Several alterations in membrane transport systems are observed in rat and human hypertension. Na+ flux changes are numerous, and cellular homeostasis to Na+ loading is impaired. Transmembrane Ca2+ movements are also numerous but clearly defined by a reduction in Ca2+ binders, a hypersensitivity of membrane phospholipase C, possible increased Ca2+ leak and reduced sensitivity of the Ca2+-pump to calmodulin. The resulting Ca2+ increase within arterial cells can be responsible for increased contractility and tone, leading to hypertension. These functional alterations in membrane transport can be secondary to a few well-defined membrane defects of genetic origin or to a diffuse structural perturbation in membranes involving lipid changes.

Semi-purified dog kidney Na+,K+-ATPase cross-linked with ovalbumin was used in batch-wise affinity chromatography for the detection of endogenous Na+,K+-ATPase inhibitor in human plasma and urine. Ammonium acetate 1 M washed off the endogenous inhibitor from the immobilized enzyme. The inhibitory activity of the eluate from hypertensive plasma and urine was significantly higher (p less than 0.0025, n = 5 and p less than 0.005, n = 6 respectively) than that of normotensive. This latter was correlated with the ability of plasma from the same subjects to compete with ouabain binding to erythrocytes. Plasma and urine extracts inhibited the activity of Na+, K+-ATPase in a dose-dependent manner as ouabain does and were shown to contain 3 or 4 active compounds by high pressure liquid chromatography. The activity of some of these compounds was lost after peptidase treatment. These data support the heterogeneity of endogenous inhibitors of Na+,K+-ATPase activity in plasma and urine.

Evidence exists which demonstrates the relationship between a Natriuretic Factor or Na+,K+-ATPase inhibitor and volemic expansion, both in man and animal. Patients having extracellular volume expansion have been studied for the effect of their plasma on erythrocytes 3H-ouabain binding. High levels of ouabain-like activity was found in plasma from acromegalic patients and patients with chronic renal failure. High levels were also observed in some hypertensive patients. A partial purification of such a compound was performed from urine of hypertensives. The partially purified compound inhibited to a greater extent the Na+,K+-ATPase semi-purified from dog kidney than that from sheep brain. The present data are consistent with the possible regulation of the activity or the secretion of plasma ouabain-like activity by extracellular volume.

A Na+-pump inhibitor was purified from 140 liters of human urine to an apparent homogeneity. Tracing of the inhibitor during the different steps of purification was achieved by simultaneous determination of its capacity to inhibit the activity of Na+,K+-ATPase and ouabain binding, and to cross-react with antidigoxin antibodies. The final purification achieved a 400,000 fold. The purification steps included flash chromatography, anionic exchange chromatography, and reversed-phase HPLC on RP18, diphenyl and phenyl packings. NMR studies indicated that the final product was a non-peptidic, possibly steroidal compound. Its molecular weight as determined by mass spectrometry was 431.

Platelet serotonin levels were measured in several psychiatric disorders to determine whether they distinguish among major depressive disorder (one or more depressive episodes and no manic episodes), dysthymic disorder (depressive neurosis), and schizophrenic and paranoid disorders. Serotonin levels in 141 subjects were determined using high performance liquid chromatography with electrochemical detection. Serotonin (5HT) levels in control subjects were significantly lower in males than in females. A marked reduction in 5HT levels, as compared to controls, was found in male and female patients with major depressive disorder, but not in dysthymic disorder. A slight but significant reduction in serotonin levels was found in female schizophrenic patients. The reduction in serotonin levels found in major depressive disorder could not be attributed to chronic antidepressant treatment. Liquid chromatography with electrochemical detection used in the present study permits a large-scale investigation.

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.

Abstract: [3H]Rauwolscine, a specific, potent, radiolabelled α2‐antagonist, binds to distinct high‐ and low‐affinity α2‐adrenoceptors in crude membrane preparations of the rat cerebral cortex. The concentration of high‐affinity α2‐adrenoceptors was increased by addition of sodium ions or guanylnucleotides. In synaptosomal plasma membrane preparations, only the low‐affinity component was found. Neither sodium or guanylnucleotides caused any increase in the concentration of these low‐affinity receptors for [3H]rauwolscine.

Experimental epidemiological and clinical studies show clearly that there exists a close relationship between primary hypertension and body sodium (Na+). The fact that the first investigators were oriented towards extracellular Na+ is not surprising when we consider the high concentration of this ion in the extracellular fluid volume, compared to the intracellular medium, as well the undisputable role of this body fluid compartment in the maintenance of arterial pressure. However, numerous studies have been unable to demonstrate any clear-cut increase in the extracellular fluid volume in essential hypertension, a result that would be expected if an increase in extracellular Na+ were involved [1].

1. The distribution and metabolism of inositol phospholipids were studied in the erythrocyte membrane of spontaneously hypertensive and normotensive Wistar-Kyoto rats. The incorporation of 32P into these lipids was considered as an index of their turnover, and a reflection of their metabolism. 2. With [γ-32P]ATP as the source of label only the polyphosphoinositides (diphosphoinositide and triphosphoinositide) incorporated 32P. The levels of labelling were lower in hypertensive than in normotensive rats. Our data indicate that, in the hypertensive rats, the kinases responsible for the labelling of polyphosphoinositides exhibited decreased Vmax. for ATP. 3. These changes may be related to those described for calcium binding and transport in the erythrocyte of spontaneously hypertensive rats.

The metabolism of phosphoinositides was investigated in the red blood cell membrane of the rat by measuring 32P-incorporation into phospholipids after incubation of membranes with [γ-32P]ATP in a medium containing magnesium. A new chromatographic procedure has been developed which facilitates the separation of triphosphoinositide, diphosphoinositide and phosphatidylinositol from the phospholipids present in lipid extracts of incubated ‘ghost’ membranes. Under our experimental conditions only two phospholipids, diphosphoinositide and triphosphoinositide, were 32P-labelled. Furthermore, the results indicate that either di- or triphosphoinositide could be labelled preferentially, depending upon the magnesium concentration of the incubation medium. This clarifies some apparent discrepancies reported in the literature between the 32P labelling of polyphosphoinositides observed in intact erythrocytes and that observed with ‘ghost’ membranes. In addition, the enzymatic pathways involved in the phosphoinositide metabolism are discussed.

Serum serotonin (5-HT) levels were measured in several patients with psychiatric disorders using high pressure liquid chromatography with electrochemical detection. A marked reduction in 5-HT levels was found in male and female patients with major depressive disorder, as compared to controls, but not in dysthymic disorder. These modifications may constitute biochemical changes suggestive of major depressive disorders; they could not be attributed to chronic antidepressant treatment.

The catecholamine concentration and dopamine-beta-hydroxylase activity were determined in several nuclei of the brain of spontaneously hypertensive rats (SHR) compared with Wistar Kyoto (WKY) controls. Catecholamines were measured by using liquid chromatography coupled with electrochemical detection. The threshold of detection was 5 X 10(-14) mole. Dopamine-beta-hydroxylase (DBH) was assayed by a sensitive radioenzymatic assay using tyramine as the substrate. The limit of detection was 5 pmoles of octopamine per sample. Significantly lower noradrenaline content was observed in 4 week-old SHR in some medullary and hypothalamic areas which are involved in cardiovascular regulation. This abnormally was no longer detectable in 12 week-old rats. The changes in catecholamine levels observed in young rats were not observed during the development of deoxycorticosterone-salt hypertension and therefore probably do not represent a compensatory mechanism tending to limit the progressive rise blood pressure. No significant difference in DBH activity was observed between the young SHR and WKY in any brain region measured. The altered relationship between noradrenaline content and DBH activity observed in young SHR when compared to WKY suggests a change in noradrenergic neurones activity and/or structure which could correspond to a genetically transmitted neurochemical abnormality associated with the initiation of hypertension in the SHR.

Abstract: A large number (about 4‐5 nmoVmg of protein) of high‐affinity (apparent dissociation constant at 37°C: KD37°C= 5 × 10−8M) calcium binding sites was characterized in synaptosomal membrane fractions enriched in plasma membranes that were isolated from rat brain. These sites were studied simultaneously in membranes from spontaneously hypertensive young rats (SHR) and their normotensive controls. No difference was observed between whole synaptosomes from these two substrains. However, plasma membraneenriched fractions from SHR exhibited a reduced calcium binding capacity without a significant change in affinity. This decrease which averaged 15‐20% was not due to any variation in the accessibility of calcium to its binding sites, as similar results were obtained in the presence of the calcium ionophore A23,R7. The reduction found in calcium binding is very similar to that previously described in erythrocyte membranes. It is envisaged that such an abnormality at nerve endings might play a role in the pathogenesis of genetic hypertension.

In rat vas deferens, (+/-)-170 150 proved to be an alpha 2-adrenoceptor blocking agent with a high alpha 2/alpha 1 selectivity ratio. In rat cerebrocortical membranes, (+/-)-170 150 was seven times more potent than yohimbine for inhibiting specific [3H]clonidine binding. However, (+/-)-170 150 appeared to be 3.5 times less selective for alpha 2-adrenoceptors sites than yohimbine. These results indicate that (+/-)-170 150 is a potent preferential alpha 2-adrenoceptor blocking agent, as had been shown by in vivo experiments.

1. Diphenylhexatriene was used as a fluorescence probe to detect structural differences between membranes from genetically hypertensive rats and those from their normotensive controls. Both isolated membranes (erythrocyte ghosts) and whole cells (platelets) were studied. 2. In the Okamoto-Aoki strain, the fluorescence polarization of diphenylhexatriene and consequently the ‘equivalent microviscosities' were altered in all membranes, even in those of young normotensive SHR. These abnormalities varied with age (in whole cells) and were not detected in females. 3. These alterations were not restricted to the SHR strain and were also observed in the Sabra hypertensive strain (SBH). The ‘equivalent microviscosity’ of hypertensive Sabra erythrocyte ghosts was higher than that of the original Sabra rats. Salt loading of the Sabra rats promoted an increase in the ‘equivalent microviscosity’ of their erythrocyte membranes, which nearly reached the Sabra hypertensive level. 4. These results support the hypothesis of a genetic change in hypertensive rats leading, directly or indirectly, to diffuse alterations of cell membrane structure, which seem to be similar to those caused by high salt intake.

A stainless steel guide was implanted in the anterior third ventricle of the anesthetized rat and an internal needle shorter than the guide was used to continuously collect cerebrospinal fluid (CSF) at a constant outflow of 1 microliter/min. Five microliter samples were injected directly into a liquid chromatographic column. The mobile phase was adjusted for selective separation of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5-HIAA). Electrochemical detection with a limit of 0.05 pmol was used, 5-HTP and 5-HT concentrations were in the 10(-8) M range in controls while DOPAC and 5-HIAA were in the 10(-7) and 10(-6) M range. Brain aromatic amino acid decarboxylase inhibition with high doses of benserazide corresponded to an increased CSF level of 5-HTP. Monoamine oxidase inhibition with tranylcypromine resulted in a diminution of DOPAC and 5-HIAA. L-Tryptophan loading associated with monoamine oxidase inhibition induced an increase in CSF level of serotonin. These pharmacologically induced changes in serotonin and dopamine metabolite levels exemplify the usefulness of these CSF determinations as indices of brain function.