Characteristics of the myocardial PM-FABP: effect of diabetes mellitus.

Properties of the myocardial PM-FABP were studied in normal and STZ-diabetic rats. The fluorescent fatty acids trans-parinaric and cis-parinaric acids were used as analogs of straight-chain (saturated) and kinked-chain (unsaturated) fatty acids respectively. Parinaric acid binding was sensitive to trypsin. Trans-parinaric acid binding was more sensitive to this protease than the binding of cis-parinaric acid. Based on the difference in sensitivity of parinaric acid binding we believe that there are two separate binding sites associated with myocardial PM-FABP; one for unsaturated fats and the other for saturated fats. Diabetes enhanced both cis- and trans-parinaric acid binding capacity in cardiomyocytes; cis-parinaric acid by 2 fold and trans-parinaric acid by 2.6 fold. In addition, there was a concomitant accumulation of free fatty acids and triglycerides in the hearts of the diabetic animals. There was a 2.2 fold increase for fatty acids and a 1.6 fold increase for trigylcerides. This association between myocardial fatty acid build-up and enhanced myocardial PM-FABP during diabetes suggest that this carrier protein might have contributed to lipid accumulation in the hearts of the diabetic rats.

Effect of insulin on fatty acid uptake and esterification in L-cell fibroblasts.

We examined the effects of insulin on fatty acid uptake in L-cell fibroblasts, using cis-parinaric acid to measure uptake rates in the absence of esterification and [3H]oleic acid to measure uptake rates in the presence of esterification. L-cells exhibited both high and low affinity insulin binding sites with Kd of 23 nM and 220 nM and a cellular density of 1.4 and 6.8 x 10(5) sites/cell, respectively. Insulin in the range 10(-9) to 10(-7) M significantly decreased both the initial rate and maximal extent of cis-parinaric acid uptake by 24 to 30%. Insulin also reduced [3H]oleic acid uptake up to 35%, depending on insulin concentration and decreased the amount of fatty acid esterified into the phospholipids and neutral lipids by 28 and 70%, respectively. In contrast, glucagon or epinephrine stimulated both the initial rate and extent of cis-parinaric acid uptake 18 and 25%, respectively. Because L-cells lack P-adrenergic receptors, the epinephrine effect was not the result of P-receptor stimulation. Hence, insulin altered not only fatty acid uptake, as determined by cis-parinaric and oleic acid uptake, but also altered the intracellular oleic acid esterification.

Fatty acid double bond orientation alters interaction with L-cell fibroblasts.

Relatively little is known of fatty acid specificity in cellular fatty acid uptake. In this study L-cells, a fibroblastic cell line with very low levels of endogenous cytosolic fatty acid binding protein, were used to examine the role of cis and trans unsaturation on fatty acid uptake. The fluorescent fatty acids, trans-parinaric acid and cis-parinaric acid, were used as analogs of straight-chain saturated, and kinked-chain unsaturated fatty acids, respectively, in order to evaluate the fatty acid specificity of the uptake system. Parinaric acid is poorly metabolizable; greater than 97% was unesterified while 3H-oleic acid was almost totally metabolized after 30 min uptake. Cis- and trans-parinaric acid uptake was saturable and dependent on the concentration of fatty acid. However, the initial rate and maximal amount of trans-parinaric acid taken up by the L-cells was greater than for cis-parinaric acid under the same conditions. The affinity of L-cell uptake for trans-parinaric acid (Km = 0.12 uM) was 35-fold higher than that for cis-parinaric acid (Km = 4.17 uM). Based on competition studies with oleic and stearic acids, it was concluded that the cis- and trans-parinaric acid were taken up by the same L-cell fatty acid uptake system. The results suggest that the L-cell fatty acid uptake system has selectivity for straight chain rather than kinked chain unsaturated fatty acids.

Effect of hydralazine on myocardial plasma membrane fatty acid binding protein (PM-FABP) during diabetes mellitus.

Chronic treatment with the antihypertensive drug hydralazine did not affect the hyperglycemic state of streptozotocin (STZ)-diabetic rats but did prevent the serum hyperlipidemia that is synonymous with these diabetic animals. After 6 weeks, untreated STZ-diabetic rats exhibited a 659% increase in serum triglycerides and 292% increase in serum cholesterol versus age-matched non-diabetic rats. Hydralazine-treated STZ-diabetic rats had serum triglyceride and cholesterol levels that did not differ from controls. Myocytes from control rats showed a preference for binding of the unsaturated fatty acid analog cis-parinaric acid vs the saturated fatty acid analog trans-parinaric acid. This preference was not altered in STZ-diabetic rat myocytes; hydralazine-treatment of STZ-diabetic rats also showed no change in fatty acid preference. STZ-diabetes caused a decrease in the affinity (Kd) for the trans, but not the cis-parinaric acid. However, total binding of both analogs was increased in STZ-diabetes. Hydralazine treatment of STZ-diabetic rats resulted in even greater total binding of both analogs. Affinity for the trans analog was further decreased in these hydralazine-treated rats, but the affinity for the cis analog was increased beyond control animals. These results suggest that the diabetic state influences the binding characteristics of the myocardial PM-FABP and that hydralazine, while reducing serum lipids in diabetes, has complex effects on the fatty acid binding by this protein.

Synaptic plasma membrane structure and polarity of long-sleep and short-sleep mice.

Membrane dielectric as a primary basis for effects of ethanol was examined in synaptic plasma membranes (SPM) of genetically selected ethanol-sensitive long-sleep (LS) and ethanol-resistant short-sleep (SS) mice. Multifrequency phase and modulation of fluorometry of diphenylhexatriene (DPH) was used to resolve structural and dielectric differences in the membrane interior core. Fluorescence spectral peak ratios, fluorescence lifetime analysis, and initial rates of photoreaction of DPH in SPM provided sensitive measures of SPM interior core dielectric properties. The membrane microenvironment sensed by DPH was more polar in SPM from SS mice than in SPM from LS mice. Physiological concentrations of ethanol in vitro (25-75 mM) increased the SPM interior core dielectric and potentiated photoreaction of DPH with other membrane components of SPM from LS, but not SS, mice. These effects of ethanol in vitro were maximal by 75 mM ethanol and/or exacerbated at higher ethanol. In addition, ethanol in vitro increased the fraction of DPH associated with photoreaction products with lipids from SPM of ethanol-sensitive LS mice. The data were consistent with ethanol in vitro increasing the polar molecules (ethanol and/or water) of SPM from LS but not SS mice. It is suggested that ethanol alters the polarity and increases reactivity of the interior core lipid-protein interface.

The distribution and half-life for retention of vanadium in the organs of normal and diabetic rats orally fed vanadium(IV) and vanadium(V).

The concentration of vanadium in organs of diabetic rats that had been fed vanadium, either as V(IV) or V(V), in their drinking water has been determined. The kidney was found to have the highest concentration, about 185 nmol/g wet tissue. This averages about three times higher than for the liver or spleen, for which concentrations were comparable. The lung, blood plasma, and blood cells tended to have the lowest accumulations of vanadium. A time-course study indicated that the half-life for elimination of vanadium from the bodies of vanadium-fed rats is about 12 d.

Effect of calmodulin on sarcoplasmic reticular Ca2+-transport in the aging heart.

Heart failure is common among the elderly and an alteration in myocardial Ca2+ transport is believed to be involved in its depressed contractile performance. Although ATP-dependent sarcoplasmic reticular (SR) Ca2+ transport has been reported to decrease in old hearts, virtually nothing appears to be known about the Ca2+ pump activity of SR in aging myocardium in the presence of calmodulin, one of its endogenous activators. In this study, the activity of the Ca2+ pump of aging cardiac SR was assessed in the presence of this endogenous stimulator. This assessment was therefore designed to give additional information about the status of this enzyme in old hearts. Male Sprague-Dawley rats were used and were divided into 3 groups: young (4-6 months old); middle-aged (15-17 months old) and old age (24-25 months old). Purified SR membranes were isolated from ventricular tissues. ATP-dependent Ca2+ accumulation by membrane vesicles of middle-aged and old hearts was significantly depressed in comparison to young hearts at all Ca2+ concentrations employed in the absence and presence of calmodulin. The activity of this Ca2+ transporter was similar in middle-aged and old hearts even in the presence of calmodulin. These results suggest that the activity of the Ca2+ pump in SR of aging hearts is depressed even in the presence of calmodulin.

Effect of myo-inositol and T3 on myocardial lipids and cardiac function in streptozocin-induced diabetic rats.

Numerous experimental studies have implied a link between diabetes-induced abnormal lipid buildup in the myocardium and the development of cardiomyopathy. Because the diabetic state in rats is associated with lowered T3 (triiodothyronine) and T4 levels and because diabetic patients excrete large amounts of myo-inositol, a lipotropic agent, we investigated the effects of myo-inositol and T3 on the elevated myocardial lipid levels and depressed cardiac performance of streptozocin (55 mg/kg i.v.)-induced diabetic (STZ-D) rats. myo-inositol (2.5 g.kg-1.day-1 in the drinking water) and T3 (30 micrograms.kg-1.day-1 s.c.) were given for an 8-wk period 3 days after diabetes induction. Untreated diabetic rats were characterized by a decreased rate of body weight gain, hyperglycemia, and hypoinsulinemia, which were not altered after myo-inositol and/or T3 treatment. Thyroid status of diabetic animals was normalized by T3 alone or in combination with myo-inositol but not by myo-inositol alone. The elevations in plasma and myocardial lipids associated with the diabetic state were prevented by myo-inositol treatment. However, the plasma lipid and myocardial cholesterol levels in diabetic rats remained elevated or were further increased with treatment with T3 or myo-inositol plus T3. myo-inositol treatment partially improved cardiac performance in STZ-D rats. T3 treatment alone did not prevent cardiac dysfunction in diabetic rats. There was, however, some improvement in heart function in the groups treated with both myo-inositol and T3, coinciding with a significant decrease in the myocardial triacylglycerol level. The data indicate that a possible correlation may exist between elevated myocardial triacylglycerol levels and cardiac dysfunction in diabetic rats.

An assessment of phospholipid methylation in sarcolemma and sarcoplasmic reticulum of the aging myocardium.

The stepwise N-methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) (phospholipid methylation) was assessed in cardiac sarcolemma and sarcoplasmic reticulum of aging rats. This phenomenon was depressed in aging hearts relative to young ones. A decrease in activity of catalytic sites appears to be involved in the depressed phospholipid methylation of aging myocardium.

Pathophysiological aspects of myocardial hypertrophy.

Heart hypertrophy in response to increased workload is a complex process in which this organ adapts to the environment by increasing the muscle mass in terms of additional contractile units and formation of different types of contractile proteins (myosin isozymes). In addition, augmentation of membrane function with respect to calcium transport activities of sarcolemma and sarcoplasmic reticulum occurs at early stages of cardiac hypertrophy associated with hyperfunction of the myocardium. However, if cardiac hypertrophy is left unattended beyond a certain period, physiological hypertrophy is converted to pathological hypertrophy whereby the cardiac muscle is unable to generate an adequate amount of contractile activity. It appears that the sympathetic nervous system is activated for producing beneficial effects at early stages but an elevated level of sympathetic tone for a prolonged period could result in dysfunction of the cardiac muscle. The transition of physiological hypertrophy to pathological hypertrophy seems to be due to the occurrence of intracellular calcium overload in the myocardial cell as a consequence of defects in the membrane calcium transport systems. It is suggested that careful attention should be paid not only to removal of the stimulus responsible for cardiac hypertrophy but also to lowering sympathetic tone. Efforts should also be made to prevent the occurrence of intracellular calcium overload due to membrane defects.

Alterations in cardiac sarcolemmal Ca2+ pump activity during diabetes mellitus.

Diabetes mellitus is frequently associated with a primary cardiomyopathy. The mechanisms responsible for this heart disease are not clear, but an alteration in myocardial Ca2+ transport is believed to be involved in its development. Even though sarcolemma plays a crucial role in cellular Ca2+ transport, little appears to be known about its Ca2+ transporting capability in the diabetic myocardium. In this regard, we have examined the status of the cardiac sarcolemmal Ca2+ pump during diabetes mellitus. Purified sarcolemmal membranes were isolated from male Wistar diabetic rat hearts 8 wk after streptozotocin injection (55 mg/kg iv). Ca2+ pump activity assessed by measuring its Ca2+-stimulated adenosine triphosphatase and Ca2+-uptake ability in the absence and presence of calmodulin was significantly depressed in the diabetic myocardium relative to controls. These results did not appear to have been influenced by the minimal sarcoplasmic reticular and mitochondrial contamination of this membrane preparation. Hence, it appears that the sarcolemmal Ca2+ pump is defective in the diabetic myocardium and may be involved in the altered Ca2+ transport of the heart during diabetes mellitus.

Effect of choline and methionine treatment on cardiac dysfunction of diabetic rats.

There is an abnormal lipid accumulation in the myocardium that might be involved in the congestive heart failure frequently associated with individuals with diabetes mellitus. Because choline and methionine have been reported to modify the incidence of myocardial necrosis in rats fed various fat diets, we decided in our study to assess their effect on the cardiac dysfunction of diabetic rats. Female Wistar rats were made diabetic with streptozocin (STZ, 55 mg/kg i.v.). One week after diabetes induction, one group received choline (0.3 mg/ml), another received methionine (0.25 mg/ml), a third received a combination of the same doses of choline and methionine, and a fourth received neither of these agents; all were administered in the drinking water. Animals were treated for 7 wk. Insulin levels were lower and glucose values higher in the serum of STZ rats relative to controls. Serum cholesterol and triglycerides were significantly elevated in all diabetic animals, and they were also elevated (P less than .05) in the hearts of untreated diabetics. In contrast, the myocardial values of the same lipids were drastically reduced in the treated diabetic animals. Cardiac performance was depressed in all STZ animals, but there was a significant improvement in heart function in treated diabetics relative to untreated ones. Thus, it appears that the buildup of cholesterol and triglycerides in the myocardium are important contributors to the cardiac dysfunction that frequently accompanies diabetes mellitus.

Subcellular distribution of cardiac 5'-nucleotidase: alteration of microsomal pool in hypertrophied pig heart.

The subcellular distribution of the 5'-nucleotidase activity was investigated in normal and hypertrophied pig hearts; normal rat hearts were used for comparison. The left ventricular hypertrophy was induced in pigs by banding the supravalvular aorta for 4, 8 and 12 weeks. By employing different procedures for the isolation of cardiac membranes, a major catalytic site for 5'-nucleotidase was found to be located at sarcolemma in rat heart and microsomes (sarcoplasmic reticulum) in pig heart. A progressive decrease in the homogenate and microsomal 5'-nucleotidase activity occurred upon the development of myocardial hypertrophy in pigs. This reduction in microsomal 5'-nucleotidase activity was characterized by a depression in both apparent Vmax and Km values. These results indicate that a primary 5'-nucleotidase pool is present in the intracellular compartment of the pig heart and is altered during the development of hypertrophy.

Sarcoplasmic reticular and mitochondrial calcium transport in cardiac hypertrophy.

To evaluate changes in Ca2+ transport activities in the cardiac sarcoplasmic reticulum (microsomes) and mitochondria, cardiac hypertrophy was induced in rabbits by constricting the abdominal aorta. The animals showed a stable non-failing left heart hypertrophy between 16-22 weeks after the operation. ATP-dependent Ca2+ uptake and Ca2+ binding activities were depressed in microsomes from hypertrophied rabbits in comparison with sham-operated controls (P less than 0.05). These changes were seen at different concentrations of free Ca2+ (10(-7) to 10(-4)M) and were accompanied by alterations in the phospholipid content of the microsomal fraction. Mitochondrial Ca2+ transport activities and phospholipid content remained unchanged in the hypertrophied heart. The results of this study identify a specific lesion in the sarcotubular membrane and suggest that the depressed Ca2+ transport activity in the microsomal fraction from the hypertrophied myocardium may be due to changes in its phospholipid composition.

Alterations in sarcolemmal Na+-Ca2+ exchange and ATP-dependent Ca2+-binding in hypertrophied heart.

In order to examine changes in Ca2+ transport in heart sarcolemma, cardiac hypertrophy was induced in rabbits by stenosis of the abdominal aorta and hearts were removed 18-20 weeks later; sham-operated normal rabbits were used as controls. Sarcolemmal vesicles were isolated from the left ventricular tissue by a sucrose density gradient method and the membrane composition as well as activities of certain marker enzymes were monitored to determine the purity of control and experimental fractions; Na+-Ca2+ exchange and Ca2+-pump activities were assessed by the Millipore filtration technique. No changes in Ca2+-influx were observed in Na+-loaded vesicles from the hypertrophied hearts when studied in the presence of different concentrations of calcium as well as at different times of incubation. In contrast, Na+-induced Ca2+-efflux from Ca2+-loaded vesicles was enhanced in the hypertrophied heart at different times of incubation and at different concentrations of sodium. ATP-dependent Ca2+-binding activity of sarcolemma from hypertrophied heart, when measured at different times of incubation and at several concentrations of calcium, was more than the control. Minimal but an equal amount of cross contamination was seen in both control and experimental preparations; however, phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid contents were increased in sarcolemma from hypertrophied hearts. These results suggest that the sarcolemmal Ca2+-transport systems may become adapted during the development of hypertrophy for augmenting Ca2+-efflux from the hypertrophied myocardial cell and this may prevent the occurrence of intracellular Ca2+ overload in a stable form of cardiac hypertrophy.

Effect of vanadate on elevated blood glucose and depressed cardiac performance of diabetic rats.

The trace element vanadium has an unclear biological function. Vanadate, an oxidized form of vanadium, appears to have an insulin-like action. The effect of vanadate on blood glucose and cardiac performance was assessed in female Wistar rats 6 weeks after they were made diabetic with streptozotocin. When vanadate was administered for a 4-week period to the diabetic rats, their blood glucose was not significantly different from that of nondiabetic controls despite a low serum insulin. In contrast, blood glucose was increased about threefold in the diabetic rats that were not treated with vanadate; these rats also had low insulin levels. Cardiac performance was depressed in the untreated diabetic animals, but the cardiac performance of the vanadate-treated diabetic animals was not significantly different from that of nondiabetic controls. Thus vanadate controlled the high blood glucose and prevented the decline in cardiac performance due to diabetes.

Sarcoplasmic reticular Ca2+-pump adaptation in cardiac hypertrophy due to pressure overload in pigs.

Ca2+-transport activities of the sarcoplasmic reticulum isolated from the non-failing hypertrophied left ventricle due to supravalvular banding of aorta for 4 and 8 weeks in pigs were compared with those from the sham operated controls. The rate of Ca2+-uptake, but not the capacity, in 4 weeks hypertrophied heart preparations was increased. On the other hand, both the rate and capacity of sarcoplasmic reticulum to accumulate Ca2+ were decreased at 8 weeks of heart hypertrophy. The Ca2+-binding and Ca2+-stimulated Mg2+ dependent ATPase activities of the reticular fractions from 4 and 8 weeks hypertrophied hearts were not significantly different from the control values. No changes in the sarcoplasmic reticular phospholipid contents were evident except that sphingomyelin was significantly decreased in 4 weeks and diphosphatidylglycerol was increased in both 4 and 8 weeks hypertrophied preparations. These results suggest that adaptation of the sarcoplasmic reticulum function in physiologic and pathologic hypertrophied hearts may occur by changing the efficiency of Ca2+-pump system during the development of cardiac hypertrophy.

Defect in the adrenergic receptor-adenylate cyclase system during development of catecholamine-induced cardiomyopathy.

We have investigated alterations in adrenergic receptors and adenylate cyclase activity in cardiac membranes from rats injected with 40 mg/kg intraperitoneal isoproterenol. Reduction in the number of beta-adrenergic and alpha-adrenergic receptors, as assessed by changes in specific binding of 3H-dihydroalprenolol (DHA) and 3H-dihydroergocryptine (DHE), was observed only at 9 and 24 hours after isoproterenol injection, respectively. On the other hand, epinephrine-stimulated, NaF-stimulated, and Gpp (NH)p-stimulated adenylate cyclase activity was decreased as early as 3 hours after isoproterenol treatment without changes in the basal adenylate cyclase activity. These results demonstrate a defect in the adrenergic receptor-adenylate cyclase system during the development of catecholamine-induced cardiomyopathy and may partly explain the attenuated inotropic adrenergic response of the heart under stressful situations.

Properties of 5'-nucleotidase in rat heart sarcolemma.

The activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was examined in membrane fractions isolated by hypotonic shock-LiBr treatment (fraction HL) and sucrose gradient separation (fraction S) of rat ventricle homogenate. The enzyme activity in these two fractions differed significantly in several respects. In fraction HL, 5'-nucleotidase had a high affinity for AMP (Km 35 microM), and ATP was a potent competitive inhibitor. In contrast, the 5'-nucleotidase displayed by fraction S showed a low substrate affinity (Km 130 microM) and less sensitivity to ATP. Treatment of membranes with trypsin and neuraminidase markedly stimulated 5'-nucleotidase in fraction HL, whereas only a modest effect was observed in fraction S. Exposure of the membranes to Triton X-100 resulted in a 60% and 10% increase in the enzyme activity in fractions HL and S, respectively. The characteristic activity ratios of 5'-nucleotidase at 200 microM relative to 50 microM AMP in fractions HL and S were modified by alamethicin in an opposite way and became identical. Although concanavalin A almost completely inhibited the 5'-nucleotidase activity in both membrane preparations at a concentration of 2 microM, Hill plots of the data on concanavalin A inhibition revealed a coefficient of 2.2 for fraction S and 1.1 for fraction HL. The differences in 5'-nucleotidase activity of the two membrane fractions are considered to be due to differences in the orientation of the vesicles of the sarcolemmal preparations. These results suggest that two distinct catalytic sites for 5'-nucleotidase are present at the intra- and extracellular surface of the rat heart sarcolemma.

Cardiac alpha- and beta-adrenergic receptor alterations in diabetic cardiomyopathy.

The effect of chronic experimental diabetes on the adrenergic receptors in the rat heart was investigated. Diabetes was induced by streptozotocin (65 mg/kg; i.v.) administration, animals were sacrificed 8 weeks later, and positive as well as negative dF/dt values were determined in isolated papillary muscle preparations. Stimulation of the contractile force generation by isoproterenol and methoxamine was attenuated in diabetic preparations. Beta- and alpha-adrenergic receptor bindings were determined in cardiac membranes by employing 3H-dihydroalprenolol and 3H-dihydroergocryptine respectively. Reduced number of beta- and alpha-receptor binding sites without changes in the affinity constants were observed in diabetic myocardium. Such a decrease in alpha- and beta-receptor density in the heart may account for the depressed contractile responsiveness to adrenergic stimuli in diabetic cardiomyopathy.