The vesicle-associated membrane proteins [VAMPs; vesicle SNAP receptors (v-SNAREs)] present on GLUT4-enriched vesicles prepared from rat adipose cells [Cain, Trimble and Lienhard (1992) J. Biol. Chem. 267, 11681–11684] have been identified as synaptobrevin 2 (VAMP 2) and cellubrevin (VAMP 3) by using isoform-specific antisera. Additional antisera identify syntaxins 2 and 4 as the predominant target membrane SNAP receptors (t-SNAREs) in the plasma membranes (PM), with syntaxin 3 at one-twentieth the level. Syntaxins 2 and 4 are enriched 5–10-fold in PM compared with low-density microsomes (LDM). Insulin treatment results in an 11-fold increase in immunodetectable GLUT4 in PM and smaller (approx. 2-fold) increases in VAMP 2 and VAMP 3, whereas the subcellular distributions of the syntaxins are not altered by insulin treatment. To determine which of the SNAP receptors (SNAREs) in PM might participate in SNARE complexes with proteins from GLUT4 vesicles, complexes were immunoprecipitated with anti-myc antibody from solubilized membranes after the addition of myc-epitope-tagged N-ethylmaleimide-sensitive fusion protein (NSF) and recombinant α-soluble NSF attachment protein (α-SNAP). These complexes contain VAMPs 2 and 3 and syntaxin 4, but not syntaxins 2 or 3. Complex formation requires ATP and is disrupted by ATP hydrolysis. When all membrane fractions are prepared from basal cells, few or no VAMPs and no syntaxin 4 are immunoprecipitated in SNARE complexes obtained from LDM alone (or from immunoisolated GLUT4 vesicles). The content of syntaxin 4 depends on the presence of PM, and participation of VAMPs 2 and 3 is enhanced 4–6-fold by the addition of solubilized GLUT4 vesicles to PM. The latter increase is greater than can be explained by the 2-fold higher levels of VAMPs added to the reaction mixture. When all membrane fractions are prepared from insulin-stimulated cells, SNARE complexes formed from PM alone contain similar levels of syntaxin 4 but 5–6-fold higher levels of VAMPs 2 and 3 compared with PM alone from basal cells. Addition of GLUT4 vesicle proteins to PM from insulin-treated cells results in a further 2-fold increase in VAMP 2 recovered in SNARE complexes. Therefore the VAMPs in PM of insulin-treated but not basal cells, and in GLUT4-vesicles from cells in either condition, are in a form that readily forms a SNARE complex with PM t-SNAREs and NSF. Insulin seems to activate PM and/or GLUT4 vesicles so as to increase the efficiency of SNARE complex formation.

Background—Angiotensin II (Ang II) has diverse actions on cardiac electrical activity. Little information is available, however, regarding immediate electrophysiological effects of Ang II on cardiac repolarization.Methods and Results—The present study investigated the immediate effects of Ang II on the slow component of delayed rectifier K⁺current (I_Ks) and action potentials in guinea pig atrial myocytes using the whole-cell patch-clamp technique. Bath application of Ang II increased the amplitude ofI_Ks(EC₅₀, 6.16 nmol/L) concentration dependently. The stable analogue Sar¹–Ang II was also effective at increasingI_Ks. The voltage dependence ofI_Ksactivation and the kinetics of deactivation were not significantly affected by these agonists. The enhancement ofI_Kswas blocked by the Ang II type 1 (AT₁) receptor antagonist valsartan (1 μmol/L) and was markedly attenuated by inclusion of GDPβS (2 mmol/L) in the pipette, indicating an involvement of G protein–coupled AT₁receptor. The stimulatory effect was also significantly reduced by the phospholipase C inhibitor compound 48/80 (100 μmol/L) and the protein kinase C inhibitors bisindolylmaleimide I (200 nmol/L) and H-7 (10 μmol/L), suggesting that AT₁receptor acts through phospholipase C–protein kinase C signaling cascade to potentiateI_Ks. As expected from its stimulatory action onI_Ks, Sar¹–Ang II markedly shortened the action potential duration, which could be reversed by valsartan.Conclusions—The potentiation ofI_Ksvia AT₁stimulation in atrial myocytes, accompanied by a shortening of the action potential duration, suggests a potential mechanism by which elevated levels of Ang II may promote atrial fibrillation in heart failure and warrants further investigation.

The mechanism of the stimulation of Na+/K+ transport by insulin in frog skeletal muscle was studied. The ouabain-binding capacity in detergent-treated plasma membranes of insulin-exposed muscles was increased 1.9-fold compared with that of controls. Na+/K(+)-ATPase activity was found in an intracellular ‘light fraction’ (fraction II) prepared by using anion-exchange chromatography. Marker enzyme activities for plasma and Golgi membranes were not detected in this fraction. The specific activity of Na+/K(+)-ATPase in fraction II from insulin-exposed muscles was 58% of that in an identical fraction from control muscles. No significant difference in the protein yield of the plasma membrane preparation was observed between these two groups. In parallel with the decrease in the Na+/K(+)-ATPase activity in fraction II from insulin-exposed muscles, the ouabain-binding capacity in this fraction was also decreased. The addition of saponin to fraction II increased both Na+/K(+)-ATPase activity and ouabain binding, indicating that some of the Na+/K(+)-ATPase is located in sealed vesicles. These findings support the view that insulin stimulates the translocation of Na+/K(+)-ATPase molecules from fraction II to the plasma membrane.