Following cervical spinal cord injury at C2 (SH hemisection model) there is progressive recovery of phrenic activity. Neuroplasticity in the postsynaptic expression of neurotransmitter receptors may contribute to functional recovery. Phrenic motoneurons express multiple serotonergic (5-HTR) and glutamatergic (GluR) receptors, but the timing and possible role of these different neurotransmitter receptor subtypes in the neuroplasticity following SH are not clear. The current study was designed to test the hypothesis that there is an increased expression of serotonergic and glutamatergic neurotransmitter receptors within phrenic motoneurons after SH. In adult male rats, phrenic motoneurons were labeled retrogradely by intrapleural injection of Alexa 488-conjugated cholera toxin B. In thin (10 μm) frozen sections of the spinal cord, fluorescently-labeled phrenic motoneurons were visualized for laser capture microdissection (LCM). Using quantitative real-time RT-PCR in LCM samples, the time course of changes in 5-HTR and GluR mRNA expression was determined in phrenic motoneurons up to 21 days post-SH. Expression of 5-HTR subtypes 1b, 2a and 2c and GluR subtypes AMPA, NMDA, mGluR1 and mGluR5 was evident in phrenic motoneurons from control and SH rats. Phrenic motoneuron expression of 5-HTR2a increased ~8-fold (relative to control) at 14 days post-SH, whereas NMDA expression increased ~16-fold by 21-days post-SH. There were no other significant changes in receptor expression at any time post-SH. This is the first study to systematically document changes in motoneuron expression of multiple neurotransmitter receptors involved in regulation of motoneuron excitability. By providing information on the neuroplasticity of receptors expressed in a motoneuron pool that is inactivated by a higher-level spinal cord injury, appropriate pharmacological targets can be identified to alter motoneuron excitability.

([Ca 2ϩ ] i ) is key to contractility of airway smooth muscle (ASM). Studies from our own laboratory and by others showed that proinflammatory cytokines such as TNF␣ and IL-13 increase agonist-induced [Ca 2ϩ ] i response in ASM (8,31,35,38). The sarcoplasmic reticulum (SR) is a specialized intracellular membrane system in smooth and striated muscle that is involved in the uptake, storage, and release of Ca . In smooth muscle (as in other muscles), the SR Ca 2ϩ ATPase (SERCA) is the sole mechanism for replenishing SR Ca 2ϩ stores, with two Ca 2ϩ ions pumped into the SR for every ATP consumed against a Ca 2ϩ gradient of ϳ1 M in the cytosol vs. 1 mM in the SR lumen (16,22). Thus, inflammation-induced increase in [Ca 2ϩ ] i may be mediated by interference with SERCA expression and function such that SR Ca 2ϩ reuptake is impaired. There is currently very limited data on the regulation of SERCA in ASM (7,17,30). In cardiac muscle, SERCA is normally modulated in an inhibitory fashion by the protein phospholamban (PLB) (33). Previous studies in bovine pulmonary artery (26) and murine gastrointestinal smooth muscle (14) have shown that, as in cardiac muscle, cyclic nucleotides, Ca 2ϩ /CaM-dependent protein kinase (CaMKII), and/or protein kinase C phosphorylate PLB, resulted in disinhibition of SERCA and accelerated SR Ca 2ϩ reuptake. However, in porcine coronary artery smooth muscle, while there is evidence of CaMKII phosphorylation of SERCA with accompanying increase in SERCA activity, PLB has not been detected in that tissue (12). In this regard, whether PLB itself regulates SERCA in ASM (as in cardiac or other smooth muscles) is not at all clear. We recently reported that porcine ASM does express PLB, that PLB regulation by CaMKII occurs, and decreased PLB expression (using small interference RNA; siRNA) increases SR Ca 2ϩ refilling following agonist exposure (30). However, in that study, we found that even with PLB suppression by siRNA, additional modulation of Ca 2ϩ reuptake can occur via CaMKII. Accordingly, the expression and contribution of PLB to SR Ca 2ϩ refilling may be tissue-and speciesspecific. In this regard, there is currently no information on SERCA regulation by PLB in human ASM. Furthermore, there is no information on alterations in SERCA with airway inflammation that could contribute to increased [Ca 2ϩ ] i and thus increased bronchoconstriction.In the present study, we hypothesized that inflammation leads to increased PLB expression and inhibition of SERCA in human ASM, thus decreasing SR Ca 2ϩ reuptake and contributing to inflammation-induced increase in [Ca 2ϩ

Unilateral denervation (Dnv) of the rat diaphragm muscle (Diam) markedly alters expression of myosin heavy chain (MHC) isoforms. After 2 wk of Diam Dnv, MHC content per half-sarcomere decreases in fibers expressing MHC(2X) and MHC(2B). We hypothesized that changes in MHC protein expression parallel changes in MHC mRNA expression. Relative MHC isoform mRNA levels were determined by Northern analysis after 1, 3, 7, and 14 days of Dnv of the rat Diam. MHC protein expression was determined by SDS-PAGE. Changes in MHC isoform protein and mRNA expression were not concurrent. Expression of MHC(Slow) and MHC(2X) mRNA isoforms decreased dramatically by 3 days of Dnv, whereas that of MHC(2A) and MHC(2B) did not change. Expression of all MHC protein isoforms decreased by 3 days of Dnv. We observed a differential effect of rat Diam Dnv on MHC isoform protein and mRNA expression. The time course of the changes in MHC isoform mRNA and protein expression suggests a predominant effect of altered protein turnover rates on MHC protein expression instead of altered transcription after Dnv.

real-time reverse transcriptase-polymerase chain reaction; electrophoresis; myosin heavy chain gene regulation; muscle plasticity IN THE RAT DIAPHRAGM MUSCLE (Dia m ), significant changes in myosin heavy chain (MHC) isoform expression occur during the first 4 wk of postnatal development (22-24, 38, 39, 41). Most fibers express protein for the MHC Neo isoform at birth (P-0), but the relative expression of this isoform gradually decreases until it disappears by postnatal day 28 (P-28). In contrast, MHC 2X and MHC 2B protein isoforms are not expressed in the rat Dia m until at least the second postnatal week. Postnatal development of the rat Dia m is also characterized by changes in muscle contractile properties and by significant growth of muscle fibers, most notably those expressing MHC 2X and MHC 2B (15,22,36,38,42,43,45,47). Thus the relative MHC isoform composition changes during postnatal development of the Dia m , and ultimately it determines Dia m function. At the present time, very little is known about the underlying mechanisms regulating MHC phenotype expression.Previous studies of postnatal development, both in limb muscles and the Dia m , have focused exclusively on changes in the relative protein expression of the different MHC isoforms in the whole muscle (9, 11, 22-24, 30, 31, 33, 38, 39, 45). However, during postnatal development of the rat Dia m , MHC content (measured by MHC mass per half-sarcomere volume and fiber cross-sectional area) increases markedly (15). This increase varies across fiber types (classified based on MHC isoform expression). At single Dia m fibers, the developmental increase in MHC content is greater in fibers expressing MHC 2X and/or MHC 2B isoforms than in those expressing MHC Slow or MHC 2A . Thus, during postnatal development, not only do Dia m fibers increase in size but also they increase their MHC protein content. This substantial increase in total MHC protein expression across Dia m fiber types is not evident if only the relative expression of MHC isoform is examined.The period from birth to adulthood provides a unique and significant opportunity to examine the mechanisms underlying rapid muscle growth and phenotype transitions. In rat limb muscles, postnatal muscle phenotype is regulated by MHC gene expression either as a result from mechanical stretch induced by skeletal growth and/or increased muscle activity (18). At present, there is no direct information regarding the transcriptional regulation of MHC isoform expression during postnatal development of the rat Dia m .An increase in MHC protein of the rat Dia m during postnatal development could be due to transcriptional, translational, and/or posttranslational mechanisms, which may vary depending on fiber type (10). Assuming transcriptional rate remains constant, an increase in mRNA expression would result in increased protein expression. For example, patterns of MHC isoform-specific mRNA and protein expression are very similar during postnatal development in pigs (25). We hypothesized that postnatal transitions ...

Oxytocin-induced Ca2+ transients play an important role in myometrial contractions. Here, using a knockout model, we found that the enzyme CD38, responsible for the synthesis of the second messenger cyclic ADP-ribose (cADPR), plays an important role in the oxytocin-induced Ca2+ transients and contraction. We also observed that CD38 is necessary for TNF-α-increased agonist-stimulated Ca2+ transients in human myometrial cells. We provide experimental evidence that the TNF-α effect is mediated by increased expression of the enzyme CD38. First, we observed that TNF-α increased oxytocin-induced Ca2+ transients and CD38 expression in human myometrial cells. Moreover, using small interference RNA technology, we observed that TNF-α stimulation of agonist-induced Ca2+ transients was abolished by blocking the expression of CD38. In control experiments, we observed that activation of the component of the TNF-α signaling pathway, NF-κB, was not affected by the treatments. Finally, we observed that the effects of TNF-α on CD38 cyclase and oxytocin-induced Ca2+ transients are abolished by progesterone. In conclusion, we provide the first experimental evidence that CD38 is important for myometrial Ca2+ transients and contraction. Moreover, CD38 is necessary for the TNF-α-mediated augmentation of agonist-induced Ca2+ transients in myometrial cells. We propose that the balance between cytokines and placental steroids regulates the expression of CD38 in vivo and cell responsiveness to oxytocin.