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Effect of preservation and transplantation on neuromuscular transmission in canine and human ileal smooth muscle
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ABSTRACTS OF PAPERS
October 1992
MECHANISMOF SUBSTANCE P (SP) INHIBITIONOF CANINE ILEAL CIRCULAR MUSCLE CONTRACTIONS IN VIVO. JET. Fox-Threlkeld, E.G. Watson, Z.Woskowska E.E.Daniel. Smooth Muscle Program, McMaster University, Hamilton, Ont. CAN. Close intraarterial injections of low concentrations of SP were shown previously to inhibit field stimulated and motilin induced contractions of circular muscle b wv& (Am J Physiol 250 G21-26, 1986) We proposed a mechanism of SP inhibition: release of acetylcholine (ACH) to act on Mi receptors inhibiting ACH release. However, there was a residual inhibition after atropine or pirenzipine suggesting release of a further inhibitory agent. Two potential inhibitors of field stimulated motility are VIP and NO. To test whether the agent was VIP, SP or the NKl agonist [Sar9,met(OZ)n]-substancc P (lO-i” to 10-M) were infused into isolated Krebs Ringer perfused ileal segments and the venous effluent was assayed for immunoreactive VIP. There was no change in the concentration of VIP during these infusions suggesting that release of VIP had no role in the SP-induced inhibition. To examine whether release of NO was involved in SP induced inhibition the NO synthase inhibitor N-o-Larginine methyl ester &NAME) was given intraarterially (lO*mols) or intravenously (lo5 mols/kg) to anaesthetized dogs prepared with local intraarterial cannulation and serosal strain gauges and electrodes. In the presence of LNAME, the inhibition of field stimulated contractions (20V/cm, OSms, and 3-5~~s) by intraarterial SP was abolished in 5/10 sites, required higher SP concentrations in 4/10 sites and was unchanged in l/IO sites. The inhibition recovered after intravenous infusion of 1O-4 mols/lcg L-arginine. These results suggest that release of NO may contribute to the mediation of SP’s inhibitory action. Supported by MRC Canada.
ROLE OF NEURALLY-DERIVED,NO-RELATEDCOMPOUNDS IN GASTROINTESTINAL MOTOR FUNCTION IN WO EFFECTS. J.E.T. Fox-Threlkeld, C. Haugh, H.-D. Allescher, G. Tougas, P. Vergara and E.E. Daniel. Smooth Muscle Program, McMaster University, Hamilton, Ontario, Canada. We tested the hypothesis that in anaesthetized, fasted dogs, lack of intestinal motility result from tonic neural release of an NOrelated compound. Isolated perfused segments of intestine tonically release VIP (Am J Physiol 256 G884) Release was markedly reduced, accompanied by increased motility by TTX, opioids and asadrenoceptor agonists (Peptides 12 1039) Hexamethonium and Ca,+-free medium turned off VIP release without increasing motility. To examine whether NO-release from nerves in a Cast-dependent manner was the source of tonic inhibition in the intestine, NO synthase was inhibited by L”-N-arginine methyl ester (L-NAME). L-Name decreased dose-dependently excitation of intestinal CM (maximal at 3x10AM). This was reversed by L-arginine (1 mM), prevented by omega-conotoxin (10-7M) or Cast-free medium, and reduced by nifedipine (10-M) and by atropine (l@‘M). L-NAME during field stimulation reduced initial inhibition and amplified excitation. L-NAME also reduced VIP output 15-30%, an effect not reversed by L-arginine. Moreover, LNAME abolished the contractile response to 6 opioids, reduced that to p opioids and somatostatin and left unchanged that to NPY or PYY. Each peptide still decreased VIP output after L-NAME. Thus tonic inhibition of intestinal motility in is partially mediated by tonic release of NOrelated mediator. If VIP and NO are co-localized in the canine ileal nerves, their release is separately regulated and the contribution of each inhibitor in the response to an agonist must be separately evaluated. Supported by the MRC Canada.
PYY AND NPY STIMULATE ISOLATED PERFUSED CANINE ILEAL CONTRACITONS BY INHIBITING VIP NOT NO RELEASE. J.E.T. Fox-Threlkeld, E.E. Daniel, F. Christinck, Z. Woskowska, S. Cipris, and T.J. McDonald. Smooth Muscle Program, MC Master University,Hamilton, Ont. CAN NPY and PYY (10-11-10-8mols)delivered to the ileum of the anaesthetized dog by close intraarterial injection produced dosedependent phasic contractions of circular muscle as measured by serosal strain gauges. These contractions were not abolished by atropinization. Infusions of NPY and PYY (1O-9to 10-7M) into isolated, Krebs Ringer-perfused ileal segments produced concentration-dependent contractions, concomitant inhibition of immunoreactive VIP in the venous outflow and amplification of field stimulated contractions at 2OV/cm, OSms and 1-3~~s. Inhibition of NO synthase by perfusion with N-o-L-arginine methyl ester (L NAME) (3X10dM) induced phasic activity and amplification of excitation to electrical field stimulation at 20V/cm, 0.5ms and 1-3~~s. L-NAME infused prior to NPY or PYY (lOaM) did not reduce contractions or the inhibition of VIP output nor further amplify field stimulated contractions. Furthermore, microelectrode studies of the myenteric plexus free ileal circular muscle demonstrated that NPY or PYY (l@sM) did not alter the resting membrane or inhibitory junction potential. These results suggest that PYY and NPY may excite motility by reducing the tonic inhibitory influence of VIP but not NO on the structures responsible for excitation in the canine ileal circular muscle. Supported by the MRC Canada.
EFFECT OF PRESERVATION AND TRANSPLANTATION ON NEUROMUSCULAR TRANSMISSION IN CANINE AND HUMAN ILEAL SMOOTH MUSCLE A.J. Bauer, D.J. Kustra and S. Todo. Departments of Medicine and Transplantation, University of Pittsburgh Medical School, Pittsburgh, PA 15261. Small intestinal transplantation (TX) is becoming a viable surgical procedure, but organ preservation remains a major obstacle. The objectives of this study were to determine the effects of harvest, preservation and transplantation on neuromuscular transmission (h’T) in canine and human small intestine. Electrical field stimulation of neural elements (l-30 Hz of 0.9 msec duration for 1 set) was used to record NT from myenteric circular smooth muscle cells. The first series of experiments investigated the effect of harvest, preservation and reperfusion of canine small intestine on NT (n=5). Electrical field stimulation (BFS) of muscles removed before surgical manipulation for TX, evoked transient, frequency dependent membrane hypcrpolarizations called inhibitory junction potentials (BPS). EFS of 5, 10 and 20 Hz evoked UPS with mean amplitudes of 6f3.6,15?2.7 and lw.8 mV, respectively. Harvesting the graft cause a slight decrease in the amplitude of the BPS. After preservation of the intact graft for 24 hours on ice in Lactate-Ringers solution (LRS), LIP amplitudes decreased significantly at all frequencies (BFS of 5, 10 and 20 Hz evoked UPS with mean amplitudes of MO, 3f2.1 and 6ti.7 mV, respectively). Re-perfusion of the TX segment by a recipient animal for a period of 1 hour caused no further decrease in BP amplitudes. The second series of experiments on human and canine investigated the effects of LRS, F-12 and University of Wisconsin (VW) preservation solutions in vitro. IP amplitudes after LR preservation were decreased to values similar to those observed after LR preservation of the intact graft. Preservation in F-12 culture media resulted in a significant improvement of NT over LRS. However, neuromuscular function was best preserved with the use of the UW solution. These data indicate that graft harvest and in viw re-perfusion have minimal effects on neuromuscular transmission and that small intestinal function is beat supported with the intracellular type UW preservation solution.
ABSTRACTS OF PAPERS
October 1992
MECHANISMOF SUBSTANCE P (SP) INHIBITIONOF CANINE ILEAL CIRCULAR MUSCLE CONTRACTIONS IN VIVO. JET. Fox-Threlkeld, E.G. Watson, Z.Woskowska E.E.Daniel. Smooth Muscle Program, McMaster University, Hamilton, Ont. CAN. Close intraarterial injections of low concentrations of SP were shown previously to inhibit field stimulated and motilin induced contractions of circular muscle b wv& (Am J Physiol 250 G21-26, 1986) We proposed a mechanism of SP inhibition: release of acetylcholine (ACH) to act on Mi receptors inhibiting ACH release. However, there was a residual inhibition after atropine or pirenzipine suggesting release of a further inhibitory agent. Two potential inhibitors of field stimulated motility are VIP and NO. To test whether the agent was VIP, SP or the NKl agonist [Sar9,met(OZ)n]-substancc P (lO-i” to 10-M) were infused into isolated Krebs Ringer perfused ileal segments and the venous effluent was assayed for immunoreactive VIP. There was no change in the concentration of VIP during these infusions suggesting that release of VIP had no role in the SP-induced inhibition. To examine whether release of NO was involved in SP induced inhibition the NO synthase inhibitor N-o-Larginine methyl ester &NAME) was given intraarterially (lO*mols) or intravenously (lo5 mols/kg) to anaesthetized dogs prepared with local intraarterial cannulation and serosal strain gauges and electrodes. In the presence of LNAME, the inhibition of field stimulated contractions (20V/cm, OSms, and 3-5~~s) by intraarterial SP was abolished in 5/10 sites, required higher SP concentrations in 4/10 sites and was unchanged in l/IO sites. The inhibition recovered after intravenous infusion of 1O-4 mols/lcg L-arginine. These results suggest that release of NO may contribute to the mediation of SP’s inhibitory action. Supported by MRC Canada.
ROLE OF NEURALLY-DERIVED,NO-RELATEDCOMPOUNDS IN GASTROINTESTINAL MOTOR FUNCTION IN WO EFFECTS. J.E.T. Fox-Threlkeld, C. Haugh, H.-D. Allescher, G. Tougas, P. Vergara and E.E. Daniel. Smooth Muscle Program, McMaster University, Hamilton, Ontario, Canada. We tested the hypothesis that in anaesthetized, fasted dogs, lack of intestinal motility result from tonic neural release of an NOrelated compound. Isolated perfused segments of intestine tonically release VIP (Am J Physiol 256 G884) Release was markedly reduced, accompanied by increased motility by TTX, opioids and asadrenoceptor agonists (Peptides 12 1039) Hexamethonium and Ca,+-free medium turned off VIP release without increasing motility. To examine whether NO-release from nerves in a Cast-dependent manner was the source of tonic inhibition in the intestine, NO synthase was inhibited by L”-N-arginine methyl ester (L-NAME). L-Name decreased dose-dependently excitation of intestinal CM (maximal at 3x10AM). This was reversed by L-arginine (1 mM), prevented by omega-conotoxin (10-7M) or Cast-free medium, and reduced by nifedipine (10-M) and by atropine (l@‘M). L-NAME during field stimulation reduced initial inhibition and amplified excitation. L-NAME also reduced VIP output 15-30%, an effect not reversed by L-arginine. Moreover, LNAME abolished the contractile response to 6 opioids, reduced that to p opioids and somatostatin and left unchanged that to NPY or PYY. Each peptide still decreased VIP output after L-NAME. Thus tonic inhibition of intestinal motility in is partially mediated by tonic release of NOrelated mediator. If VIP and NO are co-localized in the canine ileal nerves, their release is separately regulated and the contribution of each inhibitor in the response to an agonist must be separately evaluated. Supported by the MRC Canada.
PYY AND NPY STIMULATE ISOLATED PERFUSED CANINE ILEAL CONTRACITONS BY INHIBITING VIP NOT NO RELEASE. J.E.T. Fox-Threlkeld, E.E. Daniel, F. Christinck, Z. Woskowska, S. Cipris, and T.J. McDonald. Smooth Muscle Program, MC Master University,Hamilton, Ont. CAN NPY and PYY (10-11-10-8mols)delivered to the ileum of the anaesthetized dog by close intraarterial injection produced dosedependent phasic contractions of circular muscle as measured by serosal strain gauges. These contractions were not abolished by atropinization. Infusions of NPY and PYY (1O-9to 10-7M) into isolated, Krebs Ringer-perfused ileal segments produced concentration-dependent contractions, concomitant inhibition of immunoreactive VIP in the venous outflow and amplification of field stimulated contractions at 2OV/cm, OSms and 1-3~~s. Inhibition of NO synthase by perfusion with N-o-L-arginine methyl ester (L NAME) (3X10dM) induced phasic activity and amplification of excitation to electrical field stimulation at 20V/cm, 0.5ms and 1-3~~s. L-NAME infused prior to NPY or PYY (lOaM) did not reduce contractions or the inhibition of VIP output nor further amplify field stimulated contractions. Furthermore, microelectrode studies of the myenteric plexus free ileal circular muscle demonstrated that NPY or PYY (l@sM) did not alter the resting membrane or inhibitory junction potential. These results suggest that PYY and NPY may excite motility by reducing the tonic inhibitory influence of VIP but not NO on the structures responsible for excitation in the canine ileal circular muscle. Supported by the MRC Canada.
EFFECT OF PRESERVATION AND TRANSPLANTATION ON NEUROMUSCULAR TRANSMISSION IN CANINE AND HUMAN ILEAL SMOOTH MUSCLE A.J. Bauer, D.J. Kustra and S. Todo. Departments of Medicine and Transplantation, University of Pittsburgh Medical School, Pittsburgh, PA 15261. Small intestinal transplantation (TX) is becoming a viable surgical procedure, but organ preservation remains a major obstacle. The objectives of this study were to determine the effects of harvest, preservation and transplantation on neuromuscular transmission (h’T) in canine and human small intestine. Electrical field stimulation of neural elements (l-30 Hz of 0.9 msec duration for 1 set) was used to record NT from myenteric circular smooth muscle cells. The first series of experiments investigated the effect of harvest, preservation and reperfusion of canine small intestine on NT (n=5). Electrical field stimulation (BFS) of muscles removed before surgical manipulation for TX, evoked transient, frequency dependent membrane hypcrpolarizations called inhibitory junction potentials (BPS). EFS of 5, 10 and 20 Hz evoked UPS with mean amplitudes of 6f3.6,15?2.7 and lw.8 mV, respectively. Harvesting the graft cause a slight decrease in the amplitude of the BPS. After preservation of the intact graft for 24 hours on ice in Lactate-Ringers solution (LRS), LIP amplitudes decreased significantly at all frequencies (BFS of 5, 10 and 20 Hz evoked UPS with mean amplitudes of MO, 3f2.1 and 6ti.7 mV, respectively). Re-perfusion of the TX segment by a recipient animal for a period of 1 hour caused no further decrease in BP amplitudes. The second series of experiments on human and canine investigated the effects of LRS, F-12 and University of Wisconsin (VW) preservation solutions in vitro. IP amplitudes after LR preservation were decreased to values similar to those observed after LR preservation of the intact graft. Preservation in F-12 culture media resulted in a significant improvement of NT over LRS. However, neuromuscular function was best preserved with the use of the UW solution. These data indicate that graft harvest and in viw re-perfusion have minimal effects on neuromuscular transmission and that small intestinal function is beat supported with the intracellular type UW preservation solution.