Uterotropic 6-methoxybenzoxazolinone is an adrenergic agonist and a melatonin analog.

6-Methoxybenzoxazolinone (MBOA) is a compound isolated from grasses which has gonadotropic effects in a variety of animals. The weak beta-adrenergic agonist character of MBOA is shown by its in vitro stimulation of adenylate cyclase from several tissues. Tritiated MBOA bound specifically to particulate fractions from uterus is also displaced by alpha- and beta-adrenergic compounds. The adrenergic properties of MBOA suggest it may exert diverse effects including direct actions on gonadotropin synthesis and release. The mixed adrenergic agonist ephedrine and the antidepressant imipramine were also found to be uterotropic in the vole Microtus montanus following injection protocols used with MBOA. MBOA is structurally similar to melatonin (5-methoxy-N-acetyltryptamine); [3H]melatonin which binds to uterine and pineal membranes is displaced by MBOA and by other adrenergic agents. The fact that MBOA is a beta-adrenergic agonist and a melatonin analog can account for stimulatory and inhibitory effects of this compound on sexual development.

Cyclic nucleotide metabolism in pineal homogenates.

Adenylate cyclase (AC) in pineal particulate fractions from rabbit, rat, cow, and the vole Microtus montanus was stimulated by L-norepinephrine (NE) and L-isoproterenol (ISO). NE stimulation of rabbit and bovine pineal AC was biphasic, with a plateau between 0.01 microM and 1.0 microM and additional stimulation by NE above 1.0 microM. Stimulation by different ISO concentrations gave a typical hyperbolic curve, and optimal stimulation by ISO exceeded that by NE. Melatonin decreased ISO and NE stimulation of AC 10-20%. Although, alpha-adrenergic agonists increase beta-agonist-mediated adenosine-3',5'-cyclic monophosphate (cyclic AMP) accumulation in intact pinealocytes, similar amplification of AC stimulation was not seen with broken-cell preparations. Most (60-70%) pineal guanylate cyclase (GC) was recovered in supernatant fractions after centrifugation of homogenates at 110,000 x g; this soluble GC was unaffected by potential agonists. Low concentrations (0.01-1 nM) of NE, ISO, and phenylephrine (PE) stimulated GC in impure and purified membrane fractions, but each inhibited at concentrations above 10 microM. All concentrations of ISO and NE inhibited GC in the presence of the alpha-agonist PE. Melatonin alone did not affect particulate GC, but L-ISO stimulation was not seen in the presence of equivalent concentrations of melatonin. The in vitro data are consistent with both alpha- and beta-receptor regulation of cyclic nucleotide metabolism in pinealocytes. Endogenous NE may differentially regulate cyclic AMP and guanosine-3',5'-cyclic monophosphate (cyclic GMP) in pineal; low NE concentrations that stimulate GC have only a slight effect on AC, but higher NE concentrations that inhibit GC maximally stimulate AC. Particulate GC and AC also were resolved by equilibrium centrifugation, to give several discrete peaks of enzyme activity. The results support the existence of several forms of AC and GC, which have different responses to adrenergic agonists.

Beta-adrenergic binding is increased by melatonin and alpha-adrenergic compounds.

Binding of the beta-adrenergic ligands [3H]dihydroalprenolol and [125I]cyanopindolol to pineal particulate fractions was increased 1- to 3.5-fold by addition of low concentrations of melatonin, alpha-adrenergic agonists, or alpha-adrenergic antagonists. Minimum concentrations of melatonin or alpha-adrenergic compounds which increased beta-adrenergic binding were between 1 pM and 0.1 nM. The increased binding of [3H]dihydroalprenolol caused by melatonin (0.1 muM) was attributed to a major increase in Bmax, which persisted in protein fractions after removal of melatonin. Melatonin enhancement of [3H]dihydroalprenolol binding was apparent after 5 to 7 min (30(0], was was optimal between 20 and 40 min, and decreased at longer times. Alpha-Adrenergic receptors are unchanged during beta-receptor enhancement.

Adenylate cyclases in two populations of membranes purified from neuroblastoma X glioma hybrid (NG 108-15) cells.

Membranes from neuroblastoma X glioma NG108-15 hybrid cells were purified by equilibrium centrifugation on continuous and discontinuous gradients of sorbitol, using homogenates of cells which were pretreated with concanavalin A to increase membrane density. Adenylate cyclase was purified 24-fold in a heavy (H) membrane fraction from discontinuous gradients, opiate-stimulated guanosine-5'-triphosphatase was purified 3-fold, and opiate binding to receptors was increased 10-fold in this fraction. The relative purification of this membrane fraction is also verified by the fact that it contains a single protein (Mr = 58,000) which is covalently labeled by a reactive opiate analog (Klee, W. A., Simonds, W. F., Sweat, F. W., Burke, T. R., Jacobson, A. E., and Rice, K. C. (1982) FEBS Lett. 150, 125). The method of plasma membrane purification after cell treatment with concanavalin A (Lutton, J. K., Frederich, R. C. Jr., and Perkins, J. P. (1979) J. Biol. Chem. 254, 11181) appears generally applicable as established here with 3H-concanavalin A. Between 15 and 20% of the adenylate cyclase in whole-cell homogenates was recovered at low densities in continuous and discontinuous gradients and was only purified 2-fold above activity in the cell homogenate. There are significant differences between ligand binding, adenylate cyclase, and GTPase activities in light (L) and heavy (H) membrane fractions. GTPase activity in the L-membrane fraction was decreased from that in the cell homogenate and was not stimulated by opiates. Adenylate cyclase from L-membranes is only slightly inhibited by opiates in support of other data relating opiate inhibition to stimulation of GTPase (Koski, G., and Klee, W. A. (1981) Proc. Natl. Acad. Sci. 78, 4185).

Dissociation of E prostaglandin effects on liver glycogenolysis and cyclic AMP levels.

Some metabolic effects of prostaglandins have been related to their alteration of adenosine-3',5'-monophosphate (cyclic AMP) metabolism in different tissues. Prostaglandins E1 and E2 stimulate liver adenylate cyclase in vitro, but conflicting reports have been made about metabolic changes caused by E prostaglandins in hepatic tissue. We have attempted to resolve these issues by comparing the effects of PGE1 with those of glucagon using broken-cell homogenates, intact hepatocytes, liver slices and perfused liver. Prostaglandin E1 (PGE1) increased cyclic AMP in liver slices and in perfused liver without increasing glycogenolysis, but PGE1 had no discernible effect on carbohydrate or cyclic AMP metabolism in isolated hepatocytes. Glucagon caused predictable increases in cyclic AMP and glycogenolysis using hepatocytes, liver slices or perfused liver. These data can be explained by the absence of PGE effects on cyclic AMP metabolism in hepatocytes. The concentration of E prostaglandins (PGEs) increased 1.75-fold during incubations (37 degrees C) of hepatocyte suspensions, but cyclic AMP remained constant. Addition of exogenous arachidonate and indomethacin to cell suspensions increased and decreased PGEs, respectively, but cyclic AMP and glycogen metabolism were unchanged. Arachidonate and indomethacin likewise did not alter glucagon-stimulated glycogenolysis or cyclic AMP biosynthesis. The production of E prostaglandins and cyclic AMP appears to be unrelated in hepatocytes.

Identification of a Mr 58 000 glycoprotein subunit of the opiate receptor.

A Mr 58 000 subunit of the opiate receptor has been identified using tritiated fentanyl isothiocyanate, a potent opiate alkylating reagent with specificity for the delta-opiate receptor subclass. The subunit is alkylated in the presence of dextrorphan but not levorphanol. The specifically labelled protein was retained on columns of immobilized wheat germ agglutinin and is therefore presumably a glycoprotein. Partial purification of the Mr 58 000 opiate receptor subunit from neuroblastoma X glioma NG108-15 hybrid cell membranes is described.

High-density lipoprotein that supports Ureaplasma urealyticum growth.

A high-density lipoprotein with growth-promoting activity for Ureaplasma urealyticum was purified in high yield from equine serum by ammonium sulfate fractionation and molecular filtration. Fractions enriched in growth-promoting activity represented 5% of the total serum protein, and 30 micrograms of the purified protein per ml gave an activity equivalent to that from 100 micrograms of whole serum per ml. The serum was totally replaced by purified lipoprotein when tested in a soy peptone-yeast dialysate or when added to a chemically defined synthetic medium. Polyacrylamide gel electrophoresis indicated that one major protein with growth-promoting activity is present. A total of 10 proteins were distinguished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with 75% of the total contributed by two proteins with molecular weights of 160,000 and 170,000. A total of 90% of the lipoprotein was an alpha-protein with a mobility of 0.67 in two-dimensional immunoelectrophoresis (albumin = 1.0). The active component was further characterized as high-density lipoprotein by density ultracentrifugation. Two components with S = 6.4 and S = 15.8 were distinguished by velocity sedimentation. The lipid was removed from lipoprotein during its precipitation with acetone. The growth-promoting activity of delipidized protein was dependent upon the addition of exogenous cholesterol, and [14C]cholesterol was transferred to urea-plasmic cells in cultures containing the delipidized protein. A major portion of the [14C]cholesterol remained associated with the protein during filtration on Sepharose 4B columns.

Effects of prostaglandins and catecholamines on rat spleen adenylate cyclase in vitro.

The results reported here show some characteristics of adenylate cylase (EC 4.6.1.1) derived from homogenates of rat spleen, and describe the in vitro stimulation of this enzyme by prostaglandins, nucleotides, and F- under conditions where cyclic nucleotide degradative pathways are effectively inhibited. Particulate fractions from rat spleen homogenates contain high adenylate cyclase activities, and the highest specific activity is recovered in a particulate fraction prepared by low speed (1200 X g) centrifugation. Activity found in all particulate fractions is stimulated by fluoride, prostaglandins E1 and E2, catecholamines, and purine nucleotides. No stimulation is caused by prostaglandins F1 alpha and F2 alpha. Stimulation by prostaglandin E1 and E2 is augmented by GTP and other purine nucleotides, and stimulation by the combination of GTP and prostaglandin E1 is equal to that caused by optimal fluoride concentrations. Stimulation caused by L-isoproterenol is additive to that caused by GTP but is not increased by GTP.

Stimulation of adenylate cyclase from isolated hepatocytes and Kupffer cells.

Hepatocytes and Kupffer cells were separated from rat liver after prelabeling the Kupffer cells with colloidal iron and perfusion of the liver with digestive enzymes. The activity of several enzymes from Kupffer cells and hepatocytes was compared to validate this method of cell separation. The ratios of hepatocyte to Kupffer cell specific activities of glucose-6-phosphatase, 5'-nucleotidase, adenylate cyclase, and acid phosphatase were 20, 0.39, 0.18, and 0.078, respectively. Adenylate cyclases from hepatocytes and Kupffer cells were stimulated by fluoride ion, GTP, and catecholamines. Hepatocyte adenylate cyclase was also stimulated by glucagon, secretin, vasoactive intestinal polypeptide, and by prostaglandin E1, whereas, the Kupffer cell enzyme was completely insensitive to these hormones. The stimulation of hepatocyte adenylate cyclase by combinations of glucagon plus secretin, or glucagon plus vasoactive intestinal polypeptide, were equivalent to the sum of the individual stimulations. This suggests that the hepatocyte has specific receptors for glucagon and for vasoactive intestinal polypeptide and secretin. Prostaglandin E1 stimulation of hepatocyte adenylate cyclase was not additive to the stimulation caused by polypeptide hormones or catecholamines, nor did prostaglandin E1 decrease stimulation caused by these hormones. Although prostaglandin-sensitive adenylate cyclase was recovered with hepatocytes, 40 to 50% of the total liver prostaglandin-sensitive activity was recovered in a fraction of cell debris mixed with small cells which did not phagocytize colloidal iron.