Nitric oxide modulates the synthesis of extracellular matrix proteins in cultured rat mesangial cells

Nitric oxide (NO) is an important effector molecule of the inflammatory response. It is synthesized by mesangial cells and has been proposed to contribute to glomerular injury in various disease states. We studied whether NO modulates extracellular matrix production in cultured rat mesangial cells. Stimulation of rat mesangial cell NO release with gamma-interferon and lipopolysaccharide resulted in reduced production of collagen (by 35%) fibronectin (by 48%) (P < 0.05). In contrast, laminin synthesis was enhanced two-fold by the same maneuver (P < 0.05). These changes were reversed by the addition of L-NAME, a selective inhibitor of inducible nitric oxide synthase. This is the first demonstration that NO regulates the synthesis of extracellular matrix by mesangial cells. The results indicate that increased renal production of NO in glomerular diseases may attenuate the production and accumulation of matrix proteins and limit the severity of glomerulosclerosis.     

High glucose inhibits nitric oxide production in cultured rat mesangial cells

Hyperglycemia directly contributes to the development of diabetic nephropathy. A high-serum glucose concentration alters intraglomerular hemodynamics and promotes deposition of extracellular matrix in the kidney. Nitric oxide (NO) is a short-lived messenger molecule that participates in the regulation of renal blood flow, GFR, and mesangial matrix accumulation. Therefore, in this study it was tested whether high glucose directly modulates NO synthesis by rat mesangial cells in vitro by measuring the accumulation of nitrite, the stable metabolite of NO, in the incubation media. Raising the external glucose concentration to 33.3 mM for 24 to 72 h reduced nitrite levels in cell supernatants in a time-dependent manner to a nadir of 14 +/- 3% of the amount in normal glucose media (5.6 mM) (P < 0.01). The decline in NO synthesis in high glucose media was paralleled by decreased cyclic guanosine monophosphate generation; however, there was no alteration in rat mesangial cell expression of inducible NO synthase protein. The suppressive effect of high glucose on NO production by mesangial cells was not modified by inhibition of protein kinase C (H-7), the addition of antioxidants (vitamin E or superoxide dismutase), or a pan-specific anti-transforming growth factor-beta antibody. An elevated ambient glucose caused a time-dependent reduction in mesangial cell L-arginine content. Addition of L-arginine (10 to 20 mM) to external media partially reversed the inhibitory effect of high glucose on mesangial cell NO production in a dose-dependent manner. The highest dose of L-arginine (20 mM) increased mesangial cell L-arginine content to comparable levels in normal and high glucose media. These results indicate that high glucose causes depletion of L-arginine in mesangial cells and compromises NO synthesis. Limitation in the metabolic precursor and other, as yet unidentified, factors act to reduce NO production by mesangial cells in the presence of an elevated ambient glucose level, a change that may play a role in the development of diabetic glomerulosclerosis.     

Nitric oxide as a regulator of prostacyclin synthesis in cultured rat heart endothelial cells

The effects of nitric oxide (NO) and its second messenger cyclic guanosine monophosphate (cGMT) on prostacyclin (PGI2) synthesis were studied in cultured rat heart endothelial cells using three different non-enzymatic nitric oxide releasing substances as well as inhibitors of nitric oxide synthase and of soluble guanylate cyclase. Production of prostacyclin, measured as 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), was stimulated up to 1.7 fold in endothelial cells treated with the NO donors SIN-1 (3-morpholino sydnonimine), GEA 3162 (3-aryl-substituted oxatriazole imine) and GEA 3175 (3-aryl-substituted oxatriazole sulfonyl), chloride). In each case the synthesis of cGMP increase as much as 40-100 fold. An inhibitor of NO synthase, NG-nitro-L-arginine methyl ester (L-NAME), decreased the basal production of 6-keto-PGF1 alpha in non-stimulated endothelial cells, an effect that could be reversed by the NO donors SIN-1, GEA 3162 and GEA 3175. cGMP formation in the L-NAME treated endothelial cells was unaltered. The guanylate cyclase inhibitors, methylene blue (100 mumol/l) and LY83583 (100 mumol/l), caused a 1.5-10 fold increase in 6-keto-PGF1 alpha production while NO-donor-stimulated endothelial cGMP production was decreased by 10 to 90%. However, when SIN-1 was used as a stimulant, LY83583 had no significant effect on the production of cGMP. These findings support the hypothesis that NO stimulates prostacyclin production directly by activating cyclooxygenase. The results also suggest that NO could have an indirect effect on prostacyclin production via cGMP.     

Anti-mitogenic effects of sarpogrelate in cultured rat mesangial cells

We investigated the effects of the new 5-HT2A receptor antagonist, sarpogrelate, on DNA synthesis in renal mesangial cells stimulated with 5-HT in the presence and absence of platelet-derived growth factor (PDGF)-BB. Both 5-HT and PDGF-BB demonstrated a mitogenic effect on these cells. When mesangial cells were incubated in the absence of PDGF-BB, sarpogrelate inhibited DNA synthesis in these cells in a dose-dependent manner. In the presence of PDGF-BB, sarpogrelate had a weaker anti-mitogenic effect in mesangial cells stimulated with 5-HT. Sarpogrelate was cytotoxic at concentrations over 10(-5) M according to the results of LDH release assays, and it reduced the S1 phase in mesangial cells stimulated with 5-HT by a flow cytometry. These findings suggest that sarpogrelate may be effective in the treatment of some glomerulonephritis associated with mesangial cell proliferation.     

Nitric oxide inhibits Oct-1 DNA binding activity in cultured vascular smooth muscle cells

In this paper, the authors model the nonmonotonic relation between body mass index (BMI) (weight (kg)/height2 (m2)) and mortality in 13,242 black and white participants in the NHANES I Epidemiologic Follow-up Study in order to estimate the BMI at which minimum mortality occurs. The BMI of minimum mortality was 27.1 for black men (95% confidence interval (CI) 24.8-29.4), 26.8 for black women (95% CI 24.7-28.9), 24.8 for white men (95% CI 23.8-25.9), and 24.3 for white women (95% CI 23.3-25.4). Each confidence interval included the group average. Analyses conducted by smoking status and after exclusion of persons with baseline illness and persons who died during the first 4 years of follow-up led to virtually identical estimates. The authors determined the range of values over which risk of all-cause mortality would increase no more than 20% in comparison with the minimum. This interval was nine BMI units wide, and it included 70% of the population. These results were confirmed by parallel analyses using quantiles. The model used allowed the estimation of parameters in the BMI-mortality relation. The resulting empirical findings from each of four race/sex groups, which are representative of the US population, demonstrate a wide range of BMIs consistent with minimum mortality and do not suggest that the optimal BMI is at the lower end of the distribution for any subgroup.     

Nitric oxide production by mouse sponge matrix allograft-infiltrating cells. Comparison with the rat species

We have recently demonstrated in the rat species that sponge matrix allograft infiltrating cells spontaneously produce nitric oxide (.N = 0) and this .N = 0 production precedes the development of CTL. Compared with our experience in the mouse species, the CTL activity recovered from rat sponge grafts is of shorter duration and less intense. Assessment of the spontaneous .N = 0 production by mouse allograft infiltrating cells reveals a more delayed time course of production, paralleling the recovery of CTL activity from the graft. The in vitro spontaneous .N = 0 production by mouse allograft-infiltrating cells was greater than the production by syngeneic graft-infiltrating cells on all days tested. Exposure of allogeneic but not syngeneic graft infiltrating cells to the sensitizing alloantigen in vitro resulted in enhanced .N = 0 synthesis. In contrast, LPS stimulated .N = 0 production by both syngeneic and allogeneic graft cells on all days postgrafting. Culture of day-14 allograft infiltrating cells with alloantigen in the absence of NG-monomethyl-L-arginine (NMA), the competitive inhibitor of .N = 0 synthesis, resulted in elevated supernatant NO2- levels and decreased 3H-TdR uptake and CTL activity compared with cultures carried out in the presence of NMA. The supernatant NO2- levels, as well as the CTL activity and 3H-TdR incorporation of the cultured cells, was dependent on the concentration of NMA present, and these effects could be reversed by excess L-arginine. Thus, the species difference in .N = 0 synthesis (rat > mouse), observed by others, is evident in the sponge allograft model and may explain why CTL activity recovered from rat allografts is of shorter duration and less intense than that from the mouse allografts.     

Effect of serum from patients with mesangial proliferative glomerulonephritis on cultured rat mesangial cells

Adenosine, an important neuromodulatory compound in the brain and retina, is a potent vasodilator in most vascular beds throughout the body. Its actions are potentiated by inhibitors of nucleoside transport into cells. Knowledge of the existence of specific adenosine uptake systems in mammalian retina and the inhibition of the uptake by nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport, raises the possibility that the associated nucleoside transport system may be of pharmacological importance in retinal function. We have characterized the binding of the nucleoside transporter probe, [3H]NBMPR, to a cultured human retinal cell line established by transfection of SV-40 T antigen plasmid-DNA. The binding was specific, saturable and reversible. Scatchard analysis of the saturation data revealed that NBMPR binds to a homogeneous population of high affinity binding sites (KD = 0.65 +/- 0.22 nM; Bmax = 466 +/- 157 fmol/mg protein) characteristically similar to the binding sites in human retinal tissue (KD = 0.32 +/- 0.01 nM; Bmax = 292 +/- 41 fmol/mg protein). Selected compounds inhibited the binding in the cell line and retinal tissue with the same rank order of potency, suggesting that the transporters in the cell line and retinal tissue are similar. The data showed that the cell line is a useful model for the study of nucleoside transporter function in human retina.     

Nitric oxide mediates the lipopolysaccharide dependent upregulation of the heme oxygenase-1 gene expression in cultured rat Kupffer cells

Intraoperative endoscopy (IOE) is accepted as the ultimate diagnostic procedure for the complete evaluation of small bowel. Until recent years, operative endoscopy was the complement of sonde enteroscopy. The difficulties of this long and fastidious type of examination, for both the patient and the medical team are well known. It provides incomplete exploration (detubing of certain loops is too rapid), and renders impossible any diagnostic or therapeutic procedure (biopsies, electrocoagulation). The indications of IOE have diminished over recent years during the development of push enteroscopy by upper or double way. Indeed, the latter method makes it possible in a number of cases to obtain complete exploration of the small bowel with biopsies and therapeutic procedures, or an exploration enabling screening for lesions in the first jejunal loops of the lat ileal loops.     

High glucose inhibits cytosolic calcium signaling in cultured rat mesangial cells

Glomerular vasodilatation in the early stages of type I diabetes mellitus apparently results from arteriolar insensitivity to vasoconstrictors. Since cytosolic free calcium ([Ca2+]i) is a major signaling mechanism for smooth muscle contraction, we studied whether growth of smooth muscle-like rat glomerular mesangial cells in media with high glucose concentration affects [Ca2+]i responses to vasoconstrictors. In cells grown for five days in 22 mM glucose, we observed blunted responsiveness to three structurally unrelated vasoconstrictors that elevate [Ca2+]i via a phospholipase C-dependent mechanism, angiotensin II, prostaglandin F2 alpha, and arginine vasopressin. Inhibition of [Ca2+]i responses was not due to an osmotic effect of high glucose, since it was not mimicked by hypertonic mannitol. While the size of intracellular Ca2+ pools was unaffected by elevated glucose, Na+/Ca2+ exchange was markedly inhibited, thus ruling out both impaired filling of Ca2+ stores and enhanced counter-regulatory mechanisms. Impaired myoinositol transport or intracellular sorbitol accumulation were not responsible for the effects of high glucose, since supplementation of media with myo-inositol or with the aldose reductase inhibitor. Alcon 1576, failed to reverse insensitivity to vasoconstrictors. On the other hand, down-regulation or pharmacological inhibition of protein kinase C completely reversed the effects of high glucose, thus indicating involvement of this signal transduction pathway. These data suggest a possible intracellular mechanism for the impaired vascular sensitivity underlying early renal hemodynamic changes in diabetes mellitus.     

Effect of emodin on c-myc proto-oncongen expression in cultured rat mesangial cells

We report a patient with chronic asymptomatic Chagas' disease that presented Trypanosoma cruzi reactivation after kidney transplantation and immune depression. The only clinical manifestation of the disease was ulcerative skin lesions, which is unusual in Chagas' disease.     

Nitric oxide synthase activity in the gravid rat uterus decreases a day before the onset of parturition

OBJECTIVE: Our purpose was to determine the timing, tissue location, and isoform of the uterine nitric oxide synthase activity decrease at term in gravid rat uteri. STUDY DESIGN: Nitric oxide synthase specific activity was assayed in rat uteri 11 through 22 days' gestation by the difference in radiolabeled arginine to citrulline conversion with and without the cofactor reduced nicotinamide adenine dinucleotide phosphate. Nitric oxide synthase isoform was assessed by calcium sensitivity and subcellular location. RESULTS: Rat uterine nitric oxide synthase activity decreased between days 15 and 21 of gestation but did not decrease further at term (day 22), before and after the onset of labor. Decidual nitric oxide synthase activity exceeded the myometrial activity at 15 days' gestation, but then the two were equal at 18 through 22 days' gestation. The nitric oxide synthase activity was calcium insensitive except for half the decidual cytosolic activity on day 15. CONCLUSION: The decrease in pregnant rat uterine nitric oxide synthase activity coincides with the preparation of the uterus for parturition rather than the final activation of labor.     

Cytokine inducible matrix metalloproteinase expression in immortalized rat chondrocytes is independent of nitric oxide stimulation

The objective of this study was to determine if an immortalized mammalian chondrocyte cell line had a profile of matrix metalloproteinase (MMP) expression that was consistent with what has been reported for primary chondrocytes in vitro and in vivo. A combination of zymography, Western, and Northern analysis was used to examine the expression of MMPs that are relevant to cartilage degradation. Both interleukin-1beta and tumor necrosis factor alpha induced a 4- to 9-fold increase in the level of MMP-9 expression in conditioned media, and a 17- to 24-fold increase in MMP-3 mRNA. Other compounds such as basic fibroblast growth factor and staurosporine each increased MMP-9 expression individually and potentiated the effects of the two cytokines. Transforming growth factor beta had no positive or inhibitory effects. N-methyl arginine blocked the increase in nitric oxide observed following treatment with the cytokines but did not prevent the increased expression of MMPs. The pattern of metalloproteinase expression observed in IRC cells and the response to cytokines is very similar to what has been reported during the pathogenesis of osteoarthritis. The IRC cells should be useful as a model system to study basic mechanisms controlling chondrocyte MMP expression and to identify pharmacological modulators of this process.     

Intra-operative irradiation prolongs the presence of matrix metalloproteinase activity in large bowel anastomoses of the rat

There exists a growing interest in intra-operative radiation therapy as a treatment modality for large bowel cancer. In a previous experimental study we showed that high-dose intra-operative irradiation delays the healing of colonic anastomoses. However, the contribution of proteases is unknown. In the present study, the gelatinolytic and collagenolytic activity in the healing anastomoses is investigated. After a resection of a 1-cm length of colon (uninjured colon), the rats were irradiated with a single dose of 25 Gy, either to the proximal limb, referred to as the proximal group, or to both proximal and distal limbs of the bowel, referred to as the combined group, before anastomotic construction. Both groups were compared to a control group with anastomoses which were sham-irradiated. The animals were killed 1, 3 or 7 days after operation. The gelatinolytic activity in uninjured and anastomotic tissue was quantified by gelatin zymography and the collagenolytic activity by an assay using a fibrillar rat collagen substrate. Compared with resected uninjured colon, most of the gelatinolytic activities were markedly increased in anastomotic tissue of all groups during the first postoperative week, and new additional activities were detected. The additional metalloproteinases (the 95-kDa family) of both irradiated groups were significantly elevated compared to the anastomoses of the sham-irradiated control group at 7 days after operation. In anastomotic tissue of all groups, the collagenolytic activity of the tissue was also significantly increased at 1 and 3 days after construction with respect to the resected, uninjured colon. After 7 days this effect had disappeared for the sham-irradiated anastomoses, but the activity in the anastomoses in both the proximal and combined groups remained significantly elevated. The findings provide evidence that intra-operative irradiation prolongs the presence of elevated gelatinolytic and collagenolytic activities in colon anastomoses. It may contribute to a reduced or delayed accumulation of collagen and other matrix proteins that supply anastomotic strength.     

Cyclic stretching force selectively up-regulates transforming growth factor-beta isoforms in cultured rat mesangial cells

Glomerular distention from increased intraglomerular pressure stretches mesangial cells (MCs). Stretching MCs in culture stimulates extracellular matrix accumulation, suggesting that this may be a mechanism for glomerular hypertension-associated glomerulosclerosis. We examined whether mechanical stretching serves as a stimulus for the synthesis and activation of the prosclerotic molecule transforming growth factor (TGF)-beta, thus providing a potential system for auto-induction of extracellular matrix. Rat MCs cultured on flexible-bottom plates were subjected to cyclic stretching for up to 3 days and then assayed for TGF-beta mRNA, secretion of TGF-beta, and localization of active TGF-beta by immunostaining. MCs contained mRNA for all three mammalian isoforms of TGF-beta. Cyclic stretching for 36 hours increased TGF-beta1 and TGF-beta3 mRNA levels approximately twofold, without altering the levels of TGF-beta2 mRNA. This was followed at 48 to 72 hours by the increased secretion of both latent and active TGF-beta1. Latent, but not active, TGF-beta3 secretion also increased whereas the levels of TGF-beta2 were unaffected by mechanical force. The stretching force in this system is unequally distributed over the culture membrane. Localization of active TGF-beta by immunostaining demonstrated that the quantity of cell-associated cytokine across the culture was directly proportional to the zonal amplitude of the stretching force. These results demonstrate that stretching force stimulates MCs to selectively release and activate TGF-beta1. This mechanical induction of TGF-beta1 may help explain the increased extracellular matrix associated with intraglomerular hypertension.     

Negative regulation of interleukin-1beta-activated neutral sphingomyelinase by protein kinase C in rat mesangial cells

Endogenous ceramide is produced by the action of acidic or neutral sphingomyelinases (SMase) in response to stimuli such as proinflammatory cytokines or other inducers of stress. Interleukin-1beta (IL-1beta) is known to stimulate ceramide formation in rat renal mesangial cells; however, the respective subtype of SMase and its regulation have not been investigated. We found that IL-1beta induced an increase in endogenous ceramide levels via the action of a neutral SMase but not an acidic SMase in rat mesangial cells. Cytokine-induced activation of neutral SMase was inhibited by stimulation of protein kinase C (PKC) by the phorbol ester TPA which caused a reduction of ceramide back to control levels. This inhibitory effect of TPA was reversed by the specific PKC-inhibitor Ro-318220. Long-term incubation (24 h) of mesangial cells with TPA, which downregulates PKC-alpha, -delta, and -epsilon isoenzymes, resulted in a recovery of IL-1beta-stimulated neutral SMase activity as well as ceramide formation. These data implicate an important modulatory function of PKC in ceramide production in IL-1beta-activated mesangial cells.