Acute lead-induced vasoconstriction in the vascular beds of isolated perfused rat tails is endothelium-dependent

Chronic lead exposure induces hypertension in humans and animals, affecting endothelial function. However, studies concerning acute cardiovascular effects are lacking. We investigated the effects of acute administration of a high concentration of lead acetate (100 µΜ) on the pressor response to phenylephrine (PHE) in the tail vascular bed of male Wistar rats. Animals were anesthetized with sodium pentobarbital and heparinized. The tail artery was dissected and cannulated for drug infusion and mean perfusion pressure measurements. Endothelium and vascular smooth muscle relaxation were tested with acetylcholine (5 µg/100 µL) and sodium nitroprusside (0.1 µg/100 µL), respectively, in arteries precontracted with 0.1 µM PHE. Concentration-response curves to PHE (0.001-300 µg/100 µL) were constructed before and after perfusion for 1 h with 100 µΜ lead acetate. In the presence of endothelium (E+), lead acetate increased maximal response (Emax) (control: 364.4 ± 36, Pb2+: 480.0 ± 27 mmHg; P < 0.05) and the sensitivity (pD2; control: 1.98 ± 0.07, 2.38 ± 0.14 log mM) to PHE. In the absence of endothelium (E-) lead had no effect but increased baseline perfusion pressure (E+: 79.5 ± 2.4, E-: 118 ± 2.2 mmHg; P < 0.05). To investigate the underlying mechanisms, this protocol was repeated after treatment with 100 µM L-NAME, 10 µM indomethacin and 1 µM tempol in the presence of lead. Lead actions on Emax and pD2 were abolished in the presence of indomethacin, and partially abolished with L-NAME and tempol. Results suggest that acute lead administration affects the endothelium, releasing cyclooxygenase-derived vasoconstrictors and involving reactive oxygen species.

Gadolinium increases the vascular reactivity of rat aortic rings

Gadolinium (Gd) blocks intra- and extracellular ATP hydrolysis. We determined whether Gd affects vascular reactivity to contractile responses to phenylephrine (PHE) by blocking aortic ectonucleoside triphosphate diphosphohydrolase (E-NTPDase). Wistar rats of both sexes (260-300 g, 23 females, 7 males) were used. Experiments were performed before and after incubation of aortic rings with 3 µM Gd. Concentration-response curves to PHE (0.1 nM to 0.1 mM) were obtained in the presence and absence of endothelium, after incubation with 100 µM L-NAME, 10 µM losartan, or 10 µM enalaprilat. Gd significantly increased the maximum response (control: 72.3 ± 3.5; Gd: 101.3 ± 6.4%) and sensitivity (control: 6.6 ± 0.1; Gd: 10.5 ± 2.8%) to PHE. To investigate the blockade of E-NTDase activity by Gd, we added 1 mM ATP to the bath. ATP reduced smooth muscle tension and Gd increased its relaxing effect (control: -33.5 ± 4.1; Gd: -47.4 ± 4.1%). Endothelial damage abolished the effect of Gd on the contractile responses to PHE (control: 132.6 ± 8.6; Gd: 122.4 ± 7.1%). L-NAME + Gd in the presence of endothelium reduced PHE contractile responses (control/L-NAME: 151.1 ± 28.8; L-NAME + Gd: 67.9 ± 19% AUC). ATP hydrolysis was reduced after Gd administration, which led to ATP accumulation in the nutrient solution and reduced ADP concentration, while adenosine levels remained the same. Incubation with Gd plus losartan and enalaprilat eliminated the pressor effects of Gd. Gd increased vascular reactivity to PHE regardless of the reduction of E-NTPDase activity and adenosine production. Moreover, the increased reactivity to PHE promoted by Gd was endothelium-dependent, reducing NO bioavailability and involving an increased stimulation of angiotensin-converting enzyme and angiotensin II AT1 receptors.

Effect of triiodothyronine on the maxilla and masseter muscles of the rat stomatognathic system

The maxilla and masseter muscles are components of the stomatognathic system involved in chewing, which is frequently affected by physical forces such as gravity, and by dental, orthodontic and orthopedic procedures. Thyroid hormones (TH) are known to regulate the expression of genes that control bone mass and the oxidative properties of muscles; however, little is known about the effects of TH on the stomatognathic system. This study investigated this issue by evaluating: i) osteoprotegerin (OPG) and osteopontine (OPN) mRNA expression in the maxilla and ii) myoglobin (Mb) mRNA and protein expression, as well as fiber composition of the masseter. Male Wistar rats (~250 g) were divided into thyroidectomized (Tx) and sham-operated (SO) groups (N = 24/group) treated with T3 or saline (0.9%) for 15 days. Thyroidectomy increased OPG (~40%) and OPN (~75%) mRNA expression, while T3 treatment reduced OPG (~40%) and OPN (~75%) in Tx, and both (~50%) in SO rats. Masseter Mb mRNA expression and fiber type composition remained unchanged, despite the induction of hypo- and hyperthyroidism. However, Mb content was decreased in Tx rats even after T3 treatment. Since OPG and OPN are key proteins involved in the osteoclastogenesis inhibition and bone mineralization, respectively, and that Mb functions as a muscle store of O2 allowing muscles to be more resistant to fatigue, the present data indicate that TH also interfere with maxilla remodeling and the oxidative properties of the masseter, influencing the function of the stomatognathic system, which may require attention during dental, orthodontic and orthopedic procedures in patients with thyroid diseases.

PPM1B and P-IKKβ expression levels correlated inversely with rat gastrocnemius atrophy after denervation

Activated inhibitor of nuclear factor-κB kinase β (IKKβ) is necessary and sufficient for denervated skeletal muscle atrophy. Although several studies have shown that Mg2+/Mn2+-dependent protein phosphatase 1B (PPM1B) inactivated IKKβ, few studies have investigated the role of PPM1B in denervated skeletal muscle. In this study, we aim to explore the expression and significance of PPM1B and phosphorylated IKKβ (P-IKKβ) during atrophy of the denervated gastrocnemius. Thirty young adult female Wistar rats were subjected to right sciatic nerve transection and were sacrificed at 0 (control), 2, 7, 14, and 28 days after denervation surgery. The gastrocnemius was removed from both the denervated and the contralateral limb. The muscle wet weight ratio was calculated as the ratio of the wet weight of the denervated gastrocnemius to that of the contralateral gastrocnemius. RT-PCR and Western blot analysis showed that mRNA and protein levels of PPM1B were significantly lower than those of the control group at different times after the initiation of denervation, while P-IKKβ showed the opposite trends. PPM1B protein expression persistently decreased while P-IKKβ expression persistently increased for 28 days after denervation. PPM1B expression correlated negatively with P-IKKβ expression by the Spearman test, whereas decreasing PPM1B expression correlated positively with the muscle wet weight ratio. The expression levels of PPM1B and P-IKKβ were closely associated with atrophy in skeletal denervated muscle. These results suggest that PPM1B and P-IKKβ could be markers in skeletal muscle atrophy.

Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM) and a low sensitivity to vanadate (IC50 = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity.

Histone demethylase retinoblastoma binding protein 2 regulates the expression of α-smooth muscle actin and vimentin in cirrhotic livers

Liver cirrhosis is one of the most common diseases of Chinese patients. Herein, we report the high expression of a newly identified histone 3 lysine 4 demethylase, retinoblastoma binding protein 2 (RBP2), and its role in liver cirrhosis in humans. The siRNA knockdown of RBP2 expression in hepatic stellate cells (HSCs) reduced levels of α-smooth muscle actin (α-SMA) and vimentin and decreased the proliferation of HSCs; and overexpression of RBP2 increased α-SMA and vimentin levels. Treatment with transforming growth factor β (TGF-β) upregulated the expression of RBP2, α-SMA, and vimentin, and the siRNA knockdown of RBP2 expression attenuated TGF-β-mediated upregulation of α-SMA and vimentin expression and HSC proliferation. Furthermore, RBP2 was highly expressed in cirrhotic rat livers. Therefore, RBP2 may participate in the pathogenesis of liver cirrhosis by regulating the expression of α-SMA and vimentin. RBP2 may be a useful marker for the diagnosis and treatment of liver cirrhosis.

Expression of MMP-2 and MMP-9 in the rat trigeminal ganglion during the development of temporomandibular joint inflammation

Orofacial pain is a prevalent symptom in modern society. Some musculoskeletal orofacial pain is caused by temporomandibular disorders (TMDs). This condition has a multi-factorial etiology, including emotional factors and alteration of the masticator muscle and temporomandibular joints (TMJs). TMJ inflammation is considered to be a cause of pain in patients with TMD. Extracellular proteolytic enzymes, specifically the matrix metalloproteinases (MMPs), have been shown to modulate inflammation and pain. The purpose of this investigation was to determine whether the expression and level of gelatinolytic activity of MMP-2 and MMP-9 in the trigeminal ganglion are altered during different stages of temporomandibular inflammation, as determined by gelatin zymography. This study also evaluated whether mechanical allodynia and orofacial hyperalgesia, induced by the injection of complete Freund's adjuvant into the TMJ capsule, were altered by an MMP inhibitor (doxycycline, DOX). TMJ inflammation was measured by plasma extravasation in the periarticular tissue (Evans blue test) and infiltration of polymorphonuclear neutrophils into the synovial fluid (myeloperoxidase enzyme quantification). MMP expression in the trigeminal ganglion was shown to vary during the phases of the inflammatory process. MMP-9 regulated the early phase and MMP-2 participated in the late phase of this process. Furthermore, increases in plasma extravasation in periarticular tissue and myeloperoxidase activity in the joint tissue, which occurred throughout the inflammation process, were diminished by treatment with DOX, a nonspecific MMP inhibitor. Additionally, the increases of mechanical allodynia and orofacial hyperalgesia were attenuated by the same treatment.

Adrenomedullin and adrenotensin regulate collagen synthesis and proliferation in pulmonary arterial smooth muscle cells

To understand the pathophysiological mechanisms of pulmonary arterial smooth muscle cell (PASMC) proliferation and extracellular-matrix accumulation in the development of pulmonary hypertension and remodeling, this study determined the effects of different doses of adrenomedullin (ADM) and adrenotensin (ADT) on PASMC proliferation and collagen synthesis. The objective was to investigate whether extracellular signal-regulated kinase (ERK1/2) signaling was involved in ADM- and ADT-stimulated proliferation of PASMCs in 4-week-old male Wistar rats (body weight: 100-150 g, n=10). The proliferation of PASMCs was examined by 5-bromo-2-deoxyuridine incorporation. A cell growth curve was generated by the Cell Counting Kit-8 method. Expression of collagen I, collagen III, and phosphorylated ERK1/2 (p-ERK1/2) was evaluated by immunofluorescence. The effects of different concentrations of ADM and ADT on collagen I, collagen III, and p-ERK1/2 protein expression were determined by immunoblotting. We also investigated the effect of PD98059 inhibition on the expression of p-ERK1/2 protein by immunoblotting. ADM dose-dependently decreased cell proliferation, whereas ADT dose-dependently increased it; and ADM and ADT inhibited each other with respect to their effects on the proliferation of PASMCs. Consistent with these results, the expression of collagen I, collagen III, and p-ERK1/2 in rat PASMCs decreased after exposure to ADM but was upregulated after exposure to ADT. PD98059 significantly inhibited the downregulation by ADM and the upregulation by ADT of p-ERK1/2 expression. We conclude that ADM inhibited, and ADT stimulated, ERK1/2 signaling in rat PASMCs to regulate cell proliferation and collagen expression.

Effects of eugenol on resting tension of rat atria

In cardiac and skeletal muscle, eugenol (μM range) blocks excitation-contraction coupling. In skeletal muscle, however, larger doses of eugenol (mM range) induce calcium release from the sarcoplasmic reticulum. The effects of eugenol are therefore dependent on its concentration. In this study, we evaluated the effects of eugenol on the contractility of isolated, quiescent atrial trabeculae from male Wistar rats (250-300 g; n=131) and measured atrial ATP content. Eugenol (1, 3, 5, 7, and 10 mM) increased resting tension in a dose-dependent manner. Ryanodine [100 µM; a specific ryanodine receptor (RyR) blocker] and procaine (30 mM; a nonspecific RyR blocker) did not block the increased resting tension induced by eugenol regardless of whether extracellular calcium was present. The myosin-specific inhibitor 2,3-butanedione monoxime (BDM), however, reversed the increase in resting tension induced by eugenol. In Triton-skinned atrial trabeculae, in which all membranes were solubilized, eugenol did not change resting tension, maximum force produced, or the force vs pCa relationship (pCa=-log [Ca2+]). Given that eugenol reduced ATP concentration, the increase in resting tension observed in this study may have resulted from cooperative activation of cardiac thin filaments by strongly attached cross-bridges (rigor state).

Putative role of ischemic postconditioning in a rat model of limb ischemia and reperfusion: involvement of hypoxia-inducible factor-1α expression

Hypoxia-inducible factor-1α (HIF-1α) is one of the most potent angiogenic growth factors. It improves angiogenesis and tissue perfusion in ischemic skeletal muscle. In the present study, we tested the hypothesis that ischemic postconditioning is effective for salvaging ischemic skeletal muscle resulting from limb ischemia-reperfusion injury, and that the mechanism involves expression of HIF-1α. Wistar rats were randomly divided into three groups (n=36 each): sham-operated (group S), hindlimb ischemia-reperfusion (group IR), and ischemic postconditioning (group IPO). Each group was divided into subgroups (n=6) according to reperfusion time: immediate (0 h, T0), 1 h (T1), 3 h (T3), 6 h (T6), 12 h (T12), and 24 h (T24). In the IPO group, three cycles of 30-s reperfusion and 30-s femoral aortic reocclusion were carried out before reperfusion. At all reperfusion times (T0-T24), serum creatine kinase (CK) and lactate dehydrogenase (LDH) activities, as well as interleukin (IL)-6, IL-10, and tumor necrosis factor-α (TNF-α) concentrations, were measured in rats after they were killed. Histological and immunohistochemical methods were used to assess the skeletal muscle damage and HIF-1α expression in skeletal muscle ischemia. In groups IR and IPO, serum LDH and CK activities and TNF-α, IL-6, and IL-10 concentrations were all significantly increased compared to group S, and HIF-1α expression was up-regulated (P<0.05 or P<0.01). In group IPO, serum LDH and CK activities and TNF-α and IL-6 concentrations were significantly decreased, IL-10 concentration was increased, HlF-1α expression was down-regulated (P<0.05 or P<0.01), and the pathological changes were reduced compared to group IR. The present study suggests that ischemic postconditioning can reduce skeletal muscle damage caused by limb ischemia-reperfusion and that its mechanisms may be related to the involvement of HlF-1α in the limb ischemia-reperfusion injury-triggered inflammatory response.

β-Citronellol, an alcoholic monoterpene with inhibitory properties on the contractility of rat trachea

β-Citronellol is an alcoholic monoterpene found in essential oils such Cymbopogon citratus (a plant with antihypertensive properties). β-Citronellol can act against pathogenic microorganisms that affect airways and, in virtue of the popular use of β-citronellol-enriched essential oils in aromatherapy, we assessed its pharmacologic effects on the contractility of rat trachea. Contractions of isolated tracheal rings were recorded isometrically through a force transducer connected to a data-acquisition device. β-Citronellol relaxed sustained contractions induced by acetylcholine or high extracellular potassium, but half-maximal inhibitory concentrations (IC50) for K+-elicited stimuli were smaller than those for cholinergic contractions. It also inhibited contractions induced by electrical field stimulation or sodium orthovanadate with pharmacologic potency equivalent to that seen against acetylcholine-induced contractions. When contractions were evoked by selective recruitment of Ca2+ from the extracellular medium, β-citronellol preferentially inhibited contractions that involved voltage-operated (but not receptor-operated) pathways. β-Citronellol (but not verapamil) inhibited contractions induced by restoration of external Ca2+ levels after depleting internal Ca2+ stores with the concomitant presence of thapsigargin and recurrent challenge with acetylcholine. Treatment of tracheal rings with L-NAME, indomethacin or tetraethylammonium did not change the relaxing effects of β-citronellol. Inhibition of transient receptor potential vanilloid subtype 1 (TRPV1) or transient receptor potential ankyrin 1 (TRPA1) receptors with selective antagonists caused no change in the effects of β-citronellol. In conclusion, β-citronellol exerted inhibitory effects on rat tracheal rings, with predominant effects on contractions that recruit Ca2+ inflow towards the cytosol by voltage-gated pathways, whereas it appears less active against contractions elicited by receptor-operated Ca2+ channels.

Implantation of muscle satellite cells overexpressing myogenin improves denervated muscle atrophy in rats

This study evaluated the effect of muscle satellite cells (MSCs) overexpressing myogenin (MyoG) on denervated muscle atrophy. Rat MSCs were isolated and transfected with the MyoG-EGFP plasmid vector GV143. MyoG-transfected MSCs (MTMs) were transplanted into rat gastrocnemius muscles at 1 week after surgical denervation. Controls included injections of untransfected MSCs or the vehicle only. Muscles were harvested and analyzed at 2, 4, and 24 weeks post-transplantation. Immunofluorescence confirmed MyoG overexpression in MTMs. The muscle wet weight ratio was significantly reduced at 2 weeks after MTM injection (67.17±6.79) compared with muscles injected with MSCs (58.83±5.31) or the vehicle (53.00±7.67; t=2.37, P=0.04 and t=3.39, P=0.007, respectively). The muscle fiber cross-sectional area was also larger at 2 weeks after MTM injection (2.63×103±0.39×103) compared with MSC injection (1.99×103±0.58×103) or the vehicle only (1.57×103±0.47×103; t=2.24, P=0.049 and t=4.22, P=0.002, respectively). At 4 and 24 weeks post-injection, the muscle mass and fiber cross-sectional area were similar across all three experimental groups. Immunohistochemistry showed that the MTM group had larger MyoG-positive fibers. The MTM group (3.18±1.13) also had higher expression of MyoG mRNA than other groups (1.41±0.65 and 1.03±0.19) at 2 weeks after injection (t=2.72, P=0.04). Transplanted MTMs delayed short-term atrophy of denervated muscles. This approach can be optimized as a novel stand-alone therapy or as a bridge to surgical re-innervation of damaged muscles.

Adaptations of skeletal muscle pyruvate dehydrogenase kinase in response to food-restriction in mitochondrial subpopulations

University, 2006 Dr. Sandra J. Peters Pyruvate dehydrogenase (PDH) catalyses the decarboxylation of pyruvate, to form acetyl-CoA. PDH activity is down-regulated by intrinsic PDH kinases (predominantly PDK2 and PDK4 isoforms), but the understanding of the PDK isoform distribution and adaptation to nutritional stresses has been restricted to mixed mitochondrial populations, and not delineated between subsarcolemmal (SS) and intermyofibrillar (IMF) subpopulations. SS and IMF mitochondria exhibit distinct morphological and biochemical properties; however the functional differences are not well understood. This study investigated the effect of fed (FED) versus 48 h total foodrestriction (FR) on rat red gastrocnemius muscle PDK2 and 4 isoform content in SS and IMF mitochondria. PDK4 content was ~3-5 fold higher in SS mitochondria compared to IMF (p=0.001), and increased with FR -3-4- fold in both subpopulations (p<0.001). PDK2 was -2.5-4 fold higher in SS mitochondria compared to IMF (p=0.001), but PDK2 was unaltered with FR. Citrate synthase activity (|imol/min/mg mitochondrial protein) was not different between either subpopulation. As well there were no significant differences between mitochondrial subpopulations in PDH complex components in both fed and FR states. These results demonstrate that there is a markedly higher content of both PDK isofonns in SS compared to IMF mitochondria. Although PDK2 does not increase in either subpopulation in response to FR, PDK4 increases to a similar extent in both SS and IMF after 48 h food-restriction.

TIME COURSE OF SIGNALING PROCESSES INVOLVED WITH EXTRACELLULAR OSMOTIC - INDUCED GLUCOSE UPTAKE IN MAMMALIAN SKELETAL MUSCLE

Extracellular hyper-osmotic (HYPER) stress increases glucose uptake to defend cell volume, when compared to iso-osmotic (ISO) conditions in skeletal muscle. The purpose of this study was to determine a time course for changes in common signaling proteins involved in glucose uptake during acute hyper-osmotic stress in isolated mammalian skeletal muscle. Rat extensor digitorum longus (EDL) muscles were excised and incubated in a media formulated to mimic ISO (290 ± 10 mmol/kg) or HYPER (400 ± 10 mmol/kg) extracellular condition (Sigma Media-199). Signaling mechanisms were investigated by determining the phosphorylation states of Akt, AMPK, AS160, cPKC and ERK after 30, 45 and 60 minutes of incubation. AS160 was found to be significantly more phosphorylated in HYPER conditions compared to ISO after 30 minutes (p<0.01). It is speculated that AS160 phosphorylation increases glucose transporter 4 (GLUT4) content at the cell surface thereby facilitating an increase in glucose uptake under hyper-osmotic stress.

Regulation of Protein Turnover during Hyper-osmotic Stress in Skeletal Muscle

The purpose of this study was to examine the effect of hyper-osmotic stress on protein turnover in skeletal muscle tissue using an established in-vitro model. Rat EDL muscles were incubated in either hyper-osmotic (400 ± 10 Osm) or isoosmotic (290 ± 10 Osm) custom-modified media (Gibco). L-[14C]-U-phenylalanine (n=8) and cycloheximide (n=8) were used to quantify protein synthesis and degradation, respectively. Western blotting analyses was performed to determine the activation of protein synthesis and degradation pathways. During hyperosmotic stress, protein degradation increased (p<0.05), while protein synthesis was decreased (p<0.05) as compared to the iso-osmotic condition. The decline in protein synthesis was accompanied by a decrease (p<0.05) in p70s6 kinase phosphorylation, while the increase in protein degradation was associated with an increase (p<0.05) in autolyzed calpain. Therefore, hyper-osmotic extracellular stress results in an intracellular catabolic environment in mammalian skeletal muscle tissue.