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Fig. 5. Protein expression of GC-B and of endogenous and heterologously expressed VILIP-1 in hippocampal neurons. (A) Homogenates of hippocampal cells were analyzed by western blot analysis using specific antibodies against VILIP-1 (lanes 1, 3 and 4) and GC-B (lanes 2, 5 and 6). VILIP-1-expression is shown in untransfected (lane 1), control-transfected (lane 3, pOPR) and VILIP-1-transfected (lane 4, pOPR-V1) hippocampal neurons. GC-B-expression is shown in untransfected (lane 2), control-transfected (lane 5, pOPR) and VILIP-1-transfected (lane 6, pOPR-V1) hippocampal neurons. Sizes of marker proteins are indicated at the left margin. 30 µg of protein was loaded in lanes 1 and 2, 10 µg in lanes 3-6. (B) Enhancement of membrane cGMP levels following CNP-stimulation of natriuretic receptor GC-B in untransfected and VILIP-1-transfected hippocampal neurons. CNP-stimulated cGMP accumulation in hippocampal neurons transiently transfected with control vector (pOPR) and vector containing VILIP-1-cDNA (pOPR-V1). Mean values±s.d. are from seven experiments carried out in triplicate. Asterisks (***) mark a significant difference between CNP-stimulated pOPR and pOPR-V1-transfected cells (P<0.005, n=7, Student's t-test). (C) Effect of different inhibitors of membrane transport on CNP-stimulated cGMP accumulation in control-transfected and VILIP-1-transfected hippocampal neurons. Control-transfected (pOPR) and VILIP-1-transfected (pOPR-V1) hippocampal neurons were stimulated with CNP for 20 minutes in the presence or absence of monensin (+Mon) or wortmannin (+WM), which inhibit receptor recycling at different stages; or in the presence of the endocytosis inhibitor PAO (+PAO). CNP-stimulated cGMP accumulation in control-transfected (pOPR) hippocampal neurons was set to 100% and all other values were expressed as % of control (CNP, pOPR). Mean values±s.d. are from at least three experiments, except PAO (two experiments), and were carried out in triplicate. Asterisks (*,***) mark significant differences (P<0.05, P<0.005, n=<3, Student's t-test).





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