Role of N-type calcium channels in autonomic neurotransmission in guinea-pig isolated left atria

Abstract

1. Calcium entry via neuronal calcium channels is essential for the process of neurotransmission. We investigated the calcium channel subtypes involved in the operation of cardiac autonomic neurotransmission by examining the effects of selective calcium channel blockers on the inotropic responses to electrical field stimulation (EFS) of driven (4 Hz) guinea-pig isolated left atria. In this tissue, a previous report (Hong & Chang, 1995) found no evidence for N-type channels involved in the vagal negative inotropic response and only weak involvement in sympathetic responses. 2. The effects of cumulative concentrations of the selective N-type calcium channel blocker, omega-conotoxin GVIA (GVIA; 0.1-10 nM) and the non-selective N-, P/Q-type calcium channel blocker, omega-conotoxin MVIIC (MVIIC; 0.01-10 nM) were examined on the positive (with atropine, 1 microM present) and negative (with propranolol, 1 microM and clonidine, 1 microM present) inotropic responses to EFS (eight trains, each train four pulses per punctate stimulus). 3. GVIA caused complete inhibition of both cardiac vagal and sympathetic inotropic responses to EFS. GVIA was equipotent at inhibiting positive (pIC50 9.29+/-0.08) and negative (pIC50 9.13+/-0.17) inotropic responses. MVIIC also mediated complete inhibition of inotropic responses to EFS and was 160 and 85 fold less potent than GVIA at inhibiting positive (pIC50 7.08+/-0.10) and negative (pIC50 7.20+/-0.14) inotropic responses, respectively. MVIIC was also equipotent at inhibiting both sympathetic and vagal responses. 4. Our data demonstrates that N-type calcium channels account for all the calcium current required for cardiac autonomic neurotransmission in the guinea-pig isolated left atrium.

Figure 1

Representative trace recording of the inotropic response to eight trains electrical field stimulation (EFS; each train was four pulses per refractory period, see Methods) in a guinea-pig isolated left atrium in the absence of antagonists. EFS was passed across the atria as indicated by the upward and downward arrows. Second calibration bar indicates slowing of the chart speed from 25 to 5 mm min⁻¹; Insert shows a schematic representation of the analysis of the inotropic responses to EFS (see Methods). b is the baseline force of contraction immediately prior to EFS. v₁ is the vagally-mediated decrease in punctate force of contraction, calculated as the difference between b and the peak force of contraction at the end of EFS (v₂). The sympathetically-mediated positive inotropic response to EFS is measured as the difference between the maximum increase in the force of contraction following EFS (s) and b.

Figure 2

Effect of calcium channel blockers on the sympathetic response to field stimulation (EFS) of guinea-pig isolated left atria in the presence of atropine (1 μM). Curves are the positive inotropic response to eight trains EFS (each train was four pulses per refractory period; see Methods) expressed as per cent increase in baseline force (see Methods) following cumulative additions of either ω-conotoxin GVIA (GVIA; 0.1–3 nM; n=5) or ω-conotoxin MVIIC (MVIIC; 30–1000 nM; n=5). Each concentration of peptide was incubated for 60 min prior to assessing the response to EFS. The positive inotropic response to EFS with time in control atria (n=5) is shown for comparison. Vertical error bars are ±s.e.mean. Horizontal error bars are ±1 s.e.mean on the average fitted IC₅₀ for GVIA and MVIIC. C2 is the second control inotropic response to EFS immediately prior to addition of the first concentration of peptide or vehicle.

Figure 3

Representative experimental chart record of the effect of ω-conotoxin GVIA (GVIA) on inotropic responses to eight trains electrical field stimulation (EFS). EFS was passed across the atria as indicated by the upward and downward arrows. (a) is the effect of GVIA on the sympathetic nerve-mediated positive inotropic response to EFS in the presence of atropine (1 μM). Second calibration bar on (a) indicates slowing of the chart speed from 25 to 5 mm min⁻¹. (b) is the effect of GVIA on the vagal nerve mediated negative inotropic response to EFS in the presence of propranolol (1 μM) and clonidine (1 μM).

Figure 4

Effect of calcium channel blockers on the vagal response to field stimulation (EFS) of guinea-pig isolated left atria in the presence of propranolol (1 μm) and clonidine (1 μM). Curves are the negative inotropic response to eight trains EFS (each train was four pulses per refractory period; see Methods) expressed as per cent inhibition of baseline force (see Methods) following cumulative additions of either ω-conotoxin GVIA (GVIA; 0.1–3 nM; n=5) or ω-conotoxin MVIIC (10–300 nM; n=5). Each concentration of peptide was incubated for either 60 min (GVIA) or 30 min (MVIIC) prior to assessing the response to EFS. The negative inotropic response to EFS with time in control atria (n=5) is shown for comparison. Vertical error bars are ±s.e.mean. Horizontal error bars are ±1 s.e.mean on the average fitted IC₅₀ for each curve. C2 is the second control inotropic response to EFS immediately prior to addition of the first concentration of peptide or vehicle.

Figure 5

Effect of calcium channel blockade by ω-conotoxins on the positive and negative inotropic responses to field stimulation in guinea-pig isolated left atria. Curves are the inotropic response to eight trains EFS (each train was four pulses per refractory period; see Methods) expressed as per cent of the second control response to 8 trains EFS (see Methods) immediately prior to addition of cumulative concentrations of either ω-conotoxin GVIA (GVIA; 0.1–10 nM; n=5) or ω-conotoxin MVIIC (10–1000 nM; n=5). GVIA was incubated for 60 min at each concentration. MVIIC was incubated for 60 min (sympathetic responses) or 30 min (vagal responses) at each concentration. Vertical error bars are ±s.e.mean. Horizontal error bars are ±1 s.e.mean on the average fitted IC₅₀ for each curve.