Fig 1.
Binding of 3H-tiotidine to cultured neonatal rat astrocytes.
Astrocytes were incubated for 15 minutes with the indicated concentrations of 3H-tiotidine in regular phosphate buffer (A) or in phosphate buffer made in D2O (B). Results are presented as a mean value ± SEM of two experiments carried out in triplicate (n = 6).
Fig 2.
Specific 3H-tiotidine binding to cultured neonatal rat astrocytes.
3H-tiotidine binds to a single population of binding sites with a KD of = 4.7 ± 1.0 nM and Bmax of 18.6 ± 1.7 fmol/mg protein. The deuterated medium does not significantly change the binding parameters of 3H-tiotidine to astrocytic H2 receptor binding sites—KD changed to 7.4 ± 2.4 nM and Bmax dropped to 17.1 ± 2.7 fmol/mg protein.
Fig 3.
Inhibition of specific binding of 3H- tiotidine in cultured astrocytes with histamine, 2- and 4-methylhistamine; 2-MeHi and 4-MeHi, respectively.
Deuteration significantly (p < 0.0001) decreased the pIC50 of 2-methylhistamine (8.38 ± 0.13 (control) to 6.85 ± 0.16 (D2O)), whereas it significantly (p < 0.05) increased the pIC50 of histamine (7.25 ± 0.11 (control) to 7.80 ± 0.16 (D2O)) and marginally increased the pIC50 value of 4-methylhistamine from 7.31 ± 0.28 (control) to 7.67 ± 0.13 (D2O).
Fig 4.
The structure of the H2 homology model with bound histamine.
The incept shows the structure of the receptor model employed in quantum-chemical analysis, where the selected amino acid residues were truncated at the corresponding α–carbon atoms which positions were kept frozen during geometry optimization.
Fig 5.
Computational scheme of histamine monocation interacting with five water molecules to calculate the free energy of hydration.
The choice of the dielectric constant in the computations is indicated in round brackets.
Fig 6.
Computational scheme to calculate the interaction energy between the histamine monocation and the receptor site.
All the structures were obtained on the M06–2X/6–31+G(d,p) level with CPCM solvent reaction field. Please note that beside dielectric constant of 4, a value of 20 was also considered.
Table 1.
Changes in the energy of histamine hydration (ΔEHYDR), H2 receptor interaction (ΔEINTER) and total receptor binding (ΔEBIND) upon deuteration.