Enhanced L-type Ca2+ channel current density in coronary smooth muscle of exercise-trained pigs is compensated to limit myoplasmic free Ca2+ accumulation

Abstract

We hypothesized that enhanced voltage-gated Ca2+ channel current (VGCC) density in coronary smooth muscle cells of exercise-trained miniature Yucatan pigs is compensated by other cellular Ca2+ regulatory mechanisms to limit net myoplasmic free Ca2+ accumulation. Whole-cell voltage clamp experiments demonstrated enhanced VGCC density in smooth muscle cells freshly dispersed from coronary arteries of exercise-trained vs. sedentary animals. In separate experiments using fura-2 microfluorometry, we measured depolarization-induced (80 mM KCl) accumulation of myoplasmic free Ba2+ and free Ca2+. Both maximal rate and net accumulation of free Ba2+ in response to membrane depolarization were increased in smooth muscle cells isolated from exercise-trained pigs, consistent with an increased VGCC density. Depolarization also produced an enhanced maximal rate of free Ca2+ accumulation in cells of exercise-trained pigs; however, net accumulation of free Ca2+ was not significantly increased suggesting enhanced Ca2+ influx was compensated to limit net free Ca2+ accumulation. Inhibition of sarco-endoplasmic reticulum Ca2+-transporting ATPase (SERCA; 10 microM cyclopiazonic acid) and/or sarcolemmal Na+-Ca2+ exchange (low extracellular Na+) suggested neither mechanism compensated the enhanced VGCC in cells of exercise-trained animals. Local Ca2+-dependent inactivation of VGCC, assessed by buffering myoplasmic Ca2+ with EGTA in the pipette and using Ca2+ and Ba2+ as charge carriers, was not different between cells of sedentary and exercise-trained animals. Our findings indicate that increased VGCC density is compensated by other cellular Ca2+ regulatory mechanisms to limit net myoplasmic free Ca2+ accumulation in smooth muscle cells of exercise-trained animals. Further, SERCA, Na+-Ca2+ exchange and local Ca2+-dependent inactivation of VGCC do not appear to function as compensatory mechanisms. Additional potential compensatory mechanisms include Ca2+ extrusion via plasma membrane Ca2+-ATPase, mitochondrial uptake, myoplasmic Ca2+-binding proteins and other sources of VGCC inactivation.

Figure 1. Current-voltage (I–V) relationships for whole-cell VGCCs from coronary smooth muscle of sedentary (sed) and exercise-trained (ex) pigs

Currents were obtained using 10 mm Ba²⁺ as external charge carrier. Current is plotted as peak inward current measured during a 260 ms step depolarization to the membrane potential (V_m) indicated from a holding potential of −80 mV. Current is normalized to cell membrane capacitance (pA pF⁻¹). Data are means ± s.e.m.; *P < 0·05, ex (n = 4 pigs, 11 cells) vs. sed (n = 4, 8).

Figure 2. Free Ba²⁺ accumulation during membrane depolarization (80 mm KCl) using fura-2 microfluorometry

A, experimental protocol and representative recordings from single cells of both sedentary and exercise-trained pigs showing change in F₃₄₀/F₃₈₀ fluorescence ratio. Cells were superfused with PSS unless otherwise specified. Cells were exposed to 80 mm KCl (80K) and caffeine (CAF; 5 mm) in the presence of 2 mm extracellular Ca²⁺ (2Ca) for the durations indicated by the horizontal lines. Cells were then exposed to 80 mm KCl in the presence of 2 mm Ba²⁺ (2Ba; equimolar substitution for Ca²⁺) and low Na⁺ (5Na; 5 mm) for 7 min. B, maximal rate (10 s slope) and net (area under the curve, AUC) free Ba²⁺ accumulation for cells from sed (n = 6, 71) and ex (n = 5, 61) animals. Data are means ± s.e.m.; *P < 0·05, ex vs. sed.

Figure 3. Free Ba²⁺ accumulation during membrane depolarization (80 mm KCl) in the presence of the Ca²⁺ channel blocker, nifedipine

Control (-NIF; continuous line) experimental protocol is the same as presented in Fig. 2A. For experiments in the presence of nifedipine (+NIF; ○), a similar protocol was used with the addition of nifedipine (3 μm) between 13·5 and 18 min after the start of the experiment, as indicated by the arrows. Evaluation of the slope between 16 and 18 min indicates that free Ba²⁺ accumulation was abolished in the presence of nifedipine.

Figure 4. Free Ca²⁺ accumulation during membrane depolarization (80 mm KCl) using fura-2 microfluorometry

A, experimental protocol and representative recordings from single cells isolated from sedentary and exercise-trained animals showing change in F₃₄₀/F₃₈₀ ratio. Cells were superfused with PSS unless otherwise specified. Cells were exposed to 80 mm KCl and caffeine (5 mm) in the presence of 2 mm extracellular Ca²⁺ for the durations indicated by the horizontal lines. B, maximal rate (10 s slope) and net (AUC) free Ca²⁺ accumulation for cells from sed (n = 7, 84) and ex (n = 6, 76) animals. Data are means ± s.e.m.; *P < 0·05, ex vs. sed.

Figure 5. Evaluation of free Ca²⁺ accumulation in the presence of SERCA and/or Na⁺-Ca²⁺ exchange inhibition during membrane depolarization (80 mm KCl)

A, experimental protocols are similar to that used in Fig. 4A with the addition of CPA and/or low Na⁺ for the 11 min period shown by the horizontal line. Representative recordings from single cells of sedentary animals are presented for each protocol showing the change in F₃₄₀/F₃₈₀ fluorescence ratio. B, maximal rate (10 s slope). C, net free Ca²⁺ accumulation (AUC) for cells from sedentary and exercise-trained animals for all protocols. n values for control protocol as presented in Fig. 4 legend. CPA protocol: sed, n = 6, 66; ex, n = 6, 62. NCX protocol: sed, n = 5, 56; ex, n = 5, 45. Data are means ± s.e.m.: *P < 0·05, vs. respective sed; †P < 0·05, vs. control protocol.

Figure 6. Evaluation of local Ca²⁺-dependent inactivation of voltage-gated Ca²⁺ channels during membrane depolarization

A, voltage clamp template and representative current traces from a single cell using both Ca²⁺ and Ba²⁺ as charge carriers were elicited by 260 ms step depolarizations to +10 mV from a holding potential of −80 mV. Measures for calculation of T_½ decay of peak Ca²⁺ current are illustrated. B, T_½ decay of peak current was significantly shorter in the presence of Ca²⁺vs. Ba²⁺ for cells of both sed (n = 8, 36) and ex (n = 8, 29) animals. However, T_½ decay was not significantly different between cells of sed and ex animals in the presence of Ca²⁺ or Ba²⁺. Data are means ± s.e.m.; *P < 0·05 vs. respective Ca²⁺ current.