Wolfgang Sametz

Histamine-Induced release of arachidonic acid and of prostaglandins in the peripheral vascular bed

Summary1.Injection or infusion of histamine intraarterially into the isolated perfused rabbit ear dosedependently stimulated the release of prostaglandins (PGs) as measured by radioimmunoassay (PGE), bovine coronary artery strips (PGI2) and by the prelabeling technic with [1-14C]-arachidonic acid (PGI2, PGE2, PGF2α, PGD2).2.PG release was abolished by indometacin (1–3 μg/ml) and reduced by the phospholipase A2 inhibitor quinacrine (10 μg/ml) as well as by perfusing with calcium-free, 1 mM EGTA containing solution.3.The histamine H2-receptor antagonists burimamide (5μg/ml) and cimetidine (2 μg/ml) did not influence histamine-induced PG release. The H1-receptor antagonist mepyramine (0.1–1 μg/ml) abolished histamine-induced PG release.4.In the presence, but not in the absence, of bovine serum albumin there was a basal release of high amounts of arachidonic acid. Histamine tended to increase the released amount of radioactive arachidonic acid. In contrast to indometacin which only blocked PG release, mepyramine significantly reduced the histamine-stimulated release of arachidonic acid, too.5.The results show that in the peripheral vascular bed, histamine, via H1-receptors, activates a phospholipase A2 mainly by increasing a transfer of extracellular calcium into the cell. Activation of a phospholipase A2 results in the release of arachidonic acid possibly from a rather small endogenous pool which specifically provides substrate for the PG synthetase system.

Characterization of prostanoid receptors mediating actions of the isoprostanes, 8-iso-PGE2 and 8-iso-PGF2α, in some isolated smooth muscle preparations

We investigated the contracting actions of the isoprostanes (isoPs), 8‐iso‐prostaglandin (PG) F2α and 8‐iso‐PGE2, in comparison to the effects of the thromboxane (TX) A2‐mimetic U 46619 and the traditional prostaglandin PGE2 in the isolated rat aorta, isolated rat gastric fundus and the isolated guinea‐pig ileum. U 46619 and 8‐iso‐PGF2α caused contractions in the rat aorta and rat gastric fundus in a concentration‐dependent manner, whereas these agonists showed no effects in the guinea‐pig ileum. However, 8‐iso‐PGE2 and PGE2 caused contractions in all isolated organs used. The prostanoid TP‐receptor antagonist SQ 29,548 (10 nM) significantly antagonized vasoconstrictions induced by the agonists used in the rat aorta. SQ 29,548 at a final concentration of 3 μM, but not at lower concentrations, significantly inhibited contractions induced by U 46619, 8‐iso‐PGF2α and 8‐iso‐PGE2 in the rat fundus. Responses to PGE2 were unchanged. The prostanoid EP1‐receptor antagonist SC 51089 (3 μM) significantly inhibited contractions induced by 8‐iso‐PGE2 and PGE2 in the rat fundus and in the guinea‐pig ileum. SC 51089 had no effect on responses to any of the agonists tested. Our results show that 8‐iso‐PGE2, in contrast to 8‐iso‐PGF2α, can also cause contractions by activation of the EP1‐receptors in the rat gastric fundus and the guinea‐pig ileum. The findings of the present study do not support the existence of a unique isoP‐receptor in the tissues used.

Influence of epilobium extracts on prostaglandin biosynthesis and carrageenin induced oedema of the rat paw

Epilobium species have been used as remedies in folk-medicine for the treatment of pathophysiological processes of the prostata. In this paper the influence of extracts of Herba Epilobii angustifolii L. and Herba Epilobii parviflori Schreb. on prostaglandin biosynthesis and the carrageenin rat paw oedema is described. Aqueous extracts of Herba E. angustifolii reduced the release of prostaglandins I2, E2 and D2 (in the perfused rabbit ear) approximately 5 times more effectively than did similar extracts of Herba E. parviflori. Methanolic extracts were inactive. The aqueous extract of E. angustifolium strongly reduced the carrageenin-induced rat paw oedema whereas that of E. parviflorum was inactive. The chemical nature of the active compound(s) is as yet unknown but flavonoids and sitosterol derivatives can be excluded.

Dihomo-δ-linolenic acid increases the metabolism of eicosapentaenoic acid in perfused vascular tissue

The isolated rabbit ear was perfused with 14C-arachidonic acid (AA), with 14C-eicosapentaenoic acid (EPA) or with 14C-dihomo-gamma-linolenic acid (DGLA). After incorporation of 14C-AA, the ionophore A 23187 (10 micrograms) stimulated the release of products comigrating with authentic PGI2 (measured as 6-keto-PGF1 alpha), PGE2 and PGD2 on the thin layer chromatography plate. After incorporation of 14C-EPA, A 23187 did not release any trienoic 14C-PGs. After incorporation of 14C-DGLA, A 23187 stimulated the release of labeled products comigrating with 6-keto-PGF1 alpha (but not PGF1 alpha), PGE1 and PGD1. Infusion of unlabeled AA (1 and 10 micrograms/ml) did not influence the metabolism of 14C-EPA or 14C-DGLA. Infusion of unlabeled DGLA (10 micrograms/ml) strongly stimulated the release of trienoic 14C-PGs but did not significantly increase the release of bisenoic 14C-PGs. Neither DGLA nor AA influenced the release of any other labeled incorporated PG precursor, indicating that a phospholipase A2 was not affected. The results show that DGLA is able to stimulate the metabolism of incorporated 14C-EPA resulting in an increased release of antiaggregatory trienoic PGs. The mechanism of this effect is unclear but it may be mediated via the formation of a hydroperoxide derivative of DGLA. Thus, an increased generation of antithrombotic trienoic PGs may be expected under special conditions, possibly also in vivo, depending on the supply of unsaturated fatty acids or the level of various hydroperoxide derivatives.

The nonpeptide B2 receptor antagonist FR173657: inhibition of effects of bradykinin related to its role in nociception

1 The nonpeptide bradykinin B2 receptor antagonist, FR173657 ((E)‐3‐(6‐acetamido‐3‐pyridyl)‐N‐[N‐(2, 4‐dichloro‐3‐[(2‐methyl‐8‐quinolinyl) oxymethyl] phenyl]‐N‐methylaminocarbonylmethyl] acrylamide), was tested in models involving bradykinin‐induced activation of primary afferent neurones in vitro and in vivo. 2 Bradykinin‐induced contractions of the rabbit isolated iris sphincter muscle mediated by tachykinin release from trigeminal afferent neurones were inhibited in a non‐competitive manner by FR173657. A pKB value of 7.9 was calculated. Effects of substance P were unaffected by FR173657. 3 Nociceptive behavioural responses following intraplantar injection of bradykinin in unanaesthetized rats were reduced by 0.3 μmol kg−1 FR173657 s.c. (P<0.05), and completely abolished by 3 μmol kg−1 (P<0.05). Peroral administration of 5 μmol kg−1 FR173657 abolished the bradykinin effects (P<0.05); lower doses had no significant effect. 4 Shortening by intraplantar injection of bradykinin of the paw withdrawal latency in response to radiant heat was abolished by 3 μmol kg−1 FR173657 s.c. (P<0.05), while 300 nmol kg−1 had an intermediate effect. Hyperalgesia induced by prostaglandin E2 remained unaffected by FR173657. 5 Blood pressure reflexes following i.p. instillation of bradykinin in anaesthetized rats were inhibited by FR173657 s.c. with an ID50 of 1.1 μmol kg−1, while the peptidic B2 antagonist icatibant (Hoe‐140; d‐Arg0‐[Hyp3, Thi5, d‐Tic7, Oic8]‐bradykinin) caused inhibition at significantly lower doses (ID50 8.5 nmol kg−1 P<0.001). Responses to hydrochloric acid i.p. remained unaffected by FR173657. 6 FR173657 or similar nonpeptide compounds may be useful for the development of drugs for diseases involving pain induced by the release of endogenous kinins, i.e. especially in acute inflammatory conditions.

Effects of the non‐peptide B2 antagonist FR173657 on kinin‐induced smooth muscle contraction and relaxation, vasoconstriction and prostaglandin release

The non‐peptide bradykinin (BK) antagonist (E)‐3‐(6‐acetamido‐3‐pyridyl)‐N‐[N‐[2,4‐dichloro‐3‐[(2‐ methyl‐8‐quinolinyl)oxymethyl]phenyl]‐N‐methylaminocarbonylmethyl]acrylamide (FR173657) was tested in intestinal, uterine, tracheal and vascular in vitro preparations. The investigation aimed at determining the antagonistic potency, duration of action, specificity for BK receptors and apparent mode of antagonistic action of FR173657. Contractions of the isolated ileum of the guinea‐pig in response to BK were inhibited by FR173657 (10–300 nM) in a concentration‐dependent manner. The inhibition lasted for up to 90 min after wash‐out of FR173657. Cumulative concentration‐response curves to BK were shifted to the right with a concomitant decrease in the maximum effect. A pKB value of 8.7 was determined. FR173657 had no effect on contractions induced by acetylcholine, histamine, 5‐hydroxytryptamine, substance P, angiotensin II or caerulein. The concentration‐response curves for B2 receptor‐mediated relaxations of the rat isolated duodenum induced by BK were shifted to the right together with a concomitant reduction of the maximum BK effect in the presence of FR173657 (10–300 nM). A pKB of 9.0±0.2 was calculated. FR173657 had no effect on B1 receptor‐mediated relaxations in response to des‐Arg9‐BK. The concentration‐response curves for BK‐induced contractions of the rat isolated uterus were shifted to the right by FR173657 (3–300 nM) in a concentration‐dependent and parallel manner. The Schild plot for the inhibition caused by FR173657 had a slope of −0.98 indicating a competitive mode of antagonism. A pA2 value of 9.1 was determined. Contractions of the circular smooth muscles of the guinea‐pig isolated trachea in response to BK were concentration‐dependently inhibited by FR173657 (10–100 nM). An affinity estimate of 9.3 was calculated for FR173657. Contractions induced by acetylcholine and relaxations in response to isoprenaline remained completely unaffected by FR173657. In the rabbit isolated perfused ear, BK (0.01–10 nmol) produced a dose‐dependent vasoconstriction. In the presence of 30 nM FR173657, the effects of BK were reduced by at least 60%, while FR173657 completely abolished the effects of all BK doses at 300 nM. FR173657 did not affect vasoconstriction induced by noradrenaline or angiotensin II. The arterial injection of BK (10 nmol) into the rabbit isolated perfused ear caused an approximately three fold increase in the release of the prostaglandins E2 and I2 into the venous effluent. The BK‐stimulated prostaglandin release was completely abolished in the presence of FR173657 (300 nM) while the basal prostaglandin release was unchanged. In summary, FR173657 was shown to be a highly potent and selective BK antagonist which was active on B2, but not B1, receptors. FR173657 was a competitive antagonist in the rat uterus but showed a deviation from competitive inhibition in the other preparations studied similar to other second generation peptide antagonists. The inhibitory action in vitro was long‐lasting, but was fully reversible.

Influence of isoprostanes on vasoconstrictor effects of noradrenaline and angiotensin II

The isoprostanes, 8-iso-prostaglandin F2alpha and 8-iso-prostaglandin E2, which are released in vivo by free radical-catalyzed peroxidation of arachidonic acid, are potent vasoconstrictors. Increased formation of 8-iso-prostaglandin F2alpha has been detected in human cardiovascular diseases, in which enhanced plasma levels of noradrenaline and angiotensin II have harmful vasoconstrictor effects. Therefore, we investigated the influence of perfusions with the thromboxane A2 mimetic, U 46619, and with the isoprostanes, 8-iso-prostaglandin F2alpha, 8-iso-prostaglandin E2, 8-iso-prostaglandin E1 and 8-iso-prostaglandin F3alpha, on the vasoconstrictor effects of noradrenaline and angiotensin II in the isolated perfused rabbit ear. Our results demonstrate that perfusions with U 46619, 8-iso-prostaglandin E2 and 8-iso-prostaglandin F2alpha, at a subthreshold concentration (30 nM), amplified the vasoconstrictions induced by noradrenaline or angiotensin II significantly. In addition, the results show that U 46619, 8-iso-prostaglandin F2alpha, 8-iso-prostaglandin E2 and 8-iso-prostaglandin E1, which were applied as a bolus, induced much more pronounced vasoconstrictions than prostaglandin F2alpha, prostaglandin E2 and prostaglandin F3alpha. Prostaglandin E1 and 8-iso-prostaglandin F3alpha, showed no effects. In conclusion, it can be assumed that the powerful vasoconstrictions induced by 8-iso-prostaglandin E2 and 8-iso-prostaglandin F2alpha and their potentiating effects on vasoconstrictions induced by noradrenaline or angiotensin II might be of pathophysiological relevance in cardiovascular diseases.

Formation of 8-iso-PGF2α and thromboxane A2 by stimulation with several activators of phospholipase A2 in the isolated human umbilical vein

We investigated the effects of the phospholipase A2 (PLA2) activators calcium ionophore A 23187, hydrogen peroxide (H2O2), bradykinin (BK), histamine and noradrenaline (NA) on the 8‐iso‐prostaglandin (PG)F2α formation in the isolated human umbilical vein and the isolated rabbit ear. For comparison, the influence of these substances on the thromboxane A2 (TXA2) release was also investigated. The release of total (esterified as well as free) 8‐iso‐PGF2α, free 8‐iso‐PGF2α and TXB2, the stable metabolite of TXA2, was determined by specific enzyme immunoassays. The results show that bolus injections of 5.4 mmol H2O2, 30 nmol A 23187, 10 nmol BK, 50 nmol histamine and 20 nmol NA caused an increased release of total 8‐iso‐PGF2α in the umbilical vein and the rabbit ear. A perfusion with H2O2 at a final concentration of 0.3 mM also increased the release of this isoprostane. Increased formation of free 8‐iso‐PGF2α was induced by A 23187 injection and by both modes of H2O2 administration, but not by the other treatments. Bolus injections of A 23187, BK and histamine induced an increased release of TXB2 in both organs. Both modes of H2O2 administration and NA showed no releasing effects. In conclusion, our results show that the substances used are able to stimulate the formation of 8‐iso‐PGF2α concurrently with the release of PGs. This effect might be of pathophysiological relevance in inflammatory and cardiovascular diseases in which an enhanced release of free radicals, BK, histamine or NA play an important role.

Anti-inflammatory effects of a substance extracted from Epilobium angustifolium

Extracts of Epilobium species (ES) have been used in folkmedicine for the treatment of prostate diseases. The 2 best known species are E. parviflorum (Ep) and E. angustifolium (Ea). There have been only a few reports on the pharmacolo-~ gy of active agents of these plants: Several flavonoids and sitosterol derivatives have been detected in extracts [1]. Both classes of compounds are known to inhibit prostaglandin biosynthesis. Therefore, we investigated the effect of crude extracts of Ep and Ea on PG biosynthesis, the rat paw carrageenan and dextran oedemas as well as the effect of purified extracts of Ea on PG biosynthesis and the rat paw carrageenin oedema.

Influence of Polyunsaturated Fatty Acids on Vasoconstrictions Induced by 8-iso-PGF2α and 8-iso-PGE2

8-iso-PGF2α and 8-iso-PGE2, which are released in vivo by free radical catalyzed peroxidation of arachidonic acid, are equipotent vasoconstrictors in vivo and in vitro. It is assumed that they exert this effect via activation of the thromboxane A2 (TP) receptor or a TP-receptor-like isoprostane receptor. Increased levels of 8-iso-PGF2α have been detected in human cardiovascular diseases. It has been found that polyunsaturated fatty acids (PUFAs) have many beneficial effects in cardiovascular diseases, including antivasoconstrictor actions. Therefore, we investigated the influence of perfusions with eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and dihomo-γ-linolenic acid (DGLA) at final concentrations of 3 and 30 μmol/l on vasoconstriction induced by 8-iso-PGF2α, 8-iso-PGE2 and the thromboxane A2 mimetic U 46619 in the vasculature of the isolated perfused rabbit ear. Additionally, the effect of indomethacin (final concentration 3 μmol/l) on the effects of the PUFAs was investigated. Our results show that the PUFAs at a concentration of 30 μmol/l caused a significant inhibition of the vasoconstrictions induced by 8-iso-PGF2α, 8-iso-PGE2 and U 46619. Furthermore, it can be assumed that a part of the inhibitory effect of DGLA is due to the effect of a cyclooxygenase product, probably PGE1, because indomethacin reduced the inhibitory effect of DGLA.