Biomedical subjects

R A Star

Publications and source records attributed to R A Star.

At least 37 records · Page 2Linked to original sources

PubMed1998

alpha-Melanocyte-stimulating hormone and acute renal failure.

alpha-Melanocyte-stimulating hormone (MSH) is an endogenous anti-inflammatory cytokine that inhibits all major forms of inflammation, alpha-MSH level is increased at sites of inflammation in humans, and is produced in the pituitary and in macrophages. The effects of alpha-MSH are mediated by melanocortin receptors found on macrophages, neutrophils, and renal tubules. alpha-MSH inhibited ischemic acute renal failure in mice and rats, even when started 6 h after injury. alpha-MSH acts, in part, by inhibiting the maladaptive activation of genes that cause inflammatory and cytotoxic renal injury. However, alpha-MSH is effective even in the absence of neutrophils, suggesting that alpha-MSH also acts directly on renal tubules.

Acute Kidney Injury↗

PubMed1997

Alpha-melanocyte-stimulating hormone protects against renal injury after ischemia in mice and rats.

Reperfusion after ischemia induces cytokines, chemoattractant chemokines, adhesion molecules, and nitric oxide (NO). The resultant neutrophil adherence and NO potentiates renal injury. alpha-Melanocyte-stimulating hormone (alpha-MSH) is a potent anti-inflammatory agent that inhibits neutrophil migration and production of neutrophil chemokines and NO. Since neutrophils and NO promote renal ischemic injury, we sought to determine if alpha-MSH inhibits renal injury in a model of bilateral renal ischemia. alpha-MSH significantly reduced ischemia-induced renal damage, measured by changes in renal histology and plasma blood urea nitrogen and creatinine in mice. alpha-MSH significantly decreased tubule necrosis, neutrophil plugging, and capillary congestion. Delay of alpha-MSH treatment for 6 h after ischemia also significantly inhibited renal damage. alpha-MSH also significantly inhibited ischemic damage in rats. To begin to determine the mechanism of action of alpha-MSH, we measured its effects on mediators of neutrophil trafficking and induction of the inducible isoform of NO synthase-II. alpha-MSH inhibited ischemia-induced increases in mRNA for the murine neutrophil chemokine KC/IL-8. alpha-MSH also inhibited induction of mRNA for the adhesion molecule ICAM-1, which is known to be critical in renal ischemic injury. alpha-MSH inhibited nitration of kidney proteins and induction of NO synthase-II. We conclude: (a) alpha-MSH protects against renal ischemia/reperfusion injury; and (b) it may act, in part, by inhibiting the maladaptive activation of genes that cause neutrophil activation and adhesion, and induction of NO synthase.

Animals↗

PubMed1997

Intrarenal localization of nitric oxide synthase isoforms and soluble guanylyl cyclase.

1. Nitric oxide (NO) plays an important role in the regulation of renal function. To date, five isoforms of NO synthase (NOS) and four subunits of soluble guanylyl cyclase have been cloned. The kidney contains four isoforms of NOS and all subunits of soluble guanylyl cyclase. 2. This review focuses on the intrarenal location of the isoforms of NOS and the subunits of soluble guanylyl cyclase.

Animals↗

PubMed1996

Alpha-melanocyte-stimulating hormone reduces endotoxin-induced liver inflammation.

Alpha-Melanocyte-stimulating hormone (MSH) is a potent anti-inflammatory agent in many models of inflammation, suggesting that it inhibits a critical step common to different forms of inflammation. We showed previously that alpha-MSH inhibits nitric oxide (NO) production in cultured macro-phages. To determine how alpha-MSH acts in vivo, we induced acute hepatic inflammation by administering endotoxin (LPS) to mice pretreated with Corynebacterium parvum, alpha-MSH prevented liver inflammation even when given 30 min after LPS administration. To determine the mechanisms of action of alpha-MSH, we tested its influence on NO, infiltrating inflammatory cells, cytokines, and chemokines. Alpha-MSH inhibited systemic NO production, hepatic neutrophil infiltration, and increased hepatic mRNA abundance for TNF alpha, and the neutrophil and monocyte chemokines (KC/IL-8 and MCP-1). We conclude that alpha-MSH prevents LPS-induced hepatic inflammation by inhibiting production of chemoattractant chemokines which then modulate infiltration of inflammatory cells. Thus, alpha-MSH has an effect very early in the inflammatory cascade.

Animals↗

PubMed1996

Induction of inducible nitric-oxide synthase by the heterotrimeric G protein Galpha13.

While the functions of several G protein alpha subunits such as alpha(s( and alpha(q) are relatively well understood, the action of others such as alpha13 remain largely undefined. Because of recent interest in regulation of nitric-oxide synthase (NOS) by G protein-coupled signaling systems and findings that receptors for two proinflammatory substances, thrombin and thromboxane couple to alpha13, we studied the effect of alpha13 on NOS activity in a renal epithelial cell line. We found that stable overexpression of alpha13 or its GTPase-deficient mutant, alpha13Q226L, in a continuous renal epithelial cell line (MCT) increased NOS activity. The increased NOS activity was due to increased expression of the macrophage-inducible form of NOS (iNOS). iNOS protein and activity were not increased in similar cells expressing an activated alpha(s) (alpha(s)Q227L) or were minimally increased in cells expressing activated alpha(i1) (alpha-i1Q204L) and alpha(q) (alpha(q)Q209L), members of the three other G protein alpha chain families. Transient co-expression of alpha13 or alpha13Q226L increased the activity of an iNOS promoter-CAT construct demonstrating that alpha13 increases iNOS expression through transcription. Consequently, alpha13 induces iNOS through a novel mechanism that is distinct from that of other G protein alpha chains and that may mediate the actions of G protein-dependent proinflammatory agents.

Animals↗

PubMed1996

alpha-MSH production, receptors, and influence on neopterin in a human monocyte/macrophage cell line.

alpha-Melanocyte-stimulating hormone (alpha-MSH), a tridecapeptide derived from pro-opiomelanocortin, has potent antiinflammatory activity in laboratory animals. alpha-MSH inhibits nitric oxide production by murine macrophages, an influence believed to reflect activation of an autocrine circuit in these cells, one that is based on production and release of alpha-MSH and subsequent stimulation of melanocortin receptors. We found that THP-1 cells, human monocytic cells, produced alpha-MSH; this production was increased by interleukin-6, tumor necrosis factor a, or concanavalin A. These cells also expressed the gene for the human alpha-MSH receptor MC1. Unlike murine macrophages, THP-1 cells produced little nitrite in response to interferon-gamma (IFN-gamma) and lipopolysaccharide, and a-MSH inhibited this production only slightly. However, production of neopterin, a presumed primate homologue of nitric oxide in lower animals, was increased in THP-1 cells stimulated with INF-gamma plus TNF-alpha and alpha-MSH significantly inhibited this production. The evidence indicates that an autocrine regulatory circuit based on alpha-MSH occurs in human monocyte/macrophages much as in murine macrophages. alpha-MSH-induced modulation of specific inflammatory mediators/cytotoxic agents appears to differ depending on the importance of the mediators in the myelomonocytic cells of different species.

Amino Acid Sequence↗

PubMed1996

The neuropeptide alpha-MSH has specific receptors on neutrophils and reduces chemotaxis in vitro.

The proopiomelanocortin-derived peptide alpha-melanocyte stimulating hormone (alpha-MSH) has potent anti-inflammatory effects in all animal models of inflammation against which it has been tested. Understanding of the mechanism by which this occurs is incomplete, although there is recent evidence for alpha-MSH receptors in murine and human macrophages and for modulation of production of proinflammatory cytokines and related mediators by alpha-MSH. Because of the prominence of neutrophils in early stages of inflammatory reactions where alpha-MSH is effective, we examined human neutrophils for evidence of mRNA for alpha-MSH receptors and for inhibition of neutrophil chemotaxis. There was accumulation of mRNA for melanocortin receptor 1 (MC1) in RT/PCR product from neutrophils stimulated with interferon and LPS. In subsequent studies alpha-MSH inhibited migration of neutrophils from most normal volunteers when the cells were placed in FMLP or IL-8 gradients. The inhibition by alpha-MSH could be traced to alterations in cAMP in neutrophils. The presence of alpha-MSH receptor message in neutrophils is consistent with the established anti-inflammatory effects of the peptide. Direct inhibition of neutrophil chemotaxis likely contributes to the anti-inflammatory activity of alpha-MSH.

Analysis of Variance↗

PubMed1996

Differential regulation of rat glomerular and proximal tubular renin mRNA following uninephrectomy.

Angiotensin II is thought to play a role in the renal adaptations to reduced renal mass, but earlier work has shown that plasma renin activity (PRA) does not increase in this setting. To examine this paradox, we studied the effect of uninephrectomy (UNX) on circulating, juxtaglomerular, glomerular, and proximal tubular (PT) renin. PRA was unchanged 2 wk following UNX and fell slightly at 6 wk. Single kidney renin secretory capacity and cortical renin mRNA, reflecting juxtaglomerular renin, were unchanged at 2 and 6 wk. With quantitative competitive reverse transcription-polymerase chain reaction, renin mRNA in microdissected glomeruli and PT were dramatically increased 2 wk post-UNIX (for glomeruli: sham, 1.2 +/- 0.3, vs. UNX, 8.8 +/- 1.9 x 10(5) copies/glomerulus; for PT: sham, 4.6 +/- 0.9, vs. UNX, 17.7 +/- 5.1 x 10(3) copies/mm). By 6 wk, glomerular renin was unchanged, and PT renin mRNA was markedly suppressed (for glomeruli; sham, 2.9 +/- 1.2, vs. UNX, 4.2 +/- 1.1 x 10(5) copies/glomerulus; for PT: sham, 7.5 +/- 2.1, vs. UNX, 1.0 +/- 0.3 x 10(3) copies/mm). These results demonstrate differential regulation of the circulating, juxtaglomerular, glomerular, and PT renin systems. Early activation of glomerular and PT renin may result in increased local generation of angiotensin II and thereby affect renal structural and functional adaptations following UNX.

Animals↗

PubMed1995

Evidence of autocrine modulation of macrophage nitric oxide synthase by alpha-melanocyte-stimulating hormone.

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a potent inhibitory agent in all major forms of inflammation. To identify a potential mechanism of antiinflammatory action of alpha-MSH, we tested its effects on production of nitric oxide (NO), believed to be a mediator common to all forms of inflammation. We measured NO and alpha-MSH production in RAW 264.7 cultured murine macrophages stimulated with bacterial lipopolysaccharide and interferon gamma. alpha-MSH inhibited production of NO, as estimated from nitrite production and nitration of endogenous macrophage proteins. This occurred through inhibition of production of NO synthase II protein; steady-state NO synthase II mRNA abundance was also reduced. alpha-MSH increased cAMP accumulation in RAW cells, characteristic of alpha-MSH receptors in other cell types. RAW cells also expressed mRNA for the primary alpha-MSH receptor (melanocortin 1). mRNA for proopiomelanocortin, the precursor molecular of alpha-MSH, was expressed in RAW cells, and tumor necrosis factor alpha increased production and release of alpha-MSH. These results suggest that the proinflammatory cytokine tumor necrosis factor alpha can induce macrophages to increase production of alpha-MSH, which then becomes available to act upon melanocortin receptors on the same cells. Such stimulation of melanocortin receptors could modulate inflammation by inhibiting the production of NO. The results suggest that alpha-MSH is an autocrine factor in macrophages which modulates inflammation by counteracting the effects of proinflammatory cytokines.

Amino Acid Oxidoreductases↗

PubMed1995

Accuracy of urea removal estimated by kinetic models.

The most accurate method for assessing the dialysis dose delivered during high efficiency/flux hemodialysis has not been established. Most current indices of dialysis dose are based on blood-side urea measurements, and thus estimate urea removal. Unfortunately, these methods may lead to inappropriately short dialysis during high flux or high efficiency dialysis, perhaps because of inaccuracies in estimating the amount of urea removal. It is unknown whether these clearance-based approaches can accurately predict either absolute or fractional net urea removal, the latter being equivalent to the solute removal index (SRI). Therefore, we compared the urea removal calculated by five blood-side kinetic methods: (1) urea reduction ration, (2) 1-pool, (3) 2-pool models, and the (4) Smye and (5) Daugirdas formulae. These were compared with the gold standard measurement by direct dialysate quantification. Eight stable patients receiving high-flux hemodialysis were studied over four sessions each. BUN was measured at 0, 45 minutes, 90 minutes, end dialysis, one hour after dialysis (equilibrium value), and 48 hours later. Total body water was determined from the dialysate urea removal; the urea generation rate was calculated using one hour post-dialysis and 48-hour BUN values. Both the total body water and urea generation rate were provided to the 1- and 2-pool models to optimize accuracy. The urea reduction ratio overestimated SRI. The 1-pool model overestimated both absolute urea removal and SRI in 28 of 32 sessions. The 2-pool model slightly underestimated both absolute urea removal and SRI. In contrast, the Smye and Daugirdas formulas accurately estimated SRI.(ABSTRACT TRUNCATED AT 250 WORDS)

Female↗

PubMed1995

TNF-alpha and IFN-gamma induce expression of nitric oxide synthase in cultured rat medullary interstitial cells.

Cytokines increase the expression of the inducible (type II) nitric oxide synthase (NOS) in macrophages, liver, and renal epithelial cells. Previously, we found that cultured rat medullary interstitial cells (RMIC) contain high levels of soluble guanylyl cyclase. To determine whether these cells can also produce NO, we studied the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on NO production, NOS II mRNA, and NOS II protein expression. Both TNF-alpha and IFN-gamma, in the presence of a low concentration of the other cytokine, caused dose-dependent increases in NO production. Exposure to TNF-alpha and IFN-gamma stimulated the production of NOS II mRNA, as determined by Northern blotting. Restriction mapping of reverse transcription-polymerase chain reaction products indicated that normal cells contained macrophage NOS II, whereas cytokine-stimulated cells contained primarily vascular smooth muscle NOS II and some macrophage NOS II. The appearance of NOS II protein was demonstrated by Western blotting. RMIC cell guanosine 3',5'-cyclic monophosphate accumulation increased 129-fold in response to the cytokines. NOS inhibitors decreased nitrite production. We conclude that 1) TNF-alpha and IFN-gamma induce the expression of vascular smooth muscle NOS II and production of NO in RMIC, and 2) NO acts as an autocrine activator of the soluble guanylyl cyclase in RMIC.

Amino Acid Oxidoreductases↗

PubMed1995

Prolonged in vivo hypoxia enhances nitric oxide synthase type I and type III gene expression in adult rat lung.

Prolonged hypoxia in the adult rat causes a decline in endothelium-derived nitric oxide (NO) production in the pulmonary circulation. To evaluate whether this is related to a decrease in endothelial NO synthase (NOS-III) expression, we determined the effects of hypobaric hypoxia (7 or 21 days) on NOS-III gene expression in adult rat lung. Neuronal NOS (NOS-I) expression was also examined; NOS-I has been immunohistochemically localized to rat bronchiolar epithelium. NOS-III and NOS-I mRNA abundance were assessed in reverse transcription-polymerase chain reaction assays and the proteins were evaluated by immunoblot analysis. After 7 and 21 days of hypoxia, there were increases in the steady-state levels of both NOS-III and NOS-I mRNA, rising 2.7- to 3.0-fold and 2.5- to 2.8-fold, respectively. These findings were confirmed by Northern analyses. In parallel, NOS-III and NOS-I protein abundance were also increased with hypoxia by 3.0- to 3.5-fold and 2.4- to 3.0-fold, respectively. NOS activity detected by [3H]arginine to [3H]citrulline conversion rose 109%. Thus, prolonged in vivo hypoxia causes enhancement of NOS-III and NOS-I gene expression in adult rat lung, indicating that the pulmonary expression of these genes is modulated in vivo. The increase in NOS-III expression does not explain the declines in pulmonary endothelial NO production previously observed following prolonged hypoxia in this model. Alternatively, the fall in NO production may be related to diminished NOS co-factor availability.

Amino Acid Oxidoreductases↗

PubMed1994

Depletion of intracellular Ca2+ stores activates nitric-oxide synthase to generate cGMP and regulate Ca2+ influx.

The mechanism of activation of the agonist-stimulated Ca2+ entry pathway in the plasma membrane is not known. To determine the role of nitric-oxide synthase (NOS) and cGMP in the regulation of this pathway, we used intact and streptolysin O (SLO)-permeable pancreatic acini and measured the relationship between Ca2+ release from internal stores, the NO metabolic pathway, generation of cGMP, and activation of Ca2+ entry. We found that agonist- or thapsigargin (Tg)-activated Ca2+ entry is inhibited by L-NA, a specific inhibitor of NOS, and by LY83583, an inhibitor of guanylyl cyclase. Inhibition of Ca2+ entry by inhibition of NOS was reversed by the NO releasing molecules NO2- and sodium nitroprusside (SNP) and by Bt2cGMP. Inhibition of Ca2+ entry by inhibition of guanylyl cyclase was reversed by Bt2cGMP, but not by the NO releasing agents. The use of L-NA-treated cells and different concentrations of SNP revealed that cGMP has a dual effect on Ca2+ entry. Increasing cGMP up to 10-fold above control activated Ca2+ entry. Further increase in cGMP up to 80-fold above control inhibited Ca2+ entry in a concentration-dependent manner. Measurement of cellular cGMP in intact cells showed that carbachol, Tg, and NO2- increased cGMP to similar levels. The effects of carbachol and Tg were inhibited by L-NA and LY83586, whereas the effect of NO2- was inhibited only by LY83583. SLO-permeabilized cells were shown to be agonist-competent in that the agonist induced Ca2+ release from the inositol 1,4,5-trisphosphate (IP3) pool and activated a NO-dependent generation of cGMP. These cells were used to study the regulation of NOS by Ca2+ and by Ca2+ content of the internal stores. When internal stores were maintained loaded with Ca2+, increasing medium [Ca2+] up to 2.5 microM only modestly increased NOS activity. In contrast, the depletion of Ca2+ from internal stores markedly increased NOS activity independent of medium [Ca2+]. Thus, NOS senses both cytosolic [Ca2+]i and internal store Ca2+ load. We propose that activation of Ca2+ entry involves an agonist-mediated Ca2+ release from internal stores which activates a cellular pool of NOS to generate cGMP, which then modulates Ca2+ entry pathway in the plasma membrane. This mechanism can explain the capacitative nature of Ca2+ entry. The biphasic effect of cGMP provides the cells with a negative feedback mechanism which inhibits Ca2+ entry during periods of high cell [Ca2+]i. This could allow oscillatory behavior of Ca2+ entry.

Amino Acid Oxidoreductases↗

PubMed1994

Membrane localization of H+ and HCO3- transporters in the rat pancreatic duct.

The pancreatic duct secretes alkaline fluid that is rich in HCO3- and poor in Cl-. The molecular mechanisms that mediate ductal secretion and are responsible for the axial gradients of Cl- and HCO3- along the ductal tree are not well understood because H+ and HCO3- transport by duct cells have not been characterized or localized. To address these questions, we microdissected the intralobular, main, and common segments of the rat pancreatic duct. H+ and HCO3- transporters were characterized and localized by following intracellular pH while perfusing the bath and the lumen of the ducts. In intralobular ducts, Na(+)-dependent and amiloride-sensitive recovery from acid load in the absence of HCO3- was used to localize a Na+/H+ exchanger to the basolateral membrane (BLM). Modification of Cl- gradients across the luminal (LM) and BLM in the presence of HCO3- showed the presence of Cl-/HCO3- exchangers on both membranes of intralobular duct cells. Measurement of the effect of Cl- on one side of the membrane on the rate and extent of pHi changes caused by removal and addition of Cl- to the opposite side suggested that both exchangers are present in the same cell. In the presence of HCO3-, intralobular duct cells used three separate mechanisms to extrude H+: (a) BLM-located Na+/H+ exchange, (b) Na(+)-independent vacuolar-type H+ pump, and (c) BLM-located, Na(+)-dependent, amiloride-insensitive, and 4',4'-diisothiocyanatostilbene-2,2'-disulfonic acid sensitive mechanism, possibly a Na(+)-dependent HCO3- transporter. The main and common segments of the duct displayed similar mechanisms and localization of H+ and HCO3- transporters to the extent studied in the present work. In addition to the transporters found in intralobular ducts, the main and common ducts showed Na+/H+ exchange activity in the LM. Three tests were used to exclude a significant luminal to basolateral Na+ leak as the cause for an apparent luminal Na+/H+ exchange in an HCO3- secreting cells: (a) addition of amiloride and removal of Na+ from the LM had a profound effect on Na+/H+ exchange activity on the BLM and vice versa; (b) inhibition of all transporters in the BLM by bathing the duct in the inert hydrocarbon Fluorinert FC-75 did not prevent cytosolic acidification caused by removal of luminal Na+; and (c) luminal Na+ did not activate the basolateral Na(+)-dependent HCO3- transporter. An Na(+)-independent, bafilomycin-sensitive H+ pumping activity was marginal in the absence of HCO3-.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Transport and interaction of nitrogen oxides and NO2 with CO2-HCO3- transporters in pancreatic acini.

Recently, we showed that NO2- increases gap junction (GJ) permeability and synchronizes intracellular Ca2+ concentration oscillations in pancreatic acini (Loessburg et al., J. Biol. Chem. 268: 19769-19775, 1993). NO2- is also an end product of nitric oxide (NO) production and metabolism. Because of the effect of NO2- on GJ permeability and the possible importance of NO2- in NO metabolism and cytotoxicity, we used pancreatic acinar cells and intracellular pH (pHi) measurements to study the interaction of nitrogen oxides and NO2- with cellular proteins. Exposing cells to NO2- resulted in a concentration-dependent cytosolic acidification. The acidification did not require the transport of NO2- and was not mediated by diffusion of HNO2. Because the acidification was prevented by CO2-HCO3- and inhibition of carbonic anhydrase, it is possible that other nitrogen oxides present in a solution containing NO2- enter the cells by diffusion and interact with OH- or H2O to stably acidify the cytosol. NO2- itself is shown to be transported by the HCO3- transporters present in the plasma membrane. Thus manipulation of the cellular Cl- gradient and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) were used to show Cl-/NO2- exchange, whereas stimulation of external Na(+)-dependent amiloride-insensitive and DIDS-sensitive pHi increase in acidified cells was used to demonstrate a Na(+)-(NO2-)n cotransport. Hence NO2- can be a convenient substitute for HCO3- when studying HCO3- transport in an open system. The studies also show that cellular levels of nitrogen oxides and NO2- can be modulated by the cellular HCO3(-)-buffering system.(ABSTRACT TRUNCATED AT 250 WORDS)

Acids↗

PubMed1994

Nitric oxide synthase type I and type III gene expression are developmentally regulated in rat lung.

The successful transition from fetal to neonatal life involves a marked decline in pulmonary vascular resistance which is modulated in part by endothelium-derived nitric oxide. To define the molecular processes which prepare the pulmonary circulation for nitric oxide mediation of vasodilatation at the time of birth, we determined the ontogeny of endothelial nitric oxide synthase (NOS-III) gene expression in lungs from fetal and newborn rats. Maturational changes in lung neuronal NOS (NOS-I) expression were also investigated; the latter isoform has been localized to rat bronchiolar epithelium. NOS proteins were examined by immunoblot analysis, and mRNA abundance was assessed in reverse transcription-polymerase chain reaction assays. Both NOS-III and NOS-I protein were detectable in 16-day fetal lung, they increased 3.8- and 3.1-fold, respectively, to maximal levels at 20 days of gestation (term = 22 day), and they fell postnatally (1-5 days). In parallel with the findings for NOS-III protein, NOS-III mRNA increased from 16 to 20 days gestation and fell after birth. In contrast, NOS-I mRNA abundance declined during late fetal life and rose postnatally. These findings were confirmed by Northern analyses. Thus NOS-III and NOS-I gene expression are developmentally regulated in rat lung, with maximal NOS-III and NOS-I protein present near term. The regulation of pulmonary NOS-III may primarily involve alterations in transcription or mRNA stability, whereas NOS-I expression in the maturing lung may also be mediated by additional posttranscriptional processes.

Aging↗

PubMed1994

Localization and regulation of endothelial NO synthase mRNA expression in rat kidney.

Nitric oxide (NO) has effects on renal blood flow, glomerular filtration rate, renin secretion, and renal sodium excretion. Four isoforms of nitric oxide synthase (NOS) have been cloned to date. However, the molecular identity of NOS present in the renal vasculature is unknown. Endothelial NOS (NOS-III) is regulated both acutely by cell calcium and chronically by shear stress. To determine if renal blood vessels and the glomerulus express NOS-III mRNA, we used degenerate polymerase chain reaction (PCR) to clone a portion of rat NOS-III. We then assayed NOS-III mRNA in microdissected renal structures by reverse transcriptase-PCR. NOS-III mRNA was expressed at high levels in glomeruli, arcuate vessels, and interlobular artery/afferent arterioles. NOS-III mRNA was detected inconsistently in proximal tubules, thick ascending limbs, and cortical and inner medullary collecting ducts. Previous studies have shown that chronic oral treatment with the NOS inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) decreases NO synthesis and causes hypertension. To determine if the systemic blockade occurs only by competitive inhibition, we determined the effect of L-NAME on glomerular NOS-III mRNA. L-NAME administration (5 days) decreased NOS-III mRNA in the glomerulus to 25 +/- 12% of control levels. We conclude that endothelial NOS-III mRNA is preferentially expressed in the glomerulus and renal vasculature, where it can modulate renal blood flow and glomerular filtration rate. Furthermore, glomerular NOS-III may be modulated at the level of mRNA abundance in vivo by systemic L-NAME.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗

PubMed1994

Endothelial nitric oxide synthase is expressed in cultured human bronchiolar epithelium.

Nitric oxide (NO) is an important mediator of physiologic and inflammatory processes in the lung. To better understand the role of NO in the airway, we examined constitutive NO synthase (NOS) gene expression and function in NCI-H441 human bronchiolar epithelial cells, which are believed to be of Clara cell lineage. NOS activity was detected by [3H]arginine to [3H]citrulline conversion (1,070 +/- 260 fmol/mg protein per minute); enzyme activity was inhibited 91% by EGTA, consistent with the expression of a calcium-dependent NOS isoform. Immunoblot analyses with antisera directed against neuronal, inducible, or endothelial NOS revealed expression solely of endothelial NOS protein. Immunocytochemistry for endothelial NOS revealed staining predominantly in the cell periphery, consistent with the association of this isoform with the cellular membrane. To definitively identify the NOS isoform expressed in H441 cells, NOS cDNA was obtained by degenerate PCR. Sequencing of the H441 NOS cDNA revealed 100% identity with human endothelial NOS at the amino acid level. Furthermore, the H441 NOS cDNA hybridized to a single 4.7-kb mRNA species in poly(A)+ RNA isolated from H441 cells, from rat, sheep, and pig lung, and from ovine endothelial cells, coinciding with the predicted size of 4.7 kb for endothelial NOS mRNA. Guanylyl cyclase activity in H441 cells, assessed by measuring cGMP accumulation, rose 6.6- and 5.4-fold with calcium-mediated activation of NOS by thapsigargin and A23187, respectively. These findings indicate that endothelial NOS is expressed in select bronchiolar epithelial cells, where it may have autocrine effects through activation of guanylyl cyclase. Based on these observations and the previous identification of endothelial NOS in a kidney epithelial cell line, it is postulated that endothelial NOS may be expressed in unique subsets of epithelial cells in a variety of organs, serving to modulate ion flux and/or secretory function.

Amino Acid Oxidoreductases↗