Effects of excitatory amino acids on phosphoinositide metabolism in frog retina.

The effects of excitatory amino acid receptor agonists on the hydrolysis of phosphoinositides were examined using frog retinal membranes prelabeled in vitro with either 32PO4 or [3H]inositol. Glutamate stimulated release of [3H]inositol phosphates (IPs) from the retinas and altered the 32P-labeling pattern of phosphatidylinositol (PI) cycle intermediates. This indicates that glutamate affects not only the hydrolysis of phosphoinositides but possibly other steps involved in the PI cycle. Among glutamate analogs, kainate (KA), quisqualate (QA), and, to a lesser extent, N-methyl-D-aspartate (NMDA) mimicked the glutamate effect, whereas L-2-amino-4-phosphonobutyrate (L-AP4) was not effective in causing either the accumulation of [3H]IPs or the alteration of the 32P-labeling pattern of PI cycle intermediates. Among QA specific agonists, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), but not ibotenate (IBO) or trans-1-aminocyclopentane-1,3-dicarboxylate (ACPD) was active in stimulating IPs formation. KA effect on IPs formation may be due to indirect (polysynaptic) activation of receptor(s) other than L-AP4, IBO, or ACPD specific QA receptors. To avoid activating polysynaptic pathways, retinal synaptoneurosomes prelabeled with [3H]inositol were used to examine the hydrolysis of phosphoinositides. As in whole retinas, KA, carbachol (CARB), and NMDA stimulated the release of IPs while L-AP4 had minimal effect. Glycine (GLY) had no effect. Our results show CARB and KA to be the most effective in stimulating the production of IPs. Their effects were exerted directly through separate receptors and not through polysynaptic pathways. ACPD and IBO were the least effective in eliciting the release of IPs. Our studies provide evidence that ionotropic and not metabotropic glutamate receptors are involved in PI metabolism in the retina.

Unique molecular species composition of glycerolipids of frog rod outer segments.

The composition and metabolism of molecular species of glycerolipids, including phosphatidic acid (PA), phosphatidylinositol (PI) and diacylglycerol (DG), were studied in four frog retinal fractions prepared by discontinuous sucrose gradient centrifugation. Six glycerolipid classes were isolated from the lipid extracts of each fraction and converted to their corresponding 1,2-diacylglycerol acetates by acetolysis for quantitation of their molecular species by HPLC. Rod outer segments (ROS) showed a distinctive molecular species composition in all glycerolipid classes except phosphatidylcholine (PC). The relative amounts of dipolyunsaturated species in ROS were higher in phosphatidylethanolamine (PE), phosphatidylserine (PS), and PA, compared to the other retinal fractions. PI and DG of ROS had a similar molecular species composition and contained only small amounts of dipolyunsaturated species. A unique feature of the molecular species of ROS PI and DG was that they had high amounts of species containing docosahexaenoic acid (22: 6 omega 3), while PI and DG from the other retinal membranes consisted mostly of species containing arachidonic acid (20: 4 omega 6). Following in vitro incubation of frog retinas with [2-3H] glycerol, the mass and radioactivity distributions among molecular species were determined following HPLC fractionation. The unique species composition of PS in ROS is determined mainly by selective translocation from the inner segments to ROS, since the dpm %, representative of newly synthesized species composition of the same glycerolipid classes in the other membrane fractions. This suggests that the distinctive species composition of PE and PA in ROS is determined not by selective translocation from the inner segments, but by remodeling processes taking place in the ROS.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphoinositide metabolism in frog rod outer segments.

Previous studies have shown that vertebrate rod outer segments (ROS) have a light activated phospholipase C which hydrolyzes phosphatidylinositol-4,5-bisphosphonate (PIP2). Three different experimental approaches have been used to test the hypothesis that the phosphatidylinositol (PI) biosynthetic cycle is present in ROS and that PIP2 can be regenerated from DG independent of rod inner segments. In the first study, enzyme activities of the PI cycle were assayed simultaneously in the presence of CTP, myo-inositol and [gamma-32P]ATP using endogenous lipids as substrates. Under these conditions, broken (leaky) ROS prepared by continuous sucrose gradient centrifugation showed PI, PIP and DG kinase activities similar to those found in intact ROS and non-ROS membranes, whereas PI synthetase activity was much lower in the leaky ROS than in the other two fractions. The relative distribution of PI synthetase specific activity in the three membrane preparations was similar to that of the microsomal enzyme marker cytochrome c reductase. ROS prepared by discontinuous sucrose gradient centrifugation showed only 2-3% of whole homogenate PI synthetase or phosphatidyl: cytidyl transferase activities, and the distribution of activities was the same as for microsomal and mitochondrial marker enzymes. In the second study, whole retinas were incubated with myo-[2-3H]inositol or [2-3H]glycerol in vitro, and the time course of incorporation of radioactivity into PI and other phospholipids was determined for ROS and three other retinal fractions. Over a 10-hr period, the rate of incorporation of myo-[2-3H]inositol or [2-3H]glycerol into PI in ROS was lowest among the various retinal fractions. In the third study, chemical analysis of the molecular species composition of PI, DG and phosphatidic acid (PA) from ROS shows that PA is substantially different from PI and DG, the latter two being quite similar. These results are consistent with a precursor-product relationship between PI and DG, but not with the conversion of DG to PA or of PA to PI. Taken together, these three studies indicate that ROS do not have PI synthetase or phosphatidyl: cytidyl transferase activities, but do have DG, PI and PIP kinase activities. Thus, the PI in ROS lost through rapid turnover must be replaced with molecules derived from de novo synthesis in the inner segment of the photoreceptor cell.

Quantitative analysis of retinal glycerolipid molecular species acetylated by acetolysis.

A method for the quantitative analysis of molecular species of 1,2-diacylglycerol acetates (1,2-DGAC) containing polyunsaturated fatty acids is described. Phosphatidylethanolamine (PE) isolated from frog retina was used to test the method. PE was converted to 1,2-DGAC by acetolysis. The molecular species of the 1,2-DGAC were resolved by reverse-phase high performance liquid chromatography (HPLC), detected by UV absorption spectroscopy at 210 nm, and identified by gas-liquid chromatography (GLC) of the fatty acid methyl esters (FAME). Molar response curves were generated for each DGAC molecular species that eluted as a single entity from HPLC by determining the moles of fatty acids in the molecular species collected and the response (peak area unit) of the UV detector. Each molecular species response curve was linear from about 10 pmoles to 4-8 nmoles, allowing the slope of each curve to be used as a molar absorptivity. This method provides a means for quantification of most of the molecular species of all glycerolipid classes.