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.     

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.     

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.     

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.     

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.     

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.     

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^–.     

Examination of analytical methods for ions and saccharides in the extract of Phaseolus pulvini

With slight modifications, conventional assay procedures forK⁺, Na⁺, Ca²⁺, Mg²⁺, Cl^–, NO₃^–, H₂PO₄^–, fructoseand fructose-yielding saccharides, and glucose were applicableto the extract of Phaseolus pulvini. About 10 ml of a hot-waterextract from about 30 mg fresh weight of the pulvini was sufficientfor separate measurement of the ions and saccharides named above. (Received August 7, 1979; )     

Characteristic Changes in Relaxation Times of Water Protons in Vigna radiata Seedlings Exposed to Temperature Stress

Water protons in hypocotyl tissues from etiolated seedlingsof Vigna radiata that were exposed to temperature stress showedcharacteristic relaxation behaviors for ¹H-NMR. Cold stresstreatment (0C) caused gradual prolongation of NMR relaxationtimes (T₁). After exposure of tissues to cold stress for 24h, T₁ returned to the initial value as a result of subsequentincubation at normal temperature (20C). By contrast, heat stresstreatment (40C) induced a time-dependent decrease in T₁, whichdid not return completely to the initial value upon subsequentincubation at 20C after exposure to heat stress for 4 h. Weexamined changes in various physical factors that influencethe response of T₁ to temperature stress, namely, water contentand the concentrations of protein, diamagnetic (K⁺, Na⁺, Ca²⁺and Mg²⁺) and paramagnetic (Mn²⁺ and Fe²⁺) ions in the tissues.From the relationships between T₁ and these factors in vitro,we could not interpret the responses of T₁ to the temperaturestress only in terms of a change in water content. A synergisticeffect of an Mn²⁺ -protein complex and pH might be essentialfor the mechanism of changes in T₁ that are due to cold stress.The influence of heat stress on structural water in tissuesis discussed in terms of water-protein interactions. (Received December 28, 1992; Accepted May 6, 1993)     

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)     

Sodium gradient-dependent transport of magnesium in rat ventricular myocytes

Cytoplasmic concentration of Mg²⁺([Mg²⁺]_i) was measured with a fluorescentindicator furaptra in ventricular myocytes enzymatically dissociatedfrom rat hearts (25°C). To study Mg²⁺ transport acrossthe cell membrane, cells were treated with ionomycin inCa²⁺-free (0.1 mM EGTA) and high-Mg²⁺ (10 mM)conditions to facilitate passive Mg²⁺ influx. Rate of riseof [Mg²⁺]_i due to the net Mg²⁺influx was significantly smaller in the presence of 130 mMextracellular Na⁺ than in its absence. We also tested theextracellular Na⁺ dependence of the net Mg²⁺efflux from cells loaded with Mg²⁺. After[Mg²⁺]_i was raised by ionomycin and highMg²⁺ to the level 0.5-0.6 mM above the basal value(~0.7 mM), washout of ionomycin and lowering extracellular[Mg²⁺] to 1.2 mM caused rapid decline of[Mg²⁺]_i in the presence of 140 mMNa⁺. This net efflux of Mg²⁺ was completelyinhibited by withdrawal of extracellular Na⁺ and waslargely attenuated by imipramine, a known inhibitor of Na⁺/Mg²⁺ exchange, with 50% inhibition at 79 µM. The relation between the rate of net Mg²⁺ efflux andextracellular Na⁺ concentration([Na⁺]_o) had a Hill coefficient of 2 and[Na⁺]_o at half-maximal rate of 82 mM. Theseresults demonstrate the presence of Na⁺ gradient-dependentMg²⁺ transport, which is consistent withNa⁺/Mg²⁺ exchange, in cardiac myocytes.

     

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.     

Ion Effects on the H+-Translocating Adenosine Triphosphatase and Pyrophosphatase Associated with the Tonoplast of Chora corallina

H⁺-translocating ATPase and pyrophosphatase (PPase) associatedwith the tonoplast of Chara corallina were isolated with theaid of a perfusion technique, and the effects of ions on theiractivities were studied. All the alkali metal cations testedstimulated the ATPase and ATPdependent H⁺ pumping activitiesonly by 10 to 40%. Anions, on the other hand, strongly affectedthe activities. Potassium salts of Cl^- and Br^- stimulated them,while F^- and NO₃^- inhibited them. By contrast, the H⁺-translocatingPPase was insensitive to anions but sensitive to cations. Theorder of cation stimulation was Rb⁺=K⁺>Cs⁺>Na⁺=Li⁺>choline⁺.NO₃^- (50 mil), thought to be a specific inhibitor of the tonoplast-typeH⁺-ATPase, inhibited the ATPdependent H⁺ pumping almost completelybut the ATPase activity by only about 50%. Na⁺ inhibited thePP₁-dependent H⁺ pumping (I_5O=5OmM) in the presence of 50 mMKCl but not the ATP-dependent one. The PPase was more sensitiveto F^- (I₅₀=400µM) than the ATPase. Both the H⁺-ATPaseand the H⁺-PPase required Mg²⁺ for their activities, althoughan excess was inhibitory to both. The different sensitivitiesof the PP₁-dependent and the ATP-dependent H⁺- pumping enzymesto ions correspond to the tonoplast enzymes of higher plantsand may be used as "markers" to distinguish between these enzymesin characean cells (Received October 2, 1987; Accepted May 18, 1988)     

Purification and some properties of phosphomannomutase from corms of Amorphophallus konjac C. Koch

Phosphomannomutase [Glazer et al.: Biochim. Biophys. Acta 33:522–625 (1959)] was purified 1700-fold in a 39% yieldfrom cell-free extract of konjak (Amorphophallus konjac C. Koch)corms. The molecular weight of the enzyme as determined by gelfiltration was about 62,000. The enzyme required both Mg²+ and-D-glucose-l,6-bisphosphate for activity, although Mg²+ waspartially replaceable by either Co²+ or Ni²+. An apparent equilibriumconstant, K_eq=(mannose-6-phosphate) (mannose-1-phosphate), wasdetermined to be 8.5. Activity was maximal at pH 6.5 to 7.0.Activation energy was 11.1 kcal/mole. The enzyme was the moststable at pH 7.5. The addition of substrate or cofactor markedlyincreased enzyme stability toward heat denaturation. The enzymewas more labile to heat than phosphoglucomutase from konjakcorms. Treatment with various metal ions in Tris buffer inhibited theenzyme. Cu²+ and Zn²+ were the most potent inhibitors amongthe metal ions tested, while Co²+ and Ni²+ were weak. When theenzyme was treated with metal ions in the presence of histidinebuffer, Cu²+ and Zn²+ showed no inhibitory effect on the enzyme,whereas Be²+ inhibited it to an extent similar to that in Trisbuffer. Plots of 1/v versus l/(mannose-l-phosphate) at different fixedconcentrations of glucose-1,6-bisphosphate and 1/v versus 1/(glucose-1,6-bisphosphate)at different fixed concentrations of mannose-1-phosphate wereseries of converging lines. Mannose-1-phosphate at high concentrationswas found to inhibit the enzyme competitively with respect toglucose-l,6-bisphosphate. Apparent K_m and K₁ values for mannose-1-phosphatewere calculated to be 0.2 mM and 1.2 mM, respectively. The K_mvalue for glucose-1,6-bisphosphate was 1.8 µM. ¹This paper constitutes part 5 of a series of studies on konjakmannan biosynthesis. (Received May 24, 1976; )     

Effects of dihydropyridine receptor II-III loop peptides on Ca(2+) release in skinned skeletal muscle fibers

Inskeletal muscle fibers, the intracellular loop between domains II andIII of the ₁-subunit of the dihydropyridine receptor (DHPR) may directly activate the adjacent Ca²⁺ releasechannel in the sarcoplasmic reticulum. We examined the effects ofsynthetic peptide segments of this loop on Ca²⁺ release inmechanically skinned skeletal muscle fibers with functional excitation-contraction coupling. In rat fibers at physiological Mg²⁺ concentration ([Mg²⁺]; 1 mM), a20-residue skeletal muscle DHPR peptide[A_S(20);Thr⁶⁷¹-Leu⁶⁹⁰; 30 µM], shown previously toinduce Ca²⁺ release in a triad preparation, caused onlysmall spontaneous force responses in ~40% of fibers, although itpotentiated responses to depolarization and caffeine in all fibers. TheCOOH-terminal half of A_S(20)[A_S(10)] induced much larger spontaneousresponses but also caused substantial inhibition of Ca²⁺release to both depolarization and caffeine. Both peptides induced orpotentiated Ca²⁺ release even when the voltage sensors wereinactivated, indicating direct action on the Ca²⁺ releasechannels. The corresponding 20-residue cardiac DHPR peptide [A_C(20);Thr⁷⁹³-Ala⁸¹²] was ineffective, but itsCOOH-terminal half [A_C(10)] had effects similar to A_S(20). In the presence of lower[Mg²⁺] (0.2 mM), exposure to eitherA_S(20) or A_C(10) (30 µM) induced substantial Ca²⁺ release. PeptideC_S (100 µM), a loop segment reported to inhibit Ca²⁺ release in triads, caused partial inhibition ofdepolarization-induced Ca²⁺ release. In toad fibers, eachof the A peptides had effects similar to or greater than those in ratfibers. These findings suggest that the A and C regions of the skeletalDHPR II-III loop may have important roles in vivo.

     

Effects of temperature and various metal ions on the solubility of tobacco RuDP carboxylase

Tobacco RuDP carboxylase is completely soluble in 0.07 M NaClor 0.01 M Na₂SO₂, but is almost completely insoluble in salt-freesolutions at 40°C; the solubility seeming to depend on ionicstrength. Lowering the temperature increased solubility of theprotein. The solubility in 0.01–0.04 M NaCl at 0°Cwas more than double that at 40°C. RuDP solubilized theprotein even in a salt-free medium. The protein became insolubleagain on the addition of various divalent cations. Effectivenessof the metal ions was Zn⁺⁺> Ni⁺⁺>Co⁺⁺>Mn⁺⁺>Mg⁺⁺>Ca⁺⁺.Although most of the metal ions inhibited (Mg⁺⁺ activated) enzymeactivity, no direct correlation was found between the degreeof solubility depression and the degree of enzyme inhibition. (Received October 4, 1971; )     

Osmotic and Ionic Regulation in Chara L-cell Fragments

Ion absorption from rather complicated artificial pond water(APW) by cell fragments having a lower osmotic pressure thanthe intact internodal cell (L-cell fragments) was studied. L-cellfragments were prepared by taking advantage of trans cellularosmosis and ligating the cell with thread. The results wereas follows: (1) L-cell fragments absorbed more K⁺ than Na⁺ fromaKCL + NaCl mixture in the presence of Ca²⁺, Mg²⁺ and SO₂₄ inthe light; (2) the influx of KCI was larger than that of KNO₃;(3) the amount of positive charge carried by K⁺, Na⁺ and Mg²⁺across the cell membrane balanced well with the amount of negativecharge carried by Cl^– in Cl^–-containing and NO₃^–-free APW; (4) no conclusion could be made as to whether ornot the rule of electro neutrality held for the K⁺, Na⁺, Ca²⁺and NO₃^– fluxes across the cell membrane, because dilutedKNO₃ is unstable; (5) L-cell fragments in KCl-containing APWsurvived longer than those in KNO₃-containing and Cl^–-free APW; (6) after incubation in KNO₃-containing and Cl^–-freeAPW, L-cell fragments absorbed a great amount of KCI immediatelyafter being transferred to KCl-containing and NO₃^– -freeAPW; and (7) lowering the turgor pressure of the intact cellby raising the external osmotic pressure did not induce ionflux into the cell. Thus, we concluded that the L-cell fragmentsabsorbed ions from the external solution not because of theirlower turgor pressure, but because of the diluted ion concentrationof the cytoplasm and the vacuole. The electroneutrality ruleheld, at least, for K⁺, Na⁺, Mg²⁺ and Cl^– influxes acrossthe cell membrane inthe KCl-containing and NO₃^–-free APW.These results were analyzed on the basis of an extended poremodel which presumed the existence of ATP-dependent processesin the membrane, and suggested that K⁺, Na⁺ and Mg₂₊ inflowsinto an L-cell fragment are likely to be induced by active Cl^–inflow. (Received May 18, 1987; Accepted September 29, 1987)     

In squid nerves intracellular Mg(2+) promotes deactivation of the ATP-upregulated Na(+)/Ca(2+) exchanger

We investigated the role of intracellular Mg²⁺(Mg_i²⁺) on the ATP regulation ofNa⁺/Ca²⁺ exchanger in squid axons and bovineheart. In squid axons and nerve vesicles, the ATP-upregulated exchangerremains activated after removal of cytoplasmic Mg²⁺, evenin the absence of ATP. Rapid and complete deactivation of theATP-stimulated exchange occurs upon readmission ofMg_i²⁺. At constant ATP concentration, the effectof intracellular Mg²⁺ concentration([Mg²⁺]_i) on the ATP regulation of exchangeris biphasic: activation at low [Mg²⁺]_i,followed by deactivation as [Mg²⁺]_i isincreased. No correlation was found between the above results and thelevels of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] measured innerve membrane vesicles. Incorporation ofPtdIns(4,5)P₂ into membrane vesicles activates Na⁺/Ca²⁺ exchange in mammalian heart but not insquid nerve. Moreover, an exogenous phosphatase prevents MgATPactivation in squid nerves but not in mammalian heart. It is concludedthat 1) Mg_i²⁺ is an essentialcofactor for the deactivation part of ATP regulation of the exchangerand 2) the metabolic pathway of ATP upregulation of theNa⁺/Ca²⁺ exchanger is different in mammalianheart and squid nerves.

     

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.     

Stimulation of erythrocyte phosphatidylserine exposure by lead ions

Pb⁺ intoxication causes anemia that is partially due to a decreased life span of circulating erythrocytes. As shown recently, a Ca²⁺-sensitive erythrocyte scramblase is activated by osmotic shock, oxidative stress, and/or energy depletion, leading to exposure of phosphatidylserine at the erythrocyte surface. Because macrophages are equipped with phosphatidylserine receptors, they bind, engulf, and degrade phosphatidylserine-exposing cells. The present experiments were performed to explore whether Pb⁺ ions trigger phosphatidylserine exposure of erythrocytes. The phosphatidylserine exposure was estimated on the basis of annexin binding as determined using fluorescence-activated cell sorting (FACS) analysis. Exposure to Pb⁺ ions [0.1 µM Pb(NO₃)₂] significantly increased annexin binding. This effect was paralleled by erythrocyte shrinkage, which was apparent on the basis of the decrease in forward scatter in FACS analysis. The effect of Pb⁺ ions on cell volume was virtually abolished, and the effect of Pb⁺ ions on annexin binding was blunted after increase of extracellular K⁺ concentration. Moreover, both effects of Pb⁺ ions were partially prevented in the presence of clotrimazole (10 µM), an inhibitor of the Ca²⁺-sensitive K⁺ channels in the erythrocyte cell membrane. Whole cell patch-clamp experiments disclosed a significant activation of a K⁺-selective conductance after Pb⁺ ion exposure, an effect requiring higher (10 µM) concentrations, however. In conclusion, Pb⁺ ions activate erythrocyte K⁺ channels, leading to erythrocyte shrinkage, and also activate the erythrocyte scramblase, leading to phosphatidylserine exposure. The effect could well contribute to the reported decreased life span of circulating erythrocytes during Pb⁺ intoxication. cell volume; annexin; apoptosis; Gardos channel; calcium