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The role of the Ca2+-dependent phosphatase calcineurin (CaN) in the modulation of Ca2+-dependent Cl- channels (ClCa) was studied in freshly isolated rabbit coronary arterial myocytes. Immunocytochemical experiments showed that calmodulin-dependent protein kinase II (CaMKII) and CaN were distributed evenly throughout the cytoplasm of coronary myocytes at rest and translocated to the plasmalemma when intracellular Ca2+ was increased. ClCa currents (ICl(Ca)) elicited by cell dialysis with fixed intracellular Ca2+ levels up to 500 nM were inhibited by 10 microM cyclosporin A (CsA), a specific inhibitor of CaN, in a voltage-dependent manner, whereas currents evoked by 1 microM Ca2+ were not affected. Inhibition of CaN with CsA also led to a significant reduction in Ca2+ sensitivity of the channel at +50 mV; half-maximal activation increased from 363 +/- 16 nM Ca2+ in control to 515 +/- 40 nM Ca2+ in the presence of CsA. Similar effects were observed on ICl(Ca) when a specific peptide fragment inhibitor of CaN (CaN-AF, 5 microM) was dialysed into the cell via the pipette (500 nM Ca2+). Application of KN-93 (10 microM), a specific inhibitor of CaMKII, enhanced ICl(Ca) in myocytes dialysed with 1 microM Ca2+ but produced no significant effect on this current when the cells were dialysed with 350 or 500 nM Ca2+. These results are consistent with the notion that in coronary arterial cells, the activity of ClCa is enhanced by dephosphorylation of the channel or a closely associated regulatory protein. Moreover the balance of CaN and CaMKII regulating ICl(Ca) is dependent on the level of Ca2+ used to activate ICl(Ca).

Increased peripheral resistance is a hallmark of chronic heart failure and has been primarily attributed to neurohumoral pathways involving both the renin-angiotensin and sympathetic nervous systems. The increased resistance is thought to serve as a compensatory mechanism to help maintain perfusion to the vital organs by sustaining blood pressure in the fate of a failing heart. Local mechanisms, and in particular endothelial dysfunction, have also been shown to be important contributors in regulating arterial resistance and vascular remodeling in this disease. In this issue of the British Journal of Pharmacology, Gschwend et al. (2003) present new data suggesting that in the absence of a functional endothelium, myogenic constriction of small pressurized mesenteric arteries, an intrinsic property of vascular smooth muscle cells, is enhanced in a coronary artery ligation-induced myocardial infarction model of congestive heart failure (CHF) in the rat. The increased myogenic tone appears to be tightly linked to angiotensin II type 1 receptors (AT(1)). The possibility that CHF-induced stimulation of myogenic constriction is due to the local release of preformed angiotensin II or constitutive upregulation of the AT(1) receptor signaling pathways are discussed along with other potential cellular and molecular mechanisms previously suggested to play a role in myogenic reactivity.

This study investigated the enzymatic function of two putative plant GPXs, GPXle1 from Lycopersicon esculentum and GPXha2 from Helianthus annuus, which show sequence identities with the mammalian phospholipid hydroperoxide glutathione peroxidase (PHGPX). Both purified recombinant proteins expressed in Escherichia coli show PHGPX activity by reducing alkyl, fatty acid and phospholipid hydroperoxides but not hydrogen peroxide in the presence of glutathione. Interestingly, both recombinant GPXle1 and GPXha2 proteins also reduce alkyl, fatty acid and phospholipid hydroperoxides as well as hydrogen peroxide using thioredoxin as reducing substrate. Moreover, thioredoxin peroxidase (TPX) activities were found to be higher than PHGPX activities in terms of efficiency and substrate affinities, as revealed by their respective Vmax and Km values. We therefore conclude that these two plant GPX-like proteins are antioxidant enzymes showing PHGPX and TPX activities.

Background: Loss of rate-dependent action potential (AP) duration (APD) adaptation is a characteristic feature of atrial tachycardia-induced remodeling (ATR). ATR causes sarcolemmal ion-channel remodeling (ICR) and changes in Ca(2+)-handling. The present studies were designed to quantify Ca(2+)-handling changes and then to apply a mathematical AP model to assess the contributions of Ca(2+)-handling abnormalities and ICR to loss of APD rate-adaptation., Methods: Indo-1 fluorescence was used to measure intracellular Ca(2)-transients and whole-cell patch-clamp to record APs in atrial myocytes from control dogs and dogs subjected to atrial pacing at 400/min for 6 weeks. A previously developed ionic model of the canine atrial AP was modified to reproduce measured Ca(2+)-transients of control and ATR myocytes., Results: In control, APD to 95% repolarization (APD(95)) decreased by 91 ms experimentally and by 88 ms in the model over the 1-6 Hz range. In ATR myocytes, APD(95) failed to decrease over the 1-6 Hz range. Ca(2+)-handling abnormalities in ATR myocytes included slowed upstroke, decreased amplitude and strong single-beat post-rest potentiation. Unaltered Ca(2+)-handling properties included caffeine-releasable Ca(2+)-stores and Ca(2+)-transient relaxation before and after exposure to the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor cyclopiazonic acid (CPA). Including ICR alone in the model accounted for loss of APD(50) rate-adaptation; however, KR alone reduced APD(95) rate-adaptation by only 19% to 71 ms. When both ICR and Ca(2+)-handling changes were incorporated, APD(95) rate-adaptation decreased to 6 ms, accounting for experimental observations., Conclusion: ICR alone does not fully account for loss of APD rate-adaptation with atrial remodeling: Ca(2+)-handling changes appear to contribute to this clinically significant phenomenon.

Bradykinin (BK), a proinflammatory factor and vasodilator, causes functional change of the small artery. However, it is not clear whether any of these changes induced by BK are mediated by N-acetyl-D-sphingosine (ceramide). Therefore, we investigated whether BK affects the hydrolysis of sphingomyelin and generation of ceramide in the intact rat small artery. Our results suggest that BK induces sphingomyelin hydrolysis and increases ceramide production in a time- and dose-dependent manner. Relative to controls, BK causes a 50% decrease in sphingomyelin levels. Ceramide levels increase in response to BK with the highest level being obtained with 10(-8) M BK as well as similar amounts of ceramide are generated when exogenous sphingomyelinase (SMase) is added. We then determined which of the two BK receptors (BK-B(1) antagonist Lys-Des-Arg(9)-Leu(8)-BK or the BK-B(2) antagonist HOE-140) are implicated in the BK-induced generation of ceramide. The BK-B(2) antagonist did not alter the effect of BK on ceramide generation, whereas the BK-B(1) antagonist blocked the BK-induced production of ceramide. Although ceramide had no effect on KCl-induced constrictions, ceramide dilated preconstricted (phenylephrine) small pressurized rat mesenteric arteries by approximately 40%. These results suggest that the activation of the BK-B(1) receptor mediates the BK-induced activation of SMase and of the production of ceramide. In conclusion, BK-mediated effects on vascular tone may be due, at least in part, to the increased production of ceramide.

The present study investigated the effect of redox agents on Ca2+-activated Cl- currents ( ICl(Ca)) recorded in smooth muscle cells isolated from rabbit portal vein. In perforated-patch experiments on portal vein cells the amplitude of ICl(Ca) evoked by either spontaneous release of Ca2+ from internal stores or Ca2+ influx through voltage-dependent Ca2+ channels was markedly and irreversibly enhanced by the non-specific oxidant, diamide (10-200 microM). Diamide also prolonged the decay of both currents. The reductant dithiothreitol had no effect on control ICl(Ca) but reversed the increase of current amplitude produced by diamide. Diamide also increased global intracellular Ca2+ at rest but had no effect on the time-course of Ca "sparks" determined by confocal microscopy. Diamide and the endogenous oxidant hydrogen peroxide increased the amplitude and prolonged the kinetics of ICl(Ca)evoked by pipette solutions containing free Ca2+ clamped at 500 nM. Similar effects were observed with the hydrophilic thiol-reactants thimerosal and p-chloromercuriphenylsulphonic acid (PCMPS). Therefore, the gating and activation of Ca2+-activated Cl- conductance is sensitive to redox modification.

Background: An unused drug (UD) is defined as a drug which is purchased, after prescription or not, but which is not taken. Public health campaigns in France have requested people to return these drugs to their pharmacy. To data, few data have been available concerning the quantity of collected UD and their potential re-use. A study was performed in the pharmacies of the Puy-de-Dôme region in France to describe the UD circuit., Methods: A random sample of 1 out of 5 pharmacies in the Puy-de-Dôme region (France) were defined by single level stratified sampling from the list of all pharmacies operating in the region. An exhaustive record of all UD people brought back to these pharmacies was made in 1998 during three 1-week periods. The following data were recorded for each UD: the name of the drug, the pharmaceutical industry code (CIP), the price, the rate of social Security refunding, the mention of "free specimen" on the package, the inscription on the list of poisonous substances and on the list of essential drugs defined by the World Health Organization (WHO), the registration on the list of drugs reserved for hospital use, the packaging notice (opened or not), the therapeutic class, and the formulation., Results: 10,254 US (717kg) were collected during the study period. The therapeutic classes of the UD were similar to those of drugs purchased in France. According to the selling price, these UD had an economic value of 405,845FF (i.e. 3.6% of Social Security refundings paid in the Puy-de-Dôme region during this same period). Only 20% of the UD were potentially reusable for humanitarian purposes. Their estimated economic value was 87,456FF (i.e. 0.78% of the annual Puy-de-Dôme Social Security refunding). Moreover, 43.4% of the reusable drugs were on the WHO list of essential drugs., Conclusion: Although the volume of collected UD is high, use by humanitarian associations is on the decline because of the cost of collection and low economic yield. Furthermore, UD must be collected in a systematic manner to preserve the environment and prevent domestic accidents.

The auxin-binding protein 1 (ABP1) has been proposed to be involved in the perception of the phytohormone at the plasma membrane. Site-directed mutagenesis was performed on highly conserved residues at the C terminus of ABP1 to investigate their relative importance in protein folding and activation of a functional response at the plasma membrane. Detailed analysis of the dynamic interaction of the wild-type ABP1 and mutated proteins with three distinct monoclonal antibodies recognizing conformation-dependent epitopes was performed by surface plasmon resonance. The influence of auxin on these interactions was also investigated. The Cys(177) as well as Asp(175) and Glu(176) were identified as critical residues for ABP1 folding and action at the plasma membrane. On the contrary, the C-terminal KDEL sequence was demonstrated not to be essential for auxin binding, interaction with the plasma membrane, or activation of the transduction cascade although it does appear to be involved in the stability of ABP1. Taken together, the results confirmed that ABP1 conformational change is the critical step for initiating the signal from the plasma membrane.

1. Ca(2+)-activated chloride currents (I(Cl(Ca))) were recorded from smooth muscle cells isolated from rabbit pulmonary (PA) and coronary artery (CA) as well as rabbit portal vein (PV). The characteristics and regulation by Ca(2+)-calmodulin-dependent kinase II (CaMKII) were compared between the three cell types. 2. In PA and CA myocytes dialysed and superfused with K+ -free media, pipette solutions containing fixed levels of free Ca(2+) in the range of 250 nM to 1 microM evoked well sustained, outwardly rectifying I(Cl(Ca)) currents in about 90 % of cells. The CaMKII inhibitor KN-93 (5 microM) increased the amplitude of I(Cl(Ca)) in PA and CA myocytes. However, the threshold intracellular Ca(2+) concentration for detecting this effect was different in the two arterial cell types. KN-93 also enhanced the rate of activation of the time-dependent current during depolarising steps, slowed the kinetics of the tail current following repolarisation, and induced a negative shift of the steady-state activation curve. 3. In PA myocytes, the effects of KN-93 were not mirrored by its inactive analogue KN-92 but were reproduced by the inclusion of autocamtide-2-related CaMKII inhibitory peptide (ARIP) in the pipette solution. Cell dialysis with constitutively active CaMKII (30 nM) significantly reduced I(Cl(Ca)) evoked by 500 nM Ca(2+). 4. In PV myocytes, I(Cl(Ca)) was evoked by pipette solutions containing up to 1 microM free Ca(2+) in less than 40 % of cells. Application of KN-93 to cells where I(Cl(Ca)) was sustained produced a small inhibition (approximately 25%) of the current in 70 % of the cells. 5. The present study shows that regulation of Ca(2+)-dependent Cl(-) channels by CaMKII differs between arterial and portal vein myocytes.

Objectives: Indirect evidence suggests a role for Ca(2+)-overload in electrical and mechanical alterations caused by atrial tachycardia. The present study assessed the alterations in cellular [Ca(2+)] and contractile function caused by rapid atrial cellular activation., Methods: Intracellular Ca(2+) transients (CaT) and cell shortening (CS) were measured by microfluorometry (Indo-1 AM) and video edge-detection in isolated, field-stimulated canine atrial myocytes (37 degrees C)., Results: Abrupt increases in frequency (0.3-3 Hz) caused rapid increases in diastolic [Ca(2+)]i (DCa) that were maintained during rapid-pacing for up to 50 min. When short-term (3-min) rapid-pacing was imposed, CaT and CS increased initially upon returning to 0.3 Hz, but then declined rapidly to 64+/-5 and 49+/-7%, respectively, of pre-tachycardia values, returning to control after approximately 15 min. Post-tachycardia CaT and CS reductions were prevented by decreasing [Ca(2+)]o during tachycardia to prevent Ca(2+)-overload. CS reductions correlated with indices of Ca(2+) loading during tachycardia. Restoration of CaT to normal during post-tachycardia contractile dysfunction (by increasing [Ca(2+)]o) returned CS to normal, indicating that reduced Ca(2+) release, not reduced myofilament Ca(2+)-sensitivity, caused post-tachycardia contractile failure. Estimation of sarcoplasmic-reticulum Ca(2+)-stores (caffeine-induced Ca(2+)-release) confirmed tachycardia-induced Ca(2+)-loading and suggested that reduced Ca(2+)-stores decreased Ca(2+)-release post-tachycardia., Conclusions: Atrial tachycardia increases cellular Ca(2+)-loading, leading to post-tachycardia abnormalities in Ca(2+)-handling that produce contractile dysfunction. These findings are the first direct evidence for the frequently-postulated role of Ca(2+)-overload in tachycardia-induced abnormalities of atrial function.

AraC regulates transcription of the Escherichia coli arabinose operon, binding tandem DNA half-sites in the presence of arabinose and widely spaced half-sites in the absence of arabinose. In the structure of the AraC N-terminal dimerization domain with bound arabinose, the protein dimerizes via an antiparallel coiled-coil interface. The absence of bound ligand opens a second, beta-barrel interaction interface that also mediates interactions between unliganded AraC dimers in the crystal. The larger buried surface area of the beta-barrel interface, as compared with the coiled-coil interface, raised the possibility that protein-protein interactions mediated by the beta-barrel might play a role in ligand-mediated modulation of AraC DNA binding activity. For the crystallographically observed beta-barrel interaction to play a role in the cell, dimerization via this interface in the absence of arabinose would be predicted to be at least as energetically favorable as dimerization via the coiled-coil interface. In the study presented here, we use analytical ultracentrifugation to determine the oligomeric state of the AraC dimerization domain in the presence and absence of arabinose. Dimerization of the unliganded protein via the beta-barrel interface in the absence of interactions mediated by the coiled-coil interface is assayed using a mutant AraC protein with a disrupted coiled-coil interface. The results of these studies indicate that dimerization via the beta-barrel interface is substantially weaker than dimerization via the coiled-coil interface, indicating that the crystallographically observed beta-barrel interaction is not relevant to in vivo function.

Background: Ca2+-activated (I(Cl(Ca))) and swelling-induced (I(Cl(swell))) Cl- channels have, respectively, been postulated to participate in the membrane depolarization and contraction mediated by activation of alpha1-adrenoceptors and vascular wall distension during pressurization. Their respective function in generating active force in pressurized arterioles during alpha1-adrenoceptor stimulation remains unsettled., Objectives: Experimental protocols were designed to: (1) assess the relative contribution of I(Cl(Ca)) to the pressure-dependence of lumen diameter of mesenteric arterioles at different states of activation of the alpha1-adrenoceptor, and (2) investigate the potential role of I(Cl(swell)) in spontaneous and agonist-mediated myogenic reactivity., Methods: Segments of endothelium-denuded rabbit mesenteric arterioles with a lumen diameter of approximately 70 microm were cannulated at both ends and studied under isobaric conditions at 36 degrees C. Steady-state lumen diameter at each pressure step investigated (0-100 mmHg, in 20-mmHg increments) was measured by a video-microscopy edge-detection technique., Results: Under control conditions, 23% of the arterioles developed nifedipine-sensitive spontaneous myogenic tone. In the presence of 1 mM tetraethylammonium chloride (TEA) to inhibit Ca2+-dependent K+ channels, the alpha1-agonist phenylephrine (PE) contracted the vessels in a concentration-dependent manner (0.1-10 microM) and potentiated myogenic reactivity. The contraction mediated by 1 microM PE/TEA was abolished by 1 microM nifedipine, indicating that Ca2+ entry through voltage-gated Ca2+ channels was a necessary step in the cascade leading to contraction. Niflumic acid (NfA, 100 microM), a relatively selective inhibitor of I(Cl(Ca)), had no effect on myogenic tone but reversed the PE-induced contraction, varying with the concentration of PE and transmural pressure. For PE concentrations between 0.1 and 1 microM, but not for 10 microM PE, the relaxing efficacy of NfA decreased as applied pressure was raised from 0 to 100 mmHg. At all pressure steps, the NfA-induced relaxation was inversely related to the concentration of PE. DIDS (200 microM), another Cl- channel blocker, inhibited spontaneous myogenic tone, and partially suppressed a component of contraction at elevated transmural pressures in arterioles incubated in 1 microM PE/1 mM TEA/100 microM NfA., Conclusions: Our data indicate that under low to moderate stimulation of the alpha1-adrenoceptor signaling pathway, I(Cl(Ca)) channels play an important role in the sustained contraction produced. Their declining contribution to contraction with increasing transmural pressure may be explained, at least in part, by a progressive enhancement of stretch-induced ionic conductances, possibly volume-sensitive Cl- channels.

Interactions of a collection of monoclonal antibodies (mAbs) to the recombinant Nicotiana tabacum auxin-binding protein 1 (Nt-abp1) were extensively characterized using surface plasmon resonance. Dynamic interaction studies using combinations of Nt-abp1, synthetic peptides corresponding to conserved sequences within auxin-binding proteins, and the mAbs have shown that a number of the mAbs recognized discontinuous epitopes revealing the junction of distinct domains in the folded protein. In particular, the two putative auxin binding domains and the C terminus of the protein were shown to interact with each other in the folded protein. Using the auxin-induced electrical response of tobacco protoplasts as a functional assay, all the mAbs exhibited either auxin antagonist or hormonomimetic properties. These effects, measured for the first time in homologous conditions, confirm that Nt-abp1 is present at the plasma membrane and is involved in the activation of the auxin-dependent electrical response of tobacco protoplasts. Based on our surface plasmon resonance data, we propose that the key event leading to the activation of this auxin electrical response consists of a conformational change in Nt-abp1.

The Ca(2+)-activated Cl(-) current [I(Cl(Ca))] contributes to the repolarization of the cardiac action potential under physiological conditions. I(Cl(Ca)) is known to be primarily activated by Ca(2+) release from the sarcoplasmic reticulum (SR). L-type Ca(2+) current [I(Ca(L))] represents the major trigger for Ca(2+) release in the heart. Recent evidence, however, suggests that Ca(2+) entry via reverse-mode Na(+)/Ca(2+) exchange promoted by voltage and/or Na(+) current (I(Na)) may also play a role. The purpose of this study was to test the hypothesis that I(Cl(Ca)) can be induced by I(Na) in the absence of I(Ca(L)). Macroscopic currents and Ca(2+) transients were measured using the whole cell patch-clamp technique in rabbit ventricular myocytes loaded with Indo-1. Nicardipine (10 microM) abolished I(Ca(L)) at a holding potential of -75 mV as tested in Na(+)-free external solution. In the presence of 131 mM external Na(+) and in the absence of I(Ca(L)), a 4-aminopyridine-resistant transient outward current was recorded in 64 of 81 cells accompanying a phasic Ca(2+) transient. The current reversed at -42. 0 +/- 1.3 mV (n = 6) and at +0.3 +/- 1.4 mV (n = 6) with 21 and 141 mM of internal Cl(-), respectively, similar to the predicted reversal potential with low intracellular Cl(-) concentration ([Cl(-)](i)) (-47.8 mV) and high [Cl(-)](i) (-1.2 mV). Niflumic acid (100 microM) inhibited the current without affecting the Ca(2+) signal (n = 8). Both the current and Ca(2+) transient were abolished by 10 mM caffeine (n = 6), 10 microM ryanodine (n = 3), 30 microM tetrodotoxin (n = 9), or removal of extracellular Ca(2+) (n = 6). These properties are consistent with those of I(Cl(Ca)) previously described in mammalian cardiac myocytes. We conclude that 1) I(Cl(Ca)) can be recorded in the absence of I(Ca(L)), and 2) I(Na)-induced SR Ca(2+) release mechanism is also present in the rabbit heart and may play a physiological role in activating the Ca(2+)-sensitive membrane Cl(-) conductance.

The present study was undertaken to investigate the effects of specific inhibitors of calmodulin-dependent protein kinase II (CamKII) on macroscopic voltage-dependent K(+) current (K(V)) recorded from rabbit portal vein smooth muscle cells. Inhibition of L-type Ca(2+) current facilitation by 1 microM KN-62, a blocker of CamKII, was first demonstrated and provided evidence for functional CamKII activity in this preparation. KN-93, another specific and more potent inhibitor of CamKII in the rat brain, suppressed K(V) and enhanced the rate of inactivation in a dose-dependent manner, in cells dialyzed with both low (0.1 mM) and high (10 mM) EGTA pipette solution. Prolonged dialysis with 10 microM of a synthetic peptide inhibitor of CamKII (fragment 281-301) had little effect on K(V) and did not prevent the inhibitory action of KN-93 on the current. The estimated IC(50) for inhibiting peak and late currents during 250-ms steps to +60 mV (holding potential = -60 mV) were 2.9 and 0.27 microM, respectively. KN-93 also induced slight shifts of the steady-state activation (-7 mV) and inactivation (-6 mV) curves. KN-62, and KN-92, an inactive analog of KN-93, produced effects similar to those of KN-93. In current clamp experiments, 5 microM KN-93 depolarized the myocytes from a control resting membrane potential of -42.3 +/- 2.8 mV to -28.5 +/- 1.4 mV, an effect that was partially reversible after washout (-34.4 +/- 1.3 mV, n = 6). In conclusion, blockers of CamKII produce nonspecific inhibitory effects on K(V) that warrant cautious use of these compounds in physiological experiments designed to assess the role of CamKII.

Large-conductance Ca2+-dependent K+ channels (KCa), which are abundant on the sarcolemma of vascular myocytes, provide negative feedback via membrane hyperpolarization that limits Ca2+ entry through L-type Ca2+ channels (ICaL). We hypothesize that local accumulation of subsarcolemmal Ca2+ during ICaL openings amplifies this feedback. Our goal was to demonstrate that Ca2+ entry through voltage-gated ICaL channels can stimulate adjacent KCa channels by a localized interaction in enzymatically isolated rabbit coronary arterial myocytes voltage clamped in whole-cell or in cell-attached patch clamp mode. During slow-voltage-ramp protocols, we identified an outward KCa current that is activated by a subsarcolemmal Ca2+ pool dissociated from bulk cytosolic Ca2+ pool (measured with indo 1) and is dependent on L-type Ca2+ channel activity. Transient activation of unitary KCa channels in cell-attached patches could be detected during long step depolarizations to +40 mV (holding potential, -40 mV; 219 pS in near-symmetrical K+). This local interaction between the channels required the presence of Ca2+ in the pipette solution, was enhanced by the ICaL agonist Bay K 8644, and persisted after impairment of the sarcoplasmic reticulum by incubation with 10 micromol/L ryanodine and 30 micromol/L cyclopiazonic acid for at least 60 minutes. Furthermore, we provide the first direct evidence of simultaneous openings of single KCa (67 pS) and ICaL (3.9 pS) channels in near-physiological conditions, near resting membrane potential. Our data imply a novel sensitive mechanism for regulating resting membrane potential and tone in vascular smooth muscle.

Drosophila melanogaster larvae usually react against eggs of the parasitoid wasp Leptopilina boulardi by surrounding them with a multicellular melanotic capsule. The genetic determinism of this response has been studied previously using susceptible (non-capsule-forming) and resistant (capsule-forming) strains. The results suggest that differences in their encapsulation response involve a single gene, resistance to Leptopilina boulardi (Rlb), with two alleles, the resistant one being dominant. Rlb confers specific protection against Leptopilina boulardi and is thus probably involved in parasitoid recognition. Recent studies have localized this gene on the right arm of the second chromosome and our aim was to precisely determine its genetic and molecular location. Using strains bearing deletions, we demonstrated that resistance to Leptopilina boulardi is conferred by the 55C; 55F3 region and that the 55E2-E6; F3 region is particularly involved. A physical map of the 55C; 56A region was then constructed, based on a set of overlapping cosmid and P1 phage clones. Using single and double digests, cross hybridization of restriction fragments, and location of genetically mapped genes and STSs, a complete, five-enzyme restriction map of this 830-kb region was obtained.

Hyperpolarization of tobacco protoplasts is amongst the earliest auxin responses described. It has been proposed that the auxin-binding protein, ABP1, or a related protein could be involved in the first step of auxin perception at the plasma membrane. Using for the first time homologous conditions for interaction between the protein Nt-ERabp1 or a synthetic peptide corresponding to the C-terminus and tobacco protoplasts, we have demonstrated that both can induce the hyperpolarization response. The results show that Nt-ERabp1 or the C-terminal peptide alone activates the auxin pathway from the outer face of the plasma membrane.

1. The objective of this study was to determine potential post-pubertal gender-specific differences in the contractility of papillary muscles, the electrophysiological properties and Ca2+ transients of freshly dissociated ventricular myocytes from the rat heart. 2. The contractions of rat papillary muscles from 2- to 14-month-old male and female rats were studied under isometric and isotonic conditions (29 degrees C). While the hearts of young (2-4 months) male and female rats displayed a similar contractile profile, papillary muscles of female rats aged 6 months and older exhibited smaller isometric and isotonic contractions, smaller maximal rates of tension and shortening development and decline (+/-DT/dt and +/-DL/dt) velocities during both the onset and relaxation phases, and shorter contractions than age-matched males. 3. To explore the possible cellular basis accounting for these differences, action potentials and macroscopic currents were recorded from freshly dissociated myocytes using the whole-cell patch clamp technique (35 C). Action potentials from male and female myocytes of 3- and 9-month-old rats did not vary as a function of age or gender. Consistent with these results, the magnitude (expressed in pA pF-1), voltage-dependence and kinetics of the inward rectifier (IK1), transient outward (Ito) and sustained (IK) K+ currents displayed little, if any dependence on age or gender. 4. L-type Ca2+ current (ICa(L)) measured in caesium-loaded myocytes (35 C) from male and female rats of 3, 6 and 9 months of age exhibited similar characteristics. In contrast, while Ca2+ transients measured with indo-1 were similar between 3-month-old male and female rat myocytes, Ca2+ transients of 10-month-old female myocytes were significantly reduced and showed a diminished rate of relaxation in comparison with those recorded in male rats of similar age. 5. These results suggest that important gender-related changes in excitation-contraction coupling occur following puberty, probably due to differences in Ca2+ handling capabilities at the level of the sarcoplasmic reticulum.

Background: Transient atrial contractile dysfunction ("atrial stunning") follows conversion of atrial fibrillation (AF) to sinus rhythm and has significant clinical implications; however, the underlying mechanisms are poorly understood. We investigated the hypothesis that rapid atrial activation (as during AF) impairs cellular contractility and affects cellular Ca2+ handling., Methods and Results: Edge detection and indo 1 fluorescence techniques were used to measure unloaded cell shortening and intracellular Ca2+ transients in atrial myocytes from control (Ctl) dogs and dogs subjected to atrial pacing at 400 bpm for 7 (P7) or 42 (P42) days. Atrial tachycardia reduced fractional cell shortening (0.1 Hz) from 7.3+/-0.4% (Ctl) to 4.3+/-0.3% and 2.0+/-0.3% in P7 and P42 dogs, respectively (P<0.01 for each). Resting [Ca2+]i was not altered in paced dogs, but the systolic Ca2+ transient was significantly reduced. Furthermore, cells from paced dogs showed slowed relaxation and use-dependent decreases of Ca2+ transients and cell shortening compared with cells from Ctl dogs. To determine whether changes in Ca2+ transients account fully for alterations in contractility, we varied [Ca2+]o to evaluate the relation between Ca2+ transients and cell shortening. Reductions in Ca2+ transients in Ctl cells reduced shortening to the level of paced cells; however, when Ca2+ transients in P42 cells were elevated to the range of Ctl cells, a significant reduction in cell shortening remained. Similar results were obtained in dogs that maintained 1:1 capture throughout the monitoring period and dogs that developed sustained AF over the course of the study., Conclusions: Sustained atrial tachycardia causes important reductions in cellular contractility, in part by impairing cellular Ca2+ handling and decreasing systolic Ca2+ transients. These results provide direct evidence for the concept that AF induces atrial contractile dysfunction by causing a tachycardia-induced atrial cardiomyopathy.

At the request of the High Committee for Public Health (HCSP), an evaluation of the reception of its report, "Health in France" was carried out by the National Federation of Regional Health Observatories. Forty semi-directive interviews among actors of the health system from various geographic and professional backgrounds were carried out. It appears that the HCSP is an authority that is not well-known, for the most part. The report itself was mainly distributed by administrative networks; in the majority of cases, only the first part is well-known and was the particular focus of the interviewees. Overall, this document is a work instrument, a global tool, and a reference document. Several improvements are proposed concerning its form, the list of subjects covered, as well as the means of distribution.

This study was undertaken to test the hypothesis that, in the absence of extracellular Na+, Ca2+ can permeate tetrodotoxin (TTX)-sensitive Na+ channels in Cs(+)-loaded whole cell voltage-clamped guinea pig ventricular myocytes (22-24 degrees C). With 10 mM extracellular Ca2+, 50-ms step depolarizations (-50 to +25 mV) from holding potentials of -100 or -80 mV elicited fast and slow types of inward current: 1) a small (< 400 pA) dihydropyridine-insensitive inward current that exhibited similar voltage dependence to that of Na+ channels, with an activation threshold and peak near -45 mV and -30 mV, respectively; and 2) a larger and slower L-type Ca2+ current that activated and peaked at more positive potentials. Extracellular replacement of Ca2+ by Mg2+ abolished both currents. The lack of sensitivity of the low-threshold Ca(2+)-current amplitude to 50 or 200 microM Ni2+ suggests that this current is not produced by T-type Ca2+ current. In contrast, TTX dose dependently inhibited the low-threshold Ca2+ current, with a half-maximal inhibition concentration of 2.4 microM. Veratridine (10-50 microM), a plant alkaloid that alters the gating and permeability properties of Na+ current, induced an outward shift of time-dependent current during steps to -25 mV and typical slowly decaying inward tail currents after repolarization to -80 mV. Cell exposure to 10 and 50 microM extracellular Na+ inhibited the inward current by 21.2 +/- 3.9% (n = 23) and 14.0 +/- 3.0% (n = 14), respectively, whereas 1 microM Na+ (n = 14) was without effect. The application of 200 microM Na+ produced a small enhancement of the current (+6.2 +/- 4.1%; n = 14) which was just at the limit of significance. Our data support the notion that Ca2+ can permeate cardiac Na+ channels in the absence of an agonist and external Na+.

Background: The degree of skin induration (lipodermatosclerosis) around venous ulcers has prognostic significance; however, objective measurements are needed to assess the induration. The durometer, an engineering instrument used to measure the hardness of metals and plastic, has recently been adapted to assess skin induration., Objective: The purpose of this study was to measure skin induration and its relationship to skin ulceration by the use of a durometer, and to determine the influence of edema, if any, on durometer measurements., Methods: The degree of skin induration on the medial leg was determined in six sequential, nonselected patients with lipodermatosclerosis and leg ulcers, and in five normal volunteers by using a blinded observer's clinical score (0 = normal to 3 = maximal induration) and a hand-held Type 0 durometer. In addition, durometer readings in 14 patients with edema and eight control subjects were taken on the tibia, the dorsum of the foot, and behind the ankle., Results: Durometer readings in patients with leg ulcers and lipodermatosclerosis diminished as one measured from the superior edge of the ulcer to the knee (r = 0.925). The higher the clinical skin score the higher were the durometer readings (P = 0.0062). The presence of edema did not influence durometer measurements., Conclusion: The durometer is an effective and reliable instrument for measuring the degree of skin induration in venous ulceration and its readings are not affected by edema. Ulcers occur in skin most affected by lipodermatosclerosis.

We used whole cell patch-clamp and microfluorimetric (indo 1) techniques to measure Ca2+ current through L-type Ca2+ channels (I(Ca)) and Ca2+ transients in human atrial myocytes. During 1-s depolarizing pulses, I(Ca) inactivation was biexponential. The rate of rapid inactivation was slowed by ryanodine and was correlated with the rate of rise of cytoplasmic free Ca2+ concentration (r = 0.80, P < 0.01). Slower-phase I(Ca) inactivation was not affected by ryanodine but was accelerated by increasing the availability of Ca2+ to permeate the Ca2+ channel. Thus Ca2+ released from the sarcoplasmic reticulum (SR) was responsible for most I(Ca) inactivation during the first 50 ms of a depolarization to 0 mV, and thereafter inactivation by Ca2+ permeating the channel predominated. Pure voltage-dependent inactivation had a much slower time course of development (tau > 2 s) and played a smaller role than Ca2+-dependent mechanisms over a duration comparable to that of an action potential. We conclude that human atrial myocytes show both voltage- and Ca2+-dependent I(Ca) inactivation, that Ca2+-dependent mechanisms predominate over the time course of an action potential, and that although both Ca2+ released from the SR and Ca2+ permeating Ca2+ channels play a role, SR-released Ca2+ is particularly important in early, rapid I(Ca) inactivation, whereas Ca2+ permeating Ca2+ channels is more important in the slower phase of Ca2+-dependent inactivation.

The isolation and the characterization of two tobacco cDNAs, Nt-ERabp1 and Nt-ERabp2, homologous to Zm-ERabp1, encoding the major auxin-binding protein from maize coleoptiles, are described. Their predicted amino acid sequences correspond to proteins of ca. 21 kDa, in which the characteristic regions common to ABP1-related polypeptides are well-conserved. Southern analysis indicates that the genes corresponding to Nt-ERabp1 cDNA and Nt-ERabp2 cDNA derive respectively from Nicotiana tomentosiformis and Nicotiana sylvestris, the diploid progenitors of Nicotiana tabacum. Analysis of mRNA distribution in tobacco plants indicates that these two genes are preferentially expressed in flowers and growing seedlings. Whatever the tissue tested, Nt-ERabp1 mRNA is more abundant than Nt-ERabp2 mRNA. Furthermore, RT-PCR reveals developmental and organ-specific expression of these two genes in flower parts of tobacco plants. In particular, regulation of Nt-ERabp1 mRNA accumulation appears to be correlated with elongation growth of each floral organ. Recombinant Nt-ERabp1, produced in Escherichia coli, is recognized by antibodies raised against Zm-ERabp1.

1. The mechanisms involved in the 4-aminopyridine (4-AP)-induced block of delayed rectifier K+ current (IK(V)) in vascular smooth muscle cells were studied in cells enzymatically isolated from the rabbit coronary artery. 2. 4-AP inhibited slowly inactivating IK(V) in a dose-dependent manner (concentration producing half-maximal inhibition, K1/2, = 1.37 mM), and shifted the steady-state activation and inactivation curves of IK(V) by +9 and +16 mV, respectively. 3. The time constant of activation was significantly increased by 4-AP at +20 mV; deactivation kinetics were unaffected upon repolarization to -40 mV. The fast (tau f approximately 1 s) and slow (tau s approximately 5 s) time constants of inactivation (0 and +20 mV), and the recovery kinetics (tau r approximately 6 s) at -60 mV were not significantly affected by 0.5 mM 4-AP. However, tau f disappeared in the presence of 2 mM 4-AP while tau s remained unaffected. 4. Use-dependent unblock of IK(V) was revealed at potentials > or = -10 mV from analyses of the voltage dependence of 4-AP-sensitive currents and the frequency-dependent changes ('reverse use dependence') of IK(V) during the application of repetitive steps (-60 to +20 mV for 250 ms at a rate of 0.25 Hz) in control conditions, in the presence of 0.5 mM 4-AP, and after washout of the drug. These results suggested that 4-AP preferentially binds to the channel in the closed state, and unbinding is promoted by transitions to the open state. 5. The channel was modelled as a simple three-state mathematical loop model incorporating single closed, open and inactivated states. The block by 4-AP was modelled as a state-dependent interaction with 4-AP primarily binding to the closed state. Computer simulations support the hypothesis that 4-AP-induced block of the delayed rectifier K+ (KV) channel in the closed state is relieved during membrane depolarization. 6. Closed state binding of 4-AP to the KV channel depolarizes vascular smooth muscle cells by shifting the activation curve of these channels to more positive potentials.

Phenamil, a phenyl-substituted amiloride derivative and a potent inhibitor of epithelial-type sodium channels, produces significant prolongation of the action potential in isolated ventricular preparations, a tissue in which epithelial-type sodium channels have not been reported. Experiments were therefore carried out to examine the possible electrophysiological effects of phenamil on whole-cell ionic currents recorded in voltage-clamped guinea pig ventricular myocytes. At higher concentrations than those required to inhibit the epithelial sodium channels, phenamil (10-60 microM as compared to nanomoles for epithelial sodium channels) significantly increased the action potential duration in ventricular myocytes. It also induced a slow progressive depolarization which was followed eventually by oscillatory potentials near -35 mV. Higher concentrations of phenamil accelerated these changes. These effects were partially reversible. Voltage-clamp experiments using slow voltage ramps from -140 mV to +60 mV or step protocols from -120 mV to +30 mV revealed a prominent (approximately 70% at -120 mV) inhibitory effect of phenamil (50 microM) on the inwardly rectifying potassium current. Phenamil (50 microM) had little effect on the delayed rectifier potassium currents recorded by using long (5 sec) voltage steps from -40 mV to +60 mV. In the absence of K+, calcium current (L-type) was not affected by phenamil at concentrations up to 100 microM. Our data support the concept that phenamil-induced action potential prolongation in guinea pig cardiac myocytes may be produced by inhibition of inwardly rectifying potassium current.

Macroscopic currents were recorded in freshly dissociated smooth muscle cells from the rabbit portal vein using the tight seal whole cell recording mode (22 degrees C). In some experiments, the indo 1 fluorescence technique was used to simultaneously monitor the changes in the concentration of free intracellular Ca2+ ([Ca2+]i; indo 1 ratio, 400/500 nm). In cells exposed to tetraethylammonium chloride (TEA) to inhibit K+ channels and 1-10 microM nifedipine or nicardipine to inhibit L-type Ca2+ channels, cell dialysis with 30 mM Na+ increased [Ca2+]i and induced membrane current consistent with the activation of Ca(2+)-activated Cl- channels [ICl(Ca)]. From holding potential (HP) of -60 mV, high intracellular Na+ concentration ([Na+]i)-mediated current was instantaneous in response to 0.5- to 10-s voltage clamp pulses from -80 to +20 mV; steps ranging from +20 to +80 mV evoked slow time-dependent outward current (I(t); superimposed on the instantaneous current) and voltage-dependent Ca2+ transient; on return to HP, slow inward tail current appeared that reflected deactivation of I(t). Both current components 1) exhibited outward rectifying properties, 2) reversed near the predicted equilibrium potential for Cl-, 3) were stimulated by elevation of extracellular Ca2+ concentration, 4) were abolished when the cells were dialyzed with 5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 5) were inhibited by extracellular application of niflumic acid (50 microM). Complete replacement of extracellular Na+ concentration with tetramethylammonium increased both the instantaneous and time-dependent components of ICl(Ca), resting [Ca2+]i at -60 mV and Ca2+ transient at +40 mV. Cell dialysis with Na(+)-free pipette solution prevented these effects. Our results are consistent with an indirect mechanism of stimulation of ICl(Ca), which involves intracellular Ca2+ accumulation via reverse-mode electrogenic Na+/Ca2+ exchange activity.

Irregularities in K+ currents form the basis of several cardiovascular dysfunctions, among which are arrhythmias and vasospasms. The developmental regulation of voltage-gated K+ channels, however, has been difficult to study. A novel approach was therefore employed to examine these channels in muscle tissue. Primers for a PCR-based analysis were designed using published nucleic acid sequences for voltage-gated K+ channels. Final selection of the primer pairs was based on the homology present in the S4 and H5 transmembrane domains. A specific band was amplified with these primers using RNA isolated from both rat A10 vascular smooth muscle cells and rat heart tissue.

Expression and physiological effects of the root-inducing rolB gene of Agrobacterium rhizogenes T-DNA were studied simultaneously in tobacco (Nicotiana tabacum) mesophyll protoplasts. The kinetic study of the expression of rolB mRNA following exogenous auxin application showed that auxin transiently stimulated rolB expression, with mRNA levels starting to accumulate 6 to 9 h after auxin was supplied and increasing 300-fold after 12 to 18 h. The parallel study of the auxin sensitivity of rolB-transformed protoplasts, as assayed by their electrical response to the hormone, showed that the auxin treatment generated an increase in sensitivity by a factor of up to 100,000, whereas in untransformed protoplasts the same auxin treatment induced an increase in auxin sensitivity that never exceeded 30- to 50-fold. This reflects a strong cooperative effect of auxin and rolB in transformed protoplasts. Surprisingly, the maximal increase in sensitivity was observed several hours before the maximal accumulation of rolB mRNA, suggesting that the dramatic control of auxin sensitivity by auxin in rolB-transformed protoplasts requires only low levels of rolB expression. Antibodies directed against ZmER-abp1, the major auxin-binding protein from maize, differentially altered the auxin sensitivity of the electrical response of rolB-transformed and normal protoplasts. This suggests that alterations of the auxin reception-transduction pathway at the plasma membrane of rolB-transformed protoplasts may account for their increased auxin sensitivity.

Phenotypical alterations observed in rolB-transformed plants have been proposed to result from a rise in intracellular free auxin due to a RolB-catalyzed hydrolysis of auxin conjugates(J.J. Estruch, J. Schell, A. Spena [1991] EMBO J 10: 3125-3128).We have investigated this hypothesis in detail using tobacco (Nicotiana tabacum) mesophyll protoplasts isolated from plants transformed with the rolB gene under the control of its own promoter (BBGUS 6 clone) or the cauliflower mosaic virus 35S promoter (CaMVBT 3 clone). Protoplasts expressing rolB showed an increased sensitivity to the auxin-induced hyperpolarization of the plasma membrane when triggered with exogenous auxin. Because this phenotypical trait was homogeneously displayed over the entire population, protoplasts were judged to be a more reliable test system than the tissue fragments used in previous studies to monitor rolB gene effects on cellular auxin levels. Accumulation of free 1-[3H]-naphthaleneacetic acid (NAA) was equivalent in CaMVBT 3, BBGUS 6, and wild-type protoplasts, Naphthyl-[beta]-glucose ester, the major NAA metabolite in protoplasts, reached similar levels in CaMVBT 3 protoplasts, reached similar levels in CaMVBT 3 and normal protoplasts and was hydrolyzed at the same rate in BBGUS 6 and normal protoplasts. Furthermore, NAA accumulation and metabolism in BBGUS 6 protoplasts were independent of the rolB gene expression level. Essentially similar results were obtained with indoleacetic acid. Thus, it was concluded that the rolB-dependent behavior of transgenic tobacco protoplasts is not a consequence of modifying the intracellular auxin concentration but likely results from changes in the auxin perception pathway.

The properties and function of Ca(2+)-activated K+ (KCa) and voltage-dependent K+ (IK) currents of rabbit coronary myocytes were studied under whole cell voltage-clamp conditions (22 degrees C). Inhibition of KCa by tetraethylammonium chloride (1-10 mM) or charybdotoxin (50-100 nM) suppressed noisy outward rectifying current elicited by 5-s voltage steps or ramp at potentials > 0 mV, reduced the hump of the biphasic ramp current-voltage relation, and shifted by less than +5 mV the potential at which no net steady-state current is recorded (Enet; index of resting membrane potential). Inhibition of steady-state inward Ca2+ currents [ICa(L)] by nifedipine (1 microM) displaced Enet by -11 mV. Analysis of steady-state voltage dependence of IK supported the existence of a "window" current between -50 and 0 mV. 4-Aminopyridine (2 mM) blocked a noninactivating component of IK evoked between -30 and -40 mV, abolished the hump current during ramps, and shifted Enet by more than +15 mV; hump current persisted during 2-min ramp depolarizations and peaked near the maximum overlap of the steady-state activation and inactivation curves of IK (about -22 mV). A threefold rise in extracellular Ca2+ concentration (1.8-5.4 mM) enhanced time-dependent outward K+ current (6.7-fold at +40 mV) and shifted Enet by -30 mV. It is concluded that, under steady-state conditions, IK and ICa(L) play a major role in regulating resting membrane potential at a physiological level of intracellular Ca2+ concentration, with a minor contribution from KCa. However, elevation of intracellular Ca2+ concentration enhances KCa and hyperpolarizes the myocyte to limit Ca2+ entry through ICa(L).

Total protein from N. tabacum cell cytosol, partially purified by ammonium sulphate precipitation, contains a 52 kDa protein that tests positive for mineralocorticoid receptor (MCR)-like activity by Western blots, immuneprecipitation and photochemical cross-linking. Binding of RU 26752, a ligand specific to the MCR, suggests 100 fmol steroid bound per mg of tobacco protein; this is equivalent to about 6000 sites per cell assuming a 1/1 stoichiometry. Northern blots of total RNA from isolated tobacco cells, hybridized with a probe specific to mammalian MCR, revealed 2.8 kb and 2.2 kb transcripts, whereas the poly A mRNA was resolved as a prominent message of 1500 bp, preceded by a band of 2200 bp. This appears to be the first ever demonstration where a vascular plant is endowed with a pygmy protein similar to the much larger steroid hormone receptors in the animal world.

Objective: Depolarisation-induced Na+ influx through tetrodotoxin sensitive Na+ channels causes a rapid increase in intracellular Ca2+ concentration ([Ca2+]i). The Na+ current (INa) induced [Ca2+]i transients: (a) occur after blocking sarcolemmal Ca2+ channels with nisoldipine or D-600, (b) are inhibited by ryanodine, and (c) are dependent upon extracellular Ca2+. Thus the INa induced [Ca2+]i transients arise from sarcoplasmic reticular Ca2+ release triggered by Ca2+ entering the myocyte, following a transient rise in intracellular Na+ ([Na+]i), via a pathway distinct from sarcolemmal Ca2+ channels. Reverse mode Na(+)-Ca2+ exchange could provide such a pathway for Ca2+ entry. The aim of this study was to ascertain directly whether Na(+)-Ca2+ exchange mediates the INa induced release of Ca2+ from sarcoplasmic reticulum., Methods: Whole cell voltage and current clamped guinea pig ventricular myocytes dialysed with indo-1 were used; Ca2+ transients elicited upon activation of INa before and after inhibiting the exchanger were measured., Results: Following conditioning protocols to load Ca2+ stores, activation of INa during a test pulse to -50 mV from a holding potential of -80 mV elicited [Ca2+]i transients in caesium loaded myocytes superfused with solutions containing 2.5 mM Ca2+ and 5 microM nisoldipine. When extracellular Na+ was replaced with equimolar lithium, which carries current through Na+ channels but does not readily substitute for Na+ on the Na(+)-Ca2+ exchanger, or when Ni2+ (5 mM) or dichlorobenzamil (10 microM), which block the exchanger, were added to superfusion solutions, activation of INa failed to elicit [Ca2+]i transients. Lithium and Ni2+ also inhibited nisoldipine insensitive [Ca2+]i transients elicited by action potentials, indicating that INa and Na(+)-Ca2+ exchange may play a role in excitation-contraction coupling under physiological conditions., Conclusions: Activation of INa appears to promote Ca2+ entry into cardiac cells by stimulation of reverse mode Na(+)-Ca2+ exchange, triggering Ca2+ release from the sarcoplasmic reticulum.

The effect of metabolic inhibition on macroscopic and single-channel K+ currents in isolated rabbit portal vein myocytes was investigated by patch-clamp technique. Depression of adenosine triphosphate synthesis was produced by 2-deoxy-D-glucose (10 mM) and either cyanide (2 mM) or dinitrophenol (50 microM). Outward quasi-steady-state current evoked by a ramp protocol and outward time-dependent current during step depolarizations were increased during metabolic inhibition. The reversal potential for quasi-steady-state current shifted negatively toward equilibrium potential of K+ during treatment consistent with a role for K+ conductance and hyperpolarization of membrane potential. The macroscopic K+ current affected was 1) voltage dependent, 2) inhibited by intracellular Ca2+ chelation and low tetraethylammonium ion (1 mM) but unaffected by 4-aminopyridine (2 mM), and 3) associated with a rise in intracellular Ca2+ assessed by indo 1. Metabolic inhibition caused an increase in voltage-dependent large-conductance K+ channel (120-130 pS) activity in cell-attached patches of myocytes bathed in physiological solution (140 mM K+ in pipette). The channels were blocked in a flickery fashion by tetraethylammonium ion (0.5 mM) and inhibited with charybdotoxin (100 nM). We conclude that metabolic inhibition increases the activity of large-conductance Ca(2+)-activated K+ channels in vascular smooth muscle.

Corpora lutea from Sprague-Dawley rats that were orally administered 0.0 (control), 1.0, 10.0, and 100.0 mg/kg hexachlorobenzene (HCB) for 21 days were analyzed by electron microscopy. Granulosa lutein cells (GLC) from animals of the 10.0 mg group showed differences from the cells of animals that served as the controls. Golgi complexes and smooth endoplasmic reticulum appeared more conspicuous, possibly due to dilation resulting from hyperactivity. Free polysomes seemed more prominent in the cells of the 10.0 mg group. The GLC architecture from animals of the 1.0 and 100.0 mg groups was similar to that of the corresponding cells in the control group. Since smooth endoplasmic reticulum is involved in the synthesis of steroid hormones, and that free polysomes are engaged in synthesis of cytoplasmic proteins, it is suggested that HCB at a dose of 10.0 mg/kg given for 21 days may alter the synthetic activity of the GLC of the rat.

1. In order to assess the role of different ionic currents in hypoxia-induced action potential shortening, we investigated the effects of blockers of voltage-dependent and ATP-sensitive K(+)-channel on the membrane potential of hypoxic rabbit hearts and papillary muscles. The response to blocking of the inward rectifier was studied at three external K+ concentration: 2.5, 5, and 7.5 mM. 2. Hypoxia produced a progressive decline in action potential duration (APD) that levelled off after 15 to 20 min. Steady state APD values at 25% and 95% repolarization (APD25 and APD95) were 26.0 +/- 1.9% and 42.2 +/- 2.4% of controls respectively. 3. Tetraethylammonium (TEA, 10 mM) delayed but did not reduce APD shortening at the steady state. 4. Blocking of IK1 with a mixture of 0.2 mM Ba2+ and 4 mM Cs+ lengthened APD in normoxia and prevented APD95 shortening in hypoxia. The APD25 shortening was significantly attenuated at all [K]o. 5. Glibenclamide (Glib, 30 microM) did not prevent APD shortening, but produced a progressive action potential (AP) lengthening after 15 min of hypoxia. Steady levels of 48 +/- 3.5% and 62 +/- 5.0% of controls for APD25 and APD95 respectively were reached after 45 min. 6. The relation between APD25 and pacing rate was determined in normoxic and hypoxic papillary muscles and the effects of 2 mM 4-aminopyridine (4-AP) were examined. Hypoxia attenuated the APD25 shortening currently observed when the stimulation rate was lowered from 1 to 0.1 Hz without altering the plateau reduction occurring at frequencies above 2 Hz. These effects were potentiated by 4-AP.7. Our data suggest that the accelerated AP repolarization in hypoxic rabbit myocardium represents a delicate balance of several outward currents: IKI, IK-ATP. and at least one yet unidentified current component rather insensitive to changes in [K]o and to K+ channel blockers.

A mechanism capable of eliciting SR Ca2+ release independent of Ca2+ entry through voltage-gated Ca2+ channels was investigated using whole-cell voltage- and current-clamped guinea pig ventricular myocytes dialyzed with the Ca2+ indicator, Indo-1. Depolarization-induced Na+ influx through TTX-sensitive Na+ channels caused a rapid, transient increase in intracellular Ca2+ concentration ([Ca2+]i). The INa-induced [Ca2+]i transients (a) occur after blocking voltage-sensitive sarcolemmal Ca2+ channels with nisoldipine or D-600, (b) are inhibited by ryanodine, and (c) are dependent upon extracellular Ca2+. These results indicate that the INa-induced [Ca2+]i transients arise from SR Ca2+ release triggered by Ca2+ entering the myocyte, after a transient rise in [Na+]i, via a pathway distinct from sarcolemmal Ca2+ channels. One such pathway for Ca2+ entry into cardiac cells is reverse-mode Na(+)-Ca2+ exchange. Depolarization-induced Na+ influx failed to elicit Ca2+ transients when extracellular Na+ was replaced with equimolar lithium, which carries current through Na+ channels but does not readily substitute for Na+ on the exchanger. This result provides direct evidence that Ca2+ entry via reverse-mode Na(+)-Ca2+ exchange mediates the INa-induced SR Ca2+ release. Lithium also inhibited nisoldipine-insensitive [Ca2+]i transients elicited by action potentials indicating that INa and Na(+)-Ca2+ exchange may play a role in EC coupling under physiological conditions. Taken together, the results suggest that depolarization-induced Na+ influx through Na+ channels can trigger SR Ca2+ release in cardiac myocytes by activating Ca2+ influx via reverse-mode Na(+)-Ca2+ exchange. The INa-induced release of Ca2+ from SR may partially account for the positive inotropic effects of cardiac glycosides and the negative inotropic effects of antiarrhythmic drugs that block Na+ channels.

The role of sodium-calcium exchange at the sarcolemma in the release of calcium from cardiac sarcoplasmic reticulum was investigated in voltage-clamped, isolated cardiac myocytes. In the absence of calcium entry through voltage-dependent calcium channels, membrane depolarization elicited release of calcium from ryanodine-sensitive internal stores. This process was dependent on sodium entry through tetrodotoxin-sensitive sodium channels. Calcium release under these conditions was also dependent on extracellular calcium concentration, suggesting a calcium-induced trigger release mechanism that involves calcium entry into the cell by sodium-calcium exchange. This sodium current-induced calcium release mechanism may explain, in part, the positive inotropic effects of cardiac glycosides and the negative inotropic effects of a variety of antiarrhythmic drugs that interact with cardiac sodium channels. In response to a transient rise of intracellular sodium, sodium-calcium exchange may promote calcium entry into cardiac cells and trigger sarcoplasmic calcium release during physiologic action potentials.

Studies in vitro suggest that pregnancy induced hypertension, or toxemia, is associated with decreased placental prostaglandins E (PGE) levels. To validate this observation in vivo PGE were measured weekly in 24h urine collections, in a prospective cross-sectional study in 9 women from 7 to 40 weeks. In addition, urinary PGE levels were also measured in 28 hospitalized pregnant women with either chronic hypertension or toxemia. Prostaglandins E were measured by radioimmunoassay after organic extraction and silicic acid separation. Urinary PGE levels during pregnancy (normotensive and chronic hypertensive women) were significantly elevated than those of the non-pregnant state. Mean urinary PGE levels in toxemic patients were significantly decreased compared to those of normal pregnancy and patients with chronic hypertension but they were similar to the mean levels of the non-pregnant state. One fetal death attributed to aggravation of chronic hypertension and one eclampsia were associated with undetectable levels of maternal urinary PGE. In conclusion, normal pregnancy is associated with a significant increase of urinary PGE; chronic hypertension and occurrence of toxemia are associated with significant diminution of urinary PGE excretion.

The influence of external potassium Ko and tetraethylammonium on the cellular K content of hypoxic myocardium was investigated. Perfused rabbit hearts were submitted to 60 min hypoxia in medium containing 5 mM K throughout or either low (1.5 mM) or high (10 mM) K during the last 20 min of hypoxia. Paced electrical activity (2.5 Hz) was kept throughout the experiments. Tissue samples excised from the left ventricle were analyzed for total water, inulin space, and Na and K content. Lowering Ko to 1.5 mM increased both K loss and Na accumulation. Addition of 3.5 mM RbCl under these conditions reversed Na accumulation to levels found for hypoxia in normal medium but did not modify the cellular K loss. Tetraethylammonium (10 mM) did not alter Na accumulation but partly prevented the decrease in K content produced by hypoxia. A similar effect was observed by increasing Ko to 10 mM. At this high Ko prolongation of hypoxia did not enhance K loss. Abolition of electrical activity by TTX in a high K solution prevented K loss and reduced the sodium content. These results are consistent with the view that voltage-dependent channels are implicated in the K loss induced by hypoxia or ischemia. Furthermore, they indicate that the K loss may be modulated by external K because of the influence of the electrochemical gradient on passive K efflux and thus provide an explanation for the existence of a plateau in the early extracellular K accumulation observed during cardiac ischemia.

The electrophysiological and mechanical properties of the vasodilator minoxidil sulfate (MNXS) were examined in isolated smooth muscle cells and strips from rabbit portal vein. At micromolar concentrations, MNXS inhibited norepinephrine (0.1-1.0 microM)-induced contractions in isolated muscle strips. In isolated cells, norepinephrine caused a dose-dependent depolarization of the resting membrane potential, which was significantly attenuated by MNXS (5 microM); MNXS alone caused a hyperpolarization of the membrane potential. This hyperpolarization was insensitive to Na+-K+ pump blockade by ouabain, but was inhibited by the K+ channel antagonist, tetraethylammonium (20 mM). In voltage-clamp experiments, a resting (background) conductance associated with the resting membrane potential was identified. This conductance, which previously has been shown to be reduced by Ba2+ as well as tetraethylammonium, was increased by MNXS (2 microM). In additional experiments, whole-cell L-type Ca2+ currents were inhibited by micromolar concentrations of MNXS. These experiments show that concentrations of MNXS that inhibit norepinephrine-induced contractions promote K+ conductance and inhibit Ca2+ entry through voltage-dependent Ca2+ channels in vascular smooth muscle cells. These electrophysiological effects of MNXS may be responsible for the vasorelaxant effects of the drug observed in vitro and in vivo.

The upstroke of the ventricular action potential in the rabbit consists of two depolarizing components with different rates of rise. The effects of hypoxia on the resting potential (RP); the upstroke phases (I and II) and the maximum rate of rise of phase I (V max) were studied at different external K concentrations (K0). Perfused hearts were submitted to N2-equilibrated media containing 1.5 to 10 mM K0. Exposure of oxygenated hearts to different K0 changed the regenerative response from a fast rising action potential at 1.5 mM K0 to a depressed fast response at 7.5 and 10 mM K0. Hypoxia decreased the action potential amplitude (APA) at all K concentrations. In K0 less than or equal to 5 mM the reduction of APA was due to a decrease in the amplitude of phase II of the upstroke but the maximum rate of rise (V max) did not change. In contrast, phase I of the upstroke was markedly depressed by hypoxia in high K0, but phase II was unmodified and its V max compared well with values reported for other normoxic cardiac cells. Hyperkalemia per se did not slow conduction during normoxia but increased conduction time in hypoxia. The resting potential of hypoxic cells was closer to the K equilibrium potential than in the control. The RP v. Ko/Ki relation suggested that electrogenic Na extrusion persists in hypoxia. The electrogenic fraction of the resting potential as determined from pump inhibition with 10(-4) M ouabain amounted to -6 mV. Our results did not indicate whether the differential effects of hypoxia on the upstroke components were potential dependent or were related to direct effects of K+ on the ionic currents that determine the action potential. The persistence of phase II during hypoxia in partly depolarized cells may assure the maintenance of propagated electrical activity under conditions that are likely to be encountered in vivo during cardiac ischemia.

1. Single smooth muscle cells isolated from rabbit portal vein were voltage clamped at room temperature using the whole-cell configuration of the patch-clamp technique. These cells exhibited a mean resting potential of -47.9 mV and a mean input resistance of 376 M omega. 2. Using small tip diameter micropipettes (to avoid dialysis of the cells), depolarizing voltage-clamp pulses from a holding potential of -50 mV elicited two distinct outward currents: a quasi-instantaneous background current and a time-dependent current that did not appear to inactivate (delayed rectifier). Upon return to the holding potential, an outward tail current decaying back to the holding current was observed. 3. The time course of development of the tail current as estimated from envelopes of tail current protocols followed the kinetics of activation of the delayed rectifier elicited during the preceding test pulse. The tail current reversed close to the equilibrium potential for K+ ions indicating that it is mainly carried by potassium ions. 4. Using large tip diameter micropipettes to internally dialyse the cells (EGTA = 0.1 mM; ATP = 5 mM), two additional outward currents having transient kinetics were revealed: a smooth transient outward current (Ito) and spontaneous transient outward currents (STOCs). Ito was found to be mainly selective for K+ ions and exhibited voltage-dependent inactivation with half-maximal availability near -40 mV. 5. Removal of calcium from the bathing solution significantly reduced the background current and abolished both Ito and STOCs. The delayed rectifier current appeared to be insensitive to this procedure. The two types of transient outward currents were never recorded when EGTA was elevated to 5 mM inside the micropipette whereas the background and delayed rectifier currents were not affected. These results suggested that Ito and the spontaneous transient outward currents are activated by internal calcium. 6. External application of TEA (0.5-20 mM) blocked all four outward currents. Calcium replacement by barium significantly reduced the background current and Ito, and had small effects on the delayed rectifier current. When potassium was replaced with caesium (130 mM) and TEA (20 mM) inside the pipette, none of the outward currents described was ever observed. In about 60% of the cells dialysed with this solution a small inward Ca2+ current was revealed. 7. External application of caffeine (5 mM) abolished STOCs in cells in which this activity was present under control conditions. In cells lacking this type of activity under control conditions caffeine induced and later abolished this type of current.(ABSTRACT TRUNCATED AT 400 WORDS)

Blockers of the transient outward current (4-aminopyridine) and the Ca current (Co2+) as well as injection of polarizing current during the plateau were used to assess the role of these current systems as determinants of action potential duration at different pacing rates. Papillary muscles and ventricular trabecula were superfused with oxygenated Krebs solution at 33 degrees C and driven at a basic rate of 1 Hz. The effects of varying the frequency of stimulation between 0.1 and 4 Hz on action potential parameters were determined under control conditions and during exposure to 2 mM 4-aminopyridine, 1-3 mM CoCl2, or a mixture of 4-aminopyridine and CoCl2. The control relationship between action potential duration and pacing rate showed a maximum between 1 and 2 Hz. Under 4-aminopyridine, the plateau height and the action potential duration increased. The rate-dependent shortening of the action potential at frequencies below 1 Hz was reduced or abolished, and enhanced shortening was observed at rates above 1 Hz. Exposure to Co2+ reduced the action potential shortening at rates higher than 1 Hz. Both blockers, 4-aminopyridine and Co2+ were necessary to eliminate the rate-dependent changes of the action potential duration. Our results indicated that both the transient outward current and the inward calcium current determine the plateau height and duration for frequencies less than or equal to 2 Hz, whereas at higher rates, the Ca current plays a dominant role.

It has been reported that D 600 blocks the high-threshold Ca2+ channel (L-type) from the outside in isolated vascular and ileal smooth muscle cells of the rabbit (Y. Ohya, K. Terada, K. Kitamura, and H. Kuriyama. Pfluegers Arch. 408: 80-82, 1987). We have reinvestigated this hypothesis by comparing the effects of external and internal applications of D 600 and the permanently charged quaternary derivative D 890 on the whole cell Ca2+ current (Ica) recorded in vascular smooth muscle cells isolated from the rabbit portal vein. At low frequencies of stimulation (0.05 Hz), externally applied D 600 inhibited Ica in a dose-dependent fashion, with a complete block occurring at 10(-4) M. D 600 was approximately 1,000 times more potent than D 890 for causing inhibition of Ica using this protocol. During a train of stimulations at 0.5 Hz, D 600 (10(-6) M) produced a minor additional frequency-dependent block of Ica, as shown in other preparations. During superfusion with D 890 (10(-4) M), a similar protocol produced little if any decline in the amplitude of Ica. No evidence of block could be detected during intracellular dialysis of D 600 (10(-4) M). At the same concentration, intracellular application of D 890 produced a slow block of Ica. To test whether D 600 could be effectively dialysed using a patch micropipette, similar experiments were performed in cardiac ventricular myocytes. In this preparation, intracellular dialysis of D 600 induced a rapid inhibition of the Ica.(ABSTRACT TRUNCATED AT 250 WORDS)

The diffusion and electrogenic components of the resting potential of hypoxic ventricular muscle were separated by inhibition of the sodium pump with 10(-4) M ouabain. The response to varying external K concentrations (Ko) was studied. Arterially perfused rabbit hearts were submitted to 60 min hypoxia in Krebs solution containing 5 mM K throughout or to different external K concentrations during the last 20 min of hypoxia. For K concentrations between 1.5 and 10 mM, hypoxia did not change the resting potential except for a slight hyperpolarization in 7.5 mM K. The diffusion component of the resting potential did not differ from the resting potential at Ko less than 5 mM. An electrogenic potential of -3 to -6 mV was detectable at Ko values between 5 and 10 mM. The internal K concentration, Ki, was estimated from extrapolations to zero potential of the relation resting potential vs. Ko in normoxic and hypoxic hearts. These experiments revealed a decline of Ki of 16 mM with hypoxia. The variation of the diffusion potential with external K was fitted by a PNa:PK ratio five times lower than in normoxia. It has been concluded that an increase in K permeability and the persistence of electrogenic Na extrusion during hypoxia of rather short duration prevent membrane depolarization despite the myocardial K loss.