Regulation Of Adenylyl Cyclase Activity Research Articles

P-Rex1 Controls Sphingosine 1-Phosphate Receptor Signalling, Morphology, and Cell-Cycle Progression in Neuronal Cells

P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates Rac-type small G proteins in response to the stimulation of a range of receptors, particularly G protein-coupled receptors (GPCRs), to control cytoskeletal dynamics and other Rac-dependent cell responses. P-Rex1 is mainly expressed in leukocytes and neurons. Whereas its roles in leukocytes have been studied extensively, relatively little is known about its functions in neurons. Here, we used CRISPR/Cas9-mediated P-Rex1 deficiency in neuronal PC12 cells that stably overexpress the GPCR S1PR1, a receptor for sphingosine 1-phosphate (S1P), to investigate the role of P-Rex1 in neuronal GPCR signalling and cell responses. We show that P-Rex1 is required for the S1P-stimulated activation of Rac1 and Akt, basal Rac3 activity, and constitutive cAMP production in PC12-S1PR1 cells. The constitutive cAMP production was not due to increased expression levels of major neuronal adenylyl cyclases, suggesting that P-Rex1 may regulate adenylyl cyclase activity. P-Rex1 was required for maintenance of neurite protrusions and spreading in S1P-stimulated PC12-S1PR1 cells, as well as for cell-cycle progression and proliferation. In summary, we identified novel functional roles of P-Rex1 in neuronal Rac, Akt and cAMP signalling, as well as in neuronal cell-cycle progression and proliferation.

DARPP-32 in the orchestration of responses to positive natural stimuli.

Dopamine- and cAMP-regulated phosphoprotein (Mr 32kDa, DARPP-32) is an integrator of multiple neuronal signals and plays a crucial role particularly in mediating the dopaminergic component of the systems involved in the evaluation of stimuli and the ensuing elaboration of complex behavioral responses (e.g., responses to reinforcers and stressors). Dopamine neurons can fire tonically or phasically in distinct timescales and in specific brain regions to code different behaviorally relevant information. Dopamine signaling is mediated mainly through the regulation of adenylyl cyclase activity, stimulated by D1-like or inhibited by D2-like receptors, respectively, that modulates cAMP-dependent protein kinase (PKA) function. The activity of DARPP-32 is finely regulated by its phosphorylation at multiple sites. Phosphorylation at the threonine (Thr) 34 residue by PKA converts DARPP-32 into an inhibitor of protein phosphatase 1, while the phosphorylation at the Thr75 residue turns it into an inhibitor of PKA. Thus, DARPP-32 is critically implicated in regulating striatal output in response to the convergent pathways that influence signaling of the cAMP/PKA pathway. This review summarizes some of the landmark and recent studies of DARPP-32-mediated signaling in the attempt to clarify the role played by DARPP-32 in the response to rewarding natural stimuli. Particularly, the review deals with data derived from rodents studies and discusses the involvement of the cAMP/PKA/DARPP-32 pathway in: 1) appetitive food-sustained motivated behaviors, 2) motivated behaviors sustained by social reward, 3) sexual behavior, and 4) responses to environmental enrichment.

G protein signaling-biased agonism at the κ-opioid receptor is maintained in striatal neurons.

Biased agonists of G protein-coupled receptors may present a means to refine receptor signaling in a way that separates side effects from therapeutic properties. Several studies have shown that agonists that activate the κ-opioid receptor (KOR) in a manner that favors G protein coupling over β-arrestin2 recruitment in cell culture may represent a means to treat pain and itch while avoiding sedation and dysphoria. Although it is attractive to speculate that the bias between G protein signaling and β-arrestin2 recruitment is the reason for these divergent behaviors, little evidence has emerged to show that these signaling pathways diverge in the neuronal environment. We further explored the influence of cellular context on biased agonism at KOR ligand-directed signaling toward G protein pathways over β-arrestin-dependent pathways and found that this bias persists in striatal neurons. These findings advance our understanding of how a G protein-biased agonist signal differs between cell lines and primary neurons, demonstrate that measuring [35S]GTPγS binding and the regulation of adenylyl cyclase activity are not necessarily orthogonal assays in cell lines, and emphasize the contributions of the environment to assessing biased agonism.

Biochemical and Pharmacological Role of A1 Adenosine Receptors and Their Modulation as Novel Therapeutic Strategy.

Adenosine, the purine nucleoside, mediates its effects through activation of four G-protein coupled adenosine receptors (ARs) named as A1, A2A, A2B and A3. In particular, A1ARs are distributed through the body, primarily inhibitory in the regulation of adenylyl cyclase activity and able to reduce the cyclic AMP levels. Considerable advances have been made in the pharmacological and molecular characterization of A1ARs, which had been proposed as targets for the discovery and drug design of antagonists, agonists and allosteric enhancers. Several lines of evidence indicate that adenosine interacting with A1ARs may be an endogenous protective agent in the human body since it prevents the damage caused by various pathological conditions, such as in ischemia/hypoxia, epileptic seizures, excitotoxic neuronal injury and cardiac arrhythmias in cardiovascular system. It has also been reported that one of the most promising targets for the development of new anxiolytic drugs could be A1ARs, and that their activation may reduce pain signaling in the spinal cord. A1AR antagonists induce diuresis and natriuresis in various experimental models, mediating the inhibition of A1ARs in the proximal tubule which is primarily responsible for reabsorption and fluid uptake. In addition, the results of various studies indicate that adenosine is present within pancreatic islets and is implicated through A1ARs in the regulation of insulin secretion and in glucose concentrations. In the present paper it will become apparent that A1ARs could be implicated in the pharmacological treatment of several pathologies with an important influence on human health.

Gi proteins regulate adenylyl cyclase activity independent of receptor activation.

Background and purposeDespite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to Gi, some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes.Experimental approachWe used the Gs-selective (R,R)- and the Gs- and Gi-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats.Key resultsPTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response.Conclusions and implicationsTogether, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic Gi and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

Differential dopamine receptor subtype regulation of adenylyl cyclases in lipid rafts in human embryonic kidney and renal proximal tubule cells

Dopamine D1-like receptors (D1R and D5R) stimulate adenylyl cyclase (AC) activity, whereas the D2-like receptors (D2, D3 and D4) inhibit AC activity. D1R, but not the D5R, has been reported to regulate AC activity in lipid rafts (LRs). We tested the hypothesis that D1R and D5R differentially regulate AC activity in LRs using human embryonic kidney (HEK) 293 cells heterologously expressing human D1 or D5 receptor (HEK-hD1R or HEK-hD5R) and human renal proximal tubule (hRPT) cells that endogenously express D1R and D5R. Of the AC isoforms expressed in HEK and hRPT cells (AC3, AC5, AC6, AC7, and AC9), AC5/6 was distributed to a greater extent in LRs than non-LRs in HEK-hD1R (84.5±2.3% of total), HEK-hD5R (68.9±3.1% of total), and hRPT cells (66.6±2.2% of total) (P<0.05, n=4/group). In HEK-hD1R cells, the D1-like receptor agonist fenoldopam (1μM/15min) increased AC5/6 protein (+17.2±3.9% of control) in LRs but decreased it in non-LRs (−47.3±5.3% of control) (P<0.05, vs. control, n=4/group). By contrast, in HEK-hD5R cells, fenoldopam increased AC5/6 protein in non-LRs (+67.1±5.3% of control, P<0.006, vs. control, n=4) but had no effect in LRs. In hRPT cells, fenoldopam increased AC5/6 in LRs but had little effect in non-LRs. Disruption of LRs with methyl-β-cyclodextrin decreased basal AC activity in HEK-D1R (−94.5±2.0% of control) and HEK-D5R cells (−87.1±4.6% of control) but increased it in hRPT cells (6.8±0.5-fold). AC6 activity was stimulated to a greater extent by D1R than D5R, in agreement with the greater colocalization of AC5/6 with D1R than D5R in LRs. We conclude that LRs are essential not only for the proper membrane distribution and maintenance of AC5/6 activity but also for the regulation of D1R- and D5R-mediated AC signaling.

Regulation of adenylyl cyclase activity in rat testes by acylated derivatives of peptide 562–572 of a luteinizing hormone receptor

One of directions of the search of hormonal signaling systems regulators is the development of peptides that correspond to the cytoplasmic regions of G-protein-coupled receptors (GPCR). Modification of the pepti- des with hydrophobic radicals increases their efficiency and selectivity. But currently is not studied as the acti- vity of the peptide depends on the localization of the hydrophobic radicals, their number and the chemical natu- re. The aim of this work was the synthesis of modified by fatty acid radicals derivatives of peptide 562-572 corresponding to the C-terminal region of luteinizing hormone receptor (LHR), and the study of regulatory ef- fects of the acylated LHR-peptides on the basal and hormone-stimulated activity of adenylyl cyclase (AC) in the rat tissues. To elucidate the effect of localization of hydrophobic radicals and their number the modification of peptide 562-572 only at the N- or C-terminus or at both ends was carried out. To study the effect of hydrop- hobicity the residues ofpalmitic (Pal) and decanoic (Dec) acids were selected. Using a solid phase strategy we have synthesized unmodified peptide NDTKIAKK-Nle-A562-572-KA (1) and five of its acylated analogues, such as N[K(Dec)]DTKIAKK-Nle-A562-572-KA (2), NKDTKIAKK-Nle-A562-572-[K(Dec)]A (3), N[K(Dec)] DTKIAKK-Nle-A562-572-[K(Dec)]A (4), N[K(Pal)]DTKIAKK-Nle-A562-572-KA (5), and NKDTKIAKK-Nle- A562--572-[K(Pal)]A (6). Peptide 6 modified with palmitate at the C-terminus to a large extent increased the ba- sal AC activity and reduced AC stimulating effect of human chorionic gonadotropin (hCG) in the testes of rats, peptides 3 and 4 modified with decanoate at the C-terminus were less effective, but exceeded in activity the un- modified peptide 1, while peptides 2 and 5 acylated at the N-terminus were little active. The action of peptides was characterized by the tissue and the receptor specificity. Thus, the modification of LHR-peptide 562-572 with fatty acid radicals at the C-terminus increases its regulatory effect on the functional activity of the adenyla- te cyclase system in the rat testes, indicating promising the modification of GPCR-peptides with hydrophobic radicals. These data support the hypothesis that the hydrophobic radical to be localized in the locus of GPCR-peptide, where a transmembrane domain is located in the receptor.

Competition for Gβγ dimers mediates a specific cross-talk between stimulatory and inhibitory G protein α subunits of the adenylyl cyclase in cardiomyocytes

Heterotrimeric G proteins are key regulators of signaling pathways in mammalian cells. Beyond G protein-coupled receptors, the amount and mutual ratio of specific G protein α, β, and γ subunits determine the G protein signaling. However, little is known about mechanisms that regulate the concentration and composition of G protein subunits at the plasma membrane. Here, we show a novel cross-talk between stimulatory and inhibitory G protein α subunits (Gα) that is mediated by G protein βγ dimers and controls the abundance of specific Gα subunits at the plasma membrane. Firstly, we observed in heart tissue from constitutively Gαi2- and Gαi3-deficient mice that the loss of Gαi2 and Gαi3 was accompanied by a slight increase in the protein content of the nontargeted Gαi isoform. Therefore, we analyzed whether overexpression of selected Gα subunits conversely impairs endogenous G protein α and β subunit levels in cardiomyocytes. Integration of overexpressed Gαi2 subunits into heterotrimeric G proteins was verified by co-immunoprecipitation. Adenoviral expression of increasing amounts of Gαi2 led to a reduction of Gαi3 (up to 90%) and Gαs (up to 75%) protein levels. Likewise, increasing amounts of adenovirally expressed Gαs resulted in a linear 75% decrease in both Gαi2 and Gαi3 protein levels. In contrast, overexpression of either Gαi or Gαs isoform did not influence the amount of Gαo and Gαq, both of which are not involved in the regulation of adenylyl cyclase activity. The mRNA expression of the disappearing endogenous Gα subunits was not affected, indicating a posttranslational mechanism. Interestingly, the amount of endogenous G protein βγ dimers was not altered by any Gα overexpression. However, the increase of Gβγ level by adenoviral expression prevented the loss of endogenous Gαs and Gαi3 in Gαi2 overexpressing cardiomyocytes. Thus, our results provide evidence for a novel mechanism cross-regulating adenylyl cyclase-modulating Gαi isoforms and Gαs proteins. The Gα subunits apparently compete for a limited amount of Gβγ dimers, which are required for G protein heterotrimer formation at the plasma membrane.

The identification of inhibitors of Schistosoma mansoni miracidial transformation by incorporating a medium-throughput small-molecule screen

In Schistosoma mansoni, the miracidium-to-primary sporocyst transformation process is associated with many physiological, morphological, transcriptional and biochemical changes. In the present study, we use a medium-throughput small-molecule screen to identify chemical compounds inhibiting or delaying the in vitro transformation of miracidia to the sporocyst stage. The Sigma–Aldrich Library of Pharmacologically Active Compounds (LOPAC) contains 1280 well-characterized chemical compounds with various modes of action including enzyme inhibitors, antibiotics, cell-cycle regulators, apoptosis inducers and GPCR ligands. We identified 47 compounds that greatly reduce or delay this transformation process during a primary screen of live miracidia. The majority of compounds inhibiting larval transformation were from dopaminergic, serotonergic, ion channel and phosphorylation classes. Specifically, we found that dopamine D2-type antagonists, serotonin reuptake inhibitors, voltage-gated calcium channel antagonists and a PKC activator significantly reduced in vitro miracidial transformation rates. Many of the targets of these compounds regulate adenylyl cyclase activity, with the inhibition or activation of these targets resulting in increased cAMP levels in miracidia and concomitant blocking/delaying of larval transformation.

Muscarinic Inhibition of Hippocampal and Striatal Adenylyl Cyclase is Mainly Due to the M4 Receptor

The five muscarinic acetylcholine receptors (M(1)-M(5)) are differentially expressed in the brain. M(2) and M(4) are coupled to inhibition of stimulated adenylyl cyclase, while M(1), M(3) and M(5) are mainly coupled to the phosphoinositide pathway. We studied the muscarinic receptor regulation of adenylyl cyclase activity in the rat hippocampus, compared to the striatum and amygdala. Basal and forskolin-stimulated adenylyl cyclase activity was higher in the striatum but the muscarinic inhibition was much lower. Highly selective muscarinic toxins MT1 and MT2-affinity order M(1) > or = M(4) >> others-and MT3-highly selective M(4) antagonist-did not show significant effects on basal or forskolin-stimulated cyclic AMP production but, like scopolamine, counteracted oxotremorine inhibition. Since MTs have negligible affinity for M(2), M(4) would be the main subtype responsible for muscarinic inhibition of forskolin-stimulated enzyme. Dopamine stimulated a small fraction of the enzyme (3.1% in striatum, 1.3% in the hippocampus). Since MT3 fully blocked muscarinic inhibition of dopamine-stimulated enzyme, M(4) receptor would be responsible for this regulation.

Direct visualization of adenosine A2A and dopamine D2L receptor oligomers in a neuronal cell model

Dopamine D2 and adenosine A2A receptors are co‐expressed in GABAergic neurons in the basal ganglia where they antagonistically regulate adenylyl cyclase activity. Interactions between D2 and A2A are thought to play an important role in drug addiction and in the pharmacotherapy of Parkinson's disease. Molecular mechanisms may involve the formation of receptor oligomers signaling to alternative pathways. D2 and A2A have been shown to form homo‐ and heterooligomers both in recombinant cells and in native brain tissues. However, the stoichiometry of these complexes, as well as the regulation or their formation (notably by ligands) remain to be characterized.We have used bimolecular fluorescence complementation (BiFC) for direct visualization of A2A homodimers and A2A ‐ D2 heterodimers in a neuronal cell model. Initial studies revealed that the receptor‐BiFC fusion proteins remained functional. Preliminary experiments using multicolor BiFC indicated that prolonged exposure to D2 antagonist spiperone promotes A2A ‐ D2 dimerization whereas D2 agonist quinpirole treatment upregulates A2A homodimerization. In addition, studies combining BiFC with FRET provided evidence for the formation of higher‐order A2A D2 oligomers.This project was funded by Purdue University and NIMH grant award MH060397 to VJW.

Circadian clocks regulate adenylyl cyclase activity rhythms in human RPE cells

Genes and components of the circadian clock may represent relevant drug targets for diseases involving circadian dysfunctions. By exploiting an established cell line derived from human retinal pigment epithelium (HRPE), the cell constituting the blood-retinal barrier that is essential to maintain the visual functions of the sensorineural retina, we showed serum-shock induction of rhythmic changes in forskolin-evoked adenylyl cyclase (AC) activity. In the presence of Ca 2+ and protein kinase A, the forskolin-induced AC activity is significantly, but not completely inhibited, suggesting the involvement of both Ca 2+-sensitive and Ca 2+-insensitive AC isoforms in the regulation of circadian rhythmicity in these cells. Semi-quantitative RT-PCR showed circadian profile in the expression of three AC isoforms, the Ca 2+-inhibitable AC5 and AC6 and the Ca 2+-insensitive AC7, and the clock genes hPer1 and hPer2. Our results demonstrate for the first time circadian rhythmicity in a human cell line, identifying the isoforms involved in the circadian profile of AC activity and showing a rhythmicity of the clock gene mRNA expression in these cells. Therefore, the results reported here provide evidence for an intertwine between AC/[Ca 2+] i signalling pathways and Per genes in the HRPE circadian clockwork.

Short- and Long-Term Regulation of Adenylyl Cyclase Activity by δ-Opioid Receptor Are Mediated by Gαi2in Neuroblastoma N2A Cells

Activation of the opioid receptor results in short-term inhibition of intracellular cAMP levels followed by receptor desensitization and subsequent increase of cAMP above the control level (adenylyl cyclase superactivation). Using adenovirus to deliver pertussis toxin-insensitive mutants of the alpha-subunits of G(i/o) that are expressed in neuroblastoma Neuro2A cells (Galpha(i2), Galpha(i3), and Galpha(o)), we examined the identities of the G proteins involved in the short- and long-term action of the delta-opioid receptor (DOR). Pertussis toxin pretreatment completely abolished the ability of [d-Pen(2), d-Pen(5)]-enkephalin (DPDPE) to inhibit forskolin-stimulated intracellular cAMP production. Expression of the C352L mutant of Galpha(i2), and not the C351L mutants of Galpha(i3) or Galpha(o), rescued the short-term effect of DPDPE after pertussis toxin treatment. The ability of Galpha(i2) in mediating DOR inhibition of adenylyl cyclase activity was also reflected in the ability of Galpha(i2), not Galpha(i3) or Galpha(o), to coimmunoprecipitate with DOR. Coincidently, after long-term DPDPE treatment, pertussis toxin treatment eliminated the antagonist naloxone-induced superactivation of adenylyl cyclase activity. Again, only the C352L mutant of Galpha(i2) restored the adenylyl cyclase superactivation after pertussis toxin treatment. More importantly, the C352L mutant of Galpha(i2) remained associated with DOR after long-term agonist and pertussis toxin treatment whereas the wild-type Galpha(i2) did not. These data suggest that Galpha(i2) serves as the signaling molecule in both DOR-mediated short- and long-term regulation of adenylyl cyclase activity.

Lack of interaction between prostaglandin E 2 receptor subtypes in regulating adenylyl cyclase activity in cultured rat dorsal root ganglion cells

The hyperalgesic response to prostaglandin E 2 (PGE 2) is thought to be mediated by activation of the cAMP/protein kinase A pathway in primary sensory neurones. The aim of this study was to investigate the relative contribution of different PGE 2 (EP) receptor subtypes to the overall activity of adenylyl cyclase in adult rat isolated dorsal root ganglion (DRG) cells, in vitro. PGE 2 and the prostanoid EP 4 receptor agonist ONO-AE1-329 increased [ 3H]cAMP production with EC 50 values of 500 nM and 70 nM, respectively, and showed similar efficacies. No combination of prostanoid EP 1, EP 2, EP 3 or EP 4 receptor selective agonists produced synergistic increases in [ 3H]cAMP. The prostacyclin mimetic cicaprost increased [ 3H]cAMP production with an EC 50 value of 42 nM and produced a significantly greater maximal response compared with PGE 2. No evidence for prostanoid EP 3 receptor-dependent inhibition of adenylyl cyclase activity could be obtained to account for the relatively weak effect of PGE 2 compared with prostacyclin receptor agonists. Interestingly, sulprostone (prostanoid EP 3/EP 1 receptor agonist) caused a Rho-kinase-dependent retraction of neurites, suggesting an alternative role for prostanoid EP 3 receptors in DRG cells. In conclusion, PGE 2 mediated increases in adenylyl cyclase activity in primary sensory neurones is likely to be mediated by activation of prostanoid EP 4 receptors, and is not under inhibitory control by prostanoid EP 3 receptors.

The Endocannabinoid Noladin Ether Acts as a Full Agonist at Human CB2 Cannabinoid Receptors

Noladin ether (NE) is a putative endogenously occurring cannabinoid demonstrating agonist activity at CB1 receptors. Because of reported selective affinity for CB1 receptors, the pharmacological actions of NE at CB2 receptors have not been examined. Therefore, the purpose of this study was to characterize the binding and functional properties of NE at human CB2 receptors stably expressed in Chinese hamster ovary (CHO) cells as well as in HL-60 cells, which express CB2 receptors endogenously. Surprisingly, in transfected CHO cells, NE exhibits a relatively high nanomolar affinity for CB2 receptors (K(i) = 480 nM), comparable to that observed for the endocannabinoid 2-arachidonoyl glycerol (2-AG) (K(i) = 1016 nM). Furthermore, NE activates G proteins and inhibits the intracellular effector adenylyl cyclase with equivalent efficacy relative to the full cannabinoid agonists 2-AG and CP 55,940 (CP) [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol]. The rank order of potency for G protein activation and effector regulation by the three agonists is similar to their apparent affinity for CB2 receptors; CP > NE > or = 2-AG. Regulation of adenylyl cyclase activity by all agonists is inhibited by pertussis toxin pretreatment or by coincubation with AM630 [6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone], a CB2 antagonist. Chronic treatment with NE or CP results in CB2 receptor desensitization and down-regulation. All agonists also inhibit adenylyl cyclase activity in HL-60 cells. Together, these data indicate that NE acts as a full agonist at human CB2 receptors and thus might have important physiological functions at peripheral cannabinoid receptors.

OX1 Orexin Receptors Couple to Adenylyl Cyclase Regulation via Multiple Mechanisms

In this study, the mechanism of OX(1) orexin receptors to regulate adenylyl cyclase activity when recombinantly expressed in Chinese hamster ovary cells was investigated. In intact cells, stimulation with orexin-A led to two responses, a weak (21%), high potency (EC(50) approximately 1 nm) inhibition and a strong (4-fold), low potency (EC(50) = approximately 300 nm) stimulation. The inhibition was reversed by pertussis toxin, suggesting the involvement of G(i/o) proteins. Orexin-B was, surprisingly, almost equally as potent as orexin-A in elevating cAMP (pEC(50) = approximately 500 nm). cAMP elevation was not caused by Ca(2+) elevation or by Gbetagamma. In contrast, it relied in part on a novel protein kinase C (PKC) isoform, PKCdelta, as determined using pharmacological inhibitors. Yet, PKC stimulation alone only very weakly stimulated cAMP production (1.1-fold). In the presence of G(s) activity, orexins still elevated cAMP; however, the potencies were greatly increased (EC(50) of orexin-A = approximately 10 nm and EC(50) of orexin-B = approximately 100 nm), and the response was fully dependent on PKCdelta. In permeabilized cells, only a PKC-independent low potency component was seen. This component was sensitive to anti-Galpha(s) antibodies. We conclude that OX(1) receptors stimulate adenylyl cyclase via a low potency G(s) coupling and a high potency phospholipase C --> PKC coupling. The former or some exogenous G activation is essentially required for the PKC to significantly activate adenylyl cyclase. The results also suggest that orexin-B-activated OX(1) receptors couple to G(s) almost as efficiently as the orexin-A-activated receptors, in contrast to Ca(2+) elevation and phospholipase C activation, for which orexin-A is 10-fold more potent.

Isoform-Specific Regulation of Adenylyl Cyclase Function by Disruption of Membrane Trafficking

Oligomerization plays an important role in endoplasmic reticulum processing and membrane insertion (and ultimately in regulation of function) of a number of transmembrane spanning proteins. Furthermore, it is known that adenylyl cyclases (ACs), critical regulators of cellular functions, associate into higher order (dimeric) forms. However, the importance of these higher order aggregates in regulating adenylyl cyclase activity or trafficking to the cell membrane is unclear. Therefore, we examined the potential role of oligomerization in the membrane trafficking of adenylyl cyclase. For this purpose, the ability of full-length adenylyl cyclase and various truncation mutants to self-assemble and to be targeted to the cell membrane was assessed. A truncation mutant comprised of the initial six transmembrane spanning domains and half of the C1 catalytic domain coimmunoprecipitated with full-length AC VI. Using both biotinylation assays and assessment of enzyme distribution using sucrose density gradients, we demonstrate that expression of this mutant in human embryonic kidney 293 cells impaired the ability of AC VI to traffic to the plasma membrane. Furthermore, mutant expression resulted in a significant reduction in adenylyl cyclase activity. The decrease in AC VI membrane expression was not caused by alterations in enzyme transcription. The effect of the mutant was specific for the AC V and VI isoforms and expression of the transmembrane M1 domain but not the C1a domain was required for the mutant to affect adenylyl cyclase activity. In aggregate, these data suggest that alterations in the ability of adenylyl cyclases to form higher order forms regulate both enzyme trafficking and enzyme activity.

The novel antipsychotic aripiprazole is a partial agonist at short and long isoforms of D 2 receptors linked to the regulation of adenylyl cyclase activity and prolactin release

Aripiprazole is a novel antipsychotic with a unique mechanism of action, which differs from currently marketed typical and atypical antipsychotics. Aripiprazole has been shown to be a partial agonist at the D 2 family of dopamine (DA) receptors in biochemical and pharmacological studies. To demonstrate aripiprazole's action as a partial D 2 agonist in pituitary cells at the molecular level, we retrovirally transduced the short (D 2S) and the long (D 2L) form of the human DA D 2 receptor gene into a rat pituitary cell line, GH4C1. [ 3H]-raclopride saturation binding analyses revealed a B max value approximately four-fold higher at D 2S receptor-expressing GH4C1 cells than at D 2L receptor-expressing GH4C1 cells, while a K d value was similar. Aripiprazole inhibited forskolin-stimulated release of prolactin in both D 2S and D 2L receptor-expressing GH4C1 cells, whereas the maximal inhibition of prolactin release was less than that of DA. Similarly, aripiprazole partially inhibited forskolin-induced cAMP accumulation in both D 2 receptor-expressing cells. Aripiprazole antagonized the suppression attained by DA (10 −7 M) in both D 2 receptor-expressing cells and, at the maximal blockade of cAMP, yielded residual cAMP levels equal to those produced by aripiprazole alone. These results indicate that aripiprazole acts as a partial agonist at both D 2S and D 2L receptors expressed in GH4C1 cells. These data may explain, at least in part, the observations that aripiprazole shows a novel antipsychotic activity with minimal potential for adverse events including no significant increase of serum prolactin levels in clinical studies.

Novel vistas of calcium-mediated signalling in the thalamus.

Traditionally, the role of calcium ions (Ca(2+)) in thalamic neurons has been viewed as that of electrical charge carriers. Recent experimental findings in thalamic cells have only begun to unravel a highly complex Ca(2+) signalling network that exploits extra- and intracellular Ca(2+) sources. In thalamocortical relay neurons, interactions between T-type Ca(2+) channel activation, Ca(2+)-dependent regulation of adenylyl cyclase activity and the hyperpolarization-activated cation current ( I(h)) regulate oscillatory burst firing during periods of sleep and generalized epilepsy, while a functional triad between Ca(2+) influx through high-voltage-activated (most likely L-type) Ca(2+) channels, Ca(2+)-induced Ca(2+) release via ryanodine receptors (RyRs) and a repolarizing mechanism (possibly via K(+) channels of the BK(Ca) type) supports tonic spike firing as required during wakefulness. The mechanisms seem to be located mostly at dendritic and somatic sites, respectively. One functional compartment involving local GABAergic interneurons in certain thalamic relay nuclei is the glomerulus, in which the dendritic release of GABA is regulated by Ca(2+) influx via canonical transient receptor potential channels (TRPC), thereby presumably enabling transmitters of extrathalamic input systems that are coupled to phospholipase C (PLC)-activating receptors to control feed-forward inhibition in the thalamus. Functional interplay between T-type Ca(2+) channels in dendrites and the A-type K(+) current controls burst firing, contributing to the range of oscillatory activity observed in these interneurons. GABAergic neurons in the reticular thalamic (RT) nucleus recruit a specific set of Ca(2+)-dependent mechanisms for the generation of rhythmic burst firing, of which a particular T-type Ca(2+) channel in the dendritic membrane, the Ca(2+)-dependent activation of non-specific cation channels ( I(CAN)) and of K(+) channels (SK(Ca) type) are key players. Glial Ca(2+) signalling in the thalamus appears to be a basic mechanism of the dynamic and integrated exchange of information between glial cells and neurons. The conclusion from these observations is that a localized calcium signalling network exists in all neuronal and probably also glial cell types in the thalamus and that this network is dedicated to the precise regulation of the functional mode of the thalamus during various behavioural states.

Assessing opioid regulation of adenylyl cyclase activity in intact cells.

Assessing opioid regulation of adenylyl cyclase activity in intact cells.