Muscarinic Receptor Activation Modulates Ca2+Channels in Rat Intracardiac Neurons via a PTX- and Voltage-Sensitive Pathway
Authors: Jeong Seong-Woo , Wurster Robert D. 
Published: December 2017

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 (GTPγS, 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) (GDPβS, 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 GTPγS-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, ω-conotoxin (ω-CgTX) GVIA (1 μM) significantly occluded the muscarinic inhibition of the Ba2+currents. In the presence of ω-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 ω-CgTX GVIA, ω-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 M2muscarinic 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.