Chemical, experimental, and clinical studies on endogenous ouabain-like substance in hypertension.

A heat-stable, low molecular weight, anionic substance(s) capable of inhibiting 3H-ouabain binding and Na+-K+-ATPase activity could be extracted from human urine and plasma. The level of the inhibitor was elevated in 40%-50% of essential hypertensives, compared to controls, and also in some of the offspring of hypertensive parents. Higher levels of the inhibitor were measured in patients treated with beta-blocking agents than in those treated with diuretics. The inhibitor extracted from plasma also appeared capable of (1) inhibiting the uptake of serotonin in human platelets, an Na+-dependent mechanism, and (2) inducing an increase in blood pressure when injected intracerebroventricularly. From these various data, we propose that the increase in the endogenous inhibitor may play a role in essential hypertension and may modulate, at least partially, some of the various cell functions that depend on a transmembrane Na+ gradient, including cellular excitability.

Platelet serotonin in essential hypertension and in mental depression.

The similarity between the metabolic pathways of serotonin in platelets and serotoninergic nerve endings has often been emphasized. The turnover of serotonin was therefore investigated in two diseases: hypertension (as central serotoninergic neurones appear to modulate central sympathetic nervous activity) and depression (as a central 5-HT-deficiency and a low 3H-imipramine binding on platelets have been described in patients with endogenous depression). Mean platelet 5-HT level was significantly lower in essential hypertensives than in controls. A reduction in platelet 5-HT level was also observed in depression and was more marked in women than in men. Serotonin level was only weakly related to the severity of the diseases. In some hypertensive patients, administration of ketanserin resulted in a reduction of blood pressure without affecting 5-HT level. In depressive patients, maprotiline and chlorimipramine acted differently on 5-HT level but both improved the clinical symptoms.

Decreased uptake of 3H-serotonin and endogenous content of serotonin in blood platelets in hypertensive patients.

The uptake and content of serotonin in blood platelets were studied in patients with essential hypertension and in five families in which at least one member was hypertensive. Blood was obtained from male and female normotensive volunteers and hypertensive patients who were free of medication. Lineweaver-Burk plots of 3H-serotonin uptake from both control subjects and hypertensive patients were linear, which suggested simple Michaelis-Menten uptake kinetics. The maximal uptake velocity (Vmax) in hypertensive patients was significantly lower than in control subjects (control = 41.7 +/- 3.3 pmol/min/10(8) platelets, n = 17; hypertensive = 26.6 +/- 3.0 pmol/min/10(8) platelets, n = 16; p less than 0.005). The affinity constant (Km) was slightly but significantly lower in hypertensive patients (control = 0.70 +/- 0.08 microM; hypertensive = 0.46 +/- 0.08 microM; p less than 0.05). The serotonin content in blood platelets determined by high pressure liquid chromatography with electrochemical detection was significantly lower in hypertensive patients (control = 165.0 +/- 12.9 nmol/10(11) platelets, n = 29; hypertensive = 105.9 +/- 10.4 nmol/10(11) platelets, n = 27; p less than 0.001). In the five families investigated, the lowered serotonin content was observed in some normotensive members. The reduced number of carriers of serotonin uptake and the slight decrease in the affinity constant observed in platelets of patients with essential hypertension suggest that serotonin metabolism is altered in essential hypertension and that blood platelets may be a useful model in studying the serotonergic modifications at the molecular level.

Reduced Vmax of 3H-serotonin uptake but unchanged 3H-imipramine binding in the platelets of untreated hypertensive subjects.

The uptake of 3H-serotonin, endogenous serotonin content and 3H-imipramine binding were measured in platelets of subjects with essential hypertension and matched control volunteers. The uptake of 3H-serotonin and endogenous serotonin levels in platelets were significantly reduced while 3H-imipramine binding did not differ in the two experimental groups. These results provide further evidence that the uptake site for serotonin and the binding site for 3H-imipramine although associated, may be modified independently.

Clinical and biochemical approach of a circulating Na+-pump inhibitor.

Inhibition of the activity of semipurified renal Na+, K+-ATPase and of 3H-Ouabain binding to erythrocytes was used to titrate the level of a plasmatic endogenous inhibitor of the Na+, K+-pump. The inhibition effect of plasma extracts was specific and unrelated to vanadate, calcium and products of proteolysis. Similar results were obtained with the two procedures. An endogenous pump inhibitor could be detected in 28 plasmas of the 42 normotensives investigated so far. In 18 out of 21 normotensives born of hypertensive parent(s) the level was found to be higher than that observed in normotensive controls devoid of hypertensive heredity. 13 untreated essential hypertensives out of 21 also exhibited higher levels of the sodium-potassium pump inhibitor. Plasma levels of the inhibitor appear stable during several months in male subjects. No clear relationship could be established either in normotensives or hypertensives between the pump inhibitor level, the urinary output of Na+, and blood pressure. Neither could any relationship be established between the inhibitor and the intraerythrocytic Na+ concentration. The accuracy of the methodology allows a large scale clinical investigation. It can also be used in the purification of the substance, and preliminary results precised that its molecular weight was inferior to 1 000 daltons and that it was anionic. Purified fractions were obtained after gel filtration, ion exchange chromatography, and high pressure liquid chromatography. Preliminary results suggest that the purified fraction may interfere with the mechanisms of hypertension. They inhibited the Na+-dependent serotonin uptake by human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma endogenous sodium pump inhibitor in essential hypertension.

The presence of circulating Na+ pump inhibitors was investigated in hypertensive subjects using inhibition of ouabain binding to the pump and of Na+, K+-ATPase activity as tests. Plasma extracts from nearly half the normotensive subjects who were offspring of hypertensive parents as well as the essential hypertensive subjects were potent inhibitors. Three fractions, extracted from plasma, exhibited ouabain-like properties concerning competition for binding, inhibition of the Na+, K+-ATPase and of Na+-dependent serotonin uptake by platelets. When injected intracerebroventricularly, one of these fractions also induces a rise in blood pressure, as does ouabain. These results demonstrate the presence in some plasma of digitalis-like substances.

Amphetamine inhibits the electrically evoked release of [3H]dopamine from slices of the rabbit caudate.

The effects of d-amphetamine on the spontaneous and electrically evoked release of [3H]dopamine in slices of the rabbit caudate nucleus were investigated. At a concentration of 0.1 microM amphetamine did not modify the spontaneous outflow of radioactivity, but significantly inhibited the release of [3H]dopamine elicited by electrical stimulation. At a 10-fold higher concentration (1 microM) amphetamine enhanced the spontaneous outflow of radioactivity and also inhibited the stimulation-evoked release of [3H]dopamine. The inhibition by amphetamine of electrically evoked release of [3H]dopamine was also observed under conditions in which monoamine oxidase was inhibited by pargyline. At concentrations of 0.1 and 0.5 microM amphetamine there was no inhibition of neuronal uptake and retention of [3H]dopamine in slices of the rabbit caudate. In the presence of 100 microM l-3-iodotyrosine, the inhibition by amphetamine of [3H]dopamine release was still obtained. The dopamine receptor antagonists, haloperidol and sulpiride, were not able to antagonize the inhibition by amphetamine of the electrically evoked release of [3H] dopamine at concentrations which effectively blocked apomorphine-induced inhibition of stimulation-evoked release of the labeled neurotransmitter. Exposure to serotonin in the presence of an inhibitor of neuronal uptake did not modify the spontaneous outflow of radioactivity or the electrically evoked release of [3H] dopamine. Nomifensine, an inhibitor of neuronal uptake of dopamine prevented the release of [3H]dopamine induced by exposure to 10 microM amphetamine and antagonized the inhibitory effects of lower concentrations of amphetamine on the electrically evoked release of [3H]dopamine. Tyramine and amfonelic acid in low concentrations enhanced the spontaneous outflow of radioactivity and, similarly to amphetamine, inhibited the electrically evoked release of [3H]dopamine. Exposure to bretylium (1 and 10 microM) inhibited the release of [3H]dopamine elicited by electrical stimulation. In the presence of bretylium, the inhibition by amphetamine of the stimulation-evoked release of [3H]dopamine was still present. In contrast to its inhibitory action on the release of [3H]dopamine, exposure to amphetamine (0.1-1.0 microM) enhanced in a concentration-dependent manner the electrically evoked release of [3H]norepinephrine from the rabbit hypothalamus. These results indicate that the inhibition by amphetamine of the electrically evoked release of [3H]dopamine does not involve the activation of presynaptic inhibitory dopamine autoreceptors possibly located on dopaminergic nerve terminals.(ABSTRACT TRUNCATED AT 400 WORDS)

[3H]Imipramine is accumulated but not released from slices of the rabbit caudate and hypothalamus.

Slices of rabbit caudate and hypothalamus take up and accumulate [3H]imipramine. In superfused slices of both structures electrical stimulation or exposure to tyramine failed to release recently taken up [3H]imipramine. Depolarization by exposure to 30--60 mM-potassium caused only a small release of [3H]imipramine that was not concentration-dependent. The release of [3H]imipramine by high potassium was independent of the presence of calcium ions in the superfusion medium. These results contrasted with those obtained for the release of [3H]dopamine from the caudate and [3H]noradrenaline from the hypothalamus, where tyramine, electrical stimulation, and high potassium caused a significant release of the labeled neurotransmitters. The release of [3H]dopamine from the caudate and [3H]noradrenaline from the hypothalamus elicited by electrical stimulation or high potassium was entirely calcium-dependent. It is concluded that [3H]imipramine is taken up into the two brain regions and is accumulated in a nonvesicular site from which it is not released by calcium-dependent depolarizing stimuli.

Inhibition by apomorphine of the potassium-evoked release of [3H]-gamma-aminobutyric acid from the rat substantia nigra in vitro.

1 The spontaneous and potassium-evoked release of tritium from the rat substantia nigra prelabelled with [(3)H]-gamma-aminobutyric acid [(3)H]-GABA were assessed in vitro under conditions of superfusion.2 Kainic acid lesions performed in the right caudate nucleus resulted in a 70% reduction in the ability of the homolateral nigral cells to take up and retain [(3)H]-GABA when compared with the unlesioned side. The potassium-evoked release of [(3)H]-GABA remained proportional to the radioactivity retained in the tissue suggesting that the nigral GABA neurones that survived kainic acid treatment were still functional.3 The spontaneous outflow of [(3)H]-GABA was significantly increased by exposure to different concentrations of exogenous GABA (10 to 1000 muM) when amino-oxyacetic acid was present in the incubation medium.4 Apomorphine in concentrations ranging from 1 to 30 muM inhibited the calcium-dependent release of [(3)H]-GABA induced by 1 min exposure to 30 mM K(+). These concentrations of apomorphine did not affect the spontaneous outflow of radioactivity. In vivo administration of haloperidol 0.2 mg/kg antagonized the in vitro inhibition by apomorphine of the K(+)-evoked release of [(3)H]-GABA.5 The results obtained with apomorphine and haloperidol suggest the presence of presynaptic dopamine-like inhibitory receptors in gabaergic nerve terminals.6 Dopamine in concentrations ranging up to 300 muM did not modify either the spontaneous or the K(+)-evoked release of [(3)H]-GABA from the substantia nigra. These concentrations of dopamine effectively displaced [(3)H]-dopamine recently taken up into the substantia nigra.7 Our results do not support the view that dendritic release of dopamine from the substantia nigra might be involved in the physiological modulation of the spontaneous or the stimulation-evoked release of GABA.