Lipid-protein interactions in rat renal subcellular membranes: a biophysical and biochemical study.

The phase behavior of plasma membrane (PM), endoplasmic reticulum (ER), and nuclear membranes (NM) isolated from adult rat papillary cells was studied using the molecular probe Laurdan. The steady-state fluorescence data analysis was correlated with the lipid composition obtained by biochemical assays. The comparison between intact membranes and protein-free reconstituted vesicles using the whole lipid extract shows the essential role of proteins on the temperature response of natural membranes. The phospholipid (PL) and cholesterol (Cho) content was measured in the three membrane fractions, the PL/Cho molar ratio being between 1.5 and 1.9. However, Laurdan's parameters in NM show a fluid phase state pattern even at low temperature (5 degrees C), with a restricted dipole relaxation in comparison with that displayed in liquid crystalline phase state lipid model membranes. PM and ER are in a gel-like state at temperatures below 20 degrees C, showing increasing dipole relaxation with temperature. The curved fits obtained are characteristic of cholesterol-enriched membranes. The distinctive phase behavior of nuclear membranes vanishes when proteins are extracted. However, relaxation is still faster in this fraction, which correlates with the native lipid composition. NM has the lowest percentage of phosphatidylinositol and sphingomyelin-the latter being a highly saturated phospholipid- and the highest percentage of phosphatidylcholine and phosphatidylethanolamine (PE), nuclear PE being enriched in arachidonic acid. All these changes agree with the higher fluidity of NM compared with ER or PM in the conditions assayed.

Changes in rat papilla microsomal membrane fluidity during development.

During maturation, rat renal papillary microsomes suffer a rearrangement in their fatty acid phospholipid composition. The most significant changes in total phospholipids are the increase in their content of the 20:4 and a decrease in the levels of 14:0, 16:0, 18:1, 22:6 and 20:3 fatty acids. The changes in total phospholipid fatty acid content are a reflection of the variations in the individual phospholipid composition. During this period, microsomal cholesterol, phospholipid, and protein concentrations present no variations. Steady state fluorescence anisotropy obtained by using TMA-DPH (see text) as a fluorescence probe denoted higher values for 70- versus 10-day-old microsomes. Using DPH as a probe, steady state fluorescence anisotropy was determined in whole microsomes, as well as in total lipid and phospholipid vesicles, from both 10- and 70-day-old papillary cells. No differences were detected in phospholipid and total lipid vesicles between days 10 and 70. On the other hand, 10-day-old microsomes appeared to be less fluid than adult microsomes. The results indicate that these structural changes in kidney membranes during development might affect protein-lipid interaction and, therefore, the activity of many membrane enzymes.

Changes in the kinetic properties of renal arachidonoyl-CoA synthetase during development in the rat.

Changes in endogenous levels of phospholipid arachidonic acid and in its radioactive incorporation can be detected in the rat kidney medulla during maturation. In an effort to explain this phenomenon, the arachidonoyl-CoA synthetase activity in the neonatal (10 days of age) and adult kidney (70-day-old rats) were studied. The neonatal kidney enzyme showed greater affinity, but less capacity than the adult enzyme to incorporate arachidonic acid into the membrane. The apparent Km values were 27.8 and 73.9 microM while the vmax was 3.68 and 15.7 nmol/min/mg protein, for 10 and 70 days of age, respectively. Affinity for ATP was found to be almost 7-fold greater in adults when compared to neonates. The vmax for ATP also increased during development, with values of 1.04 and 2.87 at 10 and 70 days of age, respectively. Affinity for coenzyme A did not vary between the two stages studied, though the vmax increased 10-fold from 10 to 70 days of age. Since arachidonic acid availability is low in the perinatal stage because of its restricted endogenous synthesis, the higher enzyme affinity for this fatty acid at 10 days of age described here, could be of great importance in helping to capture the low but indispensable amount of arachidonic acid present in maternal milk during lactation.

Fatty acid profiles of rat renal phospholipids during development.

The fatty acid content of rat renal phospholipids was examined during development. An increase in the arachidonic acid content of a particular fraction of phosphatidylcholine (PC2), phosphatidylethanolamine and phosphatidylinositol between 10 and 20 days of age could be observed in the papilla, and these levels were maintained into adulthood, while in the 20:4 content of phosphatidylserine changes were found between 30-day-old rats and adults. In the other fraction of phosphatidylcholine (PC1), saturated fatty acids such as 16:0 and 14:0 decreased, while no changes occurred in the stearic acid (18:0) content. The pattern found for the medulla did not differ significantly from that of the papilla. In contrast, the cortex content of arachidonic acid at 10 days of age was higher than that for papilla and medulla. Levels increased between 10 and 20 days of age, returning to the original values by 30 days with no further variations in the adult. These changes in arachidonic acid content with age in addition to the differences found between the three kidney zones might explain some causes of incomplete renal function in newborns.

Compartmental study of rat renal prostaglandin synthesis during development.

The biosynthesis of prostaglandins (PGs) from the endogenous and exogenous precursor, arachidonic acid (AA), in renal papilla, medulla and cortex from neonatal to adult rats was investigated. Rat renal papilla and medulla incubated in the presence of [1-14C]AA released radioactive PGE2, PGF2 alpha and PGD2 which increased with age. No radioactive prostaglandins were found in the supernatants of renal cortex at any age studied. The amount of total prostaglandins released from the endogenous precursor also increased from 10 to 70 days of age, PGD2 being the prostaglandin that showed the most important rise. In the cortex, only PGE2 release increased with age. Cyclooxygenase (COX) activity was measured in papillary, medullary and cortical homogenates by using [1-14C]AA as substrate. Papillary and medullary COX activity increased after 10 days of age and continued to rise up to day 30 thereafter remaining unaltered until adulthood. Cortical COX activity was very low and decreased with age. These findings indicate the low capacity of the neonatal rat kidney to synthesize PGs.