Alpha 1-adrenoceptor-activated cation currents in neurones acutely isolated from rat cardiac parasympathetic ganglia

Abstract

The noradrenaline (NA)-induced cation current was investigated in neurones freshly isolated from rat cardiac parasympathetic ganglia using the nystatin-perforated patch recording configuration. Under current-clamp conditions, NA depolarized the membrane, eliciting repetitive action potentials. NA evoked an inward cation current under voltage-clamp conditions at a holding potential of -60 mV. The NA-induced current was inhibited by extracellular Ca2+ or Mg2+, with a half-maximal concentration of 13 microM for Ca2+ and 1.2 mM for Mg2+. Cirazoline mimicked the NA response, and prazosin and WB-4101 inhibited the NA-induced current, suggesting the contribution of an alpha1-adrenoceptor. The NA-induced current was inhibited by U73122, a phospholipase C (PLC) inhibitor. The membrane-permeable IP3 receptor blocker xestospongin-C also blocked the NA-induced current. Furthermore, pretreatment with thapsigargin and BAPTA-AM could inhibit the NA response while KN-62, phorbol 12-myristate 13-acetate (PMA) and staurosporine had no effect. These results suggest that NA activates the extracellular Ca2+- and Mg2+-sensitive cation channels via alpha 1-adrenoceptors in neurones freshly isolated from rat cardiac parasympathetic ganglia. This activation mechanism also involves phosphoinositide breakdown, release of Ca2+ from intracellular Ca2+ stores and calmodulin. The cation channels activated by NA may play an important role in neuronal membrane depolarization in rat cardiac ganglia.

Figure 1. Noradrenaline response in isolated cardiac ganglion neurones

A, depolarization of a rat cardiac ganglion neurone by noradrenaline (NA). Recordings were made under current-clamp conditions. Horizontal filled bar above the trace indicates the application of NA. The figure is representative of 6 reproducible experiments. B, inward currents evoked by NA (I_NA). All current traces were recorded from the same neurone. Holding potential (V_H) was −60 mV. The NA-induced current was sensitive to extracellular divalent cations. Dotted line shows the zero current level. The inset summarizes the effects of Ca²⁺ and Mg²⁺ on the NA-induced current. The current amplitude was normalized to that observed in the normal external solution containing 2 mm Ca²⁺ and 1 mm Mg²⁺. Each column shows the mean of 5–7 experiments. C, current-voltage relationship of the NA (10 μm)-induced current. The amplitude of the NA-induced current at various V_H was normalized to that observed at −60 mV in each neurone. Each point is the mean of 4–6 experiments. Vertical bars indicate ±s.e.m.D, extracellular Na⁺ dependency. The current trace is representative of 4 reproducible experiments.

Figure 2. Effects of organic and inorganic cation channel blockers

Experiments were performed in Ca²⁺-free solution containing 1 mm Mg²⁺. Each blocker was applied 1 min before the application of NA. A, effect of La³⁺ on the NA-induced current. B, effect of SK&F96365. C, concentration-dependent inhibition of the NA-induced current by La³⁺, Gd³⁺, SK&F96365 and Cd²⁺. Curves are the best non-linear fits to the Hill equation, 1/1 + (C/K_i) ^nH where C, K_i and n_H denote the concentration of each blocker applied, dissociation constant and Hill coefficient, respectively. Each point is the mean of 6–8 experiments. Vertical bars indicate ±s.e.m.

Figure 3. Effects of [Ca²⁺]_o on the NA-induced current

Experiments were performed in the solution containing 1 mm Mg²⁺. A, the effect of [Ca²⁺]_o on NA-induced current. All current traces were obtained from the same neurone. NA was applied every 6 min in various [Ca²⁺]_o. Lower panel shows the [Ca²⁺]_o dependency. Current amplitude was normalized to that observed in the solution containing 30 nm Ca²⁺. Each point is the mean of 4–6 experiments. Vertical bars indicate ±s.e.m. B, rapid inhibition of the NA-induced current by extracellular Ca²⁺. During the application of NA in Ca²⁺-free solution, the switch of extracellular solution to that containing 2 mm Ca²⁺ rapidly inhibited the current within 1 s The figure is representative of 5 reproducible experiments.

Figure 4. Effects of extracellular Mg²⁺ concentration

A, representative current traces induced by NA in the solution containing various concentrations of Mg²⁺. In this experiment, Mg²⁺ was simply added to the nominally divalent cation-free solution. NA was applied every 6 min. B, concentration-inhibition curve for the NA-induced current by Mg²⁺ in divalent cation-free extracellular solution. The amplitude of the NA-induced current in various concentrations of Mg²⁺ was normalized to that in the solution containing 1 mm Mg²⁺. Each point is the mean of 4–5 experiments. Vertical bars indicate ±s.e.m.

Figure 5. Effects of adrenoceptor agonists and antagonists

A, concentration-response curve for NA and cirazoline. The current evoked by various concentrations of NA and cirazoline was normalized to that induced by 10 μm NA. In this and subsequent figures, electrophysiological experiments were performed at a V_H of −60 mV in the Ca²⁺-free solution containing 2 mm EGTA and 1 mm Mg²⁺. Each point is the mean of 4–5 experiments. Vertical bars indicate ±s.e.m.B, effects of prazosin, WB-4101 and yohimbine on the current induced by 10 μm NA. These antagonists were applied 1 min before the application of NA. Each point is the mean of 4–6 experiments. Vertical bars indicate ±s.e.m.

Figure 6. Contribution of PLC and IP₃ receptors

A and B, inhibitory effects of PLC inhibitor U73122 (A) and membrane-permeable IP₃ receptor antagonist XeC (B) on the NA-induced current. Each inhibitor was applied 4 min before the application of NA. C, effects of 1 μm U73122, 1 μm U73343 and 2 μm XeC. Current responses in the presence of various inhibitors were normalized to that obtained just before the application of each inhibitor. Each column represents the mean of 4–6 experiments. **P < 0.01 vs. control. ***P < 0.001 vs. control. N.S., no significant difference from control.

Figure 7. Involvement of Ca²⁺ release from intracellular Ca²⁺ stores in the NA response

A and B, representative current traces showing the inhibitory effects of thapsigargin and BAPTA-AM on the NA response. Thapsigargin and BAPTA-AM were applied 4 min before the application of NA. C, inhibitory effects of thapsigargin and BAPTA-AM on the NA-induced current. Each column represents the mean of 5–6 experiments. **P < 0.01 vs. Control. ***P < 0.001 vs. Control. D, increase in [Ca²⁺]_i (measured as 340 nm/380 nm fura-2 fluorescence ratio) induced by NA even in the absence of extracellular Ca²⁺. The response is representative of 8 reproducible experiments.

Figure 8. Effects of calmodulin inhibitor, KN-62 and PKC modulators on the NA-induced current

A, inhibitory effect of W-7 on the NA-induced current. B, concentration-inhibition curve of W-7 and W-5 for the NA (1 μm)-induced current. W-7 and W-5 were applied 4 min before the application of NA. Each point is the mean of 5 experiments. Vertical bars indicate ±s.e.m. C, KN-62 had no effect on the NA response. D, effects of 3 μm KN-62, 0.3 μm staurosporine and 0.3 μm PMA on the NA-induced current. KN-62, staurosporine and PMA were applied 10 min before the application of NA.