Adenylate Cyclase Response Research Articles

(046) THE ROLE OF ADENOSINE IN THE RELAXANT MECHANISM OF RAT DISTAL VAGINA

Abstract Introduction Adenosine (ADO) is a purinergic neuromodulator that in the male exerts a positive effect on penile erection by controlling smooth muscle (SM) relaxation. However, its role in the relaxant/contractile pathways in the vagina is yet to be investigated. Objective The aim of this study was to characterize the effect of ADO stimulation on SM activity in distal vagina, evaluating its possible effect on the regulation of nitric oxide (NO) pathway using a validated animal model of female Sprague-Dawley rats. Methods The immunolocalization of ADORA2A and ADORA2B in distal vagina tissues was detected by immunohistological staining. In vitro contractility studies on noradrenaline (NA)-precontracted vaginal strips from intact Sprague-Dawley rats were performed; rat smooth muscle cells (rvSMCs) from distal vagina of intact animals were stimulated with ADO for cyclic adenosine monophosphate (cAMP) quantification. mRNA (messenger ribonucleic acid) isolated from vaginal tissue was analyzed by semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction. Results All ADORAs mRNA were expressed in the vaginal tissue, with a significant prevalence of ADORA2B; furthermore, ADORA2A and ADORA2B immunolocalization highlighted that both receptors are highly expressed in the epithelium, in blood capillaries of the vascular bed and in the smooth muscle bundles of vagina wall. In vitro ADO stimulation induced a dose-dependent relaxation (IC50=31.9±2.38 μM), which was significantly reduced by the administration of NO-synthase inhibitor L-NAME; similar effects were observed after the mechanical ablation of endothelium and in vitro administration of the soluble guanylate cyclase inhibitor ODQ, highlighting the pivotal contribution of NO signaling to the ADO relaxant activity. In vitro stimulation with increasing doses of the selective ADO receptor ADORA2A agonist CGS2-1680 induced a dose-dependent relaxation (IC50=132.6±3.47 nM); a similar but less potent relaxant effect was observed with ADORA2B selective agonist BAY60-6583. The relaxing effect induced by ADO on NA-precontracted vaginal strips was completely counteracted by the stimulation with the re-spectively selective ADORA2A and ADORA2B antagonists SHC-442416 and PSB603; a similar effect was induced by ADORA1 selective antagonist SLV320, but not by ADORA3 selective antagonists VUF5574. The mRNA relative expression of enzymes regulating the ADO turnover [adenosine deami-nase (ADA), 5′-nucleotidase (5NTD), AMP deaminase type 1 and type 2 (AMPD1, AMPD2) and adenosine kinase (ADK)] showed a higher expression of genes regulating the de novo ADO/cAMP syn-thesis (ADK, 5NTD), compared to those acting on ADO degradation (ADA, AMPD1, AMPD2). Finally, stimulation of rvSMCs with increasing doses of ADO induced a significant production of intracellular cAMP after 3 minutes (1 and 10 μM). Conclusions These data show that ADO acts as an important relaxant modulator of the vagina through the activation of its receptors, specifically ADORA2A, and the consequent cAMP-dependent adenylate cyclase re-sponse. However, in vitro studies show that ADO also acts trough the NO-dependent pathway. These results therefore demonstrate for the first time a new relaxant pathway in distal vagina, ADO signaling, that results deeply intertwined with the well-known NO pathway. This finding unravels new biological mechanisms underlying vagina physiology that need to be further studied in future research. Disclosure No.

Purification and Characterization of a Stable, Membrane-Associated Peptidoglycan Responsive Adenylate Cyclase LRR Domain from Human Commensal Candida albicans.

The evolutionarily conserved leucine rich repeat (LRR) protein domain is a unique structural motif found in many viral, bacterial, archaeal, and eukaryotic proteins. The LRR domain serves many roles, including being a signaling domain and a pathogen recognition receptor. In the human innate immune system, it serves an essential role by recognizing fragments of bacterial cell walls. Interestingly, the human fungal pathogen Candida albicans also uses an LRR domain-containing protein, Cyrp1, to sense bacterial cell wall fragments. However, the dynamics of signaling and detection of bacterial peptidoglycan fragments by the LRR of Cyr1p remains poorly characterized. Here we develop optimal recombinant expression workflows and provide characterization of the entire region of the LRR domain of Cyr1p as a peripheral membrane protein. Using a newly designed peptidoglycan enrichment bead assay, we demonstrate that this domain can bind bacterial peptidoglycan fragments under native conditions. The new membrane-associated Cyr1p-LRR construct sets the stage for the development of antifungal agents via high-throughput campaigns to inhibit cell wall-Cyr1p interactions.

Synthesis of the Mechanisms of Opioid Tolerance: Do We Still Say NO?

The use of morphine as a first-line agent for moderate-to-severe pain is limited by the development of analgesic tolerance. Initially opioid receptor desensitization in response to repeated stimulation, thought to underpin the establishment of tolerance, was linked to a compensatory increase in adenylate cyclase responsiveness. The subsequent demonstration of cross-talk between N-methyl-D-aspartate (NMDA) glutamate receptors and opioid receptors led to the recognition of a role for nitric oxide (NO), wherein blockade of NO synthesis could prevent tolerance developing. Investigations of the link between NO levels and opioid receptor desensitization implicated a number of events including kinase recruitment and peroxynitrite-mediated protein regulation. Recent experimental advances and the identification of new cellular constituents have expanded the potential signaling candidates to include unexpected, intermediary compounds not previously linked to this process such as zinc, histidine triad nucleotide-binding protein 1 (HINT1), micro-ribonucleic acid (mi-RNA) and regulator of G protein signaling Z (RGSZ). A further complication is a lack of consistency in the protocols used to create tolerance, with some using acute methods measured in minutes to hours and others using days. There is also an emphasis on the cellular changes that are extant only after tolerance has been established. Although a review of the literature demonstrates a lack of spatio-temporal detail, there still appears to be a pivotal role for nitric oxide, as well as both intracellular and intercellular cross-talk. The use of more consistent approaches to verify these underlying mechanism(s) could provide an avenue for targeted drug development to rescue opioid efficacy.

62 Peripheral transcriptome response to embryo mortality in Holstein cows

Exactly how failure through conceptus-endometrial signalling on Day 16 of pregnancy causes early embryo mortality (EM) is yet unknown. It was hypothesised that EM pregnancies are associated with an impaired endocrine action of interferon tau (IFNT) from the conceptus on the corpus luteum (CL) and peripheral blood mononuclear cells (PBMC), in addition to paracrine failure in preventing the upregulation of oestradiol-mediated luteolytic signals in the endometrium. Cows not exposed to semen served as controls on Day 16 of the oestrous cycle (EC; n=7). Conceptuses were flushed on Day 16 of pregnancy and were then sorted based on quality. Normal (N, n=9) conceptuses were elongated and translucent, whereas EM conceptuses (n=6) lacked extensive elongation and were pink, red, or opaque. The RNA extracted from conceptuses, endometrium, CL, and PBMC was submitted to RNA-Seq analysis. Trimmed and aligned sequences were analysed in R using the DESEqn 2 package. Data were submitted to ingenuity pathway analysis with fold change ≥1.5 and P ≤ 0.05. Previously, we reported that N were longer (P<0.006) than EM conceptuses. The IFNT mRNA transcript raw counts (from RNA-Seq) were greater (P<0.0001) in N than in EM conceptuses. The IFNT protein concentrations (enzyme-linked immunosorbent assay) in uterine flushings were greater (P<0.004) in N compared with EC and tended (P<0.07) to be greater in EM compared with EC cows. Similarly, interferon stimulated gene 15 (ISG15) mRNA raw counts were greater (P<0.003) in N and tended (P<0.09) to be greater in EM endometrium compared with EC endometrium. Ingenuity pathway analysis revealed that T helper (Th1) cell activation, a key canonical pro-inflammatory pathway, was upregulated in EM compared with N conceptuses. Among the 15 upregulated endometrial genes in EM compared with N, 9 were associated with oestradiol action. In the current study, 7/9 PBMC (ISG15, MX2, OAS1, OAS2, IFI44, IFI6, and DDX58) and 3/3 CL (ZBP1, OAS1, and MX1) genes (ISGs) were upregulated in N versus EC and belonged to the interferon signalling canonical pathway. Key upstream regulators were IFNAR2 and IRF7 for PBMC and type I IFNs and ISG15 for CL. There were no differences in EM compared with N genes in PBMC. Conversely, 10 genes in the CL were upregulated for EM compared with N and aligned to pro-inflammatory/calcium binding (i.e. CAPS2, S100A12) and inhibition of adenylate cyclase (HTR1B) responses. The primary canonical pathways were granulocyte adhesion and diapedesis, and hypercytokinemia. Moroever, key upstream regulators were MAPK and TNF. In conclusion, ISGs are upregulated peripherally in PBMC and CL in response to normal pregnancy. When conceptuses are compromised, pro-inflammatory and calcium-mediated responses that promote a localised “cytokine storm” immune response are upregulated and may be coupled with disruption of steroidogenesis in the CL, which is mediated by adenylate cyclase. This research was supported by USDA-Agriculture and Food Research Initiative (AFRI)-National Needs Graduate and Postgraduate Fellowship (NNF) 2016-38420-25289 and the W3112 USDA-AFRI Ruminant Reproduction Project.

Striatal ERK is Activated in Response to L‐DOPA Administration in a Knock‐in Mouse Model of DOPA‐Responsive Dystonia

Abnormal dopamine (DA) neurotransmission is associated with many dystonic disorders; however, the precise nature of the defects in DA transmission that result in dystonia are not known. To address this question, we used a knock‐in mouse model of L‐DOPA‐responsive dystonia (DRD). DRD mice display the core features of the human disorder, including reduced striatal DA levels and abnormal movement that improves in response to L‐DOPA, the precursor to DA. In the striatum, DA promotes movement by activating D1 receptors (D1Rs) expressed on direct pathway spiny projection neurons (SPNs) and inhibits movement by activating D2 receptors (D2Rs) expressed on indirect pathway SPNs. D1Rs stimulate adenylate cyclase (AC) activity and D2Rs inhibit AC; however, in DRD mice, D1Rs display an exaggerated AC response to D1 agonist stimulation and D2Rs exhibit a change in valence whereby D2R agonists increase AC. Our objective was to determine signaling alterations in both SPN subtypes downstream of AC, specifically phosphorylated ERK (p‐ERK), that contribute to the alleviation of dystonia by L‐DOPA to identify downstream targets that may serve as novel therapeutic targets. DRD and control mice were subcutaneously injected with L‐DOPA (10 mg/kg), the D1R agonist SKF 81297 (0.2 mg/kg), the D2R agonist quinpirole (0.1 mg/kg), or saline. Mice were perfused 45 min after drug treatment and striatal sections were immunostained for p‐ERK. DRD mice showed a significant and robust increase in p‐ERK levels in the dorsal striatum in response to L‐DOPA treatment compared to controls and saline‐treated mice. In contrast, SKF 81297 or quinpirole treatment evoked only modest changes in p‐ERK. The L‐DOPA specific increase in p‐ERK suggests that D1R and D2R co‐activation may regulate p‐ERK and/or that p‐ERK may serve as a coincidence detector for convergent DA and glutamatergic signaling that occurs when endogenous DA signaling is restored in the striatum. Future studies are aimed to elucidate cell‐type specific mechanisms underlying increased p‐ERK in response to L‐DOPA in DRD mice to refine downstream signaling mechanisms that are instrumental to the therapeutic effects.Support or Funding InformationThis work was supported by the United States National Institute of Health grant R01 NS088528 and T32 NS007480‐17.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intérêt de l’analgésie locorégionale continue (ALRC) dans la réhabilitation du déficit neurologique douloureux. Expérience d’une coopération anesthésiste et centre de rééducation

Antisera were raised in rabbits against synthetic peptides corresponding to sequences of the guanine nucleotide binding proteins Gi1, Gi2, Gi3 and Go. These and previously described antisera were used to identify different G-proteins in Western blots of human adipocyte plasma membranes and to quantify them using purified recombinant α subunits as standards. Go was shown to be absent or ⪡ 15 pmol/mg of protein. A band stained by a previously characterized Go antiserum is suggested to be due to nonspecific staining of Gi1. Gi1 and Gi2 were the major G-proteins. Gi1 was present at concentrations of 52 and 18 pmol/mg of protein in lean and obese subjects, respectively, and the concentration was negatively correlated with the body mass index. Gi2 concentrations averaged 64 pmol/mg of protein and there was no correlation to the body mass index. Gi3 levels were much lower (⪡ 13 pmol/mg of protein) and the presence of this protein could not be demonstrated with certainty. The concentrations of Gi1 and Gi2 are thus over two orders of magnitude higher than those of the receptors whose effects they mediate. The low concentration of Gi1 in adipocyte plasma membranes of obese subjects could in part explain the attenuated inhibitory responses of adenylate cyclase in isolated fat cells in obesity.

PGD2 and PGE2 regulate gene expression of Prx 6 in primary macrophages via Nrf2

Peroxiredoxin 6 (Prx 6) is a bifunctional enzyme with both glutathione peroxidase and acidic Ca2+-independent phospholipase A2 activities. We have recently shown that exposure of murine bone marrow-derived macrophages to LPS and IFN-γ leads to induction of COX-2 expression and secretion of PGE2, up-regulating Prx 6 mRNA levels. This study was designed to investigate various prostaglandins (PGs) for their ability to induce gene expression of Prxs, in particular Prx 6, and to determine the underlying regulatory mechanisms. We provide evidence that both conventional and cyclopentenone PGs enhance Prx 6 mRNA expression. Treatment with either activators or inhibitors of adenylate cyclase as well as cAMP analogs indicated that Prx 6 gene expression is regulated by adenylate cyclase in response to PGD2 or PGE2. Furthermore, our study revealed that JAK2, PI3K, PKC, and p38 MAPK contribute to the PGD2- or PGE2-dependent Prx 6 induction. Using stimulated macrophages from Nrf2-deficient mice or activators of Nrf2 and PPARγ, we found that Nrf2, but not PPARγ, is involved in the PG-dependent increase in Prx 6 mRNA expression. In summary, our data suggest multiple signaling pathways of Prx 6 regulation by PGs and identified Nrf2 as a critical player mediating transcriptional induction.

Chronic treatment with electroconvulsive shock prevents the salbutamol-induced hypoactivity in rats.

Several lines of evidence (binding studies, reduced responsiveness of brain adenylate cyclase to noradrenergic stimulation) indicate that chronic treatment with electroconvulsive shock (ECS) induces down-regulation of central beta-adrenoceptors. The effect of acute and chronic (10 days) treatment with ECS on salbutamol-induced suppression of exploratory activity in rats has been examined. This effect was prevented by chronic but not by acute treatment with ECS. Chronic treatment with ECS did not affect exploratory activity. The salbutamol-induced hypoactivity is mediated through central beta-adrenoceptors (antagonistic effect of (-)-propranolol but not (+)-propranolol or practolol), so the results may be regarded as functional evidence at the behavioral level for the down-regulation of beta-adrenoceptors produced by chronic treatment with ECS.

Characterization of the norepinephrine-activation of adenylate cyclase suggests a role in memory affirmation pathways: Overexposure to epinephrine inactivates adenylate cyclase, a causal pathway for stress-pathologies

Incubation with noradrenaline (norepinephrine) of isolated membranes of rat's brain corpus striatum and cortex, showed that ionic-magnesium (Mg 2+) is required for the neurotransmitter activatory response of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing) (EC 4.6.1.1)], AC. An Mg 2+-dependent response to the activatory effects of adrenaline, and subsequent inhibition by calcium, suggest capability for a turnover, associated with cyclic changes in membrane potential and participation in a short-term memory pathway. In the cell, the neurotransmitter by activating AC generates intracellular cyclic AMP. Calcium entrance in the cell inhibits the enzyme. The increment of cyclic AMP activates kinase A and their protein phosphorylating activity, allowing a long-term memory pathway. Hence, consolidating neuronal circuits, related to emotional learning and memory affirmation. The activatory effect relates to an enzyme–noradrenaline complex which may participate in the physiology of the fight or flight response, by prolonged exposure. However, the persistence of an unstable enzyme complex turns the enzyme inactive. Effect concordant, with the observation that prolonged exposure to adrenaline, participates in the etiology of stress triggered pathologies. At the cell physiological level AC responsiveness to hormones could be modulated by the concentration of chelating metabolites. These ones produce the release of free ATP 4−, a negative modulator of AC and the Mg 2+ activated insulin receptor tyrosine kinase (IRTK), thus, allowing an integration of the hormonal response of both enzymes by ionic controls. This effect could supersede the metabolic feedback control by energy charge. Accordingly, maximum hormonal response of both enzymes, to high Mg 2+ and low free ATP 4−, allows a correlation with the known effects of low caloric intake increasing average life expectancy.

Activated Alleles of the Schizosaccharomyces pombe gpa2 + Gα Gene Identify Residues Involved in GDP-GTP Exchange

The Schizosaccharomyces pombe glucose/cyclic AMP (cAMP) signaling pathway includes the Gpa2-Git5-Git11 heterotrimeric G protein, whose Gpa2 Galpha subunit directly binds to and activates adenylate cyclase in response to signaling from the Git3 G protein-coupled receptor. To study intrinsic and extrinsic regulation of Gpa2, we developed a plasmid-based screen to identify mutationally activated gpa2 alleles that bypass the loss of the Git5-Git11 Gbetagamma dimer to repress transcription of the glucose-regulated fbp1(+) gene. Fifteen independently isolated mutations alter 11 different Gpa2 residues, with all but one conferring a receptor-independent activated phenotype upon integration into the gpa2(+) chromosomal locus. Biochemical characterization of three activated Gpa2 proteins demonstrated an increased GDP-GTP exchange rate that would explain the mechanism of activation. Interestingly, the amino acid altered in the Gpa2(V90A) exchange rate mutant protein is in a region of Gpa2 with no obvious role in Galpha function, thus extending our understanding of Galpha protein structure-function relationships.

Inhibition of heat shock protein 90 attenuates adenylate cyclase sensitization after chronic morphine treatment

Cellular adaptations to chronic opioid treatment result in enhanced responsiveness of adenylate cyclase and an increase in forskolin- or agonist-stimulated cAMP production. It is, however, not known whether chaperone molecules such as heat shock proteins contribute to this adenylate cyclase sensitization. Here, we report that treatment of cells with geldanamycin, an inhibitor of heat shock protein 90 (Hsp90), led to effective attenuation of morphine-induced adenylate cyclase sensitization. In SK-N-SH human neuroblastoma cells, morphine significantly increased RNA transcript and protein levels of type I adenylate cyclase, leading to sensitization. Whole-genome tiling array analysis revealed that cAMP response element-binding protein, an important mediator for cellular adaptation to morphine, associated with the proximal promoter of Hsp90AB1 not only in SK-N-SH cells but also in rat PC12 and human embryonic kidney cells. Hsp90AB1 transcript and protein levels increased significantly during morphine treatment, and co-application of geldanamycin (0.1–10 nM) effectively suppressed the increase in forskolin-activated adenylate cyclase activation by 56%. Type I adenylate cyclase, but not Hsp90AB1, underwent significant degradation during geldanamycin treatment. These results indicate that Hsp90 is a new pharmacological target for the suppression of adenylate cyclase sensitization induced by chronic morphine treatment.

CAMP activation by PACAP/VIP stimulates IL‐6 release and inhibits osteoblastic differentiation through VPAC2 receptor in osteoblastic MC3T3 cells

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the glucagon/vasoactive intestinal peptide (VIP) superfamily, stimulates cyclic AMP accumulation initiating a variety of biological processes such as: neurotropic actions, immune and pituitary function, learning and memory, catecholamine biosynthesis and regulation of cardiopulmonary function. Both osteoclasts and osteoblasts have been shown to express receptors for PACAP/VIP implicated in their role in bone metabolism. To further understand the role of PACAP/VIP family in controlling bone metabolism, we investigated differentiation model of MC3T3-E1 cells, an osteoblastic cell line derived from mouse calvaria. Quantitative RT-PCR analysis demonstrated that MC3T3-E1 cells expressed only VPAC2 receptor and its expression was upregulated during osteoblastic differentiation, whereas VPAC1 and PAC1 receptors were not expressed. Consistent with expression of receptor subtype, both PACAP and VIP stimulate cAMP accumulation in a time- and dose-dependent manner with the similar potency in undifferentiated and differentiated cells, while Maxadilan, a specific agonist for PAC1-R, did not. Furthermore, downregulation of VPAC2-R by siRNA completely blocked cAMP response mediated by PACAP and VIP. Importantly, PACAP/VIP as well as forskolin markedly suppressed the induction of alkaline phosphatase mRNA upon differentiation and the pretreatment with 2',5'-dideoxyadenosine, a cAMP inhibitor, restored its inhibitory effect of PACAP. We also found that PACAP and VIP stimulated IL-6 release, a stimulator of bone resorption, and VPAC2-R silencing inhibited IL-6 production. Thus, PACAP/VIP can activate adenylate cyclase response and regulate IL-6 release through VPAC2 receptor with profound functional consequences for the inhibition of osteoblastic differentiation in MC3T3-E1 cells.

Cell adhesion modulates 5-HT 1D and P2Y receptor signal trafficking differentially in LTK-8 cells

In this study, we investigated adhesion-induced changes in cellular responses to serotonin 5-HT 1D and purinergic P2Y receptor stimulation. We demonstrated that detachment of LTK-8 cells increased 5-HT 1D receptor-mediated intracellular Ca 2+ and extracellular signal regulated kinase (ERK) phosphorylation responses without affecting the adenylate cyclase response. Additionally, detachment enabled 5-HT 1D receptor stimulation to inhibit P2Y receptor-induced [Ca 2+] i mobilization. Such a cross talk between the two receptor systems was not observed in attached cells. P2Y receptor-induced Ca 2+ response was insensitive to adhesion state of the cells, while ERK phosphorylation response was enhanced upon detachment. Integrity of the actin cytoskeleton did not appear to play a role in adhesion sensitivity of 5-HT 1D-mediated responses, as treatment of attached cells with cytochalasin D did not mimic detachment-induced effects. Effects of detachment were reversed immediately after re-attachment of the suspended cells on poly- l-lysine coated cover slips, suggesting that the involvement of integrins or focal adhesion complexes is unlikely. Taken collectively, our results demonstrate that not only cellular responses induced by different G protein-coupled receptors, but also different responses induced by a particular G protein-coupled receptor, can be affected differentially by the adhesion status of cells. This suggests an important role for cell adhesion in controlling the coupling of a single G protein-coupled receptor to different intracellular responses.

Protein kinase C is a common component of CGRP receptor desensitization induced by distinct agonists

The calcitonin gene-related peptide (CGRP) is a neuropeptide involved in vasodilation and other physiological functions throughout the body. The receptor for CGRP has been cloned and well studied, but the mechanism of CGRP receptor desensitization has not been fully elucidated. In the present study, we evaluated the kinetics for agonist-mediated desensitization of the adenylate cyclase response in human neuroblastoma SK-N-MC cells. Distinct CGRP receptor agonists were used, including α and β isoforms of CGRP, the linearized derivative cys(Et) 2,7αCGRP, adrenomedullin, and adrenomedullin 2. βCGRP was 4–600 times more potent at desensitizing the cAMP production as compared to the other receptor-activating ligands, and all of the desensitization effects were blocked by a CGRP receptor antagonist. Although the different agonists vary in their ability to induce functional desensitization, a pretreatment/washout sequence with each peptide was able to reduce the activation potency of the other members of the calcitonin/CGRP peptide family. Next we tested whether the desensitizing effects of the distinct peptides involve protein kinase C (PKC) or protein kinase A (PKA). A PKC inhibitor, Ro 31-8220, concentration-dependently reduced the desensitization induced by the 5 CGRP receptor agonists, while having little effect on their desensitization potencies. PKA inhibitors KT-5720 and H-89, on the other hand, showed little effect on the induced level of desensitization. The findings indicate that functional desensitization is produced by distinct peptides acting through the active site of CGRP receptors, and involves the activation of PKC as a common component necessary to achieve maximal desensitization of receptor signaling.

β-Adrenoceptor signaling in regenerating skeletal muscle after β-agonist administration

Stimulating the beta-adrenoceptor (beta-AR) signaling pathway can enhance the functional repair of skeletal muscle after injury, but long-term use of beta-AR agonists causes beta-AR downregulation, which may limit their therapeutic effectiveness. The aim was to examine beta-AR signaling during early regeneration in rat fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles after bupivacaine injury and test the hypothesis that, during regeneration, beta-agonist administration does not cause beta-AR desensitization. Rats received either the beta-AR agonist fenoterol (1.4 mgxkg(-1)xday(-1) ip) or saline for 7 days postinjury. Fenoterol reduced beta-AR density in regenerating soleus muscles by 42%. Regenerating EDL muscles showed a threefold increase in beta-AR density, and, again, these values were 43% lower with fenoterol treatment. An amplified adenylate cyclase (AC) response to isoproterenol was observed in cell membrane fragments from EDL and soleus muscles 7 days postinjury. Fenoterol attenuated this increase in regenerating EDL muscles but not soleus muscles. beta-AR signaling mechanisms were assessed using AC stimulants (NaF, forskolin, and Mn(2+)). Although beta-agonist treatment reduces beta-AR density in regenerating muscles, these muscles can produce large cAMP responses relative to healthy (uninjured) muscles. Desensitization of beta-AR signaling in regenerating muscles is prevented by altered rates of beta-AR synthesis and/or degradation, changes in G protein populations and coupling efficiency, and altered AC activity. These mechanisms have important therapeutic implications for modulating beta-AR signaling to enhance muscle repair after injury.

Effects of mood stabilizers on the inhibition of adenylate cyclase via dopamine D2‐like receptors

The mood stabilizing drugs lithium, carbamazepine and valproate modulate brain adenosine monophosphate (cAMP) levels, which are assumed to be elevated in bipolar disorder patients. The aim of this work was to investigate how these three mood stabilizing agents affect the regulation of cAMP levels by dopamine D(2)-like receptors in vitro in rat cortical neurons in culture and in vivo in the rat prefrontal cortex. The production of cAMP was measured in the cultured cortical neurons or in microdialysis samples collected from the prefrontal cortex of freely moving rats using the [8-(3)H] and [(125)I] radioimmunoassay kits. In vitro and in vivo data showed that the treatment with the mood stabilizing drugs had no effect on basal cAMP levels in vitro, but had differential effects in vivo. Direct stimulation of adenylate cyclase (AC) with forskolin increased cAMP levels both in vitro and in vivo, and this effect was significantly inhibited by all three mood stabilizers. Activation of dopamine D(2)-like receptors with quinpirole partially inhibited forskolin-induced increase in cAMP in untreated cultures, but no effect was observed in cortical neuron cultures treated with the mood stabilizing drugs. Similar results were obtained by chronic treatment with lithium and valproate in the prefrontal cortex in vivo. However, surprisingly, in carbamazepine-treated rats the activation of dopamine D(2)-like receptors enhanced the responsiveness of AC to subsequent activation by forskolin, possibly as a consequence of chronic inhibition of the activity of the enzyme. It was shown that each of these drugs affects basal- and forskolin-evoked cAMP levels in a distinct way, resulting in differential responses to dopamine D(2)-like receptors activation.

Receptor Activity-modifying Protein (RAMP) Isoform-specific Regulation of Adrenomedullin Receptor Trafficking by NHERF-1

Receptor activity-modifying proteins (RAMPs 1-3) are single transmembrane accessory proteins critical to various G-protein coupled receptors for plasma membrane expression and receptor phenotype. A functional receptor for the vasodilatory ligand, adrenomedullin (AM), is comprised of RAMP2 or RAMP3 and calcitonin receptor-like receptor (CRLR). It is now known that RAMP3 protein-protein interactions regulate the recycling of the AM2 receptor. The major aim of this study was to identify other interaction partners of RAMP3 and determine their role in CRLR-RAMP3 trafficking. Trafficking of G-protein-coupled receptors has been shown to be regulated by the Na+/H+ exchanger regulatory factor-1 (NHERF-1), an adaptor protein containing two tandem PSD-95/Discs-large/ZO-1 homology (PDZ) domains. In HEK 293T cells expressing the AM2 receptor, the complex undergoes agonist-induced desensitization and internalization. However, in the presence of NHERF-1, although the AM receptor (CRLR/RAMP3) undergoes desensitization, the internalization of the receptor complex is blocked. Overlay assays and mutational analysis indicated that RAMP3 and NHERF-1 interact via a PDZ type I domain on NHERF-1. The internalization of the CRLR-RAMP complex was not affected by NHERF-1 when CRLR was co-expressed with RAMP1 or RAMP2. Mutation of the ezrin/radixin/moesin (ERM) domain on NHERF-1 indicated that NHERF-1 inhibits CRLR/RAMP3 complex internalization by tethering the complex to the actin cytoskeleton. When examined in a primary culture of human proximal tubule cells endogenously expressing the CRLR-RAMP3 complex and NHERF-1, the CRLR-RAMP complex desensitizes but is unable to internalize upon agonist stimulation. Knock-down of either RAMP3 or NHERF-1 by RNA interference technology enabled agonist-induced internalization of the CRLR-RAMP complex. These results, using both endogenous and overexpressed cellular models, indicate a novel function for NHERF-1 and RAMP3 in the internalization of the AM receptor and suggest additional regulatory mechanisms for receptor trafficking.

Novel Function for Receptor Activity-modifying Proteins (RAMPs) in Post-endocytic Receptor Trafficking

RAMPs (1-3) are single transmembrane accessory proteins crucial for plasma membrane expression, which also determine receptor phenotype of various G-protein-coupled receptors. For example, adrenomedullin receptors are comprised of RAMP2 or RAMP3 (AM1R and AM2R, respectively) and calcitonin receptor-like receptor (CRLR), while a CRLR heterodimer with RAMP1 yields a calcitonin gene-related peptide receptor. The major aim of this study was to determine the role of RAMPs in receptor trafficking. We hypothesized that a PDZ type I domain present in the C terminus of RAMP3, but not in RAMP1 or RAMP2, leads to protein-protein interactions that determine receptor trafficking. Employing adenylate cyclase assays, radioligand binding, and immunofluorescence microscopy, we observed that in HEK293 cells the CRLR-RAMP complex undergoes agonist-stimulated desensitization and internalization and fails to resensitize (i.e. degradation of the receptor complex). Co-expression of N-ethylmaleimide-sensitive factor (NSF) with the CRLR-RAMP3 complex, but not CRLR-RAMP1 or CRLR-RAMP2 complex, altered receptor trafficking to a recycling pathway. Mutational analysis of RAMP3, by deletion and point mutations, indicated that the PDZ motif of RAMP3 interacts with NSF to cause the change in trafficking. The role of RAMP3 and NSF in AM2R recycling was confirmed in rat mesangial cells, where RNA interference with RAMP3 and pharmacological inhibition of NSF both resulted in a lack of receptor resensitization/recycling after agonist-stimulated desensitization. These findings provide the first functional difference between the AM1R and AM2R at the level of post-endocytic receptor trafficking. These results indicate a novel function for RAMP3 in the post-endocytic sorting of the AM-R and suggest a broader regulatory role for RAMPs in receptor trafficking.

Activation of a novel PKC isoform synergistically enhances D 2L dopamine receptor-mediated sensitization of adenylate cyclase type 6

Despite acutely inhibiting adenylate cyclase, prolonged activation of Gα i/o-coupled receptors leads to a subsequent heterologous sensitization of adenylate cyclase responsiveness. Recently, protein kinase signaling and phosphorylation have been implicated in the sensitization of adenylate cyclase type 6 (AC6). To examine the sensitization specifically of AC6, we constructed human embryonic kidney cells (HEK293) cells stably expressing AC6 and the Gα i/o-coupled D 2L dopamine receptor. In contrast to observations in δ-opioid-expressing Chinese hamster ovary (CHO) cells that express endogenous AC6 and AC7, neither protein kinase C (PKC) nor tyrosine kinase inhibitors attenuated D 2L receptor-mediated sensitization of AC6. Inhibition of Raf1 modestly inhibited the magnitude of D 2L receptor-induced sensitization of AC6; however, activation of PKC robustly enhanced D 2L receptor-mediated AC6 sensitization in a Raf1-dependent manner. These data indicate that, although PKC and Raf1 are not required for sensitization, activation of the PKC-Raf1 pathway robustly potentiated D 2L receptor-mediated sensitization of AC6.

Involvement of adenylate cyclase and tyrosine kinase signaling pathways in response of crayfish stretch receptor neuron and satellite glia cell to photodynamic treatment

Neuroglial interactions are most profound during development or damage of nerve tissue. We studied the responses of crayfish stretch receptor neurons (SRN) and satellite glial cells to photosensitization with sulfonated aluminum phthalocyanine Photosens. Although Photosens was localized mainly in the glial envelope, neurons were very sensitive to photodynamic treatment. Photosensitization gradually inhibited and then abolished neuron activity. Neuronal and glial nuclei shrank. Some neurons and glial cells lost the integrity of the plasma membrane and died through necrosis after the treatment. The nuclei of other glial cells but not neurons become fragmented, indicating apoptosis. The number of glial nuclei around neuron soma increased, probably indicating proliferation for enhanced neuron protection. Adenylate cyclase (AC) inhibition by MDL-12330A, or tyrosine kinase (TK) inhibition by genistein, shortened neuron lifetime, whereas AC activation by forskolin or protein tyrosine phosphatases (PTP) inhibition by sodium orthovanadate prolonged neuronal activity. Therefore, cAMP and phosphotyrosines produced by AC and TK, respectively, protected SRN against photoinactivation. AC inhibition reduced photodamage of the plasma membrane and subsequent necrosis in neuronal and glial cells. AC activation prevented apoptosis in photosensitized glial cells and stimulated glial proliferation. TK inhibition protected neurons but not glia against photoinduced membrane permeabilization and subsequent necrosis whereas PTP inhibition more strongly protected glial cells. Therefore, both signaling pathways involving cAMP and phosphotyrosines might contribute to the maintenance of neuronal activity and the integrity of the neuronal and glial plasma membranes. Adenylate cyclase but not phosphotyrosine signaling pathways modulated glial apoptosis and proliferation under photooxidative stress.