Dual signalling by different octopamine receptors converges on adenylate cyclase in Sf9 cells.

The Sf9 insect cell line, derived from Spodoptera frugiperda, was used to study the regulation of adenylate cyclase (AC) activity by octopamine receptors. The cyclic AMP (cAMP) production was stimulated in a concentration-dependent manner by octopamine. Octopamine also elicited a rise in cytosolic Ca(2+). The Ca(2+) elevation was independent of the cAMP elevation whereas the cAMP elevation was partially inhibited by removal of Ca(2+). The antagonistic effects of a series of compounds were tested on both responses. Phentolamine inhibited both responses with similar potency. Two of the tested compounds, MK-912 and RS 79948, were over 1000-fold more potent in blocking the Ca(2+) response. Ionomycin, a Ca(2+) ionophore, or activation of the heterologously expressed muscarinic M(3) receptors in the cells did not alone stimulate cAMP production. However, a Ca(2+) elevation potentiated cAMP production in the presence of a primary stimulant such as forskolin or activated G(s) proteins. This type of regulation of AC is different from previously identified Ca(2+)-sensitive AC isoforms. For comparison the Ca(2+)/calmodulin-activated type I AC was expressed and demonstrated to be stimulated directly by an increase in Ca(2+). Together the results demonstrate that octopamine can synergistically regulate the AC activity via two different receptors in Sf9 cells.     

Molecular and functional characterization of an octopamine receptor from honeybee (Apis mellifera) brain

Biogenic amines and their receptors regulate and modulate many physiological and behavioural processes in animals. In vertebrates, octopamine is only found in trace amounts and its function as a true neurotransmitter is unclear. In protostomes, however, octopamine can act as neurotransmitter, neuromodulator and neurohormone. In the honeybee, octopamine acts as a neuromodulator and is involved in learning and memory formation. The identification of potential octopamine receptors is decisive for an understanding of the cellular pathways involved in mediating the effects of octopamine. Here we report the cloning and functional characterization of the first octopamine receptor from the honeybee, Apis mellifera. The gene was isolated from a brain-specific cDNA library. It encodes a protein most closely related to octopamine receptors from Drosophila melanogaster and Lymnea stagnalis. Signalling properties of the cloned receptor were studied in transiently transfected human embryonic kidney (HEK) 293 cells. Nanomolar to micromolar concentrations of octopamine induced oscillatory increases in the intracellular Ca2+ concentration. In contrast to octopamine, tyramine only elicited Ca2+ responses at micromolar concentrations. The gene is abundantly expressed in many somata of the honeybee brain, suggesting that this octopamine receptor is involved in the processing of sensory inputs, antennal motor outputs and higher-order brain functions.     

Cyclic AMP regulates the calcium transients released from IP(3)-sensitive stores by activation of rat kappa-opioid receptors expressed in CHO cells

We analyzed intracellular Ca(2+)and cAMP levels in Chinese hamster ovary cells expressing a cloned rat kappa opioid receptor (CHO-kappa cells). Although expression of kappa(kappa)-opioid receptors was confirmed with a fluorescent dynorphin analog in almost all CHO-kappa cells, the kappa-specific agonists, U50488H or U69593, induced a Ca(2+) transient only in 35% of the cells. The Ca(2+) response occurred in all-or-none fashion and the half-maximal dosage of U50488H (812.1nM) was higher than that (3.2nM) to inhibit forskolin-stimulated cAMP. The kappa-receptors coupled to G(i/o)proteins since pertussis toxin significantly reduced the U50488H actions on intracellular Ca(2+) and cAMP. The Ca(2+) transient originates from IP(3)-sensitive internal stores since the Ca(2+) response was blocked by a PLC inhibitor (U73122) or by thapsigargin depletion of internal stores while removal of extracellular Ca(2+) had no effect. Interestingly, application of dibutyryl cAMP (+ 56.2%) or 8-bromo-cAMP (+ 174.7%) significantly increased the occurrence of U50488H-induced Ca(2+) mobilization while protein kinase A (PKA) inhibitors, Rp-cAMP (-32.3%) or myr-psi PKA (-73.9%) significantly reduced the response. Therefore, it was concluded that cAMP and PKA activity can regulate the Ca(2+) mobilization. These results suggest that the kappa receptor-linked cAMP cascade regulates the occurrence of kappa-opioid-mediated Ca(2+) mobilization.     

Parathyroid hormone potentiates nucleotide-induced [Ca2+]i release in rat osteoblasts independently of Gq activation or cyclic monophosphate accumulation. A mechanism for localizing systemic responses in bone

The regulation of tissue turnover requires the coordinated activity of both local and systemic factors. Nucleotides exist transiently in the extracellular environment, where they serve as ligands to P2 receptors. Here we report that the localized release of these nucleotides can sensitize osteoblasts to the activity of systemic factors. We have investigated the ability of parathyroid hormone (PTH), a principal regulator of bone resorption and formation, to potentiate signals arising from nucleotide stimulation of UMR-106 clonal rat osteoblasts. PTH receptor activation alone did not lead to [Ca(2+)](i) elevation in these cells, indicating no G(q) coupling, however, activation of G(q)-coupled P2Y(1) receptors resulted in characteristic [Ca(2+)](i) release. PTH potentiated this nucleotide-induced Ca(2+) release, independently of Ca(2+) influx. PTH-(1-31), which activates only G(s), mimicked the actions of PTH-(1-34), whereas PTH-(3-34), which only activates G(q), was unable to potentiate nucleotide-induced [Ca(2+)](i) release. Despite this coupling of the PTHR to G(s), cAMP accumulation or protein kinase A activation did not contribute to the potentiation. 3-Isobutyl-1-methylxanthine, but not forskolin effectively potentiated nucleotide-induced [Ca(2+)](i) release, however, further experiments proved that cyclic monophosphates were not involved in the potentiation mechanism. Costimulation of UMR-106 cells with P2Y(1) agonists and PTH led to increased levels of cAMP response element-binding protein phosphorylation and a synergistic effect was observed on endogenous c-fos gene expression following costimulation. In fact the calcium responsive Ca/cAMP response element of the c-fos promoter alone was effective at driving this synergistic gene expression. These findings demonstrate that nucleotides can provide a targeted response to systemic factors, such as PTH, and have important implications for PTH-induced signaling in bone.     

Cloning, expression and functional analysis of an octopamine receptor from Periplaneta americana

Octopamine regulates multiple physiological functions in invertebrates. The biological effects of octopamine and the pharmacology of octopamine receptors have been extensively studied in the American cockroach, Periplaneta americana. This paper reports the cloning of the first octopamine receptor from Periplaneta americana. A cDNA encoding a putative 7 transmembrane receptor was isolated from the head of Periplaneta americana. The encoded protein contains 628 amino acids and has sequence similarity to other biogenic amine receptors. This protein was expressed in COS-7 cells for radioligand binding studies using the antagonist 3H-yohimbine. Competitive binding comparing biogenic amines that could potentially function as endogenous ligands demonstrated this receptor had the highest affinity for octopamine (Ki = 13.3 microM) followed by tyramine, dopamine, serotonin and histamine. Octopamine increased both cAMP levels (EC50 = 1.62 microM) and intracellular concentrations of calcium through the receptor expressed in HEK-293 cells. Tyramine increased levels of both of these second messengers but only at significantly higher concentrations than octopamine. The cAMP increase by octopamine was independent of the increase in calcium. Competitive binding with antagonists revealed this receptor is similar to Lym oa1 from Lymnaea stagnalis. The data indicate that this cDNA is the first octopamine receptor cloned from Periplaneta americana and therefore has been named Pa oa1.     

Intracellular Ca(2+) release channels in evolution

Intracellular Ca(2+)-release channels (ICRCs) form a superfamily of genes that encompasses two distinct subfamilies: the inositol trisphosphate receptor and the ryanodine receptor genes, which encode the largest ion channels known today. During evolution from nematodes to man, mechanisms of gene duplication and divergence have increased the number of known ICRC genes, which have been gradually co-opted to contribute to the increasing complexity of intracellular Ca(2+) signalling required for regulation of specialised eukaryotic cell activities.     

Obestatin does not activate orphan G protein-coupled receptor GPR39

Recently, the ligand of the orphan G protein-coupled receptor GPR39 has been identified as obestatin, a 23-amino acid peptide derived from the ghrelin precursor protein. We used two methods to study the possible activation of GPR39 by obestatin: cAMP measurements based on a luminescent reporter gene and a fluorometric Ca(2+) flux method. The former was similar to that reported in the original publication of Zhang et al. [J.V. Zhang, P.G. Ren, O. Avsian-Kretchmer, C.W. Luo, R. Rauch, C. Klein, Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake, Science 310 (2005) 996-999]. The latter method used promiscuous as well as chimaeric G-proteins commonly used to couple orphan G protein-coupled receptors to the phospholipase C pathway, that leads to intracellular Ca(2+) rise. We could, however, not demonstrate activation of the GPR39 receptor by obestatin via any of these signal transduction pathways. We could activate GPR39 by high concentrations of Zn(2+), demonstrating cell surface expression of a functional receptor that could elicit a Ca(2+) response. The Zn(2+) response was not affected by obestatin. The identity of the native ligand for GPR39 remains to be elucidated.     

Cellular models for the study of the pharmacology and signaling of melanin-concentrating hormone receptors

Cellular models for the study of the neuropeptide melanin-concentrating hormone (MCH) have become indispensable tools for pharmacological profiling and signaling analysis of MCH and its synthetic analogues. Although expression of MCH receptors is most abundant in the brain, MCH-R(1) is also found in different peripheral tissues. Therefore, not only cell lines derived from nervous tissue but also from peripheral tissues that naturally express MCH receptors have been used to study receptor signaling and regulation. For screening of novel compounds, however, heterologous expression of MCH-R(1) or MCH-R(2) genes in HEK293, Chinese hamster ovary, COS-7, or 3T3-L1 cells, or amplified MCH-R(1) expression/signaling in IRM23 cells transfected with the G(q) protein gene are the preferred tools because of more distinct pharmacological effects induced by MCH, which include inhibition of cAMP formation, stimulation of inositol triphosphate production, increase in intracellular free Ca(2+) and/or activation of mitogen-activated protein kinases. Most of the published data originate from this type of model system, whereas data based on studies with cell lines endogenously expressing MCH receptors are more limited. This review presents an update on the different cellular models currently used for the analysis of MCH receptor interaction and signaling.     

Identification of intracellular and plasma membrane calcium channel homologues in pathogenic parasites

Ca(2+) channels regulate many crucial processes within cells and their abnormal activity can be damaging to cell survival, suggesting that they might represent attractive therapeutic targets in pathogenic organisms. Parasitic diseases such as malaria, leishmaniasis, trypanosomiasis and schistosomiasis are responsible for millions of deaths each year worldwide. The genomes of many pathogenic parasites have recently been sequenced, opening the way for rational design of targeted therapies. We analyzed genomes of pathogenic protozoan parasites as well as the genome of Schistosoma mansoni, and show the existence within them of genes encoding homologues of mammalian intracellular Ca(2+) release channels: inositol 1,4,5-trisphosphate receptors (IP(3)Rs), ryanodine receptors (RyRs), two-pore Ca(2+) channels (TPCs) and intracellular transient receptor potential (Trp) channels. The genomes of Trypanosoma, Leishmania and S. mansoni parasites encode IP(3)R/RyR and Trp channel homologues, and that of S. mansoni additionally encodes a TPC homologue. In contrast, apicomplexan parasites lack genes encoding IP(3)R/RyR homologues and possess only genes encoding TPC and Trp channel homologues (Toxoplasma gondii) or Trp channel homologues alone. The genomes of parasites also encode homologues of mammalian Ca(2+) influx channels, including voltage-gated Ca(2+) channels and plasma membrane Trp channels. The genome of S. mansoni also encodes Orai Ca(2+) channel and STIM Ca(2+) sensor homologues, suggesting that store-operated Ca(2+) entry may occur in this parasite. Many anti-parasitic agents alter parasite Ca(2+) homeostasis and some are known modulators of mammalian Ca(2+) channels, suggesting that parasite Ca(2+) channel homologues might be the targets of some current anti-parasitic drugs. Differences between human and parasite Ca(2+) channels suggest that pathogen-specific targeting of these channels may be an attractive therapeutic prospect.     

A crucial role for cAMP and protein kinase A in D1 dopamine receptor regulated intracellular calcium transients

D1-like dopamine receptors stimulate Ca(2+) transients in neurons but the effector coupling and signaling mechanisms underlying these responses have not been elucidated. Here we investigated potential mechanisms using both HEK 293 cells that stably express D1 receptors (D1HEK293) and hippocampal neurons in culture. In D1HEK293 cells, the full D1 receptor agonist SKF 81297 evoked a robust dose-dependent increase in Ca(2+)(i) following 'priming' of endogenous G(q/11)-coupled muscarinic or purinergic receptors. The effect of SKF81297 could be mimicked by forskolin or 8-Br-cAMP. Further, cholera toxin and the cAMP-dependent protein kinase (PKA) inhibitors, KT5720 and H89, as well as thapsigargin abrogated the D1 receptor evoked Ca(2+) transients. Removal of the priming agonist and treatment with the phospholipase C inhibitor U73122 also blocked the SKF81297-evoked responses. D1R agonist did not stimulate IP(3) production, but pretreatment of cells with the D1R agonist potentiated G(q)-linked receptor agonist mobilization of intracellular Ca(2+) stores. In neurons, SKF81297 and SKF83959, a partial D1 receptor agonist, promoted Ca(2+) oscillations in response to G(q/11)-coupled metabotropic glutamate receptor (mGluR) stimulation. The effects of both D1R agonists on the mGluR-evoked Ca(2+) responses were PKA dependent. Altogether the data suggest that dopamine D1R activation and ensuing cAMP production dynamically regulates the efficiency and timing of IP(3)-mediated intracellular Ca(2+) store mobilization.     

New insights concerning the glucose-dependent insulin secretagogue action of glucagon-like peptide-1 in pancreatic beta-cells.

The GLP-1 receptor is a Class B heptahelical G-protein-coupled receptor that stimulates cAMP production in pancreatic beta-cells. GLP-1 utilizes this receptor to activate two distinct classes of cAMP-binding proteins: protein kinase A (PKA) and the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs). Actions of GLP-1 mediated by PKA and Epac include the recruitment and priming of secretory granules, thereby increasing the number of granules available for Ca(2+)-dependent exocytosis. Simultaneously, GLP-1 promotes Ca(2+) influx and mobilizes an intracellular source of Ca(2+). GLP-1 sensitizes intracellular Ca(2+) release channels (ryanodine and IP (3) receptors) to stimulatory effects of Ca(2+), thereby promoting Ca(2+)-induced Ca(2+) release (CICR). In the model presented here, CICR activates mitochondrial dehydrogenases, thereby upregulating glucose-dependent production of ATP. The resultant increase in cytosolic [ATP]/[ADP] concentration ratio leads to closure of ATP-sensitive K(+) channels (K-ATP), membrane depolarization, and influx of Ca(2+) through voltage-dependent Ca(2+) channels (VDCCs). Ca(2+) influx stimulates exocytosis of secretory granules by promoting their fusion with the plasma membrane. Under conditions where Ca(2+) release channels are sensitized by GLP-1, Ca(2+) influx also stimulates CICR, generating an additional round of ATP production and K-ATP channel closure. In the absence of glucose, no "fuel" is available to support ATP production, and GLP-1 fails to stimulate insulin secretion. This new "feed-forward" hypothesis of beta-cell stimulus-secretion coupling may provide a mechanistic explanation as to how GLP-1 exerts a beneficial blood glucose-lowering effect in type 2 diabetic subjects.     

Intracellular angiotensin II activates rat myometrium

Angiotensin II is a modulator of myometrial activity; both AT(1) and AT(2) receptors are expressed in myometrium. Since in other tissues angiotensin II has been reported to activate intracellular receptors, we assessed the effects of intracellular administration of angiotensin II via microinjection on myometrium, using calcium imaging. Intracellular injection of angiotensin II increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in myometrial cells in a dose-dependent manner. The effect was abolished by the AT(1) receptor antagonist losartan but not by the AT(2) receptor antagonist PD-123319. Disruption of the endo-lysosomal system, but not that of Golgi apparatus, prevented the angiotensin II-induced increase in [Ca(2+)](i). Blockade of AT(1) receptor internalization had no effect, whereas blockade of microautophagy abolished the increase in [Ca(2+)](i) produced by intracellular injection of angiotensin II; this indicates that microautophagy is a critical step in transporting the peptide into the endo-lysosomes lumenum. The response to angiotensin II was slightly reduced in Ca(2+)-free saline, indicating a major involvement of Ca(2+) release from internal stores. Blockade of inositol 1,4,5-trisphosphate (IP(3)) receptors with heparin and xestospongin C or inhibition of phospholipase C (PLC) with U-73122 abolished the response to angiotensin II, supporting the involvement of PLC-IP(3) pathway. Angiotensin II-induced increase in [Ca(2+)](i) was slightly reduced by antagonism of ryanodine receptors. Taken together, our results indicate for the first time that in myometrial cells, intracellular angiotensin II activates AT(1)-like receptors on lysosomes and activates PLC-IP(3)-dependent Ca(2+) release from endoplasmic reticulum; the response is further augmented by a Ca(2+)-induced Ca(2+) release mechanism via ryanodine receptors activation.