Harold G. Preiksaitis

Nitric oxide mediates inhibitory nerve effects in human esophagus and lower esophageal sphincter

The effect of inhibition of nitric oxide synthase on nonadrenergic, noncholinergic nerve-mediated responses in circular smooth muscle of the human esophageal body and lower esophageal sphincter (LES) was examinedin vitro. Tissues were obtained from 10 patients (eight esophageal resection for cancer, two transplant donors). Muscle strips from the LES developed significant spontaneous tension (11.6 ± 2.1 mN/mm2,N=6) and relaxed in response to electrical stimulation. The nitric oxide synthase inhibitor,Nω-nitro-l-arginine (NNA), at 10−5 M, inhibited the relaxation, but had no significant effect on the spontaneous tension (13.0 ± 2.6 mN/mm2,P=0.07). Esophageal body strips developed little spontaneous tension, demonstrated an “off” contraction following the cessation of the electrical stimulus, and when contracted with 10−5 M carbachol, relaxed during electrical stimulation. NNA (10−5 M) inhibited the off contraction and the relaxation seen after carbachol and unmasked a prominent intrastimulus contraction. This intrastimulus contraction was enhanced by eserine and inhibited by atropine and tetrodotoxin. NNA showed similar potency in the esophageal body and LES and its effects were reversed byl-arginine, but notd-arginine. The results indicate that nitric oxide is an important mediator for nonadrenergic, noncholinergic nerve effects in the human esophagus and lower esophageal sphincter.

Cholinergic responses in the cat lower esophageal sphincter show regional variation.

BACKGROUND/AIMS The lower esophageal sphincter (LES) pressure in humans is asymmetric; the highest pressure and the most significant cholinergic contribution occurs toward the left. The basis of this asymmetry was examined using the cat as a model. METHODS The LES pressure profile was determined using a manometry catheter with four ports oriented at right angles. The LES was dissected into right and left halves with the latter including a contribution from the oblique gastric sling fibers. Isometric tension responses were studied in vitro. RESULTS In vivo, both the initial LES pressure (31.8 +/- 4.0 mm Hg) and the decrease (79.9% +/- 6.4%) after intravenous atropine (100 micrograms/kg) were greatest in the leftward direction. In vitro, both halves of the LES developed similar spontaneous tension, but the increase in tension to carbachol was twofold greater on the left than the right. Eserine increased and atropine decreased initial tension by 25%-30% in both. Strips from either side relaxed in response to electrical stimulation but the response was more complete in strips from the right, whereas sodium nitroprusside produced similar relaxation in both. CONCLUSIONS Regional differences in the LES pressure and its cholinergic component can be accounted for by differences in the in vitro properties of the LES muscle fiber groups.

Calcium Sensitization in Human Esophageal Muscle: Role for RhoA Kinase in Maintenance of Lower Esophageal Sphincter Tone

A rise in intracellular-free calcium ([Ca2+]i) concentration is important for initiating contraction of smooth muscles, and Ca2+ sensitization involving RhoA kinase can sustain tension. We previously found that [Ca2+]i was comparable in cells from the esophageal body (EB) and lower esophageal sphincter (LES) muscles, despite the fact that the LES maintains resting tone. We hypothesized that Ca2+ sensitization contributes to contraction in human esophageal muscle. Tension and [Ca2+]i were measured simultaneously in intact human EB and LES muscles using the ratiometric Ca2+-sensitive dye fura-2. Spontaneous oscillations in EB muscle tension were associated with transient elevations of [Ca2+]i. Carbachol caused a large increase in tension, compared with spontaneous oscillations, although the rise of [Ca2+]i was similar, suggesting Ca2+ sensitization. The RhoA-kinase blockers (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632) and 1-(5-isoquinolinesulfonyl)-homopiperazine hydrochloride (HA-1077) reduced carbacholand nerve-evoked contraction of the EB, accompanied by smaller reduction in the rise of [Ca2+]i. Protein kinase C inhibitors reduced force to a lesser extent. RhoA-kinase blockers caused concentration-dependent reduction of tension in spontaneously contracted LES muscles. Moreover, RhoA-kinase blockers reduced intrinsic nerve-evoked and carbachol-evoked contraction. However, there was no effect on nerve- or nitric oxide-mediated relaxation of LES. Ca2+ sensitization mediated by the RhoA-kinase pathway has an important role in contraction of human EB muscles and LES tonic contraction, a feature not previously recognized.

Tachykinin receptor expression and function in human esophageal smooth muscle.

Tachykinins are present in enteric nerves of the gastrointestinal tract and cause contraction of esophageal smooth muscle; however, the mechanisms involved are not understood. Our aim was to characterize tachykinin signaling in human esophageal smooth muscle. We investigated functional effects of tachykinins on human esophageal smooth muscle using tension recordings and isolated cells, receptor expression with reverse transcription (RT)-polymerase chain reaction (PCR) and immunoblotting, intracellular Ca2+ responses using fluorescent indicator dyes, and membrane currents with patch-clamp electrophysiology. The mammalian tachykinins [substance P and neurokinin (NK) A and NKB] elicited concentration-dependent contractions of human esophageal smooth muscle. These responses were not affected by muscarinic receptor or neuronal blockade indicating a direct effect on smooth muscle cells (SMCs). Immunofluorescence and RT-PCR identified tachykinin receptors (NK1, NK2, and NK3) on SMCs. Contraction was mediated through a combination of Ca2+ release from intracellular stores and influx through L-type Ca2+ channels. NK2 receptor blockade inhibited the largest proportion of tachykinin-evoked responses. NKA evoked a nonselective cation current (INSC) with properties similar to that elicited by muscarinic stimulation. The following paradigm is suggested: tachykinin receptor binding to SMCs releases Ca2+ from stores along with activation of INSC, which in turn results in membrane depolarization, L-type Ca2+ channel opening, rise of Ca2+ concentration, and contraction. These studies reveal new aspects of tachykinin signaling in human esophageal SMCs. Excitatory tachykinin pathways may represent targets for pharmacological intervention in disorders of esophageal dysmotility.

Intravenous pamidronate increases bone density in patients with Crohn's disease

CylextVlmmune Cell Function Assay was recently cleared by the FDA for the detection of cell-mediated immunity in inmmnosuppressed patients. The assay involves incubation of whole blood with phytohemaggiutinin (PHA). After the incubation, CD4 + cells are selected using magnetic particles, washed and lysed. The intracellular ATP from the selected cells is measured w~th [uciterin/lucikrase in a luminometer. If T cells have been activated, they will contain increased levels of intracdlular ATP. The amount of ATP is directly correlated with the degree of T cell activation During clinical trials on in:tmunosuppressed patients post solid organ tranplantation using this assay, three zones of reactivity were defined, expressed as ng/niL of ATP: low ( 225 and 525). Aim: In this study, pediatric patients with CD were tested to determine their CD4 immune cell iunction using the Cylex assay (discussed above) in response to 6-mercaptopurine therapy. Results: Those patients with CD either active or recalcitrant to 6-mercaptopurine therapy and requiring inlliximab therapy showed CD4 ATP levels in the high zone of reactivity. Patients whose disease was either not active or who were responding to 6-MP therapy had responses in the moderate to low range (Table). Conclusions: The Cylex TM Immune Cell Function Assay may provide clinicians with a new clinical tool to monitor their patients with CD on 6 MP therapy Future study are in progress to determine whether a therapeutic window of clinical efficacy based on the measurement of CD4 ATP levels can be developed in managing patients with CD on antimetabolite therapy.