Excitatory amino acid-stimulated uptake of 22Na+ in primary astrocyte cultures.

In this study we have found that L-glutamic acid, as well as being taken up by a Na+-dependent mechanism, will stimulate the uptake of 22Na+ by primary astrocyte cultures from rat brain in the presence of ouabain. By simultaneously measuring the uptake of 22Na+ and L-3H-glutamate a stoichiometry of 2-3 Na+ per glutamate was measured, implying electrogenic uptake. Increasing the medium K+ concentration to depolarize the cells inhibited L-3H-glutamate uptake, while calculations of the energetics of the observed L-3H-glutamate accumulation also supported an electrogenic mechanism of at least 2 Na+:1 glutamate. In contrast, kinetic analysis of the Na+ dependence of L-3H-glutamate uptake indicated a stoichiometry of Na+ to glutamate of 1:1, but further analysis showed that the stoichiometry cannot be resolved by purely kinetic studies. Studies with glutamate analogs, however, showed that kainic acid was a very effective stimulant of 22Na+ uptake, but 3H-kainic acid showed no Na+ -dependent uptake. Furthermore, while L-3H-glutamate uptake was very sensitive to lowered temperatures, glutamate-stimulated 22Na+ uptake was relatively insensitive. These results indicate that glutamate-stimulated uptake of 22Na+ in primary astrocytes cultures cannot be explained solely by cotransport of Na+ with glutamate, and they suggest that direct kainic acid-type receptor induced stimulation of Na+ uptake also occurs. Since both receptor and uptake effects involve transport of Na+, accurate measurements of the Na+ :glutamate stoichiometry for uptake can only be done using completely specific inhibitors of these 2 systems.

Von Willebrand factor (VIII R:Ag), fibronectin, and insulin-like growth factors I and II in diabetic retinopathy and nephropathy.

We have measured plasma von Willebrand factor (VWF) as the factor VIII-related antigen, plasma fibronectin, and two of the serum somatomedins, insulin-like growth factor I (IGF I) and IGF II, in 51 diabetic patients and 25 nondiabetic control subjects. VWF was significantly higher in the diabetic group than in the controls (173 +/- 9% SEM versus 101 +/- 9%, P less than 0.001), as has been reported by others. However, within the diabetic group there was no significant difference in VWF between those patients without retinopathy, those with background or proliferative retinopathy, or those with macular edema. There was also no difference in VWF between the diabetic subjects with and those without proteinuria. These results rule against a previously advanced hypothesis that the increase in VWF in patients with diabetes is secondary to microangiopathy. No significant difference was observed in fibronectin, IGF I, or IGF II between the diabetic and control groups, between the diabetic group without retinopathy and the retinopathic subgroups, and between the diabetic subjects with and without proteinuria. In the diabetic patients, there was no correlation between diabetic control as assessed by glycosylated hemoglobin and glycosylated serum protein, and the plasma levels of VWF, fibronectin, IGF I, or IGF II. The results of this study strongly suggest that neither plasma VWF, fibronectin, IGF I, nor IGF II plays an important primary role in the pathogenesis of diabetic microvascular disease, although one or more of these factors might play a permissive role.

Effect of bacitracin and polyamines on follicle-stimulating hormone binding to membrane-bound and detergent-solubilized bovine calf testis receptor.

We have examined the effects of bacitracin and several polyamines on the interaction of 125I-human FSH (125I-hFSH) with membrane-bound receptors (MR) or detergent-solubilized receptors (DSR) derived from bovine calf testes. Binding of 125I-hFSH to either calf testis MR or DSR was inhibited by bacitracin in a dose-related manner. Inhibition of 125I-hFSH binding to either MR or DSR was due to a decrease in the apparent affinity constant (Ka) of receptor for FSH. FSH binding inhibition by bacitracin was significantly greater with the MR (ED50 = 5.9 mM) than with the DSR (ED50 = 25 mM). Bacitracin was a more potent inhibitor of binding than the polyamines at equimolar concentrations in both MR and DSR systems. The inhibitor potency of the polyamines when using DSR was related to chain length and the number of amino groups present (spermine greater than spermidine greater than putrescine). Although it was not possible to ascertain the mechanism of binding inhibition, our results demonstrate a direct effect of bacitracin and polyamines on the interaction of FSH with testis receptors.

Mirex kinetics in the rhesus monkey. II. Pharmacokinetic model.

14C-Mirex was given iv and po to female rhesus monkeys (Macaca mulatta) and radioactivity was measured in plasma, urine, and feces at intervals after dosing and in tissues when animals were killed. Graphical analysis of plots of the logarithm of plasma concentration vs. time was used to provide estimates of the values of the first-order rate constants required by the proposed pharmacokinetic models. A BASIC-language program, FITKIN, was used to obtain numerical solutions to the differential equations for each model and to adjust the estimates to obtain a normalized, least squares fit. Of several models postulated, a mammillary, four-compartment, open-system model, providing for the urinary excretion of Mirex from a "central" compartment and for the fecal excretion of Mirex from a "fast" tissue compartment, yielded theoretical data in agreement with observed values. This model predicted that the accumulation of Mirex into fat would be retarded by the presence of a "slow" tissue compartment so that distribution equilibrium would take about half a year. From that time to the end of a 5-year projection, little decline in the quantities of Mirex was predicted for any compartment. Sequestration in fat and a lack of metabolism were responsible for the long biological half-life of Mirex in the rhesus monkey.

Studies of the hepatic excretory defects in essential fatty acid deficiency. Their possible relationship to the genesis of cholesterol gallstones.

Male hamsters were fed normal and essential fatty acid (EFA)-deficient diets for at least 12 wk before bile duct cannulation. With [32P]phosphate, hepatic synthesis of lecithin was similar, but biliary excretion of newly synthesized lecithin was significantly reduced in EFA-deficient compared to that in normal hamsters. Hepatic uptake of intravenously infused taurocholate (TC) and taurochenodeoxycholate (TCDC) were similar in both groups of animals. However, biliary excretion of intravenously infused TC was significantly reduced in EFA-deficient hamsters, whereas that of TCDC-was unchanged. The absolute rate of biliary cholesterol excretion was similar in both groups. Canalicular bile flow, as measured by [14C]erythritol clearance after functional nephrectomy, was significantly lower, with both the bile salt-dependent and independent fractions of this flow being diminished in EFA-deficient hamsters infused with TC. It is concluded that EFA deficiency leads to impaired biliary excretion of taurocholate, lecithin, and water, while cholesterol transport is unaffected, and thus results in supersaturation of bile with respect to cholesterol and production of lithogenic bile.

Studies in the biosynthesis of hepatic and biliary lecithins.

Male rats with biliary cannulae were injected with linoleate-1-(14)C, stearate-1-(14)C, palmitate-9-10-(3)H, phosphate-(32)P, l-methionine-methyl-(14)C, and choline-methyl-(3)H in various combinations and the incorporation of these isotopes into the phospholipids of liver, bile, and plasma was determined for 1-4 hr. The results summarized below favor the view (a) that exchange of saturated fatty acids plays a role in the formation of lecithins; (b) that the unsaturated fatty acids do not undergo significant exchange and determine the pathway of biosynthesis of lecithins; and (c) that there is either more than one pool of CDP-choline in liver or a pathway of biosynthesis of lecithin from choline not involving CDP-choline as an intermediate. Linoleoyl lecithin of liver attained higher specific activity with respect to phosphate-(32)P and choline-methyl-(3)H than did arachidonoyl lecithin. Lecithin in bile attained higher specific activities with respect to phosphate-(32)P, choline-methyl-(3)H, and linoleate-1-(14)C than the corresponding hepatic lecithins. Stearate-1-(14)C and palmitate-9-10-(3)H attained highest specific activities in the hepatic lecithin fraction rich in arachidonic acid. The specific activity of hepatic phosphatidyl ethanolamine was lower with respect to saturated fatty acids, but much higher with respect to (32)P than any lecithin. The ratio of specific activity of (3)H in methyl groups from choline to (14)C in methyl groups from methionine in hepatic sphingomyelin was lower than in hepatic linoleoyl lecithin.