Distribution and characterization of motilin receptors in the cat

We demonstrate binding of [¹²⁵I][Nle¹³-po]motilin to homogenates of cat gastric and small intestinal, but not to colonic smooth muscle tissue. The density was (B_max in fmol/mg protein): 0 (fundus); 12 ± 2 (corpus); 22 ± 3 (antrum); 55 ± 12 (duodenum); 44 ± 10 (jejunum); 17 ± 1 (ileum); 0 (colon). A significant (p < 0.05) difference was found between the dissociation constant for motilin in the stomach (pK_d = 8.84 ± 0.06) and in the small intestine (pK_d = 8.58 ± 0.08). The motilides erythromycin-A (EM-A), EM-523, and EM-A N-oxide displaced labeled [Nle¹³-po]motilin bound to cat duodenal receptor with potencies (pK_d) of 5.47 ± 0.23, 7.60 ± 0.24, and < 4.3, respectively. Studies with [Leu¹³-po]motilin fragments showed that the N-terminus of motilin interacts with the receptor. In the tissue bath, duodenal strips mounted in the longitudinal direction responded to motilin, EM-523, and EM-A (pEC₅₀: 8.29 ± 0.08; 7.12 ± 0.12; 5.99 ± 0.15). The compounds had a comparable intrinsic activity (83 ± 3%; 80 ± 5%; 82 ± 5% of the response to ACh), which was unaffected by atropine, TTX, hexamethonium, and zacopride but reduced by verapamil and calcium-free medium. Cat stomach and small intestine possess smooth muscle motilin receptors, which have comparable properties as those found in man and in rabbit.     

Relaxin counteracts the altered gastric motility of dystrophic (mdx) mice: functional and immunohistochemical evidence for the involvement of nitric oxide

Impaired gastric motility ascribable to a defective nitric oxide (NO) production has been reported in dystrophic (mdx) mice. Since relaxin upregulates NO biosynthesis, its effects on the motor responses and NO synthase (NOS) expression in the gastric fundus of mdx mice were investigated. Mechanical responses of gastric strips were recorded via force displacement transducers. Evaluation of the three NOS isoforms was performed by immunohistochemistry and Western blot. Wild-type (WT) and mdx mice were distributed into three groups: untreated, relaxin pretreated, and vehicle pretreated. In strips from both untreated and vehicle-pretreated animals, electrical field stimulation (EFS) elicited contractile responses that were greater in mdx than in WT mice. In carbachol-precontracted strips, EFS induced fast relaxant responses that had a lower amplitude in mdx than in WT mice. Only in the mdx mice did relaxin depress the amplitude of the neurally induced excitatory responses and increase that of the inhibitory ones. In the presence of L-NNA, relaxin was ineffective. In relaxin-pretreated mdx mice, the amplitude of the EFS-induced contractile responses was decreased and that of the fast relaxant ones was increased compared with untreated mdx animals. Responses to methacholine or papaverine did not differ among preparations and were not influenced by relaxin. Immunohistochemistry and Western blotting showed a significant decrease in neuronal NOS expression and content in mdx compared with WT mice, which was recovered in the relaxin-pretreated mdx mice. The results suggest that relaxin is able to counteract the altered contractile and relaxant responses in the gastric fundus of mdx mice by upregulating nNOS expression.     

E2 and not P4 increases NO release from NANC nerves of the gastrointestinal tract: implications in pregnancy

In women, during pregnancy, there is decreased motility of the gastrointestinal tract leading to a delay in gastric emptying and an increase in colonic transit time. Whether the rise in estradiol (E2) or progesterone (P4) is responsible for this effect is controversial. As the nitrergic component of the nonadrenergic, noncholinergic (NANC) nerves is responsible for modulating gastrointestinal motility in vivo, the purpose of this study was to evaluate whether the increased release of nitric oxide (NO) from the nitrergic component of the NANC nerves innervating the gastric fundus and colon that occurs during late pregnancy in rats is mediated by E2 or P4. Ovariectomized rats treated with E2 or P4 alone or in combination were used for our studies. We also wanted to assess the cellular and molecular mechanisms involved. The NANC activity was studied by assessing changes in tone after application of electric field stimulation (EFS). The role of NO was determined by observing the effects of EFS in the presence and absence of the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and the reversibility of the effects of L-NAME by L-arginine. Our studies indicated that there was increased magnitude of relaxation of isolated strips of rat gastric fundus and rat colon after application of EFS to tissues obtained from animals treated with E2 alone or a combination of E2 + P4 but not from those treated with P4 alone. L-NAME attenuated relaxation responses in E2- and E2 + P4-treated animals. To elucidate whether the increased NO release may be due to an increase in neuronal NOS (nNOS) protein, we used both Western blot analysis and immunohistochemistry. We also used RT-PCR to determine whether there was an increase in nNOS mRNA after treatment with sex steroids. In nonpregnant animals, nNOS was detected by Western blot in the fundus and the colon and was barely detectable in the ileum. In pregnancy, there was an increase in nNOS in both the gastric fundus and the colon. The nNOS protein was also increased in ovariectomized animals treated with either E2 alone or E2 + P4 but not P4 alone when compared with ovariectomized animals receiving vehicle. Our results indicated that there was an increase in nNOS protein that was localized to the neurons of the myenteric plexus in the gastric fundus and colon in E2- and E2 + P4-treated animals, but this increase was not observed in animals treated with P4 alone. This increase in nNOS protein was accompanied by an increase in nNOS mRNA. These results suggest the possibility that E2, rather than P4, may be responsible for the delay in gastric emptying and increase in colonic transit time observed in pregnancy.     

Acute and conditioned hypoxic tolerance augmented by endothelial nitric oxide synthase inhibition in mice.

To identify a possible role for nitric oxide (NO) in acute hypoxic tolerance (HT) we measured hypoxic survival time (HST), effect of hypoxic conditioning (HC), and survival following hypoxic conditioning while blocking or mimicking the action of nitric oxide synthase (NOS). To inhibit NOS, CD-1 mice were given supplemental endogenous NOS inhibitor asymmetrical dimethylarginine (ADMA) or a synthetic NOS inhibitor N(omega)-nitro-L-arginine (L-NNA), both of which nonselectively inhibit three of the isoforms of NOS [inducible (iNOS), neuronal (nNOS), and endothelial NOS (eNOS)]. ADMA (10 mg/kg i.p.) or saline vehicle was given 5 min before HST testing. L-NNA was given orally at 1 g/l in drinking water with tap water as the control for 48 h before testing. Both ADMA and L-NNA significantly increased HST and augmented the HC effect on HST. Neither the nNOS selective inhibitor 7-nitroindazole (7-NI) nor the iNOS selective inhibitor N-{[3-(aminomethyl)phenyl]methyl}-enthanimidamide (1400W) had a statistically significant effect on HST or HT. The NO donor, 3-morpholinosydnoeimine, when given alone did not significantly decrease HT, but it did mitigate the increased HT effect of L-NNA. These data confirm that acute hypoxic conditioning increases HT and that NOS inhibition by endogenous (ADMA) and a synthetic NOS inhibitor (L-NNA) further increases HT, whereas iNOS and nNOS inhibition does not, suggesting that it is the inhibition of eNOS that mediates enhancement of HT.     

Influence of relaxin on the neurally induced relaxant responses of the mouse gastric fundus

The peptide hormone relaxin has been reported to depress the amplitude of contractile responses in the mouse gastric fundus by upregulating nitric oxide (NO) biosynthesis at the neural level. In the present study, we investigated whether relaxin also influenced nonadrenergic, noncholinergic (NANC) gastric relaxant responses in mice. Female mice in proestrus or estrus were treated for 18 h with relaxin (1 microg s.c.) or vehicle (controls). Mechanical responses of gastric fundal strips were recorded via force-displacement transducers. In carbachol precontracted strips from control mice and in the presence of guanethidine, electrical field stimulation (EFS) elicited fast relaxant responses that may be followed by a sustained relaxation. All relaxant responses were abolished by tetrodotoxin. Relaxin increased the amplitude of the EFS-induced fast relaxation without affecting either the sustained one or the direct smooth muscle response to papaverine. In the presence of the NO synthesis inhibitor L-N(G)-nitro arginine (L-NNA), that abolished the EFS-induced fast relaxation without influencing the sustained one, relaxin was ineffective. In strips from relaxin-pretreated mice, EFS-induced fast relaxations were enhanced in amplitude with respect to the controls, while sustained ones as well as direct smooth muscle responses to papaverine were not changed. Further addition of relaxin to the bath medium did not influence neurally induced fast relaxant responses, whereas L-NNA did. In conclusion, in the mouse gastric fundus, relaxin enhances the neurally induced nitrergic relaxant responses acting at the neural level.     

Calcitonin gene-related peptide (CGRP) modulates cholinergic neurotransmission in the small intestine of man, pig and guinea-pig via presynaptic CGRP receptors.

The effect of calcitonin gene-related peptide (CGRP) on the cholinergically mediated twitch contraction in longitudinal muscle strips of the small intestine (duodenum, jejunum, ileum) of guinea-pig, pig and man was investigated. Independently of the anatomical region, CGRP inhibited the twitch response in the different specimens of all three species by about 40% with similar IC50 values (1.5-2.4 nmol/l). Only in the guinea-pig small intestine CGRP induced a contraction of the smooth muscle which was sensitive to scopolamine and tetrodotoxin. The electrically evoked [3H]acetylcholine release from jejunal longitudinal muscle strips with myenteric plexus attached of the guinea-pig, which were incubated with [3H]choline, was concentration-dependently inhibited by CGRP. A direct relaxant effect of CGRP on smooth muscle tone of carbachol precontracted preparations was only observed in specimens of the guinea-pig. In conclusion, presynaptic inhibitory CGRP receptors on cholinergic neurones modulate the release of acetylcholine in different parts of the small intestine.     

Downregulation of nNOS and synthesis of PGs associated with endotoxin-induced delay in gastric emptying

A single intraperitoneal injection of endotoxin (40 microg/kg) significantly delayed gastric emptying of a solid nutrient meal. Blockade of nitric oxide synthase (NOS) with 30 mg/kg ip N(G)-nitro-L-arginine methyl ester or 20 mg/kg ip 7-nitroindazole [neuronal NOS (nNOS) inhibitor] significantly delayed gastric emptying in control animals but failed to modify gastric emptying in endotoxin-treated rats. Administration of 2.5, 5, and 10 mg/kg ip N(6)-iminoethyl-L-lysine [inducible NOS (iNOS) inhibitor] had no effect in either experimental group. Indomethacin (5 mg/kg sc), NS-398 (cyclooxygenase-2 inhibitor; 10 mg/kg ip), and dexamethasone (10 mg/kg sc) but not quinacrine (20 mg/kg ip) significantly prevented delay in gastric emptying induced by endotoxin but failed to modify gastric emptying in vehicle-treated animals. Ca(2+)-dependent NOS activity in the antrum pylorus of the stomach was diminished by endotoxin, whereas Ca(2+)-independent NOS activity was not changed. In addition, decreased nNOS mRNA and protein were observed in the antrum pylorus of endotoxin-treated rats. Our results suggest that downregulation of nNOS in the antrum pylorus of the stomach and synthesis of prostaglandins mediate the delay in gastric emptying of a solid nutrient meal induced by endotoxin.     

Different subpopulations of cholinergic and nitrergic myenteric neurones project to mucosa and circular muscle of the guinea-pig gastric fundus

Since the stomach lacks a well-developed ganglionated submucous plexus, the somata of enteric neurones innervating the muscle or the mucosa have to be localised within the myenteric plexus. The aim of this study was to determine the projection pathways and the neurochemical coding of myenteric neurones innervating these different targets in the gastric fundus. Myenteric cell bodies projecting to the mucosa or the circular muscle were retrogradely labelled by mucosa or muscle application of the fluorescent tracer DiI and subsequently characterised by their immunoreactivity for choline acetyltransferase (ChAT), nitric oxide synthase (NOS), substance P (SP) and/or neuropeptide Y (NPY). On average 143±91 and 89±49 myenteric neurones were labelled from the mucosa and the circular muscle, respectively. DiI-labelled neurones were either ChAT- or NOS-positive. DiI-labelled ChAT-positive neurones were mainly ascending and outnumbered NOS-positive neurones, which were mainly descending (79.3±6.2% vs 20.7±6.2% for mucosa neurones; 69.3±11.1% vs 30.7±11.1% for muscle neurones). Three ChAT-positive subpopulations (ChAT/–, ChAT/SP, ChAT/NPY) and two NOS-positive subpopulations (NOS/–, NOS/NPY) were found. ChAT/SP neurones projected mainly to the circular muscle (36.1±11.9% of the cholinergic muscle neurones; mucosa projection: 8.0±2.1%), whereas ChAT/NPY neurones projected mainly to the mucosa (38.1±9.2% of the cholinergic mucosa neurones; muscle projection: 5.7±2.4%). NOS/– cells projected predominantly to the muscle. This study demonstrates polarised pathways in the myenteric plexus consisting of ascending ChAT and descending NOS cells that innervate the circular muscle and the mucosa of the gastric fundus. The ChAT/SP neurones might function as circular muscle motor neurones, whereas ChAT/NPY neurones might represent secretomotor neurones.     

Arginase attenuates inhibitory nonadrenergic noncholinergic nerve-induced nitric oxide generation and airway smooth muscle relaxation

Background

Recent evidence suggests that endogenous arginase activity potentiates airway responsiveness to methacholine by attenuation of agonist-induced nitric oxide (NO) production, presumably by competition with epithelial constitutive NO synthase for the common substrate, L-arginine. Using guinea pig tracheal open-ring preparations, we now investigated the involvement of arginase in the modulation of neuronal nitric oxide synthase (nNOS)-mediated relaxation induced by inhibitory nonadrenergic noncholinergic (iNANC) nerve stimulation.

Methods

Electrical field stimulation (EFS; 150 mA, 4 ms, 4 s, 0.5 – 16 Hz)-induced relaxation was measured in tracheal preparations precontracted to 30% with histamine, in the presence of 1 μM atropine and 3 μM indomethacin. The contribution of NO to the EFS-induced relaxation was assessed by the nonselective NOS inhibitor L-NNA (0.1 mM), while the involvement of arginase activity in the regulation of EFS-induced NO production and relaxation was investigated by the effect of the specific arginase inhibitor nor-NOHA (10 μM). Furthermore, the role of substrate availability to nNOS in EFS-induced relaxation was measured in the presence of various concentrations of exogenous L-arginine.

Results

EFS induced a frequency-dependent relaxation, ranging from 6.6 ± 0.8% at 0.5 Hz to 74.6 ± 1.2% at 16 Hz, which was inhibited with the NOS inhibitor L-NNA by 78.0 ± 10.5% at 0.5 Hz to 26.7 ± 7.7% at 8 Hz (P < 0.01 all). In contrast, the arginase inhibitor nor-NOHA increased EFS-induced relaxation by 3.3 ± 1.2-fold at 0.5 Hz to 1.2 ± 0.1-fold at 4 Hz (P < 0.05 all), which was reversed by L-NNA to the level of control airways in the presence of L-NNA (P < 0.01 all). Similar to nor-NOHA, exogenous L-arginine increased EFS-induced airway relaxation (P < 0.05 all).

Conclusion

The results indicate that endogenous arginase activity attenuates iNANC nerve-mediated airway relaxation by inhibition of NO generation, presumably by limiting L-arginine availability to nNOS.     

Modulation by NO of acetylcholine release in the ileum of wild-type and NOS gene knockout mice

Nitric oxide (NO) inhibits the release of acetylcholine and cholinergic contractions in the small intestine of several species, but no information is available about the mouse ileum. This study examines the effects of NO on the electrically evoked release of [3H]acetylcholine and smooth muscle contraction in myenteric plexus-longitudinal muscle preparations of wild-type mice and of neuronal NO synthase (nNOS) and endothelial NOS (eNOS) knockout mice. The NOS inhibitor N(G)-nitro-L-arginine (L-NNA) and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) concentration dependently increased the evoked [3H]acetylcholine release and cholinergic contractions in preparations from wild-type mice and from eNOS knockout mice. Effects of L-NNA were specifically antagonized by L-arginine. In contrast, L-NNA and ODQ did not modify the release and contractions in preparations from nNOS knockout mice. The NO donor S-nitroso-N-acetyl-DL-penicillamine inhibited the electrically evoked release of [3H]acetylcholine and longitudinal muscle contractions in a quantitatively similar manner in wild-type preparations as well as in nNOS and eNOS knockout preparations. We conclude that endogenous NO released by electrical field stimulation tonically inhibits the release of acetylcholine. Furthermore, data suggest that nNOS and not eNOS is the enzymatic source of NO-mediating inhibition of cholinergic neurotransmission in mouse ileum.     

Influence of orexin A on the mechanical activity of mouse gastric strips

The presence of orexins and orexin receptors has been revealed not only in the central nervous system but also in the gastrointestinal tract. The present study was aimed to investigate the influence of orexin A (OXA) on the mechanical activity of fundal and antral strips of the mouse stomach. In the fundus, electrical field stimulation (EFS) elicited tetrodotoxin (TTX)-sensitive, frequency-dependent contractile responses whose amplitude was markedly reduced by OXA and enhanced by the orexin-1 type receptor antagonist SB-334867. In the presence of the NO synthesis inhibitor L-N(G)-nitro arginine (L-NNA), OXA was no longer effective. Methacholine caused a sustained contracture whose amplitude was not influenced by OXA, TTX or L-NNA. In carbachol-precontracted strips, the neurally-induced relaxant responses elicited during EFS were increased in amplitude by OXA. Antral strips showed a spontaneous contractile activity that was unaffected by TTX or L-NNA and transiently depressed by EFS. OXA did not influence either the spontaneous motility or the EFS-induced effects. The results indicate that OXA exerts region-specific effects and that, in the fundus, depresses EFS-induced contractile responses by acting at the nervous level. It is likely that NO is involved in the effects of the peptide.     

Role of motilin in the control of intestinal absorption, and gastric and biliary secretions in the rat

The effects of motilin on proline absorption and gastric and biliary secretions were examined in the rat. Prolonged intravenous administration of motilin (50 pmol/kg/min) significantly inhibited (P < 0.05) proline transport across the jejunum and reduced basal acid secretion to 40% of control value. The same concentration of motilin induced choleresis and increased bile output by 32%. Incubation of intestinal strips with different concentrations of motilin produced a dose-dependent inhibitory pattern of proline accumulation in the intestinal cells.     

Effect of motilin on the discharge of rat hippocampal neurons responding to gastric distension and its potential mechanism

The study aims to find the effect of motilin on neuronal activity of gastric distension-responsive neurons in rat hippocampus and its possible mechanism. Single unit discharges in the hippocampal CA1 region were recorded extracellularly by means of four-barrel glass micropipettes in anesthetized rats and the expression of nNOS in hippocampus was observed by fluo-immunohistochemistry staining. Of the 171 recorded neurons, 76.0% were GD-excitatory (GD-E) neurons and 24.0% were GD-inhibited (GD-I) neurons. The 57.6% of GD-E neurons showed an excitatory response to motilin and the same effect was observed in 51.7% GD-I neurons. However, when NOS inhibitor nitro-l-arginine methyl ester (l-NAME) was administrated previously, the followed motilin-induced excitatory responsiveness of GD-responsive neurons was reduced. In contrast, discharge activity of GD-responsive neurons with motilin was enhanced by pretreatment of NO precursor l-arginine. The expression of nNOS-IR positive neurons was significantly increased in CA1 after administration of motilin. Our findings suggested that motilin excited the GD-responsive neurons in the hippocampal CA1 region and the excitatory effect of motilin may be mediated by the endogenous NO.     

Modulation of nitrergic relaxant responses by peptides in the mouse gastric fundus

The effects of pituitary adenylate cyclase-activating peptide (PACAP-38) and vasoactive intestinal polypeptide (VIP) were investigated in the gastric fundus strips of the mouse. In carbachol (CCh) precontracted strips, in the presence of guanethidine, electrical field stimulation (EFS) elicited a fast inhibitory response that may be followed, at the highest stimulation frequencies employed, by a sustained relaxation. The fast response was abolished by the nitric oxide (NO) synthesis inhibitor L-N(G)-nitro arginine (L-NNA) or by the guanylate cyclase inhibitor (ODQ), the sustained one by alpha-chymotrypsin. alpha-Chymotrypsin also increased the amplitude of the EFS-induced fast relaxation. PACAP-38 and VIP caused tetrodotoxin-insensitive sustained relaxant responses that were both abolished by alpha-chymotrypsin. Apamin did not influence relaxant responses to EFS nor relaxation to both peptides. PACAP 6-38 abolished EFS-induced sustained relaxations, increased the amplitude of the fast ones and antagonized the smooth muscle relaxation to both PACAP-38 and VIP. VIP 10-28 and [D-p-Cl-Phe6,Leu17]-VIP did not influence the amplitude of both the fast or the sustained response to EFS nor influenced the relaxation to VIP and PACAP-38. The results indicate that in strips from mouse gastric fundus peptides, other than being responsible for EFS-induced sustained relaxation, also exerts a modulatory action on the release of the neurotransmitter responsible for the fast relaxant response, that appears to be NO.     

Exogenous and endogenous ghrelin in gastroprotection against stress-induced gastric damage

Ghrelin, identified in the gastric mucosa has been involved in control of food intake and growth hormone (GH) release but little is known about its influence on gastric secretion and mucosal integrity. The effects of ghrelin on gastric secretion, plasma gastrin and gastric lesions induced in rats by 75% ethanol or 3.5 h of water immersion and restraint stress (WRS) were determined. Exogenous ghrelin (5, 10, 20, 40 and 80 microg/kg i.p.) increased gastric acid secretion and attenuated gastric lesions induced by ethanol and WRS and this was accompanied by the significant rise in plasma ghrelin level, gastric mucosal blood flow (GBF) and luminal NO concentrations. Ghrelin-induced protection was abolished by vagotomy and attenuated by suppression of COX, deactivation of afferent nerves with neurotoxic dose of capsaicin or CGRP(8-37) and by inhibition of NOS with L-NNA but not influenced by medullectomy and administration of 6-hydroxydopamine. We conclude that ghrelin exerts a potent protective action on the stomach of rats exposed to ethanol and WRS, and these effects depend upon vagal activity, sensory nerves and hyperemia mediated by NOS-NO and COX-PG systems.     

Cardiac cholinergic NO-cGMP signaling following acute myocardial infarction and nNOS gene transfer

Myocardial infarction (MI) is associated with oxidative stress, which may cause cardiac autonomic impairment. We tested the hypothesis that acute MI disrupts cardiac cholinergic signaling by impairing nitric oxide (NO)-cGMP modulation of acetylcholine (ACh) release and whether the restoration of this pathway following cardiac neuronal NO synthase (nNOS) gene transfer had any bearing on the neural phenotype. Guinea pigs underwent four ligature coronary artery surgery (n = 50) under general anesthesia to induce MI or sham surgery (n = 32). In a separate group, at the time of MI surgery, adenovirus encoding nNOS (n = 29) or enhanced green fluorescent protein (eGFP; n = 30) was injected directly into the right atria, where the postganglionic cholinergic neurons reside. In vitro-evoked right atrial [3H]ACh release, right atrial NOS activity, and cGMP levels were measured at 3 days. Post-MI 24% of guinea pigs died compared with 9% in the sham-operated group. Evoked right atrial [3H]ACh release was significantly (P < 0.05) decreased in the MI group as was NOS activity and cGMP levels. Tetrahydrobiopterin levels were not significantly different between the sham and MI groups. Infarct sizes between gene-transferred groups were not significantly different. The nNOS transduced group had significantly increased right atrial [3H]ACh release, right atrial NOS activity, cGMP levels, and decreased cAMP levels. Fourteen percent of the nNOS transduced animals died compared with 31% mortality in the MI + eGFP group at 3 days. In conclusion, cardiac nNOS gene transfer partially restores the defective NO-cGMP cholinergic pathway post-MI, which was associated with a trend of improved survival at 3 days.     

Effects of 1400W, a potent selective inducible NOS inhibitor, on histamine- and leukotriene D4-induced relaxation of isolated guinea pig nasal mucosa.

To clarify the role of inducible nitric oxide synthase (iNOS) in the relaxation of nasal vasculature, the effects of a potent selective iNOS inhibitor, N-[(3-aminomethyl)benzyl]acetamidine (1400W), on histamine- and leukotriene D4 (LTD4)-induced relaxations of isolated nasal septal mucosae were examined in naive guinea pigs. In addition to eNOS and nNOS, Western blots demonstrated a distinct expression of iNOS in nasal mucosal tissues of naive guinea pigs. In isolated nasal septal mucosae precontracted with norepinephrine (3 x 10(-5)M), both histamine (10(-7)-10(-3)M) and LTD4 (10(-10)-10(-7)M) exhibited relaxations, which were inhibited by a NOS inhibitor NG-monomethyl-L-arginine (L-NMMA; 10(-4)M). The inhibitory effect of L-NMMA was reversed by L-arginine (10(-3)M), indicating that the relaxations induced by histamine and LTD4 are mediated by NO. Furthermore, both the histamine- and LTD4-induced relaxations were also significantly attenuated by 1400W (10(-5)M). These findings suggest an involvement of NO generated by iNOS in agonist-induced relaxation of nasal mucosal vasculature in naive guinea pigs.     

Relations between contractile responses and beta-adrenoceptors in gastric fundus of diabetic rats.

Contractile responses to norepinephrine (NE), and the population of beta-adrenoceptors, were determined in gastric fundus smooth muscle from rats with diabetes induced by streptozotocin (STZ), and age-matched controls. Relaxation and/or contraction of fundus strips of controls and diabetics were induced by 10(-5)M NE. Responses to NE were mainly relaxation in gastric fundus isolated from controls, and contraction in fundus isolated from diabetics. Contraction was blocked by 10(-8) M prazosin and relaxation was blocked by 10(-6) M propranolol. Relaxation by isoproterenol of contraction induced by 10(-6) M acetylcholine was significantly less in fundus from diabetics than in that from controls. The number of beta-adrenoceptors, measured with [125I] iodocyanopindolol as a ligand, was significantly less in gastric fundus membrane isolated from diabetics than in that from controls, but affinity was no different. The level of plasma catecholamine was higher in diabetics than in controls. Results suggest that depression of gastric fundus relaxation and increase of contraction by NE in diabetics could be due to fewer beta-adrenoceptor binding sites caused by down-regulation by higher catecholamine level in diabetic rats.     

Relaxation of myometrium by calcitonin gene-related peptide is independent of nitric oxide synthase activity in mouse uterus

Calcitonin gene-related peptide (CGRP) inhibits myometrial contractile activity. However, the responsiveness of the mouse myometrium to CGRP is dependent on the hormonal and gestational stage. The inhibitory effect of CGRP in the myometrium is prominent during gestation and declines at parturition. The present study was undertaken to examine if nitric oxide (NO) production by nitric oxide synthase (NOS) isoforms mediates the inhibitory action of CGRP on uterine contractions as has been suggested earlier. Transgenic mice deficient in either of the three major NOS isoforms: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) were used. Isometric force measurements on myometrial strips obtained from NOS-deficient mice were carried out and the inhibitory capacity of CGRP was monitored. CGRP inhibited KCl-induced contractions of the myometrial strips obtained from eNOS(-/-), iNOS(-/-), and nNOS(-/-) mice with equal efficiency as in wild-type animals. Additionally, NOS protein expression in the mouse uterus during gestation and during the estrous cycle was examined by means of Western immunoblot analysis. No correlation between NOS expression and inhibitory activity of CGRP was evident. The results suggest that the inhibitory action of CGRP in the mouse uterus is independent of the activity of these NOS isoforms.