We investigated the intracellular route of Salmonella in macrophages to determine a plausible mechanism for their survival in phagocytes. Western blot analysis of isolated phagosomes using specific antibodies revealed that by 5 min after internalization dead Salmonellacontaining phagosomes acquire transferrin receptors (a marker for early endosomes), whereas by 30 min the dead bacteria are found in vesicles carrying the late endosomal markers cation-dependent mannose 6-phosphate receptors, Rab7 and Rab9. In contrast, live Salmonella-containing phagosomes (LSP) retain a significant amount of Rab5 and transferrin receptor until 30 min, selectively deplete Rab7 and Rab9, and never acquire mannose 6-phosphate receptors even 90 min after internalization. Retention of Rab5 and Rab18 and selective depletion of Rab7 and Rab9 presumably enable the LSP to avoid transport to lysosomes through late endosomes. The presence of immature cathepsin D (48 kDa) and selective depletion of the vacuolar ATPase in LSP presumably contributes to the less acidic pH of LSP. In contrast, proteolytically processed cathepsin D (M r 17,000) was detected by 30 min on the dead Salmonellacontaining phagosomes. Morphological analysis also revealed that after uptake by macrophages, the dead Salmonella are transported to lysosomes, whereas the live bacteria persist in compartments that avoid fusion with lysosomes, indicating that live Salmonella bypass the normal endocytic route targeted to lysosomes and mature in a specialized compartment.

To understand intracellular trafficking modulations by live Salmonella, we investigated the characteristics of in vitro fusion between endosomes and phagosomes containing live (LSP) or dead Salmonella (DSP). We observed that fusion of both DSP and LSP were time, temperature and cytosol dependent. GTPγS and treatment of the phagosomes with Rab-GDI inhibited fusion, indicating involvement of Rab-GTPases. LSP were rich in rab5, α-SNAP, and NSF, while DSP mainly contained rab7. Fusion of endosomes with DSP was inhibited by ATP depletion, N-ethylmaleimide (NEM) treatment, and in NEM-sensitive factor (NSF)–depleted cytosol. In contrast, fusion of endosomes with LSP was not inhibited by ATP depletion or NEM treatment, and occurred in NSF-depleted cytosol. However, ATPγS inhibited both fusion events. Fusion of NEM-treated LSP with endosomes was abrogated in NSF- depleted cytosol and was restored by adding purified NSF, whereas no fusion occurred with NEM-treated DSP, indicating that NSF recruitment is dependent on continuous signals from live Salmonella. Binding of NSF with LSP required prior presence of rab5 on the phagosome. We have also shown that rab5 specifically binds with Sop E, a protein from Salmonella. Our results indicate that live Salmonella help binding of rab5 on the phagosomes, possibly activate the SNARE which leads to further recruitment of α-SNAP for subsequent binding with NSF to promote fusion of the LSP with early endosomes and inhibition of their transport to lysosomes.

The present studies were undertaken to investigate the effect of C-atrial natriuretic peptide (ANP)(4-23) and several peptide fragments containing 12 amino acids from different regions of the cytoplasmic domain of natriuretic peptide receptor (NPR)-C on cell proliferation in the absence or presence of angiotensin (ANG) II, endothelin (ET)-1, and arginine vasopressin (AVP) in A-10 vascular smooth muscle cells (VSMC). The peptide fragments used have either complete G(i) activator sequences K(461)-H(472) (peptide 1) and H(481)-H(492) (peptide 3) or partial G(i) activator sequences R(469)-K(480) (peptide 2) and I(465)-H(472) (peptide Y) with truncated COOH or NH(2) terminus, respectively. The other peptide used had no structural specificity (Q(473)-K(480), peptide X) or was the scrambled peptide control for peptide 1 (peptide Z). ANG II, ET-1 and AVP significantly stimulated DNA synthesis in these cells as determined by [(3)H]thymidine incorporation that was inhibited by peptides 1, 2, and 3 and not by peptides X, Y, and Z in a concentration-dependent manner, with an apparent K(i) between 1 and 10 nM. In addition, C-ANP(4-23), which interacts with NPR-C, also inhibited DNA synthesis stimulated by vasoactive peptides; however, the inhibition elicited by C-ANP(4-23) was not additive with the inhibition elicited by peptide 1. On the other hand, basal DNA synthesis in these cells was not inhibited by C-ANP(4-23) or the peptide fragments. Furthermore, vasoactive peptide-induced stimulation of DNA synthesis was inhibited by PD-98059 and wortmannin, and this inhibition was potentiated by peptide 1. In addition, peptide 1 also inhibited vasoactive peptide-induced phosphorylation of ERK1/2 and AKT and enhanced expression of G(i)alpha proteins. These data suggest that C-ANP(4-23) and small peptide fragments containing 12 amino acids irrespective of the region of the cytoplasmic domain of NPR-C inhibit proliferative responses of vasoactive peptides through G(i)alpha protein and MAP kinase/phosphatidylinositol 3-kinase/AKT pathways.

These data suggest that C-ANP4-23 and small peptide fragments containing 12 amino acids from different regions of cytoplasmic domain of NPR-C could modulate vasoactive peptide-stimulated protein synthesis through Gqalpha/MAP kinase/P13K and AKT pathways.

These results indicate that high glucose decreased the levels and functions of Gi proteins in A10 VSMC and aorta. It may thus be suggested that decreased levels and activity of Gi proteins and adenylyl cyclase signaling induced by hyperglycemia may be one of the important mechanisms contributing to the cardiovascular complications associated with diabetes.