Muscarinic receptor activation modulates Ca2+ channels in rat intracardiac neurons via a PTX- and voltage-sensitive pathway.
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 negative logarithm of dissociation constant of 8.3 in adult rat intracardiac neurons. Internal dialysis of neurons with guanosine 5'-(thio)triphosphate (GTPgammaS, 0.5 mM) could mimic the muscarinic inhibition of the peak Ba2+ current and significantly occlude inhibitory effects of oxo-M. In addition, the internal dialysis of guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS, 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 microM) 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 GTPgammaS-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 microM) was not able to occlude any of the inhibitory effects of oxo-M, and oxo-M (3 microM) 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 microM). However, omega-conotoxin (omega-CgTX) GVIA (1 microM) significantly occluded the muscarinic inhibition of the Ba2+ currents. In the presence of omega-CgTX GVIA (1 microM) and nifedipine (10 microM), oxo-M could further inhibit approximately 20% of the total Ca2+ current. After 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 current (approximately 6-7% of the total current) was inhibited by oxo-M (3 microM). 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.
Jeong
S W
SW
Department of Physiology, Loyola Stritch School of Medicine, Maywood, Illinois 60153, USA.
Wurster
R D
RD
D000818:Animals; D001464:Barium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D003959:Diamines / Q000494:pharmacology; D004558:Electric Stimulation; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006321:Heart / Q000187:drug effects / Q000294:innervation*; D066298:In Vitro Techniques; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D018721:Muscarinic Agonists / Q000494:pharmacology*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010276:Parasympatholytics / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D037342:Pertussis Toxin; D051381:Rats; D017207:Rats, Sprague-Dawley; D011976:Receptors, Muscarinic / Q000187:drug effects*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D015220:Calcium Channels; D003959:Diamines; D018721:Muscarinic Agonists; D010276:Parasympatholytics; D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D001464:Barium; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; C054938:methoctramine