Richard A. Salvador

In vitro studies on the mechanism of action of a new antiallergic, Ro 21-7634.

The effects of Ro 21-7634 and disodium cromoglycate (cromoglycate) on the in vitro release of mediators of anaphylaxis from rat peritoneal cells and guinea pig lung tissue were compared. Ro 21-7634 was 25 fold more potent than cromoglycate as an inhibitor of antigen-induced histamine release from passively sensitized (IgE) rat peritoneal cells. Ro 21-7634 was also the more potent inhibitor of both compound 48/80- and concanavalin A-induced histamine release from rat peritoneal cells. The two drugs shared the common properties of producing the same maximal level of inhibition in each of the above releasing systems and exhibiting a time and concentration dependent loss of inhibitory activity when added to the cells prior to the releasing agent. Neither drug inhibited ionophore A23187- or ionophore X537A-induced histamine release from these cells. Ro 21-7634 inhibited antigen-induced (IgG1) histamine and SRS-A release from actively sensitized guinea pig lung fragments, whereas cromoglycate did not. The results indicate that Ro 21-7634 and cromoglycate act through a common mechanism to inhibit allergic mediator release and that Ro 21-7634 is the more potent inhibitor.

Accumulation of collagen in the uterus of the immature rat administered estradiol-17β

Abstract The total protein and collagen content of the uterus was determined in both the immature and adult ovariectomized rat administered 5 μg estradiol-17β for 4 or 5 consecutive days. In the immature rat, this treatment results in a 3-fold increase in the collagen content of the uterus. This increase is linear following the first three doses of estradiol-17β and appears to reach a maximum after approximately 5 days. The total protein of the uterus also increases uniformly throughout the experimental period; therefore, the per cent collagen in total protein remains essentially unchanged. These results suggest that the increase in the collagen content of the uterus of the immature rat administered estradiol-17β is due to a general stimulation of protein synthesis. The administration of either estrone, diethylstilbestrol or 17-ethynylestradiol-3-methyl ether to the immature rat also causes collagenous and non-collagenous protein to accumulate in the uterus. There is a significant loss of noncollagenous protein from the uterus of the adult rat 21 days after ovariectomy, which is only partially restored by the administration of estradiol-17β for 4 consecutive days. The collagen content of the uterus is also reduced markedly and does not increase upon administration of estradio l17β.

The in vivo inhibition of collagen synthesis and the reduction of prolyl hydroxylase activity by 3,4-dehydroproline.

Abstract Various proline analogs and iron chelators were tested for their effect on collagen formation which occurs in the uterus of the immature rat following the administration of estradiol-17β. dl -3,4-Dehydroproline, l -α-azetidine-2-carboxylic acid and l -pyroglutamic acid reduced the estradiol-17β stimulated formation of hydroxyproline which occurs in the uterus following administration of the hormone while l -thiazolidine-4-carboxylic acid was without effect on this response. The activity of the d - and l -isomers of 3,4-dehydroproline was compared with the racemic mixture; the l -isomer was twice as active as the latter, while the d -isomer was only half as active. l -3,4-Dehydroproline was approximately four times as potent as l -α-azetidine-2-carboxylic acid, the second most active analog of those tested. dl -3,4-Dehydroproline inhibited the incorporation of l -[ 14 C]proline into the proline and hydroxyproline of uterine collagen; it also inhibited the incorporation of [ 14 C]glycine into collagen while having less effect on the incorporation of these amino acids into noncollagen protein. These results indicate dl -3,4-dehydroproline is a fairly specific and potent inhibitor of collagen formation in vivo . These observations indicate that dl -3,4-dehydroproline reduces the hydroxylation of prolyl residues in collagen. Presumably, this occurs in part due to the incorporation of the analog into the collagen molecule in place of proline. It is probably also related to a reduction of prolyl hydroxylase activity which can be demonstrated in the tissues of animals treated with 3,4-dehydroproline. A significant reduction of prolyl hydroxylase activity was shown to persist in the uterus, lung, and heart for approximately 24 h following a single intraperitoneal dose of dl -3,4-dehydroproline (200 mg/kg).

Ro 21-7634, a new antiallergic agent with potent oral activity.

Ro 21-7634 was examined for oral antiallergic activity in two in vivo models commonly used to evaluate anti-allergics. In the rat PCA test, this drug had an oral ID50 of 1.14 mg/kg and was found to be more potent than several other antiallergics including Disodium Cromoglycate (cromoglycate), Oxatomide, Doxanthrazole, Xanoxate, 2,6-bis (ethoxyoxalylamino) pyridine, PRD-92-EA and M+B 22,948. In contrast to cromoglycate, Ro 21-7634 was found to be an orally active inhibitor of antigen-induced bronchoconstriction in passively sensitized rats (ID50=0.2 mg/kg). In addition, Ro 21-7634 inhibited antigen-induced histamine release in an in vivo passive peritoneal anaphylaxis test system, following intraperitoneal administration. Ro 21-7634 demonstrated no end organ antagonism toward histamine, methacholine or serotonin in the guinea pig.

Studies on the effect of l-3,4-dehydroproline on collagen synthesis by chick embryo polysemes

Abstract Various proline analogs have been tested in vitro for their ability to inhibit the enzymatic aminoacylation of tRNA by proline. Of these, l -3,4-dehydroproline is the most potent inhibitor. This inhibition is competitive; the K i is 100 μ m . It was shown that l -3,4-dehydroproline can serve as substrate in the aminoacylation reaction. However, the incorporation of radioactivity from l -3,4-[ 14 C]dehydroprolyl-tRNA into protein occurs at one-fifth the rate observed for l -prolyl-tRNA. The addition of l -3,4-dehydroproline in vitro inhibits the synthesis of collagen to a greater extent than non-collagen protein.

Reduction of prolyl hydroxylase activity in L-929 fibroblasts by proline analogs

Abstract Various proline analogs were examined for their effect on the activity of prolyl hydroxylase in L-929 fibroblasts. Of the analogs tested, L-3,4-dehydroproline and L-azetidine-2-carboxylic acid were effective in reducing the activity of prolyl hydroxylase. A time course on the effect of these analogs showed that the reduction of enzyme activity by L-3,4-dehydroproline occurred at a faster rate than the reduction which occurs with L-azetidine-2-carboxylic acid. The results demonstrate that L-3,4-dehydroproline is approximately four times as potent as L-azetidine-2-carboxylic acid in reducing prolyl hydroxylase activity.

Collagen proline hydroxylase activity in the uterus of the rat during rapid collagen synthesis in vivo

Abstract The activity of collagen proline hydroxylase in the 27,000 g supernatant of the uterus was compared in the normal 20-day-old rat and in the adult rat 21 days after ovariectomy. The cofactor requirements of this enzyme were shown to be qualitatively the same as the enzyme from rat liver and skin. The specific activity of collagen proline hydroxylase in the uterus of the immature rat is approximately 250% higher than that of the ovariectomized animal. Although the total protein of the uterus of the ovariectomized rat is much greater, the total activity of this enzyme is 50% higher in the uterus of the immature rat. The daily administration of 5 μg estradiol-17β for 4 consecutive days to either animal results in a significant increase in the activity of collagen proline hydroxylase. Enzyme activity increases significantly 24 hr after the first dose of estradiol-17β and remains elevated in a reproducible pattern throughout the experimental period. Other estrogens including estriol, estrone, diethylstilbestrol, and ethynylestradiol-3-methyl ether also increase significantly the activity of collagen proline hydroxylase in the uterus of the immature rat. The activity of collagen proline hydroxylase was compared in the 27,000 g supernatant of uterus of the immature and ovariectomized rat in a dose-response study with estradiol-17β and there appears to be little, if any, difference in total enzyme capacity. These results suggest that the failure of collagen to accumulate in the uterus of the ovariectomized rat administered estradiol-17β is unrelated to a low activity of collagen proline hydroxylase.

On the mechanism of the increase in prolyl hydroxylase activity in the uterus of the rat given estradiol-17β☆

Abstract The activity of prolyl hydroxylase (EC 1.14.11.2: proline. 2-oxoglutarate dioxygenase) and the protein content of the uterus were significantly increased 4hr after the administration of a single dose of estradiol-17β to either the immature rat or the adult ovariectomized rat. In contrast to results in the uterus, prolyl hydroxylase activity was decreased in heart, kidney and lung of the immature rat after estradiol-17β. Prolyl hydroxylase activity reached a maximum in 24–32 hr, and subsequently the enzyme activity decreased toward control values. In both animals, there was a 50 per cent reduction in enzyme activity 8–12hr after reaching maximum activity. The estradiol-17β-induced increase in prolyl hydroxylase activity was blocked by inhibitors of RNA and protein synthesis. In addition, an increase in total enzyme protein was demonstrated using an enzyme immunossay. Although these data provide evidence that de novo synthesis of enzyme protein occurs in the uterus after the administration of estradiol-17β. it does not rule out the possibility that other post-transcriptional effects of estradiol-17β play a role in increasing the activity of this enzyme. Efforts to stimulate uterine prolyl hydroxylase activity with either cAMP or dibutyryl cAMP were unsuccessful.