In a newly characterized cultured porcine pulmonary artery (PA) preparation, 24-h treatment with the nitric oxide (NO) donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) decreased the response to acutely applied DETA-NO compared with 24-h control (-log EC(50) 6.55 +/- 0.12 and 5.02 +/- 0.21, respectively). Treatment of PA with the cell-permeable superoxide dismutase mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride, did not change NO responsiveness in either freshly prepared or 24-h DETA-NO-treated PA. cGMP and cAMP phosphodiesterase activities were approximately equal in PA. Twenty-four-hour DETA-NO treatment did not change either cGMP or cAMP phosphodiesterase activities. Twenty-four hours in culture had no significant effect on soluble guanylyl cyclase (sGC) subunit mRNA expression, but 24-h DETA-NO treatment significantly decreased the expression of both sGCalpha(1) and sGCbeta(1). sGCbeta(1) protein expression was 42 +/- 4 ng/mg soluble protein. Twenty-four hours in culture without and with DETA-NO reduced sGCbeta(1) protein expression (36 +/- 3 and 31 +/- 3 ng/mg soluble protein, respectively, P < 0.025). Basal tissue cGMP [(cGMP)(i)] was significantly increased, and NO-induced (cGMP)(i) was significantly decreased by 24-h DETA-NO treatment. (cGMP)(i) normalized to the amount of sGC protein expressed in PA was significantly lower in PA treated for 24 h with DETA-NO compared with both freshly isolated and 24-h cultured PA. We conclude that prolonged NO treatment induces decreased acute NO responsiveness in part by decreasing both sGC expression and sGC-specific activity.
We aimed to assess intrinsic smooth muscle mechanisms contributing to greater nitric oxide (NO) responsiveness in pulmonary vascular vs. airway smooth muscle. Porcine pulmonary artery smooth muscle (PASM) and tracheal smooth muscle (TSM) strips were used in concentration-response studies to the NO donor (Z)-1-[N-2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). PASM consistently exhibited greater relaxation at a given DETA-NO concentration (NO responsiveness) than TSM NO responsiveness, with DETA-NO log EC(50) being -6.55 +/- 0.11 and -5.37 +/- 0.13 for PASM and TSM, respectively (P < 0.01). We determined relationships between tissue cGMP concentration ([cGMP](i)) and relaxation using the particulate guanylyl cyclase agonist atrial natriuretic peptide. Atrial natriuretic peptide resulted in nearly complete relaxation, with no detectable increase in [cGMP](i) in PASM and only 20% relaxation (10-fold increase in [cGMP](i)) in TSM, indicating that TSM is less cGMP responsive than PASM. Total cGMP-dependent protein kinase I (cGKI) mRNA expression was greater in PASM than in TSM (2.23 +/- 0.36 vs. 0.93 +/- 0.31 amol mRNA/mug total RNA, respectively; P < 0.01), but total cGKI protein expression was not significantly different (0.56 +/- 0.07 and 0.49 +/- 0.04 ng cGKI/mug protein, respectively). The phosphotransferase assay for the soluble fraction of tissue homogenates demonstrated no difference in the cGMP EC(50) between PASM and TSM. The maximal phosphotransferase activity indexed to the amount of total cGKI in the homogenate differed significantly between PASM and TSM (1.61 +/- 0.15 and 1.04 +/- pmol.min(-1).ng cGKI(-1), respectively; P < 0.05), suggesting that cGKI may be regulated differently in the two tissues. A novel intrinsic smooth muscle mechanism accounting for greater NO responsiveness in PASM vs. TSM is thus greater cGMP responsiveness from increased cGKI-specific activity in PASM.
Although general anesthesia allows relief from stressors such as pain, discomfort, or anxiety for patients undergoing carotid endarterectomy, neurologic assessment is less reliable than under local anesthesia. We describe a unique anesthetic management strategy for carotid endarterectomy patients incorporating the advantages of both general and local anesthesia. The technique allows thorough assessment of neurologic function during carotid cross-clamping by intraoperative wake-up, and guarantees airway management by tracheal intubation.
Cyclic methods of analysis can lead to many advantages, such as low consumption of reagents, minimization of waste, high sample throughput, simple equipment, extended running hours and no contamination of the environment. Flow-injection analysis (FIA) is considered to be a most suitable technique for cyclic methods, i.e., the outlet from the flow-through cell is fed continuously back to the reservoir using a single-line flow system. On this cyclic FIA, the chemical reaction can be easily controlled by fixing the flow rate of a circulating reagent carrier solution and by the constant introduction of a sample solution to the carrier stream. Reproducible mixing of the sample and the reagents can be achieved within the closed flow system, resulting in a highly precise and reproducible signal response. Since the reaction products accumulate in the circulated flow system, a gradual built-up of the background signal produces a continuous baseline shift that limits the useful detection scale. Therefore, elimination of the accumulated reaction products and regeneration of the main reagent are required to maintain a long-running flow system. Some reports have been published concerning cyclic FIA using kinetic (reaction rate) methods 1-4 or the skillful application of electrolysis for removing metal ions. 5,6 Other reports include the incorporation of an immobilized enzyme column for its effective use. The high cost of enzymes justifies attempts to regenerate and reuse them in the system. [7][8][9][10][11] Among the analytical methods, although spectrophotometry is the most commonly used in FIA, there appears to be little in the literature concerning cyclic FIA with spectrophotometric detection. To reuse chromogenic reagents effectively, a rapid regeneration system for them is desired under circulating flow conditions.In a rapid communication, 12 we have proposed a new concept to eliminate the reaction products in a cyclic FIA system. The adoption of a complexing agent (inhibitor) together with a chromogenic reagent makes it possible to regenerate and reuse the main reagent repeatedly. To recognize the new concept and to further test its applicability, a cyclic FIA system has been proposed for the determination of zinc. In order to improve the manipulation and the sensitivity, a water-soluble pyridylazo derivative, 2-(5-bromo-2-pyridylazo)-5-[N-n-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS), 13-17 is used as a chromogenic reagent. Hayashibe et al. 18 have shown that 5-Br-PAPS is a selective and sensitive reagent for zinc (ε = 1.4 × 10 5 dm 3 mol -1 cm -1 , at 554 nm), and successfully applied it to the determination of zinc in serum using conventional FIA without any preconcentration or separation procedure. The aim of the work described here is a cyclic FIA system for the repetitive determination and/or monitoring of zinc using 5-Br-PAPS and EDTA by spectrophotometry. Experimental ReagentsAll chemicals used were of analytical-reagent grade, and water was purified in a Millipore Milli-Q system. A zinc standard solution...
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