D. A. S. Parker

Cocaine-sensitive O-methylation of noradrenaline in dental pulp of the rabbit: Comparison with the rabbit ear artery

SummaryIncisor pulp from the rabbit metabolises exogenous noradrenaline in concentrations between 0.12 and 1.2 μmol/l mainly to NMN.Effects of chronic sympathetic denervation indicated that in incisor pulp the NMN is extraneuronal in origin, and that DOPEG and DOMA formation, as well as a major part of the noradrenaline which accumulates in the tissue, are associated with the sympathetic nerves.NMN formation was unaffected by hydrocortisone 210 μmol/l, but was strongly inhibited by cocaine 30 μmol/l. These effects contrasted with those in the rabbit ear artery, where NMN formation was increased by cocaine 30 μmol/l and decreased by hydrocortisone 210 μmol/l.In COMT-inhibited denervated pulp, cocaine inhibited the accumulation of noradrenaline.Monoamine fluorescence histochemistry of pulp exposed to noradrenaline 50 μmol/l indicated that cocaine-sensitive uptake occurred in fibroblasts.It is concluded that O-methylation of noradrenaline in dental pulp involves prior uptake of the amine by a process resembling uptake, but which is distinguished from uptake1 by its extraneuronal location.

Presynaptic control of noradrenaline release from sympathetic nerves in human dental pulp

This study was undertaken to determine whether release of noradrenaline from sympathetic nerves in human dental pulp in vitro was modulated by presynaptic adrenoceptors and by dopamine receptors. Pulp was incubated for 30 min with 3H-noradrenaline (0.6 mumol/l) and then perfused continuously with Krebs solution. Field stimulation of the sympathetic nerves at 5 Hz increased the overflow of 3H into the perfusate three-to fourfold. The stimulation-induced overflow of 3H was abolished by tetrodotoxin (0.1 mumol/l) and under Ca(2+)-free conditions, indicating that the increased 3H was derived from nerves. The stimulation-induced overflow was inhibited by noradrenaline (0.1 and 1.0 mumol/l), the alpha 2-adrenoceptor agonist UK14,304 (0.1 mumol/l), dopamine (1.0 mumol/l) and the dopamine receptor agonist, apomorphine (1.0 mumol/l). When the receptor agonists were noradrenaline or dopamine, desipramine (0.3 or 3.0 mumol/l) was present to prevent their uptake by the sympathetic nerves. Clonidine (1.0 mumol/l; tested at 2 Hz as well as 5 Hz) and the alpha 1-receptor agonist methoxamine (1.0 mumol/l) were without effect. The alpha 2-receptor antagonist/rauwolscine (0.1 mumol/l) prevented the inhibitory effects of noradrenaline and UK14,304, but had little effect on the inhibition produced by dopamine. Inhibition of the stimulation-induced overflow by apomorphine was prevented by the dopamine receptor antagonist haloperidol (0.1 mumol/l). The resting overflow of 3H was unaffected by any of the above agents except dopamine, which caused a small increase. It is concluded that the sympathetic nerves in human dental pulp possess inhibitory presynaptic alpha 2-adrenoceptors and dopamine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of [3H]-noradrenaline in human dental pulp in vitro

Slices of pulp from human maxillary and mandibular molar and promolar teeth were incubated with [3H]-noradrenaline (0.2 mumol/l) for 30 min after which the [3H]-noradrenaline and [3H]-metabolites in the tissue and medium were assayed by column chromatography. The deaminated metabolites 3,4-dihydroxy phenyl glycol (DOPEG) and 3,4-dihydroxy mandelic acid (DOMA) constituted 81% of the metabolites formed. Cocaine, an inhibitor of uptake1, decreased the formation of DOPEG and DOMA as well as the accumulation of [3H]-noradrenaline. In contrast to findings in rabbit pulp, when the disposition of exogenous noradrenaline in human pulp was examined by monoamine fluorescence histochemistry there was no evidence of extraneuronal accumulation of noradrenaline by connective tissue cells. In further experiments, pulp that had been incubated in [3H]-noradrenaline (0.6 mumol/l) for 30 min and superfused for 200 min contained [3H]-noradrenaline (183 pmol/g) and [3H]-DOMA (89 pmol/g). The 3H that overflowed into the perfusate between 85 and 90 min consisted mainly of metabolites. Stimulation of the sympathetic nerves through field electrodes increased the overflow of [3H]-noradrenaline into the perfusate threefold without affecting the overflow of metabolites. The increase was much greater (eightfold) in the presence of an alpha-adrenoceptor antagonist (rauwolscine; 0.1 mumol/l), plus inhibitors of uptake1 (desipramine; 0.3 mumol/l) and uptake 2 (corticosterone; 10 mumol/l). The results are interpreted as evidence that in human dental pulp the disposition of exogenous noradrenaline is determined largely by uptake by sympathetic nerves. After uptake, noradrenaline is deaminated by intraneuronal monoamine oxidase to DOPEG and DOMA.(ABSTRACT TRUNCATED AT 250 WORDS)

DIFFERENTIAL EFFECTS OF PHOSPHONIC ANALOGUES OF GABA ON GABAB AUTORECEPTORS IN RAT NEOCORTICAL SLICES

The effects of five phosphonic derivatives of GABA on the release of [3H]-GABA from rat neocortical slices, preloaded with [3H]-GABA, were investigated. Phaclofen and 4-aminobutylphosphonic acid (4-ABPA) increased the overflow of [3H] evoked by electrical stimulation (2Hz) in a concentration-dependent manner, with similar potencies (phaclofen EC50=0.3mmol/l, 4-ABPA EC50=0.4mmol/l). At 3mmol/l, phaclofen increased the release of [3H]-GABA by 82.6±8.6%, and 4-ABPA increased the release by 81.3±9.0%. 2-Amino-ethylphosphonic acid (2-AEPA) increased the overflow of [3H] by 46.8±10.9% at the highest concentration tested (3mmol/l). In contrast, the lower phosphonic homologue 3-aminopropylphosphonic acid (3-APPA), and 2-amino-2-(p-chlorophenyl)-ethylphosphonic acid (2-CPEPA), a baclofen analogue, did not modify the stimulated overflow. These results suggest that phaclofen, 4-ABPA and 2-AEPA are antagonists at GABAB autoreceptors, the latter being the weakest antagonist, whilst neither 3-APPA nor 2-CPEPA are active at these receptors. Since phaclofen, 4-ABPA and 2-CPEPA are antagonists and 3-APPA a partial agonist/antagonist on GABAB heteroreceptors, the lack of effect of 3-APPA and 2-CPEPA on [3H]-GABA release in this study suggests that GABAB autoreceptors may be pharmacologically distinct from the heteroreceptors.

Extraneuronal uptake of noradrenaline in rabbit dental pulp: evidence of identity with uptake

SummaryThe extraneuronal removal and disposition of noradrenaline in rabbit dental pulp was examined in view of earlier evidence that the tissue possessed an extra-neuronal uptake process resembling neuronal uptake1. Pulp, which had been depleted of sympathetic nerves by homolateral superior cervical ganglionectomy, was incubated in vitro with 3H-noradrenaline in low concentrations (0.025 or 0.18 μmol/l). When the metabolising enzymes (monoamine oxidase, catechol-O-methyl transferase) were active, 3H retention by the denervated pulp, as indicated by the 3H content after the tissue had been washed for 30 min following incubation with 3H-noradrenaline, was less than 30% of that of the innervated pulp. When the enzymes were inhibited, retention rose to approximately 30% of that of the innervated pulp. Analysis of the time course of the 3H efflux indicated that the 3H-noradrenaline in the denervated pulp had accumulated in a single compartment characterised by a t1/2 for efflux of several hours. Accumulation did not occur under Na+-free conditions, and was inhibited by desipramine (IC50 < 0.03 μmol/l) and by substrates of neuronal uptake1. Mean IC50, values of the latter were very similar to those for inhibition of neuronal uptake1 and comprised (in μmol/l): (+)amphetamine (0.29), dopamine (0.31), tyramine (0.39), (−)noradrenaline (0.70), (−)adrenaline (1.50), 5-hydroxytryptamine (20) and bretylium (35). Uptake2 inhibitors were less active (O-methyl isoprenaline, IC50 = 60 μmol/l) than uptake1 inhibitors, or were without inhibitory effects at the concentrations tested (hydrocortisone, 210 μmol/l; 2-methoxy oestrone, 10 μmol/l).The effects of Na+ omission, of (+)amphetamine, and of O-methylisoprenaline on 3H-normetanephrine formation (measured in the absence of catechol-O-methyl transferase inhibition) matched their effects on 3H-noradrenaline accumulation. The results provide strong support for the presence in rabbit dental pulp of extraneuronal uptake1 which is linked with catechol-O-methyl transferase in the removal of noradrenaline.

Inhibitory effects of adrenaline on the release of noradrenaline from sympathetic nerves in human dental pulp

The effects of adrenaline on the release of noradrenaline from sympathetic nerves in human dental pulp in vitro were examined. Sympathetic nerves were stimulated at 5 Hz for 100 sec following incubation of pulp with [3H]noradrenaline (0.6 micromol/l). In the presence of desipramine (DMI, 0.3 micromol/l), adrenaline (0.1 and 1.0 micromol/l) inhibited the release of [3H]noradrenaline, an effect which was inhibited by the alpha2-adrenoceptor antagonist rauwolscine (0.1 micromol/l) and by the alpha2-adrenoceptor agonist UK 14,304 (0.1 micromol/l). The release of [3H]noradrenaline was unaffected by adrenaline (0.001 and 0.01 micromol/l) in the presence of DMI and DMI plus rauwolscine. Although presynaptic inhibitory alpha2- and facilitatory beta-adrenoceptors are present on sympathetic nerves in human dental pulp, these results imply that adrenaline activates only the inhibitory alpha2-receptors.

Distribution of extraneuronal uptake1 in reproductive tissues: studies on cells in culture

Cultures of stromal cells from pregnant mouse uterus, and an FL cell line derived from human amnion, displayed significant capacities to O-methylate noradrenaline. O-methylation was inhibited in the stromal cells by uptake1-inhibitors, and in the FL cell line by uptake2 inhibitors. These findings are discussed in terms of the distribution and possible functional importance of catecholamine metabolising systems in the female reproductive system.

Effect of Prazosin on the Efflux of 3H-Norepinephrine and Metabolites from the Intima and Adventitia of the Rabbit Ear Artery

The spontaneous and stimulation-induced (SI) effluxes of 3H-norepinephrine (3H-NE) and its metabolites from the intimal and adventitial surfaces of perfused segments of rabbit ear arteries were determined; vessels were previously incubated with 3H-NE (0.6 microM). The total SI adventitial efflux of 3H was approximately 10-fold greater than the intimal efflux, contained a higher percentage of unchanged 3H-NE (48 vs. 12%) and a lower percentage of O-methylated metabolites (17 vs. 55%); there was little difference between the percentages of deaminated catechols (35 vs. 31%). Prazosin, at a concentration (0.24 microM) which prevented the arteries constricting during stimulation, had little effect on the composition of the SI effluxes; however, it caused 2- to 3-fold increases in the effluxes of 3H-NE and its metabolites into the lumen during the period of stimulation. This effect is attributed to the failure of the vessel wall to thicken during stimulation, thus facilitating diffusion of 3H-NE and its intraneuronally formed metabolites across the media. Prazosin decreased the percentage of unchanged 3H-NE and increased that of the deaminated catechols in the spontaneous efflux; these effects are attributed to a direct effect of prazosin on the intra-neuronal metabolism of 3H-NE.

Effects of 6-hydroxydopamine on noradrenaline metabolism linked to neuronal uptake1 and extraneuronal uptake1 in dental pulp in vitro

AbstractThe study was undertaken to determine the relative roles of neuronal and extraneuronal uptake1 in the metabolism of 3H-noradrenaline in human dental pulp. Rabbit dental pulp was used as a reference since it was already known that normetanephrine (NMN) formation in this tissue utilised extraneuronal uptake,. Slices of pulp were preincubated in the absence and presence of 6-hydroxydopamine (1.6 mmol/l, for 10 or 20 min at pH 4.5) and subsequently incubated with 3H-noradrenaline (0.18 μmol/l for 30 min at pH 7.4).The principal metabolites formed were normetanephrine in rabbit pulp and deaminated catechols (dihydroxymandelic acid and dihydroxyphenylglycol) in human pulp. In both tissues 6-hydroxydopamine strongly inhibited formation of the deaminated catechols, but was without effect on normetanephrine formation.It is concluded that:i)in vitro 6-hydroxydopamine does not influence the metabolic process which is dependent on extraneuronal uptake1, namely normetanephrine formation in rabbit dental pulp, and in human pulp. Attention is drawn to an unusual feature of the neuronal metabolism in human pulp, namely the appearance of dihydroxymandelic acid as the principal metabolite.

Evidence for uptake2-mediated O-methylation of noradrenaline in the human amnion FL cell-line

SummaryThe uptake and metabolism of 3H-noradrenaline has been examined in the FL cell-line derived originally from human amnion. Cell cultures metabolised 3H-noradrenaline (1.0 μmol/l) to 3H-normetanephrine and, to a lesser extent, to metabolites (not distinguished) of the O-methylated deaminated fraction; primary deaminated metabolites were not detected. 3(H-normetanephrine formation a) was not saturable in the noradrenaline concentration range 0.2–150 μmol/l, b was decreased to 20%–30% of control levels by uptake2 inhibitors (O-methylisoprenaline, 20 and 100 μmol/l; cimetidine, 10 μmol/l; hydrocortisone, 200 μmol/l) and c, was almost insensitive to uptake1 inhibitors (cocaine, 30 μmol/l; desipramine, 3 μmol/l).Uptake of noradrenaline was manifested after 30 minutes as a 6-fold increase in the cell content of the amine following inhibition of catechol-O-methyl transferase, either alone or in conjunction with inhibition of monoamine oxidase. Uptake was decreased maximally to 40% of control levels by O-methylisoprenaline. IC50 values for inhibition of the O-methylisoprenaline-sensitive component of uptake were (in μmol/l): corticosterone (0.3), papaverine (1.1), O-methylisoprenaline (3.0), cimetidine (6.0), (−)noradrenaline (460), and tetraethylammonium (2230). Except for the last agent, for which a comparative value is not available, the IC50s are in good agreement with those for inhibition of uptake2 in the Caki-1 cell-line reported by other investigators.The component of uptake resistant to O-methylisoprenaline was depressed by papaverine (a 50% decrease at 50 μmol/l), but was not affected by the other uptake2 inhibitors or by cocaine (30 μmol/l).It is concluded that the FL cell possesses an extraneuronal metabolising system very similar to the system in tissues such as heart and smooth muscle where transport of noradrenaline into the cell by uptake2 is followed by rapid O-methylation via catechol-O-methyl transferase. The only difference appears to be the absence of saturation of 3H-normetanephrine formation in the FL cell at low micromolar concentrations of 3H-noradrenaline. The presence of a second uptake process is suggested by the inhibitory effect of papaverine on uptake resistant to O-methylisoprenaline; lack of effect of cocaine implies that this second process is not uptake,.