Comparison of step and ramp voltage clamp on background currents in guinea-pig ventricular cells.

Isolated cardiac ventricular myocytes from guinea-pig were used to investigate the effect of voltage clamp protocols on background Na+ current (ib.Na) and inward rectifier current (i K1). Using long (4 s) clamp pulses and very long step clamps, the i-V relations showed that removal of Na+ reduces the amplitude and shifts the voltage dependence of i K1 (Spindler et al. 1998). Ramp clamps, however, gave more complicated results, with slower ramps more often giving the same results as steps and pulses. Both i K1 itself and, during faster ramps, other currents show hysteresis, so masking the steady-state changes. Using pulses, TTX had no effect on steady-state current. Small differences occurred in the ramps, but even at fast ramp speeds the effects are very much smaller than in Purkinje tissue. Only part of ib,Na is TTX sensitive and the effect does not occur in all cells.

Bee sting envenomation resulting in secondary immune-mediated hemolytic anemia in two dogs.

Immune-mediated hemolytic anemia secondary to bee envenomation developed in 2 dogs. Clinical signs included lethargy, hematuria, ataxia, and seizures; 1 dog died. Clinicopathologic data included nonregenerative anemia, spherocytosis, positive results for Coombs' test, and occult hematuria. Treatment included oral administration of corticosteroids at immunosuppressive dosages and supportive care. The surviving dog initially responded to corticosteroids, but hemolysis recurred as the dosage was tapered. Hemolysis resolved with prolonged administration of corticosteroids. Bee venom contains hyaluronidase, histamines, and hemolysins that cause toxic and hemolytic effects. Envenomation should be considered in any dog with hemolytic anemia in which other causes are ruled out and exposure to bees is known.

The effects of sodium substitution on currents determining the resting potential in guinea-pig ventricular cells.

It has recently been shown that a sodium background current, ib,Na, exists in cardiac muscle cells whose effect is to depolarize the membrane so that the resting potential, Vm, is positive to the potassium equilibrium potential, EK. In ventricular cells, where ib,Na is smallest, Vm is about 10 mV positive to EK (EK = -87 mV at 37 degrees C). Yet, replacement of Na+ ions by large impermeant cations does not cause the expected hyperpolarization. We have studied this problem in guinea-pig myocytes using a single microelectrode recording technique in combination with a rapid external solution switch. Cells depolarized < or = 0.5 mV from potentials between -80 and -73 mV and hyperpolarized up to 5 mV from potentials between -73 and -64 mV when 70 mM choline chloride or N-methyl-D-glucamine chloride were used to replace 70 mM Na+ in the bathing solution. Replacement by 70 mM lithium chloride, however, only caused hyperpolarization in very depolarized cells when the voltage change was much smaller. The changes were complete almost as soon as the solution change, i.e. within 250 ms, indicating that the actions are attributable to the external solution change rather than to secondary changes in intracellular concentrations. Patch clamp recording was used to investigate the mechanism involved. These experiments showed that the presence or absence of the inward rectifier current iK1 determines in which direction Na+ removal acts. In the absence of iK1 the changes are attributable to removal of ib,Na, whereas in the presence of iK1 the changes resemble the i(V) relation for iK1, implying that Na+ regulates iK1 in a way that can mask the changes in ib,Na. These results explain why removal of Na+ does not lead to hyperpolarization in ventricular cells as would be expected if changes in ib,Na were solely responsible. Computer reconstruction shows that the effects may be attributed to actions of sodium removal on the conductance and gating of iK1.

Hamburger-associated Escherichia coli O157:H7 infection in Las Vegas: a hidden epidemic.

OBJECTIVES: This study sought to determine whether a multistate fast food hamburger-associated outbreak of Escherichia coli O157:H7 infection involved Las Vegas residents as well and, if so, why public health officials had not detected it. METHODS: A matched case-control study was conducted among persons with bloody diarrhea and their healthy meal companions. Hamburger production, distribution, and cooking methods were reviewed. Unused hamburger patties were cultured, and E. coli O157:H7 isolates were characterized. Local laboratory stool culture practices were reviewed. RESULTS: Fifty-eight cases of bloody diarrhea were identified. Illness was associated with eating regular hamburgers (matched odds ratio [OR] = 9.0, 95% confidence interval [CI] = 1.02,433.4), but 25% of ill persons reported eating only jumbo hamburgers. Regular and jumbo hamburger patties yielded E. coli O157:H7 indistinguishable from the lone clinical isolate. No local laboratory cultured routinely for E. coli O157:H7 until after the outbreak. CONCLUSIONS: A large outbreak of E. coli O157:H7 infections escaped timely notice in Las Vegas because local laboratories did not culture for this pathogen. Health officials should encourage laboratories to screen at least all bloody stools on sorbitol-MacConkey medium.

Background inward current in ventricular and atrial cells of the guinea-pig.

Atrial and ventricular myocytes were exposed to Ca(2+)- and K(+)-free solutions containing blockers of gated channel and exchange currents. Replacement of external sodium by large organic cations revealed a background sodium current ib,Na. In atrial cells, the average conductance was 5.0 pS pF-1. In ventricular cells the conductance was 2.3 pS pF-1. Together with previous results, these figures reveal a strong gradient of background current density: sinus > atrium > ventricle. Replacement of sodium with inorganic cations showed that the channel selectivity behaves like an Eisenman group III/IV sequence, in agreement with previous results. The permeability of the channel to TMA was found to be pH dependent, suggesting that protonation of the channel is a factor determining permeation in addition to ionic size. The values of gb,Na obtained from these experiments are very similar to those assumed in computer modelling of cardiac cell electrical activity.

Sodium-calcium exchange during the action potential in guinea-pig ventricular cells.

1. Slow inward tail currents attributable to electrogenic sodium-calcium exchange can be recorded by imposing hyperpolarizing voltage clamp pulses during the normal action potential of isolated guinea-pig ventricular cells. The hyperpolarizations return the membrane to the resting potential (between -65 and -88 m V) allowing an inward current to be recorded. This current usually has peak amplitude when repolarization is imposed during the first 50 ms after the action potential upstroke, but becomes negligible once the final phase of repolarization is reached. The envelope of peak current tail amplitudes strongly resembles that of the intracellular calcium transient recorded in other studies. 2. Repetitive stimulation producing normal action potentials at a frequency of 2 Hz progressively augments the tail current recorded immediately after the stimulus train. Conversely, if each action potential is prematurely terminated at 0.1 Hz, repetitive stimulation produces a tail current much smaller than the control value. The control amplitude of inward current is only maintained if interrupted action potentials are separated by at least one full 'repriming' action potential. These effects mimic those on cell contraction (Arlock & Wohlfart, 1986) and suggest that progressive changes in tail current are controlled by variations in the amplitude and time course of the intracellular calcium transient. 3. When intracellular calcium is buffered sufficiently to abolish contraction, the tail current is abolished. Substitution of calcium with strontium greatly reduces the tail current. 4. The inward tail current can also be recorded at more positive membrane potentials using standard voltage clamp pulse protocols. In this way it was found that temperature has a large effect on the tail current, which can change from net inward at 22 degrees C to net outward at 37 degrees C. The largest inward currents are usually recorded at about 30 degrees C. It is shown that this effect is attributable predominantly to the temperature sensitivity of activation of the delayed potassium current, iK, whose decay can then mask the slow tail current at high temperatures. 5. Studies of the relationship between the tail current and the membrane calcium current, iCa, have been performed using a method of drug application which is capable of perturbing iCa in a very rapid and highly reversible manner. Partial block of iCa with cadmium does not initially alter the size of the associated inward current tail. When iCa is increased by applying isoprenaline, the percentage augmentation of the associated tail current is much greater but occurs more slowly.(ABSTRACT TRUNCATED AT 400 WORDS)

On the mechanism of isoprenaline- and forskolin-induced depolarization of single guinea-pig ventricular myocytes.

1. Isoprenaline (10 nM to 1 microM) and forskolin (0.6-100 microM) depolarized single guinea-pig myocytes studied in vitro. Under voltage clamp both agents caused an inward current to flow. 2. These effects were abolished by propranolol (100 nM) and the beta1-antagonist metoprolol (100-200 nM), but not by the beta2-agonist [corrected] salbutamol (1 microM). 3. The interaction of isoprenaline with forskolin, caffeine or isobutylmethylxanthine (IBMX) on current amplitude was as expected if all of these drugs were causing inward current by increasing intracellular levels of cyclic adenosine monophosphate (cyclic AMP). Low concentrations of forskolin (less than 600 nM) or IBMX (less than 20 microM) potentiated the effect of isoprenaline, whereas isoprenaline caused no further inward current in cells in which high concentrations of forskolin (600 nM-100 microM) or IBMX (20 microM-1 mM) were already evoking maximum inward current. 4. Isoprenaline-induced inward current was reduced 30-50% by acetylcholine (10-30 microM). This action of acetylcholine was blocked by atropine (100 nM). 5. The effect of isoprenaline on holding current was critically dependent on temperature. The onset of the current was delayed and its amplitude reduced as the myocyte was cooled from 37 degrees C to ambient temperature (22-24 degrees C). 6. Isoprenaline-induced inward current was not affected by the potassium channel blockers barium (2 mM) or tetraethylammonium (TEA; 10-20 mM). The amplitude of the inward current did not vary as a function of [K+]o. 7. The inward current was not affected by the calcium channel blockers cadmium 1 mM, or nifedipine (10 microM), or when internal calcium was reduced by including EGTA in the recording electrode filling solution. 8. The amplitude of the current was also unaffected by caesium (5 mM), which blocks the hyperpolarization-activated, non-specific channel if, or by strophanthidin (10 microM) which blocks the Na+-K+ pump. It was unchanged by substitution of external chloride by isethionate. 9. The inward current was absent when external sodium was replaced by the impermeant ion tetramethylammonium (TMA). 10. Isoprenaline- and forskolin-induced inward currents were associated with an increase in both membrane chord conductance and noise. The increase in conductance was most readily measured at potentials where the inwardly rectifying potassium channel, iK1, was small, or when iK1 was blocked by the addition of barium (2 mM).(ABSTRACT TRUNCATED AT 400 WORDS)

An isoprenaline activated sodium-dependent inward current in ventricular myocytes.

In the heart, catecholamines affect pacemaker activity by shifting the activation curve for the nonspecific inward current and increasing both the calcium current, and the delayed potassium current. We report here that in mammalian ventricle there is another mechanism that seems to involve a sodium-dependent inward current. This is elicited by agents that increase intracellular cyclic AMP concentration, such as the beta-adrenergic agonist isoprenaline, and is unaffected by agents which block the three currents listed above, but is absent when external sodium is replaced with tetramethylammonium. Most interestingly, the intracellular pathway(s) linking the beta-receptor(s) to activation of the Ca current and the Na-dependent current, which in both cases presumably involves the intracellular concentration of cAMP, differ, as isoprenaline causes a persistent augmentation of the calcium current whereas the Na-dependent current often fades. These effects of isoprenaline are antagonized by acetylcholine. In unclamped cells, the Na-dependent current depolarizes the membrane to the potential range at which repetitive firing occurs. It may therefore be involved in the generation of ventricular arrhythmias.

Acetylcholine and the mammalian 'slow inward' current: a computer investigation.

Although it is accepted that acetylcholine has two possible actions on cardiac muscle, one being the opening of time-independent potassium channels and the other a block of time-dependent calcium current, there is considerable doubt about which mechanism, if either, is chiefly responsible for modulating vagal control in any given region of the heart. It seems of particular importance to resolve this problem in the case of the mammalian sino-atrial node because this tissue not only receives the densest vagal innervation, but it is also the primary pacemaker. Recent studies on intact rabbit node (Shibata et al. 1985) have produced results which can be largely reproduced by using a computer model based on experimental studies of the sinus node current mechanisms (Noble & Noble 1984). We studied perturbations of node activity by modifying a number of these mechanisms, including an acetylcholine-activated K+ channel, iK,ACh, introduced into the model for the first time. Our findings lead us to the conclusion that the most important factor in modulating moderate chronotropic changes is a block of calcium current permeability. Complete vagal inhibition may also depend as much upon this effect as upon activation of iK,ACh current. Because the experimental studies under consideration here used beta-blockers to abolish the action of noradrenaline released by vagal stimulation, we suggest how a muscarinic block of isi might occur in vivo in the absence of beta-agonists.

The relationship between the transient inward current (TI) and other components of slow inward current in mammalian cardiac muscle.

When rabbit sino-atrial node preparations and isolated guinea-pig ventricular cells are subjected to Na-K pump blockade (either by reducing external K+ by a factor of 10: sinus node; or by the presence of 10(-7) M ouabain: ventricular cells) they develop oscillatory transient inward currents of the kind already recorded in Purkinje fibres and ventricular muscle strands. The time course of these transient currents, generally known as TI's, closely resembles that of the slow component of second inward current (isi,2) previously reported by us as occurring in rabbit sinus node when recorded near its threshold (-40 mV). Moreover, we have found that, under voltage clamp conditions, the 'envelope' of isi currents activated by depolarization from negative membrane potentials matches the outline of the iTI which develops during the initial hyperpolarization. In the sinus node, oscillations of iTI become smaller near O mV but are never flat and there is no clear cut reversal potential, whilst in ventricular cells oscillations and contractions cease at very positive membrane potentials (+35 mV) without the TI current ever becoming net outward. Replacing 75% of the external Na+ with Li+ reduces isi and iTI in the node by about the same proportion strongly suggesting that both are carried by a Na-Ca exchange mechanism. This idea is supported by reproducing the conditions of Na-K pump block in a computer model of the sinus node activity++, when oscillatory currents are generated by variations in activity of the Na-Ca exchange mechanism triggered by fluctuating levels of intracellular calcium. The same model when used to test the hypothesis that isi,2 might be carried by a non-specific ion channel showed that considerable distortion of the action potential would then occur. From the experimental and computed results it is concluded that the majority of isi,2 and iTI currents are both mediated by Na-Ca exchange.

Relationship between the transient inward current and slow inward currents in the sino-atrial node of the rabbit.

In low K+ (0.3 mM) solutions rabbit sinus node preparations show the oscillatory transient inward current, iTI, already recorded in these conditions in Purkinje and ventricular preparations. The time course of iTI closely resembles that of the slow component of the slow inward current (isi) previously reported by us (Brown, Kimura, Noble, Noble & Taupignon, 1984a) in rabbit sinus node, when recorded near its threshold (-40 mV). When the duration of voltage-clamp steps is varied there is a strong correlation between the 'envelope' of isi amplitudes on depolarization and the time course of iTI on hyperpolarization. Although oscillations of iTI become smaller near 0 mV, there is no potential at which the current records are completely flat, suggesting that there is no simple reversal potential. 75% substitution of Na+ by Li+ greatly reduces both iTI and slow isi in about the same proportion. Reducing the activity of the Na-K exchange pump by the amount expected in 0.3 mM-K+ solutions is sufficient to induce oscillatory iTI in a computer model of the sino-atrial node (Noble & Noble, 1984). The model reproduces the current as variations in the Na-Ca exchange current dependent on intracellular Ca2+ concentration ([ Ca]i). The model was also used to test the alternative hypothesis that the slow inward currents might be generated by [Ca]i-activated non-specific cation channels. It is shown that this would distort the shape of the repolarization phase of the action potential. It is concluded that the experiments and computations are consistent with the hypothesis that a large fraction of iTI and the slow component of isi could both be generated by Na-Ca exchange and that only a relatively small fraction might be generated by non-specific channels.

A model of sino-atrial node electrical activity based on a modification of the DiFrancesco-Noble (1984) equations.

DiFrancesco & Noble's (1984) equations (Phil. Trans. R. Soc. Lond. B (in the press.] have been modified to apply to the mammalian sino-atrial node. The modifications are based on recent experimental work. The modified equations successfully reproduce action potential and pacemaker activity in the node. Slightly different versions have been developed for peripheral regions that show a maximum diastolic potential near --75 mV and for central regions that do not hyperpolarize beyond --60 to --65 mV. Variations in extracellular potassium influence the frequency of pacemaker activity in the s.a. node model very much less than they do in the Purkinje fibre model. This corresponds well to the experimental observation that the node is less sensitive to external [K] than are Purkinje fibres. Activation of the Na-K exchange pump in the model by increasing intracellular sodium can suppress pacemaker activity. This phenomenon may contribute to the mechanism of overdrive suppression.

The slow inward current, isi, in the rabbit sino-atrial node investigated by voltage clamp and computer simulation.

The properties of the slow inward current, isi, in the sino-atrial (s.a.) node of the rabbit have been investigated using two microelectrodes to apply voltage clamp to small, spontaneously beating, preparations. Many of the experimental results can be closely simulated using the computer model of s.a. node electrical activity (Noble & Noble 1984) which has been developed from models of Purkinje fibre activity (Noble 1962; DiFrancesco & Noble 1984). Comparison of the computed reconstructions with experimental results provides a test of the validity of the modelling. Experiments using paired depolarizing clamp pulses show that inactivation of isi is calcium-entry dependent although, unlike the inactivation of Ca2+ currents in some other systems, it also shows some voltage-dependence. Re-availability (recovery from inactivation) of isi in s.a. node is much slower than inactivation at the same potential, showing that isi is not controlled by a single first order process. This very slow recovery from inactivation of isi in the s.a. node and the slow time course of its activation and inactivation at voltages near threshold (-40 to -50 mV) can be closely modelled by assuming that there are two components of 'total isi': a fast inward current, iCa,f' representing the 'gated' fraction and a second, slower, inward current component, iNaCa which, we propose, is caused by the sodium-calcium exchange that ensues when the initial Ca2+ -entry triggers the release of stored intracellular Ca2+. When repetitive trains of clamp pulses are given, a 'staircase' of isi magnitude is seen which can be increasing ('positive') or decreasing ('negative') according to the potential level and frequency of the pulse train given. When computer reconstructions of such staircases are made, it is found that the positive staircases (which, in contrast to negative staircases, imply that more complex processes than simple inactivation are present) can be closely simulated by a model which incorporates slower processes (suggested Na-Ca exchange current) in the total isi in addition to the gated current component.(ABSTRACT TRUNCATED AT 400 WORDS)

The ionic currents underlying pacemaker activity in rabbit sino-atrial node: experimental results and computer simulations.

The membrane currents underlying the pacemaker depolarization have been investigated in rabbit s.a. node preparations using the two-microelectrode voltage clamp technique. Many of the experimental results have been simulated using a computer model of s.a. node electrical activity. Changes of three time-dependent membrane currents which could contribute to pacemaker depolarization are found to occur in the relevant potential range: decay of the potassium current, iK, and activation of the inward current, if, and of the slow inward current, isi. The contribution of if activation to the pacemaker depolarization ranges from nil to an appreciable part depending on the preparation; when Cs (1 mM) blocks if, it nevertheless does not prevent pacemaking. In the model, holding the if activation variable at zero slows but does not stop pacemaking; doubling if conductance and shifting its activation curve by 15 mV in the positive direction causes a 15% faster rate of pacemaking. The slow time course of re-availability of isi must be allowed for when determining the isi threshold. A voltage clamp protocol designed to mimic as closely as possible an action potential followed by a pacemaker depolarization gives an estimate of isi threshold at the potential level of the last third of the pacemaker depolarization. This has been confirmed in experiments in which the voltage clamp was switched on at different points in the pacemaker depolarization. In the computer simulation, 'blocking' isi depolarizes the membrane to the zero current level (close to the potential reached at the end of a pacemaker depolarization) and stops the generation of action potentials. The decay of iK contributes to the pacemaker depolarization; with both our own model and that of K. Yanagihara, A. Noma and H. Irisawa, Jap. J. Physiol. 30, 841-857 (1980) 'blocking' iK decay abolishes pacemaker activity. Computations of extracellular K+ concentration changes compared with iK decay in a cylindrical model allow re-assessment of the interpretation of K+ concentration measurements during pacemaking made by J. Maylie, M. Morad and J. Weiss, J. Physiol., Lond. 311, 167-178 (1981).(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane currents underlying activity in frog sinus venosus.

1. The spontaneous electrical activity of small strips of muscle from the sinus venosus region of the heart of Rana catesbeiana was investigated using the double sucrose gap technique. The voltage clamp was used to record the ionic currents underlying the pace-maker depolarization and the action potential.2. The records of spontaneous electrical activity are very similar to those obtained from the sinus venosus using micro-electrodes. Moreover, the pace-maker activity is almost completely insensitive to tetrodotoxin (TTX) at 2.0 x 10(-6) g/ml., which suggests that the pace-maker responses can be classified as primary, as opposed to follower pacing.3. In response to short rectangular depolarizing voltage clamp pulses, only one inward current is activated. This current is almost completely insensitive to TTX but can be blocked by manganese ions. It appears, therefore, to be equivalent to the slow inward (Ca(2+)/Na(+)) current, I(si), of other cardiac tissues. The threshold for I(si) is near to the maximum diastolic potential, indicating that it must be activated during the pace-maker depolarization.4. Interruption of the normal pace-maker depolarization by rapid activation of the voltage clamp circuit reveals the time-dependent decay of outward current. This current reverses between -75 and -90 mV and, therefore, is probably carried mainly by potassium ions.5. Outward current decay is not a simple exponential, and Hodgkin-Huxley analysis suggests that two distinct components of outward current may be present. One of these is activated in the potential range of the pace-maker depolarization and the other at more positive potentials. Both outward currents reach full, steady-state activation at about zero mV, i.e. within the ;plateau' range of the sinus action potential.6. These results are compared with other recently published voltage clamp data from the rabbit sino-atrial node.7. A hypothesis for the generation of pace-maker activity is presented which involves (i) decay of outward current and (ii) activation of the slow inward current, I(si).

Changes in membrane currents in bullfrog atrium produced by acetylcholine.

1. A double sucrose-gap voltage-clamp technique has been used to study the effects of acetylcholine on the membrane currents in atrial trabeculae of the bullfrog, Rana catesbeiana. 2. The second, or slow inward (Ca2+/Na+) current, was found to be markedly reduced by concentrations of acetylcholine greater than approximately 2-0 X 10(-8)M. The resulting decrease in net calcium entry provides a straightforward explanation for the negative inotropic action of acetylcholine in atrial muscle. 3. Measurements of membrane resistance near the resting potential showed that relatively high doses of acetylcholine (approximately 10(-7) M) decrease membrane resistance by about twofold. This effect is shown to be the result of an increase in a time-independent background current which appears to be carried mainly by potassium ions. 4. Using appropriate pharmacological techniques, it has been possible to demonstrate: (i) that the peak slow inward current is reduced to about half its initial value before any significant increase in background current occurs; (ii) that even when a sufficient dose of acetylcholine to produce an increase in background current is used, the background current shows inward-going rectification and cannot account for the observed reduction in the slow inward current. 5. No consistent change was observed in the degree of activation of the time-dependent outward membrane currents after application of concentrations of acetylcholine which produced large decreases in the peak slow inward current. 6. These results are discussed in relation to previous electro-physiological and radioisotope studies of the mechanism of the negative inotropic effect of acetylcholine in cardiac muscle.