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Query: UNIPROT:P56851 (epididymal)
 11,273 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The mechanism by which insulin activates pyruvate dehydrogenase in rat epididymal adipose tissue was further investigated. 2. When crude extracts, prepared from tissue segments previously exposed to insulin (2m-i.u/ml) for 2min, were supplemented with Mg-2+, Ca-2+, glucose and hexokinase and incubated at 30 degrees C, they displayed an enhanced rate of increase in pyruvate dehydrogenase activity compared with control extracts. 3. When similar extracts were instead supplemented with fluoride, ADP, creatine phosphate and creatine kinase, the rate of decrease in pyruvate dehydrogenase activity observed during incubation at 30 degrees C was unaffected by insulin treatment. 4. It is suggested that insulin increases the fraction of pyruvate dehydrogenase present in the tissue in the active dephospho form by increasing the activity of pyruvate dehydrogenase phosphate phosphatase.
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PMID:Activation of pyruvate dehydrogenase in adipose tissue by insulin. Evidence for an effect of insulin on pyruvate dehydrogenase phosphate phosphatase. 16 82

Testicular and cauda epididymal sperm were obtained via catheters previously implanted in the rete testis and proximal vas deferens of bulls and were used to examine the relationships among sperm motility, cyclic adenosine 3':5'-monophosphate (cAMP) level, adenine nucleotide levels, and rates of glucose and oxygen consumption. Testicular, cauda epididymal, and ejaculated sperm contain cAMP-stimulated protein kinase, adenylate cyclase, and nucleotide phosphodiesterase. Treatment of the nonmotile testicular sperm with phosphodiesterase inhibitors resulted in a doubling of cellular cAMP concentration and a 25% increase in their glucose consumption. No change in motility, ATP level, or rate of oxygen consumption was observed. Sperm in neat cauda epididymal semen had flagellating tails but no progressive motility. Dilution of these sperm into glucose-containing buffer resulted in an increase in intracellular cAMP concentration and a decrease in ATP level with concomitant increases in ADP and AMP levels. These biochemical changes occurred within 30 s after dilution and apparently preceded the initiation of progressive motility by most cells. Since sperm in neat cauda epididymal semen became progressively motile when diluted with neat cauda epididymal plasma as well as accessory sex gland fluid or buffer, composition of the fluid surrounding the sperm is not responsible for the initiation of progressive motility upon dilution nor does cauda epididymal plasma contain an inhibitory factor. Perhaps release from contact immobilization provides the stimulation for the initial acquisition of progressive motility by cauda epididymal sperm. We conclude that during epididymal passage sperm develop from a cell physically unresponsive to changes in cAMP concentration to a form which initiates progressive motility upon changes in cAMP concentration.
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PMID:Adenine nucleotide changes at initiation of bull sperm motility. 17 61

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.
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PMID:The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds. 117 87

It has recently been shown that chimeric toxins composed of acidic fibroblast growth factor fused to mutant forms of Pseudomonas exotoxin (aFGF-PE) are cytotoxic to a variety of tumor cell lines with FGF receptors. Although aFGF-PE might be considered as a possible chemotherapeutic toxin, limited knowledge is available concerning its effect on endothelia. This study investigates whether one of the aFGF-PE fusion proteins, aFGF-PE664GluKDEL, can function as an anti-angiogenic agent. Protein synthesis studies using rat epididymal fat pad microvascular endothelial cells (RFCs) indicated that after 24 h in culture, aFGF-PE had a significant inhibitory effect on protein synthesis at concentrations greater than 100 ng/ml. In cultures incubated with 1000 ng/ml aFGF-PE, RFC protein synthesis was inhibited as much as 83%. RFCs were also cultured in a 3-dimensional type I collagen gel and incubated with either transforming growth factor beta 1, aFGF-PE, or a combination of both. Transforming growth factor beta 1 elicits in vitro angiogenesis in these 3-dimensional cultures which consist of rapid formation of complex tubular structures. Transforming growth factor beta 1-treated RFCs incubated with aFGF-PE were unable to produce this angiogenic response, nor were they able to migrate out of the 3-dimensional culture to form a monolayer as shown by controls. Cell viability analyses showed that aFGF-PE produced a dose-dependent toxic effect which ranged from 10 to 90% cell death. Competition assays in which the chimeric toxin was preincubated with antibodies to aFGF resulted in an 89% reversal of the inhibitory effects of aFGF-PE on endothelial cells. Acidic FGF-PE with a mutation in the ADP ribosylation domain of PE was inactive in both 2-dimensional and 3-dimensional cultures. These data show that aFGF-PE has specific in vitro cytotoxic, antiangiogenic, and antimigratory effects on microvascular endothelia.
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PMID:Acidic fibroblast growth factor-Pseudomonas exotoxin chimeric protein elicits antiangiogenic effects on endothelial cells. 138 Dec 75

Guanine nucleotide-binding proteins (G proteins) are important signal transducing molecules found in all cells. G proteins are associated with the plasma membrane/outer acrosomal membrane region of acrosome-intact sperm and at least one G protein is involved in the zona pellucida-induced acrosome reaction. With the goal of elucidating the functions of these proteins during spermatogenesis, we investigated the types of G proteins present in spermatogenic cells and when they first become associated with the developing acrosome. Using bacterial toxin-catalyzed [32P]ADP-ribosylation in conjunction with immunoprecipitation and immunofluorescence utilizing antibodies directed against specific regions of various G protein isotypes, the alpha subunits of Gi1, Gi2, Gi3, and G(o) were detected in mouse spermatocytes and spermatids. An antiserum recognizing a conserved sequence of G alpha i subtypes localized to the proacrosomal granules of spermatocytes and the developing acrosome of spermatids. Levels of G alpha o diminished as spermatocytes developed into spermatids such that G alpha o was not detected in cauda epididymal sperm. Immunoreactivity using G alpha o-specific antisera did not display a distinct regionalization within any of the spermatogenic cell types. G alpha s was not detected in the developing spermatogenic cells or sperm. The association of G alpha i with the developing acrosome suggests a role for G proteins may have a role in acrosome biogenesis as well as being part of a complex required later for signal transduction leading to acrosomal exocytosis.
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PMID:Developmental expression of G protein alpha subunits in mouse spermatogenic cells: evidence that G alpha i is associated with the developing acrosome. 164 27

1. Exogenous adenosine triphosphate (ATP) stimulated the short circuit current (SCC) in primary monolayer cultures of rat epididymal cells when added to the apical but not to the basolateral side of the monolayers. Half-maximal stimulation was achieved at 5 x 10(-8) M ATP. 2. The increase in SCC induced by ATP was dependent on the presence of extracellular Cl in the bathing solutions. 3. The effects of other adenosine derivatives, and purine and pyrimidine nucleotides were studied. Their orders of potency in stimulating SCC were: ATP greater than adenosine diphosphate much greater than adenosine monophosphate, adenosine, and ATP greater than inosine triphosphate greater than guanosine triphosphate greater than cytidine triphosphate. These results indicate that ATP interacts with a P2-purinoceptor at the apical membrane of the epididymal cells. 4. The SCC response to ATP was not blocked by 8-phenyltheophylline, a P1-purinoceptor antagonist or by propranolol. Although pretreatment of the cultures with piroxicam abolished the SCC response to bradykinin, it did not affect the response to ATP. This indicates that the SCC response to ATP was not mediated by an increase in the synthesis of prostaglandins. 5. Serosal to mucosal Cl flux (Js-m Cl) and net water flux were measured in the luminally perfused rat epididymis in vivo. ATP (1 microM) added to the luminal perfusion solution caused an increase in Js-m Cl and net water secretion by the epididymal duct. 6. Since spermatozoa contain a high concentration of ATP, it is proposed that ATP released from spermatozoa may affect anion and fluid secretion by the epididymis. The control of secretion via the apical purinoceptors offers a means by which spermatozoa regulate the fluidity of their own environment.
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PMID:Control of anion and fluid secretion by apical P2-purinoceptors in the rat epididymis. 321 90

The specific activity of the gamma-32P position of ATP was measured in various tissue preparations by two methods. One employed HPLC and the enzymatic conversion of ATP to glucose 6-phosphate and ADP. The other was based on the phosphorylation of histone by catalytic subunit of cAMP-dependent protein kinase (Hawkins, P.T., Michell, R.H. and Kirk, C.J. (1983) Biochem. J. 210, 717-720). The HPLC method also allowed the incorporation of 32P into the (alpha + beta)-positions of ATP to be determined. In rat epididymal fat-pad pieces and fat-cell preparations the specific activity of [gamma-32P]ATP attained a steady-state value after 1-2 h incubation in medium containing 0.2 mM [32P]phosphate. Addition of insulin or the beta-agonist isoprenaline increased this value by 5-10% within 15 min. Under these conditions the steady-state specific activity of [gamma-32P]ATP was 30-40% of the initial specific activity of the medium [32P]phosphate. However, if allowance was made for the change in medium phosphate specific activity during incubations the equilibration of the gamma-phosphate position of ATP with medium phosphate was greater than 80% in both preparations. The change in medium phosphate specific activity was a combination of the expected equilibration of [32P]phosphate with exchangeable intracellular phosphate pools plus the net release of substantial amounts of tissue phosphate. At external phosphate concentrations of less than 0.6 mM the loss of tissue phosphate to the medium was the major factor in the change in medium phosphate specific activity. It is concluded that little advantage is gained in employing external phosphate concentrations of less than 0.6 mM in experiments concerned with the incorporation of phosphate into proteins and other intracellular constituents. Indeed, a low external phosphate concentration may cause depletion of important intracellular phosphorus-containing components.
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PMID:Studies on the specific activity of [gamma-32P]ATP in adipose and other tissue preparations incubated with medium containing [32P]phosphate. 351 72

Treatment of bull spermatozoa from epididymal cauda with 5 micrograms digitonin per microliter cells removed the permeability barrier of plasma membrane for mitochondrial substrates and effectors. Such preparations yielded a high portion of coupled mitochondria characterized by ratios of active respiration to carboxyatractyloside-inhibited respiration greater than 13 in the presence of efficient substrates. Bull sperm mitochondria oxidized pyruvate and lactate in the presence of malate as well as glycerol-3-phosphate with much higher rates than succinate or palmitoyl carnitine. For the efficient substrates the respiration coupled to ADP phosphorylation amounted to 77 to 100% of the uncoupled rate. Comparable rates of active respiration were also observed with ATP indicating the high ATPase activity present in digitonin-treated spermatozoa. Uncoupled rates of respiration corresponded to rates of intact spermatozoa, but the capacity of the phosphorylating respiration exceeded the respiration rates of intact motile spermatozoa remarkably. This indicates that the spermatozoal ATP turnover at sufficient supply of substrate is mostly controlled by ATP utilizing reactions.
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PMID:Capacities of oxidative metabolism in digitonin-treated bovine epididymal spermatozoa. 356 16

The regulation of oxidative phosphorylation was studied with digitonin-treated epididymal bull spermatozoa in which mitochondria are directly accessible to low molecular compounds in the extracellular medium. Due to the high extramitochondrial ATPase activity in this cell preparation, it was possible to stimulate respiration to a small extent only by added hexokinase in the presence of glucose and adenine nucleotides. Added pyruvate kinase plus phosphoenol pyruvate, however, strongly suppressed the respiration. Under these conditions, the respiration was found to depend on the extramitochondrial [ATP]/[ADP] ratio in the range of 1-100. The contribution of the adenine nucleotide translocator to this dependence was determined by titration with the irreversible inhibitor carboxyatractyloside in the presence of ADP. Using lactate plus malate as substrate, the active state respiration was controlled to about 30% by the translocator, whereas 12 and 4% were determined in the presence of L-glycerol-3-phosphate and malate alone, respectively. In order to compare the results with those for intact cells, the adenine nucleotide patterns were determined in intact and digitonin-treated spermatozoa under conditions of controlled respiration in the presence of vanadate and carboxyatractyloside, respectively. About 21% of total cellular adenine nucleotides were found in digitonin-treated cells representing the mitochondrial compartment. While allowing for the intramitochondrial amount of adenine nucleotides, the cytosolic [ATP]/[ADP] ratio was estimated to be 6-times higher than the mitochondrial ratio in intact cells. It is concluded from the data presented that the principal mechanism by which oxidative phosphorylation in sperm mitochondria is regulated via the extramitochondrial [ATP]/[ADP] ratio is the same as that demonstrated for other isolated mitochondria.
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PMID:Regulation of oxidative phosphorylation in mitochondria of epididymal bull spermatozoa. 360 41

31P NMR signals assigned to intracellular adenine nucleotides and to inorganic phosphate were detected in dense suspensions of epididymal sperm obtained from bulls or hamsters. Similar adenine nucleotide signals and an additional large resonance peak, attributable to extracellular glycerylphosphorylcholine, were observed with whole bovine cauda epididymides. Provision of the glycolytic substrate fructose to such sperm suspensions promoted apparent conversion of intracellular ADP to ATP with a concomitant decrease in cellular inorganic phosphate (Pi) content. Subsequent treatment with the methylxanthine caffeine resulted in diminution of the intracellular gamma-P-ATP signal that was consistent with the decreased ATP and ADP contents previously demonstrated by chemical analyses of cellular extracts. Alternatively, treatment with fructose followed by the membrane-selective detergent digitonin produced loss of the nucleotide NMR signals, indicating release of ATP and Pi from the sperm cytosol with subsequent hydrolysis in the extracellular medium. Comparison of intracellular Pi and ATP resonance signals with those of ATP and Pi in vitro, in media of varied pH and cation composition, allowed calculation of a cytosolic pH of 6.5-6.6 and a cytosolic Mg2+ concentration of 0.5 mM for fresh suspensions of bovine cauda epididymal sperm. Intracellular Pi of hamster epididymal sperm reported a similar cytosolic pH. Other, more acidic compartments were not detected in these experiments. However, during prolonged incubation, the pH of the bovine sperm interior slowly decreased as the extracellular medium was acidified by extensive production of lactate. Intracellular ATP was detectable until cytosolic pH declined to approximately 5.5. Rapid intracellular acidification, resulting from exchange of internal K+ for H+, was observed after treatment with carboxylic acid ionophore nigericin. This lowering of internal pH was followed by a slower return toward initial internal pH values, probably as a consequence of secondary exchange of internal protons for other external monovalent cations, rather than as a result of the operation of a cellular homeostatic mechanism. Together, these studies utilizing noninvasive NMR techniques provide evidence that within the bovine epididymis sperm utilize an unknown energy source to phosphorylate adenine nucleotides and maintain a slightly acidic cytosolic pH.
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PMID:A 31P NMR study of the epididymis and epididymal sperm of the bull and hamster. 407 1


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