Role Of Protein Kinase A Research Articles

Vasopressin messenger ribonucleic acid regulation via the protein kinase A pathway.

Activation of vasopressin (VP) gene expression in vivo by osmotic stimuli results in an increase in both messenger RNA (mRNA) content and polyadenylate [poly(A)] tail length. VP gene transcription in vitro is stimulated by protein kinase A (PKA) activation. To examine the role of PKA in the regulation of VP mRNA poly(A) metabolism, constructs of the rat VP gene were permanently transfected into the mouse anterior pituitary cell line, AtT-20. Treatment with forskolin of cells expressing the intact VP gene resulted in increased VP gene transcription, an increase in the content of VP mRNA, and a shift toward VP mRNA species with longer poly(A) tails accompanied by the loss of VP mRNA species with shorter poly(A) tails. We uncoupled the PKA-stimulated appearance of long-tailed species from the disappearance of short-tailed species, suggesting that the size shift was caused by a coincident, but uncoupled net increase in VP mRNA species with elongated poly(A) tails and net loss of mRNA species with short poly(A) tails. These data indicate that activation of the PKA second-messenger pathway both enhances transcription of the VP gene and causes an increase in the average length of VP mRNA poly(A) tails. This latter effect, by shifting upwards the average poly(A) tail size, could result in increased translational efficiency or stability of VP mRNA, thereby providing an additional mechanism by which PKA may enhance gene expression.

Altered vascular norepinephrine responses in portal hypertensive intestine: role of PKA and guanylate cyclase.

The purpose of the present study was to determine whether selective blockade of adenosine 3',5'-cyclic monophosphate (cAMP)- or guanosine 3',5'-cyclic monophosphate (cGMP)-mediated events modulated norepinephrine responses in intestinal microvessels of normal and portal hypertensive rats. Vascular norepinephrine responses were evaluated before and after inhibition of cAMP-dependent protein kinase [protein kinase A(PKA)] with Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) or guanylate cyclase with LY-83583. Male Sprague-Dawley rats were divided into two groups: those with portal hypertension by portal vein stenosis and normal controls. The small intestine was prepared for microcirculatory studies. Arteriolar diameter and erythrocyte velocity were monitored, and microvascular flow was calculated from velocity and diameter data. The preparation was challenged with incremental concentrations of norepinephrine before and after addition of Rp-cAMPS (50 microM) or LY-83583 (30 microM). Arteriolar diameter and blood flow were significantly elevated in portal hypertensive rats; norepinephrine responses were significantly depressed. LY-83583 did not alter arteriolar diameter, blood flow, or norepinephrine responsiveness in normal or portal hypertensive rats. Rp-cAMPS did not affect arteriolar diameter, blood flow, or norepinephrine responsiveness in normal rats. However, in portal hypertensive rats, Rp-cAMPS reduced blood flow by approximately 20% (P < 0.05) and completely restored vascular norepinephrine responses to normal. The results indicate that cAMP- but not cGMP-dependent events are primarily responsible for the loss of microvascular norepinephrine responsiveness in portal hypertensive intestine.

The activation of protein kinase A pathway selectively inhibits anterograde axonal transport of vesicles but not mitochondria transport or retrograde transport in vivo

To shed light on how axonal transport is regulated, we examined the possible roles of protein kinase A (PKA) in vivo suggested by our previous work (Sato-Yoshitake et al., 1992). Pharmacological probes or the purified catalytic subunit of PKA were applied to the permeabilized-reactivated model of crayfish walking leg giant axon, and the effect was monitored by the quantitative video-enhanced light microscopy and the quantitative electron microscopy. Dibutyryl cyclic AMP caused concentration-dependent transient reduction in the number of anterogradely transported small vesicles, while the retrogradely transported organelles and anterogradely transported mitochondria showed no decrease. This transient selective inhibition of anterograde vesicle transport was reversed by the application of a specific inhibitor of PKA (KT5720) in a concentration-dependent manner, and was reproduced by the application of the purified catalytic subunit of PKA and augmented by the application of adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). Corresponding biochemical study showed that several axoplasmic proteins including kinesin were specifically phosphorylated by the activation of the PKA pathway. These findings suggest the possible roles of PKA in the regulation of the direction of the axonal transport in vivo. The finding that only vesicle transport but not mitochondria transport was inhibited also suggests that the transport of vesicles and that of mitochondria are differently regulated and might be supported by different motors.

Regulation of protein kinase A subunits during germination of Mucor rouxii sporangiospores.

Levels of protein kinase A (PKA) subunits and of cAMP have been measured during aerobic germination of the sporangiospores of the dimorphic fungus Mucor rouxii; further, the holoenzyme and its catalytic (C) and regulatory (R) subunits have been visualized through sucrose gradient centrifugation. Sporangiospores contain around 0.06 microM of a dimeric holoenzyme species of 5.5 S and a sixfold excess of a free R subunit of 2.7 S. Both these species are proposed to be derived by proteolysis from the native forms. Enzymic activity at this stage is highly inhibited, as demonstrated with permeabilized cells. Immediately upon germination, and after a transient increase in cAMP concentration from 10 microM to 90 microM, C-subunit levels fall to 30%. After the onset of germination, the specific activity and concentration of both the 5.5 S holoenzyme species and the 2.7 S species of free R subunit decrease in parallel to the increase in total protein and volume. Net synthesis of C and R subunits to form a native holoenzyme species of 8.8 S is apparent 4 h onwards after germination. A significant increase in cellular concentration is observed at 6 h. At 7 h of growth, when germ-tube emission is complete, the holoenzyme concentration is around 0.23 microM; there is almost no free R subunit and the intracellular concentration of cAMP is around 3 microM. A role for PKA during germination and morphogenesis is discussed.

Role of protein kinase-A in homologous down-regulation of parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid in human osteoblast-like SaOS-2 cells.

Homologous down-regulation of PTH/PTH-related peptide (PTHrP) receptor expression occurs in several PTH-responsive osteoblastic cell lines, but the mechanisms responsible are not well understood. We have used wild-type SaOS-2 human osteoblastic cells, in which homologous PTH/PTHrP receptor down-regulation occurs within 4 h, and a mutant cAMP-resistant subclone (Ca4A strain), to investigate the mechanisms by which PTH/PTHrP receptor mRNA is regulated. SaOS-2 cells expressed a single 2.2- to 2.5-kilobase transcript of PTH/PTHrP receptor mRNA, as assessed by Northern blot analysis of total RNA with a cDNA probe encoding the human PTH/PTHrP receptor. Homologous down-regulation of this PTH/PTHrP receptor mRNA first became significant when SaOS-2 cells had been treated with human (h) PTH-(1-34) (10(-7) M) for 8-12 h. By 24 h, steady state levels of PTH/PTHrP receptor mRNA were reduced by about 50%. This effect was mimicked by both (Bu)2cAMP (DBcAMP; 0.5 mM) and forskolin (Fsk; 10(-5) M). In contrast, down-regulation of PTH/PTHrP receptor mRNA by hPTH-(1-34), DBcAMP or Fsk was almost completely blocked in cAMP-resistant Ca4A cells. Short term (4-6 h) treatment with hPTH-(1-34), DBcAMP, or Fsk did not reduce steady state levels of PTH/PTHrP receptor mRNA in either SaOS-2 or Ca4A cells, although down-regulation was induced by 4-6 h of treatment with active phorbol esters such as 12-O-tetradecanoyl phorbol-13-acetate (200 nM) or phorbol-12,13-didecanoate (200 nM). Neither thapsigargin (1 microM) nor ionomycin (200 nM), both of which stimulate calcium transients in these cells, altered PTH/PTHrP receptor mRNA expression. Treatment with hPTH-(39-84) and hPTH-(53-84), which do not activate either cAMP-dependent protein kinase or protein kinase-C, but do stimulate 45Ca2+ uptake in these cells, did not alter PTH/PTHrP receptor mRNA expression. In the presence of actinomycin-D (1 microgram/ml), down-regulation of PTH/PTHrP receptor mRNA by hPTH-(1-34) was not observed. Cycloheximide (10 micrograms/ml) did not block down-regulation of PTH/PTHrP receptor mRNA induced by hPTH-(1-34). We conclude that homologous down-regulation of PTH/PTHrP receptor mRNA in SaOS-2 cells occurs later than the decline in functional surface receptors via a mechanism that does not involve enhanced mRNA degradation or new protein synthesis, but is dependent upon cAMP/cAMP-dependent protein kinase.

Role of protein kinase-A in homologous down-regulation of parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid in human osteoblast-like SaOS-2 cells

Homologous down-regulation of PTH/PTH-related peptide (PTHrP) receptor expression occurs in several PTH-responsive osteoblastic cell lines, but the mechanisms responsible are not well understood. We have used wild-type SaOS-2 human osteoblastic cells, in which homologous PTH/PTHrP receptor down-regulation occurs within 4 h, and a mutant cAMP-resistant subclone (Ca4A strain), to investigate the mechanisms by which PTH/PTHrP receptor mRNA is regulated. SaOS-2 cells expressed a single 2.2- to 2.5-kilobase transcript of PTH/PTHrP receptor mRNA, as assessed by Northern blot analysis of total RNA with a cDNA probe encoding the human PTH/PTHrP receptor. Homologous down-regulation of this PTH/PTHrP receptor mRNA first became significant when SaOS-2 cells had been treated with human (h) PTH-(1-34) (10(-7) M) for 8-12 h. By 24 h, steady state levels of PTH/PTHrP receptor mRNA were reduced by about 50%. This effect was mimicked by both (Bu)2cAMP (DBcAMP; 0.5 mM) and forskolin (Fsk; 10(-5) M). In contrast, down-regulation of PTH/PTHrP receptor mRNA by hPTH-(1-34), DBcAMP or Fsk was almost completely blocked in cAMP-resistant Ca4A cells. Short term (4-6 h) treatment with hPTH-(1-34), DBcAMP, or Fsk did not reduce steady state levels of PTH/PTHrP receptor mRNA in either SaOS-2 or Ca4A cells, although down-regulation was induced by 4-6 h of treatment with active phorbol esters such as 12-O-tetradecanoyl phorbol-13-acetate (200 nM) or phorbol-12,13-didecanoate (200 nM). Neither thapsigargin (1 microM) nor ionomycin (200 nM), both of which stimulate calcium transients in these cells, altered PTH/PTHrP receptor mRNA expression. Treatment with hPTH-(39-84) and hPTH-(53-84), which do not activate either cAMP-dependent protein kinase or protein kinase-C, but do stimulate 45Ca2+ uptake in these cells, did not alter PTH/PTHrP receptor mRNA expression. In the presence of actinomycin-D (1 microgram/ml), down-regulation of PTH/PTHrP receptor mRNA by hPTH-(1-34) was not observed. Cycloheximide (10 micrograms/ml) did not block down-regulation of PTH/PTHrP receptor mRNA induced by hPTH-(1-34). We conclude that homologous down-regulation of PTH/PTHrP receptor mRNA in SaOS-2 cells occurs later than the decline in functional surface receptors via a mechanism that does not involve enhanced mRNA degradation or new protein synthesis, but is dependent upon cAMP/cAMP-dependent protein kinase.

Granulocyte-macrophage colony-stimulating factor stimulates the metastatic properties of Lewis lung carcinoma cells through a protein kinase A signal-transduction pathway.

Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) by metastatic Lewis lung carcinoma cells (LLC-LN7) was previously shown to contribute to the maintenance of phenotypic characteristics associated with an increased capacity to metastasize. In the present study, pre-incubation of LLC-LN7 cells with neutralizing anti-GM-CSF antibodies diminished the capacity of the tumor cells to form experimental metastases after i.v. inoculation, while pre-incubation with recombinant GM-CSF (rGM-CSF) increased formation of metastases. In the presence of rGM-CSF, the LLC-LN7 cells exhibited an increased capacity to migrate, invade through a reconstituted basement membrane, and adhere to lung tissue. Studies to identify the signal transduction pathway through which GM-CSF enhanced the in vitro metastatic properties of the LLC-LN7 tumor cells implicated protein kinase A (PKA). Signaling through PKA was suggested by the demonstration that the stimulation of tumor-cell motility by GM-CSF was blocked in the presence of the adenylate cyclase inhibitor nicotinic acid, or the PKA inhibitors A3 or KT5720. In addition, the role of PKA as a signaling mechanism for GM-CSF was assessed by using REV-LN7 cells, which are LLC-LN7 cells that have been stably transfected with an expression vector encoding a mutant PKA RI alpha subunit and which, in turn, express a cAMP-resistant PKA. Adherence and invasion by the PKA-defective REV-LN7 cells were not stimulated by rGM-CSF, contrasting with the stimulation observed for wild-type LLC-LN7 cells. These data suggest that rGM-CSF can further enhance the in vitro metastatic characteristics of LLC-LN7 tumor cells and that this is dependent on signal transduction through PKA.

Mechanisms of desensitization to parathyroid hormone in human osteoblast-like SaOS-2 cells.

Signal transduction by the PTH receptor is now known to involve generation of multiple second messengers. Desensitization of the adenylate cyclase response to PTH is a common feature of bone- and kidney-derived target cells; however, no single mechanism appears to explain desensitization in the different cell types studied. To examine the role of protein kinase-A (PKA) in homologous desensitization to PTH, we employed human SaOS-2 osteoblast-like cells and a mutant subclone (Ca 4A), which expresses an inducible cAMP-resistant form of PKA. Pretreatment of SaOS-2 cells with PTH for 4 h reduced by 60-80% the cAMP response to subsequent rechallenge with the hormone. This homologous desensitization was significantly, but not completely, inhibited in Ca 4A cells. Desensitization was not mimicked by pretreatment of the cells with forskolin. PTH binding to its receptor was reduced 50% in both SaOS-2 and Ca 4A cells after 4-h incubation with PTH (homologous down-regulation), whereas forskolin did not cause receptor down-regulation. Pretreatment with the ionophore ionomycin for 4-24 h did not mimic desensitization to PTH. Both desensitization to PTH and receptor down-regulation were induced, however, by pretreatment with a phorbol ester (12-O-tetradecanoyl phorbol-13-acetate), and these effects were blocked completely by staurosporine. PTH-induced desensitization was not blocked by staurosporine, and receptor down-regulation was enhanced by the drug. Pertussis toxin did not prevent desensitization induced by either PTH or 12-O-tetradecanoyl phorbol-13-acetate. We conclude that homologous desensitization to PTH in SaOS-2 cells involves both cAMP-dependent and -independent mechanisms. Homologous PTH receptor down-regulation apparently is mediated by mechanisms independent of PKA activation. Neither pathway of homologous desensitization to PTH involves the action of pertussis toxin-sensitive G-proteins.

Roles of PKA and PKC in facilitation of evoked and spontaneous transmitter release at depressed and nondepressed synapses in aplysia sensory neurons

Two second messenger pathways, one that uses the cAMP-dependent protein kinase A (PKA), the other that uses protein kinase C (PKC), have been found to contribute to the short-term presynaptic facilitation of the connections between the sensory neurons in Aplysia and their target cells, the interneurons and motor neurons of the gill-withdrawal reflex. To study their relative contributions as a function of the previous history of the neuron's activity, we have examined the effects of inhibiting PKA (using Rp-cAMPS) and PKC (using H7) on the shortterm facilitation of spontaneous release as well as of the evoked release induced by serotonin at nondepressed, partially depressed, and highly depressed synapses. Our results suggest that whereas activation of PKA is sufficient to trigger the facilitation of nondepressed synapses, activation of both PKA and PKC is required to facilitate depressed synapses, with the contribution of PKC becoming progressively more important as synaptic transmission becomes more depressed.