Endogenous biogenic amine histamine is found in varying concentrations throughout an entire organism. It exerts its effects via histamine H1, H2, H3 and H4 receptors. The histamine receptors are a class of G protein–coupled receptors. After agonist binding to the receptor, G protein subunits dissociate; the ? subunit separates from the ?/? subunits. Subunit ? which is also a GTP-ase exchanges bonded guanosine diphosphate (GDP) for guanosine triphosphate (GTP). Both of the complexes (?-GTP, ?/?) subsequently activate second messenger systems in the cell.
In the master's thesis, experiments were carried out on isolated membranes of neonatal rat astrocytes, which are proven to express histamine receptors H1 and H2. By performing experiments in isolated systems, the key feature of the ligand can be determined: its affinity for binding to the receptor. In parallel, we conducted experiments in the presence of GTP and its non-hydrolyzable analogue Gpp(NH)p'. Using the radiolabelled ligands (3H mepyramine for histamine receptor H1 and 3H-tiotidine for histamine receptor H2), the presence of both histamine receptor binding sites in the isolated membrane system of astrocytes was demonstrated. The maximum densities of binding sites were 107.9 ± 37.15 and 156.50 ± 45.64 fmol/mg protein for H1 and H2 receptor, respectively. The equilibrium dissociation constants for the complex radiolabelled ligand:receptor were 12.46 ± 7.10 nM for 3H-mepyramine binding to H1 receptor binding site; and 9.37 ± 4.40 nM for 3H-tiotidine binding to H2 receptor binding site. The inhibition binding parameters of endogenous agonist histamine (IC50, Ki) displacing either 3H-mepyramine or 3H-tiotidine binding were determined experimentally in the presence or absence of GTP or Gpp(NH)p'. Ki value of 35.11 nM for histamine inhibiting 3H-mepyramine binding increased to 122.74 nM in the presence of GTP and to 221.23 nM in the presence of Gpp (NH)p'. However, the Ki values of histamine displacing 3H-tiotidine binding decreased from 95.20 nM to 14.35 nM in the presence of GTP and to 5.41 nM in the presence of Gpp(NH)p'. In both cases Gpp(NH)p', due to its non-hydrolyzable nature, has a stronger effect on Ki value change.
We confirmed that 3H-mepyramine marked the receptor binding site on the histamine H1 receptor and found that 3H-tiotidine behaved as an inverse agonist, which may bind to a different (possibly allosteric) binding site than histamine on the histamine H2 receptor. Further research is needed for definitive confirmation of the prevoius statement.
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