Dipeptidyl peptidase IV inhibition downregulates Na(+)-H(+) exchanger NHE3 in rat renal proximal tubule

In the microvillar microdomain of the kidney brush border, sodium hydrogen exchanger type 3 (NHE3) exists in physical complexes with the serine protease dipeptidyl peptidase IV (DPPIV). The purpose of this study was to explore the functional relationship between NHE3 and DPPIV in the intact proximal tubule in vivo. To this end, male Wistar rats were treated with an injection of the reversible DPPIV inhibitor Lys [Z(NO(2))]-pyrrolidide (I40; 60 mg center dot kg(-1)center dot day(-1) ip) for 7 days. Rats injected with equal amounts of the noninhibitory compound Lys[ Z(NO(2))]-OH served as controls. Na(+) -H(+) exchange activity in isolated microvillar membrane vesicles was 45 +/- 5% decreased in rats treated with I40. Membrane fractionation studies using isopycnic centrifugation revealed that I40 provoked redistribution of NHE3 along with a small fraction of DPPIV from the apical enriched microvillar membranes to the intermicrovillar microdomain of the brush border. I40 significantly increased urine output ( 67 +/- 9%; P < 0.01), fractional sodium excretion ( 63 +/- 7%; P < 0.01), as well as lithium clearance ( 81 +/- 9%; P < 0.01), an index of end-proximal tubule delivery. Although not significant, a tendency toward decreased blood pressure and plasma pH/HCO(3)(-) was noted in I40-treated rats. These findings indicate that inhibition of DPPIV catalytic activity is associated with inhibition of NHE3-mediated NaHCO(3) reabsorption in rat renal proximal tubule. Inhibition of apical Na(+) -H(+) exchange is due to reduced abundance of NHE3 protein in the microvillar microdomain of the kidney brush border. Moreover, this study demonstrates a physiologically significant interaction between NHE3 and DPPIV in the intact proximal tubule in vivo.

Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells

Carraro-Lacroix LR, Malnic G, Girardi AC. Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells. Am J Physiol Renal Physiol 297: F1647-F1655, 2009. First published September 23, 2009; doi:10.1152/ajprenal.00082.2009.-The gut incretin hormone glucagon-like peptide 1 (GLP-1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP-1 has been shown to have natriuretic actions paralleled by a diminished proton secretion. We therefore studied the role of the GLP-1 receptor agonist exendin-4 in modulating the activity of Na(+)/H(+) exchanger NHE3 in LLC-PK(1) cells. We found that NHE3-mediated Na(+)-dependent intracellular pH (pH(i)) recovery decreased similar to 50% after 30-min treatment with 1 nM exendin-4. Pharmacological inhibitors and cAMP analogs that selectively activate protein kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC) demonstrated that regulation of NHE3 activity by exendin-4 requires activation of both cAMP downstream effectors. This conclusion was based on the following observations: 1) the PKA antagonist H-89 completely prevented the effect of the PKA activator but only partially blocked the exendin-4-induced NHE3 inhibition; 2) the MEK1/2 inhibitor U-0126 abolished the effect of the EPAC activator but only diminished the exendin-4-induced NHE3 inhibition; 3) combination of H-89 and U-0126 fully prevented the effect of exendin-4 on NHE3; 4) no additive effect in the inhibition of NHE3 activity was observed when exendin-4, PKA, and EPAC activators were used together. Mechanistically, the inhibitory effect of exendin-4 on pHi recovery was associated with an increase of NHE3 phosphorylation. Conversely, this inhibition took place without changes in the surface expression of the transporter. We conclude that GLP-1 receptor agonists modulate sodium homeostasis in the kidney, most likely by affecting NHE3 activity.

Mechanisms mediating the diuretic and natriuretic actions of the incretin hormone glucagon-like peptide-1

Crajoinas RO, Oricchio FT, Pessoa TD, Pacheco BP, Lessa LM, Malnic G, Girardi AC. Mechanisms mediating the diuretic and natriuretic actions of the incretin hormone glucagon-like peptide-1. Am J Physiol Renal Physiol 301: F355-F363, 2011. First published May 18, 2011; doi: 10.1152/ajprenal.00729.2010.-Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone considered a promising therapeutic agent for type 2 diabetes because it stimulates beta cell proliferation and insulin secretion in a glucose-dependent manner. Cumulative evidence supports a role for GLP-1 in modulating renal function; however, the mechanisms by which GLP-1 induces diuresis and natriuresis have not been completely established. This study aimed to define the cellular and molecular mechanisms mediating the renal effects of GLP-1. GLP-1 (1 mu g.kg(-1).min(-1)) was intravenously administered in rats for the period of 60 min. GLP-1-infused rats displayed increased urine flow, fractional excretion of sodium, potassium, and bicarbonate compared with those rats that received vehicle (1% BSA/saline). GLP-1-induced diuresis and natriuresis were also accompanied by increases in renal plasma flow and glomerular filtration rate. Real-time RT-PCR in microdissected rat nephron segments revealed that GLP-1 receptor-mRNA expression was restricted to glomerulus and proximal convoluted tubule. In rat renal proximal tubule, GLP-1 significantly reduced Na(+)/H(+) exchanger isoform 3 (NHE3)-mediated bicarbonate reabsorption via a protein kinase A (PKA)-dependent mechanism. Reduced proximal tubular bicarbonate flux rate was associated with a significant increase of NHE3 phosphorylation at the PKA consensus sites in microvillus membrane vesicles. Taken together, these data suggest that GLP-1 has diuretic and natriuretic effects that are mediated by changes in renal hemodynamics and by downregulation of NHE3 activity in the renal proximal tubule. Moreover, our findings support the view that GLP-1-based agents may have a potential therapeutic use not only as antidiabetic drugs but also in hypertension and other disorders of sodium retention.

Dipeptidyl peptidase IV inhibition attenuates blood pressure rising in young spontaneously hypertensive rats

Objectives The present study aimed to assess the effect of the specific dipeptidyl peptidase IV (DPPIV) inhibitor sitagliptin on blood pressure and renal function in young prehypertensive (5-week-old) and adult spontaneously hypertensive rats (SHRs; 14-week-old). Methods Sitagliptin (40 mg/kg twice daily) was given by oral gavage to young (Y-SHR + IDPPIV) and adult (A-SHR R IDPPIV) SHRs for 8 days. Kidney function was assessed daily and compared with age-matched vehicle-treated SHR (Y-SHR and A-SHR) and with normotensive Wistar-Kyoto rats (Y-WKY and A-WKY). Arterial blood pressure was measured in these animals at the end of the experimental protocol. Additionally, Na(+)/H(+) exchanger isoform 3 (NHE3) function and expression in microvilli membrane vesicles were assessed in young animals. Results Mean arterial blood pressure of Y-SHR + IDPPIV was significantly lower than that of Y-SHR (104 +/- 3 vs. 123 +/- 5 mmHg, P < 0.01) and was similar to Y-WKY (94 +/- 4 mmHg, P > 0.05). Compared to Y-SHR, Y-SHR + IDPPIV exhibited enhanced cumulative urinary flow and sodium excretion and decreased NHE3 activity and expression in proximal tubule microvilli. In the A-SHR, sitagliptin treatment had no significant effect on either renal function or arterial blood pressure. Conclusion Our data suggest that DPPIV inhibition attenuates blood pressure rising in young prehypertensive SHRs, partially by inhibiting NHE3 activity in renal proximal tubule. J Hypertens 29:520-528 (C) 2011 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Essential regulatory elements for NHE3 gene transcription in renal proximal tubule cells

The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5’-flanking sequence (5’FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5’FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5’FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5’FS are used as promoter, efficient transcription only occurs with 44 bp of 5’FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5’FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.

Posttranslational mechanisms associated with reduced NHE3 activity in adult vs. young prehypertensive SHR

Crajoinas RO, Lessa LMA, Carraro-Lacroix LR, Davel APC, Pacheco BPM, Rossoni LV, Malnic G, Girardi ACC. Posttranslational mechanisms associated with reduced NHE3 activity in adult vs. young prehypertensive SHR. Am J Physiol Renal Physiol 299:F872-F881, 2010. First published July 14, 2010; doi:10.1152/ajprenal.00654.2009.-Abnormalities in renal proximal tubular (PT) sodium transport play an important role in the pathophysiology of essential hypertension. The Na(+)/H(+) exchanger isoform 3 (NHE3) represents the major route for sodium entry across the apical membrane of renal PT cells. We therefore aimed to assess in vivo NHE3 transport activity and to define the molecular mechanisms underlying NHE3 regulation before and after development of hypertension in the spontaneously hypertensive rat (SHR). NHE3 function was measured as the rate of bicarbonate reabsorption by means of in vivo stationary microperfusion in PT from young prehypertensive SHR (Y-SHR; 5-wk-old), adult SHR (A-SHR; 14-wk-old), and age-matched Wistar Kyoto (WKY) rats. We found that NHE3-mediated PT bicarbonate reabsorption was reduced with age in the SHR (1.08 +/- 0.10 vs. 0.41 +/- 0.04 nmol/cm(2)xs), while it was increased in the transition from youth to adulthood in the WKY rat (0.59 +/- 0.05 vs. 1.26 +/- 0.11 nmol/cm(2)xs). Higher NHE3 activity in the Y-SHR compared with A-SHR was associated with a predominant microvilli confinement and a lower ratio of phosphorylated NHE3 at serine-552 to total NHE3 (P-NHE3/total). After development of hypertension, P-NHE3/total increased and NHE3 was retracted out of the microvillar microdomain along with the regulator dipeptidyl peptidase IV (DPPIV). Collectively, our data suggest that the PT is playing a role in adapting to the hypertension in the SHR. The molecular mechanisms of this adaptation possibly include an increase of P-NHE3/total and a redistribution of the NHE3-DPPIV complex from the body to the base of the PT microvilli, both predicted to decrease sodium reabsorption.

Cross-Talk Between Mitochondria and NADPH Oxidase: Effects of Mild Mitochondrial Dysfunction on Angiotensin II-Mediated Increase in Nox Isoform Expression and Activity in Vascular Smooth Muscle Cells

Mitochondria and NADPH oxidase activation are concomitantly involved in pathogenesis of many vascular diseases. However, possible cross-talk between those ROS-generating systems is unclear. We induced mild mitochondrial dysfunction due to mitochondrial DNA damage after 24 h incubation of rabbit aortic smooth muscle (VSMC) with 250 ng/mL ethidium bromide (EtBr). VSMC remained viable and had 29% less oxygen consumption, 16% greater baseline hydrogen peroxide, and unchanged glutathione levels. Serum-stimulated proliferation was unaltered at 24 h. Although PCR amplification of several mtDNA sequences was preserved, D-Loop mtDNA region showed distinct amplification of shorter products after EtBr. Such evidence for DNA damage was further enhanced after angiotensin-II (AngII) incubation. Remarkably, the normally observed increase in VSMC membrane fraction NADPH oxidase activity after AngII was completely abrogated after EtBr, together with failure to upregulate Nox1 mRNA expression. Conversely, basal Nox4 mRNA expression increased 1.6-fold, while being unresponsive to AngII. Similar loss in AngII redox response occurred after 24 h antimycin-A incubation. Enhanced Nox4 expression was unassociated with endoplasmic reticulum stress markers. Protein disulfide isomerase, an NADPH oxidase regulator, exhibited increased expression and inverted pattern of migration to membrane fraction after EtBr. These results unravel functionally relevant cross-talk between mitochondria and NADPH oxidase, which markedly affects redox responses to AngII. Antioxid Redox Signal 11, 1265-1278.

NHE1, NHE2, and NHE4 contribute to regulation of cell pH in T84 colon cancer cells

The isoforms of the Na+/H+ exchanger present in T84 human colon cells were identified by functional and molecular methods. Cell pH was measured by fluorescence microscopy using the probe BCECF. Based on the pH recovery after an ammonium pulse and determination of buffering capacity of these cells, the rate of H+ extrusion (J(H)) was 3.68 mM/min. After the use of the amiloride derivative HOE-694 at 25 mu M, which inhibits the isoforms NHE1 and NHE2, there remained 43% of the above transport rate, the nature of which was investigated. Evidence of the presence of NHE1, NHE2, and NHE4 was obtained by reverse transcriptase polymerase chain reaction (RT-PCR) (mRNA) and Western blot. There was no decrease of J(H) by the NHE3 inhibitor S3226 (1 mu M) and no evidence of this isoform by RT-PCR was found. The following functional evidence for the presence of NHE4 was obtained: 25 mu M EIPA abolished J(H) entirely, but NHE4 was not inhibited at 10 mu M; substitution of Na by K increased the remainder, a property of NHE4; hypertonicity also increased this fraction of J(H). Cl--dependent NHE was not detected: in 0 Cl- solutions J(H) was increased and not reduced. In 0 Cl- cell volume decreased significantly, which was abolished by the Cl- channel blocker NPPB, indicating that the 0 Cl- effect was because of reduction of cell volume. In conclusion, T84 human colon cells contain three isoforms of the Na+/H+ exchanger, NHE1, NHE2, and NHE4, but not the Cl-dependent NHE.

Regulation of Na(+)/H(+) Exchanger Isoform 1 (NHE1) by Calmodulin-binding Sites: Role of Angiotensin II

We examined the effect of Angiotensin II (Ang II) on the interaction between the Ca(2+)/CaM complex and hNHE1. Considering that calmodulin binds to NHE1 at two sites (A and B), amino acids at both sites were modified and two mutants were constructed: SA(1K3R/4E) and SB(1K3R/4E). Wild type and mutants were transfected into PS120 cells and their activity was examined by H(+) flux (J(H+)). The basal J(H+) of wild type was 4.71 +/- 0.57 (mM/min), and it was similar in both mutants. However, the mutations partially impaired the binding of CaM to hNHE1. Ang II (10(-12) and 10(-9) M) increased the J(H+) in wild type and SB. Ang II (10(-6) M) increased this parameter only in SA. Ang II (10(-9) M) maintained the expression of calmodulin in wild type or mutants, and Ang II (10(-6) M) decreased it in wild type or SA, but not in SB. Dimethyl-Bapta-AM (10(-7) M), a calcium chelator, suppressed the effect of Ang II (10(-9) M) in wild type. With Ang II (10(-6) M), Bapta failed to affect wild type or SA, but it increased the J(H+) in SB. W13 or calmidazolium chloride (10(-5) M), two distinct calmodulin inhibitors, decreased the effect of Ang II (10(-9) M) in wild type or SB. With Ang II (10(-6) M), W13 or calmidazolium chloride decreased the J(H+) in wild type or SA and increased it in SB. Thus, with Ang II (10(-12) and 10(-9) M), site A seems to be responsible for the stimulation of hNHE1 and with Ang II (10(-6) M), site B is important to maintain its basal activity. Copyright (C) 2010 S. Karger AG, Basel

Mechanisms underlying the long-term regulation of NHE3 by parathyroid hormone

The activity of the Na(+)/H(+) exchanger NHE3 is regulated by a number of factors including parathyroid hormone (PTH). In the current study, we used a renal epithelial cell line, the opossum kidney (OKP) cell, to elucidate the mechanisms underlying the long-term effects of PTH on NHE3 transport activity and expression. We observed that NHE3 activity was reduced 6 h after addition of PTH, and this reduction persisted almost unaltered after 24 h. The decrease in activity was associated with diminished NHE3 cell surface expression at 6, 16, and 24 h after PTH addition, total cellular NHE3 protein at 16 and 24 h, and NHE3 mRNA abundance at 24 h. The lower levels of NHE3 mRNA were associated to a small, but significant, decrease in mRNA stability. Additionally, by analyzing the rat NHE3 gene promoter activity in OKP cells, we verified that the regulatory region spanning the segment -152 to +55 was mildly reduced under the influence of PTH. This effect was completely abolished by the presence of the PKA inhibitor KT 5720. In conclusion, long-term exposure to PTH results in reduction of NHE3 mRNA levels due to a PKA-dependent inhibitory effect on the NHE3 promoter and a small reduction of mRNA half-life, and decrease in the total amount of protein which is preceded by endocytosis of the apical surface NHE3. The decreased NHE3 expression is likely to be responsible for the reduction of sodium, bicarbonate, and fluid reabsorption in the proximal tubule consistently perceived in experimental models of PTH disorders.

Transcriptional regulation of the Na(+)/H(+) exchanger NHE3 by chronic exposure to angiotensin II in renal epithelial cells

Angiotensin II (Ang II) exerts an acute bimodal effect on proximal tubule NHE3: while low doses stimulate the exchanger, high doses inhibit it. In the present study, we have investigated the chronic effects of Ang II on NHE3 expression and transcriptional regulation. Treatment of a tubular epithelial cell line, OKP, with Ang II 10(-11) M significantly increased NHE protein expression and mRNA levels, without evidence of bimodal effect. No change in mRNA half-life was detected, but transient transfection studies showed a significant increase in NHE3 promoter activity. Binding sites for Sp1/Egr-1 and AP2 transcription factors of the NHE3 proximal promoter were mutated and we observed that the Sp1/Egr-1 binding site integrity is necessary for Ang II stimulatory effects. Inhibition of cytochrome P450, PI3K, PKA and MAPK pathways prevented the Ang II stimulatory effect on the NHE3 promoter activity. Taking all the results together, our data reveal that chronic Ang II treatment exerts a stimulatory effect on NHE3 expression and promoter activity. The Ang II up-regulation of the NHE3 promoter activity appears to involve the Sp1/Egr-1 binding site and the interplay of several intracellular signaling pathways. (C) 2011 Elsevier Inc. All rights reserved.

Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 µg in 50 µL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48% by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed’s lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469%). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20%), reaching the greatest increase (60%) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

Psychophysical measurements of luminance and chromatic spatial and temporal contrast sensitivity in Duchenne muscular dystrophy

In children with Duchenne muscular dystrophy, color vision losses have been related to dystrophin deletions downstream of exon 30, which affect a dystrophin isoform, Dp260, present in the retina. To further evaluate visual function in DMD children, we measured spatial, temporal, and chromatic red-green and blue-yellow contrast sensitivity in two groups of DMD children with gene deletion downstream and upstream of exon 30. Psychophysical spatial contrast sensitivity was measured for low, middle, and high spatial frequencies with achromatic gratings and for low and middle frequencies with red-green and blue-yellow chromatic gratings. Temporal contrast sensitivity was also measured with achromatic stimuli. A reduction in sensitivity at all spatial luminance contrasts was found for the DMD patients with deletion downstream of exon 30. Similar results were found for temporal luminance contrast sensitivity. Red-green chromatic contrast sensitivity was reduced in DMD children with deletion downstream of exon 30, whereas blue-yellow chromatic contrast sensitivity showed no significant differences. We conclude that visual function is impaired in DMD children. Furthermore, we report a genotype-phenotype relationship because the visual impairment occurred in children with deletion downstream but not upstream of exon 30, affecting the retinal isoform of dystrophin Dp260.

Structural basis of GC-1 selectivity for thyroid hormone receptor isoforms

Background: Thyroid receptors, TRa and TR beta, are involved in important physiological functions such as metabolism, cholesterol level and heart activities. Whereas metabolism increase and cholesterol level lowering could be achieved by TR beta isoform activation, TRa activation affects heart rates. Therefore, beta-selective thyromimetics have been developed as promising drug-candidates for treatment of obesity and elevated cholesterol level. GC-1 [ 3,5-dimethyl-4-(4'-hydroxy- 3'-isopropylbenzyl)-phenoxy acetic acid] has ability to lower LDL cholesterol with 600-to 1400-fold more potency and approximately two-to threefold more efficacy than atorvastatin (Lipitor(C)) in studies in rats, mice and monkeys. Results: To investigate GC-1 specificity, we solved crystal structures and performed molecular dynamics simulations of both isoforms complexed with GC-1. Crystal structures reveal that, in TRa Arg228 is observed in multiple conformations, an effect triggered by the differences in the interactions between GC-1 and Ser277 or the corresponding asparagine (Asn331) of TR beta. The corresponding Arg282 of TR beta is observed in only one single stable conformation, interacting effectively with the ligand. Molecular dynamics support this model: our simulations show that the multiple conformations can be observed for the Arg228 in TR alpha, in which the ligand interacts either strongly with the ligand or with the Ser277 residue. In contrast, a single stable Arg282 conformation is observed for TR beta, in which it strongly interacts with both GC-1 and the Asn331. Conclusion: Our analysis suggests that the key factors for GC-1 selectivity are the presence of an oxyacetic acid ester oxygen and the absence of the amino group relative to T(3). These results shed light into the beta-selectivity of GC-1 and may assist the development of new compounds with potential as drug candidates to the treatment of hypercholesterolemia and obesity.

Proteasome Inhibition Represses Unfolded Protein Response and Nox4, Sensitizing Vascular Cells to Endoplasmic Reticulum Stress-Induced Death

Background: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. Methodology/Principal Findings: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. Conclusion/Relevance: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.