Protein Kinase A Expression Research Articles

Matrine Treatment Blocks NogoA-Induced Neural Inhibitory Signaling Pathway in Ongoing Experimental Autoimmune Encephalomyelitis.

Myelin-associated inhibitors, such as NogoA, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp), play a pivotal role in the lack of neuroregeneration in multiple sclerosis, an inflammatory demyelinating disease of the central nervous system (CNS). Matrine (MAT), a monomer that is used in traditional Chinese medicine as an anti-inflammatory agent, has shown beneficial effects in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. However, the underlying mechanisms of MAT-induced EAE amelioration are not fully understood. In the present study, we show that MAT treatment suppressed ongoing EAE, and this effect correlated with an increased expression of growth-associated protein 43, an established marker for axonal regeneration. MAT treatment significantly reduced the levels of NogoA, its receptor complex NgR/p75NTR/LINGO-1, and their downstream RhoA/ROCK signaling pathway in the CNS. In contrast, intracellular cyclic AMP (cAMP) levels and its protein kinase (protein kinase A (PKA)), which can promote axonal regrowth by inactivating the RhoA, were upregulated. Importantly, adding MAT in primary astrocytes in vitro largely induced cAMP/PKA expression, and blockade of cAMP significantly diminished MAT-induced expression of PKA and production of BDNF, a potent neurotrophic factor for neuroregeneration. Taken together, our findings demonstrate that the beneficial effects of MAT on EAE can be attributed not only to its capacity for immunomodulation, but also to its directly promoting regeneration of the injured CNS.

Elevated levels of calcitonin gene-related peptide in upper spinal cord promotes sensitization of primary trigeminal nociceptive neurons

Orofacial pain conditions including temporomandibular disorder (TMD) and migraine are characterized by peripheral and central sensitization of trigeminal nociceptive neurons. The goal of this study was to investigate the role of calcitonin gene-related peptide (CGRP) in promoting bidirectional signaling within the trigeminal system to mediate sensitization of primary nociceptive neurons. Adult male Sprague–Dawley rats were injected intercisternally with CGRP or co-injected with the receptor antagonist CGRP8–37 or KT 5720, a protein kinase A (PKA) inhibitor. Nocifensive head withdrawal response to mechanical stimulation was investigated using von Frey filaments. Expression of PKA, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adapter molecule 1 (Iba1) in the spinal cord and phosphorylated extracellular signal-regulated kinase (P-ERK) in the ganglion was studied using immunohistochemistry. Some animals were co-injected with CGRP and Fast Blue dye and the ganglion was imaged using fluorescent microscopy. CGRP increased nocifensive responses to mechanical stimulation when compared to control. Co-injection of CGRP8–37 or KT 5720 with CGRP inhibited the nocifensive response. CGRP stimulated PKA and GFAP expression in the spinal cord, and P-ERK in ganglion neurons. Seven days post injection, Fast Blue was observed in ganglion neurons and satellite glial cells. Our results demonstrate that elevated levels of CGRP in the upper spinal cord promote sensitization of primary nociceptive neurons via a mechanism that involves activation of PKA centrally and P-ERK in ganglion neurons. Our findings provide evidence of bidirectional signaling within the trigeminal system that facilitate increased neuron-glia communication within the ganglion associated with trigeminal sensitization.

CAMP-dependent protein kinase A (PKA) regulates angiogenesis by modulating tip cell behavior in a Notch-independent manner

cAMP-dependent protein kinase A (PKA) is a ubiquitously expressed serine/threonine kinase that regulates a variety of cellular functions. Here, we demonstrate that endothelial PKA activity is essential for vascular development, specifically regulating the transition from sprouting to stabilization of nascent vessels. Inhibition of endothelial PKA by endothelial cell-specific expression of dominant-negative PKA in mice led to perturbed vascular development, hemorrhage and embryonic lethality at mid-gestation. During perinatal retinal angiogenesis, inhibition of PKA resulted in hypersprouting as a result of increased numbers of tip cells. In zebrafish, cell autonomous PKA inhibition also increased and sustained endothelial cell motility, driving cells to become tip cells. Although these effects of PKA inhibition were highly reminiscent of Notch inhibition effects, our data demonstrate that PKA and Notch independently regulate tip and stalk cell formation and behavior.

Role of HSF1-upregulated AC6 in ameliorating heart failure in mice.

Our previous studies discovered that Heat shock factor 1(HSF1) can alleviate pressure overload induced heart failure in mice. However, its molecular mechanisms are yet to be further explained. Many studies have already verified that Adenylyl Cyclase 6 (AC6) can ameliorate heart failure, but it is still unknown whether or not the pathway HSF1 is involved in the process. Our preliminary experiment showed that the expression level of AC6 is positively associated with HSF1. Therefore, in the present study, we aimed to explore whether HSF1 can play its role in ameliorating heart failure by regulating AC6, and how the potential internal mechanisms work. We applied the Transverse Aortic Constriction (TAC) for 4 weeks to develop the C57BL/6 mice pressure overload induced heart failure model. First, the mice were divided into TAC group and SHAM group. Changes in the cardiac function and morphology of the mice were observed by an ultrasonic device and Masson staining slices, expressions of AC6 mRNA were observed by RT-QPCR, expressions of HSF1 and proteinkinase A (PKA) were examined by Western Blotting, and the levels of cyclic adenosine monophosphate (cAMP) from aortic blood were measured by ELISA. Second, the TAC group were further divided into subgroups of HSF1 transgene mice, HSF1 knockout mice and wild type mice, followed by the aforesaid observations. In the SHAM group, no obvious variations of cardiac function, AC6 mRNAHSF1, PKA, cAMP and other test results were found among each of the subgroups. Compared to the SHAM group, the TAC group presented clearly weakened heart functions, while, expressions of AC6 mRNA, HSF1, PKA and cAMP all recorded obvious increases. In the TAC group, compared to the WT subgroup, the HSF1 KO subgroup presented decreases in expressions of AC6 mRNA, HSF1, PKA and cAMP, and at the same time, the heart functions were weaker, while, the HSF1 TG subgroup recorded the contrary results. In the pressure overload heart failure model, HSF1 can ameliorate heart failure by positively regulating the pathway of AC6/cAMP/PKA.

Lipolysis and thermogenesis in adipose tissues as new potential mechanisms for metabolic benefits of dietary fiber

ObjectiveDietary fiber consumption is associated with reduced risk for the development of noncommunicable diseases. The aim of the present study was to evaluate the effects of cereal dietary fiber on the levels of proteins involved in lipolysis and thermogenesis in white adipose tissue (WAT) and brown adipose tissue (BAT) of C57 BL/6 J mice fed a high-fat diet (HFD). MethodsMale C57BL/6 J mice were fed normal chow diet (Chow), HFD, HFD plus oat fiber (H-oat), or HFD plus wheat bran fiber (H-wheat) for 24 wk. Body weight and food intake were recorded weekly. Serum adiponectin was assayed by an enzyme-linked immunosorbent assay kit. Western blotting was used to assess the protein expressions of adipose triacylglycerol lipase (ATGL), cAMP protein kinase catalytic subunit (cAMP), protein kinase A (PKA), perilipin A, hormone-sensitive lipase (HSL), uncoupling protein 1 (UCP1), fibroblast growth factor 21 (FGF-21), β3-adrenergic receptor (β3AR), and proliferator-activated receptor gamma coactivator-1 α (PGC-1 α) in the WAT and BAT. ResultsAt the end of the feeding period, body and adipose tissues weight in both H-oat and H-wheat groups were lower than in the HFD group. Mice in the H-oat and H-wheat groups showed an increasing trend in serum adiponectin level. Compared with the HFD group, cereal dietary fiber increased protein expressions involved in the lipolysis and browning process. Compared with the H-wheat group, H-oat was more effective in protein expressions of PKA, PGC-1 α, and UCP1 of the WAT samples. Compared with the H-oat group, H-wheat was more effective in protein expressions of PKA, ATGL, UCP1, β3AR, and FGF-21 of the BAT samples. ConclusionsTaken together, our results suggested that cereal dietary fiber enhanced adipocyte lipolysis by the cAMP–PKA–HSL pathway and promoted WAT browning by activation of UCP1, and consequently reduced visceral fat mass in response to HFD feeding.

Changes of protein kinase A expressions in shell of accumbens nucleus during the process of chronic morphine-induced conditioned place aversion in rats

Objective To explore neurobiological mechanisms of the withdrawal-induced aversion, the changes of protein kinase A(PKA) were measured in shell of accumbens nucleus (AcbSH) of CPA model rats. Methods 1.All 36 male SD rats were divided into three groups, model group (MN group), and control group (MS group and SN group). MN group was injected with morphine, 6.5 days, 10mg/kg, intraperitoneally (IP), twice per day, naloxone injection, 0.3 mg/kg, ip, along with conditioned place aversion training, to develop the CPA model.The MS group was administrated equivalent volume of morphine and saline.Also the SN group was injected with equivalent volume of saline and naloxone.2.During the development of CPA, the expression of protein kinase A was assayed with immunohistochemistry in the AcbSH. Results Before the development of CPA, PKA expressions were no significant differences among the three groups in the AcbSH (F=2.306, P=0.130). However, after development of CPA , PKA expressions showed significant differences among the three groups(F=36.516, P=0.000). The average gray intensity of MN group (109.50±4.661) was apparently higher than the MS group(126.50±3.697, P<0.01), than the SN group (133.50±6.364, P<0.01). Conclusions 1.Protein kinase A expression, leading to the aversion in the AcbSH probably is a key pathway contributing to the development of CPA.2.The neuroadaptation mediated by PKA may be one of important molecular underpinnings of CPA. Key words: Conditioned place aversion; Protein kinase A; Immunohistochemistry

Early Intervention of Didang Decoction on MLCK Signaling Pathways in Vascular Endothelial Cells of Type 2 Diabetic Rats.

In the study, type 2 diabetic rat model was established using streptozotocin (STZ) combined with a high-fat diet, and the rats were divided into control and diabetic groups. Diabetic groups were further divided into nonintervening, simvastatin, Didang Decoction (DDD) early-phase intervening, DDD mid-phase intervening, and DDD late-phase intervening groups. The expression level of MLCK was detected using Western Blot analysis, and the levels of cyclic adenosine monophosphate (cAMP), protein kinase C (PKC), and protein kinase A (PKA) were examined using Real Time PCR. Under the electron microscope, the cells in the early-DDD-intervention group and the simvastatin group were significantly more continuous and compact than those in the diabetic group. Compared with the control group, the expression of cAMP-1 and PKA was decreased in all diabetic groups, whereas the expression of MLCK and PKC was increased in early- and mid-phase DDD-intervening groups (P < 0.05); compared with the late-phase DDD-intervening group, the expression of cAMP-1 and PKA was higher, but the level of MLCK and PKC was lower in early-phase DDD-intervening group (P < 0.05). In conclusion, the early use of DDD improves the permeability of vascular endothelial cells by regulating the MLCK signaling pathway.

Effects of lactitol and Bifidobacterium infantis on AQP3 and ICC in a rat model of constipation

Objective To analyze the roles and mechanisms of lactitol and Bifidobacterium infantis in the treatment of rat constipation and to investigate their effects on aquaporin3 (AQP3) and interstitial cells of Cajal (ICC) in colon tissues. Methods Thirty SD male rats were recruited in this study, 6 of which were randomly selected as the control and the rest were given 4 mg/kg.d of loperamide for 5 consecutive days to construct the rat model of constipation. The rats with constipation were randomly divided into four groups including model group, lactitol treatment group, Bifidobacterium infantis treatment group and lactitol in combination with Bifidobacterium infantis treatment group. General indexes including food intake, water intake, body weight, fecal water content and intestinal transit rate of each rat were measured after receiving corresponding treatments for 7 consecutive days. The levels of substance P (SP) and vasoactive intestinal peptide (VIP) in serums samples were detected by ELISA. The expression of protein kinase A (PKA) and neurokinin-1 receptor (NK-1) at mRNA level in colon tissues were detected by real-time polymerase chain reaction (real-time PCR). Western blot assay and real-time PCR analysis were used to detect the expression of AQP3 and c-kit at protein and mRNA levels, respectively. Results Compared with the rats in model group, the levels of fecal water content and intestinal transit rate, the concentrations of SP and VIP in serums samples, the expression of PKA and NK-1 at mRNA level and the expression of AQP3 and c-kit at mRNA and protein levels were significantly increased in rats from the three treatment groups (P<0.05). The most effective treatment was lactitol in combination with Bifidobacterium infantis, followed by the lactitol treatment and then the Bifidobacterium infantis treatment. Conclusion The combination therapy with lactitol and Bifidobacterium infantis increased the serum levels of SP and VIP in rats with constipation. SP could enhance the contraction of gastrointestinal smooth muscles and improve the intestinal motility by binding to the NK-1 receptor on the membrane of ICC. VIP could promote the absorption of water in intestinal tracts, soften stools and alleviate constipation by upregulating the expression of AQP3 at both protein and mRNA levels via the cyclic adenosine monophosphate-PKA (cAMP-PKA) signaling pathway. Key words: Constipation; Lactitol; Bifidobacterium infantis; Aquaporin3; Interstitial cells of Cajal

Antenatal Taurine Improves Intrauterine Growth-Restricted Fetal Rat Brain Development Which Is Associated with Increasing the Activity of PKA-CaMKII/c-fos Signal Pathway.

This study aimed to explore whether antenatal supplement of taurine can improve the brain development of fetal rats with intrauterine growth restriction (IUGR) via protein kinase A (PKA)-Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)/c-fos pathway. A total of 30 pregnant rats were randomly divided into the following three groups: control, IUGR, and IUGR with antenatal taurine supplementation groups. The expression of PKA, CaMKII, and c-fos mRNA and proteins in fetal rat brain tissues were detected by quantitative real time (qRT)-polymerase chain reaction (PCR) and Western blot, respectively; and the number of PKA-, CaMKII- and c-fos-positive cells was measured by immunohistochemistry. Western blot and qRT-PCR revealed that antenatal taurine supplementation significantly increased PKA, CaMKII, and c-fos protein and mRNA levels in fetal rats brain with IUGR (p < 0.05). Immunohistochemistry results showed that the numbers of PKA-, CaMKII-, and c-fos-positive cells were significantly decreased in the IUGR groups compared with the controls; while antenatal taurine could significantly increase the positive cell numbers (p < 0.05). So, we conclude that antenatal taurine improves IUGR fetal brain development which is associated with increasing the activity of PKA-CaMKII/c-fos signal pathway.

Inhibitory Efficacy of Dioscoreae Rhizoma on MITF, TRP-1, TRP-2, Tyrosinase, PKA and ERK Expression in Melanoma Cells (B16F10)

Objectives : The purpose of this study was to research the whitening effects and developing by cosmetics of the extract from Dioscoreae Rhizoma, which is one of the most popular health-promoting herb in herbal medications. Methods : We performed tyrosinase inhibition assay, reverse transcription-polymerase chain reaction (RT-PCR) and western blot for whitening effects. Also we measured MTT assay for cell viability. Results : The results were obtained as follows : For whitening effect, tyrosinase inhibition rate of extract from Dioscoreae Rhizoma showed more than 42.28% at 1,000 ㎍/㎖ concentration. Cell toxicity effect on melanoma cells (B16F10) of extract from Dioscoreae Rhizoma showed 81.97% with toxicity at 50 ㎍/㎖ concentration. So we were measured at a concentrations of 5, 10 and 50 ㎍/㎖ in all experiments involving cell. In addition, whitening related mRNAs including microphthalmia associated transcription factor (MITF), tyrosinase related protein-1 (TRP-1), tyrosinase related protein-2 (TRP-2), tyrosinase were reduced by Dioscoreae Rhizoma. We also found Dioscoreae Rhizoma transiently decreased protein kinase A (PKA) which is known to be upstream to the down regulation of MITF and tyrosinase. But phosphorylation of extracellular signal related kinase (pERK) were increased by Dioscoreae Rhizoma. These results imply that Dioscoreae Rhizoma decrease melanogenesis via ERK activation and subsequent down regulation of MITF and tyrosinase. Conclusions : Therefore, all these findings suggested the potent usage of Dioscoreae Rhizoma as materials of functional cosmetics by confirming whitening activity related with melanin content.

Magnesium Lithospermate B Implicates 3′-5′-Cyclic Adenosine Monophosphate/Protein Kinase A Pathway and N-Methyl-d-Aspartate Receptors in an Experimental Traumatic Brain Injury

Decreased 3'-5'-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and increased N-methyl-d-aspartate (NMDA) related apoptosis were observed in traumatic brain injury (TBI). It is of interest to examine the effect of magnesium lithospermate B (MLB) on cAMP/PKA pathway and NMDAR in TBI. A rodent weight-drop TBI model was used. Administration of MLB was initiated 1 week before (precondition) and 24 hours later (reversal). Cortical homogenates were harvested to measure cAMP (enzyme-linked immunosorbent assay), soluble guanylyl cyclases, PKA and NMDA receptor-2β (Western blot). In addition, cAMP kinase antagonist and H-89 dihydrochloride hydrate were used to test MLB's effect on the cytoplasm cAMP/PKA pathway after TBI. Morphologically, vacuolated neuron and activated microglia were observed in the TBI groups but absent in the MLB preconditioning and healthy controls. Induced cAMP, soluble guanylyl cyclase α1, and PKA were observed in the MLB groups, when compared with the TBI group (P < 0.01) Administration of H-89 dihydrochloride hydrate reversed the effect of MLB on cortical PKA and NMDA-2β expression after TBI. This study showed that MLB exerted an antioxidant effect on the enhancement of cytoplasm cAMP and PKA. This compound also decreased NMDA-2β levels, which may correspond to its neuroprotective effects. This finding lends credence to the presumption that MLB modulates the NMDA-2β neurotoxicity through a cAMP-dependent mechanism in the pathogenesis of TBI.

Yifei Xuanfei Jiangzhuo formula, a Chinese herbal decoction, improves memory impairment through inhibiting apoptosis and enhancing PKA/CREB signal transduction in rats with cerebral ischemia/reperfusion.

Apoptosis and the dysfunction of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP-responsive element binding protein (CREB) signaling pathway have a key role in memory impairment in vascular dementia (VaD), a challenging clinical problem. Yifei Xuanfei Jiangzhuo formula (YXJF), a Chinese herbal decoction, has been used to treat VaD in clinical practice and has produced positive outcomes; however, convincing evidence is currently lacking. The present study aimed to investigate the effects of YXJF on memory impairment in rats with cerebral ischemia/reperfusion and to explore the underlying mechanism. YXJF ameliorated memory impairment in rats with cerebral ischemia/reperfusion, inhibited hippocampal apoptosis in a dose-dependent manner and attenuated increases in the protein expression of B-cell lymphoma 2 (Bcl-2)-associated X protein as well as c-Jun and a reduction in Bcl-2 protein expression in the hippocampal tissue of the rats. Furthermore, administration of YXJF significantly increased the protein expression of PKA C-α and CREB, and promoted CREB phosphorylation. The results indicated that YXJF improves memory impairment through inhibiting apoptosis and enhancing PKA/CREB signal transduction in rats with cerebral ischemia/reperfusion.

Expression of AQP3 protein in hAECs is regulated by Camp-PKA-CREB signalling pathway.

Previous studies by others and our group have demonstrated the expression of AQP3 protein in human chorioamniotic membranes. Here, we investigated whether cyclic adenosine monophosphate (cAMP) up-regulation of aquaporin 3 (AQP3) protein expression in human amniotic epithelial cells (hAECs) is mediated by protein kinase A (PKA) dependent or independent pathway. Cells were treated with various concentrations of Forskolin, SP-cAMP, H-89 at various time intervals or optimal concentration of Forskolin in combination with H-89 in the blocking experiments. Forskolin significantly increased cAMP levels and the expression of PKA, p-CREB and AQP3. SP-cAMP had similar effects. H-89 inhibited PKA, p-CREB and AQP3 protein expression, and attenuated the stimulatory effect of Forskolin. These results show that the AQP3 protein expression in hAECs was regulated by cAMP-PKA-CREB signal pathway. A relatively short biological half life of AQP3 renders its rapid responsiveness to regulation.

Effects of prenatal taurine supplementation on the expression of protein kinase A-Ca2+ /calmodulin-dependent protein kinase II, c-fos in fetal rat brain with intrauterine growth restriction and its significance

Objective To explore the effects of prenatal taurine supplementation on the expression of protein kinase A(PKA) and c-fos, Ca2+ /calmodulin-dependent protein kinase Ⅱ(GaMKⅡ) in fetal rat brains with intraute-rine growth restriction and its significance. Methods Fifteen pregnant Sprague-Dawley rats were randomly divided into 3 groups: the control group, the intrauterine growth restriction (IUGR) group and the IUGR with prenatal taurine supplementation group(the taurine group), with 5 fetal rats in each group.All the fifteen fetal rat brains were detected as following: the expression of PKA, c-fos, CaMK Ⅱ mRNA in fetal rat brains was detected by way of real-time polymerase chain reaction(real-time PCR), while the expression changes of PKA, CaMK II, and c-fos protein in fetal rat brains were detected by using Western blot, and the number of PKA, CaMK II, c-fos positive cells in fetal rat brains was detected by using immunohistochemistry. Results The control group, the IUGR group and the taurine group: Comparison of the expression of PKA, CaMK Ⅱ, c-fos mRNA among 3 groups were of significant differences(F=7.934, P=0.021; F=5.568, P=0.043; F=7.332, P=0.024). Comparison of the expression of PKA, CaMK II, c-fos protein among the 3 groups were of significant differences(F=57.743, P=0.000; F=163.405, P=0.000; F=160.136, P=0.000). Comparison of the number of PKA, CaMK Ⅱ, c-fos positive cells among the 3 groups were of significant differences(F=42.903, P=0.000; F=329.123, P=0.000; F=43.674, P=0.000). Compared with the control group, the expression of mRNA, protein, positive cells of three indicators in IUGR group was less, and the difference was statistically significant (P<0.05). The expression of mRNA, protein, positive cells of three indicators in fetal rat brains of the taurine group were not different from the control group.The expression of mRNA, protein, positive cells of three indicators in fetal rat brains of the taurine group were significantly more than the IUGR group(P<0.05). Conclusions Prenatal taurine supplementation can improve PKA-CaMK II, c-fos mRNA and protein levels in fetal rat brain tissue with IUGR, and increase its number of positive cells and may enhance the regenerative capacity of the central nervous system, so as to reduce the IUGR brain injury and promote its role in brain development. Key words: Intrauterine growth restriction; Fetal rat; Taurine; Protein kinase A; Ca2+ /calmodulin-dependent protein kinase Ⅱ; c-fos

3α-diol administration decreases hippocampal PKA (II) mRNA expression and impairs Morris water maze performance in adult male rats

The effect of testosterone and its metabolites on learning and memory has been the subject of many studies. This study used the Morris water maze task to investigate the effect of intra-hippocampal injection of 3α-diol (one of the metabolites of testosterone) on acquisition stage of spatial memory in adult male rats. During the experiment we observed that 3α-diol, significantly impaired Morris water maze performance in treated rat's compared with controls. Because signaling event mediated by protein kinase A (PKA) especially PKA (II) are critical for many neuronal functions such as learning and memory, the hippocampus was analyzed for mRNA expression of PKA (II) using TaqMan real time RT-PCR. The results indicated that the transcription levels of PKA (II) were significantly decreased in animals treated with 3α-diol compared with controls. Thus, the findings suggest that administration of 3α-diol in hippocampus of adult male rats impairs memory function, possibly via down-regulation of PKA.

Glucotoxicity inhibits cAMP-protein kinase A-potentiated glucose-stimulated insulin secretion in pancreatic β-cells.

The effect of incretin is markedly blunted in patients with type 2 diabetes (T2D), and this reduced effect of incretin is correlated with a diminished insulintropic potency of glucagon-like peptide-1 (GLP-1). We reported recently that GLP-1 potentiates glucose-stimulated insulin secretion (GSIS) mainly via activation of the cAMP-protein kinase A (PKA) signaling pathway in INS-1E cells under hyperglycemic conditions. In the present study, we further explored whether glucotoxicity impairs cAMP-PKA-mediated effects and its relevance to the reduced insulinotropic action of GLP-1 in hyperglycemia. Mouse islets and INS-1E cells were cultured in 30 mmol/L glucose for 72 h. The effects of glucotoxicity on cAMP-PKA-linked pathways and its insulinotropic action were then evaluated. Chronic exposure of INS-1E cells and primary mouse islets to 30 mmol/L glucose almost abolished GSIS. The cAMP-elevating agent forskolin produced an approximate 1.9-fold increase in GSIS, significantly lower than that observed with 5.5 mmol/L glucose (~3.3-fold). Moreover, 72 h culture in the presence of 30 mmol/L glucose reduced forskolin-stimulated cAMP accumulation in β-cells. Notably, glucotoxicity reduced the expression and activity of PKA, as well as PKA-mediated effects. In contrast, glucotoxicity had no effect on the expression of Epac2, another cAMP effector. Glucotoxicity-induced reductions in PKA and its signaling account, at least in part, for the decreased incretin effect under conditions of glucotoxicity.

Effects of varying doses of simvastatin on protein kinase A in immature rabbits with chronic heart failure

Objective To investigate the effects of varying doses of simvastatin (SIM) on protein kinase A(PKA), ventricular remodeling and heart function in immature rabbits with chronic heart failure (CHF), to explore the possible mechanisms for simvastatin on CHF. Methods Sixty male immature rabbits were randomly divided into 5 groups: control (CON) group, CHF model group, small-dose simvastatin (SD-SIM) group[SIM concentration was 0.3 mg/(kg·d)], medium-dose simvastatin (MD-SIM)group[SIM concentration was 1.5 mg/(kg·d)], and the high-dose simvastatin (HD-SIM)group[SIM concentration was 3.0 mg/(kg·d)]. The general condition of immature rabbits in the experiment group was evaluated.Color doppler ultrasonography was used to detect the left ventricular structure and the function.The left ventricular myocardium was taken and fixed in paraformaldehyde (40 g/L). The expression of PKA was evaluated by immunohistochemical assay. Results In CON group, the rabbits were all alive; in the other groups, most immature rabbits were unhairing, with diet reduction, weight lose and sluggish with hyperkeratosis.Overall survival rate was 82%(49/60 cases). The mortality rate in CHF model group was significantly higher than that in the MD-SIM group(41.7% vs 8.3%, P<0.05). Compared with the CON group, left ventricular ejection fraction(LVEF) in the SD-SIM group, HD-SIM group and CHF model group all dropped sharply (all P<0.01), while the left ventricular end diastolic diameter (LVEDd) and left ventricular end systolic diameter (LVESd) increased markedly (all P<0.01). Compared with CHF model group, LVEDd and LVESd decreased and LVEF increased significantly in SD-SIM group, HD-SIM group and MD-SIM group(all P<0.05), the effects of MD-SIM group was the best(P<0.05). LVEF in MD-SIM group increased significantly compared with SD-SIM group, HD-SIM group and CHF model group (all P<0.05). PKA expression in SD-SIM group, MD-SIM group and HD-SIM group increased significantly compared to the CHF model group and CON group (all P<0.01). Compared with the SD-SIM group and HD-SIM group, the expression of MD-SIM group significantly increased (P<0.05). Conclusions Simvastatin can reverse the left ventricular remodeling and improve the heart function of immature rabbits with CHF.The effect of MD-SIM was the best.The mechanism of simvastatin may be correlated to up-regulating the expression of PKA. Key words: Simvastatin; Adriamycin; Chronic heart failure; Protein kinase A

Pituitary adenylate cyclase-activating polypeptide (PACAP) signalling enhances osteogenesis in UMR-106 cell line.

Presence of the pituitary adenylate cyclase-activating polypeptide (PACAP) signalling has been proved in various peripheral tissues. PACAP can activate protein kinase A (PKA) signalling via binding to pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1), vasoactive intestinal polypeptide receptor (VPAC) 1 or VPAC2 receptor. Since little is known about the role of this regulatory mechanism in bone formation, we aimed to investigate the effect of PACAP on osteogenesis of UMR-106 cells. PACAP 1-38 as an agonist and PACAP 6-38 as an antagonist of PAC1 were added to the culture medium. Surprisingly, both substances enhanced protein expressions of collagen type I, osterix and alkaline phosphatase, along with higher cell proliferation rate and an augmented mineralisation. Although expression of PKA was elevated, no alterations were detected in the expression, phosphorylation and nuclear presence of CREB, but increased nuclear appearance of Runx2, the key transcription factor of osteoblast differentiation, was shown. Both PACAPs increased the expressions of bone morphogenetic proteins (BMPs) 2, 4, 6, 7 and Smad1 proteins, as well as that of Sonic hedgehog, PATCH1 and Gli1. Data of our experiments indicate that activation of PACAP pathway enhances bone formation of UMR-106 cells and PKA, BMP and Hedgehog signalling pathways became activated. We also found that PACAP 6-38 did not act as an antagonist of PACAP signalling in UMR-106 cells.

CAMP/PKA‐mediated regulation of amino acid metabolism in murine T‐lymphoma cells (1095.16)

The second messenger cyclic AMP (cAMP), acting through PKA, influences gene and protein expression through CREB and other transcription factors. In order to define the proteins whose expression is regulated by cAMP and protein kinase A (PKA), we conducted quantitative proteomic studies of wild‐type (WT) and kin‐ (PKA‐null) S49 murine T lymphoma cells and compared the impact on protein expression of increases in cAMP by a cell‐permeable analog (8‐CPT‐cAMP, CPT). Numerous proteins involved in branched chain amino acid metabolism had markedly increased expression in WT S49 cells, but not kin‐ cells, treated with CPT for 16h. Expression of mRNA levels of these genes also increased in WT, but not kin‐, S49 cells in response to treatment with CPT. Consistent with this finding, incubation of WT S49 cells with CPT enhanced cell survival in growth media lacking glutamine, whereas kin‐ S49 cells show decreased viability in such media even in the presence of 8‐CPT‐cAMP. We conclude that cAMP acts via PKA to regulate expression of genes and proteins for branched chain amino acids, an action that may help protect cells from a decrease in viability in glutamine‐deficient media. Since not all of the cAMP/PKA‐regulated branched chain amino acid metabolism genes with increased expression have strong predicted binding of CREB, regulation of those genes by cAMP/PKA appears to occurs through as‐yet to be defined transcriptional pathways.Grant Funding Source: Supported by HHMI (S. S.T.), and DK54441 (S.S.T.), GM066232 (P.A.I.) and HL107200 (P.A.I.) from NIH

Determining the downstream signaling pathways through which prostaglandin E2 inhibits inflammatory mediator output in in human myometrial cells (911.6)

Objective: Prostaglandin E2 (PGE2) represses interleukin‐1β (IL‐1β) induced inflammatory mediator output in human myometrial cells via EP2/4 receptors. Downstream EP2/4 signaling involves upregulation of intracellular cAMP which may activate multiple pathways, including protein kinase A (PKA) and exchange proteins activated by cAMP (Epacs).Methods: Myometrial biopsies from term elective Caesarean deliveries prior to labour onset (n=5, 37‐40 weeks gestation) were used to isolate myometrial cells. PKA and Epac gene expression was assessed by RT‐PCR. Myometrial cells were either non‐treated or stimulated with IL‐1β +/‐ co‐treatment with cAMP analogs to selectively activate PKAI, PKAII, or Epacs. Interleukin‐8 (CXCL8) output was measured by ELISA. Statistical analysis was performed by repeated measures ANOVA followed by a Bonferroni test.Results: Expression of both Epac isoforms and six of seven PKA subunits were identified in isolated myometrial cells. IL‐1β increased CXCL8 output in human myometrial cells (p蠄0.001). Co‐treatment with PKAI or PKAII selective cAMP analogs repressed this response (p蠄0.001). No significant repression was observed when cells were co‐treated with the Epac selective cAMP analog.Conclusions: PGE2 mediated repression of IL‐1β induced inflammatory output in human myometrial cells is mediated in part by PKA, but not Epac, signaling pathways.Grant Funding Source: Supported by Canadian Institutes of Health Research and Alberta Innovates‐Health Solutions