since ob/ob mice are markedly hyperglycemic throughout their life, the increase in [3H] (+)-amphetamine binding site density in the hypothalamus may be secondary to elevated blood glucose.

Glucose regulates [3H](+)-amphetamine binding and Na+K+ ATPase activity in the hypothalamus: a proposed mechanism for the glucostatic control of feeding and satiety.

Binding sites for [3H](+)-amphetamine in the hypothalamus may mediate the anorectic actions of amphetamine and related phenylethylamines. To investigate further the role of these sites in the central control of appetite, the binding of [3H](+)-amphetamine to the hypothalamus and brainstem was measured following food deprivation and refeeding, the onset of genetic obesity, or the administration of 2-deoxy-D-glucose. Food deprivation for 24 to 72 hours reduced the Bmax for [3H](+)-amphetamine binding in the hypothalamus and brainstem but not in other brain areas or peripheral tissues. The decrease in hypothalamic and brainstem [3H](+)-amphetamine binding observed following food deprivation was time-dependent and rapidly reversed by brief refeeding with either rat chow or a 10% glucose solution. Moreover the changes in [3H](+)-amphetamine binding were highly correlated to corresponding alterations in blood glucose concentration. Furthermore, D-glucose, but not L-glucose increases the number of hypothalamic [3H](+)-amphetamine binding sites when administered in vivo or when added to hypothalamic slices in vitro. These data suggest that the [3H](+)-amphetamine binding site in the hypothalamus and (or) brainstem may be coupled to a central "glucostat."

Glucostatic regulation of (+)-[3H]amphetamine binding in the hypothalamus: correlation with Na+,K+-ATPase activity.

Preincubation of rat hypothalamic slices in glucose-free Krebs-Ringer buffer (37 degrees C) resulted in a time-dependent decrease in specific (+)-[3H]amphetamine binding in the crude synaptosomal fraction prepared from these slices. The addition of D-glucose resulted in a dose- and time-dependent stimulation of (+)-[3H]amphetamine binding, whereas incubation with L-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose failed to increase the number of (+)-[3H]amphetamine binding sites. Ouabain potently inhibited the glucose-induced stimulation of (+)-[3H]amphetamine binding, suggesting the involvement of Na+,K+-ATPase. Preincubation of hypothalamic slices with glucose also resulted in an increase in Na+,K+-ATPase activity and the number of specific "high-affinity" binding sites for [3H]ouabain, and a good correlation was observed (r = 0.89; P less than 0.02) between the glucose-stimulated increase in (+)-[3H]amphetamine and [3H]ouabain binding. Similar increases in (+)-[3H]amphetamine binding, [3H]ouabain binding, and Na+,K+-ATPase activity were observed in the hypothalamus after parenteral administration of glucose to rats. The administration of anorectic doses of amphetamine (0.1-5.0 mg/kg of body weight) also increased Na+,K+-ATPase activity in the hypothalamus. These data suggest that the (+)-[3H]amphetamine binding site in hypothalamus, previously linked to the anorectic actions of various phenylethylamines, is regulated both in vitro and in vivo by physiological concentrations of glucose. Glucose and amphetamine appear to interact at common sites in the hypothalamus to stimulate Na+,K+-ATPase activity, and the latter may be involved in the "glucostatic" regulation of appetite.