Mediators of nonadrenergic, noncholinergic relaxation in Sprague Dawley rat intestine: comparison with the mediators of other strains.

Participation of nitric oxide, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) in nonadrenergic, noncholinergic (NANC) relaxation of longitudinal muscle of various intestinal regions in Sprague Dawley rats (8-week-old) was studied in vitro. Nitric oxide was suggested to participate in NANC relaxation of every intestinal region studied. But the participation was partial and its extent varied among the regions: significant in the proximal colon and rectum, and moderate in the jejunum, ileum and distal colon. Participation of PACAP in NANC relaxation was suggested only in the distal colon, while that of VIP was not detected in any of regions. Results obtained in the present study indicate that extent of participation of nitric oxide in NANC relaxation in Sprague Dawley rat intestine is more significant than those of other strains, Wistar and Wistar-ST.

1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) inhibits cyclic GMP-PKG pathway-independent nonadrenergic, noncholinergic relaxation in longitudinal muscle of the rectum of Wistar-ST rats.

Participation of the nitric oxide-cyclic GMP pathway in nonadrenergic, noncholinergic (NANC) relaxation induced by electrical field stimulation of longitudinal muscle of the rectum of Wistar-ST rats was studied by using a selective inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). ODQ concentration dependently inhibited the relaxation and at 10 microM, maximally inhibited it by 83%. However, results obtained with N(G)-nitro-L-arginine, L-arginine and exogenously added nitric oxide excluded the participation of nitric oxide in the relaxation. An inhibitor of cyclic GMP-dependent protein kinase (PKG) partially (39%) inhibited the relaxation. ODQ also significantly inhibited the relaxation, which persisted after the PKG inhibitor-treatment, by 85%. The results strongly suggest that ODQ inhibits the NANC relaxation in a cyclic GMP-PKG pathway-independent manner.

Differences in mediator of nonadrenergic, noncholinergic relaxation of the distal colon between Wistar-ST and Sprague-Dawley strains of rats.

Participation of nitric oxide and vasoactive intestinal peptide (VIP) in electrical field stimulation-induced nonadrenergic, noncholinergic (NANC) relaxation of longitudinal muscle and in balloon distension-induced descending NANC relaxation of circular muscle were studied in the distal colon of Wistar-ST and Sprague-Dawley rats. The extent of the nitric oxide-mediated component was approximately 50% in longitudinal and circular muscle of Sprague-Dawley rats, whereas this component was absent in both muscles of Wistar-ST rats. The extent of the VIP-mediated component was approximately 40% in longitudinal muscle of Wistar-ST rats and circular muscle of Sprague-Dawley rats, whereas this component was absent in circular muscle of Wistar-ST rats and longitudinal muscle of Sprague-Dawley rats. In circular muscle of Sprague-Dawley rats, in which participation of both nitric oxide and VIP in the relaxation was suggested, inhibition of descending relaxation by N(G)-nitro-L-arginine (L-NOARG) together with VIP-(10-28) was similar to that by either of the antagonists, and exogenous VIP-induced relaxation was not affected by L-NOARG, but exogenous nitric oxide-induced relaxation was partly inhibited by VIP-(10-28). These results suggest a linkage of the pathways mediated by nitric oxide and VIP. In the immunohistochemical studies, nitric oxide synthase or VIP immunoreactive neurons were seen in the ganglia, primary internodal strands of the myenteric plexus and in the circular muscle layer. However, the overall appearance of immunoreactive cell bodies in the myenteric plexus and the numbers of immunoreactive fibers in the circular muscle layer appeared to be similar in Wistar-ST and Sprague-Dawley rats. These results suggest that mediators of NANC relaxation in the distal colon are different in different strains of rats, i.e., Wistar-ST and Sprague-Dawley, although no such difference was seen in immunohistochemical studies.

Identification of autolysosomes directly associated with proteolysis on the density gradients in isolated rat hepatocytes.

Autophagy-related vacuoles, i.e., autophagosomes (AVi), autolysosomes (AVd) and dense bodies (DB), are intracellular organelles within which macroautophagy and bulk proteolysis set out and progress. Separation of these particles in freshly isolated rat hepatocytes, monitored by beta-hexosaminidase, a lysosomal marker enzyme, was established by density gradient centrifugation. Percoll density gradients were modified and improved by adding free polyvinylpyrrolidone (PVP, 0.75%) to 60% Percoll, which made it possible to separate AVd (buoyant peak, d = 1.090) and DB (dense peak, d = 1.131) effectively. Addition of graded levels of a regulatory amino acid mixture (Reg AA) to hepatocyte incubation not only suppressed proteolysis, but also lead to a shift of vacuolar profiles on the density gradients from the buoyant to the dense region. Alterations in the vacuolar shift and proteolysis were highly proportional over a full range of regulation by Reg AA. Morphometric analysis of autophagic vacuoles by electron microscopy revealed changes in the aggregate volumes of both AVi and AVd by Reg AA, which enabled us to estimate autophagic subpopulation of the buoyant peak on the gradient profile. All the results demonstrate that AVd shifts on the density gradients in proportion to alterations in proteolysis regulated by amino acids, and thus the gradient profile can be used as a measure of macroautophagy; and in addition that AVd actively involved in proteolysis occupies only a part of the buoyant peak on the gradients.

Decrease in participation of nitric oxide in nonadrenergic, noncholinergic relaxation of rat intestine with age.

Participation of nitric oxide in the electrical field stimulation-induced nonadrenergic, noncholinergic (NANC) relaxation in various intestinal regions was studied in 2- to 50-week-old Wistar rats. In the jejunum of 2-week-old rats, the extent of the nitric oxide-mediated component of the relaxation of longitudinal muscle was approximately 60-70%, whereas the component was 40-50% in 4-week-old rats and was absent in 8- and 50-week-old rats. Thus, nitric oxide seems to be the most important mediator at young ages but its significance is lost with age. The same tendency as that in the jejunum was also shown in longitudinal muscle of the ileum, proximal and distal colon, and rectum. The tendency was also shown in the circular muscle of the rectum. Sensitivity of the longitudinal muscle of the jejunum and proximal colon to exogenously added nitric oxide was high in younger rats. Immunoreactive structures for nitric oxide synthase were observed in the circular muscle layer of the rectum. The population of the structures was denser in 4-week-old than that in 50-week-old. The results suggest that NANC relaxation in every region of the intestine at 2-week-old is almost solely mediated by nitric oxide, and its significance as an inhibitory mediator gradually or rapidly decreases with age.

Nonadrenergic, noncholinergic relaxation mediated by nitric oxide with concomitant change in Ca2+ level in rectal circular muscle of rats.

The mediators of nonadrenergic, noncholinergic (NANC) relaxation of the circular muscle of rat rectum were examined in vitro. In the circular muscle of rat rectum, NG-nitro-L-arginine (L-NOARG) at 10 microM did not affect electrical field stimulation-induced relaxation but at 100 microM it inhibited electrical field stimulation-induced relaxation by about 75% and 1-mM L-arginine reversed the inhibition. Exogenous nitric oxide (NO) (1-10 microM) concentration dependently relaxed the circular muscle. Electrical field stimulation increased the cyclic GMP content of the circular muscle to about twice its resting level. L-NOARG, even at 10 microM, completely inhibited the electrical field stimulation-induced elevation of cyclic GMP content. However, L-arginine at 1 mM did not reverse the inhibition in cyclic GMP content. Inhibitory junction potentials (i.j.ps) induced by electrical field stimulation in the circular muscle cells were not affected by L-NOARG, 100 microM. Apamin ( < or = microM) did not affect the electrical field stimulation-induced relaxation, but almost completely inhibited electrical field stimulation-induced i.j.ps. NO (0.3-10 microM) induced relaxation of the circular muscle with a concomitant decrease in intracellular Ca2+ level ([Ca2+]i). Abundant immunoreactivity of NO synthase was found in the circular muscle layer, in addition to myenteric and submucosal plexus. The results suggest that NO induces NANC relaxation with a concomitant change in [Ca2+]i in the circular muscle of rat rectum. However, the involvement of changes in cyclic GMP level and in membrane potentials in the mechanism was not shown in the present experimental conditions.

Nonadrenergic, noncholinergic relaxation in longitudinal muscle of rat jejunum.

The mediators of nonadrenergic, noncholinergic (NANC) relaxation in the longitudinal muscle of rat jejunum were studied in vitro. Electrical field stimulation (EFS) of segments of rat jejunum induced a rapid transient relaxation followed by a subsequent contraction in the presence of atropine and guanethidine. NG-Nitro-L-arginine (L-NOARG, 10 microM) inhibited the EFS-induced NANC relaxation by about 25%, and L-arginine (1 mM) completely reversed this inhibition. Exogenously added nitric oxide (0.1-10 microM) induced relaxation of the segment. Treatment of the segment with alpha-chymotrypsin resulted in about 50% inhibition of the EFS-induced relaxation. Several peptide candidates for the mediator of NANC relaxation were examined by using selective antagonists of their receptors or by a receptor-desensitization method. Results indicated that vasoactive intestinal peptide, pituitary adenylate cyclase activating peptide, peptide histidine isoleucine, atrial natriuretic peptide and neurotensin are not associated with NANC relaxation of the segments. On the other hand, apamin at 1 microM inhibited the EFS-induced relaxation by 74%. Inhibitory effects of L-NOARG and, apamin or alpha-chymotrypsin treatment on the EFS-induced relaxation were additive and almost complete. Exogenous nitric oxide-induced relaxation was not affected by apamin. Inhibitory junction potentials (i.j.p.'s) were recorded from longitudinal muscle cells of rat jejunum. Apamin at 200 nM abolished i.j.p.'s induced by two pulses of EFS. These results suggest that NANC relaxation in longitudinal muscle of rat jejunum involves two independent components: one is a nitric oxide-mediated minor component, and the other is an unknown substance-mediated apamin-sensitive major component that is inhibited by alpha-chymotrypsin treatment.