A weak electrical current enhances intracellular delivery of therapeutic substances.

Cell membranes have electrical properties which can be measured and modified. Administering a weak electrical signal across a tissue is a technique which can be used to determine the quantity of water in the intracellular and extracellular compartments. Using this real-time method of bioelectrical impedance, it was found that a variety of stimuli (including an electrical current) can enhance the passage of water and other substances into the cell. We propose an inexpensive and safe technique which could be utilized to enhance delivery of a wide variety of therapeutic agents into cells for the enhanced delivery of antibiotics, chemotherapy or other therapeutic agents.

Protein carboxyl methylation controls intracellular pH in human platelets.

OBJECTIVES: Carboxyl methylation is a reversible post-translational event which regulates the function of several cellular proteins. Because the human Na+-H+ antiporter (NHE-1) possesses a C-terminal consensus sequence for carboxyl methylation, we examined the role of protein carboxyl methylation in the regulation of intracellular pH homeostasis. DESIGN: Experiments were conducted using human platelets and N-acetyl-S-trans,trans-farnesyl-L cysteine (AFC), a specific prenylcysteine methyltransferase inhibitor. The effect of AFC on both basal intracellular pH (pHi) and on the kinetic properties of the Na+-H+ antiporter was characterized. MATERIALS AND METHODS: pHi was determined in cell suspensions using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein tetraacetoxymethyl ester, a fluorescent pH indicator. The kinetics properties of the Na+-H+ antiporter activity were determined using platelets acidified with nigericin and challenged with varying extracellular concentrations of Na+. RESULTS: AFC (20 micromol/l) decreased basal pHi significantly (7.047 +/- 0.011 versus 7.133 +/- 0.012 for control, P< 0.001). The acidification was dose-dependent and reached steady state 3 min after AFC addition. In the absence of extracellular Na+, the platelets were acidified to the same extent with AFC or with ethanol (control): 6.530 +/- 0.031 versus 6.532 +/- 0.031 (P= 0.97). However, upon addition of Na+, the platelets treated with AFC showed a significant decrease in the maximal value for initial pHi recovery compared with controls: 0.788 +/- 0.041 versus 0.983 +/- 0.047 pH/min (P< 0.02). AFC also increased the Hill coefficient (2.89 +/- 0.22 versus 2.14 +/- 0.16, P < 0.03), and tended to decrease K0.5, the [Na+] corresponding to half-maximal activation (51.3 +/- 1.8 versus 60.5 +/- 3.9 mmol/l, P = 0.06) of the antiporter. CONCLUSION: Our data indicate that inhibition of carboxyl methylation reduces basal pHi and alters the kinetic properties of the Na+-H+ antiporter in human platelets, suggesting that carboxyl methylation is implicated in the regulation of intracellular pH homeostasis.

Farnesol inhibits L-type Ca2+ channels in vascular smooth muscle cells.

Earlier experiments with animal and human arteries have shown that farnesol, a natural 15-carbon (C15) isoprenoid, is an inhibitor of vasoconstriction (Roullet, J.-B., Xue, H., Chapman, J., McDougal, P., Roullet, C. M., and McCarron, D. A. (1996) J. Clin. Invest. 97, 2384-2390). We report here that farnesol reduced KCl- and norepinephrine-dependent cytosolic Ca2+ transients in fura-2-loaded intact arteries. An effect on Ca2+ signaling was also observed in cultured aortic smooth muscle cells (A10 cells). In these cells, farnesol reduced KCl-induced [Ca2+]i transients and mimicked the inhibitory effect of Ca2+-free medium on the [Ca2+]i response to both 12,13-phorbol myristate acetate, a protein kinase C activator, and thapsigargin, a specific endoplasmic reticulum ATPase inhibitor. Perforated patch-clamp experiments further showed in two vascular smooth muscle cell lines (A10 and A7r5), a reversible, dose-dependent inhibitory effect of farnesol on L-type Ca2+ currents (IC50 = 2.2 microM). Shorter (C10, geraniol) and longer (C20, geranylgeraniol) isoprenols were inactive. L-type Ca2+ channel blockade also occurred under tight (gigaohm) seal configuration using cell-attached, single-channel analysis, thus suggesting a possible action of farnesol from within the intracellular space. We finally demonstrated that farnesol did not affect Ca2+-sensitive pathways implicated in smooth muscle contraction, as tested with alpha-toxin permeabilized arteries. Altogether, our results indicate that farnesol is an inhibitor of vascular smooth muscle Ca2+ signaling with plasma membrane Ca2+ channel blocker properties. The data have implications for the endogenous and pharmacological regulation of vascular tone by farnesol or farnesol analogues.

Farnesyl analogues inhibit vasoconstriction in animal and human arteries.

Recent studies have suggested that nonsterol, mevalonate-derived metabolites are implicated in the control of vascular tone and blood pressure. Because of the metabolic importance of farnesyl pyrophosphate, a 15-carbon (C15) intermediate of the cholesterol pathway, the vasoactive properties of the farnesyl motif were investigated. Two farnesyl analogues were used: farnesol, the natural dephosphorylated form of farnesyl pyrophosphate, and N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), a synthetic mimic of the carboxyl terminus of farnesylated proteins. Both compounds inhibited NE-induced vasoconstriction in rat aortic rings at micromolar concentration. Their action was rapid, dose dependent, and reversible. Shorter (C10) and longer (C20) isoprenols as well as N-acetyl-S-geranyl-L-cysteine (C10) did not inhibit the response to NE. In contrast, N-acetyl-S-geranylgeranyl-L-cysteine (C20), exhibited vasoactive properties similar to AFC. It was further demonstrated that AFC and farnesol inhibited KCl and NaF-induced contractions, suggesting a complex action on Ca2+ channels and G protein-dependent pathways. Finally, the effect of farnesol and AFC on the NE response was reproduced in human resistance arteries. In conclusion, mevalonate-derived farnesyl analogues are potent inhibitors of vasoconstriction. The study suggests that farnesyl cellular availability is an important determinant of vascular tone in animals and humans, and provides a basis for exploring farnesyl metabolism in humans with compromised vascular function as well as for using farnesyl analogues as regulators of arterial tone in vivo.

Mevalonate availability affects human and rat resistance vessel function.

Previous data in rat conductance vessels indicated that cellular mevalonate contributes to vascular tone and systemic blood pressure control. Using exogenous mevalonate (M) or lovastatin, a 3-hydroxy-3-methyl-glutaryl CoA (HMG-CoA) reductase inhibitor (L), we characterized the role of mevalonate availability in resistance artery function, both in experimental animals and humans. Rat mesenteric artery resistance vessels (MARV, n = 9) were incubated for 48 h with either L, M, L + M, or vehicle (V) and tested for reactivity to NE, serotonin, acetylcholine, atrial natriuretic peptide, and sodium nitroprusside (SNP). Lovastatin increased sensitivity to NE (P < 0.03) and serotonin (P < 0.003), and significantly impaired the response to all three vasodilators. These effects were reversed by co-incubation with mevalonate. Mevalonate alone had no effect. In separate experiments, intravascular free Ca2+ concentration (ivfCa2+) was determined in fura-2AM loaded MARV. Basal ivfCa2+ was increased after a 48-h exposure to L (52.7 +/- 4.6 nM, L, vs. 29.7 +/- 2.4 nM, V, n = 12, P < 0.003), as were ivfCa2+ levels following stimulation with low (100 nM) NE concentrations. Similar ivfCa2+ concentrations were achieved during maximum contraction with NE (10 mM) in both groups. Human resistance arteries of human adipose tissue were also studied. Lovastatin increased the sensitivity to NE (ED50 = 372 +/- 56 nM, V, and 99 +/- 33 nM, L, P < 0.001) and significantly decreased the relaxation to acetylcholine and SNP of human vessels. We conclude that mevalonate availability directly contribute to resistance vessel function and vascular signal transduction systems in both experimental animals and humans. The study calls for the identification of non-sterol, mevalonate-derived vasoactive metabolites, and suggests that disorders of the mevalonate pathway can alter vascular tone and cause hypertension.

In vivo effect of calcitriol on calcium transport and calcium binding proteins in the spontaneously hypertensive rat.

The abnormal intestinal Ca2+ transport reported in spontaneously hypertensive rats (SHR) has been attributed to decreased responsiveness to calcitriol. We reexamined this hypothesis by studying the calcitriol regulation of SHR duodenal calbindin-D9K and calmodulin and the relation of calcitriol to Ca2+ uptake by isolated enterocytes. SHR and normotensive Wistar-Kyoto (WKY) rats were injected with either 50 ng/d calcitriol (vit-D) or vehicle alone (control) for 3 days. Decreased calbindin-D9K (P < .001) and cellular Ca2+ flux (P < .001) were observed in control SHR. Calcitriol increased total cell and brush border calbindin-D9K (P < .0001); this variation paralleled plasma calcitriol levels in both strains. In contrast, Ca2+ flux, which increased in vit-D animals, remained lower in SHR for plasma calcitriol levels similar to those in WKY rats. Immunoreactive calmodulin was similar in both strains whether assayed in total cell or brush border membranes. In contrast, when measured by ligand blotting (45Ca), calmodulin was lower in SHR than in WKY rats (P < .01), suggesting the existence of a calmodulin pool with reduced Ca2+ binding capacity in the hypertensive strain. Calcitriol had no effect on calmodulin in either strain. In conclusion, Ca2+ binding protein regulation by calcitriol is normal in the SHR, and decreased hormone responsiveness cannot account for the defective duodenal calcium transport of this experimental model of hypertension.

Dietary calcium, defective cellular Ca2+ handling, and arterial pressure control.

The association between dietary calcium intake, calcium metabolism, and blood pressure form the basis of this review. Epidemiologic data consistently show an inverse relationship between dietary calcium and blood pressure. Clinical trials of calcium supplementation have not been as consistent in outcome. Approximately two-thirds of the supplementation studies have found a beneficial effect of calcium on blood pressure. The lack of consistency in outcome from the clinical trials relative to the epidemiological literature may be related to calcium intake. The epidemiological literature indicates an inverse relationship between calcium intake and blood pressure, with those individuals with the lowest calcium intake (< 700 mg/day) having the highest blood pressure. Clinical studies utilizing patients with high baseline calcium levels (> 700 mg/day) may not see an effect of calcium supplementation on blood pressure because of a ceiling effect. Supplemental calcium appears to correct a defect in calcium handling characterized by a renal calcium leak, increased circulating parathroid hormone, and increased intracellular calcium levels. In part, the deficit in cellular calcium homeostasis may be a consequence of abnormal calmodulin activity. Specifically, it appears that calmodulin activity is diminished in experimental hypertension and that increasing dietary calcium may improve calmodulin activity in the spontaneously hypertensive rat. The deficit in calmodulin activity has the potential to interfere with a number of cellular processes crucial to the regulation of cell function and maintenance of appropriate vascular tone. It is concluded that additional research should be directed toward understanding the ramifications of altered calmodulin activity in hypertension and the influence that dietary calcium can have on the activity of calmodulin.

Abnormal regulation of intestinal calbindin (CaBP9k) and calmodulin in the spontaneously hypertensive rat.

This review focuses particularly on abnormalities of Ca-binding proteins in transporting epithelia, which have been observed in various models of experimental hypertension. The enterocyte content of integral membrane Ca(2+)-binding protein (IMCAL) and calbindin-9k and the renal tubular calbindin-28k content have been shown to be decreased in the spontaneously hypertensive rat (SHR), compared with normotensive control rats (WKY). Similarly, calmodulin content was decreased in several tissues including the intestine; however, calmodulin activity was increased. In recent studies, the authors examined the response of intestinal calbindin-9k, calbindin-9k mRNA, and calmodulin contents to low-Ca and high-Ca diets in these two rat strains. It was shown that the SHR, unlike the WKY, was unable to augment intestinal calbindin-9k and calbindin-9k mRNA levels in response to a low-Ca diet of short duration. On the other hand, a high-Ca diet led to a similar decrease in calbindin-9k content in both SHR and WKY rats. Enterocyte calmodulin content was also diminished in the WKY, but not the SHR, fed such a high-Ca diet. Therefore, abnormal Ca-binding proteins could play a role in the disturbed Ca metabolism of arterial hypertension.

Mevalonate availability and cardiovascular functions.

Data delineating the relationship between disorders of cholesterol metabolism and elevated blood pressure (BP) do not exist. We postulated that mevalonate, the metabolic precursor of endogenous cholesterol and the direct product of 3-hydroxy-3-methylglutaryl-CoA reductase, was a contributing factor for the maintenance of vascular tone and systemic BP. We conducted in vivo, ex vivo, and in vitro experiments in normotensive and hypertensive rats, where exogenous mevalonate and lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, were used, respectively, to increase or limit mevalonate availability. Mevalonate decreased BP in the whole animal without significant change in plasma cholesterol. Incubation of aortas with mevalonate attenuated their reactivity to norepinephrine and increased their response to endothelium-dependent and -independent relaxing factors. Lovastatin, in contrast, had the opposite effect in vivo and in vitro: it increased BP, enhanced vascular response to norepinephrine, and impaired endothelium-dependent and -independent relaxations. Neither agent modified cholesterol vascular content. Alteration of vascular reactivity was also observed in resistance vessels from animals pretreated with lovastatin. Our findings suggest that mevalonate availability is an unrecognized metabolic contributor to vascular tone and BP. They imply that (i) metabolites of the mevalonate pathway other than cholesterol could potentially control vascular functions and cardiovascular hemodynamics, (ii) elevated arterial pressure could be in part the consequence of primary disorders of this pathway, and (iii) pharmacological inhibition of mevalonate production as a means to lower plasma cholesterol may have an adverse impact on other cardiovascular risk factors, such as BP.

Abnormal intestinal regulation of calbindin-D9K and calmodulin by dietary calcium in genetic hypertension.

Using isolated duodenal cells from spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar-Kyoto rats (WKY), we previously showed that cellular calcium flux was decreased in SHR and that increasing dietary calcium (from 1 to 2%) eliminated strain differences in Ca2+ fluxes. The present study was carried out to investigate the role of calbindin-D9K and calmodulin in the flux difference and dietary calcium effects. Calbindin-D9K and calmodulin were separated by sodium dodecyl sulfate (SDS) gel electrophoresis in duodenal protein extracts of SHR and WKY (12-14 and 24-26 wk old) fed either a 1 or 2% calcium diet and measured by a ligand blotting (45Ca) technique. Young SHR had a significantly lower calbindin-D9K (P less than 0.001) than did WKY on either diet. Calmodulin was significantly lower in young SHR than in WKY (P less than 0.002). There was no strain difference in calmodulin in older rats fed the normal calcium diet. Calbindin-D9K was significantly decreased by the high-calcium diet in both strains at both ages. There was a significant correlation between duodenal calbindin-D9K and plasma levels of calcitriol (r = +0.80, P less than 0.001) in WKY but not in SHR. Calmodulin was significantly decreased by dietary calcium in mature WKY (4.8 +/- 0.2 vs. 3.7 +/- 0.4 micrograms/mg cell protein, P less than 0.03), demonstrating a potential regulation by dietary calcium of this protein. Finally, there was a significant correlation between calbindin-D9K and calmodulin (r = 0.59, P less than 0.001) in WKY but not in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary calcium and blood pressure: modifying factors in specific populations.

Epidemiologic findings continue to add to the body of evidence supporting a relationship between calcium intake and blood pressure. These findings also indicate that there is a threshold of the potential protective effect of adequate calcium intake, below which the risk of hypertension increases at a greater rate. The set point of this threshold, estimated at 700-800 mg/d, may be modified by a variety of factors including dietary patterns and components, lifestyle, and genetics. This may explain, at least in part, the heterogeneous response observed in dietary-intervention studies. In animal models of hypertension it was shown that greater amounts of calcium must be given to cause a blood pressure change comparable with that in normal animals, suggesting that in high-risk human populations in which calcium metabolism may be disordered, calcium intake may have to be increased to amounts greater than 700-800 mg/d to demonstrate the blood-pressure-lowering effect. Calcium intake at or above the currently recommended daily allowance of 800 mg could be of potential benefit to certain racial groups, individuals ingesting excessive alcohol, and pregnant women, all of whom generally consume low amounts of calcium and who are at higher risk of developing hypertension.

Developmental changes of Ca2+, PO4, and calcitriol metabolism in spontaneously hypertensive rats.

We have measured Ca and P balance, serum calcitriol, and vitamin D-dependent intestinal calcium-binding protein (CaBP9k) in the spontaneously hypertensive rat (SHR) and its normotensive control, the Wistar-Kyoto rat (WKY) at 4-5 and again at 13-14 wk of age. In rats on a 1% Ca diet, P balance was significantly more positive in the 5-wk-old SHR than WKY (P less than 0.01); Ca balance tended to be greater in the 5-wk-old SHR. In contrast, in the 14-wk-old SHR, P and Ca balance were less positive than in the WKY (P was 5.8 +/- 1.3 vs. 13 +/- 1.7 mg/day, P less than 0.01, and Ca was 53 +/- 4.6 vs. 67 +/- 2.7 mg/day, P less than 0.05). On the 1% Ca diet, plasma calcitriol levels of the 5-wk-old SHR were higher than those of the WKY (58 +/- 3.2 vs. 40 +/- 2.1 pg/ml, P less than 0.002), whereas at 12 wk there was no difference. On Ca-deficient (0.1%) diets, plasma calcitriol was increased in 5-wk-old and 12-wk-old SHR and WKY, compared with the 1% Ca diet (P less than 0.001). Calcitriol metabolic clearance rate was the same in the 13-wk-old SHR and WKY on either Ca diet. Intestinal CaBP9k content of the 5-wk-old (38 +/- 2.6 vs. 40 +/- 3.9 micrograms/mg) and the 12-wk-old (11 +/- 1.6 vs. 14 +/- 1.4 micrograms/mg) SHR and WKY were similar on the 1% Ca diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium uptake by duodenal enterocytes isolated from young and mature SHR and WKY rats: influence of dietary calcium.

Intestinal calcium (Ca2+) transport was examined at the cellular level using duodenal enterocytes isolated from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Compartmental analysis of 45Ca2+ uptake was performed on enterocytes isolated from young (12- to 14-wk-old) and mature animals (24- to 26-wk-old) fed either normal (1%) or high (2%) calcium diets. Intracellular Ca2+ flux (Jc) was reduced in SHR compared with WKY for both young (0.67 +/- 0.05 vs. 1.08 +/- 0.08 nmol Ca2+.mg protein-1.min-1 P less than 0.01) and mature (0.39 +/- 0.03 vs. 0.71 +/- 0.05 nmol Ca2+.mg protein-1.min-1; P less than 0.001) animals on a normal calcium diet. On a high-calcium diet, this strain difference of Jc disappeared in the young rats (0.87 +/- 0.09 vs. 1.06 +/- 0.06 nmol Ca2+.mg protein-1.min-1, NS). In mature SHR, the high-calcium diet stimulated Jc, whereas it lowered it in mature WKY resulting in a similar flux for both strains (0.56 +/- 0.05 vs. 0.49 +/- 0.05 nmol Ca2+.mg protein-1.min-1, NS). Young SHR had a lower intracellular Ca2+ pool compared with WKY. This defect was corrected by a high-calcium diet. The membrane Ca2+ flux (Jm) was lower in mature SHR than WKY fed a normal calcium diet (P less than 0.02); Jm increased to the control value (P less than 0.05) in the SHR on a high-calcium diet. Diet-induced changes of plasma 1,25(OH)2 vitamin D levels in the SHR did not parallel the observed changes of intestinal Ca2+ fluxes. Thus duodenal enterocytes from SHR appear to have an intrinsic alteration of Ca2+ transport that can be corrected in part by a higher calcium diet.

Rapid stimulation of calcium uptake by isolated rat enterocytes by 1,25(OH)2D3.

Evidence is accumulating that 1,25(OH)2D3 may stimulate calcium transport from the intestinal lumen extremely rapidly by a mechanism which appears independent of de novo protein synthesis. To investigate this rapid action of 1,25(OH)2D3, the rate of calcium uptake by isolated enterocytes from duodena of young rats was determined in vitro as the uptake of 45Ca from 1-15 min. Prior in vitro exposure of cells to 1,25(OH)2D3 (100 pM) for 20 min significantly increased the rate of calcium uptake (p less than 0.001), an effect unaltered by 50 microM cycloheximide. Incubation with 100 pM 1-alpha-hydroxyvitamin D3 produced the same effect (p less than 0.01). In contrast, exposure to 10 pM 1,25(OH)2D3, as well as to 100 pM or to 1,000 pM 25-hydroxyvitamin D3 induced no significant change. Because both 1,25(OH)2D3 and starvation may stimulate key enzymes in polyamine metabolism, we investigated the effects of (i) difluoromethyl-ornithine (CHF2-Orn), a specific irreversible inhibitor of ornithine decarboxylase and (ii) varying the timing of feeding prior to sacrifice. Both in vitro CHF2-Orn and feeding prior to sacrifice significantly decreased the baseline rate of calcium uptake (p less than 0.05) and reduced the effect of 1,25(OH)2D3. Increased duration of starvation significantly increased the baseline rate of calcium uptake (p less than 0.02) without changing the increment in rate of calcium uptake induced by 1,25(OH)2D3. The study suggests (i) that the early action of 1,25(OH)2D3 on the influx process of intestinal calcium transport may involve a different molecular specificity from that involved in the genomic action of 1,25(OH)2D3 and (ii) that changes in polyamine metabolism may play a part in this process.

Decreased duodenal enterocyte calcium flux rates in the spontaneously hypertensive rat.

We have previously reported serum 1,25(OH)2 vitamin D3 (calcitriol) and active transduodenal calcium absorption measured in the Ussing chamber to be reduced in 12- to 14-week-old male Okamoto-Aoki spontaneously hypertensive rats (SHR). In the present study, we compared rates of calcium influx and efflux in isolated duodenal enterocytes in SHR and corresponding controls, Wistar-Kyoto rats (WKY). The early (0 to 1 minute) and the late (1 to 15 minute) phase of calcium influx rates at 1.0 mmol Ca2+ in the incubation medium were lower in the SHR than in the WKY (mean +/- SEM): 1.93 +/- 0.22 v 2.85 +/- 0.41 nmol/mg protein/min, n = 8 and n = 7 experiments, respectively, P less than .05; and 0.334 +/- 0.025 v 0.488 +/- 0.059 nmol/mg protein/min, n = 14 pairs, P less than .01. The calcium efflux rate constant of the SHR was reduced: 34.3 +/- 1.4 v 51.9 +/- 1.4% per hour, n = 11 pairs, P less than .01. However, in the absence of sodium or the presence of ouabain (4.0 mmol) in the incubation medium, a decrease in this constant was observed in the WKY but not in the SHR. These data, at the cellular level, support our previous observation in intact tissue of reduced active transduodenal calcium transport in the 12- to 14-week-old SHR. Whether the primary defect in calcium transport involves the luminal or the basolateral membrane of the enterocyte, or whether both disturbances are due to a common primary perturbation cannot be deduced from the present experiments.

The physiological surveillance of hemodialysis sessions by the continuous measurement of L.F. impedance of the circulating blood (Thomasset's method).

Everybody grants as a fact that hemodialysis disturbances are produced by volemia variations. During H-D the vascular sector is the necessary transit medium to withdraw excessive interstitial water. If this withdrawing is greater than the coming back of the interstitial water into the vascular sector, the plasmatic volume will be decreasing. Thus, to measure the variations of the plasmatic volume in circulating blood it is sufficient to effect a continuous measurement of these ones in a transducer having a definite volume. So, by the mean of an impedancemeter, 5 kHz impedance is continuously measured between the two electrodes of the transducer, because it is known (Thomasset's method) that at 5 kHz current uses only the plasma in order to pass from one electrode to another.

Determination of body fluid compartments by electrical impedance measurements.

?The ratio of the LF and HF impedances appears to be an excellent and simple tool for investigation of liquid specimens, either of the total human body, taking into consideration global impedances, or of a particular organ, taking into consideration local impedances. We have sketched out in this communication a study of the global impedance ratio variations with age, but a large number of studies still remain to be undertaken in those cases of severe denutrition, as in all cases of metabolic illnesses. We can state that, in most cases, not only does the ratio decrease but that there is extreme difficulty in reestablishing normal values. Whatever action is undertaken, it seems that the intracellular liquid compartment remains insufficient with respect to that of the extracellular liquid compartment. It is as if the water will not enter in the cells or as if it were no longer retained within the cells. Finally, with this concept of impedance ratios of Zlf/Zhf a promising experimental method has been found which will, perhaps, enable better infestigations in a field which has often been left to one side.

Investigation of electrical impedance variations of dog brain tissue during experimental metabolic disturbances.

Experiments were carried out on dogs using bielectrode probes having as adequate impedancemeter. The probe is introduced via a cranial opening into the grey matter. This gives the following: a low frequency reading which is related to the extra cellular fluid component; a high frequency reading which is related to the total overall liquids; and a proportionality ratio of these liquids in the explored volume. The impedance variations are functions of the nature and intessity of vascular disturbance. Variation of the low frequency impedance (5 kHz) is the most significant. The experimental prodedure consists of: 1) Abrupt and permanent circulatory arrest; 2) Circulatory reduction by haemorrhage followed by recovery (if necessary by means of blood transfusion); 3) Anoxia by CO inhalation, recovery affected by means of O2 inhalation; and 4) Hypoglycemic coma induced by intravenous injection of insulin. The changes in the biochemical state of the cerebral tissue give very large variations of the low frequency impedance. It is these variations which are to constitute the object of this communication.