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When erythrocyte hemoglobin (Hb) is fully saturated with O2, nitric oxide (NO) covalently binds to the cysteine 93 residue of the Hb β-chain (B93-CYS), forming S-nitrosohemoglobin. Binding of NO is allosterically coupled to the O2 saturation of Hb. As saturation falls, the NO group on B93-CYS is transferred to thiols in the erythrocyte, and in the plasma, forming circulating S-nitrosothiols. Here, we studied whether the changes in ventilation during and following exposure to a hypoxic challenge were dependent on erythrocytic B93-CYS. Studies were performed in conscious mice in which native murine Hb was replaced with human Hb (hB93-CYS mice) and in mice in which murine Hb was replaced with human Hb containing an alanine rather than cysteine at position 93 on the Bchain (hB93-ALA). Both strains expressed human γ-chain Hb, likely allowing a residual element of S-nitrosothiol-dependent signaling. While resting parameters and initial hypoxic (10% O2, 90% N2) ventilatory responses were similar in hB93-CYS mice and hB93-ALA mice, the excitatory ventilatory responses (short-term potentiation) that occurred once the mice were returned to room air were markedly diminished in hB93-ALA mice. Further, short-term potentiation responses were virtually absent in mice with bilateral transection of the carotid sinus nerves. These data demonstrate that hB93-CYS plays an essential role in mediating carotid sinus nerve-dependent short-term potentiation, an important mechanism for recovery from acute hypoxia., (Copyright © 2014 the American Physiological Society.)

S-Nitrosoglutathione (GSNO) reductase regulates cell signaling pathways relevant to asthma and protects cells from nitrosative stress. Recent evidence suggests that this enzyme may prevent human hepatocellular carcinoma arising in the setting of chronic hepatitis. We hypothesized that GSNO reductase may also protect the lung against potentially carcinogenic reactions associated with nitrosative stress. We report that wild-type Ras is S-nitrosylated and activated by nitrosative stress and that it is denitrosylated by GSNO reductase. In human lung cancer, the activity and expression of GSNO reductase are decreased. Further, the distribution of the enzyme (including its colocalization with wild-type Ras) is abnormal. We conclude that decreased activity of GSNO reductase could leave the human lung vulnerable to the oncogenic effects of nitrosative stress, as is the case in the liver. This potential should be considered when developing therapies that inhibit pulmonary GSNO reductase to treat asthma and other conditions.

Objective: To determine whether S-nitrosylation of connexins (Cxs) modulates gap junction communication between endothelium and smooth muscle., Methods and Results: Heterocellular communication is essential for endothelium control of smooth muscle constriction; however, the exact mechanism governing this action remains unknown. Cxs and NO have been implicated in regulating heterocellular communication in the vessel wall. The myoendothelial junction serves as a conduit to facilitate gap junction communication between endothelial cells and vascular smooth muscle cells within the resistance vasculature. By using isolated vessels and a vascular cell coculture, we found that Cx43 is constitutively S-nitrosylated on cysteine 271 because of active endothelial NO synthase compartmentalized at the myoendothelial junction. Conversely, we found that stimulation of smooth muscle cells with the constrictor phenylephrine caused Cx43 to become denitrosylated because of compartmentalized S-nitrosoglutathione reductase, which attenuated channel permeability. We measured S-nitrosoglutathione breakdown and NO(x) concentrations at the myoendothelial junction and found S-nitrosoglutathione reductase activity to precede NO release., Conclusions: This study provides evidence for compartmentalized S-nitrosylation/denitrosylation in the regulation of smooth muscle cell to endothelial cell communication.

S-nitrosothiols have been implicated in the etiology of various pulmonary diseases. Many of these diseases display gender preferences in presentation or altered severity that occurs with puberty, the mechanism by which is unknown. Estrogen has been shown to influence the expression and activity of endothelial nitric oxide synthase (eNOS) which is associated with increased S-nitrosothiol production. The effects of gender hormones on the expression and activity of the de-nitrosylating enzyme S-nitrosoglutathione reductase (GSNO-R) are undefined. This report evaluates the effects of gender hormones on the activity and expression of GSNO-R and its relationship to N-acetyl cysteine (NAC)-induced pulmonary hypertension (PH). GSNO-R activity was elevated in lung homogenates from female compared to male mice. Increased activity was not due to changes in GSNO-R expression, but correlated with GSNO-R S-nitrosylation: females were greater than males. The ability of GSNO-R to be activated by S-nitrosylation was confirmed by: 1) the ability of S-nitrosoglutathione (GSNO) to increase the activity of GSNO-R in murine pulmonary endothelial cells and 2) reduced activity of GSNO-R in lung homogenates from eNOS(-/-) mice. Gender differences in GSNO-R activity appear to explain the difference in the ability of NAC to induce PH: female and castrated male animals are protected from NAC-induced PH. Castration results in elevated GSNO-R activity that is similar to that seen in female animals. The data suggest that GSNO-R activity is modulated by both estrogens and androgens in conjunction with hormonal regulation of eNOS to maintain S-nitrosothiol homeostasis. Moreover, disruption of this eNOS-GSNO-R axis contributes to the development of PH.

The endogenous signaling molecule S-nitrosoglutathione (GSNO) and other S-nitrosylating agents can cause full maturation of the abnormal gene product DeltaF508 cystic fibrosis (CF) transmembrane conductance regulator (CFTR). However, the molecular mechanism of action is not known. Here we show that Hsp70/Hsp90 organizing protein (Hop) is a critical target of GSNO, and its S-nitrosylation results in DeltaF508 CFTR maturation and cell surface expression. S-nitrosylation by GSNO inhibited the association of Hop with CFTR in the endoplasmic reticulum. This effect was necessary and sufficient to mediate GSNO-induced cell-surface expression of DeltaF508 CFTR. Hop knockdown using siRNA recapitulated the effect of GSNO on DeltaF508 CFTR maturation and expression. Moreover, GSNO acted additively with decreased temperature, which promoted mutant CFTR maturation through a Hop-independent mechanism. We conclude that GSNO corrects DeltaF508 CFTR trafficking by inhibiting Hop expression, and that combination therapies--using differing mechanisms of action--may have additive benefits in treating CF.

Objective: The study examined the influence of probenecid (Pn), sildenafil (Sd) and oxidiazoloquinoxalin (ODQ) on contraction of phenylephrine (PhE) stimulated rat aortas., Methods: The study was performed at Peter Holtz Research Center of Pharmacology and Experimental Therapeutics, Ernst-Moritz-Arndt University Greifswald, Greifswald, Germany, from 1st July to 30th September 2005. Thirty-five isolated rat aortas were stimulated with 10 microM PhE or preincubated for 30 minutes with 10 microM Pn, or 10 microM ODQ, or 50 microM Sd, and then incubated with 10 microM PhE in the presence or absence of the substances. The phosphorylated myosin light chain 20 was detected by using an antibody against phosphomyosin light chain 2., Results: The ratio of PhE stimulated phosphorylation of aortas (p<0.05) to the untreated was 16.7:1 at 30 seconds and 20.4:1 at 60 seconds. The stimulation decreased significantly at 120 seconds then during the following 10 minutes. Pre-incubation with 50 microM Sd (p>0.05) or 10 microM Pn (p<0.05) reduced the phosphorylation induced by PhE that was added to each for 30 seconds. But pre-incubation with 10 microM ODQ increased the phosphorylation brought about by addition of PhE for 60 seconds, p>0.05. The washout-effect of these modulators was not significant after stimulation with PhE only., Conclusion: The study demonstrates the involvement of cyclic guanosine 3',5' monophosphate and its modulators on muscle contraction of rat aortas. Sildenafil and Pn reduced while oxidiazoloquinoxalin increased the contraction of rat aortas.

This study investigated the effect of Catha edulis (khat) on some important parameters of the metabolic syndrome in Wistar Ottawa Karlsburg W (WOKW) rat. The animals were fed with the standard chow containing 5% air dried pulverized khat leaves for 14 days; followed by the standard chow for 16 days. The khat leaves were sorted into green (khat light; KL) and crimson (khat dark; KD) leaves. The control rats were fed on standard chow. Blood glucose (G), serum insulin, serum leptin and serum lipids (triglycerides, total cholesterol, HDL-, LDL-, and VLDL cholesterol) were determined. Feeding with khat leaves reduced the body weight and the triglyceride level of the animals. The effect of KD on these parameters was stronger than that of KL. KD lowered the blood glucose concentration and the leptin content whereas KL was inactive. The khat intake had no significant influence on serum insulin, total serum cholesterol, HDL-, LDL- and VLDL-cholesterol.

Objective: The aim of the present study is to show a better short-term (2 weeks) clinical improvement in patients with heart failure (HF) who are receiving angiotensin converting enzyme inhibitors (ACEIs) (with or without digoxin) when compared to the standard therapy excluding ACEIs., Methods: The study was conducted in Al-Gamhuria Teaching Hospital, Aden, Yemen, from January to July 2003. In this study, 78 patients with HF were enrolled into 3 therapeutic groups (ACEIs alone, ACEI and digoxin and digoxin alone) and their responses within 2 weeks were recorded. Exclusion criteria were as follows: thyroid disorders, gastrointestinal disturbances (diarrhea, malabsorption), electrolyte unbalanced (unless corrected) and insufficient data. Serum creatinine was measured at the beginning and after 10 days. In addition, the patients' body weight and age were recorded. Criteria for a complete improvement within 2 weeks were the occurrence of the following: 1) The relief of pulmonary congestion, 2) Decrement in heart rate to less than 74 +/- 5, 3) Disappearance of the lower limb edema, and 5) Recorded positive electroencephalogram change. Partial amelioration was recognized if only 2 or 3 of the preceding criteria were observed., Results: Nine patients received digoxin alone, while 40 patients were treated with ACEIs and digoxin. Treatment with ACEIs without digoxin was observed in 29 patients. The discrepancy between the number of patients was necessitated by the need of patients with HF. This last category of treatment regimen produced better clinical improvement (complete with 10.1%, partial with 24.3%) compared to the digoxin group without ACEI (complete 2.5% or partial 5.1%). Nevertheless, the addition of digoxin to an ACEI increased this ratio (17.8% for complete and 28.2% for partial improvement). A 49.3% increase in serum creatinine was observed after 10 days in 25 HF patients, who were randomly selected and followed up (the baseline concentration was 99.75 +/- 9.9 umol/L, while the level after 10 days was 148.97 +/- 19.8 umol/L, p=0.005)., Conclusion: We confirmed that short-term use of ACEI regimens has a superior effect on the therapy of HF (34.4% complete and partial response) as compared to the therapy of not using ACEI (7.6% had a complete and partial response). The combination of ACEI and digoxin has resulted in the best outcome (46% had a complete and partial response). However, we also noticed a significant rise in serum creatinine by 49% concomitant with the use of ACEI (the baseline concentration was 99.75 +/- 9.9 um/L, while the level after 10 days was 148.97 +/- 19.8 umol/L, p=0.005).

In this paper, a project whose purpose was to develop and implement a workable inventory control system is discussed. Specific objectives of the project included the avoidance of out-of-stock situations, minimization of total inventory costs, and increased efficiency of the purchasing power. The initial step in the project was the determination of both fixed and variable ordering and carrying costs. Then a micro-computer was enlisted to print an inventory listing according to the total cost based on usage of the item for the past year and the inventory was subsequently classified into A, B and C categories. The economic order quantity (EOQ) was calculated for items in class A, while B and C items were purchased on a min/max basis. Also, a weekly ordering schedule for class A items was developed based on frequency of ordering. Following this schedule, all items to be ordered on a particular week are printed by the computer. The computer maintains a perpetual inventory, and a list of B and C items below minimum quantity is printed upon request. The efficiency of the inventory control increased dramatically (50%) after project implementation. Although cost savings were found, they were not substantial compared to our previous system (less than $5,000). Out-of-stock situations for B and C items occurred almost as frequently as before project implementation. This study demonstrated that the EOQ concept in conjunction with ABC inventory analysis may be an effective inventory control system in hospital pharmacy. Increased efficiency and cost savings were achieved. Protection against unpredictable demand and avoidance of out-of-stock situations can be achieved by closer adherence to stated ordering procedures for B and C items.