Stimulation Of Protein Kinase A Activity Research Articles

PKA subunit balance plays a key role in lipolysis

Protein kinase A (PKA) is a cyclic AMP (cAMP)‐dependent protein kinase composed of catalytic and regulatory subunits and involved in various physiological phenomena, including lipid metabolism. In this study, we have investigated that the stoichiometric balance between catalytic and regulatory subunits is crucial to maintain basal PKA activity and lipid homeostasis. In Caenorhabditis elegans, suppression of PKA via RNAi resulted in severe phenotypes, including shortened lifespan, decreased egg laying, reduced locomotion, and altered lipid distribution. Similarly, in mammalian adipocytes, suppression of PKA regulatory subunits via siRNAs potently stimulated PKA activity, leading to potentiated lipolysis without increasing cAMP levels. Nevertheless, insulin exerted anti‐lipolytic effects and restored lipid droplet integrity by antagonizing PKA action. Together, these data implicate the importance of subunit stoichiometry as another regulatory mechanism of PKA activity and lipid metabolism.Support or Funding InformationThis work was supported by grants from the National Creative Research Initiative Program (2011‐0018312) funded by the Ministry of Education, Science and Technology. Y.J., J.S.H., J.K. were supported by BK21 program.

Adrenomedullin and angiotensin II signaling pathways involved in the effects on cerebellar antioxidant enzymes activity.

Human adrenomedullin (AM) is a 52-amino acid peptide involved in cardiovascular control. AM has two specific receptors formed by the calcitonin-receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMP) 2 or 3, known as AM1 and AM2 receptors, respectively. In addition, AM has appreciable affinity for the calcitonin gene-1 related peptide receptor (CGRP1), composed of CRLR/RAMP1. In brain, AM and their receptors are expressed in several localized areas, including the cerebellum. AM has been reported as an antioxidant. Little is known about the role of AM in the regulation of cerebellar reactive oxygen species (ROS) metabolism. We assessed the effect of AM on three antioxidant enzymes activity: catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) and on thiobarbituric acid reactive substances (TBARS) production in rat cerebellar vermis, as well the receptor subtypes involved in AM actions. Additionally, we evaluated the role of angiotensin II (ANG II), protein kinase A (PKA) activity, and protein kinase C/nicotinamide adenine dinucleotide phosphate oxidase (PKC/NAD(P)H) (oxidase) pathway. Sprague-Dawley rats were sacrificed by decapitation and cerebellar vermis was microdissected under stereomicroscopic control. CAT, GPx, SOD activity and TBARS production was determined spectrophotometrically. Our findings demonstrated that in cerebellar vermis, AM decreased and ANG II increased CAT, GPx and SOD activity and TBARS production. Likewise, AM antagonized ANG II-induced increase antioxidant enzyme activity. AM(22-50) and CGRP(8-37) blunted AM-induced decrease of antioxidant enzymes activity and TBARS production indicating that these actions are mediated through AM and CGRP1 receptors. Further, PKA inhibitor (PKAi) blunted AM action and apocynin and chelerythrine reverted ANG II action, suggesting that AM antioxidant action is mediated through stimulation of PKA activity, while ANG II-induced stimulation through PKC/NAD(P)H oxidase pathway. Our results support the role of AM in the regulation of cerebellar antioxidant enzymes activity and suggest a physiological role for AM in cerebellum.

Protein Kinase A Subunit Balance Regulates Lipid Metabolism in Caenorhabditis elegans and Mammalian Adipocytes

Protein kinase A (PKA) is a cyclic AMP (cAMP)-dependent protein kinase composed of catalytic and regulatory subunits and involved in various physiological phenomena, including lipid metabolism. Here we demonstrated that the stoichiometric balance between catalytic and regulatory subunits is crucial for maintaining basal PKA activity and lipid homeostasis. To uncover the potential roles of each PKA subunit, Caenorhabditis elegans was used to investigate the effects of PKA subunit deficiency. In worms, suppression of PKA via RNAi resulted in severe phenotypes, including shortened life span, decreased egg laying, reduced locomotion, and altered lipid distribution. Similarly, in mammalian adipocytes, suppression of PKA regulatory subunits RIα and RIIβ via siRNAs potently stimulated PKA activity, leading to potentiated lipolysis without increasing cAMP levels. Nevertheless, insulin exerted anti-lipolytic effects and restored lipid droplet integrity by antagonizing PKA action. Together, these data implicate the importance of subunit stoichiometry as another regulatory mechanism of PKA activity and lipid metabolism.

Caffeine induces beneficial changes in PKA signaling and JNK and ERK activities in the striatum and cortex of Alzheimer's transgenic mice

Caffeine intake has been associated with a lower incidence of Alzheimer's disease (AD) in humans. In AD mouse models, caffeine significantly decreases senile plaques and amyloid beta (Aβ) levels while also protecting against or reversing cognitive impairment. To understand the mechanism(s) underlying the protective effects of caffeine against AD pathology, we investigated the effects of a two-week treatment with caffeine (3 mg/day) in transgenic (APPswe) mice and non-transgenic (NT) mice on signaling factors involved in neuronal plasticity and survival. We evaluated cAMP-dependent protein kinase A (PKA), phospho-cyclic AMP response-element binding protein (phospho-CREB), and the pro-apoptotic protein kinases extracellular signal-regulated kinase 1/2 (phospho-ERK) and phospho-c-Jun N-terminal kinase 1 (phospho-JNK) in the striatum and frontal cortex of caffeine-treated mice. In the striatum, APPswe control mice exhibited a significant decrease in phospho-CREB, as well as significant increases in phospho-JNK and phospho-ERK in comparison to NT mice. Caffeine treatment stimulated PKA activity, increased phospho-CREB levels, and decreased phospho-JNK and phospho-ERK expression in the striatum of APPswe mice, all of which are thought to be beneficial changes for brain function. Even caffeine-treated NT mice exhibited some of these changes in striatum. In the frontal cortex, caffeine did not significantly increase phospho-CREB and PKA activity, but significantly reduced phospho-JNK and phospho-ERK expression in both APPswe and NT mice. These results suggest that caffeine shifts the balance between neurodegeneration and neuronal survival toward the stimulation of pro-survival cascades and inhibition of pro-apoptotic pathways in the striatum and/or cortex, which may contribute to its beneficial effects against AD.

Deficit of complex I activity in human skin fibroblasts with chromosome 21 trisomy and overproduction of reactive oxygen species by mitochondria: involvement of the cAMP/PKA signalling pathway

DS (Down's syndrome) is the most common human aneuploidy associated with mental retardation and early neurodegeneration. Mitochondrial dysfunction has emerged as a crucial factor in the pathogenesis of numerous neurological disorders including DS, but the cause of mitochondrial damage remains elusive. In the present study, we identified new molecular events involved in mitochondrial dysfunction which could play a role in DS pathogenesis. We analysed mitochondrial respiratory chain function in DS-HSFs (Down's syndrome human foetal skin fibroblasts; human foetal skin fibroblasts with chromosome 21 trisomy) and found a selective deficit in the catalytic efficiency of mitochondrial complex I. The complex I deficit was associated with a decrease in cAMP-dependent phosphorylation of the 18 kDa subunit of the complex, due to a decrease in PKA (protein kinase A) activity related to reduced basal levels of cAMP. Consistently, exposure of DS-HSFs to db-cAMP (dibutyryl-cAMP), a membrane-permeable cAMP analogue, stimulated PKA activity and consequently rescued the deficit of both the cAMP-dependent phosphorylation and the catalytic activity of complex I; conversely H89, a specific PKA inhibitor, suppressed these cAMP-dependent activations. Furthermore, in the present paper we report a 3-fold increase in cellular levels of ROS (reactive oxygen species), in particular superoxide anion, mainly produced by DS-HSF mitochondria. ROS accumulation was prevented by db-cAMP-dependent activation of complex I, suggesting its involvement in ROS production. Taken together, the results of the present study suggest that the drastic decrease in basal cAMP levels observed in DS-HSFs participates in the complex I deficit and overproduction of ROS by DS-HSF mitochondria.

Multiple Ca2+-Dependent Mechanisms Regulate L-Type Ca2+Current in Retinal Amacrine Cells

Understanding the regulation of L-type voltage-gated Ca(2+) current is an important component of elucidating the signaling capabilities of retinal amacrine cells. Here we ask how the cytosolic Ca(2+) environment and the balance of Ca(2+)-dependent effectors shape native L-type Ca(2+) channel function in these cells. To achieve this, whole cell voltage clamp recordings were made from cultured amacrine cells under conditions that address the contribution of mitochondrial Ca(2+) uptake (MCU), Ca(2+)/calmodulin (CaM)-dependent channel inactivation (CDI), protein kinase A (PKA), and Ca(2+)-induced Ca(2+) release (CICR). Under control conditions, repeated activation of the L-type channels produces a progressive enhancement of the current. Inhibition of MCU causes a reduction in the Ca(2+) current amplitude that is dependent on Ca(2+) influx as well as cytosolic Ca(2+) buffering, consistent with CDI. Including the Ca(2+) buffer bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) internally can shift the balance between enhancement and inhibition such that inhibition of MCU can enhance the current. Inhibition of PKA can remove the enhancing effect of BAPTA suggesting that cyclic AMP-dependent phosphorylation is involved. Inhibition of CaM suppresses CDI but spares the enhancement, consistent with the substantially higher sensitivity of the Ca(2+)-sensitive adenylate cyclase 1 (AC1) to Ca(2+)/CaM. Inhibition of the ryanodine receptor reduces the current amplitude, suggesting that CICR might normally amplify the activation of AC1 and stimulation of PKA activity. These experiments reveal that the amplitude of L-type Ca(2+) currents in retinal amacrine cells are both positively and negatively regulated by Ca(2+)-dependent mechanisms.

8Cl-cAMP modifies the balance between PKAR1 and PKAR2 and modulates the cell cycle, growth and apoptosis in human adrenocortical H295R cells

Various types of protein kinase A (PKA) alterations have been observed in adrenocortical tumours and Carney complex (CNC). PKA is a heterotetramer of two regulatory and two catalytic subunits. The R1A and R2B proteins are the most abundant regulatory subunits in endocrine tissues. A decrease in R2B protein levels has been observed in adrenal adenoma, whereas tumours from patients with CNC display a decrease in R1A protein levels. Dysregulation of the balance between R1A and R2B may thus be involved in adrenal tumourigenesis. We investigated the impact of the differences in the balance of PKA subunits on cell growth using specific cAMP analogues. We assessed the effects of 8-chloroadenosine-cAMP (8Cl-cAMP), a site-selective activator of PKA R2B, in H295R adrenocortical cells. 8Cl-cAMP stimulated PKA activity, decreased R1A levels and increased R2B levels. It had no cytotoxic effects, initially stimulating DNA synthesis and then inducing apoptosis by disrupting G(2)/M progression. We observed an initial accumulation of cells in the S phase, translocation of cyclin A to the nucleus, CDK2 activation, sustained DNA synthesis and proliferating cell nuclear antigen accumulation. Cell cycle arrest in the G(2) phase was parallel with the accumulation of cyclin B and the inactivation of CDC2 kinase. The 8CPT-cAMP, which activates the R2B subunit, had similar effects. R2B silencing reduced the apoptosis induced by tumour necrosis factor alpha and transforming growth factor beta. Thus, R2B is a key regulator of proliferation/differentiation in H295R cell line along with the complex balance between the PKA subunits. Activation of PKA R2B and dysregulation of the R1A/R2B balance regulate cell cycle progression and apoptosis in adrenocortical cells by modulating cyclin production and cyclin-dependent kinase activities.

Estradiol-mediated ERK phosphorylation and apoptosis in vascular smooth muscle cells requires GPR 30

Recent studies suggest that the rapid and nongenomic effects of estradiol may be mediated through the G protein-coupled receptor dubbed GPR30 receptor. The present study examines the role of GPR30 versus a classical estrogen receptor (ERalpha) in mediating the growth regulatory effects of estradiol. GPR30 is readily detectable in freshly isolated vascular tissue but barely detectable in cultured vascular smooth muscle cells (VSMC). In freshly isolated aortic tissue, estradiol stimulated extracellular signal-regulated kinases (ERK) phosphorylation. In contrast, in cultured VSMC, where GPR30 expression is significantly reduced, estradiol inhibits ERK phosphorylation. Transfer of the genes encoding GPR30 led to estradiol stimulation of ERK phosphorylation, which is opposite the effects of estradiol in the primary culture of VSMCs. Transduction of the mineralocorticoid receptor (MR) had no effect on estradiol effects on ERK. Estradiol-mediated stimulation of ERK subsequent to heterologous GPR30 expression was pertussis toxin sensitive and phosphoinositide 3-kinase (PI3 kinase) dependent; under these conditions, estradiol also inhibited protein kinase A (PKA). In contrast, in the absence of GPR30 expression in cultured VSMC, estradiol stimulated PKA activity and inhibited ERK phosphorylation. To determine the functional effect of GPR30 (vs. estrogen receptor expression), we assessed estradiol-mediated apoptosis. In the absence of GPR30 expression, estradiol inhibited apoptosis. This effect was enhanced with ERalpha expression. In contrast, with GPR30 expression, estradiol stimulated apoptosis in an ERK-dependent manner. Thus the effect of estradiol on vascular smooth muscle cell apoptosis is likely dependent on the balance between ER-mediated PKA activation and GPR30-mediated PKA inhibition and PI3 kinase activation. Taken together, we postulate that modulation of GPR30 expression or activity may be an important determinant of the effects of estradiol in the vasculature.

Protein Kinase A Regulatory Subunits in Human Adipose Tissue

OBJECTIVE—In human adipocytes, the cAMP-dependent pathway mediates signals originating from β-adrenergic activation, thus playing a key role in the regulation of important metabolic processes, i.e., lipolysis and thermogenesis. Cyclic AMP effects are mainly mediated by protein kinase A (PKA), whose R2B regulatory isoform is the most expressed in mouse adipose tissue, where it protects against diet-induced obesity and fatty liver development. The aim of the study was to investigate possible differences in R2B expression, PKA activity, and lipolysis in adipose tissues from obese and nonobese subjects.RESEARCH DESIGN AND METHODS—The expression of the different PKA regulatory subunits was evaluated by immunohistochemistry, Western blot, and real-time PCR in subcutaneous and visceral adipose tissue samples from 20 nonobese and 67 obese patients. PKA activity and glycerol release were evaluated in total protein extract and adipocytes isolated from fresh tissue samples, respectively.RESULTS—Expression techniques showed that R2B was the most abundant regulatory protein, both at mRNA and protein level. Interestingly, R2B mRNA levels were significantly lower in both subcutaneous and visceral adipose tissues from obese than nonobese patients and negatively correlated with BMI, waist circumference, insulin levels, and homeostasis model assessment of insulin resistance. Moreover, both basal and stimulated PKA activity and glycerol release were significantly lower in visceral adipose tissue from obese patients then nonobese subjects.CONCLUSIONS—Our results first indicate that, in human adipose tissue, there are important BMI-related differences in R2B expression and PKA activation, which might be included among the multiple determinants involved in the different lipolytic response to β-adrenergic activation in obesity.

Apolipoprotein E Interrupts Interleukin-1β Signaling in Vascular Smooth Muscle Cells

Objectives— Apolipoprotein E (apoE) exerts antiatherogenic effects but precise mechanisms remain unclear. We here investigated the effect of apoE on intracellular signaling by interleukin-1β (IL-1β), a proinflammatory cytokine present in atherosclerotic lesions. Methods and Results— IL-1β-induced expression and activation of inducible nitric oxide synthase and cyclooxygenase-2 were inhibited by apoE in vascular smooth muscle cells (VSMCs). These inhibitory effects were linked to the suppression of both NF-κB and activating protein-1 (AP-1) transactivation, suggesting that the interruption of IL-1β signaling occurs upstream of transcription factors. Studies in VSMCs overexpressing IL-1β signaling intermediates revealed that NF-κB transactivation was inhibited by apoE in MyD88- and IRAK1- but not in TRAF6-transfected cells. Furthermore, apoE prevented IRAK1 phosphorylation and IRAK1-TRAF6 but not MyD88-IRAK1 complex formation. Inhibitory effects of apoE on IL-1β signaling were abolished after silencing LDL receptor-related protein-1 (LRP1) expression with siRNA. In addition, inhibitors of adenylyl cyclase and protein kinase A (PKA) restored IL-1β signaling in apoE-treated VSMCs, whereas apoE stimulated PKA activity. ApoE inhibited VSMC activation in response to IL-18 but not to tumor necrosis factor-α or polyinosinic:polycytidylic acid. Conclusion— ApoE targets IRAK-1 activation and thereby interrupts IL-1β and IL-18 signaling in VSMCs. This antiinflammatory effect represents a novel antiatherogenic activity of apoE.

Glucose triggers protein kinase A-dependent insulin secretion in mouse pancreatic islets through activation of the K+ATP channel-dependent pathway

To assess the significance of protein kinase A (PKA) in glucose triggering of ATP-sensitive K(+) (K(+)(ATP)) channel-dependent insulin secretion and in glucose amplification of K(+)(ATP) channel-independent insulin secretion. Insulin release from cultured perifused mouse pancreatic islets was determined by radioimmunoassay. In islets cultured at 5.5 mmol/l glucose, and then perifused in physiological Krebs-Ringer medium, the PKA inhibitors, H89 (10 micromol/l) and PKI 6-22 amide (30 micromol/l) did not inhibit glucose (16.7 mmol/l)-induced insulin secretion, but inhibited stimulation by the adenylyl cyclase activator, forskolin (10 micromol/l). In the presence of 60 mmol/l K(+) and 250 micromol/l diazoxide, which stimulates maximum Ca(2+) influx independently of K(+)(ATP) channels, H89 (10 micromol/l) inhibited Ca(2+)-evoked insulin secretion, but failed to prevent glucose amplification of K(+)(ATP) channel-independent insulin secretion. In the presence of 1 mmol/l ouabain and 250 micromol/l diazoxide, which cause modest Ca(2+) influx, glucose amplification of K(+)(ATP) channel-independent insulin secretion was observed without concomitant Ca(2+) stimulation of PKA activity. In islets cultured at 16.7 mmol/l glucose, glucose (16.7 mmol/l)-induced insulin secretion in physiological Krebs-Ringer medium was augmented and now inhibited by H89 (10 micromol/l), implicating that culture at 16.7 mmol/l glucose may increase Ca(2+)-sensitive adenylyl cyclase activity and hence PKA activity. In accordance, Ca(2+)-evoked insulin secretion at 60 mmol/l K(+) and 250 micromol/l diazoxide was improved, whereas glucose amplification of K(+)(ATP) channel-independent insulin secretion was unaffected. Glucose may activate PKA through triggering of the K(+)(ATP) channel-dependent pathway. Glucose amplification of K(+)(ATP) channel-independent insulin secretion, on the other hand, occurs by PKA-independent mechanisms.

Stimulation of protein kinase a activity in the rat amygdala enhances reward-related learning

Background: Drug addiction in humans is associated with abnormal metabolic activity within the amygdala and heightened control of behavior by drugs and drug-related (conditioned) stimuli. Drug-induced neuroadaptations, including activation of cAMP (cyclic adenosine monophosphate)-dependent protein kinase A (PKA), within the amygdala may contribute to the synaptic plasticity and reward-related learning that underlies pathologic behavior in addicted individuals. Methods: In this study, we tested the hypothesis that stimulation of PKA activity within the rat amygdala would facilitate the acquisition of Pavlovian approach behavior, a measure of reward-related learning. Results: Intraamygdala infusions of Sp-cAMPS (which activates PKA) produced concentration-dependent enhancements of the acquisition of approach to a conditioned stimulus that predicted water availability; intraamygdala infusions of cholera toxin (which elevates cAMP levels) produced a similar effect. Conversely, intraamygdala infusions of Rp-cAMPS, an inhibitor of PKA, impaired acquisition of approach behavior. Conclusions: Together, these data demonstrate that stimulation of PKA activity in the amygdala can facilitate reward-related learning and suggest that neuroadaptative changes in the PKA pathway within this brain region may be a mechanism by which chronic drug abuse alters the control of behavior by drug-associated stimuli.

Analysis of inhibition by H89 of UCP1 gene expression and thermogenesis indicates protein kinase A mediation of β 3-adrenergic signalling rather than β 3-adrenoceptor antagonism by H89

Although it has generally been assumed that protein kinase A (PKA) is essential for brown adipose tissue function, this has not as yet been clearly demonstrated. H89, an inhibitor of PKA, was used here to inhibit PKA activity. In cell extracts, it was confirmed that norepinephrine stimulated PKA activity, which was abolished by H89 treatment. In isolated brown adipocytes, H89 inhibited adrenergically induced thermogenesis (with an IC 50 of approx. 40 μM), and in cultured cells, adrenergically stimulated expression of the uncoupling protein-1 (UCP1) gene was abolished by H89 (full inhibition with 50 μM). However, H89 has been reported to be an adrenergic antagonist on β 1/β 2-adrenoceptors (AR). Although adrenergic stimulation of thermogenesis and UCP1 gene expression are mediated via β 3-ARs, it was deemed necessary to investigate whether H89 also had antagonistic potency on β 3-ARs. It was found that EC 50 values for β 3-AR-selective stimulation of cAMP production (with BRL-37344) in brown adipose tissue membrane fractions and in intact cells were not affected by H89. Similarly, the EC 50 of adrenergically stimulated oxygen consumption was not affected by H89. As H89 also abolished forskolin-induced UCP1 gene expression, and potentiated selective β 3-AR-induced cAMP production, H89 must be active downstream of cAMP. Thus, no antagonism of H89 on β 3-ARs could be detected. We conclude that H89 can be used as a pharmacological tool for elucidation of the involvement of PKA in cellular signalling processes regulated via β 3-ARs, and that the results are concordant with adrenergic stimulation of thermogenesis and UCP1 gene expression in brown adipocytes being mediated via a PKA-dependent pathway.

Effects of Biogenic Amines and Polypeptide Hormones on Protein Kinase A and Adenylyl Cyclase Activities in Muscles of the Mollusc Anodonta cygnea

The effects of biogenic amines, glucagon, and insulin on the cAMP-dependent protein kinase A (PKA) activity have been studied in the muscle tissue of the freshwater bivalve mollusc Anodonta cygnea. It was shown that serotonin, glucagon, and insulin both in vivo and in vitro stimulated PKA activity, whereas isoproterenol inhibited it. The stimulating effect of serotonin and inhibiting effect of isoproterenol was blocked by serotoninergic (cyproheptadine) and adrenergic (propranolol) inhibitors, which confirms specificity of the effect of biogenic amines on the PKA activity. Taking into account participation of adenylyl cyclase system in action of the above hormones, the revealed hormonal effects on the PKA activity produce metabolic effects via the following chain reaction. In the case of serotonin and glucagon: receptor → Gs-protein → AC → cAMP → PKA → phosphorylation of glycogen synthase (GS) and glucose-6-phosphate dehydrogenase (G6PDH) and inhibition of their activity; in the case of isoproterenol: β-adrenoreceptor → Gi-protein → AC inhibition → decreasing PKA → inhibition of phosphorylase and stimulation of GSI and G6PDH. A participation is suggested of the insulin-stimulated AC signaling system in the mechanism of the mitogenic insulin effect mediated, as shown in this work, via the PKA activation, but not of the metabolic effect of insulin.

DOPAMINE INHIBITS Na,H-EXCHANGER VIA D1-LIKE RECEPTOR-MEDIATED STIMULATION OF PROTEIN KINASE A IN RENAL PROXIMAL TUBULES

Dopamine causes natriuresis and diuresis via activation of D1-like receptors located in the renal proximal tubules. It is reported that this response to dopamine results from the inhibition of Na,H-exchanger and Na,K-ATPase. Earlier studies have suggested a role of protein kinase A (PKA) in the inhibition of Na,H-exchanger, however, the effect of dopamine or the dopamine receptor subtype responsible for the stimulation of PKA has not been reported. Present study was designed to examine the effect of dopamine and D1-like receptor agonist, SKF 38393, on the stimulation of PKA activity in rat renal proximal tubules. Dopamine and SKF 38393 (1 nM – 1 μM) caused stimulation of PKA activity, an effect which was antagonized by a D1-like receptor antagonist, SCH 23390 (10 μM). Stimulation of PKA activity was also seen with forskolin and di-butyryl cAMP. We also observed that dopamine and SKF 38393 inhibited Na,H-exchanger activity in the proximal tubules. This response was blocked by SCH 23390 and Rp-cAMPS triethylamine, a selective inhibitor of PKA. Similarly, forskolin and di-butyryl cAMP inhibited Na,H-exchanger activity. The data provide direct evidence showing that dopamine, through the activation of D1-like receptors stimulates PKA activity which in turn inhibits Na,H-exchanger in the proximal tubules.

Androgen-independent Induction of Prostate-specific Antigen Gene Expression via Cross-talk between the Androgen Receptor and Protein Kinase A Signal Transduction Pathways

Transcription of the prostate-specific antigen (PSA) gene escapes regulation by androgens in advanced prostate cancer. To determine the molecular mechanism(s) of androgen-independent regulation of the PSA gene, the possibility that the androgen receptor (AR) is activated in the absence of androgen by stimulation of protein kinase A (PKA) was investigated. Activation of PKA by forskolin resulted in elevated expression of the PSA gene in androgen-depleted LNCaP cells, an effect that was blocked by the antiandrogen, bicalutamide. Further evidence that induction of PSA gene expression was dependent on AR was obtained from experiments using PC3 cells devoid of AR. Neither PSA, PB, nor ARR3 androgen-responsive reporters could be induced by activation of PKA in the absence of transfected AR. In addition, when nuclear AR from forskolin-treated LNCaP cells was incubated with oligonucleotides encoding an androgen response element of the PSA promoter and examined by electromobility shift assay, an increase in AR-androgen response element complex formation was observed. Lastly, cotransfection of an expression vector for a chimeric protein encoding the amino-terminal domain of the human AR linked to Gal4 and a 5xGal4UAS reporter gene construct resulted in activation of the amino-terminal domain of the AR by stimulation of PKA activity. These results demonstrate androgen-independent induction of PSA gene expression in prostate cancer cells by an AR-dependent pathway.

Structure-function requirements of parathyroid hormone for stimulation of 1,25-dihydroxyvitamin D3 production by rat renal proximal tubules.

PTH stimulates synthesis and secretion of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in renal proximal tubule cells through activation of the protein kinase-A (PKA) or the protein kinase-C (PKC) signaling pathway. The relative contribution of the two transducing systems was explored using PTH fragments with selective activation of either PKA or PKC. Rat renal proximal tubules were isolated by Percoll centrifugation, and PKA and PKC activities were measured after treatment with synthetic fragments and analogs of PTH. Rat PTH-(1-34), [Nle8,Nle15,Tyr34]bovine PTH-(3-34), and human PTH-(13-34) increased PKC activity in a dose-dependent manner. All fragments tested stimulated PKC at physiological concentrations (10(-11)-10(-10) M). Rat PTH-(1-34) (10(-7) M) increased PKA activity 4.5-fold, but other fragments failed to stimulate PKA between 10(-12)-10(-6) M. Human PTH-(28-34) stimulation of PKC was variable from experiment to experiment. All four PTH fragments tested increased 1,25-(OH)2D3 secretion by perifused renal proximal tubules at the lowest concentrations that stimulated PKC activity. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine (10(-4) M) reduced PTH-(1-34)-stimulated PKA activity by 60%, but failed to block the rise in 1,25-(OH)2D3 secretion. The results of these studies demonstrate that PTH fragments that contain the PKC translocating domain stimulate 1,25-(OH)2D3 secretion, whereas elimination of the PKA activation domain does not alter the potency of the analogs' 1,25-(OH)2D3-stimulating activity. These results support the concept that PKC translocation may be required for PTH stimulation of 1,25-(OH)2D3 secretion.

Murine melanoma cell differentiation and melanogenesis induced by poly(ADP-ribose) polymerase inhibitors

Murine melanoma cells treated with the melanocyte-stimulating hormone (MSH) family of peptides undergo differentiation characterized by enhanced melanogenesis and altered morphology. These effects are mediated via the adenylate cyclase-cAMP pathway leading to activation of protein kinase A (PKA). We have discovered that inhibition of a post-translational modification of chromatin proteins, viz. poly(ADP-ribosylation), also induces melanogenesis and differentiation in these cells. A range of competitive inhibitors (benzamide and its derivatives) of the nuclear enzyme poly (ADP-ribose) polymerase (PADPRP; EC 2.4.2.30) was utilized, and their ability to induce melanogenesis reflected their potency as PADPRP inhibitors. These compounds induced melanogenesis at low doses (20 μ M − 2 m M) which did not affect cell growth or viability. Induction of melanogenesis was not attributable to inhibition of cyclic nucleotide phosphodiesterase by these compounds. MSH treatment caused a transient rise in cAMP levels (up to 200-fold by 5 min and returning to near basal levels by 5 h). It also stimulated PKA activity up to 5-fold, and the temporal kinetics of this activation mirrored the changes in cAMP levels. In comparison, the PADPRP inhibitors had no effect on either of these processes. These data constitute a novel demonstration of a cAMP-independent mechanism for the induction of melanoma cell differentiation, including melanogenesis.