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A de Hemptinne

Publications and source records attributed to A de Hemptinne.

At least 19 recordsLinked to original sources
PubMed1999

Chemically, mechanically, and hyperosmolarity-induced calcium responses of rat cortical capillary endothelial cells in culture.

The purpose of the present work was to characterize calcium responses of brain-capillary endothelial cells (BCEC), the cells forming the blood-brain barrier, to chemical, hyperosmolar and mechanical stimulation. Confluent BCEC cultures were grown from capillary fragments isolated from rat cerebral cortex. Intracellular free calcium ([Ca2+]i) was measured using fura-2 and digital imaging. Our experiments show large endothelial calcium responses to substance P and ATP, up to a peak value of approximately 1000 and 600 nM, respectively, and these responses were observed in 2/3 of the cells. Calcium responses to bradykinin, histamine, and hyperosmolar sucrose or mannitol were smaller, attaining a peak in the range 180-340 nM, and were observed in a smaller fraction of the cells. No calcium responses were observed to high-potassium, L-glutamate, serotonin, carbachol, noradrenaline, and nitric-oxide donors. Consecutive superfusion of the cultures with ATP, bradykinin, and histamine showed that cells with a certain response pattern were spatially grouped; the response pattern itself varied widely between experiments. Mechanical stimulation of a single cell caused a calcium response in the stimulated cell in primary cultures and triggered an intercellularly propagating calcium wave in passaged cultures. Given the important effect of endothelial [Ca2+]i on blood-brain barrier permeability and transport, we conclude that substance P and ATP are potential modulators of blood-brain barrier function. Hyperosmolarity-induced blood-brain barrier opening is probably not mediated through endothelial [Ca2+]i.

Adenosine Triphosphate↗
PubMed1996

Changes of intracellular free calcium during intracytoplasmic sperm injection.

The present experiments were undertaken to investigate whether the procedure of intracytoplasmic sperm injection (ICSI) is associated with changes in the intracellular free calcium concentration ([Ca2+]i). [Ca2+]i was measured, using the calcium-sensitive dye fura-2, during and after impalement of mouse oocytes with an ICSI pipette and injection of a small amount of medium alone or of medium containing a normal human spermatozoon. Forty-five oocytes were injected with medium. Two different responses were observed: 20 of these cells showed a large increase of [Ca2+]i upon impalement; the other 25 cells did not show any change of [Ca2+]i, neither in the acute period nor in a late period 4 hr after impalement. All the cells that responded with an increase of [Ca2+]i subsequently lysed within the first 30 min following impalement, while all the cells with no [Ca2+]i change remained intact. This observation suggests that only traumatic impalement is associated with an increase of [Ca2+]i. Thirty-one oocytes were successfully, i.e., without subsequent cell lysis, injected with a normal mouse or human spermatozoon. In none of these cells could any acute or late change of [Ca2+]i be observed. The experiments illustrate that successful performance of the ICSI procedure, i.e., ICSI not followed by cell lysis, is not associated with changes of [Ca2+]i in mouse oocytes. This suggests that the ICSI technique, by itself, does not help in activating the oocyte via manipulation-induced changes of [Ca2+]i.

Animals↗
PubMed1996

Changes of intracellular free calcium following mechanical injury in a spinal cord slice preparation.

Intracellular calcium ions are, in addition to free radicals, an important mediator of tissue destruction following traumatic injury to the spinal cord. In vivo measurements of calcium in the interstitial space and in the tissue suggest the occurrence of a posttraumatic shift of calcium from the extracellular to the intracellular compartment at the injury site. No information is, however, available on the posttraumatic changes of calcium in the intracellular compartment, where the ion exerts its crucial messenger function. We developed an in vitro model of local traumatic spinal injury, using a spinal cord slice preparation, allowing us to investigate injury-related changes of intracellular free calcium. The injury consisted of the impact of a small needle, and intracellular free calcium was measured with fura-2. Application of the injury at different places within the gray matter caused a transient and reproducible increase in the fura-2 fluorescence ratio. This injury-induced ratio increase was largely, but not completely, suppressed under zero extracellular calcium conditions. It was also largely depressed in the presence of high extracellular potassium and in the absence of extracellular sodium. It was modestly depressed by the calcium channel blocker nifedipin, by the calcium release channel blocker dantrolene, and by the gap junction blockers halothane and octanol. The calcium channel blocker flunarizine, the N-methyl D-aspartate (NMDA)-receptor-channel blocker MK-801 and the endoplasmic reticulum calcium-ATPase blocker thapsigargin had no effect. The experiments suggest that injury is associated with an increase in intracellular free calcium that is mediated by calcium influx, in part via L-type calcium channels. They furthermore give evidence that sodium influx and gap junctions are involved in these injury-associated changes of intracellular free calcium.

Animals↗
PubMed1993

Effects of flunarizine on induced calcium transients as measured in fura-2-loaded neurons of the rat dorsal root ganglion.

The effect of the calcium entry blocker flunarizine on a high-potassium induced increase of intracellular free calcium was studied. The experiments were done with neurons isolated from rat dorsal root ganglia and loaded with the calcium-sensitive dye fura-2. The increase of calcium induced by 60 mmol/l potassium was abolished after removal of extracellular calcium, was reversibly reduced by 50 mumol/l cadmium (76% inhibition), 50 mumol/l nickel (25% inhibition) and 10 mumol/l nifedipine (18% inhibition), and reversibly increased after removal of extracellular sodium (26% increase). The potassium induced increase of intracellular calcium is, therefore, mediated by transmembrane calcium influx, probably to a large extent through cadmium-sensitive calcium channels. Flunarizine (5 min incubation followed 1 min wash-out) reduced the amplitude of the high-potassium induced calcium increase in a dose-dependent manner (Kd = 370 +/- 100 nmol/l; mean +/- SEM; n = 8), causing complete inhibition at a concentration of 10 mumol/l in the majority of cells. Flunarizine (> or = 1 mumol/l) caused a reversible increase of the resting level of intracellular calcium in some cells, an effect which disappeared in the absence of extracellular calcium. The drug (1 mumol/l had no influence on the time course of recovery of intracellular calcium subsequent to a rise induced by high-potassium or by the calcium ionophore A23,187. It is concluded that flunarizine acts as an inhibitor of depolarization-mediated calcium influx. At a concentration of 1 mumol/l, the drug presumably has no effect on cellular calcium extrusion and/or sequestration mechanisms.

Animals↗
PubMed1992

Influence of KATP channel modulation on net potassium efflux from ischaemic mammalian cardiac tissue.

OBJECTIVE: The aim was to investigate the effect of pre-exposure to inhibitors or activators of the ATP regulated K+ (KATP) channels on the ischaemia induced early changes of the extracellular K+ concentration, the extracellular pH, and the action potential in mammalian cardiac tissue. METHODS: An in vitro model simulating ischaemia was applied to isolated guinea pig papillary muscles while simultaneous microelectrode measurements of the transmembrane potentials and of the pH and K+ activity at the surface of the preparation (pHs and asK respectively) were made. RESULTS: During conditions of superfusion arrest, when accumulation of metabolic acids causes acidification of pHs, the simultaneous development of a true hypoxic state of the muscle is required in order to induce shortening of the action potential duration and accumulation of extracellular K+ with associated membrane depolarisation. Glibenclamide (10-50 microM) slowed the decrease of the action potential duration in 50% of the muscles, while the increase of asK was only moderately (approximately 20%) influenced. In the presence of 200 microM of the KATP channel inhibitor, the mean K+ accumulation was not significantly different from the control. The presence of tolbutamide (1 mM) had no effect on the decrease of action potential duration, but moderately slowed the increase of asK. Superfusion with lemakalim (BRL 38227) decreased action potential duration dose dependently. In papillary muscles in which action potential duration was shortened to approximately 60% of the control duration by presuperfusion with lemakalim, a subsequent ischaemic episode did not increase but rather delayed the rise in asK. During simulated ischaemia in the presence of Ba2+ ions (1 mM), asK showed a transient decrease followed by a rise at a rate similar to that in the absence of Ba2+. CONCLUSIONS: Early ischaemic K+ accumulation and surface acidification are relatively insensitive to KATP channel inhibition or activation prior to the ischaemic insult. The rather loose coupling of the increase of asK and the decrease of action potential duration, as well as the limited effect of sulphonylureas on the K+ increase, suggests that mechanisms other than KATP channel opening could possibly contribute to the initial phase of ischaemic K+ accumulation.

Action Potentials↗
PubMed1990

Acidification and intracellular sodium ion activity during stimulated myocardial ischemia.

With the use of microelectrodes, intracellular pH (pHi), surface pH (pHs), and intracellular Na+ activity (aiNa) were measured in isolated guinea pig papillary muscles during normal superfusion and during a reversible condition of simulated ischemia. Acid loading by NH+4 prepulse or by CO2-HCO3- addition during superfusion with pH 7.4 solutions caused internal acidification followed by a recovery of pHi, which could be inhibited by amiloride. pHi recovery was associated with an amiloride-sensitive peak rise of aiNa and membrane hyperpolarization, indicative of Na(+)-H+ exchange. Peak increase of aiNa was absent if the pH of the superfusion solution was concomitantly lowered. Imposed ischemia after control superfusion caused membrane depolarization and acidification of pHi and pHs. The change of pHs consistently was larger than that of pHi. aiNa decreased from 5.5 to 4.6 mM after 10-min ischemia. Enlarging the pHi (and pHs) decrease in ischemia by prior reduction of the tissue buffer capacity (CO2-HCO3(-)-free superfusion) was unable to induce a rise of aiNa during the subsequent ischemic period. Amiloride had no significant effect on aiNa during ischemia. It is concluded that the important acidification of pHs reduces the rate of pHi regulatory Na(+)-H+ exchange and thereby contributes to a longer maintenance of the Na+ electrochemical gradient in ischemic cardiac muscle.

Amiloride↗
PubMed1988

Influence of local intracellular pH changes on pH and action potentials in adjacent parts of cardiac Purkinje fibers.

Isolated sheep cardiac Purkinje fibers were pulled through a latex membrane and both segments were independently superfused with modified Tyrode solutions. Transmembrane potentials and intracellular pH (pHi) were continuously measured in one segment (test compartment, TC), using double-barreled pH sensitive glass microelectrodes, while the internal H+ activity was altered in the adjacent part of the fiber (experimental compartment, EC). In the latter local pHi changes were produced by removal of CO2/HCO3-, by superfusing acidic (pH 6.8) solution, and by addition and subsequent withdrawal of NH4Cl. Withdrawal of CO2/HCO3- in EC was found to have no influence on pHi in TC at 0.35 to 1.0 mm distance. The action potential first shortened and later on prolonged above control duration after switching to HCO3- -free medium. Perfusing EC with an acidic solution had virtually no effect on pHi in TC while action potential duration (APD) increased. Addition and withdrawal of NH4+ in EC decreased, respectively increased, APD. In TC no change in pHi was observed at 0.8 to 1.0 mm distance. At shorter distance a slow acidification was seen, associated with the presence of NH4+ in EC. The presence of amiloride, a blocker of the pHi regulating mechanism, could not unmask a larger pHi change. It is concluded that, in cardiac muscle, large gradients of pHi are possible over a relatively short distance, while electrotonic interaction can produce changes in the time course of the action potential in neighbouring cells having a normal intracellular pH.

Action Potentials↗
PubMed1987

Surface pH and the control of intracellular pH in cardiac and skeletal muscle.

Both surface pH (pHs) and intracellular pH (pHi) were measured using single- and double-barreled pH-sensitive microelectrodes in isolated sheep cardiac Purkinje strands, rabbit and cat papillary muscle, and mouse and rat soleus muscle. Superfusion of the preparations with a relatively low buffered solution (containing 5 mM HEPES buffered to control pH) causes surface acidosis that correlates with efflux of metabolically produced acids in the unstirred layer of fluid surrounding the tissue. Acidification of the surface layer induces a slower acid change of pHi and depresses the rate of proton extrusion following an imposed intracellular acid load. In cardiac preparations, the lowering of pHi correlates with depression of twitch tension. Transient changes of pHs and pHi are seen when a weak acid or base is suddenly added to, or removed from the superfusion solution. Indirect evidence of the presence of carbonic anhydrase in the extracellular surface layer is obtained from analysis of transient pHs changes in presence and absence of acetazolamide.

Amiloride↗
PubMed1986

Influence of surface pH on intracellular pH regulation in cardiac and skeletal muscle.

The influence of the surface pH (pHs) on the intracellular pH (pHi) and the recovery of pHi after an imposed intracellular acid load was investigated in isolated sheep cardiac Purkinje fiber, rabbit papillary muscle, and mouse and rat soleus muscle. pHs and pHi, respectively, were continuously measured by use of single- and double-barreled pH-sensitive glass microelectrodes. Surface acidosis, usually obtained by superfusion with solutions of acid pH, was also produced with low buffered (5 mM N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) solutions at control pH. The pHs decrease (delta pHs) induced by low buffering was smallest (-0.08 pH unit) in Purkinje fiber and largest (-0.31 pH unit) in rat soleus muscle, which already had a more acid surface in control conditions. delta pHs was somewhat dependent on the superfusion rate. Higher superfusion rates decreased but did not abolish delta pHs. Surface acidosis was associated with a small intracellular acidification. Intracellular acid loads were produced by adding and subsequently withdrawing 20 meq/l NH4+ from the superfusate. In all preparations, the rate of recovery of pHi after NH4+ withdrawal was notably decreased at acidified pHs. This effect was amiloride sensitive. It is concluded that, in superfused multi-cellular preparations, pHs and therefore the buffer concentration of a superfusate can considerably influence steady-state pHi and pHi recovery from an imposed intracellular acid load.

Amiloride↗
PubMed1985

Intracellular pH in depolarized cardiac Purkinje strands.

In isolated sheep cardiac Purkinje strands the effect of membrane depolarization on intracellular pH (pHi) and on pHi changes produced by addition and withdrawal of NH4+ and Co2/HCO3- was investigated. pHi was continuously measured with double-barreled glass microelectrodes. Repetitive stimulation at high rate resulted in a moderate intracellular acidification (approximately 0.03 pH unit after a 3 Hz train of 2 min), whereafter pHi returned toward its pre-stimulus level. Prolonged depolarization, evoked either by current injection or by superfusion with high K+ solutions, was accompanied by a small acid shift. In the depolarized cell, addition of NH4+ to the superfusate caused intracellular alkalinization followed by re-acidification which was slower than at normal membrane potential. Following intracellular acidification caused by withdrawal of NH4+, pHi recovery also was slightly slower than in the normally polarized cell. In the depolarized fiber, removal and readdition of CO2/HCO3- produced the expected intracellular alkalinization and acidification respectively. Recovery from CO2-induced acidosis was slowed somewhat in high K+ (low Na+) superfused fibers, not in current depolarized fibers. In the depolarized cell, steady state pHi in CO2/HCO3- containing and in CO2/HCO3- free solution tended to become identical. These experiments support the hypothesis that in the normally polarized Purkinje fiber passive shuttle movement of NH4+/NH3 and CO2/HCO3- occurs and could perhaps at least be partly responsible for the lower steady state pHi as compared to that reached in NH4+-free and CO2/HCO3- -free solutions respectively.

Ammonia↗
PubMed1985

Intracellular pH and contraction of isolated rabbit and cat papillary muscle: effect of superfusate buffering.

The influence of external buffering on surface pH (pHs), intracellular pH (pHi) and developed twitch tension was investigated in rabbit and cat papillary muscle. pHs and pHi were measured using single and double-barreled microelectrodes respectively. In 20 mM HEPES buffered solution, steady state pHi is close to that in control CO2/HCO-3 (25 mM HCO-3, 5% CO2) solution. pHs and developed tension also do not differ greatly from their control values. Decreasing the HEPES concentration to 5 mM, at constant external pH, lowers pHs considerably. The surface acidosis is associated with a small intracellular acidification; steady state pHi in 5 mM HEPES is always more acid than that in control CO2/HCO-3. A significant decrease in developed tension is also seen in 5 mM HEPES. Alteration of the superfusion velocity influences pHs only slightly. Stimulation of the muscle at high frequency is shown to increase surface acidification, the extent of which is dependent on the buffer concentration. The conclusion from the present experiments is that in papillary muscle external buffering influences intracellular pH and contraction via its effect on pHs.

Acid-Base Equilibrium↗
PubMed1984

The influence of muscle respiration and glycolysis on surface and intracellular pH in fibres of the rat soleus.

Extracellular pH (pHo) and intracellular pH (pHi) of superficial fibres of the rat soleus muscle were measured in vitro using pH-sensitive glass micro-electrodes. The origin of the pH gradient existing between the bulk phase of extracellular solution and the surface of muscle fibres was investigated. The pHo decreased almost linearly over a distance of 285 microns from bulk solution to fibre surface. The magnitude of the bulk-surface pH gradient is greater in the mid region of the muscle than close to the tendon. Decreasing the superfusate velocity increased the magnitude of the pH gradient. Reducing the buffer capacity of the superfusing solution had the same effect. Inhibiting the aerobic metabolism or stimulating it acidified the fibre surface. Inhibiting glycolysis alone, or both aerobic metabolism and glycolysis, alkalinized the fibre surface. Inhibiting the membrane ionic exchange process involved in pHi regulation had no effect on surface pH. Changing the rate of aerobic or anaerobic metabolism quickly modified pHi in most cases. In conclusion the bulk-surface pH gradient seems to result mainly from diffusion of CO2 and lactic acid across an unstirred layer of fluid covering the surface of muscle fibres.

Animals↗
PubMed1984

Analysis of Cl- -HCO3(-) exchange during recovery from intracellular acidosis in cardiac Purkinje strands.

The possible role of a Cl- -HCO3(-) exchange mechanism in the recovery from intracellular acidosis of isolated cardiac Purkinje strands was investigated. Intracellular pH (pHi) was measured using double-barreled pH-sensitive microelectrodes. Acidifications were produced by withdrawing 20 meq NH+4 from the superfusate. Experiments were performed in normal CO2-HCO3(-)-buffered, in HCO3(-)free, and in Cl-free solutions and also in the presence of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), a blocker of Cl--HCO3(-) exchange. In the absence of external HCO3(-), the apparent rate of acid extrusion following induced acidification was only slightly decreased, but the observed effect does not necessarily imply the intervention of a Cl--HCO3(-) exchange mechanism. SITS had little effect on the response to acidification. In zero-Cl- solutions, recovery of pHi from acidosis was not impaired. These observations suggest that in Purkinje fibers, [Cl-]i-[HCO3(-)]o exchange plays no significant role in recovery from intracellular acidification. Moreover, additional evidence is presented in favor of a passive HCO3(-) efflux at steady-state pHi in the normal superfusate. The apparent membrane permeability to HCO-3 was estimated to be 3.2 X 10(-8) cm X s-1.

Acidosis↗
PubMed1983

Influence of organic acids on intracellular pH.

By use of double-barreled pH-sensitive microelectrodes, intracellular pH was measured in isolated sheep cardiac Purkinje strands. After equimolar substitution of 20 mmol/l Cl- by several organic anions at constant extracellular pH 6.8, the rate of induced intracellular acidification was measured. For many organic acids tested, a relation was found between the rate of intracellular acidification and the product of their dissociation constant (pK'a) and diisopropylether-to-water partition ratio (p'). L-Lactate and pyruvate, and also cyanoacetate and alpha-ketobutyrate, caused faster acidifications than anticipated from their pK'a and p'. The rate of intracellular acidification, induced by L-lactate and pyruvate, was markedly depressed in the presence of 4 mmol/l alpha-cyano-4-hydroxycinnamate, a known inhibitor of the carrier-mediated pyruvate transport. The drug also had an effect on the acidification produced by cyanoacetate, alpha-ketobutyrate, glycolate, alpha-hydroxybutyrate, and alpha-chloropropionate, but not on that produced by propionate and acetate. L-Lactate caused a faster acidification than D-lactate. Our results suggest the existence of a facilitated diffusion for L-lactate, pyruvate, and some other organic acids in sheep Purkinje cells.

Animals↗
PubMed1981

pH aspects of transient changes in conduction velocity in isolated heart fibers after partial replacement of chloride with organic anions.

Conduction velocity in isolated rabbit atrial fibers was continuously measured in solutions having a different anionic composition. When 20mmol/l of chloride was replaced by 20 mmol/l lactate or other anions of weak organic acids at constant pH 6.8, biphasic initial transient changes in conduction velocity were observed. The produced transient changes had a greater amplitude with organic acids which have a greater pK and lipid/water partition ratio. The magnitude of the transients was also greater at pH 6.8 than at pH 7.5, and also when the buffering capacity of the superfusion solution was smaller. Measurements of intracellular pH (pHi) in sheep Purkinje fibers and of pH at the surface (pHs) of sheep Purkinje and rabbit atrial fibers with pH sensitive microelectrodes, showed a transient increase of pH and a sustained decrease of pHi on replacement of 20mmol/l chloride by organic anions of weak acids (at constant pH of the superfusion solution). A combined influence of the transient pHs change and the sustained pHi modification seems to be important in the explanation of the biphasic changes in conduction velocity.

Animals↗
PubMed1980

Intracellular pH and surface pH in skeletal and cardiac muscle measured with a double-barrelled pH microelectrode.

The construction of a double-barrelled pH sensitive microelectrode for intracellular use is described. Repetitive measurements of intracellular pH were obtained in rat soleus muscle and sheep Purkinje fibres. They yielded pH values ranging between 7.1 and 7.2 in a CO2/HCO3- buffered medium at 37 degrees C. A lower pH value than that of the bulk solution was found at the surface of the cells using either double-barrelled or single-barrelled pH sensitive microelectrodes.

Animals↗