Lorenz, Robert R.

Abstract The North American Airway Collaborative (NoAAC) previously published a 3‐year multi‐institutional prospective cohort study showing variation in treatment effectiveness between 3 primary surgical techniques for idiopathic subglottic stenosis (iSGS). In this report, we update these findings to include 5 years of data evaluating treatment effectiveness. Patients in the NoAAC cohort were re‐enrolled for 2 additional years and followed using the prespecified published protocol. Consistent with prior data, prospective observation of 487 iSGS patients for 5 years showed treatment effectiveness differed by modality. Cricotracheal resection maintained the lowest rate of recurrent operation (5%), followed by endoscopic resection with adjuvant medical therapy (30%) and endoscopic dilation (50%). These data support the initial observations and continue to provide value to providers and patients navigating longitudinal decision‐making. Level of evidence: 2—prospective cohort study.

Objectives: To examine whether social determinants of health (SDH) factors are associated with time to diagnosis, treatment selection, and time to recurrent surgical intervention in idiopathic subglottic stenosis (iSGS) patients. Methods: Adult patients with diagnosed iSGS were recruited prospectively (2015-2017) via clinical providers as part of the North American Airway Collaborative (NoAAC) and via an online iSGS support community on Facebook. Patient-specific SDH factors included highest educational attainment (self-reported), median household income (matched from home zip code via U.S. Census data), and number of close friends (self-reported) as a measure of social support. Main outcomes of interest were time to disease diagnosis (years from symptom onset), treatment selection (endoscopic dilation [ED] vs cricotracheal resection [CTR] vs endoscopic resection with adjuvant medical therapy [ERMT]), and time to recurrent surgical intervention (number of days from initial surgical procedure) as a surrogate for disease recurrence. Results: The total 810 participants were 98.5% female, 97.2% Caucasian, and had a median age of 50 years (IQR, 43-58). The cohort had a median household income of $62 307 (IQR, $50 345-$79 773), a median of 7 close friends (IQR, 4-10), and 64.7% of patients completed college or graduate school. Education, income, and number of friends were not associated with time to diagnosis via multivariable linear regression modeling. Univariable multinominal logistic regression demonstrated an association between education and income for selecting ED versus ERMT, but no associations were noted for CTR. No associations were noted for time to recurrent surgical procedure via Kaplan Meier modeling and Cox proportional hazards regression. Conclusions: Patient education, income, and social support were not associated with time to diagnosis or time to disease recurrence. This suggests additional patient, procedure, or disease-specific factors contribute to the observed variations in iSGS surgical outcomes.

The effects of nitric oxide and acetylcholine (ACh) were investigated on the electrical and mechanical properties of vascular smooth muscle cells of the canine mesenteric artery. Isolated tissues with or without the endothelium were contracted with prostaglandin F2 alpha. Nitric oxide caused comparable concentration-dependent relaxations in rings with and without endothelium. ACh induced concentration-dependent relaxations only in arteries with endothelium. The relaxations to both nitric oxide and ACh were inhibited by methylene blue or oxyhemoglobin. Either in the presence or absence of prostaglandin F2 alpha, ACh caused transient hyperpolarization of the cell membrane of the vascular smooth muscle. The ACh-induced transient hyperpolarization was not observed after mechanical removal of the endothelial cells or after treatment with atropine. Nitric oxide (less than or equal to 8 X 10(-6) M) did not alter membrane potential, in either the presence or absence of the endothelium. The excitatory junction potentials generated by perivascular nerve stimulation were inhibited by ACh but not by nitric oxide. These results suggest that in the canine mesenteric artery 1) the endothelium-derived hyperpolarizing factor generated by ACh is not nitric oxide; 2) nitric oxide relaxes vascular smooth muscle by a direct effect; and 3) nitric oxide does not modify adrenergic neurotransmission.

Prior studies suggest that the mechanism of action by which halothane relaxes airway smooth muscle depends on the contractile state of the cell. We hypothesized that halothane would inhibit the influx of Ca2+ into canine airway smooth muscle cells during submaximal, but not maximal, muscarinic stimulation. This hypothesis was tested by using the rate of quenching of fura 2 fluorescence by Mn2+ in strips of canine tracheal smooth muscle as an index of Ca2+ influx. Acetylcholine (ACh) produced a dose-dependent increase in Mn2+ influx. Halothane (0.64 +/- 0.05 microM) significantly decreased Mn2+ influx and intracellular Ca2+ concentration when added to strips stimulated with a submaximal concentration of ACh (0.3 microM) but had no effect on Mn2+ influx or intracellular Ca2+ concentration during maximal stimulation with ACh (100 microM). Similar results were observed when the strips were treated with verapamil. These results demonstrate that anesthetic effects on Ca2+ homeostasis in intact canine tracheal smooth muscle cells may be critically modulated by receptor-linked mechanisms.

This study determined whether the time-dependent decline in the rate of ATP hydrolysis by actomyosin ATPase during sustained isometric force can occur in the absence of a time-dependent decline in regulatory myosin light chain (rMLC) phosphorylation in Triton X-100-permeabilized canine tracheal smooth muscle. Maximal activation with 10 μM Ca2+induced sustained increases in isometric force, stiffness, and rMLC phosphorylation; however, the increase in the ATP hydrolysis rate was initially high but then declined to a steady-state level above that of the unstimulated muscle (basal 31.8 ± 5.8 nmol ⋅ cm−3⋅ s−1; peak 81.4 ± 11.3 nmol ⋅ cm−3⋅ s−1; steady-state 62.2 ± 9.1 nmol ⋅ cm−3⋅ s−1). Activation of strips in which the rMLC was irreversibly and maximally thiophosphorylated with adenosine 5′- O-(3-thiotriphosphate) also induced sustained increases in isometric force and stiffness but a nonsustained increase in ATP hydrolysis rate. There was no significant difference in the peak or steady-state isometric force, stiffness, or ATP hydrolysis rate or in the steady-state maximum unloaded shortening velocity between strips activated by 10 μM Ca2+or rMLC thiophosphorylation (0.058 ± 0.016 and 0.047 ± 0.011 muscle lengths/s, respectively). Mechanisms other than changes in rMLC phosphorylation contribute to the time-dependent decline in actomyosin ATPase activity during sustained activation of canine tracheal smooth muscle.

The purpose of this study was to determine the mechanism by which hydrogen peroxide (H2O2), an important inflammatory mediator, relaxes canine tracheal smooth muscle (CTSM). H2O2caused concentration-dependent relaxations of CTSM strips contracted with ACh or isotonic KCl [EC50of 0.24 ± 0.04 (SE) and 0.23 ± 0.04 mM, respectively]. Indomethacin (10 μM) decreased the sensitivity of both KCl- and ACh-contracted strips to H2O2. H2O2increased intracellular cAMP levels, an increase that was abolished by indomethacin. H2O2did not affect intracellular cGMP levels. In strips treated with indomethacin and contracted with ACh or isotonic KCl, H2O2-evoked relaxations were accompanied by increases in intracellular Ca2+concentration and decreases in regulatory myosin light chain phosphorylation. We conclude that H2O2decreases Ca2+sensitivity in CTSM by decreasing regulatory myosin light chain phosphorylation through inhibition of myosin light chain kinase and/or activation of smooth muscle protein phosphatases.

Nitrovasodilators relax airway smooth muscle by both guanosine 3',5'-cyclic monophosphate (cGMP)-dependent and cGMP-independent mechanisms and by mechanisms that reduce cytosolic calcium concentration ([Ca2+]i). This study was conducted to determine the relative importance of these mechanisms in relaxation of canine tracheal smooth muscle (CTSM) induced by 3-morpholinosydnonimine (SIN-1). We measured 1) the effect of SIN-1 on force, [cGMP]i, and [Ca2+]i, and 2) the ability of methylene blue (MB) to antagonize SIN-1-induced relaxation and cGMP accumulation. The ratio of fura 2 emission fluorescence intensities due to excitation at 340- and 380-nm wavelengths (F340/F380) was used as an index of [Ca2+]i. In strips contracted with 0.3 microM acetylcholine (ACh, n = 8) or 24 mM KCl (n = 8), SIN-1 (1-100 microM) caused a concentration-dependent decrease in force which was correlated with a concentration-dependent increase in [cGMP]i. MB (10 microM) proportionally attenuated both relaxation and cGMP accumulation. In fura 2-loaded strips contracted with 0.3 microM ACh (n = 7) or 30 mM KCl (n = 7), reductions in force induced by SIN-1 (1-100 microM) were accompanied by decreases in F340/F380. These findings suggest that in CTSM contracted with ACh or KCl, SIN-1 causes relaxation which appears to be mediated by cGMP-dependent mechanisms that reduce [Ca2+]i.

Halothane is a potent bronchodilator that attenuates vagally mediated constriction of canine airways, in part by a direct action on the smooth muscle cell. In tracheal smooth muscle, acetylcholine (ACh) produces a transient peak increase in cytosolic ionized calcium concentration ([Ca2+]i), which declines to a sustained plateau concentration that is higher than that of the unstimulated muscle, and a sustained increase in force. The transient peak [Ca2+]i is caused primarily by Ca2+ release from the sarcoplasmic reticulum (SR), whereas the plateau [Ca2+]i is caused primarily by influx of extracellular Ca2+ across the plasma membrane. This study was conducted to investigate the effects of halothane on the 1) transient peak [Ca2+]i during force development (i.e., developed force) and 2) plateau [Ca2+]i during force maintenance (i.e., sustained force) induced by ACh in isolated strips of canine tracheal smooth muscle. Changes in [Ca2+]i were measured with the photoprotein, aequorin. In muscle strips contracted after incubation with 1.1 (n = 5) or 1.5 (n = 5) minimum alveolar concentration (MAC) halothane, halothane significantly attenuated the transient peak aequorin luminescence (AL) and developed force and significantly prolonged the time necessary to reach peak AL and developed force; these effects were not dose dependent. In muscle strips (n = 3) contracted with ACh, addition of halothane caused a reduction in sustained force but no decrease in plateau AL.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic receptor stimulation increases Ca2+sensitivity, i.e., the amount of force produced at a constant submaximal cytosolic Ca2+concentration ([Ca2+]i), in permeabilized smooth muscle preparations. It is controversial whether this increase in Ca2+sensitivity is in part mediated by protein kinase C (PKC). With the use of a β-escin permeabilized canine tracheal smooth muscle (CTSM) preparation, the effect of four putative PKC inhibitors {calphostin C, chelerythrine chloride, a pseudosubstrate inhibitor for PKC [PKC peptide-(19—31)], and staurosporine} on Ca2+sensitization induced by acetylcholine (ACh) plus GTP was determined. Preincubation with each of the inhibitors did not affect subsequent Ca2+sensitization induced by muscarinic receptor stimulation in the presence of a constant submaximal [Ca2+]i, neither did any of these compounds reverse the increase in Ca2+sensitivity induced by ACh plus GTP. Administration of a 1,2-diacylglycerol analog, 1-oleoyl-2-acetyl- sn-glycerol, did not induce Ca2+sensitization at a constant submaximal [Ca2+]i. Thus we found no evidence that PKC mediates increases in Ca2+sensitivity produced by muscarinic receptor stimulation in permeabilized CTSM.