Role of calcium channels and protein kinase C for release of norepinephrine and neuropeptide Y

The role of calcium for the release of norepinephrine (NE, determined by high-pressure liquid chromatography) and neuropeptide Y (NPY, determined by radioimmunoassay) was investigated in guinea pig perfused hearts with intact sympathetic innervation. In the presence of extracellular calcium (1.85 mM), electrical stimulation of the left stellate ganglion (12 Hz, 1 min) induced a closely related release of NE and NPY with the molar ratio of ~400-600 (NE) to 1 (NPY). The stimulation-evoked overflow of both transmitters was dependent from the extracellular calcium concentration and was almost completely suppressed by calcium-free perfusion. The corelease of both transmitters was not affected by the L-type calcium channel blocker felodipine (1-10 μM). However, the overflow of NE and NPY was markedly attenuated by the unselective calcium antagonist flunarizine (1-10 μM) and completely prevented by the neuronal (N-type) calcium channel blockers ω-conotoxin (1-100 nM) and cadmium chloride (10-100 μM), indicating a key role for N-type calcium channels in the exocytotic release of transmitters from cardiac sympathetic nerve fibers. Possibly due to unspecific actions, such as interference with sodium channels or uptake₁-blocking properties, the phenylalkylamines verapamil (0.01-10 μM) and gallopamil (1-10 μM) reduced NPY overflow with only a minor effect on NE overflow. The stimulated-induced transmitter release was increased up to twofold by activation of protein kinase C (phorbol 12-myristate 13-acetate, 3 nM-3 μM) and completely suppressed by inhibition of protein kinase C (polymyxin B, 100 μM). The data indicate that the stimulation-evoked exocytotic corelease of NE and NPY from guinea pig perfused hearts depends on the presence of extracellular calcium, calcium influx through N-type calcium channels, and activation of protein kinase C.