Mechanisms Responsible for the Trophic Effect of Beta-Adrenoceptors on the I-to Current Density in Type 1 Diabetic Rat Cardiomyocytes

Background/Aims: In diabetic ventricular myocytes, transient outward potassium current (I-to) amplitude is severely reduced because of the impaired catecholamine release that characterizes diabetic autonomic neuropathy. Sympathetic nervous system exhibits a trophic effect on I-to since incubation of myocytes with noradrenaline restores current amplitude via beta-adrenoceptor (beta AR) stimulation. Here, we investigate the intracellular signalling pathway though which incubation of diabetic cardiomyocytes with the beta AR agonist isoproterenol recovers I-to amplitude to normal values. Methods: Experiments were performed in ventricular myocytes isolated from streptozotocin-diabetic rats. I-to current was recorded by using the patch-clamp technique. Kv4 channel expression was determined by immunofluorescence. Protein-protein interaction was determined by coimmunoprecipitation. Results: Stimulation of beta AR activates first a G alpha s protein, adenylyl cyclase and Protein Kinase A. PKA-phosphorylated receptor then switches to the G alpha i protein. This leads to the activation of the beta AR-Kinase-1 and further receptor phosphorylation and arrestin dependent internalization. The internalized receptor-arrestin complex recruits and activates cSrc and the MAPK cascade, where Ras, c-Raf1 and finally ERK1/2 mediate the increase in Kv4.2 and Kv4.3 protein abundance in the plasma membrane. Conclusion: beta(2)AR stimulation activates a G alpha s and G alpha i protein dependent pathway where the ERK1/2 modulates the Ito current amplitude and the density of the Kv4.2 and Kv4.2 channels in the plasma membrane upon sympathetic stimulation in diabetic heart.

Human LDL Structural Diversity Studied by IR Spectroscopy

Lipoproteins are responsible for cholesterol traffic in humans. Low density lipoprotein (LDL) delivers cholesterol from liver to peripheral tissues. A misleading delivery can lead to the formation of atherosclerotic plaques. LDL has a single protein, apoB-100, that binds to a specific receptor. It is known that the failure associated with a deficient protein-receptor binding leads to plaque formation. ApoB-100 is a large single lipid-associated polypeptide difficulting the study of its structure. IR spectroscopy is a technique suitable to follow the different conformational changes produced in apoB-100 because it is not affected by the size of the protein or the turbidity of the sample. We have analyzed LDL spectra of different individuals and shown that, even if there are not big structural changes, a different pattern in the intensity of the band located around 1617 cm 21 related with strands embedded in the lipid monolayer, can be associated with a different conformational rearrangement that could affect to a protein interacting region with the receptor.