Fructose-1,6-biphosphate in rat intestinal preconditioning: involvement of nitric oxide

Abstract

Background and aims: Inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by nitric oxide (NO) in intestinal preconditioning could modify the rate of formation of glycolytic intermediates. Fructose-1,6-biphosphate (F16BP) is a glycolytic intermediate that protects tissue from ischaemia/reperfusion injury. We evaluated if F16BP may be endogenously accumulated as a consequence of GAPDH inhibition by NO during intestinal preconditioning in rats.

Methods: We assessed: (1) effect of preconditioning on F16BP content; (2) effect of NO on GAPDH activity before and during sustained ischaemia; and (3) protective effect of F16BP in control, ischaemic, and preconditioned animals with or without administration of N-nitro-L-arginine methyl ester (L-NAME), NO donor, or F16BP.

Results: Preconditioned rats showed a significant transient decrease in GAPDH activity and also maintained basal F16BP levels longer than ischaemic rats. L-NAME administration to preconditioned rats reversed these effects. F16BP administration to ischaemic rats decreased protein release in the perfusate. Administration of F16BP to L-NAME treated rats attenuated the harmful effect of L-NAME.

Conclusions: Our study indicates that F16BP may be endogenously accumulated in preconditioned rats as a consequence of inhibition of GAPDH by NO, and this may contribute to the protection observed in intestinal preconditioning.

Figure 1

Intestinal fructose-1,6-biphosphate (F16BP) levels during the different ischaemic periods (0, 2, 30, or 90 minutes) in the control, ischaemia (I), preconditioning followed by sustained ischaemia (Prec+I), and preconditioning followed by sustained ischaemia but with prior addition of L-NAME (Prec+I+NAME) groups (A) and in the control, control with previous administration of L-NAME (Control+NAME), ischaemia (I), and ischaemia with previous administration of the NO donor spermine NONOate (I+NONOS) groups (B). *p<0.05 v control;†p<0.05 v I.

Figure 2

Intestinal glucose levels during the different ischaemic periods (0, 2, 30, or 90 minutes) in the control, ischaemia (I), preconditioning followed by sustained ischaemia (Prec+I), and preconditioning followed by sustained ischaemia but with prior addition of L-NAME (Prec+I+NAME) groups (A) and in the control, control with previous administration of L-NAME (Control+NAME), ischaemia (I), and ischaemia with previous administration of the NO donor spermine NONOate (I+NONOS) groups (B). *p<0.05 v control.

Figure 3

GAPDH activity (U/mg protein) in the intestine during the different ischaemic periods (0, 2, 30, or 90 minutes) in the control, ischaemia (I), preconditioning followed by sustained ischaemia (Prec+I), preconditioning followed by sustained ischaemia but with prior addition of L-NAME (Prec+I+NAME), preconditioning (P), preconditioning with addition of L-NAME (P+NAME), and preconditioning with addition of the iNOS specific inhibitor (P+1400W) groups (A) and in the control, control with previous administration of L-NAME (Control+NAME), ischaemia (I), and ischaemia with previous administration of the NO donor spermine NONOate (I+NONOS) groups (B). *p<0.05 v control; ‡p<0.05 v preconditioned animals.

Figure 4

Nitrate and nitrite tissue production in the intestine in the following groups: control, control with previous administration of L-NAME (C+NAME), preconditioning (P: 10 minutes of ischaemia followed by 10 minutes of reperfusion), preconditioning with L-NAME (P+NAME), and preconditioning with administration of the iNOS specific inhibitor (P+1400W) (A), and control, preconditioning (P), ischaemia (I), preconditioning followed by sustained ischaemia (Prec+I), and preconditioning followed by sustained ischaemia but with prior addition of L-NAME (Prec+I+NAME) (B). *p<0.05 v control.

Figure 5

Profiles of protein release during the different ischaemic (0, 2, 30, and 90 minutes) and reperfusion (5 and 30 minutes, that is, 95 and 120 minutes in (A) and (B)) periods in the following groups: control, preconditioning followed by the ischaemia/reperfusion period (P+I/R), ischaemia/reperfusion (I/R), preconditioning followed by the ischaemia/reperfusion period with prior addition of L-NAME and with superfusion over the small intestine of 5 mM of F16BP during the process (P+N+I/R+F16BP), and preconditioning followed by the ischaemia/reperfusion period but with prior addition of L-NAME (P+N+I/R) (A) and control, ischaemia/reperfusion with superfusion over the small intestine of 5 mM of F16BP throughout the process (I/R+F16BP), ischaemia with previous administration of the NO donor spermine NONOate (I/R+NONOS), and ischaemia/reperfusion (I/R) (B). *p<0.05 v control; †p<0.05 vs I/R.

Figure 6

Intestinal fructose-1,6-biphosphate (F16BP) levels incorporated in the intestine. I, ischaemia; I+F16BP, ischaemia with superfusion over the small intestine of 5 mM of F16BP throughout the process. †p<0.05 v I.