Troglitazone enhances glucose uptake induced by alpha-adrenoceptor stimulation via phosphatidylinositol 3-kinase in rat heart.

1. Thiazolidinedione-derived agents have been reported to act as insulin sensitizers by augmenting insulin-dependent stimulation of phosphatidylinositol 3-kinase (PI3K) activity in a specific manner. It has been suggested that alpha-adrenoceptor stimulation mediates glucose uptake through PI3K in the heart. 2. To elucidate whether the thiazolidinedione-derived agent troglitazone (TRO) affects glucose uptake induced by alpha-adrenoceptor stimulation through PI3K, the rate of glucose uptake was quantified from the rate of accumulation of sugar phosphate (d[SP]/dt) using [(31)P] nuclear magnetic resonance spectroscopy after substitution of glucose with 2-deoxyglucose in rat perfused heart. Hearts were stimulated with 100 micromol/L phenylephrine plus 10 micromol/L propranolol (alpha-adrenoceptor stimulation), or 1 micromol/L isoproterenol plus 10 micromol/L phentolamine (beta-adrenoceptor stimulation). 3. The d[SP]/dt in the alpha- and beta-adrenoceptor-stimulated groups (0.45 +/- 0.06 and 0.42 +/- 0.04 micromol/min per g, respectively) was higher than that of the control group (0.27 +/- 0.02 micromol/min per g; P < 0.01). The addition of 2 microg/mL troglitazone to alpha-adrenoceptor stimulation augmented d[SP]/dt (0.72 +/- 0.08 micromol/min per g; P < 0.05 vs the alpha-adrenoceptor-stimulated group), which was effectively blocked by 3 micromol/L wortmannin (0.35 +/- 0.06 micromol/min per g; P < 0.01 vs troglitazone + alpha-adrenoceptor stimulation group). However, addition of troglitazone to beta-adrenoceptor stimulation did not alter d[SP]/dt (0.33 +/- 0.02 micromol/min per g; P = NS vs the beta-adrenoceptor-stimulated group). 4. These results indicate that troglitazone acutely enhances alpha-adrenoceptor stimulation on glucose uptake through a PI3K-dependent pathway, thus possibly improving glucose utilization in a catecholamine-released state.

Role of intracellular Na(+) kinetics in preconditioned rat heart.

To elucidate the role of intracellular Na(+) kinetics in the mechanism for ischemic preconditioning (IPC), we measured intracellular Na(+) concentration ([Na(+)](i)) using (23)Na-magnetic resonance spectroscopy in isolated rat hearts. IPC significantly delayed the initial [Na(+)](i) increase (d[Na(+)](i)/dt) compared with non-IPC control, resulting in attenuation of Na(+) accumulation (Delta[Na(+)](i)) during 27 minutes of ischemia with better functional recovery. [Na(+)](i) in IPC, but not in control, recovered to preischemic level during a 6-minute reperfusion. The Na(+)-H(+) exchange inhibitor further suppressed d[Na(+)](i)/dt in both control and IPC hearts with concomitant improvement of functional recovery, suggesting little contribution to the mechanism of IPC. The mitochondrial ATP-sensitive K(+) (mito K(ATP)) channel activator diazoxide (30 micromol/L) completely mimicked both [Na(+)](i) kinetics and functional recovery in IPC without any additive effects to IPC. The mito K(ATP) channel blocker 5-hydroxydecanoic acid (100 micromol/L) lost protective effect as well as the attenuation of d[Na(+)](i)/dt and [Na(+)](i) recovery induced by diazoxide. However, 5-hydroxydecanoic acid also lost IPC-induced protection, but incompletely abolished the alteration of d[Na(+)](i)/dt and the [Na(+)](i) recovery. The Na(+)/K(+)-ATPase inhibitor ouabain (200 micromol/L) did not change d[Na(+)](i)/dt in non-IPC hearts, but it abolished the IPC- or diazoxide-induced reduction of d[Na(+)](i)/dt and the [Na(+)](i) recovery, whereas IPC followed by ouabain treatment showed partial functional recovery with smaller Delta[Na(+)](i) than other ouabain groups. In conclusion, alteration of Na(+) kinetics by preserving Na(+) efflux via Na(+)/K(+)-ATPase mediated by mito K(ATP) channel activation mainly contributes to functional protection in IPC hearts. The contribution of mito K(ATP) channel-independent pathway relating to Na(+) kinetics including reduced Na(+) influx is limited in functional protection of IPC.

Type IV phosphodiesterase inhibitor suppresses insulin-dependent myocardial glucose uptake.

1. Phosphodiesterase (PDE) IV has been localized at cardiomyocytes and the coronary vasculature and modulates cAMP, but the effect of PDE IV on myocardial glucose uptake has not been demonstrated. 2. Glucose uptake in rat isolated hearts treated with the PDE IV inhibitor rolipram was measured by [31P] nuclear magnetic resonance spectroscopy. 3. Under non-stimulating conditions, glucose uptake was not significantly different between control and rolipram (1 micromol/L)-treated rat hearts, whereas enhanced uptake in insulin-stimulated conditions was significantly attenuated by rolipram. 4. Phosphodiesterase IV inhibitor negatively affects insulin-dependent myocardial glucose uptake.

Kinetics of a putative hypoxic tracer, 99mTc-HL91, in normoxic, hypoxic, ischemic, and stunned myocardium.

UNLABELLED: 99mTc-4,9-diaza-3,3,10,10-tetramethyldodecan-2,11-dione dioxime (HL91) was developed as a putative hypoxic reagent. This study focused on the myocardial kinetics of 99mTc-HL91 in various oxygen levels and perfusion states. METHODS: The time-activity curve of 99mTc-HL91 was measured in isolated perfused rat heart after the bolus infusion. RESULTS: 99mTc-HL91 was cleared quickly from normoxic hearts, and retention at 30 min after injection was 0.18 +/- 0.02 percentage injected dose per gram of wet weight (mean +/- SE; n = 6). When the concentration of oxygen bubbling through the perfusate was reduced from 100% to 50%, 20%, 5%, and 0%, retention of 99mTc-HL91 increased to 0.47 +/- 0.03 (n = 5), 0.48 +/- 0.03 (n = 5), 0.71 +/- 0.01 (n = 5), and 0.70 +/- 0.02 (n = 5), respectively (P < 0.05). Compartment analysis revealed that the trapping mechanism, which was dependent on tissue oxygen concentration, determined the retention rate. Although not retained in stunned myocardium (0.17 +/- 0.02, n = 5; P = not significant), 99mTc-HL91 was significantly retained when injected before ischemia (1.06 +/- 0.06, n = 5; P < 0.05). CONCLUSION: These results indicate that retention of 99mTc-HL91 correlates well with oxygen level in the perfusate, suggesting that the agent may be a useful marker of the severity of myocardial hypoxia.

Intracellular sodium accumulation during ischemia as the substrate for reperfusion injury.

To elucidate the role of intracellular Na+ kinetics during ischemia and reperfusion in postischemic contractile dysfunction, intracellular Na+ concentration ([Na+]i) was measured in isolated perfused rat hearts using 23Na nuclear magnetic resonance spectroscopy. The extension of the ischemic period from 9 minutes to 15, 21, and 27 minutes (at 37 degrees C) increased [Na+]i at the end of ischemia from 270.0+/-10.4% of preischemic level (mean+/-SE, n=5) to 348.4+/-12.0% (n=5), 491.0+/-34.0% (n=7), and 505.3+/-12.1% (n=5), respectively, whereas the recovery of developed pressure worsened with the prolongation of the ischemic period (95.1+/-4.2%, 84.3+/-1. 2%, 52.8+/-13.7%, and 16.9+/-6.4% of preischemic level). The kinetics of [Na+]i recovery during reperfusion was analyzed by the fitting of a monoexponential function. When the hearts were reperfused with low-[Ca]o (0.15 mmol/L) solution, the time constants of the recovery (tau) after 15-minute (8.07+/-0.85 minutes, n=5) and 21-minute ischemia (6.44+/-0.90, n=5) were significantly extended, with better functional recovery (98.5+/-1.4% for 15-minute [P<0.05]; 98.0+/-1.0% for 21-minute [P<0.05]) compared with standard reperfusion ([Ca]o=2.0 mmol/L, tau=3.58+/-0.28 minutes for 15-minute [P<0.0001]; tau=3.02+/-0.20 for 21-minute [P<0.0001]). A selective inhibitor of Na+/Ca2+ exchanger also decelerated the [Na+]i recovery, which suggests that the recovery reflects the Na+/Ca2+ exchange activity. In contrast, high-[Ca]o reperfusion (5 mmol/L) accelerated the [Na+]i recovery after 9-minute ischemia (tau=2.48+/-0.11 minute, n=5 [P<0.0001]) and 15-minute ischemia (tau=2.10+/-0.07, n=6 [P<0. 05]), but functional recovery deteriorated only in the hearts with 15-minute ischemia (29.8+/-9.4% [P<0.05]). [Na+]i recovery after 27-minute ischemia was incomplete and decelerated by low-[Ca]o reperfusion, with limited improvement of functional recovery (42. 5+/-7.9%, n=5 [P<0.05]). These results indicate that intracellular Na+ accumulation during ischemia is the substrate for reperfusion injury and that the [Na+]i kinetics during reperfusion, which is coupled with Ca2+ influx, also determines the degree of injury.

Lumped constant for deoxyglucose is decreased when myocardial glucose uptake is enhanced.

Quantification of myocardial glucose uptake by positron emission tomography with [18F]fluorodeoxyglucose (FDG) requires the "lumped constant" (LC), which corrects the difference of affinity between glucose and FDG to glucose transporters and phosphorylating system. Since LC was introduced, it has been considered to be constant. However, this has recently been questioned. To elucidate the constancy of LC by other than radioisotope techniques, the accumulation rate of sugar phosphates (d[SP]/dt) was measured in isolated, perfused rat hearts by 31P NMR spectroscopy with 2-deoxyglucose (DG). We postulate alpha as the affinity of DG to transporters and the phosphorylating system relative to that of glucose. Theoretically, alpha is equivalent to LC. We determined alpha by measuring d[SP]/dt at DG concentration ([DG]) = 10, 7, 5, and 3 mmol/l, keeping the total of glucose concentration ([glucose]) and [DG] to 10 mmol/l. When the glucose uptake was enhanced by insulin (10 mU/ml) or stunning, calculated alpha was reduced (insulin stimulated, 0.15; stunning, 0.19) compared with the control (0.59). These results indicate that LC can be evaluated by methods without radiolabeled tracers and is smaller when glucose uptake is augmented.

Alteration of intracellular Na+ during ischemia in diabetic rat hearts: the role of reduced activity in Na+/H+ exchange against stunning.

To elucidate the contribution of reduced activity of Na+/H+ exchange in streptozotocin-induced diabetic hearts against stunning, intracellular Na+ concentration ([Na+]i) was measured in isolated rat hearts using 23Na-MRS. The recovery of left ventricular developed pressure in hearts reperfused after 15 min global ischaemia at 37 degreesC was significantly better in diabetic ones (102.9+/-2.0% of pre-ischemic level, mean+/-s.e., n=6; P<0.05), and non-diabetic ones pre-treated with potent Na+/H+ exchange inhibitor, EIPA (1 mu mol/l; 93.8+/-2.3%, n=5; *P<0.05) than non-treated, non-diabetic hearts (75.1+/-2.5%, n=8). When diabetic hearts were pre-treated with EIPA, the recovery (101.2+/-2.6%, n=5) was identical to that of non-treated, diabetic hearts. [Na+]i in non-diabetic hearts increased to 329.1+/-8.1% of pre-ischemic level during 15 min ischemia, whereas the increase in [Na+]i in diabetic hearts significantly suppressed to 199.8+/-10.3% (P<0.001). EIPA attenuated the increase of [Na+]i during ischemia to 189.1+/-9.0% in non-diabetic hearts ( P<0.001) and to 155.3+/-4.6% in diabetic hearts (P<0.05). Thus, the EIPA-dependent Na+ accumulation during ischemia, i.e. Na+ influx probably mostly via Na+/H+ exchange was smaller in diabetic hearts by 69.7% compared with that in non-diabetic hearts. These results indicate that the cardiac protection against stunning in streptozotocin-induced diabetic hearts is mediated by the attenuation of Na+ accumulation during ischemia, which is caused by the reduced activity in Na+/H+ exchanger.