Altered expression and subcellular localization of diacylglycerol-sensitive protein kinase C isoforms in diabetic rat glomerular cells.

Protein kinase C (PKC) is implicated in the pathogenesis of diabetic nephropathy. This study was designed to identify the expression of diacylglycerol (DAG)-sensitive PKC-alpha, -betaII, -delta, and -epsilon isoforms in normal and diabetic rat glomerular cells and to determine the effects of high glucose and insulin on PKC isoform cellular compartmentalization and PKC activity. Diabetic rats treated with or without insulin and normal rats were examined 2 and 4 weeks after streptozotocin/vehicle injection. Renal cortical tissue immunogold-labeled with anti-PKC-alpha, -betaII, -delta, or -epsilon antibody was visualized by electron microscopy. From isolated glomeruli, total cell lysate and cytosol and membrane fractions were immunoblotted with the same anti-PKC isoform antibodies. PKC activity in isolated glomeruli was measured by 32P-phosphorylation of the epidermal growth factor (EGF)-receptor substrate. Immunogold labeling revealed expression of the four PKC isoforms by glomerular visceral epithelial, endothelial, and mesangial cells of both normal and diabetic rats. Immunoblot analysis of the diabetic rat glomeruli at 2 weeks demonstrated a significant increase in membrane-associated PKC-alpha, -delta, and -epsilon and a significant decrease in membrane PKC-betaII content compared with normal, which were similar at 4 weeks. Insulin treatment normalized membrane PKC isoform contents and caused a significant decrease in the cytosol content of PKC-alpha, -betaII, and -delta and total cellular PKC-alpha compared with normal. Although PKC activity in the cells of diabetic rat glomeruli was increased by 20% compared with normal, the difference did not reach statistical significance. In insulin-treated diabetic rat glomeruli, PKC activity was significantly decreased compared with non-insulin-treated diabetic rat glomeruli. In conclusion, DAG-sensitive PKC-alpha, -betaII, -delta, and -epsilon isoforms are all found in the three major glomerular cell types in rats, and the expression, compartmentalization, and activity are modulated independently by high glucose and insulin.

Dynamics of arterial and portal venous flow interactions in perfused rat liver: an intravital microscopic study.

Intravital epifluorescent microscopy was used to quantitate microvascular parameters in the single-pass, dually perfused rat liver preparation. Livers perfused via the hepatic artery (HA) and portal vein (PV) at physiological pressures and perfusion rates responded to vasoactive agents and exhibited the HA buffer response. The distribution of arterial blood was found to be highly heterogeneous, whereas PV flow was distributed uniformly. The intrasinusoidal velocity of fluorescein isothiocyanate (FITC)-labeled red blood cells (RBCs) arriving from the HA was higher than that for RBCs arriving from the PV, indicating a shorter transit time for the arterially delivered FITC-RBCs. Experiments on livers perfused simultaneously via the HA and retrogradely via the hepatic vein revealed the presence of arteriovenous shunts, with some of the arterially delivered FITC-RBCs reaching the terminal hepatic venules via direct channels without traversing the sinusoidal bed. In livers perfused portally only, changes in PV flow rate (from 8 to 20 ml/min) produced small changes in perfusion pressure but large changes in vascular diameters, while portal pressure and transit time of portal blood remained relatively constant. In experiments designed to identify the location of hepatic vascular resistance, it was observed that hepatic venular diameters measured in the preparation under identical pressure and flow conditions were greater during retrograde than during prograde perfusion, suggesting that the site of hepatic vascular resistance is presinusoidal or sinusoidal.

Endothelin-1-induced mesangial cell contraction involves activation of protein kinase C-alpha, -delta, and -epsilon.

In endothelin-1 (ET-1)-stimulated mesangial cells, to identify the independent roles of calcium and protein kinase C (PKC) causing contraction, the changes in planar surface area in response to ET-1, ionomycin, or phorbol 12-myristate 13-acetate (PMA) were compared. ET-1, PMA, and ionomycin reduced planar area to 49 +/- 3%, 56 +/- 3%, and 78 +/- 2% of basal (means +/- SE, n = 40-50 cells), respectively. ET-1 or ionomycin increased cytosolic calcium from 80 +/- 7 to 220 +/- 30 nM or 97 +/- 10 to 192 +/- 10 nM, respectively. The myosin light chain kinase inhibitor, ML-7, blunted ET-1- but not PMA-stimulated contraction (82 +/- 3% and 48 +/- 6% of time 0, respectively). Cells pretreated with 10 microM chelerythrine for 1 h or PMA for 24 h failed to contract to either ET-1 or PMA. To identify the specific PKC isoform response to ET-1, cytosolic, membrane, and particulate fractions of mesangial cell lysates were immunoblotted with PKC isoform-specific polyclonal antibodies. ET-1 increased membrane PKC-alpha, -delta, and -epsilon to 173 +/- 30%, 162 +/- 26%, and 166 +/- 11% of basal (P < 0.05 vs. basal), respectively, and decreased PKC-delta and PKC-epsilon in the cytosol to 56 +/- 11% and 37 +/- 6% of basal, respectively (P < 0.05). ET-1 increased particulate PKC-delta and PKC-epsilon to 172 +/- 15% and 187 +/- 33% of basal (P < 0.05), respectively. PKC-alpha in the cytosol and particulate fractions was not altered by ET-1, but translocation to the nucleus and cell periphery was observed by confocal immunofluorescence imaging. Ionomycin did not change PKC isoform distribution. PKC-zeta was expressed but unaltered by ET-1. Therefore, mesangial cell ET-1-stimulated contraction not only involves a calcium-dependent pathway but also includes the activation of one or more PKC-alpha, -delta, and -epsilon, but not PKC-zeta.

Effect of high glucose on mesangial cell protein kinase C-delta and -epsilon is polyol pathway-dependent.

In diabetes mellitus, enhanced activity of mesangial cell protein kinase C (PKC) may contribute to nephropathy. The purpose of this study was to determine whether high glucose alters mesangial cell diacylglycerol-sensitive PKC-alpha, -beta2, -delta, and -epsilon content, cellular distribution, and activity through polyol pathway activation. Primary cultured rat mesangial cells (passage 10) were growth-arrested in 0.5% fetal bovine serum and cultured in 5.6 mM glucose (NG) or 30 mM glucose (HG) for 48 h, with or without the aldose reductase inhibitor tolrestat or ARI-509. PKC isoform content in total cell lysates, or cytosol, membrane (Triton X-soluble), and particulate (sodium dodecyl sulfate-soluble) fractions was analyzed by immunoblotting, and band density in HG was expressed as a percentage of corresponding NG values. In HG at 48 h, increased total PKC-alpha (222 +/- 17% of NG, P < 0.001), -beta2 (209 +/- 12%, P < 0.001), and -epsilon (195 +/- 19%, P < 0.001) were observed. L-Glucose had no effect on total PKC isoform content. HG caused increased membrane- and particulate-associated PKC-alpha (257 +/- 87 and 327 +/- 66%, respectively, P < 0.05), membrane-associated PKC-delta (143 +/- 10%, P < 0.05), and membrane-associated PKC-epsilon (186 +/- 11%, P < 0.001), with no change in cytosol contents. The HG effects were not mimicked by L-glucose. In NG or HG, PKC-beta2 was not detected in the cytosol fraction, and membrane and particulate association were unchanged with phorbol ester stimulation. Confocal immunofluorescence imaging revealed that in HG, PKC-alpha, -delta, and -epsilon translocate to the nucleus and plasma membrane. Total PKC activity measured by in situ 32P-phosphorylation of the epidermal growth factor receptor substrate increased from 18 +/- 1 pmol/min per mg cell protein in NG to 33 +/- 3 pmol/min per mg cell protein in HG (P < 0.002 versus NG). In NG, tolrestat and ARI-509 exposure caused increased PKC activity, enhanced accumulation of total PKC-alpha and -beta2, with no change in total or fractional recovery of PKC-delta or -epsilon. In HG, tolrestat and ARI-509 prevented the increase in total PKC-epsilon and membrane-associated PKC-delta and -epsilon. It is concluded that within 48 h of HG, enhanced mesangial cell PKC activity is associated with accumulation and cellular redistribution of diacylglycerol-sensitive PKC isoforms, and that increased PKC-epsilon content and membrane-associated PKC-delta and -epsilon are dependent on polyol pathway activation.

Systemic hemodynamic and hepatic microvascular responses to a 33% blood volume exchange with whole blood, stroma-free hemoglobin, and oxypolyhemoglobin solutions.

Little is known about the microvascular effects of blood replacement solutions. This study was undertaken to develop an animal model suitable for studies of the microcirculatory effects of such solutions and to investigate microvascular responses to isovolemic transfusion with stroma-free hemoglobin (SFH), whole donor blood, or a new potential blood substitute solution containing oxypolyhemoglobin (OPH) as an oxygen carrier. Hamster livers were exposed and the microcirculation studied using intravital epifluorescent video microscopy. 33% blood volume replacement with SFH elevated systemic blood pressure by 25 Torr. Accompanying this increase in pressure was a 36% decrease in sinusoidal blood flow velocity and a 10% decrease in terminal hepatic venular diameters. Terminal portal venular diameters did not change. Decrease in liver sinusoidal perfusion was not due to neutrophil mediated injury, as myeloperoxidase activity in jejunum, liver, kidney, and lung remained unchanged. The reduction in perfusion was likely due to systemic vasoconstriction produced by SFH. In contrast, transfusion with whole blood did not change any of the measured parameters showing the excellent stability of the model. OPH transfused animals exhibited only a small 10 Torr transient increase in MAP 15 min post-transfusion. By 30 min MAP returned to the pre-infusion value. No significant changes were observed in either venular diameters or sinusoidal velocities in this group of animals. These results demonstrate suitability of this model for studies of the microcirculatory and hemodynamic effects of blood replacement solutions. Furthermore, OPH solution produced only minor transient disturbances in microvascular and systemic parameters.

Stretch-induced mesangial cell ERK1/ERK2 activation is enhanced in high glucose by decreased dephosphorylation.

Glomerular hypertension and hyperglycemia are major determinants of diabetic nephropathy. We sought to identify the mechanisms whereby stretch-induced activation of mesangial cell extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) is enhanced in high glucose (HG). Mesangial cells cultured on fibronectin Flex I plates in normal glucose (NG; 5.6 mM) or HG (30 mM), were stretched by 15% elongation at 60 cycles/min for up to 60 min. In HG, a 5-min stretch increased ERK1/ERK2 phosphorylation by 6.4 +/- 0.4/4.3 +/- 0.3-fold (P < 0.05 vs. NG stretch). In contrast, p38 phosphorylation was increased identically by stretch in NG and HG. Unlike many effects of HG, augmentation of ERK activity by HG was not dependent on protein kinase C (PKC) as indicated by downregulation of PKC with 24-h phorbol ester or inhibition with bisindolylmaleimide IV. In both NG and HG, pretreatment with arginine-glycine-aspartic acid peptide (0.5 mg/ml) to inhibit integrin binding or with cytochalasin D (100 ng/ml) to disassemble filamentous (F) actin, significantly reduced phosphorylation of ERK1/ERK2 and p38. To determine whether the rate of mitogen-activated protein kinase dephosphorylation is affected by HG, cellular kinase activity was inhibited by depleting ATP. Post-ATP depletion, phosphorylation of ERK1/ERK2 was reduced to 36 +/- 9/51 +/- 14% vs. 9 +/- 5/7 +/- 6% in NG (P < 0.05, n = 5). Thus stretch-induced ERK1/ERK2 and p38 activation in both NG and HG is beta(1)-integrin and F-actin dependent. Stretch-induced ERK1/ERK2 is enhanced in high glucose by diminished dephosphorylation, suggesting reduced phosphatase activity in the diabetic milieu. Enhanced mesangial cell ERK1/ERK2 signaling in response to the combined effects of mechanical stretch and HG may contribute to the pathogenesis of diabetic nephropathy.

Role of the hepatic artery in the metabolism of phenacetin and acetaminophen: intravital microscopic and multiple-indicator dilution study in perfused rat liver.

We studied the pattern of intermixing of the hepatic arterial and portal venous flows in a perfused rat liver preparation under constant flow (12 ml/min) with intravital epifluorescent microscopy; changes in the steady state extraction ratio of carbon 14-labeled phenacetin and tritiated acetaminophen, probes metabolized primarily in perivenous and periportal regions of the rat liver, respectively; and the spaces accessed by noneliminated reference indicators introduced as a bolus into the hepatic artery and portal vein at different hepatic arterial/portal venous flow regimens of 0:12, 2:10 and 4:8. The sinusoidal velocities for the hepatic arterial- and portal venous (hepatic arterial/portal venous flow at 4:8)-infused fluorescein isothiocyanate-erythrocytes (100 microliters/min) were 327 +/- 78 and 301 +/- 63 microns/sec, respectively, and the velocity for the solely portal venous-perfused liver (12 ml/min) was 347 +/- 74 microns/sec; the flow-weighted sinusoidal velocity was highly correlated to the sinusoidal volume for the dually perfused rat liver. Small but significant decreases in the extraction ratio of [14C]phenacetin (from 0.989 to 0.984 and 0.980) and tritiated acetaminophen (from 0.631 to 0.607 to 0.563), delivered simultaneously into the hepatic artery and portal vein, were observed with an increment of hepatic arterial flow within the same liver preparation; oxygen consumption rate also fell slightly, in parallel fashion. When a multiple-indicator dilution dose containing chromium 51-labeled RBCs, iodine 125-labeled albumin and tritiated water or [14C]urea was injected into the hepatic artery (which accesses both the peribiliary capillary plexus [nonsinusoidal] and the sinusoidal bed) and portal vein (which enters only the sinusoids) at 10-min intervals within each steady state, the blood volume, total albumin space, albumin Disse space, total water and parenchymal cellular water spaces were unchanged after portal venous injection for all hepatic arterial/portal venous flow ratios, suggesting that the arterial flow is ineffective in perturbing average sinusoidal flow dynamics. However, slightly larger total water spaces were obtained with hepatic arterial injection. This excess water space was almost completely accounted for by the "nonsinusoidal" extravascular space associated with the peribiliary capillary plexus; it averaged 0.03 ml/gm and was independent of flow. The anomaly, a reduced flow-weighted sinusoidal velocity for the dually perfused liver, an unchanged diameter of the terminal hepatic venule (32 microns) among the hepatic arterial/portal venous flow ratios and the reduction in the extraction ratio of the drug probes and oxygen consumption rates suggest that some of the arterial flow must have entered the sinusoids somewhat downstream.