Hideaki Nishio

VIP‐ and PACAP‐mediated nonadrenergic, noncholinergic inhibition in longitudinal muscle of rat distal colon: involvement of activation of charybdotoxin‐ and apamin‐sensitive K+ channels

1 The mediators of nonadrenergic, noncholinergic (NANC) inhibitory responses in longitudinal muscle of rat distal colon were studied. 2 An antagonist of pituitary adenylate cyclase activating peptide (PACAP) receptors, PACAP6–38, concentration‐dependently inhibited the rapid relaxation of the longitudinal muscle induced by electrical field stimulation (EFS), resulting in a maximal inhibition of 47% at 3 μm. 3 PACAP6–38 inhibited the relaxation by 75% in the presence of the vasoactive intestinal peptide (VIP) receptor antagonist, VIP10–28 at μm, which inhibited the relaxation by 44%. 4 An antagonist of large conductance Ca2+‐activated K+ channels, charybdotoxin, concentration‐dependently inhibited the rapid relaxation of the longitudinal muscle, resulting in a maximal inhibition of 58% at 100 nM. 5 An antagonist of small conductance Ca2+‐activated K+ channels, apamin, concentration‐dependently inhibited the relaxation (58% at 1 μm). 6 Treatment with both K+ channel antagonists resulted in 84% inhibition of the EFS‐induced relaxation, which is comparable to the extent of inhibition induced by PACAP6–38 plus VIP10–28. 7 The inhibitory effect of VIP10–28 and of apamin, but not of charybdotoxin was additive: the same applied to PACAP6–38 and charybdotoxin, but not apamin. 8 Exogenously added VIP (100 nM‐1 μm) induced a slow gradual relaxation of the longitudinal muscle. Charybdotoxin, but not apamin significantly inhibited the VIP‐induced relaxation. VIP10–28, but not PACAP6–38 selectively inhibited the VIP‐induced relaxation. 9 Exogenously added PACAP (10–100 nM) also induced slow relaxation. Apamin and to a lesser extent, charybdotoxin, inhibited the PACAP‐induced relaxation. PACAP6–38, but not VIP10–28 selectively inhibited the PACAP‐induced relaxation. 10 Apamin at 100 nM inhibited inhibitory junction potentials (i.j.ps) induced by a single pulse of EFS. Apamin also inhibited a rapid phase, but not a delayed phase of i.j.ps induced by two pulses at 10 Hz. VIP10–28 did not inhibit i.j.ps induced by a single pulse, but significantly inhibited the delayed phase at two pulses. A combination of apamin and VIP10–28 abolished the i.j.ps induced by two pulses. 11 Both VIP and PACAP induced slow hyperpolarization of the cell membrane of the longitudinal muscle. Apamin inhibited the PACAP‐, but not VIP‐induced hyperpolarization. 12 From these findings it is suggested that VIP and PACAP are involved in NANC inhibitory responses of longitudinal muscle of the rat distal colon via activation of charybdotoxin‐ and apaminsensitive K+ channels, respectively.

Critical role of sulfenic acid formation of thiols in the inactivation of glyceraldehyde-3-phosphate dehydrogenase by nitric oxide.

The relationship between possible modifications of the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by nitric oxide (NO) and modified enzyme activity was examined. There are 16 free thiols, including 4 active site thiols, in a tetramer of GAPDH molecule. NO donors, sodium nitroprusside (SNP), and S-nitroso-N-acetyl-DL-penicillamine (SNAP) decreased the number of free thiols with a concomitant inhibition of GAPDH activity in a concentration- and time-dependent manner. After treatment for 30 min, free thiols were maximally decreased to 8-10 per GAPDH tetramer and enzyme activity was also inhibited to 5-10% of control activity. In the presence of 30 mM dithiothreitol (DTT), these effects were completely blocked. Since similar results were obtained in the case of hydrogen peroxide (H2O2) treatment, which is known to oxidize the thiols, these effects of nitric oxide donors were probably due to modification of thiol groups present in a GAPDH molecule. On the other hand, DTT posttreatment after the treatment of GAPDH with SNP, SNAP, or H2O2 did not completely restore the modified thiols and the inhibited enzyme activity. DTT posttreatment after the 30-min-treatment with these agents restored free thiols to 14 in all treatments. In the case of SNAP treatment, all 4 active sites were restored and enzyme activity reached more than 80% of the control activity, but in two other cases one active site remained modified and enzyme activity was restored to about only 20%. Therefore, all 4 free thiols in the active site seem to be very important for full enzyme activity. DTT posttreatment in the presence of sodium arsenite, which is known to reduce sulfenic acid to thiol, almost completely restored both thiol groups and enzyme activity. These findings suggest that nitric oxide inhibits GAPDH activity by modifications of the thiols which are essential for this activity, and that the modification includes formation of sulfenic acid, which is not restored by DTT. S-nitrosylation, which is one type of thiol modification by NO, occurred when GAPDH was treated with SNAP but not SNP. Analysis of thiol modification showed that SNAP preferentially nitrosylated the active site thiols, the nitrosylation of which fully disappeared by DTT posttreatment. It seems that SNAP nitrosylates the active site thiols of GAPDH to prevent these thiols from oxidizing to sulfenic acid.

Inhibition of skeletal muscle sarcoplasmic reticulum Ca2+‐ATPase by nitric oxide

The effects of nitric oxide on the activities of thapsigargin‐sensitive sarcoplasmic reticulum Ca2+‐ATPase (SERCA) and Ca2+ uptake by sarcoplasmic reticulum (SR) membranes prepared from white skeletal muscle of rabbit femoral muscle were studied. Pretreatment of the SR preparations with nitric oxide at concentrations of up to 250 μM for 1 min decreased the SERCA activity concentration dependently, and also decreased their Ca2+ uptake. Both these effects of nitric oxide were reversible. Inhibitors of guanylyl cyclase and protein kinase G (PKG) had no significant effect on the nitric oxide‐induced inhibitions of SERCA and Ca2+ uptake. Moreover, dithiothreitol did not reverse the inhibitory effects of nitric oxide on SERCA and Ca2+ uptake. These findings suggest that nitric oxide inhibits SERCA, mainly SERCA 1, of rabbit femoral skeletal muscle by an action independent of the cyclic GMP‐PKG system or oxidation of thiols, and probably by a direct action on SERCA protein.

Nitric oxide-mediated relaxation without concomitant changes in cyclic GMP content of rat proximal colon.

1 We studied the relation of nitric oxide‐mediated relaxation of longitudinal muscle to changes in cyclic GMP content of the tissue in the proximal colon of rats. 2 Dimethylphenylpiperazinium (DMPP) and electrical field stimulation (EFS) induced nitric oxidemediated relaxation of the segments with a concomitant increase in cyclic GMP content. 3 LY 83583 and methylene blue, soluble guanylyl cyclase inhibitors, significantly inhibited the stimulatory effects of DMPP and EFS on the cyclic GMP content, but did not affect the relaxant responses of the segments to DMPP and EFS. 4 Rp‐8 bromo cyclic GMPS, an inhibitor of cyclic GMP‐dependent protein kinase had no effect on DMPP‐ and EFS‐induced relaxation. 5 These data strongly suggested that nitric oxide‐mediated relaxation of the rat proximal colon is not associated with change in cyclic GMP content of the tissue.

Possible role of orexin A in nonadrenergic, noncholinergic inhibitory response of muscle of the mouse small intestine

The effect of a novel peptide, orexin A, on longitudinal muscle of ICR mouse small intestine was examined in vitro. Exogenous orexin A induced a transient contraction in duodenal, jejunal and ileal segments. Atropine and tetrodotoxin completely inhibited the contractions. Contraction of longitudinal muscle of jejunal segments induced by electrical field stimulation was still observed after the jejunal segment had been desensitized to orexin A, suggesting that orexin A is not a final neurotransmitter to induce the contraction. On the other hand, in the presence of atropine and guanethidine, orexin A induced a transient gradual relaxation in duodenal, jejunal and ileal segments. Electrical field stimulation also induced significant relaxation of the muscle in jejunal segments. The electrical field stimulation-induced relaxation was inhibited by 55% after the desensitization of the segments to orexin A. Although the electrical field stimulation-induced relaxation was inhibited by 47% by a nitric oxide synthesis inhibitor, NG-nitro-L-arginine (L-NOARG), orexin desensitization did not affect the relaxation which persisted after L-NOARG treatment. The exogenous orexin A-induced relaxation was completely inhibited by L-NOARG. The results suggest that orexin A partially mediates nonadrenergic, noncholinergic (NANC) relaxation via activation of nitrergic neurones in longitudinal muscle of ICR mouse small intestine.

Involvement of cyclic AMP–PKA pathway in VIP-induced, charybdotoxin-sensitive relaxation of longitudinal muscle of the distal colon of Wistar-ST rats

The intracellular mechanism of vasoactive intestinal peptide (VIP)‐induced, charybdotoxin (ChTx)‐sensitive relaxation of longitudinal muscle of the distal colon of Wistar‐ST rats was studied. A single pulse or 100 pulses at 10 Hz of electrical field stimulation (EFS) induced rapid transient relaxation or that with a subsequent contraction of the longitudinal muscle in the presence of atropine and guanethidine, respectively. Rp‐8 bromo cAMPS, an inhibitor of cyclic AMP dependent protein kinase (PKA), at 30 μM inhibited the relaxations induced by EFS with a single or 100 pulses maximally by about 80 or 60%, respectively. It also inhibited VIP (300 nM)‐induced relaxation by 82%. VIP (100 nM–1 μM) increased the cyclic AMP content of longitudinal muscle myenteric plexus preparations obtained from the distal colon. ChTx at 100 nM almost completely inhibited 8 bromo cyclic AMP‐induced relaxation of the distal segments. EFS with two or three pulses at 10 Hz induced inhibitory junction potentials consisting of two phases, rapid and subsequent slow hyperpolarization in the membrane potential of longitudinal smooth muscle cells. Rp‐cAMPS, another inhibitor of PKA, inhibited the delayed slow hyperpolarization. It also inhibited the exogenously added VIP‐induced hyperpolarization of the cell membrane. Thus, the present study suggests that activation of PKA via activation of VIP receptors is associated with activation of ChTx‐sensitive K+ channels in relaxation of longitudinal muscle of the distal colon of Wistar‐ST rats.

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.

Tyrosine kinase involvement in apamin-sensitive inhibitory responses of rat distal colon

1 It has been suggested that pituitary adenylate cyclase activating peptide (PACAP) may be involved in the non‐adrenergic, non‐cholinergic (NANC) inhibitory response of longitudinal muscle of rat distal colon. In this study, we have investigated the intracellular mechanism of PACAP‐induced relaxation in this muscle. 2 PACAP induced an apamin‐sensitive relaxation of the longitudinal muscle. The tyrosine kinase inhibitors genistein at 10 μm and tyrphostin 25 at 30 μm, but not the cyclic AMP‐dependent protein kinase inhibitor Rp‐8‐bromoadenosine‐3′,5′‐cyclic monophosphorothioate at 30 μm significantly inhibited the PACAP‐induced relaxation to 60% and 25% of control values, respectively. PACAP did not increase the cyclic AMP content of the muscle. 3 Tyrphostin 25 at 10 μm significantly inhibited the relaxation of longitudinal muscle induced by electrical field stimulation (EFS), to 50% of control values. Apamin at 1 μm, an antagonist of small conductance Ca2+‐activated K+ channels, also inhibited the relaxation, to 42% of control values. The inhibitory effects of tyrphostin 25 and apamin were not additive (44% of control values). 4 PACAP induced an apamin‐sensitive, slow hyperpolarization of the cell membrane of the muscle. Tyrphostin 25 at 3 μm inhibited this PACAP‐induced hyperpolarization. Tyrphostin 25 at 10 μm and genistein at 10 μm inhibited the apamin‐sensitive inhibitory junction potentials induced by a single pulse of EFS. 5 The PACAP‐induced relaxation of longitudinal muscle occurred with a concomitant decrease in intracellular Ca2+ levels ([Ca2+]i). Tyrphostin 25 at 10 μm and apamin at 1 μm abolished these PACAP‐induced responses. 6 From these findings it is suggested that the activation of tyrosine kinase is involved in PACAP‐induced relaxation of longitudinal muscle from rat distal colon, ‘upstream of’ the activation of apamin‐sensitive K+ channels.

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.

Increase in participation of vasoactive intestinal peptide in relaxation of the distal colon of Wistar rats with age

Changes in participation of vasoactive intestinal peptide (VIP) in nonadrenergic noncholinergic (NANC) relaxation of longitudinal muscle of the distal colon with age were studied in 2‐ to 50‐week‐old Wistar rats in vitro. The extent of the VIP‐mediated component of the relaxation induced by electrical field stimulation (EFS) was determined by the effect of VIP10–28, a VIP receptor antagonist. In 2‐week‐old rats, the extent of the VIP‐mediated component of the relaxation was scarce, about 10%, whereas the component gradually increase with age and reached the maximum extent 66% at 50‐week‐old. Since our previous results suggest that VIP induces NANC relaxation via activation of charybdotoxin (ChTx, a blocker of large conductance Ca2+‐activated K+ channel)‐sensitive K+ channels with concomitant slow hyperpolarization in the muscle cells, we next studied whether ChTx‐sensitive component and slow hyperpolarization changes with age. Extent of ChTx‐sensitive component of the relaxation increased with age, showing a very similar pattern to VIP‐mediated one. EFS induced monophasic inhibitory junction potentials (i.j.ps) in longitudinal muscle cells of the distal colon of 2‐ and 4‐week‐old. EFS also induced biphasic i.j.ps in many longitudinal muscle cells of 8‐ and 50‐week‐old: rapid and subsequent slow hyperpolarization. A VIP receptor antagonist selectively inhibited the slow hyperpolarization. Exogenously added VIP induced no appreciable change in the membrane potential of longitudinal muscle cells of 2‐week‐old, whereas it induced slight slow hyperpolarization of the cell membrane in 4‐week‐old and magnitude of the hyperpolarization increased with age. On the other hand, relaxant response of the longitudinal muscle to exogenously added VIP was high in younger rats. The present results suggest that the role of VIP in mediating NANC relaxation of longitudinal muscle of the Wistar rat distal colon is very little at neonatal stage, but it increases with age.