RESUMO
The potassium (K+) ion channel KCNK13 is specifically expressed in human microglia with elevated expression observed in post-mortem human brain tissue from patients with Alzheimer's disease. Modulation of KCNK13 activity by a small-molecule inhibitor is proposed as a potential treatment for neurodegenerative diseases. Herein, we describe the evolution of a series of KCNK13 inhibitors derived from a high-throughput screening campaign, resulting in CVN293, a potent, selective, and brain permeable clinical candidate molecule. CVN293 demonstrated a concentration-dependent inhibition of the NLRP3-inflammasome mediated production of IL-1ß from LPS-primed murine microglia. Cross-species pharmacokinetic data of CVN293 are also disclosed. These findings support the advancement of CVN293 in clinical trials.
Assuntos
Bursite , Humanos , Bursite/terapia , Modalidades de Fisioterapia , Atenção Primária à SaúdeRESUMO
Fluorescence-based potassium channel assays are typically run on expensive, hard to obtain, fluorescence imaging kinetic plate readers that are uncommon in most laboratories. Here we describe the use of the Brilliant Thallium Snapshot assay to conduct an endpoint potassium channel assay, so that it can be used across multiple plate reader platforms that are more common in many labs. These methods will allow users to identify modulators of potassium channels. For this work, we have taken a kinetic mode Molecular Devices FLIPR based protocol and adapted it to be utilized on endpoint plate readers, such as the BMG Labtech PHERAstar, to identify activators of GIRK channels in CHO cells. We demonstrate that both plate readers are functionally competent at generating excellent Z' values which makes them ideally suited to finding corollary hits from the Sigma LOPAC 1,280 screening collection. Importantly, this assay has also been validated using a high content reader, demonstrating the possibility of spatially resolving signals from individual cells within a mixed cell population. The compendium of these results shows the flexibility, accessibility and functionality of endpoint-compatible potassium channel assay readouts on more common plate readers.
Assuntos
Cricetulus , Células CHO , Animais , Cinética , Canais de Potássio/metabolismo , Humanos , Bioensaio/métodos , Microscopia/métodos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Ensaios de Triagem em Larga Escala/métodosRESUMO
From our NETSseq-derived human brain transcriptomics data, we identified GPR55 as a potential molecular target for the treatment of motor symptoms in patients with Parkinson's disease. From a high-throughput screen, we identified and optimized agonists with nanomolar potency against both human and rat GPR55. We discovered compounds with either strong or limited ß-arrestin signaling and receptor desensitization, indicating biased signaling. A compound that showed minimal GPR55 desensitization demonstrated a reduction in firing frequency of medium spiny neurons cultured from rat striatum but did not reverse motor deficits in a rat hypolocomotion model. Further profiling of several desensitizing and non-desensitizing lead compounds showed that they are selective over related cannabinoid receptors CB1 and CB2 and that unbound brain concentrations well above the respective GPR55 EC50 can be readily achieved following oral administration. The novel brain-penetrant GPR55 agonists disclosed can be used to probe the role of this receptor in the brain.
Assuntos
Agonistas de Receptores de Canabinoides , Transdução de Sinais , Humanos , Ratos , Animais , Receptores de Canabinoides , beta-Arrestinas , Corpo Estriado/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptor CB2 de Canabinoide , Receptor CB1 de CanabinoideRESUMO
Nicotinic acetylcholine receptor (nAChR) α6 subunit RNA expression is relatively restricted to midbrain regions and is located presynaptically on dopaminergic neurons projecting to the striatum. This subunit modulates dopamine neurotransmission and may have therapeutic potential in movement disorders. We aimed to develop potent and selective α6-containing nAChR antagonists to explore modulation of dopamine release and regulation of motor function in vivo. High-throughput screening (HTS) identified novel α6-containing nAChR antagonists and led to the development of CVN417. This molecule blocks α6-containing nAChR activity in recombinant cells and reduces firing frequency of noradrenergic neurons in the rodent locus coeruleus. CVN417 modulated phasic dopaminergic neurotransmission in an impulse-dependent manner. In a rodent model of resting tremor, CVN417 attenuated this behavioral phenotype. These data suggest that selective antagonism of α6-containing nAChR, with molecules such as CVN417, may have therapeutic utility in treating the movement dysfunctions observed in conditions such as Parkinson's disease.
Assuntos
Dopamina , Receptores Nicotínicos , Encéfalo , Membrana Celular , Corpo Estriado , Antagonistas Nicotínicos/farmacologiaRESUMO
The low affinity metabotropic glutamate receptor mGluR7 has been implicated in numerous CNS disorders; however, a paucity of potent and selective activators has hampered full delineation of the functional role and therapeutic potential of this receptor. In this work, we present the identification, optimization, and characterization of highly potent, novel mGluR7 agonists. Of particular interest is the chromane CVN636, a potent (EC50 7 nM) allosteric agonist which demonstrates exquisite selectivity for mGluR7 compared to not only other mGluRs, but also a broad range of targets. CVN636 demonstrated CNS penetrance and efficacy in an in vivo rodent model of alcohol use disorder. CVN636 thus has potential to progress as a drug candidate in CNS disorders involving mGluR7 and glutamatergic dysfunction.
RESUMO
Neuroinflammation, specifically the NLRP3 inflammasome cascade, is a common underlying pathological feature of many neurodegenerative diseases. Evidence suggests that NLRP3 activation involves changes in intracellular K+. Nuclear Enriched Transcript Sort Sequencing (NETSseq), which allows for deep sequencing of purified cell types from human post-mortem brain tissue, demonstrated a highly specific expression of the tandem pore domain halothane-inhibited K+ channel 1 (THIK-1) in microglia compared to other glial and neuronal cell types in the human brain. NETSseq also showed a significant increase of THIK-1 in microglia isolated from cortical regions of brains with Alzheimer's disease (AD) relative to control donors. Herein, we report the discovery and pharmacological characterisation of C101248, the first selective small-molecule inhibitor of THIK-1. C101248 showed a concentration-dependent inhibition of both mouse and human THIK-1 (IC50: â¼50 nM) and was inactive against K2P family members TREK-1 and TWIK-2, and Kv2.1. Whole-cell patch-clamp recordings of microglia from mouse hippocampal slices showed that C101248 potently blocked both tonic and ATP-evoked THIK-1 K+ currents. Notably, C101248 had no effect on other constitutively active resting conductance in slices from THIK-1-depleted mice. In isolated microglia, C101248 prevented NLRP3-dependent release of IL-1ß, an effect not seen in THIK-1-depleted microglia. In conclusion, we demonstrated that inhibiting THIK-1 (a microglia specific gene that is upregulated in brains from donors with AD) using a novel selective modulator attenuates the NLRP3-dependent release of IL-1ß from microglia, which suggests that this channel may be a potential therapeutic target for the modulation of neuroinflammation in AD.
Assuntos
Doença de Alzheimer , Inflamassomos , Canais de Potássio de Domínios Poros em Tandem , Animais , Humanos , Camundongos , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Inflamassomos/metabolismo , Microglia , Doenças Neuroinflamatórias , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidoresRESUMO
We report a significant decrease in transcription of the G protein-coupled receptor GPR39 in striatal neurons of Parkinson's disease patients compared to healthy controls, suggesting that a positive modulator of GPR39 may beneficially impact neuroprotection. To test this notion, we developed various structurally diverse tool molecules. While we elaborated on previously reported starting points, we also performed an in silico screen which led to completely novel pharmacophores. In vitro studies indicated that GPR39 agonism does not have a profound effect on neuroprotection.
Assuntos
Pirimidinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Regulação Alostérica/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/química , Receptores Acoplados a Proteínas G/metabolismo , Relação Estrutura-AtividadeRESUMO
GPR6 is an orphan G-protein-coupled receptor that has enriched expression in the striatopallidal, indirect pathway and medium spiny neurons of the striatum. This pathway is greatly impacted by the loss of the nigro-striatal dopaminergic neurons in Parkinson disease, and modulating this neurocircuitry can be therapeutically beneficial. In this study, we describe the in vitro and in vivo pharmacological characterization of (R)-1-(2-(4-(2,4-difluorophenoxy)piperidin-1-yl)-3-((tetrahydrofuran-3-yl)amino)-7,8-dihydropyrido[3,4-b]pyrazin-6(5H)-yl)ethan-1-one (CVN424), a highly potent and selective small-molecule inverse agonist for GPR6 that is currently undergoing clinical evaluation. CVN424 is brain-penetrant and shows dose-dependent receptor occupancy that attained brain 50% of receptor occupancy at plasma concentrations of 6.0 and 7.4 ng/ml in mice and rats, respectively. Oral administration of CVN424 dose-dependently increases locomotor activity and reverses haloperidol-induced catalepsy. Furthermore, CVN424 restored mobility in bilateral 6-hydroxydopamine lesion model of Parkinson disease. The presence and localization of GPR6 in medium spiny neurons of striatum postmortem samples from both nondemented control and patients with Parkinson disease were confirmed at the level of both RNA (using Nuclear Enriched Transcript Sort sequencing) and protein. This body of work demonstrates that CVN424 is a potent, orally active, and brain-penetrant GPR6 inverse agonist that is effective in preclinical models and is a potential therapeutic for improving motor function in patients with Parkinson disease. SIGNIFICANCE STATEMENT: CVN424 represents a nondopaminergic novel drug for potential use in patients with Parkinson disease.
Assuntos
Corpo Estriado , Animais , Hormônios Esteroides Gonadais , RatosRESUMO
The Lysophosphatidic Acid 1 Receptor (LPA1 receptor) has been linked to the initiation and progression of a variety of poorly treated fibrotic conditions. Several compounds that have been described as LPA1 receptor antagonists have progressed into clinical trials: 1-(4-{4-[3-methyl-4-({[(1R)-1-phenylethoxy]carbonyl}amino)-1,2-oxazol-5-yl]phenyl}phenyl)cyclopropane-1-carboxylic acid (BMS-986202) and 2-{4-methoxy-3-[2-(3-methylphenyl)ethoxy]benzamido}-2,3-dihydro-1H-indene-2-carboxylic acid (SAR-100842). We considered that as LPA1 receptor function is involved in many normal physiological processes, inhibition of specific signalling pathways associated with fibrosis may be therapeutically advantageous. We compared the binding and functional effects of a novel compound; 4-({(Cyclopropylmethyl)[4-(2-fluorophenoxy)benzoyl]amino}methyl}benzoic acid (TAK-615) with BMS-986202 and SAR-100842. Back-scattering interferometry (BSI) was used to show that the apparent affinity of TAK-615 was enhanced in the presence of LPA. The binding signal for BMS-986202 was not detected in the presence of LPA suggesting competition but interestingly the apparent affinity of SAR-100842 was also enhanced in the presence of LPA. Only BMS-986202 was able to fully inhibit the response to LPA in calcium mobilisation, ß-arrestin, cAMP, GTPγS and RhoA functional assays. TAK-615 and SAR-100842 showed different inhibitory profiles in the same functional assays. Further binding studies indicated that TAK-615 is not competitive with either SAR-100842 or BMS-986202, suggesting a different site of binding. The results generated with this set of experiments demonstrate that TAK-615 acts as a negative allosteric modulator (NAM) of the LPA1 receptor. Surprisingly we find that SAR-100842 also behaves like a NAM. BMS-986202 on the other hand behaves like an orthosteric antagonist.
Assuntos
Benzamidas/farmacologia , Benzoatos/farmacologia , Ciclopropanos/farmacologia , Indenos/farmacologia , Oxazóis/farmacologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Regulação Alostérica , Animais , Benzamidas/química , Benzoatos/química , Cálcio/metabolismo , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , Ciclopropanos/química , Indenos/química , Oxazóis/química , Ratos , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidoresRESUMO
GPR81, which exhibits a high degree of homology with GPR109a, has been recently identified as a lactate receptor. Similar to GPR109a, the activation of GPR81 by lactate suppresses lipolysis, suggesting that GPR81 may be a potential drug target for treating dyslipidemia. In addition, the fact that GPR81 is expressed only in adipocytes, whereas GPR109a is expressed in various tissues and cells, including Langerhans cells, which are considered responsible for flushing, indicates that targeting GPR81 could lead to the development of antidyslipidemia agents with a reduced risk of this side effect. However, the pharmacological role of GPR81 remains largely unclear, mainly because of the lack of potent and selective surrogate GPR81 agonists suitable for in vivo studies. In the present study, we showed that lactate-induced suppression of lipolysis in explants of white adipose tissue (WAT) depends on the presence of GPR81. We also performed high-throughput screening (HTS) and identified four novel chemical clusters as GPR81 agonists. Chemical optimization of aminothiazole derivatives led to the discovery of a lead compound with improved potency. The compound inhibited lipolysis in differentiated 3T3-L1 adipocytes. Finally, intraperitoneal administration of this compound suppressed lipolysis in mice at doses that did not cause cutaneous flushing. This is the first description of a 50nM GPR81 selective agonist with in vivo efficacy, without the side effect, i.e., flushing. These results suggest that GPR81 is an attractive drug target for treating dyslipidemia without the risk of flushing.
Assuntos
Adipócitos/efeitos dos fármacos , Tecido Adiposo Branco/efeitos dos fármacos , Rubor/prevenção & controle , Hipolipemiantes/farmacologia , Lipólise/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Tiazóis/farmacologia , Células 3T3-L1 , Adipócitos/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Relação Dose-Resposta a Droga , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Hipolipemiantes/administração & dosagem , Hipolipemiantes/síntese química , Injeções Intraperitoneais , Ácido Láctico/farmacologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Relação Estrutura-Atividade , Tiazóis/administração & dosagem , Tiazóis/síntese química , TransfecçãoRESUMO
The 5-HT2A receptor (5-HT2AR) is implicated in psychotropic changes within the central nervous system (CNS). A number of polymorphisms have been reported in the 5-HT2AR gene; one of these results in a non-synonymous change, H452Y, in the carboxy-terminal tail of the receptor protein. The minor allele (9% occurrence) has been statistically associated with CNS dysfunction such as impaired memory processing and resistance to neuroleptic treatment in schizophrenic patients. We investigated the impact of H452Y mutation of the 5-HT2AR expressed in COS7 cells on distinctly coupled intracellular signalling pathways from the receptor, focusing on the heterotrimeric G protein-independent phospholipase D (PLD) pathway, compared to the conventional Gq/11-linked phospholipase C (PLC) pathway. The H452Y mutation selectively attenuated PLD signalling, which as in the wild-type receptor, was mediated by a molecular complex involving PLD1 docked to the receptor's carboxy-terminal tail domain. Co-immunoprecipitation and GST-fusion protein experiments revealed that the H452Y mutation selectively reduced PLD1 binding to the receptor. Experiments with blocking peptides to mimic short sections of the 5-HT2AR tail sequence revealed that the peptide spanning residue 452 strongly reduced PLD but not PLC responses of the receptor. Similar observations were made when assessing both PLD responses and PLD-dependent cellular proliferation elicited by activation of 5-HT2ARs natively expressed in MCF-7 cells. Overall these findings indicate that the H452Y polymorphic variant of the 5-HT2AR displays selective disruption of its PLD signalling pathway. This may potentially play a role in the CNS dysfunction associated with the H452Y allele of the 5-HT2AR.
Assuntos
Fosfolipase D/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Animais , Células COS , Chlorocebus aethiops , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Células MCF-7 , Fosfolipase D/antagonistas & inibidores , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/metabolismo , Receptor 5-HT2A de Serotonina/química , Receptor 5-HT2A de Serotonina/genética , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transfecção , Fosfolipases Tipo C/metabolismoRESUMO
The 5-HT2AR (5-hydroxytryptamine-2A receptor) is a GPCR (G-protein-coupled receptor) that is implicated in the actions of hallucinogens and represents a major target of atypical antipsychotic agents. In addition to its classical signalling though PLC (phospholipase C), the receptor can activate several other pathways, including ARF (ADP-ribosylation factor)-dependent activation of PLD (phospholipase D), which appears to be achieved through a mechanism independent of heterotrimeric G-proteins. In the present study we show that wild-type and inactive constructs of PLD1 (but not PLD2) respectively facilitate and inhibit ARF-dependent PLD signalling by the 5-HT2AR. Furthermore we demonstrate that PLD1 specifically co-immunoprecipitates with the receptor and binds to a distal site in GST (glutathione transferase) fusion protein constructs of its C-terminal tail which is distinct from the ARF-interaction site, thereby suggesting the existence of a functional ARF-PLD signalling complex directly associated with this receptor. This reveals the spatial co-ordination of an important GPCR, transducer and effector into a physical complex that is likely to reinforce the impact of receptor activation on a heterotrimeric G-protein-independent signalling pathway. Signalling of this receptor through such non-canonical pathways may be important to its role in particular disorders.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Fosfolipase D/fisiologia , Receptor 5-HT2A de Serotonina/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Fosfolipase D/químicaRESUMO
Vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase activating polypeptides (PACAPs) share 68% identity at the amino acid level and belong to the secretin peptide family. Following the initial discovery of VIP almost four decades ago a substantial amount of knowledge has been presented describing the mechanisms of action, distribution and pleiotropic functions of these related peptides. It is now known that the physiological actions of these widely distributed peptides are produced through activation of three common G-protein coupled receptors (VPAC(1), VPAC(2) and PAC(1)R) which preferentially stimulate adenylate cyclase and increase intracellular cAMP, although stimulation of other intracellular messengers, including calcium and phospholipase D, has been reported. Using a range of in vitro and in vivo approaches, including cell-based functional assays, transgenic animals and rodent models of disease, VPAC/PAC receptor activation has been associated with numerous physiological processes (e.g. control of circadian rhythms) and clinical conditions (e.g. pulmonary hypertension), which underlies on-going research efforts and makes these peptides and their cognate receptors attractive targets for the pharmaceutical industry. However, despite the considerable interest in VPAC/PAC receptors and the processes which they mediate, there is still a paucity of selective and available, non-peptide ligands, which has hindered further advances in this field both at the basic research and clinical level. This review summarises the current knowledge of VIP/PACAP and the VPAC/PAC receptors with regard to their distribution, pharmacology, signalling pathways, splice variants and finally, the utility of animal models in exploring their physiological roles.
Assuntos
Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , AMP Cíclico/metabolismo , Ligantes , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Fosfolipase D/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/agonistas , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo/agonistas , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/agonistas , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/genética , Transdução de Sinais , Peptídeo Intestinal Vasoativo/fisiologiaRESUMO
VPAC/PAC receptor activation classically results in cyclic-AMP production, with limited reports evaluating calcium signalling. These studies systematically characterise intracellular cyclic-AMP ([cAMP](i)) and calcium ([Ca(2+)](i)) responses in CHO-cells expressing recombinant human (h) VPAC/PAC receptors (hVPAC(1)R, hVPAC(2)R, hPAC(1)R), using two simple, non-radioactive, HT-amenable assays. The rank order of potency (ROP) of the agonists VIP, PACAP-27 and PACAP-38 was similar in both assays for each individual receptor subtype, although potencies (EC(50)) in the [Ca(2+)](i) assay were approximately 100-fold lower. Importantly, this shift was also evident in SHSY-5Y cells endogenously expressing hPAC(1)R. Furthermore, [Ala(11,22,28)]VIP and maxadilan were selective hVPAC(1)R and hPAC(1)R agonists, respectively, and although R3P65 had no demonstrable hVPAC(2)R selectivity, these compounds exhibited comparable reductions in [Ca(2+)](i) EC(50) values. In contrast, PG97-269 and PG99-465, putatively selective hVPAC(1)R and hVPAC(2)R antagonists, respectively, were marginally less potent in [cAMP](i) studies, whereas M65 was equipotent at hPAC(1)R. Moreover, PG99-465 alone increased [cAMP](i) at all three hVPAC/PAC receptor subtypes, with full hVPAC(1)R and hPAC(1)R agonism. With equivalent agonist ROPs generated in both assays, [Ca(2+)](i) signalling provides an alternative approach to examine hVPAC/PAC receptor pharmacology. However, these studies underscore the paucity of receptor selective compounds, complexities in comparing drug potencies across assays, and the pleiotropic nature of VPAC/PAC-receptor signalling.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , AMP Cíclico/fisiologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/efeitos dos fármacos , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Células CHO , Linhagem Celular Tumoral , Células Cultivadas , Cricetinae , Interpretação Estatística de Dados , Ensaio de Imunoadsorção Enzimática , Humanos , Microscopia de Fluorescência , Dados de Sequência Molecular , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/agonistas , Receptores Tipo II de Peptídeo Intestinal Vasoativo/antagonistas & inibidores , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/agonistas , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/antagonistas & inibidores , Transfecção , Peptídeo Intestinal Vasoativo/análogos & derivados , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
VIP/PACAP receptor activation stimulates the production of [cAMP]i and [Ca2+]i by coupling to independent G-protein subunits, although agonist potencies for the different transduction pathways appear to differ. Using CHO-K1 cells stably expressing the human VIP/PACAP receptors (hVPAC1R, hVPAC2R, and hPAC1R), functional assays ([cAMP]i and [Ca2+]i) were established and the receptor pharmacology was characterized with five peptide agonists (VIP, PACAP-27, PACAP-38, [Ala(11,22,28)]VIP, and R3P65). The rank order of potency (ROP) was consistent between assays for the individual receptor subtypes, however, higher agonist concentrations (approximately 100-fold) were required for stimulating [Ca2+]i when compared to [cAMP]i.