Abnormal regulation of adenosine 3',5'-monophosphate and corticosterone formation in an adrenocortical carcinoma.

A spontaneously occurring rat adrenocortical carcinoma which produces corticosterone was maintained by transplantation. The carcinoma appeared to utilize corticosterone biosynthetic steps similar to those of the normal adrenal, but the tumor produced only about 1-10% as much corticosterone per unit tissue weight as nontumorous adrenal glands. The tumor demonstrated little or no increase in corticosterone production in response to adrenocorticotropic hormone (ACTH) either in vivo or in vitro. In normal adrenals, ACTH increases the activity of adenyl cyclase which catalyzes the conversion of adenosine triphosphate (ATP) to adenosine-3',5'-monophosphate (cyclic AMP), the latter then serving as an intracellular regulator of steroidogenesis. ACTH failed to increase cyclic AMP levels in the tumor in vivo or in slices in vitro, conditions under which there were 50- and 20-fold increases in nontumorous adrenals. However, in homogenates fortified with exogenous ATP, adenyl cyclase activity was comparable in the tumor and adrenals, and cyclic AMP formation was increased 3-fold by ACTH in each. As measured in homogenates, the tumor did not possess a greater ability to destroy cyclic AMP than did normal adrenals. Although ATP levels in the carcinoma were found to be considerably lower than those in normal adrenals, it was not clear that this finding can explain the inability of ACTH to increase cyclic AMP levels in intact tumor cells. While the failure to normally influence cyclic AMP levels in the carcinoma cells could be an important factor in the lack of a steroid response to ACTH, several lines of evidence suggest that the tumor possesses one or more additional abnormalities in the regulation of steroidogenesis. First, in the absence of ACTH stimulation, the tissue concentrations of cyclic AMP were comparable in the tumor and in nontumorous adrenals, but these cyclic AMP levels were associated with a lower level of steroidogenesis in the tumor. Second, tumor slices failed to increase corticosterone production when incubated with cyclic AMP, in contrast to 5-fold increases observed with nontumorous adrenals.

The effects of a low extracellular concentration of potassium on the activity and numbers of Na+/K+ pumps in an EB-virus transformed human lymphocyte cell line.

The BM1A EB-virus transformed human lymphocyte cell line contains approximately 950,000 Na+/K(+)-ATPase sites per cell. The turnover number of each site is approx. 2240 molecules of rubidium per min. When cells are exposed to a low extracellular concentration of potassium the intracellular concentration of sodium rises, and the cells respond in the short term by increasing the Vmax of 86Rb+ uptake. In the longer term the cells respond by increasing both the Vmax of 86Rb+ uptake and the Bmax of [3H]ouabain binding. The suggestion that increases in the intracellular concentration of sodium is responsible for these changes is supported by the finding that monensin, which increases intracellular sodium without affecting intracellular potassium, is capable of inducing both the short- and long-term changes associated with a low external concentration of potassium.

The effects of rubidium, caesium and quinine on 5-HT-mediated behaviour in rat and mouse--3. Quinine.

It has been shown that caesium, which shares properties with quinine as a K(+)-channel blocker, enhanced 5-HT-mediated behaviour in both rats and mice. It was therefore of interest to investigate the effects of quinine on 5-HT-mediated behaviour in the rat and mouse. Quinine, dose-dependently (ED50 = 5 mg/kg), produced the 5-HT behavioural syndrome in rats pre-treated with tranylcypromine (TCP) (15 mg/kg, i.p.). p-Chlorophenylalanine (i.p., 300 mg/kg x2) or (-)-propranolol (20 mg/kg, i.p.), pindolol (4 mg/kg, i.p.) and ritanserin (0.4 mg/kg, s.c.), all prevented the behavioural syndrome induced by quinine (72 mg/kg, i.p.) plus TCP. The administration of quinine (72 mg/kg, i.p.) enhanced the 5-HT syndrome elicited by p-chloramphetamine (4 mg/kg, i.p.) and the 5-HT agonists, 8-OH-DPAT (0.5 mg/kg, s.c.), 5-MeODMT (2 mg/kg, i.p.), DOI (8 mg/kg, s.c.) and quipazine (25 mg/kg, i.p.) in rats. Pretreatment with quinine also potentiated the 5-HT2-mediated head-twitch in the mouse but had no effect on the hypothermia in the mouse, induced by 8-OH-DPAT (0.5 mg/kg, s.c.). Quinine also enhanced the rate of synthesis of 5-HT in the brain of the rat. On the basis of these findings, together with those in the preceding two papers, it is suggested that the effects of rubidium, caesium and quinine, to enhance differentially various aspects of 5-HT function are mediated by actions on 5-HT-modulated K(+)-channels. This conclusion is also discussed in relation to the actions of lithium and electroconvulsive shock on 5-HT function in brain and the treatment of manic-depressive disease.

The effects of rubidium, caesium and quinine on 5-HT-mediated behaviour in rat and mouse--1. Rubidium.

The administration of TCP (15 mg/kg, i.p.) to rats pretreated with either intraperitoneal RbCl (3 mmol/kg, twice daily for 5 days) or dietary RbCl (30 mmol/kg diet, for 14 days), resulted in the complete 5-HT behavioural syndrome. Pretreatment with p-chlorophenylalanine (i.p. 300 mg/kg x2) or (-)-propranolol (20 mg/kg, i.p.), pindolol (4 mg/kg, i.p.) and ritanserin (0.4 mg/kg, s.c.) prevented the occurrence of the 5-HT syndrome, produced by dietary RbCl plus TCP. Intraperitoneal administration of RbCl had no effect upon the 5-HT behavioural syndrome, produced by 8-OH-DPAT (0.5 mg/kg, s.c.) or 5-MeODMT (2 mg/kg, i.p.) but enhanced the 5-HT syndrome produced by quipazine (20 mg/kg, i.p.), DOI (8 mg/kg, s.c.), p-chloramphetamine (4 mg/kg, i.p.) or by TCP plus L-tryptophan (50 mg/kg, i.p.) in rats. Dietary administration of RbCl resulted in the enhancement of the 5-HT2-mediated head-twitches in the mouse and the attenuation of hypothermia in the mouse, induced by 8-OH-DPAT (0.5 mg/kg, s.c.). The accumulation of 5-HT (after inhibition of monoamine oxidase) and the rate of synthesis of 5-HT in the whole brain (minus cerebellum) were enhanced by dietary and intraperitoneal administration of RbCl, respectively. The effects of lithium and rubidium, respectively, on 5HT function in brain are compared.

The effects of rubidium, caesium and quinine on 5-HT-mediated behaviour in rat and mouse--2. Caesium.

Rats and mice were given either CsCl (3 mmol/kg, s.c.) or saline (as control), twice daily for 3 days. The administration of tranylcypromine (TCP) (15 mg/kg, i.p.) to rats pretreated with CsCl produced the 5-HT behavioural syndrome. Pretreatment with CsCl also enhanced the syndrome induced by p-chloroamphetamine (3 mg/kg, i.p.) or by TCP (15 mg/kg, i.p.) plus L-tryptophan (50 mg/kg, i.p.). p-Chlorophenylalanine (300 mg/kg, i.p., daily on 2 consecutive days) or (-)-propranolol (20 mg/kg, i.p.), pindolol (4 mg/kg, i.p.) and ritanserin (0.4 mg/kg, s.c.), all prevented the behavioural syndrome induced by CsCl and TCP in rats. Pretreatment of rats with CsCl potentiated the 5-HT syndrome, elicited by the 5-HT agonists, 8-OH-DPAT (0.5 mg/kg, s.c.), 5-MeODMT (2 mg/kg, s.c.) and quipazine (25 mg/kg, i.p.). Pretreatment with CsCl potentiated the 5-HT2-mediated head-twitches in the mouse but had no effects on hypothermia in the mouse induced by 8-OH-DPAT (0.5 mg/kg, s.c.). The rate of synthesis of 5-HT in the whole brain (excluding cerebellum) was enhanced by pretreatment with CsCl. The enhancement of 5-HT neuronal function by caesium may be related to its ability to block K(+)-channels in neuronal membranes.

The putative 5-HT1A antagonist BMY 7378 blocks 8-OH-DPAT-induced changes in local cerebral glucose utilization in the conscious rat.

It has previously been shown that the 5-HT1A agonist, 8-OH-DPAT, caused discrete changes in cerebral glucose utilization in the rat, as assessed by quantitative 2-deoxyglucose autoradiography. Here, the effect of the putative 5-HT1A antagonist, BMY 7378, on regional cerebral glucose utilization was examined, when injected alone and in rats treated with 8-OH-DPAT. In control rats, BMY 7378 (5 mg/kg, s.c.) markedly increased glucose utilization in the lateral habenular nucleus and moderately reduced glucose utilization in the hippocampal formation. Pretreatment with BMY 7378 (5 mg/kg) significantly attenuated the reductions in glucose utilization in the hippocampus, entorhinal, piriform and cingulate cortex, induced by 8-OH-DPAT (0.25 mg/kg). The 8-OH-DPAT-induced increase in glucose utilization in the copula pyramis, that is putatively associated with the appearance of the 5-HT behavioural syndrome, was also blocked by BMY 7378, as was the behavioural syndrome. In summary, BMY 7378 produced few of the discrete changes in cerebral glucose utilization that are seen with 8-OH-DPAT. However, many of the changes induced by 8-OH-DPAT were reversed by BMY 7378. These data are consistent with the hypothesis that the effects of 8-OH-DPAT on regional cerebral glucose utilization are mediated by 5-HT1A receptors.

Effect of short- and long-term administration of lithium on the release of endogenous 5-HT in the hippocampus of the rat in vivo and in vitro.

The present study determined the effect of short- (3 days) and long- (21 days) term treatment with lithium on the release of 5-hydroxytryptamine (5-HT) form the hippocampus of the rat, measured both in vivo using microdialysis and in vitro using incubated slices. In the in vivo experiments (on the chloral hydrate-anaesthetized rat) electrical stimulation of the dorsal raphe nucleus for 20 min evoked an increase of 5-HT in dialysates of hippocampus, which both lasted for the duration of the stimulus and was frequency-dependent (2-10 Hz). Electrical stimulation of the dorsal raphe nucleus, at low stimulation pulse frequencies (2 and 3 Hz), released 3-4 fold more 5-HT in rats treated for 3 days with lithium chloride (3 mmol/kg s.c. twice daily), compared to controls. However, the effect of stimulation of the dorsal raphe nucleus was not altered in rats receiving lithium in the diet for 21 days. Basal levels of 5-HT in hippocampal dialysates for rats receiving long- but not short-term treatment with lithium, were significantly lower than controls. In agreement with the in vivo experiments, the in vitro experiments showed that depolarization (high potassium)-evoked release of endogenous 5-HT from slices of hippocampus of rats treated with short- but not long-term administration of lithium was enhanced compared to controls. These experiments provide direct biochemical evidence that short-term treatment with lithium increases depolarization-evoked release of endogenous 5-HT in the hippocampus, an effect which may be related to the rapid antidepressant actions of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigation of the presynaptic effects of quinine and quinidine on the release and uptake of monoamines in rat brain tissue.

Quinine and quinidine are reported to potentiate the behavioural effects of serotonergic agents and monoamine uptake inhibitors. We have therefore investigated the presynaptic actions of quinine and quinidine on monoamine uptake and release in rat brain tissue in vitro. Quinidine evoked the release of [3H]5-HT, [3H]noradrenaline and [3H]dopamine from pre-loaded rat brain slices in a concentration dependent manner with EC50 values of 175, 486 and 150 microM, respectively. Quinine induced [3H]monoamine release with similar potencies. Both quinine and quinidine also inhibited the active uptake of [3H]5-HT, [3H]noradrenaline and [3H]dopamine into rat brain synaptosomes with IC50 values in the range 0.13-12.4 microM. The potency of each drug to inhibit [3H]5-HT uptake was significantly higher than that for [3H]noradrenaline or [3H]dopamine. The relative potency of quinidine compared to quinine was more marked in the case of [3H]5-HT (58-fold) than for [3H]noradrenaline (3-fold) or [3H]dopamine (4-fold). The inhibition of [3H]5-HT uptake by quinine and quinidine was competitive in nature and corresponded with the potencies of these drugs to inhibit [3H]paroxetine binding. No correlation was observed between the potencies of quinine and quinidine to induce the release of [3H]monoamines and to inhibit their uptake, suggesting that these effects are mediated by two distinct mechanisms. We conclude that the presynaptic actions of quinine and quinidine on monoamine uptake and release may be implicated in their potentiation of the effects of serotonergic agents and uptake blockers.

Inhibition of 5-hydroxytryptamine-mediated behaviour by the putative 5-HT2 antagonist pirenperone.

The effect of pirenperone, a putative 5-HT2 receptor antagonist, on various 5-HT-mediated behavioural responses has been examined. The head twitch response in mice, induced by administration of carbidopa (25 mg/kg) followed by 5-hydroxytryptophan (5-HTP) (200 mg/kg), was inhibited in a dose-dependent manner by pirenperone, with an ED50 of 76 micrograms/kg. The appearance of head weaving, forepaw treading and hind-limb abduction, which followed the administration of tranylcypromine (5 mg/kg) plus L-tryptophan (100 mg/kg) or quipazine (50 mg/kg) to rats, was also inhibited by pretreatment with pirenperone (100 micrograms/kg). Pirenperone did not alter the rate of 5-HT synthesis in the rat brain. Whilst pirenperone (100 micrograms/kg) did decrease methamphetamine-induced locomotor activity in rats, a dose of haloperidol producing a similar inhibition of this response did not alter the 5-HT-mediated behaviour. It is suggested, therefore, that the currently used 5-HT-induced behavioural models are 5-HT2 receptor-mediated.

Influence of thyroid hormone on 5-HT(1A) and 5-HT(2A) receptor-mediated regulation of hippocampal BDNF mRNA expression.

The aim of the present study was to determine the influence of thyroid hormone, T3, on the regulation of hippocampal BDNF expression by 5-HT receptor agonists. Chronic T3 administration prior to treatment with the 5-HT(1A) agonist, 8-OH-DPAT, significantly decreased BDNF mRNA in the dentate gyrus region of the hippocampus. Administration of 8-OH-DPAT did not alter hippocampal BDNF mRNA expression in naive, euthyroid rats. Pretreatment with the 5-HT(1A) antagonist, WAY 100635, completely blocked the 8-OH-DPAT-induced down-regulation of BDNF mRNA in chronic T3-treated rats. Acute T3 administration prior to 8-OH-DPAT treatment led to a small, but significant, decrease in hippocampal dentate gyrus BDNF mRNA. Acute or chronic administration of T3 did not alter the decrease in hippocampal BDNF mRNA induced by the 5-HT(2A/2C) receptor agonist, DOI. The influence of 8-OH-DPAT and DOI on hippocampal BDNF mRNA was also unaltered in rats rendered hypothyroid by propylthiouracil administration. Chronic T3 treatment or hypothyroidism did not influence the basal expression of hippocampal BDNF mRNA. The affinity and density of 5-HT(1A) receptors, and the hippocampal expression of 5-HT(1A) mRNA were also not influenced by chronic T3 treatment. The results of this study clearly demonstrate a powerful interaction between thyroid hormone and the 5-HT(1A) receptor in the regulation of hippocampal BDNF expression. Crosstalk between signal transduction cascades influenced by T3 and 5-HT(1A) receptors may mediate the synergistic effects of these systems on hippocampal BDNF expression.

Chronic electroconvulsive shock enhances 5-HT2 receptor-mediated head shakes but not brain C-fos induction.

Chronic electroconvulsive shock (ECS), a widely used treatment for intractable depression, increases the density of 5-HT2A receptor binding sites and mRNA in rat frontal cortex. In contrast, this treatment appears to have no significant effect on 5-HT-stimulated phosphatidyl inositol turnover in rat brain. To investigate the effect of chronic ECS on the 5-HT2 receptor family further, we determined its effects on head shakes and c-fos expression in the rat in response to the 5-HT2A/2C receptor agonist DOI [1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane]. Chronic ECS (5 electroconvulsive shocks over 10 days, via earclips under halothane anaesthesia) caused a significant enhancement in the number of head shakes counted in a 30 min period after administration of 2 or 8 mg/kg DOI. In contrast, this treatment had no effect on Fos expression, induced by either dose of DOI, in any region of rat forebrain examined. Fos expression was low-to-undetectable in the brains of animals treated with chronic ECS followed by saline and sham ECS animals that had been treated identically, but with no administration of electrocurrent. Thus the lack of any change in PI turnover, following chronic ECS administration, appears to be mirrored by the failure of this treatment to alter 5-HT2 receptor-mediated Fos expression.

BRL 46470 potently antagonizes neural responses activated by 5-HT3 receptors.

The effect of a novel 5-HT3 receptor antagonist, BRL 46470, has been studied on two electrophysiological models for 5-HT3 receptors: grease-gap recordings from rat isolated vagus nerve and whole-cell patch-clamp recordings from mouse neuroblastoma-rat glioma NG108-15 cells. Its action on the rat vagus nerve was compared to that of four other 5-HT3 receptor antagonists. On the rat vagus, BRL 46470 reduced the maximum depolarizing response to 5-HT in a concentration-dependent manner with an IC50 of 0.3-1.0 nM, but the EC50 for 5-HT was not appreciably affected. This action was similar to that of granisetron and ICS 205-930, but differed from that of GR38032F and (+)-tubocurarine which produced clear rightward shifts of the concentration-response curve to 5-HT. The 5-HT-induced fast inward current of voltage-clamped NG108-15 cells was also antagonized by 1 nM BRL 46470 in an insurmountable manner. In contrast to (+)-tubocurarine, the action of BRL 46470 on the rat vagus nerve and NG108-15 cells did not readily reverse on washing with antagonist-free medium. It is concluded that BRL 46470 is a potent, insurmountable 5-HT3 receptor antagonist on the rat vagus and NG108-15 cells.

Serotonergic regulation of mRNA expression of Arc, an immediate early gene selectively localized at neuronal dendrites.

Arc (activity regulated, cytoskeleton associated protein) is an effector immediate early gene that is selectively localized in the neuronal dendrites. Elevation of brain 5-HT by the combined administration of the monoamine oxidase inhibitor, tranylcypromine (TCP, 5 mg/kg, i.p.), and the 5-HT precursor L-tryptophan (L-TP, 100 mg/kg, i.p.), increased Arc mRNA abundance in the cingulate, orbital, frontal and parietal cortices as well as in the striatum but a reduction was observed in the CA1 region of the hippocampus. The 5-HT releasing agent p-chloroamphetamine (PCA, 5 mg/kg, s.c.) also increased Arc mRNA in the cortical and striatal areas. Depleting brain 5-HT with the tryptophan hydroxylase inhibitor, p-chlorophenylalanine (pCPA, 300 mg/kg, i.p. for two days), on the other hand, significantly attenuated the increase in Arc mRNA induced by tranylcypromine and L-tryptophan (TCP/L-TP). Pretreatment with the 5-HT2 receptor antagonist ketanserin (2 mg/kg, i.p.) significantly attenuated the effect of TCP/L-TP in the cortex but only partially in striatum and did not affect the reduction in the CA1 region. The 5-HT2 agonist DOI (0.2, 1 and 2 mg/kg, i.p.) dose-dependently increased Arc mRNA abundance in cortical areas with a pattern similar to that of TCP/L-TP and PCA. DOI, however, had much weaker effects on Arc mRNA in the striatum and did not have any significant effect in the CA1, CA3 and the dentate gyms (DG) of the hippocampus. Pretreatment with ketanserin completely blocked the effect of DOI on Arc expression. These data suggest that Arc mRNA expression can be induced in the cortex by increases in extracellular 5-HT and that 5-HT2 receptors play a major part in mediating such effects. Additional 5-HT receptors as well as other neurotransmitters may also be involved, particularly in the striatum and in CA1 subfield of the hippocampus. Overall, our data suggest that expression of Arc mRNA is highly responsive to changes in brain 5-HT functions, and may provide a sensitive marker of postsynaptic 5-HT2(2A and 2C) receptor functions.

Studies on the mechanisms by which clenbuterol, a beta-adrenoceptor agonist, enhances 5-HT-mediated behaviour and increases metabolism of 5-HT in the brain of the rat.

The head twitch response in mice produced by injection of 5-hydroxytryptophan (100 mg/kg i.p.) and carbidopa (25 mg/kg i.p.) was enhanced by administration of clenbuterol (0.5 mg/kg i.p.), a beta-adrenoceptor agonist. Clenbuterol also enhanced the hyperactivity syndrome in rats produced by quipazine (25 mg/kg i.p.), a 5-hydroxytryptamine (5-HT) agonist. This enhancement was not prevented by depletion of 5-HT in brain with p-chlorophenylalanine or after pretreatment with prazosin. The behavioural responses of the rats to administration of the alpha 2-adrenoceptor agonist, clonidine, was unaltered by acute or longer-term administration of clenbuterol. Following chronic administration of clenbuterol (5 mg/kg daily for 14 days), a procedure resulting in down-regulation of central beta-adrenoceptors, a larger dose of clenbuterol was necessary to enhance the quipazine-induced hyperactivity, suggesting that the mechanism of enhancement involved central post-synaptic beta-adrenoceptors. Further evidence for this conclusion was that a lesion of central noradrenaline pathways produced by 6-hydroxydopamine did not abolish the clenbuterol-induced enhancement of the quipazine-mediated behaviour. The binding characteristics of 5-HT2-receptors were unchanged by acute or chronic administration of clenbuterol. Clenbuterol (5 mg/kg) increased the percentage of plasma free (non-albumin bound) tryptophan, plasma free fatty acid concentration and the concentration of tryptophan and 5-hydroxyindoleacetic acid (5-HIAA) in the brain. The increase in 5-HT turnover in brain was prevented by pretreatment with the beta 1-adrenoceptor antagonist atenolol, which enters the brain poorly. It is therefore suggested that the clenbuterol-induced increase in 5-HT metabolism results from the increase in the concentration of plasma free fatty acid which increases plasma free tryptophan and thus increases the concentration of tryptophan in brain and 5-HT synthesis in brain. The clenbuterol-induced enhancement of 5-HT-mediated behaviour is therefore not associated with its effect on 5-HT metabolism. The data are discussed in relation to that obtained after administration of antidepressant drugs.

Manipulations of brain 5-HT levels affect gene expression for BDNF in rat brain.

The aim of the present study was to investigate whether changes in brain 5-HT concentrations affect the expression of BDNF mRNA in rat brain. Brain 5-HT concentration in the rat was elevated by combined treatment with tranylcypromine and L-tryptophan, tranylcypromine alone, by a single dose of the 5-HT releasing agent p-chloroamphetamine (PCA) or by the selective 5-HT reuptake inhibitor paroxetine. 5-HT was depleted by either multiple p-chlorophenylalanine (pCPA) or PCA injections. The extent of 5-HT depletion following pCPA or PCA was monitored using 5-HT immunocytochemistry. BDNF mRNA abundance in treated rats and the corresponding vehicle injected control rats was studied by in situ hybridization histochemistry (ISHH). Two hours after the combined administration of tranylcypromine and L-tryptophan BDNF mRNA abundance in the dentate gyrus was significantly decreased but increased in the frontal cortex. Tranylcypromine alone or a single injection of PCA had similar effects on BDNF mRNA expression to the combination of tranylcypromine and L-tryptophan, i.e. they caused significant reductions of BDNF mRNA expression in dentate gyrus and increased it in frontal cortex. Paroxetine also reduced BDNF mRNA in DG but was without effect in frontal cortex. Multiple injections of both pCPA or PCA resulted in marked reductions of 5-HT immunoreactive axons in the hippocampus, pCPA being more effective. Both drugs significantly increased BDNF mRNA abundances in the dentate gyrus. Multiple PCA injections also increased BDNF mRNA expression in parietal cortex, while pCPA induced 5-HT depletion was ineffective. These results suggests that 5-HT modulates BDNF mRNA levels in rat brain.

Evidence for increased in vivo sodium-potassium pump activity and potassium efflux in skeletal muscle of spontaneously hypertensive rats.

We have used 87Rb nuclear magnetic resonance spectroscopy (NMR) to study in vivo rubidium kinetics in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) controls, using rubidium as a marker for potassium. We gave 15 male, 13-week-old SHR, mean +/- s.d. blood pressure 180 +/- 10 mmHg, and 15 age-matched normotensive controls, mean blood pressure 120 +/- 9 mmHg, a daily dose of RbCl (2 mmol/kg intraperitoneally). We made repeated NMR measurements of skeletal muscle rubidium concentrations until steady state was reached. We then withdrew rubidium and made further measurements of rubidium concentrations, at intervals, for up to 1 week after the last injection. We also measured plasma and erythrocyte rubidium concentrations by flame atomic absorption spectroscopy at similar intervals after the withdrawal of rubidium. Rubidium concentrations rose at a faster rate in SHR skeletal muscle, but the steady-state muscle rubidium concentration was the same (45 mmol/l) in both SHR and WKY rats. There was also a threefold increase in the rate of rubidium efflux from both muscle and erythrocytes in SHR. These results are consistent with a marked increase in Na+,K(+)-ATPase activity and an increase in the rate of rubidium efflux in vivo in SHR. The increased rate of rubidium efflux in SHR could represent increased K+ efflux via calcium-activated K+ channels and/or result as part of cell volume regulation secondary to increased Na(+)-H+ antiporter activity.

Evidence for an altered mode of action of the sodium-lithium countertransporter in vivo in patients with untreated essential hypertension.

OBJECTIVE: To study the activity of the sodium-lithium (Na(+)-Li+) countertransport system in vivo in the erythrocytes of patients with untreated essential hypertension. DESIGN: Lithium substitutes for sodium efflux in the sodium-sodium (Na(+)-Na+) countertransport system. In essential hypertension the efflux of lithium from cells in vitro has been used as a measure of the activity of the Na(+)-Na+ countertransporter and has been shown to be increased. We administered oral lithium and used its disposition in erythrocytes to measure Na(+)-Li+ countertransporter activity in vivo. PATIENTS: Ten men with essential hypertension who had never taken any antihypertensive treatment were matched with 10 male controls for age, weight, and plasma and erythrocyte sodium and potassium concentrations. METHODS: Repeated measurements were made of plasma and intra-erythrocytic lithium concentrations during the 48h after the oral administration of 16.2 mmol lithium carbonate. Data were analysed using standard pharmacokinetic techniques. RESULTS: The rate of lithium efflux from the erythrocytes was increased in all patients with hypertension and in none of the normotensive controls. Hill plots derived from in vivo activation curves for erythrocytic Na(+)-Li+ countertransport showed that the normotensive participants had a Hill slope of 1 (SD 0.1), whereas the hypertensives had a Hill slope of 3.2 (SD 1.0). CONCLUSIONS: The activity of the Na(+)-L+ countertransport system is increased in untreated essential hypertension in vivo; this confirms in vitro findings. A new finding is that there is a change in either the stoichiometry or the co-operativity of lithium efflux via the Na(+)-L+ countertransport system, suggesting that the rate of sodium efflux may be greater than that of influx in the cells of people with hypertension.

Evidence for increased in vivo Na(+)-H+ antiporter activity and an altered skeletal muscle contractile response in the spontaneously hypertensive rat.

We have assessed the in vivo activity of the Na(+)-H+ antiporter skeletal muscle in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) controls using phosphorus (31P) nuclear magnetic resonance spectroscopy to measure changes in cytosolic acid concentrations during isometric contraction. During contraction there was a small rate of rise in skeletal muscle cytosolic acid concentration to a smaller maximum concentration in SHR. This difference in acid response was removed by amiloride and was not attributable to differences in cell buffering or the rate of production of lactic acid, suggesting that the difference in acid response in SHR skeletal muscle is due to increased in vivo Na(+)-H+ antiporter activity. Amiloride reduced resting muscle glycogen concentration and increased muscle lactate concentration in the SHR. This could be related to altered in vivo calcium metabolism. The maximum tension produced by skeletal muscle during contraction in SHR was less than in WKY rats, and relaxation between twitches was significantly greater, consistent with the finding of increased vascular smooth muscle relaxation in essential hypertension. Since increased Na(+)-H+ antiporter activity occurs in association with increased relaxation of both skeletal and vascular smooth muscle, these data are not consistent with a relationship between increased Na(+)-H+ antiporter activity and increased maximal muscle tension development. However, they show that increase Na(+)-H+ antiporter activity is associated with increased muscle relaxation.

Release of endogenous 5-hydroxytryptamine in rat ventral hippocampus evoked by electrical stimulation of the dorsal raphe nucleus as detected by microdialysis: sensitivity to tetrodotoxin, calcium and calcium antagonists.

We have utilized the brain microdialysis technique in an attempt to measure excitation-secretion coupled release of endogenous 5-hydroxytryptamine in rat brain in vivo and investigated the pharmacology of the voltage-sensitive calcium channel involved in this process. All experiments were carried out using chloral hydrate anaesthetized rats. Ascending serotoninergic neurons were electrically stimulated using an electrode implanted into the dorsal raphe nucleus. A dialysis probe was implanted into the ventral hippocampus and continuously perfused with artificial cerebrospinal fluid containing the selective 5-hydroxytryptamine uptake inhibitor citalopram (1 microM). Twenty-minute perfusates were analysed for endogenous 5-hydroxytryptamine using high performance liquid chromatography with electrochemical detection. Electrical stimulation (cathodal monophasic 1 ms pulses, 300 microA, 2-10 Hz) of the dorsal raphe nucleus for 20 min induced an immediate release of 5-hydroxytryptamine which lasted for the duration of the stimulus and was frequency-dependent. The calculated amount of 5-hydroxytryptamine release per electrical impulse was constant over the frequency range used. Addition of tetrodotoxin (10 microM) to, or omission of calcium from, the perfusion medium reduced the spontaneous output of 5-hydroxytryptamine by 60-70% and caused a near complete inhibition of the effect of low frequency (3 Hz) electrical stimulation of the dorsal raphe nucleus. Local perfusion with cadmium (30 and 300 microM), which is reported to antagonize both N- and L-type voltage-sensitive calcium channels, also caused a pronounced decrease of basal output of 5-hydroxytryptamine and a marked, but not complete inhibition of the effect of nerve stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

A short period of hypoxia produces a rapid and transient rise in [K+]e in rat hippocampus in vivo which is inhibited by certain K(+)-channel blocking agents.

Extracellular potassium concentrations, [K+]e, were measured in vivo in the rat dorsal hippocampus using valinomycin-based double-barrelled ion-selective microelectrodes. Experiments were conducted under chloral hydrate anaesthesia. The microelectrodes were implanted stereotaxically, after which different gas mixtures were administered by inhalation. Transient hypoxia was induced by changing the inspired gas from 20% O2/80% N2 to 10-0% O2/90-100% N2 for 0.5-2 min. Resting [K+]e in the dorsal hippocampus was 3.4 +/- 0.09 mM; 0.5, 1 or 2 min of 100% N2 administration caused a rapid rise of [K+]e to 0.75, 1.9 and 15 mM, respectively. Following 0.5 min of 100% N2, the switch back to 20% O2/80% N2 produced an almost instantaneous return to normal levels. The return of [K+]e to basal levels was more delayed after 1 or 2 min of 100% N2 inhalation. The rise of hippocampal [K+]e induced by hypoxia was influenced by body temperature, the increase being five-fold higher in rats whose body temperature was raised from 33 to 37 degrees C using a heating blanket. Three potassium-channel blocking agents, quinine, 4-aminopyridine and gliquidone, were tested for their action on the increase in [K+]e, induced by inhalation of 100% N2 for 0.5 min. Both 4-aminopyridine and quinine, administered systemically, attenuated the anoxia-induced rise in [K+]e by 70 and 35%, respectively. In contrast, gliquidone, given by intracerebroventricular injection, had no effect, suggesting that ATP-sensitive potassium channels are not involved in this very early change in [K+]e.