Caveolae Vesicles Research Articles

Endothelial and mural laminin-α5 contributes to neurovascular integrity maintenance

BackgroundLaminin-α5, a major component of the basal lamina, is predominantly synthesized by endothelial and mural cells (pericytes and vascular smooth muscle cells) in the CNS. Loss of laminin-α5 in either population fails to induce any abnormalities due to functional redundancy. Thus, the functional significance of laminin-α5 in neurovascular integrity remains unknown. Here, we hypothesize that ablation of laminin-α5 in both endothelial and mural cells increases neurovascular permeability.MethodsThe compound knockout mice were generated by crossing laminin-α5 floxed mice with Tie2-Cre and PDGFRβ-Cre, which target endothelial cells and mural cells, respectively. Neurovascular permeability in these mutants was determined with both exogenous and endogenous tracers. Endothelial paracellular and transcellular permeability was assessed by examining the expression of tight junction proteins and transcytosis-associated proteins. In addition, transmission electron microscopy (TEM) was used to visualize tight junction ultrastructure and endothelial caveolae vesicles. Defects in pericytes and astrocytes were investigated by examining pericyte coverage/contact and astrocyte polarity.ResultsElevated neurovascular permeability was observed in the mutants. Subsequent studies found increased Caveolin-1 and decreased major facilitator superfamily domain-containing protein 2a (MFSD2A) expression, but unaltered Claudin-5 or zonula occludens-1 (ZO-1) expression. Consistent with these results, mutant mice exhibited increased endothelial caveolae vesicle number with intact tight junction structure under TEM. Additionally, pericyte coverage and contact were also decreased in the mutant mice, while astrocyte polarity was unaffected.ConclusionsThese results strongly indicate that endothelial and mural cell-derived laminin-α5 actively maintains neurovascular integrity via the transcellular rather than paracellular mechanism.

Sound conditioning strategy promoting paracellular permeability of the blood-labyrinth-barrier benefits inner ear drug delivery.

The therapeutic effects of pharmaceuticals depend on their drug concentrations in the cochlea. Efficient drug delivery from the systemic circulation into the inner ear is limited by the blood-labyrinth-barrier (BLB). This study investigated a novel noninvasive sound conditioning (SC) strategy (90 dB SPL, 8-16 kHz, 2 h sound exposure) to temporally enhance BLB permeability in a controllable way, contributing to maximizing the penetration of pharmaceuticals from blood circulation into the cochlea. Trafficking of Fluorescein Isothiocyanate conjugated dextran and bovine serum albumin (FITC-dextran and FITC-BSA) demonstrated that paracellular leakage of BLB sustained for 6 h after SC, providing a controllable time window for systemic administration. Cochlear concentrations of dexamethasone (DEX) and dexamethasone phosphate (DEX-P), respectively transported by transcellular and paracellular pathways, showed a higher content of the latter one after SC, further confirming the key role of paracellular pathway in the SC-induced hyperpermeability. Results of high-throughput RNA-sequencing identified a series of tight junction (TJ)-associated genes after SC. The expressions of TJ (ZO-1) were reduced and irregular rearrangements of the junction were observed by transmission electron microscopy after SC. We further determined the inhibiting role of Rab13 in the recruitment of ZO-1 and later in the regulation of cellular permeability. Meanwhile, no significant change in the quantifications of endothelial caveolae vesicles after SC indicated that cellular transcytosis accounted little for the temporary hyperpermeability after SC. Based on these results, SC enhances the BLB permeability within 6 h and allows systemically applied drugs which tend to be transported by paracellular pathway to readily enter the inner ear, contributing to guiding the clinical medications on hearing loss.

Endothelial cell CD36 regulates membrane ceramide formation, exosome fatty acid transfer and circulating fatty acid levels

Endothelial cell (EC) CD36 controls tissue fatty acid (FA) uptake. Here we examine how ECs transfer FAs. FA interaction with apical membrane CD36 induces Src phosphorylation of caveolin-1 tyrosine-14 (Cav-1Y14) and ceramide generation in caveolae. Ensuing fission of caveolae yields vesicles containing FAs, CD36 and ceramide that are secreted basolaterally as small (80–100 nm) exosome-like extracellular vesicles (sEVs). We visualize in transwells EC transfer of FAs in sEVs to underlying myotubes. In mice with EC-expression of the exosome marker emeraldGFP-CD63, muscle fibers accumulate circulating FAs in emGFP-labeled puncta. The FA-sEV pathway is mapped through its suppression by CD36 depletion, blocking actin-remodeling, Src inhibition, Cav-1Y14 mutation, and neutral sphingomyelinase 2 inhibition. Suppression of sEV formation in mice reduces muscle FA uptake, raises circulating FAs, which remain in blood vessels, and lowers glucose, mimicking prominent Cd36−/− mice phenotypes. The findings show that FA uptake influences membrane ceramide, endocytosis, and EC communication with parenchymal cells.

Unique features of the arterial blood–brain barrier

CNS vasculature differs from vascular networks of peripheral organs by its ability to tightly control selective material exchange across capillary barriers. Capillary permeability is mostly defined by unique cellular components of the endothelium. While capillaries are extensively investigated, the barrier properties of larger vessels are understudied. Here, we investigate barrier properties of CNS arterial walls. Using tracer challenges and various imaging modalities, we discovered that at the mouse cortex, the arterial barrier does not reside at the classical level of the endothelium. The arterial wall’s unique permeability acts bi-directionally; CSF substances travel along the glymphatic path and can penetrate from the peri-vascular space through arteriolar walls towards the lumen. We found that caveolae vesicles in arteriole endothelial are functional transcytosis machinery components, and that a similar mechanism is evident in the human brain. Our discoveries highlight vascular heterogeneity investigations as a potent approach to uncover new barrier mechanisms.

Caveolae-Mediated Extracellular Vesicle (CMEV) Signaling of Polyvalent Polysaccharide Vaccination: A Host-Pathogen Interface Hypothesis.

We published a study showing that improvement in response to splenectomy associated defective, in regards to the antibody response to Pneumovax® 23 (23-valent polysaccharides, PPSV23), can be achieved by splenocyte reinfusion. This study triggered a debate on whether and how primary and secondary immune responses occur based on humoral antibody responses to the initial vaccination and revaccination. The anti-SARS-CoV-2 vaccine sheds new light on the interpretation of our previous data. Here, we offer an opinion on the administration of the polyvalent polysaccharide vaccine (PPSV23), which appears to be highly relevant to the primary vaccine against SARS-CoV-2 and its booster dose. Thus, we do not insist this is a secondary immune response but an antibody response, nonetheless, as measured through IgG titers after revaccination. However, we contend that we are not sure if these lower but present IgG levels against pneumococcal antigens are clinically protective or are equally common in all groups because of the phenomenon of "hyporesponsiveness" seen after repeated polysaccharide vaccine challenge. We review the literature and propose a new mechanism-caveolae memory extracellular vesicles (CMEVs)-by which polysaccharides mediate prolonged and sustained immune response post-vaccination. We further delineate and explain the data sets to suggest that the dual targets on both Cav-1 and SARS-CoV-2 spike proteins may block the viral entrance and neutralize viral load, which minimizes the immune reaction against viral attacks and inflammatory responses. Thus, while presenting our immunological opinion, we answer queries and responses made by readers to our original statements published in our previous work and propose a hypothesis for all vaccination strategies, i.e., caveolae-mediated extracellular vesicle-mediated vaccine memory.

Trans-2-Enoyl-CoA Reductase Tecr-Driven Lipid Metabolism in Endothelial Cells Protects against Transcytosis to Maintain Blood-Brain Barrier Homeostasis.

The transport and metabolism of lipids in cerebrovascular endothelial cells (ECs) have been hypothesized to regulate blood-brain barrier (BBB) maturation and homeostasis. Long-chain polyunsaturated fatty acids (LCPUFAs) as the important lipids components of cell membranes are essential for the development and function of BBB, but the direct links of lipid metabolism and ECs barrier function remain to be established. Here, we comprehensively characterize the transcriptomic phenotype of developmental cerebrovascular ECs in single-cell resolution and firstly find that trans-2-enoyl-CoA reductase (Tecr), a very-long-chain fatty acid synthesis, is highly expressed during barriergenesis and decreased after BBB maturation. EC-specific knockout of Tecr compromises angiogenesis due to delayed vascular sprouting. Importantly, EC-specific deletion of Tecr loss restrictive quality of vascular permeability from neonatal stages to adulthood, with high levels of transcytosis, but maintains the vascular tight junctions. Moreover, lipidomic analysis shows that the expression of Tecr in ECs is associated with the containing of omega-3 fatty acids, which directly suppresses caveolae vesicles formation. These results reveal a protective role for Tecr in BBB integrity and suggest that Tecr as a novel therapeutic target in the central nervous system (CNS) diseases associated with BBB dysfunction.

Fluorescent labeling in size-controlled liposomes reveals membrane curvature-induced structural changes in the KcsA potassium channel.

Biological structures with highly curved membranes, such as caveolae and transport vesicles, are essential for signal transduction and membrane trafficking. Although membrane proteins in these structures are subjected to physical stress due to the curvature of the lipid bilayers, the effect of this membrane curvature on protein structure and function remains unclear. In this study, we established an experimental procedure to evaluate membrane curvature-induced structural changes in the prototypical potassium channel KcsA. The effect of a large membrane curvature was estimated using fluorescently labeled KcsA by incorporating it into liposomes with a small diameter (<30nm). We found that a large membrane curvature significantly affects the activation gate conformation of the KcsA channel.

Pathogenesis evidence from human and animal models of detrusor underactivity

ABSTRACTDetrusor underactivity (DU) is a common urodynamic diagnosis in patients with lower urinary tract symptoms and large post-voiding residual volume. Animal and human studies showed the possible etiologies of DU include central or peripheral nerve injury, bladder outlet obstruction, chronic ischemia, aging, diabetes mellitus, and sympathetic inhibition of micturition reflex. Evidence from animal and human DU studies with various etiologies revealed highly similar gross and histological characteristics in the bladders, including increased bladder weight, bladder wall thickening, inflammation, collagen deposition, and fibrosis. In electron microscopy, smooth muscle destruction, swollen mitochondria, decreased nerve innervation, caveolae, and umbrella cell fusiform vesicles were noted in the DU bladders. Most animal DU models demonstrate detrusor contractility changes from compensatory to the decompensatory stage, and the change was compatible with human DU observation. The cystometry in the DU animal studies is characterized by impaired contractility, prolong intercontraction interval, and hyposensation, while in vitro bladder muscle strips experiment may exhibit normal detrusor contractility. Decreased bladder blood flow and increased oxidative stress in bladders had been proved in different animal DU models, suggesting they should be important in the DU pathogenesis pathway. Sensory receptors mRNA and protein expression changes in DU bladders had been observed in both animal and human studies, including muscarinic receptors M2, M3, adrenergic receptor β3, purinergic receptor P2X1, P2X3, and transient receptor potential vanilloid (TRPV) 1 and TRPV4. Although some of the sensory receptors changes remain controversial, it might be the target for further pharmacologic treatments.

PKCζ-NADPH Oxidase-PKCα Dependent Kv1.5 Phosphorylation by Endothelin-1 Modulates Nav1.5-NCX1-Cav1.2 Axis in Stimulating Ca2+ Level in Caveolae of Pulmonary Artery Smooth Muscle Cells.

Endothelin-1 (ET-1) is a potent endogenously derived vasoconstrictor, which increases pulmonary hypertension via stimulation of [Ca2+]i level in pulmonary artery smooth muscle cells (PASMCs). In this communication, we sought to investigate the mechanism by which ET-1 causes stimulation of Ca2+ concentration in caveolae vesicles of bovine PASMCs (BPASMCs). ET-1 activates PKC-α in the caveolae vesicles by O2.- derived from PKCζ-NADPH oxidase dependent pathway. PKC-α phosphorylates Kv1.5 channels leading to a marked stimulation of Na+ and Ca2+ concentration in the caveolae vesicles. The stimulation of Ca2+ concentration in the caveolae vesicles by ET-1 occurs predominantly via Cav1.2 channels. Additionally, an increase in Na+ concentration by ET-1 due to stimulation of Nav1.5 channels marginally increases Ca2+ level in the caveolae vesicles via reverse-mode Na+/Ca2+ exchanger (NCX-1) and also through "slip-mode conductance" Nav1.5 channels. 4-AP, a well-known inhibitor of Kv channels, also increases Ca2+ concentration in the caveolae vesicles via Cav1.2 channels, reverse-mode NCX-1 and Nav1.5 channels by phosphorylation independent modulation of Kv1.5 channels without the involvement of PKCζ-NADPH oxidase-PKCα signaling axis. Overall, PKCζ-NADPH oxidase-PKCα dependent phosphorylation of Kv1.5 by ET-1 modulates Nav1.5-NCX1-Cav1.2 axis for stimulation of Ca2+ concentration in caveolae vesicles of BPASMCs, which provides a crucial mechanism for better understanding of ET-1-mediated modulation of pulmonary vascular tone.

Tuning Nanoparticle Interactions with Ovarian Cancer through Layer-by-Layer Modification of Surface Chemistry.

Nanoparticle surface chemistry is a fundamental engineering parameter that governs tumor-targeting activity. Electrostatic assembly generates controlled polyelectrolyte complexes through the process of adsorption and charge overcompensation utilizing synthetic polyions and natural biomacromolecules; it can yield films with distinctive hydration, charge, and presentation of functional groups. Here, we used electrostatic layer-by-layer (LbL) assembly to screen 10 different surface chemistries for their ability to preferentially target human ovarian cancer in vitro. Our screen identified that poly-l-aspartate, poly-l-glutamate, and hyaluronate-coated LbL nanoparticles have striking specificity for ovarian cancer, while sulfated poly(β-cyclodextrin) nanoparticles target noncancerous stromal cells. We validated top candidates for tumor-homing ability with a murine model of metastatic disease and with patient-derived ovarian cancer spheroids. Nanoparticle surface chemistry also influenced subcellular trafficking, indicating strategies to target the cell membrane, caveolae, and perinuclear vesicles. Our results confirm LbL is a powerful tool to systematically engineer nanoparticles and achieve specific targeting.

Low Density Lipoproteinemia in Obese and Type-2 Diabetics from Association of IR and LDLR

Universality of association of two receptors, LDLR (LDL receptor) and IR (insulin receptor), has been explored in various cell types and intracellular compartments including caveolae vesicles in plasma membrane. The existence of association of LDLR and IR has been proved in type-2 diabetes and diabetes associated obesity. The non-functionality of LDLR in conjugation with IR has been found as of a prime reason for developing LDL related atherosclerosis in insulin defunct system. Confocal microcopy and western blots are the major techniques used to explore this study.

Fam198a, a member of secreted kinase, secrets through caveolae biogenesis pathway.

Fam198a is a member of four-jointed protein kinases, a secreted protein kinase family. It was identified as a caveolae-associated protein and colocalized with cavin-1 and caveolin-1 in both tissues and cells. The newly synthesized Fam198a precursor in endoplasmic reticulum (ER) was transported by caveolae biogenesis vesicles to Golgi apparatus in which it was proteolytically cleaved into the secreted mature form. The amino acid mutation analysis identified Arg 120 and 437 as the proteolytic sites in Fam198a precursor during maturation. In mouse embryo fibroblasts (MEFs) obtained from cavin-1-/- or caveolin-1-/- mice, Fam198a precursor was retained in ER and no mature Fam198a could be formed in these cells. Ectopic expression of exogenous cavin-1 in cavin-1-/- MEFs restored the blocked Fam198a post-translational process and secretion. Cavin-1 was also required for Fam198a secretion after its maturation in Golgi apparatus. Ectopic expression of cavin-1 in A549 cells restored the blocked Fam198a secretion. These results suggest that protein secretion is an important function for caveolae biogenesis pathway and the disruption of caveolae system will affect those functions played by the secreted proteins.

Crotamine-like from Southern Pacific rattlesnake (Crotalus oreganus helleri) Venom acts on human leukemia (K-562) cell lines and produces ultrastructural changes on mice adrenal gland

ABSTRACTCrotamine is a cationic, non-enzymatic, protein integrating a minor family of myotoxins, composed of 42 amino acid residues, described in Viperidae and Crotalidae snake’s families that has been used in neuroscience research, drug progressing and molecular diversity reports. Crotamine-like protein (CLP) from C.o.helleri venom was isolated in fraction 5 from 7 peaks obtained by sulfopropyl waters protein pak cationic exchange column. In tricine-SDS-PAGE under non-reduced conditions this CLP showed a single band of ~8 kDa molecular weight. CLP induced toxicity of K-562 cells with a CC50 of 11.09 µM. In mice adrenal gland after 24 h of CLP injection, cortical cells exhibited swollen mitochondria with scarce tubular cristae, some elements of smooth and rough endoplasmic reticula, widened nuclear envelope, slightly osmiophilic lipid droplets, and autophagic vacuoles. In some areas cortical cells plasma membrane and endothelial walls disappeared, which indicated a necrosis process. In other areas, endothelial cell cytoplasm did not present the normal caveolae and pinocytotic vesicles. To our knowledge, this is the first report on mice adrenal gland damages, caused by the injection of CLP from rattlesnakes. Our results propose that adrenal cortex lesions may be significant in the envenoming etiopathogenesis by CLP.

Adherens junctions and desmosomes are damaged by Entamoeba histolytica: Participation of EhCPADH complex and EhCP112 protease.

Entamoeba histolytica trophozoites adhere to epithelium at the cell-cell contact and perturb tight junctions disturbing the transepithelial electrical resistance. Behind tight junctions are the adherens junctions (AJs) that reinforce them and the desmosomes (DSMs) that maintain the epithelium integrity. The damage produced to AJs and DMSs by this parasite is unknown. Here, we studied the effect of the trophozoites, the EhCPADH complex, and the EhCP112 recombinant enzyme (rEhCP112) on AJ and DSM proteins. We found that trophozoites degraded β-cat, E-cad, Dsp l/ll, and Dsg-2 with the participation of EhCPADH and EhCP112. After contact of epithelial cells with trophozoites, immunofluorescence and transmission electron microscopy assays revealed EhCPADH and rEhCP112 at the intercellular space where they colocalised with β-cat, E-cad, Dsp l/ll, and Dsg-2. Moreover, our results suggested that rEhCP112 could be internalised by caveolae and clathrin-coated vesicles. Immunoprecipitation assays showed the interaction of EhCPADH with β-cat and Dsp l/ll. Besides, in vivo assays demonstrated that rEhCP112 concentrates at the cellular borders of the mouse intestine degrading E-cad and Dsp I/II. Our research gives the first clues on the trophozoite attack to AJs and DSMs and point out the role of the EhCPADH and EhCP112 in the multifactorial event of trophozoites virulence.

Internalization of the TGF-β type I receptor into caveolin-1 and EEA1 double-positive early endosomes.

Endocytosis and intracellular sorting of transforming growth factor-β (TGF-β) receptors play an important regulatory role in TGF-β signaling. Two major endocytic pathways, clathrin- and caveolae-mediated endocytosis, have been reported to independently mediate the internalization of TGF-β receptors. In this study, we demonstrate that the clathrin- and caveolae-mediated endocytic pathways can converge during TGF-β receptor endocytic trafficking. By tracking the intracellular dynamics of fluorescently-labeled TGF-β type I receptor (TβRI), we found that after mediating TβRI internalization, certain clathrin-coated vesicles and caveolar vesicles are fused underneath the plasma membrane, forming a novel type of caveolin-1 and clathrin double-positive vesicles. Under the regulation of Rab5, the fused vesicles are targeted to early endosomes and thus deliver the internalized TβRI to the caveolin-1 and EEA1 double-positive early endosomes (caveolin-1-positive early endosomes). We further showed that the caveolin-1-positive early endosomes are positive for Smad3/SARA, Rab11 and Smad7/Smurf2, and may act as a multifunctional device for TGF-β signaling and TGF-β receptor recycling and degradation. Therefore, these findings uncover a novel scenario of endocytosis, the direct fusion of clathrin-coated and caveolae vesicles during TGF-β receptor endocytic trafficking, which leads to the formation of the multifunctional sorting device, caveolin-1-positive early endosomes, for TGF-β receptors.

Anti-VEGF– and anti-VEGF receptor–induced vascular alteration in mouse healthy tissues

Systemic therapy with anti-VEGF drugs such as bevacizumab is widely used for treatment of human patients with various solid tumors. However, systemic impacts of such drugs in host healthy vasculatures remain poorly understood. Here, we show that, in mice, systemic delivery of an anti-VEGF or an anti-VEGF receptor (VEGFR)-2 neutralizing antibody caused global vascular regression. Among all examined tissues, vasculatures in endocrine glands, intestinal villi, and uterus are the most affected in response to VEGF or VEGFR-2 blockades. Thyroid vascular fenestrations were virtually completely blocked by VEGF blockade, leading to marked accumulation of intraendothelial caveolae vesicles. VEGF blockade markedly increased thyroid endothelial cell apoptosis, and withdrawal of anti-VEGF resulted in full recovery of vascular density and architecture after 14 d. Prolonged anti-VEGF treatment resulted in a significant decrease of the circulating level of the predominant thyroid hormone free thyroxine, but not the minimal isoform of triiodothyronine, suggesting that chronic anti-VEGF treatment impairs thyroid functions. Conversely, VEGFR-1-specific blockade produced virtually no obvious phenotypes. These findings provide structural and functional bases of anti-VEGF-specific drug-induced side effects in relation to vascular changes in healthy tissues. Understanding anti-VEGF drug-induced vascular alterations in healthy tissues is crucial to minimize and even to avoid adverse effects produced by currently used anti-VEGF-specific drugs.

M-Calpain-mediated cleavage of Na+/Ca2+ exchanger-1 in caveolae vesicles isolated from pulmonary artery smooth muscle

Using m-calpain antibody, we have identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of m-calpain in caveolae vesicles isolated from bovine pulmonary artery smooth muscle plasma membrane. In addition, 78, 35, and 18 kDa immunoreactive bands of m-calpain have also been detected. Casein zymogram studies also revealed the presence of m-calpain in the caveolae vesicles. We have also identified Na(+)/Ca(2+) exchanger-1 (NCX1) in the caveolae vesicles. Purification and N-terminal sequence analyses of these two proteins confirmed their identities as m-calpain and NCX1, respectively. We further sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of NCX1 in the caveolae vesicles. Treatment of the caveolae vesicles with the calcium ionophore, A23187 (1 microM) in presence of CaCl(2) (1 mM) appears to cleave NCX1 (120 kDa) to an 82 kDa fragment as revealed by immunoblot study using NCX1 monoclonal antibody; while pretreatment with the calpain inhibitors, calpeptin or MDL28170; or the Ca(2+) chelator, BAPTA-AM did not cause a discernible change in the NCX protein profile. In vitro cleavage of the purified NCX1 by the purified m-calpain supports this finding. The cleavage of NCX1 by m-calpain in the caveolae vesicles may be interpreted as an important mechanism of Ca(2+) overload, which could arise due to inhibition of Ca(2+) efflux by the forward-mode NCX and that could lead to sustained Ca(2+) overload in the smooth muscle leading to pulmonary hypertension.

Cellular Internalization of Exosomes Occurs Through Phagocytosis

Exosomes play important roles in many physiological and pathological processes. However, the exosome-cell interaction mode and the intracellular trafficking pathway of exosomes in their recipient cells remain unclear. Here, we report that exosomes derived from K562 or MT4 cells are internalized more efficiently by phagocytes than by non-phagocytic cells. Most exosomes were observed attached to the plasma membrane of non-phagocytic cells, while in phagocytic cells these exosomes were found to enter via phagocytosis. Specifically, they moved to phagosomes together with phagocytic polystyrene carboxylate-modified latex beads (biospheres) and were further sorted into phagolysosomes. Moreover, exosome internalization was dependent on the actin cytoskeleton and phosphatidylinositol 3-kinase, and could be inhibited by the knockdown of dynamin2 or overexpression of a dominant-negative form of dynamin2. Further, antibody pretreatment assays demonstrated that tim4 but not tim1 was involved in exosomes uptake. We also found that exosomes did not enter the internalization pathway involving caveolae, macropinocytosis and clathrin-coated vesicles. Our observation that the cellular uptake of exosomes occurs through phagocytosis has important implications for exosome-cell interactions and the exosome intracellular trafficking pathway.

Solubilization, purification, and reconstitution of α2β1 isozyme of Na+/K+-ATPase from caveolae of pulmonary smooth muscle plasma membrane: comparative studies with DHPC, C12E8, and Triton X-100

We identified alpha(2), alpha(1), and beta(1) isoforms of Na(+)/K(+)-ATPase in caveolae vesicles of bovine pulmonary smooth muscle plasma membrane. The biochemical and biophysical characteristics of the alpha(2)beta(1) isozyme of Na(+)/K(+)-ATPase from caveolae vesicles were studied during solubilization and purification using the detergents 1,2-heptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)), and Triton X-100, and reconstitution with the phospholipid dioleoyl-phosphatidylcholine (DOPC). DHPC was determined to be superior to C(12)E(8), whereas C(12)E(8) was better than Triton X-100 in the active enzyme yields and specific activity. Fluorescence studies with DHPC-purified alpha(2)beta(1) isozyme of Na(+)/K(+)-ATPase elicited higher E1Na-E2 K transition compared with that of the C(12)E(8)- and Triton X-100-purified enzyme. The rate of Na(+) efflux in DHPC-DOPC-reconstituted isozyme was higher compared to the C(12)E(8)-DOPC- and Triton X100-DOPC-reconstituted enzyme. Circular dichroism analysis suggests that the DHPC-purified alpha(2)beta(1) isozyme of Na(+)/K(+)-ATPase possessed more organized secondary structure compared to the C(12)E(8)- and Triton X-100-purified isozyme.

Ca 2+ influx mechanisms in caveolae vesicles of pulmonary smooth muscle plasma membrane under inhibition of α 2β 1 isozyme of Na +/K +-ATPase by ouabain

Aims We sought to determine the mechanisms of an increase in Ca 2+ level in caveolae vesicles in pulmonary smooth muscle plasma membrane during Na +/K +-ATPase inhibition by ouabain. Main methods The caveolae vesicles isolated by density gradient centrifugation were characterized by electron microscopic and immunologic studies and determined ouabain induced increase in Na + and Ca 2+ levels in the vesicles with fluorescent probes, SBFI-AM and Fura2-AM, respectively. Key findings We identified the α 2β 1 and α 1β 1 isozymes of Na +/K +-ATPase in caveolae vesicles, and only the α 1β 1 isozyme in noncaveolae fraction of the plasma membrane. The α 2-isoform contributes solely to the enzyme inhibition in the caveolae vesicles at 40 nM ouabain. Methylisobutylamiloride (Na +/H +-exchange inhibitor) and tetrodotoxin (voltage-gated Na +-channel inhibitor) pretreatment prevented ouabain induced increase in Na + and Ca 2+ levels. Ouabain induced increase in Ca 2+ level was markedly, but not completely, inhibited by KB-R7943 (reverse-mode Na +/Ca 2+-exchange inhibitor) and verapamil (L-type Ca 2+-channel inhibitor). However, pretreatment with tetrodotoxin in conjunction with KB-R7943 and verapamil blunted ouabain induced increase in Ca 2+ level in the caveolae vesicles, indicating that apart from Na +/Ca +-exchanger and L-type Ca 2+-channels, “slip-mode conductance” of Na + channels could also be involved in this scenario. Significance Inhibition of α 2 isoform of Na +/K +-ATPase by ouabain plays a crucial role in modulating the Ca 2+ influx regulatory components in the caveolae microdomain for marked increase in (Ca 2+) i in the smooth muscle, which could be important for the manifestation of pulmonary hypertension.