Charles R. Filburn

Phorbol ester and dioctanoylglycerol stimulate membrane association of protein kinase C and have a negative inotropic effect mediated by changes in cytosolic Ca2+ in adult rat cardiac myocytes.

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.

Effect of aging on striatal dopamine receptor subtypes in Wistar rats.

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.

Detection and quantitation by competitive PCR of an age-associated increase in a 4.8-kb deletion in rat mitochondrial DNA.

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.

Neurotransmitter regulation of cytosolic calcium in osteoblast-like bone cells

SummaryThe 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≫PTH>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.

Parathyroid hormone 1–34, but not 3–34 or 7–34, transiently translocates protein kinase C in cultured renal (OK) cells

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.

Enhanced Contractile Response and Protein Kinase Activation to Threshold Levels of β-Adrenergic Stimulation in Hyperthyroid Rat Heart

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.

Mitochondrial electron transport chain activities and DNA deletions in regions of the rat brain.

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.

Adenylate and guanylate cyclases of cecropia silkmoth fat body

Abstract Adenylate and guanylate cyclases were assayed in silkmoth fat body homogenates by measuring the conversion of [α- 32 P]nucleoside triphosphates to cyclic [ 32 P]nucleotides. Adenylate cyclase was dependent on dithiothreitol, required either Mg 2+ or Mn 2+ for activity, was activated by NaF, and inhibited by triton X-100. Guanylate cyclase was not dependent on dithiothreitol, was strictly dependent upon Mn 2+ , unaffected by NaF, and activated by triton X-100. Both cyclases had pH optima near 8.0 and were located chiefly in the particulate fraction of homogenates. Activities of both cyclases were maintained or elevated during the larval-pupal transformation and, in contrast to cyclic nucleotide phosphodiesterases, showed little decline in the early diapausing pupa.

Cyclic adenosine monophosphate-dependent and -independent protein kinase activity of renal brush border membranes

Abstract Kinase(s) in brush border membranes, isolated from rabbit renal proximal tubules, phosphorylated proteins intrinsic to the membrane and exogenous proteins. cAMP stimulated phosphorylation of histone; phosphorylation of protamine was cAMP independent. cAMP-dependent increases in phosphorylation of endogenous membrane protein were small, but highly reproducible. Most of the 32P incorporated into membranes represented phosphorylation of serine residues, with phosphorylthreonine comprising a minor component. cAMP did not alter the electrophoretic pattern of 32P-labeled membrane polypeptides. The small cAMP-dependent phosphorylation of brush border membrane proteins was not due to membrane phosphodiesterase or adenylate cyclase activities. Considerable cAMP was found “endogenously” bound to the membranes as prepared. However, this did not result in preactivation of the kinase since activity was not inhibited by a heat-stable protein inhibitor of cAMP-dependent protein kinases. With intrinsic membrane protein as phosphate acceptor, the relationship between rate of phosphorylation and ATP concentration appeared to follow Michaelis-Menton kinetics. With histone the relationship was complex. cAMP did not affect the apparent Km for histone. One-half maximal stimulation of the rate of histone phosphorylation was obtained with 7 × 10−8 m cAMP. The Ka values for dibutyryl cAMP, cIMP, and cGMP were one to two orders of magnitude greater. Treatment of brush border membranes with detergent greatly increased the dependency of histone phosphorylation on cAMP. Phosphorylations of intrinsic membrane protein and histone were nonlinear with time, due in part to the lability of the protein kinase, the hydrolysis of ATP, and minimally to the presence of phosphoprotein phosphatase in the border membrane. The membrane phosphoprotein phosphatase was unaffected by cyclic nucleotides. Protein kinase activity was also found in cytosolic and crude particulate fractions of the renal cortex. Activity was enriched in the brush border membrane relative to that in the crude membrane preparation. The kinase activities in the different loci were distinct both in relative activities toward different substrates and in responsiveness to cAMP.

Cyclic nucleotide phosphodiesterases of rabbit renal cortex: Characterization of brush border membrane activities

Abstract Cyclic nucleotide phosphodiesterase activity in brush border membranes, isolated from proximal tubule cells of the rabbit renal cortex, was investigated. Brush border cAMP phosphodiesterase activity was tightly bound to the membrane and was distinguished from the soluble phosphodiesterase activity of the renal cortex cytosol. Multiple forms of the brush border membrane cAMP phosphodiesterase activity, dependent on the concentration of substrate, were found. When assayed with 1 μ m or 1 m m cAMP, activities differed in pH optimum, effects of various divalent cations, inhibition by metal ion chelators and reactivation by metals, thermolability, sensitivity to inhibitors and specificity. Renal brush border membranes also possessed cGMP phosphodiesterase activity. cAMP was a relatively poor inhibitor of the hydrolysis of 1 μ m cGMP and the hydrolysis of 1 μ m cAMP was virtually insensitive to cGMP. These findings suggest that the low substrate concentration-dependent cAMP phosphodiesterase was distinct from the low substrate concentration-dependent cGMP phosphodiesterase. Heat-stable effectors of phosphodiesterase activity were found in the renal cortex. One effector activated soluble cAMP phosphodiesterase. Activation was decreased by EGTA, enhanced by Ca2+ and diminished by preincubating the effector with proteolytic enzymes. The other heat-stable effector inhibited brush border membrane phosphodiesterase activity. Inhibition was unaffected by metal ions, unaffected by preincubating the effector with proteolytic enzymes, but diminished by preincubation with phospholipase C and neuraminidase. It is suggested that changes in the activity of the enzyme (or enzymes), which in turn controls, in part, the effective concentration of cAMP at its site (or sites) of action in the renal cell, may be significant in regulating hormonal-dependent transport in the proximal tubule.