Effects of adenosine receptor antagonism on protein tyrosine phosphatase in rat skeletal muscle

Earlier studies have shown that whole body adenosine receptor antagonism increases skeletal muscle insulin sensitivity in insulin-resistant Zucker rats. To find which steps in the insulin signaling pathway are influenced by adenosine receptors, muscle from lean and obese Zucker rats, treated for 1 week with the adenosine receptor antagonist, 1,3-dipropyl-8-(4-acrylate)-phenylxanthine (BWA1433), were analyzed. All rats were first anesthetized and injected intravenously (i.v.) with 1 IU of insulin. About 3 min later the gastrocnemius was freeze clamped. Insulin receptors were partially purified on wheat germ agglutinin (WGA) columns and insulin receptor kinase activity measured in control and BWA1433-treated lean and obese Zucker rats. Protein tyrosine phosphatase (PTPase) activity was also analyzed in subcellular fractions, including the cytosolic fraction, a high-speed particulate fraction and the insulin receptor fraction eluted from WGA columns. Administration of BWA1433 increased insulin receptor kinase activity in obese but not lean Zucker rats. PTPase activities were higher in the untreated obese rat muscle particulate fractions than in the lean rat particulate fractions. The BWA1433 administration lowered the PTPase activity of the obese rats but not the lean rats. Although the PTPase activity in WGA eluate fractions containing crude insulin receptors were similar in lean and obese animals, BWA1433 administration was found to lower the PTPase activities in the fractions obtained from obese but not from the lean rats. PTPases may be upregulated in muscles from obese rats due to activated adenosine receptors. Adenosine receptor blockade, by reducing PTPase activity, may thereby increase insulin signaling.     

Protein Tyrosine Phosphatase Activity in Insulin-Resistant Rodent Psammomys Obesus

Phosphotyrosine phosphatase (PTPase) activity and its regulation by overnight food deprivation were studied in Psammomys obesus (sand rat), a gerbil model of insulin resistance and nutritionally induced diabetes mellitus. PTPase activity was measured using a phosphopeptide substrate containing a sequence identical to that of the major site of insulin receptor (IR) β-subunit autophosphorylation. The PTPase activity in membrane fractions was 3.5-, 8.3-, and 5.9-fold lower in liver, fat, and skeletal muscle, respectively, compared with corresponding tissues of albino rat.Western blotting of tissue membrane fractions in Psammomys showed lower PTPase and IR than in albino rats. The density of PTPase transmembrane protein band was 5.5-fold lower in liver and 12-fold lower in adipose tissue. Leukocyte antigen receptor (LAR) and IR were determined by specific immunoblotting and protein bands densitometry and were also found to be 6.3-fold lower in the liver and 22-fold lower in the adipose tissue in the hepatic membrane fractions. Liver cytosolic PTPase activity after an overnight food deprivation in the nondiabetic Psammomys rose 3.7-fold compared with postprandial PTPase activity, but it did not change significantly in diabetic fasted animals. Similar fasting-related changes were detected in the activity of PTPase derived from membrane fraction. In conclusion, the above data demonstrate that despite the insulin resistance, Psammomys is characterized by low level of PTPase activities in membrane and cytosolic fractions in all 3 major insulin responsive tissues, as well as in liver. PTPase activity does not rise in activity as a result of insulin resistance and nutritionally induced diabetes.     

Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function of the insulin receptor.     

Effect of tumor necrosis factor-α on the phosphorylation of tyrosine kinase receptors is associated with dynamic alterations in specific protein-tyrosine phosphatases

Tumor necrosis factor-α (TNF-α) can modulate the signalling capacity of tyrosine kinase receptors; in particular, TNF-α has been shown to mediate the insulin resistance associated with animal models of obesity and noninsulin-dependent diabetes mellitus. In order to determine whether the effects of TNF-α might involve alterations in the expression of specific protein-tyrosine phosphatases (PTPases) that have been implicated in the regulation of growth factor receptor signalling, KRC-7 rat hepatoma cells were treated with TNF-α, and changes in overall tissue PTPase activity and the abundance of three major hepatic PTPases (LAR, PTP1B, and SH-PTP2) were measured in addition to effects of TNF-α on ligand-stimulated autophosphorylation of insulin and epidermal growth factor (EGF) receptors and insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation. TNF-α caused a dose-dependent decrease in insulin-stimulated IRS-1 phosphorylation and EGF-stimulated receptor autophosphorylation to 47–50% of control. Overall PTPase activity in the cytosol fraction did not change with TNF-α treatment, and PTPase activity in the particulate fraction was decreased by 55–66%, demonstrating that increases in total cellular PTPase activity did not account for the observed alterations in receptor signalling. However, immunoblot analysis showed that TNF-α treatment resulted in a 2.5-fold increase in the abundance of SH-PTP2, a 49% decrease in the transmembrane PTPase LAR, and no evident change in the expression of PTP1B. These data suggest that at least part of the TNF-α effect on pathways of reversible tyrosine phosphorylation may be exerted through the dynamic modulation of the expression of specific PTPases. Since SH-PTP2 has been shown to interact directly with both the EGF receptor and IRS-1, increased abundance of this PTPase may mediate the TNF-α effect to inhibit signalling through these proteins. Furthermore, decreased abundance of the LAR PTPase, which has been implicated in the regulation of insulin receptor phosphorylation, may account for the less marked effect of TNF-α on the autophosphorylation state of the insulin receptor while postreceptor actions of insulin are inhibited. J. Cell. Biochem. 64:117–127. © 1997 Wiley-Liss, Inc.     

Decrease in protein tyrosine phosphatase activities in vanadate-treated obese Zucker (fa/fa) rat liver

The inhibitory action of vanadate towards protein tyrosine phosphatase (PTPase) has been considered as a probable mechanism by which it exerts insulin-like effects. In this study, we have examined thein vivo effects of vanadate on PTPases in the liver of obese Zucker rats, a genetic animal model for obesity and type II diabetes. These animals were characterized by hyperinsulinemia and mild hyperglycemia. The number of insulin receptors were significantly (p<0.01) decreased in liver. After chronic administration of vanadate in obese rats, 80% decrease in the plasma levels of insulin was observed. The insulin receptor numbers were significantly (p<0.01) higher in vanadate-treated obese rats as compared to the untreated ones. The hepatic PTPase activities in cytosolic and particulate fractions, with phosphorylated poly glu:tyr (41) and the insulin receptor peptide (residues 1142–1153) as substrates, increased in obese rats. In vanadate-treated obese rat livers, the PTPase activities in both subcellular fractions with these substrates decreased significantly (p<0.001). The decreases in PTPase activities from these groups of rats were further supported by chromatography on a Mono Q column. These data support the view that inhibition of PTPases plays a role in the insulin-mimetic action of vanadate.     

Glucagon resistance of hepatoma cells. Evidence for receptor and post-receptor defects.

Of all available liver cells in culture, only primary cultured hepatocytes are known to respond to glucagon in vitro. In the present study we investigated whether glucagon could stimulate amino acid transport and tyrosine aminotransferase (TAT;EC 2.6.1.5) activity (two well-characterized glucagon effects in the liver) in Fao cells, a highly differentiated rat hepatoma cell line. We found that glucagon had no effect on transport of alpha-aminoisobutyric acid (AIB; a non-metabolizable alanine analogue) nor on TAT activity, even though both activities could be fully induced by insulin [2-fold and 3-fold effects for AIB transport and TAT activity, respectively, after 6h; EC50 (median effective concentration) = 0.3 nM], or by dexamethasone (5-8-fold effects after 20 h; EC50 = 2 nM). Analysis of [125I]iodoglucagon binding revealed that Fao cells bind less than 1% as much glucagon as do hepatocytes, whereas insulin binding in Fao cells was 50% higher than in hepatocytes. The addition of dibutyryl cyclic AMP, which fully mimics the glucagon stimulation of both AIB transport and TAT activity in hepatocytes, induced TAT activity in Fao cells (a 2-fold effect at 0.1 mM-dibutyryl cyclic AMP) but had no effect on AIB transport. Cholera toxin stimulated TAT activity to the same extent as did dibutyryl cyclic AMP. These results indicate that the lack of glucagon responsiveness in cultured hepatoma cells results from both a receptor defect and, for amino acid transport, an additional post-receptor defect. Moreover, the results show that amino acid transport and TAT activity, which appeared to be co-induced by insulin or by dexamethasone in these cells, respond differently to cyclic AMP. This suggests that different mechanisms are involved in the induction of these activities by glucagon in liver.     

Survival of Chick Embryonic Sensory Neurons in Culture Is Supported by Phorbol Esters

Sensory neurons of the chick embryo are supported in culture by several neurotrophic factors, including the phorbol esters. Because phorbol esters are known to activate one of the second messengers, namely, protein kinase C, it was of interest to see if the neurotrophic action of phorbol 12,13-dibutyrate (PDB) was related to the activation of protein kinase C in sensory neurons. Sensory neurons were obtained from dorsal root ganglia of 10-day-old chick embryos and maintained in a serum-free medium for several days to quantify survival and analyze protein kinase C activity. PDB (30 nM) supported the survival of approximately 50% of the total number of neurons plated. This value was comparable to that supported by nerve growth factor (NGF; 40 ng/ml). If PDB and NGF were added together, there was no additive effect on the survival. The protein kinase C activity of the particulate and cytosolic fractions of sensory neurons supported by NGF for 3 days was 1.26 +/- 0.1 and 2.9 +/- 0.32 pmol/min/mg of protein, respectively. In contrast, neurons supported by PDB showed an approximately 500% increase in enzyme activity in their particulate fraction. The enzyme activity of the cytosolic fraction was decreased by approximately 40%. If NGF-supported neurons were treated with PDB (30 nM) for 15 min, protein kinase C activity increased greater than 400% in the particulate fraction, whereas an approximately 50% decrease was observed in the cytosolic fraction. The protein kinase C value, expressed as a ratio of the activities in the particulate to cytosol fractions, showed large increases after phorbol treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein tyrosine phosphatase regulation in fibroblasts from patients with an insulin receptor gene mutation

Tyrosine phosphorylation of the insulin receptor is the initial event following receptor binding to insulin, and it induces further tyrosine phosphorylation of various intracellular molecules. This signaling is countered by protein tyrosine phosphatases (PTPases), which reportedly are associated with insulin resistance that can be reduced by regulation of PTPases. Protein tyrosine phosphatase 1B (PTP1B) and leukocyte antigen-related PTPase (LAR) are the PTPases implicated most frequently in insulin resistance and diabetes mellitus. Here, we show that PTP1B and LAR are expressed in human fibroblasts, and we examine the regulation of PTPase activity in fibroblasts from patients with an insulin receptor gene mutation as an in vitro model of insulin resistance. Total PTPase activity was significantly lower in the cytosolic and membrane fractions of fibroblasts with mutations compared with controls (p<0.05). Insulin stimulation of fibroblasts with mutations resulted in a significantly smaller increase in PTP1B activity compared with stimulation of wild-type fibroblasts (p<0.05). This indicates that insulin receptor gene mutations blunt increases in PTPase activity in response to insulin, possibly via a negative feedback mechanism. Our data suggest that the PTPase activity in patients with insulin receptor gene mutation and severe insulin resistance may differ from that in ordinary type 2 diabetes.     

Elevated expression and activity of protein-tyrosine phosphatase 1B in skeletal muscle of insulin-resistant type II diabetic Goto-Kakizaki rats

We investigated the cellular mechanism(s) of insulin resistance associated with non-insulin dependent diabetes mellitus (NIDDM) using skeletal muscles isolated from non-obese, insulin resistant type II diabetic Goto-Kakizaki (GK) rats, a well known genetic rat model for type II diabetic humans. Relative to non-diabetic control rats (WKY), insulin-stimulated insulin receptor (IR) autophosphorylation and insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation were significantly inhibited in GK skeletal muscles. This may be due to increased dephosphorylation by a protein tyrosine phosphatase (PTPase). Therefore, we measured skeletal muscle total PTPase and PTPase 1B activities in the skeletal muscles isolated from control rats (WKY) and diabetic Goto-Kakizaki (GK) rats. PTPase activity was measured using a synthetic phosphopeptide, TRDIY(P)ETDY(P)Y(P)RK, as the substrate. Basal PTPase activity was 2-fold higher (P < 0.001) in skeletal muscle of GK rats when compared to WKY. Insulin infusion inhibited skeletal muscle PTPase activity in both control (26.20% of basal, P < 0.001) and GK (25.35% of basal, P < 0.001) rats. However, PTPase activity in skeletal muscle of insulin-stimulated GK rats was 200% higher than hormone-treated WKY controls (P < 0.001). Immunoprecipitation of PTPase 1B from skeletal muscle lysates and analysis of the enzyme activity in immunoprecipitates indicated that both basal and insulin-stimulated PTPase 1B activities were significantly higher (twofold, P < 0.001) in skeletal muscle of diabetic GK rats when compared to WKY controls. The increase in PTPase 1B activity in diabetic GK rats was associated with an increased expression of the PTPase 1B protein. We concluded that insulin resistance of GK rats is accompanied atleast by an abnormal regulation of PTPase 1B. Elevated PTPase 1B activity through enhanced tyrosine dephosphorylation of the insulin receptor and its substrates, may lead to impaired glucose tolerance and insulin resistance in GK rats.     

Characterization of receptors for vasoactive intestinal peptide solubilized from the lung

The zwitterionic detergent CHAPS was used to solubilize functional receptors for vasoactive intestinal peptide (VIP) from guinea pig lung. The solubilized receptors were resolved by high performance gel filtration in 3 mM CHAPS into two active fractions with apparent Stokes radii of 5.9 +/- 0.1 and 2.3 +/- 0.1 nm. The binding of 125I-VIP to the two receptor fractions was time-dependent, reversible, and saturable. Trypsin destroyed the binding activity of the receptor fractions, indicating their proteinic nature. Unlabeled VIP competitively displaced the binding of 125I-VIP to the 5.9-nm fraction (IC50 = 240 pM) and the 2.3-nm fraction (IC50 = 1.2 microM). Scatchard analysis indicated a single class of binding sites in each receptor fraction, with Kd values 300 pM and 0.97 microM for the 5.9- and 2.3-nm Stokes radii fractions, respectively. When the high affinity, 5.9-nm Stokes radius fraction was rechromatographed in 9 nM CHAPS, 46% of the binding activity eluted in the low affinity, 2.3-nm Stokes radius fraction, indicating that the latter is a product of dissociation of the high affinity receptor complex. GTP inhibited the binding of 125I-VIP to the high affinity complex but not the low affinity species. Scatchard plots of VIP binding by the high affinity receptors treated with GTP suggested the presence of two distinct binding sites (Kd 4.4 and 153 nM), compared to a single binding site (Kd = 0.3 nM) obtained in untreated receptors. The nonhydrolyzable GTP analog, guanyl-5'-yl-imidodiphosphate, inhibited VIP binding by the high affinity receptor fraction with potency nearly equivalent to that of GTP. These observations suggest that GTP-binding regulatory proteins are functionally coupled to the VIP-binding subunit in the high affinity receptor complex. The peptide specificity characteristics of the two receptor fractions were different. Peptide histidine isoleucine and growth hormone releasing factor, peptides homologous to VIP, were 87.5- and 22.9-fold less potent than VIP in displacing 125I-VIP binding by the high affinity receptor complex, respectively. On the other hand, growth hormone-releasing factor was more potent (22.7-fold) and peptide histidine isoleucine was less potent (31.3-fold) than VIP in displacing the binding by the low affinity species.     

Specific binding of okadaic acid, a new tumor promoter in mouse skin

The tumor promoter okadaic acid binds specifically to a particulate as well as a cytosolic fraction of various mouse tissues, e.g., skin, brain, lung and colon. The KD value was 21.7 nM for receptors in the particulate fraction and 1.0 nM for those in the cytosolic fraction of mouse skin. The specific binding of [3H]okadaic acid to the particulate fraction of mouse skin was inhibited dose-dependently by okadaic acid, but not okadaic acid tetramethyl ether, an inactive compound, or by other tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate and teleocidin. The results suggest a new pathway of tumor promotion mediated through the okadaic acid receptor(s).     

Functional NOS 1 in the rat mesenteric arterial bed

Previously we have demonstrated functional nitric oxide synthase (NOS) 1 in large arteries. Because resistance arteries largely determine blood pressure, this study examined whether functional NOS 1 also exists in resistance arteries. Phenylephrine (PE) contraction was measured in the absence and presence of the NOS 1 inhibitor N(5)-(1-imino-3-butenyl)-L-ornithine (VNIO) in isolated mesenteric resistance arteries (endothelium intact and denuded) from Sprague-Dawley rats. For NOS 1 activity and expression, the mesenteric arterial bed was separated into cytosolic and particulate fractions. NOS activity was assayed by measuring the conversion of [(3)H]arginine to [(3)H]citrulline inhibited by a nonselective NOS inhibitor or VNIO. VNIO increased PE sensitivity in endothelium-intact and -denuded arteries. In cytosolic and particulate fractions of the arterial bed, approximately 40% of NOS activity was inhibited by VNIO. Immunoprecipitation and Western blot analysis revealed two NOS 1 immunoreactive bands. One band corresponded to the rat brain isoform, whereas the second was of a slightly lower molecular mass. The cytosolic fraction contained both isoforms; however, the particulate fraction had only the lower molecular mass form. These studies demonstrate the existence of functional NOS 1 in resistance arteries.     

Activation of particulate guanylate cyclase by adrenomedullin in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells

We investigated the effects of adrenomedullin (ADM) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ADM increased cGMP accumulation in a time- and concentration- dependent manner. The peptide increased cGMP formation in the transformed cells by 405-fold as compared to 1. 6-fold in primary cultured CISM cells. The basal cGMP concentrations in both cell types were comparable. In addition, ADM increased cAMP accumulation in SV-CISM-2 cells and in primary cultured cells by 18. 9- and 5.8-fold, respectively. The ADM receptor antagonist, ADM(26-52), but not the atrial natriuretic peptide (ANP) receptor antagonist, anantin, inhibited ADM-induced cGMP formation. The phorbol ester, phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylate cyclases in smooth muscle, blocked ADM-stimulated cGMP accumulation. In contrast, inhibitors of the soluble guanylate cyclases, such as LY83583 and ODQ, and inhibitors of the nitric oxide cascade had little effect on ADM-stimulated cGMP production. The stimulatory effect of ADM on cGMP formation is due to activation of the guanylate cyclase system and not to a much reduced phosphodiesterase activity. ADM stimulated guanylate cyclase activity in membrane fractions isolated from SV-CISM-2 cells in a concentration-dependent manner with EC(50) value of 72 nM. Pertussis toxin, an activator of the G-protein, Gi, inhibited ADM-stimulated cGMP accumulation, whereas cholera toxin, a stimulator of the Gs G-protein and subsequently cAMP accumulation, had little effect. Pretreatment of the plasma membrane fraction with Gialpha antibody attenuated ADM-stimulated guanylate cyclase activity by 75%. We conclude that ADM increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ADM receptor and subsequent stimulation of a Gi-mediated membrane-bound guanylate cyclase.     

Insulin-receptor phosphotyrosyl-protein phosphatases.

Calmodulin-dependent protein phosphatase has been proposed to be an important phosphotyrosyl-protein phosphatase. The ability of the enzyme to attack autophosphorylated insulin receptor was examined and compared with the known ability of the enzyme to act on autophosphorylated epidermal-growth-factor (EGF) receptor. Purified calmodulin-dependent protein phosphatase was shown to catalyse the complete dephosphorylation of phosphotyrosyl-(insulin receptor). When compared at similar concentrations, 32P-labelled EGF receptor was dephosphorylated at greater than 3 times the rate of 32P-labelled insulin receptor; both dephosphorylations exhibited similar dependence on metal ions and calmodulin. Native phosphotyrosyl-protein phosphatases in cell extracts were also characterized. With rat liver, heart or brain, most (75%) of the native phosphatase activity against both 32P-labelled insulin and EGF receptors was recovered in the particulate fraction of the cell, with only 25% in the soluble fraction. This subcellular distribution contrasts with results of previous studies using artificial substrates, which found most of the phosphotyrosyl-protein phosphatase activity in the soluble fraction of the cell. Properties of particulate and soluble phosphatase activity against 32P-labelled insulin and EGF receptors are reported. The contribution of calmodulin-dependent protein phosphatase activity to phosphotyrosyl-protein phosphatase activity in cell fractions was determined by utilizing the unique metal-ion dependence of calmodulin-dependent protein phosphatase. Whereas Ni2+ (1 mM) markedly activated the calmodulin-dependent protein phosphatase, it was found to inhibit potently both particulate and soluble phosphotyrosyl-protein phosphatase activity. In fractions from rat liver, brain and heart, total phosphotyrosyl-protein phosphatase activity against both 32P-labelled receptors was inhibited by 99.5 +/- 6% (mean +/- S.E.M., 30 observations) by Ni2+. Results of Ni2+ inhibition studies were confirmed by other methods. It is concluded that in cell extracts phosphotyrosyl-protein phosphatases other than calmodulin-dependent protein phosphatase are the major phosphotyrosyl-(insulin receptor) and -(EGF receptor) phosphatases.     

Exercise promotes a subcellular redistribution of calcium-stimulated protease activity in striated muscle.

The aims of this study were (i) to investigate whether the contractile activity associated with running increases calcium-stimulated, calpastatin-inhibited protease activity (calpain-like) in a time-dependent manner and (ii) to determine whether the changes, if any, are proportionately distributed between soluble (cytosolic) and particulate (bound) fractions of striated muscle in vivo. Calcium-dependent, calpastatin-inhibited caseinolysis (i.e., calpain-like activity) was measured in control and exercised rats (25 m/min, 0% grade) at 2, 5, 15, 30, and 60 min. Total calpain-like activity in skeletal muscle increased by 26% (13.2 +/- 1.3 vs. 17.9 +/- 2.2 U/g wet wt.) (p < 0.05) after running (60 min), accompanied by an increased activity in the particulate fraction. In cardiac muscle, exercise (60 min) increased total calpain-like activity by 33% (p < 0.05), which was attributable to increases in both the cytosolic and particulate fractions. Both tissues responded with an early (2-5 min) activation of total calpain-like activity (p < 0.05), supported by early increases for particulate fractions from skeletal muscle; whereas for cardiac muscle, a noticeable early drop (p < 0.05) occurred in the particulate fraction. Minimal changes were observed for total, cytosolic, and particulate fractions of noncontracting tissue (i.e., liver). The results of this study support the hypothesis that the total calpain-like activity increases associated with level running occur early on with exercise and that the increases are accompanied by changes in the redistribution of soluble to particulate fractions. The changes would set the stage for enhanced rates of protein degradation known to occur in striated muscle with exercise.     

Nitric oxide synthase activity in human trophoblast, term placenta and pregnant myometrium

To investigate the possible role of nitric oxide (NO) produced locally or intramurally in the quiescence of the pregnant myometrium, nitric oxide synthase (NOS) activity was measured in samples from first trimester (villous, and non villous-trophoblast), term placenta and pregnant myometrium. Trophoblast tissue was obtained from psychosocial termination of pregnancy (9 – 12 weeks' gestation) whereas placenta and myometrium, from the same patient, at deliveries by Caesarean section. NOS activity was measured in both cytosolic and particulate fractions by the formation of ¹⁴C-citrulline from ¹⁴C-arginine. Western immunoblotting was used to identify the endothelial NOS (eNOS) and neuronal (nNOS) isoforms. The activity of NOS in particulate fractions from all preparations was considerably higher than the cytosolic fractions. Activity in all fractions except the myometrium was highly Ca-dependent. More than 50% of particulate NOS from the myometrium was Ca-independent. NOS activity was highest in the villous trophoblast and there was a significant difference between the villous and non-villous trophoblast. In placenta and myometrium, NOS was 2–4 fold and 20–28-fold lower than the villous trophoblast, respectively. Western blot analysis showed clearly eNOS in the particulate fraction and a weak eNOS band in the cytosolic fractions, whereas nNOS was not detectable in any of the fractions. In view of the marginal activity of NOS in the myometrium, NO produced by the trophoblast and placenta could play a significant role in maintaining uterine quiescence by paracrine effect.     

gamma-Glutamyltranspeptidase in dimethylbenz[a]anthracene-induced mammary adenocarcinomas and in livers of tumor bearing rats

A single dose of dimethylbenz[a]anthracene (DMBA) at 20 mg/kg resulted in 100% incidence of intraductal mammary adenocarcinomas in Wistar rats, the large tumors averaging 1.87 +/- 0.45 g. gamma-Glutamyltranspeptidase activities were elevated in DMBA-induced mammary adenocarcinomas relative to lactating mammary tissue in all fractions examined: 18.8-fold in homogenates; 22.1-fold in particulate fractions; and 5.7-fold in supernatant fractions. In DMBA-induced mammary adenocarcinomas, gamma-glutamyltranspeptidase was 95% particulate, 5% supernatant, whereas in lactating mammary tissue, gamma-glutamyltranspeptidase was equally distributed between particulate and supernatant fractions. Particulate gamma-glutamyltranspeptidase from DMBA-induced mammary adenocarcinomas as well as lactating mammary tissue displayed classical Michaelis-Menten characteristics: for the adenocarcinoma enzyme Km was 2.5 nM and Vmax 200 nmol mg-1 min-1; for mammary tissue enzyme Km was 2.5 nM and Vmax 11.1 nmol X mg-1 X min-1. Both particulate enzymes were activated at 50 degrees C relative to 37 degrees C to the same extent: 1.37-fold. The activities of gamma-glutamyltranspeptidase were increased 1.8-fold in the livers of rats bearing DMBA-induced mammary adenocarcinomas relative to age-matched controls. Plasma levels of gamma-glutamyltranspeptidase were also increased 1.6-fold in tumor bearing rats. There was no observable sign of liver damage in tumor bearing rats; plasma glutamic pyruvic transaminase levels were normal in these animals. Blood glucose levels were elevated 17% in rats bearing DMBA-induced mammary adenocarcinomas compared to age-matched controls, although plasma insulin levels were the same in both groups: 35.4 +/- 3.5 microIU/ml for the former; 31.9 +/- 3.1 microIU/ml for the latter.     

Dissociation of PTPase levels from their modulation of insulin receptor signal transduction

Protein tyrosine phosphatases have been implicated in the regulation of receptor tyrosine kinase signalling pathways, including that of the insulin receptor. Here, cell density-dependent changes in PTPase expression have been exploited to investigate the relationship between cellular PTPase levels and the insulin receptor signal transduction pathway. Increasing cell density (20%, 50%, and >90%) in the rat McA-RH7777 hepatoma cell line resulted in increased protein expression of the receptor-like PTPase LAR (14-fold), and the nonreceptor PTPases PTP1B (11-fold) and SHP2 (10-fold). Each of these PTPases has previously been implicated in regulating insulin receptor signal transduction. Despite these marked increases, maximum insulin receptor autophosphorylation as well as receptor expression actually increased 2-fold. MAP kinase also increased approximately 2-fold as a function of cell density and paralleled increases in expression levels. Neither sensitivity nor maximum responsiveness to insulin were decreased at increasing cell densities as assessed by activation of PI 3-kinase. Duration of response was also unimpaired. These results suggest that expression levels of relevant PTPases are not the primary determinant in their modulation of insulin receptor kinase activity. Restricted accessibility at the molecular level or involvement of accessory proteins may be more critical parameters.     

Subcellular localization and partial characterization of insulin proteolytic activity in rat liver

Using (A14-125I)-insulin as a tracer, insulin proteolytic activity in rat liver was found to be localized both to the cytosol and the endoplasmic reticulum. The membrane-associated activity was highly latent (70-80%). Both cytosolic and particulate activities had similar Km values and Mr of approx. 300 000 by gel filtration. Both were strongly inhibited by diamide (90%), but were unaffected by leupeptin or pepstatin. A comparison of the subcellular distributions with various 125I-isomers of insulin as tracers showed that both particulate and cytosolic activities were highest with (A14-125I)-insulin.