In Situ Measurement of Nitric Oxide Production in Cardiac Isografts and Rejecting Allografts by an Electrochemical Method Mahesh S. Joshi,* ,1 Jack R. Lancaster, Jr.,† Xiaoping Liu,‡ and T. Bruce Ferguson, Jr.* *Departments of Surgery and Physiology, †Department of Physiology and Medicine, and ‡Department of Pediatrics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112 Received March 20, 2001; published online July 20, 2001 A number of previous studies have indirectly (electron paramagnetic resonance, nitrite/nitrate, ribonuclease protection assay for inducible nitric oxide synthase (iNOS) mRNA, L-citrulline assay) demonstrated the production of nitrogen monoxide (NO) during early cardiac allograft rejection. This study reports the first direct, quantitative measure- ment using an electrochemical method of NO produced from rejecting allograft tissue studied in vitro. A rat heterotopic abdominal transplant prep- aration was utilized. Day 7 isograft (ACI to ACI) or allograft (Lewis to ACI) transplanted hearts were atraumatically harvested and suspended at 4°C in Ringers–Hepes solution. An electrochemical system highly sensitive and specific for NO consisting of a Nafion-coated platinum disk electrode (lower limit, 50 nM NO) coupled to an analysis system measured ongoing oxidation of NO. Measurements were car- ried out after inserting the electrode in the tissue block and warming the block to 25°C. Additional measurements were also made after incubation of tissue with aminoguanidine (AG), a relatively selec- tive iNOS inhibitor. Direct measurements (mean  SEM) from allograft tissue indicated a fourfold in- crease in NO as compared with isografts (13.41  4.40 M NO vs 3.43  2.04 M NO). Incubation of allograft tissue with AG reduced NO levels to isograft levels (13.41  4.40 M NO vs 5.94  3.14 M NO); AG had no effect on measured isograft NO levels. Direct, quantitative measurement of NO from tissue is feasible and reproducible, and discrimination between different levels of NO production can be made. These results confirm the imputed results from the previous studies us- ing this experimental model. This technology promises to be a valuable tool for evaluating spe- cific modulators of NO production studied under a variety of physiologic and pathophysiologic conditions. © 2001 Elsevier Science Key Words: nitric oxide; electrochemical sensor; allograft; isograft. The cardiac allograft rejection is mediated by al- loantigen activation and proliferation of inflamma- tory cells that infiltrate the donor organ (3). The inducible form of nitric oxide synthase (iNOS) 2 has been shown to be expressed in these inflammatory cells early in the rejection process and later in car- diac myocytes as determined by mRNA and protein expression (14, 15). Direct measurement of NO from this tissue, however, has not been previously de- scribed. The technology for the rapid detection of NO 1 To whom correspondence to be addressed at Department of Physiology, LSU Health Sciences Center, 1901 Perdido Street, New Orleans, Louisiana 70112. Fax: 504-568-6158. E-mail: mjoshi@lsuhsc.edu. 2 Abbreviations used: NO, nitrogen monoxide; iNOS, inducible form of nitric oxide synthase; EPR, electron paramagnetic resonance; NO 2 - , nitrite; NO 3 - , nitrate; AG, aminoguanidine; L-NMMA, N G -monomethyl-L-arginine; POD, postoperative day. NITRIC OXIDE: Biology and Chemistry Vol. 5, No. 6, pp. 561–565 (2001) doi:10.1006/niox.2001.0369, available online at http://www.idealibrary.com on 561 1089-8603/01 $35.00 © 2001 Elsevier Science All rights reserved.