Gαo is a major determinant of cAMP signaling in the pathophysiology of movement disorders

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Generation of mouse oocytes defective in cAMP synthesis and degradation: endogenous cyclic AMP is essential for meiotic arrest

Although it is established that cAMP accumulation plays a pivotal role in preventing meiotic resumption in mammalian oocytes, the mechanisms controlling cAMP levels in the female gamete have remained elusive. Both production of cAMP via GPCRs/Gs/adenylyl cyclases endogenous to the oocyte as well as diffusion from the somatic compartment through gap junctions have been implicated in maintaining cAMP at levels that preclude maturation. Here we have used a genetic approach to investigate the different biochemical pathways contributing to cAMP accumulation and maturation in mouse oocytes. Because cAMP hydrolysis is greatly decreased and cAMP accumulates above a threshold, oocytes deficient in PDE3A do not resume meiosis in vitro or in vivo, resulting in complete female infertility. In vitro, inactivation of Gs or downregulation of the GPCR GPR3 causes meiotic resumption in the Pde3a null oocytes. Crossing of Pde3a^−/− mice with Gpr3^−/− mice causes partial recovery of female fertility. Unlike the complete meiotic block of the Pde3a null mice, oocyte maturation is restored in the double knockout, although it occurs prematurely as described for the Gpr3^−/− mouse. The increase in cAMP that follows PDE3A ablation is not detected in double mutant oocytes, confirming that GPR3 functions upstream of PDE3A in the regulation of oocyte cAMP. Metabolic coupling between oocytes and granulosa cells was not affected in follicles from the single or double mutant mice, suggesting that diffusion of cAMP is not prevented. Finally, simultaneous ablation of GPR12, an additional receptor expressed in the oocyte, does not modify the Gpr3^−/− phenotype. Taken together, these findings demonstrate that Gpr3 is epistatic to Pde3a and that fertility as well as meiotic arrest in the PDE3A-deficient oocyte is dependent on the activity of GPR3. These findings also suggest that cAMP diffusion through gap junctions or the activity of additional receptors is not sufficient by itself to maintain the meiotic arrest in the mouse oocyte.     

SET Protein Interacts with Intracellular Domains of the Gonadotropin-releasing Hormone Receptor and Differentially Regulates Receptor Signaling to cAMP and Calcium in Gonadotrope Cells

In mammals, the receptor of the neuropeptide gonadotropin-releasing hormone (GnRHR) is unique among the G protein-coupled receptor (GPCR) family because it lacks the carboxyl-terminal tail involved in GPCR desensitization. Therefore, mechanisms involved in the regulation of GnRHR signaling are currently poorly known. Here, using immunoprecipitation and GST pull-down experiments, we demonstrated that SET interacts with GnRHR and targets the first and third intracellular loops. We delineated, by site-directed mutagenesis, SET binding sites to the basic amino acids ⁶⁶KRKK⁶⁹ and ²⁴⁶RK²⁴⁷, located next to sequences required for receptor signaling. The impact of SET on GnRHR signaling was assessed by decreasing endogenous expression of SET with siRNA in gonadotrope cells. Using cAMP and calcium biosensors in gonadotrope living cells, we showed that SET knockdown specifically decreases GnRHR-mediated mobilization of intracellular cAMP, whereas it increases its intracellular calcium signaling. This suggests that SET influences signal transfer between GnRHR and G proteins to enhance GnRHR signaling to cAMP. Accordingly, complexing endogenous SET by introduction of the first intracellular loop of GnRHR in αT3-1 cells significantly reduced GnRHR activation of the cAMP pathway. Furthermore, decreasing SET expression prevented cAMP-mediated GnRH stimulation of Gnrhr promoter activity, highlighting a role of SET in gonadotropin-releasing hormone regulation of gene expression. In conclusion, we identified SET as the first direct interacting partner of mammalian GnRHR and showed that SET contributes to a switch of GnRHR signaling toward the cAMP pathway.     

Dopamine receptor regulation of Ca2+ levels in individual isolated nerve terminals from rat striatum: comparison of presynaptic D1-like and D2-like receptors

We have directly observed the effects of activating presynaptic D1-like and D2-like dopamine receptors on Ca2+ levels in isolated nerve terminals (synaptosomes) from rat striatum. R-(+)-SKF81297, a selective D1-like receptor agonist, and (-)-quinpirole, a selective D2-like receptor agonist, induced increases in Ca2+ levels in different subsets of individual striatal synaptosomes. The SKF81297- and quinpirole-induced effects were blocked by R-(+)-SCH23390, a D1-like receptor antagonist, and (-)-sulpiride, a D2-like receptor antagonist, respectively. SKF81297- or quinpirole-induced Ca2+ increases were inhibited following blockade of voltage-gated calcium channels or sodium channels. In a larger subset of synaptosomes, quinpirole decreased baseline Ca2+. Quinpirole also inhibited veratridine-induced increases in intrasynaptosomal Ca2+ level. Immunostaining confirmed the presynaptic expression of D1, D5, D2 and D3 receptors, but not D4 receptors. The array of neurotransmitter phenotypes of the striatal nerve endings expressing D1, D5, D2 or D3 varied for each receptor subtype. These results suggest that presynaptic D1-like and D2-like receptors induce increases in Ca2+ levels in different subsets of nerve terminals via Na+ channel-mediated membrane depolarization, which, in turn, induces the opening of voltage-gated calcium channels. D2-like receptors also reduce nerve terminal Ca2+ in a different but larger subset of synaptosomes, consistent with the predominant presynaptic action of dopamine in the striatum being inhibitory.     

A cAMP/PKA/Kinesin-1 Axis Promotes the Axonal Transport of Mitochondria in Aging Drosophila Neurons

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Correlation of local changes in extracellular oxygen and pH that accompany dopaminergic terminal activity in the rat caudate-putamen

Terminal activity causes an increase in local cerebral blood flow that can be quantified by measuring the accompanying increase in tissue oxygen. Alkaline pH changes can also follow neuronal activation. The purpose of these studies was to determine whether these changes in extracellular oxygen and pH correlate. Fast-scan cyclic voltammetry was used to detect changes in dopamine, pH and oxygen levels simultaneously in the caudate-putamen after electrical stimulation of the substantia nigra in anesthetized rats. The biphasic increases in oxygen and pH followed similar time courses, and were delayed a few seconds from the immediate release and uptake of dopamine. The changes following administration of neurotransmitter receptor antagonists as well as agents that modulate blood flow were identical for oxygen and pH. Two distinct mechanisms were identified that give rise to the oxygen and pH changes: blood vessel dilatation caused by nitric oxide synthesis after muscarinic receptor activation and adenosine receptor activation. We conclude that changes in blood flow accompanying terminal activity cause alkaline pH shifts by the rapid removal of carbon dioxide, a component of the extracellular brain buffering system.     

Differential expression and signaling of the human histamine H3 receptor isoforms of 445 and 365 amino acids expressed in human neuroblastoma SH-SY5Y cells

In stably-transfected human neuroblastoma SH-SY5Y cells, we have compared the effect of activating two isoforms of 445 and 365 amino acids of the human histamine H₃ receptor (hH₃R₄₄₅ and hH₃R₃₆₅) on [³⁵S]-GTPγS binding, forskolin-induced cAMP formation, depolarization-induced increase in the intracellular concentration of Ca²⁺ ions ([Ca²⁺]i) and depolarization-evoked [^3?H]-dopamine release. Maximal specific binding (B_max) of [^3?H]-N-methyl-histamine to cell membranes was 953?±?204 and 555?±?140?fmol/mg protein for SH-SY5Y-hH₃R₄₄₅ and SH-SY5Y-hH₃R₃₆₅ cells, respectively, with similar dissociation constants (K_d, 0.86?nM and 0.81?nM). The mRNA of the hH₃R₃₆₅ isoform was 40.9?±?7.9% of the hH₃R₄₄₅ isoform. No differences in receptor affinity were found for the H₃R ligands histamine, immepip, (R)(-)-α-methylhistamine (RAMH), A-331440, clobenpropit and ciproxifan. Both the stimulation of [³⁵S]-GTPγS binding and the inhibition of forskolin-stimulated cAMP accumulation by the agonist RAMH were significantly larger in SH-SY5Y-hH₃R₄₄₅ cells ([³⁵S]-GTPγS binding, 158.1?±?7.5% versus 136.5?±?3.6% for SH-SY5Y-hH₃R₃₆₅ cells; cAMP accumulation, ?74.0?±?4.9% versus ?43.5?±?5.3%), with no significant effect on agonist potency. In contrast, there were no differences in the efficacy and potency of RAMH to inhibit [^3?H]-dopamine release evoked by 100?mM K⁺ (?18.9?±?3.0% and ?20.5?±?3.3%, for SH-SY5Y-hH₃R₄₄₅ and SH-SY5Y-hH₃R₃₆₅ cells), or the inhibition of depolarization-induced increase in [Ca²⁺]i (S2/S1 ratios: parental cells 0.967?±?0.069, SH-SY5Y-hH₃R₄₄₅ cells 0.639?±?0.049, SH-SY5Y-hH₃R₃₆₅ cells 0.737?±?0.045). These results indicate that in SH-SY5Y cells, hH₃R₄₄₅ and hH₃R₃₆₅ isoforms regulate in a differential manner the signaling pathways triggered by receptor activation.     

Intracellular cAMP determines the extent of degradation and not the synthesis of collagen by rat hepatocytes

Intracellular collagen degradation in normal rat hepatocytes was exponetially stimulated by db-cAMP (10–100 µM). The effect was manifested as a decrease (p < 0.01) in net collagen production. The extent of degradation directly co-related with the intracellular cAMP levels, only upto a threshold concentration (16.2 ± 1.3 p moles/10⁶ cells) elicited by 100 µM of db-cAMP. Higher concentrations induced no further increment. Forskolin adenylate cyclase activator (10–50 µM), produced similar effects demonstrating cAMP dependence of the phenomenon. Both db-cAMP as well as Forskolin stimulated collagen degradation (p < 0.05) in hepatocytes from rats administered CCL₄. However, the extent of stimulation was significantly (p < 0.01) less compared to that observed in normal hepatocytes. Our data demonstrates that elevated cAMP levels regulate net collagen content by signalling intracellular collagen degradation and not synthesis.Abbreviations cAMP 3,5 cyclic Adenosine Monophosphate - db-cAMP dibutyryl cyclic Adenosine Monophosphate - TCA Trichloroacetic Acid - Coll. Collagen - DMEM Dulbecoo's Minimal Essential Medium     

Role of SAP97 Protein in the Regulation of Corticotropin-releasing Factor Receptor 1 Endocytosis and Extracellular Signal-regulated Kinase 1/2 Signaling

The corticotropin-releasing factor (CRF) receptor 1 (CRFR1) is a target for the treatment of psychiatric diseases such as depression, schizophrenia, anxiety disorder, and bipolar disorder. The carboxyl-terminal tail of the CRFR1 terminates in a PDZ-binding motif that provides a potential site for the interaction of PSD-95/Discs Large/Zona Occludens 1 (PDZ) domain-containing proteins. In this study, we found that CRFR1 interacts with synapse-associated protein 97 (SAP97; also known as DLG1) by co-immunoprecipitation in human embryonic 293 (HEK 293) cells and cortical brain lysates and that this interaction is dependent upon an intact PDZ-binding motif at the end of the CRFR1 carboxyl-terminal tail. Similarly, we demonstrated that SAP97 is recruited to the plasma membrane in HEK 293 cells expressing CRFR1 and that mutation of the CRFR1 PDZ-binding motif results in the redistribution of SAP97 into the cytoplasm. Overexpression of SAP97 antagonized agonist-stimulated CRFR1 internalization, whereas single hairpin (shRNA) knockdown of endogenous SAP97 in HEK 293 cells resulted in increased agonist-stimulated CRFR1 endocytosis. CRFR1 was internalized as a complex with SAP97 resulting in the redistribution of SAP97 to endocytic vesicles. Overexpression or shRNA knockdown of SAP97 did not significantly affect CRFR1-mediated cAMP formation, but SAP97 knockdown did significantly attenuate CRFR1-stimulated ERK1/2 phosphorylation in a PDZ interaction-independent manner. Taken together, our studies show that SAP97 interactions with CRFR1 attenuate CRFR1 endocytosis and that SAP97 is involved in coupling G protein-coupled receptors to the activation of the ERK1/2 signaling pathway.     

Acute down-regulation of sodium-dependent phosphate transporter NPT2a involves predominantly the cAMP/PKA pathway as revealed by signaling-selective parathyroid hormone analogs

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR1) in cells of the renal proximal tubule mediates the reduction in membrane expression of the sodium-dependent P(i) co-transporters, NPT2a and NPT2c, and thus suppresses the re-uptake of P(i) from the filtrate. In most cell types, the liganded PTHR1 activates Gα(S)/adenylyl cyclase/cAMP/PKA (cAMP/PKA) and Gα(q/11)/phospholipase C/phosphatidylinositol 1,4,5-trisphosphate (IP(3))/Ca(2+)/PKC (IP(3)/PKC) signaling pathways, but the relative roles of each pathway in mediating renal regulation P(i) transport remain uncertain. We therefore explored the signaling mechanisms involved in PTH-dependent regulation of NPT2a function using potent, long-acting PTH analogs, M-PTH(1-28) (where M = Ala(1,12), Aib(3), Gln(10), Har(11), Trp(14), and Arg(19)) and its position 1-modified variant, Trp(1)-M-PTH(1-28), designed to be phospholipase C-deficient. In cell-based assays, both M-PTH(1-28) and Trp(1)-M-PTH(1-28) exhibited potent and prolonged cAMP responses, whereas only M-PTH(1-28) was effective in inducing IP(3) and intracellular calcium responses. In opossum kidney cells, a clonal cell line in which the PTHR1 and NPT2a are endogenously expressed, M-PTH(1-28) and Trp(1)-M-PTH(1-28) each induced reductions in (32)P uptake, and these responses persisted for more than 24 h after ligand wash-out, whereas that of PTH(1-34) was terminated by 4 h. When injected into wild-type mice, both M-modified PTH analogs induced prolonged reductions in blood P(i) levels and commensurate reductions in NPT2a expression in the renal brush border membrane. Our findings suggest that the acute down-regulation of NPT2a expression by PTH ligands involves mainly the cAMP/PKA signaling pathway and are thus consistent with the elevated blood P(i) levels seen in pseudohypoparathyroid patients, in whom Gα(s)-mediated signaling in renal proximal tubule cells is defective.     

Changes in calcineurin expression induced in the rat brain by the administration of antipsychotics

Calcineurin (CN) was recently identified as a susceptibility gene for schizophrenia as well as showing altered RNA expression levels in the post-mortem brains of individuals with schizophrenia. CN knockout mice show a number of behaviours associated with schizophrenia, including deficits in sensorimotor gating, suggesting a link between CN and psychosis. Concurrently, we found, using genome screening techniques, that antipsychotics alter CN expression levels. Therefore, western blotting, in situ hybridization, immunocytochemistry and phosphatase assays were employed to determine what effect antipsychotics have on CN. The results indicate that clozapine, risperidone and haloperidol cause substantial reductions in the A subunit of CN but not CN B at both the RNA and protein levels in the striatum and prefrontal cortex. The changes could only be observed after repeated treatment with antipsychotics but not after acute administration. The alterations in CN protein levels were specific to antipsychotics and mediated by D2 dopamine receptor antagonism. However, despite reductions in CN protein levels, the phosphatase activity of CN was significantly elevated after treatment with antipsychotics. Collectively the results suggest that CN may be a common target for antipsychotics and that antipsychotic-induced alterations in CN may represent one of the mechanisms by which antipsychotics alleviate psychosis.     

Reducing Ability of the Striatum and Cerebral Cortex in Rats following Acute Administration of Risperidone or Haloperidol: An Estimation by in Vivo Electron Paramagnetic Resonance Imaging

In vivo temporal electron paramagnetic resonance (EPR) imaging of the blood-brain barrier-permeable nitroxide radical, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy (PCAM), in the brain of rats was conducted following acute administration of risperidone (RSP) or haloperidol (HPD). The half-life of the signal intensity of PCAM was obtained from a selected area in the temporal EPR images. The half-lives in the striatum and cerebral cortex for the RSP- or HPD-treated rats were significantly longer than for the control rats (p < 0.01). This finding indicates that the reducing abilities of the striatum and cerebral cortex decreased in the rats to which either RSP or HPD had been acutely administrated because the half-life of PCAM in the selected region of the brain reflects its reducing ability.     

Activation of neutral trehalase by fermentable sugars and cAMP in the fission yeast Schizosaccharomyces pombe

Abstract Derepressed cells of Schizosaccharomyces pombe 972 h⁻ suspended in the presence of glucose or other fermentable sugars displayed a transient activation of trehalase which was not blocked by cycloheximide. Repressed cells were unable to show glucose-induced trehalase stimulation. Nitrogen sources, protonophores or uncouplers failed to produce direct trehalase activation but increased the activity of the enzyme in the presence of glucose. Exogenous cAMP induced a rapid and pronounced stimulation of trehalase in both repressed and derepressed cells suggesting that the response to glucose includes activation of adenylate cyclase as part of a cAMP signalling pathway that increases the catalytic activity of trehalase by enzyme modification.     

Interactions of glutamate and dopamine in a computational model of the striatum

A network model of simplified striatal principal neurons with mutual inhibition was used to investigate possible interactions between cortical glutamatergic and nigral dopaminergic afferents in the neostriatum. Glutamatergic and dopaminergic inputs were represented by an excitatory synaptic conductance and a slow membrane potassium conductance, respectively. Neuronal activity in the model was characterized by episodes of increased action potential firing rates of variable duration and frequency. Autocorrelation histograms constructed from the action potential activity of striatal model neurons showed that reducing peak excitatory conductance had the effect of increasing interspike intervals. On the other hand, the maximum value of the dopamine-sensitive potassium conductance was inversely related to the duration of firing episodes and the maximal firing rates. A smaller potassium conductance restored normal firing rates in the most active neurons at the expense of a larger proportion of neurons showing reduced activity. Thus, a homogeneous network with mutual inhibition can produce equally complex dynamics as have been proposed to occur in a striatal network with two neuron populations that are oppositely regulated by dopamine. Even without mutual inhibition it appears that increased dopamine concentrations could partially compensate for the effects of reduced glutamatergic input in individual neurons.     

Inhibition by ammonia of intracellular cAMP accumulation in dictyostelium discoideum: Its significance for the regulation of morphogenesis

Two compounds, ammonia (NH₃) and 3′5′ cyclic AMP (cAMP) act as specific morphogens in regulating the development of Dictyostelium discoideum [1–11]. A previous study [12] demonstrated that NH₃ at concentrations that affect the course of morphogenesis completely inhibits the extracellular release of cAMP by aggregation competent cells incubated in shaken suspension. The present study extends this finding in two respects:

• 1 Exposure of aggregation competent cells to NH₃ (supplied as ammonium carbonate) is followed within a few minutes by the complete disappearance of intracellular cAMP. Subsequent removal of NH₃ is followed by a rapid, complete restoration of the level. Neither the disappearance nor the reappearance is affected by the presence of cycloheximide, an inhibitor of protein synthesis.
• 2 In a mutant strain of D discoideum, greatly increased sensitivity to NH₃ as a regulator of morphogenesis is coupled with a correspondingly increased sensitivity to NH₃ as an inhibitor of cAMP accumulation.

These results are consistent with a recently proposed [13, 14] model of morphogenetic regulation that is based on the supposition that NH₃, by inhibiting cAMP production, restricts cAMP accumulation to specified constrained areas within the developing multicellular aggregate and thereby dictates the course of morphogenesis and cytodifferentiation.     

Deficits in dopamine clearance and locomotion in hypoinsulinemic rats unmask novel modulation of dopamine transporters by amphetamine

Insulin affects brain reward pathways and there is converging evidence that this occurs through insulin regulation of the dopamine (DA) transporter (DAT). In rats made hypoinsulinemic by fasting, synaptosomal DA uptake is reduced. Interestingly, [3H]DA uptake is increased in hypoinsulinemic rats with a history of amphetamine self-administration. The possibility that amphetamine and insulin act in concert to regulate DAT activity prompted this study. Here we show that [3H]DA uptake, measured in vitro and clearance of exogenously applied DA in vivo, is significantly reduced in rats made hypoinsulinemic by a single injection of streptozotocin. Strikingly, amphetamine (1.78 mg/kg, given every other day for 8 days) restored DA clearance in streptozotocin-treated rats but was without effect on DA clearance in saline-treated rats. Basal locomotor activity of streptozotocin-treated rats was lower compared to control rats; however, in streptozotocin-treated rats, hyperlocomotion induced by amphetamine increased over successive amphetamine injections. In saline-treated rats the locomotor stimulant effect of amphetamine remained stable across the four amphetamine injections. These results provide exciting new evidence that actions of amphetamine on DA neurotransmission are insulin-dependent and further suggest that exposure to amphetamine may cause long-lasting changes in DAT function.