Morphine and codeine are endogenous components of human cerebrospinal fluid.

We have examined cerebrospinal fluid (CSF) from twelve patients who were not on any medication and found them to contain both morphine and codeine in concentrations of 2 to 339 fmol/ml. These are comparable to the concentration of opioid peptides in spinal fluid. Both morphine and codeine are present mainly in conjugated form from which the free alkaloids can be released by acid hydrolysis.

Effect of guanethidine on collagen biosynthesis in blood vessels of hypertensive rats.

The effect of guanethidine on collagen biosynthesis in the aorta and mesenteric artery was investigated in desoxycorticosterone acetate (DOCA)-salt hypertensive rats. Prolyl hydroxylase activity (EC 1.14.11.2; proline, 2-oxoglutarate dioxygenase) and 14C-proline incorporation into collagen, two markers of collagen biosynthesis, were significantly increased in blood vessels of hypertensive rats compared with those of controls. When guanethidine (5 mg/kg, i.p.) was given daily to the hypertensive rats for 4 weeks, the blood pressure was decreased to 150 +/- 7 mm Hg, whereas the blood pressure of the untreated hypertensive rats was 218 +/- 10 mm Hg. Prolyl hydroxylase activity in the aorta and mesenteric artery and 14C-proline incorporation into aortic collagen were significantly reduced concomitant with the decrease in blood pressure. These results suggest that the decrease in vascular collagen biosynthesis in hypertensive rats treated with guanethidine is related to the lowering of their blood pressure.

The formation of hydroxyproline in collagen by cells grown in the absence of serum.

L-929 and 3T6 cells were conditioned to grow in a chemically defined medium lacking serum and ascorbate. Serum, when added, had a small stimulatory effect on the growth rate of the cells, but ascorbate had no effect either on the growth rate or on the rate of protein synthesis. These cells were also shown to lack gulonolactone oxidase activity and therefore could not synthesize their own ascorbate. Nevertheless, in the absence of serum and ascorbate both cell types were able to hydroxylate peptidyl proline to an appreciable extent. This suggests that reductants other than ascorbate can at least partially satisfy the requirement for a reductant in the prolyl hydroxylase reaction in vivo.

Prolyl hydroxylase half reaction: peptidyl prolyl-independent decarboxylation of alpha-ketoglutarate.

Prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) is a mixed-function oxygenase that hydroxylates peptidyl proline with the simultaneous and stoichiometric decarboxylation of alpha-ketoglutarate to succinate and CO2. It has been found that highly purified preparations of the enzyme can decarboxylate alpha-ketoglutarate in the absence of a peptidyl proline substrate. The uncoupled decarboxylation proceeds at only a fraction of the rate of the whole reaction and for study requires substrate quantities of the pure enzyme, as well as oxygen, ferrous ion, and ascorbate. No hydroxyproline is formed under these conditions. Immobilized antiserum to prolyl hydroxylase was found to remove both activities from enzyme preparations. However, addition of free antiserum during incubation inhibits only the complete reaction. Poly(L-proline), a specific inhibitor of prolyl hydroxylation, enhances the uncoupled decarboxylation of alpha-ketoglutarate without itself being hydroxylated. All of these findings prove that alpha-ketoglutarate can serve as substrate in the absence of peptidyl proline and is most likely the initial site of attack by oxygen. In the coupled reaction an oxidized form of the keto acid, perhaps a peroxy acid, then attacks prolyl residues in the unhydroxylated substrate.

Expression of collagen biosynthetic activities in lymphocytic cells.

Immunoglobulin-producing Merwin plasma cells, MPC-11, have been found to contain proplyl hydroxylase (prolyl-glycyl-peptide,2-oxoglutarate:oxygen oxidoreductase; EC 1.14.11.2) activity and its crossreacting protein, as well as hydroxyproline and a collagenous protein that could not be classified as type I, II, or III collagen. Friend leukemic cells, on the other hand, contained only prolyl hydroxylase. Thymus-derived (T) lymphocytes and bone-marrow-derived (B) lymphocytes freshly isolated from BALB/c mice expressed low but significant prolyl hydroxylase activity. Upon stimulation with phytohemagglutinin, the enzyme activity in T cells increased 22- to 29-fold. Crossreacting protein was also increased and appeared more stable than the prolyl hydroxylase. The effect of lipopolysaccharide stimulation on B cells uas similar but not as pronounced. Thus, even when not accompanied by other collagen biosynthetic activities, prolyl hydroxylase is present in all cells of hematologic origin.

Increased turnover of arterial collagen in hypertensive rats.

The turnover of total collagen in several tissues of 12-week-old normotensive and hypertensive rats was estimated by using tritium-labeled proline as a precursor. The effect of reutilization of the label was minimized by treatment with large doses of unlabeled proline subsequent to administering the radioactive imino acid. The collagen from skin, tail tendon, aorta, and mesenteric artery in normotensive animals had a half-life of about 60--70 days. In hypertensive animals the half-lives of skin and tail tendon collagen were unchanged but the half-lives of collagen in the aorta and mesenteric artery were reduced to 17 days.

Homology between a prolyl hydroxylase subunit and a tissue protein that crossreacts immunologically with the enzyme.

A protein, enzymatically inactive but immunologically related to prolyl hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase; EC 1.14.11.2) (cross-reacting protein), has been purified to near homogeneity from skin of newborn rats. The purified protein has a molecular weight of 60,000 on gel filtration and sodium dodecyl sulfate gel electrophoresis, corresponding to that of the smaller of the two dissimilar subunits of the enzyme. The two subunits of prolyl hydroxylase differ markedly from one another in their amino acid compositions, but crossreating protein and the smaller subunit are very similar in composition. On antibody-affinity chromatography both subunits reacted with the antibody developed against the intact enzyme. Neither crossreacting protein nor the 60,000 molecular weight subunit was adsorbed onto concanavalin A, which adsorbed the intact enzyme as well as the larger subunit. It would appear that crossreacting protein is identical to one of the subunits of prolyl hydroxylase or metabolically related to it.

Hypertension: increase of collagen biosynthesis in arteries but not in veins.

In two models of hypertension in rats, it was shown that collagen synthesis and deposition are increased in arteries where blood pressure is elevated. By contrast, there were no alterations in any of the markers of collagen synthesis in veins, where blood pressure was only slightly elevated. It would appear that the stimulus for vascular collagen synthesis is provided by a direct effect of the increased pressure on the arterial cells rather than by a humoral factor released into the general circulation.

Reduction of collagen biosynthesis in blood vessels and other tissues by reserpine and hypophysectomy.

Synthesis of collagen in the vasculature and other tissues of normotensive rats is markedly reduced by reserpine. Hypophysectomy produces similar effects. The effects of reserpine on collagen biosynthesis may be mediated through the hypothalamus.

Reduction of blood pressure and vascular collagen in hypertensive rats by beta-aminopropionitrile.

beta-Aminopropionitrile, a specific inhibitor of lysyl oxidase prevented the rise in blood pressure induced by deoxycorticosterone-salt in rats. In addition, after the onset of hypertension, administration of beta-aminopropionitrile lowered the blood pressure. Concomitant with the lowering of blood pressure, there was a reduction in the more highly crosslinked form of vascular collagen. These findings would indicate that increases in vascular connective tissue are not only sequelae of hypertension, but may also contribute to the maintenance of elevated blood pressure.

In vivo labeling and turnover of prolyl hydroxylase and a related immunoreactive protein.

Prolyl hydroxylase and an immunologically related protein (CRP) were purified from neonatal rabbit skin at various time periods following administration of 3H-leucine. The peak incorporation of label into prolyl hydroxylase was found to be 12 hours, while peak incorporation into CRP occurred within 2 hours. Semi-log plots of the loss of radioactivity from these protein pools against time indicated an apparent T 1/2 for prolyl hydroxylase of 78 hours, and a T 1/2 of CRP of 44 hours. Calculated Kd values indicate that that breakdown of active enzyme does not account for the amount of CRP found in tissues.

Increased collagen synthesis and the kinetic characteristics of prolyl hydroxylase in tissues of rabbits with experimental arteriosclerosis.

Increased aortic and liver prolyl hydroxylase activity has been suggested as an early biochemical indicator of the fibrotic changes which occur in rabbits with injury induced arteriosclerosis. Daily administration of epinephrine (0.025-0.050 mg/kg, i.v.) and thyroxine (0.050 mg/kg, i.p.) to rabbits for 3 weeks produced aortic fibrous plaques with a 4-fold increase in aortic prolyl hydroxylase and also a 5-fold increase in liver prolyl hydroxylase. Histopathologically, the livers of these rabbits show subcapsular areas of necrosis. When total prolyl hydroxylase related antigen was measured. the increase in liver prolyl hydroxylase activity accounted for only a small portion of the total prolyl hydroxylase antigen. However, in the aorta a majority of the increase in antigen is due to the increased amount of enzyme. DNA content per aorta was unchanged and RNA content increased in the aortic tissue of the arteriosclerotic rabbits. However DNA and RNA levels increased 60% in the livers of arteriosclerotic rabbits. In vitro incorporation of radioactively labeled proline into collagenase digestable protein was at least 2-fold greater in aorta and liver minces from arteriosclerotic rabbits. Michaelis--Menten kinetic parameters were obtained for the liver prolyl hydroxylase purified by affinity chromatography from arteriosclerotic rabbits. The Km for the enzyme from treated animals was not significantly different from control. However, the Vmax of the enzyme purified from diseased liver was 4-fold greater when compared to controls.

Increased collagen synthesis in blood vessels of hypertensive rats and its reversal by antihypertensive agents.

Collagen synthesis is increased in the aortas, mesenteric arteries, and to a lesser extent, in the hearts of rats either made hypertensive with desoxycorticosterone acetate-salt or that are spontaneously hypertensive. Several markers of collagen biosynthesis were shown to be increased, including prolyl hydroxylase (EC 1.14.11.2; proline, 2-oxoglutarate dioxygenase), prolyl hydroxylase-related antigen, total collagen content, and the incorporation of [(3)H]proline into total protein and into collagen. The antihypertensive agents chlorothiazide and reserpine, when administered before the onset of hypertension in the rats treated with desoxycorticosterone acetate-salt, prevented or diminished the increase in collagen biosynthesis. When reserpine was given after the onset of hypertension, prolyl hydroxylase activity was decreased concomitant with the decrease in blood pressure. Treatment with reserpine is particularly effective in diminishing arterial prolyl hydroxylase activity.

Cell-free synthesis of procollagen: L-929 fibroblasts as a cellular model for dermatosparaxis.

A cell-free system that actively synthesizes collagen was prepared from L-929 fibroblasts. Chromatographic and electrophoretic techniques were used to demonstrate that the only collagenous products are pro alpha(1) and pro alpha(2) chains. The collagen synthesized by the cell-free system was also compared to the collagen extracted from the cells. The cellular collagen was composed of aggregates of pro-alpha chains, while no alpha chains were found. Procollagen peptidase activity could not be detected in the cells, and the activity present in the medium was low, comparable to that in dermatosparaxic cell cultures. These properties indicate that L-929 cells may be a model system for dermatosparaxis.