Characterization of caveolae from rat heart: Localization of postreceptor signal transduction molecules and their rearrangement after norepinephrine stimulation

Luca, Antonio De; Sargiacomo, Massimo; Puca, Annibale Alessandro; Sgaramella, Giuseppe; Paolis, Paola De; Frati, Giacomo; Morisco, Carmine; Trimarco, Bruno; Volpe, Massimo; Condorelli, Gianluigi

doi:10.1002/(sici)1097-4644(20000615)77:4<529::aid-jcb2>3.0.co;2-x

Cited by 19 publications

(6 citation statements)

References 29 publications

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“…Several previous studies (5,9,18,33,48) had concluded that caveolae/rafts prepared from several cell types, including cardiac myocytes, do not contain Na ϩ /K ϩ -ATPase. The widely used detergent-free and carbonate-based procedure of Song et al (37) had been used successfully in numerous studies on cardiac caveolae prepared from cultured cardiac myocytes and ventricles (4,5,27,28,31,32). First, we focused on cardiac caveolae because most of our studies on the signal-transducing role of Na ϩ /K ϩ -ATPase had been done in cardiac myocytes and the intact heart (26,45).…”

Section: Resultsmentioning

confidence: 99%

Role of caveolae in signal-transducing function of cardiac Na⁺/K⁺-ATPase

Liu

Mohammadi

Aynafshar

et al. 2003

American Journal of Physiology-Cell Physiology

165

154

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Ouabain binding to Na(+)/K(+)-ATPase activates Src/epidermal growth factor receptor (EGFR) to initiate multiple signal pathways that regulate growth. In cardiac myocytes and the intact heart, the early ouabain-induced pathways that cause rapid activations of ERK1/2 also regulate intracellular Ca(2+) concentration ([Ca(2+)](i)) and contractility. The goal of this study was to explore the role of caveolae in these early signaling events. Subunits of Na(+)/K(+)-ATPase were detected by immunoblot analysis in caveolae isolated from cardiac myocytes, cardiac ventricles, kidney cell lines, and kidney outer medulla by established detergent-free procedures. Isolated rat cardiac caveolae contained Src, EGFR, ERK1/2, and 20-30% of cellular contents of alpha(1)- and alpha(2)-isoforms of Na(+)/K(+)-ATPase, along with nearly all of cellular caveolin-3. Immunofluorescence microscopy of adult cardiac myocytes showed the presence of caveolin-3 and alpha-isoforms in peripheral sarcolemma and T tubules and suggested their partial colocalization. Exposure of contracting isolated rat hearts to a positive inotropic dose of ouabain and analysis of isolated cardiac caveolae showed that ouabain caused 1) no change in total caveolar ERK1/2, but a two- to threefold increase in caveolar phosphorylated/activated ERK1/2; 2) no change in caveolar alpha(1)-isoform and caveolin-3; and 3) 50-60% increases in caveolar Src and alpha(2)-isoform. These findings, in conjunction with previous observations, show that components of the pathways that link Na(+)/K(+)-ATPase to ERK1/2 and [Ca(2+)](i) are organized within cardiac caveolae microdomains. They also suggest that ouabain-induced recruitments of Src and alpha(2)-isoform to caveolae are involved in the manifestation of the positive inotropic effect of ouabain.

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Section: Resultsmentioning

confidence: 99%

Role of caveolae in signal-transducing function of cardiac Na⁺/K⁺-ATPase

Liu

Mohammadi

Aynafshar

et al. 2003

American Journal of Physiology-Cell Physiology

165

154

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“…Heterotrimeric G proteins, receptors, and effectors reside in lipid rafts as do other signaling proteins (4,5,(14)(15)(16). G proteins transduce signals from cell surface receptors to intracellular effectors (17,18).…”

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confidence: 99%

G_s Signaling Is Intact after Disruption of Lipid Rafts

2001

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Membrane microdomains enriched in cholesterol and sphingolipids modulate a number of signal transduction pathways and provide a residence for heterotrimeric G proteins, their receptors, and their effectors. We investigated whether signaling through G(s) was dependent on these membrane domains, characterized by their resistance to detergents, by depleting cells of cholesterol and sphingolipids. For cholesterol depletion, rat salivary epithelial A5 cells were cultured under low-cholesterol conditions, and then treated with the cholesterol chelator methyl-beta-cyclodextrin. For sphingolipid depletion, LY-B cells, a mutant CHO cell line that is unable to synthesize sphingolipids, were incubated under low-sphingolipid conditions. Depletion of cholesterol or sphingolipid led to a loss or decrease, respectively, in the amount of Galpha(s) from the detergent-resistant membranes without any change in the cellular or membrane-bound amounts of Galpha(s). The cAMP accumulation in response to a receptor agonist was intact and the level slightly increased in cells depleted of cholesterol or sphingolipids compared to that in control cells. These results indicate that localization of Galpha(s) to detergent-resistant membranes was not required for G(s) signaling. Analysis of the role of lipid rafts on the kinetics of protein associations in the membrane suggests that compartmentalization in lipid rafts may be more effective in inhibiting protein interactions and, depending on the pathway, ultimately inhibit or promote signaling.

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“…Hormonal and neurotransmitter effects on composition of membrane subdomains have been intensively studied by immunoblot analysis in the recent past (de Weerd and Leeb‐Lundberg 1997; Feron et al . 1997; De Luca et al . 2000; Dessy et al .…”

Section: Discussionmentioning

confidence: 99%

“…To achieve high reproducibility of fractionation data and to limit the work associated with generating the complicated 5/10/15/20/25/30/35/40% w/v sucrose gradients, subsequent fractionations were performed on 5% (4 mL)/35% (4 mL)/40% (4 mL) w/v sucrose gradients. This simple means of fractionation has been used routinely by other authors for isolation of detergent‐insensitive membrane domains (De Luca et al . 2000; Nomura et al .…”

Section: Functional Coupling Between Dop Receptor and G Protein Is DImentioning

confidence: 99%

δ‐Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains

Bouřová

Koštrnová

Hejnova

et al. 2003

Journal of Neurochemistry

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Low-density membrane fragments (domains) were separated from the bulk of plasma membranes of human embryonic kidney (HEK)293 cells expressing a d-opioid (DOP) receptor-G i 1a fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein-linked G i 1a and endogenous pertussis-toxin sensitive G proteins was measured as D-Ala2, ]DADLE radioligand binding assay. Similar results were obtained when sodium bicarbonate was used for alkaline isolation of membrane domains. By contrast, detergentinsensitive membrane domains isolated following treatment of cells with Triton X100 exhibited no DADLE-stimulated GTPase or GTPcS binding. Functional coupling between the DOP receptor and cognate G proteins was also blocked by high-energy ultrasound and repeated freezing-thawing. Our data indicate, for the first time, that membrane domains isolated using 'detergent-free' procedures exhibit higher efficiency of coupling between a G protein-coupled receptor and its corresponding G protein(s) than bulk plasma membranes. Detergent-extraction diminishes these interactions, even when the receptor and G proteins are physically tethered together.

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Characterization of caveolae from rat heart: Localization of postreceptor signal transduction molecules and their rearrangement after norepinephrine stimulation

Cited by 19 publications

References 29 publications

Role of caveolae in signal-transducing function of cardiac Na⁺/K⁺-ATPase

Role of caveolae in signal-transducing function of cardiac Na⁺/K⁺-ATPase

G_s Signaling Is Intact after Disruption of Lipid Rafts

δ‐Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains

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Characterization of caveolae from rat heart: Localization of postreceptor signal transduction molecules and their rearrangement after norepinephrine stimulation

Cited by 19 publications

References 29 publications

Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase

Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase

Gs Signaling Is Intact after Disruption of Lipid Rafts

δ‐Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains

Contact Info

Product

Resources

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Role of caveolae in signal-transducing function of cardiac Na⁺/K⁺-ATPase

Role of caveolae in signal-transducing function of cardiac Na⁺/K⁺-ATPase

G_s Signaling Is Intact after Disruption of Lipid Rafts