In the canine gastrointestinal tract, the roles that gap junctions play in pacemaking and neurotransmission are unclear. Using antibodies to connexin (Cx)43, Cx45, and Cx40, we determined the distribution of these connexins. Cx43 was present in all locations where structural gap junctions occur. Cx40 was also widely distributed in the circular muscle of the lower esophageal sphincter (LES), stomach, and ileum. Cx45 was sparsely distributed in circular muscle of the LES. In the interstitial cells of Cajal (ICC) networks of myenteric plexus, in the deep muscular and submuscular plexuses, sparse Cx45 and Cx40 immunoreactivity was present. In colon, immunoreactivity was found only in the myenteric and submuscular plexus and nearby circular muscle cells. No immunoreactivity was found in sites lacking structural gap junctions (longitudinal muscle, inner circular muscle of the intestine, and most circular muscle of the colon). Studies of colocalization of connexins suggested that in the ICC networks, some colocalization of Cx43 with Cx40 and/or Cx45 occurred. Thus gap junctions in canine intestine may be heterotypic or heteromeric and have different conductance properties in different regions based on different connexin compositions.

Immunochemical studies with light microscopy, confocal microscopy, and electron microscopy were used to examine proteins associated with caveolin (Cav) in canine lower esophageal sphincter. The main Cav was Cav-1. It appeared to be colocalized at the cell periphery, in punctate sites, with immunoreactivity to antibodies against different COOH- and NH2-terminal epitopes of neuronal nitric oxide (NO) synthase (nNOS). One COOH-terminal-directed antibody, made in guinea pig, was used to colocalize other immunoreactivities. Those that apparently colocalized with nNOS were L-Ca2+ channels, the PM Ca2+ pump, and, in part, calreticulin and calsequestrin. The large-conductance Ca2+-activated K+ (BK(Ca)) channels were located in discrete peripheral sites, some with Cav. Immunoreactivities not fully colocalized with nNOS were to the sarcoplasmic reticulum Ca2+ pump, connexins 43, 40, and 45, and vinculin. In patch-clamp studies, NO-driven outward currents, mainly through BK(Ca) channels, were inhibited by antibodies to Cav-1 and not by calmodulin and were restored by an NO donor. Several Ca2+-handling molecules are localized at the PM with and/or near Cav. This may allow intracellular calcium concentration levels to be controlled differently than those in the cytosol near caveolae.

PYY receptors were characterized and their loci determined in canine small intestine. The density of 125I-labeled peptide tyrosine tyrosine (PYY) binding was highest in myenteric (MY) and submucosal (SUB) plexus fractions enriched in synaptosomes. Two binding sites [high affinity (H) and low affinity (L)] were found in the submucosal synaptosome-enriched membrane: dissociation constant (Kd)H = 7.6 pM, maximal binding capacity (Bmax)H = 28 fmol/mg; KdL = 0.18 nM, BmaxL = 120 fmol/mg protein. The binding of 125I-PYY reached a maximum within 30 min; dissociation was incomplete in the presence of unlabeled PYY. The rate of dissociation was enhanced after exposure of synaptosomes to guanosine 5'-O-(3-thiotriphosphate). Binding of 125I-PYY was completely inhibited by neuropeptide Y (NPY)-(13-36) (in SUB and MY) and by [Leu31,Pro34]NPY (in MY) but only partially by [Leu31,Pro34]NPY in SUB, suggesting the presence of Y2 receptor in SUB and the presence of Y1 and Y2 receptors in MY. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PYY receptor complex revealed a radioactive band at 70 kDa. The PYY receptors in the canine small intestinal myenteric and submucosal plexus correspond in location to that of PYY in synaptosomes and are coupled with G proteins. Different subtypes are present in different loci.

In canine ileum we investigated the distribution of pituitary adenylate cyclase-activating peptide (PACAP), using immunofluorescence and radioimmunoassay and the binding of 125I-PACAP-27 to membranes. Nerve profiles immunoreactive to PACAP-27, and often to vasoactive intestinal polypeptide (VIP) as well, were found in all plexi, but PACAP was present in ∼100-fold lesser amounts than VIP. High-performance liquid chromatography analysis of deep muscular plexus (DMP) synaptosomes suggested the presence of PACAP-38, PACAP-27, and a third unidentified molecular form. High- and low-affinity125I-PACAP-27 binding sites were found in DMP synaptosomes and circular smooth muscle (CM) plasma membranes. In competition studies with DMP membranes, high (H)- and low (L)-affinity dissociation constants ( K d) and maximal binding capacities (Bmax) were as follows: K d H = 66.9 pM, Bmax H = 101 fmol/mg; K d L = 2.18 nM, Bmax L = 580 fmol/mg protein. The binding of125I-PACAP-27 was fast. Dissociation was slow and incomplete in the presence of unlabeled PACAP-27 but accelerated by pretreatment with guanosine 5′- O-(3-thiotriphosphate) (GTPγS). GTPγS or cholera toxin treatment eliminated high-affinity binding in both membranes. VIP had ∼100-fold lower affinity than PACAP-27 in both membranes. Cross-linking studies identified an ∼70-kDa PACAP receptor in each membrane. Thus PACAP coexists with VIP in ileal enteric nerves and acts on PACAP-preferring, possibly Gs-coupled, receptors in DMP synaptosomes and CM membranes.