Stimulation Of Cyclic AMP Formation Research Articles

Coincidence Signaling of Dopamine D1-Like and M1 Muscarinic Receptors in the Regulation of Cyclic AMP Formation and CREB Phosphorylation in Mouse Prefrontal Cortex

In the prefrontal cortex, dopamine D₁-like and M₁ muscarinic receptors are both involved in the regulation of attentional, cognitive and emotional processes but so far no information has been provided on their functional interaction. In the present study we show that in mouse medial prefrontal cortex, concomitant activation of M₁ muscarinic receptors potentiated D₁-like receptor-induced cyclic AMP formation through a mechanism involving activation of G_q/11 and the release of G protein βγ subunits. Immunohistochemical studies indicated that the adenylyl cyclase isoforms AC2 and AC4 are expressed in mouse prefrontal cortex and that they colocalize with D₁-like receptors with a greater association for AC4. In primary cultures of frontal cortex neurons, D₁-like receptor-induced Ser133 phosphorylation of the transcription factor cyclic AMP-responsive element binding protein (CREB) was potentiated by concurrent stimulation of M₁ receptors. Suppression of AC4 expression with small interfering RNA transfection reduced D₁ stimulation of cyclic AMP formation and CREB phosphorylation and abolished the M₁ potentiation, whereas knockdown of AC2 had no significant effects. These data indicate that in mouse prefrontal cortex G_q/11-coupled M₁ receptor and G_s-coupled D₁-like receptor inputs converge on AC4 with a consequent enhancement of cyclic AMP formation and signaling to the nucleus.

Platelet aggregation pathway

Platelet activation and coagulation normally do not occur in an intact blood vessel. After blood vessel wall injury, platelet plug formation is initiated by the adherence of the platelets to subendothelial collagen [1,2]. In high shear arterial blood, platelets are first slowed down from their blood flow velocity by interacting with the collagen-bound von Willebrand factor and are subsequently stopped by binding directly to the collagen by their glycoprotein (GP) receptor complex [2,3]. The activation of these collagen receptors on platelets after their binding to the collagen activates phospholipase C-mediated cascades (Fig. 1) [1–3]. This results in the mobilization of calcium from the dense tubular system [4,5]. An increase in intracellular calcium is associated with the activation of several kinases necessary for morphologic change, the presentation of the procoagulant surface, the secretion of platelet granular content, the activation of GPs, and the activation of phospholipase A2 (see Fig. 1) [2,5–7]. The presentation of the procoagulant surface results in the colocalization of different coagulation factors on the surface of the activated platelet, which triggers a series of zymogen conversions, resulting in the release of active thrombin from prothrombin [8]. Adenosine diphosphate (ADP), adenosine triphosphate, and serotonin are released from the dense platelet granule. Activated phospholipase A2 enzymes release arachidonic acid (AA) by the cleaving of fatty acids, especially phosphatidylcholine and phosphatidylethanolamine, at their sn-2 position [9–11]. AA is a precursor for thromboxane A2 (TBXA2) synthesis. In the first step in platelets, prostaglandin (PG)-endoperoxide synthase 1 (PTGS1; also known as cyclooxygenase 1) catalyzes the transformation of AA into cyclic endoperoxide PG G2 and H2 [9]. In platelets, PGG2 and PGH2 are then mainly converted by TBXA synthase into TBXA2 [9]. Fig. 1 Effects of antiplatelet drugs in platelet aggregation pathway. (PA154444041; http://www.pharmgkb.org/do/serve?objId=PA154444041o cAMP, cyclic AMP; GNAS, guanine nucleotide binding protein a s; IP3, inositol ... The mechanism of action of aspirin is the inhibition of PTGS1, thereby preventing the production of PGs and, particularly in platelets, inhibiting TBXA2 production [10–12]. In ex vivo platelet aggregation testing, aspirin affects predominantly AA-stimulated platelet aggregation through a direct pathway, and also collagen-stimulated platelet aggregation through indirect pathways. A review by Lopez Farre et al. [12] discusses further mechanisms associated with platelet response to aspirin. The processes described above result in the local accumulation of molecules such as thrombin, TBXA2, and ADP, which are important for the further recruitment of platelets and the amplification of activation signals as described above. The secreted agonists activate their respective G protein-coupled receptors: coagulation factor II (thrombin) receptors (F2R also known as protease-activated receptor 1; F2RL3 also known as protease-activated receptor 4), TBXA2 receptor (TBXA2R), and ADP receptors (P2RY1 and P2RY12) [10,11,13–15]. The P2RY12 receptor couples to Gi, and when activated by ADP, inhibits adenylate cyclase [16]. This interaction counteracts the stimulation of cyclic AMP formation by endothelial-derived PGs, which alleviates the inhibitory effect of cyclic AMP on inositol 1,4,5-trisphosphate-mediated calcium release [14,16–20]. P2RY12 has a major role in arterial thrombosis and pharmacologic targeting of this receptor, which is an important strategy in the treatment of cardiovascular diseases [21]. Thienopyridines (ticlopidine, clopidogrel, prasugrel), a class of oral anti-platelet agents, permanently inhibit P2RY12 signaling by irreversibly binding the receptor and blocking ADP-induced platelet activation and aggregation [22]. F2R, TBXA2R, and P2RY1 couple to Gq-phospholipase C–inositol 1,4,5-trisphosphate–Ca2+ pathway, inducing shape change and platelet aggregation [14,23,24]. In addition, receptor signaling by G12/13 (F2R; TBXA2R) contributes to morphologic changes through the activation of kinases [23,24]. Platelet adhesion, cytoskeletal reorganization, secretion, and amplification loops are all different steps toward the formation of a platelet plug. These cascades finally result in the activation of the fibrinogen receptor (GPIIb/GPIIIa) expressed on platelet cells [14,25,26]. This activation results in the exposure of the binding sites for fibrinogen, which are not available in inactive platelets. The binding of fibrinogen results in the linkage of the activated platelets through fibrinogen bridges, thereby mediating aggregation [3]. The inhibition of this receptor by GPIIb/GPIIIa inhibitors blocks platelet aggregation induced by any agonist [27,28]. The individual platelet response is variable because of polymorphisms in genes involved in the activation and aggregation of platelets, in conjunction with environmental factors, and contributes to diseases such as arterial thrombosis ([29–33], and http://www.bloodomics.org/web/). In addition to the variation in platelet physiology, platelet sensitivity to drugs targeting platelet activation and aggregation is also influenced by gene polymorphisms and clinical and environmental variables [29,31,34].

PEGylated Dendritic Unimolecular Micelles as Versatile Carriers for Ligands of G Protein-Coupled Receptors

Despite its widespread application in nanomedicine, poly(ethylene glycol) (PEG) is seldom used for covalent modification of ligands for G protein-coupled receptors (GPCRs) due to potential steric complications. In order to study the influence of PEG chains on the biological activity of GPCR ligands bound to a common macromolecular carrier, we prepared a series of G3 polyamidoamine (PAMAM) dendrimers derivatized with Alexa Fluor 488, varying numbers of PEG(550)/PEG(750)/PEG(2000), and nucleoside moieties derived from the A(2A) adenosine receptor (AR) agonist CGS21680 (2-[4-(2-carboxylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine). These dendrimer conjugates were purified by size exclusion chromatography and characterized by (1)H NMR and MALDI MS. In radioligand binding assays, some PAMAM-PEG conjugates showed enhanced subtype-selectivity at the human A(2A) AR compared to monomeric ligands of comparable affinity. The functional potency was measured in the A(2A) AR-mediated activation of adenylate cyclase and inhibition of ADP-induced platelet aggregation. Interestingly, the dendrimer conjugate 10c bearing 11 PEG(750) chains (out of theoretical 32 amino end groups) and 14 nucleoside moieties was 5-fold more potent in A(2A) AR-mediated stimulation of cyclic AMP formation than 10d with 4 PEG(2000) chains and 21 nucleosides, although the binding affinities of these 2 compounds were similar. Thus, a relatively small (≤10 nm) multivalent ligand 10c modified for water solubility maintained high potency and displayed increased A(2A) AR binding selectivity over the monomeric nucleosides. The current study demonstrates the feasibility of using short PEG chains in the design of carriers that target ligand-receptor interactions.

Distribution and functional characterization of pituitary adenylate cyclase–activating polypeptide receptors in the brain of non-human primates

The distribution and density of pituitary adenylate cyclase–activating polypeptide (PACAP) binding sites have been investigated in the brain of the primates Jacchus callithrix (marmoset) and Macaca fascicularis (macaque) using [ 125I]-PACAP27 as a radioligand. PACAP binding sites were widely expressed in the brain of these two species with particularly high densities in the septum, hypothalamus and habenula. A moderate density of recognition sites was seen in all subdivisions of the cerebral cortex with a heterogenous distribution, the highest concentrations occurring in layers I and VI while the underlying white matter was almost devoid of binding sites. Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis revealed intense expression of the mRNAs encoding the short and hop-1 variants of pituitary adenylate cyclase–activating polypeptide–specific receptor (PAC1-R) in the cortex of both marmoset and macaque, whereas vasoactive intestinal polypeptide/pituitary adenylate cyclase–activating polypeptide mutual receptor, subtype 1 (VPAC1-R) and vasoactive intestinal polypeptide/pituitary adenylate cyclase–activating polypeptide mutual receptor, subtype 2 (VPAC2-R) mRNAs were expressed at a much lower level. In situ hybridization histochemistry showed intense expression of PAC1-R and weak expression of VPAC1-R mRNAs in layer IV of the cerebral cortex. Incubation of cortical tissue slices with PACAP induced a dose-dependent stimulation of cyclic AMP formation, indicating that PACAP binding sites correspond to functional receptors. Moreover, treatment of primate cortical slices with 100 nM PACAP significantly reduced the activity of caspase-3, a key enzyme of the apoptotic cascade. The present results indicate that PACAP should exert the same neuroprotective effect in the brain of primates as in rodents and suggest that PAC1-R agonists may have a therapeutic value to prevent neuronal cell death after stroke or in specific neurodegenerative diseases.

Stimulation of cyclic AMP formation and nerve electrical activity by octopamine in the terminal abdominal ganglion of the female gypsy moth Lymantria dispar

The biogenic amine octopamine is known to be present in the abdominal ganglia of some insects, but the expression of functional octopamine receptors in these neuronal structures has not yet been characterized. In the present study, we describe the presence in the female gypsy moth terminal abdominal ganglion (TAG), a key structure in the control of the insect reproductive behavior, of an octopamine receptor coupled to stimulation of adenylyl cyclase through the GTP-binding protein G s. The rank order of potency of different antagonists, which discriminate between the different classes of octopamine receptors, indicated the involvement of the neuronal type 3 receptor. The octopamine-stimulated adenylyl cyclase activity was inhibited by Ca 2+ in the low micromolar range and by activation of either protein kinase A or protein kinase C. In the isolated TAG, bath application of octopamine caused an increase of the spontaneous bursting activity of the emerging nerve of the 5th pair (V), whereas the antagonist mianserin reduced the nerve spiking activity and blocked the stimulatory effect of octopamine. These data demonstrate that the gypsy moth TAG expresses functional octopamine receptors, which may participate in the neuronal control of the insect reproductive behavior.

Functional effects of chronic electroconvulsive shock on serotonergic 5-HT 1A and 5-HT 1B receptor activity in rat hippocampus and hypothalamus

The aims of this work were to determine the influence of chronic electroconvulsive shock (ECS) on presynaptic 5-HT 1A receptor function, postsynaptic 5-HT 1A receptor function in hippocampus and hypothalamus, and presynaptic 5-HT 1B receptor function in hippocampus and hypothalamus. This represents part of an on-going study of the effects of ECS on serotonergic receptor activity in selected brain areas which may be relevant to the effects of electroconvulsive therapy (ECT) in humans. Chronic ECS reduced the ability of the 5-HT 1A receptor agonist 8-hydroxy-2(di- n-propylamino)tetraline (8-OH-DPAT) (0.2 mg/kg s.c.) to decrease 5-HT levels in hypothalamus as shown by in vivo microdialysis, indicative of a reduction in sensitivity of presynaptic 5-HT 1A autoreceptors. The ability of the 5-HT 1B receptor antagonist GR 127935 (5 mg/kg s.c.) to increase 5-HT levels in both hippocampus and hypothalamus was unaffected by chronic ECS. 8-OH-DPAT (0.2 mg/kg s.c.) increased cyclic AMP levels in hippocampus measured by in vivo microdialysis approximately 2-fold. The degree of stimulation of cyclic AMP formation was not altered by chronic ECS. However the cyclic AMP response to forskolin (50 μM) administered via the microdialysis probe, which was approximately 4-fold of basal in sham-treated rats, was almost completely abolished in ECS-treated rats. Since this indicates that either adenylate cyclase catalytic unit activity or Gs protein activity is reduced in the hippocampus after chronic ECS, the lack of change in 8-OH-DPAT-induced cyclic AMP formation may be taken as possible evidence of an increase in sensitivity of postsynaptic 5-HT 1A receptors in the hippocampus by chronic ECS. Chronic ECS increased basal plasma levels of corticosterone, ACTH and oxytocin. The ACTH response to s.c. injections of 0.2 mg/kg or 0.5 mg/kg 8-OH-DPAT was reduced by chronic ECS. Postsynaptic 5-HT 1A receptor activity in the hypothalamus, in contrast to the hippocampus, thus appears to be desensitized after chronic ECS. We conclude that chronic ECS has regionally specific effects on both pre- and post-synaptic 5-HT 1A receptors, but, in contrast to some antidepressant drugs, does not affect presynaptic 5-HT 1B receptor activity.

Metabotropic glutamate receptor mGluR5 in astrocytes: pharmacological properties and agonist regulation.

Metabotropic glutamate receptor (mGluR) agonists induce extensive phosphoinositide (PI) hydrolysis in astrocytes grown in a chemically defined medium with select growth factors. These astrocytes express mGluR5 transcripts, but none of the splice variants of mGluR1, thus permitting the characterization of mGluR5 in a native CNS cell without interference from mGluR1 activity. mGluR5 activation (1) was not associated with stimulation of cyclic AMP formation, (2) showed high sensitivity to the removal of extracellular versus intracellular Ca2+, (3) displayed high coupling efficiency relative to receptor density, and (4) induced PI hydrolysis that was suppressed by phorbol esters with low potency. The rank order of agonist potency was similar to that observed in mGluR1 and mGluR5 transfected cells. The phenylglycine antagonists tested were effective in blocking responses to 1-aminocyclopentane-1S,3R-dicarboxylic acid, but not to glutamate. Prolonged exposure to agonists induced a two-phase desensitization of mGluR5 function, an initial phase (completed by 1 h and plateaus for another 3 h) and a late phase (progressive decrease to approximately 30% of control levels by 24 h). Only the latter phase was associated with receptor down-regulation. Desensitization of mGluR5 function did not involve receptor internalization or phosphorylation mediated by protein kinase C or A; it was purely homologous, and reversible. Resensitization after short agonist treatment did not require prior receptor sequestration. Recovery after prolonged agonist exposure required new protein synthesis, but the restoration of function was more rapid than normalization of receptor protein levels, indicating that regulation also involves other components of the transduction system. The protracted desensitization of mGluR5 in astrocytes suggests that the functions mediated by this receptor are maintained under a variety of conditions ranging from repetitive stimulation to injury responses.

Involvement of calyculin A inhibitable protein phosphatases in the cyclic AMP signal transduction pathway of mouse corticotroph tumour (AtT20) cells.

1. The role of non-calcineurin protein phosphatases in the cyclic AMP signal transduction pathway was examined in mouse pituitary corticotroph tumour (AtT20) cells. 2. Blockers of protein phosphatases, calyculin A and okadaic acid, were applied in AtT20 cells depleted of rapidly mobilizable pools of intracellular calcium and activated by various cyclic AMP generating agonists. Inhibitors of cyclic nucleotide phosphodiesterases were present throughout. The accumulation of cyclic AMP was monitored by radioimmunoassay, phosphodiesterase activity in cell homogenates was measured by radiometric assay. 3. Neither calyculin A nor okadaic acid altered basal cyclic AMP levels but cyclic AMP formation induced by 41 amino acid residue corticotrophin releasing-factor (CRF) was strongly inhibited (up to 80%), 1-Norokadaone was inactive. Similar data were also obtained when isoprenaline or pituitary adenylate cyclase activating peptide1-38 were used as agonists. 4. Pertussis toxin did not modify the inhibition of CRF-induced cyclic AMP production by calyculin A. 5. Pretreatment with calyculin A completely prevented the stimulation of cyclic AMP formation by cholera toxin even in the presence of 0.5 mM isobutylmethylxanthine (IBMX) and 0.1 mM rolipram. Cholera toxin mediated ADP-ribosylation of the 45 K and 52 K molecular weight Gs alpha isoforms in membranes from calyculin A-pretreated cells was enhanced to 150-200% when compared with controls. 6. Cholera toxin-induced cyclic AMP was reduced by calyculin A within 10 min when calyculin A was applied after a 90 min pretreatment with cholera toxin. Under these conditions the effect of calyculin A could be blocked by the combination of 0.5 mM IBMX and 0.1 mM rolipram, but not by 0.5 mM IBMX alone. 7. Phosphodiesterase activity in AtT20 cell homogenates showed a significant, 2.7 fold increase after treatment with calyculin A. In control cells phosphodiesterase activity was blocked by 80% in the presence of IBMX (0.5 mM), or IBMX plus rolipram (0.1 mM). In calyculin A-treated cells phosphodiesterase activity was also strongly inhibited by IBMX, but because of the stimulating effect of calyculin A, the activity remaining was still 55% of that found in control homogenates. This activity was reduced to 5% of control by using IBMX and rolipram in combination. Assay of phosphodiesterase in Ca2+ free conditions showed that calyculin A markedly increases the activity of rolipram sensitive (type 4) phosphodiesterase. 8. Taken together, blockers of protein phosphatases (PPases) impaired signal transduction through Gs-mediated pathways and activated cyclic AMP degrading phosphodiesterase(s), indicating that PPases 1 and/or 2A are essential for agonist-mediated regulation of cyclic AMP levels in AtT20 cells, and are thus important in maintaining the secretory phenotype of the cells.

Identification of G alpha s messenger ribonucleic acid splice variants in human granulosa cells.

Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by G alpha s-linked receptors. In this paper we have investigated the expression of G alpha s mRNA splice variants in relation to expression of G alpha s protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all G alpha s mRNA isoforms as well as quantifying total amounts of G alpha s mRNA. Granulosa cells express the message for G alpha s-Large and G alpha s-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of G alpha s-Large and G alpha s-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in G alpha s variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between G alpha s variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.

Stimulation of cyclic AMP formation in the circular smooth muscle of human colon by activation of 5-HT4-like receptors.

5-HT stimulated cyclic AMP generation in human colonic circular smooth muscle in a concentration-dependent fashion (EC50 = 229.1 nM). DAU 6236 also increased cyclic AMP formation and was a partial agonist relative to 5-HT. GR 113808 inhibited the cyclic AMP formation induced by 5-HT with a -log Ki value of 9.1 and an apparent pA2 value of 9.2. Ondansetron and methysergide failed to inhibit cyclic AMP formation induced by 5-HT. These results indicate that the 5-HT4 receptors of human colonic circular muscle mediate relaxation and inhibition of spontaneous contractions via formation of cyclic AMP.

Stimulation of cyclic AMP formation by pituitary adenylate cyclase-activating polypeptide is attenuated by glutamate in rat brain slices.

In rat hippocampal slices, pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) stimulated cyclic AMP formation in dose- and time-dependent manners. The PACAP-38 action was dose-dependently attenuated by L-glutamate in the hippocampus; L-glutamate at the concentration of 1 mM attenuated PACAP-38-stimulated cyclic AMP formation by approximately 30%. The inhibitory effect of L-glutamate is also observed in rat cerebellar slices. In contrast, the inhibitory effect of a prostanoid EP3-receptor agonist on PACAP-38-stimulated cyclic AMP formation was brain region-specific; the inhibitory action was observed in the cerebellum but not in the hippocampus.

Rat Oligodendrocytes Express Muscarinic Receptors Coupled to Phosphoinositide Hydrolysis and Adenylyl Cyclase

Muscarinic receptors expressed by rat oligodendrocyte primary cultures were examined by measuring changes in second messengers following exposure to carbachol, an acetylcholine analog, and by polymerase chain reaction. Inositol phosphate levels were measured in [3H]myo-inositol-labelled young oligodendrocyte cultures following stimulation with carbachol. Atropine, a specific muscarinic antagonist, prevented the carbachol-induced accumulation of inositol phosphates. The formation of inositol trisphosphate was concentration- and time-dependent, with the peak at 100 microM carbachol and 10 min. Carbachol increased intracellular calcium levels, which were dependent both on the mobilization of intracellular stores and influx of extracellular calcium. In initial experiments with more selective antagonists, the mobilization of intracellular calcium was preferentially inhibited by pirenzepine, a selective M1 antagonist, but not methoctramine, a selective M2 antagonist, suggesting M1 muscarinic receptor involvement. A role for protein kinase C in the regulation of carbachol-stimulated inositol phosphate formation and intracellular calcium mobilization was demonstrated, as acute pretreatment with phorbol-12,13-myristate acetate abolished the formation of both second messengers. Pretreatment with 100 microM carbachol abolished the 40% increase in the cyclic AMP accumulation stimulated by isoproterenol, a specific beta-adrenergic agonist. In turn, the inhibition was alleviated by pretreatment with atropine, suggesting muscarinic receptor involvement. Polymerase chain reaction carried out with specific m1 and m2 muscarinic receptor oligonucleotide primers, confirmed that these cells express, at least, the two muscarinic receptor subtypes. Without excluding the expression of other subtypes, these results suggest that developing oligodendrocytes express m1 (M1) and m2 (M2) muscarinic receptors capable of mediating phosphoinositide hydrolysis, mobilization of intracellular calcium and the attenuation of beta-adrenergic stimulation of cyclic AMP formation.

Inhibition of dopamine agonist-induced phosphoinositide hydrolysis by concomitant stimulation of cyclic AMP formation in brain slices.

We examined the effects of cyclic AMP on dopamine receptor-coupled activation of phosphoinositide hydrolysis in rat striatal slices. Forskolin, dibutyryl cyclic AMP, and the protein kinase A activator Sp-cyclic adenosine monophosphothioate (Sp-cAMPS) significantly inhibited inositol phosphate formation stimulated by the dopamine D1 receptor agonist SKF 38393. Conversely, the protein kinase A antagonist Rp-cyclic adenosine monophosphothioate (Rp-cAMPS) dose-dependently potentiated the SKF 38393 effect. In the presence of 200 microM Rp-cAMPS, the dose-response curves of the dopamine D1 receptor agonists SKF 38393 and fenoldopam were shifted to the left and maximal agonist responses were markedly increased. The agonist EC50 values, however, were not significantly altered by protein kinase A inhibition. Neither Sp-cAMPS nor Rp-cAMPS significantly affected basal inositol phosphate accumulation. These findings demonstrate that dopaminergic stimulation of phosphoinositide hydrolysis is inhibited by elevations in intracellular cyclic AMP. Dopamine receptor agonists that stimulate adenylyl cyclase could suppress their activation of phosphoinositide hydrolysis by concomitantly stimulating the formation of cyclic AMP in striatal tissue. The interaction between dopamine D1 receptor-stimulated elevations in cyclic AMP and dopaminergic stimulation of inositol phosphate formation suggests a cellular colocalization of these dopamine-coupled transduction pathways in at least some cells of the rat striatum.

Evidence for a Distinct D1Like Dopamine Receptor that Couples to Activation of Phosphoinositide Metabolism in Brain

Dopamine and the D1 receptor agonist SKF 38393 activate the phospholipase C-mediated hydrolysis of phosphoinositides in brain slices. This action is selectively inhibited by SCH-23390, thus suggesting its mediation through the dopamine D1 receptor. To determine if the dopamine receptor that mediates phosphoinositide hydrolysis is the adenylyl cyclase-linked D1 receptor or a different subtype of the dopamine D1 receptor, 20 benzazepine compounds that were previously characterized as selective dopamine D1 receptor agonists were tested for stimulation of phosphoinositide hydrolysis in rat striatal slices and for activation of adenylyl cyclase in rat striatal membranes. The compounds displayed a range of potencies and efficacies in stimulating adenylyl cyclase or phosphoinositide hydrolysis. Compounds such as SKF 81427 and SKF 38393 were as efficacious as dopamine in stimulating phosphoinositide hydrolysis, whereas other compounds, including SKF 85174 and SKF 86284, although showing high efficacy in stimulating cyclic AMP, failed to stimulate inositol phosphate formation. There was no correlation between the potencies (r = 0.016; p > 0.95) or efficacies (r = -0.294; p > 0.24) of the tested compounds in stimulating cyclic AMP formation and phosphoinositide hydrolysis. These observations indicate that the D1-like dopamine receptor that mediates phosphoinositide hydrolysis is pharmacologically distinct from the classic D1 receptor that is coupled to stimulation of cyclic AMP formation.

Studies on the mechanism of [3H]‐noradrenaline release from SH‐SY5Y cells: the role of Ca2+ and cyclic AMP

1. The roles of both Ca2+ and adenosine 3':5'-cyclic monophosphate (cyclic AMP) in carbachol and K(+)-stimulated [3H]-noradrenaline release from SH-SY5Y human neuroblastoma cells were examined. 2. Both carbachol and K+ caused a time- and dose-related stimulation of [3H]-noradrenaline release. The release event in perfused cells was monophasic. Half-maximum stimulation measured in statically incubated (3 min) cells was 38 +/- 4 microM and 63 +/- 4 mM respectively. K+ (100 mM, added)-evoked release was greater than that produced by carbachol (1 mM). 3. Both carbachol and K+ caused a time- and dose (measured at 3 min)-related stimulation of cyclic AMP formation with half-maximum stimulation occurring at 5 +/- 1 microM and 49 +/- 2 mM respectively. In contrast to its effects on release, carbachol produced a greater stimulation of cyclic AMP formation than K+. 4. K(+)-stimulated [3H]-noradrenaline release was entirely dependent on Ca2+ entry as 2.5 mM Ni2+ abolished release. However, carbachol-evoked (1 mM) release appeared to be unaffected by Ni2+ pretreatment. 5. These data suggest that in SH-SY5Y cells, elevated cyclic AMP levels are not directly involved in [3H]-noradrenaline release. In addition, carbachol-stimulated release is largely independent of extracellular Ca2+ possibly implying a role for intracellular stored Ca2+ in the release process.

Cyclic-AMP-dependent Ca 2+ influx elicited by prostaglandin D 2 in freshly isolated nonchromaffin cells from bovine adrenal medulla

We previously reported that prostaglandin D 2 (PGD 2) specifically elevates intracellular cyclic AMP in nonchromaffin cells isolated from bovine adrenal medulla (Biochim. Biophys. Acta (1989) 1011, 75–80). Here we again found that PGD 2 increased intracellular Ca 2+ concentration ([Ca 2+] i) in freshly isolated nonchromaffin cells and investigated the cellular mechanisms of PGD 2-induced [Ca 2+] i increase using the Ca 2+ indicator fura-2 and a fluorescence microscopic imaging system. Treatment of the cells with PGD 2 receptor agonists BW245C and ZK110841 resulted in both marked stimulation of cyclic AMP formation and an increase in [Ca 2+] i. The [Ca 2+] i response was also induced by bypassing of the receptor with forskolin, a direct activator of adenylate cyclase, but not by PGE 2 or PGF 2α both of which are devoid of the ability to generate cyclic AMP in the cells. These cyclic AMP and [Ca 2+] i responses induced by PGD 2 were completely blocked by the PGD 2 receptor antagonist BWA868C. The time-course of cyclic AMP production stimulated by PGD 2 coincided with that of the [Ca 2+] i increase. While the Ca 2+-mobilizing hormone bradykinin stimulated a rapid inositol phosphate accumulation in nonchromaffin cells, PGD 2 did not stimulate it significantly. Removal of extracellular Ca 2+ markedly reduced the Ca 2+ response to PGD 2 in magnitude and duration, but did not alter the peak [Ca 2+] i response to bradykinin. These results demonstrate that PGD 2 receptor activation induces the increase in [Ca 2+] i via cyclic AMP mainly by increasing the Ca 2+ influx from the outside, unlike inositol trisphosphate which causes release of Ca 2+ from internal stores.

Neuroendocrine regulation of cyclic AMP formation in osteoblastic cell lines (UMR-106-01, ROS 17/2.8, MC3T3-E1, and Saos-2) and primary bone cells.

The effect of four different neuropeptides and norepinephrine (NE) on cyclic AMP formation in four different osteoblastic cell lines and in isolated neonatal mouse calvarial bone cells has been examined. In the rat osteosarcoma cell line UMR-106-01, vasoactive intestinal polypeptide (VIP, 0.001-1 microM), calcitonin gene-related peptide (CGRP, 0.3-30 nM), and NE (0.1-300 microM), but not neuropeptide Y (NPY, 0.001-1 microM) or substance P (SP, 0.1-10 microM), caused a dose-dependent stimulation of cyclic AMP formation. The stimulatory effects were synergistically potentiated by forskolin (0.1-3 microM). The effects of NE and VIP were time dependent, with an optimal effect seen at 5 minutes. The amount of cyclic AMP accumulated in cells stimulated with NE and VIP was in the same range. The amplitude of the cyclic AMP response induced by CGRP was smaller than that caused by VIP and NE. In the human osteosarcoma cell line Saos-2, NE (0.1 microM) and VIP (0.3 microM) stimulated cyclic AMP formation, and the effect was synergistically potentiated by forskolin. In the absence of forskolin, no effect of CGRP (30 nM) could be seen in the Saos-2 cells, but in the presence of forskolin (3 microM) a stimulatory effect was observed. SP and NPY did not change basal cyclic AMP levels in Saos-2 cells. In the osteoblastic osteosarcoma cell line of rat, ROS 17/2.8, NE (0.1 microM) caused a significant stimulatory action on cyclic AMP formation that was synergistically potentiated by forskolin (3 microM), VIP, CGRP, and SP did not affect the cellular content of cyclic AMP in ROS 17/2.8.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct linkage of three tachykinin receptors to stimulation of both phosphatidylinositol hydrolysis and cyclic AMP cascades in transfected Chinese hamster ovary cells.

The mammalian tachykinin system consists of three distinct peptides, substance P, substance K, and neuromedin K, and possesses three corresponding receptors. In this investigation we examined intracellular signal transduction of the individual tachykinin receptors by transfection and stable expression of these receptor cDNAs in Chinese hamster ovary cells. The three receptors commonly showed a rapid and marked stimulation in both phosphatidylinositol (PI) hydrolysis and cyclic AMP formation in response to tachykinin interaction. Direct linkage of the three receptors to both phospholipase C and adenylate cyclase was evidenced by the finding that tachykinin, added together with GTP, activated these enzyme activities in membrane preparations derived from tachykinin receptor-expressing cells. The stimulation of cyclic AMP formation was less efficient than that of PI hydrolysis in receptor-expressing cells as well as their membrane preparations (about 1 order of magnitude difference in the effective peptide concentrations). However, the stimulatory responses of the PI hydrolysis and cyclic AMP formation in both receptor-expressing cells and their membrane preparations were induced in complete agreement with the tachykinin binding selectivity of each subtype of the receptors. This investigation demonstrated unequivocally that the tachykinin receptors have the potential to couple directly to both phospholipase C and adenylate cyclase and to stimulate PI hydrolysis and cyclic AMP formation.

Introduction of BAPTA into intact rat submandibular acini inhibits mucin secretion in response to cholinergic and β-adrenergic agonists

Incorporation of the calcium chelator BAPTA into isolated, intact rat submandibular acini by hypotonic swelling, resulted in complete inhibition of carbamylcholine, noradrenaline and isoproterenol stimulation of mucin secretion. No effects or intracellular BAPTA on cell viability or β-adrenergic stimulation of cyclic AMP formation were observed. The data are the first to demonstrate that calcium is necessary for β-adrenergic stimulation of secretion and suggest that Ca 2+ provides a common link in the triggering of exocytosis.

Functional characteristics of calcitonin gene-related peptide receptors in human Ewing's sarcoma WE-68 cells

Calcitonin gene-related peptide (CGRP) receptor activity was studied in WE-68 human Ewing's sarcoma cells. 125I-human CGRP bound in a time-dependent, reversible and saturable manner. Scatchard plots were compatible with the presence of a homogenous population of CGRP receptors with high affinity ( K d = 15 pM, and B max = 1.9 fmol mg protein). The potency order of unlabeled peptides, in the presence of radioligand, was: human CGRP-II > human CGRP = chick CGRP > rat CGRP = rat [Tyr o]CGRP > human [Tyr o] CGRP > > salmon calcitonin (CT) > rat [Tyr o]CGRP-(28-37). Each peptide except CT and [Tyi o]CGRP-(28-37) stimulated cyclic AMP generation in a concentration-dependent manner, and the relative potencies paralleled their relative ability in inhibiting 125I-human CGRP binding. We conclude that WE-68 Ewing's sarcoma cells express genuine CGRP receptors which upon activation lead to stimulation of cyclic AMP formation. Calcitonin gene-related peptide; Calcitonin; cyclic AMP; (Human; Ewing's sarcoma cell)