Intracellular accumulation of isoproterenol enhances the lactate production in the perfused rat heart

Effects of intracellular accumulation of isoproterenol (ISO) on lactate production were examined in perfused rat heart. The lactate production during ISO perfusion in rat heart was increased and subsequent addition of an inhibitor of catechol-O-methyl transferase (COMT) further enhanced the production, and the enhanced production was significantly reduced by uptake2 inhibitor. The perfusion with ISO free-medium in the heart with high intracellular accumulation of ISO produced lactate more than that in the low intracellular accumulation. The present experiments demonstrated that the enhanced lactate production is accompanied by intracellular accumulation of ISO in the perfused rat heart, and suggested that the accumulated ISO may activate intracellular beta-adrenoceptors in the rat heart.     

Dopamine 4-sulfate: effects on isolated perfused rat heart and role of atria

We studied the effects of sulfate conjugate of dopamine on the isolated perfused rat heart (Langendorff preparation). In the experimental group, we removed atria from half number of the hearts. In the hearts with intact atria, dopamine 4-sulfate significantly improved the DT (developed tension), +dT/dt max (maximal rate of contraction), -dT/dt max (maximum rate of relaxation) over baseline values. But when atria were removed, dopamine 4-sulfate had no effect on the mechanical functions of heart. We analysed the effluent perfusate for the free and conjugated catecholamines. In the control group (no drug), and when atria were excised, the free catecholamine levels were negligible. But when the atria were kept intact, the effluent contained significant amount of free dopamine (DA), and norepinephrine (NE). These data suggested that dopamine sulfate had no direct effect on the ventricular muscle of rat heart, but was converted within the atrial tissues into free catecholamines which might be responsible for the positive inotropic actions.     

Effects of specific alpha-adrenoceptive agents on extraneuronal uptake (uptake2) of isoproterenol in perfused rat heart

Effects of specific alpha-adrenoceptive agents (alpha 1-agonist, alpha 1-antagonist, alpha 2-agonist and alpha 2-antagonist) on the extraneuronal accumulation of 3H-isoproterenol in the perfused rat heart were examined. The extraneuronal accumulation of 3H-isoproterenol in the hearts perfused with 3H-isoproterenol (10(-6)M) under COMT inhibition by tropolone (10(-4)M) was about 6 times higher than that of intact COMT. The increase in the accumulation by COMT inhibition was regarded as 100% and the effects of specific alpha-adrenoceptive agents on the accumulation was evaluated. alpha 1-agonists, methoxamine and phenylephrine, did not affect the accumulation. alpha 1-antagonists, prazosin, bunazosin and YM-12617, significantly decreased the accumulation of 3H-isoproterenol and these IC50 values were 2 x 10(-6)M, 3.5 x 10(-6)M and 2.3 x 10(-5)M, respectively. alpha 2-agonists, clonidine and guanabenz, significantly reduced the accumulation and these IC50 values were 3.4 x 10(-5)M and 2.9 x 10(-7)M, respectively. The alpha 2-antagonist, yohimbine, did not affect the accumulation. The present experiments clearly demonstrated that the tested alpha 1-antagonists and alpha 2-agonists inhibited uptake2 in rat heart but the tested alpha 1-agonists and an alpha 2-antagonist did not inhibit it.     

Electron microscopy description of cardiomyocytes from the left ventricle of rat heart after apoptosis induction by isoproterenol

Changes in cardiomyocytes from the left ventricle of rat heart were studied by light and electron microscopic and morphometric methods in the myocardial regions neighboring necrotic foci formed after the injection of 80 mg/kg β adrenomimetic isoproterenol. TUNEL assay was used to detect apoptotic cardiomyocytes. Three types of cardiomyocytes (A, B, and C) differing by the ultrastructure of the nucleus and the degree of mitochondrial changes were identified at all studied stages of necrotic focus development (4–48 h). B and C type cardiomyocytes could represent cells at different stages of apoptosis. The apoptotic changes in cardiomyocytes proved to prevail in early lesion foci (4–18 h), while cardiomyocytes at later stages were prone to necrosis; cardiomyocytes can exhibit signs of apoptosis and necrosis at the same time.     

Pseudoketogenesis in the perfused rat heart

Ketogenesis is usually measured in vivo by dilution of tracers of (3R)-hydroxybutyrate or acetoacetate. We show that, in perfused working rat hearts, the specific activities of (3R)-hydroxybutyrate and acetoacetate are diluted by isotopic exchanges in the absence of net ketogenesis. We call this process pseudoketogenesis. When hearts are perfused with buffer containing 2.3 mM of [4-3H]- plus [3-14C]acetoacetate, the specific activities of [4-3H] and [3-14C]acetoacetate decrease while C-1 of acetoacetate becomes progressively labeled with 14C. This is explained by the reversibility of reactions catalyzed by mitochondrial 3-oxoacid-CoA transferase and acetoacetyl-CoA thiolase. After activation of labeled acetoacetate, the specific activity of acetoacetyl-CoA is diluted by unlabeled acetoacetyl-CoA derived from endogenous fatty acids or glucose. Acetoacetyl-CoA thiolase partially exchanges 14C between C-1 and C-3 of acetoacetyl-CoA. Finally, 3-oxoacid-CoA transferase liberates weakly labeled acetoacetate which dilutes the specific activity of extracellular acetoacetate. An isotopic exchange in the reverse direction is observed when hearts are perfused with unlabeled acetoacetate plus [1-14C]-, [13-14C]-, or [15-14C]palmitate; here also, acetoacetate becomes labeled on C-1 and C-3. Computations of specific activities of (3R)-hydroxybutyrate, acetoacetate, and acetyl-CoA yield minimal rates of pseudoketogenesis ranging from 19 to 32% of the net uptake of (3R)-hydroxybutyrate plus acetoacetate by the heart.     

Norepinephrine-induced 1,2-diacylglycerol accumulation and change in its fatty acid composition in the isolated perfused rat heart

Phosphoinositide hydrolysis is elicited by -adrenoceptor stimulation in the myocardium, resulting in the generation of 1,2-diacylglycerol by the direct activation of phospholipase C. However, the physiological role of 1,2-diacylglycerol accumulation in the heart has been largely unexplored. Therefore, we studied the effects of norepinephrine on the accumulation of 1,2-diacylglycerol and its fatty acid composition, as well as its function in isolated perfused rat hearts. A 30 min perfusion with norepinephrine following a stabilization period of 25 min caused increases of 68% and 57% in 1,2-diacylglycerol levels in the heart at 10^–6 M and 5 × 10^–6 M, respectively, compared to controls. Analysis of its fatty acid composition showed a significant elevation in the percentages of 18:2 and 20:4 although the absolute amounts of these increases in fatty acids were relatively low when compared to the elevation in the total amount of 1,2-diacylglycerol. The change in contractility was not consistently related to an increase in 1,2-diacylglycerol. These results indicate that increase in 1,2-diacylglycerol level in response to norepinephrine perfusion was accompanied by a change in fatty acid composition of 1,2-diacylglycerol.     

Leucine enkephalin antagonizes norepinephrine-induced 45Ca++ accumulation in rat atria

Exposure of rat atrial slices to 10(-5) M norepinephrine (NE) for 10 minutes increases 45Ca++ accumulation from 1.64 +/- 0.10 to 2.23 +/- 0.06 nmol/mg tissue. In the presence of leucine enkephalin (10(-8) M), NE-stimulated 45Ca++ uptake is reduced to 1.44 +/- 0.10 nmol/mg tissue. The effect of leu-enkephalin is reversed in the presence of 10(-7) M naloxone, NE-stimulated 45Ca++ uptake being increased to 2.17 +/- 0.15 nmol/mg tissue. The results support a direct interaction of leu enkephalin with beta-agonist-stimulated Ca++ flux in rat atria, and correlate with the previously reported enkephalin antagonism of NE-induced positive chronotropy in the same tissue.     

Right atrial predominance of atrial natriuretic peptide secretion in isolated perfused rat atria.

In order to investigate the regulatory mechanism for the atrial release of atrial natriuretic peptide (ANP), a perfused rabbit atrial model was devised. In the present experiments, the effect of a reduction in atrial distension on the immunoreactive ANP (irANP) secretion was investigated and compared in the perfused right and left atria of rats. Elevations in right and left atrial pressure resulted in proportional increases in the volume of atrial distension-reduction which was larger in the right than in the left atria. The basal rate of irANP secretion was higher in the right than in the left atria. Increases in the volume of atrial distension-reduction resulted in proportional increases in irANP secretion in both atria. Increment in irANP secretion in response to a reduction in atrial distension was significantly higher in the right than in the left atria. Higher rate of irANP secretion in response to unit volume change was observed in the right atria. Increases in the volume of atrial distension-reduction resulted in accentuated irANP responses in the right atrium. IrANP content was significantly higher in the right than in the left atria. The results suggest that the right atrium is a predominant site in ANP secretion in rats.     

The subunit proportions and kinetic properties of 6-phosphofructo-1-kinase isozymes from rat heart atria and ventricle progressively change during aging

Relative to 2–3 month rats, total 6-phosphofructo-1-kinase (PFK) activity in heart atria from 12 month rats declined 31%; but, by 24 months it was decreased by only 13%. PFK activities from 12 and 24 month ventricles relative to the 2–3 month rat were decreased by 40% and 30%, respectively. This change in PFK activity in each heart region was associated with alterations of subunit composition. In heart atria from 12 and 24 month rats when compared to 3 month rats, the levels of L-type subunit were not significantly different; but the levels of the M-type subunit were decreased by 43% and 38%, respectively. With respect to levels in 2–3 month atria, the C-type subunit in 12 month atria decreased by 27%; and at 24 months it increased by 31%. Making the same comparison for the heart ventricle at 12 and 24 months, L-type subunit decreased by 30% and 24% respectively; M-type subunit decreased by approximately 47%; and the C-type subunit increased 1.9 and 4.7 fold, respectively. These age-related changes of subunit composition in atrial and ventricular PFK isozyme pools led to changes in their kinetic and regulatory properties suggesting that the aged rat could exhibit a diminished capacity to produce ATP from glucose.     

Effects of isoproterenol treatment of isolated perfused rat hearts on myofibrillar phosphorylation and ATPase activity

Perfusion of isolated rat hearts with isoproterenol resulted in increases in the level of protein-bound phosphate of the myofibrils. After perfusion of the hearts with 32P, followed by SDS-polyacrylamide gel electrophoresis of the purified myofibrils, four major 32P-containing protein bands were identified. Most of the increased 32P incorporation produced by isoproterenol was localized on the troponin I and myosin light chain bands, and, to lesser extent, on the M-protein band. ATPase activity was tested in the purified myofibrils. No changes in Ca2+ requirement for activation were found after isoproterenol perfusion. However, maximal ATPase activity was markedly reduced in the myofibrils obtained from isoproterenol-treated hearts. It would appear that the myofibrillar protein phosphorylation induced by isoproterenol perfusion results in a decrease in actomyosin ATPase activity.     

Rapid exchange of actin-bound nucleotide in perfused rat heart

In the rat heart the actin-bound nucleotide contained both ATP and ADP. The ratio of bound ATP to bound ADP depended on the functional state of the heart; it was higher in hearts stopped reversibly in diastole (low Ca(2+), high Mg(2+), or high K(+)), than in stimulated (inotropic agents or pacing) hearts. Immunoblotting and gel electrophoresis showed the existence of G-actin (30% of total actin) in the cytoplasm of the heart. Pure actin was isolated from rat hearts: in G-actin the bound nucleotide readily exchanged with ATP or ADP, and in F-actin the bound nucleotide did not exchange with ATP or ADP. The free and bound nucleotides were separated in the intact heart by extraction with 75% methanol at -15 degrees C. In rat hearts perfused with (32)P-labeled orthophosphate the actin-bound nucleotide rapidly exchanged with the cytoplasmic ATP. The full exchange of the bound ATP was immediate, whereas the full exchange of the bound ADP was slower. The full exchange of the bound ATP was independent of the heartbeat frequency, whereas the full exchange of the bound ADP was frequency dependent. The data suggest that the transformation of actin monomer-ATP to actin polymer-ADP is a part of the normal contraction-relaxation cycle of the rat heart.     

Postnatal changes in the activities of acetylcholinesterase and butyrylcholinesterase in rat heart atria

Postnatal development of the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in rat heart atria has been investigated with the use of 1.5-bis (allyldimethylammoniumphenyl) pentane-3-dibromide (BW 284 C51) as a selective inhibitor of AChE. Total cholinesterase activity (mumol acetylthiocholine hydrolysed X g-1 per hour) increased from 218 on the 1st day after birth to 426 on the 30th day and diminished to 340 in adult rats. The activity of AChE (mumol acetylthiocholine hydrolysed X g-1 per hour) underwent more dramatic changes, increasing more than 4-fold during the first month of life, from 13 on the 1st to 58 on the 30th day of life and then decreasing to 42 in adult rats. The proportion of AChE on total cholinesterase activity increased from 6% on the 1st day to 12-15% in animals aged 24 days and more. Since AChE is known to be specifically involved in the termination of the action of acetylcholine in the sinoatrial node, the observed postnatal changes in its activity are likely to play a role in the postnatal development of cardiac parasympathetic control.     

Fatty acid chain-elongation in perfused rat heart: synthesis of stearoylcarnitine from perfused palmitate

Rat hearts perfused for up to 60 min in the working mode with palmitate, but not with glucose, resulted in substantial formation of palmitoylcarnitine and stearoylcarnitine. To test whether lipolysis of endogenous lipids was responsible for the increased stearoylcarnitine content or whether some of the perfused palmitate underwent chain elongation, hearts were perfused with hexadecanoic-16,16,16-d(3) acid (M+3). The pentafluorophenacyl ester of deuterium labeled stearoylcarnitine had an M+3 (639.4 m/z) compared to the unlabeled M+0 (636.3 m/z) consistent with a direct chain elongation of the perfused palmitate. Furthermore, the near equal isotope enrichment of palmitoyl- (90.2+/-5.8%) and stearoylcarnitine (78.0+/-7.1%) suggest that both palmitoyl- and stearoyl-CoA have ready access to mitochondrial carnitine palmitoyltransferase and that most of the stearoylcarnitine is derived from the perfused palmitate.     

Glucose metabolism in the hypothermic perfused rat heart.

Although hypothermic whole organ perfusion is widely used in attempts to preserve organs for transplantation and to preserve the myocardium during cardiac surgery, little is known about substrate metabolism during hypothermia. A knowledge of metabolism utilization during hypothermic whole organ perfusion might allow optimal substrate choice for preservation of energy stores and functional capacity. Separate groups of hearts from fed rats were perfused 30 minutes with Krebs Henseleit bicarbonate buffer containing 5mM glucose-U-¹⁴C, at 37°, 25°, 20°, 15° and 10°C. From 37° to 15°C, heart rate decreased 90% and coronary flow decreased 25%. Glucose uptake decreased 5 fold from 37° to 10°C while ¹⁴CO₂ and lactate production decreased 50 fold and 28 fold, respectively. Myocardial glycogen was stable until 10°C at which point increased glycogenolysis occured. The incorporation of ¹⁴C in glycogen was stable at 37°, 30° and 25° but decreased progressively with lower temperatures. The percent recovery of glucose as ¹⁴CO₂, lactate and ¹⁴C in glycogen decreased from 73% at 37° at 10°C. Our studies indicate that metabolism of glucose is greatly reduced but significant above 15°C.     

D-3-Hydroxybutyrate metabolism in the perfused rat heart

Summary The utilization of D-3-HB and the production of acetoacetate by the perfused rat heart were investigated over a wide range of DL-3-HB concentrations. The rate of D-3-HB utilization is concentration dependent, and shows saturation kinetics. The oxidized amount of D-3-HB when D-3-HB as a sole substrate, accounts at a maximum for 50% of the total oxygen consumption, which suggest the contribution of the endogenous substrate as fuel source along with D-3-HB. The proportion of the D-3-HB consumed that is oxidized rather than released as acetoacetate increases from 70% to 93% as the concentration of D-3-HB falls from 6.99 mM to 0.30 mM.