Role of hyperpolarization-activated cyclic nucleotide-gated channel HCN2 in embryonic neural stem cell proliferation and differentiation.

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN channels) are involved in spontaneous activity in many electrically active cell types such as cardiomyocytes and neurons. In this study, the role of HCN channels in proliferation and migration of Nestin and Sox2 expressing embryonic neural progenitor cells (NPC) originating from the subventricular zone (SVZ) was examined. Immunostaining and PCR data showed that the HCN2 subtype was highly expressed in these cells. Patch clamp recordings revealed a hyperpolarization-activated current, which was sensitive to inhibitors of HCN channels. Using the fluorescence dye bis-(1,3-dibutylbarbituric acid)-trimethineoxonol (DiBAC(4)(3)) we found that a prompt reduction of the extracellular K+ concentration, or exposing the cells to acute hypoxia, induced an instant hyperpolarization in the whole cell population. Recovery from low K+ induced hyperpolarization after extracellular calcium removal, or by re-oxygenation of hypoxic cells, was sensitive to ZD7288, a HCN channel inhibitor. Treatment of neurosphere cultures from the SVZ with ZD7288 caused a significant and reversible inhibition of neurosphere formation from single cells indicating that proliferation of progenitor cells was reduced. Furthermore, the migration of neuronal cells from neurospheres was considerably retarded in the presence of ZD7288. The results suggest that HCN2 channels are involved in controlling the proliferation of NPC and that HCN2 channel-induced spontaneous electrical activity may trigger the motility response of neurosphere-derived neurons in concert with other ion channels. Furthermore, the response to hypoxia suggests that HCN2 channels may trigger the chemotactic response of NPC to ischemic brain regions seen in many studies.

Tannic acid inhibits electrogenic Na+/HCO3- co-transporter activity in embryonic neural stem cell-derived radial glial-like cells.

Self-renewing neural stem cells and progenitor cells are cell populations that generate radial glial cells and neurons through asymmetric division. Regulation of intracellular pH in stem cells with high metabolic activity is critical for both cell signaling and proliferation. We have recently found that a S0859-inhibitable electrogenic Na/HCO3 co-transporter (NBCe1, Slc4a4), is the primary pHi regulatory mechanism in stem cell-derived radial glial-like cells. Here we show, by using the voltage-sensitive fluorescent dye DiBAC4(3) and BCECF, a pH-sensitive dye, that an antioxidant, tannic acid (100 µM), can inhibit potassium- and calcium-dependent rapid changes in membrane potential and NBCe1 mediated pHi regulation in brain-derived glial-like cells in vitro. Furthermore, neural stem cell differentiation and neurosphere formation (proliferation) were completely inhibited by tannic acid. The present study provides evidence that tannic acid is a natural inhibitor of NBCe1. It is tempting to speculate that tannic acid or related compounds that inhibits NBCe1-mediated pHi regulation in glial-like cells may also have bearing on the treatment of glial neoplasms.

Regulation of intracellular pH by electrogenic Na+/HCO3- co-transporters in embryonic neural stem cell-derived radial glia-like cells.

A stroke causes a hypoxic brain microenvironment that alters neural cell metabolism resulting in cell membrane hyperpolarization and intracellular acidosis. We studied how intracellular pH (pHi) is regulated in differentiated mouse neural progenitor cells during hyperpolarizing conditions, induced by prompt reduction of the extracellular K+ concentration. We found that the radial glia-like population in differentiating embryonic neural progenitor cells, but not neuronal cells, was rapidly acidified under these conditions. However, when extracellular calcium was removed, an instant depolarization and recovery of the pHi, back to normal levels, took place. The rapid recovery phase seen in the absence of calcium, was dependent on extracellular bicarbonate and could be inhibited by S0859, a potent Na/HCO3 cotransporter inhibitor. Immunostaining and PCR data, showed that NBCe1 (SLC4A4) and NBCn1 (SLC4A7) were expressed in the cell population and that the pHi recovery in the radial glial-like cells after calcium removal was mediated mainly by the electrogenic sodium bicarbonate transporter NBCe1 (SLC4A4). Our results indicate that extracellular calcium might hamper pHi regulation and Na/HCO3 cotransporter activity in a brain injury microenvironment. Our findings show that the NBC-type transporters are the main pHi regulating systems prevailing in glia-like progenitor cells and that these calcium sensitive transporters are important for neuronal progenitor cell proliferation, survival and neural stem cell differentiation.

Extracellular acidosis is a novel danger signal alerting innate immunity via the NLRP3 inflammasome.

BACKGROUND: Local acidosis has been demonstrated in ischemic tissues and at inflammatory sites. RESULTS: Acidic extracellular pH triggers NLRP3 inflammasome activation and interleukin-1ß secretion in human macrophages. CONCLUSION: Acidic pH represents a novel danger signal alerting the innate immunity. SIGNIFICANCE: Local acidosis may promote inflammation at ischemic and inflammatory sites. Local extracellular acidification has been demonstrated at sites of ischemia and inflammation. IL-1ß is one of the key proinflammatory cytokines, and thus, its synthesis and secretion are tightly regulated. The NLRP3 (nucleotide-binding domain leucine-rich repeat containing family, pyrin domain containing 3) inflammasome complex, assembled in response to microbial components or endogenous danger signals, triggers caspase-1-mediated maturation and secretion of IL-1ß. In this study, we explored whether acidic environment is sensed by immune cells as an inflammasome-activating danger signal. Human macrophages were exposed to custom cell culture media at pH 7.5-6.0. Acidic medium triggered pH-dependent secretion of IL-1ß and activation of caspase-1 via a mechanism involving potassium efflux from the cells. Acidic extracellular pH caused rapid intracellular acidification, and the IL-1ß-inducing effect of acidic medium could be mimicked by acidifying the cytosol with bafilomycin A1, a proton pump inhibitor. Knocking down the mRNA expression of NLRP3 receptor abolished IL-1ß secretion at acidic pH. Remarkably, alkaline extracellular pH strongly inhibited the IL-1ß response to several known NLRP3 activators, demonstrating bipartite regulatory potential of pH on the activity of this inflammasome. The data suggest that acidic environment represents a novel endogenous danger signal alerting the innate immunity. Low pH may thus contribute to inflammation in acidosis-associated pathologies such as atherosclerosis and post-ischemic inflammatory responses.

Effects of acute hypoxia/acidosis on intracellular pH in differentiating neural progenitor cells.

The response of differentiating mouse neural progenitor cells, migrating out from neurospheres, to conditions simulating ischemia (hypoxia and extracellular or intracellular acidosis) was studied. We show here, by using BCECF and single cell imaging to monitor intracellular pH (pH(i)), that two main populations can be distinguished by exposing migrating neural progenitor cells to low extracellular pH or by performing an acidifying ammonium prepulse. The cells dominating at the periphery of the neurosphere culture, which were positive for neuron specific markers MAP-2, calbindin and NeuN had lower initial resting pH(i) and could also easily be further acidified by lowering the extracellular pH. Moreover, in this population, a more profound acidification was seen when the cells were acidified using the ammonium prepulse technique. However, when the cell population was exposed to depolarizing potassium concentrations no alterations in pH(i) took place in this population. In contrast, depolarization caused an increase in pH(i) (by 0.5 pH units) in the cell population closer to the neurosphere body, which region was positive for the radial cell marker (GLAST). This cell population, having higher resting pH(i) (pH 6.9-7.1) also responded to acute hypoxia. During hypoxic treatment the resting pH(i) decreased by 0.1 pH units and recovered rapidly after reoxygenation. Our results show that migrating neural progenitor cells are highly sensitive to extracellular acidosis and that irreversible damage becomes evident at pH 6.2. Moreover, our results show that a response to acidosis clearly distinguishes two individual cell populations probably representing neuronal and radial cells.

Glycosylphosphatidylinositol (GPI)-anchoring of mamba toxins enables cell-restricted receptor silencing.

Muscarinic toxins (MTs) are snake venom peptides found to selectively target specific subtypes of G-protein-coupled receptors. In here, we have attached a glycosylphosphatidylinositol (GPI) tail to three different toxin molecules and evaluated their receptor-blocking effects in a heterologous expression system. MT7-GPI remained anchored to the cell surface and selectively inhibited M(1) muscarinic receptor signaling expressed in the same cell. To further demonstrate the utility of the GPI tail, we generated MT3- and MTα-like gene sequences and fused these to the signal sequence for GPI attachment. Functional assessment of these membrane-anchored toxins on coexpressed target receptors indicated a prominent antagonistic effect. In ligand binding experiments the GPI-anchored toxins were found to exhibit similar selection profiles among receptor subtypes as the soluble toxins. The results indicate that GPI attachment of MTs and related receptor toxins could be used to assess the role of receptor subtypes in specific organs or even cells in vivo by transgenic approaches.

Selective interference with TRPC3/6 channels disrupts OX1 receptor signalling via NCX and reveals a distinct calcium influx pathway.

TRPC channels play significant roles in the regulation of neuronal plasticity and development. The mechanism by which these nonselective cation channels exert their trophic actions appears to involve entry of Ca(2+) into the cells. Using a neuronal cell model (differentiated human IMR32 neuroblastoma cells), we demonstrate a central role for sodium entry via TRPC3/6 channels in receptor-mediated increases in intracellular calcium. These Na(+)-dependent Ca(2+) influxes, which were observed in a subpopulation of cells, were efficiently blocked by protein kinase C activation, by the Na(+)/Ca(2+) exchanger inhibitors, and by molecular disruption of TRPC3/6 channel function. On the other hand, another subpopulation of cells showed a Na(+)-independent Ca(2+) entry upon stimulation of the same receptors, orexin/hypocretin and bradykinin receptors. This second type of response was not affected by the above mentioned treatments, but it was sensitive to polyvalent cations, such as ruthenium red, spermine and Gd(3+). The data suggest that a NCX-TRPC channel interaction constitutes an important functional unit in receptor-mediated Ca(2+) influx in neuronal cells.

Altered calcium signaling in an experimental model of glaucoma.

PURPOSE: To investigate calcium signaling in a rat experimental model of glaucoma. METHODS: A method for labeling ganglion cell layer (GCL) neurons with the calcium indicator Fura-2 in flat-mounted retinas of adult rats was established. Pharmacologically evoked responses in laser-induced glaucomatous and control retinas were imaged 2 weeks after the initial laser treatment. The optic nerves of the same eyes were evaluated for neurodegenerative changes. RESULTS: After laser treatment, intraocular pressure (IOP) was elevated 1.5- to 4.9-fold (24.70 ± 15.57 mm Hg) compared with control eyes (8.71 ± 1.53 mm Hg), and the area of neurodegenerative axons in optic nerve sections of laser-treated eyes was increased by 1.2- to 13.3-fold. The basal intracellular Ca(2+) level, as revealed by the Fura-2 ratio, was elevated in GCL neurons of laser-treated eyes compared with controls. This might suggest a mild degree of damage at the level of the soma in the GCL neurons of eyes with elevated IOP. Although glaucomatous GCL neurons remained functional as assessed pharmacologically, analysis of imaging data revealed that responses evoked by a brief application of ATP were slightly reduced rather than increased in the cells of laser-treated eyes compared with controls. No significant relationships were found between IOP/optic nerve damage and functional characteristics (basal intracellular Ca(2+) level or response to carbachol/elevated K(+)/ATP) within cells of laser-treated eyes. CONCLUSIONS: Ca(2+) imaging is a useful tool to map altered physiological characteristics of individual GCL neurons in the glaucomatous eye.

A role for PKD1 and PKD3 activation in modulation of calcium oscillations induced by orexin receptor 1 stimulation.

The neuropeptides orexin-A/hypocretin-1 (Ox-A) and orexin-B/hypocretin-2 play an important role in the control of energy metabolism via either of two G-protein-coupled receptors, orexin receptor 1 (Ox1R) and 2. Despite its significant physiological functions, signaling via orexin receptors is still poorly characterized. The aim of this study was to improve our understanding of early signaling events triggered by the binding of Ox-A to Ox1R. Using phosphospecific antibodies, we observed that early kinase activation by Ox-A in a HEK293 cell line stably expressing Ox1R (HEKOx1R) included ERK1/2, PKCdelta, and PKD1. Elevation of intracellular Ca(2+) is a well-characterized response to Ox1R activation. Comparison of Ox-A-induced calcium elevation and PKD1 activation demonstrated that both responses are detectable soon after stimulation and increase in a dose-dependent manner, but inhibition of protein kinase C, when low Ox-A concentrations are used, affects them differently. PKD family of protein kinases has 3 members: PKD1, 2, and 3, which are all expressed in HEKOx1R cells. In response to stimulation of the cells with 1nM Ox-A, both PKD1 and PKD3 are activated and increased in the plasma membrane, pointing at a possible role for these kinases in that cell compartment. Overexpression of either kinase-dead PKD1 or kinase-dead PKD3 disrupts Ox-A-induced calcium oscillations demonstrating the functional role of these kinases in modulating physiological responses to Ox-A.

Differentiation dependent expression of TRPA1 and TRPM8 channels in IMR-32 human neuroblastoma cells.

TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non-selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR-32 undergoes a remarkable differentiation in response to treatment with 5-bromo-2-deoxyuridine. The cells acquire a neuronal morphology with increased expression of N-type voltage gated calcium channels and neurotransmitters. Here we show using RT-PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR-32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl-isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC-030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR-32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR-32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression.

Food-induced expression of orexin receptors in rat duodenal mucosa regulates the bicarbonate secretory response to orexin-A.

Presence of appetite-regulating peptides orexin-A and orexin-B in mucosal endocrine cells suggests a role in physiological control of the intestine. Our aim was to characterize orexin-induced stimulation of duodenal bicarbonate secretion and modulation of secretory responses and mucosal orexin receptors by overnight food deprivation. Lewis x Dark Agouti rats were anesthetized and proximal duodenum cannulated in situ. Mucosal bicarbonate secretion (pH stat) and mean arterial blood pressure were continuously recorded. Orexin-A was administered intra-arterially close to the duodenum, intraluminally, or into the brain ventricles. Total RNA was extracted from mucosal specimens, reverse transcribed to cDNA and expression of orexin receptors 1 and 2 (OX1 and OX2) measured by quantitative real-time PCR. OX1 protein was measured by Western blot. Intra-arterial orexin-A (60-600 nmol.h(-1).kg(-1)) increased (P < 0.01) the duodenal secretion in fed but not in fasted animals. The OX1 receptor antagonist SB-334867, which was also found to have a partial agonist action, abolished the orexin-induced secretory response but did not affect secretion induced by the muscarinic agonist bethanechol. Atropine, in contrast, inhibited bethanechol but not orexin-induced secretion. Orexin-A infused into the brain ventricles (2-20 nmol.kg(-1).h(-1)) or added to luminal perfusate (1.0-100 nM) did not affect secretion, indicating that orexin-A acts peripherally and at basolateral receptors. Overnight fasting decreased mucosal OX1 and OX2 mRNA expression (P < 0.01) as well as OX1 protein expression (P < 0.05). We conclude that stimulation of secretion by orexin-A may involve both receptor types and is independent of cholinergic pathways. Intestinal OX receptors and secretory responses are markedly related to food intake.

The orexin OX1 receptor regulates Ca2+ entry via diacylglycerol-activated channels in differentiated neuroblastoma cells.

We studied the cellular response to orexin type 1 receptor (OX1R) stimulation in differentiated IMR-32 neuroblastoma cells. In vitro differentiation of IMR-32 cells with 5-bromo-2'-deoxyuridine leads to a neuronal phenotype with long neurite extensions and an upregulation of mainly N-type voltage-gated calcium channels. Transduction of differentiated IMR-32 cells with baculovirus harboring an OX1R-green fluorescent protein cDNA fusion construct resulted in appearance of fluorescence that was confined mainly to the plasma membrane in the cell body and to neurites. Application of orexin-A to fluorescent cells led to an increase in intracellular free Ca2+ concentration, [Ca2+]i. At low nanomolar concentrations of orexin-A, the response was reversibly attenuated by removal of extracellular Ca2+, by application of a high concentration (10 mM) of Mg2+, and by the pharmacological channel blocker dextromethorphan. A diacylglycerol, dioctanoylglycerol, but not thapsigargin or depolarization with potassium, mimicked the OX1R response with regard to Mg2+ sensitivity. A reverse transcription-PCR screening identified mRNAs for all transient receptor potential canonical (TRPC) channels, including TRPC3, TRPC6, and TRPC7, which are known to be activated by diacylglycerol. Expression of a dominant-negative TRPC6 channel subunit blunted the responses to both dioctanoylglycerol and OX1R stimulation. The results suggest that the OX1R activates a Ca2+ entry pathway that involves diacylglycerol-activated TRPC channels in neuronal cells.

Agonist potency differentiates G protein activation and Ca2+ signalling by the orexin receptor type 1.

The G protein coupling characteristics of a flag epitope-tagged orexin receptor type 1 (OX1R) was investigated in HEK293 cells. Immunoprecipitation of the OX1R and immunoblotting revealed interactions with Gq/G11 proteins as well as with Gs and Gi proteins. Stimulation with orexin-A did not affect the ability of the OX1R to coprecipitate Gq/G11 proteins, but it robustly elevated the intracellular concentration of Ca2+, [Ca2+]i. No changes in cAMP levels could be detected upon receptor stimulation. To get further insight into the functional correlation of G protein activation and Ca2+ signalling, we used baculovirus transduction to express chimeric G proteins, containing the Galphas protein backbone with various Galpha donor sequences (Galphas/x) at the N and C termini, and measured cAMP as functional output. The Galphas/x chimeric proteins with Galpha11(Galphaq) and Galpha16 structure in the C terminus were stimulated by the OX1R. Concentration-response curves with Galphas/16 revealed an agonist potency correlation between G protein activation and the elevation of [Ca2+]i via discharge of intracellular Ca2+ stores, a feature also recognized for the muscarinic M3 receptor. However, in contrast to the M3 receptor, the OX1R elevated [Ca2+]i via influx from extracellular space at about 30-fold lower agonist concentration. The results suggest that the OX1R is linked to influx of Ca2+ through a signal pathway independent of Gq/G11 protein activation.

G-protein-coupled OX1 orexin/hcrtr-1 hypocretin receptors induce caspase-dependent and -independent cell death through p38 mitogen-/stress-activated protein kinase.

We have investigated the signaling of OX(1) receptors to cell death using Chinese hamster ovary cells as a model system. OX(1) receptor stimulation with orexin-A caused a delayed cell death independently of cytosolic Ca(2+) elevation. The classical mitogen-activated protein kinase (MAPK) pathways, ERK and p38, were strongly activated by orexin-A. p38 was essential for induction of cell death, whereas the ERK pathway appeared protective. A pathway often implicated in the p38-mediated cell death, activation of p53, did not mediate the cell death, as there was no stabilization of p53 or increase in p53-dependent transcriptional activity, and dominant-negative p53 constructs did not inhibit cell demise. Under basal conditions, orexin-A-induced cell death was associated with compact chromatin condensation and it required de novo gene transcription and protein synthesis, the classical hallmarks of programmed (apoptotic) cell death. However, though the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethyl ketone (Z-VAD-fmk) fully inhibited the caspase activity, it did not rescue the cells from orexin-A-induced death. In the presence of Z-VAD-fmk, orexin-A-induced cell death was still dependent on p38 and de novo protein synthesis, but it no longer required gene transcription. Thus, caspase inhibition causes activation of alternative, gene transcription-independent death pathway. In summary, the present study points out mechanisms for orexin receptor-mediated cell death and adds to our general understanding of the role of G-protein-coupled receptor signaling in cell death by suggesting a pathway from G-protein-coupled receptors to cell death via p38 mitogen-/stress-activated protein kinase independent of p53 and caspase activation.

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.

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.

Orexin-A-induced Ca2+ entry: evidence for involvement of trpc channels and protein kinase C regulation.

The orexins are peptide transmitters/hormones, which exert stimulatory actions in many types of cells via the G-protein-coupled OX(1) and OX(2) receptors. Our previous results have suggested that low (subnanomolar) concentrations of orexin-A activate Ca(2+) entry, whereas higher concentrations activate phospholipase C, Ca(2+) release, and capacitative Ca(2+) entry. As shown here, the Ca(2+) response to subnanomolar orexin-A concentrations was blocked by activation of protein kinase C by using different approaches (12-O-tetradecanoylphorbol acetate, dioctanoylglycerol, and diacylglycerol kinase inhibition) and protein phosphatase inhibition by calyculin A. The Ca(2+) response to subnanomolar orexin-A concentrations was also blocked by Mg(2+), dextromethorphan, and tetraethylammonium. These treatments neither affected the response to high concentrations of orexin-A nor the thapsigargin-stimulated capacitative entry. The capacitative entry was instead strongly suppressed by SKF96365. An inward membrane current activated by subnanomolar concentrations of orexin-A and the currents activated upon transient expression of trpc3 channels were also sensitive to Mg(2+), dextromethorphan, and tetraethylammonium. Responses to subnanomolar concentrations of orexin-A (Ca(2+) elevation, inward current, and membrane depolarization) were voltage-dependent with a loss of the response around -15 mV. By using reverse transcription-PCR, mRNA for the trpc1-4 channel isoforms were detected in the CHO-hOX1-C1 cells. The expression of truncated TRPC channel isoforms, in particular trpc1 and trpc3, reduced the response to subnanomolar concentrations of orexin-A but did not affect the response to higher concentrations of orexin-A. The results suggest that activation of the OX(1) receptor leads to opening of a Ca(2+)-permeable channel, involving trpc1 and -3, which is controlled by protein kinase C.

Ca2+-dependent potentiation of muscarinic receptor-mediated Ca2+ elevation.

Muscarinic receptor-mediated increases in Ca(2+) in SH-SY5Y neuroblastoma cells consist of an initial fast and transient phase followed by a sustained phase. Activation of voltage-gated Ca(2+) channels prior to muscarinic stimulation resulted in a several-fold potentiation of the fast phase. Unlike the muscarinic response under control conditions, this potentiated elevation of intracellular Ca(2+) was to a large extent dependent on extracellular Ca(2+). In potentiated cells, muscarinic stimulation also activated a rapid Mn(2+) entry. By using known organic and inorganic blockers of cation channels, this influx pathway was easily separated from the known Ca(2+) influx pathways, the store-operated pathway and the voltage-gated Ca(2+) channels. In addition to the Ca(2+) influx, both IP(3) production and Ca(2+) release were also enhanced during the potentiated response. The results suggest that a small increase in intracellular Ca(2+) amplifies the muscarinic Ca(2+) response at several stages, most notably by unravelling an apparently novel receptor-activated influx pathway.

Muscarinic toxin 7 selectivity is dictated by extracellular receptor loops.

Muscarinic toxin 7 (MT7) is a mamba venom protein antagonist with extremely high selectivity for the M1 muscarinic acetylcholine receptor. To map the sites for the interaction of MT7 with muscarinic receptors we have used chimeric M1:M3 receptors and site-directed mutagenesis of the M3 and M4 receptor subtypes. Two Glu residues in M1, one in extracellular loop 2 and one in extracellular loop 3, were found to be important for the high affinity binding of MT7. Substitution of the corresponding Lys residues in the M3 receptor with Glu converted the M3 mutant to an MT7 binding receptor, albeit with lower affinity compared with M1. A Phe --> Tyr substitution in extracellular loop 2 of M3 together with the 2 Glu mutations generated a receptor with an increased MT7 affinity (apparent Ki = 0.26 nM in a functional assay) compared with the M1 receptor (apparent Ki = 1.31 nM). The importance of the identified amino acid residues was confirmed with a mutated M4 receptor constructs. The results indicate that the high selectivity of MT7 for the M1 receptor depends on very few residues, thus providing good prospects for future design and synthesis of muscarinic receptor-selective ligands.

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.