Ingeborg Dhaese

Myosin light chain phosphatase activation is involved in the hydrogen sulfide-induced relaxation in mouse gastric fundus.

The relaxant effect of hydrogen sulfide (H(2)S) in the vascular tree is well established but its influence and mechanism of action in gastrointestinal smooth muscle was hardly investigated. The influence of H(2)S on contractility in mouse gastric fundus was therefore examined. Sodium hydrogen sulfide (NaHS; H(2)S donor) was administered to prostaglandin F(2alpha) (PGF(2alpha))-contracted circular muscle strips of mouse gastric fundus, before and after incubation with interfering drugs. NaHS caused a concentration-dependent relaxation of the pre-contracted mouse gastric fundus strips. The K(+) channels blockers glibenclamide, apamin, charybdotoxin, 4-aminopyridin and barium chloride had no influence on the NaHS-induced relaxation. The relaxation by NaHS was also not influenced by L-NAME, ODQ and SQ 22536, inhibitors of the cGMP and cAMP pathway, by nerve blockers capsazepine, omega-conotoxin and tetrodotoxin or by several channel and receptor blockers (ouabain, nifedipine, 2-aminoethyl diphenylborinate, ryanodine and thapsigargin). The myosin light chain phosphatase (MLCP) inhibitor calyculin-A reduced the NaHS-induced relaxation, but the Rho-kinase inhibitor Y-27632 had no influence. We show that NaHS is able to relax PGF(2alpha)-contracted mouse gastric fundus strips. The results suggest that in the mouse gastric fundus, H(2)S causes relaxation at least partially via activation of MLCP.

Mechanisms of action of hydrogen sulfide in relaxation of mouse distal colonic smooth muscle

Hydrogen sulfide (H(2)S) has been suggested as a gaseous neuromodulator in mammals. The aim of this study was to examine the influence of H(2)S on contractility in mouse distal colon. The effect of sodium hydrogen sulfide (NaHS; H(2)S donor) on prostaglandin F(2alpha) (PGF(2alpha))-contracted circular muscle strips of mouse distal colon was investigated. In addition, tension and cytosolic calcium concentration ([Ca(2+)](cyt)) in the mouse distal colon strips were measured simultaneously in the presence of NaHS. NaHS caused concentration-dependent relaxation of the pre-contracted mouse distal colon strips. The NaHS-induced relaxation was not influenced by the K(+) channels blockers glibenclamide, apamin, charybdotoxin, barium chloride and 4-aminopyridine. The relaxation by NaHS was also not influenced by the nitric oxide inhibitor L-NAME, by the soluble guanylate cyclase respectively adenylate cyclase inhibitors ODQ and SQ 22536, by the nerve blockers capsazepine, omega-conotoxin and tetrodotoxin or by several channel and receptor blockers (ouabain, nifedipine, 2-aminoethyl diphenylborinate, ryanodine and thapsigargin). The initiation of the NaHS-induced relaxation was accompanied by an increase in [Ca(2+)](cyt), but once the relaxation was maximal and sustained, no change in [Ca(2+)](cyt) was measured. This calcium desensitization is not related to the best known calcium desensitizing mechanism as the myosin light chain phosphatase (MLCP) inhibitor calyculin-A and the Rho-kinase inhibitor Y-27632 had no influence. We conclude that NaHS caused concentration-dependent relaxations in mouse distal colon not involving the major known K(+) channels and without a change in [Ca(2+)](cyt). This calcium desensitization is not related to inhibition of Rho-kinase or activation of MLCP.

Gastric motility in soluble guanylate cyclase α1 knock‐out mice

The principal target of the relaxant neurotransmitter nitric oxide (NO) is soluble guanylate cyclase (sGC). As the α1β1‐isoform of sGC is the predominant one in the gastrointestinal tract, the aim of this study was to investigate the role of sGC in nitrergic regulation of gastric motility in male and female sGCα1 knock‐out (KO) mice. In circular gastric fundus muscle strips, functional responses and cGMP levels were determined in response to nitrergic and non‐nitrergic stimuli. sGC subunit mRNA expression in fundus was measured by real‐time RT‐PCR; in vivo gastric emptying of a phenol red meal was determined. No changes were observed in sGC subunit mRNA levels between wild‐type (WT) and KO tissues. Nitrergic relaxations induced by short trains of electrical field stimulation (EFS) were abolished, while those by long trains of EFS were reduced in KO strips; the latter responses were abolished by 1H[1,2,4,]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ). The relaxations evoked by exogenous NO and the NO‐independent sGC activator BAY 41‐2272 were reduced in KO strips but still sensitive to ODQ. Relaxations induced by vasoactive intestinal peptide (VIP) and 8‐bromo‐cGMP were not influenced. Basal cGMP levels were decreased in KO strips but NO, long train EFS and BAY 41‐2272 still induced a moderate ODQ‐sensitive increase in cGMP levels. Gastric emptying, measured at 15 and 60 min, was increased at 15 min in male KO mice. sGCα1β1 plays an important role in gastric nitrergic relaxation in vitro, but some degree of nitrergic relaxation can occur via sGCα2β1 activation in sGCα1 KO mice, which contributes to the moderate in vivo consequence on gastric emptying.

Involvement of soluble guanylate cyclase α1 and α2, and SKCa channels in NANC relaxation of mouse distal colon

In distal colon, both nitric oxide (NO) and ATP are involved in non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission. The role of the soluble guanylate cyclase (sGC) isoforms alpha(1)beta(1) and alpha(2)beta(1), and of the small conductance Ca(2+)-dependent K(+) channels (SK(Ca) channels) in the relaxation of distal colon by exogenous NO and by NANC nerve stimulation was investigated, comparing wild type (WT) and sGCalpha(1) knockout (KO) mice. In WT strips, the relaxation induced by electrical field stimulation (EFS) at 1 Hz but not at 2-8 Hz was significantly reduced by the NO-synthase inhibitor L-NAME or the sGC inhibitor ODQ. In sGCalpha(1) KO strips, the EFS-induced relaxation at 1 Hz was significantly reduced and no longer influenced by L-NAME or ODQ. The SK(Ca) channel blocker apamin alone had no inhibitory effect on EFS-induced relaxation, but combined with ODQ or L-NAME, apamin inhibited the relaxation induced by EFS at 2-8 Hz in WT strips and at 8 Hz in sGCalpha(1) KO strips. Relaxation by exogenous NO was significantly attenuated in sGCalpha(1) KO strips, but could still be reduced further by ODQ. Basal cGMP levels were lower in sGCalpha(1) KO strips but NO still significantly increased cGMP levels versus basal. In conclusion, in the absence of sGCalpha(1)beta(1), exogenous NO is able to partially act through sGCalpha(2)beta(1). NO, acting via sGCalpha(1)beta(1), is the principal neurotransmitter in EFS-evoked responses at 1 Hz. At higher stimulation frequencies, NO, acting at sGCalpha(1)beta(1) and/or sGCalpha(2)beta(1), functions together with another transmitter, probably ATP acting via SK(Ca) channels, with some degree of redundancy.

Heme deficiency of soluble guanylate cyclase induces gastroparesis

Soluble guanylate cyclase (sGC) is the principal target of nitric oxide (NO) to control gastrointestinal motility. The consequence on nitrergic signaling and gut motility of inducing a heme‐free status of sGC, as induced by oxidative stress, was investigated.

Influence of 5‐HT4 receptor activation on acetylcholine release in human large intestine with endometriosis

Background  The 5‐HT4 receptor agonist prucalopride enhances large intestinal contractility by facilitating acetylcholine release through activation of 5‐HT4 receptors on cholinergic nerves and is effective in patients with constipation. Patients with intestinal endometriosis can present with constipation. We investigated in vitro whether large intestinal endometriotic infiltration influences contractility and facilitation of acetylcholine release by prucalopride.

Small intestinal motility in soluble guanylate cyclase α1 knockout mice

Nitric oxide (NO) activates soluble guanylate cyclase (sGC) to produce guanosine-3′,5′-cyclic-monophosphate (cGMP). The aim of this study was to investigate the nitrergic regulation of jejunal motility in sGCα1 knockout (KO) mice. Functional responses to nitrergic stimuli and cGMP levels in response to nitrergic stimuli were determined in circular muscle strips. Intestinal transit was determined. Nitrergic relaxations induced by electrical field stimulation and exogenous NO were almost abolished in male KO strips, but only minimally reduced and sensitive to ODQ in female KO strips. Basal cGMP levels were decreased in KO strips, but NO still induced an increase in cGMP levels. Transit was not attenuated in male nor female KO mice. In vitro, sGCα1β1 is the most important isoform in nitrergic relaxation of jejunum, but nitrergic relaxation can also occur via sGCα2β1 activation. The latter mechanism is more pronounced in female than in male KO mice. In vivo, no important implications on intestinal motility were observed in male and female KO mice.

NO-induced motility effects in gastric fundus and pylorus of sGCβ1his105phe mutant mice

Nitric oxide (NO) is the most important relaxant neurotransmitter in the stomach. The principal target of NO is soluble guanylate cyclase (sGC), of which two physiologically prevailing isoforms exist, namely α1β1 and α2β1. The aim of this study was to investigate the role of sGC in NO-induced smooth muscle effects of gastric fundus and pylorus, and in gastric emptying, using wild type (WT) and sGCβ1his105phe mutant mice (7–14 weeks old).

Role of soluble guanylate cyclase in NO-induced effects of gastric fundus

Nitric oxide (NO) is the most important relaxant neurotransmitter in the gastric fundus. The principal target of NO is soluble guanylate cyclase (sGC). As the α1β1 isoform is most abundant in the gastrointestinal tract, the aim of this study was to investigate the role of sGC in NOinduced smooth muscle effects of gastric fundus using wild type (WT) and sGCα1 knock-out (KO) mice of both sexes (8–50 weeks old). Circular muscle strips of the gastric fundus were precontracted with prostaglandin F2α (PGF2α) in the presence of atropine and guanethidine and exposed to electrical field stimulation (EFS) and exogenous NO, before and in the presence of the sGC inhibitor ODQ or the NO synthase inhibitor L-NAME. The contractile response to PGF2α did not differ between WT and KO strips. In WT strips, EFS-induced relaxations were nearly abolished by L-NAME and ODQ indicating that NO, acting at sGC, is the neurotransmitter liberated. The responses to EFS were greatly reduced in KO strips; in the presence of ODQ or L-NAME, no electrically induced relaxations were obtained. In WT strips, NO-evoked responses were not influenced by L-NAME but reduced by ODQ; the higher the concentration of NO, the less pronounced was the inhibitory effect of ODQ. In KO strips, NO-evoked responses were minimally reduced in comparison to WT strips, while the inhibitory effect of ODQ versus NO was more pronounced. The relaxant effect of 8bromo-cGMP was not different between WT and KO strips, but the relaxant effect of BAY 41-2272 was reduced in KO strips. Cyclic guanosine monophosphate (cGMP) levels measured at maximal relaxation by EFS or NO, were increased 34–35 (male respectively female) fold by NO versus basal, but were not changed by EFS in WT strips. Basal cGMP levels were lower in KO strips, although this did not reach significance in female mice; in KO strips NO still induced a moderate increase in cGMP levels (3–5 fold versus basal) but this was significantly less pronounced. The results indicate that endogenous NO, released by EFS, mainly acts through activation of sGCα1. The observation that the relaxant response to exogenous NO is only minimally reduced in KO strips indicates that exogenous NO is able to act mainly through sGCα2, as the responses are still highly sensitive to ODQ. Obviously, the subcellular localization of cGMP raised by sGCα2 in the KO strips is different from that by activation of sGCα1 in the WT strips, as nearly the same degree of relaxation is induced by exogenous NO with a lower level of measurable cGMP. from 2nd International Conference of cGMP Generators, Effectors and Therapeutic Implications Potsdam, Germany, 10–12 June, 2005

NO-induced motility effects in distal colon of sGCα1 knockout mice

In distal colon, NO plays a less predominant role as relaxant neurotransmitter compared to the upper gastrointestinal tract. As the α1β1 isoform of soluble guanylate cyclase (sGC) is the most important one in the gastrointestinal tract, the aim of this study was to investigate the role of sGC in smooth muscle effects of endogenous and exogenous NO in distal colon using wild type (WT) and sGCα1 knock-out (KO) mice of both sexes (9–37 weeks old). Results were the same in both sexes except when indicated. Mucosa-free circular muscle strips of distal colon were precontracted with prostaglandin F2α (PGF2α) in the presence of atropine and guanethidine and exposed to electrical field stimulation (EFS) and exogenous NO, before and in the presence of the sGC inhibitor ODQ or the NO synthase inhibitor L-NAME. The contractile response to PGF2α was not different between WT and KO strips. In WT strips, only the relaxation induced by EFS at 1 Hz was significantly reduced by L-NAME and ODQ indicating that only at this stimulation frequency NO, acting at sGC, is released. The EFS-induced response at 1 Hz is decreased in KO strips when compared to WT strips but only in the set where ODQ was tested, while there was no difference in the set where L-NAME was tested. ODQ and L-NAME did not reduce the EFS-evoked responses in KO strips. In WT strips, NO-induced relaxations were not influenced by L-NAME but reduced by ODQ. The NOinduced responses were significantly lower in KO strips when compared to WT strips although this did not reach significance in the strips of male mice where L-NAME was tested. In KO strips, NO-evoked responses were not influenced by L-NAME, but reduced by ODQ. In WT strips, cyclic guanosine monophosphate (cGMP) levels measured at maximal relaxation by NO were increased 4 (male) to 11 (female) fold versus basal, but this increase did not reach significance in strips of male mice; cGMP levels were not changed by EFS at 1 Hz. Basal cGMP levels were lower in KO strips but NO still induced an increase in cGMP levels (5 – 3 fold versus basal in male respectively female). The relaxant response to endogenous NO, released by EFS at 1 Hz, is reduced in KO strips and no longer affected by ODQ, indicating that endogenous NO mainly acts through sGCα1; the non-ODQ sensitive effect of NO might be related to direct activation of small conductance Ca2+-dependent K+-channels. The relaxant response to exogenous NO is reduced in KO strips but still sensitive to ODQ, indicating that exogenous NO is able to act through sGCα2 as well as sGCα1. from 2nd International Conference of cGMP Generators, Effectors and Therapeutic Implications Potsdam, Germany, 10–12 June, 2005