Endogenous adenylyl cyclase agonists promote cell proliferation and electrolyte secretion of human ADPKD and ARPKD cells in vitro. We suggest that increased levels of cAMP may accelerate cyst growth and overall renal enlargement in patients with PKD.

Molar partition coefficients for chlorpromazine and methochlorpromazine between phospholipid vesicles or human erythrocyte ghosts and buffer are determined by ultraviolet difference spectroscopy. The partition coefficients between small unilamellar egg phosphatidylcholine vesicles and buffer at pH 7.4 are 4.4 X 10(5) for chlorpromazine and 0.8 X 10(5) for methochlorpromazine, determined with 10 microM amphiphile. An increase in the partition of chlorpromazine into vesicles is seen as the pH is increased to the pKa of chlorpromazine at 9.2. Chlorpromazine also partitions preferentially into fluid-phase phospholipid compared to solid-phase phospholipid. Molar partition coefficients between unsealed human erythrocyte ghosts and buffer at pH 8.0 with 10 microM amphiphile are determined to be 6.5 X 10(5) for chlorpromazine and 2.5 X 10(5) for methochlorpromazine. Difference spectroscopy is an equilibrium technique that does not require separation of bound from free amphiphile, as do many other methods of determining membrane-buffer partition coefficients. This method is useful for any amphiphile that has an appreciable absorbance below its critical micelle concentration and whose absorbance is sensitive to environment.

To investigate several key aspects of phosphatidylinositol transfer protein (PI‐TP) function in eukaryotic cells, rat PI‐TP was expressed in yeast strains carrying lesions in SEC14, the structural gene for yeast PI‐TP (SEC14p), whose activity is essential for Golgi secretory function in vivo. Rat PI‐TP expression effected a specific complementation of sec14ts growth and secretory defects. Complementation of sec14 mutations was not absolute as rat PI‐TP expression failed to rescue sec14 null mutations. This partial complementation of sec14 lesions by rat PI‐TP correlated with inability of the mammalian protein to stably associate with yeast Golgi membranes and was not a result of rat PI‐TP stabilizing the endogenous sec14ts gene product. These collective data demonstrate that while the in vitro PI‐TP activity of SEC14p clearly reflects some functional in vivo property of SEC14p, the PI‐TP activity is not the sole essential activity of SEC14p. Those data further identify an efficient Golgi targeting capability as a likely essential feature of SEC14p function in vivo. Finally, the data suggest that stable association of SEC14p with yeast Golgi membranes is not a simple function of its lipid‐binding properties, indicate that the amino‐terminal 129 SEC14p residues are sufficient to direct a catalytically inactive form of rat PI‐TP to the Golgi and provide the first evidence to indicate that a mammalian PI‐TP can stimulate Golgi secretory function in vivo.

Over 20 years ago, it was reported that liver cytosol contains at least two distinct proteins that transfer phosphatidylinositol in vitro, phosphatidylinositol transfer protein (PITP) and a pH 5.1 supernatant fraction containing sterol carrier protein-2 (SCP-2). In contrast to PITP, there has been minimal progress on the structural and functional significance of SCP-2 in phosphatidylinositol transport. As shown herein, highly purified, recombinant SCP-2 stimulated up to 13-fold the rapid (s) transfer of radiolabeled phosphatidylinositol (PI) from microsomal donor membranes to highly curved acceptor membranes. SCP-2 bound to microsomes in vitro and overexpression of SCP-2 in transfected L-cells resulted in the following: (i) redistribution of phosphatidylinositols from intracellular membranes (mitochondria and microsomes) to the plasma membrane; (ii) enhancement of insulin-mediated inositol-triphosphate production; and (iii) 5.5-fold down regulation of PITP. Like PITP, SCP-2 binds two ligands required for vesicle budding from the Golgi, PI, and fatty acyl CoA. Double immunolabeling confocal microscopy showed SCP-2 significantly colocalized with caveolin-1 in the cytoplasm (punctate) and plasma membrane of SCP-2 overexpressing hepatoma cells (72%), HT-29 cells (58%), and SCP-2 overexpressing L-cells (37%). Taken together, these data show for the first time that SCP-2 plays a hitherto unrecognized role in intracellular phosphatidylinositol transfer, distribution, and signaling.

The phosphatidylcholine exchange protein from beef liver catalyzes the exchange of phosphatidylcholine between single bilayer liposomes (Hellings et al. (1974), Eur. J. Biochem. 47, 601). A model has been proposed which describes the kinetics of this exchange. Steady-state equations have been derived from the model and have been used for the derivation of the theoretical rate equation. Computer analysis shows a good fit with the experimental results. It follows from the analysis that the apparent dissociation constant of the exchange protein-liposome complex decreases with an increasing phosphatidic acid content of the liposomes. This suggests that in this model system it is the phospholipid composition of the membranes involved that regulates the amount of exchange protein available to function as a carrier of phosphatidylcholine.