Helen J. Kalinsky

CGS 8216: Receptor binding characteristics of a potent benzodiazepine antagonist

CGS 8216 is a novel nonbenzodiazepine that inhibited 3H-flunitrazepam (3H-FLU) binding to rat synaptosomal membranes in vitro at subnanomolar concentrations. It prevented the in vivo labeling of brain benzodiazepine receptors by 3H-FLU with the same potency as diazepam when given orally to mice. Pharmacologic tests showed that it was devoid of benzodiazepine-like activity but it antagonized the actions of diazepam in these tests. It did not interact with alpha- or beta- adrenergic, H1-histaminergic or GABA receptors but it inhibited adenosine-activation of cyclic AMP formation. Studies with 3H-CGS 8216 demonstrated that it bound specifically and with high affinity to rat forebrain membranes and was displaced by drugs with an order of potencies similar to that observed when 3H-diazepam and 3H-FLU were used as radioligands. The regional distribution of 3H-CGS 8216 binding sites in the brain was also similar to that of 3H-FLU. Dissociation of 3H-CGS 8216 binding was slow at 0 degrees C but increased with temperature and was almost complete within 1 min at 37 degrees C. Scatchard analyses were linear, yielding KD values of 0.044, 0.11 and 0.18 nM at 0, 25 and 37 degrees C, respectively; the Bmax value did not change appreciably with temperature and was approximately 1000 fmoles/mg protein. Using 3H-FLU, the value for Bmax as well as for the KD increased with temperature. The total number of binding sites determined for 3H-FLU was greater than that for 3H-CGS 8216 at each temperature. CGS 8216 exhibited mixed-type inhibition of 3H-FLU binding. GABA did not stimulate 3H-CGS 8216 binding whereas it enhanced 3H-FLU binding. CGS 8216 may be a useful ligand for probing the antagonist properties of the benzodiazepine receptor and is likely to exhibit interesting therapeutic effects.

Basement membrane biosynthesis. Secretion without deposition of underhydroxylated basement membrane collagen by parietal yolk sacs.

1. 1. In contrast to published results with other collagen-synthesizing systems the rate of secretion of basement membrane collagen synthesized by the rat parietal yolk sac was essentially the same in the presence or absence of either dipyridyl or GPA 1734 (8,9-dihydroxy-7-methyl-benzo(b)quinolizinium bromide), with no evidence for intracellular accumulation of underhydroxylated basement membrane collagen. This was shown by measuring total radioactivity and [14C]hydroxyproline in collagenase digests of tissue and medium proteins after incubation for 3 and 6 h with [14C]proline or [14C]proline plus [3H]glycine. The secreted underhydroxylated basement membrane collagen contained 2–10% of the hydroxyproline found in controls without hydroxylation inhibitors. The identity of the non-dialyzable radioactivity in the medium after incubation with dipyridyl or GPA 1734 with underhydroxylated basement membrane collagen was established (a) by tripeptide analyses of the collagenase digests, which showed about 20% of the radioactivity to be Gly-Pro-Pro and (b) by incubation with partially purified prolyl hydroxylase from rat skin, which converted the material to hydroxyproline-containing protein with a [14C]hydroxyproline to total [14C]protein ratio close to that of control basement membrane collagen. 2. 2. Normal 14C-labelled basement membrane collagen, whether found intracellularly, secreted into the medium or deposited on Reicherts membrane, after reduction and denaturation appears to be the same molecular size on SDS-agarose chromatography. Similarly, 14-labeled basement membrane collagen from the medium synthesized in the presence of hydroxylation inhibitors and chromatographed under conditions which prevent proteolytic digestion appears as a single peak with the same molecular size as control basement membrane collagen. 3. 3. Underhydroxylated basement membrane collagen was not deposited on Reicherts membrane as evidenced by measurements of total [14C]proline and [14C]hydroxyproline in collagenase-digests of membranes free of trophoblast and epithelial cells. 4. 4. The inhibition of hydroxylation did not affect the synthesis of the glucosamine-containing moiety of basement membrane collagen. This was shown by pulsing with [14C]glucosamine in the presence of dipyridyl and chasing with 0.5 mM glucosamine in fresh medium containing Fe2+ and cycloheximide. Glucosamine incorporated intor basement membrane collagen during the pulse stage was found to deposited on Reicherts membrane after hydroxylation of proline and lysine and secretion at the chase stage.

Identification of hormonally controlled endogenous inhibitor of liver nicotinamide deamidase

Abstract A study has been carried out of the hormonally controlled endogenous inhibitor of nicotinamide deamidase, the enzyme which catalyzes the first and presumably rate-limiting step in the biosynthesis of NAD from nicotinamide. The inhibitory material has been purified and found to consist of a mixture of free fatty acids, the predominant ones being oleic, linoleic and arachidonic. A synthetic mixture of authentic fatty acids of the same composition as the purified inhibitor imitated the inhibitory potency of the latter. A variety of individual free fatty acids of varying chain-length and degree of unsaturation were tested for their ability to inhibit the purified enzyme. The more potent fatty acids caused 50% inhibition at concentrations of about 2 μM. The present results indicate that the effects of the endocrine system on the levels of free acids contribute to the mechanism of hormonal regulation of NAD synthesis from nicotinamide.

Metabolism of octanoate and its effect on glucose and palmitate utilization by isolated fat cells.

Summary Octanoate is avidly incorporated into triglycerides by isolated rat adipocytes in the presence of glucose via direct esterification without prior β-oxidation to acetyl CoA. This was shown by separation of the products formed from [1-14C] octanoate into lipid classes using Florisil columns, and after alkaline hydrolysis of the triglyceride fraction, by cochromatography with authentic fatty acids on reverse-phase Celite columns. The relative contribution of [U- 14 C] glucose and [1- 14 C] octanoate to triglyceride synthesis and CO2 formation were studied under a variety of conditions. Concentrations of octanoate below 0.5 mM have a stimulatory effect on the conversion of [U- 14 C] glucose to CO2, triglycerides and esterified fatty acids. However, a marked depression of fatty acid synthesis from [U- 14 C] glucose was observed in the presence of millimolar concentrations of octanoate. Octanoate had no effect on the esterification of palmitate, but palmitate strongly depressed the ability of rat adipocytes to esterify octanoate.

Adaptive response of hepatic acetyl-coa carboxylase to dietary alterations in genetically obese mice and their lean controls.

Abstract 1. 1. Genetically obese mice (C5 7BL/6J-ob/ob, Jackson Laboratories) have much higher levels of hepatic acetyl-CoA carboxylase activity than their lean siblings, under a variety of nutritional states. However, when these mice are fasted for 48 h and then refed a fat-free diet for 48 h, the activity of this enzyme in the lean group shows about a 9-fold increase over the measured under normal dietary conditions, while obese mice show only 1 2-fold increase. The acetyl-CoA carboxylase activity observed under the dietary conditions is thus comparable in both lean and obese animals. Oil feeding or fasting for 48 h markedly depresses the activity of this enzyme in both groups and seems to be an effective means of reducing acetyl-CoA carboxylase activity in the obese mice, particularly, to far below the values found under normal dietary conditions. 2. 2. Both acetyl-CoA carboxylase and fatty acid synthetase purified from livers of obese and lean mice show comparable specific activities and no demonstrable differences with respect to their kinetic properties. Acetyl-CoA carboxylase from the two sources is also identical with respect to sensitivity to reagents and other inhibitors (such as malonyl-CoA, palmitoyl-CoA, etc.), to heat inactivation and in its sedimentation properties. These results suggest quantitative differences rather than differences in the catalytic and regulatory properties of the obese and lean enzymes.