OX1 orexin receptors activate extracellular signal-regulated kinase in Chinese hamster ovary cells via multiple mechanisms: the role of Ca2+ influx in OX1 receptor signaling.

Activation of OX1 orexin receptors heterologously expressed in Chinese hamster ovary (CHO) cells led to a rapid, strong, and long-lasting increase in ERK phosphorylation (activation). Dissection of the signal pathways to ERK using multiple inhibitors and dominant-negative constructs indicated involvement of Ras, protein kinase C, phosphoinositide-3-kinase, and Src. Most interestingly, Ca2+ influx appeared central for the ERK response in CHO cells, and the same was indicated in recombinant neuro-2a cells and cultured rat striatal neurons. Detailed investigations in CHO cells showed that inhibition of the receptor- and store-operated Ca2+ influx pathways could fully attenuate the response, whereas inhibition of the store-operated Ca2+ influx pathway alone or the Ca2+ release was ineffective. If the receptor-operated pathway was blocked, an exogenously activated store-operated pathway could take its place and restore the coupling of OX1 receptors to ERK. Further experiments suggested that Ca2+ influx, as such, may not be required for ERK phosphorylation, but that Ca2+, elevated via influx, acts as a switch enabling OX1 receptors to couple to cascades leading to ERK phosphorylation, cAMP elevation, and phospholipase C activation. In conclusion, the data suggest that the primary coupling of orexin receptors to Ca2+ influx allows them to couple to other signal pathways; in the absence of coupling to Ca2+ influx, orexin receptors can act as signal integrators by taking advantage of other Ca2+ influx pathways.

Orexin signaling in recombinant neuron-like cells.

To assess the role of orexin receptor signaling in neuron-like cells, Neuro-2a murine neuroblastoma and PC12 human pheochromocytoma cells were stably transfected with human OX(1) or OX(2) receptors. Activation of both receptors strongly elevated cellular inositol phosphates and Ca(2+). A difference in the potency between orexin-A and -B was seen for OX(1), but not OX(2) receptors. Dependence of the orexin-mediated Ca(2+) response on extracellular Ca(2+) and the observed Ba(2+) influx indicate that in addition to phospholipase C, orexin receptors also may couple to similar non-voltage-gated Ca(2+) channels in neuronal cells as previously characterized in non-neuronal cells.

Phospholipase C activator m-3M3FBS affects Ca2+ homeostasis independently of phospholipase C activation.

In this study, we have investigated responses to the phospholipase C (PLC) activator m-3M3FBS in SH-SY5Y human neuroblastoma cells. As measured using fura-2, m-3M3FBS caused a slowly developing - full response was obtained within 4-6 min - Ca(2+) elevation both in the presence and absence of extracellular Ca(2+), indicating Ca(2+) release from intracellular stores, putatively from endoplasmic reticulum and mitochondria. PLC activity was also measured using two methods, the classical ion-exchange separation and the more novel fluorescent real-time method. In the time frame in which m-3M3FBS caused Ca(2+) elevation (up to 7 min), no PLC activation was detected. Instead, more than 20 min were required to see any inositol phosphate generation in response to m-3M3FBS. m-3M3FBS also interfered with store-operated Ca(2+) influx and Ca(2+) extrusion. In conclusion, m-3M3FBS cannot be considered either potent or specific PLC activator.

Alpha2C-adrenergic receptors exhibit enhanced surface expression and signaling upon association with beta2-adrenergic receptors.

The alpha(2C)-adrenergic receptor (alpha(2C)AR) is known to be poorly trafficked to the cell surface when expressed in a variety of cell types. We tested the hypothesis that the surface expression and signaling of alpha(2C)AR might be enhanced by heterodimerization with other G protein-coupled receptors (GPCRs). Cotransfection of alpha(2C)AR with more than 25 related GPCRs revealed that only coexpression with the beta(2)-adrenergic receptor (beta(2)AR) increased the surface localization of alpha(2C)AR in human embryonic kidney-293 cells. Coimmunoprecipitation of alpha(2C)AR with beta(2)AR confirmed a physical interaction between the two receptors. Confocal microscopy studies demonstrated that alpha(2C)AR expressed alone was mainly intracellular, whereas alpha(2C)AR coexpressed with beta(2)AR was predominantly localized to the plasma membrane. Ligand binding studies revealed a significant increase in alpha(2C)AR binding sites upon coexpression with beta(2)AR, with no apparent change in affinity for alpha(2)AR ligands. Functional assays with the alpha(2)AR-specific agonist brimonidine (UK 14,304) revealed that coexpression of beta(2)AR with alpha(2C)AR enhanced alpha(2C)AR-mediated activation of extracellular signal-regulated kinase 1/2. Furthermore, analyses of agonist-promoted receptor endocytosis demonstrated enhanced alpha(2C)AR internalization in response to alpha(2)AR agonists when alpha(2C)AR and beta(2)AR were coexpressed. In addition, substantial cointernalization of alpha(2C)AR in response to betaAR agonists was observed when alpha(2C)AR was coexpressed with beta(2)AR. These data reveal that alpha(2C)AR can interact with beta(2)AR in cells in a manner that regulates alpha(2C)AR surface expression, internalization, and functionality.

OX1 orexin receptors couple to adenylyl cyclase regulation via multiple mechanisms.

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

Different roles for Gi and Go proteins in modulation of adenylyl cyclase type-2 activity.

The effect of Gi/o protein-coupled receptors on adenylyl cyclase type 2 (AC2) has been studied in Sf9 insect cells. Stimulation of cells expressing AC2 with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) led to a twofold stimulation of cAMP synthesis that could be blocked with the protein kinase C inhibitor GF109203X. Activation of a coexpressed alpha2A-adrenoceptor or muscarinic M4 receptor inhibited the stimulation by TPA almost completely in a pertussis toxin-sensitive manner. Activation of Gs proteins switched the response of the alpha2A-adrenoceptor to potentiation of prestimulated AC2 activity. The potentiation, but not the inhibition, could be blocked by a Gbetagamma scavenger. A novel methodological approach, whereby signalling through endogenous G proteins was ablated, was used to assess specific G protein species in the signal pathway. Expression of Go proteins (alphao1 + beta1gamma2) restored both the inhibition and the potentiation, whereas expression of Gi proteins (alphai1 + beta1gamma2) resulted in a potentiation of both the TPA- and the Gs-stimulated AC2 activity. The data presented supports the view of AC2 as a molecular switch and implicates this isoform as a target for Go protein-linked signalling.

Functions of the orexinergic/hypocretinergic system.

Orexin A and orexin B are hypothalamic peptides that act on their targets via two G protein-coupled receptors (OX1 and OX2 receptors). In the central nervous system, the cell bodies producing orexins are localized in a narrow region within the lateral hypothalamus and project mainly to regions involved in feeding, sleep, and autonomic functions. Via putative pre- and postsynaptic effects, orexins increase synaptic activity in these regions. In isolated neurons and cells expressing recombinant receptors orexins cause Ca2+ elevation, which is mainly dependent on influx. The activity of orexinergic cells appears to be controlled by feeding- and sleep-related signals via a variety of neurotransmitters/hormones from the brain and other tissues. Orexins and orexin receptors are also found outside the central nervous system, particularly in organs involved in feeding and energy metabolism, e.g., gastrointestinal tract, pancreas, and adrenal gland. In the present review we focus on the physiological properties of the cells that secrete or respond to orexins.

Distinct recognition of OX1 and OX2 receptors by orexin peptides.

In this study, we have compared the abilities of orexin-A and orexin-B and variants of orexin-A to activate different Ca(2+) responses (influx and release) in human OX(1) and OX(2) receptor- expressing Chinese hamster ovary cells. Responses mediated by activation of both receptor subtypes with either orexin-A or -B were primarily dependent on extracellular Ca(2+), suggesting similar activation of Ca(2+) influx as we have previously shown for orexin-A and OX(1) receptors. Amino acid-wise truncation of orexin-A reduced its ability to activate OX(1) and OX(2) receptors, but the response mediated by the OX(2) receptor was more resistant to truncation than the response mediated by the OX(1) receptor. We also performed a sequential replacement of amino acids 14 to 26 with alanine in the truncated orexin-A variant orexin-A(14-33). Replacement of the same amino acids produced a fall in the potency for each receptor subtype, but the reduction was less prominent for the OX(2) receptor. The most marked reduction was produced by the replacement of Leu20, Asp25, and His26 with alanine. Interestingly, extracellular Ca(2+) dependence of responses to some of the mutated peptides was different from those of orexin-A and -B. The mutagenesis also suggests that although the determinants required from orexin-A for binding to and activation of the receptor are highly conserved between the orexin receptor subtypes, the OX(2) receptor requires fewer determinants. This might in part explain why orexin-B has the affinity and potency equal to orexin-A for this subtype, although it has 10- to 100-fold lower affinity and potency for the OX(1) receptor.