Temporal sequence of activation of cells involved in purinergic neurotransmission in the colon

Platelet derived growth factor receptor (PDGFR(+)) cells in colonic muscles are innervated by enteric inhibitory motor neurons. PDGFR(+) cells generate Ca2+ transients in response to exogenous purines and these responses were blocked by MRS-2500. Stimulation of enteric neurons, with cholinergic and nitrergic components blocked, evoked Ca2+ transients in PDGFR(+) and smooth muscle cells (SMCs). Responses to nerve stimulation were abolished by MRS-2500 and not observed in muscles with genetic deactivation of P2Y1 receptors. Ca2+ transients evoked by nerve stimulation in PDGFR(+) cells showed the same temporal characteristics as electrophysiological responses. PDGFR(+) cells express gap junction genes, and drugs that inhibit gap junctions blocked neural responses in SMCs, but not in nerve processes or PDGFR(+) cells. PDGFR(+) cells are directly innervated by inhibitory motor neurons and purinergic responses are conducted to SMCs via gap junctions. AbstractInterstitial cells, known as platelet derived growth factor receptor (PDGFR(+)) cells, are closely associated with varicosities of enteric motor neurons and suggested to mediate purinergic hyperpolarization responses in smooth muscles of the gastrointestinal tract (GI), but this concept has not been demonstrated directly in intact muscles. We used confocal microscopy to monitor Ca2+ transients in neurons and post-junctional cells of the murine colon evoked by exogenous purines or electrical field stimulation (EFS) of enteric neurons. EFS (1-20Hz) caused Ca2+ transients in enteric motor nerve processes and then in PDGFR(+) cells shortly after the onset of stimulation (latency from EFS was 280ms at 10Hz). Responses in smooth muscle cells (SMCs) were typically a small decrease in Ca2+ fluorescence just after the initiation of Ca2+ transients in PDGFR(+) cells. Upon cessation of EFS, several fast Ca2+ transients were noted in SMCs (rebound excitation). Strong correlation was noted in the temporal characteristics of Ca2+ transients evoked in PDGFR(+) cells by EFS and inhibitory junction potentials (IJPs) recorded with intracellular microelectrodes. Ca2+ transients and IJPs elicited by EFS were blocked by MRS-2500, a P2Y1 antagonist, and absent in P2ry1((-/-)) mice. PDGFR(+) cells expressed gap junction genes, and gap junction uncouplers, 18-glycyrrhetinic acid (18-GA) and octanol blocked Ca2+ transients in SMCs but not in neurons or PDGFR(+) cells. IJPs recorded from SMCs were also blocked. These findings demonstrate direct innervation of PDGFR(+) cells by motor neurons. PDGFR(+) cells are primary targets for purinergic neurotransmitter(s) in enteric inhibitory neurotransmission. Hyperpolarization responses are conducted to SMCs via gap junctions.