The involvement of central cholinergic system in the pressor effect of intracerebroventricularly injected U-46619, a thromboxane A2 analog, in conscious normotensive rats

The aim of this study was to determine the involvement of the central cholinergic system in the rise in blood pressure evoked by the thromboxane A2 (TxA2) analog, U-46619, given centrally. Intracerebroventricular (i.c.v.) injections of U-46619 (0.5, 1.0 and 2.0 g) caused dose- and time-related increases in blood pressure and decreased heart rate in awake rats. U-46619 (1 g; i.c.v.) also produced an approximately 65% increase in posterior hypothalamic extracellular acetylcholine and choline levels. Pretreatment with SQ-29548 (8 g; i.c.v.), selective TxA2 receptor antagonist, completely inhibited both the cardiovascular responses and the increase in acetylcholine and choline levels to subsequent injection of U-46619 (1 g; i.c.v.). Atropine (10 g; i.c.v.), nonselective muscarinic receptor antagonist, pretreatment did not affect the cardiovascular responses observed after U-46619 (1 g; i.c.v.). Pretreatment with the nonselective nicotinic receptor antagonist, mecamylamine (50 g; i.c.v.) attenuated the pressor effect of U-46619 (1 g; i.c.v.). Higher doses of mecamylamine (75 and 100 g; i.c.v.) pretreatments did not change the magnitude of the blockade of pressor response to U-46619; however, they abolished the bradycardic effect of U-46619 dose-dependently. Interestingly, pretreatment of rats with methyllycaconitine (10 g; i.c.v.) or -bungarotoxin (10 g; i.c.v.), selective antagonists of 7 subtype of nicotinic acetylcholine receptors (7nAChRs), partially abolished the pressor response to i.c.v. injection of U-46619 (1 g). Similar to the mecamylamine data, the use of higher doses of methyllycaconitine (25 and 50 g; i.c.v.) produced the same magnitude of blockade that was observed after the 10 g methyllycaconitine pretreatment, but it completely abolished the bradycardic effect of U-46619 (1 g; i.c.v.) at the dose of 25 g. The present results show that central administration of U-46619 produces pressor and bradycardic effect and increase in hypothalamic acetylcholine and choline levels by activating central TxA2 receptors. The activation of central nicotinic receptors, predominantly 7nAChRs, partially mediates the cardiovascular responses to i.c.v. injection of U-46619.     

Evidence for prejunctionally located β2-adrenoceptors in the cat spleen

Summary The number of -adrenoceptors in myocardium and spleen from 6-hydroxydopamine (6-OH-DA) treated cats was determined by radioligand binding with [¹²⁵I]-iodohydroxybenzylpindolol (IHYP). The effectiveness of 6-OH-DA pretreatment was assessed by analyses of the tissue content of catecholamines and the contractile response of isolated splenic strips to electrical stimulation. Since no effect on the splenic strip was produced by the -agonist isoprenaline, whereas noradrenaline caused contraction, it is concluded that the smooth muscle of the splenic capsule is controlled by postjunctional -adrenoceptors.The number of specific IHYP binding sites were reduced by 70% in whole spleen tissue and totally abolished in the splenic capsule by pretreatment with 6-OH-DA. Subclass analysis revealed that the reduction in total splenic -adrenoceptor number was due to a loss of ₂-adrenoceptors. However, the 6-OH-DA induced chemical sympathectomy did not produce any alteration either in -adrenoceptor density or the relative distribution of the -adrenoceptor subtype in the myocardium. It is suggested that a loss of prejunctional -adrenoceptors, due to chemical sympathectomy, might be compensated for by an increased number of postjunctional -adrenoceptors in the myocardium due to the development of denervation supersensitivity in this tissue.In conclusion, the findings provide direct biochemical evidence for existence of prejunctional ₂-adrenoceptors on the sympathetic nerve terminals of the cat spleen.     

Pharmacological characterization of A1 adenosine receptors in isolated rat ventricular myocytes

Summary The aim of the present study was the characterization of adenosine receptors in isolated rat ventricular myocytes. The CAMP-levels of rat ventricular myocytes in the presence of 1 mol/l isoprenaline were reduced by up to 48% by adenosine analogues; the rank order of potency was: R-N⁶-phenylisopropyladenosine (IC₅₀ 60 nmol/1), 5-N-ethylcarboxamidoadenosine (IC₅₀ 360 nmol/l) and S-N⁶-phenylisopropyladenosine (IC₅₀ 16 ol/l). The adenosine receptor antagonist XAC (xanthine amine congener) antagonized the effect of R-N⁶-phenylisopropyladenosine in a concentration-dependent manner with a K_i-value of 20 nmol/l. The A₁ receptor-selective radioligand R-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine bound to membranes prepared from rat ventricular myocytes in a saturable manner with a B _max of 17.7 fmol/mg protein and a K _D-value of 1.1 nmol/l. Adenosine analogues competed for the binding with the same rank order of potency as for the inhibition of the isoprenaline-induced cAMP-increase. GTP inhibited radioligand binding with an IC₅₀-value of 73 ol/l. These results suggest the presence of A₁ adenosine receptors on rat ventricular myocytes, which mediate an inhibition of adenylate cyclase. The receptors may be responsible for the effects of adenosine and its analogues on the heart.Abbreviations ¹²⁵I-HPIA R-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine - PIA N⁶-phenylisopropyladenosine - NECA 5-N-ethyl-carboxamidoadenosine - XAC 8-4-[([(2-aminoethyl)aminocarbonyl]methyl)oxy]phenyl-1,3-dipropylxanthine (xanthine amine congener) - Ro 20-1724 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone - ScAMPTME 2-O-monosuccinyladenosine-3,5-cyclic monophosphate tyrosyl methyl ester - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - GTP guanosine-5-tri-phosphate Send offprint requests to D. Martens     

Toxic effects of some “mono-n-butyl-tin compounds” on white mice

The toxic effects of mono-n-butyl-tin-trichloride, mono-n-butyl-tin-tris-(2-ethyl-hexyl-mercaptoacetate), mono-n-butyl-tin acid and mono-n-butyl-thiotin acid on white mice were investigated. These compounds were administered to white mice by means of a stomach tube in a single dose of 4000 mg/ kg b.w. at the start of the experiment. All mice were sacrificed 24 hours after the administration.The clinical course as well as the macroscopic findings in all experimental groups indicated general signs of an acute intoxication. The histological findings in the mono-n-butyl-tin-trichloride group showed pronounced changes in the digestive tract, where haemorrhages in the mucous membrane and in the inner layer of the gastric and intestinal walls had been found. In the mice of the other experimental groups, steatosis of the hepatocytes and an irregular steatosis of the renal tubular epithelium were observed.     

Induction of skeletal malformations in organ cultures of mammalian embryonic tissues

Summary Abnormalities of the cartilaginous limb bone anlagen resembling syndactyly and phocomelia can be mimicked entirely in vitro, using an organ culture system which allows the differentiation of mouse limb buds from the blastema stage (day 11.0 of gestation) to well recognizable cartilaginous anlagen of the long bones and the hand (or foot) skeleton (metacarpal anlagen) within 6 to 7 days. As reported here such malformations as triggered in vitro by 6-mercaptopurine (6-MP) should be distinguished from less specific effects, such as failure of the explant to develop typically in a proximodistal direction and to grow or form cartilage, which are produced by a variety of drugs or by altering the medium composition or other culture conditions. When culturing limb buds from embryos exposed in utero to 5-bromodeoxyuridine (BUdr) in a normal medium it was possible to obtain polydactyly in about 30% of the explants. The test system used provides another way of elucidating the mode of action of teratogenic agents, and of mechanisms involved in an abnormal mammalian development.     

Effect of the phosphodiesterase inhibitor 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711) on gastric secretion and gastric mucosal cyclic AMP

Summary The effect of the phosphodiesterase inhibitor 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711) on gastric secretion and the cyclic AMP system of the gastric mucosa was studied in rats and guinea pigs. In rats, 0.03–0.3 moles/kg ZK 62711 i.v. stimulated acid and pepsin secretion in a dose-dependent manner and 0.03 moles/kg i.v. enhanced the effect of histamine. In guinea pigs no reproducible stimulation was found after intravenous injections of ZK 62711. The stimulation of gastric secretion in the rat by 0.3 moles/kg ZK 62711 i.v. was not affected by vagotomy but was totally inhibited by 10 moles/kg cimetidine i.v. The structurally related phosphodiesterase inhibitor, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidine (Ro 20-1724), at the dose of 3.3 moles/kg i.v. stimulated gastric secretion in anaesthetised rats to a similar extent as 0.3 moles/kg ZK 62711 i.v. The content of cyclic AMP in the rat gastric mucosa in vivo was slightly increased by 10 moles/kg ZK 62711 i.v, whereas lower doses did not change it. Accumulation of cyclic AMP in the rat gastric mucosa by 50 moles/kg histamine i.v. was enhanced by simultaneous injections of 3.3 moles/kg ZK 62711 i.v. In rat gastric tissue slices in vitro 1–50 M ZK 62711 increased the level of cyclic AMP but 100 M histamine had no effect in the absence or presence of ZK 62711. In gastric mucosal slices of the guinea pig 10 and 50 M ZK 62711 increased the cyclic AMP content which effect was inhibited by 100 M cimetidine and enhanced the stimulatory effect of 100 M histamine on cyclic AMP. The activity of soluble cyclic AMP phosphodiesterase was inhibited by ZK 62711 in the rat (IC₅₀=18 M) and guinea pig gastric mucosa (IC₅₀=1.5 M). Adenylate cyclase was not affected in the homogenate of the guinea pig gastric mucosa. The results indicate that the phosphodiesterase inhibitor ZK 62711 which increases cyclic AMP levels in the gastric mucosa in vivo and in vitro, is a potent stimulator of gastric acid secretion.     

Evans blue blocks P2X-purinoceptors in rat vas deferens

Summary In rat vas deferens, Evans blue 100 M increased contractions elicited by high K⁺ and by noradrenaline but markedly reduced contractions elicited by the P_2X-purinoceptor-selective agonist ,-methylene ATP (3 M). The concentration-response curve of ,-methylene ATP was shifted to the right by Evans blue 30 M and the maximal contraction was increased. In tissues incubated with nifedipine 10 M, Evans blue 100 M tended to increase the residual contraction elicited by noradrenaline and abolished the residual response to ,-methylene ATP (3 M). The concentration-response curve of ,-methylene ATP was progressively shifted to the right by increasing concentrations of Evans blue in the presence of nifedipine; maximal contractions were increased by Evans blue 10 and 30 but not 100 M. From the shifts to the right caused by Evans blue 30 M, apparent pK_B values of 5.9 (no nifedipine) and 6.0 (nifedipine present) were calculated. It is concluded that Evans blue blocks P_2X-purinoceptors in rat vas deferens and in addition causes a non-receptor-specific enhancement of contractions.Correspondence to: R. Bültmann at the above address     

Presynaptic dopamine DA2-receptors in rabbit jejunal arteries

Summary Excitatory junction potentials (e.j.ps) evoked by nerve stimulation with 15 pulses at 1 Hz were recorded from muscle cells of rabbit isolated jejunal arteries. LY 171555 1 mol/l, SKF 38393 10 mol/l, dopamine 10 ol/l and clonidine 0.1 mol/l depressed all e j.ps in the train. The percentage inhibition was inversely related to the number of pulses. S- and R-sulpiride, 10 mol/l, domperidone 1 mol/l, SCH 23390 1 mol/l and rauwolscine 1 mol/l did not change, or even depressed the first e j.ps. Of these compounds only S- and R-sulpiride, 10 mol/l and rauwolscine 1 mol/l facilitated the late e.j.ps. The percentage facilitation increased with the number of pulses until a maximum was reached; rauwolscine 1 ol/l had the largest effect. S- and R-sulpiride, 10 mol/l, as well as domperidone 1 ol/l antagonized the action of LY 171555 1 mol/l. S-Sulpiride was more potent than its R-isomer. SCH 23390 1 mol/l and rauwolscine 1 mol/l blunted the effect of SKF 38393 10 mol/l. Rauwolscine 1 mol/l slightly reduced the inhibition by dopamine 10 mol/l; S-sulpiride 10 mol/l was antagonistic only in the presence of rauwolscine 1 mol/l. When rauwolscine 1 mol/l, prazosin 0.1 mol/l, propranolol 1 mol/l and cocaine 10 mol/l was added to the medium, dopamine 10 mol/l continued to produce the same depression of e j.ps, as in the absence of these compounds. Under such conditions S-sulpiride 10 mol/l also counteracted dopamine 10 gmol/l. Rauwolscine 1 mol/l prevented the effect of clonidine 0.1 mol/l. The antagonists were not absolutely selective against only one type of agonist. We suggest that both presynaptic DA₂- and postsynaptic DA₁-receptors are present in rabbit jejunal arteries. The activation of either receptor-type may depress the e j.ps. Dopamine interferes with neuroeffector transmission due to ₂-adrenoceptor agonist properties; its DA₂-effect is unmasked only after ₂-adrenoceptor blockade. There was no evidence for a co-transmitter function of dopamine. Send offprint requests to P. Illes at the above address     

Sympathicomimetische Wirkungen von Papaverinderivaten

Zusammenfassung In früheren Untersuchungen hatte Tetrahydropapaverolin (THP), das sich chemisch von dem musculotropen Papaverin durch Demethylierung der Methoxygruppen und Hydrierung des N-haltigen Rings unterscheidet, sich als sehr wirksames -Sympathicomimeticum erwiesen. Untersuchungen über Beziehungen zwischen chemischer Konstitution und -sympathicomimetischer Wirksamkeit von Derivaten des THP und Papaverins am Blutdruck der Katze und am elektrisch gereizten linken Vorhof des Meerschweinchenherzens haben ergeben:1. Die blutdrucksenkende Wirkung sowohl des Papaverins als auch seines partiell demethylierten (3,4-Desmethylpapaverin) und hydrierten Derivates (Tetrahydropapaverin) war, im Gegensatz zu derjenigen des THP, resistent gegen Propranolol. Das vollständig demethylierte Derivat (Papaverolin) war wirkungslos.Demgegenüber erwies sich die positiv inotrope Herzwirkung des Papaverins als eine direkte, -sympathicomimetische. Sie blieb nach Reserpinvorbehandlung bestehen und wurde durch Pronethalol kompetitiv abgeschwächt. 3,4-Desmethylpapaverin und Papaverolin waren schwächer wirksam als Papaverin. Tetrahydropapaverin wirkte nur negativ inotrop. Diese allen Papaverinderivaten zukommende chinidinartige Wirkungskomponente könnte die Ursache für ihre niedrige -adrenergische intrinsic activity (0,6) sein.2. Alle chemischen Eingriffe in das Molekül des THP, z. B. Alkylierung an C₃ oder am Stickstoff sowie Dehydroxylierung an C₃ und C₄, führten zu einer Abschwächung der -sympathicomimetischen Wirksamkeit am Herz und am Blutdruck. Eine, auch dem THP wahrscheinlich zukommende, jedoch durch die ausgeprägte -sympathicomimetische Wirksamkeit überdeckte -sympathicomimetische, blutdrucksteigernde Wirkung trat jetzt in Erscheinung.3. THP, sozusagen ein dimeres Dopamin, besitzt von allen untersuchten Papaverinderivaten die für die Ausübung -sympathicomimetischer Wirkungen optimale chemische Konfiguration. Ein großmolekularer Phenyläthyl-Substituent am Stickstoff verursacht maximale Aktivierung der sekundären Aminogruppe (+ I-Effekt). Zusammen mit den vier freien phenolischen OH-Gruppen verleiht er dem Molekül die hohe Affinität zu den adrenergischen -Receptoren.Über einen Teil der Ergebnisse wurde auf der 8. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft in Mainz (1967) berichtet (Langeneckert, et al., 1967).     

Rotational behaviour produced by intranigral injections of bovine and human β-casomorphins in rats

The biological activity of -casein derived -casomorphin peptides was evaluated by injecting bovine -casomorphin-5 (Tyr-Pro-Phe-Pro-Gly), the homologous sequence in human -casein (Tyr-Pro-Phe-Val-Glu) and the corresponding N-terminal tetrapeptides into the left substantia nigra of rats. Their ability to produce rotational behaviour was compared to that produced by three reference compounds, morphine, d-ala² d-leu⁵enkephalin and U50,488H, ligands for , and types of opioid receptors, respectively. The relative potencies of -casomorphins and morphine were compared to those tested in two in vitro assays for opioid activity: (1) inhibition of the electrically induced contraction of the isolated myenteric plexus-longitudinal muscle of the guinea-pig ileum and (2) displacement of ³H-dihydromorphine binding to brain membranes. The same ranking order of potency was found in all three assays, the peptides from human -casein being about 10-fold less potent than those from bovine -casein. The effects of both morphine and bovine -casomorphin-5 in producing rotational behaviour were antagonized by naloxone; however, approximately 10-fold more naloxone was required to antagonize the -casomorphin-5 effect than that of morphine. The present data are discussed in the light of the recent observation that high concentration of -casomorphin-like peptides are found in the cerebrospinal fluid and plasma of women with postpartum psychosis.     

Interactions of bupranolol with the polymorphic debrisoquine/sparteine monooxygenase (CYP2D6)

Summary The -adrenoceptor blocker bupranolol turned out to be a competitive inhibitor of the polymorphic cytochrome P450 CYP2D6 of which sparteine is a substrate. There was stereo-selectivity of bupranolol involved: (–)-bupranolol was the weakest inhibitor with an apparent K_i value of 1.32 M, (+)-bupranolol was the most potent with an apparent Ki value of 0.55 M, while the therapeutically used racemic bupranolol had an intermediate value of 0.88 M. A 10 min pre-incubation of 5 M bupranolol with the enzyme preparation prior to the addition of substrate, reduced the inhibition of sparteine metabolism from 52 to about 25%.This suggests that — during these inhibition studies — bupranolol was much more rapidly metabolized than was sparteine, so that the measured K_i values must represent overestimates. The enzyme catalysing bupranolol metabolism was CYP2D6: microsomes from a liver with the genetic enzyme deficiency did not metabolize bupranolol; in microsomes from livers containing the enzyme and 10 M bupranolol, 5 M quinidine caused a 72% inhibition of bupranolol metabolism.Although our methods were not sufficiently sensitive to measure the K_m of bupranolol directly, it is undoubtedly the -adrenoceptor blocker with the highest-known apparent affinity for CYP2D6. High affinity and rapid metabolism are infrequent combinations in enzymology.     

Imipramine: Clinical effects and pharmacokinetic variability

Sixty-six hospitalized depressed patients were treated for 4 weeks with imipramine (Tofranil^®) 225 mg/day. Blood samples were drawn twice weekly 15 h after the last drug intake, and IP and DMI concentrations in plasma were assayed by quantitative in situ thin-layer chromatography. Clinical rating was carried out once weekly by Hamilton's Rating Scale (HRS), Beck's Depression Inventory, WHO Depression Scale (Quantitative Part), and a side-effect scale. The patients were classified on the basis of the WHO Depression Scale (Qualitative Part) as endogenous (N=37) or non-endogenous depressions (N=29). Antidepressive effect was evaluated on the basis of the posttreatment rating scores.In patients classified as endogenous depressions all 12 responding patients (HRS7) had plasma levels of IP>45 g/l and DMI>75 g/l, whereas 11 out of 14 nonresponding patients (HRS16) had plasma levels of one or both compounds below these limits. Ten out of 12 responders had levels of IP+DMI above 240 g/l, and all nonresponders had levels of IP+DMI below this limit. Patients with partial response (HRS: 8–15) formed an overlapping group. There was no sign of an upper plasma level limit for the antidepressive effect of imipramine.The plasma level/effect relationship was less clear in patients with non-endogenous depressions, since several of them responded at low plasma levels.Some relationship between effect on blood pressure (orthostatic effect) and high plasma levels of IP and DMI was found.Using a plasma level limit of IP45 g/l and DMI75 g/l, it was possible to predict the response of the endogenous depression group for 10 out of 12 responders and 10 out of 14 nonresponders on the basis of plasma level measurements obtained after 1 week of treatment.     

Wirkung und Wirkungsmechanismus von Katecholaminen und ihren Derivaten

Zusammenfassung Zusammenfassend kann gesagt werden, daß in der Analyse der Wirkung und der Wirkungsmechanismen von Katecholaminen die Verschiedenen Typen von blockierenden Substanzen eine wichtige Rolle spielen. Dabei ist auch die sterische Struktur der verschiedenen Verbindungen in Betracht zu ziehen. Neben den -adrenergen Receptoren und den dopaminergen Receptoren gibt es die -adrenergen Receptoren. Eine chemische Differenzierung der -adrenergen Receptoren in zwei Typen, nämlich solchen, die für metabole Effekte wie Lipokinese und Lacticidämie und solche, die für die Herzwirkung verantwortlich sind, bahnt sich an. Dem Fehlen einer klaren Verwandtschaft zwischen -adrenergen und -adrenerg blockierenden Substanzen steht die weitgehende, auch die sterische Konfiguration betreffende, chemische Verwandtschaft zwischen -adrenergen und -adrenerg blockierenden Substanzen gegenüber. Die blockierenden Effekte der verschiedenen Substanzen an adrenergen Receptoren lassen sich klar unterscheiden von Wirkungen anderer Art. Dies trifft auch für die verschiedenen Herzwirkungen der -adrenerg blockierenden Substanzen zu. Rezente umfassende Artikel über Katecholamine und ihre Derivate [36a], bezüglich der klinischen Anwendungsmöglichkeiten der -adrenergen Blocker [26a] und einige Mitteilungen in Bezug auf Nebenwirkungen dieser Substanzen [26b, 82a, 95] werden als weitere Informations-Quellen empfohlen.     

α-Bungarotoxin, κ-bungarotoxin, α-cobratoxin and erabutoxin-b do not affect [3H]acetylcholine release from the rat isolated left hemidiaphragm

Endplate preparations of the rat left hemidiaphragm were incubated with [³H]choline to label neuronal transmitter stores. Nerve evoked release of newly-synthesized [³H]acetylcholine was measured in the absence of cholinesterase inhibitors to investigate whether snake venom neurotoxins by blocking presynaptic nicotinic autoreceptors affect evoked transmitter release. Contractions of the indirectly stimulated hemidiaphragm were recorded to characterize the blocking effect of -neurotoxins at the postsynaptic nicotinic receptors.Neither the long chain neurotoxins -cobratoxin (1 g ml^–1) and -bungarotoxin (5 g ml^–1) nor the short chain neurotoxin erabutoxin-b (0.1, 1 and 10 gml^–1) affected the nerve-evoked release of [³H]acetylcholine. -Bungarotoxin (1 and 5 g ml^–1), a toxin preferentially blocking neuronal nicotinic receptors, did also not affect evoked [³H]acetylcholine release, whereas (+)-tubocurarine (1 M) under identical conditions reduced the release by about 50%. -Bungarotoxin, -cobratoxin and erabutoxin-b concentration-dependently (0.01–0.6 g ml^–1)inhibited nerve-evoked contractions of the hemidiaphragm. All neurotoxins except erabutoxin-b enhanced the basal tritium efflux immediately when applied to the endplate preparation or to a non-innervated muscle strip labelled with [³H]choline. This effect was attributed to an enhanced efflux of [³H]phosphorylcholine, whereas the efflux of [³H]choline and [³H]acetylcholine was not affected.It is concluded that the -neurotoxins and -bungarotoxin do not block presynaptic nicotinic receptors of motor nerves. These nicotinic autoreceptors differ from nicotinic receptors localized at the muscle membrane and at autonomic ganglia.     

Effect of Cyclodextrins on Protein Binding of Drugs: The Diflunisal/Hydroxypropyl-β-Cyclodextrin Model Case

The binding of diflunisal to hydroxypropyl--cyclodextrin (HPCD), bovine serum albumin (BSA), human serum albumin (HSA), normal human plasma, and mixed solutions of HPCD/ protein was studied at 25°C, pH 7.4, by potentiometry using an electrode selective to diflunisal. The experimental data for diflunisal/ HPCD fit well to the 1:1 binding model. The binding of diflunisal with each of the studied proteins was compatible with a model having two independent classes of binding sites. The binding of diflunisal in mixed solutions HPCD/BSA, HPCD/HSA, and HPCD/plasma increased considerably when the HPCD concentration was increased. The binding behavior of the two biomolecules in the mixed solutions of HPCD/BSA or HPCD/ HSA was described with an additive model formulated on the basis of the estimates of the binding parameters of diflunisal derived from the separate experiments with each one of the binders tested. The lower than theoretical binding observed in HPCD/plasma solutions was ascribed to the competitive displacement of diflunisal from the HPCD cavity by plasma cholesterol.     

Cytotoxicity of diethyldithiocarbamate in human versus rodent cell lines

Summary Diethyldithiocarbamate (DDTC) and other dithiocarbamates are currently receiving attention as potential adjuncts to traditional chemotherapy. In vitro studies with rodent cancer cell lines have consistently shown that DDTC concentrations of 0.1–1.0 g/ml are highly cytotoxic. Paradoxically, however, concentrations of 10–100 g/ml have been significantly less toxic.In the present study, such a biphasic pattern was reproduced when 3 rodent cell lines were exposed for 1 hour to 0.001 to 1000 g DDTC/ml. In contrast, in 7 human cell lines survival decreased steadily with increasing DDTC concentration (in the same dose range) without evidence of a biphasic pattern. These data might have implications for studies in which rodent cell lines are used to model the effects of dithiocarbamates in human tissues.     

The cardiovascular effects of intraventricularly administered histamine in the anaesthetised rat

Summary In urethane-anaesthetised rats intraventricular (i.c.v.) injections of histamine (0.1–10.0 g) elicited dose-related rises in both the resting blood pressure and heart rate. These cardiovascular effects of histamine were antagonised in a dose-dependent manner by i.c.v. pretreatments with the histamine H₁-receptor antagonists mepyramine (10, 50 and 100 g) and diphenylpyraline (100 and 200 g). Pretreatment with the histamine H₂-receptor antagonist metiamide (100 and 200 g i.c.v.) failed to modify either of the responses. A dose-related antagonism of the hypertensive response to histamine i.c.v. was elicited by phentolamine (100 and 200 g i.c.v.) but the positive chronotropic effect was not modified by this pretreatment. The cardiovascular responses to histamine i.c.v. were abolished by mecamylamine (5.0 mg/kg i.v.) and greatly reduced by 6-hydroxydopamine (3×250 g i.c.v.), but only the tachycardia was significantly modified by atropine (100 g i.c.v.) and propranolol (1 mg/kg i.v.). Propranolol (100 g i.c.v.), bilateral vagotomy, or acute bilateral adrenal demedullation failed to modify the cardiovascular responses to histamine i.c.v. The results suggest that histamine is able to modify the resting blood pressure and heart rate by independent central modes of action, which involve central adrenergic and cholinergic mechanisms.Preliminary findings of this study were presented at the Autumn meeting of the British Pharmacological Society (Finch and Hicks, 1975).     

Two distinct adrenoceptor-biophases in the vasculature: One for α-and the other for β-agonists

Summary Strips of two canine vessels with different patterns of sympathetic innervation were used: the mesenteric artery which has an adventitio-medial plexus and the saphenous vein in which nerve terminals are distributed throughout the media.The pD₂ of (–)-adrenaline for -and -adrenoceptors was determined for both vessels (in the presence of 0.5 M propranolol and 7 M phentolamine, respectively; in the latter case the strips were contracted by 0.28 M prostaglandin F2) and found to be very similar: 6.96 and 7.01 in the saphenous vein and 6.77 and 6.91 in the mesenteric artery, respectively. The similarity of pD₂ for adrenaline acting on -and -adrenoceptors in both preparations allowed us to compare the effect of inhibition of neuronal uptake by 12 M cocaine with that of inhibition of COMT by 50 M dihydroxy-2-methyl propiophenone (U-0521) on: a) the potency of adrenaline (noradrenaline was used in the saphenous vein only) acting on -and -adrenoceptors and b) the time required by the strips to recover 50% in oil (t ₅₀) after contractions or relaxations caused by 0.23 M adrenaline. In the saphenous vein cocaine increased the potency of both adrenaline and noradrenaline for -effects more than for -effects (2.8 times vs. no increase for adrenaline; 7.1 vs. 1.9 times for noradrenaline) and U-0521 increased the potency of both adrenaline and noradrenaline for -effects more than for -effects (4.1 vs. 2.6 times for adrenaline; 1.8 times vs. no increase for noradrenaline); regarding the termination of action of adrenaline, cocaine prolonged the t ₅₀ 1.6 times after contraction and did not change it after relaxation, whereas U-0521 prolonged the t ₅₀ 6.9 times after relaxation and only 1.8 times after contraction.In the mesenteric artery only sensitivity experiments were done. Cocaine increased the potency of adrenaline by a factor of 2.1 for the -effects while there was no influence on its potency with regard to its -effects, and U-0521 increased the potency of adrenaline for the -effects more than for -effects (3.9 vs. 1.8 times, respectively).These results show that there are two different biophases for sympathomimetic agonists in the vasculature: one for -adrenoceptors which is more under the influence of the neuronal uptake and one for -adrenoceptors which is more under the influence of COMT activity. We conclude that -adrenoceptors are situated close to the nerve endings and -adrenoceptors close to COMT sites. Since these results do not differ qualitatively in two vessels with different patterns of innervation, we conclude that this asymmetry in the distribution of -and -adrenoceptors may be due to either an uneven distribution of cells with only one type of receptors each or due to an uneven distribution of receptors on the same cell.     

Reactive red 2: a P2y-selective purinoceptor antagonist and an inhibitor of ecto-nucleotidase

Effects of reactive red 2 and its parent compound acid red 33 were studied in rat vas deferens and guinea-pig taenia coli. In rat vas deferens, reactive red 2 (1 to 10 M) shifted the concentration-response curve for the P_Zx-purinoceptor-mediated contraction effect of , \-methylene ATP slightly to the right and progressively decreased the maximum (apparent antagonist K_d value 0.42 M). Acid red 33 (1000 M) shifted the curve to the right without changing the maximum (apparent K_d 386 M). The concentration-contraction curve of noradrenaline was not altered by reactive red 2. In the carbachol-precontracted guinea-pig taenia coli, reactive red 2 (0.1 to 10 M) shifted the concentration-response curve for the P_2Y-purinoceptor-mediated relaxation effect of adenosine 5-O-(2-thiodiphosphate) (ADP&S) progressively to the right; only at the highest concentration of antagonist (10 M) was the maximum slightly depressed; a pA₂ value of 7.55 (K_d 0.028 M) was derived from the shift. Acid red 33 (1000 M) shifted the concentration-relaxation curve of ADP\S to the right without changing the maximum (apparent K_d 171 M). Reactive red 2 (1 to 10 LM) also shifted the concentration-response curve for the relaxation effect of , \-methylene ATP, which is mediated by an unclassified P₂-purinoceptor, progressively to the right but simultaneously decreased the maximum (apparent K_d1.6 M). The concentration-relaxation curve of 2-chloroadenosine was not altered by reactive red 2. Pieces of vas deferens and taenia coli degraded 76 and 66 % of added ATP (10 M) within 30 min, respectively. Reactive red 2 (0.1 to 100 M) progressively reduced this degradation by up to 95%, with IC₅₀values of 3.9 ± 0.6 and 3.9 ± 2.3 M, respectively. Acid red 33 (1000 M) reduced the degradation by 30 and 20%, respectively.The results indicate that reactive red 2 is a relatively potent antagonist at both P_Zx-purinoceptors in rat vas deferens and P_2Y-purinoceptors in guinea-pig taenia coli, with a 15 fold selectivity for the P_2Y-purinoceptor. It inhibits ecto-nucleotidase in both tissues. The dichloro-triazine residue that distinguishes the compound from acid red 33 greatly enhances the potency at both receptor subtypes as well as at the nucleotidase. As regards P₂-purinoceptor subtypes, the results confirm the existence of two relaxation-mediating P₂-purinoceptors in guinea-pig taenia coli.     

ATP and endogenous agonists inhibit evoked [3H]-noradrenaline release in rat iris via A1 and P2y-like purinoceptors

Summary Effects of ATP, adenosine and purinoceptor antagonists on field stimulation-evoked (3 Hz, 2 min) [³H]-noradrenaline overflow were investigated in the rat isolated iris.ATP and adenosine inhibited the evoked overflow of [³H]-noradrenaline. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) shifted the concentration-response curve of ATP to the right in a concentration-dependent manner, but with a potency (–log K_B = 7.88) much lower than expected for an A1 adenosine receptor. In the continuous presence of DPCPX, the ATP-induced prejunctional inhibition was unaffected by suramin (100 mol/l) and DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 50 mol/l) but was antagonized by the P_2Y-receptor antagonist cibacron blue ( = reactive blue 2;30 and 100 mol/l, –log K_B = 4.7)and ,-methylene-ATP (10 mol/l). Whereas the evoked [³H]-noradrenaline overflow was unaffected by suramin and DIDS, cibacron blue and ,-methylene-ATP caused a small and transient increase. Cibacron blue at 30 mol/l failed to antagonize the inhibition of evoked [³H]-noradrenaline overflow that adenosine produced in the absence of DPCPX. Basal [³H]-noradrenaline overflow was enhanced by cibacron blue, not changed by ,-methylene-ATP and DIDS, and decreased by suramin.The results show that exogenous ATP inhibits sympathetic neurotransmission in the rat iris via A₁ and P_2Y-like purinoceptors. The latter have a low apparent affinity for cibacron blue and probably are blocked by ,-methylene-ATP. Under the present conditions, endogenous purines exert a tonic inhibition not only via A₁- but also via these P_2Y-receptors. Correspondence to: H. Fuder at the above address