William H. Baricos

Activation of purified guanylate cyclase by nitric oxide requires heme comparison of heme-deficient, heme-reconstituted and heme-containing forms of soluble enzyme from bovine lung

Bovine lung soluble guanylate cyclase was purified to apparent homogeneity in a form that was deficient in heme. Heme-deficient guanylate cyclase was rapidly and easily reconstituted with heme by reacting enzyme with hematin in the presence of excess dithiothreitol, followed by removal of unbound heme by gel filtration. Bound heme was verified spectrally and NO shifted the absorbance maximum in a manner characteristic of other hemoproteins. Heme-deficient and heme-reconstituted guanylate cyclase were compared with enzyme that had completely retained heme during purification. NO and S-nitroso-N-acetylpenicillamine only marginally activated heme-deficient guanylate cyclase but markedly activated both heme-reconstituted and heme-containing forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 microM hematin in enzyme reaction mixtures containing dithiothreitol. Preformed NO-heme activated all forms of guanylate cyclase in the absence of additional heme. Guanylate cyclase activation was observed in the presence of either MgGTP or MnGTP, although the magnitude of enzyme activation was consistently greater with MgGTP. The apparent Km for GTP in the presence of excess Mn2+ or Mg2+ was 10 microM and 85-120 microM, respectively, for unactivated guanylate cyclase. The apparent Km for GTP in the presence of Mn2+ was not altered but the Km in the presence of Mg2+ was lowered to 58 microM with activated enzyme. Maximal velocities were increased by enzyme activators in the presence of either Mg2+ or Mn2+. The data reported in this study indicate that purified guanylate cyclase binds heme and the latter is required for enzyme activation by NO and nitroso compounds.

Cerebral Phospholipid Content and Na+, K+‐ATPase Activity During Ischemia and Postischemic Reperfusion in the Mongolian Gerbil

Abstract: Using bilateral carotid artery occlusion in adult gerbils we examined the effects of ischemia and ischemia/reperfusion on cerebral phospholipid content and Na+, K+‐ATPase (EC 3.6.1.3) activity. In contrast to the large changes in phospholipid content and membrane‐bound enzyme activity that have been observed in liver and heart tissues, we observed relatively small changes in the cerebral content of total phospholipid, phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE) following ischemic intervals of up to 240 min. Following 15 min of ischemia the cerebral content of sphingomyelin (SM) was decreased to <50% of control values but returned to near‐normal levels with longer ischemic periods. Significant decreases in the cerebral content of phosphatidylinositol (PI) and phosphatidic acid (PA) were observed following shorter intervals of ischemia (15–45 min). Na+, K+‐ATPase activity of cerebral homogenates prepared from the brains of gerbils subjected to 30–240 min of ischemia was decreased but significantly different from control activity only after 30 min of ischemia (‐29%, p ≤ 0.05). With the exception of PS, reperfusion for 60 min following 60 min of ischemia resulted in marked increases in cerebral phospholipid content with PC, SM, PI, and PA levels exceeding and PE levels equal to preischemic values. Longer periods of reperfusion (180 min) resulted in decreases in cerebral phospholipid content toward (PC, SM, PI, and PA) or below (PE) preischemic levels. In contrast, the cerebral content of PS significantly decreased during reperfusion (‐51% at 60 min, p ≤ 0.05) and remained below preischemic values even after 180 min of reperfusion. The activity of cerebral Na+, K+‐ATPase increased to values slightly above preischemic values following reperfusion for either 60 or 180 min after 60 min of ischemia. These data show that major differences exist in the responses of neural and nonneural tissues to ischemia and raise important questions concerning the role of changes in membrane structure and function in irreversible ischemic injury.

Activation of coronary arterial guanylate cyclase by nitric oxide, nitroprusside, and nitrosoguanidine—Inhibition by calcium, lanthanum, and other cations, enhancement by thiols

Abstract Although reports that certain vasodilate s activate soluble guanylate cyclase, especially in the presence of thiols, and elevate cyclic GMP levels in smooth muscle suggest that cyclic GMP is involved in vascular smooth muscle relaxation, earlier reports that Ca 2+ activates guanylate cyclase and that Ca 2+ -dependent contractile agents elevate cyclic GMP levels are seemingly at odds with this hypothesis. The objective of this study was to examine the effects of Ca 2+ related cations, and thiols on bovine coronary arterial soluble guanylate cyclase. Guanylate cyclase activity was detected in the presence of Mg 2+ or Mn 2+ but not of other cations. Basal activity was greater in the presence of Mn 2+ than of Mg 2+ . Activity of guanylate cyclase stimulated by nitroprusside, nitric oxide, or nitrosoguanidine, however, was greater with Mg 2+ , although the requirement of activated enzyme for Mn 2+ was reduced about 10-fold. Ca 2+ markedly inhibited guanylate cyclase activation in the presence of Mg 2+ but not of Mn 2+ . La 2+ inhibited enzyme activation in the presence of Mg 2+ or Mn 2+ . Neither Ca 2+ nor La 3+ altered basal enzymatic activity. Results that were qualitatively similar to those indicated above were observed with partially purified, heme-free, coronary arterial soluble guanylate cyclase. Nitric oxide and nitroso compounds activated partially purified enzyme, and thiols enhanced enzyme activation by nitroprusside and nitrosoguanidine without appreciably altering basal activity. Irreversible sulfhydryl binding agents such as ethacrynic acid and gold inhibited both basal and activated guanylate cyclase. These results suggest that changes in intracellular concentrations of free Ca 2+ and sulfhydryl groups could influence the rate of formation of cyclic GMP by vasodilators and that this, in turn, could alter smooth muscle tone.

Evidence that regulation of hepatic guanylate cyclase activity involves interactions between catalytic site -SH groups and both substrate and activator.

Abstract Partially purified hepatic soluble guanylate cyclase (EC 4.6.1.2) was rapidly inactivated by molecular oxygen and by low concentrations of SH oxidants at pH 7.4, a process which was prevented and reversed by dithiothreitol. Cystamine and 5,5′-dithiobis(2-nitrobenzoic acid) inhibited enzymatic activity in a time- and temperature-dependent manner. Various disulfides, SH oxidants, and thiol alkylating agents inhibited both basal guanylate cyclase activity and activity stimulated by nitric oxide, S -nitrosocysteine, and nitrosylhemoglobin. These observations indicate that guanylate cyclase possesses one or more SH groups at its catalytic site. Preincubation of guanylate cyclase with excess MgGTP or with enzyme activators markedly protected the enzyme against inhibition by various thiol reactive agents. These findings suggest that the SH groups at the catalytic site interact also with enzyme activators. 2,3-Dimercaprol, which possesses vicinal dithiols, but not dithiothreitol markedly inhibited guanylate cyclase activity at concentrations equal to or exceeding those of MgGTP. Thus, two closely juxtaposed SH groups may be located at the catalytic site. The presence of cysteine or hematin in preincubates of guanylate cyclase, to which nitric oxide was also added, was mandatory in order to enable the activated form of the enzyme to be recovered by gel filtration. Guanylate cyclase activated by S -nitrosocysteine or nitrosyl-hemoglobin was recovered in the maximally activated state, and this was prevented by preincubation of enzyme with SH oxidants or excess MgGTP. These data imply that cysteine, hematin, and their nitrosyl derivatives bind to SH groups at the catalytic site of guanylate cyclase.

Bradykinin stimulates the ERK→Elk-1→Fos/AP-1 pathway in mesangial cells

Among its diverse biological actions, the vasoactive peptide bradykinin (BK) induces the transcription factor AP-1 and proliferation of mesangial cells (S. S. El-Dahr, S. Dipp, I. V. Yosipiv, and W. H. Baricos. Kidney Int. 50: 1850-1855, 1996). In the present study, we examined the role of protein tyrosine phosphorylation and the mitogen-activated protein kinases, ERK1/2,in mediating BK-induced AP-1 and DNA replication in cultured rat mesangial cells. BK (10(-9) to 10(-7) M) stimulated a rapid increase in tyrosine phosphorylation of multiple proteins with an estimated molecular mass of 120-130, 90-95, and 44-42 kDa. Immunoblots using antibodies specific for ERK or tyrosine-phosphorylated ERK revealed a shifting of p42 ERK2 to a higher molecular weight that correlated temporally with an increase in tyrosine-phosphorylated ERK2. Genistein, a specific tyrosine kinase inhibitor, prevented the phosphorylation of ERK2 by BK. In-gel kinase assays indicated that BK-induced tyrosine phosphorylation of ERK2 is accompanied by fourfold activation of its phosphotransferase activity toward the substrate PHAS-I (P < 0.05). Furthermore, BK stimulated a 2.5-fold increase (P < 0.05) in phosphorylation of Elk-1, a transcription factor required for growth factor-induced c-fos transcription. In accord with the stimulation of Elk-1 phosphorylation, BK induced c-fos gene expression and the production of Fos/AP-1 complexes. In addition, thymidine incorporation into DNA increased twofold (P < 0. 05) following BK stimulation. Each of these effects was blocked by tyrosine kinase inhibition with genistein or herbimycin A. Similarly, antisense oligodeoxynucleotide targeting of ERK1/2 mRNA inhibited BK-stimulated DNA synthesis. In contrast, protein kinase C inhibition or depletion had no effect on BK-induced c-fos mRNA, AP-1-DNA binding activity, or DNA synthesis. Collectively, these data demonstrate that BK activates the ERK-->Elk-1-->AP-1 pathway and that BK mitogenic signaling is critically dependent on protein tyrosine phosphorylation.Among its diverse biological actions, the vasoactive peptide bradykinin (BK) induces the transcription factor AP-1 and proliferation of mesangial cells (S. S. El-Dahr, S. Dipp, I. V. Yosipiv, and W. H. Baricos. Kidney Int. 50: 1850-1855, 1996). In the present study, we examined the role of protein tyrosine phosphorylation and the mitogen-activated protein kinases, ERK1/2,in mediating BK-induced AP-1 and DNA replication in cultured rat mesangial cells. BK (10-9 to 10-7 M) stimulated a rapid increase in tyrosine phosphorylation of multiple proteins with an estimated molecular mass of 120-130, 90-95, and 44-42 kDa. Immunoblots using antibodies specific for ERK or tyrosine-phosphorylated ERK revealed a shifting of p42 ERK2 to a higher molecular weight that correlated temporally with an increase in tyrosine-phosphorylated ERK2. Genistein, a specific tyrosine kinase inhibitor, prevented the phosphorylation of ERK2 by BK. In-gel kinase assays indicated that BK-induced tyrosine phosphorylation of ERK2 is accompanied by fourfold activation of its phosphotransferase activity toward the substrate PHAS-I ( P < 0.05). Furthermore, BK stimulated a 2.5-fold increase ( P < 0.05) in phosphorylation of Elk-1, a transcription factor required for growth factor-induced c-fos transcription. In accord with the stimulation of Elk-1 phosphorylation, BK induced c-fos gene expression and the production of Fos/AP-1 complexes. In addition, thymidine incorporation into DNA increased twofold ( P < 0.05) following BK stimulation. Each of these effects was blocked by tyrosine kinase inhibition with genistein or herbimycin A. Similarly, antisense oligodeoxynucleotide targeting of ERK1/2 mRNA inhibited BK-stimulated DNA synthesis. In contrast, protein kinase C inhibition or depletion had no effect on BK-induced c-fos mRNA, AP-1-DNA binding activity, or DNA synthesis. Collectively, these data demonstrate that BK activates the ERK→Elk-1→AP-1 pathway and that BK mitogenic signaling is critically dependent on protein tyrosine phosphorylation.

Evidence suggesting a role for cathepsin L in an experimental model of glomerulonephritis

We have utilized specific, irreversible inhibitors of cysteine proteinases to examine the role of renal cathepsin B and cathepsin L in the proteinuria which occurs in an experimental model of human glomerular disease. Administration of trans-epoxysuccinyl-L-leucylamido-(3-methyl)butane (Ep475) a specific, irreversible inhibitor of cysteine proteinases, including cathepsins B and L, significantly reduced proteinuria in rats with experimentally induced, neutrophil-independent, anti-GBM antibody disease (controls: 10 +/- 1 mg/24 h, N = 8; anti-GBM antibody disease: 203 +/- 30 mg/24 h, N = 8; anti-GBM antibody disease + Ep475: 112 +/- 13 mg/24 h, mean +/- SEM, N = 6, P less than 0.05). There was a marked reduction in the activity of both cathepsin B and cathepsin L in renal cortices obtained from Ep475-treated rats compared to either saline-treated controls or rats treated with anti-GBM IgG only. Administration of Z-Phe-Tyr(O-t-butyl)CHN2, a specific, irreversible cysteine proteinase inhibitor with a high degree of selectivity toward cathepsin L, also caused a reduction in anti-GBM antibody-induced proteinuria (90 +/- 18 mg/24 h, N = 6, P less than 0.05). This reduction in proteinuria was accompanied by a marked decrease (-84%) in the specific activity of renal cortical cathepsin L in Z-Phe-Tyr(O-t-butyl)CHN2-treated rats. However, cathepsin B activity was unchanged. There was no significant change in the renal anti-GBM antibody uptake, plasma urea nitrogen, or plasma creatinine values in the Z-Phe-Tyr(O-t-butyl)CHN2-treated rats compared to rats treated with anti-GBM IgG only or saline-treated controls. These data document the ability of cysteine proteinase inhibitors to decrease the proteinuria which occurs in a neutrophil-independent model of human anti-GBM antibody disease and suggest an important role for cathepsin L in the pathophysiology of the proteinuria which occurs in this model.

Selective alterations in responsiveness of guanylate cyclase to activation by nitroso compounds during enzyme purification.

Partially purified, heme-free, hepatic soluble guanylate cyclase was activated by NO, S-nitrosocysteine and NO-heme complexes to the extent of 19–34-fold in the presence of 3 mM Mg2+, but only up to 2-fold in the presence of 3mM Mn2+, when the GTP concentration was 1 mM. Even with Mg2+, however, nitroprusside and nitrosoguanidine failed to activate guanylate cyclase. Dithiothreitol or cysteine and, to a much lesser extent, hematin or hemoglobin restored the capacity of nitroprusside and nitrosoguanidine to activate guanylate cyclase, and enhanced enzyme activation by NO. Restoration or enhancement of enzyme activation with thiols was unrelated to their reducing potential because nonthiol reductants such as dithionite and ascorbate failed to influence guanylate cyclase activation. Instead, thiols likely reacted with the nitroso compounds to form S-nitrosothiols, which were potent activators of guanylate cyclase. Similarly, heme-containing substances reacted with nitroso compounds to form NO-heme complexes, which are known to activate guanylate cyclase. Enzyme activation by nitroprusside and nitrosoguanidine was restored, and that by NO and NO-hemoglobin was enhanced, by addition of heated hepatic soluble fraction (devoid of guanylate cyclase activity). The heated soluble fraction appears to contain a partially heat-stable, thiol-containing component(s) of molecular weight greater than 5000, which may be in part responsible for the observed effects on enzyme activation. These data suggest that partial enzyme purification results in the removal of thiol components that are required for the full expression of guanylate cyclase activation.

The cysteine proteinase inhibitor, E-64, reduces proteinuria in an experimental model of glomerulonephritis

Proteinuria is a major manifestation of glomerular disease (glomerulonephritis, GN). We examined the effect of trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a specific and irreversible cysteine proteinase inhibitor, on urinary protein excretion in a complement- and neutrophil-independent model of antiglomerular basement membrane (GBM) antibody disease. A single injection of rabbit antirat-GBM IgG produced a marked increase in urinary protein excretion 24hr after injection. In two separate studies using different pools of antiGBM IgG, administration of E-64 (5mg every 6h starting 2hr prior to induction of GN) reduced proteinuria (-45 +/- 7%, and -41 +/- 14%, Mean +/- SEM, n = 6; P less than 0.001) in the 24 hour period following induction of the disease. This reduction in urinary protein excretion was accompanied by a marked decrease in the specific activity of the cysteine proteinases cathepsins B and L in glomeruli (B: -97%; L: -84%) and renal cortex (B: -87%; L: -75%) isolated from the same E-64-treated rats compared to same saline-treated controls. These data, combined with the specificity of E-64 for cysteine proteinases, suggest a potential role for cysteine proteinases in the increased GBM permeability and proteinuria in this experimental model of glomerular disease.

The role of aspartic and cysteine proteinases in albumin degradation by rat kidney cortical lysosomes

We have investigated the degradation of 125I-labeled bovine serum albumin by lysates of rat kidney cortical lysosomes. Maximal degradation of albumin occurred at pH 3.5-4.2, with approximately 70% of the maximal rate occurring at pH 5.0. Degradation was proportional to lysosomal protein concentration (range 100-600 micrograms) and time of incubation (1-5 h). Dithioerythritol (2 mM) stimulated albumin degradation 5- to 10-fold. Albumin degradation was not inhibited by phenylmethanesulfonyl fluoride (1 mM) or EDTA (5 mM), indicating that neither serine nor metalloproteinases are involved to a significant extent. Pepstatin (5 micrograms/ml), an inhibitor of aspartic proteinases, inhibited albumin degradation by approximately 50%. Leupeptin (10 microM) and N-ethylmaleimide (10 mM), inhibitors of cysteine proteinases, decreased albumin degradation by 34 and 65%, respectively. Combinations of aspartic and cysteine proteinase inhibitors produced nearly complete inhibition of albumin degradation. Taken together, these data indicate that aspartic and cysteine proteinases are primarily responsible for albumin degradation by renal cortical lysosomes under these conditions. In keeping with the above data, we have measured high activities of the cysteine proteinases, cathepsins B, H, and L, in cortical tubules, the major site of renal protein degradation. Using the peptidyl 7-amino-4-methylcoumarin (NHMec) substrates (Z-Arg-Arg-NHMec, for cathepsin B; Arg-NHMec for cathepsin H; and Z-Phe-Phe-CHN2-inhibitable hydrolysis of Z-Phe-Arg-NHMec corrected for inhibition of cathepsin B activity for cathepsin L) values obtained were (means +/- SE, mU/mg protein, 1 mU = production of 1 nM product/min, n = 6): cathepsin B, 2.1 +/- 0.34; cathepsin H, 1.35 +/- 0.19; cathepsin L, 14.49 +/- 1.26. In comparison, the activities of cathepsins B, H, and L in liver were: 0.56 +/- 0.03, 0.28 +/- 0.04, and 1.27 +/- 0.16, respectively.

Extracellular Matrix Degradation by Cultured Mesangial Cells: Mediators and Modulators:

Decreased degradation of the glomerular extracellular matrix (ECM) is thought to contribute to the accumulation of glomerular ECM that occurs in diabetic nephropathy and other chronic renal diseases. Several lines of evidence indicate a key role for the plasminogen activator/plasminogen/plasmin system in glomerular ECM degradation. However, which of the two plasminogen activators (PAs) present in renal tissue, tissue plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA), is responsible for plasmin generation and those factors that modulate the activity of this system remain unclear. This study utilized mesangial cells isolated from mice with gene deletions for tPA, uPA, and plasminogen activator inhibitor 1 (PAI-1) to further delineate the role of the PA/plasminogen/plasmin system in ECM accumulation. ECM degradation by uPA-null mesangial cells was not significantly different from controls (92% ± 1%, n = 12). In contrast, ECM degradation by tPA-null mesangial cells was markedly reduced (–78 ± 1%, n = 12, P < 0.05) compared with controls, whereas tPA/uPA double-null mesangial cells degraded virtually no ECM. Previous studies from this laboratory have established that transforming growth factor-β1 (TGFβ1) inhibits ECM degradation by cultured mesangial cells by increasing the production of PAI-1, the major physiological PA inhibitor. In keeping with this observation, TGFβ1 (1 ng/ml) had no effect on ECM degradation by PAI-1-null MC. High glucose levels (30 mM) in the presence or absence of insulin (0.1 mM) caused a moderate increase in ECM degradation by normal human mesangial cells. In contrast, glycated albumin, whose concentration is known to increase in diabetes, produced a dose-dependent (0.2–0.5 mg/ml) inhibition of ECM degradation by normal human mesangial cells. Taken together, these results document the importance of tPA versus uPA in renal plasmin production and indicate that in contrast to elevated glucose, glycated albumin may contribute to ECM accumulation in diabetic nephropathy.