Background: Myenteric neurons, which control gut motility and absorption, are heterogeneous in structure, electrophysiology, and neuropeptide content. We hypothesized that myenteric neurons would display heterogeneous responses to neuroligands found in enteric tissue. We examined increases in intracellular calcium ([Ca2+]i), an important second messenger, evoked by selected neuroligands in cultured myenteric neurons.
Methods: Primary cultures of guinea pig myenteric neurons were characterized by using a standard immunocytochemical stain for microtubule-associated protein (MAP2). Increases in [Ca2+]i were measured by digital imaging microscopy. Results expressed as mean +/- SEM, Student's t test.
Results: Cultured myenteric neurons examined with phase contrast and Nomarsky optics extend processes and stain positively with the neuron-selective stain MAP2. Histamine did not evoke calcium mobilization in cultured neurons, although progressive increases in [Ca2+]i were seen with bradykinin, glutamate, serotonin, cholecystokinin, bombesin, adenosine triphosphate (ATP), substance P, and acetylcholine. Enteric neurons differed from one another in the ability to respond to one, two, or three of the agonist combinations tested. Time in culture did not alter the percentage of neurons responding to ATP, substance P, or acetylcholine. ATP evoked equivalent [Ca2+]i increases in neurons examined at the three time points, whereas significant decrements in neuronal calcium mobilization were seen after 8 days in culture with substance P or acetylcholine (p < 0.05 versus 1 day in vitro).
Conclusions: Cultured myenteric neurons extend processes and retain expression of neuron-specific antigens. Selected neuroligands found in enteric tissue increase [Ca2+]i, as a second messenger in subsets of myenteric neurons. Heterogeneity exists among cultured neurons in their ability to respond to ligand combinations. Changes in [Ca2+]i induced by neuroligands in myenteric neurons may be altered with time in vitro.