Chantal Roullet

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.

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)-carboxy-fluorescein 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 μmol/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 μmol/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.