Effects of the AMP-activated protein kinase inhibitor compound C on the postconditioned rat heart.

Ischemic postconditioning (IPOC) protects the myocardium from ischemic-reperfusion injury, improving functional recovery and cell viability. This protection is concurrent with stimulation of glycogen breakdown, increased mitochondrial ATP synthesis and content, maintenance of reduced-to-oxidized glutathione ratio (GSH/GSSG), and decreased oxidative damage. The present study's objective was to assess whether these effects are associated with increased resistance to mitochondrial permeability transition pore (MPTP) opening. The effects of the AMP-activated protein kinase (AMPK) inhibitor, compound C (CC), were measured to investigate association with AMPK. Mitochondria removed from postconditioned hearts required higher calcium levels to induce MPTP opening. Improved functional recovery, increased glycogen mobilization, maintenance of the GSH/GSSG ratio, decreased oxidative damage, and increased resistance to MPTP opening were abrogated when the hearts were postconditioned in the presence of CC, without affecting preservation of cell viability. Although AMPK appears to play a role in IPOC, it would not be the major cellular mediator.

Involvement of energetic metabolism in the effects of ischemic postconditioning on the ischemic-reperfused heart of fed and fasted rats.

The effects of ischemic-postconditioning (IPOC) on functional recovery and cell viability of ischemic-reperfused hearts from fed and fasted rats were studied in relation to triacylglycerol and glycogen mobilization, ATP content, glucose-6-phosphate dehydrogenase activity and reduced/oxidized glutathione (GSH/GSSG). Oxidative damage was estimated by measuring thiobarbituric acid reactive substances (TBARS). IPOC improved contractile recovery and cell viability in the fed but attenuated them in the fasted hearts. In both groups ischemia lowered glycogen. IPOC further reduced it. Triacylglycerol remained unchanged during ischemia-reperfusion in both groups, but triacylglycerol mobilization was activated by IPOC in the fasted group. ATP was increased by IPOC in the fed hearts, but lowered in the fasted ones, which appeared to be associated with the rates of ATP synthesis in isolated mitochondria. In the fed hearts IPOC raised glucose-6-phosphate dehydrogenase activity and GSH/GSSG, and lowered TBARS. These results suggest that IPOC effects are associated with changes in the ATP supply, mobilization of energy sources and glutathione antioxidant ratio.

Influence of fasting on the effects of dimethylamiloride and oxfenicine on ischaemic-reperfused rat hearts.

To assess whether glycolysis, Na+-H+ exchange and oxidation of fatty acid derived from endogenous lipolysis are involved in the beneficial effects of 24-h fasting on the ischaemic - reperfused heart, it was studied the effects of inhibiting Na+ - H+ exchange using 10 muM dimethylamiloride and fatty acid oxidation using 2 mM oxfenicine, on the functional activity, lactate production and cell viability measured with tetrazolium stain. Since fasting accelerates heart fatty acid oxidation, data were compared to those from fed rats; using Langendorff perfused (glucose 10 mM) hearts of 250-350 g Wistar rats exposed to 25 min ischaemia - 30 min reperfusion. Fasting reduced the ischaemic rise of end diastolic pressure (contracture), improved recovery of contraction and lowered lactate production in comparison with the fed whereas cellular viability was similar in both groups. Dimethylamiloride improved the recovery of contraction (fed control 24 +/- 9%, fed treated 68 +/- 11%, P < 0.05 at the end of reperfusion), attenuated the contracture (fed control 40 +/- 9%, fed treated 24 +/- 11%, P < 0.05 at the beginning of reperfusion) and reduced lactate production in the fed group and increased cellular viability in both groups (fed control 21 +/- 6%, fed treated 69 +/- 7%, P < 0.05, and fasted control 18 +/- 7%, fasted treated 53 +/- 8%, P < 0.05). Oxfenicine reduced the recovery of contraction (fasted control 88 +/- 6%, fasted treated 60 +/- 11%, P < 0.05) and increased lactate production of fasted group and attenuated the contracture in the fed. These data suggest that beneficial effects of fasting owe, at least in part, to a lowered glycolysis probably secondary to the increased fatty acid oxidation and to the accumulation of energy supplying acyl esters. Dimethylamiloride slowing of glycolysis might explain functional improvement, whereas it seems unrelated to the protection on cell viability.

Effects of 5-hydroxydecanoate and ischemic preconditioning on the ischemic-reperfused heart of fed and fasted rats.

This investigation aimed to assess whether the mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate (5-HD) could abolish the protection conferred by fasting and ischemic preconditioning (IPC) and to ascertain whether these effects are associated with glycogen breakdown and glycolytic activity. Langendorff perfused hearts of fed and 24-h fasted rats were exposed to 25 min ischemia plus 30 min reperfusion. IPC was achieved by a 3 min ischemia plus a 5 min reperfusion cycle. 5-HD (100 microM) perfusion begun 5 min before IPC or 13 min before sustained ischemia in the non preconditioned groups. Fasting improved the reperfusion recovery of contraction, decreased the contracture and the lactate production, increased glycogenolysis and did not affect the percentage of viable tissue. 5-HD abolished the effects of fasting on the contractile recovery but did not affect the contracture. 5-HD decreased the lactate production in the fed group, increased the preischemic glycogen content in both nutritional groups and did not affect the ischemic glycogen fall. IPC improved the contractile function but prevented the contracture only in the fed group, reduced lactate accumulation and glycogenolysis and evoked an increase of the viable tissue. 5-HD abolished the effects of IPC on the contractile recovery and did not affect its effect on the contracture, lactate production, glycogenolysis and viable tissue. These data suggest that the mitocondrial ATP-sensitive potassium channel is involved in the effects of fasting and IPC on the contractile function but the other cardioprotective and metabolic effects appear evoked through other mechanisms. Also suggest that besides the inhibition of the mitochondrial potassium channel, other mechanisms mediate the effects of 5-HD.

Influence of fasting on the effects of diazoxide in the ischemic-reperfused rat heart.

This investigation aimed to assess whether the mitochondrial ATP-sensitive potassium channel opener diazoxide could reproduce the protection conferred by ischemic preconditioning and to ascertain whether its effects are associated with changes in glycogen breakdown and glycolytic activity. Hearts of fed and 24-h fasted rats were perfused with 10 mM glucose containing medium and exposed to 25 min no-flow ischemia plus 30 min reperfusion. Diazoxide (10 microM) perfusion was begun 10 min before ischemia and continued throughout the experiment. Fasting accelerated reperfusion recovery of contraction, reduced the post-ischemic contracture and decreased lactate accumulation during ischemia but had no effects on glycogen levels and cellular viability. Diazoxide, did not affect glycogen catabolism but improved reperfusion recovery of contraction. Furthermore, diazoxide reduced ischemic lactate accumulation and contracture amplitude only in the fed group whereas it improved cell viability in the fed and fasted groups. These data indicate that: 1) reduced lactate production which may attenuate myocyte acidification might explain, at least in part, the beneficial effects of diazoxide on mechanical function, although data obtained with the fasted rat hearts indicate that other mechanisms must be involved as well; 2) the reduction of lactate production occurring in the fed group, does not seem to be related to glycogenolysis; and 3) since diazoxide improved cell viability in the fasted rat group where it did not reduce glycolytic activity, other mechanisms may be responsible for this cytoprotective effect.

Influence of fasting on the effects of ischemic preconditioning in the ischemic-reperfused rat heart.

The effects of fasting and ischemic preconditioning (IP) on heart function of Langendorff-perfused rat hearts exposed to 25 min global ischemia plus 30 min reperfusion (RP), were correlated with lactate release and tissue-levels of long-chain acyl carnitine (LCCa) and CoA (LCCoA). IP was achieved by a 3 min ischemia plus a 5 min reperfusion cycle. Creatine kinase leakage was measured to assess the extent of cardiac injury. Fasting reduced the ischemic-induced contracture, improved RP recovery of mechanical function, reduced lactate release and increased the end-ischemia LCCoA and LCCa levels. Both in the fed and the fasted rat hearts IP delayed the pacemaker depression, reduced the amplitude of ischemic contracture and improved the RP recovery of contraction. However, IP reduced creatine kinase and lactate release only in the fed rat hearts. IP had no effects on tissue LCCa and LCCoA in both groups. These data suggest that: 1) beneficial effects of fasting may be ascribed, at least in part, to a reduced lactate production which may attenuate ischemic myocyte acidification and to the accumulation of fatty acyl esters which would favour citric acid cycle replenishment during RP. 2) beneficial effects of IP could be in part explained by the reduction of lactate production in the fed group although data obtained with the fasted rat heart indicate that another mechanisms must also be involved in the effects of IP. 3) accumulation of LCCoA and LCCa is not involved in the noxious effects of ischemia as well as in the protection effected by IP.

Renal function in the diabetic pregnant rats.

Diabetic nephropathy is associated with increased urinary albumin and reduce kallikrein excretion. Increased activity of the renal kallikrein-kinin system has been suggested as one of the possible mechanisms underlying diabetic hyperfiltration. The present study shown that the Kallikrein-kinin system is progressively increased in the diabetic-pregnant rats at 7, 14, 21 days; 48 and 7 days after pregnancy (P < 0.05 vs Control). However, this increase during diabetic pregnancy did not reached the levels of control pregnancy. On the other hand albumin excretion shown a significant and progressive renal damage in the diabetic state. These findings suggest that the diabetic pregnancy could impair the renal hemodynamic, but, on the other side could modulate the vasodilator system at pregnancy in the attempt to protect the fetus.

Renal function in the diabetic pregnant rats.

Diabetic nephropathy is associated with increased urinary albumin and reduce kallikrein excretion. Increased activity of the renal kallikrein-kinin system has been suggested as one of the possible mechanisms underlying diabetic hyperfiltration. The present study shown that the Kallikrein-kinin system is progressively increased in the diabetic-pregnant rats at 7, 14, 21 days; 48 and 7 days after pregnancy (P < 0.05 vs Control). However, this increase during diabetic pregnancy did not reached the levels of control pregnancy. On the other hand albumin excretion shown a significant and progressive renal damage in the diabetic state. These findings suggest that the diabetic pregnancy could impair the renal hemodynamic, but, on the other side could modulate the vasodilator system at pregnancy in the attempt to protect the fetus.

Streptozotocin-induced hyperglycemia is decreased by nitric oxide inhibition.

1. Diabetes mellitus type I was induced in 3-month old male C57 BL/KS-mdb mice (N = 24) by ip injection of streptozotocin (STZ, 45 mg/kg body weight) for 5 days. 2. To determine the possible protective effects of nitric oxide inhibition against hyperglycemia, the STZ-diabetic rats received two doses of NG-nitro-L-arginine- methyl ester (L-NAME) (10 mg/kg body weight and 10 mg/mouse) dissolved in PBS for 45 consecutive days. Another group of STZ-treated rats was similarly treated with L-arginine (5 mg/mouse). 3. Blood glucose levels were 118 +/- 37 mg/dl after 8 days of L-NAME administration (10 mg/kg body weight, N = 12) and 186 +/- 22 mg/dl (N = 12) after 5 days of L-NAME administration at the 5 mg/mouse dose. Treatment with L-arginine (5 mg/mouse, N = 12) caused a significant increase in blood glucose level to 151 +/- 17.5 mg/dl, showing the relevance of nitric oxide formation in this type of diabetes. 4. In STZ-diabetic mice treated with L-NAME (N = 12), diuresis was reduced by approximately 58% compared to STZ animals, whereas in L-arginine-treated animals (N = 12) diuresis returned to STZ levels. Urinary protein excretion, which was significantly affected by STZ (123% compared to control) was significantly reduced by 66% after treatment with L-NAME for 45 days, whereas treatment with L-arginine caused a return to STZ values. 5. Urinary kallikrein excretion, which was reduced by 80% in STZ mice compared to control, returned to control levels after L-NAME treatment. 6. The present results suggest a relationship between nitric oxide levels and the reduction of diabetic state and improved renal function by L-NAME.

Streptozotocin induced hyperglycemia is decreased by nitric oxide inhibition

1. Diabetes mellitus type 1 was induced in 3-month old maleC57 BL/KS-mdb mice (N = 24)) by ip injection of streptozotocin (STZ, 45 mg/Kg body weight) for 5 days. 2. To determine the possible protective effects of nitric oxide inhibition against hyperglycemia, the STZ-diabetic rats received two doses of Ng-nitro-l-arginine-methyl ester (L-NAME) (10 mg/Kg body weight and 10 mg/mouse) dissolved in PBS for 45 consecutive days. Another group of STZ-treated rats was similarly treated with L-arginine (5 mg/mouse). 3. Blood glucose levels were 118 ñ 37 mg/dl after 8 days of L-NAME administration (10 mg/Kg body weight, N = 12) and 186 ñ 22 mg/dl (N = 12) after 5 days of L-NAME administration at the 5 mg/mouse dose. Treatment with L-arginine (5-mg/mouse, N = 12) caused a significant increase in blood glucose level to 151 ñ 17,5 mg/dl, showing the relevance of nitric oxide formation in this type of diabetes. 4. In STZ-diabetic mice treated with L-NAME (N = 12), diuresis was reduced by approximately 58 per cents compared to STZ animals, whereas in L-arginine-treated animals (N = 12) diuresis returned to STZ levels. Urinary protein excretion, which, was significantly affected by STZ (123 per cents compared to control) was significanty reduced by 66 per cents after treatment with L-NAME for 45 days, whereas treatment with-L-arginine caused a return to STZ values. 5. Urinary kallikrein excretion, which was reduced by 80 per cents in STZ mice compared to control, returned to control levels after L-NAME treatment. 6. The present results suggest a relationship between nitric oxide levels and the reduction of diabetic state improved renal function by L-name