Sieck, Gary C.

ABSTRACTLeukotoxin and endotoxin derived fromPasteurella haemolyticaserotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381–388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237–252, 1995). Furthermore, extracellular Ca2+is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca2+in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca2+by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca2+regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca2+in a concentration-dependent fashion by influx of extracellular Ca2+through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca2+even in the absence of extracellular Ca2+. Leukotoxin-induced intracellular Ca2+elevation was inhibited by pertussis toxin, inhibitors of phospholipases A2and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca2+elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca2+signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A2and C and release of arachidonic acid in bovine alveolar macrophages. Ca2+signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The differences in the Ca2+signaling mechanisms may underlie the reported temporal differences in gene expression during leukotoxin and endotoxin activation.

In aged rats, diaphragm muscle (DIAm) reduced specific force and fiber cross-sectional area, sarcopenia, is selective for vulnerable type IIx and/or IIb DIAm fibers, with type I and IIa fibers being resilient. In humans, the oxidative capacity [as measured by maximum succinate dehydrogenase (SDH max ) activity] of fast-type muscle is reduced with aging, with slow-type muscle being unaffected. We hypothesized that in aged Fischer rat DIAm exhibiting sarcopenia, reduced SDH max activity would occur in type IIx and/or IIb fibers. Rats obtained from the NIA colony (6, 18, and 24 mo old) were euthanized, and ~2-mm-wide DIAm strips were obtained. For SDH max and fiber type assessments, DIAm strips were stretched (approximately optimal length), fresh frozen in isopentane, and sectioned on a cryostat at 6 μm. SDH max , quantified by intensity of nitroblue tetrazolium diformazan precipitation, was assessed in a fiber type-specific manner by comparing serial sections labeled with myosin heavy chain (MyHC) antibodies differentiating type I (MyHC Slow ), IIa (MyHC 2A ), and IIx and/or IIb fibers. Isometric DIAm force and fatigue were assessed in DIAm strips by muscle stimulation with supramaximal pulses at a variety of frequencies (5–100 Hz) delivered in 1-s trains. By 24 mo, DIAm sarcopenia was apparent and SDH max in type IIx and/or IIb fibers activity was reduced ~35% compared with 6-mo-old control DIAm. These results underscore the remarkable fiber type selectivity of type IIx and/or IIb fibers to age-associated perturbations and suggest that reduced mitochondrial oxidative capacity is associated with DIAm sarcopenia. NEW & NOTEWORTHY We examined the oxidative capacity as measured by maximum succinate dehydrogenase activity in older (18 or 24 mo old) Fischer 344 rat diaphragm muscle (DIAm) compared with young rats (6 mo old). In 24-mo-old rats, SDH activity was reduced in type IIx/b DIAm fibers. These SDH changes were concomitant with sarcopenia (reduced specific force and atrophy of type IIx/b DIAm fibers) at 24 mo old. At 18 mo old, there was no change in SDH activity and no evidence of sarcopenia.

AbstractThe diaphragm muscle comprises various types of motor units that are recruited in an orderly fashion governed by the intrinsic electrophysiological properties (membrane capacitance as a function of somal surface area) of phrenic motor neurons (PhMNs). Glutamate is the main excitatory neurotransmitter at PhMNs and acts primarily via fast acting AMPA and N‐methyl‐D‐aspartic acid (NMDA) receptors. Differences in receptor expression may also contribute to motor unit recruitment order. We used single cell, multiplex fluorescence in situ hybridization to determine glutamatergic receptor mRNA expression across PhMNs based on their somal surface area. In adult male and female rats (n = 9) PhMNs were retrogradely labeled for analyses (n = 453 neurons). Differences in the total number and density of mRNA transcripts were evident across PhMNs grouped into tertiles according to somal surface area. A ~ 25% higher density of AMPA (Gria2) and NMDA (Grin1) mRNA expression was evident in PhMNs in the lower tertile compared to the upper tertile. These smaller PhMNs likely comprise type S motor units that are recruited first to accomplish lower force, ventilatory behaviors. In contrast, larger PhMNs with lower volume densities of AMPA and NMDA mRNA expression presumably comprise type FInt and FF motor units that are recruited during higher force, expulsive behaviors. Furthermore, there was a significantly higher cytosolic NMDA mRNA expression in small PhMNs suggesting a more important role for NMDA‐mediated glutamatergic neurotransmission at smaller PhMNs. These results are consistent with the observed order of motor unit recruitment and suggest a role for glutamatergic receptors in support of this orderly recruitment. image Cover Image for this issue: doi: 10.1111/jnc.14747.

Like all skeletal muscles, the diaphragm muscle accomplishes a range of motor behaviors by recruiting different motor unit types in an orderly fashion. Recruitment of phrenic motor neurons (PhMNs) is generally assumed to be based primarily on the intrinsic properties of PhMNs with an equal distribution of descending excitatory inputs to all PhMNs. However, differences in presynaptic excitatory input across PhMNs of varying sizes could also contribute to the orderly recruitment pattern. In the spinal cord of Sprague-Dawley rats, we retrogradely labeled PhMNs using cholera toxin B (CTB) and validated a robust confocal imaging-based technique that utilizes semiautomated processing to identify presynaptic glutamatergic (Glu) terminals within a defined distance around the somal membrane of PhMNs of varying size. Our results revealed an ~10% higher density of Glu terminals at PhMNs in the lower tertile of somal surface area. These smaller PhMNs are likely recruited first to accomplish lower force ventilatory behaviors of the diaphragm as compared with larger PhMNs in the upper tertile that are recruited to accomplish higher force expulsive behaviors. These results suggest that differences in excitatory synaptic input to PhMNs may also contribute to the orderly recruitment of diaphragm motor units.NEW & NOTEWORTHY The distribution of excitatory glutamatergic synaptic input to phrenic motor neurons differs across motor neurons of varying size. These findings support the size principle of motor unit recruitment that underlies graded force generation in a muscle, which is based on intrinsic electrophysiological properties of motor neurons resulting from differences in somal surface area. A higher density of glutamatergic inputs at smaller, more excitable motor neurons substantiates the earlier and more frequent recruitment of these units.