cAMP-PKA Cascade Research Articles

Identification of GnRH-like peptide and its potential signaling pathway involved in the oocyte meiotic maturation in the Chinese mitten crab, Eriocheir sinensis

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in reproductive regulation in vertebrates. However, GnRH was rarely isolated and its function remains poorly characterized in invertebrates. The existence of GnRH in ecdysozoa has been controversial for a long. Here, we isolated and identified two GnRH-like peptides from brain tissues in Eriocheir sinensis. Immunolocalization showed that the presence of EsGnRH-like peptide in brain, ovary and hepatopancreas. Synthetic EsGnRH-like peptides can induce germinal vesicle breakdown (GVBD) of oocyte. Similar to vertebrates, ovarian transcriptomic analysis revealed a GnRH signaling pathway in the crab, in which most genes exhibited dramatically high expression at GVBD. RNAi knockdown of EsGnRHR suppressed the expression of most genes in the pathway. Co-transfection of the expression plasmid for EsGnRHR with reporter plasmid bearing CRE-luc or SRE-luc response element into 293T cells showed that EsGnRHR transduces its signal via cAMP and Ca2+ signaling transduction pathways. In vitro incubation of the crab oocyte with EsGnRH-like peptide confirmed the cAMP-PKA cascade and Ca2+ mobilization signaling cascade but lack of a PKC cascade. Our data present the first direct evidence of the existence of GnRH-like peptides in the crab and demonstrated its conserved role in the oocyte meiotic maturation as a primitive neurohormone.

Postsynaptic burst reactivation of hippocampal neurons enables associative plasticity of temporally discontiguous inputs.

A fundamental unresolved problem in neuroscience is how the brain associates in memory events that are separated in time. Here, we propose that reactivation-induced synaptic plasticity can solve this problem. Previously, we reported that the reinforcement signal dopamine converts hippocampal spike timing-dependent depression into potentiation during continued synaptic activity (Brzosko et al., 2015). Here, we report that postsynaptic bursts in the presence of dopamine produce input-specific LTP in mouse hippocampal synapses 10 min after they were primed with coincident pre- and post-synaptic activity (post-before-pre pairing; Δt = -20 ms). This priming activity induces synaptic depression and sets an NMDA receptor-dependent silent eligibility trace which, through the cAMP-PKA cascade, is rapidly converted into protein synthesis-dependent synaptic potentiation, mediated by a signaling pathway distinct from that of conventional LTP. This synaptic learning rule was incorporated into a computational model, and we found that it adds specificity to reinforcement learning by controlling memory allocation and enabling both 'instructive' and 'supervised' reinforcement learning. We predicted that this mechanism would make reactivated neurons activate more strongly and carry more spatial information than non-reactivated cells, which was confirmed in freely moving mice performing a reward-based navigation task.

CAMP-PKA cascade: An outdated topic for depression?

Depression is a common neuropsychiatric disorder characterized by persistent depressed mood and causes serious socioeconomic burden worldwide. Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis, deficiency of monoamine transmitters, neuroinflammation and abnormalities of the gut flora are strongly associated with the onset of depression. The cyclic AMP (cAMP)/protein kinase A (PKA) cascade, a major cross-species cellular signaling pathway, is supposed as important player and regulator of depression onset by controlling synaptic plasticity, cytokinesis, transcriptional regulation and HPA axis. In the central nervous system, the cAMP-PKA cascade can dynamically shape neural circuits by enhancing synaptic plasticity, and affect K+ channels by phosphorylating Kir4.1, thereby regulating neuronal excitation. The reduction of cAMP-PKA cascade affects neuronal excitation as well as synaptic plasticity, ultimately leading to pathological outcome of depression, while activation of cAMP-PKA cascade would provide a rapid antidepressant effect. In this review, we proposed to reconsider the function of cAMP-PKA cascade, especially in the rapid antidepressant effect. Local activation or indirect activation of PKA through adjusting anchor proteins would provide new idea for acute treatment of depression.

Fungal Jasmonate as a Novel Morphogenetic Signal for Pathogenesis.

A key question that has remained unanswered is how pathogenic fungi switch from vegetative growth to infection-related morphogenesis during a disease cycle. Here, we identify a fungal oxylipin analogous to the phytohormone jasmonic acid (JA), as the principal regulator of such a developmental switch to isotropic growth and pathogenicity in the rice-blast fungus Magnaporthe oryzae. Using specific inhibitors and mutant analyses, we determined the molecular function of intrinsic jasmonates during M. oryzae pathogenesis. Loss of 12-Oxo-phytodienoic Acid (OPDA) Reductase and/or consequent reduction of jasmonate biosynthesis, prolonged germ tube growth and caused delayed initiation and improper development of infection structures in M. oryzae, reminiscent of phenotypic defects upon impaired cyclic AMP (cAMP) signaling. Chemical- or genetic-complementation completely restored proper vegetative growth and appressoria in opr1Δ. Mass spectrometry-based quantification revealed increased OPDA accumulation and significantly decreased jasmonate levels in opr1Δ. Most interestingly, exogenous jasmonate restored proper appressorium formation in pth11Δ that lacks G protein/cAMP signaling; but failed to do so in the Mitogen-activated protein (MAP) kinase mutants. Epistasis analysis placed jasmonate upstream of the cAMP pathway in rice blast. Mechanistically, intrinsic jasmonate orchestrates timely cessation of the vegetative phase and induces pathogenic development via a complex regulatory interaction with the cAMP-PKA cascade and redox signaling in rice blast.

Phosphorylation of Ser1928 mediates the enhanced activity of the L-type Ca2+ channel Cav1.2 by the β2-adrenergic receptor in neurons.

The L-type Ca2+ channel Cav1.2 controls multiple functions throughout the body including heart rate and neuronal excitability. It is a key mediator of fight-or-flight stress responses triggered by a signaling pathway involving β-adrenergic receptors (βARs), cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA). PKA readily phosphorylates Ser1928 in Cav1.2 in vitro and in vivo, including in rodents and humans. However, S1928A knock-in (KI) mice have normal PKA-mediated L-type channel regulation in the heart, indicating that Ser1928 is not required for regulation of cardiac Cav1.2 by PKA in this tissue. We report that augmentation of L-type currents by PKA in neurons was absent in S1928A KI mice. Furthermore, S1928A KI mice failed to induce long-term potentiation in response to prolonged theta-tetanus (PTT-LTP), a form of synaptic plasticity that requires Cav1.2 and enhancement of its activity by the β2-adrenergic receptor (β2AR)-cAMP-PKA cascade. Thus, there is an unexpected dichotomy in the control of Cav1.2 by PKA in cardiomyocytes and hippocampal neurons.

Unraveling the Mechanism of Dyskinesia One Transcription Factor at a Time

L-DOPA–induced dyskinesia (LID), or abnormal involuntary movements, is a common side effect of L-DOPA therapy in patients with Parkinson’s disease. When L-DOPA is initially administered, it effectively reverses akinesia caused by the loss of dopamine. However, long-term L-DOPA administration produces a hyperkinetic effect, triggering excessive movements in human patients and in animal models. The mechanisms of LID have been under intense investigation for decades, and numerous important discoveries in the past several years established the key mechanisms of LID development. Five dopamine receptor subtypes in the striatal neurons mediate the action of dopamine produced from L-DOPA. The main subtypes, D1 and D2, are expressed on the striatonigral and striatopallidal medium spiny neurons (MSNs), respectively, with a high degree of segregation. The relative contribution of the D1-dependent and D2-dependent signaling to LID has been hotly debated. However, findings demonstrating that genetic ablation of the D1, but not the D2, receptor abrogates LID (1) strongly suggest that the aberrant signaling via the D1 receptor is the main contributor. The striking molecular feature of the dopamine-depleted striatum is the hyperresponsiveness of multiple signaling pathways to acute dopaminergic stimulation. The D1 receptor in MSNs couples to the adenylyl cyclase–stimulating Golf, and in the dopamine-depleted striatum, the cyclic adenosine monophosphate (cAMP) response to L-DOPA is augmented, resulting in the increased activity of protein kinase A (PKA) and enhanced phosphorylation of PKA substrates. One of the substrates, the dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), upon phosphorylation by PKA at Thr34, acts as an inhibitor of protein phosphatase 1, further enhancing the activity of the cAMP-PKA cascade. Sensitized DARPP-32 contributes to LID because its genetic ablation or elimination of the PKA-phosphorylated residue ameliorates LID (2,3). In addition, DARPP-32 inactivation significantly attenuates the extracellular signal-regulated kinase (ERK) superactivity in the dopamine-depleted striatum (2), suggesting that the elevated cAMP signaling is an important mediator of the ERK superactivity. The ERK pathway shows very strong dopamine depletion– induced supersensitivity. The dopamine-induced hyperactivation of the ERK response is confined to the striatonigral MSNs, or direct pathway MSNs (dMSNs) (1,3), and genetic ablation of the D1 receptor prevents the supersensitive ERK response (1), reinforcing the notion of the prime role of the D1 receptor signaling in LID. The neuron-specific Ras–guanine nucleotidereleasing factor 1 (Ras-GRF1), the upstream activator of Ras, implicated in LID could serve as the mechanistic connection between the D1 receptor signaling and activation of the ERK

Role of intracellular signaling systems in regulation of erythrocyte microrheology

Erythrocyte deformability and thus the efficiency of the oxygen delivery to tissues depends on three main factors: elasticity of cell membrane, viscosity of cytoplasm, and the erythrocyte surface/volume ratio. The membrane elasticity and stability most considerably contribute to the whole cell deformability. There are experimental data suggesting that erythrocyte deformability is regulated by intracellular signaling pathways. The aim of this work was to study the role of the adenylyl cyclase–cAMP systems and intracellular calcium signaling mechanism in the regulation of erythrocyte deformability. We found that stimulator of adenylyl cyclase (AC) forskolin (10–5 M) and cell-penetrating cAMP analog dB-cAMP (5 × 10–5 M) increased erythrocyte deformability. Inhibitors of the phosphodiesterase (PDE) activity, such as isobutyl methylxanthine, vinpocetine, and cilostazol (10–5 M), also increased the erythrocyte deformability, which provides another line of evidence of the cAMP involvement in the regulation of erythrocyte deformability. On the other hand, stimulation of Ca2+ entry into erythrocytes induced by Ca2+ ionophore A23187 (3 × 10–6 M), sodium fluoride, or sodium vanadate considerably decreased the cell deformability, while blockade of the Ca2+ entry into erythrocytes by verapamil (10–5 M) increased their deformability. Mechanical stress of the erythrocyte membrane in the absence of extracellular Ca2+ did not change the deformability, while the mechanical stress applied in the presence of Ca2+ (50–200 µM) decreased deformability by 4–18%. On the whole, the obtained results suggest that for the increase in the erythrocyte deformability the AC–cAMP–PKA cascade has to be activated, while the pathway triggered by the Ca2+ entry into the cells is required to lower the erythrocyte deformability (elasticity) and hence, to increase the whole cell membrane stability. It is possible that these pathways are coordinated at the PDE level.

Wnt/β-catenin signaling modulates human airway sensitization induced by β2-adrenoceptor stimulation.

BackgroundRegular use of β2-agonists may enhance non-specific airway responsiveness. The wingless/integrated (Wnt) signaling pathways are responsible for several cellular processes, including airway inflammation and remodeling while cAMP–PKA cascade can activate the Wnt signaling. We aimed to investigate whether the Wnt signaling pathways are involved in the bronchial hyperresponsiveness induced by prolonged exposure to β2-adrenoceptor agonists in human isolated airways.MethodsBronchi were surgically removed from 44 thoracic surgery patients. After preparation, bronchial rings and primary cultures of bronchial epithelial cells were incubated with fenoterol (0.1 µM, 15 hours, 37°C), a β2-agonist with high intrinsic efficacy. The effects of inhibitors/blockers of Wnt signaling on the fenoterol-induced airway sensitization were examined and the impact of fenoterol exposure on the mRNA expression of genes interacting with Wnt signaling or cAMP–PKA cascade was assessed in complete bronchi and in cultured epithelial cells.ResultsCompared to paired controls, fenoterol-sensitization was abolished by inhibition/blockage of the Wnt/β-catenin signaling, especially the cell-surface LRP5/6 co-receptors or Fzd receptors (1 µM SFRP1 or 1 µM DKK1) and the nuclear recruitment of TCF/LEF transcriptions factors (0.3 µM FH535). Wnt proteins secretion did not seem to be involved in the fenoterol-induced sensitization since the mRNA expression of Wnt remained low after fenoterol exposure and the inactivator of Wnt secretion (1 µM IWP2) had no effect on the fenoterol-sensitization. Fenoterol exposure did not change the mRNA expression of genes regulating Wnt signaling or cAMP–PKA cascade.ConclusionsCollectively, our pharmacological investigations indicate that fenoterol-sensitization is modulated by the inhibition/blockage of canonical Wnt/β-catenin pathway, suggesting a phenomenon of biased agonism in connection with the β2-adrenoceptor stimulation. Future experiments based on the results of the present study will be needed to determine the impact of prolonged fenoterol exposure on the extra- and intracellular Wnt signaling pathways at the protein expression level.

In Vivo Study of Ethanol‐Activated Brain Protein Kinase A: Manipulations of Ca2+ Distribution and Flux

The cAMP-dependent protein kinase (PKA) signaling transduction pathway has been shown to play an important role in the modulation of several ethanol (EtOH)-induced behavioral actions. In vivo, short-term exposure to EtOH up-regulates the cAMP-signaling cascade. Interestingly, different Ca(2+) -dependent cAMP-PKA cascade mediators play a critical role in the neurobehavioral response to EtOH, being of special relevance to the Ca(2+) -dependent adenylyl cyclases 1 and 8. We hypothesize an intracellular PKA activation elicited by EtOH administration, which may be regulated by a Ca(2+) -dependent mechanism as an early cellular response. Thus, the present work aims to explore the role of Ca(2+) (internal and external) on the EtOH-activated PKA cascade. Swiss male mice received an intraperitoneal injection of EtOH (0 or 4g/kg), and brains were dissected following a temporal pattern (7, 15, 30, 45, 90, or 120minutes). Either the enzymatic PKA activity or its fingerprint was analyzed on different brain areas (cortex, hypothalamus, hippocampus, and striatum). To explore the role of Ca(2+) on the EtOH-activated PKA cascade, mice were pretreated with diltiazem (0 or 20mg/kg), dantrolene (0 or 5mg/kg), or 3,7-Dimethyl-1-(2-propynyl)xanthine (0 or 1mg/kg) 30minutes before EtOH (4g/kg) administration. After 45minutes of EtOH administration, brains were removed and dissected to measure the PKA activity or its fingerprint. Results from these experiments showed an EtOH-dependent activation of PKA in different brain areas. Manipulations involving a disruption of intracellular Ca(2+) release from the endoplasmic reticulum resulted in a decreased EtOH-induced activation of PKA. On the contrary, extracellular-to-cytoplasm Ca(2+) manipulations did not prevent the PKA activation by EtOH. Altogether, these results show the critical role of stored Ca(2+) as an intracellular mediator of different neurobiological actions of EtOH and provide further evidence of a possible new target for EtOH within the central nervous system.

PDE4B Modulates Glucocorticoid Sensitivity in Childhood Acute Lymphoblastic Leukemia

AbstractAbstract 530Although cure rates of childhood acute lymphoblastic leukemia (ALL) have improved dramatically, a substantial portion of children still relapse and the prognosis of relapsed ALL is extremely poor. Therefore, a better understanding of molecular determinants of drug resistance in ALL is imperative for the development of more efficacious and individualized therapy, particularly in the context of relapsed disease. In a recent genome-wide association study of 2,534 children with ALL, we identified that genetic variation in PDE4B– phosphodiesterase 4B–strongly influenced the risk of ALL relapse across various ALL treatment regimens (Nat Genet 2011: 43:237). While PDE4B is the predominant phosphodiesterase in lymphoid tissue and a major regulator of cyclic AMP, its role in ALL pathobiology is largely unknown. To this end, we sought to characterize the molecular mechanisms by which PDE4B modulates antileukemic drug sensitivity in ALL. We first characterized PDE4B expression in ALL blasts at diagnosis and its relationship with drug response in vivo in 3 independent cohorts of children with ALL. In 191 children with newly diagnosed ALL enrolled on the COG P9906 protocol, PDE4B expression in ALL blasts was positively correlated with minimal residual disease status at the end of remission induction (P=0.0096). Higher PDE4B expression was also associated with slower early response to induction therapy in COG 1961 (N=82, P=0.019). In 275 children with newly-diagnosed ALL enrolled on the Shanghai Children’s Medical Center ALL05 study, we determined that PDE4B2 was the predominant isoform of PDE4B in ALL blasts (P<0.0001); there was also a trend that children with poor in vivo response to the upfront single-agent prednisone treatment had higher PDE4B2 expression in the diagnostic blasts (P=0.042). In parallel, shRNA-mediated knock-down of PDE4B in a glucocorticoid-sensitive (i.e., Nalm6) and a glucocorticoid-resistant (i.e., UOCB1) ALL cell line significantly potentiated cytotoxic effects of prednisolone, whereas Nalm6 and CEM ALL cells over-expressing PDE4B2 were significantly more resistant to prednisolone compared to cells transduced with empty vectors. Sensitization to glucocorticoid was further amplified by forskolin, a stimulator of cAMP synthesis, and was concomitant with activation of PKA as determined by CREB phosphorylation, suggesting that the effects of PDE4B on glucocorticoid sensitivity involve signaling of the cAMP-PKA cascade. Importantly, PDE4B knockdown by shRNA and pharmacologic inhibition by rolipram in Nalm6 and UOCB1 cells consistently led to upregulation of BIM, a key apoptosis regulator and a critical mediator of glucocorticoid sensitivity in lymphoid cells. In both cells lines, BIM upregulation following PDE4B inhibition was enhanced by forskolin but suppressed by PKA inhibitor H89, indicating that BIM might act as a downstream effector of cAMP-PKA signaling in ALL. Finally, we evaluated effects of small molecule regulators of the PDE-cAMP pathway (namely, forskolin and rolipram) on glucocorticoid response in primary ALL cells (N=23) in vitro. Measuring IC50 by MTT assay, increased cytotoxicity was observed in 16 (70%), 8 (35%), and 17 (74%) cases, when forskolin, rolipram, or both were added to prednisolone, respectively. In conclusion, tumor expression of PDE4B at diagnosis was associated with poorer early treatment response in ALL, particularly resistance to glucocorticoids in vivo. Inhibition of PDE4B sensitized both cultured and primary ALL cells to glucocorticoids via activating the cAMP-PKA pathway and subsequent upregulation of BIM. Disclosures:No relevant conflicts of interest to declare.

High-frequency stimulation of the temporoammonic pathway induces input-specific long-term potentiation in subicular bursting cells

The subiculum (Sub) as a part of the hippocampal formation is thought to play a functional role in learning and memory. In addition to its major input from CA1 pyramidal cells, the subiculum receives input from the entorhinal cortex (EC) via the temporoammonic pathway. Thus far, synaptic plasticity in the subiculum was mainly investigated at CA1–Sub synapses. According to their spiking pattern, pyramidal cells in the subiculum were classified as bursting cells and non-bursting cells. In the present study, we demonstrate that subicular bursting cells show input-specific forms of long-term potentiation (LTP). At CA1–Sub synapses, bursting cells have been shown to express a presynaptic NMDA receptor-dependent LTP that depends on the activation of a cAMP–PKA cascade (Wozny et al., Journal of Physiology 2008). In contrast, at EC–Sub synapses the induction of LTP in bursting cells shows a high induction-threshold and relies on the activation of postsynaptic NMDA receptors, postsynaptic depolarization and postsynaptic Ca2+ influx. Each form of LTP is input-specific and fails to induce heterosynaptic plasticity. Taken together, our data suggest that distinct, input-specific mechanisms govern high frequency-induced LTP at subicular bursting cells' synapses.

Phosphorylation of GTP dissociation inhibitor by PKA negatively regulates RhoA

The cAMP-PKA cascade is a recognized signaling pathway important in inhibition of inflammatory injury events such as endothelial permeability and leucocyte trafficking, and a critical target of regulation is believed to be inhibition of Rho proteins. Here, we hypothesize that PKA directly phosphorylates GTP dissociation inhibitor (GDI) to negatively regulate Rho activity. Amino acid analysis of GDIalpha showed two potential protein kinase A (PKA) phosphorylation motifs, Ser(174) and Thr(182). Using in vitro kinase assay and mass spectrometry, we found that the purified PKA catalytic subunit phosphorylated GDIalpha-GST fusion protein and PKA motif-containing GDIalpha peptide at Ser(174), but not Thr(182). Transfection of COS-7 cells with mutated full-length GDIalpha at Ser(174) to Ala(174) (GDIalpha-Ser(174A)) abrogated the ability of cAMP to phosphorylate GDIalpha. However, mutation of Thr(182) to Ala(182) (GDIalpha-Thr(182A)) did not abrogate, and cAMP increased phosphorylation of GDIalpha to a similar extent as wild-type GDIalpha transfectants. The mutant GDIalpha-Ser(174A), but not GDIalpha-Thr(182A), was unable to prevent cAMP-mediated inhibition of Rho-dependent serum-response element reporter activity. Furthermore, the mutant GDIalpha-Ser(174A) was unable to prevent the thrombin-induced RhoA activation. Coprecipitation studies indicated that neither mutation of the PKA consensus sites nor phosphorylation alter GDIalpha binding with RhoA, suggesting that phosphorylation of Ser(174) regulated preformed GDIalpha-RhoA complexes. The findings provide strong support that the selective phosphorylation at Ser(174) by PKA is a signaling pathway in the negative regulation of RhoA activity and therefore could be a potential protective mechanism for inflammatory injury.

Effect of Cyclic Adenosine 3′,5′-Monophosphate/Protein Kinase A Pathway on Markers of Cell Proliferation in Nonfunctioning Pituitary Adenomas

Alterations in cAMP signaling have been identified as a cause of endocrine neoplasia. In particular, activating mutations of the G(s)alpha gene and protein kinase A (PKA) overactivity due to low expression of PKA regulatory subunit 1A (R1A) have been implicated in somatotroph proliferation. The objective of this study was to evaluate the effects of cAMP-PKA cascade activation in nonfunctioning pituitary adenomas (NFPA). By immunohistochemistry, R1A, R2A, and R2B expression was evaluated in cells obtained from eight surgically removed NFPA positive for gonadotropins. Cyclin D1 expression and ERK1/2 activity were analyzed under basal conditions and after cAMP-PKA cascade activation. Immunohistochemistry studies demonstrated a low R1/R2 ratio in all NFPA. Additional unbalance of R1/R2 ratio by 8-chloroadenosine cAMP (8-Cl-cAMP) and direct adenylyl cyclase stimulation by forskolin did not increase cyclin D1 expression or ERK1/2 activity in five NFPA (group 1), but even caused 74 +/- 15% and 85 +/- 13% inhibitions of cyclin D1 and ERK1/2 activity, respectively, in the remaining NFPA (group 2). Moreover, in group 2, PKA blockade by the specific inhibitor PKI increased cyclin D1 expression (96 +/- 25% over basal) and ERK1/2 activity (116 +/- 28% over basal). These data show that in contrast with what was previously observed in transformed somatotrophs, activation of the cAMP-PKA pathway did not generate proliferative signals in tumoral cells of the gonadotroph lineage, and in a subset of tumors even exerted a tonic inhibitory effect, thus confirming a different role for the cAMP-mediated pathway in promoting proliferation in the pituitary.

Dopamine-induced synaptic depression in the parabrachial nucleus is independent of CTX- and PTX-sensitive G-proteins, PKA and PLC signalling pathways

We have previously reported that dopamine (DA) depresses non-NMDA receptor-mediated glutamatergic transmission in the rat parabrachial nucleus (PBN), an interface between brainstem and forebrain that is implicated in autonomic regulation. This work examined cellular signalling pathways that might underlie this DA-induced synaptic depression. Direct activation of adenylyl cyclase with 10 μM forskolin increased the evoked EPSC but did not occlude DA-induced EPSC depression. Similarly, a preferential protein kinase A inhibitor, H-7 (10 μM), did not block DA's synaptic effects. Incubation of slices with cholera toxin (CTX; 1 μg/ml) or pertussis toxin (PTX; 0.5 μg/ml) for 20 h, procedures used to irreversibly activate or disable the G s and G i proteins, respectively, did not change DA's effects. The putative phospholipase C inhibitor, U-73122 (10 μM) and its inactive analogue U-73343 (10 μM) did not alter DA-induced reduction in the EPSCs. Alterations in signalling molecules downstream of phospholipase C including depleting internal calcium stores by thapsigargin and cyclopiazonic acid and blocking protein kinase C with chelerythrine, had no effect on DA-induced synaptic depression. Furthermore, DA's depression of the non-NMDA response was not blocked by APV, an NMDA receptor antagonist. Finally, DA depressed evoked, pharmacologically isolated NMDA receptor-mediated synaptic responses while increasing NMDA-induced inward currents in the PBN. These results indicate that DA-induced synaptic effects in the PBN are not through the activation of cholera or pertussis toxin sensitive G proteins. Furthermore, it does not employ the adenylyl cyclase–cAMP–PKA cascade, the phospholipase C signalling pathway and NMDA receptor-coupled mechanisms to depress excitatory synaptic transmission in the PBN.

Uncoupling protein 2 influences dopamine secretion in PC12h cells.

Uncoupling protein 2 (UCP2) belongs to the UCP family, and is distributed in many organs including the brain. Although UCP2 is known to be related to many functions such as the regulation of insulin secretion or the scavenging of the radicals, the role of UCP2 in the central nervous system remains unclear. In this report, rat UCP2 (rUCP2) and its mutants were overexpressed in the PC12h cells to determine the physiological roles played by UCP2 in neural cells and to elucidate the mechanisms that regulate these functions. It was found that rUCP2 was activated by the stimulation of the cAMP-protein kinase A (PKA) cascade. Moreover, the activation of rUCP2 suppressed intracellular ATP levels and inhibited the cAMP-dependent increase of dopamine secretion. Thus, UCP2 appears to be regulated by the excitatory stimulus via the cAMP-PKA cascade and serves to negatively control the synaptic output by reducing intracellular ATP levels.

Synapse Number and Synaptic Efficacy Are Regulated by Presynaptic cAMP and Protein Kinase A

The mechanisms by which neurons regulate the number and strength of synapses during development and synaptic plasticity have not yet been defined fully. This lack of fundamental knowledge in the fields of neurodevelopment and synaptic plasticity can be attributed, in part, to compensatory mechanisms by which neurons accommodate for the loss of function in their synaptic partners. This is generally achieved either by scaling up neuronal transmitter release capabilities or by enhancing the postsynaptic responsiveness. Here, we demonstrate that regulation of synaptic strength and number between identified Lymnaea neurons visceral dorsal 4 (VD4, the presynaptic cell) and left pedal dorsal 1 (LPeD1, the postsynaptic cell) requires presynaptic activation of a cAMP-PKA-dependent signal. Experimental activation of the cAMP-PKA pathway resulted in reduced synaptic efficacy, whereas inhibition of the cAMP-PKA cascade permitted hyperinnervation and an overall enhancement of synaptic strength. Because synaptic transmission between VD4 and LPeD1 does not require a cAMP-PKA pathway, our data show that these messengers may play a novel role in regulating the synaptic efficacy during early synaptogenesis and plasticity.

Distinct sensitizing effects of the cAMP-PKA second messenger cascade on rat dural mechanonociceptors.

Activation of the cAMP/protein kinase A (PKA) second messenger cascade has been implicated in the induction of mechanical hyperalgesia by inflammatory mediators. We examined the role of this cascade in mechanical sensitization of nociceptive neurons that innervate the meninges, a process thought to be involved in the pathophysiology of headache syndromes such as migraine. Single unit activity was recorded in the trigeminal ganglion from 40 mechanosensitive dural afferents (conduction velocitity: 0.3-6.6 m s(-1)) and nine mechanically insensitive dural afferents (MIAs) (conduction velocitity: 0.3-2.8 m s(-1)) while stimulating the dura with a servo force-controlled stimulator or von Frey monofilaments, respectively. Local application to the dura of dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP, 100 microM), a stable membrane-permeant cAMP analogue, produced mechanical sensitization in the majority of mechanosensitive units (19/29, 66 %). Two distinct patterns of mechanical sensitization were observed. Thirty-eight per cent of the units exhibited only a decrease in threshold (TH group), while 28 % showed only an increase in suprathreshold responses (STH group). dbcAMP also induced mechanosensitivity in the majority of MIA units (6/9, 67 %). dbcAMP-induced sensitization was blocked by the PKA inhibitors, Rp-cAMP (1 mM) and H-89 (100 microM). A mixture of inflammatory mediators induced both components of sensitization in the majority of mechanosensitive units tested. However, in each unit, PKA inhibitors blocked only one of the two effects (either TH or STH). Units that were classified as TH or STH also differed in their baseline stimulus-response slopes, thresholds and conduction velocities. These findings implicate the cAMP-PKA cascade in sensitization of dural mechanonociceptors and suggest that this cascade may produce sensitization through at least two different mechanisms operating in separate neuronal populations.

Localization and Mechanisms of Action of Cannabinoid Receptors at the Glutamatergic Synapses of the Mouse Nucleus Accumbens

Despite the role of excitatory transmission to the nucleus accumbens (NAc) in the actions of most drugs of abuse, the presence and functions of cannabinoid receptors (CB1) on the glutamatergic cortical afferents to the NAc have never been explored. Here, immunohistochemistry has been used to show the localization of CB1 receptors on axonal terminals making contacts with the NAc GABAergic neurons. Electrophysiological techniques in the NAc slice preparation revealed that cannabimimetics [WIN 55,212,2 (WIN-2) and CP55940] strongly inhibit stimulus-evoked glutamate-mediated transmission. The inhibitory actions of WIN-2 were dose-dependent (EC(50) of 293 +/- 13 nm) and reversed by the selective CB1 antagonist SR 141716A. In agreement with a presynaptic localization of CB1 receptors, WIN-2 increased paired-pulse facilitation, decreased miniature EPSC (mEPSC) frequency, and had no effect on the mEPSCs amplitude. Perfusion with the adenylate cyclase activator forskolin enhanced glutamatergic transmission but did not alter presynaptic CB1 actions, suggesting that cannabinoids inhibit glutamate release independently from the cAMP-PKA cascade. CB1 did not reduce evoked transmitter release by inhibiting presynaptic voltage-dependent Ca(2+) currents through N-, L-, or P/Q-type Ca(2+) channels, because CB1 inhibition persisted in the presence of omega-Conotoxin-GVIA, nimodipine, or omega-Agatoxin-IVA. The K(+) channel blockers 4-aminopyridine (100 micrometer) and BaCl(2) (300 micrometer) each reduced by 40-50% the inhibitory actions of WIN-2, and their effects were additive. These data suggest that CB1 receptors are located on the cortical afferents to the nucleus and can reduce glutamate synaptic transmission within the NAc by modulating K(+) channels activity.

Histamine Induces Melanogenesis and Morphologic Changes by Protein Kinase A Activation via H2 Receptors in Human Normal Melanocytes

Hyperpigmentation frequently accompanies chronic or acute inflammation. A number of inflammatory mediators have been shown to stimulate melanin synthesis in human melanocytes. Although histamine is ubiquitous as an inflammatory factor, its involvement in pigmentation remains obscure. In this work, we examined the effects of histamine on cultured human melanocytes. Treatment of human melanocytes with 0.1-10 microM histamine evoked morphologic changes and increases in tyrosinase activity. The concomitant increases in melanin content of the histamine-treated melanocytes indicated an elevation of melanin synthesis by tyrosinase activation. These stimulatory effects of histamine were completely inhibited by an H2 antagonist, famotidine, whereas H1 and H3 antagonists had no inhibitory effect whatsoever. In addition, an H2 agonist, dimaprit, induced the same degree of melanogenesis as histamine at concentrations of 0.1-10 microM. We observed an increase in the intracellular cAMP contents of human melanocytes induced by histamine via the H2 receptors. We know that this cAMP accumulation and subsequent protein kinase A activation plays a critical role in histamine-induced melanogenesis, because a specific protein kinase A inhibitor, H-89, completely suppressed these stimulatory effects of histamine, and because dibutylic cAMP, a specific protein kinase A activator, stimulated human melanocytes as potently as histamine. Taken together, we show here that histamine induces melanogenesis of human cultured melanocytes by protein kinase A activation via H2 receptors.

Multiple Facets of the Modulation of Growth by cAMP

Publisher Summary This chapter describes multiple facets of the modulation of growth by cyclic AMP (cAMP). The successes in elucidating the functions of cell-transforming proteins encoded by various oncogenes and the demonstration of antioncogenes have shed light on the assumption that cancers mostly develop from alterations of growth control mechanisms, normally involved in homeostasis, tissue repair, and development. cAMP is the first identified intracellular second messenger of hormone action. Pharmacological and genetic tools used to manipulate the CAMP-PKA cascade are summarized in the chapter. Forskolin, a plant diterpene, activates vertebrate adenylate cyclase directly and enhances its response to activated GS, thus to the receptors that regulate GS. Inhibitors of cyclic nucleotide phosphodiesterases by inhibiting the catabolism of cAMP also raise cAMP levels. It is found that due to the negative cooperativity of the phosphodiesterase system, these inhibitors are much more efficient in increasing the cAMP response to activators of adenylate cyclase than in increasing basal levels of cAMP.