Studies concerning ATP dynamics in essential hypertension.

The use of Beckmann LS-7000 liquid scintillation spectrometer enabled us to develop a rapid and reproducible technique for the enzymatic microdosage of ATP by means of the bioluminescence reaction of the luciferin-luciferase system from the firefly. Changes of ATP concentration in the plasma, erythrocytes and cerebrospinal fluid of the hypertensive subjects as compared to the control ones have been revealed by means of this method. According to our results ATP concentration significantly increases in the plasma and cerebrospinal fluid and slightly modifies in the erythrocytes of the hypertensive as compared with control subjects. Correlation of data concerning the variation of ATP distribution in the different compartments (plasma, erythrocytes, cerebrospinal fluid) with those related to Na and K content in hypertensive and control subjects seems to be useful for some remarks on the molecular mechanisms in the essential hypertension.

Dynamics and compartmentation of water in certain biosystems.

Investigation performed by nuclear magnetic resonance and by deuteration on the dynamics of water molecules in mammalian blood cells and in amphibian oocytes, muscles and nerves allowed to reveal several populations of cell water molecules. Following up relaxation and exchange processes in cell suspensions, packed cell pellets and in isolated tissues, characteristics of water dynamics and compartmentation in various biosystems are compared.

Sequential sodium-proton exchange in thrombin-induced human platelets.

Thrombin stimulation of human platelets initiates a membrane depolarization attributable to a Na+ influx into, and an alkalinization of, the cytoplasm, both of which follow a similar rapid time scale and thrombin-dose dependence. These responses precede secretion of the contents of the dense granules (serotonin) and, after 1 minute, of lysosomes (beta-glucuronidase). We have evaluated these parameters in the presence of 2H2O in order to determine if the Na+ influx and H+ efflux are sequential or simultaneous. NMR evidence indicates that 2H2O equilibration in rapid, and virtually complete within the 3 min prestimulation platelet equilibration period. In response to an 0.05 U/ml addition of thrombin, the rate of depolarization is 70-80% slower in 2H2O than in H2O. The time to reach maximal depolarization is 5 to 10 seconds longer in 2H2O, the extent of depolarization 60% inhibited, and the pH change 85% inhibited. The serotonin secretion is unaltered, while the beta-glucuronidase secretion is 130-180% enhanced. Dimethylamiloride inhibits the Na+ influx and the pH change completely. These results suggest that the Na+ and H+ fluxes across the plasma membrane are interdependent but neither simultaneous nor electroneutral. Furthermore, granule secretion, previously shown by us to be independent of the existent Na+ gradient, depends on the cytoplasmic K+ and H+ concentrations.

The structure-function relationships in the ionic channels of the axonal membranes as revealed by deuteration.

The present study has two purposes: c) to summarize our most important experimental results concerning the deuteration effects on the Ranvier node membrane as well as those obtained by others on the giant axons; ii) to reveal the significance of the macroscopic-microscopic correlations reflected in the excitation processes which underlie the ionic channel behaviour and for which deuteration has provided a new alternative of investigating the molecular mechanisms.

Structure/nerve membrane effect relationships of some local anaesthetics.

Data on electron structure and octanol-water partition coefficients of certain local anaesthetics are compared with the capacity of reversibly block the action potential of the frog sciatic nerve. Some active parts in the molecules of these substances have been shown to be primarily involved in the action of local anaesthetics.

On the electrical conductivity of rhodopsin solutions.

Measurements were made on the conductivity of digitonin extracts of frog rhodopsin with and without previous light exposure. The light-dark difference in conductivity is observed at low concentrations of rhodopsin and detergent.

Abolishment of the Ranvier node excitability under quasi-total deuteration and rhythmic stimulation.

The isolated nerve fibre, when quasi-totally deuterated, loses its excitability after about 15' of rhythmic stimulation at frequencies of 150-200 Hz. It is only on internal rehydration that excitability is recovered at high frequencies of stimulation. An excitation-energy coupling is suggested to exist, which appears to be more obvious at high frequencies of stimulation and strongly affected by the proton-deuteron exchange in the intracellular medium.

Water involvement in the mechanisms of retina electrical activity.

Retina is an excitable system containing approximately 90% water. As we found that deuteration selectively changes amplitudes and latencies of retina biopotentials, specifically the ON and OFF responses, we used it to probe the role of water in those processes. A study of the retina deuteration kinetics was simultaneously performed. This revealed the existence of at least two retinal water compartments. The data suggested a third compartment also, with a lower motional "degree of freedom," existing where H2O-D2O exchange becomes important only after saturation by D2O of the first two compartments. Correlation of the electrophysiological effects of D2O with the kinetic data suggests that the ON response is related to the first water compartment and the OFF response to the third. The results point to independence on the ON and OFF response mechanisms and, very probably, to their different morphological origins.

Computer simulation of the effect of the nodal gap resistance on ionic current measurements in the Ranvier node membrane.

Results are presented of a computer simulation of the effect of the irreducible resistance introduced by the nodal gap, in series with the impedance of the axon membrane. A clamp potential is applied to a structure modeled as an electric circuit composed of a resistance in series with the membrane impedance, and modified nerve equations describing membrane currents are solved to predict the effect of nodal series resistance on these currents. These studies reveal changes in the absolute values and kinetics of the ionic currents (errors greater than 10-20%) for selected values of series resistance.

Study of sodium and potassium intake in different categories of healthy subjects.

The usual Na+ and K+ intake was investigated in 436 apparently healthy subjects, 370 men and 66 women, ranging in age from 19 to 56 years, distributed into three lots: 219 from a metallurgical plant, 100 from a military unit and 117 from an unselected population sample. The average Na+ intake amounted to 214 mEq/24 h in the first lot of study, to 224 mEq/24 h in the second and to 203 mEq/24 h in the third, the differences among them being not significant. K+ intake in the three lots was 38 mEq/24 h, 43 mEq/24 h and 49 mEq/24 h, respectively. Na+/K+ ratio reached 5.6 in lot I, 5.2 in lot II and 4.1 in lot III, compared to the unitary ratio in natural foodstuffs. The data obtained demonstrate that Na+ intake in the general population is excessive, while K+ intake is deficient. It is assumed that this might partly account for the great incidence of arterial hypertension.

New data concerning the role of water in biosystems.

Data concerning the state and role of water (H2O) in the nerve, muscle and retina, obtained by the deuteriation technique, nuclear magnetic resonance (NMR) spectrometry and freeze-drying are reported. The existence of at least three distinct tissue water compartments is disclosed, whose sizes depend on the nature and state of the tissue. Some of these water populations appear to be indispensable to keeping up both the (supra) molecular architecture and the function of the system, while others have only a role in securing normal functioning. The mechanisms of the anesthetic action are also discussed and water involvement in this process is pointed out. Heavy water (D2O) effects on the cellular energy mechanisms are critically analysed. A change in the hydration degree of various species and the stabilization of the native conformation of biopolymers, as possible causes of heavy water effects, are remarked.

The Ranvier node as a chemo-electric pulsatory unit: a study of its structure-functions relations.

The Ranvier node of (Rana temporaria) frog nerve fibres is investigated by electron microscopy, Particular attention is given to the paranodal septate structures and to the extranodal junction of two Schwann cells. An interpretation of the functional meaning of these structures along with a quantitative analysis of the Schwann extranodal junction as regards the diffusion from/to the node is attempted. A 73 per cent reduction of the diffusion coefficient is obtained if the extranodal Schwann cell processes are considered impermeable to the diffusing vectors which indicates a protective role. Only 1 per cent reduction is obtained in the case of excitation-involved cations to which the Schwann cell membrane is considered to be permeable. This indicates the active role of the Schwann cell in the extranodal area ion diffusion, by minimizing the variations in ion concentration near the nodal membrane. Thus the nervous fibre-Schwann cell assembly may be regarded as a balanced pulsatory chemo-electric unit.

Heavy water effects on certain energetic processes in retina.

Heavy water effects on ATP concentration in frog retina were followed up. A substantial (more than 50%) decrease of ATP pools was found in the retina immersed in 2H2O-Ringer as compared to that in H2O-Ringer, which revealed that protons are strongly involved in the cellular energy processes.

Water compartments in living, glycerinated and fixed skeletal muscles of the frog.

The kinetics of water replacement with heavy water (deuterium oxide) in the gastrocnemius and sartorius muscles of the frog under isotonic conditions, studied both gravimetrically and by infrared photometry, reveals three water compartments: (i) non-exchangeable (approximately 80 ml/kg fresh weight), (ii) slowly exchanging (approximately 500 ml/kg fresh weight), (iii) rapid exchanging--extracellular (approximately 200 ml/kg fresh weight). Exposure to both glycerol and glutaraldehyde increases the permeability coefficients and the amount of rapid exchanging water; glutaraldehyde also increases the amount of non-exchangeable water. Approximately 90% of the water is kept in the tissue only by weak intermolecular forces, the energies of which amount to 1 kcal/mol. The amount of non-exchangeable water is equivalent to about six continuous adsorption layers covering the myofilaments. Approximately 70% of the tissue water appears to be replaced by glutaraldehyde during standard fixation.