Submitochondrial distribution of cAMP during incubation with rat liver mitochondria

Labeled cAMP incubated with rat liver mitochondria penetrates not only through outer mitochondrial membranes, but also into mitoplasts, where it is accumulated mainly in the matrix. Damage of mitochondrial membranes caused by single freezing-thawing treatment promotes no influx, but efflux of cAMP from mitoplasts. cAMP molecules penetrate inside mitochondria largely in an unchanged state in all submitochondrial fractions, as was demonstrated by the TLC method. cAMP transport into mitochondria can serve as a reason for: 1) stimulation of mitochondrial function by hormones whose effects are realized through activation of cytoplasmic adenylate cyclase and by extramitochondrial (cytosolic) cAMP; 2) existence of cAMP-dependent protein kinase and cAMP-phosphodiesterase in mitochondria.     

Effect of certain agents on subcellular cAMP level in different areas of rat brain

The influence was studied in vitro of certain agents (adenosine, ADP, ATP, theophylline, together with F- ions) on the cAMP concentrations in the nuclear (N) and mitochondrial (M) fractions from different areas of rat brain. F- ions caused a slight decrease of the cAMP concentrations in nuclear fractions of the thalamus with hypothalamus and a marked decrease of this cyclic nucleotide in M fractions from the cerebral cortex. After incubation with adenosine and F- ions a distinct decrease of cAMP level was observed in N fractions from the midbrain and thalamus with hypothalamus and in mitochondrial fractions obtained from all the investigated regions. The incubation with ATP and F- ions resulted in a distinct decrease of cAMP values in the nuclear fractions from all regions. The concentrations of cAMP in the mitochondria of the midbrain and thalamus with hypothalamus incubated with ATP and F- ions increased 2-3 times. The incubation of the nuclear fraction with theophylline and F- ions caused an increase of cAMP concentration in the cortex and a decrease of cAMP values in the midbrain. The level of cAMP after the incubation with theophylline and fluoride on the mitochondrial fraction is increased in the cortex and decreased in the thalamus with hypothalamus.     

Accumulation of 14C-nicotinate by membrane preparations

The incorporation of nicotinic acid (NA) into cells and mitochondria depends on temperature and the presence of glucose, is coupled with the activity of Na, K-ATPase and is disturbed by the energy exchange inhibitors. The addition of a sucrose liver extract or protein-containing fractions of the extract stimulates the 14C-NA accumulation by liver mitochondria. The stimulating effect of the extract on 14C-NA incorporation into mitochondria is retained after dialysis, but is removed upon boiling of the extract. It is assumed that the 14C-NA transport is operated by proteins: NA is incorporated (at least, partially) into liver cells, where it exists both in a free and proteinbound states. It has been found that there exist at least two protein fractions which bind 14C-NA.     

The origin and turnover of organelle membranes in castor bean endosperm

The origin and turnover of organelle membranes in castor bean (Ricinus communis L. var. Hale) endosperm was examined using choline-¹⁴C as a phospholipid precursor. On sucrose gradients three major particulate fractions were separated; a light membranous fraction (density 1.11-1.13 gram per cm³), the mitochondria (1.18 gram per cm³), and the glyoxysomes (1.24 gram per cm³). Choline-¹⁴C was readily incorporated into lecithin in all three particulate fractions, but the light membranous fraction became labeled first. Incorporation continued into all three fractions for 6 hours, at which time the available choline-¹⁴C had been completely used. Subsequently, ¹⁴C was lost from the three components at distinctly different rates. When an excess of unlabeled choline was added after 1 hour (pulse-chase experiment), incorporation of choline-¹⁴C into glyoxysomes and mitochondria continued for three hours, but at a diminishing rate. This was followed by a period in which the ¹⁴C content of the mitochondria declined at a rate expected, if the half life of lecithin in the membrane were about 50 hours and that of the glyoxysomes 10 hours. These values are close to those calculated from the experiments in which no chase was used. The labeling in the light membrane fraction behaved differently from that of the mitochondria and glyoxysomes following the chase of unlabeled choline. Incorporation continued for only 1 additional hour, and then the ¹⁴C content declined sharply in the subsequent 4 hours. The early kinetics and subsequent interrelationships are those expected if the lecithin in the membranes of mitochondria and glyoxysomes originates in components of the light membrane fraction.     

Cyclic 3',5'-adenosine monophosphate-dependent kinase and phosphoproteins in human amnion

3',5'-Cyclic adenosine monophosphate (cAMP) modulates prostaglandin production in human amnion membranes. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins following cAMP activation of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase and phosphoproteins have not previously been characterized in human amnion. Total homogenates, cytosol, and membrane fractions from human amnion were examined for [3H]cAMP binding activity and cAMP-dependent kinase activity. cAMP-dependent kinase activity was barely detectable in crude amnion fractions. Cytosol was therefore partially purified by DEAE column chromatography for further examination. Two peaks of coincident [3H]cAMP binding and cAMP-dependent kinase activity were demonstrated at 70 and 140 mM NaCl, characteristic of the Type I and Type II cAMP-dependent protein kinase isozymes. [3H]cAMP binding to the material from both peak fractions was saturable and reversible. Scatchard analysis of [3H]cAMP binding to the peak fractions was linear for peak I and curvilinear for peak II. Assuming a one-site model, [3H]cAMP binding to the Type I isozyme showed a KD = 4.17 x 10(-8) M and Bmax = 73 pmole/mg protein; using a two-site model, [3H]cAMP binding to the high-affinity site for the Type II isozyme had a KD = 3.94 x 10(-8) M and Bmax = 6.3 pmole/mg protein. Other cyclic nucleotides competed for these [3H]cAMP binding sites with a potency order of cAMP much greater than cGMP greater than (BU)2cAMP.cAMP caused a dose-dependent increase in cAMP-dependent kinase activity in the peak fractions; half-maximal activation was observed with 5.0 x 10(-8) M cAMP. The ability of cAMP to increase phosphorylation of endogenous proteins in both crude amnion cytosol and cytosol from cultures of amnion epithelial cells was assessed using [32P]ATP, SDS-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated 32P incorporation into three proteins having Mr = 80,000, 54,000, and 43,000 (P less than .01). Half-maximal 32P incorporation into these proteins occurred at 1.0 x 10(-7) M cAMP. cAMP-dependent kinase is present in human amnion; specific cAMP-enhanced phosphoproteins are also present. Hormones elevating cAMP levels in amnion may exert their effects by activating cAMP-dependent kinase and phosphorylating these phosphoproteins.     

Influence of aminophylline on the lipids in Microsporum gypseum.

The effect of aminophylline on the lipid synthesis of Microsporum gypseum has been examined. A decreased incorporation of [14C]acetate into lipids was observed when the cells were incubated for 1 h with aminophylline which was reflected in all the individual lipid fractions. However, cells grown with aminophylline in the growth medium exhibited increased levels of total phospholipids, which was probably due to a rise in intracellular cAMP as these cells exhibited 4-fold increased levels of cAMP. Decreased activity of phosphodiesterase by aminophylline accounts for the increased cAMP levels. Increased phospholipid content in aminophylline grown cells was further supported by the increased incorporation of [14C]acetate into phospholipids as well as increased activities of phospholipid biosynthetic enzymes in comparison to non-supplemented cells.     

Inhibition of protein synthesis and ATPase in mitochondria by the administration of the convulsant 3-mercaptopropionic acid.

¹⁴C leucine incorporation into proteins by cerebral cortex subcellular fractions was studied after the administration of the convulsant 3-mercaptopropionic acid (MP). It was found that MP decreased protein synthesis by isolated nerve endings and mitochondria but not by microsomal fractions. It was also observed that mitochondrial ATPase was inhibited. These findings suggest that the inhibition of protein synthesis might be an indication of a disequilibrium of the normal energy-yielding metabolism.     

Can mitochondria and synaptosomes of guinea-pig brain synthesize phospholipids?

1. The use of ;marker' enzymes for investigating the contamination by endoplasmic reticulum of mitochondrial and synaptosomal (nerve-ending) fractions isolated from guinea-pig brain was examined. NADPH-cytochrome c reductase appeared to be satisfactory. With the synaptosomal preparation there was a non-occluded enzymic activity believed to arise from contaminating microsomes and an occluded form released by detergent, which probably was derived from some type of intraterminal smooth endoplasmic reticulum. 2. Isolated brain mitochondria, both intact and osmotically shocked, could not synthesize more labelled phosphatidylcholine from CDP-[Me-(14)C]choline or phosphoryl[Me-(14)C]choline than could be accounted for by microsomal contamination. They could synthesize only phosphatidic acid and diphosphatidylglycerol from a [(32)P]P(i) precursor and not nitrogen-containing phosphoglycerides or phosphatidylinositol. 3. The synaptosomal outer membrane and the intraterminal mitochondria could not synthesize phosphatidylcholine from CDP-[Me-(14)C]choline but the synaptic vesicles and probably the intraterminal ;endoplasmic reticulum' appeared to be capable of catalysing the incorporation of label from this substrate into their phospholipids. 4. Microsomal fractions and synaptosomes from guinea-pig brain could incorporate [Me-(14)C]choline into their phospholipids by a non-energy-requiring exchange process, which was catalysed by Ca(2+). Fractionation of the synaptosomes after such an exchange had taken place revealed that the label was predominantly in the intraterminal mitochondria and not associated with membranes containing NADPH-cytochrome c reductase. 5. On the intraperitoneal injection of [(32)P]P(i) into guinea pigs, incorporation of radioactivity into phosphatidylinositol and phosphatidic acid was much faster than into the nitrogen-containing phosphoglycerides. Mitochondria and microsomal fractions showed a roughly equivalent incorporation into individual phospholipids, and that into synaptosomes was appreciably less, whereas the phospholipids of myelin showed little (32)P incorporation up to 10h.     

cAMP inhibits bile acid-induced apoptosis by blocking caspase activation and cytochrome c release

We have previously shown that cAMP protects against bile acid-induced apoptosis in cultured rat hepatocytes in a phosphoinositide 3-kinase (PI3K)-dependent manner. In the present studies, we investigated the mechanisms involved in this anti-apoptotic effect. Hepatocyte apoptosis induced by glycodeoxycholate (GCDC) was associated with mitochondrial depolarization, activation of caspases, the release of cytochrome c from the mitochondria, and translocation of BAX from the cytosol to the mitochondria. cAMP inhibited GCDC-induced apoptosis, caspase 3 and caspase 9 activation, and cytochrome c release in a PI3K-dependent manner. cAMP activated PI3K in p85 immunoprecipitates and resulted in PI3K-dependent activation of the survival kinase Akt. Chemical inhibition of Akt phosphorylation with SB-203580 partially blocked the protective effect of cAMP. cAMP resulted in wortmannin-independent phosphorylation of BAD and was associated with translocation of BAD from the mitochondria to the cytosol. These results suggest that GCDC-induced apoptosis in cultured rat hepatocytes proceeds through a caspase-dependent intracellular stress pathway and that the survival effect of cAMP is mediated in part by PI3K-dependent Akt activation at the level of the mitochondria.     

Mitochondrial DNA,RNA, and protein synthesis in different regions of developing rat brain

In vivo and in vitro (tissue slices) incorporation of labeled precursors into DNA, RNA, and proteins was measured in mitochondria obtained from cerebral hemispheres, cerebellum, and brain stem of rats at different days of postnatal development. To compare the synthesis of macromolecules in mitochondria with that in other subcellular fractions, the incorporation of labeled precursors into DNA, RNA, and proteins extracted from nuclei and into RNA and proteins extracted from microsomes and cytoplasmic soluble fractions was also measured.The results obtained showed that the incorporation of [³H]thymidine into DNA and of [¹⁴C]leucine into proteins of nuclei and mitochondria from the various brain regions examined decreased during postnatal development, however, at 30 days of age the specific radioactivity of mitochondrial DNA was higher than that of nuclear DNA. [³H]Uridine incorporation into RNA decreased from 10 to 30 days of age in nuclei while in mitochondria it was quite similar at both ages. This result may be due to a faster turnover of mitochondrial RNA compared to that of mitochondrial DNA and proteins. The results obtained suggest an active biosynthesis of macromolecules in brain mitochondria and might indicate an intense biogenesis of these organelles in rat brain during postnatal development.Preliminary reports of these results were presented at the XI FEBS Meeting, Copenhagen, August 14–19, 1977, Poster number A2-2-155-3, and at III Meeting of Italian Biochem. Soc., Siena, October 3–5, 1977, Abstract C6.     

Biochemical heterogeneity of mitochondria]

Rat liver mitochondria were fractionated on the basis of their sedimentation coefficients in the gradient of ficoll. The fractions ("heavy", "middle" and "light" mitochondria) were heterogeneous with regard to the content of protein, DNA, cytochrome a + a3 and respiratory activity. Heterogeneity of mitochondria did not result from the damage or microsomal and lysosomal contamination. The biosynthesis of DNA, RNA and proteins in the different fractions of mitochondria was studied. In vivo incorporation of radioactive precursor into RNA was highest in the fractions of "middle" mitochondria, whereas in vitro the "heavy" mitochondria showed maximum activity in the synthesis of RNA. In vitro DNA synthes was maximum in the fractions of "heavy" mitochondria, protein synthesis in "heavy" and "light" mitochondria. Activity of the synthesis of RNA, DNA and proteins in vitro depends on the content of DNA and cytochrome a + a3 in the different fractions of mitochondria. It is supposed that heterogeneity of mitochondria may be connected with their biogenesis.     

Cyclic AMP and serum arrest of the mitotic activity of human diploid fibroblasts.

Mitotic activity in confluent cultures of human diploid fibroblasts was arrested by the reduction of the serum concentration of the incubation medium to 0.5% or by the addition of 0.5 mM 6-N, 2'-O-dibutyryl-adenosine 3':5'-cyclic monophosphate (db cAMP). Under either of these conditions, cultures maintained a constant cell number for 14 days; cultures continuously exposed to medium containing 10% serum doubled their cell number during this 14-day period. The protein cotent per cell decreased by 20% when cells were maintained with 0.5% serum whereas that of cells exposed to db cAMP remained constant. Ultrastructural studies revealed that cells exposed to db cAMP exhibited a morphology typical of cells cultures with 10% serum alone, whereas cells incubated with 0.5% serum showed the ultrastructural changes in mitochondria, endoplasmic reticulum and Golgi complex previously identified with low-serum arrest. Cellular adenosine 3':5'-cyclic monophosphate (cAMP) levels remained constant during the 7-day growth period in which confluency was attained, as well as during the 14-day arrested period with 0.5% serum. These results indicated that the mitotic inhibition induced by reducing the serum concentration of the incubation medium was not mediated by increased intracellular levels of cAMP and differed from that induced by the addition of exogenous db cAMP.     

Effect of dibutyryl cyclic AMP on lipid synthesis in Microsporum gypseum

The influence of intracellular levels of cAMP on the lipid synthesis of Microsporum gypseum has been examined by exogenous supplementation of dibutyryl cAMP and its activators/inhibitors. Incorporation of [14C]acetate into various lipid fractions of M. gypseum was markedly enhanced in the presence of dibutyryl cAMP and its modulators, probably as a consequence of increased intracellular cAMP levels, which, in turn, affected the lipid biosynthesis. Increased activities of phosphatidic acid phosphatase, glycerol kinase, ethanolamine kinase and choline kinase in the presence of these additives supports the enhanced synthesis of phospholipids and suggests that lipid biosynthesis is being controlled by cAMP in M. gypseum.     

Cyclic amp and serum arrest of the mitotic activity of human diploid fibroblasts

Summary Mitotic activity in confluent cultures of human diploid fibroblasts was arrested by the reduction of the serum concentration of the incubation medium to 0.5% or by the addition of 0.5mm 6-N, 2′-O-dibutyryl-adenosine 3′:5′-cyclic monophosphate (db cAMP). Under either of these conditions, cultures maintained a constant cell number for 14 days; cultures continuously exposed to medium containing 10% serum doubled their cell number during this 14-day period. The protein content per cell decreased by 20% when cells were maintained with 0.5% serum whereas that of cells exposed to db cAMP remained constant. Ultrastructural studies revealed that cells exposed to db cAMP exhibited a morphology typical of cells cultured with 10% serum alone, whereas cells incubated with 0.5% serum showed the ultrastructural changes in mitochondria, endoplasmic reticulum and Golgi complex previously identified with low-serum arrest. Cellular adenosine 3′:5′-cyclic monophosphate (cAMP) levels remained constant during the 7-day growth period in which confluency was attained, as well as during the 14-day arrested period with 0.5% serum. These results indicated that the mitotic inhibition induced by reducing the serum concentration of the incubation medium was not mediated by increased intracellular levels of cAMP and differed from that induced by the addition of exogenous db cAMP.     

Further characterization of rat liver mitochondrial fractions. Lipid composition and synthesis, and protein profiles.

1. Heavy and light mitochondrial fractions obtained by differential centrifugation were further characterized with respect to their lipid composition and synthesis and protein profiles, as seen by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. The light mitochondrial fraction was rich in total lipids, phospholipids and cholesterol. The cardiolipin content, however, was low. 3. Rates of [3H]glycerol incorporation into phospholipids of heavy mitochondria and microsomal fractions were almost identical. On the other hand, incorporation into the individual phospholipids in light mitochondria was about 4-6 times higher. Incorporation into cardiolipin of light mitochondria was about 10-fold higher than in the heavy mitochondria. 4. Analysis of protein profiles by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed that the pattern obtained for the light mitochondria was similar to that for heavy mitochondria. However, the light fraction was relatively poor in high-molecular-weight proteins and rich in low-molecular-weight proteins. The microsomal protein profile was altogether different. 5. The significance of these findings is discussed in relation to mitochondrial biogenesis.     

The metabolism of cholesterol in the presence of liver mitochondria from normal and thyroxine-treated rats

1. [26-(14)C]- and [4-(14)C]-Cholesterol were incubated with liver mitochondria from normal and thyroxine-treated rats, and the radioactivity was measured in the carbon dioxide evolved during the incubation, in a butanol extract of the incubation mixture and in a volatile fraction containing substances of low molecular weight derived from the side chain of cholesterol. The butanol extract was separated by paper chromatography into three radioactive fractions, one of which contained the steroids more polar than cholesterol. 2. The butanol extract from incubations with [4-(14)C]cholesterol contained a radioactive substance moving with the same R(F) as cholic acid on thin-layer chromatography. 3. After incubation with [26-(14)C]-cholesterol, 60-80% of the radioactivity extracted by steam-distillation of the incubation mixture at acid pH was recovered as [(14)C]propionic acid. 4. In the presence of [26-(14)C]cholesterol, mitochondria from thyroxine-treated rats produced more radioactivity in carbon dioxide and in the volatile fraction, and less radioactivity in the fraction containing the polar steroids, than did mitochondria from normal rats. In the presence of [4-(14)C]cholesterol, mitochondria from thyroxine-treated rats produced the same amount of radioactivity in the polar steroids as did normal mitochondria. 5. Thyroxine treatment had no effect on the capacity of the mitochondria to oxidize propionate to carbon dioxide. 6. These results are best explained by supposing that thyroxine stimulates a rate-limiting reaction leading to the cleavage of the side chain of cholesterol, but has little or no influence on the hydroxylations of the ring system or on the oxidation of the C(3) fragment removed from the side chain.     

Lecithin biosynthesis in liver mitochondrial fractions

The pretreatment of rat liver mitochondrial fractions with phospholipase C preparations enhanced the incorporation of cytidine diphospho-[¹⁴C]-choline into phospholipids several-fold. Similar pretreatment of the microsomal fraction produced a similar stimulation. When the extent of microsomal contamination in the mitochondria was determined, and increments of pretreated microsomes were added to the mitochondria, the incorporation values extrapolated to zero for zero microsomal contamination. It was concluded that lecithin biosynthesis from endogenous diglycerides in the mitochondrial fractions could be ascribed to contaminating microsomes.     

Regulation by cytidine nucleotides of the acylation of sn-[14C]glycerol 3-phosphate. Regional and subcellular distribution of the enzymes responsible for phosphatidic acid synthesis de novo in the central nervous system of the rat

1. The regional and subcellular distribution of the incorporation of sn-[(14)C]glycerol 3-phosphate into rat brain lipids in vitro was investigated and compared with the relative specific activity of various chemical and enzyme markers. The similarity between the subcellular distribution of this incorporation and of NADPH-cytochrome c reductase activity indicated that the synthesis of phosphatidic acid via this route correlated with the presence of endoplasmic reticulum. 2. Experiments in which various amounts of the microsomal fraction were added to fixed amounts of nuclear, myelin, nerve-ending and mitochondrial preparations clearly demonstrated that the endoplasmic-reticulum contamination of these fractions was entirely responsible for the incorporation of sn-[(14)C]glycerol 3-phosphate. 3. The presence of CMP or CTP inhibited the incorporation of sn-[(14)C]glycerol 3-phosphate into the whole homogenate. Similar effects were observed with individual fractions, except for the mitochondria. With the mitochondrial fraction the effect of these cytidine nucleotides varied with the preparation, stimulating in some preparations and inhibiting with other preparations. The presence of CDP-choline stimulated the incorporation into the whole homogenate and to a lesser extent into the subcellular fractions. 4. These results indicate that the various organelles of the central nervous system are more dependent on endoplasmic reticulum for the production of glycerolipids de novo than has previously been appreciated.