Effects of increasing extracellular pH on protein synthesis and protein degradation in the perfused working rat heart.
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ABSTRACT:
SUBMITTER: Fuller SJ
PROVIDER: S-EPMC1138488 | biostudies-other | 1989 Apr
REPOSITORIES: biostudies-other
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Project description:In a non-recirculating system of isolated liver perfusion, stimulation of urea synthesis by NH4Cl is followed by a decrease of effluent pH by up to 0.2 pH unit. This effect is not observed when urea synthesis is inhibited by amino-oxyacetate or norvaline. When the urea formed by the liver is immediately hydrolysed with urease before the effluent perfusate reaches the pH electrode, the urea-synthesis-induced acidification is no longer observed. This indicates that accompanying alterations in hepatic metabolism after stimulation of urea synthesis, such as increased energy provision and consumption, are not responsible for the extracellular acidification, but that the effect is due to the formation of urea itself. The acidification of the extracellular space after stimulation of urea synthesis by NH4Cl is quantitatively explained by the consumption of 2 mol of HCO3-/mol of urea formed: 1 mol being incorporated into urea, the other being protonated to yield CO2 and H2O. The data match the theoretically predicted HCO3- consumption during ureogenesis and underline the role of hepatic urea synthesis for disposal of HCO3- by converting it into the excretable products CO2 and urea.
| S-EPMC1146814 | biostudies-other
Project description:1. Protein degradation was studied in the glucose (5 mM)-perfused working rat heart preparation of Taegtmeyer, Hems & Krebs [(1980) Biochem. J. 186. 701-711]. 2. The effects of cardiac workload were investigated in three different preparations: (a) control (low workload), (b) increased pressure workload (simulating conditions of aortic pressure in vivo) and (c) increased volume workload. There was no effect of increased workload on protein degradation in preparation (b) or (c) when compared with preparation (a). Insulin inhibited protein degradation in all three preparations. Significantly greater inhibition by insulin was observed in the increased-pressure-workload preparation (b). 3. Hypoxia was induced by the partial replacement of O2 in the gaseous phase by N2. Hearts maintained their cardiac output when O2 content was decreased from 95% to 55% by volume, but the stability of the preparation was less at 50% O2. Lactate output was significantly increased at O2 contents of 65% or less. The rate of protein degradation was not different from control values (95% O2) in perfusions with 65, 55 or 50% O2. 4. We conclude that acutely increased workload or acute hypoxia does not affect protein degradation in the perfused working rat heart when cardiac output is relatively stable.
| S-EPMC1154189 | biostudies-other
Project description:The synthesis rates of total heart protein and of sarcoplasmic and myofibrillar protein fractions have been determined by perfusion of isolated rat hearts with [(14)C]tyrosine at constant specific radioactivity. In hearts perfused without insulin, both myofibrillar and sarcoplasmic proteins were synthesized at a fractional rate of 10-11% per day. This corresponds to a half-life for synthesis of about 7 days. The effect of added insulin was to increase the rate of heart-protein synthesis to a half-life of 3-4 days. With hearts perfused via the left atrium and performing external work, there was a rise in the specific radioactivity of intracellular free tyrosine, and the half-life for synthesis of proteins was 3-4 days. The extent of labelling of individual myofibrillar proteins was estimated after polyacrylamide-gel electrophoresis of solubilized myofibrils in the presence of sodium dodecyl sulphate. No particular protein showed an unusually high or low specific radioactivity after labelling in perfusion. Insulin caused a general increase in labelling of all the proteins analysed.
| S-EPMC1177625 | biostudies-other
Project description:Rat hearts were perfused as working preparations by the method of Taegtmeyer, Hems & Krebs [(1980 Biochem. J. 186, 701--711]. In the presence of glucose, insulin significantly inhibited protein degradation at concentrations as low as 50 mu units/ml. Acetate or lactate, when present either as sole fuel for contraction or in combination with glucose, did not inhibit protein degradation. Insulin inhibition or protein degradation was decreased with either lactate as sole fuel. We suggest that the inhibition of protein degradation occurs over the normal range of plasma concentrations of insulin present in vivo and that the presence of glucose may be at least in part necessary for this effect of insulin.
| S-EPMC1158612 | biostudies-other
Project description:We investigated the effect of an increase in the left-atrial filling pressure on the rate of left-atrial protein synthesis in the left-side-perfused working rat heart preparation of Taegtmeyer, Hems & Krebs [(1980) Biochem. J. 186, 701-711]. An increase in filling pressure (preload) at a constant aortic pressure (afterload) increased both the intra-atrial pressure and the atrial stroke volume. The aortic pressure (afterload) was held constant. An increase in filling pressure from 5 to 20 cmH2O at an aortic pressure of 70 cmH2O, or an increase in filling pressure of 7.5 to 20 cmH2O at an aortic pressure of 100 cmH2O, significantly stimulated the rates of left-atrial protein synthesis by 30-40%. The stimulation was observed when the rates of protein synthesis were expressed relative to either protein or RNA content. Since perfusate entering the right atrium from the coronary circulation left that atrium passively, the rate of protein synthesis in this compartment can be used as an internal control. Rates of right-atrial protein synthesis were similar to those in the left atria exposed to the lower filling pressures and were unaffected by the increases in left-atrial filling pressure. We suggest that the acute effects of increased left-atrial filling pressure on protein synthesis in that compartment may be important in the development of left-atrial hypertrophy. This condition is seen in patients who have raised pulmonary venous pressures in, for example, mitral stenosis.
| S-EPMC1152543 | biostudies-other
Project description:The effects of insulin on glucose uptake and lactate release in the perfused working rat heart have been investigated in three types of preparation: (i) a control low-workload preparation; (ii) an increased-pressure-workload preparation, simulating conditions of aortic pressure encountered in vivo; (iii) an increased-volume-workload preparation, where pumping work done is approximately the same as (ii) but coronary flow is restricted because of the decreased aortic pressure. Insulin stimulated glucose uptake and lactate release in preparations (i) and (ii), but failed to do so in preparation (iii). It was considered possible that preparation (iii) was hypoxic, thus necessitating a maximal stimulation of glucose uptake. This was confirmed by improving cardiac oxygenation by addition of stroma-free haemoglobin to the perfusate in preparation (iii). Under these conditions in the absence of insulin, glucose uptake and lactate release were decreased compared with perfusions in the absence of haemoglobin. Insulin stimulation of both processes was restored. We conclude that the failure of other workers to observe insulin effects on glucose uptake and lactate release under physiological workloads [preparation (ii)] may be a consequence of intracellular hypoxia in their preparations.
| S-EPMC1158613 | biostudies-other
Project description:Rates of muscle protein synthesis and degradation measured in the perfused hindquarter were compared with those in incubated epitrochlearis muscles. With fed or starved mature rats, results without insulin treatment were identical. With insulin treatment, protein synthesis in perfused hindquarters was greater, though protein degradation was the same. Thus rates of muscle protein degradation estimated by these two methods in vitro correspond closely.
| S-EPMC1153139 | biostudies-other
Project description:BackgroundThe tumor microenvironment is characterized by regions of hypoxia and acidosis which are linked to poor prognosis. This occurs due to an aberrant vasculature as well as high rates of glycolysis and lactate production in tumor cells even in the presence of oxygen (the Warburg effect), which weakens the spatial linkage between hypoxia and acidosis.MethodsFive different human squamous cell carcinoma cell lines (SiHa, FaDuDD, UTSCC5, UTSCC14 and UTSCC15) were treated with hypoxia, acidosis (pH 6.3), or a combination, and gene expression analyzed using microarray. SiHa and FaDuDD were chosen for further characterization of cell energetics and protein synthesis. Total cellular ATP turnover and relative glycolytic dependency was determined by simultaneous measurements of oxygen consumption and lactate synthesis rates and total protein synthesis was determined by autoradiographic quantification of the incorporation of 35S-labelled methionine and cysteine into protein.ResultsMicroarray analysis allowed differentiation between genes induced at low oxygen only at normal extracellular pH (pHe), genes induced at low oxygen at both normal and low pHe, and genes induced at low pHe independent of oxygen concentration. Several genes were found to be upregulated by acidosis independent of oxygenation. Acidosis resulted in a more wide-scale change in gene expression profiles than hypoxia including upregulation of genes involved in the translation process, for example Eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2), and Ribosomal protein L37 (RPL37). Acidosis suppressed overall ATP turnover and protein synthesis by 50%. Protein synthesis, but not total ATP production, was also suppressed under hypoxic conditions. A dramatic decrease in ATP turnover (SiHa) and protein synthesis (both cell lines) was observed when hypoxia and low pHe were combined.ConclusionsWe demonstrate here that the influence of hypoxia and acidosis causes different responses, both in gene expression and in de novo protein synthesis, depending on whether the two factors induced alone or overlapping, and as such it is important for in vivo studies to take this into account.
| S-EPMC4537201 | biostudies-literature
Project description:Neuronal computations strongly depend on inhibitory interactions. One such example occurs at the first retinal synapse, where horizontal cells inhibit photoreceptors. This interaction generates the center/surround organization of bipolar cell receptive fields and is crucial for contrast enhancement. Despite its essential role in vision, the underlying synaptic mechanism has puzzled the neuroscience community for decades. Two competing hypotheses are currently considered: an ephaptic and a proton-mediated mechanism. Here we show that horizontal cells feed back to photoreceptors via an unexpected synthesis of the two. The first one is a very fast ephaptic mechanism that has no synaptic delay, making it one of the fastest inhibitory synapses known. The second one is a relatively slow (τ≈200 ms), highly intriguing mechanism. It depends on ATP release via Pannexin 1 channels located on horizontal cell dendrites invaginating the cone synaptic terminal. The ecto-ATPase NTPDase1 hydrolyses extracellular ATP to AMP, phosphate groups, and protons. The phosphate groups and protons form a pH buffer with a pKa of 7.2, which keeps the pH in the synaptic cleft relatively acidic. This inhibits the cone Ca²⁺ channels and consequently reduces the glutamate release by the cones. When horizontal cells hyperpolarize, the pannexin 1 channels decrease their conductance, the ATP release decreases, and the formation of the pH buffer reduces. The resulting alkalization in the synaptic cleft consequently increases cone glutamate release. Surprisingly, the hydrolysis of ATP instead of ATP itself mediates the synaptic modulation. Our results not only solve longstanding issues regarding horizontal cell to photoreceptor feedback, they also demonstrate a new form of synaptic modulation. Because pannexin 1 channels and ecto-ATPases are strongly expressed in the nervous system and pannexin 1 function is implicated in synaptic plasticity, we anticipate that this novel form of synaptic modulation may be a widespread phenomenon.
| S-EPMC4028192 | biostudies-literature
Project description:1. The dissimilation of a number of externally added hexose phosphates and 5'-nucleotides by the perfused rat heart is described, and non-specific esterase and 5'-nucleotidase activity associated with the superficial cell membrane or vascular system has been demonstrated. 2. The rate of production of (14)CO(2) from [U-(14)C]glucose 6-phosphate suggests that oxidation occurred after hydrolysis to glucose. The incorporation of isotope from [U-(14)C]glucose 6-phosphate into glycogen was small, and similar to that obtained with [U-(14)C]glucose as substrate. 3. Glucose 6-phosphate was also partially isomerized to fructose 6-phosphate. Similarly, fructose 6-phosphate was converted mainly into glucose 6-phosphate, but also into glucose and inorganic phosphate. When fructose 1,6-diphosphate was added to the perfusate, a mixture of glucose 6-phosphate, fructose 6-phosphate and triose phosphates accumulated in the medium approximately in the equilibrium proportions of the phosphohexose-isomerase and triose phosphate-isomerase reactions, together with inorganic phosphate and some glucose. Glucose 1-phosphate was hydrolysed to glucose, but was not converted into glucose 6-phosphate. Leakage of enzymes out into the perfusion fluid did not occur. 4. This demonstration that phosphohexose isomerase, triose phosphate isomerase and aldolase may react with extracellular substrates at an appreciable rate suggests that these enzymes are attached to the cell membrane.
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