Expression of GPR30 and GPR43 in oral tissues: deriving new hypotheses on the role of diet in animal physiology and the development of oral cancers.

Food components and salivary hormones modulate the function of various tissues in the oral cavity. However, the mechanisms underlying such interactions are poorly understood. This study aimed at the detection of GPR30 and GPR43 in oral epithelia. Although unknown yet, the expression of these receptors is hypothesized to be fundamental for the actions of salivary oestrogens, dietary isoflavones and short chain fatty acids (SCFA) in the oral environment. Either immunoblotting or RT-PCR techniques were used for receptor detection in bovine and primate oral tissues. Here we show for the first time that mRNA of the G-protein-coupled oestrogen receptor, GPR30, and the short chain fatty acid receptor, GPR43, are expressed in bovine parotid glands. Furthermore, GPR30 protein is expressed in bovine parotid gland and the tongue of the primate Theropithecus gelada. With GPR30 being a target for dietary isoflavones and GPR43 being a suggested target for short chain fatty acids, we propose new hypotheses concerning the receptors' roles in salivary gland physiology and pathology. Our findings may trigger more detailed studies on GPRs to unravel their role in regulatory mechanisms in the oral cavity as well as in cancer development in relation to diets or biologically active compounds like soy isoflavones.

Protein kinase Calpha modulates depolarizaton-evoked changes of intracellular Ca2+ concentration in a rat pheochromocytoma cell line.

Conventional protein kinase C (cPKC) isoforms are activated by a coincident rise in cytosolic Ca(2+) and membrane-bound diacylglycerol. In excitable cells, cPKC may be activated by Ca(2+) influx through voltage-gated Ca(2+) channels (VGCC). cPKCs, in turn, are known to modulate the activity of VGCC. We examined whether PKCalpha, a cPKC, could be activated by depolarization in a neuroendocrine cell line and whether activation occurred on a time scale that modulated the depolarization-evoked intracellular Ca(2+) concentration ([Ca(2+)](i)) signal. Pheochromocytoma cells (PC12 cells) were transfected with wild-type and mutant forms of PKCalpha labeled with yellow fluorescent protein to monitor kinase translocation. Simultaneously, [Ca(2+)](i) changes were monitored with fura-2. Two point mutations that render PKCalpha inactive, D187A in the Ca(2+) binding site and K368R in the ATP binding site, significantly prolonged the time-to-peak of the depolarization-evoked [Ca(2+)](i) signal. A mutation that modulates membrane insertion (W58G) and two mutations of an autophosphorylation site (S657A, S657E) had no effect on the kinetics of the [Ca(2+)](i) signal. We conclude that in PC12 cells, Ca(2+) entry through VGCC rapidly activates PKCalpha, and that PKCalpha can modulate the Ca(2+) signal on a physiologically relevant time scale. Point mutations of PKCalpha can be used as specific and potent modulators of the PKC signaling pathway.

Receptor-mediated regulation of the nonselective cation channels TRPC4 and TRPC5.

Mammalian transient receptor potential channels (TRPCs) form a family of Ca(2+)-permeable cation channels currently consisting of seven members, TRPC1-TRPC7. These channels have been proposed to be molecular correlates for capacitative Ca(2+) entry channels. There are only a few studies on the regulation and properties of the subfamily consisting of TRPC4 and TRPC5, and there are contradictory reports concerning the possible role of intracellular Ca(2+) store depletion in channel activation. We therefore investigated the regulatory and biophysical properties of murine TRPC4 and TRPC5 (mTRPC4/5) heterologously expressed in human embryonic kidney cells. Activation of G(q/11)-coupled receptors or receptor tyrosine kinases induced Mn(2+) entry in fura-2-loaded mTRPC4/5-expressing cells. Accordingly, in whole-cell recordings, stimulation of G(q/11)-coupled receptors evoked large, nonselective cation currents, an effect mimicked by infusion of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). However, depletion of intracellular Ca(2+) stores failed to activate mTRPC4/5. In inside-out patches, single channels with conductances of 42 and 66 picosiemens at -60 mV for mTRPC4 and mTRPC5, respectively, were stimulated by GTPgammaS in a membrane-confined manner. Thus, mTRPC4 and mTRPC5 form nonselective cation channels that integrate signaling pathways from G-protein-coupled receptors and receptor tyrosine kinases independently of store depletion. Furthermore, the biophysical properties of mTRPC4/5 are inconsistent with those of I(CRAC), the most extensively characterized store-operated current.

Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol.

Eukaryotic cells respond to many hormones and neurotransmitters with increased activity of the enzyme phospholipase C and a subsequent rise in the concentration of intracellular free calcium ([Ca2+]i). The increase in [Ca2+]i occurs as a result of the release of Ca2+ from intracellular stores and an influx of Ca2+ through the plasma membrane; this influx of Ca2+ may or may not be store-dependent. Drosophila transient receptor potential (TRP) proteins and some mammalian homologues (TRPC proteins) are thought to mediate capacitative Ca2+ entry. Here we describe the molecular mechanism of store-depletion-independent activation of a subfamily of mammalian TRPC channels. We find that hTRPC6 is a non-selective cation channel that is activated by diacylglycerol in a membrane-delimited fashion, independently of protein kinases C activated by diacylglycerol. Although hTRPC3, the closest structural relative of hTRPC6, is activated in the same way, TRPCs 1, 4 and 5 and the vanilloid receptor subtype 1 are unresponsive to the lipid mediator. Thus, hTRPC3 and hTRPC6 represent the first members of a new functional family of second-messenger-operated cation channels, which are activated by diacylglycerol.

Regulation of heterologously expressed transient receptor potential-like channels by calcium ions.

The Drosophila melanogaster gene product TRPL (transient receptor potential-like) is a Ca2+-permeable cation channel that contributes to the light-induced Ca2+ entry in Drosophila photoreceptors and bears homology to several recently cloned mammalian channels. Intracellular Ca2+ has been implicated to stimulate TRPL channels. This constitutes a potentially dangerous mechanism that may lead to Ca2+ overload. Therefore, we studied whether TRPL channels, like other Ca2+-permeable channels, are inhibited by intracellular Ca2+ concentrations in the micromolar range and whether this effect is mediated by calmodulin. In Sf9 cells expressing the TRPL gene along with histamine H1 receptors after infection with baculoviruses containing the corresponding complementary DNA, histamine-induced TRPL currents were inhibited by intracellular Ca2+ with an IC50 of 2.3 microM. Moreover, TRPL currents were reversibly attenuated by a preceding hyperpolarization. This attenuation reflected the action of an increased Ca2+ influx, since it was abolished in the absence of extracellular Ca2+ and enhanced by raising extracellular Ca2+ to 20 mM. Finally, the activity of TRPL channels in inside-out patches was reversibly inhibited by raising the Ca2+ concentration on the cytosolic side of the patches to 10-50 microM. Addition of calmodulin or the calmodulin inhibitor calmidazolium did not modify the inhibition of the TRPL by Ca2+. We conclude that high intracellular Ca2+ concentrations inhibit the TRPL, but no evidence was found for the requirement of calmodulin. This mechanism makes Ca2+ influx through the TRPL self-limiting. Furthermore, the TRPL may allow one to study the structural requirements for channel regulation by Ca2+.

Expression of TRPC3 in Chinese hamster ovary cells results in calcium-activated cation currents not related to store depletion.

TRPC3 (or Htrp3) is a human member of the trp family of Ca2+-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca2+ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661-671), we tested whether TRPC3 might represent a Ca2+ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca2+ over Na+. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca2+ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca2+. Likewise, infusion of Ca2+ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca2+-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca2+ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 microM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 microM). We conclude that TRPC3 codes for a Ca2+-permeable channel that supports Ca2+-induced Ca2+-entry but should not be considered store operated.

Direct activation of trpl cation channels by G alpha11 subunits.

G proteins of the Gq/11 subfamily functionally couple cell surface receptors to phospholipase C beta (PLC beta) isoforms. Stimulation of PLC beta induces Ca2+ elevation by inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release and store-dependent 'capacitative' Ca2+ entry through Ca(2+)-permeable channels. The Drosophila trp gene, as well as some human trp homologs, code for such store-operated channels. The related trp-like (trpl) gene product also forms a Ca(2+)-permeable cation channel, but is not activated by store depletion. Co-expression of the constitutively active Gq subfamily member G alpha 11 (G alpha 11) with trpl enhanced trpl currents 33-fold in comparison with co-expression of trpl with other G alpha isoforms or G beta gamma complexes. This activation could not be attributed to signals downstream of PLC beta. In particular, InsP3 infusion, modulation of protein kinase C activity or elevation of intracellular calcium concentration failed to induce trpl currents. In contrast, purified G alpha 11 (but not other G protein subunits) activated trpl channels in inside-out patches. We conclude that trpl is regulated by G11 proteins in a membrane-confined manner not involving cytosolic factors. Thus, G proteins of the Gq subfamily may induce Ca2+ entry not only indirectly via store-operated mechanisms but also by directly stimulating cation channels.

Cloning and functional expression of a human Ca2+-permeable cation channel activated by calcium store depletion.

Depletion of intracellular calcium stores generates a signal that activates Ca2+-permeable channels in the plasma membrane. We have identified a human cDNA, TRPC1A, from a human fetal brain cDNA library. TRPC1A is homologous to the cation channels trp and trpl in Drosophila and is a splice variant of the recently identified cDNA Htrp-1. Expression of TRPC1A in CHO cells induced nonselective cation currents with similar permeabilities for Na+, Ca2+, and Cs+. The currents were activated by intracellular infusion of myo inositol 1,4,5-trisphosphate or thapsigargin. Expression of TRPC1A significantly enhanced increases in the intracellular free calcium concentration induced by Ca2+ restitution after prolonged depletion. Similar results were obtained in Sf9 cells. We conclude that TRPC1A encodes a Ca2+-permeable cation channel activated by depletion of intracellular calcium stores.

Ca(2+)-permeable large-conductance nonselective cation channels in rat basophilic leukemia cells.

Spreading of Ca2+ signals in rat basophilic leukemia (RBL) cells occurs by release of ATP. Therefore we studied the effect of ATP on membrane currents. ATP (1-10 microM) activated large-conductance channels. Single channel events were resolved in the whole cell mode. Similar channel activity was observed in RBL cells transfected with the muscarinic M1 receptor after stimulation with carbachol as well as after intracellular infusion of aluminum fluoride. Activation was independent of internal Ca2+ (0-10 microM). The channels had a conductance of 250 pS in 135 mM Na+ and 70 pS in 100 mM Ca2+. The permeability (P) ratio was PCa/PNa/PCs/PMg = 16:1:0.6:0.6. These channels may contribute to secretory responses by allowing Ca2+ entry, leading to high Ca2+ concentrations in the vicinity of the channel pore.

The Drosophila cation channel trpl expressed in insect Sf9 cells is stimulated by agonists of G-protein-coupled receptors.

Structures and regulations of vertebrate channels responsible for sustained calcium elevations after hormone stimulation are largely unknown. Therefore, the Drosophila photoreceptor channels, trp and trpl, which are assumed to be involved in calcium influx, serve as model system, trpl expressed in Sf9 cells showed spontaneous activity. Hormonal stimulations of calcium influx (detected by fura-2) and of an outwardly rectifying current were observed in Sf9 cells coinfected with baculoviruses encoding trpl and various heptahelical receptors for histamine, thrombin, and thromboxane A2, all known to cause phospholipase C-beta activation in mammalian cells. Although the identity of the G-proteins and of possible second messengers involved need to be clarified, it is clear that trpl represents a receptor/G-protein regulated cation channel.

NMDA receptor agonists selectively block N-type calcium channels in hippocampal neurons.

The modulation of voltage-dependent calcium channels by various neurotransmitters has been demonstrated in many neurons. Because of the critical role of Ca2+ in transmitter release and, more generally, in transmembrane signalling, this modulation has important functional implications. Hippocampal neurons possess low-threshold (T-type) Ca2+ channels and both L- and N-type high voltage-activated Ca2+ channels. N-type Ca2+ channels are blocked selectively by omega-conotoxin and adenosine. These substances both block excitatory synaptic transmission in the hippocampus, whereas dihydropyridines, which selectively block L-type channels, are ineffective. Excitatory synaptic transmission in the hippocampus displays a number of plasticity phenomena that are initiated by Ca2+ entry through ionic channels operated by N-methyl-D-aspartate (NMDA) receptors. Here we report that NMDA receptor agonists selectively and effectively depress N-type Ca2+ channels which are involved in neurotransmitter release from presynaptic sites. The inhibitory effect is eliminated by the competitive NMDA antagonist D-2-amino-5-phosphonovalerate, does not require Ca2+ entry into the cell, and is probably receptor-mediated. This phenomenon may provide a negative feedback between the liberation of excitatory transmitter and entry of Ca2+ into the cell, and could be important in presynaptic inhibition and in the regulation of synaptic plasticity.

Cationic channels activated by extracellular ATP in rat sensory neurons.

Single channels activated by externally applied ATP were investigated in cultured sensory neurons from nodosal and spinal ganglia of rat using patch clamp and concentration clamp methods. Mean conductance of single ATP-activated channels was 17 pS when measured at a holding potential of -75 mV in saline containing 3 mM Ca2+ and 1 mM Mg2+. Sublevels of conductance were detected in some cases. The current-voltage relationship for a single channel is highly non-linear and demonstrates inwardly directed rectification. The I-V curve obtained for single channels was identical to that for macroscopic current. ATP activated the channels in the absence of divalent cations (in ethylenediaminetetra-acetate-containing medium) as well as in their presence. This indicates that ATP as a free anion can activate the receptor. Ca2+ ions decreased both macro- and microscopic ATP-activated currents. The concentration dependence of this Ca2+ effect does not fit a single site binding isotherm. The single channel current demonstrated prominent fluctuations. When measured in the 0-4 kHz frequency band the amplitude of fluctuations evaluated as a double r.m.s. was about 30% of the mean amplitude of current. The autocorrelation function for the current fluctuations in an open channel could be approximated by a single exponential with the time constant of 0.4 ms. These fluctuations did not depend on the presence of divalent cations in the external medium. The open time distribution for the investigated channels could be described by a sum of two exponentials. Presumably this reflects the existence of two subtypes of ATP-activated channels.

Receptors for ATP in rat sensory neurones: the structure-function relationship for ligands.

1. The pharmacological properties of the ATP-activated conductance in isolated sensory neurones of the rat were investigated by use of voltage clamp and concentration clamp techniques. 2. Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), cytidine 5'-triphosphate (CTP), cytidine 5'-diphosphate (CDP) and some derivatives activate these receptors, whereas adenosine 5'-monophosphate (AMP), cytidine 5'-monophosphate (CMP) and other naturally-occurring nucleotides are competitive blockers. 3. In the sequence of substances, adenosine 5'-(beta,gamma-methylene)-triphosphonate (APPCP), adenosine 5'-(beta,gamma-difluoromethylene)- triphosphonate (APPCF2P), adenosine 5'-(beta,gamma-dichloromethylene)-triphosphonate (APPCC12P) and adenosine 5'-(beta,gamma-dibromomethylene)triphosphonate (APPCBr2P), the properties of ligands depend on the radius of the atom linked to the carbon of the diphosphonate group. Thus, APPCP is an agonist, APPCF2P is a partial agonist, while dichloromethylene and dibromomethylene analogues of adenosine 5'-(beta,gamma-methylene)triphosphonate demonstrate features of competitive blockers. APPCC12P is the most effective blocker of ATP-receptors (inhibition constant Ki = 21 +/- 4 microM). An adenosyl or adenylyl radical, when connected to the terminal phosphate of ATP, converts the agonist into a partial agonist. 4. Two especially important parts of the ATP molecule are crucial for the interactions with receptors. They are: (1) the vicinity of C6 of the purine ring and (2) the polyphosphate chain. Some modifications in these regions of the molecule result in the transformation of an agonist into an antagonist.

[Effect of adenosine-5'-O-(beta, gamma-dichloromethane)triphosphate on ATP-activated currents in sensory neurons in rats]. / Vliianie adenozin-5'-O-(beta, gamma-dikhlormetan)trifosfata na ATF-aktiviruimye toki v sensornykh neironakh krysy.

The action of beta,gamma-dihalogenmethane derivatives of ATP on ATP receptors has been studied in the sensory neurons of rats. beta,gamma-Dichloromethane-ATP and beta,gamma-dibromomethane-ATP are competitive blockers and beta,gamma-difluoromethane-ATP is a partial agonist of ATP receptors. At present, beta,gamma-dichloromethane-ATP is the most efficient of the known competitive blockers of these receptors (Ki = 2.10(-5) M). The data obtained confirm that beta- and gamma-phosphates of ATP play an important role in the activation of ATP receptors.

[Effect of natural ribonucleotides on the ATP receptors of the neurons of the rat nodose ganglion]. / Vliianie prirodnykh ribonukleotidov na retseptory ATF neironov uzlovatogo gangliia krysy.

Action of natural ribonucleotides on ATP receptors of rat nodose ganglion has been studied. Nucleotides containing guanine, uracil or hypoxanthine residues as nitrogen base are competitive blockers of the receptors. Their pharmacological activity decreases along with reduction of the polyphosphate chain length. The most efficient blocker among them is guanosine-5'-triphosphate. Cytidine nucleotides as well as adenine ones are agonists of ATP receptors, if their polyphosphate chain contains two or three phosphate residues and are blockers, if it contains only one residue.

[Bis(5'-adenosyl)tetraphosphate--a partial agonist of the ATP receptors on sensory neurons of the rat]. / Bis(adenozil-5')tetrafosfat--chastichnyi agonist retseptorov ATF sensornykh neironov krysy.

Effect of bis(adenosyl-5')oligophosphates on the ATP receptors in the somatic membranes of rat sensory neurons has been studied. Bis(adenosyl-5')tetraphosphate and bis(adenosyl-5')pentaphosphate are partial agonists of these receptors. They can activate currents of about an order lower than maximum ATP-activated currents.

[Kinetics of the ATP-activated ionic currents in the sensory neurons of the rat]. / Kinetika ATF-aktiviruemykh ionnykh tokov v sensornykh neironakh krysy.

It is shown that ATP being applied to sensory neurons of the rat activates ionic currents with variable kinetics of desensitization. The rate constant of the most "rapid" component of these currents is about 30 ms. The rate constant of the most "slow" component is about 30 s. Pharmacology and ionic properties of different components of the ATP-activated current is, probably, identical.