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G Gros

Publications and source records attributed to G Gros.

At least 37 records · Page 2Linked to original sources
PubMed1998

Localization of carbonic anhydrase IV in rat and human heart muscle.

We investigated carbonic anhydrase IV (CA IV) in rat and human heart with immunohistochemical methods by both light and electron microscopy. In cryosections that were incubated with anti-CA IV/FITC, the capillaries showed a strong reaction for CA IV. In paraffin and semithin sections treated with anti-CA IV/ABC (avidin-biotin-peroxidase complex) blood vessels, capillaries, and sarcolemma (SL) were positively stained. By staining ultrathin sections with anti-CA IV/immunogold, CA IV could also be demonstrated at the latter two locations, including the specialized sarcolemmal structures intercalated discs, and T-tubules. In addition, by this method CA IV was seen to be associated with the sarcoplasmic reticulum (SR). The absence of immunostaining in SR and/or SL with some techniques probably indicates a problem of accessibility of the antigenic sites. In line with the immunohistochemical results, CA IV mRNA expression was visualized in both endothelial and muscle cells by in situ hybridization histochemistry.

Animals↗
PubMed1998

Decay of Ca2+ and force transients in fast- and slow-twitch skeletal muscles from the rat, mouse and Etruscan shrew.

Isometric single-twitch force and intracellular Ca2+ transients were recorded simultaneously, using fura-2, from slow- and fast-twitch muscle fibres of the rat, mouse and Etruscan shrew Suncus etruscus. In the slow-twitch rat soleus, force half-relaxation time was three times longer than the 50% decay time of the fura-2 signal. In contrast, in the fast-twitch extensor digitorum longus muscles of all three animals, muscle relaxation preceded Ca2+ decay. It is proposed that this surprising property of fast-twitch muscles is due to their pCa-tension curve, which is shifted to the right compared with that of slow-twitch muscle.

Animals↗
PubMed1997

Mass spectrometric determination of HCO3- permeability and carbonic anhydrase activity in intact guinea-pig colon epithelium.

1. A mass spectrometric method originally used in red blood cells was applied to suspensions of isolated colonocytes and intact colonic epithelium to measure the exchange of 18O between HCO3-, CO2 and H2O to determine intracellular carbonic anhydrase activity (Ai) and membrane bicarbonate permeability (P). 2. In suspensions of isolated guinea-pig colon epithelial cells, colonocytes, we found significantly higher values of Ai and P for cells derived from the proximal colon than for cells from the distal colon. In the case of Ai, this confirms earlier reports. 3. When the 18O exchange process was observed across the mucosal (apical) side of intact colon mucosa, the estimated values of Ai were identical to those obtained for isolated colonocytes, for both the proximal and the distal part of the colon. This is considered to be strong evidence that this method can be applied to a layer of intact epithelium as well as to cell suspensions. 4. The values of P obtained from the apical side of intact colon mucosa were 6 times higher than those estimated from measurements with isolated colonocytes. This indicates that the basolateral membrane of colon epithelium, which participates in the 18O exchange process in isolated colonocytes but not in the 18O exchange process across the apical side of intact mucosa, has a markedly lower bicarbonate permeability than the apical membrane. 5. When the 18O exchange process was observed across the serosal (basolateral) side of intact colon mucosa, the P values, as expected, were low compared with the apical side of intact mucosa. However, rather unexpectedly, the Ai values derived from these measurements were 2-3 times lower than those obtained with isolated colonocytes. It appears possible that the latter finding is an artifact due to the submucosal tissue markedly slowing down CO2 diffusion from the bathing medium into the epithelial cells, thus causing an apparent fall in Ai. 6. Ai decreased and P increased with increasing temperature, as expected, when studied on the mucosal side of intact colon. This provides additional support for the validity of the method.

Animals↗
PubMed1997

Adult fast myosin pattern and Ca2+-induced slow myosin pattern in primary skeletal muscle culture.

A primary muscle cell culture derived from newborn rabbit muscle and growing on microcarriers in suspension was established. When cultured for several weeks, the myotubes in this model develop the completely adult pattern of fast myosin light and heavy chains. When Ca2+ ionophore is added to the culture medium on day 11, raising intracellular [Ca2+] about 10-fold, the myotubes develop to exhibit properties of an adult slow muscle by day 30, expressing slow myosin light as well as heavy chains, elevated citrate synthase, and reduced lactate dehydrogenase. The remarkable plasticity of these myotubes becomes apparent, when 8 days after withdrawal of the ionophore a marked slow-to-fast transition, as judged from the expression of isomyosins and metabolic enzymes, occurs.

Animals↗
PubMed1997

A rapid electrophoretic method for separating rabbit skeletal muscle myosin heavy chains at high resolution.

An electrophoretic method using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was developed which gives a high-resolution separation of the known myosin heavy chains of rabbit skeletal muscle with excellent reproducibility. The gel of 10 cm total length consists of (i) a first stacking gel of 3.5% total gel concentration (T) and pH 6.8, (ii) a first separating gel of 6.6%T and pH 8.8, (iii) a second stacking gel of 6.6%T and pH 6.8, and (iv) a second separating gel of 8.8%T and pH 8.8. With this composition, a minigel system allows separation of six myosin heavy-chain (MHC) isoforms at room temperature without cooling and within 8h. In agreement with previous reports, the isoforms appear in the sequence MHCemb, MHC IIa, MHC IId, MHCneo, MHC IIb, MHC I. A special advantage is the detectability not only of the adult but also of the embryonic and neonatal isoforms MHCemb and MHCneo.

Animals↗
PubMed1997

Erythrocyte carbonic anhydrase activity in smokers and in diabetic patients.

Carbonic anhydrase activity was measured in lyzed erythrocytes from smoking and non-smoking young men as well as from diabetic and healthy young women. Enzyme activity was determined by a changing-pH-assay, using a stirred reaction vessel and glass pH electrode. CAA was lower in smokers than in non-smokers. Furthermore, it was lower in diabetics than in nondiabetic controls. We conclude that cigarette smoking as well as diabetes mellitus may reduce erythrocyte carbonic anhydrase activity.

Adult↗
PubMed1996

Membrane-associated carbonic anhydrase IV in skeletal muscle: subcellular localization.

Carbonic anhydrase IV (CA IV) was examined by light microscopy and electron microscopy in rat soleus muscle. Semithin sections of aldehyde-fixed Epon-embedded muscle were stained with rabbit anti-rat lung CA IV and the avidin-biotin-peroxidase complex. With this technique, capillaries and sarcolemma showed positive CA IV staining. For electron microscopy, rat soleus specimens were aldehyde-fixed, with or without subsequent osmication, and embedded in Epon. Ultrathin sections were immunostained with anti-rat lung CA IV/immunogold. Omitting osmium allowed ample antigen-antibody reactions but could not prevent the release of glycosylphosphatidylinositol-anchored CA IV from the membranes, which led to apparent background staining. Postosmication significantly reduced tissue antigenicity but kept the antigen bound to the membranes and thus allowed a very precise localization of CA IV. By electron microscopy, membrane-bound CA IV is found to be associated with capillary endothelium, sarcolemma, and sarcoplasmic reticulum (SR). Conceivably, the presence of SR staining in ultrathin sections and its absence in semithin sections reflect a problem of accessibility of the antigenic sites.

Animals↗
PubMed1995

Diffusion of myoglobin in skeletal muscle cells--dependence on fibre type, contraction and temperature.

We measured the diffusion coefficient of myoglobin (DMb) inside mammalian skeletal muscle cells with a microinjection technique. A small bolus of horse Mb was injected into a single muscle fibre and the subsequent time-dependent changes of the Mb profiles along the fibre axis were measured with a microscope-photometer. For fibres of the rat soleus muscle at 22 degrees C, a DMb of 1.3.10(-7) cm2/s was found, confirming a result obtained previously by us for rat diaphragm muscle with a photo-oxidation technique. In the extensor digitorum longus muscle of the rat, a higher value of 1.9.10(-7) cm2/s was measured. Auxotonic muscle contractions did not change the apparent DMb. For the temperature range between 22 degrees C and 37 degrees C, a temperature coefficient. Q10, of 1.5 was calculated. The implication of this result for the role of Mb in the facilitation of oxygen transport was examined. Model calculations show that with this relatively low DMb value, the intracellular oxygen supply can be improved only slightly.

Animals↗
PubMed1995

Contractile function of papillary muscles with carbonic anhydrase inhibitors.

Contractile parameters of directly stimulated rabbit papillary muscles were studied during incubation in baths containing the carbonic anhydrase inhibitors chlorzolamide (1*10(-3) M) or ethoxzolamide (1*10(-4) M). Both inhibitors caused an at least partly reversible decrease in isometric force as it has been observed in skeletal muscle, and--in contrast to the results in skeletal muscles--a decrease in time-to-peak and half-relaxation time. It is postulated that inhibition of the membrane-bound carbonic anhydrase of heart muscle might induce an intracellular acidosis and that this acidosis causes the observed effects on contractile parameters.

Animals↗
PubMed1995

Effects of carbonic anhydrase inhibitors on oxygen consumption and lactate accumulation in skeletal muscle.

In isolated rat soleus and extensor digitorum longus (EDL) muscles, the effects of carbonic anhydrase inhibitors were studied on oxygen consumption as well as lactate release and accumulation after incubation in inhibitors lasting long enough to produce marked changes in contractile parameters and in the concentrations of energy-rich phosphates. The inhibitors used were chlorzolamide (10(-3) M) and NaCNO (10(-2) M). Compared with control muscles, muscles treated with either of the two inhibitors showed a decrease in force, and an increase in time-to-peak as well as in relaxation time. Lactate content and release in soleus and in EDL were increased by factors of 2-3 with both inhibitors. With both inhibitors, oxygen consumption in the red soleus increased by approximately 27%, whereas in EDL, no significant change could be observed. The increase in aerobic metabolic rate in the red soleus only might indicate that the isozyme CA III, which is present only in this type of muscle, is in some way involved in keeping the oxygen consumption low. The increase in anaerobic metabolic rate occurring in both muscles can possibly be explained by increases in Pi and ADP.

Animals↗
PubMed1994

Functional role of bicarbonate in propionate transport across guinea-pig isolated caecum and proximal colon.

1. Unidirectional fluxes of propionate across isolated epithelia from the guinea-pig caecum and proximal colon were measured under short-circuit current conditions. In the caecum and proximal colon the serosal-to-mucosal propionate flux (JPrsm) was higher than mucosal-to-serosal flux (JPrms), resulting in a net secretory flux of propionate. 2. HCO3(-)-CO2-free solution reduced JPrms in the caecum and proximal colon markedly; JPrsm was not (caecum) or little (proximal colon) affected. The subsequent addition of acetazolamide caused a further decrease in JPrms in the proximal colon, but not in the caecum. 3. In HCO3(-)-containing solutions acetazolamide or ethoxzolamide inhibited JPrms; JPrsm was not affected. A macromolecular carbonic anhydrase inhibitor, prontosil-dextran, had no effect on propionate fluxes, indicating that the intracellular carbonic anhydrase is of importance for short-chain fatty acid transport. 4. Subsequent to carbonic anhydrase inhibition, mucosal addition of amiloride caused a slight further decrease of JPrms in the caecum and proximal colon; JPrsm was not affected. 5. Results support the view that a considerable proportion of short-chain fatty acids (SCFAs) is absorbed via a SCFA(-)-HCO3- exchange.

Acetazolamide↗
PubMed1994

Diffusivity of myoglobin in intact skeletal muscle cells.

We report a method that allows us to determine the diffusion coefficient of native myoglobin in intact and mechanically unaffected red muscle fibers. The method is based on an optical recording of intracellular diffusion of metmyoglobin, which is produced inside the cells by photooxidation of oxymyoglobin with a UV light pulse. We find a myoglobin diffusivity of 1.2 x 10(-7) cm2/s (22 degrees C), which is only 1/10th of the value measured in very dilute myoglobin solutions and 1/5th of the value obtained from measurements in solutions of myoglobin at 18 g/dl. The latter value often has been used in model calculations of oxygen transport to tissue incorporating myoglobin-facilitated oxygen diffusion. Recalculating facilitated diffusion with the value obtained by us implies that its contribution to total intracellular oxygen transport is of minor importance. Furthermore, it shows that sterical hindrance to myoglobin diffusion is dominated by the muscle-cell architecture rather than by the overall protein concentration of the muscle fiber.

Animals↗
PubMed1994

Immunohistochemical localization of carbonic anhydrase IV in capillaries of rat and human skeletal muscle.

We used polyclonal antisera raised in rabbits against membrane-bound rat lung and human lung carbonic anhydrase (CA) IV in immunofluorescence studies to stain cryosections of rat soleus and extensor digitorum longus (EDL) and several human skeletal muscles. There was strong specific staining of capillaries in all muscles investigated. Several techniques were applied to verify this result. (a) Serial sections were either incubated with anti-CA IV/FITC or processed for endothelial ATPase reaction. There was precise co-localization of antibody marked structures and ATPase stained capillaries. (b) Human muscle sections were double stained with anti-CA IV/TRITC and anti-von Willebrand factor (vWF)/FITC. vWF, a capillary marker, and CA IV were localized at identical sites. (c) The CAIV was released from capillaries by treatment with phosphatidylinositol specific phospholipase C, suggesting that the enzyme is anchored to the endothelial cell membrane via a phosphatidylinositolglycan anchor. (d) A rat hindlimb was perfused with diluted antiserum. Cryosections of perfused soleus and EDL processed for anti-rabbit IgG/FITC staining showed clear fluorescence associated with capillaries, indicating that the antigen was accessible from the capillary lumen. (e) Immune complexes formed during antiserum perfusion as described in d were precipitated from muscle homogenates. SDS-PAGE followed by immunoblotting showed that the predominant portion of total muscle CA IV was bound in these complexes and therefore must be located intravascularly.

Animals↗
PubMed1992

Rat skeletal muscle membrane associated carbonic anhydrase is 39-kDa, glycosylated, GPI-anchored CA IV.

Sarcolemmal membrane vesicle preparations from white and red muscles of rat were found to contain a carbonic anhydrase which was indistinguishable from carbonic anhydrase IV from rat lung. This isozyme appears to account for all of the carbonic anhydrase activity in the sarcolemmal vesicle preparations. Digestion of 39-kDa CA IV with endoglycosidase F reduced the Mr to 36 kDa, suggesting that it contains one N-linked oligosaccharide. Treatment of sarcolemmal vesicles with phosphatidylinositol-specific phospholipase C released all of the activity, indicating that the enzyme is anchored to membranes by a phosphatidylinositol-glycan linkage. White muscle sarcoplasmic reticulum vesicles also contain a small amount of 39-kDa CA IV-type enzyme. A 52-kDa polypeptide in sarcoplasmic reticulum membranes cross-reacts with anti-human CA II and anti-rat CA II antisera, but does not bind to the sulfonamide affinity column. This cross-reacting polypeptide has no detectable CA activity.

Animals↗
PubMed1992

Membrane-bound carbonic anhydrase in the heart.

Microsomal membranes from bovine heart homogenates were subfractionated by density gradient centrifugation. Fractions with high levels of a sarcolemmal (SL) marker are enriched in specific carbonic anhydrase (CA) activity up to ninefold compared with the microsomes. Fractions with high levels of a sarcoplasmic reticulum marker and a mitochondrial marker, respectively, exhibit specific CA activities that are similar to the one found in the microsomes. Determination of cytosolic markers reveals that the CA activity in the SL fraction is not due to contamination by cytosolic CA, and it is shown by Triton X-114 phase separation that the CA activity is due to an integral membrane protein. In cryosections from rabbit heart the SL region of cardiomyocytes is stained by the fluorescent CA inhibitor dansylsulfonamide. Intracellular staining occurs also, with a pattern suggesting the presence of CA associated with intracellular membranes. Although it cannot be excluded that there is a contribution by endothelial membranes, it appears likely that most CA of the heart is bound to the SL. The possible involvement of the enzyme in extracellular proton buffering is discussed.

Animals↗
PubMed1990

Carbonic anhydrase inhibition and calcium transients in soleus fibers.

We simultaneously measured cytoplasmic Ca2+ transients using Fura-2 and isometric force in rat soleus fiber bundles. In the presence of the carbonic anhydrase inhibitor, chlorzolamide, we observed a decreased amplitude and retarded decay of the Ca2+ signal. This corresponded with a decreased isometric force and a retarded muscle relaxation. We conclude that muscle carbonic anhydrase participates in excitation-contraction coupling, possibly by rapidly providing protons that are exchanged for Ca2+ across the sarcoplasmic reticulum membrane.

Acetazolamide↗