History of catecholamine research.

The prominence of catecholamines and their congeners in allergic diseases rests chiefly on their use in asthma and acute hypersensitivity reactions, such as anaphylaxis. They act in these indications by activating both α- and ß-adrenoceptors. Adrenaline, the prototype, was discovered in the adrenals in 1893/1894. In 1939, dopa decarboxylase was the first enzyme in the biosynthesis of catecholamines to be described. Later other catecholamines like noradrenaline and dopamine were characterized. The identification of the active chemicals went along with studies regarding catecholamine receptors. It took until 1948 before the existence of at least two different receptors for the different effects was accepted. Meanwhile, genes from all mammalian catecholamine receptors have been cloned.

Es kann die Spur von unseren Erdetagen--on pharmacologists and pharmacology.

Es kann die Spur von unseren Erdetagen is a slightly modified line from Goethe's tragedy Faust and together with the following line can be translated as "The vestiges of our path on earth/Through aeons cannot fade away". The author's festival speech at the 50th Spring Meeting of the German Pharmacological Society this year in Mainz was given under that title and is presented here in abridged form. Twelve examples are given showing that indeed our, the pharmacologists', professional vestiges cannot disappear. Many pharmacologists may share this view of their profession.

Structure-activity relationships of novel P2-receptor antagonists structurally related to Reactive Blue 2.

P2 membrane receptors for nucleotides represent significant targets for experimental pharmacology and drug research. In earlier publications, we have shown that Reactive Blue 2 (RB 2), one of the most widely used P2-receptor antagonists, displays only moderate affinity and does not discriminate between native P2X- and P2Y-receptor subtypes. In the present study we have pharmacologically evaluated a series of 15 synthesized and re-evaluated four commercially obtained and chromatographically purified RB 2 type anthraquinone derivatives on contractions of the rat vas deferens (RVD) elicited by alpha,beta-methylene ATP (alpha,beta-meATP), mediated by P2X1-receptors, and relaxations of the carbachol-precontracted guinea-pig taenia coli (GPTC) elicited by adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS), mediated by P2Y1-like receptors. Based on the structure-activity relationships (SAR) it is concluded that hydrophobic interactions of aromatic pi-electron systems, hydrogen bonds with nitrogen as donor and acceptor atoms, and, particularly, position, conformational distance and number of anionic sulfonate groups are of great importance for the blockade of the two native P2-receptor subtypes. We have also identified novel, for the most part reversible antagonists that bind with higher affinity and improved subtype selectivity in comparison to RB 2. In particular, 1-amino-4-{4-[4-chloro-6-(2-sulfonatophenylamino)-[1,3,5]triazine-2-ylamino]-2-sulfonatophenylamino}-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid trisodium salt (MG 50-3-1) is the most potent antagonist at the P2Y1-like-receptors of the GPTC reported so far (IC50=4.6 nM). It is significantly less potent as reversible antagonist at the P2X1-receptors of the RVD (IC50=2.8 microM). Thus, MG 50-3-1 represents a selective pharmacological tool and may be a lead compound for future investigations.

Distinct mixtures of muscarinic receptor subtypes mediate inhibition of noradrenaline release in different mouse peripheral tissues, as studied with receptor knockout mice.

The muscarinic heteroreceptors modulating noradrenaline release in atria, urinary bladder and vas deferens were previously studied in mice in which the M(2) or the M(4) muscarinic receptor genes had been disrupted. These experiments showed that these tissues possessed both M(2) and non-M(2) heteroreceptors. The analysis was now extended to mice in which either the M(3), both the M(2) and the M(3), or both the M(2) and the M(4) genes had been disrupted (M(3)-knockout, M(2/3)-knockout and M(2/4)-knockout). Tissues were preincubated with (3)H-noradrenaline and then stimulated electrically (20 pulses per 50 Hz). In wild-type atria, carbachol (0.01-100 microM) decreased the electrically evoked tritium overflow by maximally 60-78%. The maximum inhibition of carbachol was reduced to 57% in M(3)-knockout and to 23% in M(2/4)-knockout atria. Strikingly, the effect of carbachol was abolished in M(2/3)-knockout atria. In wild-type bladder, carbachol (0.01-100 microM) reduced the evoked tritium overflow by maximally 57-71%. This effect remained unchanged in the M(3)-knockout, but was abolished in the M(2/4)-knockout bladder. In wild-type vas deferens, carbachol (0.01-100 microM) reduced the evoked tritium overflow by maximally 34-48%. The maximum inhibition of carbachol was reduced to 40% in the M(3)-knockout and to 18% in the M(2/4)-knockout vas deferens. We conclude that the postganglionic sympathetic axons of mouse atria possess M(2) and M(3), those of the urinary bladder M(2) and M(4), and those of the vas deferens M(2), M(3) and M(4) release-inhibiting muscarinic receptors.

Modulation of the baroreceptor reflex by alpha 2A-adrenoceptors: a study in alpha 2A knockout mice.

1. Our objective was to determine whether alpha(2A)-adrenoceptors modulate the baroreceptor reflex. The efficacy of the reflex was evaluated by measuring the spontaneous blood pressure and heart rate variability at rest and the heart rate responses to evoked changes in blood pressure. Experiments were carried out in conscious, unrestrained, and anaesthetized alpha(2A)-adrenoceptor-deficient (alpha(2A)-KO) mice and WT mice. 2. In conscious alpha(2A)-KO mice, the spontaneous blood pressure variability was greater, and the spontaneous heart rate variability was lower than in conscious WT mice. This was also observed in anaesthetized animals. 3. The reflex bradycardia after intravenous injection of phenylephrine was greatly attenuated in conscious alpha(2A)-KO compared to conscious WT mice; the baroreceptor reflex gain (ratio maximal change in heart rate/maximal change in mean arterial pressure) was decreased by 40%. 4. Similar results were obtained when reflex bradycardia was elicited by intra-arterial volume loading of conscious WT and alpha(2A)-KO mice. The baroreceptor reflex gain upon volume loading was also low in anaesthetized alpha(2A)-KO mice. 5. The reflex tachycardia evoked by intravenous sodium nitroprusside injection was also significantly less in alpha(2A)-KO mice as compared to WT, conscious as well as anaesthetized; the baroreceptor reflex gains were decreased by 50 and 65%, respectively. 6. Direct stimulation of cardiac beta-adrenoceptors by the agonist isoprenaline produced similar cardioacceleration in alpha(2A)-KO and WT animals. 7. Our results show that the baroreceptor reflex function is impaired in mice lacking alpha(2A)-adrenoceptors. We conclude that central alpha(2A)-adrenoceptors facilitate the reflex response to both loading and unloading of the arterial baroreceptors.

All three alpha2-adrenoceptor types serve as autoreceptors in postganglionic sympathetic neurons.

Postganglionic sympathetic neurons and brain noradrenergic neurons use alpha(2A)- and alpha(2C)-adrenoceptors as presynaptic autoreceptors. The present experiments were carried out in order to see whether they possess presynaptic alpha(2B)-autoreceptors as well. Pieces of atria, vasa deferentia, the occipito-parietal cortex and the hippocampus were prepared from either wildtype (WT) mice or mice in which both the alpha(2A)- and the alpha(2C)-adrenoceptor gene had been disrupted (alpha(2AC)KO). The pieces were incubated with (3)H-noradrenaline and then superfused and stimulated electrically. In a first series of experiments, single pulses or brief, autoinhibition-poor pulse trains were used for stimulation. The alpha(2)-adrenoceptor agonist UK 14,304 (brimonidine) reduced the evoked overflow of tritium in all four tissues from WT mice but did not change it in any tissue from alpha(2AC)KO mice. A different pattern was obtained with medetomidine as alpha(2 )agonist. Like UK 14,304, medetomidine reduced the evoked overflow of tritium in all four tissues from WT mice and did not affect overflow in brain slices from alpha(2AC)KO mice; however, in contrast to UK 14,304, medetomidine reduced evoked overflow also in atrial and vas deferens pieces from alpha(2AC)KO mice, although with a lower maximum and potency than in WT preparations. The alpha-adrenoceptor antagonists rauwolscine, phentolamine, prazosin, spiroxatrine and WB 4101 shifted the concentration-response curve of medetomidine in alpha(2AC)KO atria and vasa deferentia to the right. The pK(d) values of the five antagonists against medetomidine in alpha(2AC)KO atria and vasa deferentia correlated with pK(d) values at prototypical alpha(2B) radioligand binding sites but not at alpha(2A) or alpha(2C) binding sites. In a second series of experiments, autoinhibition-rich pulse trains were used for stimulation. Under these conditions, rauwolscine and phentolamine increased the evoked overflow of tritium from alpha(2AC)KO atrial and vas deferens pieces but not from alpha(2AC)KO brain slices. The increase was smaller (by 40% in atria and by 70% in the vas deferens) than previously observed in WT preparations (by 200-400%). In a last series of experiments, mRNA for the alpha(2B)-adrenoceptor was demonstrated by RT-PCR in thoracolumbar sympathetic ganglia from WT, alpha(2A)KO, alpha(2C)KO and alpha(2AC)KO mice but not from alpha(2B)KO mice. The results show that brain noradrenergic neurons express only alpha(2A)- and alpha(2C)-adrenoceptors as autoreceptors. Postganglionic sympathetic neurons, however, can express alpha(2B)-adrenoceptors as presynaptic autoreceptors as well. The alpha(2B)-autoreceptors are activated by medetomidine but not by UK 14,304. They are also activated by previously released noradrenaline. The two-alpha(2)-autoreceptor hypothesis has to be replaced by a three-autoreceptor hypothesis for postganglionic sympathetic neurons.

Crosstalk between presynaptic angiotensin receptors, bradykinin receptors and alpha 2-autoreceptors in sympathetic neurons: a study in alpha 2-adrenoceptor-deficient mice.

1. In mouse atria, angiotensin II and bradykinin lose much or all of their noradrenaline release-enhancing effect when presynaptic alpha(2)-autoinhibition does not operate either because of stimulation with very brief pulse trains or because of treatment with alpha(2) antagonists. We now studied this operational condition in alpha(2)-adrenoceptor-deficient mice. Release of (3)H-noradrenaline was elicited by electrical stimulation. 2. In tissues from wild-type (WT) mice, angiotensin II and bradykinin increased the overflow of tritium evoked by 120 pulses at 3 Hz. This enhancement did not occur or was much reduced when tissues were stimulated by 120 pulses at 3 Hz in the presence of rauwolscine and phentolamine, or when they were stimulated by 20 pulses at 50 Hz. 3. In tissues from mice lacking the alpha(2A)-adrenoceptor (alpha(2A)KO) or the alpha(2B)-adrenoceptor (alpha(2B)KO), the concentration-response curves of angiotensin II and bradykinin (120 pulses at 3 Hz) were unchanged. In tissues from mice lacking the alpha(2C)-adrenoceptor (alpha(2C)KO) or both the alpha(2A)- and the alpha(2C)-adrenoceptor (alpha(2AC)KO), the concentration-response curves were shifted to the same extent downwards. 4. As in WT tissues, angiotensin II and bradykinin lost most or all of their effect in alpha(2A)KO and alpha(2AC)KO tissues when rauwolscine and phentolamine were present or trains consisted of 20 pulses at 50 Hz. 5. Rauwolscine and phentolamine increased tritium overflow evoked by 120 pulses at 3 Hz up to seven-fold in WT and alpha(2B)KO tissues, three-fold in alpha(2A)KO and alpha(2C)KO tissues, and two-fold in alpha(2AC)KO tissues. 6. Results confirm that angiotensin II and bradykinin require ongoing alpha(2)-autoinhibition for the full extent of their release-enhancing effect. Specifically, they require ongoing alpha(2C)-autoinhibition. The peptide effects that remain in alpha(2C)-autoreceptor-deficient mice seem to be because of alpha(2B)-autoinhibition. The results hence also suggest that in addition to alpha(2A)- and alpha(2C)- mouse postganglionic sympathetic neurons possess alpha(2B)-autoreceptors.

Constitutional isomers of Reactive Blue 2 - selective P2Y-receptor antagonists?

The anthraquinone derivative Reactive Blue 2 (RB 2) is one of the most widely used P2-receptor antagonists, still claimed to be P2Y-selective. RB 2 is defined as a mixture of two constitutional isomers and commercially available in different identity and purity. A sample of RB 2, offered for sale by RBI, purchased from Biotrend, Köln, Germany, was chromatographically purified and identified by 1H- and 13C-NMR studies as a 35:65 mixture of the terminal ring F meta and para constitutional isomers. The two constitutional isomers of RB 2 were synthesised and tested alongside with the ortho isomer Cibacron Blue 3GA (CB 3GA) on contractions of the rat vas deferens (RVD) elicited by alpha,beta-methylene ATP (alpha,beta-MeATP), mediated by P2X(1)-receptors, and relaxations of the carbachol-precontracted guinea pig taenia coli elicited by adenosine 5'-O-(2-thiophosphate) (ADPbetaS), mediated by P2Y(1)-like-receptors. All compounds inhibited the alpha,beta-MeATP induced contraction of the RVD and the ADPbetaS induced relaxation of the carbachol precontracted guinea-pig taenia coli. The IC(50)-values at P2X(1)-R were 9.1 microM for CB 3GA, 28.4 microM for RB 2, 19.7 microM for RB 2 meta, and 35.5 microM for RB 2 para. The IC(50)-values at P2Y(1)-like-R were 17.4 microM for CB 3GA, 7.7 microM for RB 2, 12.0 microM for RB 2 meta, and 2.6 microM for RB 2 para. The results clearly show that neither RB 2 as a mixture nor the pure ortho and meta isomer are P2Y(1)-like- versus P2X(1)-selective antagonists. In contrast the pure para-isomer of RB 2 is a moderately P2Y(1)-like- versus P2X(1)-selective antagonist.

Alpha 2-adrenoceptors in the enteric nervous system: a study in alpha 2A-adrenoceptor-deficient mice.

Mammals possess three types of alpha(2)-adrenoceptor, alpha(2A), alpha(2B) and alpha(2C). Our aim was to determine the type of alpha(2)-adrenoceptor involved in the control of gastrointestinal motility. In transmitter overflow experiments, myenteric plexus longitudinal muscle (MPLM) preparations of the ileum were preincubated with [(3)H]-choline and then superfused. The alpha(2)-adrenoceptor agonist medetomidine reduced the electrically evoked overflow of tritium from preparations taken from wild type but not alpha(2A)-adrenoceptor-knockout mice. In a second series of overflow experiments, MPLM preparations were preincubated with [(3)H]-noradrenaline and then superfused. Again medetomidine reduced the electrically evoked overflow of tritium from wild type but not alpha(2A)-knockout preparations. In organ bath experiments, medetomidine reduced electrically evoked contractions of segments of the ileum from wild type but not alpha(2A)-knockout mice. In each of these three series, phentolamine antagonized the effect of medetomidine in wild-type preparations with greater potency than rauwolscine. In conscious mice, gastrointestinal transit was assessed by means of an intragastric charcoal bolus. In alpha(2A)-knockout mice, the speed of gastrointestinal transit was doubled compared to wild-type. Medetomidine, injected intraperitoneally, slowed gastrointestinal transit in wild type but not alpha(2A)-knockout mice. We conclude that the cholinergic motor neurons of the enteric nervous system of mice possess alpha(2)-heteroreceptors which mediate inhibition of acetylcholine release, of neurogenic contractions and of gastrointestinal transit. The noradrenergic axons innervating the intestine possess alpha(2)-autoreceptors. Both hetero- and autoreceptors are exclusively alpha(2A). It is the alpha(2A)-adrenoceptor which in vivo mediates the inhibition of intestinal motility by the sympathetic nervous system.

Heterogeneity of release-inhibiting muscarinic autoreceptors in heart atria and urinary bladder: a study with M(2)- and M(4)-receptor-deficient mice.

Release-inhibiting muscarinic autoreceptors were studied in heart atria and the urinary bladder of NMRI mice, M(2)-receptor-deficient mice, M(4)-receptor-deficient mice, and wildtype mice sharing the genetic background of the knockout animals. Segments of the tissues were preincubated with (3)H-choline and then superfused and stimulated electrically. In atrial segments taken from adult mice and stimulated with 120 pulses at 1 Hz, the muscarinic receptor agonist oxotremorine-M reduced the evoked overflow of tritium. Its concentration-response curves in atria from NMRI, M(2)-wildtype, M(4)-wildtype and M(2)-knockout mice were similar, with maximal inhibition by about 75%. In atria from M(4)-knockout mice, the maximal inhibitory effect of oxotremorine-M was reduced to 57%. The concentration-response curves of oxotremorine-M were shifted to the right by ipratropium, methoctramine and pirenzepine. Methoctramine and pirenzepine were approximately equipotent antagonists in all strains except in M(4)-knockout atria in which methoctramine was more potent than pirenzepine. When atria from adult NMRI mice were stimulated by 360 pulses at 3 Hz, ipratropium increased the evoked overflow of tritium both in the absence and in the presence of physostigmine (0.1 microM). In atria taken from 1-day-old NMRI mice, oxotremorine-M failed to reduce the evoked overflow of tritium. In bladder segments taken from adult mice, superfused with medium containing oxotremorine-M (1 microM), and stimulated by 360 pulses at 3 Hz, ipratropium increased the evoked overflow of tritium. Its concentration-response curves in preparations from NMRI, M(2)-wildtype, M(4)-wildtype and M(2)-knockout mice were similar. There was one exception: ipratropium failed to cause an increase in bladder pieces from M(4)-knockout mice. Methoctramine and pirenzepine also increased the evoked overflow of tritium in all strains except the M(4)-knockout. The two antagonists were approximately equipotent in NMRI, M(4)-wildtype and M(2)-knockout preparations but methoctramine was less potent than pirenzepine in M(2)-wildtype preparations. When bladder pieces from adult NMRI mice were superfused with oxotremorine-M-free medium and stimulated by 360 pulses at 3 Hz, ipratropium increased the evoked overflow of tritium in the presence of physostigmine (0.1 microM) but not in its absence. In bladder segments taken from 1-day-old NMRI mice and superfused with medium containing oxotremorine-M (1 microM), ipratropium increased the evoked overflow of tritium in the same way as in adult tissue. It is concluded that NMRI mice and the two wildtype strains are similar in their muscarinic autoreceptors. In atria, the autoreceptors are heterogeneous. Some are M(4). The non-M(4)-autoreceptors probably are M(2). In the bladder, the autoreceptors are exclusively M(4). In both tissues, the autoreceptors are activated by previously released acetylcholine under appropriate conditions.