Angiotensin II reduces the surface abundance of KV 1.5 channels in arterial myocytes to stimulate vasoconstriction.

KEY POINTS: Several different voltage-dependent K+ (KV ) channel isoforms are expressed in arterial smooth muscle cells (myocytes). Vasoconstrictors inhibit KV currents, but the isoform selectivity and mechanisms involved are unclear. We show that angiotensin II (Ang II), a vasoconstrictor, stimulates degradation of KV 1.5, but not KV 2.1, channels through a protein kinase C- and lysosome-dependent mechanism, reducing abundance at the surface of mesenteric artery myocytes. The Ang II-induced decrease in cell surface KV 1.5 channels reduces whole-cell KV 1.5 currents and attenuates KV 1.5 function in pressurized arteries. We describe a mechanism by which Ang II stimulates protein kinase C-dependent KV 1.5 channel degradation, reducing the abundance of functional channels at the myocyte surface. ABSTRACT: Smooth muscle cells (myocytes) of resistance-size arteries express several different voltage-dependent K+ (KV ) channels, including KV 1.5 and KV 2.1, which regulate contractility. Myocyte KV currents are inhibited by vasoconstrictors, including angiotensin II (Ang II), but the mechanisms involved are unclear. Here, we tested the hypothesis that Ang II inhibits KV currents by reducing the plasma membrane abundance of KV channels in myocytes. Angiotensin II (applied for 2 h) reduced surface and total KV 1.5 protein in rat mesenteric arteries. In contrast, Ang II did not alter total or surface KV 2.1, or KV 1.5 or KV 2.1 cellular distribution, measured as the percentage of total protein at the surface. Bisindolylmaleimide (BIM; a protein kinase C blocker), a protein kinase C inhibitory peptide or bafilomycin A (a lysosomal degradation inhibitor) each blocked the Ang II-induced decrease in total and surface KV 1.5. Immunofluorescence also suggested that Ang II reduced surface KV 1.5 protein in isolated myocytes; an effect inhibited by BIM. Arteries were exposed to Ang II or Ang II plus BIM (for 2 h), after which these agents were removed and contractility measurements performed or myocytes isolated for patch-clamp electrophysiology. Angiotensin II reduced both whole-cell KV currents and currents inhibited by Psora-4, a KV 1.5 channel blocker. Angiotensin II also reduced vasoconstriction stimulated by Psora-4 or 4-aminopyridine, another KV channel inhibitor. These data indicate that Ang II activates protein kinase C, which stimulates KV 1.5 channel degradation, leading to a decrease in surface KV 1.5, a reduction in whole-cell KV 1.5 currents and a loss of functional KV 1.5 channels in myocytes of pressurized arteries.

Rab25 influences functional Cav1.2 channel surface expression in arterial smooth muscle cells.

Plasma membrane-localized CaV1.2 channels are the primary calcium (Ca(2+)) influx pathway in arterial smooth muscle cells (myocytes). CaV1.2 channels regulate several cellular functions, including contractility and gene expression, but the trafficking pathways that control the surface expression of these proteins are unclear. Similarly, expression and physiological functions of small Rab GTPases, proteins that control vesicular trafficking in arterial myocytes, are poorly understood. Here, we investigated Rab proteins that control functional surface abundance of CaV1.2 channels in cerebral artery myocytes. Western blotting indicated that Rab25, a GTPase previously associated with apical recycling endosomes, is expressed in cerebral artery myocytes. Immunofluorescence Förster resonance energy transfer (immunoFRET) microscopy demonstrated that Rab25 locates in close spatial proximity to CaV1.2 channels in myocytes. Rab25 knockdown using siRNA reduced CaV1.2 surface and intracellular abundance in arteries, as determined using arterial biotinylation. In contrast, CaV1.2 was not located nearby Rab11A or Rab4 and CaV1.2 protein was unaltered by Rab11A or Rab4A knockdown. Rab25 knockdown resulted in CaV1.2 degradation by a mechanism involving both lysosomal and proteasomal pathways and reduced whole cell CaV1.2 current density but did not alter voltage dependence of current activation or inactivation in isolated myocytes. Rab25 knockdown also inhibited depolarization (20-60 mM K(+)) and pressure-induced vasoconstriction (myogenic tone) in cerebral arteries. These data indicate that Rab25 is expressed in arterial myocytes where it promotes surface expression of CaV1.2 channels to control pressure- and depolarization-induced vasoconstriction.

Mitochondria-localized caveolin in adaptation to cellular stress and injury.

We show here that the apposition of plasma membrane caveolae and mitochondria (first noted in electron micrographs >50 yr ago) and caveolae-mitochondria interaction regulates adaptation to cellular stress by modulating the structure and function of mitochondria. In C57Bl/6 mice engineered to overexpress caveolin specifically in cardiac myocytes (Cav-3 OE), localization of caveolin to mitochondria increases membrane rigidity (4.2%; P<0.05), tolerance to calcium, and respiratory function (72% increase in state 3 and 23% increase in complex IV activity; P<0.05), while reducing stress-induced generation of reactive oxygen species (by 20% in cellular superoxide and 41 and 28% in mitochondrial superoxide under states 4 and 3, respectively; P<0.05) in Cav-3 OE vs. TGneg. By contrast, mitochondrial function is abnormal in caveolin-knockout mice and Caenorhabditis elegans with null mutations in caveolin (60% increase free radical in Cav-2 C. elegans mutants; P<0.05). In human colon cancer cells, mitochondria with increased caveolin have a 30% decrease in apoptotic stress (P<0.05), but cells with disrupted mitochondria-caveolin interaction have a 30% increase in stress response (P<0.05). Targeted gene transfer of caveolin to mitochondria in C57Bl/6 mice increases cardiac mitochondria tolerance to calcium, enhances respiratory function (increases of 90% state 4, 220% state 3, 88% complex IV activity; P<0.05), and decreases (by 33%) cardiac damage (P<0.05). Physical association and apparently the transfer of caveolin between caveolae and mitochondria is thus a conserved cellular response that confers protection from cellular damage in a variety of tissues and settings.

Role of caveolin-3 and glucose transporter-4 in isoflurane-induced delayed cardiac protection.

BACKGROUND: Caveolae are small, flask-like invaginations of the plasma membrane. Caveolins are structural proteins found in caveolae that have scaffolding properties to allow organization of signaling. The authors tested the hypothesis that delayed cardiac protection induced by volatile anesthetics is caveolae or caveolin dependent. METHODS: An in vivo mouse model of ischemia-reperfusion injury with delayed anesthetic preconditioning (APC) was tested in wild-type, caveolin-1 knockout, and caveolin-3 knockout mice. Mice were exposed to 30 min of oxygen or isoflurane and allowed to recover for 24 h. After 24 h recovery, mice underwent 30-min coronary artery occlusion followed by 2 h of reperfusion at which time infarct size was determined. Biochemical assays were also performed in excised hearts. RESULTS: Infarct size as a percent of the area at risk was reduced by isoflurane in wild-type (24.0 +/- 8.8% vs. 45.1 +/- 10.1%) and caveolin-1 knockout mice (27.2 +/- 12.5%). Caveolin-3 knockout mice did not show delayed APC (41.5 +/- 5.0%). Microscopically distinct caveolae were observed in wild-type and caveolin-1 knockout mice but not in caveolin-3 knockout mice. Delayed APC increased the amount of caveolin-3 protein but not caveolin-1 protein in discontinuous sucrose-gradient buoyant fractions. In addition, glucose transporter-4 was increased in buoyant fractions, and caveolin-3/glucose transporter-4 colocalization was observed in wild-type and caveolin-1 knockout mice after APC. CONCLUSIONS: These results show that delayed APC involves translocation of caveolin-3 and glucose transporter-4 to caveolae, resulting in delayed protection in the myocardium.

Cardiac-specific overexpression of caveolin-3 induces endogenous cardiac protection by mimicking ischemic preconditioning.

BACKGROUND: Caveolae, lipid-rich microdomains of the sarcolemma, localize and enrich cardiac-protective signaling molecules. Caveolin-3 (Cav-3), the dominant isoform in cardiac myocytes, is a determinant of caveolar formation. We hypothesized that cardiac myocyte-specific overexpression of Cav-3 would enhance the formation of caveolae and augment cardiac protection in vivo. METHODS AND RESULTS: Ischemic preconditioning in vivo increased the formation of caveolae. Adenovirus for Cav-3 increased caveolar formation and phosphorylation of survival kinases in cardiac myocytes. A transgenic mouse with cardiac myocyte-specific overexpression of Cav-3 (Cav-3 OE) showed enhanced formation of caveolae on the sarcolemma. Cav-3 OE mice subjected to ischemia/reperfusion injury had a significantly reduced infarct size relative to transgene-negative mice. Endogenous cardiac protection in Cav-3 OE mice was similar to wild-type mice undergoing ischemic preconditioning; no increased protection was observed in preconditioned Cav-3 OE mice. Cav-3 knockout mice did not show endogenous protection and showed no protection in response to ischemic preconditioning. Cav-3 OE mouse hearts had increased basal Akt and glycogen synthase kinase-3beta phosphorylation comparable to wild-type mice exposed to ischemic preconditioning. Wortmannin, a phosphoinositide 3-kinase inhibitor, attenuated basal phosphorylation of Akt and glycogen synthase kinase-3beta and blocked cardiac protection in Cav-3 OE mice. Cav-3 OE mice had improved functional recovery and reduced apoptosis at 24 hours of reperfusion. CONCLUSIONS: Expression of caveolin-3 is both necessary and sufficient for cardiac protection, a conclusion that unites long-standing ultrastructural and molecular observations in the ischemic heart. The present results indicate that increased expression of caveolins, apparently via actions that depend on phosphoinositide 3-kinase, has the potential to protect hearts exposed to ischemia/reperfusion injury.

Caveolin-3 expression and caveolae are required for isoflurane-induced cardiac protection from hypoxia and ischemia/reperfusion injury.

Volatile anesthetics protect the heart from ischemia/reperfusion injury but the mechanisms for this protection are poorly understood. Caveolae, sarcolemmal invaginations, and caveolins, scaffolding proteins in caveolae, localize molecules involved in cardiac protection. We tested the hypothesis that caveolae and caveolins are essential for volatile anesthetic-induced cardiac protection using cardiac myocytes (CMs) from adult rats and in vivo studies in caveolin-3 knockout mice (Cav-3(-/-)). We incubated CM with methyl-beta-cyclodextrin (MbetaCD) or colchicine to disrupt caveolae formation, and then exposed the myocytes to the volatile anesthetic isoflurane (30 min, 1.4%), followed by simulated ischemia/reperfusion (SI/R). Isoflurane protected CM from SI/R [23.2+/-1.6% vs. 71.0+/-5.8% cell death (assessed by trypan blue exclusion), P<0.001] but this protection was abolished by MbetaCD or colchicine (84.9+/-5.5% and 64.5+/-6.1% cell death, P<0.001). Membrane fractionation by sucrose density gradient centrifugation of CM treated with MbetaCD or colchicine revealed that buoyant (caveolae-enriched) fractions had decreased phosphocaveolin-1 and caveolin-3 compared to control CM. Cardiac protection in vivo was assessed by measurement of infarct size relative to the area at risk and cardiac troponin levels. Isoflurane-induced a reduction in infarct size and cardiac troponin relative to control (infarct size: 26.5%+/-2.6% vs. 45.3%+/-5.4%, P<0.01; troponin: 27.7+/-4.4 vs. 77.7+/-11.8 ng/ml, P<0.05). Isoflurane-induced cardiac protection was abolished in Cav-3(-/-) mice (infarct size: 53.4%+/-6.1% vs. 53.2%+/-3.5%, P<0.01; troponin: 102.1+/-22.3 vs. 105.9+/-8.2 ng/ml, P<0.01). Isoflurane-induced cardiac protection is thus dependent on the presence of caveolae and the expression of caveolin-3. We conclude that caveolae and caveolin-3 are critical for volatile anesthetic-induced protection of the heart from ischemia/reperfusion injury.

Intravascular pressure enhances the abundance of functional Kv1.5 channels at the surface of arterial smooth muscle cells.

Voltage-dependent potassium (K(v)) channels are present in various cell types, including smooth muscle cells (myocytes) of resistance-sized arteries that control systemic blood pressure and regional organ blood flow. Intravascular pressure depolarizes arterial myocytes, stimulating calcium (Ca(2+)) influx through voltage-dependent Ca(2+) (Ca(v)) channels that results in vasoconstriction and also K(+) efflux through K(v) channels that oppose vasoconstriction. We hypothesized that pressure-induced depolarization may not only increase the open probability of plasma membrane-resident K(v) channels but also increase the abundance of these channels at the surface of arterial myocytes to limit vasoconstriction. We found that K(v)1.5 and K(v)2.1 proteins were abundant in the myocytes of resistance-sized mesenteric arteries. K(v)1.5, but not K(v)2.1, continuously recycled between the intracellular compartment and the plasma membrane in contractile arterial myocytes. Using ex vivo preparations of intact arteries, we showed that physiological intravascular pressure through membrane depolarization or membrane depolarization in the absence of pressure inhibited the degradation of internalized K(v)1.5 and increased recycling of K(v)1.5 to the plasma membrane. Accordingly, by stimulating the activity of Ca(v)1.2, membrane depolarization increased whole-cell K(v)1.5 current density in myocytes and K(v)1.5 channel activity in pressurized arteries. In contrast, the total amount and cell surface abundance of K(v)2.1 were independent of intravascular pressure or membrane potential. Thus, our data indicate that intravascular pressure-induced membrane depolarization selectively increased K(v)1.5 surface abundance to increase K(v) currents in arterial myocytes, which would limit vasoconstriction.

An elevation in physical coupling of type 1 inositol 1,4,5-trisphosphate (IP3) receptors to transient receptor potential 3 (TRPC3) channels constricts mesenteric arteries in genetic hypertension.

Hypertension is associated with an elevation in agonist-induced vasoconstriction, but mechanisms involved require further investigation. Many vasoconstrictors bind to phospholipase C-coupled receptors, leading to an elevation in inositol 1,4,5-trisphosphate (IP(3)) that activates sarcoplasmic reticulum IP(3) receptors. In cerebral artery myocytes, IP(3) receptors release sarcoplasmic reticulum Ca(2+) and can physically couple to canonical transient receptor potential 3 (TRPC3) channels in a caveolin-1-containing macromolecular complex, leading to cation current activation that stimulates vasoconstriction. Here, we investigated mechanisms by which IP(3) receptors control vascular contractility in systemic arteries and IP(3)R involvement in elevated agonist-induced vasoconstriction during hypertension. Total and plasma membrane-localized TRPC3 protein was ≈2.7- and 2-fold higher in mesenteric arteries of spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto (WKY) rat controls, respectively. In contrast, IP(3)R1, TRPC1, TRPC6, and caveolin-1 expression was similar. TRPC3 expression was also similar in arteries of pre-SHRs and WKY rats. Control, IP(3)-induced and endothelin-1 (ET-1)-induced fluorescence resonance energy transfer between IP3R1 and TRPC3 was higher in SHR than WKY myocytes. IP3-induced cation current was ≈3-fold larger in SHR myocytes. Pyr3, a selective TRPC3 channel blocker, and calmodulin and IP(3) receptor binding domain peptide, an IP(3)R-TRP physical coupling inhibitor, reduced IP(3)-induced cation current and ET-1-induced vasoconstriction more in SHR than WKY myocytes and arteries. Thapsigargin, a sarcoplasmic reticulum Ca(2+)-ATPase blocker, did not alter ET-1-stimulated vasoconstriction in SHR or WKY arteries. These data indicate that ET-1 stimulates physical coupling of IP(3)R1 to TRPC3 channels in mesenteric artery myocytes, leading to vasoconstriction. Furthermore, an elevation in IP(3)R1 to TRPC3 channel molecular coupling augments ET-1-induced vasoconstriction during hypertension.

Role of decoy molecules in neuronal ischemic preconditioning.

AIMS: Decoy receptors bind with TNF related apoptosis inducing ligands (TRAIL) but do not contain the cytoplasmic domains necessary to transduce apoptotic signals. We hypothesized that decoy receptors may confer neuronal protection against lethal ischemia after ischemic preconditioning (IPC). MAIN METHOD: Mixed cortical neurons were exposed to IPC one day prior to TRAIL treatment or lethal ischemia. KEY FINDINGS: IPC increased decoy receptor but reduced death receptor expression compared to lethal ischemia. IPC-induced increase in decoy receptor expression was reduced by prior treatment with CAPE, a nuclear factor-kappa B inhibitor (NFκB). SIGNIFICANCE: Expression of decoy molecules, dependent on NFκB, may mediate neuronal survival induced by IPC.