Substance P dependence of endosomal fusion during bladder inflammation.

Urinary bladder instillation of ovalbumin into presensitized guinea pigs stimulates rapid development of local bladder inflammation. Substance P is an important mediator of this inflammatory response, as substance P antagonists largely reverse the process. Vacuolization of the subapical endosomal compartment of the transitional epithelial cells lining the bladder suggests that changes in endosomal trafficking and fusion are also part of the inflammatory response. To test directly for substance P mediation of changes in endosomal fusion, we reconstituted fusion of transitional cell endosomes in vitro using both cuvette-based and flow cytometry energy transfer assays. Bladders were loaded with fluorescent dyes by a hypotonic withdrawal protocol before endosomal isolation by gradient centrifugation. Endosomal fusion assayed by energy transfer during in vitro reconstitution was both cytosol and ATP dependent. Fusion was confirmed by the increase in vesicle size on electron micrographs of fused endosomal preparations compared with controls. In inflamed bladders, dye uptake was inhibited 20% and endosomal fusion was inhibited 50%. These changes are partly mediated by the neurokinin-1 (NK1) receptor (NK1R), as 4 mg/kg of CP-96,345, a highly selective NK1 antagonist, increased fusion in inflamed bladders but had no effect on control bladders. The receptor-mediated nature of this effect was demonstrated by the expression of substance P receptor mRNA in rat bladder lumen scrapings and by the detection of the NK1R message in guinea pig subapical endosomes by Western blot analysis. The NK1Rs were significantly upregulated following induction of an inflammatory response in the bladder. These results demonstrate that 1) in ovalbumin-induced inflammation in the guinea pig bladder, in vitro fusion of apical endosomes is inhibited, showing endocytotic processes are altered in inflammation; 2) pretreatment in vivo with an NK1R antagonist blocks this inhibition of in vitro fusion, demonstrating a role for NK1R in this process; and 3) the NK1R is present in higher amounts in apical endosomes of inflamed bladder, suggesting changes in translation or trafficking of the NK1R during the inflammatory process. This suggests that NK1R can change the fusion properties of membranes in which it resides.

Renal endosomes contain angiotensin peptides, converting enzyme, and AT(1A) receptors.

Kidney cortex and proximal tubular angiotensin II (ANG II) levels are greater than can be explained on the basis of circulating ANG II, suggesting intrarenal compartmentalization of these peptides. One possible site of intracellular accumulation is the endosomes. In the present study, we tested for endosomal ANG I, ANG II, angiotensin type 1A receptor (AT(1A)), and angiotensin converting enzyme (ACE) activity and determined whether these levels are regulated by salt intake. Male Sprague-Dawley rats were fed chow containing either high or low dietary sodium for 10-14 days. Blood and kidneys were harvested and processed for measurement of plasma, kidney, and renal intermicrovillar cleft and endosomal angiotensin levels. Kidney ANG I averaged 179 +/- 20 fmol/g and ANG II averaged 258 +/- 36 fmol/g in rats fed a high-sodium diet and were significantly higher, averaging 347 +/- 58 fmol/g and 386 +/- 55 fmol/g, respectively, in rats fed a low-salt diet. Renal intermicrovillar clefts and endosomes contained ANG I and ANG II. Intermicrovillar cleft ANG I and ANG II levels averaged 8.4 +/- 2.6 and 74 +/- 26 fmol/mg, respectively, in rats fed a high-salt diet and 7.6 +/- 1.7 and 70 +/- 25 fmol/mg in rats fed a low-salt diet. Endosomal ANG I and ANG II levels averaged 12.3 +/- 4.4 and 43 +/- 19 fmol/mg, respectively, in rats fed a high-salt diet, and these levels were similar to those observed in rats fed a low-salt diet. Renal endosomes from rats fed a low-salt diet demonstrated significantly more AT(1A) receptor binding compared with rats fed a high-salt diet. ACE activity was detectable in renal intermicrovillar clefts and was 2.5-fold higher than the levels observed in renal endosomes. Acute enalaprilat treatment decreased ACE activity in renal intermicrovillar clefts by 90% and in renal endosomes by 84%. Likewise, intermicrovillar cleft and endosomal ANG II levels decreased by 61% and 52%, respectively, in enalaprilat-treated animals. These data demonstrate the presence of intact angiotensin peptides and ACE activity in renal intermicrovillar clefts and endosomes, indicating that intact angiotensin peptides are formed and/or trafficked through intracellular endosomal compartments and are dependent on ACE activity.

Select de novo gene and protein expression during renal epithelial cell culture in rotating wall vessels is shear stress dependent.

The rotating wall vessel has gained popularity as a clinical cell culture tool to produce hormonal implants. It is desirable to understand the mechanisms by which the rotating wall vessel induces genetic changes, if we are to prolong the useful life of implants. During rotating wall vessel culture gravity is balanced by equal and opposite hydrodynamic forces including shear stress. The current study provides the first evidence that shear stress response elements, which modulate gene expression in endothelial cells, are also active in epithelial cells. Rotating wall culture of renal cells changes expression of select gene products including the giant glycoprotein scavenger receptors cubulin and megalin, the structural microvillar protein villin, and classic shear stress response genes ICAM, VCAM and MnSOD. Using a putative endothelial cell shear stress response element binding site as a decoy, we demonstrate the role of this sequence in the regulation of selected genes in epithelial cells. However, many of the changes observed in the rotating wall vessel are independent of this response element. It remains to define other genetic response elements modulated during rotating wall vessel culture, including the role of hemodynamics characterized by 3-dimensionality, low shear and turbulence, and cospatial relation of dissimilar cell types.

Differences in susceptibility to tumor necrosis factor alpha-induced apoptosis among MCF-7 breast cancer cell variants.

Widespread use of MCF-7 human breast carcinoma cells as a model system for breast cancer has led to variations in these cells between different laboratories. Although several reports have addressed these differences in terms of proliferation and estrogenic response, variations in sensitivity to apoptosis have not yet been described. Tumor necrosis factor alpha (TNF-alpha) has been shown to both induce apoptosis and inhibit proliferation in MCF-7 cells. We observed that TNF-alpha inhibited proliferation in MCF-7 cell variants from three different laboratories (designated M, L, and N). MCF-7 M cells were resistant to TNF-alpha-induced apoptosis, whereas MCF-7 L cells were moderately resistant to the effect of TNF-alpha. A third variant, MCF-7 N, underwent apoptosis when exposed to TNF-alpha. Analysis of the p55 TNF-alpha receptor (TNFR) 1 expression revealed the greatest expression in MCF-7 N cells, whereas the MCF-7 L and M cells expressed 89 and 67% of MCF-7 N cell TNFR1 levels, respectively. Ceramide generation occurred in all three variants in response to TNF-alpha treatment, with MCF-7 N cells expressing the greatest increase. Cleavage of the CPP32/caspase 3 substrate poly(ADP-ribose) was observed in MCF-7 N and L cells as early as 3 and 6 h, respectively, but poly(ADP-ribose) cleavage was not observed in MCF-7 M cells. The delayed protease activation in the L variant may represent the mechanism by which these cells display delayed sensitivity to TNF-a-induced apoptosis. Expression of the Bcl-2, Mcl-1, Bcl-X, Bax, and Bak proteins was analyzed to determine whether the differences in MCF-7 cell sensitivity to apoptosis could be correlated to the differential expression of these proteins. Whereas Bak, Bcl-X, and Mcl-1 levels were identical between variants, the levels of Bcl-2 were 3.5-3.8-fold higher and the levels of Bax were 1.5-1.7-fold lower in the resistant variants (M and L) as compared with those of the sensitive variant (N). Taken together, these results suggest that differences in susceptibility to TNF-alpha-induced apoptosis among MCF-7 breast cancer cell variants may be explained by differences in TNFR expression, ceramide generation, differential expression of the Bcl-2 family of proteins, and protease activation.

Myeloma light chains are ligands for cubilin (gp280).

Although myeloma light chains are known to undergo receptor-mediated endocytosis in the kidney, the molecular identity of the receptor has not been characterized. We examined the interaction between cubilin (gp280) and four species of light chains isolated from the urine of patients with multiple myeloma. Four lines of evidence identify cubilin, a giant glycoprotein receptor, which is restricted in distribution to endocytic scavenger pathways and which has potent effects on endosomal trafficking, as a potentially physiologically relevant binding site for light chains: 1) light chains coeluted during immunoaffinity purification of cubilin; 2) polyclonal antisera to cubilin but not control sera, displaced human light chain binding from rat renal brush-border membranes; 3) cubilin bound to multiple species of light chains during surface plasmon resonance; 4) anti-cubilin antiserum interfered with light chain endocytosis by visceral yolk sac epithelial cells. However, both binding of light chains to brush-border membranes and endocytosis of light chains by yolk sac epithelial cells were only partially inhibited by anticubilin antibodies, suggesting presence of additional or alternate binding sites for light chains. Excess light chain had a potent inhibitory effect on endosomal fusion in vitro. Binding showed dose and time-dependent saturability with low-affinity, high-capacity equilibrium binding parameters. These data demonstrate that cubilin plays a role in the endocytosis and trafficking of light chains in renal proximal tubule cells.

Membrane potential mediates H(+)-ATPase dependence of "degradative pathway" endosomal fusion.

In some epithelial cell lines, the uptake and degradation of proteins is so pronounced as to be regarded as a specialized function known as "degradative endocytosis." The endosomal pathways of the renal proximal tubule and the visceral yolk sac share highly specialized structures for "degradative endocytosis." These endosomal pathways also have a unique distribution of their H(+)-ATPase, predominantly in the subapical endosomal pathway. Previous studies provide only indirect evidence that H(+)-ATPases participate in endosomal fusion events: formation of vesicular intermediates between early and late endosomes is H(+)-ATPase dependent in baby hamster kidney cells, and H(+)-ATPase subunits bind fusion complex proteins in detergent extracts of fresh rat brain. To determine directly whether homotypic endosomal fusion is H(+)-ATPase dependent, we inhibited v-type H(+)-ATPase during flow cytometry and cuvette-based fusion assays reconstituting endosomal fusion in vitro. We report that homotypic fusion in subapical endosomes derived from rat renal cortex, and immortalized visceral yolk sac cells in culture, is inhibited by the v-type H(+)-ATPase specific inhibitor bafilomycin A1. Inhibition of fusion by H(+)-ATPase is mediated by the membrane potential as collapsing the pH gradient with nigericin had no effect on homotypic endosomal fusion, while collapsing the membrane potential with valinomycin inhibited endosomal fusion. Utilizing an in vitro reconstitution assay this data provides the first direct evidence for a role of v-type H(+)-ATPase in mammalian homotypic endosomal fusion.

Gentamicin inhibits rat renal cortical homotypic endosomal fusion: role of megalin.

Megalin, a giant glycoprotein receptor heavily concentrated in the early endosomal pathway of renal proximal tubular cells, binds gentamicin with high affinity and delivers the drug to lysosomes. Utilizing an in vitro reconstitution assay we tested whether gentamicin-induced vacuolation is associated with inhibition of early endosomal fusion, as well as whether megalin plays a role in mediating these effects. Pretreatment of rats with gentamicin inhibited rat renal proximal tubular homotypic endosomal fusion. Administered simultaneously, gentamicin and polymers of polyaspartic acid, which protect against the hemodynamic effects of gentamicin nephrotoxicity, had no net effect on fusion. Polyaspartic acid alone had no effect on fusion. Antisera to the tail of the megalin/gentamicin receptor inhibited fusion, whereas non-specific controls had no effect. Peptides matching homologous NPXY repeat sequence motifs in the cytosolic tail stimulated endosomal fusion, whereas reverse sequence control peptides had no effect. These data suggest that gentamicin inhibition of endosomal fusion in the renal proximal tubule is a damage mechanism mediated by specific peptide sequences in the cytosolic tail of the giant gentamicin-binding receptor megalin and that receptors can effect the fusion properties of membranes in which they reside.