Retardation of cation channel deactivation by mitochondrial dysfunction in adrenal medullary cells

Themechanism for cyanide (CN) activation of a nonselective cation (NS)channel coupled with a muscarinic receptor in a guinea pig chromaffincell was studied with the perforated-patch method. Bath application ofa protein kinase inhibitor resulted in a dose-dependent inhibition ofmuscarine-induced current (I_M) but had no apparent effect on the CN-induced current (I_CN). On theother hand, production of I_CN occluded muscarineactivation of NS channels in an amplitude-dependent manner.Deactivation of I_M after washout was retarded whileI_CN was also active, and the extent of theretardation increased with an increase in the relative production ofI_CN on muscarinic stimulation. Restoration ofNa⁺ pump activity from CN suppression was conspicuouslyretarded below 19-20°C, and the apparent diminution ofI_M and I_CN after washout wasretarded in parallel with a decrease in temperature. The resultssuggest that CN activation of NS channels is due to suppression ofdeactivation of the channel.

     

Maintenance Respiration and Carbon Balance of Plants at Low Levels of Sodium Chloride Salinity

The daily course of carbon influx and efflux was measured inyoung plants of Phaseolus vulgaris, Xanthium strumarium, Zeamays, and Atriplex halimus, exposed to low levels of salinity(NaCl) and varying daytime light intensities. Maintenance respiration(R_M) was calculated. In Phaseolus, Xanthium, and Atriplex, R_Mrose with increasing salinity, approximately up to those levelsof salinity above which apparent signs of toxicity appear. Athigher levels of salinity R_M declined. There was no responseof R_M to salinity in Zea. At the levels of salinity tested,salinity did not affect the ratio of growth respiration to photosynthesis. At –5 x 10⁵ Pa of NaCl salinity, the increase in R_M inXanthium was calculated to account for 24% of the growth reductioncaused by salt. The remainder could be ascribed to reduced photosynthesis.The increase of R_M is considered to be indicative of an adaptivemechanism, not present in the very salt-sensitive Zea.     

K+ transport and capacitance of the basolateral membrane of the larval frog skin

Skin from larval bullfrogs was mounted in an Ussing-type chamberin which the apical surface was bathed with a Ringer solution containing 115 mM K⁺ and thebasolateral surface was bathed with a Ringer solution containing 115 mMNa⁺. Ion transport was measured asthe short-circuit current(I_sc) with alow-noise voltage clamp, and skin resistance(R_m) wasmeasured by applying a direct current voltage pulse. Membrane impedance was calculated by applying a voltage signal consisting of 53 sine wavesto the command stage of the voltage clamp. From the ratio of theFourier-transformed voltage and current signals, it was possible tocalculate the resistance and capacitance of the apical and basolateralmembranes of the epithelium(R_a andR_b,C_a and C_b,respectively). With as the anion,R_m decreasedrapidly within 5 min following the addition of 150 U/ml nystatin to theapical solution, whereasI_sc increasedfrom 0.66 to 52.03 µA/cm² over a60-min period. These results indicate that nystatin becomes rapidlyincorporated into the apical membrane and that the increase inbasolateral K⁺ permeabilityrequires a more prolonged time course. Intermediate levels ofI_sc were obtainedby adding 50, 100, and 150 U/ml nystatin to the apical solution. Thisproduced a progressive decrease in R_a andR_b whileC_a andC_b remainedconstant. With Cl as theanion, I_sc valuesincreased from 2.03 to 89.57 µA/cm² following treatment with150 U/ml nystatin, whereas with gluconate as the anionI_sc was onlyincreased from 0.63 to 11.64 µA/cm². This suggests that theincrease in basolateral K⁺permeability produced by nystatin treatment, in the presence of morepermeable anions, is due to swelling of the epithelial cells of thetissue rather than the gradient for apicalK⁺ entry. Finally,C_b was notdifferent among skins exposed toCl,, or gluconate, despite the largedifferences inI_sc, nor didinhibition of I_scby treatment with hyperosmotic dextrose cause significant changes inC_b. These resultssupport the hypothesis that increases in cell volume activateK⁺ channels that are alreadypresent in the basolateral membrane of epithelial cells.

     

Primary production in lakes Huron and Michigan: in vitro and in situ comparisons

Oxygen- and carbon-14-based primary production estimates from9–16 h in vitro incubations were compared in lakes Huronand Michigan. For surface mixing layer compansons, gross O₂/¹⁴Cphotosynthetic quotients (gross PQ) averaged 2.2, and net O₂/¹⁴Cphotosynthetic quotients (net PQ) averaged 1.4. The mean grossPQ is consistent with a theoretical P0 based on the CO₂ andNO₃ assimilation ratio. However, within the deep chlorophylllayer, gross PQ and net PQ averaged 4.9 and 2.8 respectively.These higher values were likely due to excess NO₃ reductionat the expense of CO₂ uptake. Thus, during short experimentsunder low light conditions, oxygen evolution and CO₂ uptakemay not be tightly coupled. In vitro and in situ O₂ productionestimates were compared in four diurnal (dawn to dusk) experimentsin Lake Huron. In situ production estimates were determinedby measuring water-mass oxygen changes and oxygen transfer acrossthe air-water interface. In situ production estimates were approximatelytwice in vitro production estimates for both surface mixinglayer and deep chlorophyll layer comparisons. The differencebetween estimates was attributable to containment effects manifestin 13–16 h bottle incubations. Short-term (1–2 h)in vitro production was also compared to diurnal in vitro production.Rates of short-term production were {small tilde}1.6 times higherthan rates of diurnal production, suggesting that short-termin vitro production experiments may provide reasonable estimatesof in situ primary production.     

Leaf Growth in Cotton (Gossypium hirsutum L.) 2. The Influence of Temperature, Light, Water Stress and Root Restriction on the Growth and Initiation of Leaves

The rate parameters R₁, R₂, I/LI and I/t_0.5, which characterizethe growth in area of successive main-stem leaves, probablyall have the same temperature response. Temperature thereforeonly operates on the time scale. Water stress reduces both therelative growth rate and the advance of developmental age, thelatter however to a lesser extent than the former. The effectof root restriction is explained as resulting from mineral shortage. Gossypium hirsutum L., cotton, leaf growth, leaf initiation, relative growth rate, temperature, light, water stress, root restriction     

Chloride secretion by semicircular canal duct epithelium is stimulated via beta 2-adrenergic receptors

The ductalepithelium of the semicircular canal forms much of the boundary betweenthe K⁺-rich luminal fluid and the Na⁺-richabluminal fluid. We sought to determine whether the net ion fluxproducing the apical-to-basal short-circuit current(I_sc) in primary cultures was due to anionsecretion and/or cation absorption and under control of receptoragonists. Net fluxes of ²²Na, ⁸⁶Rb, and³⁶Cl demonstrated a basal-to-apical Clsecretion that was stimulated by isoproterenol. Isoproterenol andnorepinephrine increased I_sc with anEC₅₀ of 3 and 15 nM, respectively, and isoproterenolincreased tissue cAMP of native canals with an EC₅₀ of 5 nM. Agonists for adenosine, histamine, and vasopressin receptors had noeffect on I_sc. Isoproterenol stimulation ofI_sc and cAMP was inhibited by ICI-118551(IC₅₀ = 6 µM for I_sc) but notby CGP-20712A (1 µM) in primary cultures, and similar results werefound in native epithelium. I_sc was partially inhibited by basolateral Ba²⁺ (IC₅₀ = 0.27 mM) and ouabain, whereas responses to genistein, glibenclamide, andDIDS did not fully fit the profile for CFTR. Our findings show that thecanal epithelium contributes to endolymph homeostasis by secretion ofCl under ₂-adrenergic control with cAMP assecond messenger, a process that parallels the adrenergic control ofK⁺ secretion by vestibular dark cells. The current workpoints to one possible etiology of endolymphatic hydrops in Meniere'sdisease and may provide a basis for intervention.

     

Time of Flowering of Pea (Pisum sativum L.) as a Function of Leaf Appearance Rate and Node of First Flower

In order to assess the influence of environmental conditionson time of flowering of pea (Pisum sativum L.), a serial sowingtrial was conducted over 2 years at Dijon, France, on two wintercultivars Frisson and Frilene. Time of flowering was analysedaccording to two variables: the leaf appearance rate R_L andthe node of first flower N_I. R_L was linearly related to temperature (r² = 0·94). Thebase temperature was 2°C for both varieties. Growth rateaccounted for the residual variability of R_L . Photoperiod andtemperature acted on N_I in an additive way. Frilene, the latergenotype, was more responsive than Frisson. A model for predicting time of flowering based upon these resultsis proposed. Deviations from this model were related to N nutritionin interaction with the plant water relations. Steps for improvingthe model are then discussed.Copyright 1993, 1999 Academic Press Pisum sativum L., pea, flowering, temperature, photoperiod, phyllochron, model     

Water Import Rate in Tomato Fruit: A Resistance Model

A model of the water import rate in tomato fruit is proposed.It compares the fruit to a hollow sphere (P) with external radiusR and internal radius R_G, corresponding to pericarp, and containingan internal spherical part (G). The pathway limiting water inputrate at any point I at a distance r from the fruit centre wasassumed to be proportional: (a) in P, to the length of the arcwhich has a radius r and which goes from I to the pedicel extension;(b) in G, to r. The water input rate at I was modelled basedon a law similar to Darcy's law which takes into account thedifference between the water potential at entry of fruit andthe water potential at point I. This latter potential was thesum of fruit osmotic potential and pressure potential due toresistance of tissue to deformation. This potential was proportionalto R-r or R_G -r. The model was expressed at fruit level by alaw such that water mass imported per unit time per unit surfacearea of fruit (f_rw) was a linear function of R. The model wascompared to linear regressions of this rate in terms of R whichhad been found during fruit swelling from published results,and which were obtained at different values of nutrient solutionsalinity. The results suggested that water input in tomato fruitis conditioned by passive forces depending on fruit size.Copyright1994, 1999 Academic Press Fruit, growth, model, resistance, salinity, size, tomato, transfer, water     

Characterization of Storage Proteins in Daucus carota L.: Two Novel Proteins Display Zygotic Embryo Specificity

Although maturation-related proteins are well known in the endospermof albuminous seeds, an important question is whether the zygoticembryo possesses its own maturation proteins. We report on theisolation and partial characterization of storage proteins ofcarrot (Daucus carota L. var Nandor) dry achenes and isolatedzygotic embryos, using one- and two-dimensional electrophoresistechniques, HPLC and amino acid sequencing. The presence ofa series of abundant polypeptides showing charge heterogeneity,that are rapidly degraded upon germination, was revealed inthe endosperm. These proteins consisted of glycoproteins, themost abundant of which displayed a molecular mass (M_r) of 58,000,albumins of M_r 42,000 comprising at least one rß-1,3-glucanase,and two globulins of M_r 90,000 and 50,000–55,000 respectively,the second being an oligomer composed of three subunits of M_r13,000, 20,000 and 30,000. None of these storage proteins identifiedin the endosperm were detected in zygotic embryos. In contrast,two novel proteins were isolated from zygotic embryos, namelya globulin family of M_r 50,000 and pI 6.3–6.8, which wasnamed "daucin", and a late embry-ogenesis abundant (LEA) proteinfamily of M_r 25,000 and pI6.3–6.6, named "RAB25". Sincethe latter proteins are apparently absent of the endosperm,these results suggest that the maturation of carrot zygoticembryos requires its own specific set of storage and LEA proteins. (Received July 15, 1997; Accepted October 28, 1997)     

Properties of ATP-dependent K(+) channels in adrenocortical cells

Bovine adrenocortical zona fasciculata (AZF)cells express a novel ATP-dependent K⁺-permeable channel(I_AC). Whole cell and single-channel recordings were used to characterize I_AC channels withrespect to ionic selectivity, conductance, and modulation bynucleotides, inorganic phosphates, and angiotensin II (ANG II). Inoutside-out patch recordings, the activity of unitaryI_AC channels is enhanced by ATP in the patchpipette. These channels were K⁺ selective with nomeasurable Na⁺ or Ca²⁺ conductance. Insymmetrical K⁺ solutions with physiological concentrationsof divalent cations (M²⁺), I_ACchannels were outwardly rectifying with outward and inward chordconductances of 94.5 and 27.0 pS, respectively. In the absence ofM²⁺, conductance was nearly ohmic. Hydrolysis-resistantnucleotides including AMP-PNP and NaUTP were more potent than MgATP asactivators of whole cell I_AC currents. Inorganicpolytriphosphate (PPP_i) dramatically enhancedI_AC activity. In current-clamp recordings, nucleotides and PPP_i produced resting potentials in AZFcells that correlated with their effectiveness in activatingI_AC. ANG II (10 nM) inhibited whole cellI_AC currents when patch pipettes contained 5 mMMgATP but was ineffective in the presence of 5 mM NaUTP and 1 mM MgATP.Inhibition by ANG II was not reduced by selective kinase antagonists.These results demonstrate that I_AC is adistinctive K⁺-selective channel whose activity isincreased by nucleotide triphosphates and PPP_i.Furthermore, they suggest a model for I_AC gatingthat is controlled through a cycle of ATP binding and hydrolysis.

     

Scaling of submaximal oxygen uptake with body mass and combined mass during uphill treadmill bicycling

This study examined the scaling relationships ofnet O₂ uptake [O_2(net) = O₂  restingO₂] to body mass(M_B) andcombined mass (M_C = M_B + bicycle)during uphill treadmill bicycling. It was hypothesized thatO_2(net)(l/min) would scale proportionally withM_C [i.e.,O_2(net)  M^1.0_C] and less than proportionally withM_B [i.e.,O_2(net)  M_B].Twenty-five competitive cyclists [73.9 ± 8.8 and 85.0 ± 9.0 (SD) kg forM_B andM_C,respectively] rode their bicycles on a treadmill at 3.46 m/s andgrades of 1.7, 3.5, 5.2, and 7.0% whileO₂ was measured. Multiplelog-linear regression procedures were applied to the pooledO_2(net)data to determine the exponents forM_C andM_B afterstatistically controlling for differences in treadmill grade anddynamic friction. The regression models were highly significant (R² = 0.95, P < 0.001). Exponents forM_C (0.99, 95%confidence interval = 0.80-1.18) andM_B (0.89, 95%confidence interval = 0.72-1.07) did not differ significantly fromeach other or 1.0. It was concluded that the 0.99 M_C exponent wasdue to gravitational resistance, whereas theM_B exponent was<1.0 because the bicycles were relatively lighter for heaviercyclists.

     

Flowering of the long-day plant, Lemna gibba, under short-day schedules composed of red and far-red light

Flowering in Lemna gibba, a long-day duckweed, can be inducedunder a short-day condition when the photoperiodic regimes areR₇FR₃ (7 hr red followed by 3 hr far-red), R₅FR₅ and R₃FR₇.This indicates the necessity of a proper balance between redand far-red effects for flowering. The flowering induced bythese regimes is inhibited by a brief exposure to red givenat the start of darkness and this inhibition is reversed bysubsequent exposure to far-red. Thus, the red/far-red reversibleeffect is found only at the beginning of darkness for floweringof L. gibba. However, flowering of L. gibba is promoted by a red light breakgiven near the middle of a 14 hr dark period. The promotiveeffect is not reversed by subsequent exposure to far-red, i.e.,the effect of the red break converts from inhibition to promotionas when given later in the dark period, which suggests the involvementof a timing mechanism. (Received July 21, 1973; )     

An empirical model of primary productivity (14C) using mesocosm data along a nutrient gradient

The two parameters of the hyperbolic tangent equation, P_m and, were estimated from in situ vertical profiles of primary productionusing mesocosm data along a nutrient gradient. The parameters,derived from 4-h (around noon) ¹⁴C incubations, were used togetherwith the photosynthesis-light curve and hourly solar radiationdata to calculate daily primary production rates (P_d). Approximately40% of the daily production occurred in the 4 h around noon.Considering parameter uncertainty, there was no indication ofan increase in variation in production with increased nutrientloading, nor did biomass-specific P-I parameters increase. Annualproduction ranged from 82 to 901 g C m^–2 year^–1and was highest in the highest nutrient treatment tank. Dailyproductivity ranged from 0.02 to 9.1 g C m^–2 day^–1and was significantly correlated, in all treatments, with acomposite parameter BI₀/k (where B is phytoplankton biomass;I₀ is daily radiation and k is the extinction coefficient).Linear regressions of P_d against BI₀/k indicated that much ofthe variability (86%) in productivity was explained by lightavailability and phytoplankton biomass. Two approaches for predictingproductivity were compared: (i) predicting production directlyfrom environmental variables (i.e. BI₀/k) and (ii) predictingthe parameters of the P-I curve from environmental variablesand using these to calculate daily production.     

EFFECT OF SOME SULFHYDRYL REAGENTS ON LIGHT-INDUCED CO2-FIXATION IN CHLORELLA

1. Using intact cells of Chlorella ellipsoidea, investigationswere made on the effects of some SH-reagents (IAA, CMB and arsenite)upon various reactions pertaining to the primary photogenicagent (designated by R) formed in the mechanism of photosynthesis.
2. The pre-illumination experiments using ¹⁴CO₂ as a tracer haveled us to the inference that (i) the process of photochemicalformation of R is inhibited by IAA, that (ii) the spontaneousdecay of R in the dark is markedly accelerated by CMB and arsenite,but not at all affected by IAA, and that (iii) the participationof R in the cyclic path of carbon leading to the fixation ofCO₂ is not affected by IAA and CMB.
3. Based on the assumptionthat R is a reducing agent, it was discussedthat the fact mentionedunder (iii) is incompatible with theidea that the reductionof PGA to triose phosphate be the soleor rate-determining reductivestep in the cyclic path of carbonin photosynthesis.
4. The possibilitythat R may have a functional SH-group (s) wasinvoked to accountfor the observation that the decay of R inthe dark was markedlyaccelerated by CMB and arsenite.

(Received February 11, 1960; )     

Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport

We report, for the epithelialNa⁺ channel (ENaC) in A6 cells,the modulation by cell pH (pH_c)of the transepithelial Na⁺ current(I_Na), thecurrent through the individual Na⁺channel (i), the openNa⁺ channel density(N_o), and thekinetic parameters of the relationship betweenI_Na and theapical Na⁺ concentration. Thei andN_o were evaluatedfrom the Lorentzian I_Na noise inducedby the apical Na⁺ channel blocker6-chloro-3,5-diaminopyrazine-2-carboxamide.pH_c shifts were induced, understrict and volume-controlled experimental conditions, byapical/basolateral NH₄Cl pulses orbasolateral arrest of theNa⁺/H⁺exchanger (Na⁺ removal; block byethylisopropylamiloride) and were measured with the pH-sensitive probe2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Thechanges in pH_c were positivelycorrelated to changes inI_Na and theapically dominated transepithelial conductance. The sole pH_c-sensitive parameter underlyingI_Na wasN_o. Only thesaturation value of theI_Na kinetics wassubject to changes in pH_c.pH_c-dependent changes inN_o may be causedby influencingP_o, the ENaC openprobability, or/and the total channel number,N_T = N_o/P_o.

     

Measurement of the Carbon Dioxide Compensation Point and the Rate of Loss of 14CO2 in the Light and Dark in Some Bryophytes

The CO₂ compensation point at 25 °C and 250 µEinsteinsm^–2 s^–1 wasmeasured for 27 bryo-phyte species, andwas found to be in the range of 45–160 µl CO₂ I^–1air. Under the same conditions Zea mays gave a value of 11 µlI^–1 and Horde um vulgare 76 µI^–1. The rate of loss of photosyntheticallyfixed ¹⁴CO₂ in the light and dark in six bryophytes (three mosses,two leafy liverworts, one thalloid liverwort) was determinedin CO₂-free air and 100% O₂. The rate of ¹⁴CO₂ evolution inthe light was less than that in the dark in CL₂-free air, butin 100% O₂ the rate in the light increased, so that in all butthe leafy liverworts it was greater than that in the dark. Raisingthe temperature tended to increase the rate of 14CO₂ evolutioninto CO₂-free air both in the light and dark, so that the light/dark(L/D) ratio did not greatly vary. The lower rate of loss of¹⁴CO₂ in the light compared tothe dark could be due to partialinhibition of ‘dark respiration’ reactions in thelight, a low rate of glycolate synthesis and oxidation, or partialreassimilation of the ¹⁴CO₂ produced, or a combination of someor all of these factors.     

Joint field experiments for comparisons of measuring methods of photosynthetic production

During the 1st GAP Workshop at Konstanz in April 1982 comparativemeasurements of phytoplankton primary production by severaltechniques were conducted simultaneously at an offshore stationin Lake Konstanz and an experimental algal pond. Suspended glassbottle exposure techniques using ¹⁴C and ¹³C uptake gave P_z(mg C m^–3 h^–1) values which varied considerablynear-surface, but estimates of areal rates for the euphoticzone P_cu(mg C m^–3 h^–1) which were reasonably close.In the lake, P_z, from a vertical tube exposure (with ¹⁴C uptake)was greater than rates derived for integrated bottle samples.The oxygen bottle method permitted a good estimate of compensationdepth, corresponding to in situ growth studies. There were difficultiesin direct comparison between O₂ and carbon methods. Correlationbetween them for P_z was good in the lake but poor in the pond,both for suspended bottle and vertical tube methods. This seriesdemonstrates that despite reasonable overall estimates, comparativelyminor methodological differences in experimental technique cancause large variation. ⁺ Coordinator of the group for comparative measurements of photosyntheticproduction at the GAP Workshop, Konstanz, April 1982. ^*This paper is the result of a study made at the Group for AquaticPrimary Productivity (GAP) First International Workshop heldat the Limnological Institute, University of Konstanz, in April1982.     

A role for ERK1/2 in EGF- and ATP-dependent regulation of amiloride-sensitive sodium absorption

Receptor-mediated inhibition of amiloride-sensitive sodium absorption was observed in primary and immortalized murine renal collecting duct cell (mCT12) monolayers. The addition of epidermal growth factor (EGF) to the basolateral bathing solution of polarized monolayers reduced amiloride-sensitive short-circuit current (I_sc) by 15–25%, whereas the addition of ATP to the apical bathing solution decreased I_sc by 40–60%. Direct activation of PKC with phorbol 12-myristate 13-acetate (PMA) and mobilization of intracellular calcium with 2,5-di-tert-butyl-hydroquinone (DBHQ) reduced amiloride-sensitive I_sc in mCT12 monolayers by 46 ± 4% (n = 8) and 22 ± 2% (n = 8), respectively. Exposure of mCT12 cells to EGF, ATP, PMA, and DBHQ caused an increase in phosphorylation of p42/p44 (extracellular signal-regulated kinase; ERK1/2). Pretreatment of mCT12 monolayers with an ERK kinase inhibitor (PD-98059; 30 µM) prevented phosphorylation of p42/p44 and significantly reduced EGF, ATP, and PMA-induced inhibition of amiloride-sensitive I_sc. In contrast, pretreatment of monolayers with a PKC inhibitor (bisindolylmaleimide I; GF109203x; 1 µM) almost completely blocked the PMA-induced decrease in I_sc, but did not alter the EGF- or ATP-induced inhibition of I_sc. The DBHQ-mediated decrease in I_sc was due to inhibition of basolateral Na⁺-K⁺-ATPase, but EGF-, ATP-, and PMA-induced inhibition was most likely due to reduced apical sodium entry (epithelial Na⁺ channel activity). The results of these studies demonstrate that acute inhibition of amiloride-sensitive sodium transport by extracelluar ATP and EGF involves ERK1/2 activation and suggests a role for MAP kinase signaling as a negative regulator of electrogenic sodium absorption in epithelia. mitogen-activated protein kinase; epithelial ion transport; epithelial sodium channel     

Ionic Currents across the Plasmalemma of Chara inflata Cells: II. EFFECTS OF EXTERNAL Na+, Ca2+ AND Cl- ON K+ AND Cl CURRENTS

The electrical conductance of the plasmalemma of cells of Charainflata, due to the diffusion of ions, consists predominantlyof K⁺, Cl^– and leak components. When the membrane electricalpotential difference is stepped in a negative direction witha voltage-clamp, the resulting inward current has componentsI_K, I_Cl and I_L (leak). During such voltage-clamp steps I_K isinactivated, and Ic activated with voltage-dependent half-times.Increases in the external NaCl concentration reduce the magnitudeof I_K and increase the magnitude of I_c, but reduce the half-timeof inactivation or activation. The NaCl-induced changes in I_kand I_Cl and their kinetics were more pronounced at pH₀ =6.5than at pH₀ =9.5. When the concentration of external CaCl₂ wasincreased, I_k, I_Cl and the half-time of inactivation, (T_1/2),of I_k were all reduced. The half-time of activation of I_Cl wasincreased. The NaCI-induced changes could result from increases in bothexternal ion concentration and osmotic pressure. Previous experimentshave shown that an increase in external osmotic pressure alonealters the properties of the conductances. In this paper weattempt to separate the purely ionic effects from the osmoticones. Key words: Chara inflata, ionic effects, K⁺ and Cl^– currents     

Calcium influx through If channels in rat ventricular myocytes

The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac (I_f)/neuronal (I_h) time- and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and I_h channels in neurons. This raises the possibility of Ca²⁺ permeation in I_f, the I_h counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 Ca²⁺ signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by I_f in rat ventricular myocytes. We observed Ca²⁺ influx when HCN/I_f channels were activated. Ca²⁺ influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an I_f channel blocker, inhibited I_f and Ca²⁺ influx at the same time. Quantitative analysis revealed that Ca²⁺ flux contributed to 0.5% of current produced by the HCN2 channel or I_f. The associated increase in Ca²⁺ influx was also observed in spontaneously hypertensive rat (SHR) myocytes in which I_f current density is higher than that of normotensive rat ventricle. In the absence of EGTA (a Ca²⁺ chelator), preactivation of I_f channels significantly reduced the action potential duration, and the effect was blocked by another selective I_f channel blocker, ZD-7288. In the presence of EGTA, however, preactivation of I_f channels had no effects on action potential duration. Our data extend our previous discovery of Ca²⁺ influx in I_h channels in neurons to I_f channels in cardiac myocytes. calcium ion flux; hyperpolarization-activated, cyclic nucleotide-gated/cardiac time- and volume-dependent cation current channels