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Effects of cholesterol depletion on compartmentalized cAMP responses in adult cardiac myocytes.

ABSTRACT: ?(1)-Adrenergic receptors (?(1)ARs) and E-type prostaglandin receptors (EPRs) both produce compartmentalized cAMP responses in cardiac myocytes. The role of cholesterol-dependent lipid rafts in producing these compartmentalized responses was investigated in adult rat ventricular myocytes. ?(1)ARs were found in lipid raft and non-lipid raft containing membrane fractions, while EPRs were only found in non-lipid raft fractions. Furthermore, ?(1)AR activation enhanced the L-type Ca(2+) current, intracellular Ca(2+) transient, and myocyte shortening, while EPR activation had no effect, consistent with the idea that these functional responses are regulated by cAMP produced by receptors found in lipid raft domains. Using methyl-?-cyclodextrin to disrupt lipid rafts by depleting membrane cholesterol did not eliminate compartmentalized behavior, but it did selectively alter specific receptor-mediated responses. Cholesterol depletion enhanced the sensitivity of functional responses produced by ?(1)ARs without having any effect on EPR activation. Changes in cAMP activity were also measured in intact cells using two different FRET-based biosensors: a type II PKA-based probe to monitor cAMP in subcellular compartments that include microdomains associated with caveolar lipid rafts and a freely diffusible Epac2-based probe to monitor total cytosolic cAMP. ?(1)AR and EPR activation elicited responses detected by both FRET probes. However, cholesterol depletion only affected ?(1)AR responses detected by the PKA probe. These results indicate that lipid rafts alone are not sufficient to explain the difference between ?(1)AR and EPR responses. They also suggest that ?(1)AR regulation of myocyte contraction involves the local production of cAMP by a subpopulation of receptors associated with caveolar lipid rafts.

SUBMITTER: Agarwal SR

PROVIDER: S-EPMC3049871 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Effects of cholesterol depletion on compartmentalized cAMP responses in adult cardiac myocytes.

Agarwal Shailesh R SR MacDougall David A DA Tyser Richard R Pugh Sara D SD Calaghan Sarah C SC Harvey Robert D RD

Journal of molecular and cellular cardiology 20101127 3

β(1)-Adrenergic receptors (β(1)ARs) and E-type prostaglandin receptors (EPRs) both produce compartmentalized cAMP responses in cardiac myocytes. The role of cholesterol-dependent lipid rafts in producing these compartmentalized responses was investigated in adult rat ventricular myocytes. β(1)ARs were found in lipid raft and non-lipid raft containing membrane fractions, while EPRs were only found in non-lipid raft fractions. Furthermore, β(1)AR activation enhanced the L-type Ca(2+) current, intr ...[more]

PMID: 21115018

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Project description:Aim: Confining cAMP production to discrete subcellular locations makes it possible for this ubiquitous second messenger to elicit unique functional responses. Yet, factors that determine how and where the production of this diffusible signaling molecule occurs are incompletely understood. The fluid mosaic model originally proposed that signal transduction occurs through random interactions between proteins diffusing freely throughout the plasma membrane. However, it is now known that the movement of membrane proteins is restricted, suggesting that the plasma membrane is segregated into distinct microdomains where different signaling proteins can be concentrated. In this study, we examined what role lipid raft and non-raft membrane domains play in compartmentation of cAMP signaling in adult ventricular myocytes. Methods and Results: The freely diffusible fluorescence resonance energy transfer-based biosensor Epac2-camps was used to measure global cytosolic cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. We found that ?-adrenergic receptors, which are expressed in lipid raft and non-raft domains, produce cAMP responses near the plasma membrane that are distinctly different from those produced by E-type prostaglandin receptors, which are expressed exclusively in non-raft domains. We also found that there are differences in basal cAMP levels associated with lipid raft and non-raft domains, and that this can be explained by differences in basal adenylyl cyclase activity associated with each of these membrane environments. In addition, we found evidence that phosphodiesterases 2, 3, and 4 work together in regulating cAMP activity associated with both lipid raft and non-raft domains, while phosphodiesterase 3 plays a more prominent role in the bulk cytoplasmic compartment. Conclusion: These results suggest that different membrane domains contribute to the formation of distinct pools of cAMP under basal conditions as well as following receptor stimulation in adult ventricular myocytes.

Dataset Information

Effects of cholesterol depletion on compartmentalized cAMP responses in adult cardiac myocytes.

Publications

Effects of cholesterol depletion on compartmentalized cAMP responses in adult cardiac myocytes.

Similar Datasets

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