Methotrexate reduces HbA1c concentration but does not produce chronic accumulation of ZMP in patients with rheumatoid or psoriatic arthritis.

OBJECTIVES: The mechanism by which methotrexate (MTX) improves glucose homeostasis in patients with rheumatoid (RA) and psoriatic arthritis (PsA) remains undetermined. Animal studies indicate a role for intracellular accumulation of 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranosyl 5'-monophosphate (ZMP) but this has not been directly demonstrated in humans. We explored whether accumulation of ZMP is associated with improvements in glucose homeostasis during MTX therapy. METHOD: MTX-naïve, non-diabetic RA (n = 16) and PsA (n = 10) patients received uninterrupted MTX treatment for 6 months. To evaluate whether ZMP accumulated during MTX therapy, we measured the concentration of ZMP in erythrocytes and the concentration of its dephosphorylated derivative 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) in urine using liquid chromatography mass spectrometry (LC-MS/MS). To assess glucose homeostasis, we determined the concentration of glycated haemoglobin (HbA1c) and homeostasis model assessment of insulin resistance [HOMA-IR: fasting glucose (mmol/L) × fasting insulin (µU/mL)/22.5]. RESULTS: Erythrocyte ZMP and urinary AICAR concentrations did not increase during 6 months of MTX therapy. HbA1c concentration was reduced from 5.80 ± 0.29% at baseline to 5.51 ± 0.32% at 6 months (p < 0.001), while HOMA-IR remained unaltered. Reduction in HbA1c concentration was not associated with increased ZMP or AICAR concentrations. CONCLUSIONS: MTX therapy probably does not produce a chronic increase in erythrocyte ZMP or urinary AICAR concentrations. Collectively, our data do not support the hypothesis that MTX improves glucose homeostasis through chronic accumulation of ZMP.

Enhanced insulin action following subcutaneous co-administration of insulin and C-peptide in rats.

BACKGROUND: This study was undertaken to examine if C-peptide (C) may interact with hexameric insulin and facilitate its disaggregation into the physiologically active monomeric form. METHODS: Regular insulin (I) or an insulin analogue (IA) were injected s.c. in rats together with C or its C-terminal pentapeptide (PP). I or IA and C or PP were administered either as a physical mixture or into two separate s.c. depots. Whole body glucose utilization was evaluated using the euglycemic clamp technique. Phosphorylation of Akt/PKB and GSK in liver and skeletal muscles and 86Rb⁺ uptake by L6 cells were measured. RESULTS: S.c. injection of a mixture of I and C or I and PP resulted in a 30-55% greater (P < 0.01-0.001) and 15-27% (P < 0.05-0.001) longer stimulation of whole body glucose utilization than after separate injections. Insulin-stimulated phosphorylation of Akt/PKB in liver increased 35% more after injection of I and C in mixture compared with after separate injections. Phosphorylation of GSK3 was augmented by 50% (P < 0.05) following the injection of I and C in mixture compared with separate injections. Stimulation of myotubes with premixed I and C (1 nM) elicited 20% additional increase in ouabain-sensitive 86Rb⁺ uptake (P < 0.05) in comparison with the effect when I and C were added separately. CONCLUSIONS: Subcutaneous co-administration of insulin and C results in augmented insulin bioactivity at the level of tissue glucose uptake, intracellular signalling, and enzyme activation. These effects may be attributed to augmented C mediated disaggregation of hexameric insulin into its physiologically active monomeric form.

LXR is a negative regulator of glucose uptake in human adipocytes.

AIMS/HYPOTHESIS: Obesity increases the risk of developing type 2 diabetes mellitus, characterised by impaired insulin-mediated glucose uptake in peripheral tissues. Liver X receptor (LXR) is a positive regulator of adipocyte glucose transport in murine models and a possible target for diabetes treatment. However, the levels of LXRα are increased in obese adipose tissue in humans. We aimed to investigate the transcriptome of LXR and the role of LXR in the regulation of glucose uptake in primary human adipocytes. METHODS: The insulin responsiveness of human adipocytes differentiated in vitro was characterised, adipocytes were treated with the LXR agonist GW3965 and global transcriptome profiling was determined by microarray, followed by quantitative RT-PCR (qRT-PCR), western blot and ELISA. Basal and insulin-stimulated glucose uptake was measured and the effect on plasma membrane translocation of glucose transporter 4 (GLUT4) was assayed. RESULTS: LXR activation resulted in transcriptional suppression of several insulin signalling genes, such as AKT2, SORBS1 and CAV1, but caused only minor changes (<15%) in microRNA expression. Activation of LXR impaired the plasma membrane translocation of GLUT4, but not the expression of its gene, SLC2A4. LXR activation also diminished insulin-stimulated glucose transport and lipogenesis in adipocytes obtained from overweight individuals. Furthermore, AKT2 expression was reduced in obese adipose tissue, and AKT2 and SORBS1 expression was inversely correlated with BMI and HOMA index. CONCLUSIONS/INTERPRETATION: In contrast to murine models, LXR downregulates insulin-stimulated glucose uptake in human adipocytes from overweight individuals. This could be due to suppression of Akt2, c-Cbl-associated protein and caveolin-1. These findings challenge the idea of LXR as a drug target in the treatment of diabetes.

PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic beta cells.

Hypoglycemic sulfonylureas represent a group of clinically useful antidiabetic compounds that stimulate insulin secretion from pancreatic beta cells. The molecular mechanisms involved are not fully understood but are believed to involve inhibition of potassium channels sensitive to adenosine triphosphate (KATP channels) in the beta cell membrane, causing membrane depolarization, calcium influx, and activation of the secretory machinery. In addition to these effects, sulfonylureas also promoted exocytosis by direct interaction with the secretory machinery not involving closure of the plasma membrane KATP channels. This effect was dependent on protein kinase C (PKC) and was observed at therapeutic concentrations of sulfonylureas, which suggests that it contributes to their hypoglycemic action in diabetics.

Protein translation, proteolysis and autophagy in human skeletal muscle atrophy after spinal cord injury.

AIM: Spinal cord injury-induced loss of skeletal muscle mass does not progress linearly. In humans, peak muscle loss occurs during the first 6 weeks postinjury, and gradually continues thereafter. The aim of this study was to delineate the regulatory events underlying skeletal muscle atrophy during the first year following spinal cord injury. METHODS: Key translational, autophagic and proteolytic proteins were analysed by immunoblotting of human vastus lateralis muscle obtained 1, 3 and 12 months following spinal cord injury. Age-matched able-bodied control subjects were also studied. RESULTS: Several downstream targets of Akt signalling decreased after spinal cord injury in skeletal muscle, without changes in resting Akt Ser473 and Akt Thr308 phosphorylation or total Akt protein. Abundance of mTOR protein and mTOR Ser2448 phosphorylation, as well as FOXO1 Ser256 phosphorylation and FOXO3 protein, decreased in response to spinal cord injury, coincident with attenuated protein abundance of E3 ubiquitin ligases, MuRF1 and MAFbx. S6 protein and Ser235/236 phosphorylation, as well as 4E-BP1 Thr37/46 phosphorylation, increased transiently after spinal cord injury, indicating higher levels of protein translation early after injury. Protein abundance of LC3-I and LC3-II decreased 3 months postinjury as compared with 1 month postinjury, but not compared to able-bodied control subjects, indicating lower levels of autophagy. Proteins regulating proteasomal degradation were stably increased in response to spinal cord injury. CONCLUSION: Together, these data provide indirect evidence suggesting that protein translation and autophagy transiently increase, while whole proteolysis remains stably higher in skeletal muscle within the first year after spinal cord injury.

The α2 isoform Na,K-ATPase modulates contraction of rat mesenteric small artery via cSrc-dependent Ca2+ sensitization.

AIMS: The Na,K-ATPase is involved in a large number of regulatory activities including cSrc-dependent signalling. Upon inhibition of the Na,K-ATPase with ouabain, cSrc activation is shown to occur in many cell types. This study tests the hypothesis that acute potentiation of agonist-induced contraction by ouabain is mediated through Na,K-ATPase-cSrc signalling-dependent sensitization of vascular smooth muscle cells to Ca2+ . METHODS: Agonist-induced rat mesenteric small artery contraction was examined in vitro under isometric conditions and in vivo in anaesthetized rats. Arterial wall tension and [Ca2+ ]i in vascular smooth muscle cells were measured simultaneously. Changes in cSrc and myosin phosphatase targeting protein 1 (MYPT1) phosphorylation were analysed by Western blot. Protein expression was examined with immunohistochemistry. The α1 and α2 isoforms of the Na,K-ATPase were transiently downregulated by siRNA transfection in vivo. RESULTS: Ten micromolar ouabain, but not digoxin, potentiated contraction to noradrenaline. This effect was not endothelium-dependent. Ouabain sensitized smooth muscle cells to Ca2+ , and this was associated with increased phosphorylation of cSrc and MYPT1. Inhibition of tyrosine kinase by genistein, PP2 or pNaKtide abolished the potentiating effect of ouabain on arterial contraction and Ca2+ sensitization. Downregulation of the Na,K-ATPase α2 isoform made arterial contraction insensitive to ouabain and tyrosine kinase inhibition. CONCLUSION: Data suggest that micromolar ouabain potentiates agonist-induced contraction of rat mesenteric small artery via Na,K-ATPase-dependent cSrc activation, which increases Ca2+ sensitization of vascular smooth muscle cells by MYPT1 phosphorylation. This mechanism may be critical for acute control of vascular tone.

Phosphatidylinositol 3-kinase-mediated endocytosis of renal Na+, K+-ATPase alpha subunit in response to dopamine.

Dopamine (DA) inhibition of Na+,K+-ATPase in proximal tubule cells is associated with increased endocytosis of its alpha and beta subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na+,K+-ATPase alpha subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na+, K+-ATPase alpha subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D1 receptor subtype and the sequential activation of phospholipase A2, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na+,K+-ATPase alpha subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.

Metabolic and signaling events mediated by cardiotonic steroid ouabain in rat skeletal muscle.

The cardiac glycoside ouabain initiates a cascade of signaling events through Na+,K+-ATPase, leading to an increase in cell growth and proliferation in different cell types. We explored the effects of ouabain on glucose metabolism in skeletal muscle and clarified the mechanisms of ouabain signal transduction. In rat soleus muscle 200 microM ouabain decreased basal glucose uptake without effect on insulin-stimulated glucose uptake. Ouabain increased glycogen synthesis additively to insulin and this effect was abolished in the presence of a MEK1/2 inhibitor (PD98059) or a c-Src inhibitor (PP2). Ouabain exposure reduced glucose oxidation, and this effect was reversed in the presence of PP2. Incubation with ouabain did not affect intramuscular ATP and its metabolites; however acetyl-CoA carboxylase phosphorylation was reduced, with no effect on AMPK phosphorylation. Insulin-stimulated Akt phosphorylation was not affected by ouabain. Ouabain reduced basal and insulin-stimulated phosphorylation of PKC alpha/beta and delta isoforms, whereas phosphorylation of PKCzeta was unchanged. Ouabain exposure increased interaction of 1- and 2-subunits of Na-pump with c-Src, as assessed by co-immunoprecipitation with c-Src. Phosphorylation of ERK1/2, GSK 3 / and p90rsk activity was increased in response to ouabain, and these effects were prevented in the presence of PD98059 and PP2. In conclusion, the cardiac glycoside ouabain stimulates glycogen synthesis additively to insulin in rat skeletal muscle. This effect is mediated by activation of c-Src-, ERK1/2- p90rsk- and GSK3-dependent signaling pathway.

Insulin- and glucose-induced phosphorylation of the Na(+),K(+)-adenosine triphosphatase alpha-subunits in rat skeletal muscle.

Phosphorylation of the alpha-subunits of Na(+),K(+)-adenosine triphosphatase in response to insulin, high extracellular glucose concentration, and phorbol 12-myristate 13-acetate was investigated in isolated rat soleus muscle. All three stimuli increased alpha-subunit phosphorylation approximately 3-fold. Phorbol 12-myristate 13-acetate- and high glucose-induced phosphorylation of the alpha-subunit was completely abolished by the PKC inhibitor GF109203X, whereas insulin-stimulated phosphorylation was only partially reduced. Notably, insulin stimulation resulted in phosphorylation of the alpha-subunit on serine, threonine, and tyrosine residues, whereas high extracellular glucose or phorbol 12-myristate 13-acetate stimulation mediated phosphorylation only on serine and threonine residues. Insulin stimulation resulted in translocation of Na(+),K(+)-adenosine triphosphatase alpha(2)-subunit to the plasma membrane and increased Na(+),K(+)-adenosine triphosphatase activity in the same membrane fraction. High glucose had no effect on alpha-subunits distribution. Immunoprecipitation with antiphosphotyrosine antibody and subsequent Western blot analysis with anti-alpha(1)- and -alpha(2)-subunit antibodies revealed that both alpha(1)- and alpha(2)-subunit isoforms underwent phosphorylation on tyrosine residues in response to insulin, although with different time course and magnitude. Thus, we show that insulin-stimulated phosphorylation of Na(+),K(+)-adenosine triphosphatase alpha-subunit occurs via a PKC- and tyrosine kinase-dependent mechanism, whereas high glucose-induced phosphorylation is only PKC-dependent. Phosphorylation of Na(+),K(+)-adenosine triphosphatase alpha-subunits may be involved in regulation of Na(+),K(+)-adenosine triphosphatase activity by insulin or high extracellular glucose in skeletal muscle.

Exercise-associated differences in an array of proteins involved in signal transduction and glucose transport.

Vastus lateralis muscle biopsies were obtained from endurance-trained (running approximately 50 km/wk) and untrained (no regular physical exercise) men, and the expression of an array of insulin-signaling intermediates was determined. Expression of insulin receptor and insulin receptor substrate-1 and -2 was decreased 44% (P < 0.05), 57% (P < 0.001), and 77% (P < 0.001), respectively, in trained vs. untrained muscle. The downstream signaling target, Akt kinase, was not altered in trained subjects. Components of the mitogenic signaling cascade were also assessed. Extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase expression was 190% greater (P < 0.05), whereas p38 mitogen-activated protein kinase expression was 32% lower (P < 0.05), in trained vs. untrained muscle. GLUT-4 protein expression was twofold higher (P < 0.05), and the GLUT-4 vesicle-associated protein, the insulin-regulated aminopeptidase, was increased 4.7-fold (P < 0. 05) in trained muscle. In conclusion, the expression of proteins involved in signal transduction is altered in skeletal muscle from well-trained athletes. Downregulation of early components of the insulin-signaling cascade may occur in response to increased insulin sensitivity associated with endurance training.

Exercise training increases insulin-stimulated glucose disposal and GLUT4 (SLC2A4) protein content in patients with type 2 diabetes.

AIMS/HYPOTHESIS: Exercise enhances insulin-stimulated glucose transport in skeletal muscle through changes in signal transduction and gene expression. The aim of this study was to assess the impact of acute and short-term exercise training on whole-body insulin-mediated glucose disposal and signal transduction along the canonical insulin signalling cascade. METHODS: A euglycaemic-hyperinsulinaemic clamp, with vastus lateralis skeletal muscle biopsies, was performed at baseline and 16 h after an acute bout of exercise and short-term exercise training (7 days) in obese non-diabetic (n=7) and obese type 2 diabetic (n=8) subjects. RESULTS: Insulin-mediated glucose disposal was unchanged following acute exercise in both groups. Short-term exercise training increased insulin-mediated glucose disposal in obese type 2 diabetic (p<0.05), but not in obese non-diabetic subjects. Insulin activation of (1) IRS1, (2) IRS2, (3) phosphotyrosine-associated phosphatidylinositol-3 kinase activity and (4) the substrate of phosphorylated Akt, AS160, a functional Rab GTPase activating protein important for GLUT4 (now known as solute carrier family 2 [facilitated glucose transporter], member 4 [SLC2A4]) translocation, was unchanged after acute or chronic exercise in either group. GLUT4 protein content was increased in obese type 2 diabetic subjects (p<0.05), but not in obese non-diabetic subjects following chronic exercise. CONCLUSIONS/INTERPRETATION: Exercise training increased whole-body insulin-mediated glucose disposal in obese type 2 diabetic patients. These changes were independent of functional alterations in the insulin-signalling cascade and related to increased GLUT4 protein content.

C-peptide stimulates ERK1/2 and JNK MAP kinases via activation of protein kinase C in human renal tubular cells.

AIMS/HYPOTHESIS: Accumulating evidence indicates that replacement of C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, but the molecular mechanisms involved are incompletely understood. C-peptide shows specific binding to a G-protein-coupled membrane binding site, resulting in Ca(2+) influx, activation of mitogen-activated protein kinase signalling pathways, and stimulation of Na(+), K(+)-ATPase and endothelial nitric oxide synthase. This study examines the intracellular signalling pathways activated by C-peptide in human renal tubular cells. METHODS: Human renal tubular cells were cultured from the outer cortex of renal tissue obtained from patients undergoing elective nephrectomy. Extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and Akt/protein kinase B (PKB) activation was determined using phospho-specific antibodies. Protein kinase C (PKC) and RhoA activation was determined by measuring their translocation to the cell membrane fraction using isoform-specific antibodies. RESULTS: Human C-peptide increases phosphorylation of ERK1/2 and Akt/PKB in a concentration- and time-dependent manner in renal tubular cells. The C-terminal pentapeptide of C-peptide is equipotent with the full-length C-peptide, whereas scrambled C-peptide has no effect. C-peptide stimulation also results in phosphorylation of JNK, but not of p38 mitogen-activated protein kinase. MEK1/2 inhibitor PD98059 blocks the C-peptide effect on ERK1/2 phosphorylation. C-peptide causes specific translocation of PKC isoforms delta and epsilon to the membrane fraction in tubular cells. All stimulatory effects of C-peptide were abolished by pertussis toxin. The isoform-specific PKC-delta inhibitor rottlerin and the broad-spectrum PKC inhibitor GF109203X both abolish the C-peptide effect on ERK1/2 phosphorylation. C-peptide stimulation also causes translocation of the small GTPase RhoA from the cytosol to the cell membrane. Inhibition of phospholipase C abolished the stimulatory effect of C-peptide on phosphorylation of ERK1/2, JNK and PKC-delta. CONCLUSIONS/INTERPRETATION: C-peptide signal transduction in human renal tubular cells involves the activation of phospholipase C and PKC-delta and PKC-epsilon, as well as RhoA, followed by phosphorylation of ERK1/2 and JNK, and a parallel activation of Akt.

Intracellular mechanisms underlying increases in glucose uptake in response to insulin or exercise in skeletal muscle.

This review will provide insight on potential intracellular signalling mechanisms by which insulin and exercise/contraction increases glucose metabolism and gene expression. Glucose transport, the rate limiting step in glucose metabolism, is mediated by glucose transporter 4 (GLUT4) and can be activated in skeletal muscle by two separate and distinct signalling pathways; one stimulated by insulin and the second by muscle contractions. Impaired insulin action on whole body glucose uptake is a hallmark feature of type II (non-insulin-dependent) diabetes mellitus. Defects in insulin signal transduction through the insulin-receptor substrate-1/phosphatidylinositol 3-kinase pathway are associated with reduced insulin-stimulated glucose transporter 4 translocation and glucose transport activity in skeletal muscle from type II diabetic patients. Studies performed using glucose transporter 4-null mice show that this glucose transporter isoform plays a major role in mediating exercise-stimulated glucose uptake in skeletal muscle. Level of physical exercise has been linked to improved glucose homeostasis and enhanced insulin sensitivity. Exercise training leads to alterations in expression and activity of key proteins involved in insulin signal transduction. These changes may be related to increased signal transduction through the mitogen-activated protein kinase (MAPK) signalling cascades. Because MAPK is associated with increased transcriptional activity, these signalling cascades are candidates for these exercise-induced changes in protein expression. Understanding the molecular mechanism for the activation of signal transduction pathways will provide a link for defining new strategies to enhance glucose metabolism and improve health in the general population.

Aberrant p38 mitogen-activated protein kinase signalling in skeletal muscle from Type 2 diabetic patients.

AIMS/HYPOTHESIS: p38 mitogen activated protein kinase (MAPK) is generally thought to facilitate signal transduction to genomic, rather than metabolic responses. However, recent evidence implicates a role for p38 MAPK in the regulation of glucose transport; a site of insulin resistance in Type 2 diabetes. Thus we determined p38 MAPK protein expression and phosphorylation in skeletal muscle from Type 2 diabetic patients and non-diabetic subjects. METHODS: In vitro effects of insulin (120 nmol/l) or AICAR (1 mmol/l) on p38 MAPK expression and phosphorylation were determined in skeletal muscle from non-diabetic (n=6) and Type 2 diabetic (n=9) subjects. RESULTS: p38 MAPK protein expression was similar between Type 2 diabetic patients and non-diabetic subjects. Insulin exposure increased p38 MAPK phosphorylation in non-diabetic, but not in Type 2 diabetic patients. In contrast, basal phosphorylation of p38 MAPK was increased in skeletal muscle from Type 2 diabetic patients. CONCLUSION/INTERPRETATION: Insulin increases p38 MAPK phosphorylation in skeletal muscle from non-diabetic subjects, but not in Type 2 diabetic patients. However, basal p38 MAPK phosphorylation is increased in skeletal muscle from Type 2 diabetic patients. Thus, aberrant p38 MAPK signalling might contribute to the pathogenesis of insulin resistance.

Phosphorylation of Na,K-ATPase alpha-subunits in microsomes and in homogenates of Xenopus oocytes resulting from the stimulation of protein kinase A and protein kinase C.

The phosphorylation of the alpha-subunit of Na+/K(+)-transporting ATPase (Na,K-ATPase) by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was characterized in purified enzyme preparations of Bufo marinus kidney and duck salt gland and in microsomes of Xenopus oocytes. In addition, we have examined cAMP and phorbol esters, which are stimulators of PKA and PKC, respectively, for their ability to provoke the phosphorylation of alpha-subunits of Na,K-ATPase in homogenates of Xenopus oocytes. In the enzyme from the duct salt gland, phosphorylation by PKA and PKC occurs on serine and threonine residues, whereas in the enzyme from B. marinus kidney and Xenopus oocytes, phosphorylation by PKA occurs only on serine residues. Phosphopeptide analysis indicates that a site phosphorylated by PKA resides in a 12-kDa fragment comprising the C terminus of the polypeptide. Studies of phosphorylation performed on homogenates of Xenopus oocytes show that not only endogenous oocyte Na,K-ATPase but also exogenous Xenopus Na,K-ATPase expressed in the oocyte by microinjection of cRNA can be phosphorylated in response to stimulation of oocyte PKA and PKC. In conclusion, these data are consistent with the possibility that the alpha-subunit of Na,K-ATPase can serve as a substrate for PKA and PKC in vivo.

Receptor-mediated inhibition of renal Na(+)-K(+)-ATPase is associated with endocytosis of its alpha- and beta-subunits.

The mechanisms involved in receptor-mediated inhibition of Na(+)-K(+)-ATPase remain poorly understood. In this study, we evaluate whether inhibition of proximal tubule Na(+)-K(+)-ATPase activity by dopamine is linked to its removal from the plasma membrane and internalization into defined intracellular compartments. Clathrin-coated vesicles were isolated by sucrose gradient centrifugation and negative lectin selection, and early and late endosomes were separated on a flotation gradient. Inhibition of Na(+)-K(+)-ATPase activity by dopamine, in contrast to its inhibition by ouabain, was accompanied by a sequential increase in the abundance of the alpha-subunit in clathrin-coated vesicles (1 min), early endosomes (2.5 min), and late endosomes (5 min), suggesting its stepwise translocation between these organelles. A similar pattern was found for the beta-subunit. The increased incorporation of both subunits in all compartments was blocked by calphostin C. The results demonstrate that the dopamine-induced decrease in Na(+)-K(+)-ATPase activity in proximal tubules is associated with internalization of its alpha- and beta-subunits into early and late endosomes via a clathrin-dependent pathway and that this process is protein kinase C dependent. The presence of Na(+)-K(+)-ATPase subunits in endosomes suggests that these compartments may constitute normal traffic reservoirs during pump degradation and/or synthesis.

Phosphorylation of the Na,K-ATPase by Ca,phospholipid-dependent and cAMP-dependent protein kinases. Mapping of the region phosphorylated by Ca,phospholipid-dependent protein kinase.

Ca,phospholipid-dependent (PKC) and cAMP-dependent (PKA) protein kinases phosphorylate the alpha-subunit of the Na,K-ATPase from duck salt gland with the incorporation of 0.3 and 0.5 mol 32P/mol of alpha-subunit, respectively. PKA (in contrast to PKC) phosphorylates the alpha-subunit only in the presence of detergents. Limited tryptic digestion of the Na,K-ATPase phosphorylated by PKC demonstrates that 32P is incorporated into the N-terminal 41-kDa fragment of the alpha-subunit. Selective chymotrypsin cleavage of phosphorylated enzyme yields a 35-kDa radioactive fragment derived from the central region of the alpha-subunit molecule. These findings suggest that PKC phosphorylates the alpha-subunit of the Na,K-ATPase within the region restricted by C3 and T1 cleavage sites.

Marathon running increases ERK1/2 and p38 MAP kinase signalling to downstream targets in human skeletal muscle.

1. We tested the hypothesis that long-distance running activates parallel mitogen-activated protein kinase (MAPK) cascades that involve extracellular signal regulated kinase 1 and 2 (ERK1/2) and p38 MAPK and their downstream substrates. 2. Eleven men completed a 42.2 km marathon (mean race time 4 h 1 min; range 2 h 56 min to 4 h 33 min). Vastus lateralis muscle biopsies were obtained before and after the race. Glycogen content was measured spectrophotometrically. ERK1/2 and p38 MAPK phosphorylation was determined by immunoblot analysis using phosphospecific antibodies. Activation of the downstream targets of ERK1/2 and p38 MAPK, MAPK-activated protein kinase-1 (MAPKAP-K1; also called p90 ribosomal S6 kinase, p90rsk), MAPK-activated protein kinase-2 (MAPKAP-K2), mitogen- and stress-activated kinase 1 (MSK1) and mitogen- and stress-activated kinase 2 (MSK2) was determined using immune complex assays. 3. Muscle glycogen content was reduced by 40 +/- 6 % after the marathon. ERK1/2 phosphorylation increased 7.8-fold and p38 MAPK phosphorylation increased 4.4-fold post-exercise. Prolonged running did not alter ERK1/2 and p38 MAPK protein expression. The activity of p90rsk, a downstream target of ERK1/2, increased 2.8-fold after the marathon. The activity of MAPKAPK-K2, a downstream target of p38 MAPK, increased 3.1-fold post-exercise. MSK1 and MSK2 are downstream of both ERK1/2 and p38 MAPK. MSK1 activity increased 2.4-fold post-exercise. MSK2 activity was low, relative to MSK1, with little activation post-exercise. 4. In conclusion, prolonged distance running activates MAPK signalling cascades in skeletal muscle, including increased activity of downstream targets: p90rsk, MAPKAP-K2 and MSK. Activation of these downstream targets provides a potential mechanism by which exercise induces gene transcription in skeletal muscle.

[Tyrosine protein kinase from cattle cerebral cortex: purification, characteristics, protein substrates for phosphorylation and inhibitors of activity]. / Tirozinovaia proteinkinaza iz kory mozga byka: ochistka, kharakteristika svoistv, belkovye substraty fosforilirovaniia i ingibitory aktivnosti.

Tyrosine protein kinase present in the membrane fraction of bovine cerebral cortex were extracted and chromatographically fractionated. The activity associated with tyrosine protein kinases was fully extracted from the membranes by 1% sodium cholate and eluted in two peaks (I and II) during chromatography of protein extracts on DEAE-Toyopearl in the presence of sodium cholate. The predominant in cerebral cortex membrane tyrosine protein kinase of peak I (about 75% of the total activity) was purified 1930-fold by gel filtration on Sephacryl S-300, chromatography on hexyl- and phenyl-Sepharose and by rechromatography on DEAE-Toyopearl. The amount of the enzyme prepared from 250 g of bovine brain was 20 micrograms, the enzyme yield and specific activity being 3.8% and 3.9 nmol/mg protein/min, respectively. The purified protein kinase of peak I represents a protein with Mr of 62-63,000 (p62) capable of being autophosphorylated in the presence of [gamma-32P]. Protein kinase p62 phosphorylates enolase, tubulin and calpactin I as well as model substrates in the series: histone H5 greater than poly(G, T)n greater than or equal to histone H2A greater than poly(G, A, T)n, histone H4 greater than caseins, histones H1 and H2B, poly(G, A, L, T)n. The enzyme is specific for Mn2+ at the optimal concentration about 1 mM. The KmMn-ATP is 0.3 microM; Km for histone H5 and poly(G, T)n are 0.45 mg/ml and 0.06 mg/ml, respectively. The protein kinase p62 activity is inhibited by NaCl (IC50 approximately 75-100 mM) as well as by quercetin, adriamycin and lasalocid (IC50 approximately 14-34, 23 and 90 microM, respectively). It is concluded that protein kinase p62 is analogous to the c-src gene protein kinase.