Jones, K. A.

ABSTRACTWe describe a case of a 56‐year‐old male who developed severe, refractory hypotension after an intentional ingestion of clozapine and who became hemodynamically stable after one session of therapeutic plasma exchange (TPE). The patient, who presented after an ingestion of clozapine, was found to have altered mental status and hypotension in the emergency department. Escalating catecholamine vasoactive agents were necessary to maintain adequate hemodynamics. Angiotensin II was added and rapidly up titrated in efforts to maintain adequate blood pressure. Due to persistent hemodynamic instability despite four vasoactive agents, TPE was attempted to enhance the elimination of the highly protein bound medication. Within 2 h of completing TPE, the catecholamines and vasopressin were stopped, and angiotensin II dosing was significantly decreased. This is the first report of utilizing TPE as a method of enhanced elimination of a toxic clozapine ingestion resulting in severe hemodynamic compromise. Further study is necessary to determine if TPE should be incorporated into the care of patients experiencing clozapine toxicity.

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.

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)

Agonist-induced increases in CA2+ sensitivity are mediated in part by mechanisms that increase phosphorylation of the regulatory myosin light chain (rMLC) at constant cytosolic Ca2+ concentration ([Ca2+]i). The current study tested the hypothesis that halothane inhibits acetylcholine (ACh)-induced potentiation of rMLC phosphorylation in beta-escin-permeabilized canine tracheal smooth muscle. ACh plus GTP significantly potentiated the increase in isometric force and rMLC phosphorylation induced by 0.8 microM free Ca2+. However, whereas the potentiation of isometric force was sustained, the potentiation of rMLC phosphorylation was biphasic, peaking at 0.5 min and then declining by approximately 10 min to a steady-state level significantly above that induced by 0.8 microM free Ca2+ alone. This finding suggests that mechanisms in addition to changes in rMLC phosphorylation may mediate ACh-induced Ca2+ sensitization, as has been reported for vascular smooth muscle. Halothane (0.91 +/- 0.10 mM) significantly inhibited ACh plus GTP-induced potentiation of rMLC phosphorylation and isometric force after 2 (peak rMLC phosphorylation) and 15 (steady-state rMLC phosphorylation) min of stimulation. However, the effect of halothane on the potentiation of isometric force was significantly less than that expected from its effect on rMLC phosphorylation (i.e., halothane changed the relationship between rMLC phosphorylation and isometric force). These results demonstrate that halothane inhibits the ACh-induced increase in Ca2+ sensitivity by inhibiting the membrane receptor-coupled mechanisms that increase rMLC phosphorylation at constant submaximal [Ca2+]i. Possible additional effects of halothane on rMLC phosphorylation-independent mechanisms cannot be ruled out.

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)

This study used a beta-escin-permeabilized canine tracheal smooth muscle preparation to test the hypothesis that the volatile anesthetic halothane decreases myofilament Ca2+ sensitivity by inhibiting the membrane receptor-linked second messenger systems that regulate myofilament Ca2+ sensitivity and not by inhibiting Ca(2+)-calmodulin activation of the contractile proteins. Acetylcholine (ACh) caused a GTP-dependent increase in force at constant submaximal cytosolic Ca2+ concentration. ACh, guanosine-5'-O-(3-thiotriphosphate), and the protein kinase C agonist 12,13-phorbol dibutyrate each significantly decreased the concentration of free Ca2+ producing a half-maximal response from 0.77 +/- 0.09 microM (Ca2+ alone) to 0.16 +/- 0.01, 0.19 +/- 0.02, and 0.37 +/- 0.03 microM, respectively, demonstrating an increase in myofilament Ca2+ sensitivity. Halothane (0.92 +/- 0.12 mM) had no effect on the free Ca2+ concentration-response curves generated by Ca2+ alone. However, in the presence of 3 microM ACh plus 10 microM GTP to maximally activate muscarinic receptors, halothane significantly increased the EC50 for free Ca2+ from 0.17 +/- 0.01 microM to 0.38 +/- 0.03 microM. These findings suggest that halothane decreases myofilament Ca2+ sensitivity in beta-escin-permeabilized canine tracheal smooth muscle by inhibiting the membrane receptor-linked second messenger systems that regulate myofilament Ca2+ sensitivity.

Fredberg, J. J., K. A. Jones, M. Nathan, S. Raboudi, Y. S. Prakash, S. A. Shore, J. P. Butler, and G. C. Sieck. Friction in airway smooth muscle: mechanism, latch, and implications in asthma. J. Appl. Physiol. 81(6): 2703–2712, 1996.—In muscle, active force and stiffness reflect numbers of actin-myosin interactions and shortening velocity reflects their turnover rates, but the molecular basis of mechanical friction is somewhat less clear. To better characterize molecular mechanisms that govern mechanical friction, we measured the rate of mechanical energy dissipation and the rate of actomyosin ATP utilization simultaneously in activated canine airway smooth muscle subjected to small periodic stretches as occur in breathing. The amplitude of the frictional stress is proportional to ηE, where E is the tissue stiffness defined by the slope of the resulting force vs. displacement loop and η is the hysteresivity defined by the fatness of that loop. From contractile stimulus onset, the time course of frictional stress amplitude followed a biphasic pattern that tracked that of the rate of actomyosin ATP consumption. The time course of hysteresivity, however, followed a different biphasic pattern that tracked that of shortening velocity. Taken together with an analysis of mechanical energy storage and dissipation in the cross-bridge cycle, these results indicate, first, that like shortening velocity and the rate of actomyosin ATP utilization, mechanical friction in airway smooth muscle is also governed by the rate of cross-bridge cycling; second, that changes in cycling rate associated with conversion of rapidly cycling cross bridges to slowly cycling latch bridges can be assessed from changes of hysteresivity of the force vs. displacement loop; and third, that steady-state force maintenance (latch) is a low-friction contractile state. This last finding may account for the unique inability of asthmatic patients to reverse spontaneous airways obstruction with a deep inspiration.