Lorenz, Robert R.

Halothane relaxes airway smooth muscle in part by a direct effect on the smooth muscle cell. The purpose of this study was to investigate the possible role of cyclic nucleotides in this direct effect. Strips of canine tracheal smooth muscle in vitro were contracted with acetylcholine (ACh) and then exposed to 0.7-2.6% halothane. Isometric force and the intracellular concentrations of adenosine cyclic 3',5'-monophosphate ([cAMP]i) guanosine cyclic 3',5'-monophosphate ([cGMP]i), and free calcium ([Ca2+]i, using the fluorescent Ca(2+)-sensitive dye fura 2) were measured. ACh caused significant increases in force, [cAMP]i, [cGMP]i, and [Ca2+]i. Subsequent exposure of the strips to halothane caused an additional increase in [cAMP]i, decreases in force and [Ca2+]i, and no effect on [cGMP]i. The additional increase in [cAMP]i was similar to that produced by a concentration of isoproterenol (0.03 microM) that caused equipotent relaxation. Indomethacin abolished the increase in [cAMP]i produced by ACh and abolished the additional increase in [cAMP]i produced by halothane. In contrast, indomethacin had no effect on the decreases in force and [Ca2+]i. These findings suggest that in canine tracheal smooth muscle contracted with ACh 1) halothane increases [cAMP]i by a cyclooxygenase-dependent mechanism and 2) the increase in [cAMP]i produced by halothane is not responsible for the relaxation or the decrease in [Ca2+]i.

The mechanism of the direct relaxing effect of halothane on airway smooth muscle may involve a decrease in 1) cytosolic calcium concentration ([Ca2+]i) and/or 2) the force produced for a given [Ca2+]i (i.e., the “sensitivity” of the myofibrillar contractile system to Ca2+). This study was conducted to test the hypothesis that halothane reduces the sensitivity of the myofibrillar contractile system to Ca2+ during muscarinic receptor stimulation of canine tracheal smooth muscle. Isolated smooth muscle strips were mounted in a photometric superfusion system, stretched to their optimal length for force development, and loaded with the fluorescent Ca2+ indicator, fura 2, for simultaneous recording of fura 2 fluorescence and isometric force. Emission fluorescence intensities due to excitation at 340 (F340)- and 380 (F380)-nm wavelengths were measured and F340/F380 was used as an index of [Ca2+]i. After superfusion with Ca(2+)-free physiological salt solution (PSS) containing 1 or 100 microM acetylcholine (ACh), two consecutive cumulative concentration-response curves to CaCl2 (0.01–2.4 mM) were generated for each strip; one curve was generated in the presence of halothane. In strips stimulated with 1 (n = 6) or 100 (n = 6) microM ACh, the cumulative addition of CaCl2 to the Ca(2+)-free PSS caused concentration-dependent increases in both F340/F380 and force. In strips stimulated with 1 microM ACh, 2.4 +/- 0.3% halothane proportionally attenuated increases in both F340/F380 and force.(ABSTRACT TRUNCATED AT 250 WORDS)

Maximal relaxation of airway smooth muscle (ASM) in response to atrial natriuretic peptide (ANP), which stimulates particulate guanylyl cyclase (pGC), is less than that produced by nitric oxide (NO) and other compounds that stimulate soluble guanylyl cyclase (sGC). We hypothesized that stimulation of pGC relaxes ASM only by decreasing intracellular Ca2+concentration ([Ca2+]i), whereas stimulation of sGC decreases both [Ca2+]iand the force developed for a given [Ca2+]i(i.e., the Ca2+sensitivity) during muscarinic stimulation. We measured the relationship between force and [Ca2+]i(using fura 2) under control conditions (using diltiazem to change [Ca2+]i) and during exposure to ANP, diethylamine-NO (DEA-NO), sodium nitroprusside (SNP), and the Sp diastereoisomer of β-phenyl-1, N2-etheno-8-bromoguanosine-3′,5′-cyclic monophosphorothionate ( Sp-8-Br-PET-cGMPS), a cell-permeant analog of cGMP. Addition of DEA-NO, SNP, or Sp-8-Br-PET-cGMPS decreased both [Ca2+]iand force, causing a significant rightward shift of the force-[Ca2+]irelationship. In contrast, with ANP exposure, the force-[Ca2+]irelationship was identical to control, such that ANP produced relaxation solely by decreasing [Ca2+]i. Thus, during muscarinic stimulation, stimulation of pGC relaxes ASM exclusively by decreasing [Ca2+]i, whereas stimulation of sGC decreases both [Ca2+]iand Ca2+sensitivity.

Human umbilical artery was perfused with Krebs-Ringer bicarbonate-glucose medium at 37 C and pH 7.4. An increase in transmural pressure elicited a vasoconstriction. The artery wall contained no significant amount of norepinephrine, which supports the anatomic evidence of absence of nerve supply. Thus, the response to stretch is due to a smooth muscle cell-borne positive feedback mechanism. The myogenic reflex could still be elicited at 44 C but was reversibly abolished by cooling to 4 C. It was reversibly abolished in the absence of calcium. K2SO4-Ringer solution (50 mm in K) markedly increased the tone of the artery after an initial vasodilatation and induced rhythmicity when the perfusion pressure was increased. Bradykinin, 5-hydroxytryptamine, and angiotensin caused vasoconstriction when the artery no longer responded to stretch. Stretch could induce vasoconstriction when angiotensin no longer elicited a response because of tachyphylaxis. The stretch reflex was not blocked by phenoxybenzamine which suppressed the response to 5-hydroxytryptamine. Tachyphylaxis was not observed with bradykinin; the effect of bradykinin was not blocked by phenoxybenzamine.

To determine whether local cholinergic mechanisms evoke nitric oxide (NO)-mediated flow-induced vasorelaxation, canine coronary artery rings without endothelium were suspended beneath an organ chamber that contained a stainless steel tube and a femoral artery segment with endothelium. The rings were superfused at a basal rate of 1 ml/min with physiological salt solution that was bubbled with 95% O2-5% CO2 and maintained at 37 degrees C. They were stretched to optimal length and contracted with prostaglandin F 2 alpha (2 x 10(-6) M). When flow through the stainless steel tube (direct superfusion) was increased from the basal rate of 1 to 4 ml/min, coronary force did not change. Superfusion of the rings (n = 8) with effluent from the femoral segment (endothelial superfusion) at 4 ml/min to study flow-induced vasodilation caused a 67.3 +/- 10.8% relaxation. Treatment of the segment with the NO synthase blocker NG-monomethyl-L-arginine (10(-4) M) eliminated the relaxation seen during endothelial superfusion (P < 0.05 vs. control). Application of atropine (10(-6) M) to additional femoral segments (n = 8) abolished the coronary relaxation observed during endothelial superfusion at 1 ml/ min, and the flow-induced relaxation observed at 4 ml/min was reduced from 64 +/- 8.3 to 27 +/- 5.6% (P < 0.05 vs. control). In studies on additional segments and rings (n = 6), the flow-induced relaxations at 4 ml/min of endothelial superfusion were blunted from 86 +/- 10 to 28 +/- 13% after the segments were treated with acetylcholinesterase (0.00028 U/min for 20 min). These data indicate that basal- and flow-induced release of NO from the vascular endothelium can be mediated by local cholinergic mechanisms. It is possible that flow causes acetylcholine release from certain endothelial cells, which stimulates NO release from these cells or from neighboring endothelial cells.

The aim of the present study was to investigate the influence of inhibitors of endothelium-dependent relaxing factor on the release of atrial natriuretic factor from isolated rat atria. Electrical stimulation of the tissue produced a frequency-dependent increase in secretion of atriopeptide from basal levels. Saponin (0.3 mg/ml for 45 min) augmented the basal release of the peptide. Methylene blue (10(-5)M), oxyhemoglobin (10(-6)M), or hydroquinone (10(-5)M), agents that inhibit the effects of endothelium-derived relaxing factor by different mechanisms, caused an increase in the basal secretion of atrial natriuretic factor. Blockade of cyclooxygenase with indomethacin (10(-5)M) did not alter the release of atriopeptide, suggesting that it is not controlled by endogenous prostaglandins. These results would be explained if the inhibitors tested were to interfere with a factor released continuously from either endocardial cells or endothelial cells of the atrial blood vessels and having the same nature as endothelium-derived relaxing factor. Such release may exert a tonic negative modulatory influence on the secretion of atrial natriuretic factor.

Objectives/Hypothesis Idiopathic subglottic stenosis (iSGS) is a rare and potentially life‐threatening disease marked by recurrent and progressive airway obstruction frequently requiring repeated surgery to stabilize the airway. Unknown etiology and low disease prevalence have limited the ability to characterize the natural history of iSGS and resulted in variability in surgical management. It is uncertain how this variation relates to clinical outcomes. Study Design Medical record abstraction. Methods Utilizing an international, multi‐institutional collaborative, we collected retrospective data on patient characteristics, treatment, and clinical outcomes. We investigated variation between and within open and endoscopic treatment approaches and assessed therapeutic outcomes; specifically, disease recurrence and need for tracheostomy at last follow‐up. Results Strikingly, 479 iSGS patients across 10 participating centers were nearly exclusively female (98%, 95% confidence interval [CI], 96.1–99.6), Caucasian (95%, 95% CI, 92.2–98.8), and otherwise healthy (mean age‐adjusted Charlson Comorbidity Index 1.5; 95% CI, 1.44–1.69). The patients presented at a mean age of 50 years (95% CI, 48.8–51.1). A total of 80.2% were managed endoscopically, whereas 19.8% underwent open reconstruction. Endoscopic surgery had a significantly higher rate of disease recurrence than the open approach (chi 2 = 4.09, P = 0.043). Tracheostomy was avoided in 97% of patients irrespective of surgical approach (95% CI, 94.5–99.8). Interestingly, there were outliers in rates of disease recurrence between centers using similar treatment approaches. Conclusion Idiopathic subglottic stenosis patients are surprisingly homogeneous. The heterogeneity of treatment approaches and the observed outliers in disease recurrence rates between centers raises the potential for improved clinical outcomes through a detailed understanding of the processes of care. Level of Evidence 4. Laryngoscope , 126:1390–1396, 2016

The purpose of this study was to test the hypothesis that H2O2decreases the amount of force produced by a given intracellular Ca2+concentration (i.e., the Ca2+sensitivity) in airway smooth muscle (ASM) in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. A new preparation was developed and validated in which canine ASM strips were first exposed to H2O2and then permeabilized with 10% Triton X-100 to assess the persistent effects of H2O2on Ca2+sensitivity. Experiments in which H2O2was administered before permeabilization revealed a novel mechanism that contributed to reduced Ca2+sensitivity independently of changes in rMLC phosphorylation, in addition to an rMLC phosphorylation-dependent mechanism. The mechanism depended on factors not available in the permeabilized ASM strip or in the buffer to which the strip was exposed, since there was no effect when H2O2was added to permeabilized strips. H2O2treatment of a maximally thiophosphorylated purified myosin subfragment (heavy meromyosin) significantly reduced actomyosin ATPase activity, suggesting one mechanism by which the phosphorylation-independent reduction in Ca2+sensitivity may occur.

Experiments were performed to determine the effect of ouabain on the release of relaxing factor(s) from cultured endothelial cells, and its action on the effect of the relaxing factor(s) on arterial smooth muscle. A column of porcine aortic endothelial cells grown on microcarrier beads in suspension culture was perfused with modified Krebs-Ringer bicarbonate solution. The release of relaxing factor(s) by the endothelial cells was detected under bioassay conditions by measuring the relaxing activity of the perfusate overflowing a ring of canine coronary artery (without endothelium) contracted with prostaglandin F2 alpha. Incubation of the endothelial cells with ouabain did not affect the relaxation of the bioassay ring under basal conditions or upon stimulation of the endothelial cells with ADP but impaired the relaxation induced by bradykinin or the calcium ionophore A23187. Incubation of the bioassay ring with ouabain reduced the relaxation under basal conditions as well as the relaxation induced by ADP but did not affect the relaxation observed upon stimulation with bradykinin and A23187 and the endothelium-independent relaxations induced by nitric oxide. These experiments suggest that cultured porcine aortic endothelial cells release two endothelium-derived relaxing factors; one is released under basal conditions and upon stimulation with adenosine diphosphate and the other (which presumably is nitric oxide) upon stimulation with bradykinin and the calcium ionophore A23187.