Search

The nervous system may play a role in regulation of bone metabolism. The effects of norepinephrine(NE), vasoactive intestinal peptide(VIP), and ATP on cytosolic Ca2+ were assessed in a rat osteoblast-like osteosarcoma cell line (UMR-106) responsive to PTH. All three transmitters transiently increased Ca2+, with ATP much greater than PTH greater than NE = VIP, and then caused sustained increases in Ca2+. The ATP-induced transient resulted from mobilization of intracellular Ca2+ store, while NE and VIP-induced transients also involved influx of Ca2+. Later sustained increases by all agonists were dependent upon extracellular Ca2+. Release of intracellular Ca2+ by ATP was associated with a marked increase in IP3 but without a significant change in cAMP. NE, VIP, and ATP, through regulation of Ca2+ metabolism, may be involved in various osteoporotic conditions. [ABSTRACT FROM AUTHOR]

We sought to understand the relationship between reactive oxygen species (ROS) and the mitochondrial permeability transition (MPT) in cardiac myocytes based on the observation of increased ROS production at sites of spontaneously deenergized mitochondria. We devised a new model enabling incremental ROS accumulation in individual mitochondria in isolated cardiac myocytes via photoactivation of tetramethylrhodamine derivatives, which also served to report the mitochondrial transmembrane potential, DeltaPsi. This ROS accumulation reproducibly triggered abrupt (and sometimes reversible) mitochondrial depolarization. This phenomenon was ascribed to MPT induction because (a) bongkrekic acid prevented it and (b) mitochondria became permeable for calcein ( approximately 620 daltons) concurrently with depolarization. These photodynamically produced "triggering" ROS caused the MPT induction, as the ROS scavenger Trolox prevented it. The time required for triggering ROS to induce the MPT was dependent on intrinsic cellular ROS-scavenging redox mechanisms, particularly glutathione. MPT induction caused by triggering ROS coincided with a burst of mitochondrial ROS generation, as measured by dichlorofluorescein fluorescence, which we have termed mitochondrial "ROS-induced ROS release" (RIRR). This MPT induction/RIRR phenomenon in cardiac myocytes often occurred synchronously and reversibly among long chains of adjacent mitochondria demonstrating apparent cooperativity. The observed link between MPT and RIRR could be a fundamental phenomenon in mitochondrial and cell biology.

Deletions in human mitochondrial DNA cause various mitochondrial myopathies and increase markedly with age in highly oxidative tissues, but exhibit a differential distribution in the brain. In order to determine whether a similar pattern occurs in rat brain the levels of a 4.8 kb deletion and electron transport complex activities were measured in the striatum, hippocampus, cerebellum, and cerebral cortex of young adult and senescent male Wistar rats. Deletion-containing mtDNA was present at relatively similar levels (0.0003%) in all regions in 6 mo rats, but increased 25-, 7-, 3-, and 2-fold in the striatum, hippocampus, cerebral cortex, and cerebellum, respectively, of 22-23 mo old rats. To assess the relationship between fractional occurrence of a deletion and oxidative phosphorylation capacity, the activities of mitochondrial respiratory chain complexes I, III, IV and V, the mitochondrial ATP-ase, each of which contains subunits encoded in mtDNA, were determined in homogenates. No age-related decrements in activity were observed in any of the brain regions. Thus, while mtDNA deletions increase with age and to a large extent mirror the pattern observed in the human brain, they appear to have no effect on capacity for oxidative phosphorylation of distinct brain regions. Any reductions in capacity that may be present are likely to occur only at the level of individual cells.

Recent studies on human tissues have shown that the quantity of partially deleted mitochondrial DNA (mtDNA) increases with age. In this study, mtDNAs from the livers of young adult and old Wistar rats were analyzed by PCR. Evidence for partially deleted mtDNAs was found, with a 4834-bp deletion present in all animals and most easily detected in samples from senescent rats. The deletion breakpoint occurs at a 16-bp direct repeat present in the cytochrome oxidase I and ATPase 6 genes. This deletion in rats is similar in size and location to the 5.0-kb deletion observed in human mtDNA. The proportion of rat mtDNA with this 4.8-kb deletion was quantitated by a competitive PCR assay. The ratio of partially deleted mtDNA/total mtDNA in liver mtDNA from individual 6 month old rats ranged from 5 x 10(-6) to 3 x 10(-5), while the ratio in 24 month old rats ranged from 8 x 10(-4) to 5 x 10(-3), with a mean 100-fold increase with age. These increases are in the range observed for human mtDNA during aging. Thus senescent rats can be used as a model to study this type of mitochondrial DNA damage in aging. The method and reagents described should prove useful in studies of the mechanism(s) underlying deletions, their significance to the aging process, and testing of various compounds or interventions for their ability to slow the process.

Hexokinases are comprised of two highly homologous approximately 50-kDa halves and are product-inhibited by glucose-6-P. Four amino acid residues, Ser603, Asp657, Glu708, and Glu742, located in the C-terminal half of the tumor mitochondrial enzyme have been shown to be essential for enzyme function (Arora, K. K., Filburn, C. R., and Pedersen, P. L. (1991) J. Biol. Chem. 266, 5359-5362). Here we have assessed also the role of the N-terminal half of the same enzyme. Site-directed mutagenesis of residues predicted to interact with glucose in the N-terminal half, i.e. Ser155, Asp209, and Glu260, to Ala, have no effect on hexokinase activity. In addition, inhibition by hexose mono- and bisphosphates is unchanged for each of the mutant enzymes. Significantly, the overexpressed N-terminal polypeptide is devoid of catalytic activity but does have the capacity to bind ATP-agarose and be released with ATP and glucose-6-P. In contrast, the overexpressed C-terminal polypeptide is catalytically active and shows the same product inhibition pattern as the complete 100-kDa parent enzyme. These results emphasize that the N-terminal half of tumor hexokinase is essential neither for catalysis nor product modulation. Rather, the N-terminal half may play another role, perhaps in modulation of the ATP/glucose-6-P-dependent binding of the enzyme to tumor mitochondria or by acting as a spacer between the outer mitochondrial membrane and the C-terminal catalytic unit.

Available data indicate that insulin secretion is impaired with aging. Almost all the studies that examined insulin secretion by old animals did not take into consideration the state of renal function or the blood levels of parathyroid hormone (PTH). Old animals may have chronic renal failure (CRF) and secondary hyperparathyroidism, and both of these conditions impair insulin secretion. It is possible, therefore, that the impaired insulin secretion of aging is not due to old age per se, but rather to associated CRF and excess PTH. The present study examined this issue in adult (6 month old) and senescent rats (2 year old) with and without CRF and excess PTH. Senescent rats without CRF had normal renal function and normal blood levels of PTH, and the values were not different from those observed in adult rats. Creatinine clearance in senescent rats with CRF was significantly (P less than 0.01) lower and serum levels of PTH were significantly (P less than 0.01) higher than in senescent animals without CRF and than in the adult rats as well. Only the senescent rats with CRF displayed glucose intolerance during intravenous glucose tolerance test. For any given level of blood glucose, plasma insulin levels were lower in senescent rats with CRF than in the adult rat or senescent animals without CRF.(ABSTRACT TRUNCATED AT 250 WORDS)

We have shown that ATP increases cytosolic Ca2+ in UMR-106 cells through P2-purinergic receptor stimulation (Calcif Tissue Int 45:251-254). This response was further characterized using cells loaded with indo-1/AM or prelabeled with [3H]inositol. ATP elicited a rapid transient increase in Ca2+ from 148 to 540 nM, followed by a biphasic decline (first rapid and then slower) to basal within 1 minute and then a late slow rise to 200 nM by 4 minutes. ADP also elicited a rapid transient increase, but this was followed by a second smaller transient and a later, slow increase above basal Ca2+. These transient increases in Ca2+ induced by ATP and ADP were dose dependent, detected at 10(-6)M ATP and 10(-7)M ADP, and saturated at 10(-4)M with both nucleotides. The maximum increase in Ca2+ was 20% greater with ATP than ADP. EGTA chelation of extracellular Ca2+ abolished the biphasicity of the ATP-induced Ca2+ transient, the second ADP-induced transient, and all late slower increases in Ca2+. Desmethoxyverapamil pretreatment attenuated the biphasicity of the ATP-induced transient and the second peak elicited by ADP. Elevated extracellular Ca2+ (5 mM) prevented the return to the basal level that normally follows the ATP-induced Ca2+ transient and amplified the sustained increase in Ca2+ but had little effect on the response to ADP. IP3 and IP4 increased rapidly after addition of ATP, with I(1,4,5)P3 increasing before I(1,3,4)P3. These data indicate that P2-purinergic stimulation of UMR-106 cells causes three consecutive responses in cytosolic Ca2+: (1) a transient increase due to IP3-mediated mobilization of intracellular Ca2+; (2) a transient increase due in part to influx, probably associated with a Ca2+ channel; and (3) a later sustained increase that requires extracellular calcium.

The contractile response measured as maximum rate of force development to a near threshold concentration of isoproterenol (1 nM) was enhanced in perfused interventricular septa from hyperthyroid (128+/-4% control) compared with euthyroid rats (105+/-2%, P < 0.01). This enhanced contractile response was accompanied by a significant activation of cyclic (c)AMP-dependent protein kinase (protein kinase activity ratio increased from 0.159+/-0.008 to 0.218+/-0.019, P < 0.005, although no significant changes from base line occurred in euthyroid septa, 0.152+/-0.007-0.179+/-0.012). No difference between hyperthyroid and euthyroid hearts was observed in the contractile response to 0.1 mM dibutyryl cAMP (126.5+/-2.5% and 122.0+/-9.2% in hyperthyroid and euthyroid, respectively), and the magnitude of the response to dibutyryl cAMP was comparable with that observed in the hyperthyroid group with 1 nM isoproterenol. These results suggest that the mechanism for enhanced protein kinase activation and contractile response to low concentrations of isoproterenol in the hyperthyroid heart is at or proximal to cAMP generation. The maximum contractile response to isoproterenol (0.5 muM), however, was decreased in hyperthyroid myocardium (192+/-13%) compared with euthyroid (291+/-37%, P < 0.05). Both protein kinase activity ratio (0.356+/-0.017 and 0.344+/-0.013) and the maximum contractile response to Ca(++) (335+/-15 and 340+/-12% control in hyperthyroid and euthyroid, respectively) were similar, suggesting that the mechanism of the diminished maximum response was distal to protein kinase activation but not a function of an altered Ca(++)-troponin interaction. The diminished maximum rate of force development response in the hyperthyroid hearts was accompanied by significantly less shortening of the contraction duration that was 85.6+/-2.1% control in hyperthyroid vs. 66+/-2.8% control in euthyroid, P < 0.001. Although the basal rate of Ca(++) accumulation was greater in microsomes isolated from hyperthyroid than from euthyroid hearts, there was significantly less additional stimulation of Ca(++) accumulation in response to exogenous cAMP and protein kinase in hyperthyroid compared with euthyroid hearts. This reduction may explain the diminished effect of isoproterenol on the shortening of contraction duration in hyperthyroid compared with the euthyroid myocardium, and may explain, at least in part, the diminished maximum contractile response to isoproterenol.

Renal cortical slices were incubated with parathyroid hormone or dibutyryl cAMP and the effects on phosphate uptake and phosphorylation of proteins in brush border membranes isolated from the treated slices were determined. Na+ gradient-dependent phosphate uptake was inhibited. Phosphorylation of proteins of Mr=170 K, 135 K, 105 K, 88 K, and 68 K was increased after incubation with the hormone or the cyclic nucleotide. Phosphorylation of membrane proteins was also examined in isolated relatively intact brush border membrane vesicles and in membrane vesicles disrupted with Triton X-100. With intact membrane vesicles, total phosphorylation of the membrane was not significantly altered by cAMP. However, phosphorylation of proteins of Mr=85 K and 48 K increased whereas phosphorylation of proteins of Mr=170 K, 78 K, and 56 K decreased, relative to that found with slices. With detergent-treated membranes, which presumably were made permeable to [gamma-32P]-ATP, a cAMP-induced increase in total phosphorylation was demonstrated. Phosphorylation of proteins of Mr=135 K, 78 K, 65 K, and 56 K was markedly enhanced. These findings suggest that proteins of Mr=135 K, 78 K, 65 K, and 56 K are localized on the cytosolic side of the membrane whereas proteins of Mr=85 K and 48 K are present on the luminal surface of the membrane. Incubation of the isolated brush border membrane vesicles with Ca2+, or Ca2+ plus cAMP, also affected the phosphorylation of membrane proteins. The phosphorylation of proteins of Mr=105 K, 68 K, and 20 K was increased by Ca2+. The Mr=20 K protein may be myosin light chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Both D1 and D2 dopamine receptor subtypes are lost from striata as Wistar rats age. The magnitude of loss differs slightly for the two subtypes (approximately 30% for D1, approximately 40% for D2) as does the temporal pattern (progressive loss from 3 to 24 months for D2, no decrease in D1 after 12 months) although most D2 loss also occurs in the first half of the lifespan. Dopamine stimulated adenylate cyclase activity also declines during striatal aging in a manner roughly proportioned to D1 receptor loss.

The age dependence of contractile and associated biochemical parameters of basal- and catecholamine-stimulated myocardial contractile performance was investigated using isolated perfused septa from adult and senescent rats. Base-line maximum rate of force development (dF/dt), beta-receptor number and affinity, cAMP levels, and cAMP-dependent protein kinase activity were not different in the two age groups. During maximal isoproterenol stimulation, the increase in dF/dt was 40% less in the senescent hearts, and the cAMP levels and cAMP activation of protein kinase increased two fold but to the same extent in both age groups. The maximum contractile response to dibutyryl cAMP (DBcAMP) in the senescent was half that observed in the adult hearts. However, adult and senescent septa responded equally to an increase in perfusate [Ca2+] to 1.0 mM, which enhanced contractility to the same extent as that obtained with isoproterenol and DBcAMP in adult septa. These data taken together suggest that the factors that limit the contractile response to catecholamines in the senescent heart act subsequent to protein kinase activation but proximal to the Ca2+-troponin interaction.

Two soluble forms of 3':5'-cyclic-nucleotide phosphodiesterase (o':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) were found in the larval fat body of the silkmoth Hyalophora cecropia. These differ in elution profile on Sephadex G-200, solubility in ammonium sulfate, metal ion requirements and kinetic properties. Phosphodiesterase I has Km values of 11 muM and 1.8 muM for cyclic AMP and cyclic GMP, respectively, has 5-fold greater maximal activity with cyclic AMP than with cyclic GMP, and is activated by Mg2+ and Co2+, and inhibited by EDTA. phosphodiesterase II has Km values of 625 muM and 125 muM for cyclic AMP and cyclic GMP, respectively, has similar maximal activity with both substrates, and is not activated by divalent metal ions or inhibited by EDTA. Cyclic nucleotides and methylxanthines competitively inhibit both enzymes. Phosphodiesterase is found in both soluble and particulate fractions of homogenates. Total activity is highest during the larval stage of the insect, drops markedly following pupation, and rises again during pharate adult development.

The possible role of cyclic AMP-mediated phosphorylation events in the regulation of exocrine secretion after beta-adrenergic stimulation was examined in vitro in dispersed acinar cell aggregates from rat parotid gland. l-Isoproterenol, a beta-adrenergic agonist, stimulated endogenous activity of cyclic AMP-dependent protein kinase, alterations in the 32P content of 3 parotid phosphoproteins (increased 32P in 2, Mr = 27,000 and 14,000; decreased 32P in the remaining, Mr = 13,600), and amylase secretion in a dose-dependent manner. All responses were half-maximal within a range of l-isoproterenol concentrations of approximately 4 X 10(-8) to 5 X 10(-7) M. Examination of the time course of these 3 processes revealed that by 30 s after addition of l-isoproterenol, significant elevations in cyclic AMP-dependent protein kinase activity and alterations in the 32P content of the 3 parotid proteins had occurred, whereas secretion of amylase from cells was first detected 1-2 1/2 min after hormonal stimulation. Dibutyryl cyclic AMP (2 mM) elicited the same changes in parotid protein 32P content as l-isoproterenol. Our results support the concept of a role for cyclic AMP-regulated protein phosphorylation in the sequence of cellular events leading to exocrine protein secretion from the rat parotid gland following beta-adrenergic stimulation.

While protein kinase C (PKC) appears to play a role in the action of PTH in renal cells, direct evidence of activation by PTH is lacking. Rat PTH (1-34) caused a rapid, transient translocation of PKC in opossum kidney (OK) cells from a basal value of 0.09 to maximum of 0.24 at 10-15 sec. Both the time course and dose-response relationship of translocation matched a corresponding increase in cytosolic Ca2+. In contrast, PTH activation of cAMP-dependent protein kinase (PKA), while also rapid, was greater in magnitude (0.10 to 0.50), persistent, and occurred at a threshold level of 3 x 10(-10)M PTH, compared to 10(-8)M for PKC. Neither bPTH(3-34) nor bPTH(7-34) activated either protein kinase, while both antagonized rPTH(1-34)-induced PKC translocation more effectively than PKA activation. These differential effects of PTH agonist and antagonists further support the suggestion that PTH acts through two signal transduction mechanisms in which one or more receptors is linked in distinct ways to adenylate cyclase and phospholipase C.