Dehydroepiandrosterone prevents the aggregation of platelets obtained from postmenopausal women with type 2 diabetes mellitus through the activation of the PKC/eNOS/NO pathway.

The steroid hormone dehydroepiandrosterone (DHEA), suggested to be a cardioprotector, prevents platelet aggregation in healthy humans. This hormone is reduced in postmenopausal women by 60% of its normal value. Platelets in patients with type 2 diabetes (T2D) are more sensitive to aggregation, which has been attributed to a reduced ability to produce nitric oxide (NO). In light of these precedents and considering that DHEA is able to increase the production of NO in cultured endothelial cells, we suggest that DHEA prevents the aggregation of platelet from postmenopausal women with T2D through the activation of PKC/eNOS/NO/cGMP pathway. To determine the effect of DHEA in platelet aggregation, platelet-rich plasma (PRP) obtained from postmenopausal women with T2D was preincubated with DHEA, and aggregation induced by ADP was determined in the presence or absence of L-NNA (LNG-nitroarginine), Rottlerin, NOS, or PKC delta inhibitors, respectively. Platelet NO production was measured with the fluorescent probe DAF2DA and eNOS activation was determined by Western blot, using an anti-p-eNOS (ser 1177) antibody. DHEA 1) prevented platelet aggregation by 40% compared to control, 2) increased NO production by 63%, 3) increased p-eNOS (phosphorylated endothelial nitric oxide synthase) levels, and 4) increased cGMP production. These effects were reduced in the presence of L-NNA or Rottlerin. DHEA prevents platelet aggregation induced by ADP. This effect is mediated by the activation of the PKCδ/eNOS/NO/cGMP pathway. Our results suggest that DHEA could be considered to be a potential therapeutic tool in the prevention of atherothrombotic processes in postmenopausal women with T2D.

Induction of B(1)-kinin receptors in vascular smooth muscle cells: cellular mechanisms of map kinase activation.

Vascular smooth muscle cell (VSMC) proliferation is a prominent feature of the atherosclerotic process that occurs after endothelial injury. Although a vascular wall kallikrein-kinin system has been described, its contribution to vascular disease remains undefined. Because the B(1)-kinin receptor subtype (B1KR) is induced in VSMCs only in response to injury, we hypothesize that this receptor may be mediating critical events in the progression of vascular disease. In the present study, we provide evidence that des-Arg(9)-bradykinin (dABK) (10(-8) M), acting through B1KR, stimulates the phosphorylation of mitogen-activated protein kinase (MAPK) (p42(mapk) and p44(mapk)). Activation of MAPK by dABK is mediated via a cholera toxin-sensitive pathway and appears to involve protein kinase C, Src kinase, and MAPK kinase. These findings demonstrate that the activation of B1KR in VSMCs leads to the generation of second messengers that converge to activate MAPK and provide a rationale to investigate the mitogenic actions of dABK in vascular injury.

Activation of MAPK by modified low-density lipoproteins in vascular smooth muscle cells.

A high concentration of circulating low-density lipoproteins (LDL) is a major risk factor for atherosclerosis. Native LDL and LDL modified by glycation and/or oxidation are increased in diabetic individuals. LDL directly stimulate vascular smooth muscle cell (VSMC) proliferation; however, the mechanisms remain undefined. The extracellular signal-regulated kinase (ERK) pathway mediates changes in cell function and growth. Therefore, we examined the cellular effects of native and modified LDL on ERK phosphorylation in VSMC. Addition of native, mildly modified (oxidized, glycated, glycoxidized) and highly modified (highly oxidized, highly glycoxidized) LDL at 25 microg/ml to rat VSMC for 5 min induced a fivefold increase in ERK phosphorylation. To elucidate the signal transduction pathway by which LDL phosphorylate ERK, we examined the roles of the Ca(2+)/calmodulin pathway, protein kinase C (PKC), src kinase, and mitogen-activated protein kinase kinase (MEK). Treatment of VSMC with the intracellular Ca(2+) chelator EGTA-AM (50 micromol/l) significantly increased ERK phosphorylation induced by native and mildly modified LDL, whereas chelation of extracellular Ca(2+) by EGTA (3 mmol/l) significantly reduced LDL-induced ERK phosphorylation. The calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonamide (40 micromol/l) significantly decreased ERK phosphorylation induced by all types of LDL. Downregulation of PKC with phorbol myristate acetate (5 micromol/l) markedly reduced LDL-induced ERK phosphorylation. Pretreatment of VSMC with a cell-permeable MEK inhibitor (PD-98059, 40 micromol/l) significantly decreased ERK phosphorylation in response to native and modified LDL. These findings indicate that native and mildly and highly modified LDL utilize similar signaling pathways to phosphorylate ERK and implicate a role for Ca(2+)/calmodulin, PKC, and MEK. These results suggest a potential link between modified LDL, vascular function, and the development of atherosclerosis in diabetes.

Regulation of B(2)-kinin receptors by glucose in vascular smooth muscle cells.

The development of vascular disease is accelerated in hyperglycemic states. Vascular injury plays a pivotal role in the progression of atherosclerotic vascular disease in diabetes, which is characterized by increased vascular smooth muscle cell (VSMC) proliferation and extracellular matrix accumulation. We previously reported that diabetes alters the activity of the kallikrein-kinin system and results in the upregulation of kinin receptors in the vessel wall. To determine whether glucose can directly influence the regulation of kinin receptors, the independent effect of high glucose (25 mM) on B(2)-kinin receptors (B2KR) in VSMC was examined. A threefold increase in B2KR protein levels and a 40% increase in B2KR surface receptors were observed after treatment with high glucose after 24 h. The mRNA levels of B2KR were also significantly increased by high glucose as early as 4 h later. To elucidate the cellular mechanisms by which glucose regulates B2KR, we examined the role of protein kinase C (PKC). High glucose increased total PKC activity and resulted in the translocation of conventional PKC isoforms (beta(1) and beta(2)), novel (epsilon), and atypical (zeta) PKC isoforms into the membrane. Inhibition of PKC activity prevented the increase in B2KR levels induced by ambient high glucose. These findings provide the first evidence that glucose regulates the expression of B(2) receptors in VSMC and provide a rationale to further study the interaction between glucose and kinins on the pathogenesis of atherosclerotic vascular disease in diabetes.

Native and modified LDL activate extracellular signal-regulated kinases in mesangial cells.

Glycation and/or oxidation of LDL may promote diabetic nephropathy. The mitogen-activated protein kinase (MAPK) cascade, which includes extracellular signal-regulated protein kinases (ERKs), modulates cell function. Therefore, we examined the effects of LDL on ERK phosphorylation in cultured rat mesangial cells. In cells exposed to 100 microg/ml native LDL or LDL modified by glycation, and/or mild or marked (copper-mediated) oxidation, ERK activation peaked at 5 min. Five minutes of exposure to 10-100 microg/ml native or modified LDL produced a concentration-dependent (up to sevenfold) increase in ERK activity. Also, 10 microg/ml native LDL and mildly modified LDL (glycated and/or mildly oxidized) produced significantly greater ERK activation than that induced by copper-oxidized LDL +/- glycation (P < 0.05). Pretreatment of cells with Src kinase and MAPK kinase inhibitors blocked ERK activation by 50-80% (P < 0.05). Native and mildly modified LDL, which are recognized by the native LDL receptor, induced a transient spike of intracellular calcium. Copper-oxidized (+/- glycation) LDL, recognized by the scavenger receptor, induced a sustained rise in intracellular calcium. The intracellular calcium chelator (EGTA/AM) further increased ERK activation by native and mildly modified LDL (P < 0.05). These findings demonstrate that native and modified LDL activate ERKs 1 and 2, an early mitogenic signal, in mesangial cells and provide evidence for a potential link between modified LDL and the development of glomerular injury in diabetes.

Mechanisms by which bradykinin promotes fibrosis in vascular smooth muscle cells: role of TGF-beta and MAPK.

Accumulation of extracellular matrix (ECM) is a hallmark feature of vascular disease. We have previously shown that hyperglycemia induces the expression of B(2)-kinin receptors in vascular smooth muscle cells (VSMC) and that bradykinin (BK) and hyperglycemia synergize to stimulate ECM production. The present study examined the cellular mechanisms through which BK contributes to VSMC fibrosis. VSMC treated with BK (10(-8) M) for 24 h significantly increased alpha(2)(I) collagen mRNA levels. In addition, BK produced a two- to threefold increase in alpha(2)(I) collagen promoter activity in VSMC transfected with a plasmid containing the alpha(2)(I) collagen promoter. Furthermore, treatment of VSMC with BK for 24 h produced a two- to threefold increase in the secretion rate of tissue inhibitor of metalloproteinase 1 (TIMP-1). The increase in alpha(2)(I) collagen mRNA levels and alpha(2)(I) collagen promoter activity, as well as TIMP-1 secretion, in response to BK were blocked by anti-transforming growth factor-beta (anti-TGF-beta) neutralizing antibodies. BK (10(-8) M) increased the endogenous production of TGF-beta1 mRNA and protein levels. Inhibition of the mitogen-activated protein kinase (MAPK) pathway by PD-98059 inhibited the increase of alpha(2)(I) collagen promoter activity, TIMP-1 production, and TGF-beta1 protein levels observed in response to BK. These findings provide the first evidence that BK induces collagen type I and TIMP-1 production via autocrine activation of TGF-beta1 and implicate MAPK pathway as a key player in VSMC fibrosis in response of BK.

Role of reactive oxygen species in bradykinin-induced mitogen-activated protein kinase and c-fos induction in vascular cells.

Bradykinin stimulates proliferation of aortic vascular smooth muscle cells (VSMCs). We investigated the action of bradykinin on the phosphorylation state of the mitogen-activated protein kinases p42(mapk) and p44(mapk) in VSMCs and tested the hypothesis that reactive oxygen species (ROS) might be involved in the signal transduction pathway linking bradykinin activation of nuclear transcription factors to the phosphorylation of p42(mapk) and p44(mapk). Bradykinin (10(-8) mol/L) rapidly increased (4- to 5-fold) the phosphorylation of p42(mapk) and p44(mapk) in VSMCs. Preincubation of VSMCs with either N-acetyl-L-cysteine and/or alpha-lipoic acid significantly decreased bradykinin-induced cytosolic and nuclear phosphorylation of p42(mapk) and p44(mapk). In addition, the induction c-fos mRNA levels by bradykinin was completely abolished by N-acetyl-L-cysteine and alpha-lipoic acid. Using the cell-permeable fluorescent dye dichlorofluorescein diacetate, we determined that bradykinin (10(-8) mol/L) rapidly increased the generation of ROS in VSMCs. The NADPH oxidase inhibitor diphenylene iodonium (DPI) blocked bradykinin-induced c-fos mRNA expression and p42(mapk) and p44(mapk) activation, implicating NADPH oxidase as the source for the generation of ROS. These findings demonstrate that the phosphorylation of cytosolic and nuclear p42(mapk) and p44(mapk) and the expression of c-fos mRNA in VSMCs in response to bradykinin are mediated via the generation of ROS and implicate ROS as important mediators in the signal transduction pathway through which bradykinin promotes VSMC proliferation in states of vascular injury.

Calcium-calmodulin mediates bradykinin-induced MAPK phosphorylation and c-fos induction in vascular cells.

The vasoactive peptide bradykinin (BK) has been implicated in the pathophysiology of a number of vascular wall abnormalities, but the cellular mechanisms by which BK generates second messengers that alter vascular function are as yet undefined. Exposure of vascular smooth muscle cells (VSMC) to BK (10(-7) M) produced a rapid and transient rise in intracellular calcium, which preceded an increase in tyrosine phosphorylation of mitogen-activated protein kinase (MAPK). MAPK activation by BK was observed as early as 1 min, peaked at 5 min, and returned to baseline by 20 min. Treatment of cells with the intracellular calcium chelator EGTA-acetoxymethyl ester inhibited BK-stimulated MAPK activation, suggesting that intracellular calcium mobilization contributes to the activation of MAPK. The calmodulin inhibitor W-7 also markedly inhibited BK-induced MAPK phosphorylation in the cytoplasm as well as in the nucleus. Moreover, the BK-induced increase in c-fos mRNA levels was significantly inhibited by the calmodulin inhibitor, indicating that calmodulin is required for BK signaling leading to c-fos induction. These results implicate the calcium-calmodulin pathway in the mechanisms for regulating MAPK activity and the resultant c-fos expression induced by BK in VSMC.

Mechanisms of MAPK activation by bradykinin in vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) proliferation is a prominent feature of the atherosclerotic process occurring after endothelial injury. A vascular wall kallikrein-kinin system has been described. The contribution of this system to vascular disease is undefined. In the present study we characterized the signal transduction pathway leading to mitogen-activated protein kinase (MAPK) activation in response to bradykinin (BK) in VSMC. Addition of 10(-10)-10(-7) M BK to VSMC resulted in a rapid and concentration-dependent increase in tyrosine phosphorylation of several 144- to 40-kDa proteins. This effect of BK was abolished by the B(2)-kinin receptor antagonist HOE-140, but not by the B(1)-kinin receptor antagonist des-Arg(9)-Leu(8)-BK. Immunoprecipitation with anti-phosphotyrosine antibodies followed by immunoblot revealed that 10(-9) M BK induced tyrosine phosphorylation of focal adhesion kinase (p125(FAK)). BK (10(-8) M) promoted the association of p60(src) with the adapter protein growth factor receptor binding protein-2 and also induced a significant increase in MAPK activity. Pertussis and cholera toxins did not inhibit BK-induced MAPK tyrosine phosphorylation. Protein kinase C downregulation by phorbol 12-myristate 13-acetate and/or inhibitors to protein kinase C, p60(src) kinase, and MAPK kinase inhibited BK-induced MAPK tyrosine phosphorylation. These findings provide evidence that activation of the B(2)-kinin receptor in VSMC leads to generation of multiple second messengers that converge to activate MAPK. The activation of this crucial kinase by BK provides a strong rationale to investigate the mitogenic actions of BK on VSMC proliferation in disease states of vascular injury.

Cellular distribution of exogenous aprotinin in the rat kidney.

Aprotinin, an inhibitor of the enzymatic activity of kallikrein in vitro, has been used to study the possible contributions of the kallikrein-kinin systems to physiological and pathological conditions. Pharmacokinetic studies indicate that aprotinin is concentrated in the kidney; however, there is little information with regard to its cellular distribution. The purpose of the present work was to study the cellular distribution of aprotinin, which would be valuable for a better understanding of its intrarenal effects. Sprague-Dawley rats (200-250g, n = 36) received aprotinin (50000 KIU/rat) and were killed at different intervals after its administration. The kidneys were examined histologically and the cellular distribution of aprotinin was studied by immunohistochemistry. Aprotinin was localized at 30 min concentrated within vesicles in the apical border of the proximal tubule cells. Later (2 h) it was observed distributed over the cytoplasm, where it remained for the 24 h studied. Aprotinin was also detected in connecting tubule cells colocalized with kallikrein, and in the basal portion of collecting tubule cells. No evidence of endogenous aprotinin was observed. The binding of aprotinin to the connecting tubule cells and collecting ducts offers a partial explanation of its renal effects.

Induction of renal kallikrein and renin gene expression by insulin and IGF-I in the diabetic rat.

The renal kallikrein-kinin system and the renin-angiotensin system are implicated in the pathogenesis of diabetic nephropathy. We have shown that renal kallikrein and renin gene expression are altered by diabetes. To investigate the cellular mechanisms responsible for these changes, we examined the effects of acute insulin and insulin-like growth factor I (IGF-I) treatment on renal kallikrein-kinin and renin-angiotensin system components. Three weeks after induction of diabetes, we measured renal kallikrein and renin mRNA levels, renal kallikrein and renal renin activity, and plasma renin activity in control and diabetic rats and diabetic rats treated with insulin or IGF-I for 2 or 5 h. In diabetic rats, kallikrein and renin mRNA levels were reduced >50% compared with control rats. Renal tissue kallikrein levels and plasma renin activity were decreased, whereas renal renin content was unchanged. Insulin increased kallikrein and renin mRNA levels after 2 h. IGF-I, at a dosage that stimulated kallikrein mRNA levels in control rats, had no effect on renal kallikrein and renin content or mRNA levels in diabetic rats. However, infusion of a fivefold higher IGF-I dosage resulted in a two- to threefold increase in kallikrein and renin mRNA levels in 2 h. These data suggest that 1) diabetes suppresses kallikrein and renin gene expression, and these abnormalities are reversed by insulin or IGF-I; and 2) the diabetic state produces resistance to IGF-I induction of kallikrein and renin gene expression. These changes in regulated synthesis of kallikrein and renin in the kidney may underlie renal vascular changes that develop in diabetes.

Bradykinin induces tubulin phosphorylation and nuclear translocation of MAP kinase in mesangial cells.

Glomerular hypertension and glomerular hypertrophy act early and synergistically to promote glomerular injury in diabetes. We have previously shown that increased renal kinin production contributes to the glomerular hemodynamic abnormalities associated with diabetes. Glomerulosclerosis, characterized by mesangial cell proliferation and matrix expansion, is the final pathway leading to renal failure. The signal(s) initiating mesangial cell proliferation is ill defined. In the present study, we utilized immunofluorescence, immunoprecipitation, and immunoblotting techniques to identify substrates that are tyrosine phosphorylated in response to bradykinin action in mesangial cells. Immunofluorescence microscopy of mesangial cells stained with anti-phosphotyrosine (anti-PY) antibodies following bradykinin treatment (10(-9)-10(-6) M) revealed a dose-dependent increase in the labeling of cytoplasmic and nuclear proteins. Immunoprecipitation with anti-PY, followed by immunoblot revealed bradykinin-induced tyrosyl phosphorylation of tubulin and mitogen-activated protein kinase (MAPK). Confocal microscopy of mesangial cells stained for MAPK indicated that bradykinin stimulation resulted in translocation of MAPK from the cytoplasm to the nucleus by 2 h. These data demonstrate that bradykinin action results in the tyrosine phosphorylation of cellular proteins in mesangial cells and suggest a role for tubulin and MAPK in the signaling cascade of bradykinin leading to altered mesangial function.

Postnatal maturation of tissue kallikrein-producing cells (connecting tubule cells) in the rat kidney: a morphometric and immunohistochemical study.

The mature, fully differentiated connecting tubule (CNT) cell plays an important role in the regulation of serum potassium levels and synthesizes the enzyme tissue kallikrein, a main component of a renal vasoactive system, the kallikrein-kinin system. To characterize the growth of CNT cells (tissue kallikrein-producing cells), we studied the rat kidney at three different time points of postnatal development: at day 5, day 15, and day 30. The CNT cells were identified on tissue sections by a standardized immunohistochemical procedure. The tissue kallikrein content was determined by radioimmunoassay and the activity of the enzyme in kidney homogenates was measured using a selective synthetic substrate. The number of immunolabeled CNT and CNT cells per cortex area gradually increased from day 5 to day 30. A similar rise in the content and activity of tissue kallikrein was observed when the enzyme levels were determined by radioimmunoassay or by the enzymatic method. In addition, the morphometric analysis showed that the distal end of CNT had larger cells that displayed a more intense tissue kallikrein staining than those present in the proximal end, suggesting that the postnatal development of CNT is induced from its juxtamedullary portion. Our results show that tissue kallikrein expression is very low in the newborn rat, increasing gradually with age to reach adult levels at day 30. This finding, together with the morphometric data, suggests immaturity of CNT cells in newborn rats, a fact that could contribute to explaining the high serum potassium levels reported at this stage. In addition, the contrasting behavior of kallikrein and renin in the postnatal development (kallikrein increasing and renin decreasing) could explain the gradual decrease in renal vascular resistance and increase in renal blood flow observed after birth.

[G proteins]. / Proteínas G.

G proteins play a central role in the mechanism of action of most hormones and neurotransmitters. They act as signal transducers between membrane receptors activated by extracellular stimuli on the one hand and intracellular effectors which control the concentrations of cytosolic messenger molecules (cAMP, cGMP, inositol phosphates, Ca2+) on the other. G proteins form a highly conserved family of membrane-associated proteins composed of alpha, beta and subunits. The alpha subunit, which is unique for each G protein, determines its biological activity and binds GDP and GTP. A number of diseases are already known to involve structural and/or quantitative changes of G proteins in plasma membranes. Interestingly, proteins encoded by some oncogenes show a high degree of homology with G proteins, which suggests that certain malignancies may be caused by alterations of transmembrane signaling.