Christine A. Parker

Potentiation of barbiturate‐induced alterations in presynaptic noradrenergic function in rat frontal cortex by imidazol(in)e α2‐adrenoceptor agonists

1 In order to resolve the extent to which presynaptic noradrenergic mechanisms contribute to the anaesthetic‐sparing effects of α2‐adrenoceptor agonists in vivo microdialysis was used to investigate the combined effects of sodium pentobarbitone and imidazol(in)e α2‐adrenoceptor agonists on extracellular levels of noradrenaline (NA) in the rat frontal cortex. 2 Dialysate levels of NA were markedly reduced by the addition of TTX (2u2003μM) or by the removal of calcium in the perfusate. These data imply that dialysate NA levels are ultimately dependent on exocytotic release mechanisms from afferent coeruleo‐cortical neurones. 3 Systemic administration of sodium pentobarbitone (85u2003mgu2003kg−1, i.p.) induced general anaesthesia and reduced NA levels by 92% after 30u2003min. The restoration of basal levels 90u2003min later was closely associated with a return of the corneal blink reflex. 4 Basal NA levels in conscious animals were not affected by an intravenous infusion of equally radioactive solutions of either imidazoline (clonidine) or imidazole (mivazerol) α2‐adrenoceptor agonists. The dose rate employed for each compound was 2u2003μgu2003kg−1u2003h−1 over 2u2003h. 5 The co‐administration of intravenous clonidine or mivazerol, each at 2u2003μgu2003kg−1u2003h−1 for 2u2003h, with sodium pentobarbitone (85u2003mgu2003kg−1, i.p.), produced a marked and prolonged reduction in NA efflux. After 2u2003h, NA levels remained suppressed by 95% (clonidine) and 80% (mivazerol) and animals remained deeply anaesthetized. 6 The accumulation of tritium in brain tissue was 42–73% lower across all brain regions examined after [3H]‐mivazerol administration than after [3H]‐clonidine administration. Sodium pentobarbitone did not alter the accumulation of tritium in brain tissue after the administration of either α2‐adrenoceptor agonist. 7 These data demonstrate that α2‐adrenoceptor agonists potentiate the inhibitory effects of sodium pentobarbitone on extracellular levels of NA in the frontal cortex. Further studies will be necessary to establish a causal role of noradrenergic mechanisms in the potentiation of anaesthesia by selective α2‐adrenoceptor agonists.

Comparative effects of efaroxan and b-carbolines on the secretory activity of rodent and human b cells

Abstract: The pancreatic b‐cell expresses an imidazoline‐binding site that is involved in the regulation of insulin secretion. This site is pharmacologically atypical in comparison with the I1 and I2 sites described in other tissues, and it has been classified as I3. The structural requirements for binding of ligands to the I3 site have not been fully defined, although a range of synthetic I3 ligands have been characterized in functional terms. Evidence has been presented that an endogenous I3 ligand may exist, because extracts of brain contain an active principle that stimulates insulin secretion in a manner consistent with the involvement of I3 sites. The active component has not been identified but has been equated with the long‐sought clonidine displacing substance (CDS) that is proposed as the endogenous ligand for imidazoline‐binding sites. Recent evidence has indicated that one active component of CDS may be a b‐carboline, but it is not known whether b‐carbolines can stimulate insulin secretion. Thus, we have studied the effects of b‐carbolines on insulin secretion and cytosolic Ca21 levels in rodent and human islet cells. The results reveal that harmane, pinoline, and norharmane cause a dose‐ and glucose‐dependent increase in insulin secretion but show that this response differs in a number of ways from that elicited by the well‐characterized I3‐agonist, efaroxan. Thus, b‐carbolines represent a new class of insulin secretagogues, although it remains unclear whether their action is mediated solely by I3 sites in the b cell.

Extraction of active clonidine-displacing substance from bovine lung and comparison with clonidine-displacing substance extracted from other tissues

Crude methanolic clonidine-displacing substance (CDS) extracted from bovine lung competed for radioligand binding from alpha2-adrenoceptors and I2-sites present in rat brain membranes, and from I1-sites present in rat brain and kidney membranes. There was no difference in the competition of [3H]clonidine binding to alpha2-adrenoceptors present in either rat or rabbit brain membranes by the crude CDS extract and therefore either tissue could be used to estimate the number of units of CDS present in extracts. Further purification by reverse phase high performance liquid chromatography (RP-HPLC), with UV detection, of extracts obtained from bovine lung, brain and rat brain exhibited similar three-peak profiles, previously reported. Corresponding fractions competed for radioligand binding to alpha2-adrenoceptors present in rat brain membranes, eluting between 19 and 23 min, which corresponded with the middle peak of the three-peaks. Therefore, we propose the CDS-like material eluting from all these tissues to be similar. Interestingly, CDS extracted from bovine adrenal glands under the same conditions showed a similar three-peak profile, but did not repeat the displacement of binding just at 19-23 min, but at every time point after 4 min. This suggests this tissue could represent a source of CDS in this species.

Isolation of RP-HPLC pure clonidine-displacing substance from NG108-15 cells

A crude extract of clonidine-displacing substance (CDS) has previously been extracted from the NG108-15 cell line. This study aimed to purify CDS extracted from this cell line further, by the technique of reverse phase-HPLC (RP-HPLC), and subsequently determine whether this refined CDS bears any similarity to CDSs extracted from other tissues. Crude CDS was extracted from NG108-cells and fractionated by RP-HPLC eluting with a linear gradient of methanol (5-65%; 1 ml min(-1) flow rate) over 50 min., and collected at 1 min. intervals. The pharmacological activities of the CDS fractions were determined by their abilities to displace bound [3H]clonidine to alpha(2)-adrenoceptors in rat brain membranes. RP-HPLC analysis of CDS revealed a pharmacologically active fraction distinct from agmatine, eluting at 24 min, corresponding to an absorbance peak observed at this time. Collectively, these results confirmed that CDS was present in the NG108-15 cell line. However, the RP-HPLC analysis showed the pharmacological activity to elute at a more hydrophobic gradient than previously observed with CDSs extracted from bovine tissues. These results support the notion of the existence of several CDSs.

Evaluation of 11C-BU99008, a positron emission tomography ligand for the Imidazoline2 binding site in human brain

The imidazoline2 binding site (I2BS) is thought to be expressed in glia and implicated in the regulation of glial fibrillary acidic protein. A PET ligand for this target would be important for the investigation of neurodegenerative and neuroinflammatory diseases. 11C-BU99008 has previously been identified as a putative PET radioligand. Here, we present the first in vivo characterization of this PET radioligand in humans and assess its test–retest reproducibility. Methods: Fourteen healthy male volunteers underwent dynamic PET imaging with 11C-BU99008 and arterial sampling. Six subjects were used in a test–retest assessment, and 8 were used in a pharmacologic evaluation, undergoing a second or third heterologous competition scan with the mixed I2BS/α2-adrenoceptor drug idazoxan (n = 8; 20, 40, 60, and 80 mg) and the mixed irreversible monoamine oxidase type A/B inhibitor isocarboxazid (n = 4; 50 mg). Regional time–activity data were generated from arterial plasma input functions corrected for metabolites using the most appropriate model to derive the outcome measure VT (regional distribution volume). All image processing and kinetic analyses were performed in MIAKAT. Results: Brain uptake of 11C-BU99008 was good, with reversible kinetics and a heterogeneous distribution consistent with known I2BS expression. Model selection criteria indicated that the 2-tissue-compartment model was preferred. VT estimates were high in the striatum (105 ± 21 mL⋅cm−3), medium in the cingulate cortex (62 ± 10 mL⋅cm−3), and low in the cerebellum (41 ± 7 mL⋅cm−3). Test–retest reliability was reasonable. The uptake was dose-dependently reduced throughout the brain by pretreatment with idazoxan, with an average block across all regions of about 60% (VT, ∼30 mL⋅cm−3) at the highest dose (80 mg). The median effective dose for idazoxan was 28 mg. Uptake was not blocked by pretreatment with the monoamine oxidase inhibitor isocarboxazid. Conclusion: 11C-BU99008 in human PET studies demonstrates good brain delivery, reversible kinetics, heterogeneous distribution, specific binding signal consistent with I2BS distribution, and good test–retest reliability.