The research will focus on the roles and mechanisms of neurogenic Ca 2+ signals in autonomic nervous system control of arterial diameter, particularly the Ca2+ signals generated in smooth muscle cells (SMC) by the concerted action of the sympathetic 'triad' of co-transmitters, ATP, norepinephfine (NE) and neuropeptide Y (NPY). Although components of neurogenic vasoconstriction are attributable to ATP, NE and NPY, the underlying Ca 2+ signals in SMC are not known. Preliminary confocal imaging of Ca 2+ within the SMC of intact pressurized rat mesenteric small arteries during low frequency nerve stimulation has revealed novel Ca2+ transients, putatively associated with excitatory junction potentials (EJPs) and provisionally called """"""""junctional Ca 2+ transients"""""""" (jCaTs). With higher frequency stimulation, Ca 2+ 'flashes' (may be due to SMC action potentials) and Ca 2+waves are generated. Stimulation of perivascular sensory nerves elicits vasodilation; preliminary results implicate an increase in the frequency of Ca 2+ sparks. Specific Experimental goals include: 1) Test the hypothesis that jCaTs arise from Ca 2+entering SMCs through P2X1 receptors, which are activated by the ATP contained in one or a few quanta of transmitter, 2) Test the hypothesis that neurally released ATP elicits Ca 2+ influx, but not Ca 2+ waves, 3) Test the hypothesis that neurally released NE elicits Ca 2+ waves that function to activate contraction, 4) Test the hypothesis that neurally released NPY activates the Y1 receptor on SMCS to modulate the effects of NE by changing the frequency of Ca 2+ waves, and 5) Test the hypothesis that calcitonin gene related peptide (CGRP) released from sensory nerves decrease Ca 2+ spark frequency; thus causing vasodilation. Mesenteric small arteries from rats and mice (including a P2Xz-receptor deficient transgenic mouse) will be loaded with fluo-4 and mounted on a confocal myograph for simultaneous Ca2+ imaging, recording of isometric force, and measurement of membrane potential. A real-time confocal microscope will be used to provide 2D images fast enough to resolve jCaTs, flashes and waves. The research will test a comprehensive hypothesis on sympathetic neuromuscular transmission and the control of smooth muscle contraction by the autonomic nervous system: Neurally released ATP activates P2X1 receptors. The resulting Ca 2+ influx activates a small contraction, generates EJPs and is visualized as jCaTs. EJPs may summate to trigger action potentials, visualized as Ca 2+ 'flashes' Neurally released NE and NPY (as metabotropic or G protein-lined receptors) increase the frequency of Ca 2+ waves, which activate strong contraction. By visualizing novel, physiological neurogenic Ca 2+ signals and contraction of intact arteries, the research seeks to provide new information on the concerted action of neurotransmitters in controlling arterial function.
