Polyphloretin Phosphate Research Articles

Blockade by polyphloretin phosphate of the prostaglandin E1-induced human cutaneous reaction

The effect of polyphloretin phosphate (PPP) on the human cutaneous reaction induced by prostaglandin E1 (PGE1) has been studied in thirty subjects. Pre-treatment of the skin with three intradermal injections of 1oo μg PPP reduced by 37% the magnitude of the erythema induced by intradermal injection of 1 μg PGE1. The PGE1induced weal, which is probably mainly due to PGE1-induced endogenous histamine release, was not reduced. The specific nature of the PG-blocking activity of PPP was supported, since PPP did not block the erythema and weal induced by intradermal injection of histamine and bradykinin. Intravenous injection of 100 mg PPP did not reduce the erythema due to intradermal injection of PGE1 probably because of too low tissue concentration of PPP.

Effect of polyphloretin phosphate on the response of non-gravid rat uterus to ‘folkloric’ and ‘standard’ oxytocics in vitro

Polyphloretin phosphate (PPP) has been reported by previous workers to be a specific antagonist of prostaglandin (PGE 1, PGE 2 & PGF 2 α )-induced contractions of isolated jird colon, gerbil colon, guinea pig ileum, and rabbit jejunum. In the present study, we examined the effect of PPP on uterotonic activities of crude papaya latex (a folkloric oxytocic), PGF 2 α , oxytocin, acetylcholine, and 5-hydroxytryptamine (standard oxytocics) on non-gravid, oestrogen-primed (50 μg/kg) rats in vitro. The effect of PPP on the oxytocics was evaluated qualitatively by incubating the tissues in PPP (25 – 400 μg/ml) for 20 min prior to the addition of a constant concentration of each oxytocic. PPP concentration dependently inhibited the contractile response of the uterine muscles to all the oxytocics. The inhibition was reversible after washing out the drugs. Results of the present study suggest that PPP is a non-specific and reversible antagonist of the response of non-gravid rat uterine smooth muscle to oxytocics in vitro. The specificity of PPP as a prostaglandin antagonist could therefore be species/tissue dependent.

Effects of prostaglandin antagonist phloretin derivatives on mouse ear edema induced with different skin irritants

Edema was induced in one ear of male mice of the CFLP strain with solutions of different skin irritants (croton oil 10 μL/35 μg, dithranol 10 μL/30 μg, capsaicin 10 μL/40 μg or arachidonic acid to 10 μL/2 mg per ear). Edema, determined by the edema-disk gravimetric technique, was inhibited in a dose-dependent manner by the intraperitoneally administered prostaglandin antagonists polyphloretin phosphate (PPP) or di-4-phloretin phosphate (DPP). With croton oil-induced mouse ear edema, DPP 10 mg/kg caused a 38% inhibition, PPP 25 mglkg a 33% inhibition. With dithranol-induced edema DPP 0.5 mglkg caused a 57% inhibition, while PPP 25 mglkg was needed to exert a similar effect. Doses of DPP and PPP needed to cause a >40% inhibition of edema were 10 mglkg and 25 mglkg, respectively, for capsaicin, and 25 mg/kg and 100 mglkg for arachidonic acid. The inhibition of the ear edema by the phloretin derivatives was: dithranol > croton oil > capsaicin > arachidonic acid. This probably reflects the different contributions of prostaglandins to the inflammation.

Polyphloretin phosphate (PPP) antagonists of prostaglandin action also inhibit prostaglandin biosynthesis in-vitro

Several polyphloretin phosphate (PPP) fractions (low mol. wt LC1259; high mol. wt LC1261; crude mixture, LC101) were confirmed in their established property as antagonists of the pharmacological actions of prostaglandins in a preparation of guinea-pig isolated ileum stimulated by prostaglandin (PG)E2. Further samples of the same material were then compared in-vitro with indomethacin in their ability to inhibit prostaglandin biosynthesis from arachidonic acid by a microsomal enzyme preparation. All three PPP fractions potently inhibited prostaglandin generation, with the rank order of potency LC1259 = LC101 = indomethacin greater than LC1261. The oral LD50 in mice was 25 mg kg-1 for indomethacin and greater than 1 g kg-1 for LC101. PPP fractions (especially LC101) may therefore have therapeutic potential as anti-inflammatory agents.

Angiotensin Stimulates Adenylate Cyclase Activity via Prostaglandins in the Adrenal Glomerulosa Membrane.

The effects of angiotensin II (A II) on adenylate cyclase activities in membranes of the zona glomerulosa (the capsular fraction) and the zona fasciculata (the decapsulated fraction) from rat adrenocortical glands were investigated. A time- and GTP-dependent stimulation by A II of adenylate cyclase activity was observed in the capsular fraction but not in the decapsulated fraction. The activation of adenylate cyclase by ACTH and A II was additive. Stimulation by A II was inhibited by an angiotensin antagonist, DD-3487 (DD). Moreover, the addition of a prostaglandin antagonist, a mixture of polyesters of polyphloretin phosphate (PPP) and an inhibitor of prostaglandin synthesis, indomethacin, was effective in inhibiting A II-induced stimulation of the capsular adenylate cyclase activity, suggesting that the activation of A II receptors located on the capsular membrane leads to the release of prostaglandins, which in turn stimulates the adenylate cyclase.

In vitro penetration of hamster oocyte-cumulus complexes using physiological numbers of sperm

We have compared the ability of uncapacitated, capacitated acrosome intact, and acrosome-reacted hamster sperm to penetrate the cumulus and corona radiata of fresh hamster oocyte-cumulus complexes (OCC) in vitro. This was done using physiological numbers (1–20) of sperm so that cumulus and corona radiata cells did not disperse during challenge. Uncapacitated sperm did not penetrate to the zona pellucida surface; most (74%) uncapacitated sperm bound to cumulus cells at the periphery of the OCC. Capacitated acrosome-intact sperm penetrated to the zona pellucida surface; a significant percentage of these sperm arrived at the zona pellucida without showing evidence of initiating an acrosome reaction. Most capacitated acrosome-reacted sperm did not enter the extracellular matrix between cumulus and corona radiata cells; those which did penetrated to the zona surface with difficulty, if at all. These results suggest that the changes which occur in the sperm surface during capacitation are more important than the acrosome reaction in enabling hamster sperm to penetrate the cumulus and corona radiata. The effects of gold sodium thiomalate (GST) and polyphloretin phosphate (PPP) (inhibitors of hyaluronidase) on penetration of the OCC by capacitated sperm were also examined. Both synthetic inhibitors blocked sperm penetration to the zona pellucida, but the effective concentrations of inhibitors were far in excess of what was needed to block hyaluronidase activity. Reasons for concluding that the action of these inhibitors is nonspecific are discussed. These data show that hamster sperm with intact acrosomes can penetrate the cumulus and corona radiata cell layers of fresh OCC in vitro and support the hypothesis that the acrosome reaction occurs on the zona pellucida surface.

Polyphloretin phosphate, an antagonist of thyrotropin action: evidence for an interaction with the hormone rather than the receptor.

Polyphloretin phosphate (PPP) is known to be an inhibitor of bovine TSH (bTSH)-induced stimulation of the thyroid in both in vivo and in vitro assays. The present studies were undertaken to delineate the mechanism of these effects. A high molecular weight PPP preparation strongly inhibited both the binding of 125I-labeled bTSH [( 125I]bTSH) to human thyroid membranes and the stimulation of adenylate cyclase evoked by bTSH therein. Inhibition of bTSH-induced adenylate cyclase activity by PPP was evident both in the absence and the presence of NaCl (150 mM) in the incubation medium. Incubation of membranes with PPP, followed by its removal, did not affect subsequent binding of [125I]bTSH, indicating that PPP did not bind firmly to or damage the TSH receptor. Gel chromatography on Sephadex G-100 revealed that [125I]bTSH incubated with PPP eluted earlier than [125I]bTSH alone, indicating that PPP had formed a higher molecular weight complex with [125I]bTSH. This effect could be prevented by the addition of an excess of unlabeled bTSH to the incubation mixture. Binding of [125I] bTSH in the higher molecular weight peak generated by incubation with PPP was less than half that in control specimens of [125I]bTSH. Studies with PPP were also conducted in a highly sensitive assay that employs cultured porcine thyroid cells and measures the cAMP response induced by bTSH. The inhibitory effect of PPP on bTSH-induced cAMP accumulation was also evident in this assay. However, the presence of divalent cations Ca++ and Mg++ in the assay medium greatly diminished the inhibitory effect of PPP. Similarly, addition of Ca++ and Mg++ to the incubation medium greatly reduced or abolished the inhibitory effect of PPP on [125I]bTSH binding. Both effects of these salts to lessen the inhibitory response to PPP were overcome by increasing the PPP concentration. Gel chromatographic studies revealed that Ca++ and Mg++ acted by inhibiting the formation of the high molecular weight complex of bTSH and PPP. From these findings, we conclude that PPP exerts its inhibitory effect on TSH-induced stimulatory responses in the thyroid, in vivo as well as in vitro, by forming a complex with the hormone. The complex either does not bind to TSH receptors or does so with much lower affinity.

Contractile responses of the guinea-pig esophageal muscularis mucosae in vitro to arachidonic acid and its metabolites

The responsiveness of the guinea-pig esophageal muscularis mucosae to arachidonic acid (AA) and its cyclooxygenase and lipoxygenase metabolites was examined in vitro. AA (0.1–30 μM) produced a concentration-dependent contraction of the muscularis mucosae (ean EC 50 ± S.E.M. = 5.1 ± 1.0 μM). The contractions in response to low concentrations of AA (0.1–3 μM) were prevented by pretreatment of the tissue with indomethacin (1–10 μM), while those in response to high concentrations (10–100 μm) were prevented by BW755C (10–100 μM). The contractile response to AA was antagonized by polyphloretin phosphate (PPP, 1–10 μg/ml) and by FPL 55712 (1–10 μM). All cyclooxygenase and lipoxygenase metabolites of AA tested also produced a sustained contraction of the muscularis mucosae with the following order of sensitivity; leukotriene (LT) D 4 > LTC 4 > prostaglandin (PG) E 2 > PGF 2α > PGI 2 > thromboxane B 2. The responses to LTC 4 and LTD 4 were antagonized by FPL 55712 (0.1–1 μM), while those to PGE 2 and PGF 2α were antagonized by PPP (3–100 μg/ml). The present results indicate that exogenously applied AA contracts the isolated muscularis mucosae of the guinea-pig esophagus by an indirect action via its metabolism to both PGs and LTs. The putative PG and LT receptors located in this tissue are probably similar to those in the ileal longitudinal muscle, but differ from those in the airway smooth muscle.

Sow (Sus scrofa) follicular fluid: Prostaglandin content and effect on the motility of isolated oviducts

The present study provides information regarding the effects of the sow follicular fluid (FF) on the motility of isolated segments of swine and rabbit oviducts. In addition, the concentration of prostaglandins (PGs) F 2 α, E 2 and E 1 in the follicular fluid of sow ovaries isolated at different stages of the sex cycle as well as the generation of the same PGs by walls of ovarian follicles in early and late proestrus, in estrus, in metestrus and in diestrus, were explored. The stimulatory contractile effect of proestrous FF in isolated segments of sow fimbria was antagonized by polyphloretin phosphate (PPP), a PG receptor blocker and by indomethacin, an inhibitor of PG synthesis. The positive inotropism evoked by the FF was mimiked by bradykinin and the influences of both interventions were similarly antagonized by PPP. It appears plausible that the inotropic effect of the preovulatory FF on the sow fimbria could be not only by PGs already present in the fluid, but also by the stimulation of the synthesis of tubal PGs by follicular fluid bradykinin. The FF also stimulated the ampullary tubal segments isolated from proestrous sows whereas the same volume of FF depressed significantly the isometric developed tension of rabbit ampulla. The total concentration of the three PGs in the FF from late proestrous follicles was significantly greater than that of the same PGs in the other two stages of the sex cycle (early proestrus and diestrus), whereas the concentration of each PG (PGE 2, PGF 2 α or PGE 1), did not differ within any of the stages of the cycle. Furthermore, the total amount of the three PGs produced by the walls of follicles from late proestrous ovaries was also significantly greater than that generated by ovarian follicles from early proestrus, estrus, metestrus and diestrus.

Rat mammary preadipocytes in culture produce a trophic agent for mammary epithelia-prostaglandin E2.

Cell strains and cell lines rat mammary (Rama) 350-353 have been isolated from the slowly adherent stromal fraction of enzymatically digested rat mammary glands. Primary cultures of this fraction yield fat cels on extended culture. Their proportion can be increased with horse serum or growth hormone in the medium, and this increase is associated with a 100-fold or more increase in the release of radioimmunoassayable prostaglandins of the E type (PGE). The stromal cell strains and lines that are capable of yielding fat cells also secrete elevated levels (greater than 100 ng/mg/24 hr) of PGE; the fast-sticking epithelial fraction in primary cultures and the epithelial cell lines derived from it secrete 10-100 times less. Chromatography and radioisotopic labeling of the culture media from Rama 352 cells identify the PG as PGE2. PGE2 with insulin and hydrocortisone maximally stimulates [3H]DNA synthesis of epithelial cell lines and primary cultures from normal and tumorous glands by 2-4-fold at concentrations (10-20 ng/ml) well below those released by the preadipocytic stromal cells (20-100 ng/ml). Medium exposed to most cultured cells stimulates [3H]DNA synthesis of one epithelial cell line, Rama 25, by 2-4-fold. Prevention of the synthesis of PGE2 in Rama 352 cultures with indomethacin or flurbiprofen abolishes the mitogenic activity present in the culture medium, and the PG receptor antagonist polyphloretin phosphate inhibits completely the mitogenic activity for Rama 25 cells. Myoepithelial-like cell lines normally secrete moderate levels of PGE (10-100 ng/mg/24 hr) but the mitogenic activity for Rama 25 cells released from one such line, Rama 29, is not abolished by preventing the synthesis of PG's nor by PG-receptor antagonists.

Suppression of laxative action of phenolphthalein by orally-administered indomethacin or aspirin.

Oral administration of phenolphthalein produced a dose-dependent increase of wet faeces excreted by mice. The response to phenolphthalein was reduced by pretreatment with indomethacin, aspirin or polyphloretin phosphate (PPP), but not with benoxaprofen. These findings support the view that the effect of phenolphthalein may be suppressed by PG synthetase inhibitors (indomethacin, aspirin) and by PPP.

Role of intrinsic arachidonate metabolites in the vascular action of erythrocyte breakdown products.

In helically-cut strips of dog basilar and mesenteric arteries, the isometric tension developed by application of ghost-free hemolysate from dog erythrocytes was recorded. The hemolysate contracted basilar arteries in a concentration-dependent fashion, the response being attenuated by treatment with either aspirin or polyphloretin phosphate, a prostaglandin antagonist. Mesenteric arteries were contracted only slightly by high concentrations of hemolysate. When the mesenteric arteries had partially been contracted with prostaglandin F2 alpha or norepinephrine, the hemolysate induced relaxations, which were abolished by aspirin in approximately half the preparations used. Studies on rat stomach strips exposed to superfusate of dog cerebral arteries showed a release of prostaglandin-like substance by the hemolysate application. It may be concluded that the hemolysate contracts basilar arteries and relaxes mesenteric arteries, mainly through prostaglandins synthesized in and released from the vascular wall. Such a mechanism may be involved in the pathogenesis of cerebral vasospasm following a subarachnoid hemorrhage.

Vasoactive intestinal peptide stimulation of human thyroid cell function.

Neurons containing vasoactive intestinal peptide (VIP) have been found to reach thyroid follicular cells in man and other mammals. Human thyroid cells in culture were exposed to different concentrations of VIP and TSH, and thyroid cell activation was assessed by measurements of cAMP accumulation. VIP exerted a dose-dependent activation, and this thyroid-stimulating effect was more rapid and short-acting than that of TSH. At low concentrations of the two agents, their effects were additive, but they seemed to initiate their effects via different receptors, since polyphloretin phosphate inhibited the effect of TSH but not that of VIP. Moreover, antiadrenergic and anticholinergic agents did not influence thyroid activation by VIP. In addition to cAMP activation in cell cultures, VIP stimulated T4 release from human thyroid slices. It is possible that, in addition to sympathetic-adrenergic and parasympathetic-cholinergic nerves, VIPergic nerves may participate in the autonomic nervous control of thyroid function.

Effect of polyphloretin phosphate on the contraceptive action of a polyethylene intrauterine device in rats

A polyethylene intrauterine device (IUD) inserted in one horn of the rat uterus on Day 2 of pregnancy prevented implantation in 9 out of 12 rats in the horn with the device. When polyphloretin phosphate (PPP), a substance known to inhibit alkaline phosphatase, was administered to such rats from Day 1 till Day 5 of pregnancy, the device prevented implantation in only 3 out of 12 rats in the horn with the device. PPP also significantly reduced the raised levels of endometrial alkaline phosphatase observed in the presence of the device. A significant rise in endometrial alkaline phosphatase was seen in such animals not administered PPP. It is suggested that there is a correlation between the anti-implantation effect of an IUD and raised levels of endometrial alkaline phosphatase in rats.

Effect of prostaglandin E1 and polyphloretin phosphate on hemolysis of human erythrocytes.

Prostaglandin E1 was found to reduce the hemolysis rate induced by various factors, such as frequent shakings, treatment with hog pancreatic phospholipase A2, the addition of active oxygens generated by a xanthine oxidase system, and the addition of a prostaglandin antagonist, polyphloretin phosphate (PPP). Prostaglandin E1 was found to act on the erythrocytes in such a way as to cause the phospholipids in the membrane to become more compactly arranged thus becoming less susceptible to the attack of hemolytic reagents. It was observed that the extents of hemolysis were different between erythrocytes from males and females and furthermore, it was shown that prostaglandin E1 clearly reduced the rate of hemolysis of erythrocytes from males, while, in females, the effects of prostaglandin E1 were less than those in erythrocytes from males.

PH dependent inhibition of serum oxytocinase activity by prostaglandins and cyclic GMP

The effect of pH in the range 6.2 to 7.7 on the inhibition of serum oxytocinase (EC 3.4.11.3) activity by various compounds was studied using S-benzyl-L-cysteine-p-nitroanilide (BCN) as substrate. Prostaglandins E 1, E 2, F 2α, 8-bromo-cGMP, cGMP, indomethacin and polyphloretin phosphate (PPP) produced a dose related pH dependent inhibition of serum oxytocinase. Their effect was maximum at pH 6.2 and minimum at pH 7.7. Hypertonic urea and saline also caused pH dependent inhibition; saline being most active at pH 6.2, and urea at pH 7.7. A similar pH dependent inhibition was found when these compounds were examined for their effect on the hydrolysis of L-leucine-p-nitroanilide (LN) by serum aminopeptidases at pH between 6.2 and 7.7. Although the inhibitors were more effective against the hydrolysis of LN than BCN substrate, cGMP and its 8-bromo derivative were more active against the BCN hydrolysis. cAMP, 8-bromo-cAMP, dibutyryl (db)-cAMP, db-cGMP, AMP, ADP, ATP, GDP, GTP, aspirin, sodium salicylate, paracetamol, theophylline and isobutylmethylxanthine (IBMX) at comparable concentrations and within the same pH range had no effect on the hydrolysis of either substrate. It is concluded that in serum obtained during pregnancy, the hydrolysis of LN may largely be attributed to oxytocinase activity. Thus, inhibition of LN hydrolysis by prostaglandins and other substances may be regarded as inhibition of oxytocinase activity.

Different responsiveness of a variety of isolated dog arteries to prostaglandin D 3

The addition of prostaglandin (PG) D 2 contracted helical strips of dog cerebral, coronary, renal and femoral arteries; the contraction was greatest in cerebral arteries. The contractile response of cerebral arteries was potentiated by aspirin and attenuated by polyphloretin phosphate. In the arterial strips contracted with PGF 2α, PGD 2 elicited a concetration-related relaxation; the relaxation was greatest in mesenteric arteries. In mesenteric arterial strips contracted with norepinephrine, a lesser degree of relaxation was induced, and in the K +-contracted arteries, only a contraction was induced. Treatment with PGD 2 attenuated the contractile responses of cerebral and mesentric arteries to PGF 2α or PGE 2; this inhibitory effect was approximately 10 times greater in mesenteric arteries. However, the response to serotonin (for cerebral arteries) or norepinephrine (for mesenteric) was unaffected. It may be concluded that the heterogeneity of response to PGD 2 of a variety of dog arteries is due to different contributions of vasoconstrictor and vasodilator mechanisms. PGD 2 appears top share the mechanism underlying arterial contraction with PGF 2α and PGE 2, and interferes with the effect of these PG's possibly on receptor sites.

Enhancement by prostacyclin of the contractility of the guinea-pig airways smooth muscle

Aerosolized prostacyclin (PGI 2) potentiated the increase in pulmonary resistance to inflation induced by serotonin, prostaglandin F 2α (PGF 2α), acetylcholine and histamine in the guinea-pig. This was not due to a reflex, nor to further production of PG cyclooxygenase derivatives. PGI 2 and PGF 2α induced contraction of the parenchyma lung strip of the guinea-pig, which could be inhibited by polyphloretin phosphate and by PGE 1. Since PGF 2α failed to potentiate the bronchial responses to acetylcholine, histamine or serotonin, under conditions where PGI 2 was effective, the in vitro similarities between the two PGs cannot explain the in vivo results. The ability of PGI 2 to potentiate bronchial responses was not shared by the other PGs. Since the latter are either bronchoconstrictor agents by themselves (PGF 2α and PGD 2), or bronchodilators (PGE 1, PGE 2), our hypothesis is that PGI 2 potentiates the responses of the bronchi to various agonists by a mechanism similar to that which accounts for the potentiation of acute inflammation and pain by PGE 1 and PGE 2, the latter being ineffective in enhancing the bronchial responses because of the associated bronchodilator activity.

Inhibition of serum oxytocinase activity by prostaglandins

The effect of prostaglandins (PGs) and other compounds on human serum oxytocinase (EC 3.4.11.3) activity in vitro was studied by a sensitive assay using S-berizyl- l-cysteine-p-nitroanilide as substrate. PGE 1, PGE 2 and PGF 2α significantly inhibited serum oxytocinase activity in a dose-related manner and in decreasing order of potency. cGMP, 8-bromo-cGMP, indomethacin, polyphloretin phosphate, hypertonic saline and urea were also active. cAMP, db-cAMP, 8-bromo-cAMP, db-cCMP, 5′-AMP, 5′-ADP, 5′-ATP, 5′-GDP, 5′-GTP, aspirin, sodium salicylate, paracetamol, theophylline and IBMX did not inhibit the enzyme activity. The results suggest that the oxytocic action of prostaglandins may be due, at least in part, to an inhibition of oxytocinase activity.

Effects of polyphloretin phosphate (PPP) and quinterenol on cyclic nucleotide phosphodiesterase activities

On the basis of previously-observed synergism with noradrenaline in hormone-stimulated lipolysis in isolated fat cells, polyphloretin phosphate (PPP) and quinterenol were compared with papaverine for inhibitory effects on cyclic AMP and cyclic GMP phosphodiesterase activities from guinea-pig heart, guinea-pig lung and rat epididymal adipose tissue. PPP was a more potent inhibitor than papaverine of both enzyme activities from all three sources. Quinterenol was without inhibitory activity against the cyclic nucleotide phosphodiesterase activities examined.