Carsten Leander Buus

Small Artery Remodeling Depends on Tissue-Type Transglutaminase

Remodeling of small arteries is essential in the long-term regulation of blood pressure and blood flow to specific organs or tissues. A large part of the change in vessel diameter may occur through non–growth-related reorganization of vessel wall components. The hypothesis was tested that tissue-type transglutaminase (tTG), a cross-linking enzyme, contributes to the inward remodeling of small arteries. The in vivo inward remodeling of rat mesenteric arteries, induced by low blood flow, was attenuated by inhibition of tTG. Rat skeletal muscle arteries expressed tTG, as identified by Western blot and immunostaining. In vitro, activation of these arteries with endothelin-1 resulted in inward remodeling, which was blocked by tTG inhibitors. Small arteries obtained from rats and pigs both showed inward remodeling after exposure to exogenous transglutaminase, which was inhibited by addition of a nitric oxide donor. Enhanced expression of tTG, induced by retinoic acid, increased inward remodeling of porcine coronary arteries kept in organ culture for 3 days. The activity of tTG was dependent on pressure. Inhibition of tTG reversed remodeling, causing a substantial increase in vessel diameter. In a collagen gel contraction assay, tTG determined the compaction of collagen by smooth muscle cells. Collectively, these data show that small artery remodeling associated with chronic vasoconstriction depends on tissue-type transglutaminase. This mechanism may reveal a novel therapeutic target for pathologies associated with inward remodeling of the resistance arteries.

Activation of resistance arteries with endothelin-1: From vasoconstriction to functional adaptation and remodeling

Remodeling of resistance arteries is a key feature in hypertension. We studied the transition of vasoconstriction to remodeling in isolated rat skeletal muscle arterioles. Arterioles activated with 10 nM endothelin-1 showed functional adaptation when kept at low distension in a wire myograph setup, where contractile properties shifted towards a smaller lumen diameter after 1 day. Pressurized arteries kept in organoid culture showed physical inward remodeling after 3-day activation with 10 nM endothelin-1, characterized by a reduction in relaxed diameter without a change in the wall cross-sectional area (eutrophic remodeling). The relaxed lumen diameter (at 60 mm Hg) decreased from 169 ± 5 (day 0) to 155 ± 4 µm (day 3). An antibody directed to the β3-integrin subunit (but not one directed to the β1-integrin subunit) enhanced remodeling, from a reduction in relaxed diameter at 60 mm Hg of 15 ± 2.4 to 22 ± 1.8 µm (both on day 3). Collagen gel contraction experiments showed that the antibody directed to the β3-integrin subunit enhanced the compaction of collagen by smooth muscle cells, from 83 ± 1.5 to 68 ± 1.5% of the initial gel diameter. In conclusion, these data show that inward eutrophic remodeling is a response to sustained contraction, which may involve collagen reorganization through β3-integrins.

MECHANISMS OF CA2+ SENSITIZATION OF FORCE PRODUCTION BY NORADRENALINE IN RAT MESENTERIC SMALL ARTERIES

1 Mechanisms of Ca2+ sensitization of force production by noradrenaline were investigated by measuring contractile responses, intracellular Ca2+ concentration ([Ca2+]i) and phosphorylation of the myosin light chain (MLC) in intact and α‐toxin‐permeabilized rat mesenteric small arteries. 2 The effects of noradrenaline were investigated at constant membrane potential by comparing fully depolarized intact arteries in the absence and presence of noradrenaline. Contractile responses to K‐PSS (125 mM K+) and NA‐K‐PSS (K‐PSS + 10 μM noradrenaline) were titrated to 30 and 75 %, respectively, of control force, by adjusting extracellular Ca2+ ([Ca2+]o). At both force levels, [Ca2+]i was substantially lower with NA‐K‐PSS than with K‐PSS. With K‐PSS, the proportion of MLC phosphorylated (≈30 %) was similar at 30 and 75 % of control force; with NA‐K‐PSS, MLC phosphorylation was greater at the higher force level (40 vs. 34 %). 3 In α‐toxin‐permeabilized arteries, the force response to 1 μM Ca2+ was increased by 10 μM noradrenaline, and MLC phosphorylation was increased from 35 to 45 %. The protein kinase C (PKC) inhibitor calphostin C (100 nM) abolished the noradrenaline‐induced increase in MLC phosphorylation and contractile response, without affecting the contraction in response to Ca2+. Treatment with ATPγS in the presence of the MLC kinase inhibitor ML‐9 increased the sensitivity to Ca2+ and abolished the response to noradrenaline. 4 The present results show that in rat mesenteric small arteries noradrenaline‐induced Ca2+ sensitization is associated with an increased proportion of phosphorylated MLC. The results are consistent with a decreased MLC phosphatase activity mediated through PKC. Furthermore, while MLC phosphorylation is a requirement for force production, the results show that other factors are also involved in force regulation.

Neuropeptide Y regulates intracellular calcium through different signalling pathways linked to a Y1-receptor in rat mesenteric small arteries

Simultaneous measurements of intracellular calcium concentration ([Ca2+]i) and tension were performed to clarify whether the mechanisms which cause the neuropeptide Y (NPY)‐elicited contraction and potentiation of noradrenaline contractions, and the NPY inhibition of forskolin responses are linked to a single or different NPY receptor(s) in rat mesenteric small arteries. In resting arteries, NPY moderately elevated [Ca2+]i and tension. These effects were antagonized by the selective Y1 receptor antagonist, (R)‐N2‐(diphenacetyl)‐N‐[(4‐hydroxyphenyl)methyl]‐D‐arginineamide (BIBP 3226) (apparent pKB values of 8.54±0.25 and 8.27±0.17, respectively). NPY (0.1 μM) caused a near 3 fold increase in sensitivity to noradrenaline but did not significantly modify the tension‐[Ca2+]i relationship for this agonist. BIBP 3226 competitively antagonized the contractile response to NPY in arteries submaximally preconstricted with noradrenaline (pA2 7.87±0.20). In arteries activated by vasopressin, the adenylyl cyclase activator forskolin (3 μM) induced a maximum relaxation and a return of [Ca2+]i to resting levels. NPY completely inhibited these effects. The contractile responses to NPY in arteries maximally relaxed with either sodium nitroprusside (SNP) or nifedipine were not significantly higher than those evoked by the peptide at resting tension, in contrast to the contractions to NPY in forskolin‐relaxed arteries. BIBP 3226 competitively antagonized the contraction to NPY in forskolin‐relaxed arteries with a pA2 of 7.92±0.29. Electrical field stimulation (EFS) at 8–32 Hz caused large contractions in arteries relaxed with either forskolin or noradrenaline in the presence of phentolamine. These responses to EFS were inhibited by BIBP 3226. Similar EFS in resting, non‐activated arteries did not produce any response. The present results suggest that different intracellular pathways are linked to a single NPY Y1 receptor in intact rat mesenteric small arteries, and provide little support for involvement of other postjunctional NPY receptors in the contractile responses to NPY. Neurally released NPY also seems to act through Y1 receptors, and may serve primarily as an inhibitor of vasodilatation.

Chronic cystamine treatment inhibits small artery remodelling in rats.

Background/Aims: We investigated whether the tissue transglutaminase inhibitor cystamine is able to inhibit remodelling of small arteries in vivo, a possibility suggested by recent in vitro experiments. Methods: Using osmotic minipumps, phenylephrine, cystamine and/or amlodipine were infused for 1–2 weeks into 9-week-old Wistar rats. Small arteries were then removed for pressure myograph investigation. Results: Phenylephrine infusion caused inward remodelling of the small arteries compared to vehicle infusion. The remodelling was abolished by concomitant infusion with cystamine; blood pressure was unaffected. Second, we investigated whether cystamine was able to inhibit outward remodelling. Rats were first infused with phenylephrine for 1 week, and some were infused for a further week with amlodipine with or without cystamine. Amlodipine caused 24% outward remodelling compared to vessels from rats at completion of the phenylephrine infusion. The outward remodelling was attenuated 86% by concomitant cystamine infusion. A series of in vitro experiments supported the inhibitory action of cystamine on tissue transglutaminase. Conclusion: The ability of cystamine to inhibit inward remodelling independent of blood pressure is consistent with a role of tissue transgluaminase in this process. It remains to be determined if the ability of cystamine to inhibit outward remodelling also involves inhibition of tissue transglutaminase.

Interactions between neuropeptide Y and the adenylate cyclase pathway in rat mesenteric small arteries: role of membrane potential

1 Simultaneous measurements of membrane potential and tension were performed to investigate the intracellular mechanisms of neuropeptide Y (NPY) in rat mesenteric small arteries. 2 NPY (0.1 μM)depolarized arterial smooth muscle cells from −55 to −47 mV and increased wall tension by 0.22N m−1, representing 11% of the contraction elicited by a high potassium solution. Isoprenaline (1 μM) and acetylcholine (1 μM) evoked hyper polarizations of 11 and 17 mV, resupectively. NPY inhibited the isoprenaline ‐induced effects on membrane potential without affecting those of acetylcholine. 3 Forskolin evoked sustained concentration‐dependent hyper polarizations of small mesenteric arteries. NPY (0.1 mM) inhibited the resuponses to 1 μM forskolin, but did not alter the stable hyperpolarization elicited by the supecific activator of protein kinase A (PKA) SP‐5,6‐DCl‐cBIMPS (0.1 mm). Forskolin increased the cyclic AMP (cAMP) content of the arteries 21‐fold, and NPY inhibited the forskolin‐evoked increase in cAMP levels by 91 %. 4 The hyperpolarization produced by 1 μM forskolin was not affected by either charybdotoxin (0.1 μM) or 4‐aminopyridine (0.5mM), but glibenclamide (5 μM) inhibited the hyperpolarization by 70%. Glibenclamide also inhibited the hyperpolarization evoked by SP‐:5,6‐DCl‐cBIMPSuby59%. 5 Neither depolarization nor contraction caused by NPY were significantly affected by either glibenclamide (5 μM) or nifedipine (1 μM), but they were reduced by gadolinium (10 μM). However, the blocking effect of NPY on forskolin‐elicited hyperpolarization was not affected by gadolinium. 6 Charybdotoxin (O.1 μM) and 4‐aminopyridine (0.5 mM) strongly enhanced the depolarization and contraction caused by NPY (O.1μM), and nifedipine (1 μM) prevented the enhanced resuponses to NPY in the presence of charybdotoxin. 7 These findings suggest that NPY acts through at least two different intracellular mechanisms in mesenteric small arteries: a depolarization of arterial smooth muscle which is probably due to activation of non‐selective cation channels, and a marked inhibition of adenylate cyclase activity, which in turn inhibits the hyperpolarization produced by cAMP accumulation in these arteries.

Neuropeptide Y2 receptors are involved in enhanced neurogenic vasoconstriction in spontaneously hypertensive rats

1 The present study addressed the role of neuropeptide (NPY) Y2 receptors in neurogenic contraction of mesenteric resistance arteries from female spontaneously hypertensive rats (SHR). Arteries were suspended in microvascular myographs, electrical field stimulation (EFS) was performed, and protein evaluated by Western blotting and immunohistochemistry. 2 In vasopressin‐activated endothelium‐intact arteries, NPY and fragments with selectivity for Y1 receptors, [Leu31,Pro34]NPY, Y2 receptors, NPY(13–36), and rat pancreatic polypeptide evoked more pronounced contractions in segments from SHR than in Wistar Kyoto (WKY) arteries, even in the presence of the Y1 receptor antagonist, BIBP3226 (0.3 μM, (R)‐N(2)‐(diphenacetyl)‐N‐[(4‐hydroxyphenyl)methyl]D‐arginineamide). 3 In the presence of prazosin and during vasopressin activation, EFS‐evoked contractions were larger in arteries from SHR compared to WKY. EFS contractions were enhanced by the Y2 receptor selective antagonist BIIE0246TF (0.5 μM, (S)‐N2‐[[1‐[2‐[4‐[(R,S)‐5,11‐dihydro‐6(6h)‐oxodibenz[b,e]azepin‐11‐y1]‐1‐piperazinyl]‐2‐oxoethyl]cyclo‐pentyl‐N‐[2‐[1,2‐dihydro‐3,5 (4H)‐dioxo‐1,2‐diphenyl‐3H‐1,2,4‐triazol‐4‐yl]ethyl]‐argininamide), reduced by BIBP3226, and abolished by the combination of BIBP3226 and BIIE0246TF. 4 Immunoblotting showed NPY Y1 and Y2 receptor expression to be similar in arteries from WKY and SHR, although a specific Y2 receptor band at 80 kDa was detected only in arteries from WKY. 5 Immunoreaction for NPY was enhanced in arteries from SHR. In contrast to arteries from WKY, BIIE0246TF increased NPY immunoreactivity in EFS‐stimulated arteries from SHR. 6 The present results suggest that postjunctional neuropeptide Y1 and Y2 receptors contribute to neurogenic contraction of mesenteric small arteries. Moreover, both enhanced NPY content and altered neuropeptide Y1 and Y2 receptor activation apparently contribute to the enhanced neurogenic contraction of arteries from SHR.

Thanks to the Reviewers

CHRISTOPHER ADAMSON MICHAEL P. ALLEN WILLIAM BAILEY STEVE BALKIN RICHARD E. BALL JOYCE BARAKETT ROBERT A. BEAUREGARD E. M. BECK HOWARD S. BECKER LAWRENCE BENNETT BENNETT BERGER LEONARD BERKEY MICHAEL BETZ EGON BITTNER LEONARD BLUMBERG PHILIP BLUMSTEIN ROBERT BOGDEN EDNA BONACICH PEG BORTNER VERN BULLOUGH LEONARD CAIN KITTY CALAVITA JOHN CARSLEY JOAN CASSELL PAUL CHALFANT RANDALL COLLINS MARK COLVIN JOHN A. CONLEY ELAINE CUMMING ARLENE KAPLAN DANIELS JON DARLING JOAN DEBARDELEBEN CHARLES DERBER STEVEN DEUTSCH IRWIN DEUTSCHER PAUL DIMAGGIO JOAN EAKIN WILLIAM EATON SUSAN ECKSTEIN SHELDON EKLAND-OLSON ROBERT EMERSON PAULA S. ENGLAND DAVID M. ERMANN DARYL EVANS WILLIAM FALK SAMUEL FARBER ROB FAULKNER JOE FEAGIN ROSLYN FELDBERG SANDY FELDHEIM MARK FISHMAN NANCY FRANK ELIOT FREIDSON PETER FREITAG EDGAR Z. FRIEDENBERG WILLIAM FRIEDLAND SAMUEL FRIEDMAN JOHN GALLIHER DAVID GARTMAN GILBERT GEIS JAMES GESCHWENDER DON C. GIBBONS PEGGY GIORDANO DANIEL GLASER FRED GOLDNER ROBERT A. GORDON WALTER GOVE SUSAN GRAY DAVID GREENBERG ALLEN D. GRIMSHAW EDWARD GROSS BRUCE HACKETT JOHN HAGAN RICHARD F. HAMILTON SUE KIEFER HAMMERSMITH SHARON HARLAN CLAYTON HARTJEN JAMES HENSLIN JOHN P. HEWITT BARBARA HEYL SALLY T. HILLSMAN LYNDA LYTLE HOLMSTROM RANDY HODSON ALLEN HORWITZ JOAN HUBER DREW HUMPHRIES ALLEN W. IMERSHEIN JAMES INCIARDI DAVID JACOBS GARY JENSEN CAROLE JOFFE JOHN JOHNSON PAUL JOSEPH RACHEL KAHN-HUT DEBRA KALMUSS JACK KATZ RONALD KESSLER JOSEPH A. KOTARBA RONALD KRAMER NANCY KUTNER BARBARA R. LASLETT PAT LAUDERDALE RONALD LAWSON JEFFREY LEITER RHONDA F. LEVINE CYRIL LEVITT ELLIOT LIEBOW CLARENCE LO ULI LOCHER MARGARET LOCK HELENA Z. LOPATA DAVID E. LOPEZ DAVID LUCKENBILL PETER LYMAN LARRY LYON STEVE LONGSTAFF CHARLES McCAGHY JOHN D. McCARTHY SCOTT G. McNALL LINDA C. MAJKA GERALD MARKLE JOHN MARKOFF CORA MARRETT TONY MASI DOUGLAS MAYNARD ROBERT MEIER JANET E. MICKISH TERANCE MIETHE S.M. MILLER BETH MINTZ MERRY ANN MORASH DAVID L. MORGAN CAROL KIAPERMAN MORROW CHARLES C. MOSCOS JR MARTHA MYERS JOANE NAGEL DOROTHY NELKIN MARGARET K. NELSON LINDA B. NILSON STEPHEN NORLAND MELVIN OLIVER MARVIN OLSEN SUSAN OLZAK ANN L. PAGE TOBY PARCEL DOROTHY PAWLUCH HAROLD E. PEPINSKY CHARLES PERROW ROBERT PERRUCCI SUZANNE PETERS KAREN J. PETERSON MICHAEL PETRUNIK MARK PEYROT GERALD PLATT ALPHONSO PINKNEY HENRY PONTELL ROBERT C. PRUS RICHARD QUINNEY NICOLE FISCHER RAFTER PRUDENCE RAINS DONNA RANDALL STEVEN RANDALL JOSEPH RANKIN RICHARD RATCLIFF PAMELA RICHARDS JAMES RICHARDSON RAY RIST JACK ROACH JANET ROACH JIM ROBBINS E. BURKE ROCHFORD JOSEPH W. ROGERS ROBIN ROOM RACHEL ROSENFELD BARBARA KATZ ROTHMAN LILLIAN RUBIN JOSEPHINE A. RUGGIERO WILLIAM A. RUSHING SHERYL RUZEK SASKIA SASSEN-KOOB SANDRA P. SCHOENBERG WILLIAM SHAFFIR NANCY S. SHAW CLIFFORD D. SHEARING JAMES F. SHORT JR. ROBERTA SIMMONS DOUGLAS SMITH MICHAEL SMITH DAVID A. SNOW LEE SODERSTROM JACK W. SPENCER STEVEN SPITZER DARRELL J. STEFENMEIER RONALD TAYLOR JIM THOMAS ROBERT THOMAS GARY TIEDEMAN KATHLEEN TIERNEY RONALD TROYER JAMES D. UNNEVER BERT USEEM PAULINE VAILLANCOURT ARTHUR J. VIDICH DIANA VAUGHAN EDWARD J. WALSH VIVIENNE WALTERS CATHERINE WATSON J. ALLEN WHITT SIDNEY WILLHELM JUDITH G. WITTNER PETER YIN MAYER N. ZALD DAVID ZARET