Pathophysiological Role of Caveolae in Hypertension

Caveolae, flask-shaped cholesterol-, and glycosphingolipid-rich membrane microdomains, contain caveolin 1, 2, 3 and several structural proteins, in particular Cavin 1-4, EHD2, pacsin2, and dynamin 2. Caveolae participate in several physiological processes like lipid uptake, mechanosensitivity, or signaling events and are involved in pathophysiological changes in the cardiovascular system. They serve as a specific membrane platform for a diverse set of signaling molecules like endothelial nitric oxide synthase (eNOS), and further maintain vascular homeostasis. Lack of caveolins causes the complete loss of caveolae; induces vascular disorders, endothelial dysfunction, and impaired myogenic tone; and alters numerous cellular processes, which all contribute to an increased risk for hypertension. This brief review describes our current knowledge on caveolae in vasculature, with special focus on their pathophysiological role in hypertension

Oxidative Regulation spannungsgesteuerter Natriumkanäle

Current through voltage-gated sodium (NaV) channels initiates action potentials and influences the duration of their plateau phase. Reactive oxygen species (ROS) may modify NaV channels and alter their function possibly leading to impairment of signal transduction or to apoptosis of cells. The drastically increased ROS concentration in extreme situations (stroke, cardiac infarction) and long-term effects of moderately increased ROS concentration (several chronical diseases, age) are critical. In this dissertation changes of sodium currents by oxidative stress were characterized. Recombinant ion channels were expressed in a heterologous system and functionally analyzed by the patch-clamp method. Molecular targets for the oxidation sensitivity were identified. At skeletal muscle sodium channels (NaV1.4) the oxidant chloramine-T caused irreversible loss of inactivation and irreversible reduction of peak current. With mutant channels of rNaV1.4, which have the less facile oxidizable leucine in the position of methionine, it has been shown that M1305 from the inactivation motif and the double methionine motif M1469/M1470 from the putative inactivation linker acceptor essentially influence the oxidation sensitivity of inactivation. By kinetic analyses it was acknowledged that several targets have to be oxidized to derive loss of rNaV1.4 inactivation. The reduction of peak current due to oxidant action was significantly higher in cardiac muscle channel NaV1.5 compared to skeletal muscle channel. The current loss is due to oxidative modification of a cysteine in domain 1 within the pore and structures in domain 2. Alterations of the voltage-dependence of inactivation and the reduction of single channel current and open probability were observed. The intense current loss in the cardiac sodium channel may be protective together with potassium channels and in securing energy metabolism, if the ATP production via the electron transport chain is inhibited by lack of oxygen.Ströme durch spannungsgesteuerte Natriumkanäle (NaV-Kanäle) initiieren Aktionspotenziale und beeinflussen die Dauer von deren Plateauphase. Reaktive Sauerstoffverbindungen (ROS) können NaV-Kanäle modifizieren und ihre Funktion beeinflussen, was zu Störungen der Erregungsleitung und zur Apoptose von Zellen führen kann. Kritisch ist die drastisch erhöhte ROS-Konzentration bei extremen Ereignissen (Schlaganfall, Herzinfarkt) und die Langzeitwirkung moderat erhöhter ROS-Konzentration (zahlreiche chronische Erkrankungen, Alter). In der vorliegenden Arbeit wurden Veränderungen der Natriumströme bei oxidativem Stress charakterisiert. Rekombinante Ionenkanäle wurden in einem heterologen System exprimiert und funktionell mit der Patch-Clamp-Methode analysiert. Molekulare Zielstellen für die Oxidationssensitivität wurden ermittelt. Bei Skelettmuskelnatriumkanälen (NaV1.4) führte das Oxidationsmittel Chloramin T zu irreversiblem Inaktivierungsverlust und irreversibler Reduktion des Spitzenstroms. Mit Mutanten, bei denen Methionin durch das weniger leicht oxidierbare Leucin ersetzt war, wurde gezeigt, dass bei rNaV1.4 M1305 aus dem Inaktivierungsmotiv und das dem Akzeptor zugerechnete Doppel-Methionin-Motiv M1469/M1470 wesentlichen Einfluss auf die Oxidationssensitivität der Inaktivierung haben. Kinetische Analysen bestätigten, dass für den Inaktivierungsverlust von rNaV1.4 mehrere Zielstellen oxidiert werden müssen. Der Herzmuskelkanal NaV1.5 war bezüglich des Spitzenstroms deutlich oxidationssensitiver als der Skelettmuskelkanal. Der Stromverlust geht auf die oxidative Modifikation eines Cysteins in Domäne 1 innerhalb der Pore sowie Strukturen in Domäne 2 zurück. Veränderungen bei der Spannungsabhängigkeit der Inaktivierung und die Verringerung von Einzelkanalstrom und Offenwahrscheinlichkeit traten auf. Der starke Stromverlust des Herzmuskelkanals könnte im Zusammenspiel mit Kaliumkanälen und bei der Sicherung des Energiehaushaltes bei Sauerstoffmangel protektiv wirken

Role of Ryanodine Type 2 Receptors in Elementary Ca 2+ Signaling in Arteries and Vascular Adaptive Responses

Background: Hypertension is the major risk factor for cardiovascular disease, the most common cause of death worldwide. Resistance arteries are capable of adapting their diameter independently in response to pressure and flow-associated shear stress. Ryanodine receptors (RyRs) are major Ca2+-release channels in the sarcoplasmic reticulum membrane of myocytes that contribute to the regulation of contractility. Vascular smooth muscle cells exhibit 3 different RyR isoforms (RyR1, RyR2, and RyR3), but the impact of individual RyR isoforms on adaptive vascular responses is largely unknown. Herein, we generated tamoxifen-inducible smooth muscle cell-specific RyR2-deficient mice and tested the hypothesis that vascular smooth muscle cell RyR2s play a specific role in elementary Ca2+ signaling and adaptive vascular responses to vascular pressure and/or flow. Methods and Results: Targeted deletion of the Ryr2 gene resulted in a complete loss of sarcoplasmic reticulum-mediated Ca2+-release events and associated Ca2+-activated, large-conductance K+ channel currents in peripheral arteries, leading to increased myogenic tone and systemic blood pressure. In the absence of RyR2, the pulmonary artery pressure response to sustained hypoxia was enhanced, but flow-dependent effects, including blood flow recovery in ischemic hind limbs, were unaffected. Conclusions: Our results establish that RyR2-mediated Ca2+-release events in VSCM s specifically regulate myogenic tone (systemic circulation) and arterial adaptation in response to changes in pressure (hypoxic lung model), but not flow. They further suggest that vascular smooth muscle cell-expressed RyR2 deserves scrutiny as a therapeutic target for the treatment of vascular responses in hypertension and chronic vascular diseases

Amelioration of tubular damage score of I/R-induced AKI by pretreatment of mice with capsaicin, but not with capsazepine or genetic ablation of TRPV1.

<p><b>Panel A</b>: Tubular damage scores of ipsilateral kidneys (I/R Control) and kidneys after I/R (I/R) of control mice. Tubular damage scores of ipsilateral kidneys (Capsaicin C) and kidneys after I/R injury (Capsaicin I/R) of mice pretreated with capsaicin. Tubular damage scores of ipsilateral kidneys (Capsazepine C) and kidneys after I/R injury (Capsazepine I/R) of mice pretreated with capsazepine. Tubular damage scores of ipsilateral kidneys (<i>Trpv1−/−</i> C) and kidneys after I/R injury (<i>Trpv1−/−</i> I/R) of <i>Trpv1−/−</i> mice. Scores of ipsilateral control kidneys without I/R injury were all 0. *, <i>P</i><0.05. <i>P</i>≥0.05; not significant. <b>Panel B:</b> Histological sections. (<b>a</b>) I/R Control, histology of ipsilateral kidney before renal I/R injury of a control mouse. (<b>b</b>) I/R, histology of kidney after I/R injury of a control mouse. The section shows acute tubular necrosis characterized by loss of tubular epithelial cells (arrow) and shedding of the brush border (asterisk). (<b>c</b>) Capsaicin I/R, histology of kidney after I/R injury of a mouse pretreated with capsaicin. Acute tubular necrosis (arrow). (<b>d</b>) <i>Trpv1−/−</i> I/R, histology of kidney after I/R injury of a <i>Trpv1−/−</i> mouse. Acute tubular necrosis (arrow) and shedding of the brush border (asterisk). Scale bar 100 µm. Magnification × 200.</p

Eicosanoids and anandamide levels in non-ischemic (control) and ischemic (I) kidneys of (WT) wild-type mice after I-induced AKI.

<p>5,6-EET, 5,6-epoxyeicosatrienoic acid; 8,9-EET, 8,9-epoxyeicosatrienoic acid; 11,12-EET, 11,12-epoxyeicosatrienoic acid; 14,15-EET, 14,15-epoxyeicosatrienoic acid; 5,6-DHET, 5,6-dihydroxyeicosastrienoic acid; 8,9-DHET, 8,9-dihydroxyeicosastrienoic acid; 11,12-DHET, 11,12-dihydroxyeicosastrienoic acid; 14,15-DHET, 14,15-dihydroxyeicosastrienoic acid; 20-HETE, 20-hydroxyeicosatetraenoic acid; 9,10-EPOME, 9(10)epoxy-9Z-octadecenoic acid; 12,13-EPOME, 12(13)epoxy-9Z-octadecenoic acid; 9,10-DIHOME, 9(10)-dihydroxy-12Z-octadecenoic acid; 9,10-DIHOME, 9(10)-dihydroxy-12Z-octadecenoic acid. n = 5 in each group.</p

Lower Ly-6B.2 positive cells in the kidneys after I/R-induced AKI in mice treated with capsaicin, but not with capsazepine or in <i>Trpv1</i>−/− mice.

<p>Ly-6B.2 positive cells were analyzed in the medulla (red color in panels B, marked by arrows). <i>Panels A:</i> Ly-6B.2 positive cells per field view in ipsilateral kidneys (I/R Control) and kidneys after I/R (I/R) injury of control mice. Ly-6B.2 positive cells in ipsilateral kidneys (Capsaicin C) and kidneys after I/R injury (Capsaicin I/R) of mice pretreated with capsaicin. Ly-6B.2 positive cells in ipsilateral kidneys (Capsazepine C) and kidneys after I/R injury (Capsazepine I/R) of mice treated with capsazepine. Ly-6B.2 positive cells in ipsilateral kidneys (<i>Trpv1</i>−/− C) and kidneys after I/R injury (<i>Trpv1</i>−/− I/R) of <i>Trpv1</i>−/− mice. *, P<0.05. P>0.05; not significant. <b>Panels B:</b> Immunohistological sections. (a) I/R Control, images of Ly-6B.2 positive cells in ipsilateral kidney before renal I/R injury of a control mouse. (b) I/R, Ly-6B.2 positive cells in a kidney after I/R injury of a control mouse. (c) Capsaicin I/R, Ly-6B.2 positive cells in a kidney after I/R injury of a mouse pretreated with capsaicin. (d) Trpv1−/− I/R, Ly-6B.2 positive cells in a kidney after I/R injury of a <i>Trpv1</i>−/− mouse. Scale bar 50 µm. Magnification ×400.</p

Serum creatinine levels in mice subjected to renal I/R injury were ameliorated by pretreatment with capsaicin, but not capsazepine or genetic ablation of TRPV1.

<p>Control, control mice that underwent sham surgery (nephrectomy) without I/R. I/R Control, control mice that underwent renal I/R injury. Capsaicin I/R, wild-type mice pretreated with capsaicin followed by renal I/R injury. Capsazepine I/R, wild-type mice pretreated with capsazepine followed by renal I/R injury. <i>Trpv1−/−</i> mice that underwent renal I/R injury. *<i>P</i><0.05. <i>P</i>>0.05; not significant.</p

Eicosanoids and anandamide levels in non-ischemic (I/R Control) and ischemic (I/R) kidneys of wild-type mice after I/R-induced AKI, in non-ischemic (Capsaicin C) and ischemic (Capsaicin I/R) kidneys of wild-type mice after I/R-induced AKI, and in non-ischemic (<i>Trpv1</i>−/− C) and ischemic (<i>Trpv1</i>−/− I/R) kidneys of <i>Trpv1</i>−/− mice after I/R-induced AKI. n>8 in each group.

<p>Eicosanoids and anandamide levels in non-ischemic (I/R Control) and ischemic (I/R) kidneys of wild-type mice after I/R-induced AKI, in non-ischemic (Capsaicin C) and ischemic (Capsaicin I/R) kidneys of wild-type mice after I/R-induced AKI, and in non-ischemic (<i>Trpv1</i>−/− C) and ischemic (<i>Trpv1</i>−/− I/R) kidneys of <i>Trpv1</i>−/− mice after I/R-induced AKI. n>8 in each group.</p

NGAL abundance in the kidney was lower in I/R-induced AKI in mice treated with capsaicin, but not with capsazepine or in <i>Trpv1−/−</i> mice.

<p><b>Panels A:</b> NGAL abundance of ipsilateral kidneys (I/R Control) and kidneys after I/R (I/R) injury of control mice. NGAL abundance of ipsilateral kidneys (Capsaicin C) and kidneys after I/R injury (Capsaicin I/R) of mice pretreated with capsaicin. NGAL abundance of ipsilateral kidneys (Capsazepine C) and kidneys after I/R injury (Capsazepine I/R) of mice treated with capsazepine. NGAL abundance of ipsilateral kidneys (<i>Trpv1</i>−/− C) and kidneys after I/R injury (<i>Trpv1−/−</i> I/R) of <i>Trpv1−/−</i> mice. *, <i>P</i><0.05. <i>P</i>>0.05; not significant. <b>Panels B:</b> Immunohistological sections. (<b>a</b>) I/R Control, images of NGAL negative staining of ipsilateral kidney before renal I/R injury of a control mouse. (<b>b</b>) I/R, intense renal NGAL staining after I/R injury of a control mouse. (<b>c</b>) Capsaicin I/R, renal NGAL staining after I/R injury of a mouse pretreated with capsaicin. (<b>d</b>) <i>Trpv1−/−</i> I/R, renal NGAL staining after I/R injury of a <i>Trpv1−/−</i> mouse. Scale bar 100 µm. Magnification ×200.</p

Age attenuates the T‐type CaV3.2‐RyR axis in vascular smooth muscle

Abstract Caveolae position CaV3.2 (T‐type Ca2+ channel encoded by the α‐3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca2+ influx to trigger Ca2+ sparks and large‐conductance Ca2+‐activated K+ channel feedback in vascular smooth muscle. We hypothesize that this mechanism of Ca2+ spark generation is affected by age. Using smooth muscle cells (VSMCs) from mouse mesenteric arteries, we found that both Cav3.2 channel inhibition by Ni2+ (50 µM) and caveolae disruption by methyl‐ß‐cyclodextrin or genetic abolition of Eps15 homology domain‐containing protein (EHD2) inhibited Ca2+ sparks in cells from young (4 months) but not old (12 months) mice. In accordance, expression of Cav3.2 channel was higher in mesenteric arteries from young than old mice. Similar effects were observed for caveolae density. Using SMAKO Cav1.2−/− mice, caffeine (RyR activator) and thapsigargin (Ca2+ transport ATPase inhibitor), we found that sufficient SR Ca2+ load is a prerequisite for the CaV3.2‐RyR axis to generate Ca2+ sparks. We identified a fraction of Ca2+ sparks in aged VSMCs, which is sensitive to the TRP channel blocker Gd3+ (100 µM), but insensitive to CaV1.2 and CaV3.2 channel blockade. Our data demonstrate that the VSMC CaV3.2‐RyR axis is down‐regulated by aging. This defective CaV3.2‐RyR coupling is counterbalanced by a Gd3+ sensitive Ca2+ pathway providing compensatory Ca2+ influx for triggering Ca2+ sparks in aged VSMCs