Biomedical subjects

Gary C Sieck

Publications and source records attributed to Gary C Sieck.

At least 19 recordsLinked to original sources

PubMed2007

Pkd2+/- vascular smooth muscles develop exaggerated vasocontraction in response to phenylephrine stimulation.

Vascular complications are the leading cause of morbidity and mortality in autosomal dominant polycystic kidney disease. Although evidence suggests an abnormal vascular reactivity, contractile function in Pkd mutant vessels has not been studied previously. Contractile response to phenylephrine (PE; 10(-10) to 10(-4)M), an alpha1-adrenergic receptor agonist, was examined. De-endothelialized Pkd2(+/-) aortic rings generated a higher maximum force (F(max)) than that in wild-type (wt; 5.78 +/- 0.73 versus 2.69 +/- 0.43 mN; P < 0.001) and a significant left shift in PE dosage-response curve. On simultaneous recordings, Pkd2(+/-) aortic helical strips also responded to PE with a greater F(max) but a lesser [Ca(2+)](i) rise, resulting in a greatly enhanced Deltaforce/DeltaCa(2+) ratio than that in wt. At F(max), a higher elevation in the phosphorylated regulatory myosin light chain was observed in Pkd2(+/-) strips. Ca(2+)-dependent calmodulin/myosin light-chain kinase-mediated contraction was examined by direct Ca(2+) (pCa8-5) stimulation to beta-escin permeabilized aortic strips; the pCa-force curve in Pkd2(+/-) strips was not shifted, thereby indicating that PE induced dosage-response alteration that resulted from Ca(2+)-independent mechanisms. Quantitative analyses of contractile proteins demonstrated elevated expressions in smooth muscle alpha-actin and myosin heavy chain in Pkd2(+/-) arteries, changes that likely contribute to the higher F(max). Similar to those in aortas, de-endothelialized Pkd2(+/-) resistance (fourth-order mesenteric) arteries responded to PE with a stronger contraction but a lesser [Ca(2+)](i) rise than in wt. Taken together, the arterial vasculature in Pkd2(+/-) mice exhibits an exaggerated contractile response and increased sensitivity to PE. An enhanced Ca(2+)-independent force generation and elevated contractile protein expression likely contribute to these abnormalities.

Animals↗

PubMed2006

Endovascular treatment of experimental aneurysms by use of fibroblast-coated platinum coils: an angiographic and histopathologic study.

BACKGROUND AND PURPOSE: The purpose of this study was to determine whether implanting exogenous fibroblasts on platinum coils could enhance intra-aneurysmal fibrosis. Hypotheses included: (1) fibroblast-coated (FBC) platinum coils can improve angiographic results after embolization; and (2) FBC platinum coils can accelerate histological healing of embolized aneurysms. METHODS: Experimental aneurysms in rabbits were embolized with control platinum coils (n=18) or FBC coils (n=18). Subjects were euthanized at 14 days, 1 month, 3 months and 6 months after implantation. Digital subtraction angiography was used to evaluate stability after embolization. Histological samples were examined with a grading system (range, 0 to 12) based on neck and dome healing. RESULTS: Histology total scores and fibrosis ratio at 14 days were significantly greater in the FBC coil group compared with controls (6.6+/-1.9 versus 2.5+/-1.1, 1.2+/-0.6% versus 0.2+/-0.3%, respectively; P=0.0090). Cavities embolized with FBC coils showed cellular proliferation and thrombus organization, with an endothelialized membrane bridging the neck. There were no differences between groups in the later timepoints. The FBC coil group showed radiographic stability in 11 (61%) cases, coil compaction in 2 (11%) cases, and progressive occlusion in 5 (28%) cases. No progressive occlusion was seen in controls; 3 (17%) of 18 control cases exhibited coil compaction (P=0.0546). CONCLUSIONS: FBC coils can accelerate early histological healing compared with control coils in the rabbit aneurysm model.

Actins↗

PubMed2006

Mechanisms underlying myosin heavy chain expression during development of the rat diaphragm muscle.

During early postnatal development in rat diaphragm muscle (Dia(m)), significant transitions in myosin heavy chain (MHC) isoform expression occur that are associated with fiber growth and increased MHC protein. At present, there is no direct information regarding the transcriptional regulation of MHC isoform expression during postnatal Dia(m) development. We hypothesized postnatal changes in MHC isoform mRNA expression are followed by concomitant changes in MHC protein expression. The Dia(m) was removed at postnatal days 0, 14, 28, and 84 (adult). MHC mRNA expression was determined by real-time RT-PCR. MHC protein expression was determined by SDS-PAGE. There was a significant effect of postnatal age on MHC isoform mRNA and protein expression. At birth, the MHC(Neo) isoform accounted for 28% of MHC mRNA and 54% of total MHC protein. By postnatal day 14, MHC(Neo) mRNA and protein increased significantly, and both decreased significantly by day 28, consistent with transcriptional control of the expression of this developmental isoform. By postnatal day 28, there were minimal changes in mRNA expression for MHC(Slow) and MHC(2X), yet protein expression increased significantly. MHC(2A) mRNA and protein expression did not change during this time. Thus changes in MHC protein expression did not follow (or parallel) changes in MHC mRNA for the adult MHC isoforms. The present findings indicate that changes in MHC expression in the developing rat Dia(m) are not driven solely by changes in mRNA expression. Knowledge of isoform-specific MHC mRNA expression only yields predictive information on MHC protein expression for the MHC(Neo) isoform.

Aging↗

PubMed2006

Agonist-induced cyclic ADP ribose production in airway smooth muscle.

Cyclic ADP-ribose (cADPR) triggers sarcoplasmic reticulum (SR) Ca(2+) release in airway smooth muscle (ASM). SR Ca(2+) release is an important component of the intracellular Ca(2+) ([Ca(2+)](i)) response of ASM to agonists. Whether cADPR is endogenously produced in ASM during agonist stimulation has not been established. In this study, cADPR production was examined in acutely dissociated porcine ASM cells. ACh stimulation (> or = 1 microM) significantly increased cADPR levels, peaking between 30s and 1 min. This effect was inhibited by M(2) and M(3) muscarinic receptor antagonists. Histamine ((> or = 5 microM) increased cADPR levels to a greater extent than ACh, while diphenhydramine blocked histamine-induced cADPR elevation. Both bradykinin (100 nM) and endothelin-1 (100 nM) also increased cADPR levels to a greater extent than ACh or histamine. These results indicate that in porcine ASM, certain agonists acting via receptors increase cADPR levels. Furthermore, the extent of cADPR responses to agonist varies, possibly reflecting differences in G-protein coupling.

Acetylcholine↗

PubMed2006

Neuregulin-dependent protein synthesis in C2C12 myotubes and rat diaphragm muscle.

The nerve-derived trophic factor neuregulin (NRG) is a prime candidate molecule for modulating muscle fiber growth. NRG regulates signal transduction in skeletal muscle through activation of ErbB receptors present at the neuromuscular junction. In this study, we hypothesize that NRG increases protein synthesis in maturing muscle via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. NRG signal transduction and its ability to stimulate protein synthesis (measured by incorporation of [(3)H]phenylalanine into the protein pool) were investigated in differentiated C(2)C(12) myotubes and rat diaphragm muscle (DIAm). In C(2)C(12) myotubes, NRG dose dependently increased phosphorylation of ErbB3 and recruitment of the p85 subunit of PI3K. NRG also increased phosphorylation of Akt, a downstream effector of PI3K. NRG treatment increased total protein synthesis by 35% compared with untreated control myotubes. This NRG-induced increase in Akt phosphorylation and protein synthesis was completely blocked by wortmannin, an inhibitor of PI3K but was unaffected by PD-98059, an inhibitor of MEK. In DIAm obtained from 3-day-old rat pups, Akt phosphorylation increased approximately 30-fold with NRG treatment (vs. untreated DIAm). NRG treatment also significantly increased protein synthesis in the DIAm by 29% after 3 h of incubation with [(3)H]phenylalanine (vs. untreated DIAm). Pretreatment with wortmannin abolished the NRG-induced increase in protein synthesis, suggesting a critical role for PI3K in this response. The results of the present study support the hypothesis that nerve-derived NRG contributes to the regulation of skeletal muscle mass by increasing protein synthesis via activation of PI3K.

Androstadienes↗

PubMed2006

Role of transient receptor potential C3 in TNF-alpha-enhanced calcium influx in human airway myocytes.

Previous studies have suggested that the proinflammatory cytokine, TNF-alpha, contributes to airway hyperresponsivness by altering airway smooth muscle (ASM) Ca(2+) responses to agonist stimulation. The present study examined the effects of TNF-alpha on Ca(2+) influx pathways in cultured human ASM cells (HASMCs). Proteins encoded by the transient receptor potential (TRP) gene family function as channels through which receptor-operated and store-operated Ca(2+) entry (SOCE) occur. In the present study, the presence of TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 mRNA and protein expression was confirmed in cultured HASMCs using RT-PCR and Western blot analysis. TNF-alpha treatment significantly increased TRPC3 mRNA and protein levels in HASMCs as well as SOCE. TNF-alpha treatment also increased both the peak and plateau intracellular Ca(2+) concentration responses in HASMCs elicited by acetylcholine and bradykinin. The effects of TNF-alpha treatment on SOCE and agonist-induced intracellular Ca(2+) concentration responses were attenuated using small interfering RNA transfection, which knocked down TRPC3 expression. Thus, in inflammatory airway diseases, TNF-alpha treatment may result in increased myocyte activation due to altered Ca(2+) influx pathways. These results suggest that TRPC3 may be an important therapeutic target in inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease.

Bronchi↗

PubMed2006

Denervation effects on myonuclear domain size of rat diaphragm fibers.

Denervation (DNV) of rat diaphragm muscle (DIAm) leads to selective atrophy of type IIx and IIb fibers, whereas the cross-sectional area of type I and IIa fibers remains unchanged or slightly hypertrophied. DIAm DNV also increases satellite cell mitotic activity and myonuclear apoptosis. Similar to other skeletal muscles, DIAm fibers are multinucleated, and each myonucleus regulates the gene products in a finite fiber volume, i.e., myonuclear domain (MND). MND size varies across DIAm fiber types in rank order, I < IIa < IIx < IIb [fiber type based on myosin heavy chain isoform expression]. We hypothesized that, after DNV, the total number of myonuclei per fiber does not change and, accordingly, that MND changes proportionately to the change in fiber size regardless of fiber type. Adult rats underwent unilateral (right side) DIAm DNV, and after 2 wk single fibers were dissected. Fiber cross-sectional area, myonuclear number, and MND were measured by confocal microscopy, and these values in DNV DIAm were compared with those obtained in controls. After DNV, type I fibers hypertrophied, type IIa fiber size was unchanged, and type IIx and IIb fibers atrophied compared with control. The total number of myonuclei per fiber was not affected by DNV. Accordingly, after DNV, type I fiber MND increased by 25%, whereas it decreased in type IIx and IIb fibers by 50 and 70%, respectively. These results suggest that MND is not maintained after DNV-induced DIAm fiber hypertrophy or atrophy. These results are interpreted with respect to consequent effects of DNV on myonuclear transcriptional activity and protein turnover.

Animals↗

PubMed2006

Store-operated Ca2+ influx in airway smooth muscle: Interactions between volatile anesthetic and cyclic nucleotide effects.

BACKGROUND: Volatile anesthetics produce bronchodilation in part by depleting sarcoplasmic reticulum Ca stores in airway smooth muscle (ASM). Other bronchodilatory drugs are known to act via cyclic nucleotides (cyclic adenosine 3',5'-cyclic monophosphate, cyclic guanosine 3',5'-cyclic monophosphate). Intracellular Ca regulation in ASM involves plasma membrane Ca influx, including that triggered by sarcoplasmic reticulum Ca depletion (store-operated Ca entry [SOCE]). The authors hypothesized that anesthetics and bronchodilatory agents interact in inhibiting SOCE, thus enhancing ASM relaxation. METHODS: In enzymatically dissociated porcine ASM cells imaged using fluorescence microscopy, sarcoplasmic reticulum Ca was depleted by 1 microm cyclopiazonic acid in 0 extracellular Ca, nifedipine, and potassium chloride (preventing Ca influx through L-type channels and SOCE). Extracellular Ca was rapidly reintroduced to selectively activate SOCE in the presence or absence of 1 minimum alveolar concentration (MAC) halothane, isoflurane, or sevoflurane. Anesthetic interference with SOCE regulation by cyclic nucleotides was examined by activating SOCE in the presence of (1) 1 microm acetylcholine, (2) 100 microm dibutryl cyclic adenosine 3',5'-cyclic monophosphate, or (3) 100 microm 8-bromo-cyclic guanosine 3',5'-cyclic monophosphate. RESULTS: SOCE was enhanced by acetylcholine, whereas volatile anesthetics and both cyclic nucleotides partially inhibited Ca influx. Preexposure to 1 or 2 MAC anesthetic (halothane > isoflurane > sevoflurane) inhibited SOCE. Only halothane and isoflurane inhibited acetylcholine-induced augmentation of Ca influx, and significantly potentiated cyclic nucleotide inhibition such that no influx was observed in the presence of anesthetics and cyclic nucleotides. CONCLUSIONS: These data indicate that volatile anesthetics prevent sarcoplasmic reticulum refilling by inhibiting SOCE and enhancing cyclic nucleotide blunting of Ca influx in ASM. Such interactions likely result in substantial airway relaxation in the presence of both anesthetics and bronchodilatory agents such as beta agonists or nitric oxide.

Acetylcholine↗

PubMed2005

Elevated blood pressure and cardiac hypertrophy after ablation of the gly96/IEX-1 gene.

gly96/IEX 1 is a growth- and apoptosis-regulating, immediate early gene that is widely expressed in epithelial and vascular tissues. In vascular tissues, expression of the gene is induced by mechanical stretch, and overexpression of the gene prevents injury-induced vascular smooth muscle hypertrophy and neointimal hyperplasia. We now show that deletion of the gly96/IEX-1 gene in mice is associated with development of elevated blood pressure, cardiac hypertrophy, and diminished fractional shortening of the left ventricle. Systolic blood pressure in conscious male gly96/IEX-1-/- mice is 20-25 mmHg higher than in gly96/IEX-1+/+ mice. Serum and/or urine concentrations of sodium, potassium, creatinine, angiotensin II, corticosterone, aldosterone, epinephrine, norepinephrine, prostaglandin E2, thromboxane B2, prostaglandin-6-keto-1alpha, nitrites and nitrates, cAMP, and cGMP are normal in gly96/IEX-1-/- mice. Alterations in dietary sodium intake do not alter blood pressure in gly96/IEX-1-/- mice. Aortic mRNAs for endothelial nitric oxide synthase, guanylate cyclase-alpha, and cGMP kinase-1 are increased in gly96/IEX-1-/- mice. Treatment with Nomega-nitro-L-arginine methyl ester or L-arginine does not alter blood pressure in gly96/IEX-1-/- mice. Gly96/IEX-1-/- mice respond to infused sodium nitroprusside with decrements in blood pressure similar to those seen in wild-type littermate mice. In contrast to gly96/IEX-1 transgenic mice that have abnormalities in immune function, gly96/IEX-1-/- mice have normal lymphoid tissue architecture and a normal complement of T and B cells in lymphoid tissues. Ablation of the gly96/IEX-1 gene results in hypertension and cardiac hypertrophy, suggesting a novel role for this gene in cardiovascular physiology.

Animals↗

PubMed2005

Cyclic nucleotide regulation of store-operated Ca2+ influx in airway smooth muscle.

Sarcoplasmic reticulum (SR) Ca2+ release and plasma membrane Ca2+ influx are key to intracellular Ca2+ ([Ca2+]i) regulation in airway smooth muscle (ASM). SR Ca2+ depletion triggers influx via store-operated Ca2+ channels (SOCC) for SR replenishment. Several clinically relevant bronchodilators mediate their effect via cyclic nucleotides (cAMP, cGMP). We examined the effect of cyclic nucleotides on SOCC-mediated Ca2+ influx in enzymatically dissociated porcine ASM cells. SR Ca2+ was depleted by 1 microM cyclopiazonic acid in 0 extracellular Ca2+ ([Ca2+]o), nifedipine, and KCl (preventing Ca2+ influx through L-type and SOCC channels). SOCC was then activated by reintroduction of [Ca2+]o and characterized by several techniques. We examined cAMP effects on SOCC by activating SOCC in the presence of 1 microM isoproterenol or 100 microM dibutryl cAMP (cell-permeant cAMP analog), whereas we examined cGMP effects using 1 microM (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO nitric oxide donor) or 100 microM 8-bromoguanosine 3',5'-cyclic monophosphate (cell-permeant cGMP analog). The role of protein kinases A and G was examined by preexposure to 100 nM KT-5720 and 500 nM KT-5823, respectively. SOCC-mediated Ca2+ influx was dependent on the extent of SR Ca2+ depletion, sensitive to Ni2+ and La3+, but not inhibitors of voltage-gated influx channels. cAMP as well as cGMP potently inhibited Ca2+ influx, predominantly via their respective protein kinases. Additionally, cAMP cross-activation of protein kinase G contributed to SOCC inhibition. These data demonstrate that a Ni2+/La3+-sensitive Ca2+ influx in ASM triggered by SR Ca2+ depletion is inhibited by cAMP and cGMP via a protein kinase mechanism. Such inhibition may play a role in the bronchodilatory response of ASM to clinically relevant drugs (e.g., beta-agonists vs. nitric oxide).

Animals↗

PubMed2005

Respiratory muscle plasticity.

Plasticity of respiratory muscles must be considered in the context of their unique physiological demands. The continuous rhythmic activation of respiratory muscles makes them among the most active in the body. Respiratory muscles, especially the diaphragm, are non-weight-bearing, and thus, in contrast to limb muscles, are not exposed to gravitational effects. Perturbations in normal activation and load known to induce plasticity in limb muscles may not cause similar adaptations in respiratory muscles. In this review, we explore the structural and functional properties of the diaphragm muscle and their response to alterations in load and activity. Overall, relatively modest changes in diaphragm structural and functional properties occur in response to perturbations in load or activity. However, disruptions in the normal influence of phrenic innervation by frank denervation, tetrodotoxin nerve block and spinal hemisection, induce profound changes in the diaphragm, indicating the substantial trophic influence of phrenic motoneurons on diaphragm muscle.

Adaptation, Physiological↗

PubMed2005

Diaphragm dysfunction in chronic obstructive pulmonary disease.

RATIONALE: Hypercapnic respiratory failure because of inspiratory muscle weakness is the most important cause of death in chronic obstructive pulmonary disease (COPD). However, the pathophysiology of failure of the diaphragm to generate force in COPD is in part unclear. OBJECTIVES: The present study investigated contractile function and myosin heavy chain content of diaphragm muscle single fibers from patients with COPD. METHODS: Skinned muscle fibers were isolated from muscle biopsies from the diaphragm of eight patients with mild to moderate COPD and five patients without COPD (mean FEV(1) % predicted, 70 and 100%, respectively). Contractile function of single fibers was assessed, and afterwards, myosin heavy chain content was determined in these fibers. In diaphragm muscle homogenates, the level of ubiquitin-protein conjugation was determined. RESULTS: Diaphragm muscle fibers from patients with COPD showed reduced force generation per cross-sectional area, and reduced myosin heavy chain content per half sarcomere. In addition, these fibers had decreased Ca2+ sensitivity of force generation, and slower cross-bridge cycling kinetics. Our observations were present in fibers expressing slow and 2A isoforms of myosin heavy chain. Ubiquitin-protein conjugation was increased in diaphragm muscle homogenates of patients with mild to moderate COPD. CONCLUSIONS: Early in the development of COPD, diaphragm fiber contractile function is impaired. Our data suggest that enhanced diaphragm protein degradation through the ubiquitin-proteasome pathway plays a role in loss of contractile protein and, consequently, failure of the diaphragm to generate force.

Adult↗

PubMed2005

[Ca2+]i reduction increases cellular proliferation and apoptosis in vascular smooth muscle cells: relevance to the ADPKD phenotype.

Cardiovascular complications are the leading cause of morbidity and mortality in autosomal dominant polycystic kidney disease. Pkd2+/- vascular smooth muscle cells (VSMCs) have an abnormal phenotype and defective intracellular Ca2+ ([Ca2+]i) regulation. We examined cAMP content in vascular smooth muscles from Pkd2+/- mice because cAMP is elevated in cystic renal epithelial cells. We found cAMP concentration was significantly increased in Pkd2+/- vessels compared with wild-type vessels. Furthermore, reducing the wild-type VSMC [Ca2+]i by Verapamil or BAPTA-AM significantly increased cellular cAMP concentration (mainly by phosphodiesterase [PDE] inhibition), the rate of VSMC proliferation (determined by direct cell counting, 3H-incorporation, FACS analysis of cells entering S phase, and quantitative Western PCNA and ERK1/2 analyses), and the rate of apoptosis (by Hoechst staining, FACS analysis of the Annexin-V positive cells, and quantitative Western Bax, cytochrome c, and activated caspase 9 and 3 analyses). The low [Ca2+]i induced VSMC proliferation was independent of cAMP/B-Raf signaling, while that of apoptosis was promoted by cAMP. In summary, Pkd2+/- VSMCs have elevated cAMP levels. This elevation can also be induced by reducing [Ca2+]i in wild-type VSMCs. The [Ca2+]i reduction and cAMP accumulation can cause an increase in both cellular proliferation and apoptosis, resembling Pkd mutant phenotype.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2005

Caveolae targeting and regulation of large conductance Ca(2+)-activated K+ channels in vascular endothelial cells.

The vascular endothelium is richly endowed with caveolae, which are specialized membrane microdomains that facilitate the integration of specific cellular signal transduction processes. We found that the large conductance Ca(2+)-activated K+ (BK) channels are associated with caveolin-1 in bovine aortic endothelial cells (BAECs). OptiPrep gradient cell fractionation demonstrated that BK channels were concentrated in the caveolae-rich fraction in BAECs. Immunofluorescence imaging showed co-localization of caveolin-1 and BK channels in the BAEC membrane. Immunoprecipitation and glutathione S-transferase pull-down assay results indicated that caveolin-1 and BK channels are physically associated. However, whole cell patch clamp recordings could not detect BK (iberiotoxin-sensitive) currents in cultured BAECs under baseline conditions, even though the presence of BK mRNA and protein expression was confirmed by reverse transcription-PCR and Western blots. Cholesterol depletion redistributed the BK channels to non-caveolar fractions of BAECs, resulting in BK channel activation (7.3 +/- 1.6 pA/picofarad (pF), n = 5). BK currents were also activated by isoproterenol (ISO, 1 microM, 6.9 +/- 2.4 pA/pF, n = 6). Inclusion of a caveolin-1 scaffolding domain peptide (10 microM) in the pipette solution completely abrogated the effects of ISO on BK channel activation, whereas inclusion of the scrambled control peptide (10 microM) did not inhibit the ISO effects. We have also found that caveolin-1 knockdown by small interference RNA activated BK currents (5.3 +/- 1.4 pA/pF, n = 6). We conclude that: 1) BK channels are targeted to caveolae microdomains in vascular endothelial cells; 2) caveolin-1 interacts with BK channels and exerts a negative regulatory effect on channel functions; and 3) BK channels are inactive under control conditions but can be activated by cholesterol depletion, knockdown of caveolin-1 expression, or ISO stimulation. These novel findings may have important implications for the role of BK channels in the regulation of endothelial function.

Amino Acid Sequence↗

PubMed2004

Role of CD38 in myometrial Ca2+ transients: modulation by progesterone.

Oxytocin-induced Ca(2+) 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 Ca(2+) transients and contraction. We also observed that CD38 is necessary for TNF-alpha-increased agonist-stimulated Ca(2+) transients in human myometrial cells. We provide experimental evidence that the TNF-alpha effect is mediated by increased expression of the enzyme CD38. First, we observed that TNF-alpha increased oxytocin-induced Ca(2+) transients and CD38 expression in human myometrial cells. Moreover, using small interference RNA technology, we observed that TNF-alpha stimulation of agonist-induced Ca(2+) transients was abolished by blocking the expression of CD38. In control experiments, we observed that activation of the component of the TNF-alpha signaling pathway, NF-kappaB, was not affected by the treatments. Finally, we observed that the effects of TNF-alpha on CD38 cyclase and oxytocin-induced Ca(2+) transients are abolished by progesterone. In conclusion, we provide the first experimental evidence that CD38 is important for myometrial Ca(2+) transients and contraction. Moreover, CD38 is necessary for the TNF-alpha-mediated augmentation of agonist-induced Ca(2+) 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.

ADP-ribosyl Cyclase↗

PubMed2004

Influence of corticosteroids on myonuclear domain size in the rat diaphragm muscle.

Skeletal muscle fibers are multinucleated. Each myonucleus regulates gene products and protein expression in only a restricted portion of the muscle fiber, the myonuclear domain (MND). In the rat diaphragm muscle (DIAm), corticosteroid (CoS) treatment causes atrophy of fibers containing myosin heavy chain (MHC): MHC2X and/or MHC2B. We hypothesized that DIAm fiber MND size is maintained during CoS-induced atrophy. Adult male rats received methylprednisolone for 11 days at 1 (CoS-Low, n = 8) or 8 mg x kg(-1) x day(-1) (CoS-High, n = 8). Age-matched (CTL-AgeM, n = 8), sham-operated (SHAM-AgeM, n = 8), and weight-matched (CTL-WtM, n = 8) animals served as controls. In single DIAm fibers, cross-sectional area (CSA), MND size, and MHC expression were determined. Fiber CSA and MND size were similar in CTL-AgeM and SHAM-AgeM groups. Only fibers containing MHCslow or MHC2A displayed smaller CSA in CTL-WtM than in CTL-AgeM and SHAM-AgeM groups, and MND size was reduced in all fibers. Thus fibers containing MHCslow and MHC2A maintain the number of myonuclei, whereas MHC2X or MHC2B fibers show loss of myonuclei during normal muscle growth. Both CoS groups displayed smaller CSA and MND size than CTL-AgeM and SHAM-AgeM groups. However, compared with CTL-WtM DIAm fibers, only fibers containing MHC2X or MHC2B displayed reduced CSA and MND size after CoS treatment. Thus little, if any, loss of myonuclei was associated with CoS-induced atrophy of MHC2X or MHC2B DIAm fibers. In summary, MND size does not appear to be regulated during CoS-induced DIAm atrophy.

Animals↗

PubMed2004

Neurotrophins improve neuromuscular transmission in the adult rat diaphragm.

Neurotrophins modulate acute and sustained synaptic plasticity. In cultured Xenopus laevis neuromuscular junctions, neurotrophins improve neuromuscular transmission. Whether this influence exists at the mammalian neuromuscular junction is unknown. We hypothesized that neurotrophins improve neuromuscular transmission at neuromuscular junctions of adult rat diaphragm muscle fibers. A diaphragm muscle-phrenic nerve preparation was used to determine the effects of brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4) and K252a [tyrosine kinase (Trk) receptor inhibitor] on the extent of neuromuscular transmission failure induced by repetitive nerve stimulation. We found significant enhancement of neuromuscular transmission with BDNF or NT-4 treatment, whereas K252a treatment worsened neuromuscular transmission. In contrast, diaphragm muscle contractile and fatigue properties were unaffected by neurotrophin or K252a treatment. These results demonstrate that BDNF and NT-4 improve synaptic transmission in the adult rat diaphragm muscle, likely in a Trk-dependent fashion. Neurotrophins may constitute a novel therapeutic target to improve neuromuscular function in the diaphragm.

Animals↗

PubMed2004

Synaptic vesicle cycling at type-identified diaphragm neuromuscular junctions.

Differences in neuromuscular transmission and neuromuscular junction morphology exist across muscle fiber types. We hypothesized that these fiber-type differences are reflected in the size of the cycling synaptic vesicle pool. Synaptic vesicle cycling at type-identified rat diaphragm neuromuscular junctions was examined by fluorescently labeling presynaptic vesicles with FM4-64. We found that FM4-64 fluorescence uptake was higher at presynaptic terminals of type I/IIa fibers than type IIx/IIb fibers. However, no fiber-type differences in the rate of FM4-64 destaining were found with repetitive nerve stimulation. Synaptic vesicle density at active zones was examined by transmission electron microscopy. In accordance with FM4-64 uptake, synaptic vesicle density was greater at type I/IIa than IIx/IIb fibers. These results demonstrate differences in synaptic vesicle cycling across diaphragm muscle fiber types, which may underlie previously observed differences in neuromuscular transmission across diaphragm muscle fiber types. In the diaphragm, motor units comprising type I and type IIa fibers are most frequently recruited with a duty cycle of approximately 40%. Motor units comprising IIx/IIb fibers are infrequently recruited and only for short durations. The capacity for synaptic vesicle release and cycling at different muscle fiber types matches the functional requirements of these motor units. If the demand for recruitment of motor units comprising IIx/IIb fibers increases, for example, with mechanical loading, there is an increased risk for neuromuscular transmission failure that my relate to the capacity for synaptic vesicle release and cycling. Muscle fiber type-specific adaptations should be considered when examining neuromuscular disorders.

Animals↗