Antagonism of 5-hydroxytryptamine2 receptor-mediated phosphatidylinositol turnover by d-lysergic acid diethylamide

The interactions of the indolealkylamine hallucinogen d-lysergic acid diethylamide (d-LSD) and two phenalkylamine hallucinogens, 2,5-dimethoxy-4-bromoamphetamine (DOB) and 2,5-dimethoxy-4-iodoamphetamine (DOI), with 5-hydroxytryptamine2 (5-HT2) receptors were analyzed in rat cortex using both radioligand binding techniques and biochemical measurements of phosphatidylinositol (PI) turnover. 5-HT2 binding sites were labeled by [3H]ketanserin. DOB and DOI displayed decreased affinity for 5-HT2 sites in the presence of 10(-4) M GTP, whereas the ability of d-LSD to compete for these sites was not affected by the presence of 10(-4) M GTP. Moreover, the Hill slope of the d-LSD competition curve was unity in both the absence and presence of 10(-4) M GTP. These findings suggest that d-LSD is an antagonist at 5-HT2 receptors. PI turnover studies in rat cortex showed that at 10(-5) M concentrations d-LSD, DOB and DOI display partial agonist activity in comparison to 10(-5) 5-HT. Stimulation of PI turnover by 5-HT, DOB and DOI was inhibited by the 5-HT2 antagonist ketanserin (10(-6) M). The d-LSD PI signal was not affected by the presence of ketanserin. In addition, nanomolar concentrations of d-LSD did not stimulate PI turnover in rat cortex. Moreover, nanomolar concentrations of d-LSD are able to significantly antagonize the stimulatory effect of 10(-5) M 5-HT on PI turnover. These data suggest that d-LSD acts as an antagonist at 5-HT2 receptors in rat cortex. At high concentrations (greater than 1 microM) d-LSD stimulates low-level PI turnover via a non-5-HT2 receptor-mediated mechanism.     

Differential agonist-mediated internalization of the human 5-hydroxytryptamine 7 receptor isoforms

The human 5-hydroxytryptamine 7 (5-HT(7)) serotonin receptor is a class A G-protein coupled receptor that has three isoforms, 5-HT(7(a)), 5-HT(7(b)), and 5-HT(7(d)), which are produced by alternative splicing. The 5-HT(7) receptors are expressed in discrete areas of the brain and in both vascular and gastrointestinal smooth muscle. Central nervous system 5-HT(7) receptors may play a role in mood and sleep disorders. 5-HT(7) receptors show high affinity for a number of antidepressants and typical and atypical antipsychotics. We report here that the human 5-HT(7(d)) isoform expressed in human embryonic kidney (HEK) 293 cells exhibits a pattern of receptor trafficking in response to agonist that differ from 5-HT(7(a)) or 5-HT(7(b)) isoforms. We employed a modification of a live cell-labeling technique to demonstrate that surface 5-HT(7(d)) receptors are constitutively internalized in the absence of agonist. This is in contrast to 5-HT(7(a)) and 5-HT(7(b)) isoforms, which do not show this profound agonist-independent internalization. Indeed, the 5-HT(7(d)) isoform displays this internalization in the presence of a 5-HT(7) -specific antagonist. In addition, the human 5-HT(7) isoform shows a diminished efficacy in stimulation of cAMP-responsive reporter gene activity in transfected cells compared with 5-HT(7(a)) or 5-HT(7(b)) receptors expressed at comparable levels. Thus, the carboxy-terminal tail of 5-HT(7(d)), which is the longest among known human 5-HT(7) isoforms, may contain a motif that interacts with cellular transport mechanisms that is distinct from 5-HT(7(a)) and 5-HT(7(b)).     

Elevated brain tryptophan and enhanced 5-hydroxytryptamine turnover in acute hepatic heme deficiency: clinical implications

Administration of 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine, a suicide inhibitor of hepatic cytochrome P-450, to phenobarbital-pretreated rats rapidly causes a marked and sustained hepatic heme depletion and results in porphyria. We have shown that this event results in marked impairment of hepatic tryptophan pyrrolase activity and consequently in elevated tryptophan content and enhanced 5-hydroxytryptamine (5-HT) turnover in the brain of such porphyric rats. All these effects were reversed by administration of exogenous heme. Using an indirect assay of 5-HT-dependent function, we now show that this elevated 5-HT turnover in porphyric animals is associated with enhanced serotonergic tone. We also show that it can be potentiated by tryptophan administration, reversed by administration of exogenous heme, alleviated by treatment with p-chlorophenylalanine, an inhibitor of 5-HT synthesis, and attenuated by administration of valine, an amino acid that is known to compete with tryptophan uptake in the brain. In patients with hepatic porphyria, acute hepatic heme depletion results in severe, often life-threatening attacks. These attacks are hallmarked by neuropsychiatric symptoms of unknown etiology, but that can often be successfully treated by i.v. administration of heme. Because acute hepatic heme depletion may also be expected to compromise hepatic tryptophan metabolism in such individuals, our findings raise the possibility that elevated tryptophan content and 5-HT turnover in the brain may play a role in the neurological dysfunction associated with acute attacks of hepatic porphyria.     

The effects of subhypnotic doses of ethanol on regional catecholamine turnover

A 2 g/kg dose of ethanol given intraperitoneally to rats significantly reduced the turnover of dopamine in the substantia nigra and caudate nucleus, increased dopamine turnover in the olfactory tubercle and had no effect on dopamine turnover in the nucleus accumbens, amygdala and hypothalamus. The same dose of ethanol decreased the probenecid-induced homovanillic acid accumulation in the caudate nucleus. The turnover of norepinephrine was also decreased in hypothalamus and increased in the pons medulla region. No change in norepinephrine turnover was observed in frontal cortex, parietal cortex, cerebellum, amygdala, hippocampus and locus ceruleus region. The distribution of ethanol was similar in cortex, caudate nucleus, hypothalamus and pons-medulla. Catecholamine turnover in different brain regions seems to be differentially sensitive to the effects of ethanol, with most regions being unaffected by ethanol.     

Effects of 5-hydroxytryptamine and 5-hydroxytryptamine receptor agonists on ion transport across mammalian airway epithelia.

1. 5-Hydroxytryptamine has been suggested as a candidate for an endogenous inhibitor of airway sodium transport. Amiloride, an inhibitor of epithelial sodium channels, has therapeutic potential in disorders of airway ion transport such as cystic fibrosis, but its duration of action in vivo is short. 5-Hydroxytryptamine and related compounds have been studied to investigate whether any might be a useful alternative to amiloride for clinical use, and to further assess the possible physiological role of 5-hydroxytryptamine in the regulation of airway ion transport. 2. Sheep tracheal epithelium was mounted in Ussing chambers under short-circuit conditions. Mucosal application of 5-hydroxytryptamine resulted in an immediate, reversible, concentration-related decrease in the short-circuit current, maximal with 38% inhibition of the short-circuit current at 25 mmol/l. This response was completely inhibited by pretreatment of tissues with mucosal amiloride (100 mumol/l). These features are consistent with a direct effect of 5-hydroxytryptamine on amiloride-sensitive sodium channels. Similar results were obtained in a limited number of studies using human bronchial epithelium. 3. The effects of mucosal addition of a range of 5-hydroxytryptamine agonists were studied to determine if any was a more potent blocker of amiloride-sensitive sodium transport than 5-hydroxytryptamine. The 5-HT3 agonist 2-methyl-5-hydroxytryptamine had no effect on the short-circuit current at concentrations of up to 5 mmol/l. The 5-HT1D agonist sumatriptan had no effect at concentrations below 5 mmol/l and at 5 mmol/l had only a transient effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of St John's wort on catecholamine turnover and cardiovascular regulation in humans

BACKGROUND: St John's wort (Hypericum perforatum) is a popular over-the-counter antidepressant. Its antidepressive effect has been attributed in part to inhibition of monoamine transporters and monoamine oxidase, on the basis of in vitro studies. METHODS: In a double-blind, randomized, placebo-controlled, crossover study, 16 healthy subjects (11 men and 5 women; mean age, 31 +/- 5 years) ingested either St John's wort (300 mg three times daily) or placebo for 7 days. Imipramine treatment (50 mg three times daily) in 7 subjects served as a positive control. After treatment, physiologic and biochemical tests included cardiovascular reflex testing, graded head-up tilt testing, and plasma catecholamine determinations. RESULTS: St John's wort had no effect on blood pressure, heart rate, heart rate variability, or blood pressure variability, regardless of the test condition. St John's wort had no effect on plasma concentrations of norepinephrine and its main metabolite, dihydroxyphenylglycol, whereas plasma dihydroxyphenylacetic acid (DOPAC; the main metabolite of dopamine) concentrations increased in every subject (1661 +/- 924 pg/mL versus 1110 +/- 322 pg/mL with placebo, P=.04). In contrast, imipramine increased resting blood pressure (124 +/- 10 mmHg/71 +/- 5 mmHg versus 110 +/- 8 mmHg/61 +/- 6 mmHg with placebo, P=.005 for systolic values and P=.003 for diastolic values) and heart rate (74 +/- 7 beats/min versus 62 +/- 6 beats/min with placebo, P=.005) and elicited a marked orthostatic tachycardia (increase in heart rate of 43 +/- 17 beats/min versus 26 +/- 8 beats/min with placebo, P=.006). CONCLUSIONS: Our findings challenge the concept that St John's wort elicits a major change in norepinephrine uptake or monoamine oxidase activity in vivo. The consistent increase in plasma DOPAC concentrations might suggest a novel mode of action or an inhibitory effect on dopamine beta-hydroxylase that should be followed up. We propose that a combination of physiologic and biochemical profiling may help better define the mode of action and potential side effects of herbal remedies.     

(+)-Norfenfluramine-induced arterial contraction is not dependent on endogenous 5-hydroxytryptamine or 5-hydroxytryptamine transporter

(+)-Norfenfluramine, the major metabolite of fenfluramine, causes vasoconstriction dependence on the 5-hydroxytryptamine (5-HT)(2A) receptor in rat. (+)-Norfenfluramine was reported as a 5-hydroxytryptamine transporter (5-HTT) substrate and 5-HT releaser. Because the arterial 5-HTT exists and is functional in the rat, we hypothesized that (+)-norfenfluramine causes vasoconstriction by releasing 5-HT from vascular smooth muscle via 5-HTT. The released 5-HT, in turn, activates the 5-HT(2A) receptor. Isometric contractility experiments showed that (+)-norfenfluramine-induced mouse aortic contraction was reduced by the 5-HTT inhibitor fluoxetine (1 micriM) but not by fluvoxamine (1 microM). Tryptophan hydroxylase (TPH)-deficient (Tph1-/-) mice lack peripheral 5-HT. (+)-Norfenfluramine (10 nM-100 microM)-contracted aorta from wild-type and Tph1-/- mice with equivalent potency (-log EC(50) [M], wild type = 5.73 +/- 0.02, Tph1-/- = 5.62 +/- 0.09), and these contractions were inhibited by the 5-HT(2A) receptor antagonist ketanserin (3 nM) by a similar magnitude in aorta from wild-type and Tph1-/- mice (wild type = 19.4, Tph1-/- = 15.4-fold rightward shift versus control), as did fluoxetine (1 microM) (wild type = 22.4, Tph1-/- = 28.8-fold rightward shift versus control). To further test the role of 5-HTT in (+)-norfenfluramine-induced aortic contraction, the 5-HTT-targeted mutation mouse was used. (+)-Norfenfluramine induced similar aortic contraction in wild-type and 5-HTT-targeted mutation mice, and these contractions were inhibited by fluoxetine (1 microM). Thus, (+)-norfenfluramine vasoconstriction is not dependent on 5-HTT-mediated release of endogenous 5-HT but by activating membrane 5-HT(2A) receptors directly. Understanding of the mechanism by which (+)-norfenfluramine induces vasoconstriction is important to characterize and understand the function of the serotonergic system in peripheral arterial vasculature.     

The effect of propranolol on catecholamine clearance

Normal subjects given propranolol increased their plasma t1/2 for infused isoproterenol from 2.68 to 6.25 minutes. Propranolol increased plasma norepinephrine (NE) levels only slightly. Propranolol increased the t1/2 of isoproterenol but not that of NE in men with autonomic nervous system degeneration. This suggests that propranolol acts on nonneuronal uptake-2 processes, rather than on uptake-1 mechanisms. alpha-Blockers slow uptake-1 and beta-blockers slow uptake-2 processes. When 27 subjects exercised, those who attained the highest plasma levels of the alpha- and beta-receptor agonist NE also had the briefest apparent t1/2 for NE. Adrenergic receptor blocking drugs slow catecholamine clearance. NE may stimulate its own clearance.     

Pharmacological characterization of 5-hydroxytryptamine2 and 5-hydroxytryptamine3 receptors in rat dorsal root ganglion cells

5-Hydroxytryptamine (5-HT) depolarized 87% of the rat dorsal root ganglion cells recorded. 5-HT increased the input resistance (Rin) in 50%, decreased Rin in 41% and produced both responses in 9% of the responding cells. When 5-HT increased the Rin, the response was mimicked by the 5-HT2 agonists alpha-methyl-5-HT, (+/-)-1-(2,5-dimethoxy-4 iodophenyl)-2-aminopropane HCl, quipazine and MK 212 (6-chloro-1-[1-piperazinyl]-pyrazine), but not by 2-methyl-5-HT or carboxamidotryptamine. The response to 5-HT was antagonized by ketanserin, spiperone and methiothepin. The unsurmountable blockade induced by higher concentrations of ketanserin was not explained by pseudo-irreversible antagonism or multiple receptor subtypes, but could result from a two-state receptor model or multiple subtypes of the 5-HT2 receptor. This conclusion is supported by the partial agonist action of DOI. Cells responding to 5-HT with depolarization and decreased Rin responded similarly to 2-methyl-5-HT and phenylbiguanide, but not to alpha-methyl-5-HT or carboxyamidotryptamine. This response was surmountably blocked by ICS 205-930 (3-tropanyl-indole-3-carboxylate) (pA2 = 10.3) and MDL 72222 (3-tropanyl-3,5-dichlorobenzoate)(pA2 = 7.8). The arylpiperazines, quipazine and MK 212, antagonized the action of 2-methyl-5-HT with IC50 values of 8 and 4 nM, respectively. These data indicate that 5-HT2 receptors mediate the increased Rin and 5-HT3 receptors mediate the decreased Rin.     

The effect of serotonin (5-hydroxytryptamine) and derivatives on guanosine 3'',5''-monophosphate in human monocytes.

The effects of several putative mediators of inflammatory responses on the cyclic nucleotide content of mononuclear leukocytes from human peripheral blood were investigated. Incubation of mononuclear cells with 100 muM serotonin (a maximally effective concentration) for 5 min caused a five- to eightfold increase in their content of guanosine 3',5'-monophosphate (cAMP)...     

Pulmonary extraction of [3H]bupivacaine: modification by dose, propranolol and interaction with [14C]5-hydroxytryptamine

The authors studied the single-pass pulmonary extraction of the potent local anesthetic, bupivacaine, in 21 anesthetized rabbits. Pulmonary extraction of [3H]bupivacaine and [14C]5-hydroxytryptamine (5-HT) was quantified from multiple indicator-dilution outflow curves using indocyanine green as the intravascular reference substance. Pulmonary extraction at control (n = 15; mean +/- S.D.) was 81 +/- 6 and 78 +/- 9% for [3H]bupivacaine and [14C]5-HT, respectively. The apparent volume of distribution of bupivacaine was 38 +/- 9 ml/kg compared with 12 +/- 4 ml/kg for indocyanine green. Simultaneous administration of up to 300 micrograms/kg of bupivacaine did not affect the disposition of either radiolabeled amine; however, injection of 1000 micrograms/kg of bupivacaine significantly (P less than .01) depressed pulmonary extraction of both [3H]bupivacaine and [14C]5-HT. In the presence of 1000 micrograms/kg bupivacaine, the apparent volume of distribution of [3H]bupivacaine decreased to 24 +/- 9 ml/kg (P less than .01). Fifteen minutes after administration of propranolol (100-250 micrograms/kg i.v.), [14C]5-HT removal was unchanged, but the pulmonary extraction of [3H]bupivacaine was significantly decreased to 70 +/- 12% (n = 6; P less than .01). These data suggest that bupivacaine is extensively removed as it enters the lung and that the removal process is a combination of passive diffusion and a small component of saturable specific binding. The interaction of bupivacaine with 5-HT may be part of this specific binding (i.e., endothelial cell uptake) or may have been secondary to direct effects of large concentrations of bupivacaine on membrane function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of insulin- and proinsulin-induced hypoglycemia on pituitary hormone and catecholamine secretion

The effects of insulin- and proinsulin-induced hypoglycemia on pituitary hormone and catecholamine secretion were compared in normal men to search for possible hypothalamic or pituitary inhibitory effects of proinsulin on glucocounterregulatory responses. When subjects received 0.1 U/kg i.v. human insulin and 25-38 micrograms/kg i.v. human proinsulin on separate occasions, plasma glucose decreased more rapidly after insulin, and the nadir was slightly lower, but integrated hypoglycemic responses were similar. Cortisol, growth hormone (GH), prolactin, epinephrine, and norepinephrine responses occurred more rapidly after insulin than after proinsulin. Peak and integrated cortisol, GH, and catecholamine responses to insulin and proinsulin were similar, but those of prolactin were reduced after proinsulin when compared with insulin by 42% (P less than .01) and 34% (P less than .05), respectively. When euglycemia was maintained by a variable glucose infusion rate after the injection of insulin and proinsulin, no differences were observed in plasma levels of any of the hormones. The intravenous injection of a dose of proinsulin (6 micrograms/kg), which did not produce hypoglycemia but was the molar equivalent of insulin used in the first protocol, failed to modify the GH or prolactin responses to a combined injection of GH-releasing hormone (1 microgram/kg) and thyrotropin-releasing hormone (500 micrograms). Our results indicate that the onset of pituitary hormone and catecholamine responses to hypoglycemia are related to the rate of plasma glucose decline, with the slower responses to proinsulin reflecting a more gradual onset of hypoglycemia. The magnitude of the cortisol, GH, and catecholamine responses, however, was comparable with proinsulin- and insulin-induced hypoglycemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effect of plasma protein binding of bupivacaine on its in vivo transfer into the brain and salivary gland of rats

The effect of plasma protein binding of bupivacaine on its transfer into brain and salivary gland was studied using bovine serum albumin, human alpha 1-acid glycoprotein (AAG) and human serum. The in vivo brain extraction and salivary gland extraction of [3H] bupivacaine relative to [14C]butanol were determined with an intracarotid injection technique used on rats. The brain extraction varied inversely with the bovine serum albumin (0-7.5%) and AAG (0-3.0 mg/ml) concentrations. The salivary extraction only slightly varied inversely with the AAG concentration, whereas no significant effects of bovine albumin binding on salivary gland uptake were observed. The in vivo percentage of exchangeable drug in brain or salivary gland capillaries was severalfold greater than the in vitro percentage of unbound drug. The percent values of free drug, brain exchangeable drug and salivary exchangeable drug were 12 +/- 1, 81 +/- 7 and 93 +/- 18% for umbilical cord serum, 8.6 +/- 1.1, 73 +/- 6 and 103 +/- 3% for normal human serum, 5.9 +/- 0.5, 60 +/- 4 and 89 +/- 3% for serum of rheumatoid arthritis patients and 5.0 +/- 0.2, 45 +/- 2 and 83 +/- 3% for serum of metastatic cancer patients. These data indicate that bupivacaine is not transported through the brain capillary wall, i.e., the blood-brain barrier, from the bovine albumin-bound pool, but bupivacaine is partially available for transfer from the circulating AAG-bound pool. However, both bovine albumin-bound and AAG-bound bupivacaine are readily available for transport through salivary gland capillaries.(ABSTRACT TRUNCATED AT 250 WORDS)