J. M. Midgley

Activities of octopamine and synephrine stereoisomers on α‐adrenoceptors

1 The activities of the (‐)‐ and (+)‐forms of m‐ and p‐octopamine and m‐ and p‐synephrine on α1‐adrenoceptors from rat aorta and anococcygeus and α2‐adrenoceptors from rabbit saphenous vein were compared with those of noradrenaline (NA). 2 The rank order of potency of the (‐)‐ forms on α1‐adrenoceptors from rat aorta and α2‐adrenoceptors was NA > m‐octopamine = m‐synephrine > p‐octopamine = p‐synephrine. The two m‐compounds were 6 fold less active than NA on α1‐adrenoceptors from rat aorta and 150 fold less active on α2‐adrenoceptors. The two p‐compounds were 1,000 fold less active than NA on both α1‐adrenoceptors from rat aorta and α2‐adrenoceptors. The rank order of potency of the (‐)‐ forms on α1‐adrenoceptors from rat anococcygeus was NA = m‐synephrine > m‐octopamine > p‐octopamine = p‐synephrine. m‐Octopamine was 4 fold less active than NA and (‐)‐m‐synephrine. The two p‐compounds were 30 fold less active than NA. 3 The rank order of potency of the (+)‐ forms was NA > m‐octopamine > m‐synephrine > p‐octopamine > p‐synephrine on both α1‐ and α2‐adrenoceptors. The potency of each (+)‐ form was 1–2 orders of magnitude less than that of the (‐) counterpart, the differences being greater for the stereoisomers of synephrine than for those of octopamine on both α1‐ and α2‐adrenoceptors. 4 The yohimbine diastereoisomer antagonists, rauwolscine and corynanthine, were tested against (‐)‐NA and (‐)‐m‐octopamine‐induced contractions in both preparations. Based upon the known selectivities of these isomers for α‐adrenoceptor subtypes, it is concluded that the rat aorta contains only α1‐adrenoceptors while the rabbit saphenous vein possesses predominantly α2‐adrenoceptors. 5 Ligand binding data for the octopamine and synephrine stereoisomers at α1‐ and α2‐binding sites from rat cerebral cortex was also obtained. (‐)‐Forms were more active than (+)‐forms. The rank order of affinity of the (‐)‐forms for both α1‐ and α2‐binding sites was NA > m‐octopamine = m‐synephrine > p‐synephrine > p‐octopamine. The relative affinities of the members of the series against α1‐binding sites were very similar to their relative functional activities on rat aorta. However, the affinities of both m‐ and p‐compounds relative to that of (‐)‐NA were much greater at the α2‐binding sites than were the relative activities in rabbit saphenous vein, possibly suggesting low intrinsic efficacy. Functional antagonist responses to NA by the (‐)‐octopamine and synephrines could not, however, be demonstrated on rat aorta or rabbit saphenous vein. 6 The activities of m‐octopamine and m‐synephrine were not significantly different from each other on either α1‐adrenoceptors from rat aorta or α2‐adrenoceptors; however, m‐synephrine is more active than m‐octopamine on α1‐adrenoceptors from rat anococcygeus. Both m‐octopamine and m‐synephrine can be considered to be naturally occurring α1‐selective amines. However, if m‐ and p‐octopamine are co‐released with NA in amounts proportional to their concentration, it is concluded that their activities on α1‐ and α2‐adrenoceptors are too low to be physiologically significant.

Penetration of synthetic corticosteroids into human aqueous humour

The penetration of prednisolone acetate (1%) and fluorometholone alcohol (0.1%) into human aqueous humour following topical application was determined using the very sensitive and specific technique of Gas Chromatography with Mass Spectrometry (GCMS). Prednisolone acetate afforded peak mean concentrations of 669.9 ng/ml within two hours and levels of 28.6 ng/ml in aqueous humour were detected almost 24 hours post application. The peak aqueous humour level of fluorometholone was 5.1ng/ml. The results are compared and contrasted with the absorption of dexamethasone alcohol (0.1%), betamethasone sodium phosphate (0.1%) and prednisolone sodium phosphate (0.5%) into human aqueous humour.

Selective inhibition of adenylyl cyclase by octopamine via a human cloned α2A-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 α2A‐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 α2A‐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 μM. 3 The phenolamines, octopamine and synephrine were only able to couple the α2A‐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 μM. 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 α2A‐adrenoceptor can be coupled selectively by different endogenous agonists to G‐protein pathways mediating the regulation of adenylyl cyclase activity.

The expression of a cloned Drosophila octopamine/tyramine receptor in Xenopus oocytes

The expression of a cloned Drosophila octopamine/tyramine receptor (OctyR99AB) is described in Xenopus oocytes. Agonist stimulation of OctyR99AB receptors increased intracellular Ca2+ levels monitored as changes in the endogenous inward Ca2+-dependent chloride current. The receptor is preferentially sensitive to biogenic amines with a single hydroxyl on the aromatic ring. The G-protein, Galphai, appears to be involved in the coupling of the receptor to the production of intracellular calcium signals, since the effect is pertussis-toxin sensitive and is blocked or substantially reduced in antisense knockout experiments using oligonucleotides directed against Galphai but not by those directed against Galphao, Galphaq and Galpha11. The increase in intracellular calcium levels induced by activation of the OctyR99AB receptor can potentiate the ability of activation of a co-expressed beta2-adrenergic receptor to increase oocyte cyclic AMP levels. A comparison of the pharmacological coupling of OctyR99AB to different second messenger systems when expressed in Xenopus oocytes with previous studies on the expression of the receptor in a Chinese hamster ovary cell line suggests that the property of agonist-specific coupling of the receptor to different second messenger systems may be cell-specific, depending upon the G-protein environment of any particular cell type.

β-Adrenergic activities of octopamine and synephrine stereoisomers on guinea-pig atria and trachea

The activities of the (‐)‐ and (+)‐forms of m‐ and p‐octopamine and m‐ and p‐synephrine on β1‐ and β2‐ adrenoceptors in guinea‐pig atria and trachea have been compared with that of noradrenaline. The rank order of potency of the (‐)‐forms on β1‐adrenoceptors was noradrenaline > m‐synephrine. m‐octopamine = p‐octopamine > p‐synephrine. m‐Synephrine was 100‐fold, m‐and p‐octopamine about 6000‐fold, and p‐synephrine about 40 000‐fold less active than noradrenaline. The (+)‐forms were 1‐2 orders of magnitude less active than their (‐)‐counterparts. The four (‐)‐compounds were more than four orders of magnitude less active than noradrenaline on β2,‐adrenoceptors, and the (+)‐forms had no detectable activity in concentrations as high as 10−4 M. If m‐and p‐octopamine are co‐released with noradrenaline in amounts proportional to their concentration, their activities at these structures are too low to be physiologically significant.

m‐Octopamine: Normal Occurrence with p‐Octopamine in Mammalian Sympathetic Nerves

The development of a radiochemical enzyme assay for p‐octopamine in 1969 led to its identification in a large number of invertebrate nerve systems and in mammalian sympathetic nerves. The original method by which p‐octopamine was measured has now been found to be nonspecific; however, modifications of this procedure can determine both m‐ and p‐octopamine. We recently developed a new specific method for the unequivocal identification and quantitative determination in tissue of the six octopamine and synephrine isomers. With this method—negative chemical ionization gas chromatography‐mass spectrometry—the more physiologically active m‐octopamine has been found in association with p‐octopamine in 10 organs of the rat. m‐Octopamine is present in concentrations equal to those of p‐octopamine in heart, spleen, and liver and in concentrations from 30 to 60% of p‐octopamine in adrenals, vas deferens, brain, kidney, large intestine, bladder, and lungs. In vivo inhibition of monoamine oxidase markedly increased the concentrations of both m‐ and p‐octopamine in all organs examined. Both amines were virtually absent from all organs except the adrenals following chemical sympathectomy with 6‐hydroxydopamine, thereby establishing that m‐ and p‐octopamine are localized within sympathetic nerve endings.

Chapter 26 Agonist-specific coupling of G-protein-coupled receptors to second-messenger systems

Publisher Summary This chapter presents evidences for the concept of agonist-specific coupling of G-protein-coupled receptors to multiple second-messenger systems. It discusses the related concept of synthetic ligand-specific coupling of G-protein-coupled receptors to second-messenger systems. The important general implications of these concepts for pharmacology and signalling mechanisms are examined in the chapter. Agonist-specific coupling of G-protein-coupled receptors to second-messenger systems may be a general property shared by a range of different receptors. A cloned type 1 pituitary adenylyl cyclase- activating polypeptide (PACAP) receptor and four spliced variant forms of the receptor have been reported to be differentially coupled to adenylate cyclase and phospholipase C by two naturally occurring forms of PACAP—namely, PACAP-27 and PACAP-38. Studies on several vertebrate cloned G-protein- coupled receptors have also demonstrated that a range of synthetic ligands that can act as agonists of these receptors may also be capable of coupling the receptors differentially to different second-messenger systems.

Octopaminergic neurons in the locust brain: morphological, biochemical and electrophysiological characterisation of potential modulators of the visual system

The two Protocerebral-Medulla 4 neurons (PM4a and b) in the locust brain have adjacent cell bodies in the medial deutocerebrum. They project through the posterior protocerebrum, forming limited arborisations en route, and enter the lobula and medulla of the ipsilateral optic lobe, where they form extensive, overlapping arborisations. The PM4a and b neurons are octopamine immunoreactive. Their octopamine content (approximately 25 pg per cell) is confirmed by gas chromatography-mass spectrometry; each cell contains approximately 25 pg p-octopamine. Simultaneous intracellular recording from exposed PM4a and b cell bodies reveals that the two cells are physiologically indistinguishable. They receive multimodal sensory inputs. Tactile/mechanosensory stimuli to much of the animals body and head, acoustic stimuli, and simple visual stimuli all give rise to e.p.s.p.s and action potentials in the PM4 cell body. Simultaneous recording from the cell body in the deutocerebrum and the axon in the lobula demonstrates that action potentials are predominantly initiated in the deutocerebrum and propagate centrifugally, towards the optic lobe. Occasionally, bright light flashes will initiate an action potential in the axon in the optic stalk, which probably propagates bidirectionally: centripetally to the cell body, and centrifugally into the optic lobe. The extensive arborisations in the lobula and medulla are therefore likely to be sites of octopamine release. Because PM4 neurons are octopaminergic, project to the optic lobe, and receive modalities of sensory input known to dishabituate the Descending Contralateral Movement Detector (DCMD) visual interneuron, it is proposed that PM4 neurons are neuromodulatory — mediating dishabituation or arousal of the visual system.

Analysis of unconjugated morphine, codeine, normorphine and morphine as glucuronides in small volumes of plasma from children

A sensitive method for the analysis of unconjugated morphine, codeine, normorphine and total morphine after hydrolysis of glucuronide conjugates is described. The method was applicable to 50-microliters volumes of plasma. The analytes were converted to heptafluorobutyryl (HFB) derivatives before analysis by gas chromatography-negative ion chemical ionization mass spectrometry. Morphine and codeine were quantified against their [2H3]-isotopomers. Linearity, precision and accuracy were quite acceptable (in the 10(-10)-10(-9) g range), and the absolute limits of detection were < 1 pg.

Penetration of topically applied prednisolone sodium phosphate into human aqueous humour

Gas Chromatography with Mass Spectrometry (GCMS) was utilised to determine the penetration of prednisolone sodium phosphate into the aqueous humour of human volunteers undergoing routine cataract extraction. Detectable levels of prednisolone were measured in the aqueous humour within 15 minutes. Peak concentrations occurred between 90 and 240 minutes, and the steroid could not be detected in samples taken 10 hours or more after topical administration.