The induction and detection in vitro of iNOS in the porcine basilar artery

The expression of iNOS in vascular tissues has an adverse effect on vascular responses to vasoconstrictors and NO-mediated vasodilators. The development of a simple method for detecting the iNOS expression by functional means would be extremely useful. Here we describe a method for inducing iNOS in the porcine basilar artery followed by the detection of iNOS protein by immunocytochemical means and the characterisation of functional responses to U46619 and L-arginine. Porcine basilar arteries were treated with LPS (1, 10 and 100 microg/ml) for between 5 and 18 h at 37 degrees C. Inducible NOS protein was expressed in a concentration-dependent manner in the endothelial and smooth muscle cells after 5 h and persisted for 18 h. Vessels treated with LPS showed a time-dependent reduction in contractile function in response to U46619 (10 nM) reaching significance at the 18-h time point. Moreover, a similar time-dependent increase in the vasodilator response to exogenously applied L-arginine (30 microM) was observed at both 5- and 18-h time points. These effects of LPS at the 18-h time point were prevented by the incubation of vessels with dexamethasone (100 microM) in addition to LPS. The vasodilator response to L-arginine was prevented with the incubation with and in the presence of the inhibitor of inducible NOS, 1400W (10 microM) in addition to LPS. These results show that iNOS protein can be expressed in porcine cerebral arteries and that the iNOS is functional. The assessment of contractile function and responses to L-arginine using single concentrations is a rapid and effective method for establishing whether functional iNOS is present in porcine cerebral arteries.