Biomedical subjects

C M Roullet

Publications and source records attributed to C M Roullet.

8 recordsLinked to original sources

PubMed1998

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.

Acetylcysteine↗

PubMed1997

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.

Animals↗

PubMed1996

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.

Animals↗

PubMed1995

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.

Adult↗

PubMed1994

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.

Animals↗

PubMed1991

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)

Alkaline Phosphatase↗

PubMed1989

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.

Animals↗

PubMed1989

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.

Aging↗