Effect of halothane on metabolism of 5-hydroxytryptamine by rat lungs perfused in situ

The effect of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on the uptake of 14C-labelled 5-hydroxytryptamine (5-HT) and its metabolism to 5-hydroxyindol-3-ylacetic acid (5-HIAA) was investigated in rat lungs perfused in situ. The rate of accumulation of 14C-labelled 5-HIAA in the tissue, monitored as an index of 5-HT metabolism, was linear with time, displayed saturation kinetics and remained stable for at least 180 min of perfusion. Exposure of the lungs to halothane (4%) for 60 min reversibly reduced production of 5-HIAA through an increase in the apparent Km for metabolism of the amine from 1.45 to 3.52 microM (P less than 0.001); the anaesthetic had no effect on the Vmax. of the process. The magnitude of the inhibition increased with time of exposure to the anaesthetic. Halothane exposure did not alter the distribution of [3H]sorbitol or [14C]5-HT, pulmonary vascular resistance, levels of ATP or the kinetics of amino acid transport in the tissue. Inhibition of protein synthesis by cycloheximide did not mimic the effect of the anaesthetic. These observations, together with those made in lungs exposed to inhibitors of 5-HT uptake and metabolism, were consistent with a halothane-mediated inhibition of 5-HT uptake, which did not appear to involve non-specific changes in membrane permeability.     

Exogenous Tryptophan Increases Synthesis, Storage, and Intraneuronal Metabolism of 5-Hydroxytryptamine in the Rat Hypothalamus

The effects of tryptophan administration on neurochemical estimates of synthesis [5-hydroxytryptophan (5-HTP) accumulation following administration of a decarboxylase inhibitor], storage [5-hydroxytryptamine (5-HT) concentrations], and metabolism [5-hydroxyindoleacetic acid (5-HIAA) concentrations] of 5-HT in selected regions of the hypothalamus were determined using HPLC coupled to an electrochemical detector. Tryptophan methyl ester HCl (30-300 mg/kg i.p.) produced a dose-dependent increase in the rate of 5-HTP accumulation throughout the hypothalamus but had no effect on the rate of accumulation of 3,4-dihydroxyphenylalanine. Peak 5-HTP levels were attained by 30 min following administration of tryptophan (100 mg/kg i.p.) and were maintained for an additional 60 min. Tryptophan also produced concomitant dose-dependent increases in 5-HT and 5-HIAA concentrations in these same regions without changes in the 5-HIAA/5-HT ratio. These results indicate that exogenous tryptophan administration selectively increases the synthesis, storage, and metabolism of 5-HT in the hypothalamus without altering the synthesis of catecholamines. Inhibition of 5-HT uptake with chlorimipramine or fluoxetine produced modest (10-40%) reductions in 5-HIAA concentrations throughout the hypothalamus, revealing that only a minor portion of 5-HIAA is derived from released and recaptured 5-HT, whereas the major portion of this metabolite reflects intraneuronal metabolism of unreleased 5-HT. In both chlorimipramine- and fluoxetine-treated rats, 5-HIAA concentrations were significantly increased by tryptophan administration, indicating that the increase in synthesis of 5-HT following precursor loading is accompanied by an increase in the intraneuronal metabolism of 5-HT.     

Comparative evaluation of sequestration of polyamines by perfused rabbit and rat lungs

Differences were observed in the sequestration of polyamines putrescine, spermidine and spermine by isolated, ventilated, perfused rat and rabbit lungs, former being able to accumulate more polyamines compared to the latter. Steady state equilibrium was reached earlier for spermine in rat. Isolated ventilated lungs were perfused with harmaline and ouabain, inhibitors known to inhibit the sodium pump at a maximum concentration of 1 mM for rabbit lungs and 0.4 and 0.2 mM for rat lungs, respectively. They did not affect the uptake of polyamines by rat lung but decreased the uptake of putrescine by rabbit lung. Decreased sodium (50 meq/L) in the perfusate increased the uptake of spermine and spermidine by rabbit lung but again showed no effect with rat lung. However, the uptake of polyamines by isolated ventilated rat and rabbit lungs perfused for 60 min with these compounds was linear over the entire range of high concentrations studied. These results suggest that the major uptake process of polyamines by intact lungs of both animal species is primarily by simple diffusion. HPLC analysis of the perfusate and lungs from both animal species post-perfusion indicated no detectable metabolites of the polyamines.     

The occurrence, synthesis and metabolism of 5-hydroxytryptamine and 5-hydroxytryptophan in the cestode Hymenolepis diminuta: a high performance liquid chromatographic study

The biosynthesis and metabolism of 5-hydroxytryptamine (serotonin; 5-HT) in the cestode Hymenolepis diminuta was investigated by High Performance Liquid Chromatography (HPLC). Incubation of intact H. diminuta in [3H]tryptophan resulted in substantial radioactivity recovered in 5-HT, 5-hydroxytryptophan (5-HTP), and 5-hydroxyindoleacetic acid (5-HIAA). Furthermore, the tissue levels of 5-HT and 5-HTP, as determined by HPLC with electrochemical detection, were significantly depressed when the animals were deprived of tryptophan. On the other hand, the tissue levels of 5-HTP were significantly increased following incubation with the 5-HTP decarboxylase inhibitor m-hydroxybenzylhydrazine. The synthesis and metabolism of 5-HT are discussed in the light of 5-HT as a physiological transmitter in H. diminuta.     

Endogenous Release of Neuronal Serotonin and 5-Hydroxyindoleacetic Acid in the Caudate-Putamen of the Rat as Revealed by Intracerebral Dialysis Coupled to High-Performance Liquid Chromatography with Fluorimetric Detection

Extracellular levels of endogenous serotonin (5-HT) and its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were measured in the caudate-putamen of anesthetized and awake rats using intracerebral microdialysis coupled to HPLC with fluorimetric detection. A dialysis probe (of the loop type) was perfused with Ringer solution at 2 microliters/min, and samples collected every 30 or 60 min. Basal indole levels were followed for up to 4 days in both intact and 5,7-dihydroxytryptamine (5,7-DHT) lesioned animals. Immediately after the probe implantation, the striatal 5-HT levels were about 10 times higher than the steady-state levels that were reached after 7-8 h of perfusion. The steady-state baseline levels, which amounted to 22.5 fmol/30 min sampling time, remained stable for 4 days. In 5,7-DHT-denervated animals, the steady-state levels of 5-HT, measured during the second day after probe implantation, were below the limit of detection (less than 10 fmol/60 min). However, during the first 6 h post-implantation, the 5-HT output was as high as in intact animals, which suggests that the high 5-HT levels recovered in association with probe implantation were blood-derived. As a consequence, all other experiments were started after a delay of at least 12 h after implantation of the dialysis probe. In awake, freely moving animals, the steady-state 5-HT levels were about 60% higher than in halothane-anesthetized animals, whereas 5-HIAA was unaffected by anesthesia. KCl (60 and 100 mM) added to the perfusion fluid produced a sharp increase in 5-HT output that was eight-fold at the 60 mM concentration and 21-fold at the 100 mM concentration. In contrast, 5-HIAA output dropped by 43 and 54%, respectively. In 5,7-DHT-lesioned animals, the KCl-evoked (100 mM) release represented less than 5% of the peak values obtained for the intact striata. Omission of Ca2+ from the perfusion fluid resulted in a 70% reduction in baseline 5-HT output, whereas the 5-HIAA levels remained unchanged. High concentrations of tetrodotoxin (TTX) added to the perfusion medium (5-50 microM) resulted in quite variable results. At a lower concentration (1 microM), however, TTX produced a 50% reduction in baseline 5-HT release, whereas the 5-HIAA output remained unchanged. The 5-HT reuptake blocker, indalpine, increased the extracellular levels of 5-HT sixfold when added to the perfusion medium (1 microM), and threefold when given intraperitoneally (5 mg/kg). By contrast, the 5-HIAA level remained unaffected during indalpine infusion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Retrograde axonal transport after radioactive hydroxyindole injections into the olfactory bulb—an autoradiographic study

Light microscope autoradiography was used to study the retrograde transport of labelled material after injection of [³H]serotonin ([³H]5-HT), [³H]5-hydroxytryptophan ([³H]5-HTP) and [¹⁴C]5-hydroxyindoleacetic acid ([¹⁴C]5-HIAA) into the olfactory bulb (OB) of rat. A perikaryal labelling was clearly visualized in the Raphe Dorsalis (RD) and the Raphe Centralis (RC) 24 h after injection of [³H]5-HT (but not after injection of [³H]5-HTP or [¹⁴C]5-HIAA) into the OB of rats without monoamine-oxidase inhibitor (MAOI). In the OB, the labelled cells (mitral, granular, periglomerular and tufted cells) and the varicosities (dispersed in granular, plexiform and glomerular layers) were greater in number and intensity at 8 h than at 24 h after [³H]5-HT (10⁻³ M) injection. Five hours after injection of [¹⁴C]5-HIAA (10⁻³ M) some mitral, granular and tufted cells were labelled in the cytoplasm, nuclei and dendrites. A few varicosities were also observed. In contrast, after [³H]5-HTP injection no clear labelling was visualized in axonal processes. A net autoradiographic reaction was seen, however, in the capillary walls and some granular cells.

After injection of [³H]5-HT at various concentrations (10⁻² M to 10⁻⁵ M) into the OB of rats pretreated with MAOI, a selectivity in the pattern of labelling in the injection site and the afferent cell bodies was found at 10⁻⁴ M and 10⁻⁵ M. At these concentrations, the serotoninergic RD and RC neurons were clearly labelled, but the non-serotoninergic neurons such as those originating in the Locus Coeruleus, prepiriform cortex were devoid of label. In the OB, only varicosities and fiber-like structures were reactive. In the RD cell bodies, the intensity of labelling as well as the number of labelled cells were greater at higher concentrations of injected [³H]5-HT and when rats were pretreated with a MAOI.     

N-oxidation of imipramine by isolated perfused rat and rabbit lung

Pulmonary uptake and metabolism of imipramine (IMP) was investigated in isolated perfused rat (IPrL) and rabbit (IPRL) lung preparations. Perfusate containing ¹⁴C-IMP (1.2 μmole/g lung) was recirculated through the pulmonary artery in artificially ventilated lungs. The radioactivity in the perfusate declined rapidly and about 80% of the dose was taken up by the lungs within 10 minutes in both IPrL and IPRL preparations. A steady-state was apparently reached thereafter in the IPRL, while a portion of the radiolabel effluxed into the perfusate of the IPrLs, thus reducing the net lung content to 54% of added IMP by 60 minutes. After 60 minutes perfusion, metabolites of IMP accounted for the major radioactivity (80%) in the perfusate, while the lung contained mainly (83%) the unchanged parent compound. The principal metabolite was identified as IMP-N-oxide (IMP-NO) which was found in the perfusate after 5 minutes of perfusion. Only 3% of the added IMP was metabolized by IPRL in 60 minutes. SKF-525A, an inhibitor of cytochrome P-450-mediated monooxygenase system, did not inhibit but enhanced the metabolism of IMP by IPrL to IMP-NO. IMP was principally metabolized to IMP-NO by incubations of 9,000 g supernatant fractions of rat lungs to a significantly higher extent than similar rabbit lung preparations. Including SKF-525A significantly accelerated the metabolism of IMP to IMP-NO in accordance with the perfusion experiments. These results suggest that in contradiction to publishedd reports, IMP is appreciably metabolized by the rat lung $\text{via}$ N-oxidation by non-cytochrome P-450 pathway and the metabolite formed in the lung is released into the circulation indicating its low affinity for the lung tissue.     

Uptake, metabolism and release of carnitine and acylcarnitines in the perfused rat liver

The uptake and release of carnitine and isovalerylcarnitine have been studied in the perfused rat liver. Labelled carnitine accumulates in rat livers perfused with 50 or 500 microM [3H]carnitine. When alpha-ketoisocaproate (5 mM) is added to the perfusate after 30 min of perfusion, the net uptake of carnitine in the liver stops, and there is even a decrease in liver radioactivity. The decrease in liver carnitine can be attributed to an enhanced formation and efflux to the perfusate of short-chain acylcarnitines. Thin-layer chromatography of liver and perfusate extracts showed that efflux rates for branched-chain acylcarnitines (isovalerylcarnitine) formed are at least 2.5-fold the efflux rate for carnitine. Acetylcarnitine is released about twice as fast as carnitine from the liver. Perfusion with 50 microM [3H]isovalerylcarnitine showed that the influx rate of isovalerylcarnitine exceeds that of carnitine 1.5-fold. Since the efflux rate is still higher, a net loss of carnitine from the liver to the perfusate will result when branched-chain acylcarnitines are formed in the perfused liver. The addition of 500 microM unlabelled carnitine to the perfusate does not influence the release of labelled carnitine or acylcarnitines from the liver, showing that uptake and release are independent processes. Isovalerylcarnitine accumulates faster than carnitine does, also in the perfused rat heart. A mechanism for the development of secondary carnitine deficiencies associated with organic acidemia is proposed.     

The regulation of 5-hydroxytryptamine release from superfused synaptosomes by 5-hydroxytryptamine and its immediate precursors

The effect of L-tryptophan, 5-hydroxy-L-tryptophan (5-HTP), and 5-hydroxytryptamine (5-HT) on the K+-evoked release of [3H]5-HT from superfused rat brain synaptosomes was studied. 5-HT at concentrations above 10 nM significantly inhibited the K+-evoked release of [3H]5-HT. A slight enhancement of [3H]5-HT release was observed at a concentration of 5nM. In contrast tryptophan at a concentration of 10 nM significantly enhanced [3H]5-HT release with little effect at higher concentrations. 5-HTP did not significantly effect [3H]5-HT release. The results confirm previous findings that 5-HT inhibits its own release from nerve endings, and demonstrate that low concentrations of tryptophan in the synaptic region may act as a positive feedback regulator of 5-HT release.     

Comparison of the cardiovascular responses to intracerebroventricular administration of tryptamine, 5-hydroxytryptamine, tryptophan and 5-hydroxytryptophan in rats

General characteristics of the cardiovascular responses to intracerebroventricular (i.c.v.) injection of tryptamine, 5-hydroxytryptamine (5-HT), tryptophan and 5-hydroxytryptophan (5-HTP) were compared. Relatively small doses of tryptamine and 5-HT (0.005-0.1 microM) produced considerable, long-lasting and dose-dependent pressor effects, which sometimes were followed by prolonged depressor effects. Tryptophan (0.02-0.5 microM) and 5-HTP (0.02-0.2 microM) caused variable and usually slight, but long-lasting, vascular responses or no vascular response A large dose of tryptamine (0.5 microM) evoked variable vascular effects, while the same dose of 5-HT and 5-HTP evoked marked and prolonged depressor effects. The vascular responses to the drugs were accompanied by variable changes in heart rate. Tryptamine, 5-HT and 5-HTP, in the majority of rats, produced a bradycardia. The present study provides evidence that the cardiovascular response to i.c.v. administration of tryptamine is similar to that of 5-HT, supporting the idea that tryptamine, in addition to 5-HT, participates in the central physiological regulation of the rat cardiovascular system. The role of tryptophan and 5-HTP by themselves in this regulation, if any is of secondary importance.     

Absorption of 5-methyltetrahydrofolate in rat jejunum with intact blood and lymphatic vessels

Transport results from in vitro studies may not be applicable to in vivo situations. In this study, we extended our previous in vitro observations regarding the intestinal transport of 5-methyltetrahydrofolate to in vivo studies in the unanesthetized rat and examined the effect of the unstirred water layer on the absorption process. We used a well defined intestinal perfusion technique. Absorption of 0.5 and 5 microM 5-methyltetrahydrofolate proceeded in a linear manner for 40 min of perfusion at 0.31 and 1.74 nmol/100 cm per min, respectively. Absorption of 0.5 microM 5-methyltetrahydrofolate increased with increasing perfusate flow-rate from 0.5 to 2 to 4 ml/min, indicating an unstirred water layer influence on the absorption rate. Absorption of the substrate was saturable with an apparent Kt of 5.7 microM and Vmax of 3.45 nmol/100 cm per min. Absorption was pH-dependent, and was inhibited by structural analogues. In contrast to the in vitro data, addition of glucose (20 mM) to the perfusate was unnecessary for in vivo absorption to proceed. Unconjugated cholic (5 mM) and deoxycholic (1 mM) acids and the organic anion rose bengal (0.1 mM) inhibited the absorption of 0.5 microM 5-methyltetrahydrofolate when added to the perfusate. Conclusions: the results of previous in vitro studies of 5-methyltetrahydrofolate intestinal transport are applicable to in vivo situations, except that luminal glucose was found to be unnecessary in the latter. The unstirred water layer modulated the absorption of 5-methyltetrahydrofolate, while unconjugated bile acids and rose bengal inhibited it.     

Pulmonary disposition and effects of drugs on pulmonary removal of endogenous substances

Pulmonary sequestration of drugs and other xenobiotics is known to affect respiratory and nonrespiratory functions of the lung and may be causally related to drug-induced pulmonary disease. We employed chlorphentermine (CP), an anorexic drug of amphiphilic physicochemical properties, to unravel possible mechanisms underlying drug-induced pulmonary hypertension associated with this class of drugs. In isolated perfused lungs, CP interferes with pulmonary clearance of 5-hydroxy tryptamine (5-HT) and norepinephrine. Pulmonary uptake and metabolism of 5-HT are inhibited by CP in rabbit and rat lungs. In in vitro incubations, CP is a powerful inhibitor of pulmonary monoamine oxidase activity. When pulmonary metabolism of 5-HT was blocked by pargyline, CP also inhibited pulmonary uptake of 5-HT. These effects of CP can be demonstrated in vivo in rabbits and rats during a single pulmonary passage of radio-labeled 5-HT. Not all pneumophilic drugs interfere with pulmonary clearance of 5-HT, as illustrated by the lack of effects by chlorpromazine and propranolol despite their pulmonary accumulation to a significant extent. Pulmonary accumulation of the chlorinated analogs such as chlomipramine and CP is greater than their nonchlorinated congenors, and this is in agreement with their ability to impede pulmonary clearance of 5-HT. These studies suggest that the electrophilic chlorine substitution increases the affinity of these chemicals for lung tissue, which increases the likelihood of their interference with the pulmonary disposition of 5-HT.     

Production of 5-hydroxyindoleacetic acid from serotonin by cultured endothelial cells

Endothelial cells cells from bovine aorta and human umbilical vein and fibroblasts from human foreskin were cultured and subsequently evaluated for ability to metabolize serotonin (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA). Cells were incubated for three hours with 4 X 10(-6) M [14C] 5-HT creatinine sulfate. [14C] 5-HIAA was separated from labeled 5-HT by column chromatography and measured for scintillation counting. Production of 5-HIAA by bovine aorta cells was 39.0+/-7.5 (S.E.M., n=6) nmoles per 10(9) cells per hour. Production of 5-HIAA was markedly inhibited by the presence of 10(-4) M iproniazid (an inhibitor of monoamine oxidase) or 10(-4) M imipramine (an inhibitor of amine transport). 5-HIAA was the only product of 5-HT metabolism detected by thin layer chromatography. Production of 5-HIAA by human umbilical vein endothelial cells was 5.4+/-2.0 nmoles per 10(9) cells per hour (n=5) and by human foreskin fibroblasts was 3.9+/-1.4 nmoles per 10(9) cells per hour (n=5). The results obtained during incubation in the presence and absence of inhibitors indicate that bovine aorta endothelial cells maintained in tissue culture are able to transport serotonin with subsequent production of 5-HIAA. By contrast, human umbilical vein endothelial cells and fibroblasts exhibited relatively low rates of 5-HT uptake and metabolism.     

In Vivo Brain Dialysis Study of the Somatodendritic Release of Serotonin in the Raphe Nuclei of the Rat: Effects of 8-Hydroxy-2-(Di-n-Propylamino)tetralin

Abstract: The characteristics of the serotonin (5-HT) output in the dorsal and median raphe nuclei of the rat were studied using in vivo microdialysis. The basal output of 5-HT increased after KC1 was added to the perfusion fluid. In contrast, neither the omission of calcium ions nor the addition of 0.5 nM tetrodotoxin affected dialysate 5-HT or 5-hy-droxyindoleacetic acid (5-H1AA). Reserpine did not decrease the output of 5-HT and 5-HIAA 24 h later and p-chloroamphetamine increased 5-HT in both vehicle- and reserpine-treated rats severalfold. 8-Hydroxy-2-(di-n-pro-pylamino)tetralin (8-OH-DPAT), at 1 or 10 μM, perfused into the raphe did not change the outputs of 5-HT or 5-HIAA. Higher doses (0.1, Land 10 mM) increased extracellular 5-HT in the raphe, probably via an inhibition of uptake. In animals bearing two probes (raphe nuclei and ventral hippocampus), only the 10 vaM dose of 8-OH-DPAT perfused into the raphe decreased the hippocampal output of 5-HT and 5-HIAA. The systemic injection of 0.1 mg/kg 8-OH-DPAT decreased dialysate 5-HT and 5-HIAA in the raphe and hippocampus. These results suggest that extracellular 5-HT in raphe nuclei originates from a cytoplasmic pool and is not dependent on either nerve impulse of 5-HT neurons or local activation of 5-HT_1A receptors.     

Effects ofp-chlorophenylalanine on the metabolism of serotonin from 5-hydroxytryptophan

The effects ofD,L--chlorophenylalanine methyl ester (PCPA-methyl ester) and two of its metabolites, 2-(-chlorophenyl)-ethylamine (PCPEA) and -chlorophenylacetic acid (PCPAA), on the metabolism of serotonin (5-HT) fromD,L-5-hydroxytryptophan (5-HTP) ware studied in vitro and in vivo using the telencephalon and brainstem of the rat. For in vivo studies and some in vitro experiments, rats were injected with either 100 mg/kg PCPA-methyl ester or saline alone on days 1, 2, and 3, and were killed on day 15. When the in vivo metabolism of 5-HT was to be studied, the saline group and the PCPA group of animals were injected with 75 g/kg [³H]D,L-5-HTP 20 min before sacrificing. With respect to the values found for the saline-injected animals, the specific activity (S.A.; dpm/nmol) of 5-HIAA was significantly greater in the telencephanol and brainstem of the animals injected with PCPA-methyl ester. The S.A. of 5-HTP was the same in both groups; the S.A. of 5-HT was lower in the telencephalon of the PCPA group than in the saline group; in the brainstem, there was no difference. In both the saline- and PCPA-injected animals, the S.A. of 5-HIAA was greater than the S.A. of 5-HT. There was no difference between the saline- and PCPA-injected animals with regard to: (1)L-5-HTP decarboxylase activity; (2)L-5-HTP-induced release of [³H]5-HT in vitro from crude nerve ending fractions (P₂); or (3) in vitro uptake of [³H]D,L-5-HTP and its conversion to [³H]5-HT using the P₂ fraction. In vitro studies demonstrated that the PCPEA could directly cause a large increase in the release of [³H]5-HT from the P₂ fraction, whereas PCPA and PCPAA had little or no apparent effect. The data were interpreted to suggest that in the telencephalon of the animals treated with PCPA-methyl ester, there was a higher turnover of 5-HT than was found in the saline-treated group.     

5-HT uptake blockade prevents the increasing effect of K(ATP) channel blockers on electrically evoked [3H]-5-HT release in rat and mouse neocortical slices

To explore if prolonged--as opposed to acute--5-HT uptake blockade can lead to changes in the function of ATP-dependent potassium (K(ATP)) channels, we investigated in rat and mouse neocortical slices the effects of K(ATP) channel blockers on electrically evoked [3H]-serotonin ([3H]-5-HT) release after short- and long-term exposure to 5-HT uptake blockers. Glibenclamide (1 microM), a K(ATP) channel blocker, enhanced the electrically evoked [3H]-5-HT release by 66 and by 77%, respectively, in rat and in mouse neocortex slices. This effect was confirmed in the rat by tolbutamide (1 microM), another K(ATP) channel antagonist. After short-term blockade (45 min) of 5-HT uptake, glibenclamide still increased the release of [3H]-5-HT in the rat. Glibenclamide, however, failed to enhance [3H]-5-HT release after long-term uptake blockade (210 min). In the mouse, however, both short- and long-term inhibition of 5-HT reuptake by citalopram (1 microM) prevented the facilitatory effect of glibenclamide. The Na(+)/K(+)-ATPase inhibitor ouabain (3.2 microM) abolished the glibenclamide-induced increase in [3H]-5-HT release in both rat and mouse, suggesting that an operative Na(+)/K(+)-ATPase is a prerequisite for activation of K(ATP) channels. The terminal 5-HT(1B) autoreceptor-mediated feedback control was involved in the glibenclamide-induced increase in [(3)H]-5-HT release only in mouse neocortical tissue, as evident from the use of the 5-HT(1B) autoreceptor ligands metitepin (1 microM) and cyanopindolol (1 microM). These results suggest that in the rat long-term uptake blockade leads to an impaired activity of the Na(+)/K(+)-ATPase, which increases intracellular ATP and consequently closes K(ATP) channels. In the mouse, however, short-term uptake blockade seems to already reduce the activity of the Na(+)/K(+)-ATPase and thereby the consumption of ATP. Blockade of 5-HT transporters thus may close K(ATP) channels through increased intracellular ATP. The following slight depolarisation seems to facilitate 5-HT release. These results may contribute to a better understanding of the mechanisms involved in the clinical time latency of antidepressant efficacy of monoamine uptake blockers.     

On the uptake and storage of 5-hydroxytryptamine, 5-hydroxytryptophan and catecholamines by adrenal chromaffin cells and nerve endings

Summary Light-microscopic autoradiographs of the adrenal medulla at various intervals after the intravenous injection of [³H] 5-HTP, [³H] 5-HT, [³H] noradrenaline and [³H] adrenaline have been studied. The distribution of silver grains following [³H] 5-HTP uptake was found to be uniform over each of the two main cell populations, adrenaline-storing (A) cells and noradrenaline-storing (NA) cells in the adrenal medulla, but A cells were twice as active as NA cells in incorporating the isotope, a situation very similar to that found after [³H] dopa uptake. 5-HT administration resulted in a pattern resembling the distribution of [³H] noradrenaline uptake, with A cells being 4 or 5 times more active than NA cells and a gradient of activity from the periphery of the medulla inwards. However, the time-course for the loss of radioactivity was not the same for both amines: levels of 5-HT activity were not significantly reduced after one week whereas the degree of [³H] noradrenaline labelling after one week was less than 10% of that at one hour. Thus 5-HT may be bound to sites in the adrenal medulla normally occupied by noradrenaline but it would appear that the release mechanism is different. There was no evidence of 5-HT uptake by adrenal nerve endings.     

Selective effect of phorbol ester on serotonin removal and ACE activity in rabbit lungs

The effect of phorbol myristate acetate (PMA) on pulmonary removal of [14C]serotonin (5-[14C]HT) and metabolism of [3H]benzoyl-phenylalanyl-alanyl-proline (BPAP), a synthetic substrate for angiotensin-converting enzyme (ACE), was evaluated in isolated rabbit lungs perfused in situ with Krebs-albumin. Metabolic functions were assessed before, during, and after perfusion with 80 nM PMA (n = 11), or PMA plus 133 microM papaverine (n = 10) or PMA diluent (dimethyl sulfoxide, n = 11). Organ kinetic parameters (apparent Vmax, Km) were calculated by use of indicator-dilution techniques and by a mathematical model of whole-organ metabolism. PMA treatment resulted in a significant decline in Vmax for BPAP metabolism (from 52 +/- 4 to 30 +/- 4 nmol/s) and 5-HT removal (from 2.1 +/- 0.2 to 1.1 +/- 0.1 nmol/s). Km for BPAP was not significantly altered, whereas Km for 5-HT removal was higher after treatment (before treatment, 1.1 +/- 0.1 microM; after treatment, 2.3 +/- 0.6 microM). Coperfusion with papaverine, which attenuated the pressor response to PMA, abolished PMA-induced changes in Vmax for BPAP metabolism and in Km for 5-HT removal but left PMA-induced changes in Vmax for 5-HT removal intact. We conclude that PMA alters endothelial metabolic function by both hemodynamic and biochemical mechanisms that are independent of circulating blood cells. Pulmonary capacity for BPAP metabolism may largely reflect perfused surface area, and capacity for 5-HT removal may be more sensitive to frank endothelial cell dysfunction in this model.     

EFFECT OF THE ADMINISTRATION OF L-5-HYDROXYTRYPTOPHAN AND A MONOAMINE OXIDASE INHIBITOR ON GLUCOSE METABOLISM IN RAT BRAIN

The effects of the presence of large amounts of 5-HT and of its precursor 5-HTP in brain on cerebral utilization of glucose were studied. [U-¹⁴C]Glucose was injected to fed rats that had previously been treated with L-5-HTP, L-5-HTP and an inhibitor—N-[β-(2-chlorophenoxy)-ethyl]-cyclopropylamine hydrochloride (Lilly-51641)-of MAO, or Lilly-51641 alone. Such treatment increased the concentrations of 5-HTP and 5-HT in the brain. After treatment with 5-HTP and Lilly-51641, and to a lesser extent with Lilly-51641 alone, the concentration of glucose in plasma was increased. However, the uptake of glucose by the brain did not appear to be proportionately increased, and this suggested an impairment in this mechanism. After the administration of Lilly-51641 alone and more especially of Lilly-51641 plus 5-HTP, the concentration of glucose in the brain was increased. This increase was thought to be due to an impairment of glucose utilization, because the flux of ¹⁴C from glucose to amino acids in the brain was reduced. The concentrations of most major amino acids in the brain were not greatly affected by these treatments. GABA and alanine concentrations in the brain were modestly increased after treatment with 5-HTP alone or in combination with Lilly-51641. The present results suggest that the metabolism of glucose to amino acids in the brain is altered when the concentration of 5-HTP, or more especially that of 5-HT, in the brain is increased.     

Inhibitory effects of cis- and trans-resveratrol on noradrenaline and 5-hydroxytryptamine uptake and on monoamine oxidase activity

This study investigated for the first time the potential effects of cis- and trans-resveratrol (c-RESV and t-RESV) on noradrenaline (NA) and 5-hydroxytryptamine (5-HT) uptake by synaptosomes from rat brain, on 5-HT uptake by human platelets, and on monoamine oxidase (MAO) isoform activity. Both c-RESV and t-RESV (5-200 microM) concentration-dependently inhibited the uptake of [3H]NA and [3H]5-HT by synaptosomes from rat brain and the uptake of [3H]5-HT by human platelets. In both experimental models, t-RESV was slightly more efficient than c-RESV. Furthermore, in synaptosomes from rat brain, the RESV isomers were less selective against [3H]5-HT uptake than the reference drug fluoxetine (0.1-30 microM). On the other hand, both c-RESV and t-RESV (5-200 microM) concentration-dependently inhibited the enzymatic activity of commercial (human recombinant) MAO isoform (MAO-A and MAO-B) activity, c-RESV being slightly less effective than t-RESV. In addition, both RESV isomers were slight but significantly more selective against MAO-A than against MAO-B. Since the principal groups of drugs used in the treatment of depressive disorders are NA/5-HT uptake or MAO inhibitors, under the assumption that the RESV isomers exhibit a similar behaviour in humans in vivo, our results suggest that these natural polyphenols may be of value as structural templates for the design and development of new antidepressant drugs with two important biochemical activities combined in the same chemical structure: NA/5-HT uptake and MAO inhibitory activity.