Else Müller-Schweinitzer

2-[125Iodo]LSD, a new ligand for the characterisation and localisation of 5-HT2 receptors.

Summary1.LSD was iodinated with Na125I and chloramine T, to get the radioligand [125I]LSD (125IOL) and with N-I-succinimide to obtain the nonradioactive compound 2-I-LSD (IOL) for comparative pharmacological studies.2.The introduction of iodine in position 2 of LSD leads to an increase in selectivity for 5HT2 receptors. In rat cortex membranes, 125IOL possesses a KD=0.9±0.1 nmol/l, Bmax=240±20 fmoles/mg, and a nonspecific binding of 30–40% in presence of 100 nmol/l ketanserin.3.In competition experiments, 5HT antagonists showed monophasic displacement curves. Their KI-values correlate well with pD′2-values for inhibition of 5HT-induced contraction of canine basilar artery. It can be concluded that the sites labelled by 125IOL have pharmacological properties in common with central 5HT2 receptors, which are identical with vascular postjunctional 5HT receptors.4.The high specific radioactivity of 125IOL permits detection of even small 5HT2 receptor densities which exist in the guinea pig ileum. These 125IOL binding sites are pharmacologically different to those found in the brain or on the vessels and might be a special subpopulation of 5HT2 sites. For example, ketanserin has a high affinity to the sites labelled by 125IOL in the brain and a 100 times lower affinity to the sites labelled in the ileum.5.In a routine binding screen with various ligands, the inhibition constants of IOL for α1, α2, β, histamine and muscarinic receptors are >100 nmol/l with the exception for dopamine receptors, 40 nmol/l.6.125IOL was employed for the autoradiographic localisation of its binding sites after in vitro labelling of microtome rat brain sections. 125IOL labelled 5HT2 sites in the cortex and dopamine receptors in the nucleus caudatus. The exposure times required were very short, compared to those of other 5HT2 ligands available.

Evidence for mediation by 5-HT2 receptors of 5-hydroxytryptamine-induced contraction of canine basilar artery

SummaryThe agonist potencies of 8 indole derivatives and the potencies of 19 recognized antagonists to inhibit constrictor responses to 5-hydroxytryptamine (5-HT) of canine basilar artery were established. In addition the affinities of the indole derivatives for [3H]5-hydroxytryptamine ([3H]5-HT) binding sites and the affinities of the antagonists for [125Iodo]LSD ([125I]LSD) binding sites in rat brain cortex membranes were determined. Comparison was also made between the potencies of the antagonists on canine basilar artery and the KD values published for displacement of [3H]ketanserin binding (Leysen et al. 1982).There was a good correlation between the affinities of the antagonists for 5-HT2 binding sites labelled by both [125I]LSD and [3H]ketanserin and the affinity parameters calculated for inhibition of constrictor responses to 5-HT of canine basilar artery. No correlation could be found between the affinities of the indole derivatives for 5-HT1 binding sites labelled by [3H]5-HT and their potencies to constrict canine basilar artery.It is concluded that constrictor responses to 5-HT of canine basilar artery are mediated by 5-HT2-like receptors.

Alpha-adrenoceptors, 5-hydroxytryptamine receptors and the action of dihydroergotamine in human venous preparations obtained during saphenectomy procedures for varicose veins

SummaryChanges in tension were monitored isometrically on spiral strips from human saphenous veins obtained during surgical removal of varicose veins. Concentration-response curves for noradrenaline and 5-hydroxytryptamine (5-HT) were established by cumulative administrations, curves for dihydroergotamine were constructed from the mean responses to single concentrations. The use of the antagonists prazosin, yohimbine and pizotifen provided evidence for the existence of both postjunctional α1- and α2-adrenoceptors and for the existence of 5-HT receptors. The venoconstrictor effects of dihydroergotamine were unchanged by prazosin. Yohimbine antagonized both dihydroergotamine and 5-HT at about 60 times higher concentrations than required against noradrenaline whereas pizotifen inhibited responses to both dihydroergotamine and 5-HT at about 100 times lower concentrations than those to noradrenaline.These new results are in contrast to conclusions drawn from animal studies and do not support the suggestion that in man the venoconstrictor activity of dihydroergotamine is mediated through stimulation of α-adrenoceptors. The present results strongly suggest that in human saphenous veins the constrictor activity of dihydroergotamine is mediated at least in part through stimulation of 5-HT receptors.

Tissue specific susceptibility of alpha-adrenoceptor mediated vasoconstriction to nifedipine

SummaryThe influence of the calcium antagonist nifedipine on α1- and α1-adrenoceptor vasoconstrictor effects was investigated in vitro. Changes in tension were monitored isometrically on helical strips of canine circumflex coronary and saphenous arteries suspended in 10 ml organ baths and of saphenous veins superfused with Krebs-Henseleit solution. Distinction between α1- and α2-adrenoceptor was made by using selective α-adrenoceptor blocking drugs such as rauwolscine, yohimbine, corynanthine and prazosin, and the agonists noradrenaline, phenylephrine and guanfacine. In venous and both arterial vascular smooth muscles, the contractile process could be triggered by stimulation of both α1- and α2-like adrenoceptors. Nifedipine inhibited the venoconstrictor response to the α2-agonist guanfacine, leaving that to the α1-agonist phenylephrine unchanged. In saphenous arteries, nifedipine in addition to guanfacine also antagonized constrictor responses to phenylephrine, though to a significantly weaker extent. In circumflex coronary arteries, nifedipine was equally potent in antagonizing responses to both α1- and α2-adrenoceptor stimulation.It is suggested that the susceptibility of α-adrenoceptormediated vasoconstrictor effects to blockade by calcium antagonists depends not only on the subtype of α-adrenoceptor but, in addition, on the type and origin of vascular smooth muscle and may be a reflection of tissue variations in intracellular calcium stores.

Responsiveness of isolated canine cerebral and peripheral arteries to ergotamine.

SummaryChanges in tension of spiral strips from dog saphenous, external carotid and basilar arteries were monitored isometrically. Serotonin (5-HT) contracted the 3 arterial preparations in about the same concentration range. Compared with noradrenaline (NA) (100%) the efficacy of 5-HT was about 60% on saphenous, 150% on external carotid and about 500% on basilar arteries. Ergotamine (E) stimulated the 3 vascular preparations in concentrations about 100 times lower than 5-HT. Compared with NA the efficacy of E was about 20% on saphenous, 50% on external carotid and 200% on basilar arteries. Compared with 5-HT (100%) however, E had similar constrictor activities in the 3 arteries. It seemed likely therefore that 5-HT receptors are involved in the vasoconstrictor activity of E. Evidence for this was obtained in experiments in which cyproheptadine (Cy), a 5-HT antagonist, was used. For antagonism of E concentrations of Cy about 6 times higher than those required to inhibit 5-HT were necessary, whereas for antagonism of NA about 500 times higher Cy concentrations were necessary.The results suggest that on canine arterial vascular smooth muscle the constrictor activity of E is mediated mainly through serotoninergic receptor sites.

Dihydroergotamine: Pharmacokinetics, Pharmacodynamics, and Mechanism of Venoconstrictor Action in Beagle Dogs

Dihydroergotamine (DHE) elicits selective and long-lasting venoconstrictor activity, although the drug disappears rapidly from the blood. Therefore, a comparative study on the pharmacokinetic and pharmacodynamic properties of DHE was performed in beagle dogs. In addition, the mechanism of the venoconstrictor activity of DHE was investigated in vivo. Changes in the diameter of the saphenous vein and plasma level–time curves of DHE and its metabolites were determined in conscious beagle dogs. After both intravenous and oral administrations of DHE, the venoconstrictor response is of markedly longer duration than would be expected on the basis of the half-life for elimination of DHE from blood. The experimental data support the suggestion that the long duration of the DHE-induced venoconstriction is due (a) to an extremely slow dissociation of the drug from its receptor sites on the venous smooth muscle cell, and (b) to the formation of active metabolites. Using the antagonists ketanserin, pizotifen, and rauwolscine, evidence is presented that the venoconstrictor activity of DHE is mediated through stimulation of 5-HT receptors; there is no evidence of involvement of a-adrenoceptors.

Pharmacological studies on frozen stored canine saphenous veins and basilar arteries.

SummaryCanine saphenous veins were either placed in Krebs-Henseleit solution and stored for 24 h at +4°C, or immersed in FCS (fetal calf serum) containing 1.8 mol/l DMSO (dimethyl sulfoxide), slowly frozen to-70°C and stored for 4 weeks at-70°C or-190°C. Canine basilar arteries were either stored in Krebs-Henseleit solution for 24 h at +4°C or slowly frozen and stored for 3 months in FCS plus 1.8 mol/l DMSO at-70°C. Subsequent pharmacological investigations revealed a considerable attenuation of the contractile force of frozen-stored vessels but the evidence suggests that there may be a very good preservation of the main biochemical properties, such as monoamine oxidase activity, endogenous prostaglandin synthesis and uptake1 mechanisms in veins stored at-190°C and there is an excellent correlation of thepD2 values for various tryptamine derivatives on canine basilar arteries stored for 3 months at-70°C with those calculated on fresh preparations. It is concluded that freezing isolated blood vessels may be considered an effective means of preserving and storing vascular tissues for pharmacological investigations.

Evidence for stimulation of 5-HT receptors in canine saphenous arteries by ergotamine

SummaryChanges in tension of spiral strips from dog saphenous arteries were monitored isometrically. Dose-response curves for noradrenaline, 5-HT and ergotamine were established without and after a 30 min incubation with phentolamine or pizotifen. Phentolamine was about 15 times more potent in antagonizing responses to noradrenaline (pA2 value=7.4) than those to 5-HT (pA2 value=6.2) but it was nearly equipotent in antagonizing responses to ergotamine (pA2 value=6.5) and those to 5-HT. Pizotifen was about 500 times less potent in antagonizing noradrenaline effects than 5-HT but again nearly equipotent when tested against ergotamine and 5-HT.It is suggested that in canine saphenous arteries the stimulant activity of ergotamine is mediated mainly through 5-HT receptors.

Enhanced prostaglandin synthesis contributes to the venoconstrictor activity of ergotamine.

Spiral strips from dog saphenous veins were contracted by 2.5 X 10(-9) M ergotamine, a concentration giving about 50% of maximal response. Phentolamine (3.6 X 10(-6) M) reduced these effects by 60% and indomethacin (2.8 X 10(-7) M) by 30%. Indomethacin was more effective in reducing ergotamine-induced increase of prostaglandin E-like activity in the bathing fluid than those induced by noradrenaline or potassium chloride. Arachidonic acid was more effective in increasing tension in ergotamine-stimulated than in noradrenaline- or potassium-stimulated veins. These findings suggest that the long-lasting venoconstrictor activity of ergotamine is mediated mainly by alpha-adrenoceptors but that enhanced formation of prostaglandin E-like substance(s) may also contribute.

Changes in the venous compliance by bradykinin and angiotensin II and its significance for the vascular effects of cyclosporine-A

SummaryLocal infusion of both bradykinin and enalaprilic acid, the active metabolite of the converting enzyme inhibitor enalapril, elicited dose-dependent reduction of the compliance of the saphenous vein in conscious dogs. The competitive bradykinin B1-receptor antagonist, des-Arg9-Leu8-bradykinin antagonized venous responses to both bradykinin and enalaprilic acid effectively and with similar potency whereas the bradykinin B2-receptor antagonist Thi5,8, d-Phe7-bradykinin was about 100 times less potent when tested against bradykinin. Under control conditions local infusion of angiotensin II had no contractile activity but it elicited considerable venodilation after blockade of endogenous thromboxane A2 synthesis by dazoxiben. Blockade of the converting enzyme by enalapril (3 mg/kg i.v.) enhanced the maximal responses to bradykinin. Under these conditions concomitant local infusion of angiotensin II attenuated the venoconstrictor effects of bradykinin. Venous responses to bradykinin were inhibited after oral treatment of the dogs with the thromboxane A2 receptor antagonist BM 13,177 and nearly completely abolished after i.v. administration of the thromboxane synthesis inhibitor dazoxiben. In contrast, venous responses to enalaprilic acid were unchanged by thromboxane AZ receptor blockade and enhanced after dazoxiben. Following oral administration of cyclosporine-A (10 and 30 mg/kg), venous responses to bradykinin were attenuated while those to enalaprilic acid remained unchanged. Concomitant local infusion of the angiotensin II receptor antagonist saralasin (1 μg/min) reversed completely the cyclosporine-A-induced reduction of the venoconstrictor effects of bradykinin.It is suggested that (1) bradykinin acts through stimulation of bradykinin B1 receptors inducing local formation of angiotensin II and enhanced synthesis of both venoconstrictor (thromboxane A2-like) and venodilator (prostaglandin E2- and/or prostacyclin I2-like) material, and (2) cyclosporine-A intensifies the venodilator component presumably through stimulation of the circulating renin-angiotensin system.