A MATE family multidrug efflux transporter pumps out fluoroquinolones in Bacteroides thetaiotaomicron.

We cloned a gene, bexA, that codes for a multidrug efflux transporter from the chromosomal DNA of Bacteroides thetaiotaomicron ATCC 29741 by using an Escherichia coli DeltaacrAB DeltaacrEF mutant as a host. Although the initial recombinant construct contained other open reading frames, the presence of bexA alone was sufficient to confer to the E. coli host elevated levels of resistance to norfloxacin, ciprofloxacin, and ethidium bromide. Disruption of bexA in B. thetaiotaomicron made the strain more susceptible to norfloxacin, ciprofloxacin, and ethidium bromide, showing that this gene is expressed in this organism and functions as a multidrug efflux pump. The deduced BexA protein sequence was homologous to the protein sequence of Vibrio parahaemolyticus NorM, a multidrug efflux transporter, and thus, BexA belongs to the multidrug and toxic compound extrusion (MATE) family.

Active efflux of norfloxacin by Bacteroides fragilis.

Norfloxacin was actively pumped out by Bacteroides fragilis, which is intrinsically resistant to most fluoroquinolones. Reserpine moderately inhibited the efflux. A one-step spontaneous mutant had increased resistance to norfloxacin, ethidium bromide, and puromycin, a result suggesting that the efflux is catalyzed by a multidrug pump with specificity similar to that of NorA/Bmr.

Hyperpolarization induced by sodium removal in rabbit sinoatrial node cells. Possible role of electrogenic sodium-calcium exchange.

Spontaneously active rabbit sinoatrial node (SAN) cells were bathed in K-free solution or in K-free ouabain (20 microM)-containing solution to depress the electrogenic Na(+)-K+ pump activity. In SAN cells exposed to K-free solution, the automatic action potentials ceased with gradual depolarization, followed by an eventual steady-state membrane potential of -32 +/- 1 mV. Under conditions where the Na(+)-K+ pump was blocked, removal of external Na+ produced a large and rapid hyperpolarization in the membrane potential and the membrane was hyperpolarized by 23 +/- 0.5 mV. When the external Na+ was lowered, Na+ was replaced by Li+. The Na-free hyperpolarization was not affected by applications of verapamil (4 microM), lidocaine (1 mM), and quinidine (50 microM), but was inhibited by either quinacrine (50 microM) or Cd2+ (10 mM), which are blockers of Na(+)-Ca2+ exchange. In the absence of external K+, replacement of external NaCl by sucrose produced a hyperpolarization similar to that seen in the replacement of external Na+ by Li+. In the K-free ouabain (20 microM)-containing solution, removal of external Na+ also produced a hyperpolarization, and the membrane potential dropped from -29 +/- 1 to -48 +/- 1 mV. The intracellular acidification due to NH4Cl removal after exposure to NH4Cl (20 mM) produced a decrease in Na-free hyperpolarization, which in the presence of ouabain was inhibited by the application of Cd2+ (10 mM). Removal of external Ca2+ nearly completely blocked Na-free hyperpolarization. It can be concluded that Na-free hyperpolarizations are related to the functioning of an electrogenic Na(+)-Ca2+ exchange mechanism.

Effect of strontium on action potential repolarization in rabbit sinoatrial node cells.

Using standard microelectrode techniques, the effects of strontium (Sr++) ions on action potential (AP) repolarizations in rabbit sinoatrial node (SAN) cells were studied. In a nominally magnesium-free, calcium-free solution, Sr++ (0.5-3.55 mmol) produced an early afterdepolarization (EAD) and a prolongation of the AP. Until now, no experimental evidence has been presented in favor of an EAD mechanism in SAN cells. Superfusion of the SAN cells with 0.5 mmol Sr++ solution containing 1-5 mmol magnesium (Mg++) or 0.5 mmol calcium (Ca++) did not induce the EAD. Increasing the extracellular Mg++ to 1-5 mmol did not affect the EAD induced by superfusion with 3.55 mmol Sr++ solution. Increasing the extracellular Ca++ to 0.5 mmol suppressed the 3.55 mmol Sr(++)-induced EAD. The presence of 0.3 mmol manganese (Mn++) suppressed the EAD induced by superfusion with 0.5 mmol Sr++ solution. The EAD did not occur in the presence of 1 x 10(-6) mol nitrendipine or 2 x 10(-6) mol diltiazem hydrochloride. The presence of 0.03 mmol Mn++ did not significantly alter the 0.5 mmol Sr(++)-induced EAD. These results suggest that a slow inward Sr++ current through the Ca++ channel has an important role in the development of the EAD.

Effects of lidocaine and verapamil on early afterdepolarizations in isolated rabbit sinoatrial node.

The effects of local anaesthetic anti-arrhythmic agents (lidocaine) and Ca antagonists (Verapamil) have been examined on the early afterdepolarizations (EADs) in isolated rabbit sinoatrial (SA) node. In a nominally calcium free and magnesium free solution, strontium (0.5-4.5 mM) produced an EAD in small pieces isolated from the SA node. The additional presence of 0.02-0.6 mM lidocaine did not abolish the strontium (0.5 mM)-induced EAD. 0.6 mM lidocaine produced an increase in EAD amplitude and then abolished a prolonged action potential (AP) associated with repetitive EADs. On the other hand, the addition of 4 micro M verapamil abolished the strontium (0.5 mM)-induced EAD but did not abolish the AP.It is concluded that under conditions when the AP is not abolished, EAD blockade by lidocaine is less effective than that by verapamil.

Magnesium as the regulator of electrical activity in rabbit sinoatrial node cells treated with ouabain.

Recent advances in the understanding of the role of magnesium (Mg2+) as a regulator of electrical activity in sinoatrial node cells are discussed. In the presence of 10 microM ouabain or in the absence of extracellular potassium (K+), spontaneous pacemaker activity ceased rapidly. Further reduction of extracellular calcium (Ca2+) produced a rhythmic activity. The slow regenerative potential (SRP) has been used to define the rhythmic activity. To determine the ionic mechanism of inducing SRP, the effects of blocker of an inward sodium (Na+) current mediated through Ca2+ channels, Mg2+ at 5.2 mM, were studied in a preparation of rabbit sinoatrial node. The increase in extracellular Mg2+ completely blocked the SRP. In conclusion, it is suggested that the slow inward Na+ current is the most important factor for the generation of the SRP depolarization and that Mg2+ is a major extracellular regulator of the SRP.

Influence of magnesium and extracellular calcium reduction on ouabain-treated sinoatrial node cells in rabbit heart.

The influence of the extracellular Mg on the slow regenerative potential induced by Ca removal was investigated by using rabbit SA node tissue treated with ouabain. When the SA node tissue was superfused with ouabain (10(-5) M), the preparation became quiescent. Further superfusion with Mg-free and Ca-free solution produced a small regenerative potential reaching 21 +/- 3 mV (n = 5). Superfusion with high Mg, Ca-free solution containing 5.19 mM Mg inhibited the regenerative potential. Superfusion of the ouabain-treated SA node tissue with Ca-free (2 mM EGTA) solution containing normal Mg or Mg-free, Ca-free (2 mM EGTA) solution produced a slow regenerative potential reaching 45 +/- 2 mV (n = 5) or 44 +/- 3 mV (n = 5). Increasing the external Mg to 3.6 mM or 6.18 mM in the Ca-free, EGTA solution caused a significant inhibition of slow regenerative potential. The addition of diltiazem (0.08 mM) blocked both normal action potential and slow regenerative potential induced by superfusion with Mg-free, Ca-free (2 mM EGTA) solution. These results suggest that Mg inhibits the development of the slow regenerative potential resulting from inward currents through the Ca channel.

Effects of ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid on rabbit sinoatrial node cells treated with cardiotonic steroids.

The effect of ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) was studied in rabbit sinoatrial node cells treated with ouabain. When isolated sinoatrial node preparations were bathed in the presence of 10(-5) M ouabain in order to depress electrogenic Na-K pump activity, action potentials ceased rapidly. Thereafter, spontaneous miniature fluctuations of less than 2 mV were observed at the resting potential level, while the membrane potential reached a value of about -31 mV. After exposure to the Ca-free 2 mM EGTA solutions, the miniature fluctuations decreased in frequency as well as in amplitude. However, successive exposure to the Ca-free EGTA solutions produced a short series of slow regenerative potentials. Peak amplitude of the slow regenerative potential increased from reduced miniature fluctuations to a maximum value of 46 +/- 3 mV (mean +/- S.E.; n = 10). Resting potential before initiation of the slow regenerative potential reached a value of about -34 mV. Also, Ca-free perfusion produced a small regenerative potential reaching 19 +/- 2 mV (n = 3). In Ca-free EGTA solution, reduction of external Na produced a decrease in amplitude of the slow regenerative potential when choline chloride or LiCl replaced NaCl. Replacement of 25% of the external Na with choline suppressed the slow regenerative potential. Replacement of 60% of the external Na with Li did not affect the slow regenerative potential. Further reduction of the external Na to 0% of the control condition produced a small regenerative potential. Replacement of Na with Li did not abolish the regenerative potential. In the presence of 4 x 10(-6) M verapamil Ca-free EGTA perfusion did not induce a slow regenerative potential. In the presence of 0.7 x 10(-6) M nifedipine Ca-free EGTA perfusion induced a small regenerative potential reaching 27 +/- 5 (n = 3). For low external pH (6.3) obtained with 5% CO2, the slow regenerative potential induced by Ca-free, EGTA perfusion was suppressed, and the regenerative potential amplitude decreased by about 16 mV. Application of 2.5 mM SrCl2 caused a decrease in slow regenerative potential, and the potential decreased by about 21 mV. The slow regenerative potential is not altered noticeably by 10(-5) M tetrodotoxin.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of sodium-calcium exchange current in single ventricular cells of guinea-pig.

1. The Na-Ca exchange current was investigated in single ventricular cells from guinea-pig hearts by combining the techniques of whole-cell voltage clamp and intracellular perfusion. 2. The membrane conductance was minimized by blocking Ca and K channels as well as the Na-K pump. Under these conditions, when Ca2+ was loaded internally by a pipette solution containing 430 nM-Ca2+, changing the Li+-rich external solution to a Na+-rich one induced a significant inward current. Applying external Na+ in the absence of internal Ca2+ did not appreciably change the current. 3. In contrast, perfusing 1 mM-external Ca2+ in the presence of internal Na+ which was loaded by a 20 mM-Na+ pipette solution, induced a marked outward current. Ca2+ superfusion in the absence of internal Na+ caused only a small current change. 4. The current-voltage relation of external-Ca2+- and external-Na+-induced current showed almost exponential voltage dependence as given by the equation i = a exp (rEF/RT), where a is a scaling factor that determines the magnitude of the current and r is a partition parameter used in the rate theory and represents the position of the energy barrier in the electrical field, which indicates the steepness of the voltage dependence of the current. E, F, R and T have their usual meanings. The value of a was 1-2 microA/microF and r about 0.35 for the Ca2+-induced outward current. At very positive or negative potentials, the current magnitude became smaller than expected from an exponential relation. 5. The current was blocked by heavy metal cations, such as La3+, Cd2+, Mn2+ and Ni2+ and partially blocked by amiloride and D600. 6. The temperature coefficient (Q10) value of the Ca2+-induced outward current was 3.6 +/- 0.4 (n = 4) at 0 mV and 4.0 +/- 0.9 at 50 mV in the range between 21 and 36 degrees C. 7. The outward current magnitude showed a sigmoidal dependence upon the external Ca2+ concentration with a half-maximum concentration, K1/2 of 1.38 mM and a Hill coefficient of 0.9 +/- 0.2 (n = 5). 8. Sr2+ could replace Ca2+ with K1/2 of 7 mM. Mg2+ and Ba2+, however, did not replace Ca2+. 9. The inward current component also showed a sigmoidal external Na+ dependence with K1/2 of 87.5 +/- 10.7 mM and a Hill coefficient of 2.9 +/- 0.4 (n = 6). 10. The reversal potential of the current was obtained near the values expected for 3 Na+:1 Ca2+ exchange.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of extracellular calcium removal on sino-atrial node cells treated with potassium-depleted solutions.

The effect of extreme calcium reduction was studied in rabbit sino-atrial node cells treated with K-free media. Ca removal (Ca = 0, EGTA = 6 mM) suppressed spontaneous miniature fluctuations elicited by exposure to the K-free media. Further, successive exposure to the Ca-free, EGTA solutions produced a propagated action potential and suppressed a contracture. The action potential was not noticeably altered by tetrodotoxin (TTX). In the presence of normal Ko, reducing the bathing calcium concentration with EGTA prolonged the duration of the action potential. The potential induced by Ca removal was similar to that observed in the K-free media. From these results, we have concluded that a drastic reduction in the extracellular Ca concentration produces a decrease in the overloaded Ca in the cell exposed to the K-free media, resulting in an increase in the amplitude of action potentials.

Effects of Rb and Cs on the electrogenic Na-pump in rabbit sinoatrial node cells.

The effects of Rb and Cs on the electrogenic Na-pump of the rabbit sinoatrial node cell were studied, using conventional microelectrode techniques. Hyperpolarizations induced by K or its substitutes were recorded from the preparations which were perfused previously with an extracellular K-free solution. The K-induced hyperpolarizations after K-depletion depended linearly on K-concentration in the range of 1.35 to 10.8 mM. When 2 mM Rb was used as an activator, a subthreshold oscillation appeared and the amplitude of the spontaneous action potentials in the Rb solution was larger than with the standard solution. As the concentration of Rb increased, the Rb-activated hyperpolarization increased, but the amplitude of the action potential decreased. In the case of Cs, the induced hyperpolarization was smaller than with K or Rb, and a transient recovery of pacemaker activity and gradual hyperpolarization preceded the true recovery in the standard solution. Since these hyperpolarization effects were not observed after the application of ouabain-containing K-free solution, the hyperpolarizations were considered to be electrogenic. A small hyperpolarization induced by activation of the external site of the Na-pump appears to be necessary for the immediate recovery of automatism from arrest at low resting potentials.