Interaction of cimetidine with equine hemoglobin

Cimetidine (CIM) is a proven inhibitor of cytochrome P-450 (CYP) (RendicÂ et al., 1984; Ioannoni et al., 1986). The inhibition of CYP is mediated through this heme containing protein by binding as a sixth axial ligand with iron. This binding results in a Type II optical difference spectrum (Schenkman et al., 1967; Lin & Lu, 1998) and is associated with the direct binding of CIM to the heme moiety of CYP. Both the cyano and imidazole groups of CIM have been shown to bind to the heme-iron at the sixth axial position (RendicÂ et al., 1983). Cimetidine does not interact with all heme-containing proteins as it has been shown not to form a ligand with the heme-iron of mouse catalase (Baird et al., 1987). We have previously reported that CIM decreases both local and systemic NO3 ) production in an equine soft-tissue in ammation model (Hunter et al., 1999). In an attempt to determine the mechanism by which CIM decrease NO3 ) in this model, two experiments were undertaken. The objective of this project was to determine if CIM would bind to equine Hb and if CIM binding to equine Hb would alter NO3 ) formation. The ®rst experiment was conducted to determine if spectral changes occurred with CIM in the presence of equine Hb. Hemoglobin is necessary for the conversion of NO to NO3 ) (Doyle & Hoekstra, 1981; Wennmalm et al., 1992; Moncada & Higgs, 1993) and as a heme-containing protein, may bind with CIM, decreasing NO3 ) production. Equine Hb (Sigma Chemical Company, St Louis, MO, USA) was used at concentrations of 0.1 and 1 mg/mL in phosphate buffered saline (PBS). Cimetidine was added to the sample cuvette to yield ®nal concentrations of 0, 0.1, 0.5 and 1.0 mM. Zaltidine, a guanidine derivative H2-receptor antagonist, interaction was also investigated at concentrations of 0, 1, 5 and 10 mM. Equal volumes of PBS were added to the reference cuvette when spectra were obtained. The second study was conducted in an attempt to determine where in the metabolism of L-Arg to NO3 ) CIM inhibits the formation of NO3 . Based on the results of the ®rst experiment, studies were conducted to evaluate the ability of CIM to inhibit the conversion of NO to NO3 . The effect of CIM on the conversion of NO to NO3 ) catalysed by Hb was investigated using a carbon ®ber microsensor to detect NO (ISO-NOP30; World Precision Instruments Inc., Sarasota, FL, USA). The microsensor was connected to an isolated NO meter (ISO-NO; World Precision Instruments, Inc.) which was serially connected to a personal computer using an 18-bit data recording system (Duo18 TM ; World Precision Instruments Inc.). All studies were conducted at 37 °C. The microsensor was calibrated using S-nitroso-N-acetyl-D,L-penicillamine (SNAP). A stock solution of SNAP was prepared each day that studies were conducted. Fifty milligram of ethylenediamine tetra-acetic acid (EDTA) was added to 250 mL of H2O (pH 9.0). Approximately 5 mg of SNAP was added to the EDTA solution and the molarity of SNAP calculated. Next, 10 mL of 0.1 M CuSO4 (pH 4.0) was pipetted into a 20 mL scintillation vial and placed in a H2O bath (37 °C). The microsensor was placed in the solution and allowed to equilibrate. A baseline reading was obtained and then 10, 20, 40 and 80 lL aliquots of the SNAP solution were added and the current (pA) recorded after each addition. This was repeated twice to obtain a standard curve for NO determination. As SNAP requires subphysiological pH (» 4) and CuSO4 as a catalyst to produce NO at a constant rate, it was not appropriate for generating NO under physiological conditions. For these reasons diethylamine (DEA/NO) was used as a NO source. It spontaneously degrades to NO at physiological pH (7.4) in PBS. The ®rst study was conducted using 10 mL of PBS to which 50 lL of a 0.3-mg/mL solution (pH 9.0) of DEA/NO was added. Nitric oxide production was measured for 10 min and 100 lL aliquots were removed every 2 min and placed in 100 lL of 10% TCA and frozen at )70 °C for triplicate NO3 analysis. Next, CIM was added to a ®nal concentration of 1.5 mM to determine if CIM effected the production of NO from DEA/NO. An additional group of studies were conducted in the same manner, except that a 1 mg/mL solution of equine Hb (» 0.015 mM) in PBS was used. Cimetidine was added to the solution to give ®nal concentrations of 0, 0.1, 0.5, 1.0 and 1.5 mM. Nitrate analysis was conducted using the method of Hunter et al. (1999). Spectra resulting from CIM (0.1 and 1 mg/mL Hb) and Zaltidine (ZAL) (1 mg/mL Hb) interaction with equine Hb are shown in Figs 1±3, respectively. Cimetidine, at a concentration of 1.0 mM in 1 mg/mL Hb, produced a spectra different from that observed at the lower concentrations of CIM, Hb, or at any concentration for ZAL. This indicates that CIM interacts with Hb in some manner to induce a spectral change, but there is no indication of direct interaction of CIM with the iron-heme of Hb. J. vet. Pharmacol. Therap. 24, 299±302, 2001. SHORT COMMUNICATION