Cardiovascular effects of alpha-adrenergic drugs: Differences between clonidine and guanabenz

Summary Guanabenz induced a pressor effect in pithed rats through postsynaptic ₂-adrenoceptors whereas clonidine activated both vascular ₁ and ₂-adrenoceptors. Previous treatment with prazosin, and ₁-antagonist, or depletion of the noradrenergic stores by reserpine produced supersensitivity to the pressor response to clondine only, probably through postsynaptic ₁-adrenoceptors.The hypotension and bradycardia developed in normotensive rats after intravenous guanabenz administration were abolished by prazosin, whereas the central effects of clonidine were antagonized by both prazosin and yohimbine.Selective destruction of central noradrenergic neurons by [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine] (DSP 4) or reserpine plus blockade of catecholamine synthesis by -methyl-p-tyrosine abolished the hypotension and bradycardia produced by guanabenz but merely reduced the bradycardia from clonidine.The present results suggest that, in rats, guanabenz is a selective stimulant of central -autoadrenoceptors antagonized by prazosin whereas at a vascular level guanabenz preferentially activates -adrenoceptors antagonized by yohimbine. The differences observed between the mechanisms by which guanabenz and clonidine produce their central cardiovascular responses might be attributed to their acting on different nuclei.     

Effect of K+ channel blockers on the α2-adrenoceptor-coupled regulation of electrically evoked noradrenaline release in hippocampus

Summary The question whether presynaptic ₂-adrenoceptors regulating noradrenaline release in hippocampus directly couple to tetraethylammonium chloride (TEA) or -dendrotoxin (-DTX)-sensitive K⁺ channels was investigated. Hippocampal slices, prelabelled with [³H] noradrenaline, were superfused in the presence of (+)-oxaprotiline and electrically stimulated with 4 pulses delivered at 100 Hz, in order to avoid autoinhibition due to released noradrenaline.TEA enhanced the evoked [³H]noradrenaline release in rabbit hippocampus in a concentration-dependent manner, yielding an approximately 4-fold increase at 30 mmol/l, whereas the spontaneous outflow of tritium was only slightly affected at this concentration. The ₂-adrenoceptor agonist clonidine, at 10–100 nmol/l inhibited the evoked [³H]noradrenaline release between 77% and 96%. The inhibitory effect of the ₂-agonist was distinctly diminished in the presence of 30 mmol/l TEA but was restored in low Ca²⁺/high Mg²⁺ buffer. Therefore, the diminution of the ₂-agonist effect by TEA observed in experiments with normal Ca²⁺ can be explained by an increase of the Ca²⁺ availability for the release process due to the prolongation of action potentials. In rabbit hippocampus -DTX (10–200 nmol/l) did neither affect the evoked release of [³H]noradrenaline nor its ₂-agonist-induced modulation. However, in rat hippocampus -DTX significantly increased the evoked transmitter release and diminished the effect of clonidine.Taken together, the present data for the rabbit hippocampus exclude the possibility that activation of presynaptic ₂-adrenoceptors inhibits depolarization-evoked [³H]noradrenaline release by inducing an outward K⁺ current through TEA- or -DTX-sensitive K⁺ channels. However, there are species differences between the rabbit and the rat so that in the rat the ₂-adrenoceptors could actually be coupled to K⁺ channels — provided that the release-enhancing properties of -DTX do not account for the ₂-antagonism observed.Correspondence to C. Allgaier at the above address     

Inhibitory α2-Adrenergic mechanism regulating gastric secretion in pylorus-ligated rats

Summary Several -adrenoceptor agonists given intracerebroventricularly or subcutaneously to rats were assessed for their effects on gastric secretion under condition of pylorus ligation. Intracerebroventricular injection of -adrenoceptor agonists reduced gastric secretion, in the following order (relative potency, clonidine = 1): oxymetazoline (100) > clonidine (1) > methoxamine (0.024) > phenylephrine (0.003). The antisecretory effects of oxymetazoline and clonidine given intracerebroventricularly were antagonized with yohimbine administered by the same route. Subcutaneous injection of -adrenoceptor agonists also reduced gastric secretion, in the following order (relative potency, clonidine = 1): clonidine (1) > oxymetazoline (0.3) phenylephrine (0.001) methoxamine (0.0006). Oxymetazoline, when given intracerebroventricularly, was most effective in decreasing the volume and titratable acidity of gastric secretion. Pretreatment with 6-hydroxydopamine intracerebroventricularly (250 g/rat × 2; 72 and 120 h before) reduced the antisecretory effect of clonidine given intracerebroventricularly. Thus, gastric secretion appears to be regulated in an inhibitory manner by ₂-, but not by ₁- in the central and peripheral nervous systems, in pylorus-ligated rats.     

Presynaptic imidazoline receptors and α2-adrenoceptors in the human heart: discrimination by clonidine and moxonidine

The involvement of presynaptic ₂-autoreceptors and imidazoline receptors in the modulation of noradrenaline release was investigated in strips from human atrial appendages preincubated with [³H]noradrenaline and superfused with medium containing desipramine and corticosterone. Electrical impulses were applied transmurally at 2 Hz. The imidazoline derivatives moxonidine and clonidine reduced the evoked tritium overflow in a concentration-dependent manner. Moxonidine was 18-fold more potent than clonidine. The concentration-response curve for moxonidine, but not for clonidine was shifted to the right by the ₂-adrenoceptor antagonist rauwolscine. The apparent pA₂ value of rauwolscine against moxonidine was 8.41. An inhibitory effect was also observed for the imidazoline derivative BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline), a mixed ₂-adrenoceptor antagonist/imidazoline receptor agonist; BDF 6143 was about 2-fold more potent than clonidine. Rauwolscine (1 M) did not substantially shift the concentration-response curve of BDF 6143.It is concluded that noradrenaline release in the human atrium is inhibited not only via presynaptic ₂-autoreceptors but also via presynaptic non-I₁, non-I₂ imidazoline receptors. The failure of rauwolscine to antagonize the inhibitory effect of clonidine suggests that clonidine preferentially stimulates the presynaptic imidazoline receptors; the ₂-adrenoceptor component of its action is probably suppressed by an inhibitory interaction between the two receptors. In contrast, moxonidine acts via presynaptic ₂-autoreceptors, leaving the presynaptic imidazoline receptor unaffected.     

Characterization of α- and β-adrenergic receptors linked to human platelet adenylate cyclase

Summary Adrenaline and noradrenaline cause aggregation of human platelets through -adrenergic receptors, whereas isoprenaline through -adrenergic receptors can inhibit aggregation. Either type of adrenergic receptors is coupled to platelet adenylate cyclase. Stimulation and inhibition of adenylate cyclase by -and -adrenergic stimulants, respectively, had been demonstrated in human platelet lysates. These effects were characterized with regard to the effectiveness of various agonists and antagonists.Reduction of platelet adenylate cyclase activity was observed only with L-adrenaline and L-noradrenaline. This inhibitory effect, which was increased in the presence of a -adrenergic blocking agent, was half-maximal at about 1 to 2×10^–6 M adrenaline, and maximal inhibition (by 50–60%) was observed at about 3×10^–5M. Various other catecholamine and imidazoline derivatives that act as -adrenergic agonists in other cell types neither induced aggregation nor affected the enzyme activity.Adrenaline-induced inhibition of platelet adenylate cyclase was prevented by -adrenergic blocking agents. These compounds inhibited the effects of adrenaline on aggregation and on adenylate cyclase with similar efficacies. Dihydrogenated ergot alkaloids were more effective than phentolamine and yohimbine; phenoxybenzamine, tolazoline and azapetine were least effective. Adrenaline-induced inhibition of platelet adenylate cyclase was reversed by phentolamine without apparent lag phase.In the presence of -adrenergic blocking agents, adrenaline was capable of increasing adenylate cyclase activity between 20 and 50%. Only adrenaline and isoprenaline stimulated adenylate cyclase activity; other compounds that stimulate -adrenergic receptors in other cell types, including -adrenergic stimulants, had no effect on the activity of the platelet enzyme. The stimulatory effect of adrenaline was prevented by various -adrenergic blocking agents including pindolol and propranolol. Preferentially -adrenergic receptor blocking agents such as practolol and atenolol were without effect.These findings indicate that the spectrum of compounds capable of exhibiting intrinsic activity through - and -adrenergic receptors of human platelets is very narrow and that either type of platelet adrenergic receptors appears to differ from those found in other cell types.     

Bedeutung der N- und α-Methylierung für die Affinität von Brenzcatechinaminen zu den adrenergischen Receptoren

Summary 1. N-methylation of dopamine yielding epinine means potentiation of the -adrenergic pressor action in the cat. It is only N-methylation which renders dopamine to a direct acting -sympathomimetic amine: the positive inotropic effect of epinine on the isolated electrically driven guinea pig auricle remained uninfluenced by pretreatment of the animals with reserpine, whereas the dose-response curve of dopamine was shifted to the right.The blood pressure lowering effect of epinine after phenoxybenzamine was—in contrast to that of dopamine—abolished by pronethalol.—N-methylation of noradrenaline doubled the affinity for the -receptors of the heart.2. Also by -methylation dopamine gains affinity to the adrenergic -receptors (heart and vessels): d--methyldopamine, an exclusively directly acting catecholamine, had a 40 times stronger inotropic action than dopamine on reserpinized auricles; the action of l--methyldopamine, a mainly indirectly acting amine, was only 4 times stronger than dopamine.-methylnoradrenaline, although having the same inotropic activity as noradrenaline, had a 10 times stronger depressor action than noradrenaline in the cat pretreated with phenoxybenzamine.By -methylation, the -adrenergic pressor effect of dopamine as well as that of noradrenaline was enhanced only in the range of lower doses since the dose-response curves of the -methylated compounds were generally less steep than those of the non-methylated catecholamines, indicating a lower intrinsic activity of the -methylated derivatives.3. The N- and -methylated catecholamines -methylepinine and -methyladrenaline resp. were blood pressure lowering agents per se, because both methylgroups additively enhanced the affinity to the vascular -receptors. -methylepinine was the most potent -sympathomimetic on the heart in the dopamine series (dopamine < d--methyldopamine epinine < dl--methylepinine). However, in the noradrenaline series the twofold methylated compound -methyladrenaline had the lowest positive inotropic action (d(–)-adrenaline > d(–)-noradrenaline (–)erythro--methylnoradrenaline > (–) erythro--methyladrenaline).4. From the results the following conclusions are drawn: The N- as well as the -methyl-group exerts and +I-effect on the ammonium group. Thereby, protonation will be increased which leads to an enhanced affinity of the resp. catecholamine to the adrenergic - and -receptor. Since the -CH₃ group—dependant upon its steric configuration—also causes a steric hindrance at the receptor site, the increase in affinity due to the +I-effect is partially neutralized. By that the lower intrinsic affinity of the -methylated compounds as indicated by the different slope of their pressor dose-response curves, is also readily explained. Furtheron it is intelligible why -methylation enhanced the -adrenergic activity in the less potent dopamine series (preponderance of the +I-effect), whereas it lowered the affinity to the cardiac -receptors in the noradrenaline series (preponderance of the steric hindrance).5. Although -methyladrenaline was the least potent -sympathomimetic of the noradrenaline series in the guinea pig heart, it was the most potent compound in lowering the cat's blood pressure. Therefore, it seems to be questionable whether the cardiac and the vascular -receptors and/or the mechanisms by which they induce the pharmacodynamic actions are identical.

Über einen Teil der Ergebnisse wurde auf der 7. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft in Mainz, 24.–27. April 1966, berichtet (Palm, Langeneckert u. Holtz, 1966).     

An insulin-releasing property of imidazoline derivatives is not limited to compounds that block α-adrenoceptors

Summary As we have demonstrated previously phentolamine stimulates the release of additional insulin from isolated mouse islets and raises plasma insulin levels in the whole rat. This effect was independent of the well known property of phentolamine to block -adrenoceptors.In experiments on isolated pancreatic islets from mice we now demonstrate that tolazoline and antazoline which are chemically closely related to phentolamine, share its ability to potentiate insulin release.The following results were taken as evidence that this effect does not result from an a-adrenoceptor blocking action of imidazoline compounds.More than 10 times higher concentrations of phentolamine were required to liberate additional insulin from isolated islets than were effective in counteracting the inhibitory effect of clonidine on insulin release.The newly introduced ₂-adrenoceptor antagonist BDF 8933, which is an imidazoline derivative, stimulates insulin release as well, while the irreversible -adrenoceptor blocking agent benextramine of different structure failed to do so, even when being present in concentrations blocking the ₂-adrenoceptor-mediated effects of clonidine.Antazoline shared the ability of phentolamine to stimulate insulin release despite having no or only very little -adrenoceptor blocking activity. When used under our conditions, it almost entirely failed to alleviate the inhibition of insulin release induced by clonidine.We conclude that the response of the islet cells to imidazoline derivatives is not limited to those capable of blocking -adrenoceptors. On the other hand, -adrenoceptor blocking agents of different chemical structure fail to induce the release of additional insulin. We take this as evidence that in our experiments the islet cells respond to imidazoline derivatives and not to -adrenoceptor blockade. Send offprint requests to A. Schulz at the above address     

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.     

Modulation of motor activity by α1 and α2 stimulation in mice

Summary The influence of two -adrenoceptor agonists, clonidine and B-HT 920, on motor activity was tested in mice. Both, clonidine and B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-[4,5-d]-azepine) in the dose range 30–300 g/kg s.c. equieffectively inhibited exploratory activity. On the other hand only clonidine, which stimulates ₂- and ₂-adrenoceptors increased locomotor activity in mice treated with reserpine (5 mg/kg) and apomorphine (3 mg/kg) in the doses of 0.3 and 1 mg/kg i.p. The highly selective ₂-agonist B-HT 920 was ineffective under these conditions up to 30 mg/kg i.p. It is concluded, that in mice sedative -adrenoceptors are of the ₂- and excitatory of the ₁-type.     

Comparative behavioral characterization of the neuroactive steroids 3α-OH,5α-pregnan-20-one and 3α-OH,5β-pregnan-20-one in rodents

Pregnan steroids have been shown to possess anesthetic, hypnotic, anticonvulsant and anxiolytic properties. In this study, two endogenous neuroactive steroid isomers, 3-hydroxy-5-pregnan-20-one (3,5-P) and 3-hydroxy-5-pregnan-20-one 3,5-P), were studied for differences in their pharmacological properties using behavioral assays. 3,5-P and 3,5-P were similar in their potencies and efficacies in blocking pentylenetetrazol-induced seizures in mice (ED₅₀: 3,5-P=2.8 mg/kg and 3,5-P=3.0 mg/kg). Similarly, both neuroactive steroids produced roto-rod deficits within the same range of potency (TD₅₀:3,5-P=18.8 mg/kg and 3,5-P=21.2 mg/kg). However, in animal models of anxiety, subtle differences were observed between the two isomers. In both the light/dark transition test and elevated plus-maze, 3,5-P was more efficacious than 3,5-P, though both compounds had similar potencies. In the Geller-Seifter test, 3,5-P was more potent and efficacious than 3,5-P. Neither compound had significant effects on unpunished responding within the dose range tested. Both compounds produced similar biphasic curves in the locomotor test. All together, the data indicate that 3,5-P and 3,5-P have similar anticonvulsant activity, but the 5-isomer possesses more potent and efficacious anxiolytic properties than the 5-isomer.     

The stimulatory effect of clonidine through imidazoline receptors on locus coeruleus noradrenergic neurones is mediated by excitatory amino acids and modulated by serotonin

Clonidine and other imidazoline/oxazoline drugs, such as cirazoline and rilmenidine, have been shown to stimulate the activity of noradrenergic neurones in the locus coeruleus (NA-LC) by an ₂-adrenoceptor-independent mechanism through the activation of I-imidazoline receptors. The endogenous modulation of the stimulatory effect of clonidine on NA-LC neurones was further investigated after inactivation of ₂-adrenoceptors with N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (EEDQ). In EEDQ-pretreated rats (6 mg/kg, i.p., 6h), clonidine caused a rapid and dose-dependent (320–5120 g/kg, i.v.) increase in the firing rate of NA-LC neurones (ED₅₀ = 809 g/kg, E_max = 90%). The stimulatory effect of clonidine on NA-LC neurones was completely blocked by pretreatment of rats with the excitatory amino acid receptor antagonist kynurenic acid (1–3 mol in 10–30 mol i.c.v., 2–5 min before clonidine). In contrast, the stimulatory effect of clonidine on NA-LC neurones was potentiated by pretreatment with reserpine (5 mg/kg, s.c., 18 h) (E_max increased by 63%). Pretreatment with -methyl-p-tyrosine (250 mg/kg, i.p., 24 h) did not alter the stimulatory effect of clonidine, but pretreatment with p-chloro-phenylalanine (400 mg/kg, i.p., 24 h) markedly enhanced the stimulatory effect of clonidine on NA-LC neurones (E_max increased by 139%). The present results indicate that the imidazoline receptor-mediated stimulatory effect of clonidine on NA-LC neurones is an indirect effect dependent on an excitatory amino acid pathway and modulated by an inhibitory serotonin mechanism.     

α2-Adrenoceptor modulation of rat ventral hippocampal 5-hydroxytryptamine release in vivo

Summary The putative existence of a functional ₂-adrenoceptor-mediated modulation of 5-HT release in vivo from serotonergic neuronal terminals in the ventral hippocampus was investigated using intracerebral microdialysis in chloral hydrate-anaesthetised rats. The ₂-adrenoceptor agonist clonidine (0.01–0.3 mg/kg, SC) dose-dependently decreased the 5-HT output. The response to clonidine was antagonized by systemic or local administration of the ₂-adrenoceptor antagonist idazoxan (0.1 mg/kg, SC, or 10 µmol/l, via the dialysis perfusion medium). Similarly, the 5-HT release-suppressing response to the thiazole ₂-adrenoceptor agonist jingsongling (0.1 mg/kg, SC) was blocked by idazoxan (0.1 mg/kg, SC). The mixed -adrenoceptor/5-HT₁ receptor antagonist pindolol (8.0 mg/kg, SC) did not affect the clonidine-induced reduction of 5-HT release. Tyrosine hydroxylase inhibition by means of -methyl-para-tyrosine (-MT; 250 mg/kg, IP) caused a drastic reduction (>80%) in dialysate 3,4-dihydroxyphenyl acetic acid (DOPAC) output but did not affect the 5-HT output per se over 3 h post-injection. Nor did the -MT pretreatment prevent, but instead significantly enhanced, the 5-HT release-suppressing effect of clonidine. The results demonstrate that the release of 5-HT from serotonergic nerve terminals in rat ventral hippocampus in vivo is modulated by ₂-adrenoceptors, probably both by heteroreceptors on the axon terminals of the serotonergic neurones and by other ₂-adrenoceptor sites situated pre- and/or postsynaptic to the noradrenergic terminals. Our results also suggest that while functionally operative, these sites may receive little physiological tone, at least in chloral hydrate-anaesthetised rats. Send offprint requests to S. Hjorth at the above address     

B-HT 920 acts as an α 1-adrenoceptor agonist in the rabbit aorta under certain in vitro conditions

Summary We have investigated the interaction of the ₂-adrenoceptor agonist B-HT 920 with the -adrenoceptors in the rabbit aorta using various experimental conditions.In standard Krebs-Henseleit solution B-HT 920 behaved as an antagonist when tested against the ₁-agonist phenylephrine. However, it behaved as a partial agonist at -receptors when subcontractile concentrations of various spasmogens (angiotensin II, serotonin, prostaglandin F₂ ) were applied, although no change in the affinity of the receptor to B-HT 920 was observed. By means of the selective antagonists prazosin and rauwolscine it was established that B-HT 920 activated ₁-renoceptors. The same agonistic effects of B-HT 920 were obtained after pretreatment of the animal with reserpine or in the presence of ouabain. The various treatments used (except reserpine) did not influence the contractile response to phenylephrine.The contractile response to B-HT 920 was found to be highly susceptible to the calcium entry blocker nitrendipine whereas the response to phenylephrine was not.It is concluded that spasmogens modulate the responsiveness of -receptors to certain agonists, possibly by causing a depolarization of the cell membrane and, thereby, sensitization of a mechanism involved in excitation-contraction coupling, conceivably a calcium gating mechanism.This study was supported by a grant of the Deutsche Forschungsgemeinschaft     

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.     

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.     

Binding of 3H-clonidine to an α-adrenoceptor in membranes of guinea-pig ileum

Summary The binding of ³H-clonidine to membrane particles from guinea-pig ileum was investigated. The specific binding, i.e. the binding that could be inhibited by high concentrations of unlabeled clonidine or noradrenaline, was of high affinity, K _D3 nM. The number of sites was approximately 25 fmol/mg protein. Rate constants of association and dissociation were 5.3×10⁷ M^–1 min^–1 and 0.18 min^–1, respectively. Affinites of various drugs to the binding site were determined by measuring their effect on the binding of ³H-clonidine. The affinity of adrenergic agonists decreased in the order clonidine = tramazoline > (–)-erythro--methylnoradrenaline > (–)-noradrenaline (–)-phenylephrine. (–)-Noradrenaline had about 20 times more affinity than the (+)-isomer. The affinity of -adrenoceptor antagonists decreased in the order phentolamine > rauwolscine = yohimbine > WB 4101 > pseudoyohimbine > prazosin = corynanthine. Yohimbine and rauwolscine had about 100 times more affinity than their stereoisomer corynanthine. Serotonin 10 M and metiamide 10 M did not affect the binding, and propranolol inhibited it only at high concentrations. — The results indicate that ³H-clonidine labels an ₂-adrenoceptor in guinea-pig ileum. The orders of affinity of -adrenoceptor agonists and antagonists agree well with their orders of potency in functional tests, namely as modulators of cholinergic transmission in the guinea-pig ileum and as modulators of noradrenaline release in the rabbit pulmonary artery. An -adrenoceptor should be classified as ₂ when the affinities of clonidine, tramazoline and -methylnoradrenaline greatly exceed the affinity of phenylephrine, and when the affinities of rauwolscine and yohimbine exceed those of prazosin and corynanthine.     

Further studies on (±)-YM-12617, a potent and selective α1-adrenoceptor antagonist and its individual optical enantiomers

Summary YM-12617, 5-[2-[[2-(o-ethoxyphenoxy)ethyl]amino]propyl]-2-methoxybenzenesulfonamide HCl is structurally novel, an extremely potent and highly selective ₁-adrenoceptor antagonist. An asymmetric center exists at the -carbon atom in the phenethylamine portion of YM-12617, therefore two optical enantiomers exist. -Adrenoceptor blocking properties and hypotensive activities of YM-12617 and its enantiomers have been compared in vitro and in vivo. 1. In the isolated rabbit aorta, R(–)- and S(+)-YM-12617 competitively antagonized phenylephrine-induced contraction with pA₂ values of 9.95 and 7.69, respectively. Although R(–)- and S(+)-YM-12617 were also competitive antagonists toward UK-14,304 at prejunctional ₂-adrenoceptors in the isolated guinea-pig ileum, the affinities of R(–)-YM-12617 (pA₂ = 6.18) and S(+)-YM-12617 (pA₂ = 5.64) for these receptors were 5,900 and 110 times lower than those displayed for postjunctional ₁-adrenoceptors in the isolated rabbit aorta. 2. R(–)- and S(+)-YM-12617 displaced both ³H-prazosin and ³H-idazoxan binding to rat brain membranes; however, the affinities of the R(–)- and S(+)-enantiomers for ₁-adrenoceptors (pK_i = 9.95 and 7.83, respectively) were 21,000 and 72 times higher than those for ₂-adrenoceptors (pK _i = 5.62 and 5.97), respectively. 3. Based on pA₂ values obtained in the isolated tissues and pK _i values in the binding assays, R(–)-YM-12617 was 132–182 times more potent than S(+)-YM-12617 as an antagonist at ₁-adrenoceptors. In contrast, the R(–)- and S(+)-enantiomers were similar in potency at blocking ₂-adrenoceptors. 4. In normotensive pithed rats, R(–)- and S(+)-YM-12617 preferentially antagonized the ₁-adrenoceptor mediated pressor effect of phenylephrine with DR₁₀ values of 1.38 and 705 g/kg i. v., respectively, although a high dose (3,000 g/kg i.v.) also inhibited the effect of UK-14,304 at postjunctional ₂-adrenoceptors. R(–)-YM-12617 exhibited an over 2,000-fold selectivity for postjunctional ₁-adrenoceptors, and R(–)-YM-12617 was over 500 times more potent than S(+)-YM-12617 in antagonizing postjunctional ₁-adrenoceptors based on DR₁₀ values. 5. In anesthetized rats, R(–)- and S(+)-YM-12617 dose-dependently produced hypotension with ED₂₀ values, doses required decreased mean blood pressure by 20%, of 0.64 and 61 g/kg i. v., respectively. R(–)-YM-12617 exerted a 95 times more potent hypotensive activity than S(+)-YM-12617, and its isomeric activity ratio was consistent with that for ₁-adrenoceptors but not ₂-adrenoceptors. 6. The present results suggest that the high stereoselectivity of the optical enantiomers of YM-12617 is in the ₁-adrenoceptor, but not in the ₂-adrenoceptor, and their antagonist potency for ₁-adrenoceptors is likely to contribute to the hypotensive effect. Send offprint requests to K. Honda     

Development and Use of Enzyme-Linked Immunosorbent Assays (ELISA) for the Detection of Protein Aggregates in Interferon-Alpha (IFN-α) Formulations

Purpose. Protein aggregates are thought to be involved in the immunogenicity of recombinant proteins in humans. To probe human IFN- formulations for the presence of soluble protein aggregates, enzyme-linked immunosorbent assays (ELISA) were developed. Methods. For the detection of IFN--IFN- and HSA-IFN- aggregates, sandwich ELISAs were developed using a monoclonal anti-IFN- antibody as a capture antibody and the same anti-IFN- antibody and an anti-human serum albumin (HSA) antibody (HRP-labeled), respectively. Results. Marketed freeze-dried, HSA-containing IFN- formulations tested in the ELISAs all contained IFN--IFN- and/or HSA-IFN- protein aggregates, although in varying amounts. These aggregates were predominantly IFN- dimers and 1:1 conjugates of HSA with IFN-. Test formulations revealed that aggregation of IFN- was strongly affected by the presence of pharmaceutical excipients, pH of the formulation, lyophilisation procedure, and storage temperature and time. Conclusions. The ELISAs are rapid, highly specific for aggregates in the presence of both IFN- and HSA monomers and allow the direct detection of both types of aggregates in formulations in the nanogram range. The new assays will assist the monitoring of the aggregate-inducing processes during IFN- formulation and storage in an early phase and the development of aggregate-free IFN- formulations.     

In vivo studies on alpha-adrenergic receptor subtypes in human veins

Summary We studied in vivo responsiveness of venous ₁ and ₂-adrenoceptors, measuring the diameter changes in superficial veins in response to -adrenergic agonists and antagonists in healthy human volunteers. The dorsal hand vein technique was used because it permits complete dose-response studies of venous constriction without confounding reflex alterations.Local infusions of all agonists studied induced dose-dependent contraction of the hand vein; the maximal effects (Emax) were: norepinephrine (88% ± 10%), methox amine (97% ± 5%), phenylephrine (95% ± 6%), clonidine (54% ± 12%), and azepexole (68% ± 26%). Clonidine reduced the norepinephrine-induced venoconstriction by 11% ± 10%. Oral doses of 1 mg prazosin antagonized the venoconstriction induced by norepinephrine, methoxamine, and clonidine, but not by azepexole. Yohimbineantagonism was observed against all agonists studied. Inhibition by yohimbine of clonidine-induced venoconstriction was irreversible over 60–180 min.Results show that the in vivo effects on veins of -adrenergic agonists are in good agreement with results from in vitro experiments. Agonists with ₁- and ₂-adrenoceptor subtype selectivity cause venoconstriction in vivo, but ₂-receptor mediated constriction is intrinsically weaker. Clonidine acts as a partial antagonist against norepinephrine, presumably on postsynaptic ₂-receptors. At high doses, ₂-adrenoceptor subtype selectivity of clonidine and yohimbine appear to be partially lost in vivo. Send offprint requests to H. G. Eichler at the above address     

Effects of ring substitution on the pre- and postjunctional alpha-adrenergic activity of aryliminoimidazolidines

Summary The pre- and postjunctional -adrenergic agonist potency of a series of aryliminoimidazolidines was determined in the isolated rabbit ear artery. This series included clonidine, an antihypertensive agent thought to act by stimulating brainstem -receptors and known to be a preferentially prejunctional -adrenergic agonist. Although all of the compounds acted preferentially on the prejunctional -adrenoceptor, ring substitution had a dramatic effect on both potency and the degree of selectivity. 2-(3,4-Dihydroxyphenylimino) imidazolidine was both the most potent and most selective prejunctional -agonist in this series.