Hemal H. Patel

Interaction of membrane/lipid rafts with the cytoskeleton: impact on signaling and function: membrane/lipid rafts, mediators of cytoskeletal arrangement and cell signaling.

The plasma membrane in eukaryotic cells contains microdomains that are enriched in certain glycosphingolipids, gangliosides, and sterols (such as cholesterol) to form membrane/lipid rafts (MLR). These regions exist as caveolae, morphologically observable flask-like invaginations, or as a less easily detectable planar form. MLR are scaffolds for many molecular entities, including signaling receptors and ion channels that communicate extracellular stimuli to the intracellular milieu. Much evidence indicates that this organization and/or the clustering of MLR into more active signaling platforms depends upon interactions with and dynamic rearrangement of the cytoskeleton. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to MLR and help regulate lateral diffusion of membrane proteins and lipids in response to extracellular events (e.g., receptor activation, shear stress, electrical conductance, and nutrient demand). MLR regulate cellular polarity, adherence to the extracellular matrix, signaling events (including ones that affect growth and migration), and are sites of cellular entry of certain pathogens, toxins and nanoparticles. The dynamic interaction between MLR and the underlying cytoskeleton thus regulates many facets of the function of eukaryotic cells and their adaptation to changing environments. Here, we review general features of MLR and caveolae and their role in several aspects of cellular function, including polarity of endothelial and epithelial cells, cell migration, mechanotransduction, lymphocyte activation, neuronal growth and signaling, and a variety of disease settings. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

Microtubules and Actin Microfilaments Regulate Lipid Raft/Caveolae Localization of Adenylyl Cyclase Signaling Components

Microtubules and actin filaments regulate plasma membrane topography, but their role in compartmentation of caveolae-resident signaling components, in particular G protein-coupled receptors (GPCR) and their stimulation of cAMP production, has not been defined. We hypothesized that the microtubular and actin cytoskeletons influence the expression and function of lipid rafts/caveolae, thereby regulating the distribution of GPCR signaling components that promote cAMP formation. Depolymerization of microtubules with colchicine (Colch) or actin microfilaments with cytochalasin D (CD) dramatically reduced the amount of caveolin-3 in buoyant (sucrose density) fractions of adult rat cardiac myocytes. Colch or CD treatment led to the exclusion of caveolin-1, caveolin-2, β1-adrenergic receptors (β1-AR), β2-AR, Gαs, and adenylyl cyclase (AC)5/6 from buoyant fractions, decreasing AC5/6 and tyrosine-phosphorylated caveolin-1 in caveolin-1 immunoprecipitates but in parallel increased isoproterenol (β-AR agonist)-stimulated cAMP production. Incubation with Colch decreased co-localization (by immunofluorescence microscopy) of caveolin-3 and α-tubulin; both Colch and CD decreased co-localization of caveolin-3 and filamin (an F-actin cross-linking protein), decreased phosphorylation of caveolin-1, Src, and p38 MAPK, and reduced the number of caveolae/μm of sarcolemma (determined by electron microscopy). Treatment of S49 T-lymphoma cells (which possess lipid rafts but lack caveolae) with CD or Colch redistributed a lipid raft marker (linker for activation of T cells (LAT)) and Gαs from lipid raft domains. We conclude that microtubules and actin filaments restrict cAMP formation by regulating the localization and interaction of GPCR-Gs-AC in lipid rafts/caveolae.

Inhibition of p75 Neurotrophin Receptor Attenuates Isoflurane-mediated Neuronal Apoptosis in the Neonatal Central Nervous System

Background:Exposure to anesthetics during synaptogenesis results in apoptosis and subsequent cognitive dysfunction in adulthood. Probrain-derived neurotrophic factor (proBDNF) is involved in synaptogenesis and can induce neuronal apoptosis via p75 neurotrophic receptors (p75NTR). proBDNF is cleaved into mature BDNF (mBDNF) by plasmin, a protease converted from plasminogen by tissue plasminogen activator (tPA) that is released with neuronal activity; mBDNF supports survival and stabilizes synapses through tropomyosin receptor kinase B. The authors hypothesized that anesthetics suppress tPA release from neurons, enhance p75NTR signaling, and reduce synapses, resulting in apoptosis. Methods:Primary neurons (DIV5) and postnatal day 5-7 (PND5-7) mice were exposed to isoflurane (1.4%, 4 h) in 5% CO2, 95% air. Apoptosis was assessed by cleaved caspase-3 (Cl-Csp3) immunoblot and immunofluorescence microscopy. Dendritic spine changes were evaluated with the neuronal spine marker, drebrin. Changes in synapses in PND5-7 mouse hippocampi were assessed by electron microscopy. Primary neurons were exposed to tPA, plasmin, or pharmacologic inhibitors of p75NTR (Fc-p75NTR or TAT-Pep5) 15 min before isoflurane. TAT-Pep5 was administered by intraperitoneal injection to PND5-7 mice 15 min before isoflurane. Results:Exposure of neurons in vitro (DIV5) to isoflurane decreased tPA in the culture medium, reduced drebrin expression (marker of dendritic filopodial spines), and enhanced Cl-Csp3. tPA, plasmin, or TAT-Pep5 stabilized dendritic filopodial spines and decreased Cl-Csp3 in neurons. TAT-Pep5 blocked isoflurane-mediated increase in Cl-Csp3 and reduced synapses in PND5-7 mouse hippocampi. Conclusion:tPA, plasmin, or p75NTR inhibition blocked isoflurane-mediated reduction in dendritic filopodial spines and neuronal apoptosis in vitro. Isoflurane reduced synapses and enhanced Cl-Csp3 in the hippocampus of PND5-7 mice, the latter effect being mitigated by p75NTR inhibition in vivo. These data support the hypothesis that isoflurane neurotoxicity in the developing rodent brain is mediated by reduced synaptic tPA release and enhanced proBDNF/p75NTR-mediated apoptosis.

G-protein-coupled receptor-signaling components in membrane raft and caveolae microdomains.

The efficiency of signal transduction in cells derives in part from subcellular, in particular plasma membrane, microdomains that organize signaling molecules and signaling complexes. Two related plasma membrane domains that compartmentalize G-protein coupled receptor (GPCR) signaling complexes are lipid (membrane) rafts, domains that are enriched in certain lipids, including cholesterol and sphingolipids, and caveolae, a subset of lipid rafts that are enriched in the protein caveolin. This review focuses on the properties of lipid rafts and caveolae, the mechanisms by which they localize signaling molecules and the identity of GPCR signaling components that are organized in these domains.

Caveolae and Lipid Rafts G Protein-Coupled Receptor Signaling Microdomains in Cardiac Myocytes

Abstract: A growing body of data indicates that multiple signal transduction events in the heart occur via plasma membrane receptors located in signaling microdomains. Lipid rafts, enriched in cholesterol and sphingolipids, form one such microdomain along with a subset of lipid rafts, caveolae, enriched in the protein caveolin. In the heart, a key caveolin is caveolin‐3, whose scaffolding domain is thought to serve as an anchor for other proteins. In spite of the original morphologic definition of caveolae (“little caves”), most work related to their role in compartmenting signal transduction molecules has involved subcellular fractionation or immunoprecipitation with anti‐caveolin antibodies. Use of such approaches has documented that several G protein‐coupled receptors (GPCR), and their cognate heterotrimeric G proteins and effectors, localize to lipid rafts/caveolae in neonatal cardiac myocytes. Our recent findings support the view that adult cardiac myocytes appear to have different patterns of localization of such components compared to neonatal myocytes and cardiac fibroblasts. Such results imply the existence of multiple subcellular microdomains for GPCR‐mediated signal transduction in cardiac myocytes, in particular adult myocytes, and raise a major unanswered question: what are the precise mechanism(s) that determine co‐localization of GPCR and post‐receptor components with lipid rafts/caveolae in cardiac myocytes and other cell types?

Mechanisms of cardiac protection from ischemia/reperfusion injury: a role for caveolae and caveolin-1

Caveolae, small invaginations in the plasma membrane, contain caveolins (Cav) that scaffold signaling molecules including the tyrosine kinase Src. We tested the hypothesis that cardiac protection involves a caveolin‐dependent mechanism. We used in vitro and in vivo models of ischemia‐reperfusion injury, electron microscopy (EM), transgenic mice, and biochemical assays to address this hypothesis. We found that Cav‐1 mRNA and protein were expressed in mouse adult cardiac myocytes (ACM). The volatile anesthetic, isoflurane, protected ACM from hypoxia‐induced cell death and increased sarcolemmal caveolae. Hearts of wild‐type (WT) mice showed rapid phosphorylation of Src and Cav‐1 after isoflurane and ischemic preconditioning. The Src inhibitor PP2 reduced phosphorylation of Src (Y416) and Cav‐1 in the heart and abolished isoflurane‐induced cardiac protection in WT mice. Infarct size (percent area at risk) was reduced by isoflurane in WT (30.5±4 vs. 44.2±3, n=7, P<0.05) but not Cav‐1−/− mice (46.6±5 vs. 41.7±3, n=7). Cav‐1−/−mice exposed to isoflurane showed significant alterations in Src phosphorylation and recruitment of C‐terminal Src kinase, a negative regulator of Src, when compared to WT mice. The results indicate that isoflurane modifies cardiac myocyte sarcolemmal membrane structure and composition and that activation of Src and phosphorylation of Cav‐1 contribute to cardiac protection. Accordingly, therapies targeted to post‐translational modification of Src and Cav‐1 may provide a novel approach for such protection.—Patel, H. H., Tsutsumi, Y. M., Head, B. P., Niesman, I. R., Jennings, M., Horikawa, Y. Huang, D., Moreno, A. L., Patel, P. M., Insel, P. A., Roth, D. M. Mechanisms of cardiac protection from ischemia/reperfusion injury: a role for caveolae and caveolin‐1. FASEB J. 21, 1565–1574 (2007)

Morphine enhances pharmacological preconditioning by isoflurane: Role of mitochondrial KATP channels and opioid receptors

Background Adenosine triphosphate–regulated potassium channels mediate protection against myocardial infarction produced by volatile anesthetics and opioids. We tested the hypothesis that morphine enhances the protective effect of isoflurane by activating mitochondrial adenosine triphosphate–regulated potassium channels and opioid receptors. Methods Barbiturate-anesthetized rats (n = 131) were instrumented for measurement of hemodynamics and subjected to a 30 min coronary artery occlusion followed by 2 h of reperfusion. Myocardial infarct size was determined using triphenyltetrazolium staining. Rats were randomly assigned to receive 0.9% saline, isoflurane (0.5 and 1.0 minimum alveolar concentration [MAC]), morphine (0.1 and 0.3 mg/kg), or morphine (0.3 mg/kg) plus isoflurane (1.0 MAC). Isoflurane was administered for 30 min and discontinued 15 min before coronary occlusion. In eight additional groups of experiments, rats received 5-hydroxydecanoic acid (5-HD; 10 mg/kg) or naloxone (6 mg/kg) in the presence or absence of isoflurane, morphine, and morphine plus isoflurane. Results Isoflurane (1.0 MAC) and morphine (0.3 mg/kg) reduced infarct size (41 ± 3%; n = 13 and 38 ± 2% of the area at risk; n = 10, respectively) as compared to control experiments (59 ± 2%; n = 10). Morphine plus isoflurane further decreased infarct size to 26 ± 3% (n = 11). 5-HD and naloxone alone did not affect infarct size, but abolished cardioprotection produced by isoflurane, morphine, and morphine plus isoflurane. Conclusions Combined administration of isoflurane and morphine enhances the protection against myocardial infarction to a greater extent than either drug alone. This beneficial effect is mediated by mitochondrial adenosine triphosphate–regulated potassium channels and opioid receptors in vivo.

Compartmentation of G-protein-coupled receptors and their signalling components in lipid rafts and caveolae

G-protein-coupled receptors (GPCRs) and post-GPCR signalling components are expressed at low overall abundance in plasma membranes, yet they evoke rapid, high-fidelity responses. Considerable evidence suggests that GPCR signalling components are organized together in membrane microdomains, in particular lipid rafts, enriched in cholesterol and sphingolipids, and caveolae, a subset of lipid rafts that also possess the protein caveolin, whose scaffolding domain may serve as an anchor for signalling components. Caveolae were originally identified based on their morphological appearance but their role in compartmentation of GPCR signalling has been primarily studied by biochemical techniques, such as subcellular fractionation and immunoprecipitation. Our recent studies obtained using both microscopic and biochemical methods with adult cardiac myocytes show expression of caveolin not only in surface sarcolemmal domains but also at, or close to, internal regions located at transverse tubules/sarcoplasmic reticulum. Other results show co-localization in lipid rafts/caveolae of AC (adenylyl cyclase), in particular AC6, certain GPCRs, G-proteins and eNOS (endothelial nitric oxide synthase; NOS3), which generates NO, a modulator of AC6. Existence of multiple caveolin-rich microdomains and their expression of multiple modulators of signalling strengthen the evidence that caveolins and lipid rafts/caveolae organize and regulate GPCR signal transduction in eukaryotic cells.

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-3β 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-3β 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.

The cyclic AMP effector Epac integrates pro- and anti-fibrotic signals

Scar formation occurs during the late stages of the inflammatory response but, when excessive, produces fibrosis that can lead to functional and structural damage of tissues. Here, we show that the profibrogenic agonist, transforming growth factor β1, transcriptionally decreases expression of Exchange protein activated by cAMP 1 (Epac1) in fibroblasts/fibroblast-like cells from multiple tissues (i.e., cardiac, lung, and skin fibroblasts and hepatic stellate cells). Overexpression of Epac1 inhibits transforming growth factor β1-induced collagen synthesis, indicating that a decrease of Epac1 expression appears to be necessary for the fibrogenic phenotype, an idea supported by evidence that Epac1 expression in cardiac fibroblasts is inhibited after myocardial infarction. Epac and protein kinase A, a second mediator of cAMP action, have opposite effects on migration but both inhibit synthesis of collagen and DNA by fibroblasts. Epac is preferentially activated by low concentrations of cAMP and stimulates migration via the small G protein Rap1 but inhibits collagen synthesis in a Rap1-independent manner. The regulation of Epac expression and activation thus appear to be critical for the integration of pro- and anti-fibrotic signals and for the regulation of fibroblast function.