Responses to catecholamines of the rat isolated uterus throughout the natural oestrous cycle

Summary The effects of noradrenaline (NA), adrenaline (Adr) and isoprenaline (Iso) on rat isolated uterus were studied, throughout the natural oestrous cycle. The -inhibitory effects of the catecholamines were measured as a percentage inhibition of a standard acetylcholine (ACh)-induced contraction. Iso produced approximately 80% maximum inhibition of the standard ACh-contraction in all 4 stages of the oestrous cycle. Adr and NA produced 80% maximum inhibition in oestrus only, and 50–60% maximum inhibition in proestrus, metoestrus and dioestrus. The differences in the degree of inhibition produced by the catecholamines were overcome (i.e. the maximum inhibition produced by Adr and NA was increased to become equal to that produced by Iso) when experiments were repeated in the presence of the uptake inhibitors desmethylimipramine (DMI) and normetanephrine (NMN), but not in the presence of an -adrenoceptor antagonist (azapetine 10^–7 M). Variations in the uptake of³H-Iso and³H-NA were observed in the different stages of the oestrous cycle. Small -adrenoceptor mediated motor responses to NA and Adr were observed only in the presence of a -antagonist (propranolol 10^–5 M), in uteri from rats in oestrus, metoestrus and dioestrus, but not proestrus. It is concluded that in the rat isolated uterus, -inhibitory receptors predominate throughout the natural oestrous cycle, although the existence of -excitatory receptors has been shown in 3 of the 4 stages.     

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     

Depression of exploratory activity by clonidine in rats as a model for the detection of relative pre- and postsynaptic central noradrenergic receptor selectivity of α-adrenolytic drugs

Summary The effects of various -adrenoceptorblocking drugs on the depression of exploratory activity (ambulation and rearing) induced by 0.1 mg/kg i.p. clonidine were investigated in the rat. In parallel experiments, the effects of the same drugs on pre- and postsynaptic -receptors were determined in vitro (field-stimulated cortex slices and isolated vas deferens of the rat, respectively). Tolazoline, esproquine, yohimbine and piperoxan distinctly antagonized the inhibition of exploration produced by clonidine. All these drugs were found to possess relatively higher selectivity for the presynaptic -receptors, as judged by the ratios of the concentrations inducing a 50% increase in field-stimulated ³H-noradrenaline-overflow and the concentrations required to shift the EC₅₀ for the antagonism of noradrenaline-induced contractions of the vas deferens to the right by a factor of 2 (pA₂, ratio <1): In contrast, phentolamine and phenoxybenzamine which showed preferential postsynaptic -receptor blocking activity (ratio>1), potentiated rather than antagonized the effects of clonidine. Mianserin, although preferentially blocking the postsynaptic receptors, had no effect on clonidine-induced hypoactivity up to the high dose of 100 mg/kg i.p., probably because of its additional NA-uptake-inhibiting properties. The antagonism of clonidine by the selective presynaptic -receptor blockers was observed within a limited dose-range. Increasing the doses above an optimal level, which varied from one compound to another, resulted in a decrease in the effect. It is suggested that this phenomenon reflects the counter-balancing postsynaptic -adrenoceptor blockade occuring at higher concentrations of these drugs. In general, the results show a fairly good correlation between antagonism of clonidine in vivo and preferential blockade of presynaptic -receptors in vitro. Clonidine-induced suppression of exploration therefore seems to be a valuable model for the investigation of drug interactions with -adrenergic receptors in the central nervous system.These results were presented in part at the Spring Meeting of German Pharmacological Society, Mainz, 16–18 March, 1977     

The effectiveness of yohimbine in blocking rat central dopamine autoreceptors in vivo

Summary The influence of various -adrenoceptor antagonists (10 mg/kg i.p.) upon the rate of turnover of dopamine (DA) in the rat brain was investigated. Taking the levels of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) as a measure of the rate of DA turnover, it was found that prazosin and phenoxybenzamine decreased, whereas piperoxane and yohimbine increased the turnover rate both in the corpus striatum and in the tuberculum olfactorium. Azapetine, phentolamine and tolazoline as well as the -adrenoceptor antagonist propranolol were without a significant effect, whereas the DA antagonist haloperidol increased DOPAC and HVA levels and decreased the levels of DA itself.The possibility that the yohimbine-induced increase in the DA turnover rate was produced by a direct blockade of DA autoreceptors, was investigated under conditions where influences other than those elicited via DA autoreceptors are thought to be eliminated, i.e. in rats treated with reserpine or -butyrolactone (GBL). In rats that were pretreated with reserpine, yohimbine (10 mg/kg i.p.) was found to be ineffective in antagonizing the reduction of the accumulation of 3,4-dihydroxyphenylalanine (DOPA) following decarboxylase inhibition, that was produced by the DA agonist apomorphine (2.0 mg/kg i.p.). In rats pretreated with reserpine, yohimbine (10 mg/kg i.p.) was also ineffective in antagonizing the reduction of the DOPAC and HVA levels produced by apomorphine (2.0 mg/kg i.p.), but it was effective in antagonizing the reduction of the HVA level that was produced by the selective DA autoreceptor agonist N,N-di-n-propyl-7-hydroxy-2-aminotetralin (DP-7-AT, 1.0 mg/kg i.p.). In rats treated with GBL and a decarboxylase inhibitor, apomorphine (2.0 mg/kg i.p.) and DP-7-AT (1.0 mg/kg i.p.) induced a maximal suppression of the GBL-induced increase in the accumulation of DOPA. The effects of both apomorphine and DP-7-AT were partially inhibited by yohimbine (10 mg/kg i.p.). In inhibiting the effect of DP-7-AT, yohimbine appeared to be 100–200 times less effective than the DA antagonist haloperidol when both antagonists were administered at a fixed pretreatment time (1 h). It is concluded that yohimbine does indeed possess direct central DA autoreceptor blocking properties in vivo, and that this has to be taken into consideration if yohimbine is used as a pharmacological tool in order to achieve a selective blockade of ₂-adrenoceptors.     

Effect of hypophysectomy on pregnenolone-16α-carbonitrile-induced ultrastructural changes in rat liver

Summary Pregnenolone-16-carbonitrile (PCN) given orally to rats induces proliferation of the smooth-surfaced endoplasmic reticulum (SER) in hepatocytes, without producing marked alterations in other cell organelles. It appears that this steroid carbonitrile has a similar effect on the liver of hypophysectomized animals. Hypophysectomy alone causes a decrease of the SER.Abbreviations N Nucleus - RER rough surfaced endoplasmic reticulum - SER smooth surfaced endoplasmic reticulum - M mitochondrion - Mb microbody Fellow of the Medical Research Council of Canada.     

Alpha2-adrenoceptor-mediated responses to so-called selective alpha1-adrenoceptor agonists after partial blockade of alpha1-adrenoceptors

Summary In dog saphenous vein — a tissue possessing both postsynaptic ₁- and ₂-adrenoceptors — the effects of two selective ₁-adrenoceptor agonists (phenylephrine and methoxamine) were compared with that of the selective ₂-adrenoceptor agonist, UK-14,304, before and after phenoxybenzamine. Furthermore, the influence exerted by prazosin, yohimbine and verapamil on the effects of these agonists was also studied before and after phenoxybenzamine. In the absence of phenoxybenzamine, prazosin (56 nmol/l) caused a parallel shift of the concentration-response curves of both phenylephrine and methoxamine to the right (by 0.94 and 1.1 log units, respectively) and had no effect on the concentration-response curve of UK-14,304, while 20 nmol/l yohimbine caused a marked parallel shift of the concentration-response curve of UK-14,304 to the right (by 1.18 log units) and caused only minor displacements of those of phenylephrine and methoxamine (by 0.2 and 0.33 log units, respectively). After exposure of the strips to 30 nmol/l phenoxybenzamine, prazosin (56 nmol/l) caused small shifts of the concentration-response curves of both phenylephrine (by 0.36 log units) and methoxamine (by 0.31 log units) and did not change that of UK-14,304, while yohimbine (20 nmol/l) caused pronounced parallel shifts of the concentration-response curves (to the right) of all the agonists: phenylephrine (by 1.0 log units), methoxamine (by 0.93 log units) and UK-14,304 (by 1.28 tog units). When UK-14,304 was added to the bath during a sub-maximal contraction to phenylephrine it caused a further contraction almost up to the maximum; if this procedure was repeated after phenoxybenzamine (30 nmol/1), there was no further contraction to UK-14,304.In the absence of phenoxybenzamine, verapamil (5 mol/l) caused a parallel shift of the concentration-response curve of phenylephrine (or methoxamine) to the right and a non-parallel shift (with marked depression of the maximal effect) of that of UK-14,304. However, after phenoxybenzamine (30 nmol/l), the same concentration of verapamil caused non-parallel shifts of the concentration-response curves of the three agonists to the right with about equal depression of the maximal effects. We conclude that, after removal of ₁-adrenoceptor reserve by phenoxybenzamine, the responses to selective ₁-adrenoceptor agonists are predominantly ₂-adrenoceptor-mediated. This may explain why under these conditions, the selective ₁-and ₂-adrenoceptor agonists are equally antagonized by calcium entry blockers.This work was supported by a grant from the University of Porto (Subsidio para acção de investigação no. 36/85) Send offprint requests to S. Guimarães at the above address     

Pre- and postsynaptic effects of yohimbine stereoisomers on noradrenergic transmission in the pulmonary artery of the rabbit

Summary Effects on noradrenergic neurotransmission of five stereoisomers of yohimbine and of the closely related compound yohimbol were studied in strips of the pulmonary artery of the rabbit. In some experiments the tissue was preincubated with ³H-noradrenaline. Three effects were observed. Firstly, antagonism to the contractile effect of noradrenaline and of sympathetic nerve stimulation; the antagonism reflected competitive blockade of postsynaptic -adrenoceptors. Secondly, an increase in the stimulation-evoked overflow of total tritium and ³H-noradrenaline; the increase appeared to be due to blockade of presynaptic -adrenoceptors. Thirdly, an increase in the basal outflow of ³H-3,4-dihydrophenylglycol, presumably by impairment of the vesicular storage of ³H-noradrenaline. According to their relative potencies in eliciting these effects, the drugs could be divided into three groups. Rauwolscine, -yohimbine and yohimbol preferentially blocked the presynaptic -adrenoceptor; rauwolscine and -yohimbine, like yohimbine, at low concentrations increased the contractile response to sympathetic nerve stimulation. Corynanthine preferentially blocked the postsynaptic -adrenoceptor. Pseudoyohimbine and 3-epi--yohimbine were very weak antagonists at either receptor; they mainly accelerated the basal outflow of ³H-3,4-dihydroxyphenylglycol.From these results and those of a previous study it is concluded that, in a series of twelve -adrenolytic drugs, rauwolscine shows the greatest preference for presynaptic and corynanthine the greatest preference for postsynaptic -adrenoceptors. In view of the chemical similarity of the two compounds these opposite properties are striking. Corynanthine and rauwolscine might be useful tools for the subclassification of -adrenoceptors.     

Distinct targets for tetanus and botulinum A neurotoxins within the signal transducing pathway in chromaffin cells

Summary Tetanus and botulinum A neurotoxins inhibited exocytosis evolved by various secretagogues in intact and permeabilized chromaffin cells. The block of exocytosis in intact chromaffm cells due to botulinum A neurotoxin could partially be overcome by enhancing nicotine- and veratridine-induced stimulation, whereas the block due to tetanus toxin persisted under the same conditions. The receptor-mediated restoration of ³H-noradrenaline release was specific for nicotinic stimulation, because exocytosis did not occur during muscarinic stimulation. Depolarization of intact chromaffin cells with increasing concentration of K⁺ failed to restore exocytosis that had been blocked by either toxin.When chromaffin cells, treated with tetanus or botulinum A neurotoxins, were exposed to the Ca²⁺-ionophore A 23187 or permeabilized by staphylococcal -toxin, Ca²⁺-stimulated exocytosis was also inhibited. The inhibition was unaffected by increasing concentrations of free Ca²⁺. Activation of proteinkinase C and of G-proteins by phorbolester and GMPPNHP, respectively, increased Ca²⁺-induced exocytosis in control cells as well as in cells treated with tetanus and botulinum A neurotoxins. The block, however, could not be relieved by these manipulations, and it could not be relieved by activating the cGMP or cAMP pathways with analoga of cyclic nucleotides, phosphodiesterases inhibitors, and forskolin either.It is concluded that nicotine and veratridine trigger a mechanism within the sequence of events leading to exocytosis that is located beyond the increase in intracellular Ca²⁺-concentration. This pathway may not be affected by botulinum A neurotoxin. The target of tetanus toxin is probably located even closer to the fusion process, i. e. beyond the step upon which botulinum A neurotoxin acts. Send offprint requests to H. Bigalke at the above address     

Effect of benzo(α)pyrene treatment on the benzo(α)pyrene hydroxylase activity in maternal liver,placenta, and fetus of the rat during day 13 to day 18 of gestation

Summary Pregnant rats were treated with an oral dose of 40.0 mg benzo() pyrene (BP) per kg body weight and were sacrificed 24 h later. Maternal liver, placenta, and fetus were assayed for BP-hydroxylase activity. The enzyme activity was measured from day 13 to day 18 of gestation. Pretreatment of rats with BP results in an approximately 15-fold increase of BP-hydroxylase activity in the maternal liver compared to the enzyme activity in the liver of control animals. These enzyme levels were maintained throughout the duration of gestation.No BP-hydroxylase activity could be measured in the fetus and the placenta of control animals. After treatment with BP maximal induction of BP-hydroxylase activity in the placenta was not achieved before day 15 of gestation. BP treatment resulted in a detectable enzyme activity in the fetus. However, the degree of induction increased with the age of the fetus. Electron microscopic pictures demonstrate a swelling and enlargement of the endoplasmic reticulum (ER) in the trophoblast cell of the placenta after BP treatment. In the fetal liver the ER is characterized by a moderate enlargement while there is no effect in the maternal liver after treatment of the animals with BP.This work was supported by a grant from the Deutsche Forschungsgemeinschaft given to the Sonderforschungsbereich 29, Embryonale Entwicklung und Differenzierung (Embryonal-Pharmakologie).Parts of this work were presented at the Spring Meeting of the German Pharmacological Society held in Mainz, March 1971.     

Effects of alpha-adrenoceptor agonists and antagonists in a maze-exploration model of ‘fear’-motivated behaviour

Summary An elevated X-maze with alternating open and enclosed arms was investigated as a model for the study of fear-induced behaviour. As predicted, the anxiolytics diazepam and amylobarbitone increased, and the putative anxiogenics ACTH and picrotoxin decreased the proportion of open arm entries. The ₁-adrenoceptor agonists phenylephrine and ST587, and the ₂-adrenoceptor antagonists idazoxan, piperoxane, RS-21361 and yohimbine decreased relative open-arm entries, thus resembling the putative anxiogenics. On the other hand, azepexole, clonidine and guanabenz, agonists at ₂-adrenoceptors, and the ₁-adrenoceptor antagonists prazosin and thymoxamine, enhanced the proportion of open arm entries at low doses, suggesting anxiolytic-like properties. A paradoxical fall in open arm entries occurred with these agents at higher doses. These results provide further evidence for the involvement of noradrenergic systems in fear-motivated behaviour.     

2,2′,3′,4,4′,5,5′-Heptachlorobiphenyl (PCB 180) — on its toxicokinetics,biotransformation and porphyrinogenic action in female rats

The toxicokinetics and biotransformation of 2,2,3,4,4,5,5-heptachlorobiphenyl, as well as its influence on the activity of microsomal and cytosolic enzymes and on the porphyrin pathway in the liver were studied in female rats following oral treatment with 7 mg/kg every other day for 3 months. One day after cessation of treatment the concentration of the compound in liver, spleen, CNS and blood was 100–500 times and in the trachea it was only 5 times less than in the adipose tissue. The daily excretion with the feces and urine amounted to 35 and 1.5 g, respectively. In both excreta, heptachlorobiphenylol was identified as a metabolite. The biotransformation rate was estimated to be about 5%. Investigations of the liver revealed increases in the relative liver weight, total cytochrome P-450 content, O-deethylation of 7-ethoxycoumarin and in the activity of glutathione S-transferases. Disturbances of the hepatic porphyrin pathway were not detected. Only at the end of a post-dosing period of 12 months did the hepatic uroporphyrinogen decarboxylase show diminished activity. Only one of these animals with diminished enzyme activity showed drastically elevated porphyrins. In these animals, the fecal and urinary porphyrins did not differ from controls. At no time did heptachlorobiphenyl influence the urinary excretion of delta-aminolevulinic acid and porphobilinogen. The results indicate 1) that this congener shows expected toxicokinetics with the exception of being accumulated in the trachea and 2) that this congener induces disturbances of the hepatic porphyrin pathway several months after cessation of treatment.     

Effect of phenothiazine neuroleptic drugs and tricyclic antidepressants on phosphodiesterase activity in rat cerebral cortex

The activity of phosphodiesterase (PDE) of rat cerebral cortex following the administration in vitro and in vivo of various concentrations of neuroleptic phenothiazine drugs and tricyclic antidepressive drugs has been investigated. It has been shown that PDE activity is inhibited by phenothiazine neuroleptic drugs (fluphenazine > trifluperazine > thioproperazine > chlorpromazine = thioridazine). Tricyclic antidepressants nortriptyline, chlorimipramine, protiptyline, imipramine and desipramine at a concentration of 10^–3 M caused 60–80% inhibition of PDE activity. It has also been found that the investigated phenothiazine compounds inhibit the high affinity PDE activity more than the PDE activity of low affinity to the substrate.The results obtained suggest that the mechanism of the neuroleptic action of phenothiazine drugs is partially connected with their influence on cyclic 3,5-AMP metabolism.Supported by Polish Academy of Sciences, 09.4.1.5.     

Pharmacological characterization of the postjunctional alpha-adrenoceptors of the rat isolated seminal vesicle

Summary In an attempt to define the pharmacological characteristics of the postjunctional -adrenoceptors of the rat seminal vesicle, responses to certain phenylethanolamine and imidazoline agonists were investigated, in vitro, under experimental conditions outlined by Furchgott (1972), using ₁-selective, non-selective and ₂-selective adrenoceptor antagonists. Adrenaline (ADR), noradrenaline (NA) and phenylephrine (PE) produced concentration-dependent contractions. In many experiments the concentration-response (C-R) curves had a distinct shoulder at the level of 60–80% of the maximum response (E_max), a situation reminiscent of the rat anococcygeus muscle and the rat basilar artery. The relative potencies of ADR:NA:PE, derived from their EC₅₀ values, were 4.07:1:0.26. In contrast clonidine, oxymetazoline and naphazoline failed to contract the tissue even in concentrations up to 1 × 10^–3 M. In fact the imidazoline derivatives prevented responses to the phenylethanolamines. The antagonist action of clonidine, against phenylephrine, was studied in detail.Prazosin, phentolamine, yohimbine, corynanthine and clonidine all caused a rightward displacement of the C-R curves for NA without depressing E_max. The Arunlakshana and Schild plots of the data were linear and had slopes not significantly different from unity. The pA₂ estimates obtained were 9.17 (9.13–9.21) for prazosin, 8.58 (8.07–9.09) for phentolamine, 6.70 (6.44–6.98) for yohimbine and 7.05 (6.81–7.30) for corynanthine. Clonidine had a pA₂ value of 6.60 (6.55–6.67) against phenylephrine.On the basis of results obtained with antagonists, the postjunctional -adrenoceptors of the rat seminal vesicle could be firmly placed in the gross category of ₁. The concept of heterogeneity of ₁-adrenoceptors is discussed in the light of the profiles of the phenylethanolamine and imidazoline agonists as well as the antagonist potencies of prazosin and yohimbine. Send offprint requests to S. I. Sharif at the above address     

Effect of chronic lithium administration on adrenoceptor binding and adrenoceptor regulation in rat cerebral cortex

Summary Lithium (Li) at a concentration, which exerts prophylactic effects in affective disorders is known to alter noradrenaline turnover and the -adrenoceptor-dependent cAMP accumulation. In the present study the action of chronic Li administration (at least 5 weeks) on agonist and antagonist binding to adrenoceptors and on the regulation of adrenoceptors was investigated in rat cerebral cortex. Li treatment caused a small but significant decrease in the number of -adrenoceptor binding sites by 10% (³H-dihydroalprenolol binding) leaving the number of ₁- and ₂-adrenoceptor binding sites (³H-prazosin and ³H-rauwolscine, respectively) unchanged. The affinity of the radioligands as well as the affinity of agonists to these binding sites were not altered. The up-regulation of -adrenoceptor binding sites produced by repeated reserpine injections was inhibited by 32% in rats treated concomitantly with Li, although the noradrenaline depleting effect of reserpine was not impaired. In contrast, Li treatment had no effect on the up-regulation of -adrenoceptor binding induced by 6-OH-dopamine, nor did it alter the -adrenoceptor down-regulation following chronic administration of desipramine. The up-regulation of ₁-adrenoceptor binding sites caused by reserpine or 6-OH-dopamine also remained unaffected by Li. It is concluded that chronic Li has limited effects on cortical adrenoceptors and their regulation. The inhibition of -adrenoceptor up-regulation caused by reserpine may reflect an action of Li on non-adrenergic systems rather than a general stabilizing effect on adrenoceptors proposed previously.Send offprint requests to: G. Gross     

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.     

A coupled assay for histidine decarboxylase: in vivo turnover of this enzyme in mouse brain

Summary A sensitive coupled assay for histidine decarboxylase has been developed. This method involved conversion of [³H]histidine into [³H]histamine by the enzyme sample, with methylation of this product in situ, catalysed by the enzyme histamine N-methyltransferase, to yield [³H]N-tele-methylhistamine. The radioactive product was separated from the substrate by (i) extraction into chloroform, (ii) ion-exchange chromatography and (iii) liquid cationexchange extraction. The no tissue assay blank comprised 0.0007% of the substrate radioactivity. Sample material with a histidine decarboxylase activity of as little as 0.14 fmol/ min/ml (measured at 1 M histidine) gave double the blank value. More than 50 assays could be performed in one day.This assay was used to determine the in vivo changes in mouse brain histidine decarboxylase activity following irreversible inhibition with (+)-fluoromethylhistidine (-FMH). From the time course of recovery of enzyme activity the half-life of histidine decarboxylase in vivo was calculated to be 53 h.Nomenclature of Black and Ganellin (1974)     

Effects of suramin and α,β-methylene ATP indicate noradrenaline-ATP co-transmission in the response of the mouse vas deferens to single and low frequency pulses

Summary Vasa deferentia from mice were field-stimulated by trains of 10 pulses delivered at 0.5 Hz. The pulses elicited separate twitches, the first of which (corresponding to a single pulse) exceeded the following ones in height and width and often was clearly biphasic. , -Methylene-ATP 1 mol/1 and suramin 100 mol/1 caused almost identical changes. They reduced the height of the first twitch in the train by about one half and also reduced its width in a manner indicating that only the second, slow phase remained, but reduced much more markedly the following twitches in which now a small second, slow phase also became detectable. Idazoxan 0.1 mol/1 or yohimbine 0.1 mol/l, when added in the presence of a, -methylene-ATP or suramin, further decreased the first twitch but enhanced twitches No. 2 to 10. These responses were then almost abolished by prazosin 0.1 mol/l. Successive addition of prazosin 0.1 mol/l and idazoxan 0.1 mol/1 to previously untreated vasa deferentia depressed the response to the first pulse by about one half in a manner indicating that only the first, rapid phase remained, but had comparatively little effect on the responses to the subsequent pulses. Suramin 100 mol/l almost abolished the contractions remaining in the presence of prazosin and idazoxan. The results indicate that the first, rapid phase of the neurogenic contractions elicited by single or low frequency pulses is mediated by ATP which substantially contributes to all responses in a train. The second, slow phase is mediated by noradrenaline which substantially contributes to the response to the first pulse only. The adrenergic contribution seems to be mediated by postjunctional ₁- as well as a ₂-adrenoceptors. Prejunctional ₂-adrenergic autoinhibition depresses the release of both ATP and noradrenaline. Send offprint request to I. v. Kügelgen at the above address     

Bis(carbamoyloxymethyl) Esters of 2′,3′-Dideoxyuridine 5′-monophosphate (ddUMP) as Potential ddUMP Prodrugs

No HeadingPurpose. We previously reported the synthesis of bis(pivaloyloxymethyl) 2,3-dideoxyuridine 5-monophosphate (POM₂-ddUMP) (1a) as a membrane-transport prodrug formulation of the free parent nucleotide, ddUMP. Although successful at delivering ddUMP into cells in culture, POM₂-ddUMP was rapidly degraded by plasma carboxylate esterases after intravenous administration to experimental animals, and therefore has limited therapeutic potential as a systemically administered prodrug. We now report the synthesis of bis(N,N-dimethylcarbamoyloxymethyl)- and bis(N-piperidinocarbamoyloxymethyl) 2,3-dideoxyuridine 5-m onophosphate [DM₂-ddUMP (1b) and DP₂-ddUMP (1c), respectively], analogues of POM₂-ddUMP that were designed to be more resistant to degradation by plasma esterases..Methods. After entering cell by passive diffusion, it was anticipated that loss of one of the carbamoyloxymethyl groups of 1b and 1c would occur by spontaneous chemical hydrolysis to give the intermediate phosphodiesters, 2b and 2c. Cleavage of the remaining carbamoyloxymethyl groups by cellular phosphodiesterase I would generate ddUMP. 1b and 1c were prepared by condensation of 2,3-dideoxyuridine (ddU) with the appropriate bis(N-alkylcarbamoyloxymethyl) phosphate in DMA in the presence of triphenylphosphine and diethyl azodicarboxylate (the Mitsunobo reagent).Results. The half-lives of 1b and 1c when incubated at a concentration of 10^–4 M in human plasma at 37°C were 3.5 h and 3.7 h, respectively, similar to the half-lives observed under the same temperature conditions in 0.05 M aqueous phosphate buffer, pH 7.4. By contrast, the half-life of the POM₂ prodrug, 1a, in plasma was only 5 min. The initial products of degradation of 1b and 1c were the phosphodiesters 2b and 2c. The latter compounds gave rise to ddUMP when incubated with snake venom phosphodiesterase I.Conclusions. These findings support the premise inherent in the design of 1b and 1c, namely that the carbamate prodrugs are far more resistant to hydrolysis by plasma carboxylate esterases than their POM counterparts and can revert to the free parent 5-mononucletides by successive chemical and enzymatic hydrolysis. Further studies of 1b and 1c as membrane-permeable prodrugs of ddUMP are in progress.     

Characterisation of postjunctional α-adrenoceptors in isolated human femoral veins and arteries

Summary In order to characterise the pharmacological properties of postjunctional -adrenoceptors, both the contractile effects of -adrenoceptor agonists and the blocking potencies of selective -adrenoceptor antagonists were studied in isolated human femoral veins and arteries.The veins were more sensitive to noradrenaline than the arteries. Guanfacine had a higher intrinsic activity in veins than in arteries, whereas the reverse was true for phenylephrine.The antagonists rauwolscine and yohimbine were more potent against noradrenaline in the veins than in arteries, while corynanthine was equally potent in either tissue. They antagonised the noradrenaline response in a competitive manner. Prazosin proved to be the most potent competitive antagonist in arteries, while in veins it exerted weak and noncompetitive antagonism.The results suggest that the -adrenoceptor population at the postjunctional site differs between human femoral veins and arteries. The veins seem to contain more ₂- than ¹-adrenoceptors postjunctionally, whereas in the arteries the ₂-subtype prevails. The results indicate the possibility of influencing selectively adrenergic reactions in the capacitance and resistance vessels.