Histamine is an important neurotransmitter in the central nervous system, as it regulates sensory, motor, vegetative and hormonal functions in the organism. Its numerous effects in the central nervous systems are mediated by activation of histamine H1, H2, H3 in H4 receptors. Important targets of histaminergic neurons are astrocytes, by far most numerous glial cells, which have an important role in physiological and pathophysiological processes in the brain. Previous studies have indicated that astrocytes express histamine H1 and H2 receptors.
Histamine H2 receptors contain seven hydrophobic transmembrane domains. It is assumed the third-transmembrane Asp98 is the primary binding site for the positively charged nitrogen of the side chain of histamine. Asp186 and Thr190 of the fifth-transmembrane domain form hydrogen bonds with imidazole nitrogen atoms of histamine. Therefore, hydrogen bonds are crucial for the interaction of histamine with the receptor. Histamine receptors form interactions with various ligands - agonist and antagonists. When protons, involved in hydrogen bonds, are replaced by deuterium, which weights twice as much, intermolecular and intramolecular distances change. This affects the structure and stability of the receptor and ligands, which reflects in modification of ligand binding characteristics. In the present study, our goal was to determine binding characteristics of histamine H2 receptor subtype on newborn rat cortical astrocytes in primary culture, and to study the influence of hydrogen bonds on the binding of ligands to receptor. With saturation binding experiments using radioligand, we identified binding sites of histamine H2 receptor in astrocytes and determined its molecular-pharmacological properties in physiological conditions. Binding of 3H-tiotidine revealed the existence of a homogeneous population of binding sites with maximal binding capacity 23.9 ± 4.9 fmol/mg protein and dissociation constant 5.6 ± 2.3 nM. Binding of 3H-tiotidine in the concentration range 1-11 nM was saturable and reversible. Specific binding of 3H-tiotidine to the functional H2 receptor was further confirmed by competition binding studies using different concentrations of various inhibitors. Cimetidine, a specific H2-receptor antagonist, showed higher affinity in displacing 3H-tiotidine than mepyramine, which is a specific H1-receptor antagonist. The deuterated water (D2O) in incubation medium caused that protons, involved in hydrogen bonds, were replaced by atoms of deuterium. This changed intramolecular and intermolecular distances in the receptor molecule and its environment, which resulted in lower affinity of 3H-tiotidine for the binding sites of histamine H2 receptor. Therefore, it led to a lower affinity of cimetidine, but higher of histamine. The affinity of mepyramine has not been changed. Inhibition curves have been altered – inhibition curve of histamine became gentler, by cimetidine steeper and by mepyramine it stayed unchanged. In conclusion, we showed the significance of hydrogen bonds regarding the binding of ligands to histamine H2 receptor. Deuteration changes the distance and therefore the strength of hydrogen bonds, which affects the binding characteristics of histamine H2 receptor.
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