Endothelial prostaglandin D2 opposes angiotensin II contractions in mouse isolated perfused intracerebral microarterioles.

HYPOTHESIS: A lack of contraction of cerebral microarterioles to Ang II ("resilience") depends on cyclooxygenase (COX) and lipocalin type prostaglandin D sythase L-PGDS producing PGD2 that activates prostaglandin D type 1 receptors (DP1Rs) and nitric oxide synthase (NOS). MATERIALS & METHODS: Contractions were assessed in isolated, perfused vessels and NO by fluorescence microscopy. RESULTS: The mRNAs of penetrating intraparenchymal cerebral microarterioles versus renal afferent arterioles were >3000-fold greater for L-PGDS and DP1R and 5-fold for NOS III and COX 2. Larger cerebral arteries contracted with Ang II. However, cerebral microarterioles were entirely unresponsive but contracted with endothelin 1 and perfusion pressure. Ang II contractions were evoked in cerebral microarterioles from COX1 -/- mice or after blockade of COX2, L-PGDS or NOS and in deendothelialized vessels but effects of deendothelialization were lost during COX blockade. NO generation with Ang II depended on COX and also was increased by DP1R activation. CONCLUSION: The resilience of cerebral arterioles to Ang II contractions is specific for intraparenchymal microarterioles and depends on endothelial COX1 and two products that are metabolized by L-PGDS to generate PGD2 that signals via DP1Rs and NO.

Adenosine, type 1 receptors: role in proximal tubule Na+ reabsorption.

Adenosine type 1 receptor (A1 -AR) antagonists induce diuresis and natriuresis in experimental animals and humans. Much of this effect is due to inhibition of A1 -ARs in the proximal tubule, which is responsible for 60-70% of the reabsorption of filtered Na(+) and fluid. Intratubular application of receptor antagonists indicates that A1 -AR mediates a portion of Na(+) uptake in PT and PT cells, via multiple transport systems, including Na(+) /H(+) exchanger-3 (NHE3), Na(+) /PO4(-) co-transporter and Na(+) -dependent glucose transporter, SGLT. Renal microperfusion and recollection studies have shown that fluid reabsorption is reduced by A1 -AR antagonists and is lower in A1 -AR KO mice, compared to WT mice. Absolute proximal reabsorption (APR) measured by free-flow micropuncture is equivocal, with studies that show either lower APR or similar APR in A1 -AR KO mice, compared to WT mice. Inhibition of A1 -ARs lowers elevated blood pressure in models of salt-sensitive hypertension, partially due to their effects in the proximal tubule.

Comparison of inhibitors of superoxide generation in vascular smooth muscle cells.

BACKGROUND AND PURPOSE: We compared the dose-dependent reductions in cellular superoxide anion (O(2)(-)) by catalytic agents: superoxide dismutase (SOD), polyethylene glycol (PEG)-SOD and the nitroxide 4-hydroxy-2,2,6,6,-tetramethylpiperidine-1-oxyl (tempol) with uncharacterized antioxidants: 5,10,15,20-tetrakis (4-sulphonatophenyl) porphyrinate iron (III)(Fe-TTPS), (-)-cis-3,3',4',5,7-pentahydroxyflavane (2R,3R)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol (-epicatechin), 2-phenyl-1,2-benzisoselenazol-3(2H)-one (ebselen) and N-acetyl-L-cysteine (NAC) with the spin trap nitroblue tetrazolium (NBT) and with the vitamins or their analogues: ascorbate, alpha-tocopherol and 6-hydroxy-2,5,7,8-tetramethylkroman-2-carboxy acid (trolox). EXPERIMENTAL APPROACH: O(2)(-) was generated in primary cultures of angiotensin II-stimulated preglomerular vascular smooth muscle cells from spontaneously hypertensive rats and detected by lucigenin-enhanced chemiluminescence. KEY RESULTS: SOD, PEG-SOD, NAC and tempol produced a similar maximum inhibition of O(2)(-) of 80-90%. -Epicatechin, NBT, ebselen and Fe-TTPS were significantly (P < 0.0125) less effective (50-70%), whereas trolox, alpha-tocopherol and ascorbate had little action even over 24 h of incubation (<31%). Effectiveness in disrupted and intact cells was similar for the permeable agents, PEG-SOD and tempol, but was enhanced for SOD. Generation of O(2)(-) was increased by NAC and NBT at low concentrations but reduced at high concentrations. CONCLUSIONS AND IMPLICATIONS: Maximum effectiveness against cellular production of O(2)(-) requires cell membrane permeability and catalytic action as exemplified by PEG-SOD or tempol. NAC and NBT have biphasic effects on O(2)(-) production. Vitamins C and E or analogues have low efficacy.

Polyglutamine protein aggregation and toxicity are linked to the cellular stress response.

Chronic exposure of cells to expanded polyglutamine proteins results in eventual cell demise. We constructed mouse cell lines expressing either the full-length androgen receptor (AR), or truncated forms of AR containing 25 or 65 glutamines to study the cellular consequences of chronic low-level exposure to these proteins. Expression of the polyglutamine-expanded truncated AR protein, but not the full-length expanded protein, resulted in the formation of cytoplasmic and nuclear aggregates and eventual cell death. Nuclear aggregates preferentially stained positive for heat shock protein (hsp)72, a sensitive indicator of a cellular stress response. Biochemical studies revealed that the presence of nuclear aggregates correlated with activation of the c-jun NH2-terminal kinase (JNK). Different metabolic insults, including heat shock treatment, and exposure to sodium arsenite or menadione, proved more toxic to those cells expressing the polyglutamine-expanded truncated protein than to cells expressing the non-expanded form. Cells containing cytoplasmic polyglutamine-protein aggregates exhibited a delayed expression of hsp72 after heat shock. Once expressed, hsp72 failed to localize normally and instead was sequestered within the protein aggregates. This was accompanied by an inability of the aggregate-containing cells to cease their stress response as evidenced by the continued presence of activated JNK. Finally, activation of the cellular stress response increased the overall extent of polyglutamine protein aggregation, especially within the nucleus. Inclusion of a JNK inhibitor reduced this stress-dependent increase in nuclear aggregates. Abnormal stress responses may contribute to enhanced cell vulnerability in cells expressing polyglutamine-expanded proteins and may increase the propensity of such cells to form cytoplasmic and nuclear inclusions.

Stress preconditioning attenuates oxidative injury to the alveolar epithelium of the lung following haemorrhage in rats.

Inhibition of cAMP-dependent stimulation of vectorial fluid transport across the alveolar epithelium following haemorrhagic shock is mediated by reactive nitrogen species released within the airspaces of the lung. We tested here the hypothesis that the prior activation of the cellular heat shock or stress response, via exposure to either heat or geldanamycin, would attenuate the release of airspace nitric oxide (NO) responsible for the shock-mediated failure of the alveolar epithelium to respond to catecholamines in rats. Rats were haemorrhaged to a mean arterial pressure of 30-35 mmHg for 60 min, and then resuscitated with a 4 % albumin solution. Alveolar fluid clearance was measured by change in concentration of a protein solution instilled into the airspaces 5 h after the onset of haemorrhage. Stress preconditioning restored the cAMP-mediated upregulation of alveolar liquid clearance after haemorrhage. The protective effect of stress preconditioning was mediated in part by a decrease in the expression of iNOS in the lung. Specifically, stress preconditioning decreased the production of nitrite by endotoxin-stimulated alveolar macrophages removed from haemorrhaged rats or by A549 and rat alveolar epithelial type II cell monolayers stimulated with cytomix (a mixture of TNF-alpha, IL-1beta and IFN-gamma) for 24 h. In summary, these results provide the first in vivo evidence that stress preconditioning restores a normal fluid transport capacity of the alveolar epithelium in the early phase following haemorrhagic shock by attenuating NO-mediated oxidative stress to the lung epithelium.

Glucocorticoid modulation of androgen receptor nuclear aggregation and cellular toxicity is associated with distinct forms of soluble expanded polyglutamine protein.

Spinobulbar muscular atrophy is a progressive motor neuron disease caused by abnormal polyglutamine tract expansion in the androgen receptor (AR) gene, and is part of a family of central nervous system (CNS) neurodegenerative diseases, including Huntington's disease (HD). Each pathologic protein is widely expressed, but the cause of neuronal degeneration within the CNS remains unknown. Many reports now link abnormal polyglutamine protein aggregation to pathogenesis. A previous study reported that activation of the wild-type glucocorticoid receptor (wtGR) suppressed the aggregation of expanded polyglutamine proteins derived from AR and huntingtin, whereas a mutant receptor containing an internal deletion, GRDelta108-317, increased polyglutamine protein aggregation, in this case primarily within the nucleus. In this study, we use these two forms of GR to study expanded polyglutamine AR protein in different cell contexts. Using cell biology and biochemical approaches, we find that wtGR promotes soluble forms of the protein and prevents nuclear aggregation in NIH3T3 cells and cultured neurons. In contrast, GRDelta108-317 decreases polyglutamine protein solubility, and causes formation of nuclear aggregates in non-neuronal cells. Nuclear aggregates recruit hsp72 more rapidly than cytoplasmic aggregates, and are associated with decreased cell viability. Limited proteolysis and chemical cross-linking suggest unique soluble forms of the expanded AR protein underlie these distinct biological activities. These observations provide an experimental framework to understand why expanded polyglutamine proteins may be toxic only to certain populations of cells, and suggest that unique protein associations or conformations of expanded polyglutamine proteins may determine subsequent cellular effects such as nuclear localization and cellular toxicity.

Geldanamycin inhibits NF-kappaB activation and interleukin-8 gene expression in cultured human respiratory epithelium.

Geldanamycin is a benzoquinone ansamycin with multiple pharmacologic properties. Recent data demonstrated that geldanamycin conferred protection in an animal model of inflammation-associated acute lung injury. In the current study, we investigated the effects of geldanamycin on interleukin (IL)-8 gene expression and nuclear factor (NF)-kappaB activation. Geldanamycin inhibited tumor necrosis factor (TNF)-alpha-mediated IL-8 gene expression in A549 human respiratory epithelial cells as measured by enzyme-linked immunosorbent assay and Northern blot analyses. In cells transiently transfected with an IL-8 promoter-luciferase reporter plasmid, geldanamycin inhibited TNF-alpha-mediated luciferase activity. Geldanamycin inhibited TNF-alpha-mediated NF-kappaB activation as measured by electromobility shift assays and transient transfections with a NF-kappaB-dependent luciferase reporter plasmid. In contrast, geldanamycin did not affect TNF-alpha-mediated degradation of the NF-kappaB inhibitory protein IkappaBalpha and did not block nuclear translocation of the NF-kappaB p65 subunit as measured by Western blot analyses. Geldanamycin added directly to nuclear extracts of TNF-alpha-treated cells reduced the formation of the NF-kappaB/DNA complex. These results demonstrate that geldanamycin inhibits TNF-alpha-mediated IL-8 gene expression in A549 cells by inhibiting activation of the IL-8 promoter. The mechanism of inhibition involves inhibition of NF-kappaB activation, which is independent of IkappaBalpha degradation or p65 nuclear translocation. Geldanamycin appears to directly inhibit the ability of NF-kappaB to bind DNA. The observed in vitro effects could account, in part, for the anti-inflammatory properties of geldanamycin observed in vivo.

Reactive nitrogen species inhibit alveolar epithelial fluid transport after hemorrhagic shock in rats.

Our recent experimental work demonstrated that a neutrophil-dependent inflammatory response in the lung prevented the normal up-regulation of alveolar fluid clearance by catecholamines following hemorrhagic shock. In this study, we tested the hypothesis that the release of NO within the airspaces of the lung was responsible for the shock-mediated failure of the alveolar epithelium to respond to catecholamines in rats. Hemorrhagic shock was associated with an inducible NO synthase (iNOS)-dependent increase in the lung production of NO and a failure of the alveolar epithelium to up-regulate vectorial fluid transport in response to beta-adrenergic agonists. Inhibition of iNOS restored the normal catecholamine-mediated up-regulation of alveolar liquid clearance. Airspace instillation of dibutyryl cAMP, a stable analog of cAMP, restored the normal fluid transport capacity of the alveolar epithelium after prolonged hemorrhagic shock, whereas direct stimulation of adenyl cyclase by forskolin had no effect. Pretreatment with pyrrolidine dithiocarbamate or sulfasalazine attenuated the iNOS-dependent production of NO in the lung and restored the normal up-regulation of alveolar fluid clearance by catecholamines after prolonged hemorrhagic shock. Based on in vitro studies with an alveolar epithelial cell line, A549 cells, the effect of sulfasalazine appeared to be mediated in part by inhibition of NF-kappaB activation, and the protective effect was mediated by the inhibition of IkappaBalpha protein degradation. In summary, these results provide the first in vivo evidence that NO, released within the airspaces of the lung probably secondary to the NF-kappaB-dependent activation of iNOS, is a major proximal inflammatory mediator that limits the rate of alveolar epithelial transport after prolonged hemorrhagic shock by directly impairing the function of membrane proteins involved in the beta-adrenergic receptor-cAMP signaling pathway in alveolar epithelium.

Effects of dietary salt intake on plasma arginine.

Because L-arginine is degraded by hepatic arginase to ornithine and urea and is transported by the regulated 2A cationic amino acid y(+) transporter (CAT2A), hepatic transport may regulate plasma arginine concentration. Groups of rats (n = 6) were fed a diet of either low salt (LS) or high salt (HS) for 7 days to test the hypothesis that dietary salt intake regulates plasma arginine concentration and renal nitric oxide (NO) generation by measuring plasma arginine and ornithine concentrations, renal NO excretion, and expression of hepatic CAT2A, and arginase. LS rats had lower excretion of NO metabolites and cGMP, lower plasma arginine concentration (LS: 83 +/- 7 vs. HS: 165 +/- 10 micromol/l, P < 0.001), but higher plasma ornithine concentration (LS: 82 +/- 6 vs. HS: 66 +/- 4 micromol/l, P < 0.05) and urea excretion. However, neither the in vitro hepatic arginase activity nor the mRNA for hepatic arginase I was different between groups. In contrast, LS rats had twice the abundance of mRNA for hepatic CAT2A (LS: 3.4 +/- 0.4 vs. HS: 1.6 +/- 0.5, P < 0.05). The reduced plasma arginine concentration with increased plasma ornithine concentration and urea excretion during LS indicates increased arginine metabolism by arginase. This cannot be ascribed to changes in hepatic arginase expression but may be a consequence of increased hepatic arginine uptake via CAT2A.

AT1 receptor antagonist combats oxidative stress and restores nitric oxide signaling in the SHR.

The tubuloglomerular feedback (TGF) responses of the spontaneously hypertensive rat (SHR) are under exaggerated regulation by angiotensin II (Ang II) type 1 receptors (AT1-R). Since AT1-Rs enhance oxygen radical (O2-) generation, we tested the hypothesis that the exaggerated TGF was due to a diminished blunting by macula densa (MD)-derived nitric oxide (NO) because of excessive AT1-R-dependent generation of O2-. Groups of SHR and control Wistar-Kyoto (WKY) rats received vehicle (Veh), the AT1-R antagonist candesartan (Cand; 3 mg. kg-1. day-1), or nonspecific therapy with hydralazine + hydrochlorothiazide + reserpine (HHR) for two weeks. Compared with WKY rats, the elevated mean arterial pressure of SHR (WKY 125 +/- 2 vs. SHR 163 to 779 mm Hg, P < 0.001) was reduced (P < 0.001) similarly in SHR by Cand and HHR (121 +/- 5 and 116 +/- 5 mm Hg, P = NS). The SHR had an increased maximal TGF response (change in stop flow pressure during luminal perfusion of fluid: SHR 11.2 +/- 0.5 vs. WKY 8.3 +/- 0.4 mm Hg, P < 0.01) and a reduced TGF response to blockade of neuroneal NO synthase (nNOS) in the MD with luminal 7-nitroindazole (7-NI: DeltaTGF in WKY 2.8 +/- 0.4 vs. SHR 1.1 +/- 0.6 mm Hg, P < 0.05). Although the elevated TGF responses of SHR were normalized by both HHR and Cand, only Cand restored a normal TGF response to luminal perfusion of the MD with 7-NI (DeltaTGF with 7-NI in SHR: Veh + 1.8 +/- 0.4 vs. Cand + 3.4 +/- 0.5 mm Hg, P < 0.05). To abrogate the local effects of O2-, tempol (a membrane-permeable superoxide dismutase mimetic) was perfused into the efferent arteriole. During tempol, SHR given vehicle or HHR had a much increased response to blockade of nNOS with 7-NI (DeltaTGF in SHR with 7-NI during tempol: Veh 6.3 +/- 1.0 and HHR 4.5 +/- 0.8 mm Hg, P < 0.01 vs. no tempol for both), implying that the effects of NO had been prevented because of excessive O2-. In contrast, the TGF response to 7-NI in SHR given Cand was unaffected by tempol (DeltaTGF with 7-NI during tempol: 2.9 +/- 0.9, P = NS, compared with no tempol). In conclusion, TGF responses of SHR are exaggerated because of the effects of hypertension and AT1-R. AT1-R blockade specifically diminishes oxidative stress and restores NO signaling in the juxtaglomerular apparatus of the SHR.

Nephron pO2 and renal oxygen usage in the hypertensive rat kidney.

BACKGROUND: The kidney has a high rate of oxygen usage (QO2) that is closely dependent on tubular Na+ transport (TNa). However, little is known concerning the regulation of the cortical partial pressure of oxygen (pO2). METHODS: First, the pO2 was measured in the outer cortical proximal (PT) and distal tubules (DT), efferent arterioles (EA), and superficial (SC) and deep cortical (DC) tissues in normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHRs) using an ultramicrocoaxial O2 electrode. We next assessed the determinants of QO2 and tubular reabsorption of sodium (TNa) for whether they could account for any differences in renal cortical pO2 in SHRs. RESULTS: The pO2 in the EA was reduced 40 to 50% compared with arterial values but was similar in the two strains (WKY rats 45 +/- 2 vs. SHRs 41 +/- 1 mm Hg, P = NS). The pO2 value in the PT, DT, and SC did not differ within strains. All were significantly (P < 0. 001) lower in SHRs (for example, pO2 in PT of WKY rats 39 +/- 1 vs. SHRs, 30 +/- 1 mm Hg). The pO2 in the renal vein was above that at any site in the EA or the cortex, implying a precapillary shunting of O2 from the artery to vein. SHRs had reduced renal blood flow (RBF) leading to a reduced (P < 0.05) rate of O2 delivery (WKY rats 42 +/- 6 vs. SHRs 30 +/- 1 micromol. min-1. g-1) and a reduced glomerular filtration rate, leading to a lower (P < 0.001), TNa (WKYs 115 +/- 9 vs. SHRs 66 +/-8 micromol. min-1. g-1). However, despite the 43% reduction in TNa, the renal O2 usage was not significantly different between strains (WKY rats 7.6 +/- 0.8 vs. SHRs 9.0 +/- 1.0 micromol. min-1. g-1). Therefore, the SHRs had a sharp reduction (P < 0.001) in the O2 efficiency for Na+ reabsorption (TNa/QO2; WKY rats 15.1 +/- 1.6 vs. SHRs 7.3 +/-1.0 micromol-1). CONCLUSIONS: A precapillary O2 shunt reduces the pO2 of cortical nephrons. The pO2 is reduced further in SHRs because of less efficient O2 usage for Na+ transport.

Co-translational interactions of apoprotein B with the ribosome and translocon during lipoprotein assembly or targeting to the proteasome.

Hepatic lipoprotein assembly and secretion can be regulated by proteasomal degradation of newly synthesized apoB, especially if lipid synthesis or lipid transfer is low. Our previous studies in HepG2 cells showed that, under these conditions, newly synthesized apoB remains stably associated with the endoplasmic reticulum (ER) membrane (Mitchell, D. M., Zhou, M., Pariyarath, R., Wang, H., Aitchison, J. D., Ginsberg, H. N., and Fisher, E. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14733-14738). We now show that independent of lipid synthesis, apoB chains that appear full-length are, in fact, incompletely translated polypeptides still engaged by the ribosome and associated with the ER translocon. In the presence of active lipid synthesis and transfer, translation and lipoprotein assembly are completed, and the complexes exit the ER. Upon omitting fatty acids from, or adding a microsomal triglyceride transfer protein inhibitor to, culture media to reduce lipid synthesis or transfer, respectively, apoB was degraded while it remained associated with the ER and complexed with cytosolic hsp70 and proteasomes. Thus, unlike other ER substrates of the proteasome, such as major histocompatibility complex class I molecules, apoB does not fully retrotranslocate to the cytosol before entering the ubiquitin-proteasome pathway. Although, upon immunofluorescence, apoB in proteasome-inhibited cells accumulated in punctate structures similar in appearance to aggresomes (cytosolic structures containing molecules irreversibly lost from the secretory pathway), these apoB molecules could be secreted when lipid synthesis was stimulated. The results suggest a model in which 1) apoB translation does not complete until lipoprotein assembly terminates, and 2) assembly with lipids or entry into the ubiquitin-proteasome pathway occurs while apoB polypeptides remain associated with the translocon and attached to the ribosome.

Expression of heat shock proteins in a linear rodent wound.

Heat shock proteins (hsps) are ubiquitous and known to be expressed in all organisms. These stress proteins are likely to be induced in the wound environment and may play a critical role in the overall process of wound repair. Linear incisions were made in Sprague-Dawley rats. Serial skin biopsies were taken, the dermis and epidermis were separated and a protein lysate made. The expression of hsp 72, 47, and 32 were analyzed by Western blotting and immunohistochemistry. There were distinct patterns of expression of hsp 72, 47, and 32 in the wound. In unwounded dermis, there was no constitutive expression of any of the heat shock proteins studied. In the epidermis, there was constitutive expression of hsp 32 and 72, but not hsp 47. With wounding, all hsps exhibited increased expression both in the dermis and epidermis. These patterns of protein expression are suggestive of the individual heat shock proteins' molecular function, such as hsp 72's role as an indicator of cellular stress and injury, hsp 47's role in collagen synthesis, and hsp 32's role as an antioxidant.

The pathophysiology of renin release in renovascular hypertension.

Renovascular hypertension (RVH) results from occlusion of blood flow to either kidney, which stimulates renin release. Increased renin leads to a series of actions that rapidly leads to increased systemic blood pressure. Experimental renovascular hypertension is developed in animals by placement of a clip that occludes more than 50% of renal blood flow to that kidney. The major stimulus for renin release in renovascular hypertension is the severe drop in hydrostatic pressure in the afferent arteriole, the location of the juxtaglomerular renin-secreting granular cells. The pressure drop changes the degree of stretch of these cells which leads to baroreceptor-mediated renin release. The level of renin released can be modified by sympathetic nerves and to a lesser degree by the macula densa. Several hormone or vasoactive agents may augment renin released during RVH, but nearly all are secondary to changes in the pressure receptor mechanism.

TP receptor-mediated vasoconstriction in microperfused afferent arterioles: roles of O(2)(-) and NO.

Thromboxane A(2) (TxA(2)) preferentially constricts the renal afferent arteriole. Nitric oxide (NO) modulates vasoconstriction and is rapidly degraded by superoxide radical (O(2)(-)). We investigated the roles of NO and O(2)(-) in rabbit isolated, perfused renal afferent arteriole responses to the TxA(2)/prostaglandin H(2) (TP) receptor agonist U-46,619. U-46,619 (10(-10)-10(-6) M) dose-dependently reduced afferent arteriolar luminal diameter (ED(50) = 7.5 +/- 5.0 nM), which was blocked by the TP receptor antagonist ifetroban (10(-6) M). Tempol (10(-3) M) pretreatment, which prevented paraquat-induced vasoconstriction in afferent arterioles, blocked the vasoconstrictor responses to U-46,619. To test whether U-46,619 stimulates NO and whether tempol prevents U-46, 619-induced vasoconstriction by enhancing the biological activity of NO, we examined the luminal diameter response to U-46,619 in arterioles pretreated with N(w)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) or L-NAME + tempol. During L-NAME, the sensitivity and maximal responses of the afferent arteriole to U-46, 619 were significantly (P < 0.05) enhanced. Moreover, L-NAME restored a vasoconstrictor response to U-46,619 in vessels pretreated with tempol. In conclusion, in isolated perfused renal afferent arterioles TP receptor activation stimulates NO production, which buffers the vasoconstriction, and stimulates O(2)(-) production, which mediates the vasoconstriction, in part, through interaction with NO.

Extracellular matrix regulates the hepatocellular heat shock response.

BACKGROUND: Cirrhosis is characterized by the accumulation of collagen within the extracellular matrix (ECM) of the liver and progressive hepatocellular dysfunction. Since recent studies have shown that the ECM can modulate cellular function, we examined whether the ECM could contribute to hepatocellular dysfunction. To address this question we examined hepatocyte behavior in two different ECM environments. MATERIALS AND METHODS: Primary rat hepatocytes were cultured as a monolayer on collagen or as multicellular aggregates (spheroids) within a laminin-rich ECM. Hepatocytes were then compared for viability, response to proinflammatory cytokines, and their capacity to activate a heat shock response and adopt a thermotolerant phenotype. In addition, we compared the ability of prior heat shock exposure to protect hepatocytes from tumor necrosis factor (TNF) alpha/actinomycin-D-induced apoptosis in the two different ECM environments. RESULTS: Hepatocytes cultured as a monolayer on collagen exhibited decreased viability, underwent spontaneous apoptosis, and displayed an attenuated cytokine-stimulated nitric oxide production compared to hepatocytes cultured as spheroids. In response to heat, hepatocytes in both ECM environments expressed inducible heat shock protein 70 (hsp72). But, only the hepatocyte spheroids exhibited thermotolerance in response to a subsequent thermal challenge. In contrast to previous reports, induction of the heat shock response failed to protect hepatocytes against TNFalpha-induced apoptosis. CONCLUSIONS: These data demonstrate that the ECM can play an integral role in specific hepatocellular behaviors. Furthermore, the progressive deposition of collagen within the ECM, which is characteristic of fibrotic liver diseases, may directly contribute to the progressive hepatocellular dysfunction observed in cirrhosis. Hepatocellular viability, response to proinflammatory cytokines, heat shock response, and thermotolerance were all altered depending on the composition of the ECM. In contrast, TNFalpha-induced apoptosis was independent of the composition of the ECM.

Roles of NO and oxygen radicals in tubuloglomerular feedback in SHR.

The spontaneously hypertensive rat (SHR) has enhanced tubuloglomerular feedback (TGF) responses and diminished buffering by juxtaglomerular apparatus (JGA)-derived nitric oxide (NO) despite enhanced expression of NO synthase (NOS) isoforms in the JGA. We tested the hypothesis that the enhanced TGF response is due to inactivation of NO by oxygen radicals (O(-)(2)). SHR had significantly (P<0.05) greater expression of the peroxynitrate reaction product, nitrotyrosine, in renal cortex. A membrane-permeant, metal-independent superoxide dismutase mimetic, tempol, was used to test the functional role of O(-)(2). Maximum TGF responses, assessed from changes in proximal stop-flow pressure (P(SF)) during orthograde loop of Henle (LH) perfusion of artificial tubular fluid (ATF), were enhanced in SHR [Wistar-Kyoto rat (WKY) 8.8+/-0.4 (n = 30 nephrons) vs. SHR 10.8+/-0.4 mm Hg (n = 39 nephrons), P<0.001]. TGF responses of SHR were unresponsive to microperfusion of 7-nitroindazole (7-NI, 10(-4) M), which is an inhibitor of neuronal NOS (nNOS) [WKY 8.3+/-0.3 to 10.8+/-0.4 (n = 8, P<0.001) vs. SHR 10.0+/-0.7 to 10.5+/-0.8 mm Hg (n = 8; not significant)]. Microperfusion of tempol (10(-4) M) into the efferent arteriole (EA) supplying the peritubular capillaries (PTC) blunted TGF. The response to tempol was significantly (P< 0.05) greater in SHR [DeltaTGF in WKY 19+/-6% (n = 10) vs. SHR 32+/-3% (n = 10)]. Microperfusion of the NO donor compound S-nitroso-N-acetyl-penicillamine (SNAP, 10(-7)-10(-4) M) via the LH blunted TGF, but the sensitivity of the response was impaired significantly (P<0.05) in SHR nephrons. PTC perfusion of tempol (10(-4) M) normalized the response to loop perfusion of both SNAP and 7-NI in SHR nephron to levels in WKY (during tempol, DeltaP(SF) with 7-NI in WKY 8.9+/-0.6 to 11.4+/-0.8; n = 12 vs. SHR 9.5+/-0.5 to 12.5+/-0.4 mm Hg; n = 16). In conclusion, TGF responses are enhanced in SHR, in part due to a diminished role for NO from nNOS in blunting TGF due to enhanced O(-)(2) formation. O(-)(2) in the JGA enhances TGF responses by inactivation of locally generated NO.