Protective effect of nitric oxide on development of acute pancreatitis in rats

Nitric oxide (NO) has been implicated to regulate pancreatic circulation, promote capillary integrity, and inhibit leukocyte adhesion. We investigated the role of NO in the development of pancreatitis. Nitro-l-arginine, an inhibitor of NO synthase, in total dose of 35 mg/kg body wt was infused in the rats with edematous pancreatitis induced by two intraperitoneal injections of cerulein (20 g/kg).l-Arginine (125 or 250 mg/kg), a NO donor was intravenously administered twice in the rats with hemorrhagic pancreatitis induced by waterimmersion stress plus two intraperitoneal injections of cerulein (40 g/kg). The degree of pancreas edema, serum amylase levels, and histologic alterations were investigated. Nitro-l-arginine exacerbated cerulein-induced pancreatitis and caused a decrease in pancreatic blood flow.l-Arginine ameliorated the severity of hemorrhagic pancreatitis dose dependently and improved the pancreatic blood flow. These findings suggest that NO could confer protection against the development of hemorrhagic pancreatitis, probably through improvement of the pancreatic microcirculation.This study was supported by the research fund of the Research Committee on Intractable Diseases of the Pancreas of the Japanese Ministry of Health and Welfare, and by a Grant-in-Aid for General Scientific Research from the Ministry of Education, Science and Culture, Japan.     

Insulin secretion in rats with chronic nitric oxide synthase blockade

Summary Nitric oxide, which is produced from l-arginine by a nitric oxide-synthase enzyme, has been shown to be a ubiquitous messenger molecule. Recently, it has been suggested that nitric oxide might influence insulin secretion by activating the soluble guanylate cyclase and generating cyclic guanosine monophosphate (cGMP). We have investigated the role of the nitric oxide pathway in insulin secretion by evaluating the insulin response to several secretagogues in rats in which nitric oxide-synthase was chronically inhibited by oral administration of the l-arginine analogue, N^G-nitro-l-arginine methyl ester (l-NAME). Blood pressure and aortic wall cGMP content were used as indices of nitric oxide-synthase blockade. Insulin secretion was evaluated after an intravenous bolus of d-glucose, l-arginine or d-arginine. Chronic l-NAME administration induced a 30% increase in blood pressure and a seven-fold drop in arterial cGMP content. Body weight, fasting plasma glucose and insulin were not influenced by l-NAME administration. First-phase insulin secretion (1+3 min) in response to glucose was not significantly different in l-NAME and control rats. The areas under the insulin curve were similar in both groups. Insulin secretion in response to d-arginine or l-arginine in l-NAME-treated and control rats were also similar. In conclusion, chronic nitric oxide-synthase blockade increases blood pressure and decreases aortic cGMP content, but does not alter insulin secretion in response to several secretagogues. Chronic oral administration of l-NAME in the rat provides an adequate animal model for studying the l-arginine nitric oxide-pathway.Abbreviations NO Nitric oxide - cGMP guanosine 3: 5 cyclic monophosphate - l-NMMA N^G-monomethyl-l-arginine - l-NAME N^G-nitro-l-arginine-methyl-ester - NOD mice non obese diabetic mice     

Cyclic GMP-Mediated Activation of a Glibenclamide-Sensitive Mechanism in the Rabbit Sphincter of Oddi

We investigated whether glibenclamide-sensitive potassium channels are involved in cyclic GMP (cGMP)-mediated relaxation of the rabbit Oddi's sphincter. Changes in isometric tension were measured in the presence of atropine (1 M) and guanethidine (4 M). Concentration–response curves for nitroglycerin, vasoactive intestinal polypeptide (VIP), and sodium nitroprusside (SNP) were shifted to the right in the presence of (p-chloro-d-Phe⁶, Leu¹⁷)-VIP (VIPa), a VIP receptor antagonist. Glibenclamide (1 M) attenuated the relaxations to VIP, nitroglycerin, or 8-bromo cGMP. In the presence of tetrodotoxin (TTX), glibenclamide attenuated relaxations to VIP without effect on those to nitroglycerin. Furthermore, nitroglycerin increased both cAMP and cGMP concentrations, however, it failed to increase the tissue cAMP concentration in the presence of TTX. VIPa also blocked the increase in content of either cyclic nucleotide. VIP increased cAMP with a TTX-sensitive increase in cGMP content. 8-Bromo cGMP (1 M) significantly increased the tissue cAMP content. This was blocked by either TTX or VIPa (both 1 M). We conclude that ATP-sensitive potassium channel (K_ATP) activation contributes to cGMP-mediated relaxation of the Oddi's sphincter of the rabbit. Activation of K_ATP results from a cyclic AMP-mediated process due to cGMP-dependent VIP release from neurons.     

Klinisch-chemische Ultramikromethoden

Zusammenfassung Es werden Probleme und Möglichkeiten bei Arbeiten im Größenordnungsbereich von Mikrolitern (1 l=0.001 ml) dargestellt und eine Grundausrüstung beschrieben, welche es gestattet, klinisch-chemische Analysen ausgehend von kleinsten Probenmengen (5–20 l) einfach, rasch und zuverlässig auszuführen. Die Arbeitsweise von halbautomatischen Pipettenflaschen, einer Ultramikrobürette, eines speziellen Spektro-Kolorimeters und anderer apparativer Hilfsmittel wird dargestellt.

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Summary The problems and practicability of working with microliters (1 l=0.001 ml.) are presented. A basic apparatus is described, which permits the simple, rapid and reliable clinical-chemical analysis of very small samples (5–20 l.). Directions are given for the operation of semiautomatic pipettes, an ultramicroburette, a special spectrocolorimeter and other auxilliary apparatus.

     

Deleterious effects of digitalis on reperfusion-induced arrhythmias and myocardial injury in ischemic rat hearts: possible involvements of myocardial Na+ and Ca2+ imbalance

Summary Isolated rat hearts were made ischemic for 25 min after an initial recirculating perfusion, followed by 30 min of reperfusion. In some hearts, interventions including administration of ouabain and/or high [K⁺] in the buffer were performed during the first 10 min of reperfusion.During ischemia, intracellular Na⁺ (Na_i) increased from 15 to 64 [mol/g dry weight (dwt). During reperfusion, Na_i declined rapidly (at 10 min of reperfusion: 48 nol/g dwt, at 30 min: 25 mol/g dwt) and regular rhythm was recovered within 10 min in hearts without any intervention during reperfusion.⁴⁵Ca²⁺ uptake increased from 0.8 to 7.5 mol/g dwt after 30 min of reperfusion. Ventricular function recovered by 45 %.A 10-min perfusion with 10 or 50 M of ouabain increased Na_i (17 to 21 or 27 mol/g dwt) with increased left-ventricular (LV) contractile function, but these effects were reversed by combination of high perfusate [K⁺] (20 mM) in non-ischemic hearts.A 10-min reperfusion with ouabain retarded or stopped the decline in Na_i (at 10 min of reperfusion: 54 or 63 mol/g dwt, at 30 min: 32 or 40 mol/g dwt). These amounts of ouabain also increased the incidence of ventricular tachyarrhythmias during reperfusion to 30 % or 50 %, and increased the duration of ventricular fibrillation from 6.5 to 11.5 or 18.0 min.⁴⁵Ca²⁺ uptake reached to 8.8 or 10.0 mol/g dwt, and function recovered only 35 % or 28 %. When high perfusate [K⁺] was combined with ouabain during reperfusion, the retarded decline in Nai, augmented⁴⁵Ca²⁺ uptake, and reduced recovery of function caused by ouabain alone were attenuated. These results suggest that digitalis has toxic effects on reperfused ischemic hearts by inhibition of rapid active outward transport of previously elevated Na_i and potentiation of Ca²⁺ overload.The work was supported in part by grant HL 37936 from the National Heart, Lung and Blood Institute. J. R. Neely was deceased on November 29, 1988     

Gastric diversion or pylorus ligation for gastric mucosal integrity and acid secretion studies in the rat?

The advantages of gastric diversion over pylorus ligation in rat gastric mucosal integrity and acid secretion studies over 6 hr were investigated. Mucosal injury developed in 80% of pylorus-ligation controls. Atropine (5 mg/kg) or cimetidine (40 mg/kg) had no effect on this injury (2.9 mm²±0.9 and 2.8 mm²±0.7, respectively, vs 3.1 mm²±1, mean±sem, N=10; however vagotomy increased it (13.7 mm²±Pylorus-ligation H⁺ output was higher than that of gastric diversion (390.5 mol±54.8 vs 61 mol±2.5, mean±sem, N=10, P<0.001). Cimetidine (40 mg/kg) depressed H⁺ output of gastric diversion (21.3 mol±1.2 vs 61 mol±2.5, mean±sem, N=10, P<0.001), but not of pylorus ligation (424 mol±74.2 vs 390.5 mol±54.8, mean±sem, N=10). Vagotomy or atropine depressed pylorus-ligation H⁺ output (P<0.001), but each allowed an output (36.6 mol±5.5 and 120 mol±29, respectively, mean±sem, N=10) significantly (P<0.001) higher than that associated with it in gastric diversion (16 mol±1.4 and 17.1 mol±1.6, respectively, mean±sem, N=10). This study demonstrates that in the rat pylorus ligation, in contrast to gastric diversion, injures the gastric mucosa, stimulates H⁺ secretion, and overshadows the efficacy of antisecretory agents.     

Alterations in the heart sarcolemmal Ca2+ transport activity by some β-adrenergic antagonists

Summary The effects of some -adrenergic antagonists such as acebutolol, propranolol, pindolol and oxprenolol (1–1000 M) were studied on the rat heart sarcolemmal Ca²⁺ transport activities. Pindolol enhanced sarcolemmal ATP-dependent Ca²⁺ binding and Ca²⁺-stimulated ATPase whereas acebutolol had no effect. Both propranolol and oxprenolol had biphasic actions on the sarcolemmal Ca²⁺ pump activities; the lower concentrations (1 and 10 M) were stimulatory, but the higher concentrations (100 and 1000 M) were inhibitory. None of the drugs used in this study had any effect on Mg²⁺ ATPase and non-specific Ca²⁺ binding activities of heart sarcolemma except that 1000 M propranolol decreased Mg²⁺ ATPase activity significantly. Mitochondrial and microsomal ATP-dependent Ca²⁺ binding activities were unaffected by these drugs (1–1000 M), except that 1000 M propranolol was inhibitory. These results suggest differences among various -adrenergic blocking drugs with respect to their actions on sarcolemmal Ca²⁺ pump in the myocardium.     

Mechanisms in rabbit aorta for hyperglycaemia-induced alterations in angiotensin II and norepinephrine effects

Summary The (Na⁺, K⁺)-ATPase activity operative in rabbit aortic intima-media incubated with normal plasma levels of glucose and myo-inositol (70 mol/l) is decreased when the glucose content of the medium is raised from 5 to 10 mmol/l or higher; this effect is prevented by aldose reductase inhibitors and by raising the myo-inositol content of the medium to 500 mol/l. The decrease in (Na⁺, K⁺)-ATPase activity results from the loss of a component normally regulated (stimulated) by endogenously released adenosine through a receptor that stimulates phosphatidylinositol turnover in a discrete pool. The replenishment of this phosphatidylinositol pool selectively requires myo-inositol transport and is inhibited when increased polyol pathway activity impairs myo-inositol transport at a normal plasma level. Adenosine is a vasodilator, some endothelium-released vasodilators modulate the responses to vasoconstrictors by stimulating an increase in (Na⁺, K⁺)-ATPase activity in vascular smooth muscle. Whether adenosine mediates this effect in angiotensin II or norepinephrine-stimulated aorta was examined. Angiotensin II (100 nmol/l) and norepinephrine (1 mol/l) evoked marked increases in (Na⁺, K⁺)-ATPase activity in aortic intima-media incubated with 5 mmol/l glucose and 70 mol/l myo-inositol, which were inhibited when adenosine deaminase was added or the medium myo-inositol omitted to inhibit myo-inositol transport. Raising the medium glucose to 30 mmol/l inhibited the angiotensin II and norepinephrine evoked increases in (Na⁺, K⁺)-ATPase activity, and this was prevented when tolrestat (10 mol/l) was added or the myo-inositol content of the medium was raised from 70 to 500 mol/l. Hyperglycaemia causes decreased (Na⁺, K⁺)-ATPase activity prevented by aldose reductase inhibitors and by raising plasma myo-inositol by a mechanism which inhibits an adenosine-(Na⁺, K⁺)-ATPase regulatory system, which modulates the responses to angiotensin II and norepinephrine in some blood vessels.     

Nitric oxide-mediated neurotransmission is attenuated in the anococcygeus muscle from diabetic rats

Summary The effect of STZ-induced diabetes of 8-weeks duration was examined on nitric oxide-mediated neurotransmission in the rat anococcygeus muscle. In the presence of noradrenergic blockade and raised tissue tone, relaxant responses to nerve stimulation (0.5–5 Hz, for 10 s), sodium nitroprusside (5 and 10 nmol/l) and nitric oxide (1 and 3 mol/l) were significantly reduced in anococcygeus muscles from diabetic rats compared to responses from control rats (p <0.05). In contrast, relaxations to papaverine (3 and 10 mol/l were not reduced in tissues from diabetic rats. The nitric oxide synthesis inhibitor NOLA (100 mol/l) abolished relaxant responses to nerve stimulation but had no effect on responses to any of the relaxant agents used. Exposure to NOLA at 10 mol/l reduced stimulation-induced relaxations; this reduction was significantly greater in tissues from the diabetic group than from the control group (p <0.05), probably as a consequence of the smaller relaxant responses in muscles from diabetic rats. Contractile responses to nerve stimulation (1–10 Hz, for 10 s), but not noradrenaline (0.03–30 mol/l), were significantly greater in anococcygeus muscles from diabetic rats than from control rats (p <0.05). NOLA (100 mol/l) significantly enhanced stimulation-induced contractions (p <0.05), however the enhancement was significantly less in tissues from diabetic rats (p <0.05). The results suggest that STZ-induced diabetes impairs smooth muscle reactivity to nitric oxide in the rat anococcygeus muscle.Abbreviations STZ Streptozotocin - NOLA N^G-nitro-l-arginine - NANC nonadrenergic noncholinergic - ANOVA analysis of variance     

Ascorbic acid stimulates the resorption of canine articular cartilage induced by a factor derived from activated rabbit macrophages

Summary Articular cartilage explants from the knees of mongrel dogs release 5–10% of their proteoglycan content spontaneously when cultured for 4 days in serum-free modified Bigger's medium. A factor synthesized and secreted by lipopolysaccharide-stimulated rabbit macrophages can stimulate this release of proteoglycan by 2 to 3-fold. The release of proteoglycan in response to macrophage factor is maximal in the presence of 1.5–50 g/ml l-ascorbic acid. In the absence of ascorbate, or with high levels of ascorbate (150 g/ml), the effect of the factor is diminished by 50%. d-isoascorbate, reduced glutathione, or dithiothreitol cannot substitute for l-ascorbate in producing this effect, while dehydroascorbate can.     

The effect of dbcAMP on the lysolecithin induced hemolysis of calf and adult cattle erythrocytes

Summary The calf erythrocytes have an increased sensitivity against lysolecithin as compared to their adult counterparts. 10^–3M dbcAMP increases the hemolysis induced by 5g of lysolecithin in 0.15 M NaCl containing 10 mM phosphate buffer (pH 7.4). By increasing the level of phosphate buffer (75 mM) in the incubation mixture, 10^–3M dbcAMP decreases the hemolysis induced by 5g of lysolecithin. These data suggest a dual effect exerted by dbcAMP: the relatively labilizing or stabilizing effect prevails as a function of exogenous inorganic phosphate level.10-⁶M dbcAMP also has a relative protective effect against lysolecithin.The combined addition of cAMP (10^–3) and theophyllin (10^–4M) does not stabilize the membrane.By increasing the level of lysolecithin to 20g/ml the stabilizing effect of dbcAMP disappears.DbcAMP (10^–3) as well as cAMP (10^–3M) and theophyllin (10^–4M) have a minimum increasing effect on hemolysis in the absence of lysolecithin, too.

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Abbreviations dbcAMP N⁶-20-dibutyryladenosine 35-monophosphate - cAMP cyclic 35-adenosine monophosphate     

Relaxant effect of omeprazole and lansoprazole in guinea pig gallbladder muscle strips in vitro

Background. The aim of this study was to investigate the role of omeprazole and lansoprazole, H⁺-K⁺ ATPase inhibitors, in gallbladder smooth muscle contractility in vitro. Methods. Gallbladder muscle strips obtained from guinea pigs were mounted in an organ bath. The responses of both precontracted strips and strips under basal tension to omeprazole and lansoprazole were determined. Results. Spontaneous contractile activity was blocked following omeprazole and lansoprazole administration. The agents also caused concentration-dependent relaxation in carbachol- and KCl-precontracted gallbladder muscle strips. Pretreatment with atropine (1µM), N^W-nitro l-arginine methyl ester (l-NAME; 30µM), indomethacin (10µM), ammonium chloride (7.5mM), sodium acetate (7.5mM), tetraethylammonium chloride (0.5mM), glibenclamide (1µM), 4-aminopyridine (0.1mM), or clotrimazole did not inhibit this relaxation. Gallbladder strips were placed in high-concentrtion potassium (80mM), calcium-free solution. The contraction produced with the addition of Ca²⁺ (2.5mM) was completely relaxed by omeprazole, lansoprazole, and nifedipine separately. Conclusions. These results demonstrate that omeprazole and lansoprazole have potent inhibitory effects on spontaneous contractions and cause dose-dependent relaxation in precontracted gallbladder smooth muscle strips of guinea pig in vitro. This effect could be due to blockade of the calcium channels.     

Regional glucose andβ-Hydroxybutyrate use by developing rat brain

Rates of glucose andd--hydroxybutyrate use were determined in five brain regions of 20-day-old rats. The regions studied were cerebral cortex, thalamus, striatum, cerebellum, and brain stem. The tracers for determining rates of substrate use were [³H]fluorodeoxyglucose and [3-¹⁴C]-d--hydroxybutyrate. Two or five minutes after isotope administration the animals were sacrificed in a 6-kW, 2450-MHz focused microwave device. Ten minutes prior to isotope administration the animals were injected intraperitoneally with normal saline or DL--hydroxybutyrate (10mmol/kg). Bloodd--hydroxybutyrate levels averaged 0.21 mol/ml in saline-injected and 3.13 mol/ml in hyperketonemic rats. Rates of glucose utilization were significantly heterogeneous between regions in both groups: thalamus > cerebral cortex striatum > brain stem > cerebellum. These rates were 20–35% lower in hyperketonemic rats. Rates ofd--hydroxybutyrate use varied significantly between regions only in the saline group, with the brain stem rate being significantly lower than that in cortex or cerebellum. Regional rates ofd--hydroxybutyrate use did not correlate significantly with regional rates of glucose use in either the saline or the hyperketonemic groups. Regional rates of glucose use were strongly and positively correlated between conditions, as were regional rates ofd--hydroxybutyrate use. Thus, in 20-day-old rats, the regional heterogeneity of brain glucose use is similar to that in adult rats.d--Hydroxybutyrate use is much less regionally heterogeneous. When brain regional rates ofd--hydroxybutyrate use are increased seven- to ninefold in acute hyperketonemia, there are compensatory decreases in regional rates of glucose use sufficient to keep regional rates of energy production unchanged.     

Role of histamine H3 receptors in control of mouse intestinal motility in vivo and in vitro: comparison with alpha2-adrenoceptors

We tested drugs acting at histamine H₃ receptors in mice on the gastrointestinal transit of a charcoal meal in vivo and on neurogenic contractions of isolated ileal preparations. The agonist (R)--methylhistamine (100 mol/kg) caused a maximum 25% reduction of gastrointestinal transit, an effect mimicked by immepip (100 mol/kg) and antagonized by thioperamide (20 mol/kg) or clobenpropit (20 mol/kg). In the isolated ileum, (R)--methylhistamine (10–100 M) caused a slight, thioperamide-insensitive, reduction (maximum 15%) of electrically evoked cholinergic contractions. In comparison, the ₂-adrenoceptor agonist clonidine (0.1 mol/kg) caused a 35.2% inhibition of the gastrointestinal transit and almost completely reduced (maximum 82% at 1 M) the cholinergic contraction of the isolated ileum, both effects being antagonized by idazoxan (0.4 mol/kg and 1 M, respectively). These results suggest that histamine H₃ receptors, located outside the myenteric plexus, mediate an inhibition of the gastrointestinal transit in vivo. Conversely, the presence of ₂-adrenoceptors in the cholinergic nerve endings and their inhibitory role in the control of gastrointestinal propulsion is confirmed.     

Evaluation of rat insulin messenger RNA in pancreatic and extrapancreatic tissues

Summary The purpose of these studies was to determine whether insulin detected immunochemically in extrapancreatic tissues of the adult rat is synthesized in situ by quantitating mRNA in these tissues. A blot hybridization assay was utilized with cloned ³²P-proinsulin cDNA. The lower limit of detection was estimated to be 3pg. Proinsulin mRNA concentration was found to be 1000–1500 g in isolated pancreatic islets and was easily detected in total pancreatic RNA at 10–15 pg/ g. Proinsulin mRNA was quantitated in rat insulinoma cells adapted to culture at levels 150 those in normal islets. Samples of RNA (20–50 g) enriched about 50-fold for mRNA sequences by repeated oligo-deoxythymidylate chromatography were assayed. No insulin mRNA was detected in 50 g samples of RNA from brain or in 20 g samples from subsections of brain or other extrapancreatic tissues. RNA samples were undegraded as assessed by ability to stimulate protein synthesis in a cell-free system. Proinsulin mRNA from pancreas was added to brain homogenates and recovered intact. Brain RNA samples with insulin mRNA levels 1:1000 that of pancreas would be predicted to have 50–75 pg proinsulin mRNA/50 g sample assayed if present. Because none was found, brain must have a concentration <1:6,000 that of pancreas. These findings suggest that immunoassayable insulin detected in extrapancreatic tissues of the adult rat is synthesized by the pancreas.     

Reversal of multidrug resistance by novel cyclosporin A analogues and the cyclopeptolide SDZ 214-103 biosynthesized in vitro

It was shown that cyclopeptolide SDZ 214-103 (10 M) is more active in rhodamine-123 accumulation in actinomycin-d-resistant human lymphoma cells CCRF/ACTD400 than cyclosporin A (10 M), but equipotent in the doxorubicin-resistant Friend erythroleukemia cell line F4-6/ADR. In F4-6/ADR cells, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay showed comparable cytotoxic effects of doxorubicin at various concentrations in the presence of SDZ 214-103 and cyclosporin A. For the other novel cyclosporin A analogues minor multidrug-resistance-modulating potency was demonstrated. At equipotent modulating doses of verapamil (10 M) and cyclosporin A (10 M) in the MTT assay regarding doxorubicin cytotoxicity, cyclosporin A was efficient in the rhodamine-123-uptake assay while verapamil was not active when identical incubation times were used.Abbreviations MDR multidrug resistance - Pgp-170 P-glycoprotein with a molecular mass of 170 kDa - D-Hiv D-2-hydroxyisovaleric acid     

Inhibition of basal and stimulated gastric H+ and pepsin secretion in duodenal ulcer patients by metiamide,an H-2 histamine antagonist

Metiamide was given orally in one dose of 200 mg in 23 studies in patients with duodenal ulcer, 4 in the basal state, 11 during histamine infusion, and 8 before insulin hypoglycemia stimulation. In the latter 8 patients insulin was given at another time without metiamide. In 17 studies acid secretion was suppressed by metiamide—up to 75% in the basal state, 53% after histamine, and 80% after insulin. Pepsin secretion was reduced to the same extent as H⁺ in the histamine studies but not in the basal (57%) or insulin (44%) studies, so that in the latter pepsin/acid ratios were 3-fold greater than in controls. Blood levels of metiamide were measured in 17 studies. In 10 out of 11 who showed inhibition of 40% or more, peak blood levels of metiamide were 0.45 g/ml to 1.25 g/ml. In 5 of 6 who did not show inhibition, blood levels were 0.05–0.4 g/ml; in the sixth it was 0.8 g/ml. Therefore a critical blood level for suppression of basal or stimulated secretion appears to be 0.45 g/ml.Supported by Public Health Service Grants AM09260 and AM05286; and by a Grant-in-Aid from Smith Kline & French Laboratories; Veterans Administration Hospital, Birmingham, Alabama, Research Project 3649-01.     

Autocrine growth inhibition of IL-1β-treated cultured human aortic smooth muscle cells: Possible role of nitric oxide

Summary This study was designed to investigate whether interleukin (IL)-1 would stimulate nitric oxide (NO) production in cultured human aortic vascular smooth muscle cells (VSMCs), and to determine the basic effect of the liberated NO on VSMC proliferation. NO production was estimated from nitrite concentration of culture medium in multi-well plates, determined by the Griess method. VSMCs were IL-1-pretreated in insert cups, and co-cultured with untreated VSMC in the wells.³H-thymidine (³H-Tdr) incorporation into the VSMC in wells was evaluated for VSMC proliferative activity. IL-1 stimulated NO production in VSMCs in a concentration-dependent manner. This effect was further enhanced by the addition of a membrane-permeable cyclic adenosine monophosphate derivative, dibutyryl cyclic AMP (db-cAMP), and was significantly reduced by concomitant use of an NO synthase inhibitor, N^G-nitro-l-arginine methyl ester (l-NAME). IL-1-pretreated VSMCs significantly inhibited³H-Tdr incorporation of the co-cultured VSMC. This inhibitory effect was significantly enhanced by the addition of db-cAMP, while this inhibition was significantly decreased by preincubation withl-NAME, and was abolished in thel-arginine-free medium. These results suggest that, in human VSMC, IL-1 stimulates NO production that is enhanced by intracellular cAMP accumulation, and that the liberated NO inhibits further VSMC proliferation in an autocrine fashion.     

Nitric oxide mediates inhibitory nerve effects in human esophagus and lower esophageal sphincter

The effect of inhibition of nitric oxide synthase on nonadrenergic, noncholinergic nerve-mediated responses in circular smooth muscle of the human esophageal body and lower esophageal sphincter (LES) was examinedin vitro. Tissues were obtained from 10 patients (eight esophageal resection for cancer, two transplant donors). Muscle strips from the LES developed significant spontaneous tension (11.6 ± 2.1 mN/mm²,N=6) and relaxed in response to electrical stimulation. The nitric oxide synthase inhibitor,N -nitro-l-arginine (NNA), at 10^–5 M, inhibited the relaxation, but had no significant effect on the spontaneous tension (13.0 ± 2.6 mN/mm²,P=0.07). Esophageal body strips developed little spontaneous tension, demonstrated an off contraction following the cessation of the electrical stimulus, and when contracted with 10^–5 M carbachol, relaxed during electrical stimulation. NNA (10^–5 M) inhibited the off contraction and the relaxation seen after carbachol and unmasked a prominent intrastimulus contraction. This intrastimulus contraction was enhanced by eserine and inhibited by atropine and tetrodotoxin. NNA showed similar potency in the esophageal body and LES and its effects were reversed byl-arginine, but notd-arginine. The results indicate that nitric oxide is an important mediator for nonadrenergic, noncholinergic nerve effects in the human esophagus and lower esophageal sphincter.This research was supported in part by an ICI Pharma/Medical Research Council of Canada Research Fellowship grant awarded to H.G. Preiksaitis and a Medical Research Council Program Grant PG8.     

Inhibition of insulin-stimulated xylose uptake in denervated rat soleus muscle: a post-receptor effect

Summary Insulin (100 U/l) stimulated xylose uptake in rat soleus muscle from a basal value of 2.3±0.5 to 11.6±2.1 mol · g^-1 · h^-1. Denervation (section of the sciatic nerve) markedly reduced the stimulatory action of insulin (basal 1.3 ±0.4 mol · g^-1 · h^-1; insulin-stimulated 4.5±0.6 mol · g^-1 · h^-1). This effect appeared 3 days after denervation and was maximal after 5 days. Denervation also affected the insulin dose response curve; there was no effect of insulin in denervated muscle until the concentration exceeded 1 U/l. Denervation inhibited insulin-stimulated -aminoisobutyrate uptake by 77% but did not affect ¹²⁵I-insulin binding or glucose-independent activation of glycogen synthase by insulin. There was no effect of denervation on the insulinomimetic effects of concanavalin A, hydrogen peroxide, vitamin K₅, anoxia, 24-dinitrophenol, cooling, hyperosmolarity or EDTA, but the effect of diamide was inhibited. It is concluded [1] that denervation inhibits insulin-stimulated sugar transport at some early post-receptor step, and [2] that the mechanism whereby insulin activates glycogen synthase is different from the activation of the membrane transport of sugars and amino acids.