Clearance Mechanisms of Atrial and Brain Natriuretic Peptides in Rats

To assess clearance mechanisms of atrial and brain natriuretic peptides in the circulation, we examined the effects of a neutral endopeptidase (NEP) inhibitor and a clearance receptor ligand on plasma concentrations of the peptides in normal rats. Plasma concentrations of endogenous -rat atrial natriuretic peptide (-rANP) were not significantly elevated by intravenous infusion of a NEP inhibitor, phosphoramidon, but were elevated threefold by intravenous infusion of a clearance receptor ligand, des(Gln¹⁸-Gly²²)-rANP_(4–23)-NH₂ [C-ANF_(4–23)]. On the other hand, the clearance of -rANP given intravenously at the pharmacological dose, 600 pmol/ min/kg for 2 min, was decreased to one-third by the administration of phosphoramidon, although the administration of C-ANF_(4–23) did not significantly decrease the clearance. The clearance of rat brain natriuretic peptide (rBNP) given at 600 pmol/min/kg for 2 min was approximately 38% lower than that of -rANP. The effect of phosphoramidon on the clearance of rBNP was not significant and was similar to that of C-ANF_(4–23). These results suggest that clearance receptor is involved in the clearance of the physiological levels of -rANP and that NEP plays a major role in the clearance of a pharmacological dose of -rANP, at which clearance receptors are thought to be saturated, and also indicate a pharmacokinetic difference between -rANP and rBNP.     

Natriuretic Peptide-Potentiating Actions of Neutral Endopeptidase Inhibition in Rats with Experimental Heart Failure

We developed a rat model of heart failure induced by myocardial infarction (MI) which preserves responsiveness to exogenously administered natriuretic peptide, and investigated the potentiating action of neutral endopeptidase (NEP) inhibition on the renal response to endogenous natriuretic peptide in MI rats, comparing with that in the established cardiac-failing model with arterio-venous fistula (AVF). The endogenous plasma concentration of -rat atrial natriuretic peptide (-rANP) in the MI rat was 6.4-fold higher than that in the normal rat, and intravenous infusion of phosphoramidon (165 nmol/min/kg), an NEP inhibitor, induced larger increases in circulating -rANP levels and natriuresis in MI rats than in normal controls. The maximal natriuretic effect of phosphoramidon (165 nmol/ min/kg) was equal to that of exogenously administered -rANP (100 pmol/min/kg) in MI rats, whereas plasma -rANP concentration under NEP inhibition was much lower than that after administration of -rANP. The endogenous -rANP levels in AVF rats were as high as those in MI rats. However, the natriuretic effect of phosphoramidon was less in AVF rats than in MI rats, which was consistent with the decreased natriuretic activity observed with administration of exogenous to -rANP in the AVF rat. These results indicate that the natriuretic effect of NEP inhibition is dependent on elevated endogenous -rANP levels in cardiac-failing rats, but cannot be accounted for simply in terms of the increase in circulating -rANP levels. Endogenous natriuretic peptide-mediated natriuresis under NEP inhibition also appears to correlate with the responsiveness to the exogenously administered peptide.     

DOPA decarboxylase inhibition does not influence the diuretic and natriuretic response to exogenous α-atrial natriuretic peptide in man

Summary The role of dopamine synthesis in the renal actions of human -atrial natriuretic peptide (ANP) was investigated in six dehydrated volunteers using the DOPA decarboxylase inhibitor carbidopa.Each subject received oral placebo or carbidopa (100 mg) followed by an infusion of ANP 10 pmol · kg^–1 · min^–1 for 1 h. The responses to placebo alone and to carbidopa alone were investigated on separate occasions. ANP produced a similar increase in plasma immunoreactive ANP whether placebo or carbidopa pretreatment had been given. Urinary dopamine excretion was increased by ANP. Carbidopa pretreatment substantially attenuated this increase without affecting the natriuretic or water-diuretic response to ANP. Carbidopa also failed to alter the change in filtration fraction produced by ANP.The results suggest that increased synthesis of intrarenal dopamine is not required for the renal effects of ANP in man.     

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.     

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.     

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     

An Eastern Cooperative Oncology Group phase I trial of all-trans-retinoic acid and interferon-alpha: E2Y92

The Eastern Cooperative Oncology Group conducted a Phase I trial to determine the maximally tolerated doses of combination therapy with alpha interferon (IFN-) and all-trans-retinoic acid (tRA). Fifty patients with incurable malignancies received IFN- administered subcutaneously three times weekly, and tRA administered by mouth at bedtime. Doses were escalated between patient groups, starting at tRA dose level of 45 mg/m² and 3 million units of IFN-.Major, dose-limiting toxicities were attributable to either the tRA (rash, chelitis) or IFN (constitutional symptoms), and were observed only at tRA dose levels of 224 mg/m² and 291 mg/m², or 6 million units of IFN-. The maximally tolerated dose level of 172.5 mg/m² of tRA and 3 million units of IFN- was well-tolerated, with no grade 3 or 4 toxicities attributable to therapy. One patient at the third dose level (75 mg/m² of tRA and 3 million units of IFN-) developed acute hepatic and renal failure and a metabolic encephalopathy of unclear etiology.We conclude that tRA and IFN- may be safely administered together at the maximally tolerated dose of tRA as a single agent without unexpected side effects. The recommended doses of IFN- and tRA for Phase II trials are 3 million units of IFN- and 172.5 mg/m² of tRA.     

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.     

Effects of α-methyl-Dopa on conditioned behaviour in the cat

Summary After -methyl-DOPA (-MD) administration to cats a significant decrease of a conditioned avoidance response (CAR) was observed 8 hours later. In reserpine-pretreated cats -MD caused a pronounced reduction of CAR which was not reversed by (+)-amphetamine. A subsequent administration of l-DOPA resulted in a dose-dependent CAR reversal. Different possibilities of the central action exerted by -MD are discussed.     

The effect of α-amanitin on passive and active avoidance acquisition in mice

-Amanitin, a specific and potent inhibitor of form II DNA-dependent RNA polymerase, produced greater than 98% inhibition of the enzyme in mouse brain within 2 h of intracerebroventricular (icv.) injection. Mice were given one trial passive avoidance training and retested on the task 4 h later. Mice treated with -amanitin 2 h before training or immediately after training demonstrated a retention deficit when compared to non-injected or saline injected controls.Active avoidance was trained for 1 h using a Sidman schedule with a drumturning response. Performance during the last 15 min of training was compared to performance in the first 15 min of a retesting session, 4 h after training. -Amanitin, 2 h prior to training reduced the number of responses, per cent escapes and per cent avoidances in the retesting session. Post-training injection of -amanitin significantly reduced the number of responses and per cent avoidances.Rotarod and spontaneous motor activity were not affected by -amanitin. Whole body temperature was slightly and transiently reduced in icv. administration of -amanitin.     

Brain catecholamines modifications. The effects on memory facilitation induced by oxotremorine in mice

The immediate posttrial injection of oxotremorine (0.250 Mol/kg, IP) can facilitate the retention of a passive-avoidance response in mice. After the administration of alfa-methyl-p-tyrosine methylester (-MPT) by intracerebroventricular injection at doses that had no effect on retention (100 g, 10 l, 60 min before trial), the immediate posttrial injection of oxotremorine did not enhance retention.The employed dose of -MPT reduced brain levels of norepinephrine by about 40% and those of dopamine by about 25%.Pretreatment with nialamide (30 mg/kg, 20 h IP), which prevents the catecholamine depletion induced by -MPT, counteracted the effects of -MPT on the actions of oxotremorine on retention.These results suggest a participation of brain catecholamines on the actions of oxotremorine on retention and a possible interaction of cholinergic neurons with catecholaminergic system in memory processes.     

Plasma concentration of α-methyldopa and sulphate conjugate after oral administration of methyldopa and intravenous administration of methyldopa and methyldopa hydrochloride ethyl ester

Summary The plasma concentrations of free -methyldopa and methyldopa sulphate conjugate were measured in 7 hypertensive patients with normal renal function following -methyldopa (1 g) orally. Five of these patients subsequently received -methyldopa ethyl ester (250 mg) (methyldopate) intravenously and two further patients received 250 mg of -methyldopa intravenously. After oral administration a large amount of total plasma -methyldopa was present as sulphate conjugate. There were wide interindividual differences in the ratio of free: conjugated -methyldopa in plasma (ratio at 4 hours ranged from 3.73 – 0.83) suggesting that individual differences in the extent of sulphate conjugation may occur. There was no close correlation between the degree of conjugation and the fall in arterial pressure. At all time intervals examined, plasma concentrations were higher following intravenous -methyldopa than -methyldopate. The plasma concentration of -methyldopa (free and esterified) 60 minutes after i.v. -methyldopate was 1.7±0.3 µg/ml wile at the same time after the same dose of methyldopa by the same route the mean concentration was 5.9 µg/ml. Although small amounts of sulphate conjugate were detected after i.v. -methyldopate, insignificant quantities of conjugate were found after i.v. -methyldopa. The average fall in mean arterial pressure was 27 mm Hg following i.v. -methyldopa but only 2.7 mm Hg following -methyldopate. These results suggest that sulphate conjugation of -methyldopa occurs in the gastrointestinal tract during absorption. Hydrolysis of -methyldopa ethyl ester does not appear to be instantaneous and pharmacokinetic differences between the ester and free -methyldopa have been demonstrated.     

Inhibition by opiate narcotics of rat flexor α-motoneurones

Summary Specific effects of opiate narcotics on rat flexor -motoneurones were studied in ventral roots of laminectomized rats under halothane anaesthesia. The -motoneurones were activated by tetanic stimulation of the cut ipsilateral common peroneal nerve, exciting up to group II- but not group III- and C-afferents. Morphine (0.5–3.0 mg/kg i.v.) reduced or completely suppressed the discharge rate of flexor -motoneurones in a dose-dependent manner. This effect was antagonized by naloxone (0.5 mg/kg i.v.) and mimicked by levorphanol (1.0 mg/kg i.v.), but not by an equal dose of its steroisomer dextrorphan, suggesting that the effect described is a specific one. After spinalization, the inhibitory effect of morphine was abolished. Previous studies had shown that opiates (e.g. morphine, given in a dose of 2 or 4 mg/kg i.v.) excite rat extensor -motoneurones, an effect opposite to the opiate narcotic action on flexor -motoneurones. The action of opiates leading to an inhibition of flexor -motoneurones may contribute to akinesia and catalepsy, and opioid-induced muscular rigidity. From the results presented it appears that morphine produces a reciprocal change in the activity evoked in extensor and flexor reflex pathways.Some of these results were presented at the 18th Spring Meeting of the German Pharmacological Society, Mainz, March 16–18, 1977These studies were supported by a grant (B-3) from the Sonder-forschungsbereich 33 Nervensystem und biologische Information of the Deutsche Forschungsgemeinschaft     

One-Dimensional and Two-Dimensional 1H- and 13C-Nuclear Magnetic Resonance (NMR) Analysis of Vitamin E Raw Materials or Analytical Reference Standards

Two-dimensional spectral analysis (COSY, HETCOR) was utilized to make the complete ¹³C- and ¹H-NMR assignments for -, -, --, and -tocopherol as well as for the acetate and succinate esters of -tocopherol. ¹³C-NMR was found to be especially useful in distinguishing between the various tocopherols and distinguishing between the d-isomer and the d,l-racemic mixture. HETCOR spectra were also found to be useful for the qualitative identification of mixtures of the tocopherols and sesame oil. Using a procedure designed to minimize errors arising from spin relaxation and nuclear Overhauser effects, ¹³C-NMR peak integrals were used to quantitate -tocopherol and -tocopherol in the presence of sesame oil using benzoic acid as the standard for calibration of the quantitation. The NMR results were compared to a capillary column gas chromatographic analysis of the individual -tocopherol and -tocopherol reference materials.Chris W. Myers: Recipient of the Analysis and Pharmaceutical Quality Section Undergraduate Award in Pharmaceutical Analysis from the APQ Section of the American Association of Pharmaceutical Scientists.     

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.     

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     

Bildung und Speicherung von α-Methylnoradrenalin

Zusammenfassung An Meerschweinchen wurde die Wirkung von Reserpin auf die Bildung von -Methylnoradrenalin aus -Methyldopa untersucht. Im Herzen und Vas deferens verursacht Reserpin eine starke Verminderung der -Methylnoradrenalin-Bildung, im Gehirn hat es nur einen geringen Einfluß.Bei der Perfusion isolierter Meerschweinchenherzen mit Dopamin bzw. -Methyldopamin wird wesentlich mehr -Methyldopamin in das Herz aufgenommen als Dopamin. Reserpin-Vorbehandlung vermag weder die Dopamin- noch die -Methyldopamin-Aufnahme zu hemmen; es vermindert jedoch die Bildung von Noradrenalin bzw. -Methylnoradrenalin aus ihren Vorstufen, wobei die Bildung von Noradrenalin am stärksten reduziert wird.Bei der Inkubation isolierter Meerschweinchenherz-Granula mit -Methyldopamin oder -Methylnoradrenalin vermindert Reserpin die Aufnahme der beiden Amine in die Granula.Nach Perfusion von Kaninchenherzen mit Dopamin bzw. -Methyldopamin wurde die subcelluläre Verteilung dieser beiden Amine untersucht. Während -Methyldopamin hauptsächlich partikulär gebunden vorliegt, befindet sich Dopamin vorwiegend im Cytoplasma.

---

Summary Experiments have been carried out in order to study the influence of reserpine pretreatment on the storage of -methylnoradrenaline and its synthesis from -methyldopa (3×400 mg/kg).Reserpine diminishes in heart and vas deferens of guinea-pigs the synthesis of -methylnoradrenaline from -methyldopa while its effect in brain is not significant.During infusion of isolated guinea-pig hearts with -methyldopamine or dopamine (0.6 moles/min, 30 min) much more -methyldopamine is taken up into the hearts than dopamine. Pretreatment with reserpine (4×25 g/kg) has no effect on the uptake of these amines; it causes, however, a decrease of synthesis of noradrenaline and -methylnoradrenaline from their precursors. The inhibitory effect on the synthesis of noradrenaline is much more pronounced than that on the synthesis of -methylnoradrenaline.In incubation experiments with storage granules isolated from guinea-pig hearts the uptake of -methyldopamine and -methylnoradrenaline (0.25 moles/ml) is significantly reduced by reserpine (40 g/ml).After infusion of rabbit hearts with -methyldopamine or dopamine (1.5 moles/min, 60 min) the subcellular distribution of these amines was studied. The main part of -methyldopamine is located in the particulate fractions whereas dopamine is predominantly found in the cytoplasma.

---

---

Herrn Prof. Dr. Peter Holtz zum 65. Geburtstag am 6.2.1967 gewidmet.

---

Ausgeführt mit der Unterstützung der Deutschen Forschungsgemeinschaft.     

Effect of haloperidol on reflex activation of rat α-motoneurones

Summary Effects of haloperidol on rat flexor and extensor -motoneurones were studied in ventral roots of laminectomized rats under halothane anesthesia. The -motoneurones were activated by tetanic stimulation of low-threshold afferents (group I and II), either of the ipsilateral peroneal nerve (flexor -motoneurones) or gastrocnemius-soleus nerve (extensor -motoneurones).Haloperidol, given in the doses of 0.075, 0.15 and 0.30 mg/kg i.p. inhibited the reflex activation of flexor -motoneurones; higher doses seemed to be more effective than lower ones. Apomorphine (2 mg/kg s.c.) partially antagonized the inhibitory action of haloperidol with some latency. Higher doses of haloperidol (0.15–0.60 mg/kg i.p.) also inhibited the reflex activation of extensor -motoneurones; this inhibitory effect was, at least for a short time, antagonized by apomorphine (2 mg/kg s.c.).The threshold for reflex activation both of flexor and extensor -motoneurones was raised by haloperidol and lowered by a subsequent administration of apomorphine.Our results suggest that akinesia and catalepsy, induced in rats by haloperidol might be, at least in part, due to a decrease in sensitivity of -motoneurones to proprioceptive stimuli.     

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).     

Contraction-mediating α2-adrenoceptors in the mouse vas deferens

Summary The question of the existence of postjunctional, contraction-mediating ₂-adrenoceptors, in addition to the known ₁-adrenoceptors, was studied in the mouse isolated vas deferens. Both the ₁-selective agonist phenylephrine and the ₂-selective agonist 5-bromo-6-(2imidazolin-2-ylamino)-quinoxaline (UK 14,304) caused contraction of the vas deferens. In the presence of the ₁-selective antagonist prazosin (added in order to prevent an ₁ component in the effect of high concentrations of UK 14,304), the ₂-selective antagonists yohimbine and idazoxan shifted the concentration—response curve of UK 14,304 to the right in a manner compatible with competitive antagonism and with dissociation constants K_B indicating the involvement of ₂-adrenoceptors. The maximal contraction elicited by UK 14,304 (in the presence of prazosin) was much lower than the maximal contraction elicited by phenylephrine. The effect of UK 14,304 was not changed by the P₂-purinoceptor agonist ,-methylene-ATP and was reduced by neuropeptideY, but was markedly enhanced by relatively low concentrations of phenylephrine. When the sympathetic fibres of the vas deferens were stimulated by trains of ten widely spaced (0.5 Hz) electric pulses, the tissue responded with ten separate twitches in which purinergic and adrenergic components were isolated by prazosin and suramin, respectively. Prazosin reduced the first adrenergic twitch in these trains at concentrations close to its K_B value at ₁-adrenoceptors, whereas yohimbine and idazoxan reduced the first adrenergic twitch at concentrations far lower than their K_B values at ₁-adrenoceptors. The results indicate that the smooth muscle of the mouse vas deferens possesses contraction-mediating ₂-adrenoceptors. They are activated by UK 14,304 and probably also by noradrenaline of neural origin. Responses mediated by the ₂-adrenoceptors are enhanced by simultaneous ₁-receptor activation, an interaction that may increase the contribution of the ₂-adrenoceptors to the adrenergic phase of neurogenic contractions. Send offprint requests to R. Bültmann at the above address