A 4-AMINO ANALOGUE OF TETRAHYDROBIOPTERIN ATTENUATES ENDOTOXIN-INDUCED HEMODYNAMIC ALTERATIONS AND ORGAN INJURY IN RATS Florian Fitzal,* Heinz Redl,* Wolfgang Strohmaier,* Ernst R. Werner, † and Soheyl Bahrami* *Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, A-1200 Vienna, Austria; † Institute for Med. Chemie & Biochemie, University of Innsbruck, A-6020 Innsbruck, Austria Received 15 Jan 2001; first review completed 16 Feb 2001; accepted in final form 5 Dec 2001 ABSTRACT—Most recently we have shown that 4-aminotetrahydrobiopterin (4-ABH 4 ), an analogue of tetrahydrobiopterin (cofactor of NO synthase), even administered 2 h after endotoxin challenge, improves survival rate in rats. The following experiment was performed to examine the effects of 4-ABH 4 with respect to endotoxin-induced hemodynamic alterations and organ failure. At 2 h after endotoxic challenge (10 mg kg -1 body weight) animals received 4-ABH 4 at a dose of 1, 10, or 100 mg kg -1 body weight. The controls were treated similarly but received saline at the same volume. Eight hours after endotoxin challenge cardiac index and stroke volume were significantly increased in animals treated with 10 mg 4-ABH 4 compared to controls (0.23 ± 0.06 vs. 0.16 ± 0.04 mL min -1 kg -1 and 0.29 ± 0.05 vs. 0.22 ± 0.03 mL beat -1 ) while mean arterial pressure and peripheral vascular resistance index did not significantly differ among the groups. Plasma alanine aminotransferase (ALT) and creatinine levels were significantly increased in endotoxin controls compared with laboratory controls (ALT: 1643 ± 1436 vs. 74 ± 17 U L -1 ; Creatinine: 91 ± 29 vs. 42± 3 μmol L -1 ) which was attenuated in animals treated with 10 mg kg -1 4-ABH 4 (ALT: 417 ± 318 U L -1 ; Creatinine: 78 ± 26 μmol L -1 ). Moreover, endotoxin-induced lung edema and intestinal necrosis were significantly reduced by 4-ABH 4 . Our study provides information that tetrahydrobi- opterin analogue, 4-ABH 4 , improves LPS induced hemodynamic conditions and organ injury. This may, at least in part, account for the previously observed protection of rats by 4-ABH 4 against endotoxin-induced mortality in the same endo- toxic shock model. KEYWORDS—Blood pressure, cardiac index, nitric oxide, nitric oxide synthase INTRODUCTION There is growing evidence that either endotoxin itself or mediators secondary to endotoxin stimulation, including tumor necrosis factor (TNF) and interleukin 1 (IL-1), can activate the L-arginine NO pathway in a variety of cells. The resulting enhanced formation of NO, in turn, is largely responsible for the severe hypotension and the loss of vascular responsiveness that occurs in endotoxemia or sepsis (1–3). Nitric oxide over- production during septic shock increases oxygen radical forma- tion (3, 4), induces apoptosis (5, 6), decreases left ventricular contractility (7), and is detrimental to the blood flow in small intestine (8). The excessive formation of NO has been thought to contrib- ute to the hyporeactivity to vasopressor therapy resulting in peripheral vascular failure, and to the development of multiple organ failure (MOF) during septic shock (2, 9, 10). Similarly, animal studies have suggested that NO overproduction may mediate vascular hyporeactivity and decompensation follow- ing endotoxic shock (11, 12). To improve the hemodynamic derangement during septic shock, efforts have been made to regulate excessive formation of NO attributed to the inducible form of NO synthase (iNOS). Conflicting results, however, have been reported concerning the role of iNOS in the cardiac dysfunction early after endotoxemia. Cardiac contractile dysfunction has been reported to occur without involvement of NOS, but complete inhibition of NOS with a high dose of 30 mg kg -1 N(G)-nitro-L-arginine methylester (L-NAME) was noted to deteriorate coronary perfusion in endotoxemic heart (13). In parallel, the beneficial effects of mercaptoethylquani- dine (MEG), that more selectively inhibits iNOS in vivo, were found to be related to the decreases in leukocyte and free radical-mediated myocardial dysfunction during early endo- toxemia (14). The intracellular concentration of tetrahydrobiopterin (BH 4 ), one of several cofactors of NOS (15), is known to affect the amount of NO formed from L-arginine by cultured cells (16, 17). Treatment of rats with bacterial lipopolysaccharide (LPS) has been shown to induce biosynthesis of BH 4 in various organs (18). Cytokines that induce NO formation concurrently induce the biosynthesis of BH 4 (19). Furthermore, the inhibi- tion of GTP cyclohydrolase I, the first and rate limiting enzyme for tetrahydrobiopterin synthesis, has been shown to abolish the rise in kidney tetrahydrobiopterin levels, to reduce the NO formation in kidney, and to ameliorate endotoxin-induced hypotension in experimental endotoxic shock (20). Most recently we have shown that the pterin antagonist 4-ABH 4 , even administered 2 h after endotoxin challenge, improves survival rate in rats (21). This compound designed to inhibit potential recycling reactions inside the active NOS dimer (22), has previously been shown to be a potent pteridin antagonist of recombinant rat neuronal NOS (23) and recom- binant murine iNOS (24). The continuous infusion of 4-ABH 4 significantly and effectively inhibited the endotoxin-induced iNOS activity in lung tissues and lowered the plasma nitrite and nitrate concentrations to the levels of laboratory control Address reprint requests to Soheyl Bahrami, PhD, L. Boltzmann Institute for Experimental and Clinical Traumatology, Donaueschingenstr. 13, A-1200 Vienna, Austria. SHOCK, Vol. 18, No. 2, pp. 158–162, 2002 158