The Journal of Neurophysiology Vol. 78 No. 3 September 1997, pp. 1476-1490
Copyright ©1997 The American Physiological Society

Muscarinic Receptor Activation Modulates Ca²⁺ Channels in Rat Intracardiac Neurons via a PTX- and Voltage-Sensitive Pathway

Seong-Woo Jeong¹ and Robert D. Wurster²

¹ Department of Physiology and ² Departments of Physiology and Neurological Surgery, Loyola Stritch School of Medicine, Maywood, Illinois 60153

Jeong, Seong-Woo and Robert D. Wurster. Muscarinic receptor activation modulates Ca²⁺ channels in rat intracardiac neurons via a PTX- and voltage-sensitive pathway. J. Neurophysiol. 78: 1476-1490, 1997. With use of the whole cell patch-clamp technique, effects of the potent muscarinic agonist oxotremorine methiodide (oxo-M) on voltage-activated Ca²⁺ channel currents were investigated in acutely dissociated adult rat intracardiac neurons. In all tested neurons oxo-M reversibly inhibited the peak Ba²⁺ current. Inhibition of the peak Ba²⁺ current by oxo-M was associated with slowing of activation kinetics and was concentration dependent. The concentration of oxo-M necessary to produce a half-maximal inhibition of current and the maximal inhibition were 40.8 nM and 75.9%, respectively. Inhibitory effect of oxo-M was completely abolished by atropine. Among different muscarinic receptor antagonists, methoctramine (100 and 300 nM) significantly antagonized the current inhibition by oxo-M, with a negativelogarithm of dissociation constant of 8.3 in adult rat intracardiac neurons. Internal dialysis of neurons with guanosine 5-(thio)triphosphate (GTPS, 0.5 mM) could mimic the muscarinic inhibition of the peak Ba²⁺ current and significantlyocclude inhibitory effects of oxo-M. In addition, the internal dialysis of guanosine-5-O-(2-thiodiphosphate) (GDPS, 2 mM) also significantly reduced the muscarinic inhibition of the peak Ba²⁺ current by oxo-M. Inhibitory effects of oxo-M were significantly abolished by pertussis toxin (PTX, 200 and 400 ng/ml) but not by cholera toxin (400 ng/ml). Furthermore, the bath application of N-ethylmaleimide (50 µM) significantly reduced the inhibition of the peak Ba²⁺ current by oxo-M. The oxo-M shifted the activation curve derived from measurments of tail currents toward more positive potentials. A strong conditioning prepulse to +100 mV significantly relieved the muscarinic inhibition of peak Ba²⁺ currents by oxo-M and the GTPS-induced current inhibition. In a series of experiments, changes in intracellular concentration of bis-(o-aminophenoxy)-N,N,N,N-tetraacetic acid and protein kinase activities failed to mimic or occlude the current inhibition by oxo-M. The dihydropyridine antagonist nifedipine (10 µM) was not able to occlude any of the inhibitory effects of oxo-M, and oxo-M (3 µM) failed to reduce the slow tail currents induced by the L-type agonist methyl 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate (FPL 64176; 2 µM). However, -conotoxin (-CgTX) GVIA (1 µM) significantly occluded the muscarinic inhibition of the Ba²⁺ currents. In the presence of -CgTX GVIA (1 µM) and nifedipine (10 µM), oxo-M could further inhibit ~20% of the total Ca²⁺ current. After complete removal of N-, Q-, and L-type currents with use of -CgTX GVIA, -agatoxin IVA, and nifedipine, 70% of the R-type current (~6-7% of the total current) was inhibited by oxo-M (3 µM). In conclusion, the M₂ muscarinic receptor activation selectively inhibits N-, Q-, and R-type Ca²⁺ channel currents, sparing L-type Ca²⁺ channel currents mainly via a PTX- and voltage-sensitive pathway in adult rat intracardiac neurons.

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