The distribution of (-)-propranolol in the isolated rabbit iris

It has been postulated that propranolol lowers the intraocular pressure by adrenergic neurone block. However, in the isolated iris of albino rabbits, there was only a small degree of cocaine-sensitive (i.e., neuronal) accumulation of ³H-(-)-propranolol, and none at all after pretreatment of the animals with reserpine. Moreover, after preloading of the iris with ³H-(-)-propranolol, veratridine failed to release any labelled material. Hence, any adrenergic neurone blocking action of propranolol is highly unlikely.Albino and pigmented irides were first exposed to ³H-(-)-propranolol and then washed out. The results and their compartmental analysis indicated an extensive binding of ³H-(-)-propranolol in or at pigment cells; the binding is characterized by a low dissociation constant. It is very likely that the initial binding and the subsequent slow dissociation from pigment cells explains the long duration of action of beta-adrenoceptor antagonists in human therapy.     

Agonist-mediated conformational changes of β-adrenoceptors could occur independent of functional coupling to Ns

Summary Agonist and antagonist binding characteristics of -adrenoceptors in turkey erythrocyte ghosts were determined at different temperatures ranging between 7°C and 42°C. [³H]-DHA saturation binding experiments revealed that the antagonist-receptor interaction is entropy-driven with a small enthalpic contribution. Isoproterenol/[³H]-DHA competition binding followed the law of mass action at all the investigated temperatures. The agonist-receptor interaction is enthalpy driven with a small unfavorable decrease in entropy. This is consistent with the agonist's ability to favor an endoenergetic transconformation of the receptors.Only part of the agonist-bound receptors can undergo functional coupling to the stimulatory component of the adenylate cyclase system (Ns). This coupling process is associated with locking-in of the agonist and becomes persistent in the presence of the alkylating reagent N-ethylmaleimide. The number of agonist/N-ethylmaleimide-sensitive sites (i.e. coupling-prone receptors) increases with the temperature until it reaches a plateau value of 50% between 27–32°C. Qualitatively similar data were obtained for rat lung and turkey erythrocyte membranes. These observations suggest that the whole receptor population can undergo agonist-mediated conformational changes but that only part of them can couple to Ns.     

L-654,284 a new potent and selective α2-adrenoceptor antagonist

Summary L-654,284 ((2R, 12bS)-N-(1,3,4,6,7,12b-hexahydro-2H-benzo[b]-furo[2,3-a] quinolizine-2-yl)-N-methyl-2-hydroxyethanesulfonamide) was tested in several in vitro and in vivo models for ₂-adrenoceptor antagonist activity and compared to several reference agents. In vitro L-654,284 competed for the binding of ³H-clonidine or ³H-rauwolscine (K _i's 0.8 nM, 1.1 nM) and blocked the presynaptic effects of clonidine in the rat isolated vas deferens (pA₂, 9.1). L-654,284 exhibited marked ₂- vs. ₁-adrenoceptor selectivity in vitro, inhibiting ³H-prazosin binding with a K _i of 110 nM and blocking the effects of methoxamine on the vas deferens with a pA₂ of 7.5. In vivo L-654,284 at 22 nmoles/kg i.v. doubled the ED₅₀ of clonidine to produce mydriasis in rats. Given orally, the potency of L-654,284 in this test was reduced by a factor of 5.5. L-654,284 also potently increased cerebrocortical NE turnover in the rat, another in vivo index of ₂-adrenoceptor blockade in the central nervous system. In the periphery, L-654,284 demonstrated ₂-adrenoceptor selectivity by preferentially blocking the pressor effects of UK 14304 versus those of methoxamine in the pithed rat. Overall, L-654,284 was generally a more potent ₂-adrenoceptor antagonist than RX 781094 with comparable ₂/₁ selectivity and was several times more potent and ₂-selective than WY 26703 or yohimbine. In addition, L-654,284 had better (5–6 times) oral bioavailability than RX 781094 or WY 26703.     

Effect of endothelium on basal and α-adrenoceptor stimulated calcium fluxes in rat aorta

Summary The rate of unstimulated influx of Ca²⁺ into rat aorta smooth muscle, measured as uptake of ⁴⁵Ca, was inhibited in the presence of endothelium as compared to influx in the absence of endothelium. Efflux of ⁴⁵Ca from unstimulated prelabelled tissues was also reduced in the presence of endothelium. In normal physiological solution the rate of influx and efflux of Ca²⁺ stimulated by B-HT 920 (1 and 10 M), but not that stimulated by phenylephrine (30 nM and 1 M), was also reduced in the presence of endothelium. In the presence of the calcium entry blocker flunarizine (3 M), phenylephrine (1 M) stimulated efflux of Ca²⁺ was inhibited by the presence of endothelium. A correlation between inhibition of Ca²⁺ influx and modulation of -adrenoceptor agonist-induced contractions by endothelium could not be demonstrated, and methylene blue, an antagonist of endothelium mediated inhibition of B-HT 920 contractions, did not affect Ca²⁺ influx stimulated by the agonist. The effects of endothelium on Ca²⁺ influx and efflux are unlikely to be due to alterations by endothelium of diffusion of ⁴⁵Ca or the agonists in the vessel. The results demonstrate that an endothelial derived factor or factors can reduce calcium influx into smooth muscle cells and also modulate the release of calcium from cells, perhaps by affecting intracellular calcium pumping mechanisms. A reduction of calcium influx cannot be the sole explanation for the modulatory effect of endothelium on -adrenoceptor agonist-induced contractions but an effect on intracellular calcium metabolism may be important.     

Sodium regulation of agonist and antagonist binding to β-adrenoceptors in intact and Ns-deficient membranes

Summary Agonist binding to various hormone receptors mediating adenylate cyclase inhibition is decreased by sodium ions. We studied the influence of Na⁺ on agonist and antagonist binding to -adrenoceptors in membrane preparations of guinea pig lung, S49 lymphoma wild-type cells (WT) and their N_s-deficient cyc^– variants by measuring binding of the antagonist, [¹²⁵I]iodocyanopindolol ([¹²⁵I]CYP). At 37° C, sodium decreased the receptor affinity for the agonist, isoproterenol, in all three membrane preparations. In lung and WT membranes, Na⁺ steepened the shallow agonist competition curves in a manner similar to and synergistic with guanine nucleotides. When binding was performend at 4° C, sodium regulation but not guanine nucleotide regulation of agonist binding was preserved. At the low temperature, [¹²⁵I]CYP affinity was reduced, and sodium increased [¹²⁵I]CYP binding in both N_s-containing and N_s-deficient membranes by increasing the antagonist affinity without significant change in total receptor number. Compared to Na⁺, Li⁺ and K⁺ were much less potent and efficient in decreasing agonist and increasing antagonist binding. Na⁺ and Mg²⁺ had opposite effects on agonist binding in the N_s-containing lung and WT membranes but not in the N_s-deficient cyc^– membranes. The data indicate that sodium not only regulates binding of inhibitory hormone receptors but also agonist and antagonist binding to the adenylate cyclase stimulatory -adrenoceptor. The finding that sodium regulation of -adrenoceptor binding is also observed in the N_{s 2} cyc^– membranes, furthermore, indicates that the target of sodium is not the -subunit of N_s but possibly a component common to both types of receptor systems regulating adenylate cyclase activity.     

Species differences in the relative analgesic potencies of some classical opiates and opioid peptides

The analgesic ED₅₀ values of some classical morphine congeners (morphine, methadone, fentanyl, azidomorphine) in the rat and mouse tail-flick tests were found to be similar. However, several synthetic derivatives of the natural enkephalins were more potent in mice than in rats. (These analogs contain d-amino acid in position 2 and d- or l-sulfonic (or phosphonic) acid residue in position 5). -Endorpin, d-Met², Pro⁵-enkephalinamide and two partial agonists showed intermediate interspecies relative potencies. According to the data obtained, similar opiate receptors might mediate the analgesic action of classical opiates in rats and in mice. However, the opiate receptors responsible for the antinociceptive effects of the above mentioned enkephalin analogues must be dissimilar in the two species examined. The results are discussed in terms of the role of - and -receptors in mediation of the analgesic effect induced by different types of opioids.     

ZK 91296, a partial agonist at benzodiazepine receptors

ZK 91296 (ethyl 5-benzyloxy-4-methoxymethyl--carboline-3-carboxylate) is a potent and selective ligand for benzodiazepine (BZ) receptors. Biochemical investigations indicate that ZK 91296 may be a partial agonist at BZ receptors. Such partial agonism may explain to some extent why ZK 91296 needs higher BZ receptor occupancy than diazepam for the same effect against chemical convulsants and for behavioural effects. The lack of sedatiye effects, and the very potent inhibition of reflex epilepsy, spontaneous epilepsy and DMCM-induced seizures suggest, furthermore, that ZK 91296 may possess pharmacological selectivity for a particular type of BZ receptor interaction, perhaps including topographic as well as receptor subtype differentiation.     

Retardation of cerebral dopamine turnover after morphine withdrawal and its enhanced acceleration by acute morphine administration in rats

Summary To clarify the effects of withdrawal from chronic morphine treatment on cerebral dopamine (DA) turnover, we have measured the -methyl-p-tyrosine (MT)-induced depletion of DA in five brain areas of male Wistar rats given morphine twice daily for 40 or 60 days. After the last morphine dose (50 or 70 mg/kg) the rats were withdrawn for 1, 2 or 4 days. In order to study the development of tolerance some of the rats were challenged with 10 mg/kg of morphine.Withdrawal of morphine retarded the MT-induced DA depletion in the limbic forebrain and after long enough chronic treatment in the striatum, too. The challenge dose of morphine accelerated the cerebral DA depletion slightly less in rats withdrawn for 1 day from 60-day chronic morphine treatment than in rats treated chronically with saline, but it enhanced the DA depletion more in rats withdrawn from morphine for 2 and 4 days than in chronic saline rats. This enhancement was clearest in rats withdrawn for 4 days from 60-day treatment. Thus withdrawal from morphine seems to sensitize the rats to the DA depletion accelerating effect of morphine.Our results show that repeated administration of morphine creates no marked tolerance to the DA depletion accelerating effect of morphine. In contrast, the dopaminergic neurones of the chronically treated rats seem to depend on continuous morphine administration for their normal functioning. Furthermore, the retarded DA turnover after discontinuation of morphine treatment seems to sensitize the dopaminergic neurones to the DA depletion accelerating effect of morphine. The limbic dopaminergic neurones are more easily affected by both acute and chronic morphine treatment than the striatal ones.     

Alpha-adrenoceptors, 5-hydroxytryptamine receptors and the action of dihydroergotamine in human venous preparations obtained during saphenectomy procedures for varicose veins

Summary Changes in tension were monitored isometrically on spiral strips from human saphenous veins obtained during surgical removal of varicose veins. Concentration-response curves for noradrenaline and 5-hydroxytryptamine (5-HT) were established by cumulative administrations, curves for dihydroergotamine were constructed from the mean responses to single concentrations. The use of the antagonists prazosin, yohimbine and pizotifen provided evidence for the existence of both postjunctional ₁- and ₂-adrenoceptors and for the existence of 5-HT receptors. The venoconstrictor effects of dihydroergotamine were unchanged by prazosin. Yohimbine antagonized both dihydroergotamine and 5-HT at about 60 times higher concentrations than required against noradrenaline whereas pizotifen inhibited responses to both dihydroergotamine and 5-HT at about 100 times lower concentrations than those to noradrenaline.These new results are in contrast to conclusions drawn from animal studies and do not support the suggestion that in man the venoconstrictor activity of dihydroergotamine is mediated through stimulation of -adrenoceptors. The present results strongly suggest that in human saphenous veins the constrictor activity of dihydroergotamine is mediated at least in part through stimulation of 5-HT receptors.     

Teratological studies on the TCDD congener 3,3′,4,4′-tetrachloroazoxybenzene in sensitive and nonsensitive mouse strains: Evidence for direct effect on embryonic tissues

The teratogenicity of 3,3,4,4-tetrachloroazoxybenzene (TCAOB), a TCDD congener, was studied in Ah-responsive (C57BL and NMRI) and non-responsive (DBA/2J and AKR/NBom) strains of mice. In the responsive strains, the TCAOB produced cleft palate and hydronephrosis in 50–90% of the offspring at a dose level of 6–8 mg/kg b.w. in the absence of apparent maternal toxicity. Day 11 was shown to be the day of highest sensitivity (palatal closure occurs at day 14) in the C57BL strain. Higher doses (16 mg/kg b.w.) produced high rate of fetal death both in responsive (C57BL; 60%) and non-responsive (DBA; 40%) strains. These doses induced cleft palate in 95% of the surviving C57BL fetuses but failed to do so in the DBA strain. The non-sensitivity of the DBA and AKR strains appeared to segregate as a dominant trait. Backcrosses between NMRI x DBA F₁ generation and NMRI showed an intermediate sensitivity. It was shown that the genotype of the embryo was of ultimate importance for the development of cleft palate. There appeared however to be an additional host (maternal) factor as well, because the offspring of NMRI females mated with NMRI x DBA F₁ males showed a higher rate of cleft palate as compared to those of the crossing between NMRI x DBA F₁ females and NMRI males. Light and scanning electron microscopy indicated that the apical epithelial cells of the secondary palates failed to follow the normal pattern of programmed cell death, suggesting a similar mechanism of pathogenesis as previously described for TCDD.