Caveolae are plasma membrane invaginations that may play an important role in numerous cellular processes including transport, signaling, and tumor suppression. By targeted disruption of caveolin-1, the main protein component of caveolae, we generated mice that lacked caveolae. The absence of this organelle impaired nitric oxide and calcium signaling in the cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic tone. In addition, the lungs of knockout animals displayed thickening of alveolar septa caused by uncontrolled endothelial cell proliferation and fibrosis, resulting in severe physical limitations in caveolin-1-disrupted mice. Thus, caveolin-1 and caveolae play a fundamental role in organizing multiple signaling pathways in the cell.

Virtually all blood vessels are surrounded by adventitial fat. Adipocytes produce a host of vasoactive substances that may influence vascular contraction. We tested whether or not perivascular adipose tissue modulates contraction of aortic ring preparations. We studied aortic rings surrounded by periadventitial adipose tissue from adult Sprague-Dawley rats. At a maximum concentration of 300 nM angiotensin II, 6.5 microM serotonin, and 5 microM phenylephrine, the contractile response of intact rings was 95%, 80%, and 30% lower than that of vessels without periadventitial fat. The anticontractile effect of periadventitial fat was reduced by inhibition of ATP-dependent K+ channels with glibenclamide (3 microM) and by the tyrosine kinase inhibitor genistein (10 microM). Blocking NOS, cyclo-oxygenase, cytochrome P450, or adenosine receptors did not restore the vascular response in intact vessels. The anticontractile effect of perivascular fat was present in Zucker fa/fa rats, suggesting that leptin receptors were not responsible. Transferring the bath solution from intact vessels, isolated periadventitial tissue, and cultured rat adipocytes to precontracted vessels lacking periadventitial fat resulted in a rapid relaxation. We suggest that perivascular adventitial adipose tissue releases a transferable adventitium-derived relaxing factor that acts by tyrosine kinase-dependent activation of K+ channels in vascular smooth muscle cells.

Large-conductance potassium (BK) channels in vascular smooth muscle cells (VSMCs) sense both changes in membrane potential and in intracellular Ca(2+) concentration. BK channels may serve as negative feedback regulators of vascular tone by linking membrane depolarization and local increases in intracellular Ca(2+) concentration (Ca(2+) sparks) to repolarizing spontaneous transient outward K(+) currents (STOCs). BK channels are composed of channel-forming BKalpha and auxiliary BKbeta1 subunits, which confer to BK channels an increased sensitivity for changes in membrane potential and Ca(2+). To assess the in vivo functions of this ss subunit, mice with a disrupted BKbeta1 gene were generated. Cerebral artery VSMCs from BKbeta1 -/- mice generated Ca(2+) sparks of normal amplitude and frequency, but STOC frequencies were largely reduced at physiological membrane potentials. Our results indicate that BKbeta1 -/- mice have an abnormal Ca(2+) spark/STOC coupling that is shifted to more depolarized potentials. Thoracic aortic rings from BKbeta1 -/- mice responded to agonist and elevated KCl with a increased contractility. BKbeta1 -/- mice had higher systemic blood pressure than BKbeta1 +/+ mice but responded normally to alpha(1)-adrenergic vasoconstriction and nitric oxide-mediated vasodilation. We propose that the elevated blood pressure in BKbeta1 -/- mice serves to normalize Ca(2+) spark/STOC coupling for regulating myogenic tone. The full text of this article is available at http://www.circresaha.org.

Abstract-Hypertension and kidney damage in the double transgenic rat (dTGR) harboring both human renin and human angiotensinogen genes are dependent on the human components of the renin angiotensin system. We tested the hypothesis that monocyte infiltration and increased adhesion molecule expression are involved in the pathogenesis of kidney damage in dTGR. We also evaluated the effects of long-term angiotensin-converting enzyme (ACE) inhibition, AT 1 blockade, and human renin inhibition on monocyte recruitment and inflammatory response in dTGR. Systolic blood pressure and 24-hour albuminuria were markedly increased in 7-week-old dTGR as compared with age-matched normotensive Sprague Dawley rats. We found a significant monocyte/macrophage infiltration in the renal perivascular space and increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the interstitium, intima, and adventitia of the small renal vessels. ␣ L ␤ 2 integrin and ␣ 4 ␤ 1 integrin, the corresponding ligands for ICAM-1 and VCAM-1, were also found on infiltrating monocytes/macrophages. The expression of plasminogen activator inhibitor-1 and fibronectin in the kidneys of dTGR were increased and distributed similarly to ICAM-1. In 4-week-old dTGR, long-term treatment with ACE inhibition (cilazapril), AT 1 receptor blockade (valsartan), and human renin inhibition (RO 65-7219) (each drug 10 mg/kg by gavage once a day for 3 weeks) completely prevented the development of albuminuria. However, only cilazapril and valsartan were able to decrease blood pressure to normotensive levels. Interestingly, the drugs were all equally effective in preventing monocyte/macrophage infiltration and the overexpression of adhesion molecules, plasminogen activator inhibitor-1, and fibronectin in the kidney. Our findings indicate that angiotensin II causes monocyte recruitment and vascular inflammatory response in the kidney by blood pressure-dependent and blood pressure-independent mechanisms. ACE inhibition, AT 1 receptor blockade, and human renin inhibition all prevent monocyte/macrophage infiltration and increased adhesion molecule expression in the kidneys of dTGR. Key Words: angiotensin II Ⅲ intercellular adhesion molecule-1 Ⅲ vascular cell adhesion molecule-1 Ⅲ plasminogen activator inhibitor-1 Ⅲ fibronectin Ⅲ renin H ypertension is a major risk factor for renal injury. However, the mechanisms underlying the development and progression of hypertension-induced kidney damage are incompletely understood. There is growing evidence that vascular inflammatory responses and interstitial accumulation of extracellular matrix proteins are involved in the pathogenesis. 1,2 Moreover, both experimental and clinical studies revealed that angiotensin II (Ang II), the key effector of the local and circulating renin-angiotensin system (RAS), plays a central role in the pathogenesis of hypertensioninduced end-organ damage (for reviews see References 3 and 4). The mechanisms of Ang II-induced hypertension and renal damage are...

Abstract-Ca2ϩ sparks are localized intracellular Ca 2ϩ events released through ryanodine receptors (RyRs) that control excitation-contraction coupling in heart and smooth muscle. Ca 2ϩ spark triggering depends on precise delivery of Ca 2ϩ ions through dihydropyridine (DHP)-sensitive Ca 2ϩ channels to RyRs of the sarcoplasmic reticulum (SR), a process requiring a very precise alignment of surface and SR membranes containing Ca 2ϩ influx channels and RyRs. Because caveolae contain DHP-sensitive Ca 2ϩ channels and may colocalize with SR, we tested the hypothesis that caveolae are the structural element necessary for the generation of Ca 2ϩ sparks. Using methyl-␤-cyclodextrin (dextrin) to deplete caveolae, we found that dextrin dose-dependently decreased the frequency, amplitude, and spatial size of Ca 2ϩ sparks in arterial smooth muscle cells and neonatal cardiomyocytes. However, temporal characteristics of Ca 2ϩ sparks were not significantly affected. We ruled out the possibility that the decreases in Ca 2ϩ spark frequency and size are caused by changes in DHP-sensitive L-type channels, SR Ca 2ϩ load, or changes in membrane potential. Our results suggest a novel signaling model that explains the formation of Ca 2ϩ sparks in a caveolae microdomain. The transient elevation in [Ca 2ϩ ] i at the inner mouth of a single caveolemmal Ca 2ϩ channel induces simultaneous activation and thus opens several RyRs to generate a local Ca 2ϩ release event, a Ca 2ϩ spark. Alterations in the molecular assembly and ultrastructure of caveolae may lead to pathophysiological changes in Ca 2ϩ signaling. Thus, caveolae may be intimately involved in cardiovascular cell dysfunction and disease. Materials and MethodsSingle SMCs were isolated enzymatically from myogenic cerebral (100 to 800 m in diameter posterior and basilar) arteries from adult Sprague-Dawley rats (12 to 14 weeks; 200 to 280 g), as previously described. 14 Single cardiomyocytes were isolated enzymatically from newborn rats. 21 For Ca 2ϩ imaging, the cells were incubated with the Ca 2ϩ indicator fluo-3-AM (5 m) and pluronic acid (0.005% wt/vol) for 30 minutes at room temperature in Ca 2ϩ -free Hanks solution. 3,14 SMCs and cardiomyocytes were imaged using a BioRad laser scanning confocal microscope attached to a Nikon Diaphot microscope. Whole-cell membrane currents and potentials in freshly isolated cerebral artery myocytes were measured using the perforated patch configuration of the patch-clamp technique configuration with amphotericin B or nystatin. 22 Currents were recorded from holding potentials of Ϫ80 mV (Ϫ100 mV) during lineage voltage ramps at 0.67 V/s from Ϫ100 to ϩ100 mV or 300-ms step pulses to different potentials; pulse frequency 0.2 Hz. 22,23 An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org. ResultsWe used a laser scanning confocal microscope and the Ca Figure 1 online, available at http:// www.circresaha.org) by membrane depolarization (using 60 mmol/L external K ϩ ) or by th...

BACKGROUND AND PURPOSEThe cation channel transient receptor potential canonical (TRPC) 6 has been associated with several pathologies including focal segmental glomerulosclerosis, pulmonary hypertension and ischaemia reperfusion-induced lung oedema. We set out to discover novel inhibitors of TRPC6 channels and investigate the therapeutic potential of these agents. EXPERIMENTAL APPROACHA library of potential TRPC channel inhibitors was designed and synthesized. Activity of the compounds was assessed by measuring intracellular Ca 2+ levels. The lead compound SAR7334 was further characterized by whole-cell patch-clamp techniques. The effects of SAR7334 on acute hypoxic pulmonary vasoconstriction (HPV) and systemic BP were investigated. KEY RESULTS SAR7334 inhibited TRPC6, TRPC3 and TRPC7-mediated Ca2+ influx into cells with IC50s of 9.5, 282 and 226 nM, whereas TRPC4 and TRPC5-mediated Ca 2+ entry was not affected. Patch-clamp experiments confirmed that the compound blocked TRPC6 currents with an IC50 of 7.9 nM. Furthermore, SAR7334 suppressed TRPC6-dependent acute HPV in isolated perfused lungs from mice. Pharmacokinetic studies of SAR7334 demonstrated that the compound was suitable for chronic oral administration. In an initial short-term study, SAR7334 did not change mean arterial pressure in spontaneously hypertensive rats (SHR). CONCLUSIONS AND IMPLICATIONSOur results confirm the role of TRPC6 channels in hypoxic pulmonary vasoregulation and indicate that these channels are unlikely to play a major role in BP regulation in SHR. SAR7334 is a novel, highly potent and bioavailable inhibitor of TRPC6 channels that opens new opportunities for the investigation of TRPC channel function in vivo.

Abstract-Intracellular

The heme-enzyme soluble guanylyl cyclase (sGC) is an ubiquitous NO receptor, which mediates NO downstream signaling by the generation of cGMP. We studied the mechanism of action of the anthranilic acid derivatives 5-chloro-2-(5-chloro-thiophene-2-sulfonylamino-N-(4-(morpholine-4-sulfonyl)-phenyl)-benzamide sodium salt (HMR1766) (proposed international nonproprietary name, ataciguat sodium) and 2-(4-chloro-phenylsulfonylamino)-4,5-dimethoxy-N-(4-(thiomorpholine-4-sulfonyl)-phenyl)-benzamide (S3448) as a new class of sGC agonists. Both compounds activated different sGC preparations (purified from bovine lung, or crude from human corpus cavernosum) in a concentrationdependent and quickly reversible fashion (EC 50 ϭ 0.5-10 M), with mixed-type activation kinetics. Activation of sGC by these compounds was additive to activation by NO donors, but instead of being inhibited, it was potentiated by the heme-iron oxidants 1H-[1,2,4]-oxdiazolo[3,4-a]quinoxalin-1-one (ODQ) and 4H-8-bromo-1,2,4-oxadiazolo(3,4-d) benz(b)(1,4)oxazin-1-one (NS2028), suggesting that the new compounds target the ferric heme sGC isoform. Protoporphyrin IX acted as a competitive activator, and zinc-protoporphyrin IX inhibited activation of hemeoxidized sGC by HMR1766 and S3448, whereas heme depletion of sGC by Tween 20 treatment reduced activation. Both compounds increased cGMP levels in cultured rat aortic smooth muscle cells; induced vasorelaxation of isolated endothelium-denuded rat aorta, porcine coronary arteries, and human corpus cavernosum (EC 50 1 to 10 M); and elicited phosphorylation of the cGMP kinase substrate vasodilator-stimulated phosphoprotein at Ser239. HMR1766 intravenous bolus injection decreased arterial blood pressure in anesthetized pigs. All of these pharmacological responses to the new compounds were enhanced by ODQ and NS2028. Our findings suggest that HMR1766 and S3448 preferentially activate the NO-insensitive heme-oxidized form of sGC, which exists to a variable extent in vascular tissues, and is a pharmacological target for these new vasodilator drugs.The heterodimeric heme-protein soluble guanylyl cyclases (E.C. 4.6.1.2. pyrophosphate lyase, cyclizing; sGC) function as a receptor for the ubiquitous signaling molecule NO. Binding of NO to the ferrous heme activates the enzyme for rapid catalysis of cGMP formation from GTP (Koesling and Friebe, 1999). The second-messenger cGMP triggers several biological processes, such as a decrease in vascular tone and platelet activity, through an interaction with cGMP-specific protein kinases, phosphodiesterases, and ion channels (Munzel et al., 2003). A unifying concept of the molecular requisites for sGC activation has been put forward (Ignarro et al., 1984; Ballou A.M. was supported by grants from Hoechst-Marion-Roussel/Aventis and the German Research Council (SFB 553, project C10).Part of this work was published previously as a thesis (by A.T.) titled Kontrolle der glattmuskulä ren GC-Aktivitä t via NO-Sensitivitä t, Hä m-RedoxStatus und Proteinexpression, 2001 (ISBN 3-930...