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1.
J Vet Pharmacol Ther ; 30(2): 132-8, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17348898

ABSTRACT

Liver dysfunction often is associated with an imbalance in the production and removal of free radicals derived from oxygen and nitrogen and has been managed clinically with antioxidant supplements, including silymarin extract derived from milk thistle. The potential for enhanced bioavailability of a phytosome complex containing phosphatidylcholine and silybin, the primary active flavonolignan in silymarin extract, was tested in dogs. A group of eight beagles (four males, four females) were dosed orally with a silybin-phosphatidylcholine complex (SPC) and a commercially available standardized silymarin extract containing equivalent levels of silybin. Dosing with the SPC resulted in Cmax, Tmax, and AUC0-24 h values (mean+/-SD) for total silybin of 1310+/-880 ng/mL, 2.87+/-2.23 h, and 11,200+/-6520 ng.h/mL, respectively; corresponding values for a standardized silymarin extract were 472+/-383 ng/mL, 4.75+/-2.82 h, and 3720+/-4970 ng.h/mL. A second, separate group of beagles were also dosed with the extract alone, yielding values of 449+/-402 ng/mL, 6.87+/-7.43 h, and 2520+/-2976 ng.h/mL. These data show that a phytosome complex of phosphatidylcholine and silybin markedly enhances bioavailability in dogs.


Subject(s)
Antioxidants/pharmacokinetics , Dogs/metabolism , Phosphatidylcholines/pharmacokinetics , Phytotherapy , Silymarin/pharmacokinetics , Administration, Oral , Animals , Antioxidants/administration & dosage , Area Under Curve , Biological Availability , Female , Male , Phosphatidylcholines/administration & dosage , Phosphatidylcholines/blood , Plant Extracts/administration & dosage , Plant Extracts/blood , Plant Extracts/pharmacokinetics , Silybin , Silymarin/administration & dosage , Silymarin/blood
2.
J Exp Med ; 192(7): 1001-14, 2000 Oct 02.
Article in English | MEDLINE | ID: mdl-11015441

ABSTRACT

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.


Subject(s)
Heart/physiology , Mitochondria, Heart/physiology , Myocardium/metabolism , Reactive Oxygen Species/metabolism , Animals , Calcium/metabolism , Cells, Cultured , Fluorescent Dyes , Mitochondria, Heart/metabolism , Myocardium/cytology , Oxidation-Reduction , Permeability , Proteins/metabolism , Rats , Solubility , Sulfhydryl Compounds/metabolism
3.
Mech Ageing Dev ; 87(1): 35-46, 1996 May 24.
Article in English | MEDLINE | ID: mdl-8735905

ABSTRACT

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.


Subject(s)
Aging/genetics , Aging/metabolism , Brain/metabolism , DNA, Mitochondrial/genetics , Electron Transport , Mitochondria/metabolism , Animals , Humans , Male , Oxidative Phosphorylation , Rats , Rats, Wistar , Sequence Deletion , Tissue Distribution
4.
Mutat Res ; 316(2): 69-78, 1994 Aug.
Article in English | MEDLINE | ID: mdl-7521004

ABSTRACT

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.


Subject(s)
Aging/genetics , DNA, Mitochondrial/genetics , Sequence Deletion , Adenosine Triphosphatases/genetics , Animals , Base Sequence , DNA Primers , Electron Transport Complex IV/genetics , Molecular Sequence Data , Polymerase Chain Reaction , Rats , Rats, Wistar , Repetitive Sequences, Nucleic Acid
5.
J Biol Chem ; 268(24): 18259-66, 1993 Aug 25.
Article in English | MEDLINE | ID: mdl-8349702

ABSTRACT

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.


Subject(s)
Hexokinase/metabolism , Mutagenesis, Site-Directed , Protein Structure, Secondary , Amino Acid Sequence , Animals , Base Sequence , Computer Graphics , Glucose/metabolism , Hexokinase/chemistry , Hexokinase/genetics , Kinetics , Liver Neoplasms, Experimental/enzymology , Mammals , Mice , Models, Molecular , Molecular Sequence Data , Molecular Weight , Oligodeoxyribonucleotides , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Restriction Mapping , Saccharomyces cerevisiae/enzymology , X-Ray Diffraction
6.
Kidney Int ; 40(4): 662-7, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1745015

ABSTRACT

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)


Subject(s)
Aging/physiology , Insulin/metabolism , Aging/pathology , Animals , Glucose/pharmacology , Hyperparathyroidism, Secondary/pathology , Hyperparathyroidism, Secondary/physiopathology , In Vitro Techniques , Insulin Secretion , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Islets of Langerhans/pathology , Kidney Failure, Chronic/pathology , Kidney Failure, Chronic/physiopathology , Male , Parathyroid Hormone/blood , Perfusion , Rats , Rats, Inbred Strains
7.
J Bone Miner Res ; 6(7): 697-708, 1991 Jul.
Article in English | MEDLINE | ID: mdl-1659120

ABSTRACT

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.


Subject(s)
Calcium/metabolism , Osteoblasts/metabolism , Phosphatidylinositols/metabolism , Adenosine Diphosphate/pharmacology , Adenosine Triphosphate/pharmacology , Animals , Cytosol/metabolism , Extracellular Space/metabolism , Nucleotides/pharmacology , Osteoblasts/drug effects , Osteosarcoma/metabolism , Rats , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism
8.
J Biol Chem ; 266(9): 5359-62, 1991 Mar 25.
Article in English | MEDLINE | ID: mdl-2005085

ABSTRACT

Recent studies from this and other laboratories have resulted in the cloning and sequencing of hexokinases from a variety of tissues including yeast, human kidney, rat brain, rat liver, and mouse hepatoma. Significantly, studies on the hepatoma enzyme conducted in this laboratory (Arora, K.K., Fanciulli, M., and Pedersen, P.L. (1990) J. Biol. Chem. 265, 6481-6488) resulted also in its overexpression in Escherichia coli in active form. We have now used site-directed mutagenesis for the first time in studies of hexokinase to evaluate the role of amino acid residues predicted to interact with either glucose or ATP. Four amino acid residues (Ser-603, Asp-657, Glu-708, and Glu-742) believed to interact with glucose were mutated to alanine or glycine, whereas a lysine residue (Lys-558) thought to be directly involved in binding ATP was mutated to either methionine or arginine. Of all the mutations in residues believed to interact with glucose, the Asp-657----Ala mutation is the most profound, reducing the hexokinase activity to a level less than 1% of the wild type. The relative Vmax values for Ser-603----Ala, Glu-708----Ala, and Glu-742----Ala enzymes are 6, 10, and 6.5%, respectively, of the wild-type enzyme. Glu-708 and Glu-742 mutations increase the apparent Km for glucose 50- and 14-fold, respectively, while the Ser-603----Ala mutation decreases the apparent Km for glucose 5-fold. At the putative ATP binding site, the relative Vmax for Lys-558----Arg and Lys-558----Met enzymes are 70 and 29%, respectively, of the wild-type enzyme with no changes in the apparent Km for glucose. No changes were observed in the apparent Km for ATP with any mutation. These results support the view that all 4 residues predicted to interact with glucose from earlier x-ray studies may play a role in binding and/or catalysis. The Asp-657 and Ser-603 residues may be involved in both, while Glu-708 and Glu-742 clearly contribute to binding but are not essential for catalysis. In contrast, Lys-558 appears to be essential neither for binding nor catalysis.


Subject(s)
Glucose/metabolism , Hexokinase/metabolism , Amino Acids/metabolism , Animals , Base Sequence , Catalysis , Electrophoresis, Polyacrylamide Gel , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Hexokinase/genetics , Hydrolysis , Liver Neoplasms, Experimental/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligonucleotide Probes , Phosphorylation
9.
Circ Res ; 66(4): 1143-55, 1990 Apr.
Article in English | MEDLINE | ID: mdl-2317891

ABSTRACT

We used left ventricular myocytes from adult rats to investigate the effect of 4 beta-phorbol 12-myristate 13-acetate (PMA) and of sn-1,2-dioctanoylglycerol (DiC-8) on the membrane association of protein kinase C (PKC), cytosolic [Ca2+], (Cai) homeostasis, and the contractile properties of single cardiac cells. Because PKC activity is known to be highly Ca2+ sensitive, the K+ concentration of the bathing medium was raised from 5 to 30 mM in some experiments, a perturbation known to depolarize the cell and increase Cai. In cell suspensions both PMA (3 x 10(-10) and 3 x 10(-7) M) and DiC-8 (10(-5) and 10(-4) M) increased membrane association of PKC. The effect of PMA (10(-7) M) on PKC translocation was enhanced in 30 mM KCl compared with 5 mM KCl. During steady field stimulation at 1 Hz in 1 mM bathing [Ca2+], both PMA (10(-7) M) and DiC-8 (10(-5) M) decreased twitch amplitude to approximately 60% of control in 5 mM KCl, and the negative inotropic effect of either drug was more pronounced in 30 mM KCl than in 5 mM KCl. In single cardiac myocytes loaded with the Ca2+ indicator indo-1 and bathed in 5 mM KCl, we simultaneously measured cell length and Cai. The myofilament responsiveness to Ca2+ was assessed by the relation between contraction amplitude and the peak of the Cai transient. The negative inotropic effect of both PMA and DiC-8 was related to a diminished amplitude of the Cai transient and not to a decreased myofilament responsiveness to Ca2+. In the absence of electrical stimulation, PMA (10(-7) M) and DiC-8 (10(-5) M) decreased the frequency of contractile waves due to spontaneous Ca2+ release from the sarcoplasmic reticulum, and DiC-8 also decreased resting Cai. Thus, activation of PKC, which is thought to occur as part of the response of cardiac muscle to alpha 1-adrenergic stimulation, is associated with a negative inotropic action due to a smaller Cai transient rather than to a decrease in the myofilament responsiveness to Ca2+. These effects on the membrane association of PKC and on contractility are enhanced by cell depolarization achieved by raising [KCl] in the bathing medium.


Subject(s)
Calcium/metabolism , Cytosol/metabolism , Diglycerides/pharmacology , Glycerides/pharmacology , Myocardial Contraction/drug effects , Myocardium/metabolism , Protein Kinase C/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Animals , Biological Transport , Cell Membrane/enzymology , Male , Myocardium/ultrastructure , Rats , Rats, Inbred Strains
10.
Am J Physiol ; 258(3 Pt 2): F545-52, 1990 Mar.
Article in English | MEDLINE | ID: mdl-2156447

ABSTRACT

The effect of parathyroid hormone (PTH) on cytosolic Ca2+ was studied on suspensions of purified rat renal proximal tubules using the fluorescent indicator quin-2. Rat PTH-(1-34) produced a transient 40% increase in apparent cytosolic Ca2+ at 20 s, followed by a rapid return toward the basal level. The half-maximal dose for both the rate of rise and peak apparent Ca2+ was 3 X 10(-8) M for rat PTH-(1-34). Unlike PTH, forskolin and dibutyryl adenosine 3',5'-cyclic monophosphate had no immediate effect. Bovine PTH-(3-34) blocked the effect of PTH in a concentration-dependent manner. Acute reduction of medium Ca2+ to less than 10(-6) M had no effect on either PTH- or angiotensin II (ANG II)-induced transients, but prevented any sustained increases. Washing tubules in nominally Ca2(+)-free medium followed by ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid reduced both transients by 50%. PTH at 2 X 10(-7) caused small (6-9%) increases in accumulation of [3H]inositol phosphates comparable with that produced by norepinephrine at 10(-7) M. At 10(-7) M, norepinephrine produced increases in Ca2+ and inositol phosphates similar to PTH; at 10(-5) M much larger increases in inositol phosphates occurred. Exposure to high levels of either norepinephrine or ANG II before PTH administration prevented any subsequent stimulation by PTH or other agonists. A submaximal dose of norepinephrine only slightly blunted the effect of PTH or ANG II.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Calcium/metabolism , Cytosol/metabolism , Kidney Tubules, Proximal/metabolism , Parathyroid Hormone/physiology , Angiotensin II/pharmacology , Animals , Bucladesine/pharmacology , Colforsin/pharmacology , Dose-Response Relationship, Drug , Drug Interactions , Inositol Phosphates/metabolism , Norepinephrine/pharmacology , Parathyroid Hormone/pharmacology , Peptide Fragments/pharmacology , Rats , Rats, Inbred Strains
11.
Calcif Tissue Int ; 45(4): 251-4, 1989 Oct.
Article in English | MEDLINE | ID: mdl-2553224

ABSTRACT

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.


Subject(s)
Adenosine Triphosphate/pharmacology , Calcium/metabolism , Norepinephrine/pharmacology , Osteoblasts/metabolism , Vasoactive Intestinal Peptide/pharmacology , Animals , Colforsin/pharmacology , Cyclic AMP/metabolism , Cytosol/metabolism , Inositol Phosphates/metabolism , Norepinephrine/antagonists & inhibitors , Osteoblasts/drug effects , Parathyroid Hormone/pharmacology , Prazosin/pharmacology , Propranolol/pharmacology , Rats , Tumor Cells, Cultured
12.
Biochem Biophys Res Commun ; 159(3): 1352-8, 1989 Mar 31.
Article in English | MEDLINE | ID: mdl-2930565

ABSTRACT

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.


Subject(s)
Kidney/enzymology , Parathyroid Hormone/pharmacology , Peptide Fragments/pharmacology , Protein Kinase C/metabolism , Animals , Cell Line , Kinetics , Opossums , Protein Kinases/metabolism , Structure-Activity Relationship , Teriparatide
13.
J Immunol ; 139(5): 1472-8, 1987 Sep 01.
Article in English | MEDLINE | ID: mdl-3114367

ABSTRACT

Interleukin 2 (IL-2) production and recognition are clearly involved in the age-associated proliferative defect of mitogen-stimulated T lymphocytes. The external signal delivered by mitogens is transmitted across the membrane via the release of two messenger molecules, diacylglycerol and inositol 1,4,5-trisphosphate (IP3), involved in the activation of protein kinase C (PK-C) and the elevation of cytosolic free Ca2+. In that Ca2+ mobilization and PK-C activation appear to be crucial events in the production of IL-2 and the expression of IL-2 receptors, a defect in transmembrane signaling would result in decreased synthesis and response to IL-2. We therefore examined PK-C activity and translocation, generation of inositol 1,4,5-trisphosphate, and cytosolic Ca2+ levels as a function of age in murine G0 T lymphocytes before and after exposure to mitogenic doses of concanavalin A (Con A). The basal levels and distribution of PK-C before and after direct activation of the enzyme by 2 or 20 nM phorbol myristate acetate were comparable in both age groups indicating no inherent age-associated functional defect in the enzyme. However, the Con A-induced PK-C translocation was reduced by 50% in cells from 24-mo-old animals. The Con A stimulation of G0 T lymphocytes increased free cytoplasmic Ca2+ concentration ([Ca2+]i) and the production of inositol phosphates to the same level, irrespective of the age of the donor. However, basal levels of both of these second messengers were consistently higher in lymphocytes derived from old mice. As a result, the net increase in inositol phosphates and [Ca2+]i was reduced by approximately the same extent as that observed for the translocation of PK-C. These results clearly point to an age-associated defect in the generation of phosphoinositide-derived second messengers and indicate that an alteration in signal transduction plays a primary role in the age-related impairment of the mitogen-induced, IL-2-mediated proliferative response of T lymphocytes.


Subject(s)
Aging/immunology , Concanavalin A/pharmacology , Lymphocyte Activation/drug effects , T-Lymphocytes/immunology , Animals , Biological Transport , Calcium/metabolism , Female , Inositol 1,4,5-Trisphosphate , Inositol Phosphates/biosynthesis , Interleukin-2/biosynthesis , Mice , Mice, Inbred C57BL/immunology , Mice, Inbred C57BL/physiology , Protein Kinase C/metabolism , Receptors, Immunologic/biosynthesis , Receptors, Interleukin-2 , T-Lymphocytes/drug effects , T-Lymphocytes/physiology , Tetradecanoylphorbol Acetate/pharmacology
14.
Neurobiol Aging ; 7(5): 357-61, 1986.
Article in English | MEDLINE | ID: mdl-2946969

ABSTRACT

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.


Subject(s)
Aging/metabolism , Corpus Striatum/analysis , Receptors, Dopamine/analysis , Adenylyl Cyclases/metabolism , Animals , Corpus Striatum/enzymology , Male , Mice , Mice, Inbred C57BL , Rats , Rats, Inbred Strains , Receptors, Dopamine D1 , Receptors, Dopamine D2
15.
J Biol Chem ; 259(20): 12311-4, 1984 Oct 25.
Article in English | MEDLINE | ID: mdl-6238025

ABSTRACT

An affinity column, prepared by immobilizing phosphatidylserine and cholesterol in polyacrylamide, was utilized in the purification of protein kinase C. Protein kinase activity and phorbol ester binding were monitored by assaying Ca2+ plus phosphatidylserine-dependent phosphorylation of histone H1 and [3H]phorbol dibutyrate binding, respectively. Both activities were present in a cytosolic extract of rabbit renal cortex, eluted together from a DEAE-cellulose column, bound to the affinity column in the presence of Ca2+, and eluted symmetrically upon application of EGTA. Recovery from the affinity column was high (30-50%) and resulted in as much as a 6000-7700-fold purification, depending on the region of the DEAE-cellulose peak that was applied. Following affinity column purification, protein kinase and phorbol ester binding activity eluted symmetrically upon gel filtration, with a molecular weight of approximately 80 kDa. A protein of the same size was present in silver-stained gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity column purified samples from the DEAE-cellulose peak. From 2-4 other, smaller proteins were also present, their number and relative amounts depending on the region of the DEAE-cellulose peak used. These data indicate that Ca2+-dependent/binding to a polyacrylamide-immobilized phospholipid provides a useful technique for purification of protein kinase C as well as other, unidentified proteins exhibiting a Ca2+ plus phospholipid-dependent interaction.


Subject(s)
Caenorhabditis elegans Proteins , Kidney Cortex/metabolism , Protein Kinases/isolation & purification , Receptors, Drug , Receptors, Immunologic/isolation & purification , Animals , Carrier Proteins , Chromatography, Affinity/methods , Chromatography, DEAE-Cellulose/methods , Electrophoresis, Polyacrylamide Gel , Phorbol Esters/metabolism , Phosphatidylserines , Protein Kinase C , Protein Kinases/metabolism , Rabbits , Receptors, Immunologic/metabolism
16.
Miner Electrolyte Metab ; 10(2): 103-12, 1984.
Article in English | MEDLINE | ID: mdl-6321935

ABSTRACT

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)


Subject(s)
Calcium/pharmacology , Cyclic AMP/pharmacology , Kidney Cortex/metabolism , Membrane Proteins/metabolism , Parathyroid Hormone/pharmacology , Animals , Bucladesine/pharmacology , Kidney Cortex/drug effects , Microvilli/metabolism , Octoxynol , Phosphates/metabolism , Phosphorylation , Polyethylene Glycols/pharmacology , Rabbits
17.
Mol Pharmacol ; 21(1): 128-32, 1982 Jan.
Article in English | MEDLINE | ID: mdl-6127617

ABSTRACT

The regulation of phosphatidylinositol turnover by alpha-adrenergic agonists in rat parotid acinar cell aggregates was examined with respect to kinetics and agonist-antagonist interactions. Phosphatidylinositol turnover was followed by the changes in the specific activities of [32P]phosphatidic acid and [32P]phosphatidylinositol. The specific activity of phosphatidic acid increased rapidly (within 1 min) after addition of epinephrine (10(-5) M), reached a maximal level within 12-16 min, and then decreased. Incorporation of 32P into phosphatidylinositol exhibited a lag phase of about 5 min and then increased continuously for an additional 40 min. The absolute amounts of phosphatidic acid and phosphatidylinositol did not change. The concentrations of epinephrine needed to stimulate 32P incorporation into phosphatidic acid and phosphatidylinositol, measured at 15 and 30 min, respectively, were similar; Ka values of 2.05 +/- 0.46 X 10(-6) M for phosphatidic acid and 2.98 +/- 0.30 X 10(-6) M for phosphatidylinositol were found. The effects of agonists on 32P labeling of phosphatidylinositol, in order of potency, were epinephrine greater than or equal to norepinephrine greater than phenylephrine much greater than normetanephrine. When various adrenergic antagonists were evaluated for their ability to inhibit 10(-5) M epinephrine-stimulated 32P incorporation into both phosphatidic acid and phosphatidylinositol, the order of antagonist potency was prazosin greater than or equal to phenoxybenzamine greater than phentolamine greater than or equal to yohimbine greater than much greater than propranolol. These findings indicate that phosphatidylinositol-phosphatidic acid turnover in the rat parotid gland is mediated by the alpha 1-adrenergic receptor system.


Subject(s)
Parotid Gland/metabolism , Phosphatidic Acids/metabolism , Phosphatidylinositols/metabolism , Receptors, Adrenergic, alpha/physiology , Receptors, Adrenergic/physiology , Adrenergic alpha-Agonists/pharmacology , Animals , Cells, Cultured , Epinephrine/pharmacology , Kinetics , Male , Potassium/metabolism , Prazosin/metabolism , Rats , Rats, Inbred Strains
18.
J Biol Chem ; 256(18): 9731-6, 1981 Sep 25.
Article in English | MEDLINE | ID: mdl-6270099

ABSTRACT

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.


Subject(s)
Parotid Gland/metabolism , Phosphoproteins/metabolism , Protein Kinases/metabolism , Receptors, Adrenergic, beta/physiology , Receptors, Adrenergic/physiology , Animals , Bucladesine/pharmacology , Cyclic AMP/metabolism , Enzyme Activation , Isoproterenol/pharmacology , Kinetics , Male , Molecular Weight , Parotid Gland/cytology , Phosphorylation , Rats , Rats, Inbred Strains
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