Uranium mining in relation to toxicological impacts on inland waters

Protection of tropical rivers from metal pollution requires that mining wastewaters be biologically tested for aquatic toxicity before release from the site into natural ecosystems occurs, and that a safe dilution which incorporates a minimum 10-fold safety factor applied to the lowest NOEC threshold value be utilized. Application of these test methods to wastewaters from an operating uranium mine has shown that pre-release toxicity testing provides accurate information on the toxicity of metal-containing wastewaters with a high degree of confidence. Field validation of the laboratory results was obtained when wastewaters which were field diluted through a release into a billabong gave similar results to laboratory-diluted wastewaters. No one species is always the most sensitive to exposure to complex wastewaters. Changes with time in wastewater chemistry, toxicity, and in the physiological capacity of specific organisms to survive in a contaminated environment (tolerance), can result in different species having varying sensitivities over time to exposure to complex wastewaters collected from the same location. As a result of the remote likelihood of finding the most sensitive species, it is necessary to test the toxicity of complex wastewaters to a battery of organisms, representing different trophic levels of the ecosystem, under physical conditions representative of the specific environment needing protection. Use of a natural billabong as a biological filter for releasing mine wastewaters did not result in toxicity mitigation and prevented controlled dilution from occurring during periods of high creek flow.     

Releasing activities of d-fenfluramine and fluoxetine and fluoxetine on rat hippocampal synaptosomes preloaded with [3H]serotonin

Summary Rat hippocampal synaptosomes preloaded with [³H]serotonin and maintained in a superfusion apparatus were exposed for 3 min to d-fenfluramine or fluoxetine. Both drugs evoked a tritium overflow which was reserpine-sensitive requiring the presence of intact synaptic vesicles. However the two drugs displayed different characteristics: 1) the overflow was immediate with dfenfluramine whereas the releasing activity of fluoxetine showed a delay of about 2 min; 2) d-fenfluramine-induced overflow was already apparent at 0.15 mol/l whereas the minimal effective concentration of fluoxetine was 2.5 mol/l. Their concentration-effect curves were differently shaped, the effect of d-fenfluramine being saturable at 5–20 mol/l (EC₅₀ about 1 gmol/l) while no saturation was observed with fluoxetine up to 10 mol/l; 3) only 1907o of the tritium overflow evoked by fluoxetine (2.5–10 mol/l) consisted of true [³H]serotonin, compared with 7001o when 0.5 mol/l d-fenfluramine was used; 4) the releasing action of 0.5 mol/l d-fenfluramine was completely Ca⁺⁺-dependent, while at higher dfenfluramine concentrations the Ca⁺⁺-independent overflow became more important. The fluoxetine induced overflow was mainly. (70010) Ca⁺⁺-independent; 5) the releasing acitvity of d-fenfluramine was mainly (80%) blocked by the serotonin uptake blockers indalpine, midalcipram and also fluoxetine whereas fluoxetine-induced overflow was insensitive to inhibition of the serotonin carrier.In conclusion, the releasing activity of d-fenfluramine is already present at a very low concentration (0.5 mol/l) and at this concentration its mechanism of action was Ca⁺⁺-dependent, together with the requirement of a functional serotonin carrier. These data therefore do not support the hypothesis of a simple. displacement of 5-HT from its storage vesicles but suggest an exocytotic release possibly triggered by interaction of d-fenfluramine with intracellular receptors. A direct releasing activity is also shown for fluoxetine, very marked at 5–10 mol/l; such effect is different from that of d-fenfluramine and is probably due to the overflow of 5-hydroxyindoleacetic acid, formed in the synaptosomes after the fluoxetine-induced displacement of serotonin from its storage vesicles. The active concentrations of fluoxetine on serotonin release are compatible with those found in rat brain at doses inducing an anorectic activity. Send offprint requests to M. Gobbi at the above address     

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

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

Protein Aggregates Seem to Play a Key Role Among the Parameters Influencing the Antigenicity of Interferon Alpha (IFN-α) in Normal and Transgenic Mice

Purpose. During long-term treatment of various malignant or viral diseases with IFN- up to 20% of patients develop anti-IFN- antibodies for as yet unknown reasons. Methods. To address this issue, a mouse model using Balb/C mice was established and the relevance of several potentially anti-IFN- antibodies inducing factors was studied. Results. The model revealed that both a higher frequency of injections and a higher dosage of IFN- were more immunogenic and that the route of administration affected the antibody response to IFN-. The intrinsic immunostimulatory activity of IFN- itself also enhanced the immune response. IFN- protein aggregates (IFN--IFN- and human serum albumin (HSA)-IFN- aggregates), which were recently identified in all marketed IFN- products, were significantly more immunogenic than IFN- monomers. These aggregates broke the tolerance against human IFN- monomers in human IFN- transgenic mice. Conclusions. Based on these animal studies it is proposed that the immune response to IFN- in humans is most probably elicited by a combination of several factors among which IFN- protein aggregates seem to play a key role.     

Wirkung von cyclischem Adenosin-3′,5′-Monophosphat (3′,5′-AMP) und seinem Dibutyrylderivat (DBA) auf Lipolyse,Glykogenolyse und Corticosteronsynthese

Zusammenfassung 1. Am isolierten Fettgewebe von Ratten hatte das Dibutyrylderivat des cyclischen Adenosin-3,5-Monophosphat (DBA) eine etwa 100 mal stärkere lipolytische Wirkung als das nicht substituierte cyclische Adenosin-3,5-Monophosphat (3,5-AMP). Hormone (ACTH, Noradrenalin) waren an diesem Testobjekt 10000 mal wirksamer als DBA. Durch Hemmung der Phosphodiesterase mit Theophyllin ließ sich auch die Wirkung des DBA verstärken.2. An isolierten Nebennieren von Ratten stimulierte DBA die Corticosteronsynthese etwa 100 mal stärker als 3,5-AMP; ACTH war aber 500 mal wirksamer als DBA. Durch Theophyllin ließ sich die Wirkung von ACTH, DBA bzw. 3,5-AMP nicht verstärken. Hohe Konzentrationen des Xanthinderivates hemmten die Corticosteronsynthese.3. An Ratten war die hyperglykämische Wirkung des DBA wesentlich stärker als diejenige des 3,5-AMP: Für eine Erhöhung des Blutzuckerspiegels um 40 mg/100 ml benötigten wir von DBA weniger als 1 mol/kg, von 3,5-AMP aber 30 mol/kg. Diese Wirkung der Nucleotide ließ sich durch Theophyllin nicht verstärken. Der Fettsäuren- und Glyceringehalt des Plasmas wurde durch Injektion von DBA bzw. 3,5-AMP nicht erhöht, sondern erniedrigt. — Die Ergebnisse wurden im Zusammenhang mit dem Second Messenger Concept von Sutherland u. Mitarb. diskutiert.Über einen Teil der Ergebnisse wurde auf der 8. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft (Stock u. Westermann, 1967; Bieck u. Westermann, 1967) sowie in einer kurzen Mitteilung (Bleck et al., 1968) berichtet.     

Fenofibrate decreases asymmetric dimethylarginine level in cultured endothelial cells by inhibiting NF-κB activity

Previous investigations have demonstrated that endogenous inhibitors of nitric oxide synthase (NOS), such as asymmetric dimethylarginine (ADMA), contribute importantly to endothelial dysfunction, and that fenofibrate has a protective effect on the endothelium in rats treated with low-density lipoprotein (LDL) by reducing ADMA levels. In the present study, we explored further the possible mechanism underlying inhibition of ADMA generation by fenofibrate in cultured human umbilical vein endothelial cells (HUVECs). Endothelial injury was induced in cultured HUVECs by incubation with oxidative LDL (ox-LDL) and the levels of ADMA, lactate dehydrogenase (LDH), NO and tumour necrosis factor- (TNF-) in the conditioned medium were measured. Cell viability and the activity of dimethylarginine dimethylaminohydrolase (DDAH) and nuclear factor-B (NF-B) in the cultured HUVECs were also determined. Incubation of HUVECs with ox-LDL (100 g/ml) for 24 h markedly elevated ADMA, LDH and TNF- in the conditioned medium and significantly increased the activity of NF-B, concomitantly with a significant decrease in the activity of DDAH and the content of NO. Pretreatment with fenofibrate (3, 10 or 30 M) significantly inhibited the increases in ADMA, LDH and TNF-, attenuated the decreased levels of NO and the decreased activity of DDAH and prevented the activation of NF-B. Similar effects were observed in the presence of pyrrolidine dithiocarbamate (PDTC, 10 M), an antagonist of NF-B. The beneficial effects of fenofibrate on cultured endothelial cells were abolished by MK-886, a specific peroxisome proliferator-activated receptor- (PPAR) antagonist. The present results suggest that fenofibrate inhibits ox-LDL-induced endothelial cell damage by decreasing ADMA and increasing DDAH activity, and the protective effects of fenofibrate on endothelial cells may be related to reduction of NF-B activity by activation of the PPAR receptor.     

Increase of serum very low density lipoproteins in rats after administration of α-hexachlorocyclohexane

Summary After enteral administration of 200 mg/kg -hexachlorocyclohexane (-HCH) female Wistar rats develop a hyperlipemia. 48 h after administration of -HCH, serum triglycerides are increased by 300%, whereas both serum cholesterol and serum total phospholipids only increase by about 45%. Serum free fatty acids are not significantly altered. Fractionation of the serum lipoproteins by ultracentrifugation shows that the hyperlipemia is due to a fivefold increase in serum very low density lipoproteins.Hepatic triglyceride secretion, calculated after i.v. injection of Triton WR 1339, is increased in animals pretreated with -HCH. Corresponding to this observation, drugs known to diminish the triglyceride secretion of the liver, such as actinomycin D, cycloheximide; glucagon, orotic acid, CFT 1201, and CFT 1042 reduce the -HCH-induced hyperlipemia.We concluded from the results that hyperlipoproteinemia after -HCH is due to an increased hepatic very low density lipoprotein secretion.At the same time, the blood sugar level was decreased in fasting animals after treatment with -HCH. Earlier experiments suggest that this effect is due to a decreased gluconeogenesis in the liver.The results have already been presented in part at the Pharmacology Meeting Graz 1974 (Grajewski and Oberdisse, 1974)     

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.     

A Candidate for Cancer Gene Therapy: MIP-lα Gene Transfer to an Adenocarcinoma Cell Line Reduced T\imorigenicity and Induced Protective Immunity in Immunocompetent Mice

Purpose. To evaluate the possibility of cancer gene therapy by the gene delivery of chemokine, the effects of human macrophage inflammatory protein l (hu-MIP-l), murine-macrophage inflammatory protein l (mu-MIP-l), and human-interleukin 8 (hu-IL-8) on tumor progression and immunization were studied. Methods. Cachexia-inducing and highly tumorigenic adenocarcinoma cells (cell line colon 26, clone 20) were transfected with either a control plasmid, hu-MIP-l, mu-MIP-l, or hu-IL-8 expression vector. The production of hu-MIP-1 reached >1.5 ng/ml in vitro when transfectant cells were cultured at a cell density of 2 × 10⁵ cells in 7 ml for 3 days. Immunocompetent BALB/c mice were inoculated into the footpad with the tumor cells, and then primary tumor growth, morphological analyses, and tumor immunogenicity were studied. Results. The secretion of hu-MIP-l, mu-MIP-l, and hu-IL-8 did not affect the growth rate in vitro. Reduced tumorigenicities in vivo were observed in transfected cells with hu-MIP-l and mu-MIP-l. Morphologic observation of the site of inoculation of cells transfected with hu-MIP-l showed infiltration of macrophages and neutrophils on the 5th day after the inoculation. Mice that had rejected cells transfected with hu-MIP-l gene were immune to a subsequent challenge with the parental cells. Conclusions. The rejection of the cells depends on cytolysis and generates potent and long lasting antitumor immunity. These data suggest that tumor cells transfected with the MIP-l gene might be useful as an effective therapy for the treatment of certain tumors.     

Effects of deuterium substitution on the chronotropic responses to some sympathomimetic amines in the isolated rat atria

Summary In spontaneously beating rat atria the potencies for the chronotropic effects of the following deuterated phenylethylamine derivatives were higher than the potencies of the corresponding non-substituted (protio-) amines: ,,d₂--phenylethylamine; ,,,-d₄-p-tyramine; ,,,-d₄-m-tyramine; ,,-d₃-p-octopamine. In contrast, ,,-d₃-noradrenaline and ,,-d₃-m-octopamine were equipotent with the corresponding protio-amines. Experiments performed in atria depleted of endogenous noradrenaline by pretreatment with reserpine and in atria exposed to the monoamine oxidase (MAO) inhibitor pargyline indicated: a. p-octopamine had both direct and indirect effects, but the chronotropic responses to p-octopamine in tissues with normal MAO activity depended mostly on the direct action of the amine; deuterium substitution enhanced the indirect component of action of p-octopamine; b. m-octopamine possessed considerable indirect effects while d₃-m-octopamine behaved as an amine of direct action. The substitution of deuterium for hydrogens in the -carbon of the alkyl-side chain of phenylethylamines decreases the rate of deamination by MAO. Therefore, the results obtained with all the amines, except for m-octopamine and ,,p-d₃-m-octopamine, could be interpreted in terms of the direct, indirect or mixed action of those compounds and/or of the influence that MAO activity has on the chronotropic responses to these amines. The results obtained with protio-and deuterio-m-octopamine suggested that deuterium substitution, either at the - or the -carbon, can alter some other mechanisms in addition to the enzymatic deamination.Career Investigator on leave of absence from the Consejo de Investigaciones Cientificas y Técnicas, ININFA, Junín 956, 5°P, RA-1113 Buenos Aires, Argentina Send offprint requests to S. M. Celuch     

Incorporation of the biomarker concept in ecotoxicology calls for a redefinition of terms

Biomarkers have received a lot of attention but the biomarker concept has not yet been defined properly. As a consequence, various interpretations of the term biomarker exist, some of which overlap with well-established ecotoxicological concepts. To allow incorporation of the biomarker concept within the framework of modern ecotoxicology, a clarification of terms is needed. In this paper, definitions are presented for the terms biomarker, bioindicator and ecological indicator, which do not overlap and are linked to different levels of biological organization. The use of these concepts for ecological effects assessment is discussed.     

Opposite interactions between α- and β-endorphin fragments with dopamine mediated responses on the rat rectum in vitro

Summary Dopamine causes a dose-dependent contraction of the rat rectum in vitro followed by a relaxation. This contraction can be inhibited by apomorphine and phenylephrine. This inhibition can be attenuated by the -endorphin (E) fragments 2–17 (des-Tyr¹--endorphin, DTE) and 6-17 (des-enkephalin--endorphin, DEE). E 6-17 seems to be the shortest sequence with full activity in this respect since a shorter fragment (E 10-17) was less effective. The atypical neuroleptics oxypertine, sulpiride, and clozapine, the classic neuroleptic haloperidol and metoclopramide have a similar action to DEE. The peptides and atypical neuroleptics do not affect the dopamine response per se while the classic neuroleptics haloperidol and metoclopramide enhance the dopamine response.The effects of the -type endorphins are opposite to those of the -type endorphins, since des-Tyr¹--endorphin (DTE, E 2-16) and des-enkephalin--endorphin (DEE, E 6-16) enhance the phenylephrine-induced decreased responsiveness to dopamine. Structure-activity studies revealed that the active moiety of the -endorphin fragments probably resides in the 6–9 region. In addition the -type endorphins directly inhibit the dopamine response.It is concluded that the rat rectum may be used to analyse neuroleptic-like action. In this model - and -endorphin fragments may directly or indirectly influence the interaction of dopamine with the rectum. Because of the strong similarities between the effects of -type endorphins and that of neuroleptics the results support the purported neurolepticlike action of -type endorphins. The influence of -type endorphins and -type endorphins on the apomorphine or phenylephrine induced decreased responsiveness to dopamine, although opposite, seems to be mediated by an influence on different dopamine sensitive systems.     

Relationship of metabolism of 2′-, 3′- and 5′-adenine nucleotides to presynaptic inhibition of transmitter release in rat vas deferens

Summary In the isolated rat vas deferens stimulated at 0.2 Hz, a series of 2, 3-, and 5-substituted adenine nucleotides all inhibited the twitch responses, their actions being potentiated by the nucleoside transport inhibitors, HNBTGR, NBMPR and dipyridamole.The metabolism of these nucleotides was examined utilising HPLC analysis of the bathing medium after exposure to 30 M nucleoside or nucleotide for 5 min. 5-AMP, 5-ADP, 5-ATP, and NAD⁺ were all partially hydrolysed to adenosine, the relative extent of this being 5-AMP>5-ADP=5-ATPNAD⁺. However, the other nucleotides examined were not detectably converted to adenosine or to adenosine deamination products.These results indicate that the 2-, 3- and 5-substituted nucleotides studied act at a P₁-purinoceptor in rat vas deferens to inhibit neurotransmission and, with the exception of 5-AMP, 5-ADP, 5-ATP and NAD⁺, all appear to act directly at this receptor. However, the 5-adenine nucleotides (AMP, ADP and ATP) and NAD⁺ all appear to act at least partially indirectly subsequent to their hydrolysis to adenosine.Abbreviations. The following abbreviations are used ADA adenosine deaminase (EC 3.5.4.4) - 5-ADP adenosine 5-diphosphate - 2,5-ADP adenosine 2,5-diphosphate - 3 5-ADP, adenosine 3,5-diphosphate - 2-, 3 or 5-AMP adenosine 2-, 3-, or 5-monophosphate - 5-ATP adenosine 5-triphosphate - cNADP⁺ -nicotinamide dinucleotide 2,3-cyclic monophosphate - CoA coenzyme A - HNBTGR 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine - NAD⁺ -nicotinamide adenine dinucleotide - NADP⁺ -nicotinamide adenine dinucleotide phosphate - NBMPR 6-(4-nitrobenzylthio)-purine riboside     

Effects of adenosine derivatives on cAMP accumulation and lipolysis in rat adipocytes and on adenylate cyclase in adipocyte plasma membranes

Summary N⁶-monosubstituted adenosine (Ad)-derivatives and Ad-derivatives altered in the adenine-or ribose-moiety have been compared with Ad in their effects on noradrenaline (NA)-stimulated cAMP accumulation, on lipolysis stimulated by NA or theophylline (THEO) and on adenylate cyclase (AC) activity of adipocyte plasma membranes.In isolated adipocytes about 0.01 M Ad caused a 50% inhibition of cAMP accumulation stimulated maximally by 1M NA. Depending upon the structure of substituent, the Ad-N⁶-derivatives were up to one order of magnitude either more or less active than Ad itself. 2-fluoro-Ad was nearly as active as Ad, whereas 2,5-dideoxy-Ad and 2-deoxy-Ad were practically ineffective as inhibitors of NA-stimulated cAMP accumulation. All compounds showed the same order of potency relative to Ad, when tested against lipolysis stimulated maximally by 1 mM THEO or submaximally by 0.3 M NA.In adipocyte plasma membranes a 50% inhibition of AC activity stimulated by 10M NA was observed at about 10 M Ad. None of the N⁶-substituted derivatives had any effect on either basal or NA-stimulated AC activity, whereas 2,5-dideoxy-Ad proved to be about 40 times more potent than Ad. 2-deoxy-Ad and 2-fluoro-Ad were nearly equipotent to Ad. Similar results were obtained, if AC was stimulated with 5-guanylylimidodiphosphate or NaF. Neither the N⁶-derivatives nor THEO could reverse the inhibitory effect of Ad on AC in plasma membranes.It is concluded, that different mechanisms are involved in the inhibitory effects of Ad on cAMP accumulation and lipolysis in intact cells and on AC activity of adipocyte plasma membranes.     

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%     

Development and Use of Enzyme-Linked Immunosorbent Assays (ELISA) for the Detection of Protein Aggregates in Interferon-Alpha (IFN-α) Formulations

Purpose. Protein aggregates are thought to be involved in the immunogenicity of recombinant proteins in humans. To probe human IFN- formulations for the presence of soluble protein aggregates, enzyme-linked immunosorbent assays (ELISA) were developed. Methods. For the detection of IFN--IFN- and HSA-IFN- aggregates, sandwich ELISAs were developed using a monoclonal anti-IFN- antibody as a capture antibody and the same anti-IFN- antibody and an anti-human serum albumin (HSA) antibody (HRP-labeled), respectively. Results. Marketed freeze-dried, HSA-containing IFN- formulations tested in the ELISAs all contained IFN--IFN- and/or HSA-IFN- protein aggregates, although in varying amounts. These aggregates were predominantly IFN- dimers and 1:1 conjugates of HSA with IFN-. Test formulations revealed that aggregation of IFN- was strongly affected by the presence of pharmaceutical excipients, pH of the formulation, lyophilisation procedure, and storage temperature and time. Conclusions. The ELISAs are rapid, highly specific for aggregates in the presence of both IFN- and HSA monomers and allow the direct detection of both types of aggregates in formulations in the nanogram range. The new assays will assist the monitoring of the aggregate-inducing processes during IFN- formulation and storage in an early phase and the development of aggregate-free IFN- formulations.     

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.     

Involvement of α-adrenoceptors in the excitatory effect of the A2 adenosine receptors agonist 5′-N-ethylcarboxamidoadenosine (NECA) on cardiac automaticity in the isolated right ventricle of the rat

The effects of the non-selective A₂ adenosine receptor agonist 5-N-ethyl-carboxamidoadenosine (NECA) were studied on ventricular automaticity induced by a local injury in the isolated right ventricle of the rat. In concentrations ranging from 0.1 to 100 nM, NECA significantly increased ventricular automaticity. This effect was not apparent when the nonselective -adrenoceptor blocker phenoxybenzamine was present at a concentration of 10 M, which antagonizes both ₁-and ₂-adrenoceptors, as well as when rats were pretreated with reserpine. In non-reserpinized rats, the excitatory effect of NECA was also abolished in the presence of the selective ₁-adrenoceptor antagonist prazosin, but not in the presence of the ₂-adrenoceptor antagonist idazoxan. In reserpinized rats, the excitatory effect of NECA was restored in the presence of the non specific -adrenoceptor agonist phenylephrine as well as in the presence of the selective ₁-adrenoceptor agonist amidephrine but not in the presence of the selective ₂-adrenoceptor agonist clonidine. These results suggest that the excitatory effect of NECA on ectopic ventricular automaticity is dependent on endogenous catecholamines and that -adrenoceptors of type 1 are, in some way, involved in this effect.     

A comparison of the effects of TMB-8 and nifedipine on pressor responses to α1- and α2-adrenoceptor agonists in pithed rats

TMB-8 has been characterized as an inhibitor of the release of Ca⁺ from intracellular pools. We have studied the modification of the pressor responses to selective _l-adrenoceptor agonists (methoxamine and phenylephrine), and to selective ₂-adrenoceptor agonists (B-HT 920 and B-HT 933) in pithed rats, produced by TMB-8. We have compared this modification with that produced by the calcium antagonist nifedipine. Nifedipine (100 g/kg, 300 g/kg, and 1000 g/kg) inhibited in a dose-dependent manner the pressor responses to the ₁- and ₂-adrenoceptor agonists, the dose-response curves to the ₂-adrenoceptor agonists being shifted further to the right. TMB-8 at a dose of 3000 g/kg did not modify the pressor effects of the _l-adrenoceptor agonists, and neither did it reinforce the inhibition of such responses produced by nifedipine. By contrast, TMB-8 pretreatment (0.03 g/kg, 0.3 g/kg, 3 g/kg, 30 g/kg, 300 g/kg and 3000 g/kg) inhibited the responses to both ₂-adrenoceptor agonists, the inhibition being more pronounced with B-HT 920. A similar effect was obtained with 0.03 g/kg TMB-8 and 0.3 g/kg TMB-8, particularly in the case of B-HT 920. It was stronger with higher doses, but similar for all doses over 3 g/kg. The inhibition of the pressor responses mediated by the stimulation of ₂-adrenoceptors by TMB-8 was less in rats treated with the Ca²⁺ entry promoter BAY K 8644 (300 g/kg), and could also be reduced by the continuous infusion of CaCl₂ (0.25 g/min). These results suggest that in pithed rats TMB-8 may also behave as an inhibitor of the Ca⁺ influx into vascular smooth muscle.     

Metabolism and presynaptic inhibitory activity of 2′,3′ and 5′-adenine nucleotides in rat vas deferens

Summary In the isolated rat vas deferens stimulated at 0.2 Hz, [¹⁴C]labelled 5-AMP, 5-ADP and 5-ATP (10 M) inhibited twitch responses, were broken down to [¹⁴C]adenosine in the medium and incorporated into [¹⁴C]adenine ribonucleotides in the tissue. Pretreatment of tissues with 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine (NBTGR), a potent inhibitor of adenosine transport, potentiated the presynaptic inhibitory action of these 5 nucleotides and reduced their incorporation in [¹⁴C]adenine nucleotides, but did not alter the appearance of [¹⁴C]adenosine in the medium.A series of 2, 3 and 5-substituted adenine nucleotides (10 M) inhibited the twitch responses of the vas deferens stimulated at 0.2 Hz. This effect was potentiated by NBTGR. Addition of exogenous adenosine deaminase very significantly reduced the inhibitory actions of adenosine, 5-AMP, 5-ADP and 5-ATP and also reduced those of 2, 5-ADP, NAD⁺ and dePCoA. The inhibitory actions of the other 2, 3 and 5 adenine nucleotides studied were not altered by exogenous adenosine deaminase.These results indicated that the presynaptic inhibitory actions of 5-AMP, 5-ADP and 5-ATP in rat vas deferens predominantly result from their prior hydrolysis to adenosine whereas the 2, 3 and 5-substituted adenine nucleotides appear to act mainly directly to inhibit transmitter release.Abbreviations. The following abbreviations are used 5-ADP 5-adenosine diphosphate - 2,5-ADP 2,5-adenosine diphosphate - 3,5-ADP 3,5-adenosine diphosphate - 2,3 or 5-AMP 2,3 or 5-adenosine monophosphate - 5-ATP 5-adenosine triphosphate - CoA coenzyme A - 2,3-cAMP 2,3-cyclic adenosine monophosphate - cNADP⁺ -nicotinamide dinucleotide 2,3-cyclic monophosphate - dePCoA dephosphocoenzyme A - NAD⁺ -nicotinamide adenine dinucleotide - NADP⁺ -nicotinamide adenine dinucleotide phosphate - NBTGR 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine - oxid CoA oxidized-coenzyme A