The interaction of vinyl chloride with rat hepatic microsomal cytochrome P-450 in vitro.

In the presence of hepatic microsomes, vinyl chloride produces a ‘type I’ difference spectrum and stimulates carbon monoxide inhibitable NADPH consumption. A comparison of the binding and Michaelis parameters for the interaction of vinyl chloride with uninduced, phenobarbital and 3-methylcholanthrene induced microsomes indicates that the binding and metabolism of vinyl chloride is catalyzed by more than one type P-450 cytochrome, but predominantly by cytochrome P-450. Metabolites of vinyl chloride from this enzyme system decrease the levels of cytochrome P-450 and microsomal heme, but not cytochrome $\text{b}$₅ or NADPH-cytochrome $\text{c}$ reductase $\text{in vitro}$.     

Uterine vein prostaglandin levels in late pregnant dogs

We studied the uterine venous plasma concentrations of prostaglandins E₂, F_2α, 15 keto 13,14 dihydro E₂ and 15 keto 13,14 dihydro F_2α in late pregnant dogs in order to evaluate the rates of production and metabolism of prostaglandin E₂ and F_2α in pregnancy $\text{in vivo}$. We used a very specific and sensitive gas chromatography-mass spectrometry assay to measure these prostaglandins. The uterine venous concentrations of prostaglandin E₂ and 15 keto 13,14 dihydro E₂ were 1.35±.27 ng/ml and 1.89±.37 ng/ml, respectively; however, we could not find any prostaglandin F_2α and very little of its plasma metabolite in uterine venous plasma. Since uterine microsomes can generate prostaglandin F_2α and E₂ from endoperoxides, prostaglandin F_2α production $\text{in vivo}$ must be regulated through an enzymatic step after endoperoxide formation. Prostaglandin E₂ is produced by pregnant canine uterus in quantities high enough to have a biological effect in late pregnancy; however, prostaglandin F_2α does not appear to play a role at this stage of pregnancy.     

OKY-1581: A selective inhibitor of thromboxane synthesis invivo and invitro

OKY-1581 is an effective inhibitor of thromboxane synthesis $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$ caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.     

A nitroxide-sterol derivative potently modifies cholesterol biosynthesis by normal and neoplastic guinea pig lymphocytes

Leukemic guinea pig lymphocytes (L₂ C) synthesise cholesterol in vitro at a forty-fold greater rate than normal cells. Equilibration (18 h) with lecithin or lecithin-cholesterol liposomes, respectively, enhances or suppresses sterol manufacture by normal lymphocytes but does not influence sterol production by L₂ C cells. In contrast, $\text{> 5·10}{}^9$ molecules/cell of a nitroxide-derivative of androstane, (17 β-hydroxy-4′,4′-dimethylspiro [5 α-androstan-3,2′-oxazolidin]-3′-yloxyl), commonly used as a membrane spin-probe, drastically inhibit sterol production by both normal and leukemic cells (maximum within 2 h). At $\text{< 5·10}{}^9$ molecules/cell, this sterol stimulates cholesterol synthesis. 25-Hydroxycholesterol at low concentrations also stimulates sterol manufacture, whereas high concentrations are also inhibitory in both cell types.     

Electron spin resonance detection of free radicals in the mercaptan-activation and UV-inactivation of neocarzinostatin

Differentiation of chick embryo myoblasts $\text{in}\text{,}\text{vitro}$ requires both cell-cell recognition and cell-cell fusion. Prostaglandin E₁ is known to play a role in controlling fusion, and a specific receptor has been postulated. We demonstrate two peaks of specific binding activity for prostaglandin E₁ during myoblast differentiation $\text{in}\text{,}\text{vitro}$: one at 36 hours and one at 44 hours of culture. The prostaglandin binding activity of both peaks is sensitive to the inhibitors of prostaglandin synthesis, indomethacin and aspirin, and to the antibiotic tunicamycin. The 36 hour peak of binding activity occurs at the same time as the process of cell-cell recognition (24–36 hours) and recognition and prostaglandin binding exhibit similar sensitivity to indomethacin, aspirin and tunicamycin.     

Prostaglandin F2α receptors in bovine corpus luteum cell membranes. Effect of enzymes and protein reagents

Various enzymes and proteins reagents inhibited [³H]prostaglandin F₂α binding to bovine corpus luteum cell membranes. Studies were undertaken (a) to explore further on the dose response relationships with the above agents, (b) to investigate the mechanism of inhibition of binding with respect to receptor affinities and number and (c) to assess whether decreased binding reflected changes in receptors and/or other membrane components.Preincubation of membranes with phoshpolipase A, trypsin, pronase, lipase, tetranitromethane, dinitrofluorobenzene, acetic anhydride and $\text{N-}\text{ethylmaleimide}$ resulted in moderate to drastic inhibitions of [³H]prostaglandin F₂α binding. The dose-dependent inhibition of binding by enzymes, but not by protein reagents (except for $\text{N-}\text{ethylmaleimide}$), exhibited a biphasic pattern: at lower concentrations, the loss of binding was low and relatively plateaued, but at higher concentrations, the losses were dramatic. The drastic reduction in binding by trypsin was due to destruction rather than solubilization of receptors from membranes. Phospholipase A was intrinsically more effective than phospholipases C and Ca²⁺ was not required for its inhibition of [³H]prostaglandin F₂α binding. Protein reagents inhibition of binding was differently influenced by added Ca²⁺ i.e., loss of binding increased with some ($\text{N-}\text{ethylmaleimide}$), decreased with others (tetranitromethane, dinitrofluorobenzene and azobenzene sulfenylbromide). These results are interpreted to indicate that Ca²⁺ induced conformational changes in membranes which may result in exposure of new groups and burying of already exposed modifiable groups.Treatment of membranes wiht trypsin and $\text{N-}\text{ethylmaleimide}$ selectively abolished high affinity prostaglandin F₂α receptors. The low affinity receptors were present but their numbers as well as their affinity were decreased. Lipase, phospholipase A, acetic anhydride, dinitrofluorobenzen and tetranitromethane appear to decrease binding by totally abolishing all prostaglandin F₂α receptors or by severely reducing their affinities.The occupancy of receptors by prostaglandin F₂α afforded considerable protection against trypsin, phospholipase A, lipase and dinitrofluorobenzene. These data indicated that the inhibition of binding by the above agents, at least in part, can be attributable to changes in receptor sites alone.     

Clearance of prostaglandin F2ALPHA during circulatory shock.

The rate of disappearance of total circulating radioactivity following an intravenous bolus injection of 3HPGF_2α was determined during splanchnic artery occlusion (SAO) shock in the dog. In addition, the pattern of PGF_2α metabolite formation was assessed in both shocked and nonshocked animals. Although the clearance of circulating prostaglandin metabolites is significantly impaired during SAO shock as a result of decreased renal function, neither the pattern nor the time course of PGF_2α metabolite formation appears to be altered. Thus, increases in circulating prostaglandin concentrations during SAO shock reflect an increase in the rate of $\text{de novo}$ synthesis and release of these materials, and are not the result of decreased prostaglandin degradation.     

Lack of dependence of amine or prostaglandin biosynthesis on absolute cerebral level of pteridine cofactor

Using 2-amino-6-(5'-2'-deoxyphosphoribosyl)-amino-5- or -6-formamido-6-hydroxypyrimidine (dFPyd-P₃), a specific inhibitor of tetrahydrobiopterin (BH₄) synthesis, cerebral pools of BH₄ were reduced to half of that of controls; while, simultaneously, the biosynthesis $\text{de novo}$ of L-erythrodihydroniopterin (BH₂) from GTP was inhibited by about 98%. Nevertheless, there was no effect on the cerebral levels of serotonin, dopamine, norepinephrine or on the biosynthesis of prostaglandin E₂ or F₁. The data are presented in evidence that the absolute level of the cofactor (BH₄) is not regulatory of amine or protaglandin biosynthesis $\text{in vivo}$. Amine and prostaglandin biosynthesis proceeded even at cofactor concentrations of 9×10⁷ M nsuggesting that their biosynthesis is dependent on the rate of H⁺ + e shuttle between BH₂ and BH₄.     

Modulators of monoamine oxidase in plasma

Addition of small amounts of plasma activated the deamination of tryptamine by platelet monoamine oxidase (MAO). At higher concentrations, plasma inhibited the deamination instead. The inhibition was increased with increasing amounts of plasma added. The inhibition was uncompetitive in nature, partially reversed by prior ultrafiltration of the plasma through PM30 membranes and completely reversed by protein precipitation of plasma with perchloric acid. Addition of high amounts of plasma $\text{in}$$\text{vitro}$ also inhibited the activity of bovine striatal MAO. The inhibition of striatal deamination of tryptamine by plasma was noncompetitive in nature, completely reversed by ultrafiltration through PM30 membranes and partially reversed by perchloric acid treatment. The inhibition of striatal deamination of serotonin was noncompetitive in nature, not reversed by ultrafiltration but completely reversed by perchloric acid treatment. The pattern of inhibition of platelet or striatal MAO by plasma was different from that induced by addition of bovine serum albumin (BSA). Low concentrations of BSA added $\text{in}$$\text{vitro}$ activated the deamination of tryptamine or serotonin by platelet or striatal MAO by decreasing the K_m, while higher concentrations also decreased the V_max. The presence of protein, non-albumin circulating modulators of platelet or striatal MAO in plasma is discussed.     

Synthesis of a trans-hydrindane nuclear analog of 11-desoxy-dihydro-prostaglandin E1

Stereosecific synthesis of $\text{trans}$-hydrindanone $\text{2a}$, a bicyclic analog of prostaglandin E₁, $\text{via}$ the $\text{trans}$-hydrindane β-keto ester $\text{8}$, is described. When tested in the guinea pig, $\text{2a}$ exhibited no effects on blood pressure and no broncho-constriction or dilation activity. Additionally, $\text{2a}$ failed to inhibit both ADP $\text{and collagen induced blood platelet aggregation}$.     

Lipid metabolism and in vitro production of progesterone and prostaglandin F during induced regression of porcine corpora lutea

The effects of Cloprostenol administration on porcine luteal lipid and arachidonic acid accumulation were examined in relation to luteal $\text{in vitro}$ progesterone and prostaglandin F synthesis in 18 mature gilts at day 12 of the estrous cycle. Basal and net $\text{in vitro}$ release of progesterone from luteal tissue was depressed at 8 hr after treatment whereas net $\text{in vitro}$ release of prostaglandin F was elevated at 8 hr. Inclusion of copper dithiothreitol or reduced glutathione in the incubation media resulted in minor alterations of $\text{in vitro}$ release of progesterone and prostaglandin F and no changes in composition of luteal lipids or fatty acids. Luteal contents of triglyceride had increased by 8 hr after treatment whereas contents of free and esterified cholesterols had increased by 32 hr after Cloprostenol administration. Luteal contents of phospholipid and free fatty acids were not affected by Cloprostenol administration. At 32 hr after treatment, percentages and content of arachidonic acid had increased in luteal cholesterol esters and triglycerides. Although arachidonic acid percentages increased in luteal free fatty acids and phospholipids, calculated arachidonic acid contents did not change following Cloprostenol administration. Induced luteal regression was associated with decreased $\text{in vitro}$ progesterone release, increased $\text{in vitro}$ prostaglandin F release, and accelerated lipid and arachidonic acid accumulation within the corpus luteum. The effects of altered lipid metabolism on release of prostaglandin F could not be defined. However, availability of arachidonic acid did not appear to be rate-limiting in relation to luteal $\text{in vitro}$ prostaglandin F synthesis.     

Intraperitoneal injection of zymosan in mice induces pain,inflammation and the synthesis of peptidoleukotrienes and prostaglandin E2

Intraperitoneal injection of zymosan in mice induced rapid extravasation and accumulation of plasma proteins in the peritoneal cavity. Neutrophils began to appear in the peritoneal cavity after a lag period of approximately 3 hours. The injected mice exhibited a pain response (writhing) during the first 30 minutes after injection, but writhing ceased before protein or cell accumulation had reached maximum levels. The injection of zymosan induced synthesis of PGE₂ (measured by RIA) which reached maximum levels of 30 minutes, then declined slowly. Peptido-leukotriene levels (detected by bioassay, RIA and HPLC) increased rapidly after injection, reached a peak within an hour of injection and declined to undetectable levels within 4 hours. The early peptido-LT was predominantly LTC₄, while later, LTE₄ was the major component. LTD₄ levels remained low throughout and no LTB₄ was detected at any time. Indomethacin treatment elevated levels of peptido-LTs, recued PGE₂ levels and inhibited writhing. Phenidone reduced peptido-LT levels. $\text{In}$$\text{vitro}$ studies demonstrated that zymosan stimulates LTC4 synthesis by peritoneal cells whereas LTE₄, LTD₄, LTB₄ or monoHETES were not detectable (using HPLC methods). The source of enzymes responsible for the $\text{in}$$\text{vivo}$ metabolism of LTC₄ to LTD₄ and LTE₄ could not be identified.     

Effects of hydrocortisone on the hypercalcemia and plasma levels of 13,14-dihydro-15-keto-prostaglandin E2 in mice bearing the HSDM1 fibrosarcoma

In mice bearing the prostaglandin-producing HSDM₁ fibrosarcoma, the plasma concentration of 13,14-dihydro-15-keto-PGE₂ was elevated before the development of hypercalcemia, and the magnitude of the rise was greater than that of PGE₂. When hydrocortisone, which inhibits synthesis of PGE₂ by HSDM₁ cells in culture, was administered to tumor-bearing mice, the steroid hormone prevented the rises in plasma PGE₂ metabolite and calcium concentrations. At the dose levels used, hydrocortisone did not inhibit the calcium-mobilizing action of parathyroid hormone $\text{in vivo}$ or the bone resorption-stimulating activity of $\text{PGE}{}_2\text{in vitro}$. These findings are consistent with our hypothesis that the hypercalcemic syndrome in HSDM₁ tumor-bearing mice is due to the secretion of PGE₂ by the tumor.     

Effect of prostaglandin E2 on vascular responses of the rabbit kidney to nerve stimulation and noradrenaline, in vitro and in situ

The effect of prostaglandin E₂ on vascular responses of the rabbit kidney to renal nerve stimulation and noradrenaline was examined $\text{in}$$\text{vitro}$ and $\text{in}$$\text{situ}$ as a test of the hypthesis that prostaglandins of the E series may be involved in the regulation of adrenergic neuroeffector transmission. Intraarterial administration of prostaglandin E₂ to the $\text{in}$$\text{vitro}$ kidney caused marked inhibition of vascular responses to nerve stimulation whereas the responses to noradrenaline were not significantly altered. In the $\text{in}$$\text{situ}$ preparation, vascular responses to both nerve stimulation and noradrenaline were inhibited by prostaglandin E₂ infusion, although its effect on responses to nerve stimulation was approximately twice that observed on responses to noradrenaline.It is concluded that prostaglandin E₂ acts primarily at a prejunctional level of adrenergic neuroeffector transmission in the kidney, although a postjunctional effect has also been observed.     

Uteroplacental dysfunction and prostaglandin metabolism in zinc deficient pregnant rats

Relationships between perinatal mortality, disrupted uteroplacental function and prostaglandin metabolism have been studied in Zn-deficient rats. Uterine contractility $\text{in vitro}$, placental blood flow $\text{in viro}$, and uterine and placental prostaglandin synthesis from [1?¹⁴C] arachidonic acid $\text{in vitro}$ were investigated at day 22 of pregnancy. High amplitude uterine contractions were almost completely eliminated and utero-placental blood flow was decreased by 85% by Zn deficiency. Synthesis of [1?¹⁴C]-prostaglandin E₂, F_2α and 6-keto-F_1α from [1?¹⁴C] arachidonic acid decreased significantly in uterine tissue but increased in placentae. These possibly inter-related effects may contribute to the high perinatal mortality observed in Zn deficiency.     

The role of protein synthesis in the stimulation by LH of prostaglandin accumulation in rat preovulatory follicles in vitro

Preovulatory follicles isolated from immature rats, treated $\text{in vivo}$ with pregnant mare's serum gonadotropin, were incubated $\text{in vitro}$ and the accumulation of prostaglandin E measured. The addition of luteinizing hormone (5 μg/ml) increased this accumulation, after a lag period of 3 hours. This delay suggested the involvement of macromolecular synthesis in the mechanism of prostaglandin stimulation by luteinizing hormone. When the synthesis of protein was inhibited by the addition of puromycin (100 μM), the luteinizing hormone stimulation of prostaglandin E in these follicles was completely abolished. This inhibition was not seen with an analogue of puromycin, which does not inhibit protein synthesis, puromycin amino-nucleoside. These data suggest that concomitant protein synthesis is required for the luteinizing hormone stimulation of prostaglandin accumulation in rat follicles.     

Prostaglandin D2, a potential antineoplastic agent

Cytotoxic actions of various prostaglandins were examined on L1210 mouse leukemia and several human leukemia cell lines, and prostaglandin D₂ (PGD₂) was found most active. PGD₂ exerted a dose dependent inhibition of L1210 cell growth over 3.6 μ$\text{M}$. At 14.3 μ$\text{M}$ growth was completely inhibited, and the number of viable cells remarkably decreased during culture. Microscopically the remaining cells showed degenerative changes with many vacuoles in their cytoplasm. The IC₅₀ value of PGD₂ on L1210 cell growth was calculated to be 6.9 μ$\text{M}$ (2.4 μg/ml), and at this concentration the DNA synthesis in 24 hr cultured cells was also decreased to a half of the level in the control cells. Such growth inhibition by PGD₂ was also found at similar concentrations with several human leukemia cell lines such as NALL-1, RPMI-8226, RPMI-8402, and Sk-Ly-16. Among other prostaglandins tested, PGA₂ showed a comparable, and PGE₂ a less but significant growth inhibitory activity, while PGB₂, PGF_2α and PGI₂ had no such effects on cell proliferation at 14.3 μ$\text{M}$ concentration. These results suggest a potential antineoplastic activity of PGD₂.     

Stimulation of prostaglandin synthetase activity in rat granulosa cells by gonadotropins in vivo

The effect of $\text{in vivo}$ exposure to gonadotropin on prostaglandin synthetase activity in rat granulosa cells was examined in two experimental settings. The first setting was immature rats treated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). The second was mature rats on the day of proestrus. In the experiments using immature rats, the administration of hCG (20 I.U.) at noon of the second day after the PMSG (20 I.U.) injection led to large (more than 5 fold) increases in granulosa cell prostaglandin synthetase activity 5 and 10 h later. Follicular fluid PGE levels were also markedly increased at 5 and 10 h after hCG. Similar results were also found in experiments performed with mature proestrus rats. Granulosa cell prostaglandin synthetase activity was elevated at approximately 4 and 8 h after the endogenous LH surge (about 4 p.m. on proestrus), in comparison with the activity at midnight of diestrus, or noon and 4 p.m. on proestrus. In these experiments the changes in prostaglandin synthetase activity (10 fold) also paralleled the increases in follicular fluid PGE concentrations. Thus the exposure to gonadotropin $\text{in vivo}$ produced essentially the same effect as we had reported earlier for isolated granulosa cells incubated with LH $\text{in vitro}$. The stimulation of prostaglandin synthetase activity must therefore be ascribed an important role in the physiological regulation of granulosa cell prostaglandin synthesis by LH.     

Prostaglandin E2 rather than lymphocyte-activating factor produced by activated human mononuclear cells stimulates increases in murine thymocyte cAMP

Culture supernatants (SUPS) of endotoxin (LPS)-activated human mononuclear cells (MNL) stimulated greater production of cAMP by thymocytes than by spleen cells of C3H/HeJ or nude ($\text{nu}\text{nu}$) mice. Similarly, the addition of prostaglandin E₂ (PGE₂) stimulated higher levels of cAMP in thymocytes and progressively lower levels in spleen cells from C3H/HeJ mice and $\text{nu}\text{nu}$ spleen cells, respectively. Partial purification on Bio-Gel P100 of the LPS-induced MNL SUPS yielded peaks of thymocyte proliferative activity characteristic of lymphocyte activation factor (LAF) but these fractions failed to stimulate cAMP levels in thymocytes. Moreover, MNL SUPS induced with LPS in the presence of indomethacin retained their LAF activity but no longer increased thymocyte cAMP levels. Radioimmunoassay of the SUPS for PGE₂ revealed significantly higher levels of PGE₂ in the media of those MNL cultures stimulated by LPS than when stimulated by phorbol myristic acetate, phytohemagglutin, or extracted cell wall fraction of Actinomyces viscosus. Thus, PGE₂ is produced by human MNL and may exert considerable immunoregulatory effects mediated by elevation of lymphocyte cAMP levels.     

Comparative metabolic clearance rates of beta-endorphin and beta-lipotropin in humans

In normal human subjects under basal conditions, we have reported that molar concentrations of immunoreactive β-lipotropin (IR-β-LPH) are approximately threefold greater than those of IR-β-endorphin (β-Ep). Following acute stimulation, there is a further two- to threefold disproportionate rise in plasma concentrations of IR-β-LPH as compared to those of IR-β-Ep. To begin to assess the possible factors involved in such altered IR-β-LPH/IR-β-Ep ratios in plasma, the metabolic clearance rate (MCR), volume of distribution (V_d), fractional rate of disappearance (K_d), and half-life ($\text{t}\text{1}\text{2}$) of these peptides were determined by means of bolus injection of highly purified human β-LPH and synthetic human β-Ep in normal human subjects. β-Ep was found to have an MCR and a K_d greater than that of β-LPH, and a shorter $\text{t}\text{1}\text{2}$. These differences, however, although they may in part be contributory, cannot solely account for the greater ratio of IR-β-LPH to IR-β-Ep in plasma, or for the disproportionate rise in plasma concentrations of these peptides after acute stimulation.