Primary Muscle Cell Cultures Research Articles

Massive cell vacuolization induced by organic amines such as procainamide.

Procaine and some other basic drugs reportedly induce vacuolization of various cell types. We addressed the concentration-effect and structure-activity relationships as well as the mechanism of this effect using three cell lines. Massive vacuolization occurs over several hours in primary cultures of rabbit pulmonary artery smooth muscle cells (SMCs) and COS-1 cells in response to procaine and loosely related amine compounds (procainamide, N-acetyl-procainamide, metoclopramide, lidocaine, triethylamine, nicotine) used at 2.5 mM. Furthermore, chloroquine, propranolol, diphenhydramine, and neutral red are active in this respect at 100 to 250 microM in SMCs and COS-1 cells. Human embryonic kidney 293 cells mildly responded to triethylamine, nicotine, and propranolol only. Tetraethylammonium was uniformly inactive, as well as many other drugs in all three cell types (concentrations up to 2.5 mM). Procainamide does not induce apoptosis in SMCs treated for up to 48 h, although the vacuolization is sustained and proliferation and migration are reduced during this period. Procainamide-induced vacuolization is reversible on drug washing, largely prevented by bafilomycin A1 cotreatment, and has a tentatively identified Golgi origin (uptake of ceramide-C5). Procainamide and neutral red are concentrated in SMCs in a bafilomycin A1-sensitive manner. The preventive effect of bafilomycin A1 suggests that the vacuoles originate from the osmotic swelling of acidic organelles in which the charged basic drugs are trapped at low pH. Drug transport at the plasma membrane may be limiting for this type of response, as suggested by the cell type selectivity of agents and the inhibitory effect of some drugs such as quinidine.

Cyclic adenosine monophosphate (cAMP) increases natriuretic peptide receptor C (NPR-C) expression in human aortic smooth muscle cells

Activation of the intracellular cAMP-signaling pathway by either forskolin or the cAMP-mimetic dibutyryl cAMP significantly increased transcript levels of NPR-C in primary cultures of human aortic smooth muscle cells. The time course of the increase was rapid, with significant differences from control occurring within 3 h of treatment and reaching ∼6 times control value after 24 h of exposure to 10 μM forskolin. Expression levels of the natriuretic peptide receptor B (NPR-B), but not the natriruetic peptide receptor A (NPR-A) were also increased by forskolin, rising to a level of ∼2 times control at 96 h. NPR-B transcript levels in the presence of dibutyryl cAMP were unaltered by the protein kinase A (PKA) inhibitor KT-5720, suggesting a PKA-independent pathway to NPR-B up-regulation. In contrast, KT-5720 reduced NPR-C transcript to a lower level that was not significantly different from control. Partial re-differentiation of AOSMC by culture in growth factor-reduced matrix (Matrigel) did not significantly change NPR-C transcript levels compared with cells grown on plastic, and the dibutyryl cAMP-induced increase in NPR-C (∼eight–nine-fold control value) was retained. The dibutyryl cAMP/forskolin effect on NPR-C transcript was not reproduced by the β2-selective adrenergic agonist isoproterenol (10 μM), but was replicated by incubation with the phosphodiesterase inhibitor isobutylmethylxanthine (0.5 mM). Up-regulated NPR-B and NPR-C transcript levels were reflected, respectively, in a two-fold increase in CNP-stimulated cGMP and an increase in 125 I -ANF binding competed by the NPR-C-specific natriuretic peptide, C-ANF(4–23) following a 4-day treatment with 0.125 mM dbcAMP. The present data suggest that elevation of cAMP in human vascular smooth muscle may potentiate the vasoactive effects of natriuretic peptides acting through the NPR-B and NPR-C receptors.

Regulated expression and subcellular localization of HMGB1, a chromatin protein with a cytokine function.

High mobility group box protein 1 (HMGB1) has been considered as a ubiquitous nuclear protein with an architectural function, but even early reports have described its presence outside of the nucleus. Today, we have only started to understand the extranuclear and extracellular functions of HMGB1: we know that it participates in developmental and differentiation processes, triggers and modulates many of the inflammatory cascades in the body, and may even be involved in the metastatic invasion programme of cancer cells. Given such diverse roles, it is important to know which cells express HMGB1, where, and how much. The present review deals with the expression pattern of HMGB1 and provides evidence that, far from being housekeeping, the HMGB1 gene is tightly regulated. This can have implications for therapeutic intervention on inflammatory diseases as well as cancer.

IgA sensitizes airway smooth muscle to histamine

Rationale IgA is an important defense against infectious pathogens, which are common triggers of asthma symptoms. We recently reported that primary cell cultures of human airway smooth muscle (ASM) cells express the IgA receptor (pIgR) that binds polymeric IgA (pIgA) but not monomeric IgA (mIgA), and that pIgA increases cytosolic calcium - a necessary prequel to smooth muscle contraction and therefore bronchoconstriction. In this study, we asked if pIgA sensitizes ASM to histamine-induced responses. Methods Cytosolic calcium was measured in Fura2-loaded human ASM cells stimulated first with histamine 10 −6M and then treated for 2h with buffer, pIgA or mIgA. Post-incubation histamine-induced cytosolic calcium concentrations were compared to the pre-incubation response. Tension was measured in isolated canine tracheal ASM strips first in response to histamine 10 −5M and then treated for 4h with buffer, pIgA or mIgA. Post-incubation histamine-induced tension was compared to the pre-incubation response. Results Incubation with pIgA (0.12 μg/ml) significantly augmented the histamine-induced increase in cytosolic calcium in human ASM cells compared to mIgA (12 μg/ml) or buffer (p<0.05). Similarly, incubation with pIgA (6 μg/ml) appreciably enhanced histamine-induced increases in tension (by 2g) in canine tracheal ASM strips compared to mIgA 3 μg/ml (0.36g) or buffer (0.35g; p<0.05). Conclusions Incubation with pIgA sensitizes human ASM cells and canine tracheal ASM strips to histamine. Because IgA is an important epithelial defense against viral and bacterial attachment, functional responses to IgA in ASM may account, at least in part, for the worsening of symptoms during upper respiratory tract infections in patients with allergic asthma.

Determination of depolarisation- and agonist-evoked calcium fluxes on skeletal muscle cells in primary culture

Changes in intracellular calcium concentration ([Ca 2+] i) evoked by prolonged depolarisation (120 mM KCl) or by the application of 15 mM caffeine were measured on skeletal muscle cells in primary culture. The extrusion rate (PV max) of calcium from the myoplasm was determined, which in turn enabled the calculation of the calcium flux (Fl) underlying the measured calcium transients. PV max was found to increase during differentiation, from 107±10 μM/s at the early myotube stage to 596±36 μM/s in secondary myotubes. This was paralleled by a decrease in resting [Ca 2+] i from 99±4 to 51±2 nM. The depolarisation-evoked Fl rose to peak and then ceased despite the continuous presence of KCl. In contrast, the caffeine-induced Fl showed a peak and a clear steady-level with a peak-to-steady ratio of 5.6±1.2. Removal of external calcium suppressed the depolarisation–induced flux by 88±5% indicating that both an influx and a release from the SR underlie the K +-evoked calcium transients. Subsequent applications of caffeine resulted in essentially identical fluxes indicating an efficient refilling of the internal stores. Moreover, if a depolarisation-induced calcium transient preceded the second caffeine-evoked release, the latter was significantly larger than the first suggesting that much of the calcium that entered was stored in the SR rather than extruded.

Circadian Rhythm of Glucose Uptake in Cultures of Skeletal Muscle Cells and Adipocytes in Wistar-Kyoto, Wistar, Goto-Kakizaki, and Spontaneously Hypertensive Rats

Hypertension and noninsulin-dependent diabetes mellitus are usually associated with marked glucose intolerance. Hypertensive and even nonhypertensive diabetic individuals display disturbances of the normal circadian blood pressure rhythm. However, little is known about circadian changes of the glucose uptake in muscle and fat cells, the major glucose utilizing tissues. Therefore, we investigated circadian rhythms of glucose uptake in primary muscle and fat cell cultures of hypertensive and type II diabetic rats and their respective control strains. 2-Deoxy-d-(1-3H)glucose uptake was measured over 48 h after synchronization of cells by means of medium change with and without addition of insulin, phloretine, and/or staurosporine. The circadian changes of glucose uptake were assessed by fitting cosine curves to the uptake values. Insulin stimulation of deoxyglucose uptake was only present in control animals, not in hypertensive and diabetic rats. Deoxyglucose uptake displayed a circadian rhythm in control animals, and was markedly disturbed in hypertensive and diabetic animals. Blocking of glucose transporters by phloretine abolished the circadian pattern of deoxyglucose uptake indicating a role of glucose transporters in its generation. Inhibition of kinases by staurosporine inhibited the insulin-stimulated deoxyglucose uptake, but did not dampen the circadian rhythmicity of basal deoxyglucose uptake. The generation of the circadian rhythm of glucose uptake in muscle and fat cell cultures is therefore probably insulin independent and independent of protein kinases. In summary, our results show for the first time: (a) a circadian rhythm of deoxyglucose uptake in glucose utilizing muscle and fat cells in vitro, (b) a disruption of this rhythm in cells of hypertensive and diabetic rats.

Glia cell line-derived neurotrophic factorregulates the distribution of acetylcholine receptors in mouse primary skeletal muscle cells

It was recently reported that glia cell line-derived neurotrophic factor (GDNF) facilitates presynaptic axonal growth and neurotransmitter release at neuromuscular synapses. Little is known, however, whether GDNF can also act on the postsynaptic apparatus and its underlying mechanisms. Using biochemical cold blocking of existing membrane acetylcholine receptors (AchRs) and biotinylation of newly inserted receptors we demonstrate that GDNF increases the insertion of AChRs into the surface membrane of mouse primary cultured muscle cells and that this does not require protein synthesis. Quantitative data from double-label imaging indicate that GDNF induces a quick and substantial increase in AchR insertion as well as lateral movement into AchR aggregates, relative to a weak effect on reducing the loss of receptors from pre-existing AchR aggregates, which in contrast to the effect of PMA. These effects occur in both innervated and un-innervated muscles, and GDNF affects nerve-muscle co-cultures more than it affects muscle-only cultures. Neurturin, another member of GDNF-family ligands has similar effects on AchRs as GDNF but the unrelated growth factor, EGF does not. Studies on protein phosphorylation and specific inhibitors of cell signal transduction indicate that GDNF function is mediated by receptor GFRα1 and involves MAPK, cAMP/cAMP responsive element-binding factor and Src kinase activities. GDNF may signal through c-Ret as well as NCAM-140 pathways since both the signaling receptors are expressed in the neuromuscular junction (NMJ). These data suggest that GDNF is an autocrine regulator of NMJ to promote the insertion and stabilization of postsynaptic AchRs. In vivo, GDNF may function as a synaptotrophic modulator for both pre- and postsynaptic differentiation to strengthen the functional and structural connections between nerve and muscle, and contribute to the synaptogenesis and plasticity of neuromuscular synapses.

Rat strain differences in the ectopic osteogenic potential of recombinant human BMP adenoviruses

Different animal strains have different genetic backgrounds that influence their physiological function and pathological process. The differences in genetic background may affect the efficiency of adenoviral infection and target gene expression and further cause different gene therapy results when target genes are delivered with adenoviral vectors. In this study, ectopic bone was not seen in ADCMVBMP4 injection sites, but was formed in ADCMVBMP9 injection sites in all rat strains. The mean volumes of bone induced with ADCMVBMP9 were 0.87 ± 0.2 cm3 in Wistar, 0.26 ± 0.1 cm3 in Long-Evans, 0.34 ± 0.2 cm3 in Sprague-Dawley, 0.44 ± 0.1 cm3 in ACI, 0.66 ± 0.2 cm3 in PVG, and 0.58 ± 0.1 cm3 in Fischer344 rats. This indicates that ADCMVBMP9 has different bone formation potentials in different immunocompetent rat strains (P = 0.02). The basic levels of CD4+ and CD8+ T cells in blood before viral infection and titers of adenoviral neutralizing antibodies 30 days post-viral infection were significantly different among rat strains (P < 0.01). The efficiencies of target gene expression delivered with adenovirus were also significantly different in primary muscle cell cultures from different rat strains (P < 0.01). The different osteogenic potentials of ADCMVBMP9 among rat strains may be, in part, due to the differences in immune factors and target gene expression efficiency in muscle tissue.

Mirk/dyrk1B Is a Rho-induced Kinase Active in Skeletal Muscle Differentiation

The Rho family of small GTPases regulates numerous signaling pathways that control the organization of the cytoskeleton, transcription factor activity, and many aspects of the differentiation of skeletal myoblasts. We now demonstrate that the kinase Mirk (minibrain-related kinase)/dyrk1B is induced by members of the Rho-family in myoblasts and that Mirk is active in skeletal muscle differentiation. Mirk is an arginine-directed serine/threonine kinase which is expressed at elevated levels in skeletal muscle compared with other normal tissues. A Mirk promoter construct was activated when C2C12 myoblasts were switched from growth to differentiation medium and was also activated by the Rho family members RhoA, Cdc42, and to a lesser degree Rac1, but not by MyoD or Myf5. Mirk protein levels increased following transient expression of constitutively active Cdc42-QL, RhoA-QL, or Rac1-QL in C2C12 cells. High concentrations of serum mitogens down-regulated Mirk through activation of the Ras-MEK-Erk pathway. As a result, Mirk transcription was induced by the MEK1 inhibitor PD98059 and by the switch from growth to differentiation medium. Mirk was induced with similar kinetics to another Rho-induced differentiation gene, myogenin. Mirk protein levels increased 10-fold within 24-48 h after primary cultured muscle cells; C2C12 mouse myoblasts or L6 rat myoblasts were induced to differentiate. Thus Mirk was induced following the commitment stage of myogenesis. Stable overexpression of Mirk enabled myoblasts to fuse more rapidly when placed in differentiation medium. The function of Mirk in muscle differentiation was established by depletion of endogenous Mirk by small interfering RNA, which prevented myoblast fusion into myotubes and inhibited induction of markers of differentiation, including myogenin, fast twitch troponin T, and muscle myosin heavy chain. Other members of the dyrk/minibrain/HIPK family of kinases in lower organisms have been shown to regulate the transition from growth to differentiation, and Mirk is now shown to participate in skeletal muscle development.

In vitro construction of urinary bladder wall using porcine primary cells reseeded on acellularized bladder matrix and small intestinal submucosa.

Partial or radical cystectomy requires replacement of the urinary reservoir normally achieved by using small or large bowel segments. Our aim was to establish tissue engineering of an bioartificial bladder wall using primary cultures of porcine urothelial (pUC) and bladder smooth muscle cells (pSMC) to be reseeded on different acellular biological matrices. Primary porcine cultures of pUC and pSMC were established from open bladder biopsy material 25 mm2 in size. Acellular matrix was generated either from a) porcine bladder wall segments or b) tubular small intestinal submucosa with the still attached decellularized muscularis layer. Reseeding of these matrices with primary cells was done in a two-dimensional static model and in a three-dimensional rotating bioreactor perfused with cell culture medium for a period of 6 weeks. Prior to reseeding the cultured cells were characterized as pUC and pSMC by immunohistochemical staining with either anti-keratin 7 or anti-alpha actin. For both matrices a reseeded double layer cell system of pUC and pSMC could be identified after incubation in the described systems for 6 weeks. Our results document successful generation of tissue engineered urinary bladder wall, which can be used in further large animal transplantation experiments.

Promyelocytic leukaemia zinc finger protein (PLZF) is a glucocorticoid- and progesterone-induced transcription factor in human endometrial stromal cells and myometrial smooth muscle cells.

The promyelocytic leukaemia zinc finger (PLZF) protein belongs to the family of Krüppel-like zinc finger proteins. It is a transcriptional repressor involved in cell cycle control and has been implicated in limb development, differentiation of myeloid cells, and spermatogenesis. Little is known about the regulation of PLZF expression. In search for mediators of progesterone signalling in the female reproductive tract, we discovered induction of PLZF mRNA in primary cultures of human endometrial stromal cells and myometrial smooth muscle cells (SMC) in response to progesterone. Surprisingly, dexamethasone was a more potent inducer of PLZF expression than progesterone and elicited a sustained up-regulation of PLZF mRNA levels within 2 h. Immunofluorescence showed localization of PLZF to the nuclei of dexamethasone-treated SMC. In uterine biopsies, nuclear staining for PLZF was found in myometrial cells and endometrial stromal cells of the secretory phase. The transcriptional start site of the PLZF gene was located to position -5801 in SMC. Transfected promoter constructs containing up to 4.1 kb of 5'-flanking DNA were not induced by activated glucocorticoid or progesterone receptor. In contrast, co-transfection of c-jun and c-fos expression vectors resulted in stimulation of reporter gene activity, indicating an involvement of AP-1 transcription factors in PLZF expression.

Rat strain differences in the ectopic osteogenic potential of recombinant human BMP adenoviruses

Different animal strains have different genetic backgrounds that influence their physiological function and pathological process. The differences in genetic background may affect the efficiency of adenoviral infection and target gene expression and further cause different gene therapy results when target genes are delivered with adenoviral vectors. In this study, ectopic bone was not seen in ADCMVBMP4 injection sites, but was formed in ADCMVBMP9 injection sites in all rat strains. The mean volumes of bone induced with ADCMVBMP9 were 0.87 ± 0.2 cm3 in Wistar, 0.26 ± 0.1 cm3 in Long-Evans, 0.34 ± 0.2 cm3 in Sprague-Dawley, 0.44 ± 0.1 cm3 in ACI, 0.66 ± 0.2 cm3 in PVG, and 0.58 ± 0.1 cm3 in Fischer344 rats. This indicates that ADCMVBMP9 has different bone formation potentials in different immunocompetent rat strains (P = 0.02). The basic levels of CD4+ and CD8+ T cells in blood before viral infection and titers of adenoviral neutralizing antibodies 30 days post-viral infection were significantly different among rat strains (P < 0.01). The efficiencies of target gene expression delivered with adenovirus were also significantly different in primary muscle cell cultures from different rat strains (P < 0.01). The different osteogenic potentials of ADCMVBMP9 among rat strains may be, in part, due to the differences in immune factors and target gene expression efficiency in muscle tissue.

Phenotypic diversity of smooth muscle cells isolated from human intracranial basilar artery

The present work examined heterogeneity of vascular smooth muscle cells cultured from human cerebral arteries that has not been previously reported. Primary smooth muscle cell cultures were isolated from human intracranial basilar arteries. Using a ring isolation method, multiple clones were generated from the cell cultures. These clones had two distinctly different morphologies: (1) fusiform; and (2) stellate. At confluence the fusiform-shaped clones grew in compact clusters with overlapping cells while the stellate-shaped clones were contact-inhibited growing in a monolayered pattern. The smooth muscle differentiation markers, α-smooth muscle-actin, calponin and smooth muscle-myosin heavy chains were expressed in all these clones. In response to serum stimulation, the stellate-shaped clones had a higher growth rate than the fusiform clones. This study reports that smooth muscle cells derived from human basilar arteries are heterogeneous.

Intracellular alkalinization induces Ca 2+ influx via non-voltage-operated Ca 2+ channels in rat aortic smooth muscle cells

In smooth muscle, the cytosolic Ca 2+ concentration ([Ca 2+] i) is the primary determinant of contraction, and the intracellular pH (pH i) modulates contractility. Using fura-2 and 2′,7′-biscarboxyethyl-5(6) carboxyfluorescein (BCECF) fluorometry and rat aortic smooth muscle cells in primary culture, we investigated the effect of the increase in pH i on [Ca 2+] i. The application of the NH 4Cl induced concentration-dependent increases in both pH i and [Ca 2+] i. The extent of [Ca 2+] i elevation induced by 20 mM NH 4Cl was approximately 50% of that obtained with 100 mM K +-depolarization. The NH 4Cl-induced elevation of [Ca 2+] i was completely abolished by the removal of extracellular Ca 2+ or the addition of extracellular Ni 2+. The 100 mM K +-induced [Ca 2+] i elevation was markedly inhibited by a voltage-operated Ca 2+ channel blocker, diltiazem, and partly inhibited by a non-voltage-operated Ca 2+ channel blocker, SKF96365. On the other hand, the NH 4Cl-induced [Ca 2+] i elevation was resistant to diltiazem, but was markedly inhibited by SKF96365. It is thus concluded that intracellular alkalinization activates the Ca 2+ influx via non-voltage-operated Ca 2+ channels and thereby increases [Ca 2+] i in the vascular smooth muscle cells. The alkalinization-induced Ca 2+ influx may therefore contribute to the enhancement of contraction.

Betacellulin and amphiregulin induce upregulation of cyclin D1 and DNA synthesis activity through differential signaling pathways in vascular smooth muscle cells.

Activation of EGF receptors is closely involved in vascular proliferative diseases. The signaling mechanisms of EGF ligands, including betacellulin (BTC) and amphiregulin (AR), are poorly understood. We examined how BTC and AR induced DNA synthesis activity in primary cultures of human thoracic aortic smooth muscle cells (HTASMCs). BTC induced phosphorylation of all four EGF receptors present on HTASMCs: ErbB1, ErbB2, ErbB3, and ErbB4. BTC rapidly induced the phosphorylation of Akt, GSK3alpha/beta, and two FoxO factors, FKHR and AFX, in a dose- and time-dependent manner. BTC increased nuclear beta-catenin accumulation. BTC increased cyclin D1 mRNA, cyclin D1 protein, and DNA synthesis activity. Pretreatment with the phosphatidylinositol 3'-kinase (PI 3'-kinase) inhibitor wortmannin suppressed BTC-induced cyclin D1 mRNA and protein and DNA synthesis activity. In contrast, AR, a specific ErbB1 ligand, induced sustained ERK1/2 and Elk1 phosphorylation, increased cyclin D1 mRNA and protein, and increased DNA synthesis activity. AR did not produce any changes in Akt phosphorylation. Pretreatment with PD98059 suppressed AR-induced cyclin D1 mRNA and protein. Thus, the PI 3'-kinase/Akt/GSK/FoxO/beta-catenin pathway could be the major signaling cascade for BTC-induced upregulation of cyclin D1 protein, whereas a sustained ERK/Elk1 activation could be the major signaling cascade for AR-induced upregulation of cyclin D1 protein in HTASMCs. Moreover, immunohistochemical staining revealed that that BTC, ErbB1, and ErbB4 are upregulated in the plaques of human atherosclerotic coronary arteries. Taken together, BTC and AR could be potent growth factors in proliferative vascular diseases.

Heme oxygenase is expressed in human pulmonary artery smooth muscle where carbon monoxide has an anti-proliferative role

Heme oxygenase is the rate-limiting enzyme in the catabolism of heme to carbon monoxide, bilirubin and free iron. Many cell types express heme oxygenase-2 constitutively while heme oxygenase-1 is induced at sites of inflammation and oxidative stress. In systemic blood vessels, carbon monoxide may have an important homeostatic role where, like its better-studied counterpart nitric oxide, it is emerging as a vasodilator and an inhibitor of proliferation. However, much less is known regarding the role of heme oxygenase and carbon monoxide in the pulmonary circulation where vascular responses are very different. Here, using primary cultures of human pulmonary artery smooth muscle cells, we present novel data showing that this cell type expresses heme oxygenase-2 constitutively and, in the presence of oxidants, can induce heme oxygenase-1. We also show that the carbon monoxide-releasing molecule, tricarbonyldichlororuthenium (II) dimer, potently and profoundly inhibits proliferation of human pulmonary artery smooth muscle cells. Pulmonary hypertension is a disease characterised by abnormal vascular smooth muscle cell growth and remodelling of the pulmonary vasculature. Our observations support the growing evidence that the heme oxygenase/carbon monoxide system may play a role in the pathology of pulmonary hypertension.

Protein turnover and sensory traits of longissimus muscle from implanted and nonimplanted heifers.

Primary bovine muscle cell culture studies were conducted to determine whether implanting heifers had a direct effect on in vitro protein synthesis and degradation and to determine the effect of implanting heifers on longissimus muscle palatability. Feedlot heifers (n = 96) were administered one of six implant regimens to characterize their effect on in vitro amino acid uptake and protein degradation. Treatments consisted of: 1) a nonimplanted control (NI/NI); 2) no implant on d 1 and Revalor-H administered on d 84 of the experiment (NI/Rev); 3) Revalor-H on d 1, but no implant given at d 84 (Rev/NI); 4) Revalor-H administered on d 1 and d 84 (Rev/Rev); 5) Revalor-IH administered on d 1 and Revalor-H at d 84 (RIH/Rev); and 6) Synovex-H given at d 1 and Revalor-H administered at d 84 (Syn/Rev). Blood and longissimus lumborum muscle were collected 20 min postmortem, and serum and muscle extracts were incubated with primary bovine muscle cells. Implant treatments had minimal effects on shear force and sensory traits; however, steaks from Rev/Rev heifers were 0.31 kg more tender (P < 0.05) than steaks from NI/NI heifers. Serum protein synthesis and degradation were not affected (P > 0.10) by any implant treatment. When primary bovine muscle cells were treated with muscle extract, amino acid uptake was greater for heifers implanted with Rev/ Rev than for the average of all other treatments (P < 0.01). The Rev/Rev implant regimen also increased (P < 0.05) amino acid uptake compared with heifers treated with RIH/Rev, Syn/Rev, NI/NI, NI/Rev, or Rev/NI. Cellular protein degradation of the muscle cell culture treated with muscle extract tended (P < 0.10) to be higher in NI/NI-treated cells compared with the average of all implant treatments. In addition, cells treated with muscle extract from heifers implanted with Rev/Rev had lower (P < 0.05) protein degradation than the NI/NI control heifers. These results indicate that anabolic implant strategies can directly affect both muscle protein synthesis and degradation via effects that seem to be more autocrine than paracrine in nature.

Motorcycle exhaust particulates enhance vasoconstriction in organ culture of rat aortas and involve reactive oxygen species.

The effects of motorcycle exhaust particulate on vasoconstriction were determined using rat thoracic aortas under organ culture conditions treated with organic extracts of motorcycle exhaust particulate from a two-stroke engine. The motorcycle exhaust particulate extract (MEPE) induced a concentration-dependent enhancement of vasoconstriction elicited by phenylephrine in the organ cultures of both intact and endothelium-denuded aortas for 18 h. Nifedipine (an L-type Ca2+ channel blocker), manganese acetate (an inorganic Ca2+ channel blocker), and staurosporine (a nonselective protein kinase C inhibitor), but not the selective protein kinase C inhibitor chelerythrine, inhibited the enhancement of vasoconstriction by MEPE. Staurosporine has also been reported as a myosin light chain kinase (MLCK) inhibitor, so we tested whether the MLCK pathway was involved in the effect of MEPE. The results showed that ML-9 (a selective MLCK inhibitor) could inhibit the enhancement of vasoconstriction by MEPE. The phosphorylation of a 20-kDa myosin light chain in a primary culture of rat vascular smooth muscle cells was also enhanced by MEPE. Moreover, we also examined the role of reactive oxygen species (ROS) in the stimulatory effect of MEPE on vasoconstriction. The antioxidant N-acetylcysteine significantly inhibited the enhancement of vasoconstriction by MEPE. A time-dependent increase in ROS production by MEPE was also detected in primary cultures of vascular smooth muscle cells. These results indicate that MEPE induces a marked enhancement of vasoconstriction in aortas under organ culture conditions and imply that a ROS-Ca2+-MLCK pathway may be involved in this MEPE-induced response.

Reduced activation of phosphatidylinositol-3 kinase and increased serine 636 phosphorylation of insulin receptor substrate-1 in primary culture of skeletal muscle cells from patients with type 2 diabetes.

To understand better the defects in the proximal steps of insulin signaling during type 2 diabetes, we used differentiated human skeletal muscle cells in primary culture. When compared with cells from control subjects, myotubes established from patients with type 2 diabetes presented the same defects as those previously evidenced in vivo in muscle biopsies, including defective stimulation of phosphatidylinositol (PI) 3-kinase activity, decreased association of PI 3-kinase with insulin receptor substrate (IRS)-1 and reduced IRS-1 tyrosine phosphorylation during insulin stimulation. In contrast to IRS-1, the signaling through IRS-2 was not altered. Investigating the causes of the reduced tyrosine phosphorylation of IRS-1, we found a more than twofold increase in the basal phosphorylation of IRS-1 on serine 636 in myotubes from patients with diabetes. Concomitantly, there was a higher basal mitogen-activated protein kinase (MAPK) activity in these cells, and inhibition of the MAPKs with PD98059 strongly reduced the level of serine 636 phosphorylation. These results suggest that IRS-1 phosphorylation on serine 636 might be involved in the reduced phosphorylation of IRS-1 on tyrosine and in the subsequent alteration of insulin-induced PI 3-kinase activation. Moreover, increased MAPK activity seems to play a role in the phosphorylation of IRS-1 on serine residue in human muscle cells.

Side population cells and Bcrp1 expression in lung.

Side population (SP) cells are a rare subset of cells found in various tissues that are highly enriched for stem cell activity. SP cells can be isolated by dual-wavelength flow cytometry because of their capacity to efflux Hoechst dye, a process mediated by the ATP-binding cassette transporter breast cancer resistance protein (Bcrp) 1. By performing flow cytometry of enzymedigested mouse lung stained with Hoechst dye, we found that SP cells comprise 0.03-0.07% of total lung cells and are evenly distributed in proximal and distal lung regions. By RT-PCR, we found that lung SP cells express hepatocyte nuclear factor-3beta, but not thyroid transcription factor-1. Surface marker analysis revealed lung SP cells to be stem cell antigen 1 positive, Bcrp1 positive, lineage marker negative, and heterogeneous at the CD45 locus. As expected, we did not detect lung SP cells in Bcrp1-deficient animals. We, therefore, employed nonisotopic in situ hybridization and immunostaining for Bcrp1 as a strategy to localize these cells in vivo. Expression was observed in distinct lung cell types: bronchial and vascular smooth muscle cells and round cells within the distal air space. We confirmed the expression of Bcrp1 in primary bronchial smooth muscle cell cultures (BSMC) and in lavaged distal airway cells, but neither possessed the capacity to efflux Hoechst dye. In BSMC, Bcrp1 was localized to an intracellular compartment, suggesting that the molecular site of Bcrp1 expression regulates SP phenotype.