Sustained major molecular response on interferon alpha-2b in two patients with polycythemia vera.

Quantitative assessment of the JAK2 V617F allele burden during disease evolution and ongoing myelosuppressive treatment is likely to be implemented in the future clinical setting. Interferon alpha has demonstrated efficacy in treatment of both chronic myeloid leukemia and the Philadelphia chromosome negative chronic myeloproliferative disorders. Reductions in the JAK2 V617F allele burden in patients treated with pegylated interferon alpha-2a (Peg-IFN-2a) have been demonstrated, although follow-up was relatively short. We report here the first profound and sustained molecular responses with a JAK2 V617F allele burden below 1.0% in two patients with polycythemia vera treated with interferon alpha-2b (IFN-2b). Discontinuation of IFN-2b in one of the patients was followed by a sustained long-lasting (12 months of follow-up) major molecular response.

Laparoscopic colonic surgery in Denmark 2004-2007.

OBJECTIVE: Laparoscopic colonic surgery was introduced about 15 years ago and has together with the evidence-based 'fast-track' methodology improved early postoperative outcome. The purpose of this study was to asses the organization and early outcome after laparoscopic colonic surgery in Denmark from 2004 to 2007. METHOD: Based upon the National Patient Register, all laparoscopic colonic operations performed in Denmark between January 2004 and December 2006 were analysed regarding number of hospital departments and procedures, hospital stay, readmissions and mortality. RESULTS: One thousand one hundred and forty-nine laparoscopic colonic resections without simultaneous stoma formation were performed in the study period. Twenty-five departments performed the procedures but only four departments performed more than 100 procedures. The median length of primary stay was 4 days (mean 7.7 days). One hundred and twenty-five (10.9%) patients were re-admitted within 30 days and total length of stay (primary plus readmissions) was a median of 5 days (mean 8.5 days). Thirty-day mortality was 2.6% and hospital mortality 3.5%. CONCLUSION: This nationwide study has shown an increased implementation of laparoscopic colonic surgery but probably performed in too many low volume departments. Laparoscopic colonic surgery should be monitored and further advances secured by adjustment of perioperative care to fast-track care.

Reduced coronary reserve in response to short-term ischaemia and vasoactive drugs in ex vivo hearts from diabetic mice.

AIM: The aim of the present study was to compare the coronary flow (CF) reserve of ex vivo perfused hearts from type 2 diabetic (db/db) and non-diabetic (db/+) mice. METHODS: The hearts were perfused in the Langendorff mode with Krebs-Henseleit bicarbonate buffer (37 degrees C, pH 7.4) containing 11 mmol L(-1) glucose as energy substrate. The coronary reserve was measured in response to three different interventions: (1) administration of nitroprusside (a nitric oxide donor), (2) administration of adenosine and (3) production of reactive hyperaemia by short-term ischaemia. RESULTS: Basal CF was approximately 15% lower in diabetic when compared with non-diabetic hearts (2.1 +/- 0.1 vs. 2.6 +/- 0.2 mL min(-1)). The maximum increase in CF rate in response to sodium nitroprusside and adenosine was significantly lower in diabetic (0.6 +/- 0.1 and 0.9 +/- 0.1 mL min(-1) respectively) than in non-diabetic hearts (1.2 +/- 0.1 and 1.4 +/- 0.1 mL min(-1) respectively). Also, there was a clear difference in the rate of return to basal CF following short-term ischaemia between diabetic and non-diabetic hearts. Thus, basal tone was restored 1-2 min after the peak hyperaemic response in non-diabetic hearts, whereas it took approximately 5 min in diabetic hearts. CONCLUSION: These results show that basal CF, as well as the CF reserve, is impaired in hearts from type 2 diabetic mice. As diabetic and non-diabetic hearts were exposed to the same (maximum) concentrations of NO or adenosine, it is suggested that the lower coronary reserve in type 2 diabetic hearts is, in part, because of a defect in the intracellular pathways mediating smooth muscle relaxation.

Measurement of coronary flow reserve in isolated hearts from mice.

AIM: Langendorff-perfused murine hearts are increasingly used in cardiovascular research, but coronary cardiovascular haemodynamics vary considerably from one research group to another. The aim of this study was to establish an isolated, retrogradely perfused mouse heart preparation for the simultaneous measurement of left ventricular haemodynamics and of coronary flow (CF). METHODS: Heart rate was controlled by right atrial pacing (480 beats min(-1)) and heart temperature was kept constant. Accurate flow values of <0.5 mL min(-1) could be determined, and this methodology was then used to study the stability of this preparation, as well as coronary response to vasoactive drugs and to short-term ischaemia. RESULTS: The CF and maximum systolic pressure were well maintained over a 2-h perfusion period, both showing a 10% decline per hour. Sodium-nitroprusside (endothelium-independent) and adenosine (endothelium-dependent) increased CF relatively modest (30-50% above baseline values). Short-term no-flow ischaemia caused a transient 40-50% increase in CF on reperfusion. Peak reflow occurred approximately 15 s after start of reperfusion and flow returned to baseline during the following 1-2 min. Increased coronary blood flow following infusion of vasoactive drugs (nitroprusside or adenosine) or short-term ischaemia were associated with minor changes in ventricular pressure development. CONCLUSIONS: Blood flow and haemodynamics can readily be determined in this isolated perfused mouse heart model, but CF reserve is relatively small, compared with blood-perfused organs.

Myocardial metabolism and efficiency after warm continuous blood cardioplegia.

BACKGROUND: Warm continuous blood cardioplegia (WCBCP) has been recommended during prolonged cardiac arrest to minimize functional deterioration. Myocardial metabolism and efficiency after this cardioplegic modality are not well described. METHODS: Substrate oxidation, blood flow, and myocardial function were measured before, during, and after 3 hours of WCBCP in 7 pigs. RESULTS: Free fatty acid and glucose oxidation decreased by 60% +/- 3.8% and 94% +/- 1.2%, respectively, during cardioplegia (both p < 0.05) and increased to 62% +/- 28% and 122% +/- 62% of baseline during the early recovery phase (p < 0.05 for glucose). One hour after WCBCP oxidation rates were similar to baseline. The transient postcardioplegic increase in substrate oxidation was associated with a 43% +/- 23% elevation of oxygen consumption (MVO2) compared with baseline and a 62% +/- 18% increase in myocardial blood flow. Cardiac output and mean arterial pressure did not change significantly after WCBCP, although myocardial function (stroke work, left ventricular end-systolic pressure, end-diastolic pressure, contractility, and efficiency) was depressed (p < 0.05). End-diastolic pressure and contractility improved from early to late phase of recovery, whereas the other indicators of ventricular function remained depressed. CONCLUSIONS: Myocardial substrate oxidation was preserved after 3 hours of WCBCP, although ventricular function was moderately impaired. Thus, WCBCP with a seemingly normal substrate and oxygen supply was associated with a reduced cardiac efficiency.

Different tolerance to hypothermia and rewarming of isolated rat and guinea pig hearts.

We examined the effect of hypothermia and rewarming on myocardial function and calcium control in Langendorff-perfused hearts from rat and guinea pig. Both rat and guinea pig hearts demonstrated a rise in myocardial calcium ([Ca]total) in response to hypothermic perfusion (40 min, 10 degrees C), which was accompanied by an increase in left ventricular end diastolic pressure (LVEDP). The elevation in [Ca]total was severalfold higher in guinea pig than in rat hearts, reaching 12.9 +/- 0.8 and 3.1 +/- 0.6 micromol.g dry wt-1, respectively. The rise in LVEDP, however, was comparable in the two species: 62.5 +/- 2.5 (guinea pig) and 52.5 +/- 5.1 mm Hg (rat). Following rewarming, [Ca]total remained elevated in guinea pig, whereas a moderate decline in [Ca]total was observed in the rat (13.6 +/- 1.9 and 2.2 +/- 0.3 micromol.g dry wt-1, respectively). Posthypothermic values of LVEDP were also significantly higher in guinea pig compared to rat hearts (42.5 +/- 6.8 vs 20.5 +/- 5.1 mm Hg, P < 0.027). Furthermore, whereas rat hearts demonstrated a 78 +/- 7% recovery of left ventricular developed pressure, there was only a 15 +/- 7% recovery in guinea pig hearts. Measurements of tissue levels of high energy phosphates and glycogen utilization indicated a higher metabolic requirement in guinea pig than in rat hearts in order to oppose the hypothermia-induced calcium load. Thus, we conclude that isolated guinea pig hearts are more sensitive to a hypothermic insult than rat hearts.

Glucose and fatty acid oxidation by the in situ dog heart during experimental cooling and rewarming.

BACKGROUND: Reduced myocardial function after hypothermia may be metabolic in origin, but the relationship between myocardial metabolism and the various components of hypothermia-mediated dysfunction has not been thoroughly investigated. METHODS: In the present study we measured myocardial uptake and oxidation of glucose and oleate in mongrel dogs undergoing cooling to 25 degrees C followed by rewarming to 37 degrees C, using radiolabeled substrates. RESULTS: Segment work index declined from 39.3 +/- 5.1 to 15.1 +/- 2.4 mm Hg in response to cooling from 37 degrees to 25 degrees C and did not recover completely on rewarming (27.2 +/- 4.2 mm Hg, p < 0.05). Oleate uptake declined from 3,251 +/- 619 to 1,043 +/- 356 nmol.min-1.100 g-1 (p < 0.05) when the dogs were cooled from 37 degrees to 25 degrees C. Simultaneously, oxidation rate fell from 1,089 +/- 158 to 354 +/- 83 nmol.min-1.100 g-1 (p < 0.05). On rewarming, oleate uptake was restored to prehypothermic values, whereas its rate of oxidation remained depressed (480 +/- 129 nmol.min-1.100 g-1; p < 0.05). Uptake and oxidation of glucose also declined significantly during cooling. However, both uptake and oxidation of glucose recovered fully on rewarming. CONCLUSIONS: The results of the present study demonstrate a reduced capacity to oxidize fatty acids by the myocardium during rewarming after hypothermia.

Stimulation of carbohydrate metabolism reduces hypothermia-induced calcium load in fatty acid-perfused rat hearts.

In the present study we examined the impact of glycolysis and glucose oxidation on myocardial calcium control and mechanical function of fatty acid-perfused rat hearts subjected to hypothermia rewarming. One group (control) was given glucose (11.1 mM) and palmitate (1.2 mM) as energy substrates. In a second group glycolysis was inhibited by iodoacetate (IAA, 100 microM) and replacement of glucose with pyruvate (5 mM), whereas in the third group glucose oxidation was stimulated by administration of dichloroacetate (DCA, 1 mM) and insulin (500 microU/ml). All groups showed a rise in myocardial calcium ([Ca]total in response to hypothermia (10 degrees C). However, [Ca]total was significantly lower both in IAA- and DCA-treated hearts, as compared to controls (2.20 +/- 0.22 and 2.94 +/- 0.20 v 3.83 +/- 0.29 nmol/mg dry wt., P < 0.025). The reduced calcium load in the treated hearts was correlated with higher levels of high energy phosphates. Following rewarming control and DCA-treated hearts still showed elevated [Ca]total, whereas IAA-treated hearts [Ca]total was not different from the pre-hypothermic value. All groups showed a reduction in cardiac output following rewarming. Furthermore, the control group, in contrast to both IAA- and DCA-treated hearts, showed a significant reduction in systolic pressure. These results show that hypothermia-induced calcium uptake in glucose and fatty acid-perfused rat hearts was reduced by two different metabolic approaches: (1) inhibition of glycolysis by IAA while simultaneously by-passing the glycolytic pathway by exogenous pyruvate: and (2) stimulation of glucose oxidation by DCA. Thus, glycolytic ATP is not an essential regulator of sarcolemmal calcium transport under the present experimental conditions. Instead, we suggest that a change in oxidative substrate utilization in favour of carbohydrates may improve myocardial calcium homeostasis during hypothermia and rewarming.

Myocardial substrate oxidation during warm continuous blood cardioplegia.

BACKGROUND: Although long-chain fatty acids are a major energy substrate utilized by the myocardium, changes in the substrate balance toward a predominating fatty acid utilization could jeopardize the myocardium during cardiac operative procedures. METHODS: In the present study myocardial substrate utilization was examined during warm continuous blood cardioplegia (4 hours, 37 degrees C), using pigs undergoing cardiopulmonary bypass. Hearts were perfused antegradely in a closed extracorporeal circuit in which cardioplegic donor blood (hematocrit, 22%) containing 14C-glucose and 3H-oleate was delivered to the heart. Arterial and coronary sinus blood samples were taken at intervals for determination of plasma concentrations of energy substrates, as well as glucose and oleate oxidation rates (14CO2 and 3HOH production). RESULTS: The concentration of fatty acids in the cardioplegic perfusate did not change significantly during the cardiac arrest period. The mean concentration of glucose showed a 30% decline (not significant), whereas the lactate concentration increased from a starting value of 3.12 +/- 0.27 to 6.31 +/- 0.72 mmol/L at the end (mean +/- standard error of the mean; n = 8; p < 0.05). Only fatty acid levels showed a significant (positive) arterial-coronary sinus difference. Myocardial oxidation of oleate varied between 302 +/- 71 and 650 +/- 66 nmol.min-1.heart-1, whereas the range of variation for glucose oxidation was 144 +/- 64 to 355 +/- 107 nmol.min-1.heart-1. However, the changes in fatty acid levels and glucose oxidation rates during the cardiac arrest period were not statistically significant. We calculated that overall glucose oxidation accounted for less than 5% of the total aerobic energy production. CONCLUSIONS: The present results demonstrate overreliance on fatty acids as a source of energy during warm continuous blood cardioplegia, consistent with a condition of myocardial insulin resistance.

Normotherm continuous blood cardioplegia for 4 hours in an in vivo pig model.

Warm, continuous blood cardioplegia should theoretically maintain cardiac arrest for hours without ischaemic or hypothermic injury. In the absence of in vivo studies of myocardial metabolism and ultrastructural and/or functional preservation during and after more than 2 hours of cardiac arrest and after weaning from bypass, we devised a porcine model with a closed extracorporeal circuit for the heart alone. Normothermic blood cardioplegia was administered antegrade and recirculated for 2 or 4 hours, each in seven pigs. After aortic declamping all were successfully weaned from bypass and reperfused for 1 hour. Thereafter we found no significant intergroup difference in haemodynamic characteristics (average fall in mean arterial pressure 31.7 +/- 3.2% and 26.9 +/- 2.6%) or blood analyses. After 5 and 60 minutes of cardiac arrest there was minimal lactate production (5.7 +/- 10.7 and 0.5 +/- 10.5 nmol/l, respectively), whereas in the remainder of the arrest period there was lactate uptake, indicating aerobic heart metabolism. Our setup avoids systemic hyperkalaemia, gives good cardiac protection with no deterioration between 2 and 4 hours and is well suited for studies on the quiescent, blood-perfused oxygenated heart.

Phospholipid degradation in hypoxic/reoxygenated cardiomyocytes in response to phospholipase C from Bacillus cereus.

In the present study, we investigated possible mechanisms behind exogenous phospholipase C-induced glycerol production in irreversibly damaged myocytes. Rat ventricular myocytes were preincubated for 60 min in substrate-free Krebs-Henseleit bicarbonate buffer equilibrated with 95% N2-5% CO2 (37 degrees C, pH = 7.4), resulting in exhaustion of cellular high energy phosphates and loss of rod-shaped morphology. At the end of the preincubation period, the incubation vials were divided into two groups; one receiving 10 mU/ml phospholipase C (PC-PLC), whereas the other received an equivalent volume of buffer (control incubations). Incubation was then continued for another 60 min under 95% air-5% CO2 atmosphere. Samples for measurement of metabolite levels were taken immediately after cell isolation, at the end of the preincubation period and at the end of the normoxic incubation period. During the 60 min incubation period following reoxygenation, glycerol output was markedly higher from PC-PLC treated than from control myocytes. However, the elevated glycerol output from these cells was not accompanied by a simultaneous rise in glycerol-3-phosphate, nor was it inhibited by inclusion of pyruvate in the incubation buffer. On the other hand, glycerol output from PC-PLC treated myocytes was effectively inhibited by a diacylglycerol lipase inhibitor (U-57908, The Upjohn Company). Analysis of cellular lipids revealed a 22% reduction of phospholipid in PC-PLC treated myocytes (P < 0.02), while the content of triacylglycerol, diacylglycerol and unesterified fatty acids increased by 76, 261 and 103%, respectively (P < 0.02). No significant changes were observed for these parameters in control myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of fatty acids on myocardial calcium control during hypothermic perfusion.

Although hypothermia is regarded as providing protection of the myocardium during cardiac operations, rapid cooling of the myocardium in the nonarrested state may have detrimental effects on the function of the myocardial cell membrane as a permeability barrier. We therefore measured total cellular calcium in isolated working rat hearts, receiving either glucose (11.1 mmol/L) or glucose plus palmitate (1.2 mmol/L), before, during, and after a 40-minute hypothermic arrest (10 degrees C, Langendorff perfusion). In both groups a rise in total cellular calcium, measured by 45Ca2+ technique, was observed during hypothermia, followed by a decline on rewarming. However, the rise in total cellular calcium during hypothermia was significantly (p < 0.05) higher in hearts perfused with palmitate (from 1.0 +/- 0.2 to 3.5 +/- 0.2 nmol/mg dry weight) compared with that in glucose-perfused hearts (from 1.1 +/- 0.13 to 2.6 +/- 0.2 nmol/mg dry weight). Palmitate-perfused, but not glucose-perfused, hearts showed arrhythmias and delayed pressure development 1 to 2 minutes after rewarming. In addition cardiac output of these hearts was significantly lower (p < 0.025) than that of glucose-perfused hearts 5 to 10 minutes after rewarming. These data show that hypothermia per se causes a net calcium uptake in isolated rat hearts and that this effect is aggravated by high concentrations of fatty acids. Thus the impaired recovery of myocardial function in palmitate-perfused hearts can possibly be related to a distorted calcium handling.

Substrate preference of isolated perfused rat hearts during hypothermia and rewarming.

Fatty acid and glucose oxidation rates were measured in isolated rat hearts undergoing hypothermia and rewarming. The hearts were perfused in the Langendorff mode with Krebs-Henseleit bicarbonate buffer containing 11.1 mM glucose plus 0.6 mM albumin-bound oleic acid as energy substrates. The hearts were stabilized at 37 degrees C and thereafter cooled progressively to 15 degrees C over a period of 60 min. The hearts were kept at this temperature for 10 min and then rewarmed to 37 degrees C during the next 30 min. Control hearts were perfused at 37 degrees C throughout the whole perfusion period. Trace amounts of [14C]glucose or [14C]oleic acid were included in the perfusate, and the rate of substrate oxidation was determined on the basis of the radioactive CO2 production. In normothermic hearts steady state oxidation rates of glucose and oleate were found to be 0.17 +/- 0.01 and 0.51 +/- 0.07 mumol min-1 g-1 dry wt, respectively (mean +/- SEM). In response to hypothermia (15 degrees C) glucose oxidation was reduced by 76% (from 0.17 +/- 0.01 to 0.04 +/- 0.01 mumol min-1 g-1 dry wt) and oleate oxidation by 47% (from 0.51 +/- 0.07 to 0.27 +/- 0.02 mumol min-1 g-1 dry wt). Upon rewarming glucose and fatty acid oxidation rates returned to essentially the same values (0.12 +/- 0.02 and 0.45 +/- 0.04 mumol min-1 g-1 dry wt) as those observed under steady state normothermic conditions. The molar ratio between glucose and fatty acid oxidation was, however, significantly (P < 0.05) lower in hypothermic than in normothermic hearts.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphatidylcholine metabolism in hypoxic and phospholipase C exposed rat ventricular myocytes.

A phospholipase C specific for choline and ethanolamine acyl and plasmalogen glycerophospholipids (PC-PLC) has been described in myocardial tissue. In the present study we investigated whether an endogenous PC-PLC is activated in hypoxic, substrate-free incubations of rat ventricular myocytes. The phosphatidylcholine pool of the myocytes was prelabelled with [14C]choline during a 4-h preincubation (pulse) period. The myocytes were subsequently washed and incubated for another 2 h (chase period) in normoxic, hypoxic, or hypoxic buffer supplemented with PC-PLC from Bacillus cereus. We hypothesized that an increase in the total (intracellular plus extracellular) content of [14C]phosphocholine (one of the products resulting from PC-PLC action on phosphatidylcholine) throughout the chase period would indicate PC-PLC activity. Instead, an apparent decrease was observed for this parameter in all myocyte groups (17-29%), even in the one exposed to exogenous PC-PLC. However, 60 min after the start of the chase period, the level of total [14C]phosphocholine was higher in hypoxic (p = 0.022) and hypoxic + PC-PLC exposed (p = 0.013) myocytes compared with normoxic controls. The total content of [14C]choline increased significantly (p < 0.017) in all myocyte groups during the incubation period (98-153%) as a result of an increment of this metabolite in the buffer. Furthermore, the values measured in hypoxic and hypoxic + PC-PLC exposed myocytes during the first hour of the chase period were significantly (p < 0.017) higher than the corresponding values in normoxic myocytes. The present results do not allow firm conclusions regarding endogenous PC-PLC activation in energy-depleted rat cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipolytic effect of low-molecular-weight-heparin (Fragmin) and heparin/dihydroergotamine in thromboprophylactic doses during total hip replacement.

Eighteen patients admitted for implantation of a primary hip prosthesis were randomized to one of the following thromboprophylactic regimens: (1) dextran 500 ml 12 h-1 on the day of surgery and 500 ml on days 1, 3, and 5 postoperatively, (2) heparin 5000 IU and dihydroergotamine 0.5 mg 8 h-1, or (3) Fragmin (Kabi-Pharmacia, Sweden) 2500 IU 12 h-1 on the day of surgery and 5000 IU 24 h-1 throughout the observation period. Fragmin is a low molecular-weight heparin which has proven both efficient and safe as a thromboprophylactic drug. The purpose of the study was to elucidate whether Fragmin had a lower lipolytic (fatty acid-mobilizing) effect than standard heparin. The plasma level of lipoprotein lipase (LPL) was increased on the first post-operative day both in the heparin/dihydroergotamine (preoperative 217 +/- 79 microU ml-1 vs. 1289 +/- 318, n = 6, p < 0.05) and the Fragmin-groups (136 +/- 16 vs. 668 +/- 123, n = 6, p < 0.05). This increase in plasma LPL was significantly higher in the heparin/dihydroergotamine-group compared to the Fragmin-group (p < 0.05). In both groups, the enzyme activity gradually decreased during the next 2 days. The circulating level of non-esterified fatty acids increased post-operatively only in the heparin/dihydroergotamine-group. Thus, the lower lipolytic effect of Fragmin compared to standard heparin is detectable using thromboprophylactic doses during surgery in patients on a standard hospital diet. From a theoretical viewpoint, the use of Fragmin is advantageous in patients with myocardial ischaemia and patients risking respiratory distress problems due to a low circulating level of non-esterified fatty acids.

Myocardial cell vulnerability to exogenous phospholipase attack.

Myocardial cell vulnerability to phospholipase C (PC-PLC) attack was investigated in three different preparations of rat myocardial cells: triacylglycerol (TG)-loaded, hypothermic/rewarmed and energy depleted myocytes. The attack by PC-PLC was evaluated as PC-PLC induced glycerol output due to the combined action of phospholipase C and intracellular lipases. PC-PLC induced glycerol output was significantly higher (p < 0.05) in all three myocyte preparations, compared to their respective controls. Cell morphology (% rod shaped myocytes) of TG-loaded or hypothermic/rewarmed myocytes was not different from their controls, whereas energy depleted myocytes almost exclusively were rounded up, due to hypercontraction of the myofilaments. Hypothermic/rewarmed and energy depleted myocytes showed a significantly higher release of lactate dehydrogenase (LDH), compared to their controls although the difference was much more pronounced in the latter. Finally, the cellular contents of ATP were maintained both in TG-loaded and hypothermic rewarmed myocytes, while energy depleted myocytes contained only about 25% of the normal ATP level. These results demonstrate that attack from exogenously added phospholipases can occur, not only in seriously damaged cardiac myocytes, but in myocytes with a more subtle damage as well.

Effects of hypothermia and rewarming on phospholipase C-evoked glycerol output in rat myocardial cells.

The combined action of phosphatidylcholine preferring phospholipase C (PC-PLC) and intracellular lipases has recently been shown to cause glycerol output in energy deprived rat cardiomyocytes. In the present study we examined the effect of hypothermia and rewarming on PC-PLC evoked glycerol output in freshly isolated, calcium-tolerant myocytes. The cells were preincubated for 60 min at hypothermic (5 degrees C) or normothermic (37 degrees C) conditions in Krebs-Henseleit bicarbonate buffer (pH 7.4) supplemented with 1 mM DL-carnitine, 1% B.S.A. and 5 mM glucose. Addition of PC-PLC resulted in a significantly higher (P less than 0.05) output of glycerol in myocytes undergoing rewarming than in myocytes kept constantly at 5 degrees C or 37 degrees C. The values obtained for PC-PLC induced glycerol output (difference in glycerol output between incubations with and without PC-PLC) were 6.77 +/- 2.6 (37 degrees C), 4.54 +/- 1.7 (5 degrees C) and 22.85 +/- 5.9 (5-37 degrees C) nmol/10(6) cells.h. Rewarming in addition caused a significantly higher (P less than 0.05) leakage of lactate dehydrogenase (LDH) from the rewarmed cells as compared to cells at constant temperatures (5 degrees C or 37 degrees C). However, there was no additional effect of PC-PLC on LDH leakage. The elevated PC-PLC induced glycerol output in rewarmed myocytes was not related to a fall in the percentage of rod-shaped cells or a reduced cellular content of ATP, since no differences could be detected between the various myocyte preparations with respect to these parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycogen consumption in hypoxic rat cardiomyocytes.

Glycogen consumption was investigated in isolated adult rat myocytes incubated for 2 h (37 degrees C) in substrate-free, hypoxic Krebs-Henseleit bicarbonate buffer. No consumption of glycogen occurred after 1 h of incubation, and the residual glycogen after 2 h was 23% despite an 89% reduction of the initial ATP content (from 27.1 +/- 1.8 to 3.1 +/- 0.5 nmol/mg dry weight, n = 12). The residual glycogen was not due to lactate inhibition of glycolytic enzymes, since myocytes incubated in the presence of 5 mM glucose maintained high energy phosphates throughout the incubation period despite a considerable lactate accumulation (1740 +/- 43 nmol/mg dry weight in glucose-supplemented vs. 138 +/- 14 nmol/mg dry weight in substrate-free incubations, n = 12). We have previously shown that the content of cyclic AMP in myocytes is not altered in response to hypoxia, thereby excluding activation of glycogen phosphorylase a. In the present study, the fall in myocyte ATP content was not followed by a rise in AMP, possibly preventing allosteric activation of glycogen phosphorylase b. However, addition of cyanide to the hypoxic incubations increased cellular AMP (initial level 2.1 +/- 0.4 nmol/mg dry weight vs. 9.8 +/- 0.7 after 30 min, n = 12) without increasing the amount of glycogen consumed, also ruling out the lack of glycogen phosphorylase b activation in the myocytes. Therefore, the glycogen rest was probably confined to the 17% of myocytes hypercontracted at the start of incubations.

Regulation of lipolysis in normoxic and hypoxic rat myocytes.

The mechanism involved in ischemia-induced myocardial lipolysis is still a matter of controversy. To elucidate the regulation of lipolysis at the cellular level, we incubated isolated rat myocytes in normoxic or hypoxic medium containing 11.1 mM glucose. Rates of lipolysis (glycerol output) were significantly (P less than 0.05, n = 12) higher in hypoxic than in normoxic myocytes (34.9 +/- 3.9 vs. 17.7 +/- 3.4 nmol/10(6) cells.30 min). However, there was no change in the content of cellular triacylglycerol (TG) in normoxic myocytes whereas it fell slightly (8 +/- 2 nmol/10(6) cells.30 min, P less than 0.05, n = 12) in hypoxic myocytes. On a molar basis glycerol output was significantly higher than the corresponding fall in TG (P less than 0.05, n = 12, both normoxic and hypoxic myocytes). This difference (glycerol output--TG reduction) amounted to 17.1 +/- 3.4 nmol/10(6) cells.30 min in normoxic myocytes and 27.6 +/- 5.1 nmol/10(6) cells.30 min in hypoxic myocytes (P less than 0.05, n = 12, normoxic vs. hypoxic). The hypoxia-induced rise in glycerol output was paralleled by an increased intracellular level of glycerol-3-phosphate. Both these responses were, however, dose-dependently inhibited by addition of pyruvate to the incubation medium, giving rise to a close correlation between cellular glycerol-3-phosphate and glycerol output (r = 0.75, P less than 0.05). This indicates mass action of glycerol-3-phosphate on fatty acid-TG cycling under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)