Cellular signaling with nitric oxide and cyclic GMP

During the past two decades, nitric oxide signaling has been one of the most rapidly growing areas in biology. This simple free radical gas can regulate an ever growing list of biological processes. In most instances nitric oxide mediates its biological effects by activating guanylyl cyclase and increasing cyclic GMP synthesis. However, the identification of effects of nitric oxide that are independent of cyclic GMP is also growing at a rapid rate. The effects of nitric oxide can mediate important physiological regulatory events in cell regulation, cell-cell communication and signaling. Nitric oxide can function as an intracellular messenger, neurotransmitter and hormone. However, as with any messenger molecule, there can be too much or too little of the substance and pathological events ensue. Methods to regulate either nitric oxide formation, metabolism or function have been used therapeutically for more than a century as with nitroglycerin therapy. Current and future research should permit the development of an expanded therapeutic armamentarium for the physician to manage effectively a number of important disorders. These expectations have undoubtedly fueled the vast research interests in this simple molecule.

Guanylin peptides: cyclic GMP signaling mechanisms

Guanylate cyclases (GC) serve in two different signaling pathways involving cytosolic and membrane enzymes. Membrane GCs are receptors for guanylin and atriopeptin peptides, two families of cGMP-regulating peptides. Three subclasses of guanylin peptides contain one intramolecular disulfide (lymphoguanylin), two disulfides (guanylin and uroguanylin) and three disulfides (E. coli stable toxin, ST). The peptides activate membrane receptor-GCs and regulate intestinal Cl- and HCO3- secretion via cGMP in target enterocytes. Uroguanylin and ST also elicit diuretic and natriuretic responses in the kidney. GC-C is an intestinal receptor-GC for guanylin and uroguanylin, but GC-C may not be involved in renal cGMP pathways. A novel receptor-GC expressed in the opossum kidney (OK-GC) has been identified by molecular cloning. OK-GC cDNAs encode receptor-GCs in renal tubules that are activated by guanylins. Lymphoguanylin is highly expressed in the kidney and heart where it may influence cGMP pathways. Guanylin and uroguanylin are highly expressed in intestinal mucosa to regulate intestinal salt and water transport via paracrine actions on GC-C. Uroguanylin and guanylin are also secreted from intestinal mucosa into plasma where uroguanylin serves as an intestinal natriuretic hormone to influence body Na+ homeostasis by endocrine mechanisms. Thus, guanylin peptides control salt and water transport in the kidney and intestine mediated by cGMP via membrane receptors with intrinsic guanylate cyclase activity.

A pulsatile flow model for in vitro quantitative evaluation of prosthetic valve regurgitation

A pulsatile pressure-flow model was developed for in vitro quantitative color Doppler flow mapping studies of valvular regurgitation. The flow through the system was generated by a piston which was driven by stepper motors controlled by a computer. The piston was connected to acrylic chambers designed to simulate "ventricular" and "atrial" heart chambers. Inside the "ventricular" chamber, a prosthetic heart valve was placed at the inflow connection with the "atrial" chamber while another prosthetic valve was positioned at the outflow connection with flexible tubes, elastic balloons and a reservoir arranged to mimic the peripheral circulation. The flow model was filled with a 0.25% corn starch/water suspension to improve Doppler imaging. A continuous flow pump transferred the liquid from the peripheral reservoir to another one connected to the "atrial" chamber. The dimensions of the flow model were designed to permit adequate imaging by Doppler echocardiography. Acoustic windows allowed placement of transducers distal and perpendicular to the valves, so that the ultrasound beam could be positioned parallel to the valvular flow. Strain-gauge and electromagnetic transducers were used for measurements of pressure and flow in different segments of the system. The flow model was also designed to fit different sizes and types of prosthetic valves. This pulsatile flow model was able to generate pressure and flow in the physiological human range, with independent adjustment of pulse duration and rate as well as of stroke volume. This model mimics flow profiles observed in patients with regurgitant prosthetic valves.

Cyclic AMP increases the survival of ganglion cells in mixed retinal cell cultures in the absence of exogenous neurotrophic molecules, an effect that involves cholinergic activity

Natural cell death is a well-known degenerative phenomenon occurring during development of the nervous system. The role of trophic molecules produced by target and afferent cells as well as by glial cells has been extensively demonstrated. Literature data demonstrate that cAMP can modulate the survival of neuronal cells. Cultures of mixed retinal cells were treated with forskolin (an activator of the enzyme adenylyl cyclase) for 48 h. The results show that 50 µM forskolin induced a two-fold increase in the survival of retinal ganglion cells (RGCs) in the absence of exogenous trophic factors. This effect was dose dependent and abolished by 1 µM H89 (an inhibitor of protein kinase A), 1.25 µM chelerythrine chloride (an inhibitor of protein kinase C), 50 µM PD 98059 (an inhibitor of MEK), 25 µM Ly 294002 (an inhibitor of phosphatidylinositol-3 kinase), 30 nM brefeldin A (an inhibitor of polypeptide release), and 10 µM genistein or 1 ng/ml herbimycin (inhibitors of tyrosine kinase enzymes). The inhibition of muscarinic receptors by 10 µM atropine or 1 µM telenzepine also blocked the effect of forskolin. When we used 25 µM BAPTA, an intracellular calcium chelator, as well as 20 µM 5-fluoro-2'-deoxyuridine, an inhibitor of cell proliferation, we also abolished the effect. Our results indicate that cAMP plays an important role controlling the survival of RGCs. This effect is directly dependent on M1 receptor activation indicating that cholinergic activity mediates the increase in RGC survival. We propose a model which involves cholinergic amacrine cells and glial cells in the increase of RGC survival elicited by forskolin treatment.

Effect of pilocarpine mouthwash on salivary flow

Pilocarpine is a cholinergic agonist that increases salivary flow and has been used to treat xerostomia. Oral intake is the most frequent route of administration. Adverse effects are dose-dependent and include sudoresis, facial blushing and increased urinary frequency. The objective of the present study was to evaluate the effects of topical pilocarpine solutions as mouthwashes on salivary flow and their adverse effects on healthy subjects. Forty volunteers received 10 ml 0.5, 1 and 2% pilocarpine solutions or 0.9% saline in a randomized, double-blind, placebo-controlled manner. Salivation was measured before and 45, 60 and 75 min after mouth rinsing for 1 min with 10 ml of saline or pilocarpine solutions. Vital signs were measured and ocular, gastrointestinal and cardiovascular symptoms, anxiety and flushing were estimated using visual analog scales. There was a dose-dependent increase in salivation. Salivation measured after 1 and 2% pilocarpine (1.4 ± 0.36 and 2.22 ± 0.42 g, respectively) was significantly (P<0.001) higher than before (0.70 ± 0.15 and 0.64 ± 0.1 g), with a plateau between 45 and 75 min. Cardiovascular, visual, gastrointestinal and behavioral symptoms and signs were not changed by topical pilocarpine. Mouth rinsing with pilocarpine solutions at concentrations of 1 to 2% induced a significant objective and subjective dose-dependent increase in salivary flow, similar to the results reported by others studying the effect of oral 5 mg pilocarpine. The present study revealed the efficacy of pilocarpine mouthwash solutions in increasing salivary flow in healthy volunteers, with no adverse effects. Additional studies on patients with xerostomia are needed.

Responsiveness of glycogen breakdown to cyclic AMP in perfused liver from rats with insulin-induced hypoglycemia

The responsiveness of glycogen breakdown to cAMP was investigated in isolated perfused liver from male Wistar fed rats (200-220 g) with insulin-induced hypoglycemia. The activation of glycogenolysis by 3 µM cAMP was decreased (P<0.05) in livers from rats with hypoglycemia induced by the administration of insulin or during the direct infusion of insulin into the isolated liver. The direct effect of insulin on glycogen catabolism promoted by 3 µM cAMP occurred as early as 3 min after starting insulin infusion. In contrast, the cAMP agonists resistant to phosphodiesterases, 8Br-cAMP and 6MB-cAMP, used at the same concentration as cAMP, i.e., 3 µM, did not modify the effect of insulin. The data suggest that the decreased hepatic responsiveness of glycogen breakdown during insulin-induced hypoglycemia is a direct effect of insulin decreasing the intracellular levels of cAMP.

Role of nitric oxide of the median preoptic nucleus (MnPO) in the alterations of salivary flow, arterial pressure and heart rate induced by injection of pilocarpine into the MnPO and intraperitoneally

We investigated the effect of L-NAME, a nitric oxide (NO) inhibitor and sodium nitroprusside (SNP), an NO-donating agent, on pilocarpine-induced alterations in salivary flow, mean arterial blood pressure (MAP) and heart rate (HR) in rats. Male Holtzman rats (250-300 g) were implanted with a stainless steel cannula directly into the median preoptic nucleus (MnPO). Pilocarpine (10, 20, 40, 80, 160 µg) injected into the MnPO induced an increase in salivary secretion (P<0.01). Pilocarpine (1, 2, 4, 8, 16 mg/kg) ip also increased salivary secretion (P<0.01). Injection of L-NAME (40 µg) into the MnPO prior to pilocarpine (10, 20, 40, 80, 160 µg) injected into the MnPO or ip (1, 2, 4, 8, 16 mg/kg) increased salivary secretion (P<0.01). SNP (30 µg) injected into the MnPO or ip prior to pilocarpine attenuated salivary secretion (P<0.01). Pilocarpine (40 µg) injection into the MnPO increased MAP and decreased HR (P<0.01). Pilocarpine (4 mg/kg body weight) ip produced a decrease in MAP and an increase in HR (P<0.01). Injection of L-NAME (40 µg) into the MnPO prior to pilocarpine potentiated the increase in MAP and reduced HR (P<0.01). SNP (30 µg) injected into the MnPO prior to pilocarpine attenuated (100%) the effect of pilocarpine on MAP, with no effect on HR. Administration of L-NAME (40 µg) into the MnPO potentiated the effect of pilocarpine injected ip. SNP (30 µg) injected into the MnPO attenuated the effect of ip pilocarpine on MAP and HR. The present study suggests that in the rat MnPO 1) NO is important for the effects of pilocarpine on salivary flow, and 2) pilocarpine interferes with blood pressure and HR (side effects of pilocarpine), that is attenuated by NO.

The L-arginine/nitric oxide/cyclic-GMP pathway apparently mediates the peripheral antihyperalgesic action of fentanyl in rats

There are only a few studies on the molecular mechanisms underlying the peripheral antihyperalgesic effect of opioids. The aim of this study was to investigate the molecular bases of the peripheral antihyperalgesic effect of fentanyl in a model of prostaglandin-induced chemical hyperalgesia. Prostaglandin E2 (1.4 nmol) injected into one hind paw of male Wistar rats (200-250 g, N = 6 in each experimental or control group) pretreated with indomethacin (2.5 mg/kg) potentiated the nocifensive response to formalin (1%) injection made 60 min later. Drugs applied locally 30 min after prostaglandin E2 induced the following effects: fentanyl (0.1-1.0 nmol) caused a dose-dependent reversal of the hyperalgesic state, naloxone (2 nmol) co-injected with fentanyl (1 nmol) completely reversed the antihyperalgesic effect, Nomega-nitro-L-arginine (NOARG, 0.05-0.2 µmol) in combination with fentanyl (1.0 nmol) caused a dose-dependent inhibition of the antihyperalgesic effect of fentanyl, co-administration of L-arginine (0.5 µmol) with NOARG (0.2 µmol) plus fentanyl (1.0 nmol) fully restored the antihyperalgesic effect, and the cyclic-GMP phosphodiesterase inhibitor UK-114,542-27 (5-[2-ethoxy-5-(morpholinylacetyl) phenyl]-1,6-dihydro-1-methyl-3-propyl-7H-pyrazolo [4,3-d]-pyrimidin-7-one methanesulfonate monohydrate; 0.5-2.0 µmol) potentiated a subeffective dose of fentanyl (0.1 nmol) in a dose-dependent manner. However, UK-114,542-27 (2.0 µmol) injected alone did not produce this antihyperalgesic effect. Systemically administered fentanyl (1.0 nmol, sc) did not cause antinociception. Taken together, these results support the view that fentanyl reverses prostaglandin E2-induced hyperalgesia, probably by activating an opioid receptor at the periphery, and furthermore the L-arginine/nitric oxide/cyclic-GMP pathway may mediate this peripheral effect of fentanyl.

Blood flow measurements in rats using four color microspheres during blockade of different vasopressor systems

The use of colored microspheres to adequately evaluate blood flow changes under different circumstances in the same rat has been validated with a maximum of three different colors due to methodological limitations. The aim of the present study was to validate the use of four different colors measuring four repeated blood flow changes in the same rat to assess the role of vasopressor systems in controlling arterial pressure (AP). Red (150,000), white (200,000), yellow (150,000), and blue (200,000) colored microspheres were infused into the left ventricle of 6 male Wistar rats 1) at rest and 2) after vasopressin (aAVP, 10 µg/kg, iv), 3) renin-angiotensin (losartan, 10 mg/kg, iv), and 4) sympathetic system blockade (hexamethonium, 20 mg/kg, iv) to determine blood flow changes. AP was recorded and processed with a data acquisition system (1-kHz sampling frequency). Blood flow changes were quantified by spectrophotometry absorption peaks for colored microsphere components in the tissues evaluated. Administration of aAVP and losartan slightly reduced the AP (-5.7 ± 0.5 and -7.8 ± 1.2 mmHg, respectively), while hexamethonium induced a 52 ± 3 mmHg fall in AP. The aAVP injection increased blood flow in lungs (78%), liver (117%) and skeletal muscle (>150%), while losartan administration enhanced blood flow in heart (126%), lungs (100%), kidneys (80%), and gastrocnemius (75%) and soleus (94%) muscles. Hexamethonium administration reduced only kidney blood flow (50%). In conclusion, four types of colored microspheres can be used to perform four repeated blood flow measurements in the same rat detecting small alterations such as changes in tissues with low blood flow.

Assessment of tidal volume and thoracoabdominal motion using volume and flow-oriented incentive spirometers in healthy subjects

The objective of the present study was to evaluate incentive spirometers using volume- (Coach and Voldyne) and flow-oriented (Triflo II and Respirex) devices. Sixteen healthy subjects, 24 ± 4 years, 62 ± 12 kg, were studied. Respiratory variables were obtained by respiratory inductive plethysmography, with subjects in a semi-reclined position (45º). Tidal volume, respiratory frequency, minute ventilation, inspiratory duty cycle, mean inspiratory flow, and thoracoabdominal motion were measured. Statistical analysis was performed with Kolmogorov-Smirnov test, t-test and ANOVA. Comparison between the Coach and Voldyne devices showed that larger values of tidal volume (1035 ± 268 vs 947 ± 268 ml, P = 0.02) and minute ventilation (9.07 ± 3.61 vs 7.49 ± 2.58 l/min, P = 0.01) were reached with Voldyne, whereas no significant differences in respiratory frequency were observed (7.85 ± 1.24 vs 8.57 ± 1.89 bpm). Comparison between flow-oriented devices showed larger values of inspiratory duty cycle and lower mean inspiratory flow with Triflo II (0.35 ± 0.05 vs 0.32 ± 0.05 ml/s, P = 0.00, and 531 ± 137 vs 606 ± 167 ml/s, P = 0.00, respectively). Abdominal motion was larger (P < 0.05) during the use of volume-oriented devices compared to flow-oriented devices (52 ± 11% for Coach and 50 ± 9% for Voldyne; 43 ± 13% for Triflo II and 44 ± 14% for Respirex). We observed that significantly higher tidal volume associated with low respiratory frequency was reached with Voldyne, and that there was a larger abdominal displacement with volume-oriented devices.

Modulation of store-operated Ca2+ entry by cyclic-ADP-ribose

Store-operated Ca2+ entry plays an important role in Ca2+ homeostasis in cells but the mechanisms of control of these channels are not completely understood. We describe an investigation of the role of the CD38-cyclic-ADP-ribose (cADPR)-ryanodine-channel (RyR) signaling pathway in store-operated Ca2+ entry in human smooth muscle. We observed that human myometrial cells have a functional store-operated Ca2+ entry mechanism. Furthermore, we observed the presence of transient receptor potential 1, 3, 4, 5, and 6 ion channels in human myometrial cells. Store-operated Ca2+ transient was inhibited by at least 50-70% by several inhibitors of the RyR, including ryanodine (10 µM), dantrolene (10 µM), and ruthenium red (10 µM). Furthermore, the cell permeable inhibitor of the cADPR-system, 8-Br-cADPR (100 µM), is a potent inhibitor of the store-operated entry, decreasing the store operated entry by 80%. Pre-incubation of cells with 100 µM cADPR and the hydrolysis-resistant cADPR analog 3-deaza-cADPR (50 µM), but not with ADP-ribose (ADPR) leads to a 1.6-fold increase in the store-operated Ca2+ transient. In addition, we observed that nicotinamide (1-10 mM), an inhibitor of cADPR synthesis, also leads to inhibition of the store-operated Ca2+ transient by 50-80%. Finally, we observed that the transient receptor potential channels, RyR, and CD38 can be co-immunoprecipitated, indicating that they interact in vivo. Our observations clearly implicate the CD38-cADPR-ryanodine signaling pathway in the regulation of store-operated Ca2+ entry in human smooth muscle cells.

Correlations between pulse oximetry and peak expiratory flow in acute asthma

Few studies are available concerning correlations between pulse oximetry and peak expiratory flow in children and adolescents with acute asthma. Although the Global Initiative for Asthma states that measurements of lung function and oximetry are critical for the assessment of patients, it is not clear if both methods should necessarily be included in their evaluation. Since there is a significant difference in cost between pulse oximetry equipment and peak expiratory flow devices, we determined whether clinical findings and peak expiratory flow measurements are sufficient to determine the severity of acute asthma. The present prospective observational study was carried out to determine if there is correlation between pulse oximetry and peak expiratory flow determination in 196 patients with acute asthma aged 4 to 15 years diagnosed according to the Global Initiative for Asthma criteria. Patients experiencing their first or second wheezing episode, with fever, related acute or chronic diseases, and unable to perform the peak expiratory flow maneuver were excluded. Measurements of peak expiratory flow and pulse oximetry were performed at admission and after 15 min of each inhaled salbutamol cycle. Correlations obtained by linear regression using the Pearson correlation coefficients (r) were 0.41 (P < 0.0001), 0.53 (P < 0.0001), 0.51 (P < 0.0001), and 0.61 (P < 0.0001) at admission and after the first, second and third cycles of salbutamol, respectively. These correlations showed that one measure cannot substitute the other (Pearson's coefficient <0.7), probably because they evaluate different aspects in the airways, suggesting that peak expiratory flow should not be used alone in the assessment of acute asthma in children and adolescents.

Role of nitric oxide and prostaglandin in the maintenance of cortical and renal medullary blood flow

This study was undertaken in anesthetized dogs to evaluate the relative participation of prostaglandins (PGs) and nitric oxide (NO) in the maintenance of total renal blood flow (TRBF), and renal medullary blood flow (RMBF). It was hypothesized that the inhibition of NO should impair cortical and medullary circulation because of the synthesis of this compound in the endothelial cells of these two territories. In contrast, under normal conditions of perfusion pressure PG synthesis is confined to the renal medulla. Hence PG inhibition should predominantly impair the medullary circulation. The initial administration of 25 µM kg-1 min-1 NG-nitro-L-arginine methyl ester produced a significant 26% decrease in TRBF and a concomitant 34% fall in RMBF, while the subsequent inhibition of PGs with 5 mg/kg meclofenamate further reduced TRBF by 33% and RMBF by 89%. In contrast, the initial administration of meclofenamate failed to change TRBF, while decreasing RMBF by 49%. The subsequent blockade of NO decreased TRBF by 35% without further altering RMBF. These results indicate that initial PG synthesis inhibition predominantly alters the medullary circulation, whereas NO inhibition decreases both cortical and medullary flow. This latter change induced by NO renders cortical and RMBF susceptible to a further decrease by PG inhibition. However, the decrease in medullary circulation produced by NO inhibition is not further enhanced by subsequent PG inhibition.

Anti-cyclic citrullinated peptide antibodies and rheumatoid factor in leprosy patients with articular involvement

The objective of the present research was to evaluate the usefulness of anti-cyclic citrullinated peptide (anti-CCP) antibodies and the IgM rheumatoid factor (IgM RF) test for the differential diagnosis of leprosy with articular involvement and rheumatoid arthritis (RA). Anti-CCP antibodies and IgM RF were measured in the sera of 158 leprosy patients (76 with and 82 without articular involvement), 69 RA patients and 89 healthy controls. Leprosy diagnosis was performed according to Ridley and Jopling classification criteria and clinical and demographic characteristics of leprosy patients were collected by a standard questionnaire. Leprosy patients with any concomitant rheumatic disease were excluded. Serum samples were obtained from all participants and frozen at _20°C. Measurement of anti-CCP antibodies and IgM RF were performed by ELISA, using a commercial second-generation kit, and the latex agglutination test, respectively. Anti-CCP antibodies and IgM RF were detected in low frequencies (2.6 and 1.3%, respectively) in leprosy patients and were not associated with articular involvement. Among healthy individuals both anti-CCP antibodies and IgM RF were each detected in 3.4% of the subjects. In contrast, in the RA group, anti-CCP antibodies were present in 81.2% and IgM RF in 62.3%. In the present study, both anti-CCP antibodies and IgM RF showed good positive predictive value for RA, helping to discriminate between RA and leprosy patients with articular involvement. However, anti-CCP antibodies were more specific for RA diagnosis in the population under study.

Normal flow-mediated vasodilatation of the brachial artery and carotid artery intima-media thickness in subclinical hypothyroidism

Subclinical hypothyroidism (SHT) is a disease for which exact therapeutic approaches have not yet been established. Previous studies have suggested an association between SHT and coronary heart disease. Whether this association is related to SHT-induced changes in serum lipid levels or to endothelial dysfunction is unclear. The aim of this study was to determine endothelial function measured by the flow-mediated vasodilatation of the brachial artery and the carotid artery intima-media thickness (IMT) in a group of women with SHT compared with euthyroid subjects. Triglycerides, total cholesterol, HDL-C, LDL-C, apoprotein A (apo A), apo B, and lipoprotein(a) were also determined. Twenty-one patients with SHT (mean age: 42.4 ± 10.8 years and mean thyroid-stimulating hormone (TSH) levels: 8.2 ± 2.7 µIU/mL) and 21 euthyroid controls matched for body mass index, age and atherosclerotic risk factors (mean age: 44.2 ± 8.5 years and mean TSH levels: 1.4 ± 0.6 µIU/mL) participated in the study. Lipid parameters (except HDL-C and apo A, which were lower) and IMT values were higher in the common carotid and carotid bifurcation of SHT patients with positive serum thyroid peroxidase antibodies (TPO-Ab) (0.62 ± 0.2 and 0.62 ± 0.16 mm for the common carotid and carotid bifurcation, respectively) when compared with the negative TPO-Ab group (0.55 ± 0.24 and 0.58 ± 0.13 mm, for common carotid and carotid bifurcation, respectively). The difference was not statistically significant. We conclude that minimal thyroid dysfunction had no adverse effects on endothelial function in the population studied. Further investigation is warranted to assess whether subclinical hypothyroidism, with and without TPO-Ab-positive serology, has any effect on endothelial function.