Rates of Photosynthesis in Characean Cells: I. PHOTOSYNTHETIC 14CO2 FIXATION BY NITELLA TRANSLUCENS

A study has been made of photosynthetic ¹⁴CO₂ fixation by isolated‘mature’ internodes of Nitella translucens. Experimentalconditions were similar to those used in studies of the ionicrelations of these cells. Maximum rates of photosynthesis were33–40µµmoles CO₂, fixed per cm² of surfacearea per second (equivalent to 12–15 /xmoles fixed permg chlorophyll per hour). ^l4CO₂ fixation was inhibited to thedark level by 3(3,4,dichlorophenyl)-1, 1-dimethylurea (at 0-6µM or 10µM) and by the uncoupler carbonyl cyanide-m-chlorophenylhydrazone(SµM). The presence of imidazole or ammonium sulphate(both of which uncouple ATP production in vitro) did not resultin an inhibition of 14CO2 fixation. These results are discussedin relation to published work on solute uptake by Nitella translucens.During photosynthesis there was rapid movement of ¹⁴C-labelledorganic compounds out of the chloroplasts. ¹⁴C-labelled sucrose,ammo-acids, and sugar phosphates were found in samples of vacuolarsap.     

Photosynthetic Carbon Sources of Stream Macrophytes

Rates of photosynthesis of four submerged stream macrophyteswere examined under varying pH and composition of inorganiccarbon species. Callitriche stagnatis and Sparganium simplexused only CO₂ for photosynthesis. Potamogeton crispus and P.pectinatus used HCO₃ in addition to CO₂, but with much lowerefficiency. The photosynthetic rates at air equilibrium anda total inorganic carbon concentration of 5.0 mM were 2–3times lower than maximum rates at CO2 saturation for the HCO₃users and 10–14 times lower for the CO₂ users. The CO₂compensation point of entire plants of Callitriche (2.5 µM)and Sparganium (6.0µM) was well below the equilibriumconcentration (15 µM). and the low saturation points (250–500µM) also pointed to efficient use of CO2. Callitricheand Sparganium compete successfully with HCO₃^– users inhardwater streams, which have a higher exchange and generationcapacity of CO2 than stagnant and more soft waters. Rates ofphotosynthesis of Potamogeton crispus and P. pectinatus decreasedat high pH. Depending on the two alternative hypotheses forHCO₃use, this decline can be explained by CO₃^––inhibition of HCO₃^– uptake or by increasing capacity tobuffer H+efflux from the plant. Habitats subject to high pH,e. g. small ponds with dense vegetation, may have a strong selectionfor efficient mechanisms of HCO₃ use. Key words: Photosynthesis, Macrophytes, Carbon-source     

DCEBIO stimulates Cl- secretion in the mouse jejunum

We investigated the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one(DCEBIO) on the Cl^– secretory response of the mouse jejunum using the Ussing short-circuit current (I_sc) technique. DCEBIO stimulated a concentration-dependent, sustained increase in I_sc (EC₅₀ 41 ± 1 µM). Pretreating tissues with 0.25 µM forskolin reduced the concentration-dependent increase in I_sc by DCEBIO and increased the EC₅₀ (53 ± 5 µM). Bumetanide blocked (82 ± 5%) the DCEBIO-stimulated I_sc consistent with Cl^– secretion. DCEBIO was a more potent stimulator of Cl^– secretion than its parent molecule, 1-ethyl-2-benzimidazolinone. Glibenclamide or NPPB reduced the DCEBIO-stimulated I_sc by >80% indicating the participation of CFTR in the DCEBIO-stimulated I_sc response. Clotrimazole reduced DCEBIO-stimulated I_sc by 67 ± 15%, suggesting the participation of the intermediate conductance Ca²⁺-activated K⁺ channel (IK_Ca) in the DCEBIO-activated I_sc response. In the presence of maximum forskolin (10 µM), the DCEBIO response was reduced and biphasic, reaching a peak response of the change in I_sc of 43 ± 5 µA/cm² and then falling to a steady-state response of 17 ± 10 µA/cm² compared with DCEBIO control tissues (61 ± 6 µA/cm²). The forskolin-stimulated I_sc in the presence of DCEBIO was reduced compared with forskolin control tissues. Similar results were observed with DCEBIO and 8-BrcAMP where adenylate cyclase was bypassed. H89, a PKA inhibitor, reduced the DCEBIO-activated I_sc, providing evidence that DCEBIO increased Cl^– secretion via a cAMP/PKA-dependent manner. These data suggest that DCEBIO stimulates Cl^– secretion of the mouse jejunum and that DCEBIO targets components of the Cl^– secretory mechanism. 1-ethyl-2-benzimidazolinone; forskolin; glibenclamide; clotrimazole; H89     

Interactions of NH4+ and L-glutamate with NO3- transport processes of non-mycorrhizal Fagus sylvatica roots

The processes of NO₃^– uptake and transport and the effectsof NH₄⁺ or L-glutamate on these processes were investigatedwith excised non-mycorrhizal beech (Fagus sylvatica L.) roots.NO₃^– net uptake followed uniphasic Michaelis-Menten kineticsin a concentration range of 10µM to 1 mM with an apparentK_m of 9.2 µM and a V_max of 366 nmol g^–1 FW h^–1.NH₄⁺, when present in excess to NO₃^–, or 10 mM L-glutamateinhibited the net uptake of NO₃^– Apparently, part of NO₃^–taken up was loaded into the xylem. Relative xylem loading ofNO₃^– ranged from 3.21.6 to 6.45.1% of NO₃^– netuptake. It was not affected by treatment with NH₄⁺ or L-glutamate.¹⁶N/¹³N double labelling experiments showed that NO₃^–efflux from roots increased with increasing influx of NO₃^–and, therefore, declined if influx was reduced by NH₄⁺ or L-glutamateexposure. From these results it is concluded that NO₃^–net uptake by non-mycorrhizal beech roots is reduced by NH₄⁺or L-glutamate at the level of influx and not at the level ofefflux. Key words: Nitrate transport, net uptake, influx, efflux, ammonium, Fagus, Fagaceae     

A Comparison between the Uptake of Potassium by Plants from Solutions of Constant Potassium Concentration and during Depletion

A comparison was made between two methods of measuring the relationshipbetween the external [K⁺] and the flux of K⁺ into whole plantsof Lolium perenne and Raphanus sativus. The values of flux obtainedfrom solutions of 1.2 µM K⁺ held constant around the rootswere three and six times greater for Lolium and Raphanus respectivelythan the values obtained at the same concentration in a depletionexperiment in which the solutions, initially 100 µM K⁺,were depleted to below 1.2 µM K⁺ by plant uptake. In thedepletion experiment with Lolium, the flux was higher into plantsgrown at low [K⁺] than into plants grown at 100 µM eventhough [K⁺] within the plant was about the same for all groupsof plants. It is suggested that Lolium grown at low [K⁺] hasan efficient mechanism for K⁺ uptake which continues to operatefor some time after the plants have been transferred to a higherconcentration. With both species, K_m was 15–20 µMin the depletion experiment and below 1 µM when concentrationswere held constant.     

Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart

Isolated rat heart perfused with 1.5-7.5µM NO solutions or bradykinin, which activates endothelial NOsynthase, showed a dose-dependent decrease in myocardial O₂uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 µM NO, n = 18,P < 0.05) and to 1.2 ± 0.1 µM O₂ · min¹ · gtissue¹ (10 µM bradykinin, n = 10,P < 0.05). Perfused NO concentrations correlated with aninduced release of hydrogen peroxide (H₂O₂) inthe effluent (r = 0.99, P < 0.01). NO markedlydecreased the O₂ uptake of isolated rat heart mitochondria(50% inhibition at 0.4 µM NO, r = 0.99,P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b andcytochrome c, which accounts for the effects in O₂uptake and H₂O₂ release. Most NO was bound tomyoglobin; this fact is consistent with NO steady-state concentrationsof 0.1-0.3 µM, which affect mitochondria. In the intact heart,finely adjusted NO concentrations regulate mitochondrial O₂uptake and superoxide anion production (reflected byH₂O₂), which in turn contributes to thephysiological clearance of NO through peroxynitrite formation.

     

SH oxidation coordinates subunits of rat brain ryanodine receptor channels activated by calcium and ATP

We have reported that ryanodine receptor (RyR) channels display three different responses to cytoplasmic free Ca²⁺ concentration ([Ca²⁺]) depending on their redox state (Marengo JJ, Hidalgo C, and Bull R. Biophys J 74: 1263–1277, 1998), with low, moderate, and high maximal fractional open times (P_o). Activation by ATP of single RyR channels from rat brain cortex was tested in planar lipid bilayers with 10 or 0.1 µM cytoplasmic [Ca²⁺]. At 10 µM [Ca²⁺], low-P_o channels presented lower apparent affinity to activation by ATP [[ATP] for half-maximal activation (K_aATP) = 422 µM] than moderate-P_o channels (K_aATP = 82 µM). Oxidation of low-P_o channels with thimerosal or 2,2'-dithiodipyridine (DTDP) gave rise to moderate-P_o channels and decreased K_aATP from 422 to 82 µM. At 0.1 µM cytoplasmic [Ca²⁺], ATP induced an almost negligible activation of low-P_o channels. After oxidation to high-P_o behavior, activation by ATP was markedly increased. Noise analysis of single-channel fluctuations of low-P_o channels at 10 µM [Ca²⁺] plus ATP revealed the presence of subconductance states, suggesting a conduction mechanism that involves four independent subchannels. On oxidation the subchannels opened and closed in a concerted mode. subconductance states; calcium ion release channels; calcium ion regulation; thimerosal; 2,2'-dithiodipyridine     

Effect of Sodium on Phosphate Uptake in Unicellular and Filamentous Cyanobacteria

The effect of Na⁺ on phosphate uptake was studied in four strainsof cyanobacteria: Synechococcus PCC 7942, Gloeothece PCC 6501,Phormidium sp. and Chlorogloeopsis PCC 6912. Phosphate uptakewas stimulated by Na⁺ in all cases. Li⁺ and K⁺ acted as partialanalogues for Na⁺. Half-saturation [K_1/2(Na⁺)] of phosphateuptake was reached with Na⁺ concentrations ranging from 317µM in Chlorogloeopsis to 659 µM in Phormidium. Theconcentration of phosphate required to reach half-saturationof phosphate uptake [K_1/2(P_i)]was not changed by the presenceof Na⁺. (Received April 11, 1994; Accepted July 5, 1994)     

Partial agonists and antagonists reveal a second permeability state of human lymphocyte P2Z/P2X7 channel

Extracellular ATP is known to trigger apoptosis of thymocytesand lymphocytes through a P2Z receptor at which ATP is a partial agonist, giving only 70% of the maximum response of3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (BzATP), a full agonist. This cytolytic receptor and its associated ion channel areCa²⁺ (andBa²⁺) selective but also passmolecules up to the size of ethidium cation (314 Da).RT-PCR showed identity between lymphocyte P2Z and thehP2X₇ gene recently cloned fromhuman monocytes. When human leukemic B lymphocytes were incubated withATP and¹³³Ba²⁺,an immediate influx of isotope occurred. It was augmented by 45% whenATP was added 10 min before isotope. Time-resolved flow cytometry wasused to examine kinetics of ethidium uptake in cells incubated withBzATP or the partial agonists ATP, 2-methylthioadenosine 5'-triphosphate, or adenosine5'-O-(3-thiotriphosphate).Maximally effective concentrations of BzATP (50 µM) induced immediateuptake of ethidium at a rate linear with time. In contrast, a delay was observed (30 s) before ethidium uptake commenced after addition ofmaximally effective ATP concentrations (500 µM) at 37°C, and thedelay was longer at 24°C. ATP addition 2-10 min beforeethidium abolished the delay. The delay was longer with other partialagonists and inversely related to maximal flux produced by agonist. Adelay was also observed for submaximal BzATP concentrations (10-20µM). P2Z/P2X₇ inhibitors, KN-62and5-(N,N-hexamethylene)-amiloride, reduced the rate of agonist-induced ethidium uptake and lengthened thedelay. The results support a model in which agonists forP2Z/P2X₇ receptor mediate animmediate channel opening allowing passage of small inorganic cations,followed by a slow further permeability increase allowing passage oflarger permeant cations like ethidium. The rate of the second stepdepends on time and temperature and the efficacy and concentration ofagonist and is slowed by antagonists, suggesting it depends on thefraction of P2Z/P2X₇ channels held in the initial openstate.

     

Characterization of vectorial chloride transport pathways in the human pancreatic duct adenocarcinoma cell line HPAF

Pancreatic duct cells express a Ca²⁺-activated Cl^- conductance (CaCC), upregulation of which may be beneficial to patients with cystic fibrosis. Here, we report that HPAF, a human pancreatic ductal adenocarcinoma cell line that expresses CaCC, develops into a high-resistance, anion-secreting epithelium. Mucosal ATP (50 µM) caused a fourfold increase in short-circuit current (I_sc), a hyperpolarization of transepithelial potential difference (from -4.9 ± 0.73 to -8.5 ± 0.84 mV), and a fall in resistance to less than one-half of resting values. The effects of ATP were inhibited by mucosal niflumic acid (100 µM), implicating an apical CaCC in the response. RT-PCR indicated expression of hClC-2, hClC-3, and hClC-5, but surprisingly not hCLCA-1 or hCLCA-2. K⁺ channel activity was necessary to maintain the ATP-stimulated I_sc. Using a pharmacological approach, we found evidence for two types of K⁺ channels in the mucosal and serosal membranes of HPAF cells, one activated by chlorzoxazone (500 µM) and sensitive to clotrimazole (30 µM), as well as one blocked by clofilium (100 µM) but not chromanol 293B (5 µM). RT-PCR indicated expression of the Ca²⁺-activated K⁺ channel KCNN4, as well as the acid-sensitive, four transmembrane domain, two pore K⁺ channel, KCNK5 (hTASK-2). Western blot analysis verified the expression of CLC channels, as well as KCNK5. We conclude that HPAF will be a useful model system for studying channels pertinent to anion secretion in human pancreatic duct cells. Ussing chamber; short-circuit current; RT-PCR; immunoblot     

Vasopressin and PGE(2) regulate activity of apical 70 pS K(+) channel in thick ascending limb of rat kidney

Vasopressin and prostaglandinE₂ (PGE₂) are involved in regulating NaClreabsorption in the thick ascending limb (TAL) of the rat kidney. Inthe present study, we used the patch-clamp technique to study theeffects of vasopressin and PGE₂ on the apical 70 pSK⁺ channel in the rat TAL. Addition of vasopressinincreased the channel activity, defined asNP_o, from 1.11 to 1.52 (200 pM) and 1.80 (500 pM),respectively. The effect of vasopressin can be mimicked by eitherforskolin (1-5 µM) or 8-bromo-cAMP/dibutyryl-cAMP (8-Br-cAMP/DBcAMP) (200-500 µM). Moreover, the effects of cAMP and vasopressin were not additive and application of 10 µM H-89 abolished the effect of vasopressin. This suggests that the effect ofvasopressin is mediated by a cAMP-dependent pathway. Applying 10 nMPGE₂ alone had no significant effect on the channelactivity. However, PGE₂ (10 nM) abolished thestimulatory effect of vasopressin. The PGE₂-inducedinhibition of the vasopressin effect was the result of decreasing cAMPproduction because addition of 200 µM 8-Br-cAMP/DBcAMPreversed the PGE₂-induced inhibition. In addition toantagonizing the vasopressin effect, high concentrations of PGE₂ reduced channel activity in the absence of vasopressinby 33% (500 nM) and 51% (1 µM), respectively. The inhibitory effect of high concentrations of PGE₂ was not the result ofdecreasing cAMP production because adding the membrane-permeant cAMPanalog failed to restore the channel activity. In contrast, inhibiting protein kinase C (PKC) with calphostin C (100 nM) abolished the effectof 1 µM PGE₂. We conclude that PGE₂ inhibitsapical K⁺ channels by two mechanisms: 1) lowconcentrations of PGE₂ attenuate the vasopressin-inducedstimulation mainly by reducing cAMP generation, and 2) highconcentrations of PGE₂ inhibit the channel activity by aPKC-dependent pathway.

     

Modulation of mitochondrial Ca2+ by nitric oxide in cultured bovine vascular endothelial cells

In the present study, we used laser scanning confocal microscopy in combination with fluorescent indicator dyes to investigate the effects of nitric oxide (NO) produced endogenously by stimulation of the mitochondria-specific NO synthase (mtNOS) or applied exogenously through a NO donor, on mitochondrial Ca²⁺ uptake, membrane potential, and gating of mitochondrial permeability transition pore (PTP) in permeabilized cultured calf pulmonary artery endothelial (CPAE) cells. Higher concentrations (100–500 µM) of the NO donor spermine NONOate (Sper/NO) significantly reduced mitochondrial Ca²⁺ uptake and Ca²⁺ extrusion rates, whereas low concentrations of Sper/NO (<100 µM) had no effect on mitochondrial Ca²⁺ levels ([Ca²⁺]_mt). Stimulation of mitochondrial NO production by incubating cells with 1 mM L-arginine also decreased mitochondrial Ca²⁺ uptake, whereas inhibition of mtNOS with 10 µM L-N⁵-(1-iminoethyl)ornithine resulted in a significant increase of [Ca²⁺]_mt. Sper/NO application caused a dose-dependent sustained mitochondrial depolarization as revealed with the voltage-sensitive dye tetramethylrhodamine ethyl ester (TMRE). Blocking mtNOS hyperpolarized basal mitochondrial membrane potential and partially prevented Ca²⁺-induced decrease in TMRE fluorescence. Higher concentrations of Sper/NO (100–500 µM) induced PTP opening, whereas lower concentrations (<100 µM) had no effect. The data demonstrate that in calf pulmonary artery endothelial cells, stimulation of mitochondrial Ca²⁺ uptake can activate NO production in mitochondria that in turn can modulate mitochondrial Ca²⁺ uptake and efflux, demonstrating a negative feedback regulation. This mechanism may be particularly important to protect against mitochondrial Ca²⁺ overload under pathological conditions where cellular [NO] can reach very high levels. nitric oxide synthase; permeability transition pore; endothelium     

UPTAKE OF ACETATE BY NEOTRICULA APERTA (GASTROPODA: POMATIOPSIDAE), THE SNAIL HOST OF SCHISTOSOMA MEKONGI IN THE LOWER MEKONG BASIN

All three races of Neotricula aperta, an epilithic, schistosometransmitting, snail of the Mekong and Mul rivers of NortheastThailand and southern Laos, were found to take up acetate froma dilute solution. After 48 h incubation the mean specific netuptake rates (µmol^–1 g^–1 h^–1), from750 µM acetate, were: 1.86, -race; 1.39, ß-raceand 3.25, y-race. Over 48 h the snails were able to achievereductions in the ambient acetate concentration of up to 60%.Incubations under bacteriostatis suggested that bacteria arenot involved in the uptake of acetate by N. aperta. The uptakecharacteristics conform to the Michaelis-Menten model. The transportconstants, J_max (µmol^–1 g^–1 h^–1) andK_t (µM) were 1.10 and 102 respectively (-race). Racialdifferences in uptake characteristics are discussed in relationto micro-habitat differences. HPLC indicated concentrations of acetate in y-N. aperta microhabitatsof around 325 µM. This suggests a pool size sufficientto satisfy only 6% of the snail's basal metabolic rate (BMR).Levels within the epilithic aufwuchs, however, are probablyhigh enough to provide for more than 50% of the BMR. The possible role of acetate in the energy metabolism of N.aperta is discussed. Short-chain carboxylic acids (such as acetate),arising from the decomposition of the aufwuchs, could representsources of fermentable organics that may be taken up by N. apertasnails and used to supplement their nutrition during times offood shortage. However, further investigations involving ¹⁴C-labellingtechniques are required. The findings of this investigationhave implications for the chemical ecology and life-cycle ofN. aperta. (Received 16 June 1994; accepted 28 July 1994)     

Effect of Plant Growth Regulators on Nitrate Utilization by Roots of Nitrogen-Depleted Dwarf Bean

We examined the effect of pretreatments (18 h at 5 µmoldm^–3) with abscisic acid, the ethylene-releasing substance‘Ethephon’, gibberellic acid, indoleacetic acid,kinetin and zeatin on nitrate uptake and in vivo nitrate reductaseactivity (NRA) in roots of nitrogen-depleted Phaseolus vulgarisL. Nitrate uptake showed an apparent induction pattern witha steady state after about 6 h, in all treatments. The nitrateuptake rate after 6 h was unaffected or at most 30% lower aftertreatments with the plant growth regulators. Gibberellic acid, kinetin and zeatin induced substantial NRAin roots in the absence of nitrate, whereas Ethephon enhancedNRA only during nitrate nutrition. Kinetin-induced NRA (Ki-NRA)was maximal after a pretreatment at 1 µmol dm^–3,and showed a lag phase of 6–8 h. Ki-NRA was additive tonitrate-induced NRA (NO^–₃-NRA) for at least 24 h, independentof the induction sequence. After full induction, Ki-NRA approximated20% of NO-₃-NRA. Abscisic acid counteracted the developmentof Ki-NRA, but not of NO^–₃-NRA. Cycloheximide and tungstatewere equally effective to suppress the development of nitratereductase activity after supply of kinetin or NO^–₃. Our data are consistent with the operation of two independentenzyme fractions (Ki-NRA and NO^–₃-NRA) with apparentlyidentical properties but with separate control mechanisms. Theabsence of major effects of plant growth regulators on the time-courseand rate of nitrate uptake suggests that exogenous regulators,and possibly endogenous phytohormones are of minor importancefor initial nitrate uptake. The differential effect of someregulators on nitrate uptake and root NRA furthermore indicatesthat the processes of uptake and reduction of NO^–₃ arenot obligatory or exclusively coupled to each other.     

Sodium-Stimulation of Phosphate Uptake in the Cyanobacterium Anabaena PCC 7119

Anabaena PCC 7119 showed higher rates of phosphate uptake whencells were under P-starvation. Phosphate uptake was energy-dependentas indicated the decrease observed when assays were performedin the dark or in the presence of inhibitors of photosyntheticelectron transport, energy transfer and adenosine triphosphataseactivity. Phosphate uptake was stimulated by Na⁺ both in P-sufficientcells and P-starved cells. Li⁺ and K⁺ acted as partial analoguesfor Na⁺. The Na⁺-stimulation of phosphate uptake followed Michaelis-Mentenkinetics, half-saturation (K) of phosphate uptake was reachedwith a Na⁺ concentration of 212 µM. The absence of Na⁺reduced the rates of phosphate uptake at all phosphate concentrationsassayed (1–20 µM). The maximum uptake rates (V_max)decreased from 658 nmol P (mg dry wt)^-1 h^-1 in the presenceof Na⁺ to 149 nmol P (mg dry wt)^-1 h^-1 in the absence of Na⁺.The absence of Na⁺ did not change significantly the concentrationof phosphate required to reach half-saturation (K) (3.01 µMin the presence of Na⁺ vs 3.21 µM in the absence of Na⁺).In the presence of Na⁺ the rate of phosphate uptake was affectedby the pH; optimal rates were observed at pH 8. In the absenceof Na⁺ phosphate uptake was not affected by the pH; low rateswere observed in all cases. Monensin, an ionophore which collapsesNa⁺-gradients, reduced the rate of phosphate uptake in Na⁺-supplementedcells. These results indicated the existence of a Na⁺-dependentphosphate uptake in Anabaena PCC 7119. (Received September 8, 1992; Accepted November 17, 1992)     

Intermediate-conductance Ca2+-activated K+ channel is expressed in C2C12 myoblasts and is downregulated during myogenesis

We report here the expression in C₂C₁₂ myoblasts of the intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channel. The IK_Ca current, recorded under perforated-patch configuration, had a transient time course when activated by ionomycin (0.5 µM; peak current density 26.2 ± 3.7 pA/pF; n = 10), but ionomycin (0.5 µM) + 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (100 µM) evoked a stable outward current (28.4 ± 8.2 pA/pF; n = 11). The current was fully inhibited by charybdotoxin (200 nM), clotrimazole (2 µM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (300 µM), but not by tetraethylammonium (1 mM) or D-tubocurarine (300 µM). Congruent with the IK_Ca channel, elevation of intracellular Ca²⁺ in inside-out patches resulted in the activation of a voltage-insensitive K⁺ channel with weak inward rectification, a unitary conductance of 38 ± 6 pS (at negative voltages), and an IC₅₀ for Ca²⁺ of 530 nM. The IK_Ca channel was activated metabotropically by external application of ATP (100 µM), an intracellular Ca²⁺ mobilizer. Under current-clamp conditions, ATP application resulted in a membrane hyperpolarization of 35 mV. The IK_Ca current downregulated during myogenesis, ceasing to be detectable 4 days after the myoblasts were placed in differentiating medium. Downregulation was prevented by the myogenic suppressor agent basic FGF (bFGF). We also found that block of the IK_Ca channel by charybdotoxin did not inhibit bFGF-sustained myoblast proliferation. These observations show that in C₂C₁₂ myoblasts the IK_Ca channel expression correlates inversely with differentiation, yet it does not appear to have a role in myoblast proliferation. ATP; cell proliferation     

Beta-adrenergic stimulation does not activate Na+/Ca2+ exchange current in guinea pig, mouse, and rat ventricular myocytes

The effect of -adrenergic stimulation on cardiac Na⁺/Ca²⁺ exchange has been controversial. To clarify the effect, we measured Na⁺/Ca²⁺ exchange current (I_NCX) in voltage-clamped guinea pig, mouse, and rat ventricular cells. When I_NCX was defined as a 5 mM Ni²⁺-sensitive current in guinea pig ventricular myocytes, 1 µM isoproterenol apparently augmented I_NCX by 32%. However, this increase was probably due to contamination of the cAMP-dependent Cl^– current (CFTR-Cl^– current, I_CFTR-Cl), because Ni²⁺ inhibited the activation of I_CFTR-Cl by 1 µM isoproterenol with a half-maximum concentration of 0.5 mM under conditions where I_NCX was suppressed. Five or ten millimolar Ni²⁺ did not inhibit I_CFTR-Cl activated by 10 µM forskolin, an activator of adenylate cyclase, suggesting that Ni²⁺ acted upstream of adenylate cyclase in the -adrenergic signaling pathway. Furthermore, in a low-extracellular Cl^– bath solution, 1 µM isoproterenol did not significantly alter the amplitude of Ni²⁺-sensitive I_NCX at +50 mV, which is close to the reversal potential of I_CFTR-Cl. No change in I_NCX amplitude was induced by 10 µM forskolin. When I_NCX was activated by extracellular Ca²⁺, it was not significantly affected by 1 µM isoproterenol in guinea pig, mouse, or rat ventricular cells. We concluded that -adrenergic stimulation does not have significant effects on I_NCX in guinea pig, mouse, or rat ventricular myocytes. cystic fibrosis transmembrane conductance regulator; nickel ion     

Characterization of regulatory mechanisms and states of human organic cation transporter 2

Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37°C. After subtraction of unspecific uptake determined in WT at 37°C or in hOCT2 at 8°C saturable specific uptake of both substrates was measured. K_m values of hOCT2-mediated uptake of 95 µM amiloride and 24 µM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC₅₀ (in µM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca²⁺/CaM complex (–55 ± 5%, n = 10 and –63 ± 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (–54 ± 5%, n = 14, and –31 ± 6%, n = 26) and PKA (–16 ± 5%, n = 16, and –18 ± 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 ± 6%, n = 8, and +55 ± 17%, n = 16). Inhibition of Ca²⁺/CaM complex resulted in a significant decrease of V_max (160–99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (–22 ± 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca²⁺/CaM complex causes changes in transport capacity via hOCT2 trafficking. organic cation transport; fluorescence measurement; 4-[4-(dimethylamino)-styryl]-n-methylpyridinium; amiloride     

Relation between Ultra-Violet Spectral Change and Nucleotide Binding of the Chloroplast Coupling Factor 1

Properties of the nucleotide binding sites on chloroplast couplingfactor 1 (CF₁) were studied by equilibrium dialysis and UV spectroscopy.From our direct binding studies, we identified at least fourkinds of ADP binding sites on CF₁; a barely dissociable ADPbinding site (site A), a slowly exchangeable high affinity sitewith dissociation constant (Kd) 0.021 µM (site B), anotherslowly exchangeable high affinity site with Kd 1.6 µM(site C) and several low affinity (Kd {small tilde}30 µM)sites. The Kd values for sites B and C of the other nucleotidestested were 0.5 µM and 16 µM (GDP), 8 µM and34 µM (CDP), 17 µM and 20 µM (UDP) and 1.4µM and 1.4 µM (PP₁). From a comparison of the observed UV spectral change and theamount of nucleotide bound to these sites, as calculated fromthe above Kd values, we concluded that the nucleotide bindingto site B or G induces UV spectral changes that are almost thesame in shape and magnitude. The estimated difference molarabsorption coefficient () was 3.4?10³M^–1_ADP cm^–1for ADP at 278 nm. Our conclusions were strengthened by thegood agreement between the observed spectra and the calculatedspectra (derived from the Kd and values of ADP and GDP) whenADP and GDP were added together to CF₁. The cause of the unusual behavior of GDP in the UV differencespectrum which was unexplained in our previous report was shownto be competition between the GDP added and previously boundADP at sites B and C; this distorted the real spectrum inducedby GDP. (Received October 3, 1983; Accepted February 13, 1984)     

p38 mitogen-activated protein kinase inhibits calcium-dependent chloride secretion in T84 colonic epithelial cells

We havepreviously shown that Ca²⁺-dependent Clsecretion across intestinal epithelial cells is limited by a signalingpathway involving transactivation of the epidermal growth factorreceptor (EGFR) and activation of ERK mitogen-activated protein kinase (MAPK). Here, we have investigated a possible role for p38 MAPK inregulation of Ca²⁺-dependent Cl secretion.Western blot analysis of T₈₄ colonic epithelial cells revealed that the muscarinic agonist carbachol (CCh; 100 µM)stimulated phosphorylation and activation of p38 MAPK. The p38inhibitor SB-203580 (10 µM) potentiated and prolonged short-circuitcurrent (I_sc) responses to CCh acrossvoltage-clamped T₈₄ cells to 157.4 ± 6.9% of thosein control cells (n = 21; P < 0.001).CCh-induced p38 phosphorylation was attenuated by the EGFR inhibitortyrphostin AG-1478 (0.1 nM-10 µM) and by the Src family kinaseinhibitor PP2 (20 nM-2 µM). The effects of CCh on p38phosphorylation were mimicked by thapsigargin (TG; 2 µM), whichspecifically elevates intracellular Ca²⁺, and wereabolished by the Ca²⁺ chelator BAPTA-AM (20 µM), implyinga role for intracellular Ca²⁺ in mediating p38 activation.SB-203580 (10 µM) potentiated I_sc responses toTG to 172.4 ± 18.1% of those in control cells (n = 18; P < 0.001). When cells were pretreated withSB-203580 and PD-98059 to simultaneously inhibit p38 and ERK MAPKs,respectively, I_sc responses to TG and CCh weresignificantly greater than those observed with either inhibitor alone.We conclude that Ca²⁺-dependent agonists stimulate p38 MAPKin T₈₄ cells by a mechanism involving intracellularCa²⁺, Src family kinases, and the EGFR. CCh-stimulated p38activation constitutes a similar, but distinct and complementary,antisecretory signaling pathway to that of ERK MAPK.