Aging Reduces L-Type Calcium Channel Current and the Vasodilatory Response of Small Mesenteric Arteries to Calcium Channel Blockers

Abstract

Calcium channel blockers (CCBs) are widely used to treat cardiovascular disease (CVD) including hypertension. As aging is an independent risk factor for CVD, the use of CCBs increases with increasing age. Hence, this study was designed to evaluate the effect of aging on the sensitivity of small mesenteric arteries to L-type voltage-gated calcium channel (LTCC) blockers and also to investigate whether there was a concomitant change in calcium current density. Third order mesenteric arteries from male F344 rats, aged 2.5-3 months (young) and 22-26 months (old) were mounted on wire myograph to measure the tension during isometric contraction. Arteries were contracted with 100 mM KCl and were then relaxed in a cumulative concentration-response dependent manner with nifedipine (0.1 nM-1 μM), verapamil (0.1 nM-10 μM), or diltiazem (0.1 nM-10 μM). Relaxation-concentration response curves produced by cumulative concentrations of three different CCBs in arteries of old rats were shifted to the right with statistically significant IC50s. pIC50 ± s.e.m: (8.37 ± 0.06 vs. 8.04 ± 0.05, 7.40 ± 0.07 vs. 6.81 ± 0.04, and 6.58 ± 0.07 vs. 6.34 ± 0.06) in young vs. old. It was observed that the maximal contractions induced by phenylephrine and reversed by sodium nitroprusside were not different between young and old groups. However, Bay K 8644 (1 μM) increased resting tension by 23 ± 4.8% in young arteries and 4.7 ± 1.6% in old arteries. LTCC current density were also significantly lower in old arteries (-2.77 ± 0.45 pA/pF) compared to young arteries (-4.5 ± 0.40 pA/pF); with similar steady-state activation and inactivation curves. Parallel to this reduction, the expression of Cav1.2 protein was reduced by 57 ± 5% in arteries from old rats compared to those from young rats. In conclusion, our results suggest that aging reduces the response of small mesenteric arteries to the vasodilatory effect of the CCBs and this may be due to, at least in part, reduced current density of LTCC.

Keywords: F344 rats; aging; calcium channel blockers; mesenteric arteries; voltage-gated calcium channel.

Figure 1

L-type voltage-gated calcium channel currents. (A) Representative traces of inward Ba²⁺ currents generated by 8 mV steps from a holding potential of −70 to +58 mV in vascular smooth muscle cells (VSMCs) isolated from mesenteric arteries of young and old rats. Membrane capacitances were 32 and 48 pF, respectively. Currents from both animal groups (cont) were equally blocked by 1 μM nifedipine (nif). (B) Averaged current-voltage (I-V) relationships showing current density in VSMCs of arteries from young compared to old. Steady state activation (C), and inactivation (D) curves are represented as I/I_max and G/G_max; respectively.

Figure 2

Western immunoblots comparing the expression of Ca_v1.2 channel protein in arteries from old and young rats. (A) Representative band densities of Ca_v1.2 channel protein isolated from mesenteric arteries of old and young rats. Band densities for the internal standard, β-actin, of the same blot is shown in the lower panel. (B) Average densitometric values of normalized Ca_v1.2 to β-actin densities from four Western blots, indicating reduced Ca_v1.2 channel protein expression in mesenteric arteries of old compared to young rats. Bars represent means ± S.E.M. ^*Significantly different from young (P < 0.0001).

Figure 3

Effect of Bay K 8644 on basal tension. Representative tension recording traces of segments of mesenteric arteries isolated from young and old rats that were treated with cumulative concentrations of L-type calcium channel agonist, Bay K 8644 (A). Average concentration-response curves of Bay K 8644 induced-contraction plotted as percentage of phenylephrine (PE) contraction (B) for arteries from young and old rats. Values are mean ± SEM, ^*denotes significance at P ≤ 0.05.

Figure 4

Effect of calcium channel blockers on KCl-induced contraction. Responses of mesenteric arteries isolated from young and old rats that were pre-contracted with 100 mM KCl and relaxed with cumulative concentrations of three calcium channel blockers, nifedipine (A), verapamil (B), and diltiazem (C). (D) Shows the relationship between cumulative concentrations of sodium nitroprusside (SNP) and % relaxation of phenylephrine (PE; 4 μM)-induced contraction.

Figure 5

Effect of nifedipine on diameter and relative intracellular calcium level changes. Average data of diameter changes (A) and intracellular calcium level as indicated by R340/380 (B) of small mesenteric arteries isolated from young and old rats that were pre-contracted with 60 mM KCl and then relaxed with 1 μM nifedipine. Values are mean ± SEM of % changes from KCl contraction. ^*denotes significance at P ≤ 0.05.