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%     

Inhibitors of α-glucosidase and α-amylase from Cyperus rotundus

Context: A methanol extract of Cyperus rotundus L. (Cyperaceae) rhizomes showed inhibitory activity against α-glucosidase and α-amylase, two enzymes involve in carbohydrate digestion.

Objective: Identification of compounds from C. rotundus rhizomes responsible for the inhibition of α-glucosidase and α-amylase.

Materials and methods: Compounds were identified by a phytochemical investigation using combined chromatographic and spectroscopic methods. α-glucosidase and α-amylase inhibitory activities were evaluated by in vitro enzyme inhibition assays.

Results: A new (2RS,3SR)-3,4′,5,6,7,8-hexahydroxyflavane (1), together with three known stilbene dimers cassigarol E (2), scirpusin A (3) and B (4) were isolated. Compound 2 inhibited both α-glucosidase and α-amylase activities while the flavane 1 only showed effect on α-amylase, and compounds 3 and 4 were active on α-glucosidase. All four compounds showed significant 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity.

Discussion: The inhibitory activities against α-amylase and α-glucosidase of the C. rotundus rhizomes were reported for the first time. Stilbene dimers are considered as potent inhibitors of α-glucosidase and promising antihyperglycemic agents.

Conclusion: The isolated compounds may contribute to the antidiabetic property of C. rotundus.     

Binding sites of α1- and α2-adrenoreceptors

Conclusions The models constructed for the binding sites of rat brain ₁-AR and ₂-AR satisfy all the steric requirements and energy characteristics of interaction with known ligands, cited in [5–7, 14]. The differences detected in the arrangement and orientation of the functional groups of the binding sites permit an explanation of a whole series of typical differences in the interaction of adrenoactive substances with both subtypes of-AR.Our analysis showed that the greatest contribution to the interaction with the receptor is made by ionic, donor-acceptor, and hydrophobic bonds. The role of van der Waals forces in the interactions examined is evidently extremely negligible. The most effective and specific preparations prove to be compounds that not only form the maximum number of donor-acceptor bonds with the receptor but also orient their own hydrophobic fragments in such a way that the ionic and donor-acceptor bonds formed between the molecule and the receptor are shielded from contact with the aqueous phase. The energy effects of hydrophobic interactions of this type may be rather substantial (3.9–3.12).The production of new synthetic preparations, for which the conditions of complementarity to the-AR will be most fully satisfied, can be carried out taking the requirements of structural correspondence of the topography of the binding sites into account.Translated from Khimiko-farmatsevticheskii Zhurnal, Vol. 18, No. 8, pp. 904–912, August, 1984.     

Stimulation frequency-noradrenaline release relationships examined in α2A-, α2B- and α2C-adrenoceptor-deficient mice

The stimulation frequency-noradrenaline release relationship was studied in the vas deferens and the cerebral cortex of NMRI mice, mice in which the alpha2A-, the alpha2B-, the alpha2C- or both the alphaCA- and the alpha2C-adrenoceptor gene had been disrupted (alpha2AKO, alpha2BKO, alpha2CKO and alpha2ACKO), and the wildtype mice from which the knockout animals had been generated. Tissue pieces were preincubated with 3H-noradrenaline and then superfused and stimulated electrically with a constant number of pulses (30 in vas deferens and 50 in brain cortex) at frequencies between 0.03 and 100 Hz. The frequency-evoked tritium overflow curves ascended monophasically in the vas deferens of wildtype and NMRI mice. Disruption of the alpha2B-adrenoceptor gene caused no change. In the vas deferens of alpha2CKO mice, the overflow evoked by low frequencies (0.3 and 1 Hz) was slightly increased. In the vas deferens of alpha2AKO and alpha2ACKO mice, the evoked overflow was increased to a greater extent. Rauwolscine (1 microM) caused a marked increase of the evoked overflow of tritium from the vas deferens of NMRI, wildtype, alpha2BKO and alpha2CKO mice. Rauwolscine also increased the evoked overflow of tritium from the vas deferens of alpha2AKO and alphaC2ACKO mice, but to a smaller extent. The gene disruptions and rauwolscine slightly steepened the slope of the vas deferens frequency-overflow curve. In the brain cortex of wildtype and NMRI mice, the frequency-evoked tritium overflow curves were U-shaped. In the brain cortex of alpha2BKO and alpha2CKO mice, the evoked overflow was slightly reduced. In the brain cortex of alpha2AKO and alpha2AcKO mice, in contrast, the evoked overflow was increased. Rauwolscine (1 microM) caused a marked increase of the evoked overflow of tritium from the brain cortex of NMRI, wildtype, Q2BKO and alpha2CKO mice. Rauwolscine also increased the evoked overflow of tritium from the brain cortex of alpha2AKO and alpha2ACKO mice, but to a smaller extent. The gene disruptions and rauwolscine flattened the U shape of the brain cortex frequency-overflow curve. It is concluded that alpha2-autoinhibition is one factor that shapes the frequency-noradrenaline release relationships in the mouse vas deferens and cerebral cortex. The autoreceptors are mainly alpha2A and to a minor extent, and well detectable in the vas deferens only, alpha2C. When both the alpha2A- and the alpha2C-adrenoceptor have been deleted, alpha2B-adrenoceptors may be expressed as autoreceptors in noradrenergic neurons. It seems possible that alpha2C-autoreceptors depress mainly release at low (around 1 Hz) whereas alpha2A-autoreceptors depress mainly release at high (around 10 Hz) frequencies.     

Biodegradation of α-hexachlorocyclohexane

1. Urine and stools were collected daily of 4 adult rats kept in single cages and injected once i.p. with alpha-hexachlorocyclohexane (alpha-HCH; dose per animal 126-150 mumoles), labelled uniformly with 14-C (10.8-14.0 X 10(6) dpm per animal). 2. In 4 weeks, 65% of the label was excreted through the kidneys and 16% by way of the intestine, with an estimated 8% being retained in depot fat. 3. GLC-analysis of the pooled urine showed it to contain very little unchanged drug, on average 0.05% of the dose. The time-course of the renal excretion of 14-C-labelled substance corresponded rather closely to the excretion of organically bound 36-Cl seen in earlier experiments with 36-Cl-labelled drug, indicating that the majority of urinary metabolites, presumably, still bear chlorine. 4. All or nearly all of the faecal 14-C was found by GLC to be accounted for by the stool's content of unchanged alpha-HCH. 5. Taken together, the results indicate a mean extent of alpha-HCH-degradation in the rat in the order of 80-85% of a dose. 6. Two rats were given 400 mg/kg of "cold" alpha-HCH by mouth 4 days before i.p. application of 14-C-labelled drug and were found to excrete more label through the kidneys in the first week than did the non-pretreated rats. This indicates that the drug stimulates its own degradation.     

New α-chloralose derivatives

Chloralose is an easily available carbohydrate derivative bearing biological properties. It constitutes a convenient starting material for various synthetic developments. Herein we describe the preparation of hydroxylamino derivatives of α-chloralose using well-established synthetic procedures.     

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.     

Agonist pharmacology at recombinant α1A- and α1L-adrenoceptors and in lower urinary tract α1-adrenoceptors

BACKGROUND AND PURPOSE

Two distinct α₁-adrenoceptor phenotypes (α_1A and α_1L) have recently been demonstrated to originate from a single α_1A-adrenoceptor gene. Here, we examined the agonist profiles of recombinant α_1A and α_1L phenotypes and of lower urinary tract (LUT) α₁-adrenoceptors.

EXPERIMENTAL APPROACH

A series of drugs (A61603, Ro 115–1240, NS-49, MK017 and ESR1150) originally developed for stress urinary incontinence (SUI) therapy were used to stimulate recombinant α_1A- and α_1L-adrenoceptor phenotypes, and their potencies and intrinsic activity estimated from Ca²⁺ responses. Agonist-induced contractions were also examined in LUT tissues of rats and humans and in human mesenteric artery and rat tail artery.

KEY RESULTS

All the drugs were potent agonists of the α_1A-adrenoceptor compared with the α_1L-adrenoceptor phenotype. Among them, Ro 115–1240 was shown to be an α_1A-specific partial agonist that produced partial contractions through α_1A-adrenoceptors in rat prostate and tail artery, but not in the other LUT tissues and human mesenteric artery. In contrast, P-come 102 showed full agonist activity at α_1A- and α_1L-adrenoceptors, but was less selective than noradrenaline for α_1A-adrenoceptors. Like noradrenaline, P-come 102 was highly potent at inducing contractions in all of the LUT tissues tested. However, the potency and intrinsic activity of P-come 102 were significantly lower than those of noradrenaline in human mesenteric artery.

CONCLUSIONS AND IMPLICATIONS

The α_1A- and α_1L-adrenoceptor phenotypes and LUT α₁-adrenoceptors were demonstrated to have distinct agonist profiles. As adrenergic contractions in LUT are predominantly mediated through α_1L-adrenoceptors, the development of α_1L-selective agonists may provide clinically useful drugs for SUI therapy.     

Synthesis and Tuberculostatic Activity of Arylhydrazides of α-Functionalized α-Alkylcarboxylic acids

Pharmaceutical Chemistry Journal -     

Inhibition of microsomal lipid peroxidation by α-tocopherol and α-tocopherol acetate

1. The antioxidant effects of α-tocopherol and α-tocopherol acetate were assayed for the (a) oxygen uptake, (b) chemiluminescence and (c) malondialdehyde formation, of tert-butyl hydroperoxide-supplemented rat liver microsomes.

2. Oxygen uptake was inhibited 60% by both α-tocopherol and α-tocopherol acetate with the half-maximal effect at 5 nmol tocopherol/mg protein. Chemiluminescence and malondialdehyde formation were equally inhibited 35% by both tocopherols with half-maximal effects at 2 nmol tocopherol/mg protein.

3. The rate of O₂ uptake by tocopherol-supplemented microsomes was dependent on O₂ concentration. A 60% inhibition by 5 nmol tocopherol/mg protein at 0.2 mM O₂ is decreased to 5% inhibition at 0.6 mM O₂.

4. The inhibition of O₂ uptake, chemiluminescence and malondialdehyde formation indicate that both α-tocopherol and α-tocopherol acetate have similar effects as free radical traps in the hydrophobic domain of biomembranes. The different inhibition observed at different O₂ concentrations indicate competition between vitamin E and O₂ by unoxygenated lipid radicals.     

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.     

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.     

Presynaptic α2-autoreceptors in brain cortex: α2D in the rat and α2A in the rabbit

Summary Presynaptic ₂-autoreceptors in rat and rabbit brain cortex were compared by means of antagonists and agonists. Brain cortex slices were preincubated with [³H]-noradrenaline and then superfused and stimulated by 3 (rat) or 4 (rabbit) pulses at a frequency of 100 Hz.The ₂-adrenoceptor agonist bromoxidine (UK 14 304) reduced the electrically evoked overflow of tritium with EC₅₀ values of 4.5 nmol/l in the rat and 0.7 nmol/l in the rabbit. The antagonists phentolamine, 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole (BRL 44408), rauwolscine, 1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo(c,f)imidazo(1,5-a)azepine (BRL 41992), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4, 5-tetrahydro-3-benzazepine (SKF 104078), imiloxan, prazosin and corynanthine did not per se increase the evoked overflow of tritium but shifted the concentration-inhibition curve of bromoxidine to the right in a manner compatible with competitive antagonism. Up to 4 concentrations of each antagonist were used to determine its dissociation constant K_D. The K_D values correlated only weakly between the rat and the rabbit. Dissociation constants K_A of bromoxidine were calculated from equieffective concentrations in unpretreated brain slices and slices in which part of the ₂-adrenoceptors had been irreversibly blocked by phenoxybenzamine. The K_A value was 123 nmol/l in the rat and 7.2 nmol/l in the rabbit.The results confirm the species difference between rat and rabbit brain presynaptic ₂-autoreceptors. Comparison with data from the literature indicates that the rat brain autoreceptors can be equated with the _2D subtype as defined by radioligand binding, whereas the rabbit brain autoreceptors conform to the _2A subtype. For example, the antagonist affinities for the rat autoreceptors correlate with their binding affinities for the gene product of ₂-RG20, the putative rat _2D-adrenoceptor gene (r = 0.97; P<0.01), but not with their binding affinities for the gene product of ₂-C10, the putative human _2A-adrenoceptor gene. Conversely, the rabbit autoreceptors correlate with the ₂-C10 (r = 0.98; P<0.001) but not with the ₂-RG20 gene product. Since presynaptic ₂-autoreceptors are also _2D in rat submaxillary gland and perhaps vas deferens and _2A in rabbit pulmonary artery, the possibility arises that the majority of ₂-autoreceptors generally are _2D in the rat and _2A in the rabbit. Moreover, receptors of the _2A/D group generally may be the main mammalian ₂-autoreceptors.Correspondence to: N. Limberger at the above address     

On the role of α-methyldopamine in the antihypertensive effect of α-methyldopa

Summary In renal hypertensive rats the cerebral concentration of -methyldopa, -methyldopamine, -methylnoradrenaline, dopamine and noradrenaline as well as the blood-pressure were determined simultaneously. The antihypertensive effect followed a time course identical to that of the increase in the cerebral concentration of -methyldopamine and of the decrease in the concentration of dopamine, whereas lowering of blood pressure on the one hand, and changes in the levels of -methylnoradrenaline and noradrenaline, on the other, were not related to each other. Dose-response relationships showed the same correlations and lack of correlations, respectively.These results suggest that non--hydroxylated catecholamines play a major role in mediating the antihypertensive effect of -methyldopa or, alternatively, that only the newly biosynthesized -methyl-noradrenaline is effective in lowering blood pressure.A preliminary communication has been presented at the Spring Meeting 1973 of the German Pharmacological Society at Mainz (Waldmeier et al., 1973).     

Anti-angiogenic effect of α-mangostin

Eleven known prenyl xanthones, isolated from the pericarp of Garcinia mangostana, were tested for their ability to inhibit the phosphorylation of kinase domain receptor (KDR) tyrosine kinase. α-Mangostin was found to inhibit phosphorylation of KDR. α-Mangostin also showed to inhibit phosphorylation of the Y1175 residue of KDR (10 μM). This is the first report that α-mangostin inhibited the phosphorylation of KDR tyrosine kinase and also the Y1175 residue of KDR. α-Mangostin also showed inhibitory effects on proliferation of human umbilical vein endothelial cells (HUVECs) (IC₅₀ 1.2 μM) and human umbilical artery endothelial cells (IC₅₀ 2.4 μM), as well as the migration (IC₅₀ 0.034 μM) and tubule formation (at the concentrations of 0.6 and 1.2 μM) of HUVECs. These results suggest that the inhibition of the phosphorylation of KDR tyrosine kinase is concerned in the anti-angiogenic activity of α-mangostin.     

Effects of α2-receptors

Summary Clonidine has a dual action on naloxone-precipitated morphine withdrawal symptoms in rats: a suppressive action on body shakes and body weight loss and a potentiating action on jumping and aggression.It has been suggested that this potentiating, excitatory action is mediated by ₁-receptors. More specific ₂-agonists therefore should have a less excitatory effect on the latter symptoms. This hypothesis has been studied in rats dependent on morphine. Withdrawal was precipitated using naloxone. Prior to naloxone the ₂-agonists clonidine, guanfacine, azepexole, BHT-920, UK 14304 or the centrally acting ₁-agonist ST 587 were administered. All ₂-agonists but not the ₁-agonist potentiated the jumping and decreased body shakes and body weight loss.The effects of clonidine and azepexole were characterized pharmacologically using the -antagonists yohimbine and prazosin. Jumping potentiated by clonidine was antagonized by yohimbine whereas prazosin had no effect. Azepexole induced jumping was decreased by yohimbine both with respect to incidence and frequency, whereas prazosin only lowered the frequency. The suppressive actions of clonidine and azepexole on body shakes were reversed by yohimbine and not by prazosin. The data indicate that the potentiation of jumping by ₂-agonists as well as the suppression of body shakes in morphine withdrawal behaviour is mediated by ₂-receptors.     

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.     

Aralast: an α1-protease inhibitor for the treatment of α-antitrypsin deficiency

α-Antitrypsin (AAT) is a serine protease inhibitor, which inhibits the proteolytic enzyme elastase. Individuals with a deficiency of AAT may develop clinical manifestations that include a decline in lung function. Deficiency of AAT can lead to many clinical manifestations, most commonly chronic obstructive pulmonary disease in the form of emphysema. However, patients with this genetic disorder may also develop dysfunctions of other organs such as the liver and/or skin. There are ～ 100 alleles associated with the gene encoding for AAT, where the estimated prevalence of this disorder is approximately as common as cystic fibrosis; however, misdiagnosis continues to be a problem. Augmentation therapy using intravenous AAT has been shown to reduce the forced expiratory volume in one second decline, associated with AAT deficiency. Restoration of serum AAT concentrations above 11 μM have correlated with a reduced level of disease progression. The normal dosing regimen of intravenous AAT is 60 mg/kg given every week. Although a dosage consolidation of 250 mg/kg given every 28 days has been explored, long-term efficacy has not been determined. Aralast is one of three approved human plasma-derived treatment options used to prevent the progression of emphysema associated with AAT deficiency disorder.