MUSCARINIC RECEPTOR ACTIVATION MODULATES CA2-SENSITIVE AND VOLTAGE-SENSITIVE PATHWAY( CHANNELS IN RAT INTRACARDIAC NEURONS VIA A PTX)

With use of the whole cell patch-clamp technique, effects of the poten t muscarinic agonist oxotremorine methiodide (oxo-M) on voltage-activa ted Ca2+ channel currents were investigated in acutely dissociated adu lt rat intracardiac neurons. In all tested neurons oxo-M reversibly in hibited the peak Ba2+ current. Inhibition of the peak Ba current by ox o-M was associated with slowing of activation kinetics and was concent ration dependent. The concentration of oxo-M necessary to produce a ha lf-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 negative logarithm of dissociation consta nt of 8.3 in adult rat intracardiac neurons. Internal dialysis of neur ons with guanosine 5'-(thio) triphosphate (GTP gamma S, 0.5 mM) could mimic the muscarinic inhibition of the peak Ba2+ current and significa ntly occlude inhibitory effects of oxo-M. In addition, the internal di alysis of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S, 2 mM) also s ignificantly reduced the muscarinic inhibition of the peak Ba2+ curren t by oxo-M. Inhibitory effects of oxo-M were significantly abolished b y pertussis toxin (PTX, 200 and 400 ng/ml) but not by cholera toxin (4 00 ng/ml). Furthermore. the bath application of N-ethylmaleimide (50 m u M) 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 conditioni ng prepulse to +100 mV significantly relieved the muscarinic inhibitio n of peak Ba2+ currents by oxo-M and the GTP gamma S-induced current i nhibition. In a series of experiments, changes in intracellular concen tration of bis-(o-aminophenoxy) -N,N,N',N'-tetraacetic acid and protei n kinase activities failed to mimic or occlude the current inhibition by oxo-M. The dihydropyridine antagonist nifedipine (10 mu M) was not able to occlude any of the inhibitory effects of oxo-M, and oxo-M (3 m u M) failed to reduce the slow tail currents induced by the L-type ago nist methyl [2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate (FPL 64 176; 2 mu M). However, omega-conotoxin (omega-CgTX) GVIA (1 mu M) sign ificantly occluded the muscarinic inhibition of the Ba2+ currents. In the presence of omega-CgTX GVIA (1 mu M) and nifedipine (10 mu M), oxo -M could further inhibit. similar to 20% of the total Ca2+ current. Af ter complete removal of N-, Q-, and L-type currents with use of omega- CgTX GVIA, omega-agatoxin IVA, and nifedipine, 70% of the R-type curre nt (similar to 6-7% of the total current) was inhibited by oxo-M (3 mu M). In conclusion, the M-2 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 a dult rat intracardiac neurons.