The role of glycolysis in myocardial calcium control.

Because glycolysis is thought to be important for maintenance of cellular ion homeostasis, the aim of the present study was to examine the role of glycolysis in the control of cytosolic calcium ([Ca2+]i) and cell shortening during conditions of increased calcium influx. Thus, [Ca2+]i and unloaded cell shortening were measured in fura-2/AM loaded rat ventricular myocytes. All cells were superfused with Tyrode's solution containing glucose and pyruvate (to preserve oxidative metabolism), and glycolysis was inhibited by iodoacetate (IAA, 100 microM). Calcium influx was increased, secondary to an increase in intracellular sodium, by addition of veratrine (1 microgram/ml), or directly by either elevating [Ca2+]o from 2 to 5 mM or by exposing the cells to isoproterenol (1 to 100 nm). Veratrine exposure caused a time-dependent increase in both diastolic and systolic [Ca2+]i that resulted in cellular calcium overload and hypercontraction. The rate of increase in [Ca2+]i was more rapid in IAA-treated than in untreated myocytes, leading to a 13+/-3 v 5+/-2% increase (P<0.05) in diastolic [Ca2+]i after 5 min of exposure. The corresponding increases in systolic [Ca2+]i were 43+/-6 and 24+/-5% (P<0.05). Elevated [Ca2+]o resulted in increased [Ca2+]i transient amplitudes and cell shortening. These responses were each attenuated by inhibiting glycolysis, so that the increase was 38+/-5 v 68+/-9% ([Ca2+]i transient amplitude, P<0.05) and 41+/-11 v 91+/-18% (cell shortening, P<0.05). Inhibition of glycolysis did not, however, affect the increase in calcium transient or cell shortening during addition of isoproterenol. We conclude that glycolysis plays an essential role in the maintenance of intracellular calcium homeostasis during severe calcium overload. Glycolysis was also essential for signalling the inotropic effect that accompanied elevation in extracellular calcium, while the changes in intracellular calcium following administration of isoproterenol were not influenced by glycolysis in the present model.

Measurements of leukotrienes in human plasma by solid phase extraction and high performance liquid chromatography.

Leukotrienes (LTs) are biologically active compounds derived from lipoxygenase catalyzed metabolism of arachidonic acid in mammalian tissues. The present report describes a simple method for extraction and isolation of dihydroxy-LTs; LTB4, LTB5 and the peptido-LTs; LTC4, LTD4 and LTE4 from human plasma, using a pretreatment cartridge which utilizes both hydrophobic and ion-exchange interactions. 5 ml acidified plasma or acetate buffer containing commercially available LT standards were passed through the cartridges under suction, and the absorbed LTs were subsequently eluted in a stepwise manner with acetate buffer containing increasing amounts of methanol. The eluted LTs were analyzed by reversed-phase high performance liquid chromatography (HPLC) on octadecylsilyl (ODS)-silica, using a Waters HPLC unit. Both with plasma and acetate buffer the present methodology resulted in good separation of the LTs with a total run-time of less than 32 min. Recovery of dihydroxy-LTs was approximately 80% (range 73-82%) both when the standards were dissolved in plasma and in acetate buffer. Recovery of the peptido-LTs was, however somewhat lower (47-50%). It should be noted that the present method has the advantage that exposure to chemicals of high toxicity is avoided.

Effect of stimulation and veratrine on total cellular calcium in rat and guinea-pig ventricular myocytes.

In rat cardiac myocytes, calcium efflux by Na+/Ca(2+)-exchange is expected only during ventricular systole following initial action potential repolarization. In contrast, in guinea-pigs, calcium influx via Na+/Ca(2+)-exchange is expected only during the initial portion of the action potential. Thus electrical stimulation is expected to result in reduced intracellular calcium ([Ca2+]i) in rat and an increase in guinea pig. We tested this hypothesis by measuring total cellular calcium ([Ca]tot) using 45Ca following stimulation of isolated rat and guinea-pig ventricular myocytes. Many studies have also emphasized that the rate and the direction of Na+/Ca(2+)-exchange across the sarcolemma are in part dependent on the magnitude of the transsarcolemmal sodium gradient. Thus, increasing intracellular sodium ([Na+]i) is expected to result in an increased [Ca2+]i. This hypothesis was also tested by measuring [Ca]tot following veratrine administration. Enzymatically isolated rat and guinea-pig ventricular myocytes were divided into two groups; non-stimulated and stimulated (1 Hz). The concentration-dependent effects of veratrine (1,10,100 micrograms/ml) on [Ca]tot were determined in both these groups. In the absence of veratrine, non-stimulated rat myocytes had a significantly higher [Ca]tot than did stimulated ones. Non-stimulated guinea-pig myocytes had a significantly lower [Ca]tot when compared with stimulated ones. Veratrine increased [Ca]tot in both species in a concentration-dependent fashion. In addition, following veratrine the difference between [Ca]tot in non-stimulated and stimulated rat myocytes was no longer significant. These results support those of others who have demonstrated that stimulation is associated with a gain of cellular calcium in both rabbit and guinea-pig ventricle and a calcium loss in rat ventricle.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-associated reduction in pineal beta-adrenergic receptor density is prevented by life-long food restriction in rats.

At 28 months of age (old rats), male Fisher 344 rats which had been fed 40% of the ad libitum food intake since they were 6 months old, had a similar beta-adrenergic receptor density (Bmax) in their pineal gland as young, 3-month-old rats. In contrast, old rats which had been fed ad libitum for the same period had approximately 50% of the Bmax value compared to that of young rats. The beta-receptor density of cerebral cortical tissue and the beta-receptor affinity (Kd) of both the cortex and the pineal gland did not decline with age and was not affected by food restriction. The reduction in pineal beta-receptor density with age may be casually related to the concurrent age-associated decline in pineal production of melatonin.

Effects of fasting and glucose-insulin-potassium on glycogen contents in heart, skeletal muscle and liver.

The effect of intravenous infusion of glucose, insulin and potassium (GIK) on the glycogen contents of the heart, skeletal muscle and liver was investigated in rats. One group of animals received intravenous infusion of GIK continuously for 3 days, and two other groups of rats were given the same amount of saline intravenously. The animals receiving GIK, and a control group of the rats, were fasted for 8 h before sacrifice, while a second control group of animals was given free access to water and pellets until sacrifice. The heart, rectus femoris muscle and liver were then analysed for glycogen contents. It was found that fasting caused a depletion of glycogen in skeletal muscle and liver, while it did not cause any changes in myocardial glycogen which remained similar to that in the fed animals. Infusion of GIK prevented the depletion of glycogen in skeletal muscle and liver, but, unexpectedly, it did not cause any significant changes in myocardial glycogen content.

A longitudinal study of the biological variability of plasma lipoproteins in healthy young adults.

The fasting serum concentrations of total cholesterol, low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), total triglyceride and apoprotein A1 were measured at intervals of 12-18 weeks for 60 weeks in 17 male and 11 female healthy young adults in order to assess the variability of these risk factors for coronary disease. No statistically significant seasonal changes were detected in any variable in either sex, although a progressive rise in apoprotein A1 concentration was observed. The coefficients of variation for random fluctuations with time were in the rank order: total cholesterol less than HDL-C less than apoprotein A1 less than LDL-C less than triglyceride. These differences were attributable to biological, rather than to methodological, factors. Within subjects, HDL cholesterol concentration varied inversely with triglyceride concentration and directly with apoprotein A1 concentration. The marked differences which exist in the biological variability of lipid risk factors for atherosclerosis need to be taken into account when making comparisons in epidemiological studies of the predictive powers of single on-entry measurements for future disease. Fluctuations of HDL-C with time appear to be related in part to variations in triglyceride-rich lipoprotein metabolism.

Increased plasma cyclic AMP concentrations in fasting man.

Plasma concentrations of adenosine 3', 5' monophosphate (cyclic AMP), free fatty acids (FFA), glycerol and glucose were measured after 24, 48 and 72 h fasting in healthy male students. There was a marked rise in plasma concentrations of cyclic AMP, FFA and glycerol from 24 to 48 h fasting with no further changes from 48 to 72 h fasting. Plasma glucose concentrations fell. After 72 h fasting, inhibition of adipose tissue lipolysis with a nicotinic acid analogue effected no significant change in plasma cyclic AMP concentrations, indicating that the elevated plasma nucleotide concentrations during fasting were not related to stimulation of lipolysis.

The effect of glucagon on plasma cyclic AMP and glucose concentrations in patients with alcoholic cirrhosis.

Glucagon was infused intravenously into four patients with alcoholic cirrhosis and five healthy subjects and serial measurements were made of plasma cyclic AMP and glucose concentrations. The results in the cirrhotic patients did not differ significantly from those in healthy subjects.