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.     

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.     

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.     

Taste responses to divalent cations in the frog glossopharyngeal nerve: competitive inhibition of the Mg2+ response by Ca2+

In the frog glossopharyngeal nerve, single water fibers respondto low CaCl₂ (1–2 mM) and relatively high MgCl₂ (100 mM).In the present study, it was found that stimulation by a mixtureof low CaCl₂ and relatively high MgCl₂ led to a small response.This suggests that the Ca⁺ response is inhibited by the presenceof Mg²⁺ and the Mg²⁺ response is inhibited by the presence ofCa²⁺. Hence, it is suggested that there are different receptorsites for divalent cations in single water fibers of the frogglossopharyngeal nerve, a calcium receptor site (X_Ca) responsiblefor the Ca²⁺ response and a magnesium receptor site (X_Mg) responsiblefor the Mg²⁺ response. It has been reported that Mg²⁺ inhibitsthe Ca²⁺ response by competing with Ca²⁺ for X_Ca (Kitada andShimada, 1980). In the present study, the inhibition of theMg²⁺ response by Ca²⁺ was examined quantitatively under theassumption that the magnitude of the neural response is proportionalto the amount of MgX_Mg complex minus a constant (the thresholdconcentration of the MgX_Mg complex). The results obtained indicatethat Ca²⁺ competes with Mg²⁺ for X^Mg. The apparent dissociationconstants for MgX_Mg complex and CaX_Mg complex, which were obtainedfrom the present study, were 8.0 x 10^–2 M and 7.2 x 10^–4M, respectively. Thus, competition between Ca⁺ and Mg²⁺ forthe distinct receptor sites involved in taste reception wasdemonstrated by the results described in this paper. Since thedivalent cations do not always bring about activation of tastereceptors, the responses to salts in the frog glossopharyngealnerve cannot be explained in terms of changes in the surfacepotential outside the taste cells. The present results suggestthat there exist multiple specific receptor sites for cationsinvolved in salt taste responses, and only the binding of eachseparate cation to its appropriate receptor sites leads to activationof the receptor and the initiation of impulses in sensory nerveendings.     

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.     

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.     

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.     

Stimulatory Effect of Chloride Ions on NADP Malic Enzyme from Leaves of two C4 Species of Cyperus

NADP malic enzyme (EC 1.1.1.40 [EC] ) from leaves of two C₄ speciesof Cyperus (C. rotundus and C. brevifolius var leiolepis) exihibiteda low level of activity in an assay mixture that contained lowconcentrations of Cl^–. This low level of activity wasmarkedly enhanced by increases in the concentration of NaClup to 200 mM. Since the activity of NADP malic enzyme was inhibitedby Na₂SO₄ and stimulated by relatively high concentration ofTris-HCl (50–100 mM, pH 7–8), the activation ofthe enzyme by NaCl appears to be due to Cl^–. Variationsin the concentration of Mg²⁺ affected the K_A (the concentrationof activator giving half-maximal activation) for Cl^–,which decreased from 500 mM to 80 mM with increasing concentrationsof Mg²⁺ from 0.5 mM to 7 mM. The K_m for Mg²⁺ was decreased from7.7 mM to 1.3 mM with increases in the concentration of NaClfrom zero to 200 mM, although the increase of V_max was not remarkable.NADP malic enzyme from Cyperus, being similar to that from otherC₄ species, was able to utilize Mn²⁺. The K_m for Mn²⁺ was 5mM, a value similar to that for Mg²⁺. The addition of 91 mMNaCl markedly decreased the K_m for Mn²⁺ to 20 +M. NADP malicenzyme from Setaria glauca, which contains rather less Cl^–than other C₄ species, was inactivated by concentrations ofNaCl above 20 mM, although slight activation of the enzyme wasobserved at low concentrations of NaCl at pH7.6. (Received February 20, 1989; Accepted June 12, 1989)     

Ion Composition of the Chara Internode

Ion compositions of the cytoplasm and the vacuole of Chara australiswere analyzed according to Kishimoto and Tazawa (1964) and Kiyosawa(1979a). The ions in the cytoplasm and the vacuole analyzedwere K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl^–, NO₃^– and H₂PO₄^–.Assuming that the volume of the cytoplasm V_p is 10% of thatof the whole cell V, the concentrations of K⁺, Na⁺, Ca²⁺, Mg²⁺,Cl^–, NO₃^– and H₂PO₄^– in the cytoplasm averaged70, 15, 13, 4.6, 31, 2.2 and 16 mM, respectively. If the volumeof the cytoplasm was assumed to be 5% of that of the whole cell,their averaged concentrations were 139, 31, 25, 9.2, 62, 4.4and 33 mM, respectively. The averaged ion compositions of thecell sap were K⁺, 111; Na⁺, 47; Ca²⁺, 4.4; Mg²⁺, 8.9; Cl^–,91; NO^–₃, 3.3 and H₂PO₄^–, 6.0 mM. These values,taking the concentrations and the charges of the protein (Kiyosawa1979b) and amino acids (Sakano and Tazawa 1984) into accountand assuming the presence of some uni- or oligovalent anionsand/or small nonelectrolyte molecules, could explain fairlywell both the electroneutrality and the osmotic pressure ofthe cell, except when V_p/V = 5%. (Received May 18, 1987; Accepted September 29, 1987)     

Ion Content of the Halotolerant Alga Dunaliella salina

The intracellular concentration of the major ions in Dunaliellasalina cells were determined, following the removal of extracellularions by ion-exchange minicolumns. Log phase cells, grown inmedia containing 1–4 molar NaCl, contained 30–50mM chloride and 200–350 mM magnesium (5 mM in medium).Phosphorus, which is present intracellularly mostly as polyphosphate,was present in amounts of 60–100 fmoles per cell, equivalentto a concentration of 600–1,000 mM (0.2 mM in medium).Previous data indicated that such cells contained 20–40mM Na⁺, 150–300mM K⁺, 20mM SO^2–₄, and very low concentrationsof Ca2⁺ and charged nitrogenous compounds. Mg²⁺ and K⁺ seemto serve as the major counter ions for the intracellular negativecharge present in the massively accumulated polyphosphates.The former accounts for about 2/3 of the required positive charge.This is supported by the observation that limitation in thephosphate or K⁺ supply in the medium lead to a parallel decreasein the accumulation of intracellular phosphorus, Mg²⁺ or K⁺. ¹Present address: Department of Vegetables, The Volcani Center,Bet-Dagan 50250, Israel. (Received June 13, 1988; Accepted August 25, 1988)     

A sulfite-dependent adenosine triphosphatase of Thiobacillus thiooxidans. Its partial purification and some properties

A sulfite-dependent ATPase [EC 3.6.1.3 [EC] ] of Thiobacillus thiooxidanswas activated and solubilized by treatment with trypsin [EC3.4.4.4 [EC] ], and purified 84-fold with a 32% recovery. It requiredboth Mg²⁺ and SO₃^2– for full activity, and its optimumpH was found at 7.5–8.0. Mn²⁺, Co²⁺, and Ca²⁺ could partiallysubstitute for Mg²⁺, while SeO₃^2– and CrO₄^2– couldpartially substitute for SO₃^2–. The enzyme hydrolyzed ATP and deoxy-ATP most rapidly and otherphosphate esters were poorer substrates. The apparent Km valuefor ATP was 0.33 mM. The enzyme activity was strongly inhibitedby 0.2 mM NaN₃ and 10 mM NaF. (Received July 27, 1977; )     

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)     

Participation of Inorganic Orthophosphate in Regulation of the Ribulose-1,5-bisphosphate Carboxylase Activity in Response to Changes in the Photosynthetic Source-Sink Balance

Sink-limited conditions, defined as treatment with continuousillumination, cause a reduction in the rate of photosyntheticfixation of CO₂ in single-rooted leaves of soybean (Glycinemax. Merr. cv. Turunoko). We suggested previously that thisreduction is due to a deactivation of ribulose-1,5-bisphosphatecarboxylase (RuBPcase, E.C. 4.1.1.39 [EC] ) that is caused by a decreasein the level of P_i in the leaves [Sawada et al. (1989) PlantCell Physiol. 30: 691, Sawada et al. (1990) Plant Cell Physiol.31: 697]. In the present study, the mechanism of regulationof RuBPcase activity by P_i was examined. The activity of RuBPcasein the sink-limited leaves, exposed for 6 or 7 d to continuousillumination to alter the source/sink balance, was enhancedwith increasing concentrations of P_i, in a CO₂-free preincubationmedium in the presence of 5 mM MgCl₂ The maximum value [6.3µmole CO₂ (mg Chl)^–1 min^–1] was obtained atapproximately 5 mM P_i after a 5 min incubation, being 3 timesof the activity without the preincubation. The activity of acrude preparation of RuBPcase that had been deactivated by removalof CO₂ and Mg²⁺ ions by the gel filtration was 5.2–9.3nmole CO₂ (mg protein)^–1 min^–1 and was also enhancedby P_i plus Mg²⁺ ions. The maximum value [147–151 nmoleCO₂ (mg protein)^–1 min^–1] was attained at 5 mM P_iafter a 5 min incubation. The cycle of activation and inactivationof deactivated crude RuBPcase was perfectly reversible by additionof P_i to the enzyme and removal of P_i from the enzyme. Levelsof free P_i and of esterified phosphate in the sink-limited leaveswere 69% and 31% of the total phosphate, respectively. By contrast,in the control leaves, these values were 87% and 13%, respectively.These results support our previously stated hypothesis and indicatean important role for free P_i in the regulation of RuBPcaseactivity, in particular in sink-limited plants. (Received February 21, 1992; Accepted July 23, 1992)     

Movement of Ions and Electrogenesis in Higher Plant Cells

During the past 10 years considerable information has accumulatedon the electrochemical relationships of higher plant cells duringtransport of mineral ions. Using the Nernst equation as a criterion,none of eight ions (K⁺, Na⁺, Ca⁺⁺, Mg⁺⁺, NO₃^–, Cl^–,H₂PO₄^–, and SO₄^–) is in a passive equilibrium. Na⁺,Ca⁺⁺, and Mg⁺⁺ are subject to an exclusion mechanism, and allof the anions appear to be pumped inwardly. K⁺ apparently approachesan electrochemical balance under certain conditions but probablyis actively accumulated. Compartmental analyses giving estimatesof amounts in the cytoplasm and vacuole and of unidirectionalfluxes permit application of the Ussing flux-ratio equation.The criterion in oat coleoptile cells suggests that at the plasmalemmaNa⁺ is pumped out while K⁺ and Cl^– are pumped in. K⁺ andCl^– appear to be coupled in active transport across thetonoplast into the vacuole. Good evidence has been found thatthe cell's electropotential arises from an electrogenic pump:CN^– (cyanide) and DNP (dinitrophenol) reversibly blockthe potential and ionic transport; cell potentials are higherthan can be accounted for by diffusion; the responses of respirationand potential to the concentration of CN^– are nearly parallel;and CN^– inhibited tissue approaches a fit to the Goldmanconstant field equation. Future objectives should be identificationof the ion, or ions, subject to the electrogenic pump and discoveryof the immediate energy source.     

Effects of Ions and Membrane Potential on the Elongation of the Unicellular Green Alga Closterium

Cells of the unicellular green alga Closterium ehrenbergii elongatedexclusively at septa and for 4–5 hours after cell division.Cell elongation was strongly inhibited by a decrease in eitherthe external concentration of Ca²⁺ or pH, and was also inhibitedby several competitive Ca²⁺ channel blockers. Changes in concentrationsof other external ions had no effect on the elongation. Theaverage concentrations of ions in the intracellular fluid ofthe interphase cell before cell division was as follows (inmM): K⁺=56.5, Na⁺=4.8, Ca²⁺=2.4, Mg²⁺=1.3, Cl^–=59.5; thepH was 7.4. The levels of K⁺, Na⁺ and Cl^– ions decreasedsignificantly with cell elongation, suggesting that this process,which proceeds with water uptake, surpasses ion absorption.The plasma membrane potential (Vm) in both the interphase cellsand in the elongating cells was in the range of –90 to–105 mV (interior negative). The Vm was entirely determinedby the simple diffusion of K⁺. A decrease in the external concentrationof Ca²⁺ caused depolarization, probably by an indirect effectof low Ca²⁺. Changes in the extracellular level of H⁺ and othercations barely affected Vm. Thus, external Ca²⁺ and H⁺ are concludedto affect cell elongation but not via a change in the Vm acrossthe plasma membrane. (Received February 29, 1988; Accepted June 8, 1988)     

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     

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     

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     

Magnesium and Calcium Inhibition of Squash Leaf NADH Nitrate Reductase

This paper describes the first experimental evidence that theinhibition of nitrate reductase by Mg²⁺ or Ca²⁺ is related tothe hysteretic properties of the enzyme. The low activity formof nitrate reductase, i.e. the form of nitrate reductase showinghysteretic behaviour, was inhibited 70–90% by 5 mM Ca²⁺or Mg²⁺. However, no inhibition by Ca²⁺ or Mg²⁺ was seen afterthe enzyme was converted to its high activity form by preincubationwith substrates. Addition of thiol compounds or certain aminoacids to the assay mixture also prevented the Mg²⁺ or Ca²⁺ inhibition. (Received June 28, 1993; Accepted August 11, 1993)     

Chloride-dependent calcium transients induced by angiotensin II in vascular smooth muscle cells

Cl^– is essential for the vasoconstrictive response to angiotensin II (ANG II). In vascular smooth muscle cells (VSMC), we determined whether ANG II-induced transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is Cl^– dependent. After incubating the cells at different extracellular Cl^– concentration ([Cl^–]_e) for 40 min, the ANG II-induced Ca²⁺ transients at 120 meq/l Cl^– were more than twice those at either 80 or 20 meq/l Cl^–. Replacing Cl^– with bicarbonate or gluconate yielded similar results. In addition, after removal of extracellular Ca²⁺, ANG II-induced as well as platelet-derived growth factor-induced Ca²⁺ release exhibited Cl^– dependency. The difference of Ca²⁺ release with high vs. low [Cl^–]_e was not affected by acutely altering [Cl^–]_e 1 min before administration of ANG II when [Cl^–]_i was yet to be equilibrated with [Cl^–]_e. Pretreatment of a Cl^– channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid, increased ANG II-induced Ca²⁺ release and entry at 20 meq/l Cl^– but did not alter those at 120 meq/l Cl^–. However, after equilibration, a reduced [Cl^–]_e did not affect thapsigargin-induced Ca²⁺ release, suggesting that Cl^– may not affect the size of intracellular Ca²⁺ stores. Nevertheless, at high [Cl^–], the peak increase of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] induced by ANG II was approximately sixfold that at low [Cl^–]. Thus the Cl^–-dependent effects of ANG II on Ca²⁺ transients may be mediated, at least in part, by a Cl^–-dependent Ins(1,4,5)P₃ accumulation in VSMC. anion; inositol 1,4,5-trisphosphate; Ca²⁺ release