Y. S. Prakash

Adaptations of Diaphragm Neuromuscular Junction following Inactivity

We hypothesized that differences exist in the morphological adaptations of neuromuscular junctions (NMJs) on different fiber types in response to prolonged inactivation. Two weeks of inactivity of both phrenic motoneurons and diaphragm muscle was induced by spinal cord hemitransection at C2 (spinal isolation; SI). A three-color fluorescent immunocytochemical technique, combined with laser-scanning confocal microscopy, was used to create two- (2D) and three-dimensional (3D) images of NMJs and obtain morphological information concerning: (1) innervating axons and presynaptic nerve terminals; (2) motor endplates (postsynaptic apparatus consisting of acetylcholine receptors), and (3) myosin heavy chain (MHC) phenotype of muscle fibers. In both sham controls (CTL) and SI animals, planar (2D) and surface (3D) areas of motor endplates and nerve terminals on type II muscle fibers (anti-fast MHC immunoreactive) were smaller than on type I (nonimmunoreactive to anti-fast MHC) fibers, when normalized for fiber diameter. The number of branches, total branch length and perimeter of both motor endplates and nerve terminals were greater for NMJs on type II fibers than on type I fibers. The extent of overlap between nerve terminal and endplate was greater on type I fibers than on type II fibers. After SI, there was a significant expansion of NMJs on type II fibers. Planar and surface areas of motor endplates and nerve terminals, number of endplate and nerve terminal branches, total branch length, and perimeter were all increased on type II fibers following SI. The extent of overlap of nerve terminal and endplate increased on type II fibers, approaching that observed in type I fiber NMJs. These results indicate that neuromuscular inactivation leads to a selective expansion of type II fiber NMJs through addition of new terminal area, and elongation of existing terminal branches. These changes may represent a compensatory effort to improve neuromuscular transmission.

Effects of volatile anesthetics on store-operated Ca2+ influx in airway smooth muscle

Background:In airway smooth muscle (ASM), volatile anesthetics deplete sarcoplasmic reticulum (SR) Ca2+ stores by increasing Ca2+ “leak.” Accordingly, SR replenishment becomes dependent on Ca2+ influx. Depletion of SR Ca2+ stores triggers Ca2+ influx via specific plasma membrane channels, store-operated Ca2+ channels (SOCC). We hypothesized that anesthetics inhibit SOCC triggered by increased SR Ca2+ “leak,” preventing SR replenishment and enhancing ASM relaxation. Methods:In porcine ASM cells, SR Ca2+ was depleted by cyclopiazonic acid or caffeine in 0 extracellular Ca2+, nifedipine and KCl (preventing Ca2+ influx through L-type and SOCC channels). Extracellular Ca2+ was rapidly introduced to selectively activate SOCC. After SOCC activation, SR was replenished and the protocol repeated in the presence of 1 or 2 minimum alveolar concentration halothane, isoflurane, or sevoflurane. In other cells, characteristics of SOCC and interactions between acetylcholine (Ach) and volatile anesthetics were examined. Results:Cyclopiazonic acid produced slow SR leak, whereas the caffeine response was transient in ASM cells. Reintroduction of extracellular Ca2+ rapidly increased [Ca2+]i. This influx was insensitive to nifedipine, SKF-96365, and KBR-7943, inhibited by Ni2+ and blockade of inositol 1,4,5-triphosphate-induced SR Ca2+ release, and enhanced by ACh. Preexposure to 1 or 2 minimum alveolar concentration halothane completely inhibited Ca2+ influx when extracellular Ca2+ was reintroduced, whereas isoflurane and sevoflurane produced less inhibition. Only halothane and isoflurane inhibited ACh-induced augmentation of Ca2+ influx. Conclusion:Volatile anesthetics inhibit a Ni2+/La3+-sensitive store-operated Ca2+ influx mechanism in porcine ASM cells, which likely helps maintain anesthetic-induced bronchodilation.

Comparison of Volatile Anesthetic Effects on Actin–Myosin Cross-bridge Cycling in Neonatal versus Adult Cardiac Muscle

Background: The neonatal myocardium is more sensitive to volatile anesthetics compared with adults. The greater myocardial sensitivity of neonates may be attributable to greater anesthetic effect on force regulation at the level of the cross-bridge. In the current study, the authors compared the effects of 1 and 2 minimum alveolar concentration (MAC) halothane and sevoflurane on cardiac muscle from 0- to 3-day-old (neonate) and 84-day-old (adult) rats. Methods: Triton X-100–skinned muscle strips were maximally activated at pCa (negative logarithm of the Ca2+ concentration) of 4.0, and the following were measured in the presence or absence of anesthetic: Rate of force redevelopment after rapid shortening and restretching (ktr) and isometric stiffness at maximal activation and in rigor. The fraction of attached cross-bridges (&agr;fs) and apparent rate constants for cross-bridge attachment (fapp) and detachment (gapp) were calculated assuming a two-state model for cross-bridge cycling. Anesthetic-induced changes in the mean stiffness per cross-bridge were also estimated from values in rigor versus maximum activation in the presence or absence of anesthetic. Results: Neonatal cardiac muscle displayed significantly smaller &agr;fs, slower ktr, and slower fapp compared with adult cardiac muscle; however, gapp was not significantly different. Halothane, and sevoflurane to a significantly lesser extent, decreased &agr;fs, fapp, and the mean force per cross-bridge and increased gapp to a greater extent in neonates. Conclusions: These data indicate that weaker force production in neonatal cardiac muscle involves, at least in part, less efficient cross-bridge cycling kinetics. The authors conclude that the greater myocardial sensitivity of neonates to volatile anesthetics reflects, at least in part, a direct inhibition of cross-bridge cycling, especially the rates of cross-bridge attachment and detachment.

Effects of Halothane on Sarcoplasmic Reticulum Calcium Release Channels in Porcine Airway Smooth Muscle Cells

BackgroundVolatile anesthetics relax airway smooth muscle (ASM) by altering intracellular Ca2+ concentration ([Ca2+]i). The authors hypothesized that relaxation is produced by decreasing sarcoplasmic reticulum Ca2+ content via increased Ca2+ “leak” through both inositol trisphosphate (IP3) and ryanodine receptor channels. MethodsEnzymatically dissociated porcine ASM cells were exposed to acetylcholine in the presence or absence of 2 minimum alveolar concentration (MAC) halothane, and IP3 levels were measured using radioimmunoreceptor assay. Other cells were loaded with the Ca2+ indicator fluo-3 and imaged using real-time confocal microscopy. ResultsHalothane increased IP3 concentrations in the presence and absence of acetylcholine. Inhibition of phospholipase C blunted the IP3 response to halothane. Exposure to 2 MAC halothane induced a transient [Ca2+]i response, suggesting depletion of sarcoplasmic reticulum Ca2+. Exposure to 20 &mgr;m Xestospongin D, a cell-permeant IP3 receptor antagonist, resulted in a 45 ± 13% decrease in the [Ca2+]i response to halothane compared with halothane exposure alone. In permeabilized cells, Xestospongin D or 0.5 mg/ml heparin decreased the [Ca2+]i response to halothane by 65 ± 13% and 68 ± 22%, respectively, compared with halothane alone. In both intact and permeabilized cells, 20 &mgr;m ryanodine blunted the [Ca2+]i response to halothane by 32 ± 13% and 39 ± 21%, respectively, compared with halothane alone. Simultaneous exposure to Xestospongin D and ryanodine completely inhibited the [Ca2+]i response to halothane. ConclusionsThe authors conclude that halothane reduces sarcoplasmic reticulum Ca2+ content in ASM cells via increased Ca2+ leak through both IP3 receptor and ryanodine receptor channels. Effects on IP3 receptor channels are both direct and indirect via elevation of IP3 levels.

Effect of halothane on cADP-ribose-induced Ca2+ release system in tracheal smooth muscle

release from intracellular stores; however, cells possess other intracellular Ca releasing systems, including the so-called Ca -induced Ca release system, mediated by the ryanodine receptor–channel (RyR). Recently it was found that the endogenous nucleotide cADP-ribose (cADPR) is a potent activator of the RyR, and this nucleotide has been proposed to be a second messenger in several intracellular signaling pathways. Biosynthesis of cADPR from -NAD is catalyzed by adenosine diphosphate (ADP)-ribosyl cyclase, and cADPR is hydrolysis is mediated by the cADPR hydrolase to ADP-ribose (ADPR). Volatile anesthetics have multiple actions on intracellular Ca homeostasis, including activation of the RyR and sensitization of this channel to pharmacologic agonists such as caffeine and ryanodine. Recently, we reported that halothane can sensitize the RyR to cADPR in sea urchin egg homogenates. It has been previously shown that the cADPR system is functional in porcine smooth muscle cells. In fact, in porcine airway smooth muscle cells cADPR has been shown to be a second messenger responsible for intracellular Ca increase induced by acetylcholine. In the current study, we found that halothane potentiates the cADPR-induced Ca release through the RyR in porcine airway smooth muscle cells. We propose that modulation of the cADPR signaling system by halothane may be an important component of the complex effect of this volatile anesthetic on intracellular Ca homeostasis.