Glucose Induced H+ Influx and Transient Currents in Excised Roots: particularly those of Zea mays

The application of D-glucose to solutions bathing excised maize,wheat, pea and bean roots causes a rapid depolarization of theelectrical potentials between the cut tops of the roots andthe bathing solutions. Similar effects are observed for theplasma membrane potentials of maize lateral roots. A flow cell apparatus was used to demonstrate qualitative andquantitative relations between glucose induced H⁺ influx andthe transient decrease in current through the root. The currentchanges appear to be due entirely to H⁺ fluxes. Current andH⁺ fluxes are strongly influenced by external pH, the optimumpH for glucose induced current change being about 4.0. A similarpH optimum was found for 3-O-methyl-D-glucopyranoside but 1-O-methyl--D-glucopyranosidedid not significantly affect the trans-root potential at anypH, suggesting a significant role for the anomeric hydroxylgroup of glucose. Compounds which depolarize the trans-root potential also inhibitthe glucose induced depolarization. Surface -SH groups are probablynot involved in the glucose/H⁺ cotransport. Eadie-Hofstee plots relating the depolarization of trans-rootpotential to the concentrations of D-glucose or 3-O-methyl-D-glucopyranosidehave shown that K_m values increase with increasing monosaccharideconcentration and are very similar to reported values of 3-O-methyl-D-glucopyranosideuptake in maize root segments. K_m values for a similar rangeof D-glucose concentrations do not vary significantly with pHor with membrane depolarization due to a 10-fold increase ofKCl concentration. However, V_max is lowered by an increase inexternal pH or a decrease in trans-root potential. It appearsthat both proton and electrical gradients can affect glucoseinduced H⁺ influx. The auxin herbicide, 2, 4-dichlorophenoxyethanoic acid (0.01mM) stimulates the glucose induced depolarizations in a mannerconsistent with an increase in cytoplasmic pH. This is discussedin relation to the reported action of indole-3-acetic acid andfusicoccin on maize root tissue.     

Changes of action potentials and force at lowered [Na+]o in mouse skeletal muscle: implications for fatigue

We examined 1) whether the effects of lowered trans-sarcolemmal Na⁺ gradient on force differed between nonfatigued fast- and slow-twitch muscles of mice and 2) whether effects on action potentials could explain the decrease of force. The Na⁺ gradient was reduced by lowering the extracellular [Na⁺] ([Na⁺]_o). The peak force-[Na⁺]_o relationships for the twitch and tetanus were the same in nonfatigued extensor digitorum longus and soleus muscles: force was maintained over a large range of [Na⁺]_o and then decreased abruptly over a much smaller range. However, fatigue was significantly exacerbated at a lowered [Na⁺]_o that had little effect in nonfatigued soleus muscle. This finding suggests that substantial differences exist in the Na⁺ effect on force between nonfatigued and fatigued muscle. The reduced contractility in nonfatigued muscles at lowered [Na⁺]_o was largely due to 1) an increased number of inexcitable fibers and threshold for action potentials, 2) a reduction of action potential amplitude, and 3) a reduced capacity to generate action potentials throughout trains. sodium gradient; muscle contraction; action potential train; extensor digitorum longus; soleus     

The Inhibition of Growth by Gravitropic Stimulation in Darkness in Agravitropic Primary Roots of Zea and the Role of Calcium

The primary roots of the "Golden Cross Bantam 70" cultivar ofZea mays are agravitropic in darkness and their orthogravitropismis light-dependent. Analysis of the agravitropic roots providesimportant information about the mechanism of orthogravitropism.However, the underlying mechanism of the agravitropic responsein darkness is unknown. We found that the growth of intact primaryroots was inhibited by gravitropic stimulation (i.e., changingthe orientation of the roots from vertical to horizontal) indarkness, but that of detipped roots was not. The role of calciumin this gravistimulation-dependent inhibition of growth wasinvestigated using apical 5-mm segments of the primary roots.The gravistimulation-dependent inhibition of growth was preventedby applying 10 mM MES-KOH buffer at pH 6.0 to the root cap.By contrast, the application of 0.1–1 mM buffer at pH6.0 and 10 mM buffer at pH 4.5–5.0 allowed the gravistimulation-dependentinhibition of growth. Furthermore, when the buffer of 10 mM(pH 6.0) contained 1–5 mM CaCl₂, the gravistimulation-dependentinhibition of growth was apparent. By contrast, when weak (1mM) buffer at pH 6.0 or 10 mM buffer at pH 4.5 contained 5 mMEGTA, no gravistimulation-dependent inhibition of growth wasobserved. Thus, the gravistimulation-dependent inhibition ofgrowth in darkness seemed to be mediated by an increase in thelevel of free Ca²⁺ in the root tip. These results suggest thatfree Ca²⁺ in the apoplast of the root tip plays an importantrole in the agravitropic response in darkness as well as inorthogravitropism under light of the roots of this cultivarof Zea mays. (Received March 21, 1994; Accepted July 25, 1994)     

Nucleotide regulation of the voltage-dependent nonselective cation conductance in murine colonic myocytes

ATP is proposed to be a major inhibitory neurotransmitter in the gastrointestinal (GI) tract, causing hyperpolarization and smooth muscle relaxation. ATP activates small-conductance Ca²⁺-activated K⁺ channels that are involved in setting the resting membrane potential and causing inhibitory junction potentials. No reports are available examining the effects of ATP on voltage-dependent inward currents in GI smooth muscle cells. We previously reported two types of voltage-dependent inward currents in murine proximal colonic myocytes: a low-threshold voltage-activated, nonselective cation current (I_VNSCC) and a relatively high-threshold voltage-activated (L-type) Ca²⁺ current (I_L). Here we have investigated the effects of ATP on these currents. External application of ATP (1 mM) did not affect I_VNSCC or I_L in dialyzed cells. ATP (1 mM) increased I_VNSCC and decreased I_L in the perforated whole-cell configuration. UTP and UDP (1 mM) were more potent than ATP on I_VNSCC. ADP decreased I_L but had no effect on I_VNSCC. The order of effectiveness was UTP = UDP > ATP > ADP. These effects were not blocked by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS), but the phospholipase C inhibitor U-73122 reversed the effects of ATP on I_VNSCC. ATP stimulation of I_VNSCC was also reversed by protein kinase C (PKC) inhibitors chelerythrine chloride or bisindolylmaleimide I. Phorbol 12,13-dibutyrate mimicked the effects of ATP. RT-PCR showed that P2Y₄ is expressed by murine colonic myocytes, and this receptor is relatively insensitive to PPADS. Our data suggest that ATP activates I_VNSCC and depresses I_L via binding of P2Y₄ receptors and stimulation of the phospholipase C/PKC pathway. inhibitory junction potentials; smooth muscle; enteric nervous system     

光学活性拟除虫菊酯对棉铃虫神经细胞钠通道电流的影响

用全细胞膜片钳技术对比分析了alpha体氯氰菊酯与theta体氯氰菊酯对棉铃虫Helicoverpa armigera幼虫离体培养中枢神经细胞Na⁺通道门控过程的影响。结果表明,alpha体氯氰菊酯作用后,神经细胞Na⁺通道电流（I_Na）先增大,同时通道的激活电压向负电位方向移动约10 mV,提示alpha体氯氰菊酯使通道激活电位降低,通道更容易被激活。药剂作用约10 min后,I_Na又迅速降低,表明alpha体氯氰菊酯对开放状态的Na⁺通道有抑制作用。另外,alpha体氯氰菊酯使I_Na到达峰值的时间缩短,但对失活时间无明显影响。Theta体氯氰菊酯也使I_Na激活电位左移,幅值降低,但降低速率较慢。总的结果表明alpha体氯氰菊酯与theta体氯氰菊酯对棉铃虫中枢神经细胞处于关闭和开放状态的钠通道均有作用,且alpha体氯氰菊酯对钠通道电流的抑制作用强于theta体氯氰菊酯。     

Characterization of the rat Na+/nucleoside cotransporter 2 and transport of nucleoside-derived drugs using electrophysiological methods

The Na⁺-dependent nucleoside transporter 2 (CNT2) mediates active transport of purine nucleosides and uridine as well as therapeutic nucleoside analogs. We used the two-electrode voltage-clamp technique to investigate rat CNT2 (rCNT2) transport mechanism and study the interaction of nucleoside-derived drugs with the transporter expressed in Xenopus laevis oocytes. The kinetic parameters for sodium, natural nucleosides, and nucleoside derivatives were obtained as a function of membrane potential. For natural substrates, apparent affinity (K_0.5) was in the low micromolar range (12–34) and was voltage independent for hyperpolarizing membrane potentials, whereas maximal current (I_max) was voltage dependent. Uridine and 2'-deoxyuridine analogs modified at the 5-position were substrates of rCNT2. Lack of the 2'-hydroxyl group decreased affinity but increased I_max. Increase in the size and decrease in the electronegativity of the residue at the 5-position affected the interaction with the transporter by decreasing both affinity and I_max. Fludarabine and formycin B were also transported with higher I_max than uridine and moderate affinity (102 ± 10 and 66 ± 6 µM, respectively). Analysis of the pre-steady-state currents revealed a half-maximal activation voltage of about –39 mV and a valence of about –0.8. K_0.5 for Na⁺ was 2.3 mM at –50 mV and decreased at hyperpolarizing membrane potentials. The Hill coefficient was 1 at all voltages. Direct measurements of radiolabeled nucleoside fluxes with the charge associated showed a ratio of two positive inward charges per nucleoside, suggesting a stoichiometry of two Na⁺ per nucleoside. This discrepancy in the number of Na⁺ molecules that bind rCNT2 may indicate a low degree of cooperativity between the Na⁺ binding sites. two-electrode voltage clamp; concentrative nucleoside transport; presteady-state currents     

Role of extracellular [Ca2+] in fatigue of isolated mammalian skeletal muscle

The possiblerole of altered extracellular Ca²⁺concentration([Ca²⁺]_o)in skeletal muscle fatigue was tested on isolated slow-twitch soleusand fast-twitch extensor digitorum longus muscles of the mouse. Thefollowing findings were made. 1) Achange from the control solution (1.3 mM[Ca²⁺]_o)to 10 mM[Ca²⁺]_o,or to nominally Ca²⁺-freesolutions, had little effect on tetanic force in nonfatigued muscle.2) Almost complete restoration oftetanic force was induced by 10 mM[Ca²⁺]_oin severely K⁺-depressed muscle(extracellular K⁺ concentration of10-12 mM). This effect was attributed to a 5-mV reversal of theK⁺-induced depolarization andsubsequent restoration of ability to generate action potentials(inferred by using the twitch force-stimulation strength relationship).3) Tetanic force depressed bylowered extracellular Na⁺concentration (40 mM) was further reduced with 10 mM[Ca²⁺]_o.4) Tetanic force loss at elevatedextracellular K⁺ concentration (8 mM) and lowered extracellular Na⁺concentration (100 mM) was partially reversed with 10 mM[Ca²⁺]_oor markedly exacerbated with low[Ca²⁺]_o.5) Fatigue induced by using repeatedtetani in soleus was attenuated at 10 mM[Ca²⁺]_o(due to increased resting and evoked forces) and exacerbated at low[Ca²⁺]_o.These combined results suggest, first, that raised[Ca²⁺]_oprotects against fatigue rather than inducing it and, second, that aconsiderable depletion of[Ca²⁺]_oin the transverse tubules may contribute to fatigue.

     

Membrane Potentials in Excitable Cells of Aldrovanda vesiculosa Trap-Lobes

The resting membrane potential in excitable cells of Aldrovandatrap-lobes is composed of diffusion and electrogenic potentials.The diffusion potential, about –100 mV in artificial pondwater, was determined from the external K⁺ and Na⁺ concentrations.The permeability ratio, P_Na/P_K of the membrane was estimatedto be about 0.3. The electrogenic potential hyperpolarized themembrane to about –140 mV. The peak value of the actionpotential increased by +26 mV with a tenfold increase in theexternal Ca²⁺ concentration. The action potential was blockedby an application of the Ca²⁺ chelater or the Ca channel blocker,LaCl₃. Cells showed additional Ca²⁺ influx (7.8 pmole/cm² impulse)during membrane excitation. These facts suggest that the transientincrease in Ca²⁺ influx causes the action potential presentin cells of Aldrovanda trap-lobes. ¹ Present address: Jerry Lewis Neuromuscular Research Center,School of Medicine, University of California Los Angeles, LosAngeles, CA90024, U.S.A. ² Present address: Biological Laboratory, Kyoritsu Women's University,Hachioji 193, Japan. (Received September 21, 1983; Accepted September 7, 1984)     

Regulation of the Cytoplasmic pH of Chara corallina: Response to Changes in External pH

Effects of external pH (pH_o) on the cytoplasmic pH (pH_c) ofChara corallina have been measured with the weak acid 5, 5-dimethyloxazolidine-2,4-dione (DMO) following standardized pretreatment of cells insolutions at pHo 4.5, 6.3 and 8.3. Irrespective of pH_c duringpretreatment, pH_o responded to pHo during the experimental periodsof 150–180 min or (in one experiment) 90–110 min.There were increases or decreases of about 0.5 in pHo when cellswere transferred from pH_o 4.5 to 8.3 or vice versa. In the darkpH_c was 0.2–0.3 units lower than the corresponding valuein the light. The results are discussed in relation to the factorsinvolved in the regulation of pH_c in C. corallina, which maybegin to break down below about pH_o4.5, as indicated by relativelylarge decreases in pH_c at low pH_o. Key words: Chara corallina, Cytoplasmic pH, External pH, DMO     

Ion Levels and Membrane Potential in Chick Heart Tissue and Cultured Cells

Intracellular concentrations of sodium and potassium as well as resting potentials and overshoots have been determined in heart tissue from chick embryos aged 2–18 days. Intracellular potassium declined from 167 mM at day 2 to 117–119 mM at days 14–18. Intracellular sodium remained nearly constant at 30–35 mM during the same period. The mean resting potential increased from -61.8 mV at day 3 to about -80 mV at days 14–18. The mean overshoot during the same period increased from 12 to 30 mV. P_Na/P_K calculated from the ion data and resting potentials declined from 0.08 at day 3 to 0.01 at days 14–18. Thus, the development of embryonic chick heart during days 2–14 is characterized by a declining intracellular potassium concentration and an increasing resting potential and overshoot. Heart cells from 7- to 8-day embryos, cultured either in monolayer or reassociated into aggregates, were compared with intact tissue of the same age. The intracellular concentrations of sodium and potassium were similar in the three preparations and cultured cells responded to incubation in low potassium medium or treatment with ouabain in a manner similar to that of intact tissue. Resting potentials and overshoots were also similar in the three preparations.     

CO2 chemosensitivity in Helix aspersa: three potassium currents mediate pH-sensitive neuronal spike timing

Elevated levels of carbon dioxide increase lung ventilation in Helix aspersa. The hypercapnic response originates from a discrete respiratory chemosensory region in the dorsal subesophageal ganglia that contains CO₂-sensitive neurons. We tested the hypothesis that pH-dependent inhibition of potassium channels in neurons in this region mediated the chemosensory response to CO₂. Cells isolated from the dorsal subesophageal ganglia retained CO₂ chemosensitivity and exhibited membrane depolarization and/or an increase in input resistance during an acid challenge. Isolated somata expressed two voltage-dependent potassium channels, an A-type and a delayed-rectifier-type channel (I_KA and I_KDR). Both conductances were inhibited during hypercapnia. The pattern of voltage dependence indicated that I_KA was affected by extracellular or intracellular pH, but the activity of I_KDR was modulated by extracellular pH only. Application of inhibitors of either channel mimicked many of the effects of acidification in isolated cells and neurons in situ. We also detected evidence of a pH-sensitive calcium-activated potassium channel (I_KCa) in neurons in situ. The results of these studies support the hypothesis that I_KA initiates the chemosensory response, and I_KDR and I_KCa prolong the period of activation of CO₂-sensitive neurons. Thus multiple potassium channels are inhibited by acidosis, and the combined effect of pH-dependent inhibition of these channels enhances neuronal excitability and mediates CO₂ chemosensory responses in H. aspersa. We did not find a single "chemosensory channel," and the chemosensitive channels that we did find were not unique in any way that we could detect. The protein "machinery" of CO₂ chemosensitivity is probably widespread among neurons, and the selection process whereby a neuron acts or does not act as a respiratory CO₂ chemosensor probably depends on the resting membrane potential and synaptic connectivity. carbon dioxide     

Electrical Potentials and Na, K, and Cl Concentrations in the Vacuole and Cytoplasm of Nitella translucens

The potential differences across the tonoplast and plasmalemmamembranes have been measured in the single cells of Nitellatranslucens, the cells being immersed in an artificial pondwater (composition: NaCl _1.0 mM., KC1 _0.1 mM., CaCl₂, _0.1 mM.).The potential of the cytoplasm is –138 m V with respectto the bathing medium and –18 mV with respect to the vacuole.The concentrations of Na, K, and Cl have been measured in thetwo cell fractions. The concentrations in the flowing cytoplasmare: Na 14 mM., K 119 mM., and Cl 65 mM.; the vacuolar concentrationsare: Na 65 mM., K 75 mM.,and Cl 160 mM. The observed potential differences across the two membranesare compared with the Nernst potentials for all three ions.This analysis shows that all three ions are actively transportedat the plasmalemma: Na is pumped outwards while K and Cl arepumped inwards. At the tonoplast Na is pumped into the vacuolewhile K and Cl are close to electrochemical equilibrium. The inhibitor, ouabain, has no effect on the cell resting potential.     

Autolysis Promotes the Extension Capacity of Zea mays Coleoptile Cell Walls in Response to Acid pH Solutions

The relationship between autolytic degradation of ß(1–3),(1–4)-D-glucanand acid pH-induced extension of isolated Zea mays cell wallshas been investigated using a constant-load extension technique.Acidic buffer (4.5) was able to induce an additional extension(E_a) on cell walls already extended at pH 6.8 buffer under a20 g-mass load, indicating that the additional extension (E_a)was the parameter that better represented the effect of thedifferent treatments on the mechanical properties of maize coleoptilecell walls. The additional extension in response to acidic pHwas higher when cell walls had been previously autolysed for24 h at pH 5.5. Furthermore, the acid-pH effect was dependenton the presence during the constant load extension of some thermo-labilefactors, suggesting the participation of expansins. Acid pHincreased E_a of native cell walls through an increase in theplastic extension (E_p) in agreement with a one step mechanismleading directly to irreversible (plastic) wall extension assuggested by Cosgrove (1977). The autolytic degradation of ß(1–3),(1–4)-D-glucan was also able to modify the mechanicalproperties of maize coleoptile cell walls increasing its elasticextension (E_e) in response to pH 4.5 buffer but that modificationonly leads to an increase in wall extension when expansins areactive, suggesting a cooperation between ß-glucanturnover and expansin action. (Received August 5, 1998; Accepted March 16, 1999)     

KATP channels depress force by reducing action potential amplitude in mouse EDL and soleus muscle

Although ATP-sensitive K⁺ (K_ATP) channel openers depress force, channel blockers have no effect. Furthermore, the effects of channel openers on single action potentials are quite small. These facts raise questions as to whether 1) channel openers reduce force via an activation of K_ATP channels or via some nonspecific effects and 2) the reduction in force by K_ATP channels operates by changes in amplitude and duration of the action potential. To answer the first question we tested the hypothesis that pinacidil, a channel opener, does not affect force during fatigue in muscles of Kir6.2^-/- mice that have no cell membrane K_ATP channel activity. When wild-type extensor digitorum longus (EDL) and soleus muscles were stimulated to fatigue with one tetanus per second, pinacidil increased the rate at which force decreased, prevented a rise in resting tension, and improved force recovery. Pinacidil had none of these effects in Kir6.2^-/- muscles. To answer the second question, we tested the hypothesis that the effects of K_ATP channels on membrane excitability are greater during action potential trains than on single action potentials, especially during metabolic stress such as fatigue. During fatigue, M wave areas of control soleus remained constant for 90 s, suggesting no change in action potential amplitude for half of the fatigue period. In the presence of pinacidil, the decrease in M wave areas became significant within 30 s, during which time the rate of fatigue also became significantly faster compared with control muscles. It is therefore concluded that, once activated, K_ATP channels depress force and that this depression involves a reduction in action potential amplitude. Kir6.2^-/- mice; pinacidil; action potential train; M wave     

Sodium Dependent Photosynthetic Oxygen Evolution in a Marine Diatom

Photosynthetic oxygen evolution in air-equilibrated culturesof Phaeodactylum tricornutum is dependent on the presence ofsodium, but not potassium; sodium cannot be replaced by eitherpotassium, lithium or ammonium. Respiration is not sodium dependent.At constant CO₂ concentrations the depression of oxygen evolutionin the absence of sodium is more pronounced at pH 8.0 than atpH 6.5 and it is concluded that sodium facilitates the utilizationof bicarbonate. Sodium increases the affinity of Phaeodactylumfor inorganic carbon as does growth at low inorganic carbonconcentrations. Key words: Sodium, Photosynthesis, Marine diatom, Phaeodactylum     

The effects of modified ringer's solutions on the membrane potentials of innervated and denervated frog sartorius muscle fibers

Comparison has been made between innervated and chronically denervated frog sartorius muscle fibers for resting potentials and a number of features of the action potential. Muscles were obtained from force-fed frogs maintained at room temperature for periods up to one year, and were studied with intracellular microelectrodes. Denervated muscles increased in sensitivity to acetylcholine by 100–400-fold. Studies were made in normal Ringer's solution, and in media in which concentrations of K⁺, Na⁺, Ca⁺⁺, and Cl? were altered. The only significant differences noted between the denervated and the innervated fibers were a reduction in the maximum rate of fall of the action potential (ca. 20%) and an increase in the fall time of the active membrane potential (ca. 25%). These differences were present in normal Ringer's solution and remained when the bathing medium was modified. The resting membrane potential of denervated and innervated muscles varied with log [K⁺]_o in exactly the same manner, and followed the theoretical relation proposed by Hodgkin (Proc. Roy. Soc., B, 148: 1–37, ′58), with the term representing the ratio of the sodium to potassium permeabilities assigned a value of 0.01. The results suggest that (a) the resting sodium and potassium permeabilities are reduced proportionately after denervation, since it is known that denervated frog muscle has a smaller potassium permeability, and (b) the mechanism controlling the increase in potassium conductance during the action potential is less available after denervation. Data indicate that the system controlling the sodium permeability is capable of activation to the same extent as in innervated muscles. Muslces which had been allowed to reinnervate did not show the differences presented by the denervated muscles. Innervated and denervated muscles did not show any significant changes in maximum rates of rise or fall of the action potential, nor of the active membrane potential amplitude over a 30 mV range of resting membrane potentials, indicating that the sodium and potassium permeability systems are fully available in frog muscle at membrane potentials larger than ?80 mV.     

Regulation of water permeability in rabbit conjunctival epithelium by anisotonic conditions

Effects of unilateral exposure to anisotonic conditions on diffusional water permeability of the isolated rabbit conjunctiva were determined. A segment of the bulbar-palpebral conjunctiva was mounted between Ussing-type hemichambers under short-circuit conditions. Unidirectional water fluxes (J_dw) were measured in either direction by adding ³H₂O to one hemichamber and sampling from the other. Electrical parameters were measured simultaneously. J_dw were determined under control isosmotic conditions and after introduction of either hyper- or hypotonic solutions against the tear or stromal sides of the preparations. In each of these four separate conditions, the anisotonic medium produced an 20–30% reduction in J_dw across the tissue, with the exception that to obtain such reduction with increased tonicity from the stromal side (medium osmolality increased by adding sucrose), conditions presumptively inhibiting regulatory volume increase mechanisms (e.g., pretreatment with amiloride and bumetanide) were also required. All reductions in J_dw elicited by the various anisotonic conditions were reversible on restoration of control tonicity. In experiments in which preparations were pretreated with the protein cross-linking agent glutaraldehyde, anisotonicity-elicited reductions in J_dw were not observed. Such reductions were also not observed in the presence of HgCl₂, implying the involvement of aquaporins. However, it is possible that the mercurial may be toxic to the epithelium, preventing the tonicity response. Nevertheless, from concomitant changes in transepithelial electrical resistance, as well as [¹⁴C]mannitol fluxes, [¹⁴C]butanol fluxes, and Arrhenius plots, arguments are presented that the above effects are best explained as a cell-regulated reduction in membrane water permeability that occurs at the level of water-transporting channels. Presumably both apical and basolateral membranes can downregulate their water permeabilities as part of a protective mechanism to help maintain cell volume. unidirectional water fluxes; net water fluxes; Ussing chamber; short-circuit current; electrolyte transport; cell volume regulation; paracellular mannitol permeability     

Effects of Anion Channel Inhibitors on Light-Induced Potential Changes in the Liverwort Conocephalum conicum

The effect of three different anion channel inhibitors, namely(5-nitro-2-3-phenylpropyl-amino)benzoic acid (NPPB), Zn²⁺ andanthracene-9-carboxylic acid (A-9-C) on the action potentialin the liverwort Conocephalum conicum were tested. All threecaused an increase of the excitability threshold and a decreaseof action potential amplitudes. This confirms the involvementof anion channels in the action potentials in Conocephalum.In plants treated with 1 or 2 mM A-9-C but not with NPPB (50or 100 µM) and Zn²⁺ (100 or 500 µM), a light-inducedtransient depolarization occurred. In contrast to action potentials,the amplitude of this voltage transient depended on the lightintensity and on the duration of preceding dark period. Alsoin contrast to action potentials, which are blocked by TEA,when applied together with A-9-C, TEA even increased the amplitudesof the light-induced voltage transients to up to 170 mV. Thedepolarization was obviously limited by the voltage-dependentopening of K⁺ channels in the absence of TEA. The amplitudeof the light-induced voltage transients (in the presence ofTEA) increased in elevated CaCl₂ concentrations pointing toa Ca²⁺ permeability giving rise to the depolarization. However,none of the Ca²⁺ channel blockers tested, La³⁺, Gd³⁺, nifedipine,verapamil or diltiazem, had an effect. The light-induced voltagetransients in A-9-C treated plants are quite different fromlight- and electrically triggered action potentials but sharesome similarities with light-induced generator potentials. (Received July 9, 1996; Accepted February 20, 1997)     

Effects of Pressure and Temperature on the Catalytic and Regulatory Properties of Muscle Pyruvate Kinase from an Off-Shore Benthic Fish

SYNOPSIS. Muscle pyruvate kinase from an abyssal Coryphaenoidesspecies occurs as a single electrophoretic form with an isoelectricpoint of about pH 6.0. Maximum catalytic rates are dramaticallyreduced by pressure. For catalysis at 3°C, the volume changeof activation, V*, is about 44 cm³/mole (calculated between14.7 and 8000 psi). The value ot V* decreases at higher temperaturesbut is pH independent. The activation energy for rattail musclepyruvate kinase at 14.7 psi is about 13 Kcal/mole and doublesat 12,000 psi. Mg²⁺ saturation kinetics involve positive site-siteinteractions. Hill plots yield n values of about 2.4 and K_avalues of about 2 mM (at 3°C), and these constants are pressureindependent. The K_m values for ADP increase slightly with pressure.PEP saturation curves are complex: at high PEP concentrations,the n values are about 2–2.5, while at low PEP levels,values for the Hill constant are about 1.0. The Hill constantlor PEP is not affected by pressure, but the apparent K_m increasessomewhat with pressure. FDP dramatically activates rattail musclepyruvate kinase (500% activation with 0.1 mM FDP) by (1) reducingthe K_mPEP, (2) increasing the maximum velocity, and (3) overridingnegative ATP modulation of the enzyme. The latter control featureis strictly dependent upon pressure and is not observed at lowpressure. In the presence of FDP, the K_m for PEP decreases athigh pressures, in this way counteracting the inhibitory effectsof pressure. Under low concentrations of substrates, pyruvatekinase activity is probably determined by its kinetic propertiesand not by energy-volume relationships.     

A computational analysis of central CO2 chemosensitivity in Helix aspersa

We created a single-compartment computer model of a CO₂ chemosensory neuron using differential equations adapted from the Hodgkin-Huxley model and measurements of currents in CO₂ chemosensory neurons from Helix aspersa. We incorporated into the model two inward currents, a sodium current and a calcium current, three outward potassium currents, an A-type current (I_KA), a delayed rectifier current (I_KDR), a calcium-activated potassium current (I_KCa), and a proton conductance found in invertebrate cells. All of the potassium channels were inhibited by reduced pH. We also included the pH regulatory process to mimic the effect of the sodium-hydrogen exchanger (NHE) described in these cells during hypercapnic stimulation. The model displayed chemosensory behavior (increased spike frequency during acid stimulation), and all three potassium channels participated in the chemosensory response and shaped the temporal characteristics of the response to acid stimulation. pH-dependent inhibition of I_KA initiated the response to CO₂, but hypercapnic inhibition of I_KDR and I_KCa affected the duration of the excitatory response to hypercapnia. The presence or absence of NHE activity altered the chemosensory response over time and demonstrated the inadvisability of effective intracellular pH (pH_i) regulation in cells designed to act as chemostats for acid-base regulation. The results of the model indicate that multiple channels contribute to CO₂ chemosensitivity, but the primary sensor is probably I_KA. pH_i may be a sufficient chemosensory stimulus, but it may not be a necessary stimulus: either pH_i or extracellular pH can be an effective stimuli if chemosensory neurons express appropriate pH-sensitive channels. The lack of pH_i regulation is a key feature determining the neuronal activity of chemosensory cells over time, and the balanced lack of pH_i regulation during hypercapnia probably depends on intracellular activation of pH_i regulation but extracellular inhibition of pH_i regulation. These general principles are applicable to all CO₂ chemosensory cells in vertebrate and invertebrate neurons. hypercapnia; potassium channels; computer modeling; central chemoreceptors