[Impact of empagliflozin on peak oxygen uptake in HFmrEF patients: a randomized controlled trial].

Objective: To evaluate the impact of empagliflozin on peak oxygen uptake (VO2peak) in patients with heart failure with mildly reduced ejection fraction (HFmrEF). Methods: In this randomized controlled trial, consecutive HFmrEF patients admitted to the Department of Cardiology of China-Japan Friendship Hospital from September 2019 to October 2020 were screened, and randomly assigned to empagliflozin group (EG) or conventional group (CG) using a random number table. The enrolled patients were treated according to the guidelines, and patients in the empagliflozin group received additional empagliflozin (10 mg, once a day, orally) on top of the conventional treatment. The primary end points were VO2peak at 6 months after treatment, and the secondary end points included other parameters of cardiopulmonary exercise test (CPET), 6-minute walking distance, N-terminal B-type pro-natriuretic peptide (NT-proBNP) level, and Kansas City Cardiomyopathy Questionnaire (KCCQ) score. Results: A total of 112 patients were included (mean age 69 (57, 78) years, 84 male (75.0%)). There were 55 cases in CG group and 57 cases in EG group. There were no significant differences in baseline data including age, sex, body mass index, left ventricular ejection fraction, systolic blood pressure, heart rate, estimated glomerular filtration rate, glycosylated hemoglobin, hemoglobin, NT-proBNP, daily dose of tolasemi, combined medication, CPET parameters, the proportion of New York Heart Association heart function Ⅲ/Ⅳ, history of coronary heart disease, history of hypertension, history of diabetes (all P>0.05). At 6 months after treatment, VO2peak was significantly higher in EG group than in CG group(P=0.023). VE/VCO2 slope was significantly lower in EG group than in CG group(P=0.034). Oxygen uptake efficiency slope was significantly higher in EG group than in CG group(P=0.038). The level of NT-proBNP was significantly lower in EG group than in CG group(P=0.020). Six-minute walking distance was significantly higher in EG group than in CG group(P=0.037). KCCQ score was significantly higher in EG group than in CG group(P=0.048). Exercise oscillatory ventilation decreased in both groups (1 case in each group, P>0.05). Conclusion: Empagliflozin can significantly improve VO2peak in patients with HFmrEF.

Nitric oxide is involved in phosphorus deficiency-induced cluster-root development and citrate exudation in white lupin.

*White lupin (Lupinus albus) forms specialized cluster roots characterized by exudation of organic anions under phosphorus (P) deficiency. Here, the role of nitric oxide (NO) in P deficiency-induced cluster-root formation and citrate exudation was evaluated. *White lupin plants were treated with the NO donor sodium nitroprusside (SNP) and scavenger or inhibitor of NO synthase under conditions of P deficiency (0 muM) or P sufficiency (50 muM). *Phosphorus deficiency enhanced NO production in primary and lateral root tips, with a greater increase in cluster roots than in noncluster roots. NO concentrations decreased with cluster root development from the pre-emergent stage, through the juvenile stage, to the mature stage. The P deficiency-induced increase in NO production was inhibited by antagonists of NO synthase and xanthine oxidoreductase, suggesting the involvement of these enzymes in NO production. SNP markedly increased the number of cluster roots. Citrate exudation from different root segments in P-deficient roots was positively correlated with endogenous root NO concentrations. *These findings demonstrate differential patterns of NO production in white lupin, depending on root zone, developmental stage and P nutritional status. NO appears to play a regulatory role in the formation of cluster roots and citrate exudation in white lupin under conditions of P deficiency.

Citrate exudation from white lupin induced by phosphorus deficiency differs from that induced by aluminum.

Both phosphorus (P) deficiency and aluminum (Al) toxicity induce root exudation of carboxylates, but the relationship between these two effects is not fully understood. Here, carboxylate exudation induced by Al in Lupinus albus (white lupin) was characterized and compared with that induced by P deficiency. Aluminum treatments were applied to whole root systems or selected root zones of plants with limited (1 microM) or sufficient (50 microM) P supply. Aluminum stimulated citrate efflux after 1-2 h; this response was not mimicked by a similar trivalent cation, La(3+). P deficiency triggered citrate release from mature cluster roots, whereas Al stimulated citrate exudation from the 5- to 10-mm subapical root zones of lateral roots and from mature and senescent cluster roots. Al-induced citrate exudation was inhibited by P limitation at the seedling stage, but was stimulated at later growth stages. Citrate exudation was sensitive to anion-channel blockers. Al treatments did not affect primary root elongation, but inhibited the elongation of lateral roots. The data demonstrate differential patterns of citrate exudation in L. albus, depending on root zone, developmental stage, P nutritional status and Al stress. These findings are discussed in terms of possible functions and underlying mechanisms.

Carbachol inhibits the L-type Ca2+ current augmented by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in guinea pig ventricular myocytes: calcium-sensitivity hypothesis for muscarinic inhibition.

The L-type Ca2+ current [I(Ca(L))] increases with time after patch rupture in guinea pig ventricular myocytes dialyzed with pipette solutions containing > or =20 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid ([BAPTA]pip). I(Ca(L)) progressively increases because BAPTA chelates subsarcolemmal Ca2+ to disinhibit cardiac adenylyl cyclase (AC) activity. We studied inhibition by carbachol (CCh) of I(Ca(L)) (22-24 degrees C). At 40 mM [BAPTA]pip, 100 microM CCh reversibly suppressed I(Ca(L)) maximally by 42%; half-maximal inhibition (20%) required 1 microM. Atropine antagonized the CCh effect on BAPTA-stimulated I(Ca(L),) as did dialysis with 50 microM guanosine-5'-O-(3-thio)triphosphate. At 20, 30, and 40 mM [BAPTA]pip, I(Ca(L)) increased by 6.7 +/- 1.8, 10.1 +/- 1.4, and 11.3 +/- 1.2 pA/pF, respectively. Inhibition by 100 microM CCh averaged -1.8 +/- 0.6, -2.3 +/- 0.4, and -4.1 +/- 0.4 pA/pF at 20, 30, and 40 mM [BAPTA](pip), respectively. Dialysis of the AC inhibitor 2'-dAMP (100 microM) suppressed I(Ca(L)) run up in 40 mM BAPTA and its inhibition by CCh. Replacing 1.8 mM external Ca2+ with Ba2+, which lacks high-affinity regulatory sites on AC, suppressed CCh-induced inhibition. Neither I(Ca(L)) run up nor its inhibition by CCh occurred when 40 mM EGTA, a slower chelator, replaced BAPTA. Our results support the AC disinhibition hypothesis for BAPTA. We propose that CCh inhibits I(Ca(L)) in BAPTA by increasing either AC sensitivity to inhibition by ambient Ca2+ or the activity of the inhibitory guanine nucleotide binding protein.

Comparison of L-type calcium channel blockade by nifedipine and/or cadmium in guinea pig ventricular myocytes.

We tested the assumption that nifedipine blocks L-type calcium current [I(Ca(L))] at +10 mV and unmasks Na(+)/Ca(2+) exchange-triggered contractions in guinea pig isolated ventricular myocytes. Voltage-clamp pulses elicited I(Ca(L)) at +10 mV and evoked contractions in myocytes superfused with Tyrode's solution (35 degrees C). Nifedipine blocked I(Ca(L)) with an IC(50) of 0.3 microM; this decreased to 50 nM at a holding potential of -40 mV, indicating preferential block of inactivated L-type Ca(2+) channels. Use-independent block of I(Ca(L)) increased with concentration (10-100 microM) and application time when nifedipine was rapidly applied (t(1/2) = approximately 0.2 s) during rest intervals (5-30 s). The fraction of use-dependent block of I(Ca(L)) diminished with increasing drug concentration. Nifedipine also accelerated I(Ca(L)) inactivation on the first test pulse. The combination of 30 microM nifedipine/30 microM Cd(2+) (Nif 30/Cd 30) was as effective as 100 microM nifedipine to suppress I(Ca(L)) on the first test pulse at +10 mV. The incidence of complete block of contractions, as for complete block of I(Ca(L)), increased as a function of nifedipine concentration and application time. Neither nifedipine nor Nif 30/Cd 30 affected Na(+)/Ca(2+) exchange current at +10 to +100 mV. Contractions at +100 mV, although as large as those at +10 mV, were delayed in onset and resistant to nifedipine or Nif 30/Cd 30. We conclude that nifedipine-sensitive I(Ca(L)) triggers contractions at +10 mV, whereas nifedipine-resistant Na(+)/Ca(2+) exchange current initiates those at +100 mV.

Inositol-1,4,5-trisphosphate increases contractions but not L-type calcium current in guinea pig ventricular myocytes.

OBJECTIVE: We studied the effects of intracellularly applied inositol-1,4,5-trisphosphate (InsP3) to test the hypothesis that InsP3 is a messenger for stimulation of L-type calcium current (ICa(L)) and contractions by muscarinic agonists. METHODS: Voltage clamp pulses elicited ICa(L) that evoked contractions recorded with an edge detector in single guinea pig ventricular myocytes superfused with Tyrode's solution (36 degrees C). InsP3 or cyclic AMP (cAMP) was dialyzed into the cell at selected times via the patch electrode. RESULTS: InsP3 (1-10 microM) transiently increased isotonic contractions when applied for 4-5 min; higher concentrations (50-300 microM) caused a sustained decrease in contractions. InsP3 had no effect on ICa(L) at any concentration tested. Caffeine (10 mM)-induced contractures were increased and decreased, respectively, at 3 and 100 microM InsP3. Pentosan polysulfate (50 micrograms/ml), an InsP3 receptor antagonist, opposed the increased contractions by InsP3. Intrapipette cyclic AMP (10-300 microM) caused sustained increases of ICa(L) and contractions. Cyclic AMP, but not InsP3, also increased ICa(L) when intrapipette Cs+ suppressed K+ currents. CONCLUSIONS: Increased myocyte shortening at low InsP3 concentrations accords with receptor-initiated sarcoplasmic reticulum Ca2+ release. The transient stimulation of contractions at low concentrations and the sustained reduction of contractions at high concentrations are not consistent with a role for InsP in the persistent increase of contractions by muscarinic agonist in ventricular muscle and myocytes. The failure of InsP3 to change ICa(L) when contractions were increased or decreased militates against the L-type calcium channel being an effector of InsP3.

Elevated cAMP suppresses muscarinic inhibition of L-type calcium current in guinea pig ventricular myocytes.

We investigated the effect of carbachol (CCh) on L-type Ca2+ current (ICa(L)) enhanced by dialyzed adenosine 3',5'-cyclic monophosphate (cAMP) and/or bath-applied 3-isobutyl-1-methylxanthine (IBMX) in guinea pig isolated ventricular myocytes. At pipette concentrations ([cAMP]pip) from 30 microM to 1 mM, cAMP increased ICa(L) to 25.8 +/- 0.9 microA/cm2 (682 +/- 24.8% increase above control). CCh (100 microM) did not inhibit ICa(L) at any [cAMP]pip. IBMX, a nonselective phosphodiesterase (PDE) inhibitor, increased ICa(L) maximally at 300 microM IBMX (17.9 +/- 0.7 microA/cm2; 449 +/- 20% increase). CCh (100 microM) inhibited ICa(L) by 92 +/- 9.5% at 30 microM IBMX and 78 +/- 4.6% at 100 microM IBMX; this effect was reduced or absent at higher IBMX concentrations (300 and 1,000 microM). Coadministration of cAMP and IBMX also progressively suppressed inhibition by CCh. CCh had a negligible effect on ICa(L) at 750 microM IBMX in the absence of pipette cAMP and at 50 microM IBMX in the presence of 100 microM [cAMP]pip. ACh-activated K+ current (IK(ACh)) was unchanged in atrial myocytes dialyzed with 100 microM cAMP; this excludes a phosphorylation-dependent desensitization of the muscarinic receptor (mAChR) or Gi by cAMP. LY83583 (100 microM), an inhibitor of cyclic guanosine monophosphate (cGMP) production, attenuated inhibition of ICa(L) by CCh in the presence of IBMX. 8-Bromo-cGMP (8-Br-cGMP), an activator of cGMP-dependent protein kinase (PKG), mimicked CCh in its actions on ICa(L) raised by both cAMP (no significant change) and IBMX (49 +/- 5.1% inhibition). Okadaic acid, an inhibitor of type 1 and 2A phosphatases, blocked inhibition of IBMX-stimulated ICa(L) by either CCh or 8-Br-cGMP. Thus the ability of CCh to inhibit ICa(L) appears caused by cGMP/PKG activation of an okadaic acid-sensitive protein phosphatase, and elevated levels of cAMP protect against this action.

Lack of effect of McN-A-343 on membrane current and contraction in guinea pig ventricular myocytes.

We asked whether agonist occupancy of M(1) muscarinic receptor (mAChR) causes increased L-type Ca(2+) [I(Ca(L))] and contractions in ventricular myocytes. Voltage-clamp pulses evoked I(Ca(L)) in guinea pig ventricular myocytes superfused with Tyrode's solution (22-24 degrees C). The mAChR agonists carbachol (Cch, nonselective), McN-A-343 (McN, M(1)-selective), and oxotremorine (Oxo, M(2)-selective) were tested at 0.1 mM. None of these agonists affected basal I(Ca(L)). McN did not change isoproterenol-stimulated I(Ca(L)) in 13 of 15 cells. The slight decrease in two cells was not muscarinic because atropine (1 microM) did not antagonize it. Carbachol or Oxo decreased isoproterenol-stimulated I(Ca(L)) by 87 +/- 6.7 (n = 8 cells) and 49 +/- 9.0% (n = 4 cells), respectively. Atropine blocked inhibition by Cch or Oxo. External stimulation evoked contractions of single myocytes (35 degrees C). McN increased contraction in 1 of 22 cells stimulated at 0.2 Hz and in 0 of 16 cells stimulated at 1.0 Hz. Carbachol significantly increased contraction in 10 of 15 cells at 0.2 Hz and in 8 of 10 cells at 1.0 Hz stimulus frequency. Summarily, the M(1)-selective agonist McN had a negligible role to regulate I(Ca(L)). The antiadrenergic effect of mAChR agonists is attributable to M(2) receptor occupancy. That Cch, but not McN, increased cell shortening excludes participation of M(1) mAChR in the stimulant effect of Cch on guinea pig ventricular myocyte contractions.

On the mechanism of cesium-induced voltage and current tails in single ventricular myocytes.

The mechanisms by which different concentrations of cesium modify membrane potentials and currents were investigated in guinea pig single ventricular myocytes. In a dose-dependent manner, cesium reversibly decreases the resting potential and action potential amplitude and duration, and induces a diastolic decaying voltage tail (Vex), which increases at more negative and reverses at less negative potentials. In voltage-clamped myocytes, Cs+ increases the holding current, increases the outward current at plateau levels while decreasing it at potentials closer to resting potential, induces an inward tail current (Iex) on return to resting potential and causes a negative shift of the threshold for the inward current. During depolarizing ramps, Cs+ decreases the outward current negative to inward rectification range, whereas it increases the current past that range. During repolarizing ramps, Cs+ shifts the threshold for removal of inward rectification negative slope to less negative values. Cs+-induced voltage and current tails are increased by repetitive activity, caffeine (5 mM) and high [Ca2+]O (8.1 mM), and are reduced by low Ca2+ (0.45 mM), Cd2+ (0.2 mM) and Ni2+ (2 mM). Ni2+ also abolishes the tail current that follows steps more positive than ECa. We conclude that Cs+ (1) decreases the resting potential by decreasing the outward current at more negative potentials, (2) shortens the action potential by increasing the outward current at potentials positive to the negative slope of inward rectification, and (3) induces diastolic tails through a Ca2+-dependent mechanism, which apparently is an enhanced electrogenic Na-Ca exchange.

Carbachol increases contractions and intracellular Ca++ transients in guinea pig ventricular myocytes.

We tested hypotheses concerning the muscarinic receptor subtype and the involvement of L-type Ca current (ICa) in the stimulation of contractions by carbachol (CCh) in single guinea pig ventricular myocytes. When superfused with Tyrode's solution (36 degrees C, 5.4 mM [Ca++]o) and stimulated at 0.2 Hz, CCh (EC50 approximately 18 microM) increased the early component of isotonic contractions by acting at muscarinic receptors indistinguishable from the M2 subtype because AF-DX 116 (M2-selective) was more potent than pirenzepine (M1-selective) as an antagonist of the CCh effect. Action potential duration decreased slightly and ICa was not increased when CCh increased contractions. Carbachol increased intracellular Ca++ transients and contractions reversibly, which indicated an effect via sarcoplasmic reticulum (SR) Ca stores. Ryanodine (1-10 microM) blocked the early contraction component increased by CCh, another indication that CCh action depends on SR Ca stores. We previously found that CCh increased a background Na+ current by occupancy of M2 receptors. We now report that the increased contractions by CCh can also originate at M2 receptors and that SR Ca stores are involved in the CCh effect. Because CCh did not significantly increase ICa, the initial increase of intracellular Na+ by CCh may eventually act through Na-Ca exchange to enhance excitation-contraction coupling.

Carbachol promotes Na+ entry and augments Na/Ca exchange current in guinea pig ventricular myocytes.

The effect of carbachol (CCh) on the Na/Ca exchange current (I(Na/Ca)) was studied in voltage-clamped ventricular myocytes isolated from guinea pig hearts and superfused with Tyrode solution at 35 degrees C. CCh (100 microM) increased outward current during depolarizations (10-200 ms) from -45 mV and tail current amplitude on repolarization; CCh had no effect on the L-type Ca2+ current. Amplitudes of the outward and tail currents declined with increasing duration of the depolarizing clamp pulse. Ouabain produced similar current changes that are suppressed by intrapipette ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and are characteristic of I(Na/Ca). Depolarization from -80 to -30 mV elicited the rapid Na+ current followed by a slowly decaying inward I(Na/Ca) (J. C. Gilbert, T. Shirayama, and A. J. Pappano. Circ. Res. 69: 1632-1639, 1991.) that was reversibly increased by CCh. Atropine (1-3 microM) prevented the CCh effect. All procedures that suppressed I(Na/Ca) also suppressed the CCh effect. Sarcoplasmic reticulum (SR) Ca2+ release participated in generating I(Na/Ca) because 10 mM caffeine or 1 microM ryanodine blocked I(Na/Ca) and the effect of CCh. Rapid superfusion of 10 mM caffeine induced inward I(Na/Ca) at -75 mV; a caffeine-induced charge transfer gives an SR Ca2+ content of 67 microM. CCh increased caffeine-induced current; SR Ca2+ content rose to 98 microM. CCh also augmented the amplitude of steady-state intracellular Ca2+ transients and contractions during a train of voltage-clamp pulses (-75 to 30 mV for 200 ms) at 1 Hz. CCh elevated intracellular Na+ (M. Korth and V. Kühlkamp. Pflügers Arch. 403: 266-272, 1985) by inducing a background Na+ current [K. Matsumoto and A. J. Pappano. J. Physiol. (Lond.) 415: 487-502, 1989]. Together with these data, the present results are consistent with the hypothesis that CCh, via muscarinic receptors, eventually promotes I(Na/Ca) at the sarcolemma through a mechanism that requires the SR and that this action accounts for the increased contractions.

Mechanisms for depolarization by l-palmitoylcarnitine in single guinea pig ventricular myocytes.

INTRODUCTION: The changes of the resting potential (RP) and of the current-voltage (I-V) relationship induced by l-palmitoylcarnitine (l-PC) in the presence of the IKI blocker, cesium, or in the presence of the INa/K blocker, ouabain, were tested in guinea pig ventricular myocytes to ascertain the relative contributions of IKI and INa/K suppression to the membrane depolarization caused by this amphiphile. METHODS AND RESULTS: Ramp voltages were applied to myocytes with the whole cell, patch clamp technique. l-PC (10 microM) produced additional membrane depolarization in the presence of either 10 mM Cs+ or 30 microM ouabain. In the presence of Cs+, l-PC, like 3 microM ouabain, shifted current inward at potentials negative to -20 mV as a result of INa/K blockade. In the presence of 30 microM ouabain, l-PC, like Cs+, shifted current inward at potentials between -27 and -88 mV and outward at potentials negative to -88 mV. This is attributed to IKI block because the current was inwardly rectifying, with a reversal potential near EK. When l-PC or ouabain inhibited INa/K, the presence of an Ni(2+)-sensitive component attributed to INa/Ca distorted the membrane I-V relationship, particularly in the presence of Cs+. The relative contributions of IKI and INa/K block by l-PC were voltage dependent. At the RP, l-PC produced a greater block of INa/K than of IKI. CONCLUSION: l-PC depolarizes the resting membrane by inhibiting both IKI and INa/K. It is concluded that suppression of INa/K by l-PC predominates over block of IKI to depolarize the membrane at the RP.

Barium-induced diastolic depolarization and controlling mechanisms in guinea pig ventricular muscle.

Barium-induced diastolic depolarization (Ba(2+)-DD) and its modulation were studied in guinea pig papillary muscle and single ventricular myocytes. In papillary muscles, Cs+ (4 mM) abolished the DD induced by Ba2+ (0.05-0.2 mM) by abolishing the undershoot at the end of the action potential (AP; consistent with a block of an outward current). Acetylcholine (ACh 1 microM) had little effect on Ba(2+)-DD, whereas norepinephrine (NE 1 microM) enhanced it by increasing the undershoot and by inducing an oscillatory potential. Low [Ca2+]o (0.54 mM) decreased the resting potential and increased Ba(2+)-DD amplitude. High [Ca2+]o (8.1 mM) had opposite effects. Cs+ also reduced Ba(2+)-DD in low [Ca2+]o. In isolated myocytes, Ba(2+)-DD and the pacemaker current induced by Ba2+ (Ba(2+)-IKdd) increased on depolarization and reversed on hyperpolarization. Although not significantly, high [Ca2+]o slightly decreased and low [Ca2+]o slightly increased Ba(2+)-IKdd. Cd2+ markedly reduced the slow inward current ICa and the AP duration (APD), but did not affect Ba(2+)-IKdd. We conclude that Ba(2+)-DD (a) is entirely due to a voltage- and time-dependent decrease in gK1, since it is abolished by Cs+ (no contribution of a nonblocked decaying IK) by eliminating the undershoot (no If), (b) is potentiated by NE through an increased undershoot and an oscillatory potential, (c) is modified by high and low [Ca2+]o mostly through changes in the resting potential, and (d) is not affected by the block of the slow channel by Cd2+.

Palmitoyl-L-carnitine acts like ouabain on voltage, current, and contraction in guinea pig ventricular cells.

We previously showed that palmitoyl-L-carnitine (L-PC) inhibits the Na/K pump current (INa/K). In the present report, we test the hypothesis that L-PC, like ouabain, should increase myocyte shortening. Membrane potentials or ionic currents were recorded simultaneously with cell shortening in single guinea pig ventricular myocytes at room temperature (22 degrees C). Like ouabain, L-PC (1 microM) reversibly depolarized the resting membrane, decreased action potential duration, and increased the amplitude of myocyte contractions. Neither L-PC nor ouabain had a significant effect on Ca current (ICa). When L-PC increased cell shortening during ramp voltage clamp, membrane current shifted inward at voltages negative to -20 mV and shifted outward at more positive voltages. Similar to toxic concentrations of ouabain, L-PC induced transient inward currents and aftercontractions. At concentrations that inhibit INa/K, L-PC acted like ouabain to produce characteristic effects on membrane potentials, currents, and cell contractions that were unrelated to significant changes in ICa. L-PC reduces surface negative charge of erythrocytes and myocytes (C. Gruver and A. J. Pappano, J. Mol. Cell. Cardiol. 25: 1275-1284, 1993), and we speculate that L-PC inhibits INa/K by this mechanism.

Cesium abolishes the barium-induced pacemaker potential and current in guinea pig ventricular myocytes.

INTRODUCTION: The ability of cesium to block barium-induced diastolic depolarization ("Ba-DD") and pacemaker current was tested in isolated ventricular myocytes. Because Ba-DD is due to decreasing K+ conductance and there is no I(f) at the resting potential, this approach permits verification of whether Cs+ is a specific blocker of i(f) or if it instead also blocks a K+ pacemaker current. METHODS AND RESULTS: Guinea pig isolated ventricular myocytes were studied by a discontinuous, single electrode, voltage clamp method. During hyperpolarizing voltage clamp steps from -80 up to -140 mV in Tyrode's solution, the inward current increased as a function of voltage but did not change as a function of time (no I(f) or K+ depletion). Cesium (4 mM) reduced the current size during the hyperpolarizing steps but did not induce or unmask time-dependent currents. Barium (0.05 to 0.1 mM) induced diastolic depolarization, and, in its presence, depolarizing voltage clamp steps were followed by an outward tail current that reversed at -92.0 +/- 1.3 mV. Outward tail currents were larger at -50 mV than at the resting potential, and inward tail currents decayed more rapidly and to a larger extent during larger hyperpolarizing steps. In the presence of Ba2+, Cs+ (4 mM) had little effect on the steady-state current but markedly reduced or abolished undershoot, Ba-DD, and time-dependent tail currents at potentials both positive and negative to the resting potential. Cs+ had a smaller effect on the steady-state current-voltage (I-V) relation in the presence than in the absence of Ba2+, as part of the IK1 channels were already blocked by Ba2+ and the time-dependent changes induced by Ba2+ were not present. Both Ba2+ and Cs+ had little blocking effect on the steady-state current positive to the negative slope region of the I-V relation. CONCLUSION: In ventricular myocytes, Cs+ abolishes the Ba(2+)-induced pacemaker current by blocking the time-dependent change in K+ conductance, not by blocking I(f). Because Cs+ can also block a decaying K+ pacemaker current, the abolition of a pacemaker current by Cs+ in other cardiac tissues cannot be taken as unequivocal proof that the blocked current is I(f).

Brassica anther-specific genes: characterization and in situ localization of expression.

A cDNA library of Brassica napus (cv. Westar) was constructed using poly(A)+ RNA isolated from developing anthers of flower buds 2-3 mm in length. Differential hybridization, using cDNA probes complementary to poly(A)+ RNA from developing anthers or seedlings, was used for initial screening. In addition to Southern and Northern blot analyses of selected clones, RNA-PCR assays and in situ hybridization were used to study the temporal and spatial gene regulation in anthers at the transcriptional level. Five independent cDNA clones, showing no cross-hybridization to one another, were characterized, and their expression patterns could be grouped into three distinct categories. Two cDNA clones, BA112 and BA158, are tapetum-specific: the corresponding mRNAs accumulate in young anthers and decline as the tapetum cells degenerate later in anther development. The transcripts represented by BA54 and BA73 accumulate late in anther development and reach a maximum level in mature anthers prior to anthesis; BA54 has been confirmed to be pollen-specific. The third category, represented by BA42, is found to encode a protein sharing 64-67% amino acid similarity with chalcone synthase (CHS) from various plant species; the transcript is localized in the peripheral cells of the vascular bundle, tapetum, and developing microspores.

Alteration of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase activase activities by site-directed mutagenesis.

Site-directed mutagenesis was performed on the 1.6 and 1.9 kilobase spinach (Spinacea oleracea) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase cDNAs, encoding the 41 and 45 kilodalton (kD) isoforms of the enzyme, to create single amino acid changes in the putative ATP-binding site of Rubisco activase (Lys-107, Gln-109, and Ser-112) and in an unrelated cysteine residue (Cys-256). Replacement of Lys-107 with Met produced soluble protein with reduced Rubisco activase and ATPase activities in both isoforms. Substituting Ala or Arg for Lys-107 produced insoluble proteins. Rubisco activase activity increased in the 41-kD isoform when Gln-109 was changed to Glu, but activity in the 45-kD isoform was similar to the wild-type enzyme. ATPase activity in the Glu-109 mutations did not parallel the changes in Rubisco activase activity. Rather, a higher ratio of Rubisco activase to ATPase activity occurred in both isoforms. The mutation of Gln-109 to Lys inactivated Rubisco activase activity. Replacement of Ser-112 with Pro created an inactive protein, whereas attempts to replace Ser-112 with Thr were not successful. The mutation of Cys-256 to Ser in the 45-kD isoform reduced both Rubisco activase and ATPase activities. The results indicate that the two activities of Rubisco activase are not tightly coupled and that variations in photosynthetic efficiency may occur in vivo by replacing the wild-type enzyme with mutant enzymes.

[Insight into the mechanism of torsade de pointes (TDP) induced by cesium chloride].

Standard intracellular potential recording technique was used in isolated guinea pig ventricular muscle and monophasic action potential (MAP) recording technique was performed on both the epicardium and endocardium of dog hearts in vivo. The results showed that CsCl lengthened action potential durations of ventricular muscle as well as QT intervals of dog hearts. The incidence of early after depolarization (EAD) induction was 71.4%. Ventricular tachycardia induced by CsCl manifested as typical TdP in 44.4% with EAD shown on epicardial MAP recording. This suggested that EAD and triggered activity might be an important mechanism of TdP. As verapamil could depress the induction of EAD, we believe that Ca++ inward currents was responsible for the induction of EAD and TdP by CsCl.

Expression of the two isoforms of spinach ribulose 1,5-bisphosphate carboxylase activase and essentiality of the conserved lysine in the consensus nucleotide-binding domain.

The two isoforms of ribulose 1,2-bisphosphate carboxylase activase (Rbu-P2 carboxylase) from spinach (Spinacea oleracea L.) were individually purified from Escherichia coli transformed with expression vectors for the appropriate cDNAs. Both isoforms catalyzed activation of Rbu-P2 carboxylase (ribulose 1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) and ATP hydrolysis. The kinetics of the two isoforms with respect to ATP concentration were different, in that the 45-kDa polypeptide exhibited a sigmoidal response while a rectangular response was observed with the 41-kDa isoform. These observations suggest that the additional domain at the C terminus of the 45-kDa isoform modulates the ATP regulation of activity. Lysine 169, at the putative ATP-binding site of the 41-kDa form of Rbu-P2 carboxylase activase, was changed to arginine, isoleucine, and threonine by directed mutagenesis. These mutations abolished Rbu-P2 carboxylase activase and ATPase activities, as well as the capability of the protein to bind ATP. These results confirm that lysine 169 is an essential residue.

[Detection of carcinoembryonic antigen of endometrial carcinoma and hyperplasia].

The amount and distribution of carcinoembryonic antigen (CEA) was determined in 41 cases of endometrial carcinoma and 23 cases of endometrial hyperplasia using the Avidin-biotin-peroxidase-complex technique. The surface location and amount of CEA in cancer specimens were found to be related to the histological differentiation of tumor. In endometrial hyperplasia, the content of CEA was the same as that of cancer, but the surface location was different.