Selective stimulation of venous prostaglandin E 9-ketoreductase by bradykinin

The effects of bradykin on prostaglandin metabolism in canine mesenteric vessels were examined. Bradykinin stimulated microsomal prostaglandin synthesis in both artery and vein; this stimulation was more pronounced when [¹⁴C]phosphatidylcholine rather than [¹⁴C]arachidonate was used as the substrate for prostaglandin synthetase. This suggested that bradykinin enhanced a membrane phospholipase. In addition, bradykinin selectively stimulated prostaglandin E 9-ketoreductase activity from veins but not arteries. This may explain the finding that bradykinin induces the release of prostaglandin E compounds from arteries but prostaglandin F compounds from veins.     

Interactions of prostaglandin E1, bradykinin and histamine and the increase of plasma fibrinogen in rats

Considering that tissue injury caused by laparotomy significantly increases the liver synthesis of plasma fibrinogen, and that PGE1, bradykinin and histamine are released into the injured tissues, the effect of above mentioned inflammatory agents and of the adrenal medulla on plasma fibrinogen levels in rats was studied. The subcutaneous administration of PGE1, bradykinin or histamine does not modify plasma fibrinogen levels acting independently comparing with non-injected animals or injected with the drug vehicle. Bradykinin + histamine did not modify plasma fibrinogen levels either. However the administration of prostaglandin E1 + bradykinin + histamine reproduced the increase of fibrinogen characteristics of laparotomy. This increase was partially but significantly inhibited in rats that had undergone bilateral removal of the adrenal medulla or administration of PGE1 + bradykinin + histamine + bupivacaine (a local anesthetic), but it was not modified when the adrenal medullectomy was unilateral. It is concluded that plasma fibrinogen increase is obtained only when PGE1 acts in presence of bradykinin or histamine and the adrenal medulla should be partially responsible for said increase.     

Selective stimulation of venous prostaglandin E 9-ketoreductase by bradykinin.

The effects of bradykin on prostaglandin metabolism in canine mesenteric vessels were examined. Bradykinin stimulated microsomal prostaglandin synthesis in both artery and vein; this stimulation was more pronounced when [14C] hosphatidylcholine rather than [14C] arachidonate was used as the substrate for prostaglandin synthetase. This suggested that bradykinin enhanced a membrane phospholipase. In addition, bradykinin selectively stimulated prostaglandin E 9-ketoreductase activity from veins but not arteries. This may explain the finding that bradykinin induces the release of prostaglandin E compounds from arteries but prostaglandin F compounds from veins.     

Cardiovascular reactivity to arachidonic acid, prostaglandin E2, prostacyclin and bradykinin in carrageenan treated rats

In the rat, during carrageenan-induced peritonitis, the hypotensive activity of arachidonic acid, which depends on PG biosynthesis, is increased during the first hours, and thereafter is not modified. The vasodilator action of PGE2 is reduced during the first day while the actions of PGI2 and bradykinin are not modified. The concentrations of plasma albumin and haptoglobin, two inhibitors of PG biosynthesis are reduced during the first hours. Thereafter the concentration of haptoglobin is increased by 100% while the concentration of albumin remains diminished. During this peritonitis, no plasmatic inhibitory influence on PG synthesis is seen. The anti-inflammatory action of carrageenans does not depend on PG synthesis inhibition.     

Microglia-specific expression of microsomal prostaglandin E2 synthase-1 contributes to lipopolysaccharide-induced prostaglandin E2 production

Microsomal prostaglandin E2 synthase (mPGES)-1 is an inducible protein recently shown to be an important enzyme in inflammatory prostaglandin E2 (PGE2) production in some peripheral inflammatory lesions. However, in inflammatory sites in the brain, the induction of mPGES-1 is poorly understood. In this study, we demonstrated the expression of mPGES-1 in the brain parenchyma in a lipopolysaccharide (LPS)-induced inflammation model. A local injection of LPS into the rat substantia nigra led to the induction of mPGES-1 in activated microglia. In neuron-glial mixed cultures, mPGES-1 was co-induced with cyclooxygenase-2 (COX-2) specifically in microglia, but not in astrocytes, oligodendrocytes or neurons. In microglia-enriched cultures, the induction of mPGES-1, the activity of PGES and the production of PGE2 were preceded by the induction of mPGES-1 mRNA and almost completely inhibited by the synthetic glucocorticoid dexamethasone. The induction of mPGES-1 and production of PGE2 were also either attenuated or absent in microglia treated with mPGES-1 antisense oligonucleotide or microglia from mPGES-1 knockout (KO) mice, respectively, suggesting the necessity of mPGES-1 for microglial PGE2 production. These results suggest that the activation of microglia contributes to PGE2 production through the concerted de novo synthesis of mPGES-1 and COX-2 at sites of inflammation of the brain parenchyma.     

Binding of prostaglandin E1 to human erythrocyte membrane

Prostaglandin E1 is known to alter the structural and functional characteristics of red blood cells, yet, little is understood about the membrane receptors mediating this process. We therefore studied the binding of tritium-labeled prostaglandin E1 to the intact human erythrocyte membrane and demonstrated that the interaction is highly specific, rapid, saturable and reversible. Scatchard analysis of prostaglandin E1 binding to the membrane preparations showed the presence of two independent classes of prostaglandin E1 binding sites which differed in their affinity for the autacoid. The high-affinity class had Kd = 3.6 X 10(-9) M and the low-affinity class had Kd = 5.6 X 10(-5) M. The optimum pH for the binding of [3H]prostaglandin E1 to the erythrocyte membrane was found to be around 7.5 and maximum specific binding occurred at a concentration of 5 mM Mg2+ in the incubation mixture. [3H]Prostaglandin E1 bound to the membrane preparation could not be displaced by GTP or by its stable derivative Gpp[NH]p. However, prostaglandin E1 bound to the erythrocyte membrane preparation could be rapidly displaced by cyclic AMP. The IC50 (concentration of the nucleotide displacing 50% bound [3H]prostaglandin E1 from the membrane) was 75 nM. Other adenine nucleotides or cyclic GMP could not substitute for cyclic AMP. Unlike the right-side-out erythrocyte membrane, the inside-out membrane preparations do not bind [3H]prostaglandin E1. Treatment of right-side-out erythrocyte membrane preparation with neuraminidase markedly decreases the binding of prostaglandin E1. Incubation of the erythrocyte membrane preparation with trypsin resulted in total loss of the binding activity. These results indicate that the prostaglandin E1 binding sites located on the cell surface and sialic acid residues are required for prostaglandin E1 binding to the human erythrocytes. These results also indicated that the binding sites are glycoprotein in nature.     

Cardiovascular actions of python bradykinin and substance P in the anesthetized python, Python regius

The cardiovascular actions of python bradykinin (BK) and substance P (SP) have been investigated in the anesthetized ball python, Python regius. Bolus intra-arterial injections of python BK (0.03-3 nmol/kg) produced concentration-dependent increases in arterial blood pressure, heart rate (HR), and cardiac output concomitant with small decreases in systemic resistance and stroke volume. Intra-arterial injection of 3 nmol/kg python BK produced a tenfold increase in circulating concentration of norepinephrine, but epinephrine levels did not change. BK-induced tachycardia was attenuated (>90%) by the beta-adrenergic receptor antagonist sotalol, and the hypertensive response was attenuated (>70%) by the alpha-adrenergic receptor antagonist prazosin, indicating that effects of python BK are mediated at least in part by activation of the extensive network of adrenergic neurons present in vascular tissues. Bolus intra-arterial injections of python SP in the range 0. 01-30 pmol/kg produced concentration-dependent decreases in arterial blood pressure and systemic peripheral resistance concomitant with increases in cardiac output and stroke volume but with only minor effects on HR. The data suggest that kinins play a physiologically important role in cardiovascular regulation in the python.     

A calcitonin-like action of prostaglandin E1

The pharmacological effects of PGE1 (6 and 9 days, 2-1,250 micrograms/kg per day subcutaneously) upon the growth and the bone resorption of mammals were studied using the proximal tibia and upper incisor of immature rats along with lead acetate as a time marker, and upon the serum calcium and inorganic phosphorus levels. The following results were obtained. 1. PGE1 hardly affected the body weight or the weight of organs of the rats but apparently inhibited the longitudinal growth of proximal tibia in a dose related manner. 2. PGE1 clearly inhibited not only the longitudinal growth (incisor growth) but also the appositional growth (dentin formation) of incisal dentin. 3. The grade of the inhibitory effect on the growth was in the order of bone growth greater than dentin formation greater than incisor growth. 4. The occurrence of osteoporosis due to a low calcium diet was inhibited by the simultaneous administration of PGE1, the mechanism being considered to be mainly due to the inhibitory effect on the bone resorption. 5. PGE1 lowered the level of serum calcium and the lowering effect was not observed in the thyro-parathyroidectomized rat. From the facts that the above effects were exactly the same as those of calcitonin (1), the possibility that the subcutaneous injection of PGE1 may induce a calcitonin-like action, a part of which may dependent on the calcitonin secretion is suggested.     

Synthesis of tritium-labelled prostaglandin E1 and its binding to human platelets

A method has been developed that makes it possible to obtain [5,6-3H2]PGE1 with a yield of 35% and a molar radioactivity of 1.7-1.8 TBq/mmol. The binding of [5,6-3H2]PGE1 to native platelets proved to be specific, saturating and reversible. It is characterized by low values (approximately 10(-9) M) of dissociation constants for high-affinity sites, correlates with the inhibition of ADP-induced aggregation of platelets and can be considered as receptor binding. Specific binding of 10 +/- 2 molecules of PGE1 with one platelet was found to cause 50% inhibition of the ADP-induced aggregation.     

Effect of prostaglandin E1 on chemiluminescence response and adherence of human polymorphonuclear leukocytes to human cultured endothelial cells of prostaglandin E1 treated polytraumatized patients

We investigated the effect of prostaglandin E1 on human polymorphonuclear leukocytes, in vivo. Polymorphonuclear leukocytes of a prostaglandin E1 and placebo study group were harvested and their function, as production of oxygen-derived metabolites and adherence to human cultured endothelial cells, was compared. Additionally, data obtained from polymorphonuclear leukocytes of a prostaglandin E1 and placebo group were compared with data obtained from polymorphonuclear leukocytes from 28 blood donors, who served as a control group. Production of oxygen-derived metabolites by polymorphonuclear leukocytes during contact with endothelial cells was measured by chemiluminescence. Chemiluminescence was significantly (p < 0.01) increased in the placebo group in comparison to the control group decreasing to values of control group after 6 d (post-trauma). Chemiluminescence response was not significantly suppressed in patients treated with prostaglandin E1 in comparison to the placebo group. Adherence of polymorphonuclear leukocytes (placebo group) to endothelial cells was significantly increased (p < 0.01) within the first 6 d post-trauma Following day 6, values were in the same range as values for the control group. Adherence was not significantly suppressed in patients treated with prostaglandin E1 in comparison to the placebo group. In conclusion, prostaglandin E1 at a dose of 20 ng/kg bw/min does not influence production of oxygenderived metabolites and adherence in polytraumatized patients in comparison to a placebo group. Additionally, production of oxygen-derived metabolites by polymorphonuclear leukocytes in response to endothelial cells is shown and it is evident that endothelial cells might influence production of oxygen derived metabolites by polymorphonuclear leukocytes.     

Use of substance P fragments to differentiate substance P receptors of different tissues

The C- and N-terminal fragments of substance P were compared to the parent molecule with respect to their ability to: (a) contract the isolated guinea pig ileum, (b) induce salivation in the rat, (c) excite single cat dorsal horn neurones, and (d) induce scratching by intracranial injections in mice. C-terminal fragments as small as the heptapeptide were potent SP agonists on all assay systems. C-terminal fragments containing five amino acids or less were, at most, only weakly active. The C-terminal hexapeptide was a potent SP receptor stimulant on the isolated guinea pig ileum and, when directly applied by microiontophoresis, on cat dorsal horn neurons. However, the same compound was only 2-5% as potent as substance P in eliciting salivation and scratching in vivo, an indication that this fragment may be especially labile to enzymatic degradation. N-terminal fragments were totally inactive on the isolated guinea pig ileum. On the rat salivation and central nervous system assays, however, N-terminal fragments were capable of weak SP-like activity. It is concluded that SP receptors exist in multiple forms which we have labelled SP1 and SP2 receptors for those insensitive or sensitive to N-terminal fragments, respectively.     

Effect of forskolin on alterations of vascular permeability induced with bradykinin, prostaglandin E1, adenosine, histamine and carrageenin in rats

The effect of the diterpene forskolin on vascular permeability alone and in combination with bradykinin, prostaglandin E1, adenosine or histamine has been investigated in rats. Vascular permeability in rat skin was measured using [125I]-labelled bovine serum albumin ([125I]BSA) as a tracer. In addition, the effect of forskolin on footpad edema induced by the injection of a mixture of 2% carrageenin was determined. Forskolin caused a marked potentiation of the increase in vascular permeability in rat skin elicited by the intradermal injection of histamine or bradykinin. However, forskolin caused a significant suppression of the prostaglandin E1-induced vascular permeability response and at a low concentration suppressed the response to adenosine. Forskolin greatly potentiated the footpad edema induced with carrageenin in rats. Intravenous administration of the enzyme bromelain, which reduces plasma kininogen levels, inhibited the footpad edema induced with carrageenin or with a mixture of carrageenin and forskolin. Parenteral administration of a prostaglandin synthetase inhibitor, indomethacin, suppressed the footpad edema induced with carrageenin, but did not inhibit the footpad edema induced with a mixture of carrageenin and forskolin. An antihistamine, cyproheptadine, had no effect on carrageenin-induced footpad edema either in the presence or absence of forskolin. These results suggest that both bradykinin and prostaglandins are essential for the development of carrageenin-induced footpad edema and that bradykinin plays an important role in the potentiative effect of forskolin on footpad edema induced with carrageenin in rats.     

Properties of substance P aggregates. Application to the synthesis and purification of substance P

By 1H-NMR spectroscopy it has been shown that Substance P is largely aggregated at basic and acid pH and in saline solutions. These SP polymers dissociate rapidly by addition of pyridine and acetonitrile and slowly by addition of methanol. The difficulties previously encountered in the purification of SP and SP analogs may be attributed to this aggregation and can be overcome under disaggregating conditions. As a first application of our study we propose a reliable method for obtaining SP with good yield.     

Regulation of prostaglandin E synthases: effects of siRNA-mediated inhibition of microsomal prostaglandin E synthase-1

Prostaglandin E2 (PGE2) is a key mediator involved in several inflammatory conditions. In this study, we investigated the expression and regulation of the terminal PGE2 synthesizing enzyme prostaglandin E synthases (mPGES-1, mPGES-2 and cPGES) in gingival fibroblasts stimulated with pro-inflammatory cytokines. We used siRNA knockdown of mPGES-1 to elucidate the impact of mPGES-1 inhibition on mPGES-2 and cPGES expression, as well as on PGE2 production. The cytokines TNFalpha and IL-1beta increased protein expression and activity of mPGES-1, accompanied by increased COX-2 expression and PGE2 production. The isoenzymes mPGES-2 and cPGES, constitutively expressed at mRNA and protein levels, were unaffected by the pro-inflammatory cytokines. We show for the first time that treatment with mPGES-1 siRNA down-regulated the cytokine-induced mPGES-1 protein expression and activity. Interestingly, mPGES-1 siRNA did not affect the cytokine-stimulated PGE2 production, whereas PGF(2alpha) levels were enhanced. Neither mPGES-2 nor cPGES expression was affected by siRNA silencing of mPGES-1. Dexamethasone and MK-886 both inhibited the cytokine-induced mPGES-1 expression while mPGES-2 and cPGES expression remained unaffected. In conclusion, mPGES-1 siRNA down-regulates mPGES-1 expression, and neither mPGES-2 nor cPGES substituted for mPGES-1 in a knockdown setting in gingival fibroblasts. Moreover, mPGES-1 siRNA did not affect PGE2 levels, whereas PGF(2alpha) increased, suggesting a compensatory pathway of PGE2 synthesis when mPGES-1 is knocked down.