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Synthase Activity (synthase + activity)
Kinds of Synthase Activity Selected AbstractsPhysiological role of phosphatidylcholine in the Pseudomonas putida A ATCC 12633 response to tetradecyltrimethylammonium bromide and aluminiumLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2009P.S. Boeris Abstract Aims:, To evaluate the effect of tetradecyltrimethylammonium bromide (TTAB) and aluminium stresses on the phospholipid (PL) composition of Pseudomonas putida A ATCC 12633. Methods and Results:,Pseudomonas putida were grown with TTAB in the presence or absence of AlCl3, and the PL composition was analysed. The presence of TTAB resulted in an increase in phosphatidylglycerol and phosphatidic acid levels (6- and 20-fold, respectively) with respect to the levels in cells grown without the surfactant. With AlCl3, phosphatidylcholine (PC) increased (threefold) and cell-free extracts contained approximately threefold more phosphatidylcholine synthase activities than extracts without AlCl3, indicating that the PC level is dependent upon activation of this enzyme. Conclusions:, The negative charges of the headgroups of PL are the primary membrane-associated factors for the response to TTAB. PC are involved in cellular responses to binding Al3+ and should be viewed as a temporary reservoir of available Al3+ to allow a more efficient utilization of TTAB by Ps. putida. Significance and Impact of the Study:, The changes in the PL of Ps. putida in the presence of TTAB and AlCl3 indicate that different responses are utilized by bacteria to maintain optimal PL composition in the presence of such environmental pollutants. [source] MOUSE STRAIN-SPECIFIC DIFFERENCES IN CARDIAC METABOLIC ENZYME ACTIVITIES OBSERVED IN A MODEL OF ISOPROTERENOL-INDUCED CARDIAC HYPERTROPHYCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 1-2 2007Michael D Faulx SUMMARY 1Alterations in myocardial energy metabolism accompany pressure overload-induced hypertrophy. We previously described a novel model of catecholamine-induced hypertrophy in which A/J mice exhibit more robust cardiac hypertrophy than B6 mice. Accordingly, we assessed the influence of mouse strain on the activities of key myocardial metabolic enzymes and whether there are strain-related metabolic adaptations to short-term, high-dose isoproterenol (ISO) administration. 2Thirty-nine male mice (19 A/J mice, 20 B6 mice), aged 12,15 weeks, were randomly assigned to receive either ISO (100 mg/kg, s.c.) or vehicle (sterile water) daily for 5 days. On Day 6, all hearts were excised, weighed, freeze clamped and assayed for pyruvate dehydrogenase (PDH), medium chain acyl-CoA dehydrogenase, carnitine palmitoyl transferase I and citrate synthase activities. Plasma fatty acids (FA) were also measured. 3The ISO-treated A/J mice demonstrated greater percentage increases in gravimetric heart weight/bodyweight ratio than ISO-treated B6 mice (24 vs 3%, respectively; P < 0.001). All enzyme activities were significantly greater in vehicle-treated B6 mice than in A/J mice, illustrating a greater capacity for aerobic metabolism in B6 mice. Administration of ISO reduced PDHa (active form) activity in B6 mice by 47% (P < 0.001), with no significant change seen in A/J mice. Free FA levels were not significantly different between groups; thus, the differences in PDHa were not due to changes in FA. 4The basal activity of myocardial metabolic enzymes is greater in B6 mice than in A/J mice and ISO alters myocardial PDH activity in a mouse strain-dependent manner. Compared with A/J mice, B6 mice demonstrate less ISO-induced cardiac hypertrophy, but greater activity of key enzymes regulating FA and carbohydrate oxidation, which may protect against the development of hypertrophy. The metabolic adaptations associated with ISO-induced hypertrophy differ from those reported with pressure overload hypertrophy. [source] Androgen replacement therapy improves function in male rat muscles independently of hypertrophy and activation of the Akt/mTOR pathwayACTA PHYSIOLOGICA, Issue 4 2009C. Hourdé Abstract Aim:, We analysed the effect of physiological doses of androgens following orchidectomy on skeletal muscle and bone of male rats, as well as the relationships between muscle performance, hypertrophy and the Akt/mammalian target of rapamycin (mTOR) signalling pathway involved in the control of anabolic and catabolic muscle metabolism. Methods:, We studied the soleus muscle and tibia from intact rats (SHAM), orchidectomized rats treated for 3 months with vehicle (ORX), nandrolone decanoate (NAN) or dihydrotestosterone (DHT). Results:, Orchidectomy had very little effect on the soleus muscle. However, maximal force production by soleus muscle (+69%) and fatigue resistance (+35%) in NAN rats were both increased when compared with ORX rats. In contrast, DHT treatment did not improve muscle function. The relative number of muscle fibres expressing slow myosin heavy chain and citrate synthase activity were not different in NAN and ORX rats. Moreover, NAN and DHT treatments did not modify muscle weights and cross-sectional area of muscle fibres. Furthermore, phosphorylation levels of downstream targets of the Akt/mTOR signalling pathway, Akt, ribosomal protein S6 and eukaryotic initiation factor 4E-binding protein 1 were similar in muscles of NAN, DHT and ORX rats. In addition, trabecular tibia from NAN and DHT rats displayed higher bone mineral density and bone volume when compared with ORX rats. Only in NAN rats was this associated with increased bone resistance to fracture. Conclusion:, Physiological doses of androgens are beneficial to muscle performance in orchidectomized rats without relationship to muscle and fibre hypertrophy and activation of the Akt/mTOR signalling pathway. Taken together our data clearly indicate that the activity of androgens on muscle and bone could participate in the global improvement of musculoskeletal status in the context of androgen deprivation induced by ageing. [source] Splice-isoform specific immunolocalization of neuronal nitric oxide synthase in mouse and rat brain reveals that the PDZ-complex-building nNOS, ,-finger is largely exposed to antibodiesDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2007Kristina Langnaese Abstract Knock out mice deficient for the splice-isoform ,, of neuronal nitric oxide synthase (nNOS,,) display residual nitric oxide synthase activity and immunosignal. To attribute this signal to the two minor neuronal nitric oxide synthase splice variants, ,, and ,,, we generated isoform-specific anti-peptide antibodies against the nNOS,, specific ,,-finger motif involved in PDZ domain scaffolding and the nNOS,, specific N-terminus. The nNOS,, ,,-finger-specific antibody clearly recognized the 160-kDa band of recombinant nNOS,, on Western blots. Using immunocytochemistry, this antibody displayed, in rats and wild-type mice, a labeling pattern similar to but not identical with that obtained using a commercial pan-nNOS antibody. This similarity indicates that the majority of immunocytochemically detectable nNOS is not likely to be complexed with PDZ-domain proteins via the ,,-finger motif. This conclusion was confirmed by the inhibition of PSD-95/nNOS interaction by the nNOS,, ,,-finger antibody in pull-down assays. By contrast, nNOS,, ,,-finger labeling was clearly reduced in hippocampal and cortical neuropil areas enriched in NMDA receptor complex containing spine synapses. In nNOS,, knock out mice, nNOS,, was not detectable, whereas the pan-nNOS antibody showed a distinct labeling of cell bodies throughout the brain, most likely reflecting ,,/,,-isoforms in these cells. The nNOS,, antibody clearly detected bacterial expressed nNOS,, fusion protein and nNOS,, in overexpressing HEK cells by Western blotting. Immunocytochemically, individual cell bodies in striatum, cerebral cortex, and in some brain stem nuclei were labeled in knock out but not in wild-type mice, indicating an upregulation of nNOS,, in nNOS,, deficient animals. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007 [source] Effects of dietary fatty acids on insulin sensitivity and secretionDIABETES OBESITY & METABOLISM, Issue 6 2004Melania Manco Globalization and global market have contributed to increased consumption of high-fat, energy-dense diets, particularly rich in saturated fatty acids( SFAs). Polyunsaturated fatty acids (PUFAs) regulate fuel partitioning within the cells by inducing their own oxidation through the reduction of lipogenic gene expression and the enhancement of the expression of those genes controlling lipid oxidation and thermogenesis. Moreover, PUFAs prevent insulin resistance by increasing membrane fluidity and GLUT4 transport. In contrast, SFAs are stored in non-adipocyte cells as triglycerides (TG) leading to cellular damage as a sequence of their lipotoxicity. Triglyceride accumulation in skeletal muscle cells (IMTG) derives from increased FA uptake coupled with deficient FA oxidation. High levels of circulating FAs enhance the expression of FA translocase the FA transport proteins within the myocites. The biochemical mechanisms responsible for lower fatty acid oxidation involve reduced carnitine palmitoyl transferase (CPT) activity, as a likely consequence of increased intracellular concentrations of malonyl-CoA; reduced glycogen synthase activity; and impairment of insulin signalling and glucose transport. The depletion of IMTG depots is strictly associated with an improvement of insulin sensitivity, via a reduced acetyl-CoA carboxylase (ACC) mRNA expression and an increased GLUT4 expression and pyruvate dehydrogenase (PDH) activity. In pancreatic islets, TG accumulation causes impairment of insulin secretion. In rat models, ,-cell dysfunction is related to increased triacylglycerol content in islets, increased production of nitric oxide, ceramide synthesis and ,-cell apoptosis. The decreased insulin gene promoter activity and binding of the pancreas-duodenum homeobox-1 (PDX-1) transcription factor to the insulin gene seem to mediate TG effect in islets. In humans, acute and prolonged effects of FAs on glucose-stimulated insulin secretion have been widely investigated as well as the effect of high-fat diets on insulin sensitivity and secretion and on the development of type 2 diabetes. [source] Effects of short-term training on insulin sensitivity and skeletal muscle glucose metabolism in Standardbred horsesEQUINE VETERINARY JOURNAL, Issue S36 2006L. STEWART-HUNT Summary Reasons for performing study: Increased insulin sensitivity occurs after a period of exercise training, but the mechanisms underlying this training-associated increase in insulin action have not been investigated. Objective: To examine the effects of short-term endurance training (7 consecutive days) and a subsequent period of inactivity (5 days) on whole body insulin sensitivity and GLUT-4 protein and the activities of glycogen synthase (GS) and hexokinase (HK) in skeletal muscle. It was hypothesised that training would increase insulin sensitivity in association with increased GLUT-4 protein and activities of GS and HK, but that these changes would be transient, returning to baseline after 5 days of inactivity. Methods: Seven mature Standardbred horses completed training consisting of 7 consecutive days of 45 min of treadmill exercise at a speed that elicited 55% of pretraining maximal aerobic capacity (VO2peak). Insulin sensitivity was determined by rate of glucose disposal (M) during the last 60 min of a 120 min euglycaemic-hyperinsulinaemic clamp (EHC) performed before (-2 days) and at 1 and 6 days following training. VO2peak was measured before (UT) and after (TR) training and the period of inactivity (IA). Results: Training resulted in a 9% increase in mean VO2peak (P<0.05) that was maintained following inactivity (IA). Mean M values were more than 2-fold higher (P<0.05) in TR than in UT. Mean M was also higher (P<0.05) in IA when compared to UT. GLUT-4 protien abundancewas more than 10-fold higher in TR and IA (P<0.001) than in UT. Pre-EHC GS activity and GS fractional velocity were increased (P<0.05) in TR when compared to UT and IA. Pre-EHC HK activity was increased (P<0.05) in IA when compared to UT and TR. Muscle glycogen was 66% lower (P<0.05) in TR than in UT and IA. Conclusions: Short-term training resulted in increases in whole body insulin sensitivity, and GLUT-4 protein content and glycogen synthase activity in skeletal muscle. The enhancements in insulin sensitivity, GLUT-4 protein and glycogen synthase activity were still evident after 5 days of inactivity. Potential relevance: Insulin resistance in equids has been associated with obesity and predisposition to laminitis. Regular physical activity may mitigate risk of these conditions via enhancement of insulin sensitivity and/or control of bodyweight. [source] Increased expression of VEGF following exercise training in patients with heart failureEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 4 2001T. Gustafsson Background and aims During the last decades several angiogenic factors have been characterized but so far it is unknown whether local muscle exercise training increases the expression of these factors in patients with moderate heart failure. Expression of the major putative angiogenic factor vascular endothelial growth factor (VEGF) at the level of messneger RNA (mRNA) and/or protein was therefore studied before and after 8 weeks of training in patient with chronic heart failure. Methods VEGF mRNA and protein concentrations were determined in skeletal muscle biopsies before and after 8 weeks of one-legged knee extension training in patients with chronic heart failure (New York Heart Association II,III). Results Exercise training increased the citrate synthase activity and peripheral exercise capacity by 46% and 36%, respectively, in parallel with a two-fold increase in VEGF at both the mRNA (P = 0·03) and protein (P = 0·02) levels Conclusion The increase in VEGF gene expression in response to exercise training indicates VEGF to be one possible mediator in exercise-induced angiogenesis and may therefore regulate an important and early step in adaptation to increased muscle activity in patient with chronic heart failure. [source] Heat Shock Protein Expression is Increased in Cardiac and Skeletal Muscles of Fischer 344 Rats After Endurance TrainingEXPERIMENTAL PHYSIOLOGY, Issue 1 2000T. R. Samelman Heat shock proteins (HSPs) are expressed when cells are exposed to various types of stress and they may provide protection against cellular insult. Previous data have shown increases in HSP expression following acute exhaustive exercise in rats (Locke et al. 1990, 1995; Salo et al. 1991) and humans (Liu et al. 1999); however, it is not known if chronic exercise will increase resting levels of HSPs. The purpose of this study was to determine if basal protein levels of HSP 72/73 and HSP 60 are increased in cardiac and skeletal muscle of endurance trained Fischer 344 rats. Heart, soleus (SOL) and lateral gastrocnemius (LG) muscles were removed and hearts were sectioned into left ventricle (LV), right ventricle (RV) and atria (AT). Endurance training improved myocardial citrate synthase activity by 88, 90 and 77% and cytochrome c oxidase activity by 58, 51 and 89% in LV, RV and AT, respectively. LV and RV oxidative enzyme activities were greater when compared to AT for both trained and untrained rats (P < 0.05). HSP 72/73 expression was significantly greater (P < 0.05) in LV, RV and SOL from endurance trained versus from control rats (26, 45 and 67%, respectively). HSP 60 was also increased (P < 0.05) in LV, RV and SOL in trained relative to untrained rats. HSP 72/73 and HSP 60 were unchanged in AT and LG after training. These results indicate that endurance training increases the basal expression of stress proteins and this observation is consistent with the hypothesis that endurance training may activate a protective mechanism to stress. [source] The N -acetylglutamate synthase/N -acetylglutamate kinase metabolon of Saccharomyces cerevisiae allows co-ordinated feedback regulation of the first two steps in arginine biosynthesisFEBS JOURNAL, Issue 5 2003Katia Pauwels In Saccharomyces cerevisiae, which uses the nonlinear pathway of arginine biosynthesis, the first two enzymes, N -acetylglutamate synthase (NAGS) and N -acetylglutamate kinase (NAGK), are controlled by feedback inhibition. We have previously shown that NAGS and NAGK associate in a complex, essential to synthase activity and protein level [Abadjieva, A., Pauwels, K., Hilven, P. & Crabeel, M. (2001) J. Biol. Chem.276, 42869,42880]. The NAGKs of ascomycetes possess, in addition to the catalytic domain that is shared by all other NAGKs and whose structure has been determined, a C-terminal domain of unknown function and structure. Exploring the role of these two domains in the synthase/kinase interaction, we demonstrate that the ascomycete-specific domain is required to maintain synthase activity and protein level. Previous results had suggested a participation of the third enzyme of the pathway, N -acetylglutamylphosphate reductase, in the metabolon. Here, genetic analyses conducted in yeast at physiological level, or in a heterologous background, clearly demonstrate that the reductase is dispensable for synthase activity and protein level. Most importantly, we show that the arginine feedback regulation of the NAGS and NAGK enzymes is mutually interdependent. First, the kinase becomes less sensitive to arginine feedback inhibition in the absence of the synthase. Second, and as in Neurospora crassa, in a yeast kinase mutant resistant to arginine feedback inhibition, the synthase becomes feedback resistant concomitantly. We conclude that the NAGS/NAGK metabolon promotes the co-ordination of the catalytic activities and feedback regulation of the first two, flux controlling, enzymes of the arginine pathway. [source] Analyses of the CYP11B gene family in the guinea pig suggest the existence of a primordial CYP11B gene with aldosterone synthase activityFEBS JOURNAL, Issue 15 2002Hannes E. Bülow In this study we describe the isolation of three genes of the CYP11B family of the guinea pig. CYP11B1 codes for the previously described 11,-hydroxylase [Bülow, H.E.,Möbius, K., Bähr, V. & Bernhardt, R. (1996) Biochem. Biophys. Res. Commun. 221, 304,312] while CYP11B2 represents the aldosterone synthase gene. As no expression for CYP11B3 was detected this gene might represent a pseudogene. Transient transfection assays show higher substrate specificity for its proper substrate for CYP11B1 as compared to CYP11B2, which could account for the zone-specific synthesis of mineralocorticoids and glucocorticoids, respectively. Thus, CYP11B2 displayed a fourfold higher ability to perform 11,-hydroxylation of androstenedione than CYP11B1, while this difference is diminished with the size of the C17 substituent of the substrate. Furthermore, analyses with the electron transfer protein adrenodoxin indicate differential sensitivity of CYP11B1 and CYP11B2 as well as the three hydroxylation steps catalysed by CYP11B2 to the availability of reducing equivalents. Together, both mechanisms point to novel protein intrinsic modalities to achieve tissue-specific production of mineralocorticoids and glucocorticoids in the guinea pig. In addition, we conducted phylogenetic analyses. These experiments suggest that a common CYP11B ancestor gene that possessed both 11,-hydroxylase and aldosterone synthase activity underwent a gene duplication event before or shortly after the mammalian radiation with subsequent independent evolution of the system in different lines. Thus, a differential mineralocorticoid and glucocorticoid synthesis might be an exclusive achievement of mammals. [source] The effect of modulation of , -glutamyl transpeptidase and nitric oxide synthase activity on GSH homeostasis in HepG2 cellsFUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 1 2007Inga Kwiecie Abstract High glutathione (GSH) level and elevated , -glutamyl transpeptidase (,GT) activity are hallmarks of tumor cells. Toxicity of drugs and radiation to the cells is largely dependent on the level of thiols. In the present studies, we attempted to inhibit ,GT activity in human hepatoblastoma (HepG2) cells to examine whether the administration of ,GT inhibitors, acivicin (AC) and 1,2,3,4-tetrahydroisoquinoline (TIQ) influences cell proliferation and enhances cytostatic action of doxorubicin (DOX) and cisplatin (CP) on HepG2 cells. The effects of these inhibitors were determined by 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), BrdU and lactate dehydrogenase (LDH) tests and by estimation of GSH level. Additionally, we investigated the changes in caspase-3 activity, which is a marker of apoptosis. The obtained results showed that the ,GT inhibitors introduced to the medium alone elicited cytotoxic effect, which was accompanied by an increase in GSH level in the cells. TIQ concomitantly increased caspase-3 activity. Doxorubicin and CP proved to be cytotoxic, and both inhibitors augmented this effect. As well DOX as CP radically decreased GSH levels, whereas ,GT inhibitors had diverse effects. Therefore, the obtained results confirm that ,GT inhibitors can enhance pharmacological action of DOX and CP, which may permit clinicians to decrease their doses thereby alleviating side effects. Aminoguanidine (nitric oxide synthase inhibitor) given alone was little cytotoxic to HepG2 cells, while its introduction to the medium together with DOX and CP significantly increased their cytotoxicity. Aminoguanidine on its own did not show any effect on GSH level in HepG2 cells, but markedly and significantly elevated its concentration when added in combination with CP but not with DOX. This indicates that when CP was used as a cytostatic, GSH level rose after treatment with its combination with both AC and aminoguanidine. [source] Increasing dimethylarginine levels are associated with adverse clinical outcome in severe alcoholic hepatitis,HEPATOLOGY, Issue 1 2007Rajeshwar P. Mookerjee Previous studies suggest reduced hepatic endothelial nitric oxide synthase activity contributes to increased intrahepatic resistance. Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, undergoes hepatic metabolism via dimethylarginine-dimethylamino-hydrolase, and is derived by the action of protein-arginine-methyltransferases. Our study assessed whether ADMA, and its stereo-isomer symmetric dimethylarginine (SDMA), are increased in alcoholic hepatitis patients, and determined any relationship with severity of portal hypertension (hepatic venous pressure gradient measurement) and outcome. Fifty-two patients with decompensated alcoholic cirrhosis were studied, 27 with acute alcoholic hepatitis and cirrhosis, in whom hepatic venous pressure gradient was higher (P = 0.001) than cirrhosis alone, and correlated with ADMA measurement. Plasma ADMA and SDMA were significantly higher in alcoholic hepatitis patients and in nonsurvivors. Dimethylarginine-dimethylamino-hydrolase protein expression was reduced and protein-arginine-methyltransferase-1 increased in alcoholic hepatitis livers. ADMA, SDMA and their combined sum, which we termed a dimethylarginine score, were better predictors of outcome compared with Pugh score, MELD and Maddrey's discriminant-function. Conclusion: Alcoholic hepatitis patients have higher portal pressures associated with increased ADMA, which may result from both decreased breakdown (decreased hepatic dimethylarginine-dimethylamino-hydrolase) and/or increased production. Elevated dimethylarginines may serve as important biological markers of deleterious outcome in alcoholic hepatitis. (HEPATOLOGY 2007;45:62,71.) [source] Regional specialization of rat quadriceps myosin heavy chain isoforms occurring in distal to proximal parts of middle and deep regions is not mirrored by citrate synthase activityJOURNAL OF ANATOMY, Issue 1 2007Tertius Abraham Kohn Abstract Myosin heavy chain (MHC) isoform content and citrate synthase (CS) activities were measured in the Quadriceps femoris (QF) muscle of 18 female rats. The muscle group was divided into superficial, middle and deep, distal, central and proximal parts. MHC IIb and IIx were more abundant in superficial regions (P < 0.05) with low CS activities compared with deeper parts. The deeper parts expressed all four isoforms (MHC IIb, MHC IIx, MHC IIa and MHC I), with a concomitantly higher CS activity. MHC I, MHC IIa and MHC IIb isoform content varied significantly along the length of the deep regions. Only MHC IIb and CS activity in the proximal middle part correlated (negatively) with each other. This study showed that the QF has regional specialization and that standardization of sampling site is important. Furthermore, CS activity and MHC isoforms are only loosely associated, or not at all. [source] Modulation of the cGMP signaling pathway by melatonin in pancreatic , -cellsJOURNAL OF PINEAL RESEARCH, Issue 2 2009Ina Stumpf Abstract:, Melatonin influences the second messenger cyclic guanosine 3,,5,-monophosphate (cGMP) signaling pathway in pancreatic , -cells via a receptor-mediated mechanism. In the present study, it was determined how the regulation of cGMP concentrations by melatonin proceeds. The results provide evidence that melatonin acts via the soluble guanylate cyclase (sGC), as molecular investigations demonstrated that long-term incubation with melatonin significantly reduced the expression levels of the sGC mRNA in rat insulinoma , -cells (INS1) cells, whereas mRNA expression of membrane guanylate cyclases was unaffected. Incubation with melatonin abolished the S-nitrosoacetyl penicillamine-induced increase of cGMP concentrations in INS1 cells. In addition, the cGMP-inhibitory effect of melatonin was reversed by preincubation with the sGC inhibitors 1H-(1,2,4)oxadiazolo(4,3- ,)quinoxalin-1-one and 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one. Nitric oxide (NO) production was not influenced after 1 hr of melatonin application, but was influenced after a 4 hr incubation period. Preincubation of INS1 cells with the NO synthase inhibitor NG -monomethyl- l -arginine did not abolish the cGMP-inhibitory effect of melatonin. Transcripts of cyclic nucleotide-gated (CNG) channels were significantly reduced after melatonin treatment in a dose-dependent manner, indicating the involvement of these channels in mediating the melatonin effect in INS1 cells. The results of this study demonstrate that melatonin mediates its inhibitory effect on cGMP concentrations in pancreatic , -cells by inhibiting the sGC, but does not influence NO concentration or NO synthase activity in short-term incubation experiments. In addition, it was demonstrated that melatonin is involved in modulation of CNG channel mRNA. [source] Effects of some synthetic kynurenines on brain amino acids and nitric oxide after pentylenetetrazole administration to ratsJOURNAL OF PINEAL RESEARCH, Issue 4 2004Leila Bikjdaouene Abstract:, We have previously proven that some synthetic kynurenines behave as antagonists of the N-methyl- d -aspartate receptor inhibiting neuronal subtype of nitric oxide synthase activity. We now investigate the anticonvulsant activity of four of these kynurenines in pentylenetetrazole (PTZ)-treated rats. The rats were treated with each kynurenine (10,160 mg/kg, s.c.) 30 min before PTZ administration (100 mg/kg, s.c.). Then, latency, duration and intensity of the first seizure and the percent animal survival were noted. PTZ-induced death was counteracted by high doses of kynurenines. Latency of the first seizure was significantly increased and its intensity reduced at the same doses, whereas the duration of the first seizure significantly decreased with doses of 20 mg/kg in most of the kynurenines tested. Three hours after PTZ administration, the surviving animals were sacrificed and the levels of brain amino acids and nitrite were measured. PTZ administration increased glutamate, glutamine, serine and taurine levels in different brain areas. High doses of kynurenines generally counteracted the effects of PTZ on excitatory amino acids, but they also reduced inhibitory aminoacids. However, the most consistent effect of kynurenines was the dose-dependent reduction of brain nitrite levels induced by PTZ. These results reveal a new family of anticonvulsant drugs that affect mainly to nitric oxide production in the brain. [source] Ethanol Consumption Increases Nitric Oxide Production in Rats, and Its Peroxynitrite-Mediated Toxicity Is Attenuated by PolyenylphosphatidylcholineALCOHOLISM, Issue 6 2002Enrique Baraona Background: Nitric oxide generally mediates beneficial responses but becomes deleterious when coexistence with enhanced superoxide formation leads to the synthesis of peroxynitrite, a potent oxidant and nitrating agent. Methods: To study the effects of ethanol and polyenylphosphatidylcholine on nitric oxide metabolism and toxicity, 36 rats were pair-fed liquid diets with 36% of energy either as ethanol or as additional carbohydrate for 24 days and were killed 90 min after intragastric feeding. Half received polyenylphosphatidylcholine in the diet (3 g/liter), and the other half equivalent amounts of essential fatty acids and choline. Nitric oxide was measured by chemiluminescence in arterial blood and liver cytosol and as a product of the inducible nitric oxide synthase activity. Peroxynitrite formation was assessed by the increase in nitrotyrosine protein residues, measured immunochemically. Results: In blood, administration of ethanol with or without polyenylphosphatidylcholine doubled nitric oxide levels. In the liver, ethanol increased nitric oxide by 52% (p < 0.01), and polyenylphosphatidylcholine attenuated this effect. Ethanol consumption increased the cytosolic activity of the inducible nitric oxide synthase and induced microsomal cytochromes P-450 capable of producing both nitric oxide and superoxide. This was associated with an 18% (p < 0.01) increase in nitrotyrosine protein residues, products of peroxynitrite toxicity, which occurred predominantly in steatotic hepatocytes. Polyenylphosphatidylcholine attenuated these changes by decreasing the ethanol effect on both the cytosolic and the microsomal activities, in addition to acting as a powerful antioxidant. Acute administration of the same ethanol dose increased nitric oxide levels, but did not affect nitrotyrosine protein residues. Conclusions: Chronic, but not acute, ethanol administration increases peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide, both of which are prevented by polyenylphosphatidylcholine. [source] Biosynthesis and Compositional Regulation of Poly[(3-hydroxybutyrate)- co -(3-hydroxyhexanoate)] in Recombinant Ralstonia eutropha Expressing Mutated Polyhydroxyalkanoate Synthase GenesMACROMOLECULAR BIOSCIENCE, Issue 3 2004Takeharu Tsuge Abstract Summary: A new strategy for bacterial polyhydroxyalkanoate (PHA) production by recombinant Ralstonia eutropha PHB,4 harboring mutated PHA synthase genes (phaCAc) from Aeromona caviae was investigated. The strain harboring wild-type phaCAc gene produced a PHA copolymer consisting of (R)-3-hydroxybutyrate and (R)-3-hydroxyhexanoate [P(3HB- co -3HHx)] with 3.5 mol-% of 3HHx fraction from soybean oil. When the mutants of phaCAc gene were applied to this production system, 3HHx fraction in copolymers was varied in the range of 0,5.1 mol-%. Thus, the regulation of PHA copolymer compositions has been achieved by the use of mutated PHA synthase genes. Relationship between PHA synthase activity and 3HHx fraction in P(3HB- co -3HHx). [source] Rga5p is a specific Rho1p GTPase-activating protein that regulates cell integrity in Schizosaccharomyces pombeMOLECULAR MICROBIOLOGY, Issue 2 2003Teresa M. Calonge Summary Schizosaccharomyces pombe Rho1p regulates (1,3),- d -glucan synthesis and is required for cell integrity maintenance and actin cytoskeleton organization, but nothing is known about the regulation of this protein. At least nine different S. pombe genes code for proteins predicted to act as Rho GTPase-activating proteins (GAPs). The results shown in this paper demonstrate that the protein encoded by the gene named rga5+ is a GAP specific for Rho1p. rga5+ overexpression is lethal and causes morphological alterations similar to those reported for Rho1p inactivation. rga5+ deletion is not lethal and causes a mild general increase in cell wall biosynthesis and morphological alterations when cells are grown at 37°C. Upon mild overexpression, Rga5p localizes to growth areas and possesses both in vivo and in vitro GAP activity specific for Rho1p. Overexpression of rho1+ in rga5, cells is lethal, with a morphological phenotype resembling that of the overexpression of the constitutively active allele rho1G15V. In addition (1,3),- d -glucan synthase activity, regulated by Rho1p, is increased in rga5, cells and decreased in rga5 -overexpressing cells. Moreover, the increase in (1,3),- d -glucan synthase activity caused by rho1+ overexpression is considerably higher in rga5, than in wild-type cells. Genetic interactions suggest that Rga5p is also important for the regulation of the other known Rho1p effectors, Pck1p and Pck2p. [source] On the mechanism of selectivity of the corn herbicide BAS 662H: a combination of the novel auxin transport inhibitor diflufenzopyr and the auxin herbicide dicambaPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 10 2002Klaus Grossmann Abstract BAS 662H, a 1:2.5 combination of the semicarbazone-type auxin transport inhibitor diflufenzopyr and the auxin herbicide dicamba, is used as a post-emergence herbicide in corn. The combination has been observed to provide more effective broadleaf weed control and improved tolerance in corn than typical rates of dicamba used alone. In order to analyze this phenomenon, the uptake, translocation, metabolism and action of both compounds, applied alone and in combination, were investigated in Amaranthus retroflexus L, Galium aparine L and corn (Zea mays L). When plants at the third-leaf stage were foliarly treated with diflufenzopyr and dicamba equivalent to field rates of 100 and 250,gha,1, respectively, diflufenzopyr synergistically increased dicamba-induced 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activity and ethylene formation in G aparine and even more in A retroflexus, followed by accumulations of (+)-abscisic acid (ABA) in the shoot tissue within 20,h. This correlated with subsequent growth inhibition, hydrogen peroxide overproduction and progressive tissue damage. Diflufenzopyr also enhanced the activity of other auxin herbicides, such as quinclorac and picloram, and of the synthetic auxin, 1-naphthaleneacetic acid. After foliar and root application of [14C]diflufenzopyr, alone or as BAS 662H, considerably lower tissue concentrations and systemic translocation of radioactivity beyond treated plant parts were found in corn, compared to G aparine and particularly A retroflexus. Furthermore, diflufenzopyr decreased foliar uptake of [14C]dicamba by c,50% selectively in corn, compared to the treatment alone. Metabolism of [14C]diflufenzopyr was more rapid in corn than in the weed species. In combination, the two compounds had no mutual effect on their metabolic degradation. In BAS 662H, diflufenzopyr synergizes the herbicidal activity of dicamba in sensitive weed species. In corn this effect is prevented by a more rapid metabolism of diflufenzopyr, coupled with lower uptake and translocation. Selectivity of BAS 662H is additionally favoured by a higher crop tolerance to dicamba because of reduced foliar uptake of this herbicide in corn under the influence of diflufenzopyr. © 2002 Society of Chemical Industry [source] Naphthoquinones and bioactive compounds from tobacco as modulators of neuronal nitric oxide synthase activityPHYTOTHERAPY RESEARCH, Issue 12 2009Priya Venkatakrishnan Abstract Studies were conducted with extracts of several varieties of tobacco in search of neuronal nitric oxide synthase (nNOS) inhibitors which may be of value in the treatment of stroke. Current therapies do not directly exploit modulation of nNOS activity due to poor selectivity of the currently available nNOS inhibitors. The properties of a potentially novel nNOS inhibitor(s) derived from tobacco extracts, and the concentration-dependent, modulatory effects of the tobacco-derived naphthoquinone compound, 2,3,6-trimethyl-1,4-naphthoquinone (TMN), on nNOS activity were investigated, using 2-methyl-1,4-naphthoquinone (menadione) as a control. Up to 31 µM, both TMN and menadione stimulated nNOS-catalysed l -citrulline production. However, at higher concentrations of TMN (62.5,500 µM), the stimulation was lost in a concentration-dependent manner. With TMN, the loss of stimulation did not decrease beyond the control activity. With menadione (62.5,500 µM), the loss of stimulation surpassed that of the control (78 ± 0.01% of control activity), indicating a true inhibition of nNOS activity. This study suggests that potential nNOS inhibitors are present in tobacco, most of which remain to be identified. Copyright © 2009 John Wiley & Sons, Ltd. [source] Impact of rising CO2 on emissions of volatile organic compounds: isoprene emission from Phragmites australis growing at elevated CO2 in a natural carbon dioxide spring,PLANT CELL & ENVIRONMENT, Issue 4 2004P. A. SCHOLEFIELD ABSTRACT Isoprene basal emission (the emission of isoprene from leaves exposed to a light intensity of 1000 µmol m,2 s,1 and maintained at a temperature of 30 °C) was measured in Phragmites australis plants growing under elevated CO2 in the Bossoleto CO2 spring at Rapolano Terme, Italy, and under ambient CO2 at a nearby control site. Gas exchange and biochemical measurements were concurrently taken. Isoprene emission was lower in the plants growing at elevated CO2 than in those growing at ambient CO2. Isoprene emission and isoprene synthase activity (IsoS) were very low in plants growing at the bottom of the spring under very rich CO2 and increased at increasing distance from the spring (and decreasing CO2 concentration). Distance from the spring did not significantly affect photosynthesis making it therefore unlikely that there is carbon limitation to isoprene formation. The isoprene emission rate was very quickly reduced after rapid switches from elevated to ambient CO2 in the gas-exchange cuvette, whereas it increased when switching from ambient to elevated CO2. The rapidity of the response may be consistent with post-translational modifications of enzymes in the biosynthetic pathway of isoprene formation. Reduction of IsoS activity is interpreted as a long-term response. Basal emission of isoprene was not constant over the day but showed a diurnal course opposite to photosynthesis, with a peak during the hottest hours of the day, independent of stomatal conductance and probably dependent on external air temperature or temporary reduction of CO2 concentration. The present experiments show that basal emission rate of isoprene is likely to be reduced under future elevated CO2 levels and allow improvement in the modelling of future isoprene emission rates. [source] Responses of CAM species to increasing atmospheric CO2 concentrationsPLANT CELL & ENVIRONMENT, Issue 8 2000P. M. Drennan ABSTRACT Crassulacean acid metabolism (CAM) species show an average increase in biomass productivity of 35% in response to a doubled atmospheric CO2 concentration. Daily net CO2 uptake is similarly enhanced, reflecting in part an increase in chlorenchyma thickness and accompanied by an even greater increase in water-use efficiency. The responses of net CO2 uptake in CAM species to increasing atmospheric CO2 concentrations are similar to those for C3 species and much greater than those for C4 species. Increases in net daily CO2 uptake by CAM plants under elevated atmospheric CO2 concentrations reflect increases in both Rubisco-mediated daytime CO2 uptake and phosphoenolpyruvate carboxylase (PEPCase)-mediated night-time CO2 uptake, the latter resulting in increased nocturnal malate accumulation. Chlorophyll contents and the activities of Rubisco and PEPCase decrease under elevated atmospheric CO2, but the activated percentage for Rubisco increases and the KM(HCO3,) for PEPCase decreases, resulting in more efficient photosynthesis. Increases in root:shoot ratios and the formation of additional photosynthetic organs, together with increases in sucrose-Pi synthase and starch synthase activity in these organs under elevated atmospheric CO2 concentrations, decrease the potential feedback inhibition of photosynthesis. Longer-term studies for several CAM species show no downward acclimatization of photosynthesis in response to elevated atmospheric CO2 concentrations. With increasing temperature and drought duration, the percentage enhancement of daily net CO2 uptake caused by elevated atmospheric CO2 concentrations increases. Thus net CO2 uptake, productivity, and the potential area for cultivation of CAM species will be enhanced by the increasing atmospheric CO2 concentrations and the increasing temperatures associated with global climate change. [source] A proteomic analysis of 14-3-3 binding proteins from developing barley grainsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2006Ross D. Alexander Abstract 14-3-3 proteins are important eukaryotic regulatory proteins. Barley (Hordeum vulgare,L.) 14-3-3A was over-expressed, immobilised and used to affinity purify 14-3-3 binding proteins from developing barley grains. Binding was shown to be phosphorylation-dependent. These proteins were fractionated by PAGE and identified by MALDI-TOF MS. In total, 54,14-3-3 binding proteins were identified, 49,of these interactions are novel to plants. These proteins fell into a number of functional categories. The largest category was for carbohydrate metabolism, including plastidic enzymes for starch synthesis and modification. 14-3-3 was shown to be present in isolated plastids. Four of five enzymes involved in sucrose biosynthesis from triose phosphates were identified, suggesting co-ordinated regulation of this pathway. Invertase and sucrose synthase, which break down sucrose to hexoses, were found. Sucrose synthase activity was shown to be inhibited by exogenous 14-3-3 in a dosage-dependent manner. The second-largest functional group was for proteins involved in stress and defence responses; for example, RGH2A, closely related to the MLA powdery mildew resistance protein, was found. This work illustrates the broad range of processes in which 14-3-3 may be involved, and augments previous data demonstrating key roles in carbohydrate metabolism and plant defence. [source] Panax notoginseng saponins attenuate acute lung injury induced by intestinal ischaemia/reperfusion in ratsRESPIROLOGY, Issue 6 2009Ling RONG ABSTRACT Background and objective: Acute lung injury remains a challenge for both clinicians and scientists. The effects of Panax notoginseng saponins (PNS) on acute lung injury induced by intestinal ischaemia/reperfusion (II/R) were studied in rats. Methods: Forty-eight Wistar rats were randomly assigned to four groups: (1) a sham-operated group that received laparotomy without II/R (n= 12); (2) a sham + PNS group, which was identical to group 1 except for PNS treatment (n= 12); (3) an II/R group that had 1 h of intestinal ischaemia followed by 3 h of reperfusion (n= 12); and (4) an II/R + PNS group that received 100 mg/kg of PNS, i.v., 15 min before reperfusion (n= 12). The effects of PNS administration on lung tissue histology, activities of oxidant and antioxidant enzymes, levels of malondialdehyde, nitric oxide and inducible nitric oxide synthase activity were examined. Levels of surfactant protein B, cell numbers in BAL fluid and plasma levels of pro-inflammatory cytokines were also examined. Results: Compared with the II/R group, pulmonary parenchymal damage, activities of oxidant enzymes, levels of malondialdehyde and nitric oxide, inducible nitric oxide synthase activity in lung tissue, and plasma levels of pro-inflammatory cytokines were significantly reduced by PNS treatment. In addition, the decreases in antioxidant enzyme activities were prevented in the II/R + PNS group. Total leukocyte and neutrophil counts were significantly decreased by PNS treatment. The decline in surfactant protein B levels in BAL fluid was reduced in the II/R + PNS group compared with the II/R group. Conclusions: Administration of PNS before reperfusion injury alleviates acute lung injury induced by II/R, and this is attributable to the antioxidant and anti-inflammatory effects of PNS. [source] Phosphatidylinositol Synthase of Tetrahymena: Inositol Isomers as Substrates in Phosphatidylinositol Biosynthesis and Headgroup Exchange ReactionsTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2 2007BRIDGET M. RIGGS ABSTRACT. Phosphatidylinositol (PtdIns) synthase in microsomal fractions derived from Tetrahymena vorax was studied to determine its activity requirements. The suitability of inositol isomers as substrates for the synthase and in headgroup exchange reactions also was investigated. Tetrahymena PtdIn synthase activity was optimum in the presence of 2 mM MgCl2 plus 2 mM MnCl2, a pH of 7.8, and a temperature of 30°C. The enzyme retained approximately 80% of its activity after incubation at 70°C for 10 min. PtdIns headgroup exchange activity was maximal in the presence of cytidine monophosphate. By following either the accumulation of radiolabeled reaction products or the loss of radiolabel from precursors, each of the inositol isomers tested appeared to serve as substrates for both the PtdIns synthase and PtdIns:inositol phosphatidyl transferase activities. In each case, myo -inositol and scyllo -inositol were the preferred substrates. The data suggest two routes for the formation of phosphatidyl-non- myo -inositols in Tetrahymena and the potential for the production of novel, non- myo -inositol-containing second messengers. [source] Role of leptin in the regulation of growth and carbohydrate metabolism in the ovine fetus during late gestationTHE JOURNAL OF PHYSIOLOGY, Issue 9 2008Alison J. Forhead Leptin is an important regulator of appetite and energy expenditure in adulthood, although its role as a nutritional signal in the control of growth and metabolism before birth is poorly understood. This study investigated the effects of leptin on growth, carbohydrate metabolism and insulin signalling in fetal sheep. Crown,rump length-measuring devices and vascular catheters were implanted in 12 sheep fetuses at 105,110 days of gestation (term 145 ± 2 days). The fetuses were infused i.v. either with saline (0.9% NaCl; n= 6) or recombinant ovine leptin (0.5,1.0 mg kg,1 day,1; n= 6) for 5 days from 125 to 130 days when they were humanely killed and tissues collected. Leptin receptor mRNA and protein were expressed in fetal liver, skeletal muscle and perirenal adipose tissue. Throughout infusion, plasma leptin in the leptin-infused fetuses was 3- to 5-fold higher than in the saline-infused fetuses, although plasma concentrations of insulin, glucose, lactate, cortisol, catecholamines and thyroid hormones did not differ between the groups. Leptin infusion did not affect linear skeletal growth or body, placental and organ weights in utero. Hepatic glycogen content and activities of the gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase in the leptin-infused fetuses were lower than in the saline-infused fetuses by 44, 48 and 36%, respectively; however, there were no differences in hepatic glycogen synthase activity or insulin signalling protein levels. Therefore, before birth, leptin may inhibit endogenous glucose production by the fetal liver when adipose energy stores and transplacental nutrient delivery are sufficient for the metabolic needs of the fetus. These actions of leptin in utero may contribute to the development of neonatal hypoglycaemia in macrosomic babies of diabetic mothers. [source] Effect of exercise training on endothelium-derived nitric oxide function in humansTHE JOURNAL OF PHYSIOLOGY, Issue 1 2004Daniel J. Green Vascular endothelial function is essential for maintenance of health of the vessel wall and for vasomotor control in both conduit and resistance vessels. These functions are due to the production of numerous autacoids, of which nitric oxide (NO) has been the most widely studied. Exercise training has been shown, in many animal and human studies, to augment endothelial, NO-dependent vasodilatation in both large and small vessels. The extent of the improvement in humans depends upon the muscle mass subjected to training; with forearm exercise, changes are restricted to the forearm vessels while lower body training can induce generalized benefit. Increased NO bioactivity with exercise training has been readily and consistently demonstrated in subjects with cardiovascular disease and risk factors, in whom antecedent endothelial dysfunction exists. These conditions may all be associated with increased oxygen free radicals which impact on NO synthase activity and with which NO reacts; repeated exercise and shear stress stimulation of NO bioactivity redresses this radical imbalance, hence leading to greater potential for autacoid bioavailability. Recent human studies also indicate that exercise training may improve endothelial function by up-regulating eNOS protein expression and phosphorylation. While improvement in NO vasodilator function has been less frequently found in healthy subjects, a higher level of training may lead to improvement. Regarding time course, studies indicate that short-term training increases NO bioactivity, which acts to homeostatically regulate the shear stress associated with exercise. Whilst the increase in NO bioactivity dissipates within weeks of training cessation, studies also indicate that if exercise is maintained, the short-term functional adaptation is succeeded by NO-dependent structural changes, leading to arterial remodelling and structural normalization of shear. Given the strong prognostic links between vascular structure, function and cardiovascular events, the implications of these findings are obvious, yet many unanswered questions remain, not only concerning the mechanisms responsible for NO bioactivity, the nature of the cellular effect and relevance of other autacoids, but also such practical questions as the optimal intensity, modality and volume of exercise training required in different populations. [source] Disruption of ATCSLD5 results in reduced growth, reduced xylan and homogalacturonan synthase activity and altered xylan occurrence in ArabidopsisTHE PLANT JOURNAL, Issue 5 2007Adriana Jimena Bernal Summary Members of a large family of cellulose synthase-like genes (CSLs) are predicted to encode glycosyl transferases (GTs) involved in the biosynthesis of plant cell walls. The CSLA and CSLF families are known to contain mannan and glucan synthases, respectively, but the products of other CSLs are unknown. Here we report the effects of disrupting ATCSLD5 expression in Arabidopsis. Both stem and root growth were significantly reduced in ATCSLD5 knock-out plants, and these plants also had increased susceptibility to the cellulose synthase inhibitor isoxaben. Antibody and carbohydrate-binding module labelling indicated a reduction in the level of xylan in stems, and in vitro GT assays using microsomes from stems revealed that ATCSLD5 knock-out plants also had reduced xylan and homogalacturonan synthase activity. Expression in Nicotiana benthamiana of ATCSLD5 and ATCSLD3, fluorescently tagged at either the C- or the N-terminal, indicated that these GTs are likely to be localized in the Golgi apparatus. However, the position of the fluorescent tag affected the subcellular localization of both proteins. The work presented provides a comprehensive analysis of the effects of disrupting ATCSLD5 in planta, and the possible role(s) of this gene and other ATCSLDs in cell wall biosynthesis are discussed. [source] Activity of nitric oxide synthase in mature and immature human spermatozoaANDROLOGIA, Issue 2 2010C. Roessner Summary Nitric oxide (NO) is known to be involved in multiple signal transduction pathways of male germ cells, including sperm capacitation. In somatic cells, NO production was found to be part of apoptosis signalling. The aim of our study was to further clarify the role of NO in spermatozoa by investigation of NO synthase activity with regard to sperm maturity and sperm apoptosis signalling. Semen specimens from 19 healthy donors were subjected to density gradient centrifugation to separate the predominantly mature and immature sperm fraction. NO synthase activity was evaluated using diaminofluoresceine-2-diacetate by FACS. Apoptosis signalling was monitored by flowcytometric analyses of caspase-3 (CP3) and integrity of the transmembrane mitochondrial potential (TMP). TUNEL assay was used to detect DNA fragmentations. Maturity of human spermatozoa was associated with increased NO synthase activity and inactivated apoptosis signalling (lower levels of disrupted TMP, active CP3 and DNA fragmentations, P < 0.05). Activation of apoptosis signalling was significantly negatively correlated to NO production, indicating a rather anti-apoptotic effect of NO. This might underline the recently proposed role of NO in physiological sperm signal transduction, e.g. during capacitation. [source] Neuronal nitric oxide synthase activity in rat urinary bladder detrusor: participation in M3 and M4 muscarinic receptor functionAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 3 2005B. Orman Summary 1,The aim of this paper was to determine the different signalling cascades involved in contraction of the rat urinary bladder detrusor muscle mediated via muscarinic acetylcholine receptors (muscarinic AChR). Contractile responses, phosphoinositides (IPs) accumulation, nitric oxide synthase (NOS) activity and cyclic GMP (cGMP) production were measured to determine the reactions associated with the effect of cholinergic agonist carbachol. The specific muscarinic AChR subtype antagonists and different inhibitors of the enzymatic pathways involved in muscarinic receptor-dependent activation of NOS and cGMP were tested. 2,Carbachol stimulation of M3 and M4 muscarinic AChR increased contractility, IPs accumulation, NOS activity and cGMP production. All of these effects were selectively blunted by 4-DAMP and tropicamide, M3 and M4 antagonists respectively. 3,The inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), neuronal NOS (nNOS) and soluble guanylate cyclase, but not of protein kinase C and endothelial NOS (eNOS), inhibited the carbachol action on detrusor contractility. These inhibitors also attenuated the muscarinic receptor-dependent increase in cGMP and activation of NOS. 4,In addition, sodium nitroprusside and 8-bromo-cGMP, induced negative relaxant effect. 5,The results obtained suggest that carbachol activation of M3 and M4 muscarinic AChRs, exerts a contractile effect on rat detrusor that is accompanied by an increased production of cGMP and nNOS activity. The mechanism appears to occur secondarily to stimulation of IPs turnover via PLC activation. This in turn, triggers cascade reactions involving CaM, leading to activation of nNOS and soluble guanylate cyclase. They, in turn, exert a modulator inhibitory cGMP-mediated mechanism limiting the effect of muscarinic AChR stimulation of the bladder. [source] | |||||||||||||||||||||||||||||||