Oxido-reductive titrations of cytochrome c oxidase followed by EPR spectroscopy.

Experiments are described on oxido-reductive titrations of cytochrome c oxidase as followed by low-temperature EPR and reflectance spectroscopy. The reductants were cytochrome c or NADH and the oxidant ferricyanide. Experiments were conducted in the presence and absence of either cytochrome c or carbon monoxide, or both. An attempt is made to provide a complete quantitative balance of the changes observed in the major EPR signals. During reduction, the maximal quantity of heme represented in the high-spin ferric heme signals (g approximately 6; 2) is 25% of the total heme present, and during reoxidation 30%. With NADH reduction there is little difference between the pattern of disappearance of the low-spin ferric heme signals in the absence or presence of cytochrome c. The copper and high-spin heme signals, however, disappear at higher titrant concentrations in the presence of cytochrome c than in its absence. In these titrations, as well as in those with ferrocytochrome c, the quantitative balance indicates that, in addition to EPR-detectable components, EPR-undetectable components are also reduced, increasingly so at higher titrant concentrations. The quantity of EPR-undectable components reduced appears to be inverely related to pH. A similar inverse relationship exists between pH and appearance of high-spin signals during yhe titration. At pH 9.3 the quantity of heme represented in the high-spin signals is less than 5%, whereas it approximately doubles from pH 7.4 to pH 6.1. In the presence of CO less of the low-spin heme and copper signals disappears for the same quantity of titrant consumed, again implying reduction of EPR undetectable components. At least one of these components is represented in a broad absorption band centered at 655 nm. The stoichiometry observed on reoxidation, particularly in the presence of CO, is not compatible with the notion that the copper signal represents 100% of the active copper of the enzyme as a pair of interacting copper atoms.     

Cyclic AMP phosphodiesterases and Ca2+ current regulation in cardiac cells.

At least four different isoforms of phosphodiesterases (PDEs) are responsible for the hydrolysis of cAMP in cardiac cells. However, their distribution, localization and functional coupling to physiological effectors (such as ion channels, contractile proteins, etc.) vary significantly among various animal species and cardiac tissues. Because the activity of cardiac Ca2+ channels is strongly regulated by cAMP-dependent phosphorylation, Ca(2+)-channel current (ICa) measured in isolated cardiac myocytes may be used as a probe for studying cAMP metabolism. When the activity of adenylyl cyclase is bypassed by intracellular perfusion with submaximal concentrations of cAMP, effects of specific PDE inhibitors on ICa amplitude are mainly determined by their effects on PDE activity. This approach can be used to evaluate in vivo the functional coupling of various PDE isozymes to Ca2+ channels and their differential participation in the hormonal regulation of ICa and cardiac function. Combined with in vitro biochemical studies, such an experimental approach has permitted the discovery of hormonal inhibition of PDE activity in cardiac myocytes.     

Reduction of Muscarinic Acetylcholine Receptor Number and Affinity by an Endogenous Substance

We examined the soluble fraction from homogenates of 12-day embryonic chick heart for the presence of an endogenous modulator of muscarinic acetylcholine receptors (mAChR). Homogenates were separated into 100,000 g soluble and crude membrane fractions by differential centrifugation. Aliquots of membranes were incubated in the presence or absence of the soluble fraction and the muscarinic antagonist, [3H]quinuclidinyl benzilate ( [3H]QNB), and the data subjected to Scatchard analysis. In the presence of the soluble fraction, mAChR number decreased up to 70% and the affinity for [3H]QNB decreased six- to eightfold. These results suggested that an endogenous soluble factor (ESF) affected cholinergic ligand binding to the receptor. The amount of ESF extracted from less than 10 mg of brain was sufficient to reduce by 50% [3H]QNB binding to 50 fmol mAChR. ESF activity was partially purified by heat and acid treatment. The loss of receptors was dependent upon the amount of ESF added and was time dependent. QNB protected some receptors from loss due to ESF. The change in mAChR affinity for [3H]QNB was observed only if ESF was present continuously during the [3H]QNB binding assay. Ultrafiltration and gel filtration showed that ESF was less than 10,000 daltons and probably less than 700 daltons. ESF activity was blocked by EDTA. However, ESF was not a divalent cation since it was base labile, and removal of divalent cations with Chelex-100 did not inhibit ESF activity. ESF activity was also blocked by catechol, catecholamines, ascorbate, and dithiothreitol. ESF was present in embryonic but not in adult heart.     

Glucocorticoid stimulation of metabolism and glycerol-3-phosphate dehydrogenase activity in cultured heart cells

The direct effects of the glucocorticoids hydrocortisone and corticosterone on myocardial metabolism were studied in cultured heart cells by assessing several parameters previously unreported. Hormone and growth factor concentrations were carefully controlled by using a serum-free medium, which also allowed maintenance of cells in the absence of glucocorticoids. Heart cell beating rate, glucose uptake rate, and CO2 evolution from radioactively labeled glucose were increased by the addition of 0.03 microM corticosterone to the medium of cells maintained in culture for 11 days. There were no further changes in these parameters as steroid concentration was increased to 14.43 microM. The activity of NAD-linked sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) was increased by both corticosteroids and was dose dependent between 0.06 and 1.44 microM corticosterone. The difference between glycerol-3-phosphate dehydrogenase activity in cells maintained with hydrocortisone as compared to cells maintained without hydrocortisone increased with days in culture. The protein and DNA contents of dishes maintained with corticosteroid were depressed, demonstrating an inhibitory effect on cellular replication. Glucocorticoids have numerous direct effects on cardiac cell metabolism, and the nature of these effects suggests that secondary responses of the cell to chronic exposure are significant.     

Preferential distribution of non-esterified fatty acids to phosphatidylcholine in the neonatal mammalian myocardium.

Non-esterified fatty acids are used to a limited extent as an energy source in the newborn-mammalian heart. Therefore additional roles for palmitic and oleic acids during this early period of growth and development were investigated in the cultured neonatal-rat heart cell model system. Our results indicate significant differences in nonesterified-fatty-acid metabolism exist in this system in comparison with the adult rat or embryonic chick heart. Initial rates of depletion of palmitate and oleate from serum-free growth medium by heart cells obtained from 2-day-old rats and maintained in culture for 10 or 11 days were 111 +/- 2 and 115 +/- 3 pmol/min per mg of protein respectively. In serum-containing medium, the initial depletion rates were 103 +/- 3 and 122 +/- 4 pmol/min per mg of protein respectively, when endogenous serum nonesterified-fatty-acid concentrations were included in rate calculations. Less than 1% of the intracellularly incorporated fatty acids were found in aqueous products at any time. After 25 h, 15.5% of the initial palmitate was deposited intracellularly in the phosphatidylcholine lipid fraction, 4.2% in the triacylglycerol + fatty-acid-ester fraction and 3.1% in the sphingomyelin fraction. These results contradict the classical view, based on findings with the lipid-dependent adult heart, that exogenous nonesterified fatty acids are directed intracellularly primarily to pathways of oxidation or to storage as triacylglycerol. More importantly, it underscores the significance of exogenous non-esterified fatty acids in membrane biosynthesis of the developing mammalian heart. Included here is a new method for one-dimensional t.l.c. separation of metabolically important polar lipids.     

Heme models. I. Solution behavior of a water soluble iron porphyrin.

A well-behaved water soluble iron-porphyrin system, meso-tetra-(4-carboxyphenyl) porphinato iron (III) was synthesized. Its solution behavior is described using visable and electron paramagnetic resonance (EPR) spectroscopy. The complex exists in solution as three distinct forms of bridged dimers, oxo, hydroxo and aquo, with the following pK's: oxo + H+ in equilibrium hydroxo, pK = 9.58; hydroxo + H+ in equilibrium aquo, pK = 6.72. In the presence of excess imidazole the second pK is found to be 7.05. Detailed analysis of the interaction of the hydroxo-bridged form with imidazole is presented. It is found that one dimer unit simultaneously binds two imidazole molecules, with an over-all equilibrium constant log Keq = -1.22. EPR spectra are presented for the various forms of iron-porphyrin discussed.     

Olfactory memory: the long and short of it

It has been proposed that memory for odors does not have a short-term (or working) memory system. The distinction between short- and long- term memory in other sensory modalities has been generally supported by three main lines of evidence: capacity differences between the proposed systems, evidence of differential coding, and differential memory losses in neuropsychological patients. The present paper examines these issues in an effort to establish a similar distinction for the memory of olfactory stimuli. Each of these lines of evidence is examined in relation to the literature on olfactory memory. Based on this examination, it seems that there is at least preliminary support from each of these lines of evidence to advocate a distinction between a long- and short-term memory for olfactory stimuli. Emphasis is placed upon the qualitative similarity of olfactory memory to other memory systems. This similarity is further highlighted through an examination of the literature pertinent to serial position effects in memory for olfactory stimuli.