Postjunctional α-adrenoceptors

Summary The postsynaptic -adrenoceptors in rat aorta and in pithed rat were investigated according to their sensitivity to nine -adrenergic agonists and to the selective antagonists yohimbine (₂) and prazosin (₁) and the non-selective one, phentolamine. In addition, in radioligand binding studies, the affinity and selectivity of the drugs were determined on rat cerebral cortex using [³H] yohimbine and [³H] prazosin.On rat aorta, prazosin is 1,000 times more potent than yohimbine against each -adrenoceptor agonist, whether ₁- or ₂-selective. Rat aorta probably contains only ₁-adrenoceptors.Pressor effects in pithed rats are mediated by post-junctional ₁- and ₂-adrenoceptors. The dose-response curve for -methylnorepinephrine in the presence of prazosin, using Hofstee's plots, revealed ₁- and ₂-adrenoceptors, respective proportions being 80.5 and 19.5%     

Influence of continuous ambulatory peritoneal dialysis on serumα 1-acid glycoprotein concentration and drug binding

Summary The influence of continuous ambulatory peritoneal dialysis (CAPD) on the concentrations of ₁-acid glycoprotein in serum and dialysate and on the serum binding of oxprenolol, propranolol and phenytoin has been studied.Before starting CAPD treatment, the serum binding of oxprenolol and propranolol was higher and that of phenytoin lower than in healthy volunteers, and the serum ₁-AGP concentration was higher. During the first days to weeks after starting CAPD, the serum ₁-AGP concentration rose with a concomitant increase in the binding of oxprenolol and propranolol. Subsequently, the ₁-AGP level and the binding of oxprenolol and propranolol decreased to the values found before starting CAPD. The binding of phenytoin showed little change. The concentration of ₁-AGP in dialysate was 2 to 5% of that in serum.     

Evidence in favour of a selective α1-adrenoceptor blocking action of WB 4101 in vivo

Summary The -adrenoceptor blocking potency of WB 4101 at ₁- and ₂-adrenoceptors has been investigated in pithed rats.WB 4101 was approximately 97 times more potent at antagonizing the vasopressor responses produced by the selective ₁-adrenoceptor agonist phenylephrine, than those produced by the selective ₂-adrenoceptor agonist M-7.A dose of WB 4101 (3 mg/kg) that caused extensive blockade of vascular ₁-adrenoceptors, but little or no blockade of vascular ₂-adrenoceptors, exerted no significant blockade of the presynaptic ₂-adrenoceptors in the rat heart.The results support the view that WB 4101 is a highly selective antagonist at ₁-adrenoceptors in vivo.     

Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors and α2D-adrenoceptors modulating release of acetylcholine in guinea-pig ileum

The study was designed to classify in terms of _2A, _2B, _2C and _2D the presynaptic ₂-autoreceptors, as well as the ₂-receptors modulating the release of acetylcholine, in the myenteric plexus-longitudinal muscle (MPLM) preparation of the guinea-pig ileum. A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the MPLM preparation were preincubated with ³H-noradrenaline or ³H-choline and then superfused and stimulated electrically.The stimulation periods used (3H-noradrenaline: 3 trains of 20 pulses, 50 Hz, train interval 60 s; ³H-choline: single trains of 30 pulses, 0.2 Hz) did not lead to ₂-autoinhibition or inhibition of ³H-acetylcholine release by endogenous noradrenaline. The ₂-selective agonist 5-bromo6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced the evoked overflow of tritium in both ³H-noradrenaline and ³H-choline experiments. Most (³H-noradrenaline) or all (³H-choline) of the 10 antagonists shifted the concentration-inhibition curves of UK 14,304 to the right. pK_d values of the antagonists were calculated from the shifts. pK_d values from ³H-noradrenaline experiments correlated with pK_d values from ³H-choline experiments (r = 0.981).It is concluded that ₂-autoreceptors and ₂-heteroreceptors modulating the release of acetylcholine in the MPLM preparation are of the same subtype. Comparison with antagonist affinities for prototypic native ₂ binding sites, binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that both are _2D. The results are consonant with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree, across mammalian or at least across rodent and lagomorph species. The same may hold true for ₂-adrenoceptors on non-noradrenergic neurones.     

Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors in mouse brain

The study was devised to classify, by means of antagonist affinities, the presynaptic ₂-autoreceptors in mouse cerebral cortex in terms of _2A, _2B, _2C and _2D. A set of antagonists was chosen that was able to discriminate between the four subtypes. Slices of the cortex were preincubated with ³H-noradrenaline and then superfused and stimulated electrically.The stimulation periods used (4 pulses, 100 Hz) did not lead to ₂-autoinhibition as shown by the lack of an increase by rauwolscine of the evoked overflow of tritium. The ₂-selective agonists 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) and -methylnoradrenaline reduced the evoked overflow. All 10 antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. Rauwolscine also shifted the concentration-inhibition curve of -methylnoradrenaline to the right. pK_d values of the antagonists were calculated from the shifts. The pK_d values of rauwolscine against UK 14,304 and -methylnoradrenaline were very similar (8.0 and 7.9, respectively).Comparison with antagonist affinities for prototypic native ₂ binding sites, ₂ binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that the ₂-autoreceptors in mouse brain cortex are _2D. This is the first subtype determination of ₂-autoreceptors in the mouse. It supports the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree.     

Pharmacologic differentiation between pre- and postjunctional α2-adrenoceptors by SK & F 104078

Summary Most ₂-adrenoceptor antagonists do not discriminate between pre- and postjunctional ₂-adrenoceptors, and this has led to the commonly held belief that pre- and postjunctional ₂-adrenoceptors may represent one homogeneous population of receptors. SK&F 104078 has been shown to be a potent antagonist at postjunctional ₂-adrenoceptors at concentrations that do not block prejunctional ₂-adrenoceptors. Thus, SK & F 104078 is a competitive postjunctional ₂-adrenoceptor antagonist in canine and rabbit saphenous veins, canine saphenous artery and human platelet with a dissociation constant of approximately 100 nmol/l. Conversely, SK & F 104078 is inactive as a prejunctional ₂-adrenoceptor antagonist in atria from dog, guinea pig, rabbit and rat, and in guinea-pig ileum at concentrations up to 10,000 nmol/l. Likewise, SK & F 104078 has the ability to block postjunctional arterial ₂-adrenoceptors in vivo in the pithed rat at doses that do not inhibit prejunctional ₂-adrenoceptors in the same model. The results suggest that pre- and postjunctional ₂-adrenoceptors may not represent one homogeneous class, but rather are discrete subtypes of the ₂-adrenoceptor that may be differentiated by SK & F 104078. Send offprint requests to R. R. Ruffolo, Jr.     

Stimulation of prostaglandin E2-synthesis by noradrenaline in primary cell cultures from rabbit splenic pulpa is mediated by atypical α-adrenoceptors

Summary In primary cell cultures originating from rabbit splenic pulpa the effects of various adrenoceptor agonists on prostaglandin (PG)-synthesis were studied. The cells-microscopically identified as fibroblasts-released PGs into the medium: especially PGE₂ besides small amounts of PGF₂ and PGD₂.Noradrenaline increased dose-dependently the amount of PGs released into the medium. Besides noradrenaline, only the catecholamines adrenaline and -methylnoradrenaline strongly activated PG-synthesis. Other -adrenoceptor agonists like the phenylethylamine and imidazoline derivatives were only weak agonists or completely ineffective. All adrenoceptor agonists without intrinsic activity in these cells antagonized the noradrenaline effect on PG-synthesis, the imidazolines being more potent antagonists than the phenylethylamines.The -adrenoceptor agonist isoprenaline stimulated PG-synthesis at high concentrations only. The effects of both noradrenaline and isoprenaline were inhibited by low concentrations of phentolamine and phenoxybenzamine, but not by propranolol. The preferential ₂-adrenoceptor antagonists yohimbine and rauwolscine were about 50 times more potent in blocking the noradrenaline effect on PG-synthesis than the more ₁-specific antagonist corynanthine. However, prazosin, another ₁-adrenoceptor antagonist, was about equipotent with yohimbine.It is concluded that noradrenaline elicits PG-synthesis in rabbit splenic fibroblasts via -adrenoceptor stimulation. The -adrenoceptor involved has properties which are different from those reported so far for ₁- or ₁-adrenoceptors.     

α1-adrenoceptor subtype affinities of drugs for the treatment of prostatic hypertrophy

Summary We have used radioligand binding and inositol phosphate accumulation studies to determine the affinity at mixed _1A- and _1B-adrenoceptors (rat cerebral cortex and kidney), _1A-adrenoceptors (rat cerebral cortex and kidney following inactivation of _1B-adrenoceptors by chloroethylclonidine treatment) and _1B-adrenoceptors (rat spleen) for drugs currently under investigation for the treatment of benign prostatic hypertrophy, alfuzosin, naftopidil and (–)- and (+)-tamsulosin. Alfuzosin and naftopidil had similar affinities in all model systems (approximately 10 nM and 130 nM, respectively) and lacked relevant selectivity for ₁-adrenoceptor subtypes. Their potency to inhibit noradrenaline-stimulated inositol phosphate formation in cerebral cortex matched their affinities as determined in the binding studies. Tamsulosin had higher affinity at _1A- than at _1B-adrenoceptors, and was slightly more potent than alfuzosin and naftopidil at _1B- and considerably more potent at _1A-adrenoceptors. However, the interaction of the tamsulosin isomers with chloroethylclonidine-insensitive (_1A-like) adrenoceptors was complex. A detailed analysis of the tamsulosin data and those obtained with other drugs, most notably noradrenaline and oxymetazoline, suggested that chloroethylclonidine-insensitive ₁-adrenoceptors may be heterogenous and that this heterogeneity may differ between cerebral cortex and kidney of the rat.     

Antagonists that differentiate between α2A- and α2D-adrenoceptors

Four antagonists were examined for their ability to differentiate _2A from the orthologous _2Dadrenoceptors. The antagonists were (2S,12bS) 1, 3-di-methylspiro(1, 3, 4, 5, 6, 6, 7,12b-octahydro-2H-benzo[b]furo[2,3-a]quinolizine)-2,4-pyrimidin-2-one (MK 912), 2-[2-(methoxy-1, 4-benzodioxanyl)imidazoline (RX 821002), efaroxan and benoxathian. The ₂-autoreceptors in rabbit brain cortex were chosen as _2A- and the a₂-autoreceptors in guinea-pig brain cortex as _2D-adrenoceptors. Slices of the brain cortex were preincubated with ³H-noradrenaline and then superfused and stimulated electrically by brief pulse trains (4 pulses, 100 Hz) that led to little, if any, ₂-autoinhibition. 5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK 14,304) was used as an ₂-adrenoceptor agonist.UK 14, 304 decreased the stimulation-evoked overflow of tritium. The antagonists shifted the concentration-inhibition curve of UK 14, 304 to the right in an apparently competitive manner. Dissociation constants of the antagonists were calculated from the shifts. MK 912, RX 821002 and efaroxan had markedly higher affinity for (guinea-pig) _2D-adrenoceptors (pK _d values 10.0, 9.7 and 9.1, respectively) than for (rabbit) _2A-adrenoceptors (pK _d 8.9, 8.2 and 7.6, respectively). Benoxathian had higher affinity for _2A- (pK _d 7.4) than for _2D-adrenoceptors (pK _d 6.9). Ratios calculated from the K _d values of the four compounds differentiated between _2A and _2D up to 100 fold. It is concluded that MK 912, RX 821002, efaroxan and benoxathian are antagonists with high power to differentiate _2A- from _2D-adrenoceptors.     

Exploring the pharmacology of the pro-convulsant effects of α2-adrenoceptor antagonists in mice

The effects of selective and specific ₂-adrenoceptor antagonists on electroshock seizure threshold in mice were investigated. Idazoxan, at low doses, efaroxan, RX811059 and RX821002 significantly lowered seizure threshold. The ₁-agonist St 587 and the -agonist isoprenaline were also pro-convulsant. On the other hand the ₂-agonists clonidine and UK 14,304 produced small increases in seizure threshold. Anticonvulsant effects were also produced by low doses of the noradrenaline uptake inhibitor desipramine. This compound increases levels of noradrenaline in the synaptic cleft which could subsequently act at post-synaptic ₂-adrenoceptors. The pro-convulsant action of ₂-adrenoceptor antagonists could be explained in terms of two mechanisms: a) blockade of endogenous noradrenaline which may normally exert a tonic anti-convulsant influence on seizure threshold, through post-synaptic ₂-receptors and/or b) increased activation of ₁- and -adrenoceptors by elevated synaptic noradrenaline levels following blockade of pre-synaptic ₂-adrenoceptors. Of the ₂-antagonists tested, idazoxan was unusual in that high doses were not pro-convulsant; this difference may be explained by ₁-adrenoceptor mediated actions or be related to its recently described affinity at a non-adrenoceptor site — a function for which is currently unknown.     

Comparison of cloned and pharmacologically defined rat tissue α1-adrenoceptor subtypes

Multiple ₁-adrenoceptor subtypes have been defined by pharmacological and receptor cloning techniques, but the precise alignment of cloned and pharmacologically-defined subtypes is still unclear. We have compared the affinities of 8 subtype-selective compounds at three cloned ₁-adrenoceptor subtypes (rat _1B, bovine _1C rat _1A/D) with those previously determined by the same methods in rat spleen, cerebral cortex, and kidney (Naunyn-Schmiedeberg's Arch. Pharmacol. 348: 385–395, 1993). Among all compounds tested to date at cloned ₁-adrenoceptor subtypes (+)-tamsulosin appears to be the most selective with a rank order of potency _1C > _{1A/D 1B}. Affinities for the _1A-selective 5-methyl-urapidil, methoxamine, oxymetazoline, phentolamine and (–)- and (+)-tamsulosin and for noradrenaline and SDZ NVI-085 at the splenic _1B-adrenoceptors and at their low affinity sites in cerebral cortex and kidney correlated best with those at the cloned _1B-adrenoceptor. Affinities of these drugs at their high affinity sites in cerebral cortex (pharmacologically-defined _1A-adrenoceptor) were matched best by those at the cloned _1C-adrenoceptor. Rat kidney appears to contain two chloroethylclonidine-resistant ₁-adrenoceptor subtypes one of which is similar to the cloned at _1C- and one to the cloned _1A/D-adrenoceptor. We conclude that the cloned _1B-adrenoceptor is the genetic correlate of the pharmacologically-defined _1B-adrenoceptor. An ₁-adrenoceptor subtype corresponding to the cloned _1A/D-adrenoceptor appears to exist in rat kidney. Among cloned ₁-adrenoceptor subtypes, the bovine _1C-adrenoceptor bears the closest resemblance to the pharmacologically-defined _1A-adrenoceptor in rat cortex and to one of the chloroethylclonidine-insensitive subtypes in rat kidney.     

All three α<Subscript>2</Subscript>-adrenoceptor types serve as autoreceptors in postganglionic sympathetic neurons

Postganglionic sympathetic neurons and brain noradrenergic neurons use _2A- and _2C-adrenoceptors as presynaptic autoreceptors. The present experiments were carried out in order to see whether they possess presynaptic _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 _2A- and the _2C-adrenoceptor gene had been disrupted (_2ACKO). The pieces were incubated with ³H-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 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 _2ACKO mice. A different pattern was obtained with medetomidine as 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 _2ACKO mice; however, in contrast to UK 14,304, medetomidine reduced evoked overflow also in atrial and vas deferens pieces from _2ACKO mice, although with a lower maximum and potency than in WT preparations. The -adrenoceptor antagonists rauwolscine, phentolamine, prazosin, spiroxatrine and WB 4101 shifted the concentration-response curve of medetomidine in _2ACKO atria and vasa deferentia to the right. The pK_d values of the five antagonists against medetomidine in _2ACKO atria and vasa deferentia correlated with pK_d values at prototypical _2B radioligand binding sites but not at _2A or _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 _2ACKO atrial and vas deferens pieces but not from _2ACKO 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 _2B-adrenoceptor was demonstrated by RT-PCR in thoracolumbar sympathetic ganglia from WT, _2AKO, _2CKO and _2ACKO mice but not from _2BKO mice. The results show that brain noradrenergic neurons express only _2A- and _2C-adrenoceptors as autoreceptors. Postganglionic sympathetic neurons, however, can express _2B-adrenoceptors as presynaptic autoreceptors as well. The _2B-autoreceptors are activated by medetomidine but not by UK 14,304. They are also activated by previously released noradrenaline. The two-₂-autoreceptor hypothesis has to be replaced by a three-autoreceptor hypothesis for postganglionic sympathetic neurons.     

Subclassification of presynaptic α2-adrenoceptors: α2A-autoreceptors in rabbit atria and kidney

The study was devised to classify, by means of antagonist affinities, the presynaptic ₂-autoreceptors of rabbit atria and kidney in terms of _2A, _2B, _2C and _2D-A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the left atrium and slices of the kidney cortex were preincubated with ³H-Noadrenaline and then superfused and stimulated electrically.In one series of experiments, tissue pieces were stimulated by relatively long pulse trains (1 or 2 min) leading to ₂-autoinhibition. All 11 (atria) or 10 (kidney) antagonists increased the evoked overflow of tritium. pEC_30% values (concentrations causing 30% increase) were interpolated from concentration-response curves. In a second series of experiments, tissue pieces were stimulated by brief pulse trains (0.4 s) that did not lead to ₂-autoinhibition, and concentration-inhibition curves of the ₂-selective agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) were determined. Most of the 11 (atria) or 8 (kidney) antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. pK_d values of the antagonists were calculated from the shifts. pEC_30% values correlated with pK_d values, both in atria (r = 0.728) and in the kidney (r = 0.930). pEC_30% values in atria correlated with pEC_30% values in the kidney (r = 0.988) and pK_d values in atria correlated with pK_d values in kidney (r = 0.923).It is concluded that the ₂-autoreceptors in atria and the kidney are the same. Comparison with antagonist affinities for prototypic native ₂ binding sites, ₂ binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that both autoreceptors are _2A. This conclusion is reached with either of the two independent estimates of autoreceptor affinity, pEC_30% and pK_d. The results are compatible with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree, across mammalian or at least rodent and lagomorph species.     

Possible subdivision of postsynaptic α-adrenoceptors mediating pressor responses in the pithed rat

Summary Additional evidence has been obtained indicating a possible subclassification of postsynaptic -adrenoceptors into ₁ and ₂-subtypes. The pressor responses to the -adrenoceptor agonists L-phenylephrine and guanfacine were quantified after i.v. administration to pithed rats. The -sympatholytic drug yohimbine (1 mg/kg) displaced both dose-response curves to the right, but the effect was greatest for guanfacine. After prazosin (0.1 mg/kg) a 53-fold shift to the right was noticed for the dose-response characteristic of L-phenylephrine. Prazosin antagonized the effect of only the higher doses of guanfacine. The findings indicate that L-phenylephrine and prazosin preferentially interact with ₁-adrenoceptors as agonist and antagonist, respectively. Yohimbine proved less selective than prazosin, but preferentially blocks postjunctional ₂-adrenoceptors in the vascular wall. The results obtained with guanfacine may be interpreted to indicate that this drug acts on ₂-adrenoceptors at lower doses and additionally stimulates ₁-adrenoceptors at higher ones. Preliminary findings with corynanthine and rauwolscine support this interpretation.     

Differential serum protein binding of benzidine- and benzidine-congener based dyes and their derivatives

Environmental dyes and their derivatives, some of which are genotoxic, must be transported within the body to the tissues which they affect. One mechanism for this can be observed directly by crossed immunoelectrophoresis (X-IEP). Binding of these chemicals to certain serum proteins changes electrophoretic and immunoprecipitation morphology in X-IEP patterns. This is demonstrated here for four azo dyes derived from benzidine, 3,3-dimethylbenzidine, and 3,3-dimethoxybenzidine, and their parent aromatic amines. Direct Red 2 (a 3,3-dimethylbenzidine-based dye), Direct Blue 15 (a 3,3-dimethoxybenzidine-based dye), Direct Black 38 (a benzidinebased dye), and Evans Blue (a 3,3-dimethylbenzidine-based dye) all bound to albumin, ₁-lipoprotein, -lipoprotein, and hemopexin. Direct Red 2 only slightly affected the mobilities of these proteins. Direct Blue 15 bound also to prealbumin and ₁-antichymotrypsin, and degraded C₃ globulin. Direct Black 38 and Evans Blue bound to numerous additional proteins. Evans Blue bound variably to proteins of sera from different individuals, suggesting that there are individual differences in serum protein binding capabilities for these chemicals. Of the three derivatives of the benzidine dyes, only 3,3-dimethylbenzidine caused changes in X-IEP patterns, indicating its binding to the serum proteins. This chemical differentially affected sub-populations of ₁-lipoprotein, either by altering its electrophoretic mobility or inhibiting its recognition by antibodies. Autoradiographic analyses demonstrated the binding of benzidine and 3,3-dimethylbenzidine to both ₁- and -lipoproteins.Supported by the Bal F. Swan Foundation, Denver, CO     

Über den nachweis und die bedeutung von α-methylnoradrenalin im harn von hypertonikern bei verabreichung von α-methyldopa

Zusammenfassung An 13 Hypertonikern wurde die Harnausscheidung von -Methyldopa, -Methyldopamin und Katecholammen während einer Dauerbehandlung oder nach einer Einzeldosis von -Methyldopa untersucht. Die im Harn enthaltenen Substanzen werden durch Ionenaustausch- und Papierchromatographie voneinander getrennt und fluorometrisch oder biologisch bestimmt. — Von alien Patienten wurde außer freiem und konjugiertem -Methyldopa und -Methyldopamin auch -Methylnoradrenalin ausgeschieden. Gleichzeitig nahm die Ausscheidung von Noradrenalin gegenüber der Vor-und Nachperiode bei fast allen Patienten ab. Es kann ausgeschlossen werden, daß die bei der Behandlung durch die Nieren ausgeschiedenen Mengen von -Methyldopa, -Methyldopamin oder -Methylnoradrenalin die renale Ausscheidung von Noradrenalin herabsetzten. Die während der Behandlung ausgeschiedene Menge von Noradrenalin, Adrenalin und -Methylnoradrenalin zusammengenommen unterschied sich nicht von der Katecholamin-Ausscheidung vor und nach der Behandlung. — Das biosynthetisch vom Menschen gebildete -Methylnoradrenalin verhielt sich nach dem Ergebnis chemischer und pharmakologischer Tests wie Corbadrin, welches die Erythro-Konfiguration aufweist; -Methylnoradrenalin ließ sich eindeutig von dem Diastereomeren von Corbadrin differenzieren, welches die Threo-Konfiguration besitzt. Im Harn der Patienten konnte -Methyladrenalin nicht nachgewiesen werden. Gegen eine Beteiligung von -Methyladrenalin, welches nach Tierversuchen durch Erregung von adrenergen -Receptoren blutdrucksenkend wirkt, an der hypotensiven Wirkung von -Methyldopa spricht ferner, daß die Blutdruckabnahme nach -Methyldopa durch Propranolol nicht aufgehoben, sondern eher sogar verstärkt wurde. — Die systolische Blutdrucksenkung war sowohl der Abnahme der renalen Ausscheidung von Noradrenalin als auch dem Prozentsatz von -Methylnoradrenalin an der gesamten Katecholamin-Ausscheidung significant korreliert; auch im Zeitverlauf ergab sich bei drei Patienten nach einer Einzeldosis von -Methyldopa eine Korrelation zwischen hypotensivem Effekt einerseits und Abnahme der Noradrenalin- bzw. Zunahme der -Methylnoradrenalin-Ausscheidung andererseits. — Die Ergebnisse machen es wahrscheinlich, daß der früher in Tierversuchen nach Verabreichung von -Methyldopa nachgewiesene teilweise Ersatz des sympathischen Überträgerstoffs Noradrenalin durch -Methylnoradrenalin auch unter therapeutischer Dosierung am Menschen stattfindet. Die Korrelationen von hypotensiven Effekten und Veränderungen im Aminstoffwechsel lassen den SchluB zu, daß -Methyldopa den Blutdruek von Hypertonikern durch Freisetzung von -Methylnoradrenalin als falscher Überträgersubstanz und entsprechende Verminderung der Noradrenalin-Freisetzung aus sympathischen Nervenfasern herabsetzt.

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Detection and significance of -Methylnoradrenaline in the urine of hypertensive patients after administration of -Methyldopa

Summary In 13 hypertensive patients the urinary excretion of -methyldopa, -methyldopamine und catechol amines was analyzed either during continuous administration or after a single dose of -methyldopa. The compounds contained in the urine samples were separated by ion exchange and paper chromatography and determined fluorimetrically and biologically. — All patients excreted both free and conjugated -methyldopa, -methyldopamine, and small amounts of -methylnoradrenaline. Compared with the pre- and postdrug period, in nearly all patients the noradrenaline excretion was decreased while -methyldopa was administered. The experimental data obtained exclude the possibility that the noradrenaline excretion was diminished by the simultaneous excretion of -methylnoradrenaline or of excessive amounts of -methyldopa and -methyldopamine. The combined quantities of noradrenaline, adrenaline and -methylnoradrenaline excreted daily during administration of -methyldopa did not differ significantly from the amount of noradrenaline and adrenaline excreted daily before drug treatment was started and after it was discontinued. —Evidence was obtained that -methylnoradrenaline isolated from urine of patients given -methyldopa behaved chemically and biologically in a way similar to (–)-corbadrine (erythro-configuration) but differed markedly from the diastereomer of corbadrine (threo-configuration). Hence, biosynthesis of -methylnoradrenaline leads to the levorotatory erythro-isomer. — In animal experiments carried out previously -methyladrenaline was found as another metabolite of -methyldopa which decreased blood pressure by activation of adrenergic -receptors. The patients given -methyldopa did not excrete -methyladrenaline. The method employed was sensitive enough to detect amounts of -methyladrenaline less than 3 per cent of the -methylnoradrenaline present. Furthermore, involvement of a -adrenergic component in the response of the blood pressure to -methyldopa in hypertensive patients was made unlikely by the observation that propranolol did not antagonize but rather enhanced the hypotensive effect of -methyldopa. — There was a significant correlation between the fall of systolic blood pressure and both the decrease in urinary excretion of noradrenaline and the increase in the percentage of -methylnoradrenaline of the total catecholamine output. Likewise, the time course of the depressor response to a single dose of -methyldopa closely corresponded to the decrease in noradrenaline and the increase in -methylnoradrenaline excretion. Conversely, the return of the blood pressure to the initial level was reflected by an increase in noradrenaline and a decrease in -methylnoradrenaline excretion. — In animal experiments it was previously found that administration of -methyldopa caused a partial displacement of noradrenaline by -methylnoradrenaline which subsequently was released by sympathetic nerve stimulation. The present findings demonstrate that -methylnoradrenaline is formed in man as well. It is concluded that -methyldopa lowers the blood pressure of hypertensive patients by the release of -methylnoradrenaline as a false neurotransmitter and the concomitant decrease in noradrenaline liberation from sympathetic nerve fibres.

     

Involvement of α-adrenoceptors in the excitatory effect of the A2 adenosine receptors agonist 5′-N-ethylcarboxamidoadenosine (NECA) on cardiac automaticity in the isolated right ventricle of the rat

The effects of the non-selective A₂ adenosine receptor agonist 5-N-ethyl-carboxamidoadenosine (NECA) were studied on ventricular automaticity induced by a local injury in the isolated right ventricle of the rat. In concentrations ranging from 0.1 to 100 nM, NECA significantly increased ventricular automaticity. This effect was not apparent when the nonselective -adrenoceptor blocker phenoxybenzamine was present at a concentration of 10 M, which antagonizes both ₁-and ₂-adrenoceptors, as well as when rats were pretreated with reserpine. In non-reserpinized rats, the excitatory effect of NECA was also abolished in the presence of the selective ₁-adrenoceptor antagonist prazosin, but not in the presence of the ₂-adrenoceptor antagonist idazoxan. In reserpinized rats, the excitatory effect of NECA was restored in the presence of the non specific -adrenoceptor agonist phenylephrine as well as in the presence of the selective ₁-adrenoceptor agonist amidephrine but not in the presence of the selective ₂-adrenoceptor agonist clonidine. These results suggest that the excitatory effect of NECA on ectopic ventricular automaticity is dependent on endogenous catecholamines and that -adrenoceptors of type 1 are, in some way, involved in this effect.     

Isoprenaline-induced increase in mRNA levels of inhibitory G-protein α-subunits in rat heart

Summary Long-term -adrenergic stimulation has been shown to desensitize the -adrenoceptor/adenylyl cyclase signalling pathway at both the receptor and the G-protein level. To further elucidate the cellular mechanism of G-protein regulation we investigated the influence of prolonged infusion of isoprenaline (2.4 mg/kg·d) on myocardial mRNA levels of different G-protein -subunits in rats. For comparison rats were treated with triiodothyronine (T₃; 0.5 mg/kg·d) which induces cardiac hypertrophy like isoprenaline but has different effects on the adenylyl cyclase system. Isoprenaline- and T₃-treated animals developed an increase in heart/body weight ratio of 41±3% and 27±4%, respectively (P<0.05). Isoprenaline increased myocardial total RNA concentration by 39±6% (P<0.05). Hybridization with ³²P-labeled rat cDNAs demonstrated an expression rank order of G_s-mRNA>G_i-2-mRNA>G_i–3-mRNA and no detectable expression of G_i–1-mRNA in rat myocardium. mRNA levels of G_s G_i–2 and G_i–3 were 36.9±1.28, 10.7±1.07 and 3.7±0.19 pg/g total RNA, respectively. Isoprenaline increased G_i–2 ^– and G_i–3-mRNA concentrations per g total RNA by 49±18% and 27±710, respectively (P<0.05). This effect was abolished by simultaneously administered propranolol (9.9 mg/kg·d), indicating a,-adrenoceptor-mediated mechanism. In contrast, T₃-induced cardiac hypertrophy was not accompanied by changes in G_i-mRNA expression. G_sa-mRNA levels were unaffected by either treatment.In conclusion, long-term stimulation with isoprenaline in vivo induces a -adrenoceptor-mediated increase in myocardial G_i–2 ^– and G_i–3-mRNA without affecting G_s-mRNA. These results suggest that similar increases in myocardial G_i–2-mRNA in end-stage human heart failure may be at least partly explained by increased -adrenergic stimulation due to increased sympathetic activity.Parts of this work were presented at the wintermeeting of the Deutsche Gesellschaft fur Pharmakologie und Toxikologie in Hannover, 1990 (Eschenhagen et al.), Naunyn-Schmiedebergs Arch Pharmacol 342 (Suppl):R8. The work was supported by the Deutsche Forschungsgemcinschaft Send offprint requests to: T. Eschenhagen at the above address     

Binding of thioridazine and thioridazine metabolites to serum proteins

Summary The binding constants of the binding between thioridazine and its metabolites, side-chain sulfoxide, sidechain sulfone and ring sulfoxide, on the one hand, and the plasma proteins, ₁-acid glycoprotein, albumin and total serum proteins, on the other, were determined. The binding constants between the drug substances and ₁-acid glycoprotein were found to be about a thousand times higher than the binding constants between the drug substances and albumin. The binding constants of whole serum were close to those of ₁-acid glycoprotein. Analysis of the binding data indicated competition between thioridazine and its metabolites.A number of drug substances were screened for possible binding interaction with thioridazine and its metabolites. Tricyclic antidepressants and propranolol significantly increased the free concentration of thioridazine. Also salicyclic acid and the plasticizing agent, TBEP, had this effect.     

Pharmacological profiles of a novel α1-adrenoceptor agonist,PNO-49 B,at α1-adrenoceptor subtypes

The effects of a newly synthesized compound, PNO-49B, (R)-(-)-3-(2-amino-l-hydroxyethyl)-4-fluo-romethanesulfonanilide hydrochloride, on ₁-adrenocepfor subtypes were examined in various tissues in which the following distribution of ₁-adrenoceptor subtypes has been suggested: dog carotid artery (_1B), dog mesenteric artery (_1N), rabbit thoracic aorta (_1B + _1L), rat liver (₁B), rat vas deferens (_1A + _1L), rat cerebral cortex (_1A + _1B) and rat thoracic aorta (controversial subtype).PNO-49 B (0.1–100 M) produced concentration-dependent contractions in dog mesenteric artery, rabbit thoracic aorta, rat thoracic aorta and rat vas deferens; and the maximal amplitudes of contraction were almost the same as or slighly less than those of noradrenaline. By contrast, the maximal response to PNO-49 B in dog carotid artery was markedly smaller than the response to noradrenaline. In rabbit thoracic aorta, the contractile response to PNO-49 B was not affected by inactivation of the _1B subtype with chloroethylclonidine (CEC), although the response to noradrenaline was attenuated by that treatment. The dissociation constants (K_A) of PNO-49 B were not different among the rat thoracic aorta, dog carotid and mesenteric arteries and rabbit thoracic aorta (CEC-pretreated). The contractile responses to PNO-49 B were inhibited competitively by prazosin, HV723 (-ethyl-3,4,5-trimethoxy--(3-((2-(2-methoxy-phenoxy)-ethyl)))-amino(propyl)benzeneacetonitrile fumarate) and by WB4101 (2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane). The estimated pA₂ values were high for prazosin and WB4101 in rat thoracic aorta and for HV 723 in dog mesenteric artery, whereas the pA₂ values for these three antagonists in rabbit thoracic aorta were low and were not altered by pretreatment with CEC. The binding of [³H]-prazosin to membranes prepared from rat vas deferens and liver was inhibited by PNO-49 B in a concentration-dependent manner. The resulting pK₁ value for the liver was approximately 1.5 log units lower (one thirtieth in affinity) than the values for the epididymal and prostatic portions of the vas deferens. PNO-49B also inhibited biphasically [³H]prazosin binding to prazosin-high affinity sites of rat cerebral cortex membranes, and the low but high affinity sites for PNO-49B was abolished by CEC-pretreatment. PNO-49B had no effect on the prejunctional ₂-adrenoceptors in rat vas deferens (prostatic portion) nor on the -adrenoceptors in rat atria. The contractile response to PNO-49 B in rat thoracic aorta was not inhibited by cimetidine, pyrilamine or ketanserin.These results indicate that PNO-49B is an ₁-adrenoceptor agonist with a lower affinity and/or efficacy at the _1B subtype as compared with other ₁-subtypes.