Tilo Görnemann

Pharmacological properties of a wide array of ergolines at functional alpha1-adrenoceptor subtypes

Ergot alkaloids act as (partial) agonists or antagonists at serotonergic, dopaminergic and α-adrenergic receptors. In contrast to their affinity at serotonergic (5-HT) and dopaminergic receptor subtypes, only limited information is available concerning their interaction with α-adrenoceptor subtypes. This especially holds true for native α-adrenoceptors. Therefore, we studied the pharmacological profile of 25 ergolines at α1A-, α1B- and α1D-adrenoceptors in vascular rings or strips of rat and guinea pig endowed with these receptors. Contractile responses were studied by measurement of isometric tension changes in rat tail artery (α1A, α1B), guinea pig spleen (α1B) and rat thoracic aorta (α1D). The anti-migraine drugs ergotamine and dihydroergotamine, the anti-parkinsonian drug lisuride and the anti-hyperprolactinemic drug terguride behaved as antagonists or low-efficacy partial agonists at all three α1-adrenoceptor subtypes with nanomolar receptor affinity. Derivatives of these drugs showed lower affinity at α1-adrenoceptors than the parent compounds. Each individual ergoline derivative tested showed low discriminatory capability at the subtypes, α1A, α1B, α1D. A low discriminatory power between the subtypes (α1A, α1B, α1D) seems to be a class effect of the ergolines. The nanomolar affinities of ergotamine, dihydroergotamine and lisuride for α1-adrenoceptors may affect their effectiveness as anti-migraine and anti-parkinsonian drugs, respectively.

Pharmacological profile of the clonidine‐induced inhibition of vasodepressor sensory outflow in pithed rats: correlation with α2A/2C‐adrenoceptors

Background and purpose: Resistance blood vessels are innervated by sympathetic and primary sensory nerves, which modulate vascular tone through the release of noradrenaline and calcitonin gene‐related peptide (CGRP), respectively. Moreover, electrical stimulation of the perivascular sensory outflow in pithed rats results in vasodepressor responses which are mainly mediated by CGRP release. The present study has investigated the role of α2‐adrenoceptors in the inhibition of these vasodepressor responses.

Search for influence of spatial properties on affinity at α1-adrenoceptor subtypes for phenylpiperazine derivatives of phenytoin.

A series of phenylpiperazine derivatives of phenytoin was evaluated for their affinity at α(1)-adrenoceptor subtypes in functional bioassays (rat tail artery: α(1A) and/or α(1B); guinea pig spleen: α(1B); rat aorta: α(1D)). The most potent compounds at α(1A)-, α(1B)- and α(1D)-adrenoceptors, 11, 18 and 8, showed affinities in the submicromolar range. The role of a hydrogen bond donor group for affinity and selectivity at α(1B)-adrenoceptors, postulated by Bremners pharmacophore model, was confirmed by functional and molecular modelling studies.

Striking differences of action of lisuride stereoisomers at histamine H1 receptors

This study has characterised the pharmacological profile of some dopaminergic agents of the ergoline family including the antiparkinsonian drug 8S-lisuride at native guinea pig histamine H1 receptors and recombinant human and guinea pig H1 receptors. We used segments of guinea pig ileum to study contractile responses, Sf9 insect cell membranes expressing the guinea pig H1 receptor (gpH1R) and the human H1 receptor (hH1R) to analyse GTPase activity of Gq-proteins and we conducted [3H]mepyramine binding studies using recombinant gpH1Rs and hH1Rs. 8S-Lisuride acted as a potent partial agonist at H1Rs of guinea pig ileum (pD2 7.6, Emax 28% of histamine-induced maximum response) and as a silent antagonist at recombinant gpH1Rs (pA2 7.5) and hH1Rs (pA2 7.7) in GTPase studies. In contrast, its epimeric counterpart, 8R-lisuride, lacked efficacy and showed much lower affinity for H1Rs of both species than 8S-lisuride. High affinity of 8S-lisuride and low affinity of 8R-lisuride was also estimated for gpH1Rs and hH1Rs in radioligand binding studies. The 1-allylated derivative of 8S-lisuride, 1-allyl-8S-lisuride, was equipotent with its parent compound (pD2 7.7) and showed enhanced efficacy in guinea pig ileum and at recombinant gpH1Rs in GTPase studies (Emax 53%, 32%). Other antiparkinsionian drugs such as 8S-terguride, pergolide, cabergoline and bromocriptine displayed lower affinities for H1Rs than 8S-lisuride. In conclusion, our results show that the antiparkinsonian drug 8S-lisuride is dramatically more potent than its epimeric counterpart 8R-lisuride in all assays used. 8S-Lisuride behaved as a partial agonist at gpH1Rs and as a silent antagonist at hH1Rs. Thus 8S-lisuride may act as an antagonist in vivo. This may be of potential importance since H1Rs modulate dopaminergic transmission in the brain.

Characterization of the Molecular Fragment That Is Responsible for Agonism of Pergolide at Serotonin 5-Hydroxytryptamine2B and 5-Hydroxytryptamine2A Receptors

Cardiac-valve regurgitation observed in Parkinson patients treated with the ergoline dopamine receptor agonist 8β-methylthiomethyl-6-propylergoline (pergolide) has been associated with the agonist efficacy of the drug at 5-hydroxytryptamine2B (5-HT2B) receptors. 5-HT2A receptors may also play a role in pergolide-induced cardiac-valve regurgitation. We studied the pharmacological profile of pergolide and eight derivatives in porcine vascular rings endowed with 5-HT2B and 5-HT2A receptors to detect the molecular fragment of the pergolide molecule that may be responsible for agonism at these receptors. Pergolide derivatives showed a different substitution pattern at N(6), and the side chain at C(8) was modified by replacement of the sulfur against an oxygen atom. We demonstrate that the potent agonism of pergolide both at 5-HT2B and 5-HT2A receptors is retained when the N(6) propyl substituent is replaced by ethyl. However, agonism can be converted into antagonism if N(6) propyl is replaced by methyl. The N(6)-unsubstituted derivative was a low efficacy 5-HT2B receptor partial agonist and a 5-HT2A receptor antagonist. This pharmacological pattern was also applicable for pergolide congeners with an oxygen in the side chain at C(8). 6-Methylpergolide retained agonist efficacy and potency compared with pergolide at human (h) D2LONG(L) and hD2SHORT(S) receptors as determined by guanosine 5′-O-(3-[35S]thio)triphosphate binding. Based on the ability of pergolide to produce potent agonism at 5-HT2B receptors and the failure of 6-methylpergolide to act as an agonist but as a potent antagonist, we conclude that the N(6) propyl substituent of pergolide is crucial for 5-HT2B receptor agonism and thus a determinant of valvular regurgitation.

Antiserotonergic properties of terguride in blood vessels, platelets, and valvular interstitial cells.

Serotonin (5-hydroxytryptamine; 5-HT) is involved in heart valve tissue fibrosis, pulmonary arterial fibrosis, and pulmonary hypertension. We aimed at characterizing the antiserotonergic properties of the ergot alkaloid derivative terguride [1,1-diethyl-3-(6-methyl-8α-ergolinyl)urea] by using functional receptor assays and valvular interstitial cell culture. Terguride showed no vasoconstrictor effect in porcine coronary arteries (5-HT2A receptor bioassay) and no relaxant effect in porcine pulmonary arteries (5-HT2B receptor bioassay). Terguride behaved as a potent antagonist at 5-HT2A receptors (noncompetitive antagonist parameter pD′2 9.43) and 5-HT2B receptors (apparent pA2 8.87). Metabolites of terguride (N″-monodeethylterguride and 6-norterguride) lacked agonism at both sites. N″-monodeethylterguride and 6-norterguride were surmountable antagonists at 5-HT2A receptors (pA2 7.82 and 7.85, respectively) and 5-HT2B receptors (pA2 7.30 and 7.11, respectively). Kinetic studies on the effects of terguride in pulmonary arteries showed that the rate to reach drug-receptor equilibrium for terguride was fast. Washout experiments showed that terguride easily disappeared from the receptor biophase. Pretreatment with terguride inhibited 5-HT-induced amplification of ADP-stimulated human platelet aggregation (IC50 16 nM). In porcine valvular interstitial cells, 5-HT-induced activation of extracellular signal-regulated kinase (ERK) 1/2, an initiator of cellular proliferation and activity, was blocked by terguride as shown by Western blotting. In these cells, the stimulatory effect of 5-HT on [3H]proline incorporation (index of extracellular matrix collagen) was blocked by terguride. Because of the inhibition of both 5-HT2A and 5-HT2B receptors, platelet aggregation, and cellular proliferation and activity (ERK1/2 phosphorylation and collagen production) terguride may have therapeutic potential in the treatment of fibrotic disorders.

Pharmacological characterization of ergotamine-induced inhibition of the cardioaccelerator sympathetic outflow in pithed rats

Ergotamine inhibits the sympathetically-induced tachycardia in pithed rats. The present study identified the pharmacological profile of this response. Male Wistar rats were pithed and prepared to stimulate the preganglionic (C7–T1) cardiac sympathetic outflow. Intravenous continuous infusions of ergotamine dose-dependently inhibited the tachycardic responses to sympathetic stimulation, but not those to exogenous noradrenaline. Using several antagonists, the sympatho-inhibition to ergotamine was: (1) partially blocked by rauwolscine (α2), haloperidol (D1/2-like) or rauwolscine plus GR127935 (5-HT1B/1D); (2) abolished by rauwolscine plus haloperidol; and (3) unaffected by either saline or GR127935. In animals systematically pretreated with haloperidol, this sympatho-inhibition was: (1) unaffected by BRL44408 (α2A), partially antagonized by MK912 (α2C); and (3) abolished by BRL44408 plus MK912. These antagonists failed to modify the sympathetically induced tachycardic responses per se. Thus, the cardiac sympatho-inhibition by ergotamine may be mainly mediated by α2A/α2C-adrenoceptors, D2-like receptors and, to a lesser extent, by 5-HT1B/1D receptors.

Dopamine/serotonin receptor ligands. Part 17: A cross-target SAR approach: Affinities of azecine-styled ligands for 5-HT2A versus D1 and D2 receptors

Dibenzo- and benzindolo-azecines represent a novel class of high-affinity dopamine receptor antagonists. To further characterize these drugs as potential neuroleptics, we selected a set of azecine derivatives and ring expanded homologues and we measured their antagonist activity at the 5-HT(2A) receptor in the porcine coronary artery. SARs found for the 5-HT(2A) receptor resemble those for the D1 but not the D2 receptor. The protein-ligand interactions were discussed with respect to the different binding pockets.

Phenylephrine contracts porcine pulmonary veins via α1B-, α1D-, and α2-adrenoceptors

We have recently shown that the postjunctional alpha(2)-adrenoceptor mediating contraction of porcine pulmonary veins is of the alpha(2C)-subtype. We could also demonstrate that alpha(1)-adrenoceptors might contribute to the contraction in that blood vessel. In the present study, we aimed at characterising the alpha(1)-adrenoceptor subtype(s) involved using pharmacological and molecular biological methods. In isolated rings of porcine pulmonary veins the typical alpha(1)-adrenoceptor agonist phenylephrine caused a concentration-dependent contraction that was inhibited by the alpha(1B)-adrenoceptor selective antagonists 1-[4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl]-2-[2-(isopropyl)-6-methoxyphenoxy]ethan-1-one (Rec15/2615; pA(2) 8.96+/-0.13) and 4-amino-2-[4-[1-(benzyloxycarbonyl)-2(S)-[[(1,1-dimethylethyl)amino]carbonyl]-piperazinyl]-6,7-dimethoxyquinazoline (L-765,314; pA(2) 7.22+/-0.05), as well as the alpha(1D)-adrenoceptor selective antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY7378; pA(2) 8.29+/-0.15, slope of the Schild plot 0.75+/-0.09, significantly different from unity, P<0.05), but not by the alpha(1A)-adrenoceptor selective antagonists (+/-)-1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-benzodioxin-5-yl)-1-piperazinyl]propyl]amino]-2,4(1H,3H)-pyrimidinedione (B8805-033) and N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha,alpha-dimethyl-1H-indole-3-ethanamine (RS-17053). These findings suggest that phenylephrine activates both alpha(1B)- and alpha(1D)-adrenoceptors. The observation was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR) in porcine pulmonary veins, where mRNA signals for alpha(1B)- and alpha(1D)-adrenoceptors could be detected. However, the antagonist properties of rauwolscine and yohimbine (non-subtype selective alpha(2)-adrenoceptor antagonists) against phenylephrine showed that this agonist also activates alpha(2)-adrenoceptors in pulmonary veins. This was strengthened in experiments using tissues that were stimulated with forskolin (cell permeable activator of adenylyl cyclase). Phenylephrine mimicked the effect of the selective alpha(2)-adrenoceptor agonist UK14304 by causing an inhibition of forskolin-stimulated cAMP accumulation that was blocked by rauwolscine. It is concluded that, in addition to alpha(1B)- and alpha(1D)-adrenoceptors, phenylephrine can stimulate alpha(2)-adrenoceptors in porcine pulmonary veins.