Role of mitochondrial permeability transition pore and mitochondrial ATP-sensitive potassium channels in the protective effects of ischemic preconditioning in isolated hearts from fed and fasted rats.

The aim of the present study was to assess whether the protective effects of ischemic preconditioning (PC) are associated with activation of the mitochondrial ATP-sensitive potassium channels (mitoKATP) and if there is any relationship between the activity of these channels and the mitochondrial permeability transition pore (MPTP) opening in ischemic-reperfused rat hearts under different nutritional conditions. Langendorff-perfused hearts of fed and 24-h fasted rats were exposed to 25 min of no-flow global ischemia plus 30 min of reperfusion. Fasting accelerated functional recovery and attenuated MPTP opening. The mitoKATP blocker, 5-hydroxydecanoic (HD), did not influence functional recovery and MPTP opening induced by ischemia-reperfusion in the fed hearts but partially reversed the beneficial effects of fasting. PC and the mitoKATP opener, diazoxide (DZ), improved functional recovery, preserved cell viability, and inhibited MPTP opening in both fed and fasted hearts. The protection elicited by PC and DZ on contractile recovery and MPTP opening was reversed by HD, which did not affect cell viability. Altogether, these results argue for a role of mitoKATP and its impact on preservation mitochondrial inner membrane permeability as a relevant factor in the improvement of contractile function in the ischemic-reperfused rat heart. They also suggest that the functional protection elicited by PC may be related to this mechanism.

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.

Effects of fasting and hypoxic preconditioning on the hypoxic-reoxygenated ventricular strips of the rat heart.

The investigation aimed to assess the effects of hypoxic preconditioning in right ventricle strips of fed and 24-h fasted rats, which display a fast fatty acid catabolism, and to ascertain whether these effects are associated with changes in the tissue levels of long-chain acylCoA and acyl carnitine and glycolytic activity. Strips were mounted isometrically in Krebs-bicarbonate solution with 10 mM dextrose and paced at 1 Hz. Strips were exposed to 30 min hypoxia and 60 min reoxygenation with or without a previous preconditioning cycle of 5 min hypoxia followed by a 10 min reoxygenation. During hypoxia the fasted rat strips underwent a greater contracture with respect to the fed group. Preconditioning reduced the contracture strength and accelerated the post-hypoxic recovery only in the fasted rat strips. Hypoxia evoked an increase in the acylCoA and acyl carnitine tissue-contents of the strips which reached higher levels in the fasted than in the fed rat groups. Preconditioning had no effects on the content of these metabolites. During hypoxia lactate output was lower in the fasted than in the fed rat strips and preconditioning abolished this decrease. These data suggest that the protective effects of hypoxic preconditioning occur in the heart tissue predisposed to the oxidation of fatty acid and can not be ascribed to changes in the accumulation of acylCoA and acyl carnitine but could be due, at least in part, to an activation of glycolysis.

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.

Effects of hypoxic preconditioning on the hypoxic-reoxygenated atria from fed and fasted rats.

Hypoxic preconditioning (PC) was studied using rat atria set up isometrically in 10 mM dextrose medium and paced at 1 Hz, applying three different protocols wherein fed and 24-h fasted rats were used in protocols 1 and 2 and only the fed in protocol 3. In protocol 1, PC was achieved applying a 5 min hypoxia followed by 10 min of reoxygenation before the onset of a 60 min hypoxia and 60 min reoxygenation. In protocol 2 the 5 min and a posterior 45 min hypoxia were applied in the absence of dextrose whereas in the 10 min and 60 min reoxygenation periods dextrose was present. In protocol 3, two cycles of 5 min dextrose-free hypoxic periods were applied before the sustained hypoxia (dextrose-free) and reoxygenation periods (10 min and final 45 min, both in the presence of dextrose). In the control groups of all protocols, the equilibration periods were prolonged to compensate the duration of PC. In the control groups of protocols 1 and 2, the sustained hypoxia evoked greater disturbances of contractility and a smaller post-hypoxic recovery in the fasted than in the fed rat atria. In protocol 1, PC markedly reduced the rise in resting tension and improved the post-hypoxic recovery in the fasted rat atria whereas in the fed rat atria protective effects were small and brief. In protocol 2, PC evoked a small reduction of contracture only in the atria from fasted rats and in protocol 3, PC exacerbated the hypoxic disturbances. These data suggest that PC effects depend both on the severity of the PC stress and the sustained hypoxia; and that PC does not require coronary flow.

Lack of protective effects of adenosine on the hypoxic and reoxygenated atria and ventricular strips of the rat.

The aim of the investigation was to assess whether adenosine would ameliorate the hypoxic-induced disturbances of the isolated atria and ventricular strips from fed and 24 h fasted rats. Adenosine 100-50 microM exerted negative inotropic and chronotropic effects on the aerobic atria whereas 10 microM was ineffective. During hypoxia the atria underwent a decline of the peak developed tension and pacemaker frequency. Adenosine 50 microM was detrimental for the performance of hypoxic atria whereas a 10 microM neither affected the fall of peak tension nor the post-hypoxic recovery. Adenosine 100 microM did not affect the peak developed tension of the aerobic ventricular strips. Under hypoxia the ventricular strips from fed and fasted rats exhibited a pronounced depression of their peak developed tension together with the development of a strong contracture and a partial recovery after reoxygenation, which attained a similar extent in both nutritional states. Lactate output during hypoxia was lower in the group of fasted rats. Adenosine 100 microM did not exhibit any effect on the ventricular functions and glycolytic activity in both experimental groups. Results suggests that adenosine has no beneficial effects on rat isolated atria and ventricular strips in hypoxic conditions

Effects of pentanoate, 4-pentenoate, 3-hydroxybutyrate and insulin on the tissue-levels of long-chain acyl CoA and acylcarnitine in the oxygenated and hypoxic rat atria.

Under hypoxic conditions the atrial contents of long-chain acyl CoA (LCCoA) and long-chain acylcarnitine display a close correlation with the noxious effects of fasting on the atrial functions as well as with the amelioration effected by inhibitors of carnitine palmitoyltransferase I. These findings suggested that fatty acid oxidation was detrimental for the hypoxic atria. However, since changes of the LCCoA and LCCa levels which may occur together with the hypoxic disturbances attained under some other metabolic interventions had not been assessed yet, present investigation aimed to provide information about this issue. At the end of the prehypoxic equilibration period, all the treatments tested evoked a fall of the free-CoA levels whereas free-carnitine, LCCoA and LCCa remained unchanged. In the hypoxic atria, 4-pentenoate, an inhibitor of fatty acid beta-oxidation that also can be oxidized, did not change LCCoA and LCCa levels whereas the readily oxidizable pentanoate evoked a drop of LCCoA. These effects may be due to the trapping of CoA as the short-chain acyl esters of both substances. Since 4-pentenoate and pentanoate were noxious on the hypoxic atria even though they did not increase LCCoA and LCCa contents, it may be inferred that short-chain acyl esters might be deleterious during oxygen shortage. The exposure to 3-hydroxybutyrate, an oxidizable substrate whose availability increases during fasting, did not alter the LCCoA and LCCa contents, agreeing with the weak detrimental effects that it exerts on the hypoxic atria. On the other hand, insulin elicited a rise in the LCCoA and a fall in the LCCa contents. Inasmuch insulin had been shown to improve the performance of the hypoxic atria, these findings suggest that LCCoA might not be involved in the noxious effects of fatty acid oxidation whereas LCCa would be the major toxic catabolite.

Influence of moderate cooling (37 degrees C-25 degrees C) on the reactivity of isolated rat tail artery.

The aim of the investigation was to examine the effects of cooling on the tail artery regarding the scarceness of such studies in spite of the essential thermoregulatory role played by this vessel. Segments of the proximal portion were suspended isometrically in medium containing 1.25 mM Ca. Lowering the temperature to 25 degrees C increased the sensitivity and maximum strength of the adrenaline concentration-effect curves. These changes were reversed by warming back to 37 degrees C. Cocaine attenuated the increase of sensitivity without changing the increase of the maximum response. Either the sensitivity and strength of the responses to phenylephrine and serotonin were increased by cooling. Clonidine evoked weak contractions in 18 out of 38 experiments. After cooling, the responses persisted only in 7 arteries and the strength was almost halved. Responses to field electric stimulation at 25 degrees C exhibited a pronounced increase of strength and a small increase of sensitivity. -log Kb for prazosin against adrenaline was increased by cooling (8.7 and 9.1 at 37 degrees C and 25 degrees C, P < 0.01). After partial receptor inactivation using phenoxybenzamine, the dissociation-constant (KA) indicated a moderate affinity for phenylephrine that was not changed by cooling (4.1 and 4.2 x 10(-6) at 37 degrees and 25 degrees C respectively). Receptor reserve and occupancy at EC50 also remained unchanged at 25 degrees C. It can be concluded that: 1) cooling increases the tail artery reactivity, partly as a consequence of the inhibition of adrenergic neuronal uptake; 2) responsiveness to alpha 2-agonists is not involved in the effects of cooling whereas the role of alpha 1-adrenoceptor could not be properly clarified; 3) cooling may facilitate some steps of the contractile activation beyond the agonist-receptor interaction.

Influence of moderate cooling (37§C-25§C) on the reactivity of isolated rat tail artery

The aim of the investigation was to examine the effects of cooling on the tail artery regarding the scarceness of such studies in spite of the essential thermoregulatory role played by this vessel. Segments of the proximal portion were suspended isometrically in medium containig 1.25 mM Ca. Lowering the temperature to 25 degrees Celsius increased the sensitivity and maximum strength of the adrenaline concentration-effect curves. These changes were reversed by warming to 37 degrees Celsius. Cocaine attenuated the increase of sensitivity without changing the increase of the maximum response. Either the sensitivity and strength of the responses to phenylephrine and serotomin were increased by cooling. Clonidine evoked weak contractions in 18 out of 38 experiments. After cooling, the responses persisted only in 7 arteries and the strength was almost halved. Responses to field eletric stimulation at 25 degrees Celsius exhibited a pronounced increase of strength and a small increase of sensitivity. -log Kb for prazosin against adrenaline was encreased by cooling (8.7 and 9.1 at 37 degrees Celsius and 25 degrees Celsius C, P<0.01). After partial receptor inactivation using phenoxybenzamine, the dissociation-constant (KA) indicated a moderate affinity for phenylephrine that was not changed by cooling (4.1 and 4.2 x 10(-6) at 37 degrees Celsius respectively). Receptor reserve and occupancy at EC(50) also remained unchanged at 25 degrees Celsius. It can be concluded that: 1) cooling increases the tail artery reactivity, partly as a consequence of the inhibition of adrenergic neuronal uptake; 2) responsiveness to alpha 2-agonists is not in volved in the effects of cooling whereas the role of alpha 1-adrenoceptor could not be properly clarified; 3) cooling may facilitate some steps of the contractile activation beyond the agonist-receptor interaction.

Influence of moderate cooling (37 degrees C-25 degrees C) on the reactivity of isolated rat tail artery.

The aim of the investigation was to examine the effects of cooling on the tail artery regarding the scarceness of such studies in spite of the essential thermoregulatory role played by this vessel. Segments of the proximal portion were suspended isometrically in medium containing 1.25 mM Ca. Lowering the temperature to 25 degrees C increased the sensitivity and maximum strength of the adrenaline concentration-effect curves. These changes were reversed by warming back to 37 degrees C. Cocaine attenuated the increase of sensitivity without changing the increase of the maximum response. Either the sensitivity and strength of the responses to phenylephrine and serotonin were increased by cooling. Clonidine evoked weak contractions in 18 out of 38 experiments. After cooling, the responses persisted only in 7 arteries and the strength was almost halved. Responses to field electric stimulation at 25 degrees C exhibited a pronounced increase of strength and a small increase of sensitivity. -log Kb for prazosin against adrenaline was increased by cooling (8.7 and 9.1 at 37 degrees C and 25 degrees C, P < 0.01). After partial receptor inactivation using phenoxybenzamine, the dissociation-constant (KA) indicated a moderate affinity for phenylephrine that was not changed by cooling (4.1 and 4.2 x 10(-6) at 37 degrees and 25 degrees C respectively). Receptor reserve and occupancy at EC50 also remained unchanged at 25 degrees C. It can be concluded that: 1) cooling increases the tail artery reactivity, partly as a consequence of the inhibition of adrenergic neuronal uptake; 2) responsiveness to alpha 2-agonists is not involved in the effects of cooling whereas the role of alpha 1-adrenoceptor could not be properly clarified; 3) cooling may facilitate some steps of the contractile activation beyond the agonist-receptor interaction.

Influence of moderate cooling (37ºC-25ºC) on the reactivity of isolated rat tail artery

The aim of the investigation was to examine the effects of cooling on the tail artery regarding the scarceness of such studies in spite of the essential thermoregulatory role played by this vessel. Segments of the proximal portion were suspended isometrically in medium containig 1.25 mM Ca. Lowering the temperature to 25 degrees Celsius increased the sensitivity and maximum strength of the adrenaline concentration-effect curves. These changes were reversed by warming to 37 degrees Celsius. Cocaine attenuated the increase of sensitivity without changing the increase of the maximum response. Either the sensitivity and strength of the responses to phenylephrine and serotomin were increased by cooling. Clonidine evoked weak contractions in 18 out of 38 experiments. After cooling, the responses persisted only in 7 arteries and the strength was almost halved. Responses to field eletric stimulation at 25 degrees Celsius exhibited a pronounced increase of strength and a small increase of sensitivity. -log Kb for prazosin against adrenaline was encreased by cooling (8.7 and 9.1 at 37 degrees Celsius and 25 degrees Celsius C, P<0.01). After partial receptor inactivation using phenoxybenzamine, the dissociation-constant (KA) indicated a moderate affinity for phenylephrine that was not changed by cooling (4.1 and 4.2 x 10(-6) at 37 degrees Celsius respectively). Receptor reserve and occupancy at EC(50) also remained unchanged at 25 degrees Celsius. It can be concluded that: 1) cooling increases the tail artery reactivity, partly as a consequence of the inhibition of adrenergic neuronal uptake; 2) responsiveness to alpha 2-agonists is not in volved in the effects of cooling whereas the role of alpha 1-adrenoceptor could not be properly clarified; 3) cooling may facilitate some steps of the contractile activation beyond the agonist-receptor interaction. (AU)

Effects of fasting, hypoxia, methylpalmoxirate and oxfenicine on the tissue-levels of long-chain acyl CoA and acylcarnitine in the rat atria.

During hypoxia the atria from fasted rats exhibit a faster decline in the pacemaker and contractile activities than those from fed rats. Oxfenicine and methylpalmoxirate, inhibitors of carnitine palmitoyltransferase 1 (CPT 1), ameliorate these disturbances. Since the fasted rat atria have greater triacylglycerol stores and a faster lipolysis, and CPT 1 funnels fatty acid into beta-oxidation, the effects of fasting could be ascribed to the accumulation of amphipathic metabolites such as long-chain acyl CoA (LCCoA) and long-chain acylcarnitine (LCCa). Hence, this investigation aimed to assess whether the levels of these metabolites correlate with the effects of fasting and CPT 1 inhibitors. At the end of the prehypoxic equilibration period the fasted rat atria had a 6.5-fold greater content of LCCa than those of the fed rats and methylpalmoxirate impeded the increase. During hypoxia the LCCoA content increased 9-fold in the fasted rat atria, LCCa levels were 3.6-fold greater in the fasted than in the fed group, and free-CoA and free-carnitine showed a significant fall. The increases of LCCoA and LCCa as well as the fall in free-CoA were abolished by both inhibitors. The decrease of free-carnitine was impeded by methylpalmoxirate, but oxfenicine unexpectedly decreased its concentration in both nutritional groups. These data suggest that: (1) the atrial CPT 1 activity is enhanced during fasting, (2) in the hypoxic atria levels of LCCoA and LCCa were closely correlated with the noxious effects of fasting and the amelioration effected by CPT 1 inhibitors, and (3) the effects of amphipathic metabolites during oxygen deprivation can be attenuated by pharmacological interventions.

Effects of 3-hydroxybutyrate on the hypoxic and reoxygenated atria from fed and fasted rats.

When exposed to hypoxia, the isolated atria from fed rats released lactate into the medium and underwent a decline of the peak developed tension and pacemaker frequency. The atria from 24-h fasted rats showed a rise in the resting tension together with a greater decline of the pacemaker rate and a lower lactate output than those from fed rats. The exposure to 5 mM 3-hydroxybutyrate caused only a small and brief decline in the pacemaker rate in the fed rats atria indicating that ketone bodies are able to exert only a minor detrimental effect on the hypoxic atria. Since the lactate output remained unaffected, this effect cannot be ascribed to a lowering in the energy supply from anaerobic glycolysis. On the contrary, 3-hydroxybutyrate improved the post-hypoxic recovery of the peak tension in the atria from fasted rats. This finding may be reflecting an anaplerotic role of 3-hydroxybutyrate, thus suggesting that in addition to glucose a second substrate is needed to meet the energy demand in the reoxygenated atria from fasted rats.

Exogenous substrates as energy source for the contractile activity of the isolated rat tail artery.

The isolated rat tail artery underwent a pronounced depression of the contractile responses to adrenaline during the incubation in a glucose-free medium containing 2-deoxyglucose and/or oxfenicine in order to inhibit the utilization of glycogen and/or endogenous triacylglycerol. When glucose was returned after 90 min of exposure to oxfenicine, the contraction strength recovered completely. In the medium with 2-deoxyglucose the addition of palmitate or hexanoate produced a recovery level 28% and 16% below the control values respectively. The effect of palmitate was nearly abolished and that of hexanoate partially decreased in the medium containing both inhibitors. Under this condition pyruvate reestablished the extent of the contraction to about 80% of the control value whereas beta-hydroxybutyrate produced a weak and transient recovery. These data suggest that in the tail artery the major portion of the energy needed to sustain the contractile activity is supplied by the oxidation of the more important plasmatic substrates with the exception of ketone bodies. However the Embden-Meyerhof pathway seems necessary to maintain at least a fraction of the contraction strength.

Assessment of the energetic role of endogenous substrates in the tail artery of rat by means of enzyme inhibitors.

The rat tail artery during a 180 min incubation period in a medium containing glucose plus oxfenicine (an inhibitor of fatty acid oxidation) did not show changes in the contractile responses to adrenaline. In a substrate-free medium the extent of the contractions underwent a slight decrease during the last 60 min of incubation. When the substrate-free medium contained 2-deoxyglucose (an inhibitor of glycolysis and glycogenolysis) or oxfenicine, the decline of the contractile activity developed faster and attained a similar extent with each inhibitor. When the substrate-free medium contained 2-deoxyglucose together with oxfenicine or methylpalmoxirate (an inhibitor of fatty acid oxidation) the arteries displayed a pronounced and early fall in the contraction strength. These data suggest that in the presence of glucose the reserve substrates are not necessary as fuel source for the arterial contractions. However, in substrate-free conditions they constitute an important energy source. Furthermore, glycogen and triacylglycerol share the supply of energy and there does not seem to be any other reserve material in the smooth muscle of the rat tail artery.

Effects of L-carnitine on the hypoxic atria from fed and fasted rats.

The aim of the investigation was to assess whether L-carnitine, an essential cofactor in the mitochondrial transfer of fatty acids, would ameliorate the hypoxic-induced disturbances in the isolated rat atria. During hypoxia, the atria released lactate into the bathing medium and underwent a rise in resting tension and a decline of the peak developed tension and pacemaker frequency. The atria from 24-h fasted rats, which oxidize faster their endogenous triacylglycerol stores>> exhibited greater functional disturbances during hypoxia and a smaller recovery after reoxygenation, with respect to the fed rats' atria. Furthermore, at the end of the hypoxic incubation the fasted rats atria displayed a reduction of the free CoA content together with a 3-fold increase in the content of long-chain fatty-acyl CoA, in comparison with those of fed rats. The addition of 5 mM L-carnitine 60 min before the onset of hypoxia did not exert any effect on the hypoxic atria. In contrast, 20 mM L-carnitine accelerated the decline of the pacemaker activity in the fasted rat atria and worsened the contracture development in both nutritional states. The fall of the peak tension and the posthypoxic recovery as well as the levels of free CoA and long-chain fatty-acyl CoA, and lactate output, were not affected by 20 mM L-carnitine treatment. These data suggest that L-carnitine is not beneficial for the hypoxic rat atria, even in the fasted state, wherein the atrial fatty acid catabolism is increased.