In vitro test system for the screening of H 3 agonists and antagonists

This protocols describes a standard in vitro test based on the relaxant response of agonists on electrically driven guinea pig duodenum.

1. Tissue preparation

• Male Dunkin-Hartley guinea pig (300-400 g), killed by cervical dislocation. • Rapidly remove and clean the duodenum in a dissection disc with Krebs-Heinseleit solution. • Cut segments of the entire duodenum (15-20 mm long). • From each animal prepare two/four segments of the whole duodeno-jejunal tract.

• Set up the tissue in 10 ml organ bath at 37°C containing Krebs-Henseleit solution gassed with 95% O2 and 5% CO 2 (pH 7.4) under a constant load of 0.7±0.2 g. • Insert a pair of coaxial platinum electrodes 10 mm apart from the longitudinal axis of preparation and connected to an electrical stimulator delivering to the tissue single square-wave pulse (0.5 msec duration, 200-250 mA intensity, supramaximum voltage) every 10 sec. • By the use of an isotonic transducer, connected with a pen-writing polygraph, record the electrical field stimulation (EFS)-evoked contraction. • Equilibration period: 60 min. Wash the preparation 4 x 15 min with fresh Krebs-Henseleit solution. • Alternatively, isolate and remove a 25-cm portion of the ileum proximal to the cecum after discarding the terminal 10 cm. Gently uncoil the tissue, avoiding to stretch it. Free the segment from mesentery and cut the ileum into 20-25 mm long segments, to obtain four preparations from each animal. Set-up ileal segments according to the procedure previously described for duodenum .

2a. Histamine H 3 agonists screening

• At the end of the equilibration period, EFS-induced twitch contractions began stable in their amplitude and last without fade up to 3 to 4 hours. These contractions are blocked by 1 µM tetrodotoxin, partially (20%) reduced by 0.1 mM hexamethonium and abolished by 10 nM atropine, suggesting a predominant involvement of postganglionic excitatory cholinergic fibers. In these experimental conditions, the histamine H 3 agonists are able to induce a presynaptic inhibitory effect on EFS-induced twitch contractions, without affecting contractions evoked by exogenous muscarinic agonists.

• Administer cumulatively the H 3 agonist R-(α)-methylhistamine (MHA) by adding logarithmically increasing concentration to the bath fluid. To avoid desensitization phenomena, construct only a single concentration-response curve to the H 3 agonist in the same preparation. • To compare the effects of different H 3 agonists, for each duodenum segments use one (or two) for standard compound MHA and the other(s) for experiments with different compounds able to activate the presynaptic histamine H 3-receptor subtype. • Construct the concentration-response curve (CRC) of the agonist, histamine (HA): 10 nM - 1µM; MHA: 1 nM - 1 µM; methimepip: 1 nM - 0.1 µM. Perform experiments with HA or MHA in presence of (1 µM: add 10 µl of mepyramine 1 mM 30 min before histamine administration) and (1 µM: add 10 µl of famotidine 1 mM 30 min before histamine administration) to minimize possible H 1- and H 2-mediated contraction. • Express tissue responses to H3 agonists as the percentage of reduction of the predrug level of EFS-induced response. MHA- and methimepip-induced maximal relaxant effect (in duodenum): ~ 60%. Tissue responses measured as a percentage-inhibition of the plateau contraction or of the EFS-induced twitch contraction display a smaller coefficient of variation between replicates than those expressed as the change in tissue length (cm). • For each drug concentration, express the results as mean ± SEM of N values (eight replicates are suitable). From the individual log-concentration-response curve of each agonist calculate the EC 50 by the use of a non-linear regression analysis program (Graph Pad Prism 5.0, San Diego, CA, USA). Express the agonist potency as pD 2 value (–Log EC50 ) ± SEM. MHA: pD 2 ± SEM = 7.76 ± 0.07; methimepip: pD 2 ± SEM = 8.26 ± 0.18.

2b. Histamine H 3 antagonist screening

• At the end of the equilibration period, administer the H 3 antagonist at the desired concentration, 30 min before the construction of CRC to the H 3 agonist immediately before the administration of mepyramine and famotidine (if necessary). • Use different segments from the same animal for constructing the CRCs to the agonist alone or to the agonist in presence of the antagonist.

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• When testing competitive antagonists, parallel rightward displacement of the agonist CRC with no change of the maximal response is obtained. Then, use linear regression to calculate pA 2 value and the slope of the Schild plot. Use at least three antagonist concentrations.

• In presence of antagonist causing surmountable antagonism only at lower concentrations, calculate pA 2 values from single CRC which produces surmountable antagonism, using the Gaddum’s equation:

pA 2 = -Log[B] + Log[CR-1]

where [B] represents the concentration of antagonist and CR is the concentration-ratio at the EC 50 level.

3. Materials and reagents

• Male Dunkin-Hartley guinea pig (300-400 g) . . • Krebs-Henseleit solution: NaCl 113.0 mM; KCl 4.7 mM; MgSO 4 7 H2O 1.2 mM; CaCl 2 2 H 2O 1.8 mM; KH 2PO 4 1.2 mM; NaHCO 3 25.0 mM; dextrose 11.2 mM. • Mepyramine solutions: 10 mM - 1 µM in distilled water. • Dissolve famotidine 10 mM in HCl 0.1 M and make further dilutions in distilled water. • Histamine dihydrochloride solutions: 100 mM - 1 µM in distilled water. • MHA dihydrobromide solutions: 10 mM - 100 nM.

• Dilute all available H 3 antagonists (10 mM - 10 µM) in distilled water or in the appropriate solvent as indicated by suppliers. If you use DMSO, prepare 10 mM stock solution, in 100% DMSO. Dilute 1 mM in 50% DMSO and further dilutions in distilled water.

References

Arunlakshana O, Schild HO. Some quantitative uses of drug antagonism. Br J Pharmacol 1959; 14: 48-58. Coruzzi G, Poli E, Bertaccini G. Histamine receptors in isolated guinea pig duodenal muscle: H3 receptors inhibit cholinergic neurotransmission. J Pharmacol Exp Ther 1991; 258: 325-31. Poli E, Menozzi A, Pozzoli C, Coruzzi G, Kitbunnadaj R, Timmermann H, Leurs R. Functional characterisation of the novel histamine H(3) receptor agonist, VUF 5810, on the guinea-pig isolated ileum. Inflamm Res 2004; 53 Suppl 1: S77-8. Pozzoli C, Poli E. Assessment of gastrointestinal motility using three different assays in vitro. (2010) Curr Protoc Toxicol 2010 Nov; Chapter 21: Unit 21.8. doi: 10.1002/0471140856.tx2108s46.

CONTRIBUTED BY: Cristina Pozzoli ([email protected]) LAST MODIFIED: 2013-02-11

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