Circulating platelet-derived endothelial cell growth factor increases in hepatocellular carcinoma patients

BACKGROUND: Platelet-derived endothelial cell growth factor (PD-ECGF) is an angiogenic factor that is expressed in various cancer tissues. Little is known regarding plasma PD-ECGF levels in patients with chronic liver disease such as chronic hepatitis (CH), cirrhosis, and hepatocellular carcinoma (HCC) with cirrhosis. The expression of PD-ECGF in HCC tissues also remains to be clarified. METHODS: Plasma PD-ECGF levels in patients with chronic liver disease were determined with an enzyme-linked immunoadsorbent assay system using the mouse monoclonal antibodies specific to PD-ECGF. These were cross-sectionally compared among groups of normal persons, CH, cirrhosis, and HCC patients. The HCC patients were classified into two groups based on TNM stage: early and advanced stage disease groups. PD-ECGF expressions in HCC tissues were immunohistologically examined. RESULTS: The plasma PD-ECGF levels from the normal individuals and those with CH, cirrhosis, and HCC specimens were 4.2+/-0.5, 4.3+/-0.6, 4.6+/-1.1, and 6.0 +/-2.5 U/mL, respectively. The plasma PD-ECGF concentration was highest in HCC (P < 0.05). No significant difference was found among the normal subjects, CH, and cirrhosis specimens. Plasma PD-ECGF concentrations were significantly higher in the advanced stage disease HCC group compared with the early stage disease group (6.75+/-2.62 U/mL vs. 4.19+/-0.34 U/mL) (P < 0.05). Immunohistochemical expression of PD-ECGF in HCC cells increased significantly compared with normal liver cells (P < 0.05). CONCLUSIONS: Circulating PD-ECGF plasma level might be a new tumor marker for progression in patients with HCC. Immunohistological findings correspond to elevation of the plasma PD-ECGF in HCC patients. It is possible that increased production of PD-ECGF in HCC cells causes abundant neovascularization.     

Increase of lipid hydroperoxides in the rat liver and kidney after administering ferric nitrilotriacetate

Two hours after an intraperitoneal injection of ferric nitrilotriacetate to rats at a dose of 15 mg of Fe/kg of body weight, the level of lipid hydroperoxides, which was determined by a specific method involving chemical conversion into 1-naphthyldiphenylphosphine oxide and HPLC, had increased significantly in the liver (from 190.1 +/- 58.8 of the control to 467.1 +/- 175.8 pmol/mg of protein) and kidney (from 181.8 +/- 52.3 of the control to 405.9 +/- 22.7 pmol/mg of protein). These results demonstrate that oxidative stress was transiently increased by an iron overload in these tissues.     

Chemolipiodolization and prostaglandin E1 administration with use of hepatic arterial infusion port for the treatment of hepatocellular carcinoma and liver cirrhosis

Frequent chemolipiodolization and prostaglandin E1 (PGE1) administered through a hepatic arterial infusion port were used for treatment in 2 cases of hepatocellular carcinoma (HCC) with liver cirrhosis. Chemolipiodolization was performed every 4 weeks with 6 ml lipiodol, 3 ml Optilay and 30 mg Epirubicin or 10 mg Mytomycin C. PGE1 (10 ug) was administrated to the hepatic artery once every week after the first 7 days administration. The treatment resulted in a decrease of the AFP level, an arrest of HCC growth and a reduction in ascites with an improvement of clinical and biochemical parameters in both cases. These encouraging preliminary results show that frequent lipiodolization is effective for unresectable HCC and frequent PGE1 administration via the hepatic artery is a safe and efficient treatment for liver cirrhosis.     

Glucuronidation of retinoids by rat recombinant UDP: glucuronosyltransferase 1.1 (bilirubin UGT)

Rat liver recombinant BR1UGT1.1 was found to have significant activity toward retinoid substrates. UGT1.1 glucuronidation activity was 91 +/- 18 pmol/mg x min for atRA and 113 +/- 19 pmol/mg x min for 5,6-epoxy-atRA. The apparent K(M) and V(max) of atRA acid glucuronidation by UGT1.1 were 59.1 +/- 5.4 microM and 158 +/- 43 pmol/mg x min, respectively. SDS-PAGE and Western blot analysis of UGT1.1-transfected HK293 membrane proteins photolabeled with [11,12-3H]atRA revealed a protein of approximately 56 kDa that was labeled by [3H]atRA, detected by anti-pNP UGT antibody and not present in membranes from nontransfected HK293 cells. Liver microsomes from Gunn rats, which lack UGT1.1, had significant activity toward atRA (111 +/- 28 pmol/mg x min).     

Myenteric neurons with different projections have different dendritic tree patterns: a morphometric study in the pig ileum

Species differences in the biotransformation of the antiemetic tropisetron, a potent 5-hydroxytryptamine type 3 (5-HT3) receptor antagonist, were evident in liver slice incubates of human, rat and dog, and reflected the species differences observed in vivo with respect to the relative importance of individual pathways. The dominant biotransformation pathway of tropisetron (10 microM) in human liver slices was formation of 6-hydroxy-tropisetron, whereas in rat liver slices it was 5-hydroxy-tropisetron, and in dog liver slices N-oxide formation. Initial rates of tropisetron metabolite formation in the liver slices (8 mm in diameter, 200 +/- 25 microns thickness) of human (83 +/- 61 pmol/h/mg slice protein), rat (413 +/- 98 pmol/h/mg slice protein) and dog (426 +/- 38 pmol/h/mg slice protein) would predict less of a first-pass effect in humans compared to the rat or the dog. For human and rat, the prediction matched well with the species ranking of tropisetron bioavailability; however, for dog the in vitro data overestimated the apparent first-pass effect. The jejunum is not expected to contribute to the first-pass effect in humans, since human jejunum microsomes did not metabolize tropisetron. The major organ of excretion for tropisetron and its metabolites is the kidney, but the contribution of the kidney to the overall metabolism of tropisetron would be small. Species independent N-oxide formation (2-12 pmol/h/mg slice protein) was the major pathway in human, rat and dog kidney slices, and was comparable to N-oxide formation in the rat and human liver slices but was 1/10 the rate in dog liver slices. This study has demonstrated that the liver is the primary site of tropisetron biotransformation, and the usefulness of organ slices to characterize cross species differences in the dominant biotransformation pathways.     

Inducibility of enzyme activities associated with the cytochrome P-450 1A family, ethoxyresorufin O-deethylase, and methoxyresorufin O-demethylase in human hepatocyte lines derived from normal liver tissue

Three human hepatocyte cultures have been developed from specimens of normal human liver, in each case from an infant or child, by coculture with liver epithelial cells from 6-day-old rat pups in a complex growth medium. In the established cultures hepatocytes predominate and maintain typical hepatocellular morphology by light microscopy and albumin secretion into supernatant medium. The activity of ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) basally and after treatment with polycyclic aromatic hydrocarbons was measured spectrofluorometrically in cell homogenates from each culture. Very low levels of EROD and MROD activity were found in each culture without induction [EROD: 1.78 +/- 0.71 (mean +/- SE) pmol/min/mg protein; MROD: 1.33 +/- 0.10 pmol/min/mg protein]. After treatment with 10 microM dibenz (a,h)anthracene x 48 hr, EROD and MROD activities rose approximately 20- to 50-fold. When the basal and induced enzyme activities were remeasured 2 months later, results were essentially the same. Incubation with 10 microM benz(a)anthracene x 48 hr also led to induction of EROD and MROD activities. We believe that these cultures can be regarded as human hepatocyte lines, which conserve human hepatic polycyclic aromatic hydrocarbon-inducible P-450s, most likely including P-450 1A2.     

Cirrhosis of the liver as a precancerous condition

Cirrhosis of the liver can be regarded as premalignant state, since more than 80 percent of hepatocellular carcinoma (HCC) in the western world develop in a cirrhotic liver. The risk to develop this malignancy depends on the activity of the underlying cirrhosis, its etiology and the duration of the disease. Patients suffering from cirrhosis of the liver due to HBV-, HCV- or HDV-infection and patients with genetic hemochromatosis exhibit a high risk for HCC. This risk is further increased by cocarcinogens, such as alcohol, nicotine and toxins. Ultrasound and AFP-studies aim to diagnose HCC early. The sensitivity of AFP in the serum is remarkably low (about 64%). In contrast a normal AFP-concentration (< 20 ng/ml) carries a high negative prognostic value (> 90%). Patients suspected to suffer from HCC according to the results of screening procedures should be subjected to additional radiologic investigations, such as CT-arterioportography or lipiodol-angiography.     

Activity of cathepsin B and collagenase in urine and excretion of fibronectin and TGF-beta 1 in urine of patients with membranous glomerulonephritis

In 30% cases nephrotic syndrome is due to membranous glomerulonephritis (MG). Fifty percent of patients reveal end stage renal disease in 15 years follow-up. The another 50% gain persistent remission. The pathogenesis of disease is not known. Protein accumulation in glomeruli leads to progressive loss of kidney structure and function in MG. Also the role of tissue proteolytic systems and growth factors in this process is not known. We aimed to estimate urine cathepsin B, collagenase activity and urine excretion of TGF-beta 1 and fibronectin in MG. MG patients revealed increased urine cathepsin B activity (10.58 +/- 8.73 pmol AMC/mg creatinine/min. vs. control 7.11 +/- 2.05 pmol AMC/mg creatinine/min. [p < 0.05]), urine collagenase activity (8.59 +/- 4.26 pmol AMC/mg creatinine/min. vs. control 3.84 +/- 2.09 pmol AMC/mg creatinine/min. [p > 0.02]) and increased urine excretion of fibronectin (214 +/- 335 ng/mg creatinine vs. control 12.7 +/- 6.7 ng/mg creatinine [p < 0.05]) and increased urine excretion of TGF-beta 1 (283.55 +/- 248.13 pg/ml vs. control 36.11 +/- 48.01 pg/ml [p < 0.05]). The results indicates on glomerular overproduction of TGF-beta 1 and urinary leak of proteolytic enzymes which may exacerbate glomerular proteolytic activity in MG. This may lead to glomerular protein accumulation and progressive loss of kidney function and structure in MG. Increased urine fibronectin excretion in MG patients seems to confirm the hypothesis.     

Acute suppurative parotitis with spread to the deep neck spaces

The occurrence of hepatocellular carcinoma (HCC) in renal transplant recipients has typically been associated with hepatitis B or C infection. We encountered two cases of HCC in renal transplant recipients with negative hepatitis B and C markers and no underlying liver pathology, in whom immunosuppression therapy consisted of prednisone and azathioprine (AZA). Patient no. 1 is a 66-year-old man with diabetes who underwent cadaveric renal transplantation 13 years before presentation. An ultrasound obtained for evaluation of a prolonged prothrombin time and decreased serum albumin level showed a suspicious nodular lesion in the left lobe of the liver. A computed tomographic (CT) scan confirmed a 4- x 5- x 5-cm mass that, on biopsy, was determined to be well-differentiated HCC. There was no evidence of metastasis, and the results of random biopsies of the surrounding parenchyma were normal. The patient underwent a left lateral segmentectomy, did well, and an initial alpha-fetoprotein (AFP) level of 85995 ng/mL decreased to 9 ng/mL. Approximately 20 months postoperatively, however, a surveillance CT scan showed three hypervascular lesions in the right lobe of the liver and the AFP level increased to 28,370 ng/mL. Subsequent percutaneous alcohol injections yielded good results, and the patient is alive and well 13 months later. Patient no. 2 is a 57-year-old man who underwent cadaveric renal transplantation 24 years earlier. A CT scan of the abdomen obtained for evaluation of lower abdominal pain showed a 4- x 4- x 6.5-cm mass in the right lobe of the liver that, on biopsy, was found to be poorly differentiated HCC. Multiple biopsies of adjacent liver parenchyma showed no evidence of cirrhosis, AFP level was normal, and imaging studies showed no evidence of tumor spread. The patient underwent a right hepatic lobectomy and is doing well without evidence of recurrence 27 months postoperatively. Our two patients had no evidence of viral hepatitis, cirrhosis, or metabolic liver disease, yet both developed HCC. The use of AZA may have had a role in the development of HCC. In renal transplant recipients on long-term immunosuppression therapy, particularly AZA, it is prudent to maintain a high index of suspicion for HCC when liver enzyme level or function abnormalities are encountered.     

Circulating vascular endothelial growth factor (VEGF) is a possible tumor marker for metastasis in human hepatocellular carcinoma

Vascular endothelial growth factor (VEGF) is closely related to angiogenesis in various human cancers. However, little is known of its circulating levels in hepatocellular carcinoma (HCC). We examined circulating VEGF levels in chronic liver disease to assess their clinical significance. Plasma VEGF concentrations were determined, by enzyme immunoassay, in patients with chronic hepatitis (CH; n = 36), liver cirrhosis (LC; n = 77), and HCC (n = 86) for a cross-sectional study. Plasma VEGF levels in healthy controls (n = 20) and CH, LC, and HCC patients were 17.7 +/- 5.4 (mean +/- SD), 30.6 +/- 22.8, 34.4 +/- 27.0, and 51.1 +/- 71.9 pg/ml, respectively. The levels were significantly elevated in the HCC group, compared with the control, CH, and LC groups. Plasma VEGF levels in stage I, II, III, IVA, and IVB HCC patients were 27.6 +/- 16.1, 26.5 +/- 13.7, 35.8 +/- 15.3, 45.4 +/- 39.4, and 103.1 +/- 123.2 pg/ml, respectively. The stage IVB patients with remote metastasis showed significantly marked elevation compared with the patients at the other stages. Platelet numbers were weakly correlated with plasma VEGF levels in the HCC group. Plasma VEGF level was highly elevated in patients with HCC, particularly those with metastatic disease. We consider that plasma VEGF is a possible tumor marker for metastasis of HCC. Circulating VEGF may be derived mainly from the large burden of tumor cells, and partly from platelets activated by the vascular invasion of HCC cells.     

Development of a homologous radioimmunoassay for mouse growth hormone receptor

A RIA for mouse GH receptor (mGHR) was developed. A synthetic peptide corresponding to the carboxyl-terminal 14 amino acids of the mGHR (GHR-2 peptide) was used as the antigen for antiserum production. The synthetic peptide was also used as the standard and radioligand in the RIA. The ability of the antiserum to recognize the mGHR was demonstrated by quantitating receptor concentrations in liver and mammary gland from virgin and 15-day-pregnant mice. Serial dilutions of these samples yielded displacement curves parallel to the synthetic peptide. No significant cross-reactivity was seen with serum from virgin or 15-day-pregnant mice, mGH, recombinant mGH-binding protein (mGHBP), a synthetic peptide identical to the hydrophilic tail of mGHBP, or a 14-amino acid synthetic peptide corresponding to amino acids 338-351 of mGHR (GHR-1 peptide). The concentration range of the mGHR RIA was 0.5-200 nM, and the intra- and interassay coefficients of variation were 6.5% and 6.1%, respectively. The concentration of liver GHR increased significantly during pregnancy compared with that in virgin mice, from 0.246 +/- 0.045 pmol/mg protein (mean +/- SEM; n = 5) in the virgin animals to 1.015 +/- 0.159 pmol/mg protein (n = 5) in pregnant mice. In contrast, the mGHR concentration in the mammary gland decreased significantly during pregnancy from 0.606 +/- 0.201 pmol/mg protein (mean +/- SEM; n = 5) to 0.299 +/- 0.027 pmol/mg protein (n = 5). Comparison of the total number of binding sites in livers from virgin and pregnant mice using the GH RRA and the combined results of the mGHR and mGHBP RIAs showed that the two methods gave almost identical results for livers from virgin animals, or 0.363 +/- 0.063 pmol/mg protein (mean +/- SEM; n = 3) and 0.371 +/- 0.008 pmol/mg protein (n = 3) for the GH RRA and the mGHR plus mGHBP RIAs, respectively. However, in livers from pregnant animals, the combined results from the mGHR and mGHBP RIAs were approximately 1.8 times higher than those obtained by the GH RRA, or 6.732 +/- 0.612 pmol/mg protein (mean +/- SEM; n = 3) and 3.693 +/- 0.67 pmol/mg protein (n = 3) for the mGHR plus the mGHBP RIAs and the GH RRA, respectively. The increase in the total GH binding capacity in livers from pregnant mice compared with those from virgin animals was largely due to an increase in the GHBP content. The increase in GHR was only 2.4-fold, or from 0.153 +/- 0.01 pmol/mg protein (mean +/- SEM; n = 3) in virgin mice to 0.364 +/- 0.03 pmol/mg protein (n = 3) in the 15-day-pregnant mice, whereas GHBP increased almost 30-fold during pregnancy, or from 0.218 +/- 0.003 pmol/mg protein (mean +/- SEM; n = 3) in virgin animals to 6.369 +/- 0.607 pmol/mg protein (n = 3) in pregnant mice.     

Interindividual variability in expression and activity of human beta-glucuronidase in liver and kidney: consequences for drug metabolism

Glucuronidation of drugs represents a major pathway of human drug metabolism. Numerous studies show that the glucuronides formed can accumulate during chronic therapy and/or have direct pharmacological activity. In both cases, cleavage of the glucuronide by human beta-glucuronidase (beta-Gluc) would release the parent compound, thereby modifying drug disposition. Variability in expression of beta-Gluc could therefore be a confounding factor for interindividual variability in drug disposition both in the setting of accumulating glucuronides or for the use of glucuronides as prodrugs, such as the nontoxic glucuronide-spacer derivative of doxorubicin (Dox-S-G). We therefore investigated expression and function of beta-Gluc in human liver (n = 30) and human kidney (n = 18). Cleavage of the model compound 4-methylumbelliferyl-beta-D-glucuronide (MUG) revealed a wide range of activities in liver (0.32-1.85 mumol/mg/h, mean value 0.87 +/- 0.34 mumol/mg/h) and kidney (0.07-1.00 mumol/mg/h, mean 0.39 +/- 0.21 mumol/mg/h), which followed a log normal distribution. Variable enzyme activity was closely correlated to enzyme expression as assessed by Western blotting (r = 0.80, P < .001 and r = 0.71, P < .05 for liver and kidney, respectively). Glycyrrhizin (Ki = 470 and 570 microM), estradiol 3-glucuronide (Ki = 0.9 and 1.2 mM) and paracetamol glucuronide (Ki = 1.6 and 2 mM) were found to inhibit beta-Gluc activity competitively in liver and kidney, respectively. Enzyme kinetics were investigated in detail for MUG and Dox-S-G. Whereas MUG followed monophasic Michaelis-Menten kinetics in liver (K(m) = 1.32 +/- 0.25 mM, Vmax = 1201 +/- 462 nmol/mg/h, n = 3) and kidney (K(m) = 1.04 +/- 0.05 mM, Vmax = 521 +/- 267 nmol/mg/h, n = 3), cleavage of Dox-S-G was best described by the Hill equation, which indicated a cooperative substrate binding pattern of Dox-S-G. In summary, beta-Gluc function shows wide interindividual variability in human liver and kidney that is due to different steady-state levels of the enzyme. Moreover, enzyme kinetics are substrate-dependent, with Dox-S-G showing a cooperative binding. These data indicate the possibility of wide interindividual variability in beta-Gluc-mediated cleavage of drug glucuronides in the human.     

Birthmarks to worry about. Cutaneous markers of dysraphism

CYFRA 21-1 is a fragment of cytokeratin 19 (CK 19). Four patients with large intrahepatic (or peripheral) cholangiocarcinoma (CC) and high serum levels of CYFRA 21-1 (normal, < or = 2 ng/ml) are reported. CYFRA 21-1 levels exceeded 9 ng/ml in all 4 patients. Carcinoembryonic antigen (CEA), was high in 1 (CEA; normal range, < or = 5.0 ng/ml) and carbohydrate antigen 19-9 (CA 19-9) was high in 3 (CA19-9; normal range, < or = 36 U/ml). We also measured serum levels of CYFRA 21-1 in 13 patients with hepatocellular carcinoma (HCC) more than 5 cm in diameter. Levels of CYFRA 21-1 exceeded 2 ng/ml in 9 of the HCC patients and were higher than 9 ng/ml in 2 of the HCC patients. Levels of alpha fetoprotein (AFP) and/or protein induced by vitamin K absence or antagonist II (PIVKA II) were elevated in all HCC patients (AFP, PIVKA II, respectively; normal range, < or = 10.0 ng/ml and < or = 0.1 AU/ml) CYFRA 21-1 levels were measured twice or three times during the clinical course in 2 CC patients and in 6 HCC patients, and increased gradually with tumor growth in the 2 CC patients and in 3 of the 6 HCC patients. Marked increases in serum CYFRA 21-1 levels in patients with large liver cancers, particularly in those with normal levels of AFP and PIVKA II, would suggest the existence of intrahepatic CC rather than HCC.     

Gender differences in the activities of aspirin-esterases in rat tissues

The activities of aspirin (acetylsalicylic acid)-esterases were measured in several tissues (liver, kidney, adrenal glands, brain and serum) from adult male and female Wistar rats. In males, both aspirin-esterase I (assayed at pH 5.5) and II (assayed at pH 7.4) activities were higher in liver homogenates when compared to females (aspirin-esterase I: males 48.9 +/- 4.8 (N = 8) and females 29.3 +/- 4.2 (N = 8) nmol of salicylic acid formed min-1 mg protein-1; aspirin-esterase II: males 41.4 +/- 4.1 (N = 8) and females 26.1 +/- 4.5 (N = 8) nmol of salicylic acid formed min-1 mg protein-1, P < 0.001). In serum, enzyme activity was higher in females than in males (aspirin-esterase I: males 0.85 +/- 0.06 (N = 6) and females 1.18 +/- 0.11 (N = 6) nmol of salicylic acid formed min-1 mg protein-1, aspirin-esterase II: males 1.03 +/- 0.13 (N = 6) and females 1.34 +/- 0.11 (N = 6) nmol of salicylic acid formed min-1 mg protein-1, P < 0.001). In the other tissues assayed, no statistically significant difference between males and females was found. There were no statistically significant differences when the enzymes were assayed in different phases of the estrous cycle in liver and serum. These results show that the differences in aspirin-esterase activity observed between males and females are not due to the estrous cycle. The gender difference obtained in our study may indicate an involvement of gonadal hormones in the control of the hydrolysis of aspirin. This possibility is currently under investigation.     

Increased serum levels of monosialo-alpha-fetoprotein in hepatocellular carcinoma and other malignancies

Serum alpha-fetoprotein (AFP) from cord blood and from patients with hepatitis, cirrhosis, hepatocellular carcinoma, gastrointestinal tumors and yolk sac tumor was analyzed by extended agarose gel electrophoresis coupled with our sensitive detection method of antibody-affinity blotting. AFP was separated into AFP-A, AFP-B and AFP-C from the anode to the cathode, corresponding to disialo-, monosialo- and asialo-AFP, respectively, as revealed by the results of neuraminidase digestion of serum AFP. AFP-Af, which migrated ahead of AFP-A as band or leading smear and varied widely in intensity, was eliminated in calculating the proportions of other band intensities. Disialo-AFP was the major component and monosialo-AFP the minor one in benign conditions, the latter being 4.2 +/- 6.0% in chronic hepatitis and 9.0 +/- 8.9% in cirrhosis. Monosialo-AFP in hepatocellular carcinoma was increased significantly, the proportion being 29.9 +/- 11.2%. AFP in other malignancies was further characterized by the appearance of asialo-AFP.     

Neonatal de-afferentation of capsaicin-sensitive sensory nerves increases in vivo insulin sensitivity in conscious adult rats

Sensory neuropeptides, released from the peripheral nervous system, might modulate glucose homeostasis by antagonizing insulin action. The effects of de-afferentation of functional small diameter unmyelinated C-fibres (sensory nerves) on in vivo insulin-mediated intracellular glucose metabolism were investigated by using euglycaemic insulin (6 and 18 mU/kg x min) clamps with [3-(3)H]-glucose infusion in 24 adult rats, treated neonatally with either capsaicin (CAP) (50 mg/kg) or vehicle (CON). Following the clamp, skeletal muscle groups, liver and adipose tissue were freeze-clamped. At plasma insulin levels of approximately 90 mU/l, CAP-rats showed a 21% increase in whole body glucose uptake compared with CON (24.4 +/- 1.6 vs 20.1 +/- 0.8 mg/kg min, p < 0.02), which was paralleled by a 20% increase in whole body glycolysis (12.6 +/- 0.8 vs 10.5 +/- 0.5 mg/ kg.min p < 0.05) (concentration of 3H2O in plasma). Whole body skeletal muscle glycogenesis was increased by 80% in CAP-rats (5.7 +/- 0.7 vs 3.1 +/- 0.7 mg/kg x min, p < 0.05) with increased muscle glycogen synthase activity. Whole body (muscle, liver and adipose tissue combined) de novo lipogenesis also was increased in CAP-rats compared with CON (0.69 +/- 0.10 vs 0.44 +/- 0.06 mg/kg x min, p < 0.05) (incorporation of [3-(3)H]-glucose counts into glycogen or fat). Hepatic glucose production was lower in CAP-rats compared with CON (0.6 +/- 0.6 vs 2.1 +/- 0.7 mg/kg x min, p < 0.05). Plasma glucagon, corticosterone, epinephrine and norepinephrine levels were reduced in CAP-rats: 43 +/- 2 compared with 70 +/- 6 pg/ml, 855 +/- 55 compared with 1131 +/- 138 nmol/l, 513 +/- 136 compared with 1048 +/- 164 pmol/l and 928 +/- 142 compared with 1472 +/- 331 pmol/l, respectively, p < 0.05. At plasma insulin levels of approximately 400 mU/l, CAP-rats showed no differences in peripheral and hepatic insulin action compared with CON. We conclude that the removal of endogenous sensory neuropeptides, by de-afferentation of capsaicin-sensitive sensory nerves, increases in vivo insulin sensitivity, but not responsiveness: 1) primarily through an increased sensitivity of skeletal muscle glycogen synthesis to insulin; 2) through a reduction in the levels of counter-regulatory hormones, thereby creating a milieu which favours overall in vivo insulin sensitivity with respect to glucose uptake, glucose production, glycolysis, glycogenesis and lipogenesis.     

Effect of endogenous L-arginine on the measurement of nitric oxide synthase activity in the rat kidney

The endogenous level of L-arginine in renal cortex, outer medulla, and inner medulla and its effect on the measurement of nitric oxide synthase (NOS) activity in these regions was determined. L-Arginine was measured with an amino acid analyzer and total NOS activity was measured as the rate of formation of radiolabeled L-citrulline from L-[14C]arginine. Total NOS activity was that activity inhibited by NG-nitromonomethyl-L-arginine (300 microM). The endogenous L-arginine concentration was 452 +/- 45 (mean +/- SEM), 313 +/- 25, and 95 +/- 8 pmol/mg wet weight in the cortex, outer medulla, and inner medulla, respectively (n = 12). Total NOS activity was 61 +/- 19, 709 +/- 94, and 1347 +/- 76 pmol.h-1.mg protein-1 in the cortex, outer medulla, and inner medulla, respectively, when endogenous L-arginine was not considered in the calculation. Correcting for endogenous L-arginine gave values of 185 +/- 61, 1714 +/- 239, and 1707 +/- 104 pmol.h-1.mg protein-1, respectively. Dowex extraction to remove endogenous L-arginine from samples also increased NOS activity in cortex and medulla. The data indicate that there is a differential distribution of endogenous L-arginine in the kidney and that these levels must be taken into account when measuring NOS activity. NOS activity is also distributed differentially, with activity in the medulla being nearly 10-fold higher than in the cortex.     

Guanine nucleotide-binding inhibitory protein-mediated inhibition of adenylyl cyclase is enhanced in spontaneously hypertensive rat preglomerular arteriolar smooth muscle cells

The purpose of our study was to determine whether Gi-mediated control over adenylyl cyclase in preglomerular arteriolar smooth muscle cells (PGASMC) is enhanced in the spontaneously hypertensive rat (SHR). PGASMC were cultured from preglomerular microvessels isolated from adult SHR (14-15 wk of age) and age-matched WKY rats. Confluent monolayers of cells in third passage were used for the experiments. cAMP released into the media (30 min) as well as cellular levels of cAMP were measured in the presence of a phosphodiesterase inhibitor, 1-isobutyl-3-methyl-xanthine (IBMX; 100 microM) and expressed as pmol/mg protein. Total (released + cellular) cAMP was significantly lower in SHR (14.19 +/- 2.30 pmol/mg protein) as compared with WKY (28.3 +/- 3.04 pmol/mg protein). Correspondingly, the released (4.6 +/- 0.4 pmol/mg protein) as well as cellular (9.78 +/- 2.18 pmol/mg protein) cAMP levels were also significantly lower in SHR when compared with WKY (8.85 +/- 1.26 and 18.86 +/- 2.0 pmol/mg protein, respectively). The steady-state levels of none of the Gi alpha subunits, namely Gi alpha 1, Gi alpha 2 and Gi alpha 3, were higher in the SHR PGASMC. Pertussis toxin treatment (PTX; 100 ng/ml; 24 hr) caused complete ADP-ribosylation of Gi alpha subunits in both WKY and SHR PGASMC. The same treatment of PTX also produced a significant increase in total cAMP in SHR, but not in WKY, such that the total cAMP levels after PTX treatment were not significantly different between the two strains. Interestingly, PTX significantly increased the released (20.26 +/- 0.90 pmol/mg protein) but not the cellular (13.63 +/- 1.63 pmol/mg protein) cAMP in SHR. Forskolin (1 microM) induced similar increases in total cAMP and isoproterenol (1 microM) caused greater increases in total cAMP in SHR cells compared with WKY cells. These data strongly suggest that in SHR PGASMC total adenylyl cyclase activity is not altered. Furthermore, steady-state expressions of Gi alpha-1, Gi alpha-2 and Gi alpha-3 are not increased whereas Gi-mediated inhibition of adenylyl cyclase is augmented in SHR PGASMC.