Y.K. Mao

Myogenic nitric oxide synthase activity in canine lower oesophageal sphincter: morphological and functional evidence

1 Studies on canine lower oesophageal sphincter (LOS) evaluated the existence and function of a myogenic, nitric oxide synthase (NOS) by use of immunocytochemistry for NOS isozymes, NADPH‐d histochemistry, [3H]‐L‐arginine to [3H]‐L‐citrulline transformation. In addition, functional studies in the muscle bath were performed. 2 Smooth muscle bundles or freshly isolated smooth muscle cells of LOS were NADPH‐d reactive but did not recognize some antibodies against neural, endothelial or inducible NOS. NADPH‐d reactivity and immunoreactivity to a neural NOS antibody were colocalized in LOS enteric nerves. Muscle plasma membrane‐enriched fractions from fresh and cultured LOS cells converted [3H]‐L‐arginine to [3H]‐L‐citrulline; activity was mostly Ca2+/calmodulin‐dependent. 3 N‐Nitro‐L‐arginine (L‐NOARG) persistently increased tone (blocked by L‐arginine) in muscle strips despite blockade of nerve function. Nifedipine prevented or abolished L‐NOARG‐induced, but not carbachol‐induced, contraction showing that tone increase by L‐NOARG required functional L‐Ca channels. 4 Membrane‐bound, myogenic NOS in canine LOS may release NO continuously when Ca2+ entry through L‐Ca channels occurs under physiological conditions and thereby modulate tone in LOS.

Myogenic NOS in canine lower esophageal sphincter: enzyme activation, substrate recycling, and product actions.

Depolarization elicited outward K+ currents from canine lower esophageal sphincter (LES) muscle cells, primarily through iberiotoxin (IbTX)- and tetraethylammonium-sensitive Ca2+-dependent K+ channels. Current magnitudes varied with pipette Ca2+concentration (EC50 = 108.5 nM). N G-nitro-l-arginine (l-NNA, 10-4 M), IbTX (10-8 M), or buffering intracellular Ca2+ to 8 nM decreased outward currents >80%. Sodium nitroprusside (NaNP, 10-4 M) restoredl-NNA-inhibited or low intracellular Ca2+ concentration (not IbTX)-inhibited currents.l-NNA or IbTX application depolarized LES cells from -43 to -35 mV. NaNP restored the membrane potential to -46 mV afterl-NNA but not after IbTX application. Nifedipine (30 μM) reduced outward currents and abolished or reduced l-NNA or NaNP effects, respectively. Immunocytochemistry revealed the presence of both argininosuccinate synthetase and argininosuccinate lyase in LES muscle cells. l-Citrulline, likel-arginine, reversedl-NNA inhibition of outward currents; only l-arginine reversed inhibition of outward currents by an antibody to argininosuccinate synthetase. Therefore, endogenous nitric oxide production, activated by Ca2+entrance involving l-type Ca2+ channels, may continuously enhance outward currents to modulate LES muscle cell membrane potential and excitability.

Colocalization of inhibitory mediators, NO, VIP and galanin, in canine enteric nerves.

The colocalization of three putative inhibitory mediators of enteric nerves, vasoactive intestinal peptide (VIP), galanin (GAL) and nitric oxide synthase (nNOS), was examined in the myenteric plexus of canine antrum, intestine and colon. Many ileal and colonic neurons contained nNOS-immunoreactive (nNOS-IR) activity with some also containing VIP-IR; only a few neurons also contained GAL-IR. Ileal and colonic VIP-IR nerves often appeared to be interneurons innervating nNOS nerves. Many antral neurons contained VIP-IR with nearly all also containing GAL-IR. A few also contained nNOS-IR. The predominance of nNOS-IR neurons relative to VIP-IR and GAL-IR neurons in the ileal and colonic, but not the antral, myenteric plexus is consistent with NO being the primary inhibitory mediator in the intestine but not in the antrum.

Distribution of galanin-immunoreactive nerves in the canine gastrointestinal tract

The distribution of nerves containing galanin immunoreactivity (GAL-IR) in the canine gastrointestinal tract was determined by immunohistochemistry. GAL-IR was observed in nerve cell bodies and nerve fibers in all layers of the lower esophagus, gastric antrum, pylorus, ileum, and colon, and in the sphincters of lower esophagus and pylorus. There were numerous GAL-IR-containing nerve cell bodies in the myenteric plexus of gastric antrum. GAL-IR nerve somata were also common in the myenteric plexus of lower esophagus, ileum, pylorus, and colon, and in the submucous plexus of stomach, ileum, and colon. GAL-IR-positive nerve fibers were also observed around submucous blood vessels of the stomach. GAL-IR nerves are distributed more widely in the canine enteric nervous system than previously recognized.

An endothelial cell-line contains functional vasoactive intestinal polypeptide receptors : they control inwardly rectifying K+ channels

Bovine endothelial cells cultured from pulmonary artery (ATCC cell line No. 209) were found to contain a high density of 125I-VIP (vasoactive intestinal polypeptide) binding sites. These were found to be saturable and to be fit by a single binding site model (Kd 1.8 nM; Bmax 534 fmol/mg protein). Studies of association and dissociation of 125I-VIP to this site revealed that binding was fully reversible and yielded a Kd value similar to that from equilibrium binding. However competition studies showed that VIP competed for binding at two sites (Ki1 1.2 x 10(-11) M, Ki2 4.7 x 10(-9) M; N1 = 21%, N2 = 77%; Ki a dissociation constant for inhibitor; N percentage of occupied receptors). [Phe1]VIP also competed at two sites, but VIP-(10-28), PHM, [4-Cl-D-Phe6,Leu17]VIP and [D-Ala4]VIP displaced all specific VIP binding in a simple competitive manner. These VIP binding sites were shown to be functional. In patch clamp studies VIP 10(-8)-10(-7) M inhibited opening of inwardly rectifying K+ channels on hyperpolarization. These channels were affected appropriately by alteration in the K(+)-gradient and by Ba2+ or Cs+. The VIP antagonist [4-Cl-D-Phe6, Leu17]VIP prevented or reversed the effects of VIP. These results show that functional VIP receptors are present in high density in a endothelial cell line and provide a possible model for analysis of the molecular biology of these receptors.

Characterization of neurokinin type 1 receptor in canine small intestinal muscle.

The binding of [3H]substance P (SP) was localized and characterized in canine small intestine. The highest density of [3H]SP binding occurred in the fraction enriched in circular muscle membranes (Bmax 148 +/- 14 fmol/mg; Kd 0.81 +/- 0.01 nM). In the kinetics studies binding was reversible, yielding a similar Kd value. NK1 agonists and antagonists fully displaced, but ligands at the NK2 and NK3 receptor only partially displaced, [3H]SP binding. The potency order of displacement resembled that for the human NK1 receptor ortholog rather than the rat NK1 receptor. Densities of [3H]SP binding were lower in myenteric plexus and longitudinal muscle membrane fractions, but suggested binding in both neural and muscle membranes. The conclusion is that NK1 receptors occur primarily in canine intestine circular muscle plasma membrane.

The distribution of NPY-containing nerves and the catecholamine contents of canine enteric nerve plexuses.

Our previous study demonstrated that PYY was a major neuropeptide in the canine enteric nervous system, the present study defines the locations of NPY-containing enteric neurons. NPY-positive nerve cell bodies and fibers were numerous in gastric and pyloric myenteric plexuses as were positive nerve fibers in antral and pyloric muscle layers, pyloric sphincter muscle layers, and surrounding blood vessels. In contrast to findings for PYY, there were considerably fewer NPY-positive nerve cell bodies and fibers in the canine ileum and colon. Noradrenaline was the predominant catecholamine in all plexuses, the rank order of its contents being: deep muscular > submucous > myenteric plexus. The dopamine/noradrenaline ratio was constant in all plexuses; adrenaline was present in minor amounts. PYY is the more abundant neuropeptide in ileum and colon, and NPY has a greater presence in the gastric antrum, pylorus, and surrounding blood vessels. PYY and NPY may play different functional roles in the GI tract.

Characterization of cholecystokinin receptors in canine intestinal circular muscle

We localized and characterized the binding of [3H](+/-)-L364,718 in canine small intestine circular muscle. The highest densities of [3H]L364,718 binding were located in the fraction enriched in deep muscular plexus synaptosomal membranes. In this fraction [3H]L364,718 binding was of high density (Bmax 136.78 +/- 53.66 fmol/mg) and high affinity (Kd 1.67 +/- 0.74 nM). Kinetics studies revealed that binding was reversible and yielded a similar Kd value. L364,718, CCK-8-S, and L365,260 fully displaced [3H]L364,718 binding, but ligands at CCKB receptors, gastrin-17, and YM022 did not. Therefore, CCKA receptors in canine intestine circular muscle are located on nerve endings.