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This study examines the role of sphingolipids in mediating the apoptosis of PC12W cells induced by the angiotensin II type 2 (AT2) receptor. PC12W cells express abundant AT2 receptor but not angiotensin II type 1 receptor and undergo apoptosis when stimulated by angiotensin II. AT2receptor-induced ceramide accumulation preceded the onset of caspase 3 activation and DNA fragmentation. AT2 receptor-induced ceramide accumulation did not result from the degradation of complex sphingolipids (SL) such as sphingomyelin or glycosphingolipids, as no changes in neutral or acidic sphingomyelinase activities, sphingomyelin level, nor in cellular glycolipid composition were observed. AT2 receptor activated serine palmitoyltransferase with a maximum time of 24 h after angiotensin II stimulation. The AT2 receptor-induced accumulation of ceramide was blocked by inhibitors of the de novo pathway of SL synthesis, β-chloro-l-alanine and fumonisin B1. Inhibition of the de novo biosynthesis of SLs by fumonisin B1 and β-chloro-l-alanine completely abrogated the AT2 receptor-mediated apoptosis. Pertussis toxin and orthovanadate blocked AT2 receptor-mediated ceramide production. Taken together our data demonstrate that in PC12W cells the stimulation of AT2 receptor induces the activation of de novo pathway, and a metabolite of this pathway, possibly ceramide, mediates AT2 receptor-induced apoptosis.

The carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 (AT1) receptor has recently been shown to interact with several classes of cytoplasmic proteins that regulate different aspects of AT1 receptor physiology. Employing yeast two-hybrid screening of a mouse kidney cDNA library with the carboxyl-terminal cytoplasmic domain of the murine AT1a receptor as a bait, we have isolated a novel protein with a predicted molecular mass of 18 kDa, which we have named ATRAP (for AT1 receptor-associated protein). ATRAP interacts specifically with the carboxyl-terminal domain of the AT1a receptor but not with those of angiotensin II type 2 (AT2), m3 muscarinic acetylcholine, bradykinin B2, endothelin B, and beta2-adrenergic receptors. The mRNA of ATRAP was abundantly expressed in kidney, heart, and testis but was poorly expressed in lung, liver, spleen, and brain. The ATRAP-AT1a receptor association was confirmed by affinity chromatography, by specific co-immunoprecipitation of the two proteins, and by fluorescence microscopy, showing co-localization of these proteins in intact cells. Overexpression of ATRAP in COS-7 cells caused a marked inhibition of AT1a receptor-mediated activation of phospholipase C without affecting m3 receptor-mediated activation. In conclusion, we have isolated a novel protein that interacts specifically with the carboxyl-terminal cytoplasmic domain of the AT1a receptor and affects AT1a receptor signaling.

Resonance energy transfer was measured between egg phosphatidylcholine liposomes containing the intramolecular excimer forming pyrene-labelled phospholipid analogue 1,2-bis[pyren-1-(-yl)]decanoyl-sn-glycero-3-phosphocholine (bisPDPC) as a donor and DNA-bound adriamycin as an acceptor. Membrane association of DNA turned out to be critically dependent on the presence of sphingosine in the liposomes. Identical result was obtained by measuring the extent of quenching of the fluorescent DNA-bound dye Hoechst 33258 due to energy transfer to the lipophilic stain Nile Red incorporated in egg phosphatidylcholine liposomes containing varying amounts of sphingosine. The attachment of DNA to sphingosine-containing membranes could be reversed by the further inclusion of the negatively charged phosphatidic acid up to approximately 1:2 PA/sphingosine molar ratio in the liposomes, thus suggesting the involvement of electrostatic interactions. Differential scanning calorimetry measurements confirmed a lack of association between DNA and dimyristoylphosphatidylcholine liposomes. Instead drastic changes were produced by DNA in the heat capacity scans measured for liposomes also incorporating sphingosine. Fluorescence microscopy revealed an extensive aggregation of sphingosine containing pyrene-phosphatidylcholine-labelled egg phosphatidylcholine liposomes in the presence of DNA. Together with other available data on the effects of sphingosine, the present findings suggest that sphingosine could directly alter the chromatin structure. Accordingly, such alterations may contribute to the control of replication and gene expression.

A 48-year-old male patient presented with dyspnea on exertion. Patient was found to have pulmonary hypertension. Myocardial biopsy showed amyloidosis and further work-up revealed Salmon-Durie stage 1A multiple myeloma. Patient had no other clinical manifestations of amyloidosis. It is possible that the pulmonary hypertension is caused by amyloid deposition into pulmonary arteries as the arterial amyloid deposition is common in AL amyloidosis. Treatment with sildenafil led to hemodynamic and symptomatic improvement.