A comparison of agonist-specific coupling of cloned human alpha(2)-adrenoceptor subtypes.

The agonist-specific coupling properties of the three cloned human alpha(2)-adrenoceptor subtypes have been compared, when expressed at similar levels in Chinese hamster ovary (CHO) cell lines, using noradrenaline and (+/-)-meta-octopamine as agonists. Noradrenaline can couple the receptor to both the inhibition and stimulation of forskolin-stimulated cyclic AMP production in all three receptor subtypes, with the relative strength of the coupling to the pathways varying for each of the receptor subtypes. meta-Octopamine selectively couples the alpha(2A)-adrenoceptor only to the inhibition of forskolin-stimulated cyclic AMP production. However, meta-octopamine couples the alpha(2B)- and alpha(2C)-adrenoceptors to both the inhibition and stimulation of forskolin-stimulated cyclic AMP production. The relative potency of meta-octopamine to noradrenaline varies between the different alpha(2)-adrenoceptor subtypes. The effects of meta-octopamine are around two orders of magnitude less potent than those of noradrenaline on both the alpha(2A)- and alpha(2B)-adrenoceptor subtypes. In contrast, in the case of the alpha(2C)-adrenoceptor, meta-octopamine is only one order of magnitude less potent than noradrenaline in the stimulation of forskolin-stimulated cyclic AMP production and, in addition, is equipotent with noradrenaline in the inhibition of forskolin-stimulated cyclic AMP production and has an increased maximal response. This raises the possibility that meta-octopamine may have physiologically important actions via alpha(2C)-adrenoceptors in vivo. The results show that the modulation of cyclic AMP production occurs in both a subtype- and agonist-specific manner for alpha(2A)-adrenoceptors and in a subtype specific manner for alpha(2B)- and alpha(2C)-adrenoceptors.

The effect of site-directed mutagenesis of two transmembrane serine residues on agonist-specific coupling of a cloned human alpha2A-adrenoceptor to adenylyl cyclase.

1. The effects of substitution of the Ser200 and Ser204 residues with alanine on the signalling properties of the cloned human alpha2A-adrenoceptor, stably expressed in Chinese hamster ovary (CHO) cell lines, have been investigated using noradrenaline and the structural isomers of octopamine. 2. The Ser-->Ala200 or the Ser-->Ala204 mutant forms of the alpha2A-adrenoceptor, when expressed in cells in the absence of pertussis toxin pretreatment, are two orders of magnitude more sensitive to inhibition of cyclic AMP production by (+/-)-para-octopamine and (+/-)-meta-octopamine, respectively, than cells expressing the wild-type receptor. Binding studies indicate that the effects are not due to an increased agonist affinity for the mutant receptors and that they are likely to be due to agonist-mediated conformational changes in receptor structure. 3. After incubation with pertussis toxin, (+/-)-meta-octopamine (100 microM and above) produced a stimulation of cyclic AMP levels in cells expressing the Ser-->Ala204 mutant form of the alpha2A-adrenoceptor but showed no stimulation in cells expressing the Ser-->Ala200 mutant receptor. Under these conditions (+/-)-para-octopamine did not produce any increases in cyclic AMP production in cells expressing either of the mutant receptor forms or the wild-type receptor. 4. The results emphasise the importance of the Ser200 and Ser204 residues of the alpha2A-adrenoceptor in exerting an inhibitory influence on the ability of (+/-)-para-octopamine and (+/-)-meta-octopamine respectively, to induce a receptor-agonist conformation capable of inhibiting forskolin-stimulation of cyclic AMP levels. 5. It is clear that Ser204 also prevents meta-octopamine from generating a receptor-agonist conformation that can increase cyclic AMP levels, emphasising the importance of this residue in the agonist-specific coupling of this receptor to different second messenger systems.

Selective inhibition of adenylyl cyclase by octopamine via a human cloned alpha 2A-adrenoceptor.

1. In this study we have compared the abilities of the enantiomers of the structural isomers of the phenolamines, octopamine and synephrine, and the catecholamines, noradrenaline and adrenaline, to couple selectively a human cloned alpha 2A-adrenoceptor, stably expressed in a Chinese hamster ovary (CHO) cell line, to G-protein linked second messenger pathways mediating an increase and a decrease in cyclic AMP production. 2. The catecholamines couple the alpha 2A-adrenoceptor to both an increase and a decrease in the rate of cyclic AMP production. In the absence of pertussis toxin pretreatment both catecholamines tested showed a dose-dependent decrease with a maximum at 100 nM. After pertussis toxin pretreatment they both produced a dose-dependent increase in cyclic AMP production with a maximum at 10 microM. 3. The phenolamines, octopamine and synephrine were only able to couple the alpha 2A-adrenoceptor to a dose-dependent decrease in cyclic AMP production at concentrations up to 1 mM, with the synephrine isomers being more potent than the corresponding octopamine isomers. The meta-isomers of both phenolamines were more potent than the corresponding para-isomers and the (-)-enantiomers were more potent than the (+)-enantiomers. Thus, (-)-meta-synephrine [(-)-phenylephrine] was the most effective isomer tested with an observable decrease occurring between 100 nM and 1 microM. 4. The effects of octopamine and the catecholamines on the decrease in cyclic AMP production were additive at submaximal concentrations, whilst octopamine reduced the stimulant effect of submaximal concentrations of noradrenaline on cyclic AMP production after pertussis toxin pretreatment. 5. The time courses of the inhibitory effects of both meta-octopamine and noradrenaline were parallel and peaked after a 1 min exposure to the agonist. In contrast, the stimulant effects of noradrenaline after pertussis toxin pretreatment were of a much slower time course with a maximum effect occurring after a 5 min incubation period. 6. Since octopamine and synephrine occur naturally in, and are co-released with catecholamines from, mammalian tissues, the results of the present study suggest that the human cloned alpha 2A-adrenoceptor can be coupled selectively by different endogenous agonists to G-protein pathways mediating the regulation of adenylyl cyclase activity.

Improvement of activated carbon for air sampling.

In order to improve the charcoal tube method, activated carbon was treated with benzyl chloride (5 and 20%) in methylene chloride and heated (300 degrees C). The carbons were evaluated with regards to water uptake, adsorption of toluene and carbon tetrachloride, desorption characteristics for polar compounds and storage stability of ketones. Carbons treated with benzyl chloride picked up less moisture, showed higher desorption efficiencies for polar compounds and lower catalytic activity compared with untreated carbons and those heat-treated with helium at 800 degrees C. The best results were obtained with 20% benzyl chloride.

A filter method for the active and passive monitoring of sulfur dioxide in workplace air.

A method where sulfur dioxide is collected on impregnated filters (glycerol/potassium hydroxide solution) is described. Sampling can be done either with a pump or by the use of two different passive monitors available on the market. Analysis is made by ion chromatography. The methods have been evaluated and compared with a colorimetric air monitoring badge system (ProTek). Laboratory tests show that the accuracy of the filter methods is acceptable and that samples can be stored. Water vapor does not interfere, but hydrogen sulfide causes a minor decrease in recovery. ProTek has high accuracy but storage tends to decrease the recovery. Field tests in a steel rolling mill and a sulfate pulp mill showed a fairly good correspondence between the methods.

Development and evaluation of field methods for ammonia in air.

Three methods for measuring ammonia in air have been evaluated. Filters impregnated with 10% (volume/volume) phosphoric acid in methanol were used for two methods. Sampling was done either with a filter cassette connected to a pump or with the filters placed in a passive monitor (Gasbadge). The filters were leached with distilled water after the sampling and analyzed with ion chromatography or colorimetry (Nessler). The third method tested was a colorimetric air-monitoring badge system (ProTek). The tests showed that the accuracy of the filter methods is good and that the results are not affected by humidity. If ion chromatography is used for the analysis, amines do not interfere. The Gasbadge monitors increased their uptake when the air velocity over the sampler was raised from 0.2 to 1.0 m/s. The accuracy of the ProTek method was poor, the method was biased, and blank samples showed high values. In field tests carried out in a foundry and at a fertilizer plant, the agreement between the filter methods was good, whereas the results of the ProTek method deviated drastically from those of the other methods.