Differential localization of Ca2+ channel alpha1 subunits in the enteric nervous system: presence of alpha1B channel-like immunoreactivity in intrinsic primary afferent neurons.

Abstract

Immunocytochemistry was employed to locate calcium (Ca2+) channel proteins in the enteric nervous system (ENS) of the rat and guinea pig. Anti-peptide antibodies that specifically recognize the alpha1 subunits of class A (P/Q-type), B (N-type), C and D (L-type) Ca2+ channels were utilized. Alpha1B channel-like immunoreactivity was abundant in both enteric plexuses, the mucosa, and circular and longitudinal muscle layers. Immunoreactivity was predominantly found in cholinergic varicosities, supporting a role for Ca2+ channels, which contain the alpha1B subunit, in acetylcholine release. Immunoreactivity was also associated with the cell soma of calbindin-immunoreactive submucosal and myenteric neurons, cells that have been proposed to be intrinsic primary afferent neurons. Alpha1C channel-like immunoreactivity was distributed diffusely in the cell membrane of a large subset of neuronal cell bodies and processes, whereas alpha1D was found mainly in the cell soma and proximal dendrites ofvasoactive intestinal polypeptide-immunoreactive neurons in the guinea pig gut. Alpha1A channel-like immunoreactivity was found in a small subset of cell bodies and processes in the rat ENS. The differential localization of the alpha1 subunits of Ca2+ channels in the ENS implies that they serve distinct roles in neuronal excitation and signaling within the bowel. The presence of alpha1B channel-like immunoreactivity in putative intrinsic primary afferent neurons suggested that class B Ca2+ channels play a role in enteric sensory neurotransmission; therefore, we determined the effects of the N-type Ca2+ channel blocker, omega-conotoxin GVIA (omega-CTx GVIA), on the reflex-evoked activity of enteric neurons. Demonstrating the phosphorylation of cyclic AMP (cAMP)-responsive element-binding protein (pCREB) identified neurons that became active in response to distension. Distension elicited hexamethonium-resistant pCREB immunoreactivity in calbindin-immunoreactive neurons in each plexus; however, in preparations stimulated in the presence of omega-CTx GVIA, pCREB immunoreactivity was found only in calbindin-immunoreactive neurons in the submucosal plexus and not in myenteric ganglia. These data confirm that intrinsic primary afferent neurons are located in the submucosal plexus and that N-type Ca2+ channels play a role in sensory neurotransmission.