Enhancing effects of transition metals on the salt taste responses of single fibers of the frog glossopharyngeal nerve: specificity of and similarities among Ca2+, Mg2+ and Na+ taste responses

Fibers of the frog glossopharyngeal nerve (water fibers) thatare sensitive to water also respond to CaCl₂, MgCl₂ and NaCl.In the present study, interaction among cations (Ca²⁺, Mg²⁺and Na⁺) on taste cell membrane in frogs was studied using transitionmetals (NiCl₂, CoCl₂ and MnCl₂), which themselves are barelyeffective in producing neural response at concentrations below5 mM. Unitary discharges from single water fibers were recordedfrom fungiform papillae with suction electrode. Transition metalions (0.05–5.0 mM) had exclusively enhancing effects onthe responses to 50 mM Ca²⁺, 100 mM Mg²⁺ and 500 mM Na⁺. Theeffects of transition metal ions were always reversible. Therank order of effectiveness of transition metals at 1 mM inthe enhancement of the responses to 50 mM CaCl₂, 100 mM MgCl₂and 500 mM NaCl was NiCl₂ > CoCl² > MnCl₂. The concentrationof transition metal ions effective to enhance salt responsewas almost the same among Ca²⁺, Mg²⁺ and Na⁺ responses. Theresults suggest that a common mechanism is involved in the enhancementof Ca²⁺, Mg²⁺ and Na⁺ taste responses. The enhanced Mg²⁺ responseand the enhanced Na⁺ response were greatly inhibited by theaddition of Ca²⁺ ions, and the enhanced Ca²⁺ response was inhibitedby the addition of Mg²⁺ or Na⁺ ions, suggesting that competitiveantagonism occurs between Ca²⁺ and Mg²⁺ ions and between Ca²⁺and Na⁺ ions in the presence of Ni²⁺ ions. Ni²⁺ ions had a dualeffect on the Ca²⁺ response induced by low concentration (0.1mM) of CaCl₂: enhancement at lower concentrations (0.02–0.1mM) of NiCl₂ and inhibition at higher concentrations (0.5–5mM)of NiCl₂. The present results suggest that transition metalions do not affect the receptor-antagonist complex, but affectonly the receptor-agonist complex.     

Competitive inhibition of the response to Na+ by Ca2+ in water fibers of the frog glossopharyngeal nerve

Single water fibers of the frog glossopharyngeal nerve respondto low concentrations of CaCl₂ (1–2 mM) and to relativelyhigh concentrations of NaCl(>80 mM). However, stimulationby a mixture with a low concentration of CaCl₂ and relativelyhigh concentration of NaCl gives rise to only a small response,suggesting that the effects of Ca²⁺ and Na⁺ are mutually antagonistic.It has been reported that Na⁺ inhibits the response to Ca²⁺by competing with Ca²⁺ for a calcium receptor site (X_Ca; Kitadaand Shimada, 1980). In the present study, it was found tha Ca²⁺inhibited the response to Na⁺. Therefore, the sodium receptorsite (X_Na) responsible for the response to Na is different fromX_Ca. The inhibition of the response to Na⁺ by Ca²⁺ was examinedquantitatively on the assumption that the magnitude of the neuralresponse is proportinal to the amount of NaX_Na complex minusa constant (the threshold concentration of the NaX_Na complex).The results obtained indicate that Ca²⁺ competes with Na₊ forX_Na. The apparent dissociation constants for the NaX_Na complexand the CaX_Na complex obtained from the present study were 1.0M and 1.2 x 10^-3 M, respectively, X_Na as proposed here, doesnot represent simply a binding site for cations since therecan be competition for X_Na by an antagonistie cation. The highaffinity of X_Na for Ca²⁺ suggests that X_Na is a specific receptorsite involved in salt-taste reception. Since Mg²⁺ did not affectthe response to Na⁺, the affinity of X_Na for cations is notcharge-specific but is, rather, chemically specific. The presentresults indicate that both Ca²⁺ and Na⁺ have a dual action,being involved both in excitation and in inhibition, in waterfibers of the frog glossopharyngeal nerve.     

Enhancing Effect of Nickel Ions on the Response to Magnesium Ions of Single Fibers of the Frog Glossopharyngeal Nerve: Competitive Inhibition by Calcium Ions of the Nickel-enhanced Response to Magnesium Ions

Single fibers of the frog glossopharyngeal nerve respond toMgCl₂ at concentrations exceeding 10 mM. NiCl₂ at 1 mM enhancedthe Mg²⁺ response. CaCl₂ at 0.5–2 mM induced an inhibitionof the Ni²⁺-enhanced response to Mg²⁺ ions. A quantitative explanationfor these results is provided by the hypothesis that Ni²⁺ ionssecondarily affect a magnesium receptor (designated X^*_Mg) thatis responsible for the Mg²⁺ response and that Ca²⁺ ions inhibitthe Ni²⁺-enhanced response to Mg²⁺ ions by competing with Mg²⁺ions for X^*_Mg. Double-reciprocal plots of the experimental dataindicate that Ni²⁺ ions do not affect the affinities of X^*_Mgfor both Mg²⁺ ions (agonist) and Ca²⁺ ions (competitive antagonist)appreciably, and that Ni²⁺ ions at 1 mM enhanced the maximalresponse to Mg²⁺ ions by 270%. It appears that a magnesium receptorinteracts with an Ni²⁺-binding element that is affected by Ni²⁺ions and, thus, Ni²⁺ ions can induce an enhancement of the Mg²⁺response. Chem. Senses 22: 613–622,1997.     

Competitive inhibition of the nickel-induced response to choline by calcium ions in single water fibers of the frog glossopharyngeal nerve

NiCl₂ induces a response to cboline Cl and enhances the responseto CaCl₂ in water-sensitiv fibers (water fibers) of the frogglossopharyngeal nerve. The Ni²⁺-induced choline⁺ response wasinhibited by Ca²⁺ ions and, conversely, the enhanced Ca²⁺ responseby Ni²⁺ ions was inhibited by choline⁺ ions. Hence, there existsa mutual antagonism between Ca²⁺ and choline⁺ ions. In the presentstudy, the inhibition of the Ni²⁺-induced choline⁺ responseby Ca²⁺ ions was investigated quantitatively. The assumptionwas made that receptors for choline (X_Ch) exist and that bindingof a choline⁺ ion to X_Ch, brings about a neural response. Itwas further assumed that the magnitude of the neural responseis proportional to the amount of choline-X_Ch, complex minussome constant (the threshold concentration of the choline-X_Ch,complex). The results from analysis of double-reciprocal plotwere consistent with the hypothesis that Ca²⁺ ions compete withcholine⁺ ions for X_Ch,. The dissociation constants for the choline-X_Ch,complex and the CaX_Ch, complex were obtained to be 0.6 M and7.4 x 10^-5 M, respectively. This result indicates that the affinitiesof X_Ch, for choline⁺ and Ca²⁺ ions are very different. Furthermore,Mg²⁺ ions did not affect the Ni²⁺-induced choline⁺ response,an indication that the affinity of X_Ch, is not charge-specific,but is chemically specific. The identification of a competitiveinhibitor of the choline⁺ response provide* evidence for existenceof a choline-specific receptor at the surface of taste cellsthat are innervated by the water fibers of the frog glossopharyngealnerve. Differences between the features of the response to cholineCl in the chorda tympani nerve of the rat and those in the frogglossopharyngeal nerve are discussed.     

A quantitative study of dual action of nickel ions on the taste response to calcium ions of single fibers of the frog glossopharyngeal nerve: inhibition and enhancement by nickel ions

Unitary discharges from single water fibers of the frog glossopharyngealnerve, caused by stimulation with 0.02–5 mM CaSO₄, wererecorded from fungiform papillae with a suction electrode. NiSO₄at concentrations of 0.2–2 mM, namely, at concentrationsthat are barely effective in producing impulses, had a dualaction on the Ca²⁺ response: NiSO₄ caused both inhibition andenhancement of the Ca²⁺ response. In the present study, thisdual action of Ni²⁺ ions on the Ca²⁺ response was investigatedin detail. Single water fibers yielded a saturation type ofconcentration-response curve for CaSO₄, which suggested thatsulfateions do not affect the Ca²⁺ response. Thus, sulfateswere used as test salts in the present study. At low concentrationsof Ca²⁺ ions, Ni²⁺ ions inhibited the Ca²⁺ response, but athigher concentrations of Co²⁺ ions they enhanced it. The resultscan be explained quantitatively by the hypothesis that Ni²⁺ions inhibit the Ca²⁺ response by competing with Ca²⁺ ions forthe Ca²⁺ receptor (X_ca) that is responsible for the Ca²⁺ responseand that Ni²⁺ ions enhance the Ca²⁺ response by acting on amembrane element that interacts with X_ca. Double-reciprocalplots of the data indicate that the enhancing action of Ni²⁺ions is saturated at 1–2 mM Ni²⁺ ions and that Ni²⁺ ionsat these concentrations increase the maximal response of theCa²⁺ response by 182%. Dissociation constants for the Ca-X_cacomplex and the Ni-X_ca, complex were 4.2 x 10^–5 M and7.6 x 10^–5 M, respectively. The analysis suggests thatNi²⁺ ions enhance the Ca²⁺ response by affecting the Ca-X_cacomplex without altering the affinity of X_ca, for Ca²⁺ ions.     

A Quantitative Study of the Enhancing Effect of Nickel Ions on the Taste Response to Sodium Ions of Single Fibers of the Frog Glossopharyngeal Nerve: Competitive Inhibition by Calcium Ions of the Nickel-Enhanced Response to Sodium Ions

Single water fibers of the frog glossopharyngeal nerve respondto relatively high concentrations of NaCl (>80 mM). NiCl₂at 1 mM enhanced the Na⁺ response and reduced the thresholdconcentration for NaCl to 20 mM. CaCl₂ at 0.5–1 mM inducedan inhibition of the Ni²⁺-enhanced response to Na⁺ ions. A quantitativeexplanations for these results is provided by the hypothesisthat Ni²⁺ ions secondarily affect a sodium receptor or channel(designated X_Na^*) that is responsible for the Na⁺ response andthat Ca²⁺ ions inhibit the Ni²⁺-enhanced response to Na⁺ ionsby competing with Na⁺ ions for X_Na^*. Double-reciprocal plotsof the experimental data indicate that the affinity of X_Na^*for both Na⁺ ions (agonist) and Ca²⁺ ions (competitive antagonist)in the presence of 1 mM NiCl₂ was five times higher than thepreviously reported values obtained in the absence of NiCl₂(Kitada, 1991). Ni²⁺ ions at 1 mM enhanced the maximal responseto Na⁺ ions by 190%. It appears that a sodium receptor (or channel)interacts with a Ni²⁺-binding element that is affected by Ni²⁺ions and, thus, Ni²⁺ ions can induce both an increase in theaffinity of the sodium receptor for the respective cations andan enhancement of the Na⁺ response. Chem Senses 21: 65–73,1996.     

RNA interference targeted to multiple P2X receptor subtypes attenuates zinc-induced calcium entry

A postulated therapeutic avenue in cystic fibrosis (CF) is activation of Ca²⁺-dependent Cl^– channels via stimulation of Ca²⁺ entry from extracellular solutions independent of CFTR functional status. We have shown that extracellular zinc and ATP induce a sustained increase in cytosolic Ca²⁺ in human airway epithelial cells that translates into stimulation of sustained secretory Cl^– transport in non-CF and CF human and mouse airway epithelial cells, cell monolayers, and nasal mucosa. On the basis of these studies, the Ca²⁺ entry channels most likely involved were P2X purinergic receptor channels. In the present study, molecular and biochemical data show coexpression of P2X₄, P2X₅, and P2X₆ subtypes in non-CF (16HBE14o^–) and CF (IB3-1) human bronchial epithelial cells. Other P2X receptor Ca²⁺ entry channel subtypes are expressed rarely or not at all in airway epithelia, epithelial cell models from other CF-relevant tissues, or vascular endothelia. Novel transient lipid transfection-mediated delivery of small interference RNA fragments specific to P2X₄ and P2X₆ (but not P2X₅) into IB3-1 CF human airway epithelial cells inhibited extracellular zinc- and ATP-induced Ca²⁺ entry markedly in fura-2 Ca²⁺ measurements and "knocked down" protein by >65%. These data suggest that multiple P2X receptor Ca²⁺ entry channel subtypes are expressed in airway epithelia. P2X₄ and P2X₆ may coassemble on the airway surface as targets for possible therapeutics for CF independent of CFTR genotype. purinergic receptors; zinc receptors; airway epithelia; cystic fibrosis; therapy     

Effect of Ions on the Orientation of Cortical Microtubules in Spirogyra Cells

Effects of ions on the orientation of cortical micro-lubules(MTs) in Spirogyra cells were studied. After depo-lymerizalionwith amiprophos-methyl (APM), MTs were allowed to reorganizein NaCI solutions of various concentrations. As the concentrationof NaCI increased, the frequency of cells that had oblique MTsincreased. When cells in NaCI solution were transferred intoartificial pond water (APW) and incubated for 6 h, all the MTschanged to become transverse to the longitudinal axis of thecell. KC1 and MgCl₂ also had effects on the orientation of MTs.However, NH₄Cl, CaCl₂;, CoCl₂, and Co(NO₃)₂ did not show anyeffect. These results suggest that Na⁺, K⁺, and Mg²⁺have effectson MT orientation and that NH⁺₄, Ca²⁺, Co²⁺, Cl^–, andNO^–₃ have little effect. When MTs were reorganized ineither NaCl or KCl solutions, all the oblique MTs were organizedinto an S-helix. In contrast, some of the oblique MTs were foundas a Z-helix in the cells incubated in MgCl₂ or mannitol solutions.These results suggest that effects of Na⁺ and K⁺ on the orientationof MTs are not the same as those of Mg²⁺ and mannitol. Theseresults provide the first evidence that ions are involved inthe orientation of MTs in algae. (Received January 27, 1998; Accepted August 10, 1998)     

Effects of ATP, Mg2+, and redox agents on the Ca2+ dependence of RyR channels from rat brain cortex

Despite their relevance for neuronal Ca²⁺-induced Ca²⁺ release (CICR), activation by Ca²⁺ of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg²⁺], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg²⁺], and RyR redox state on the Ca²⁺ dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg²⁺] up to 1 mM inhibited vesicular [³H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg²⁺ inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca²⁺ dependencies, defined by low, moderate, or high maximal fractional open time (P_o), that depend on RyR redox state, as we have previously reported. In all cases, cis-ATP addition (3 mM) decreased threshold [Ca²⁺] for activation, increased maximal P_o, and shifted channel inhibition to higher [Ca²⁺]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg²⁺] up to 1 mM inhibited low activity channels more than moderate activity channels but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg²⁺] induced a right shift in Ca²⁺ dependence for all channels so that [Ca²⁺] <30 µM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca²⁺ at physiological [ATP] and [Mg²⁺]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR. Ca²⁺-induced Ca²⁺ release; Ca²⁺ release channels; endoplasmic reticulum; thimerosal; 2,4-dithiothreitol; ryanodine receptor     

Mg2+ activates the ryanodine receptor type 2 (RyR2) at intermediate Ca2+ concentrations

To clarify whether activity of the ryanodine receptor type 2 (RyR2) is reduced in the sarcoplasmic reticulum (SR) of cardiac muscle, as is the case with the ryanodine receptor type 1 (RyR1), Ca²⁺-dependent [³H]ryanodine binding, a biochemical measure of Ca²⁺-induced Ca²⁺ release (CICR), was determined using SR vesicle fractions isolated from rabbit and rat cardiac muscles. In the absence of an adenine nucleotide or caffeine, the rat SR showed a complicated Ca²⁺ dependence, instead of the well-documented biphasic dependence of the rabbit SR. In the rat SR, [³H]ryanodine binding initially increased as [Ca²⁺] increased, with a plateau in the range of 10–100 µM Ca²⁺, and thereafter further increased to an apparent peak around 1 mM Ca²⁺, followed by a decrease. In the presence of these modulators, this complicated dependence prevailed, irrespective of the source. Addition of 0.3–1 mM Mg²⁺ unexpectedly increased the binding two- to threefold and enhanced the affinity for [³H]ryanodine at 10–100 µM Ca²⁺, resulting in the well-known biphasic dependence. In other words, the partial suppression of RyR2 is relieved by Mg²⁺. Ca²⁺ could be a substitute for Mg²⁺. Mg²⁺ also amplifies the responses of RyR2 to inhibitory and stimulatory modulators. This stimulating effect of Mg²⁺ on RyR2 is entirely new, and is referred to as the third effect, in addition to the well-known dual inhibitory effects. This effect is critical to describe the role of RyR2 in excitation-contraction coupling of cardiac muscle, in view of the intracellular Mg²⁺ concentration. [³H]ryanodine binding; CICR; stimulation by physiological Mg²⁺, excitation-contraction coupling in the heart     

A P2X7 receptor stimulates plasma membrane trafficking in the FRTL rat thyrocyte cell line

Thyroid cells express a variety of P2Y and P2X purinergic receptor subtypes. G protein-coupled P2Y receptors influence a wide variety of thyrocyte-specific functions; however, functional P2X receptor-gated channels have not been observed. In this study, we used whole cell patch-clamp recording and fluorescence imaging of the plasma membrane marker FM1-43 to examine the effects of extracellular ATP on membrane permeability and trafficking in the Fisher rat thyroid cell line FRTL. We found a cation-selective current that was gated by ATP and 2',3'-O-(4-benzoylbenzoyl)-ATP but not by UTP. The ATP-evoked currents were inhibited by pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, adenosine 5'-triphosphate-2',3'-dialdehyde, 100 µM Zn²⁺, and 50 µM Cu²⁺. Fluorescence imaging revealed pronounced, temperature-sensitive stimulation of exocytosis and membrane internalization by ATP with the same pharmacological profile as observed for activation of current. The EC₅₀ for ATP stimulation of internalization was 440 µM in saline containing 2 mM Ca²⁺ and 2 mM Mg²⁺, and 33 µM in low-Mg²⁺, nominally Ca²⁺-free saline. Overall, the results are most consistent with activation of a P2X₇ receptor by ATP^4–. However, low permeability to N-methyl-D-glucamine⁺ and the propidium cation YO-PRO-1 indicates absence of the cytolytic pore that often accompanies P2X₇ receptor activation. ATP stimulation of internalization occurs in Na⁺-free, Ca²⁺-free, or low-Mg²⁺ saline and therefore does not depend on cation influx through the ATP-gated channel. We conclude that ATP activation of a P2X₇ receptor stimulates membrane internalization in FRTL cells via a transduction pathway that does not depend on cation influx. purinergic receptor; internalization; patch clamp     

The responses to choline ions induced by transition metal ions in single water fibers of the frog glossopharyngeal nerve

In single water-sensitive fibers (water fibers) of the frogglossopharyngeal nerve, application of a solution of 500 mMcholine Cl to the tongue elicited responses of varying magnitude.Some water fibers (plain choline-insensitive water fibers) barelyresponded to the solution, while some water fibers (plain choline-sensitivewater fibers) exhibited a considerable response to this solution.NiCl₂. which is barely effective in producing neural responseat concentrations below 5 mM, induced the response of plaincholine-insensitrve water fibers to choline⁺ ions. It was confirmed,in a collision test, that the Ni²⁺-induced responses to choline⁺ions were derived from water fibers. However, NiCl₂ did notaffect the magnitude of me response generated by choline⁺ ionsin plain choline-sensitive water fibers. The concentration-responsecurve for choline Cl in the presence of 1 mM NiCl₂ for plaincholine-insensitive water fibers was similar to the curves obtainedin the absence of NiCl₂ for plain choline-sensitive water fibers.Other organic salts, such as tris(hydroxymethyl)arrdnomethane-HCl,triethanotamine-HCl and tetraethylammonium Cl, elicited no responseor only a very small response from water fibers, and NiCl₂ didnot affect these responses. It is suggested that there existsa choline receptor for the response to choline⁺ ions in theapical membrane of frog taste cells and that Ni²⁺ ions exposethe sites of such choline receptors, which are deeply embeddedin the receptor membrane, to the outside medium. The effectof Ni²⁺ ions results in an increase in the number of the cholinereceptor sites available for binding of choline⁺ ions. The rankorder of effectiveness of transition metal ions in elicitingthe appearance or enhancement of the response to choline Clwas Ni²⁺ > Co²⁺ > Mn²⁺. Mg²⁺ ions had no effect on theresponse to choline⁺ ions. A similar rank order was previouslyobtained in enhancement of the responses to Ca²⁺, Mg²⁺ and Na²⁺ions (Kitada, 1994a). It seems likely that the mechanism forenhancement or elicitation of the response to choline⁺ ionsby the transition metal ions has features in common with thatfor enhancement of the responses to Ca²⁺, Mg²⁺ and Na⁺ ions.     

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A     

Effects of cations on the sugar receptor-site of the blowfly, Phormia

1) Ca^{+ +} (1 to 10 mM) lowered the binding affinity of sugarreceptor-site for sucrose in the labellar sugar receptor ofthe blowfly, Phormia regina, without changing the maximum-responseamplitude. It also elevated the values of the Hill coefficient(n_H) in some degrees. 2) Other divalent cations such as Mg^{+ +}, Ba^{+ +} or Cd^{+ +} alsoshowed almost the same property as above. The sequence of theeffect is as follows: Ba^{+ +}, Mg^{+ +} x Ca^{+ +} x Cd^{+ +}. Trivalentcation, La^{+ + +} (1 mM), changed the value of n_H from 1 (La^{++ +}-free) to 2. 3) On the contrary, the action of monovalent cations such asK⁺ or Na⁺, of which ionic strength was made the same as thatof the divalents hardly suppressed the response. 4) The results obtained do not support the hypothesis, at leaston the sugar receptor of the fly, that the receptor potentialis attributable to a change of the surface potential (zeta potential)as is proposed for the frog sugar receptor.     

Polyunsaturated fatty acids mobilize intracellular Ca2+ in NT2 human teratocarcinoma cells by causing release of Ca2+ from mitochondria

In a variety of disorders, overaccumulation of lipid in nonadipose tissues, including the heart, skeletal muscle, kidney, and liver, is associated with deterioration of normal organ function, and is accompanied by excessive plasma and cellular levels of free fatty acids (FA). Increased concentrations of FA may lead to defects in mitochondrial function found in diverse diseases. One of the most important regulators of mitochondrial function is mitochondrial Ca²⁺ ([Ca²⁺]_m), which fluctuates in coordination with intracellular Ca²⁺ ([Ca²⁺]_i). Polyunsaturated FA (PUFA) have been shown to cause [Ca²⁺]_i mobilization albeit by unknown mechanisms. We have found that PUFA but not monounsaturated or saturated FA cause [Ca²⁺]_i mobilization in NT2 human teratocarcinoma cells. Unlike the [Ca²⁺]_i response to the muscarinic G protein-coupled receptor agonist carbachol, PUFA-mediated [Ca²⁺]_i mobilization in NT2 cells is independent of phospholipase C and inositol-1,4,5-trisphospate (IP₃) receptor activation, as well as IP₃-sensitive internal Ca²⁺ stores. Furthermore, PUFA-mediated [Ca²⁺]_i mobilization is inhibited by the mitochondria uncoupler carboxyl cyanide m-chlorophenylhydrozone. Direct measurements of [Ca²⁺]_m with X-rhod-1 and ⁴⁵Ca²⁺ indicate that PUFA induce Ca²⁺ efflux from mitochondria. Further studies show that ruthenium red, an inhibitor of the mitochondrial Ca²⁺ uniporter, blocks PUFA-induced Ca²⁺ efflux from mitochondria, whereas inhibitors of the mitochondrial permeability transition pore cyclosporin A and bongkrekic acid have no effect. Thus PUFA-gated Ca²⁺ release from mitochondria, possibly via the Ca²⁺ uniporter, appears to be the underlying mechanism for PUFA-induced [Ca²⁺]_i mobilization in NT2 cells. arachidonic acid; mitochondrial Ca²⁺ uniporter; G protein-coupled receptor; IP₃ receptor     

Maitotoxin and P2Z/P2X7 purinergic receptor stimulation activate a common cytolytic pore

The effects ofmaitotoxin (MTX) on plasmalemma permeability are similar to thosecaused by stimulation of P2Z/P2X₇ionotropic receptors, suggesting that1) MTX directly activatesP2Z/P2X₇ receptors or2) MTX andP2Z/P2X₇ receptor stimulationactivate a common cytolytic pore. To distinguish between these twopossibilities, the effect of MTX was examined in1) THP-1 monocytic cells before andafter treatment with lipopolysaccharide and interferon-, a maneuverknown to upregulate P2Z/P2X₇receptor, 2) wild-type HEK cells andHEK cells stably expressing theP2Z/P2X₇ receptor, and3) BW5147.3 lymphoma cells, a cellline that expresses functional P2Z/P2X₇ channels that are poorlylinked to pore formation. In control THP-1 monocytes, addition of MTXproduced a biphasic increase in the cytosolic freeCa²⁺ concentration([Ca²⁺]_i);the initial increase reflects MTX-inducedCa²⁺ influx, whereas the secondphase correlates in time with the appearance of large pores and theuptake of ethidium. MTX produced comparable increases in[Ca²⁺]_iand ethidium uptake in THP-1 monocytes overexpressing theP2Z/P2X₇ receptor. In bothwild-type HEK and HEK cells stably expressing theP2Z/P2X₇ receptor, MTX-inducedincreases in[Ca²⁺]_iand ethidium uptake were virtually identical. The response of BW5147.3cells to concentrations of MTX that produced large increases in[Ca²⁺]_ihad no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- andBz-ATP-induced pores activate with similar kinetics and exhibit similarsize exclusion. Last, MTX-induced pore formation, but not channelactivation, is greatly attenuated by reducing the temperature to22°C, a characteristic shared by theP2Z/P2X₇-induced pore. Together,the results demonstrate that, although MTX activates channels that aredistinct from those activated byP2Z/P2X₇ receptor stimulation, thecytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.

     

Passive Interactions of Ca2+ and other Multivalent Cations with the Membranes of Isolated Corn Mitochondria

The presence of Ca²⁺ in a hypo-osmotic reaction medium reducessuccinate: cytochrome c reductase activity and the release ofouter membrane-specific antimycin A-insensitive NADH: cytochromec reductase. The action of Ca²⁺ is non-competitive and approximately30 mmol m^–3 Ca²⁺ affords half-maximal (I₅₀) protection.The effect of a range of inorganic and organic multivalent cationson succinate: cytochrome c reductase activity suggests thatthe action of Ca²⁺ is non-specific and probably involves Ca²⁺binding to outer membrane component(s) which may be proteins. Valinomycin- or gramicidin-induced passive swelling of isolatedcorn mitochondria in isotonic K.C1 is also non-competitivelyinhibited by up to 50% with Ca²⁺. Half-maximal inhibition (I₅₀)occurs at 0-35 mol m^–3 Ca²⁺ for valinomycin and 1-0 molm^–3 Ca²⁺ for gramicidin. Other divalent cations, Mg²⁺,Sr²⁺ and Ba²⁺, seem to inhibit similarly while the trivalentcations La³⁺ and Ho³⁺ show a maximum inhibition of up to 85%,with an I₅₀ of 0.1 mol m^–3 for valinomycin. It is suggestedthat non-specific cation binding may reduce membrane fluiditythereby slowing down the rate of ionophore penetration throughthe inner membrane. Key words: Calcium, Mitochondria, Membranes     

Increase in cytosolic Ca2+ induced by elevation of extracellular Ca2+ in skeletal myogenic cells

Cytoplasmic Ca²⁺concentration ([Ca²⁺]_i) variation is akey event in myoblast differentiation, but the mechanism by which itoccurs is still debated. Here we show that increases of extracellular Ca²⁺ concentration ([Ca²⁺]_o)produced membrane hyperpolarization and a concentration-dependent increase of [Ca²⁺]_i due to Ca²⁺influx across the plasma membrane. Responses were not related toinositol phosphate turnover and Ca²⁺-sensing receptor.[Ca²⁺]_o-induced[Ca²⁺]_i increase was inhibited byCa²⁺ channel inhibitors and appeared to be modulated byseveral kinase activities. [Ca²⁺]_i increasewas potentiated by depletion of intracellular Ca²⁺ storesand depressed by inactivation of the Na⁺/Ca²⁺exchanger. The response to arginine vasopressin (AVP), which inducesinositol 1,4,5-trisphosphate-dependent[Ca²⁺]_i increase in L6-C5 cells, was notmodified by high [Ca²⁺]_o. On the contrary,AVP potentiated the [Ca²⁺]_i increase in thepresence of elevated [Ca²⁺]_o. Other clones ofthe L6 line as well as the rhabdomyosarcoma RD cell line and thesatellite cell-derived C2-C12 line expressed similar responses to high[Ca²⁺]_o, and the amplitude of the responseswas correlated with the myogenic potential of the cells.

     

Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels

Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca²⁺, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca²⁺ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca²⁺ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X₃R > P2X_2b + X₄R > P2X_2bR > P2X_2a + X₄R > P2X₄R > P2X_2aR > P2X₇R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca²⁺ influx because of the coactivation of endogenously expressed Ca²⁺-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca²⁺ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca²⁺ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca²⁺ signals were not affected by voltage-gated Ca²⁺ influx and removal of extracellular sodium. Ca²⁺ signals reflected well the receptor-specific EC₅₀ values for ATP and the extracellular Zn²⁺ and pH sensitivities of P2XRs. These results indicate that intracellular Ca²⁺ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors. ATP-gated receptor channels; inward currents; intracellular calcium signals; desensitization-inactivation; voltage-gated calcium influx; localized and global calcium signals     

Salivary ions and neural taste responses in the hamster

Saliva is a chemically complex fluid that bathes oral surfacesand may affect early events in mammalian gustation. We measuredchorda tympani responses to taste stimuli in hamsters (Mesocricetusauratus) while their tongues were adapted to either water, artificialsaliva or natural saliva. Artificial saliva on the tongue loweredneural responses to taste stimuli that were present in the artificialsaliva and to those stimuli that cross-adated with salivarycomponents. Changing from a water-adapted tongue to one soakedwith pilocarpine-stimulated saliva from donor hamsters led tosignificantly smaller responses to NaCl. Responses to sucrose,NH₄Cl and quinine were unaffected. Chemical analysis of hamstersaliva revealed ‘normal’ mammalian levels of K⁺,Ca²⁺ and Mg²⁺, but unexpectedly low levels of Na⁺ and Cl^–.