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D DiFrancesco

Publications and source records attributed to D DiFrancesco.

At least 73 records · Page 4Linked to original sources
PubMed1988

Muscarinic control of the hyperpolarization-activated current (if) in rabbit sino-atrial node myocytes.

1. The mechanism by which acetylcholine (ACh), by stimulation of muscarinic receptors, acts to inhibit activation of the hyperpolarization-activated 'pacemaker' current, if was investigated in isolated rabbit sino-atrial (SA) node myocytes. 2. Intracellular loading with GTP gamma S, a non-hydrolysable analogue of GTP, did not impair the ACh action on if, but made it irreversible. On the other hand, the ACh action on if disappeared after a few minutes of cell loading with GDP beta S, a GDP analogue known to bind to G-proteins and prevent their receptor-stimulated action. Furthermore, incubation of cells in a solution containing pertussis toxin (PTX) led to abolition of the if response to ACh. These results indicate that the inhibitory effect of ACh on if is mediated by G-proteins activated by muscarinic receptors. 3. Intracellular loading with phosphodiesterase (PDE) increased the rate of if current run-down, but did not abolish the inhibitory action of ACh on if. 4. Extracellular perfusion with isobutylmethylxanthine (IBMX), a PDE inhibitor, increased if activation by shifting the current activation range to more positive voltages, as inferred by a three-pulse protocol analysis; in the presence of IBMX, the inhibition of if by ACh was not abolished. 5. The ACh-induced if depression persisted also in cells loaded with cyclic GMP. In these cells, as in those loaded with PDE, the if run-down was fast. 6. Oxotremorine, a muscarinic agonist coupled to adenylate cyclase but not to phosphoinositide turnover in cardiac cells, simulated ACh in its inhibitory action on if. The above results rule against the ACh action being mediated by PDE or by phosphoinositide turnover. 7. To investigate the possible involvement of cyclic AMP as a second messenger in the ACh action on if, we loaded cells with cyclic AMP and IBMX; under these conditions the action of ACh disappeared within a few minutes of whole-cell recording. 8. In cells where the slow inward Ca2+ current (isi) was measured together with if, ACh was seen to depress both currents. 9. In cells superfused with forskolin, the if amplitude on stepping to the half-activation voltage range was enhanced as a consequence of a depolarizing shift of the activation curve; ACh was not effective on if following stimulation by forskolin, but strongly depressed in the same cell the if current stimulated to a similar degree by isoprenaline.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1987

Acetylcholine inhibits activation of the cardiac hyperpolarizing-activated current, if.

Acetylcholine (ACh) in low doses (0.1-1 microM) reversibly inhibits voltage-dependent activation of the "pacemaker" current, if, in isolated sino-atrial node cells. This action is brought about by a negatively-directed shift of the current activation curve, opposite to that due to catecholamines on the same current. The if inhibition is antagonized by atropine, indicating the involvement of muscarinic receptors. In cells incubated in pertussis toxin-containing solutions, if does not respond to ACh, suggesting that G-proteins mediate the ACh-induced if depression. Further, ACh can inhibit if following catecholamine-induced stimulation, but has a negligible effect on if stimulated by forskolin, a direct activator of adenylate-cyclase. Our results indicate that ACh acts on if by inhibiting basal adenylate-cyclase activity.

Acetylcholine↗
PubMed1987

The pacemaker current in the sinus node.

The cardiac 'pacemaker' current is recorded in isolated sino-atrial node cells during hyperpolarizations at voltages from -40/-50 mV to -100/-110 mV, which corresponds to the range where diastolic depolarization occurs. if is a hyperpolarizing-activated current, carried by Na and K, and in the pacemaker voltage range is inward. These properties allow if to serve as a tool to generate and control the 'pacemaker' depolarization phase of the action potential in sino-atrial node cells. The current if has long been shown to mediate the accelerating action of catecholamines in the heart. More surprisingly, recent experiments show that low doses of acetylcholine exert on if a strong inhibitory action. This new finding modifies the view that the slowing of pacemaker activity caused by acetylcholine is essentially due to activation of a K-current.

Action Potentials↗
PubMed1986

Properties of the hyperpolarizing-activated current (if) in cells isolated from the rabbit sino-atrial node.

Individual cells were isolated from the sino-atrial node area of the rabbit heart using an enzyme medium containing collagenase and elastase. After enzymatic treatment the cells were placed in normal Tyrode solution, where beating resumed in a fraction of them. Isolated cells were studied in the whole cell configuration. Action potentials as well as membrane currents under voltage-clamp conditions were similar to those in multicellular preparations. Pulses to voltages more negative than about -50 mV caused activation of the hyperpolarizing-activated current, if. Investigation of the properties of this current was carried out under conditions that limited the influence of other current systems during voltage clamp. The if current activation range usually extended approximately from -50 to -100 mV, but varied from cell to cell. In several cases, pulsing to the region of -40 mV elicited a sizeable if. Both current activation and deactivation during voltage steps had S-shaped time courses. A high variability was however observed in the sigmoidal behaviour of if kinetics. Plots of the fully-activated current-voltage (I-V) relation in different extracellular Na and K concentrations showed that both ions carry the current if. While changes in the external Na concentration caused the current I-V relation to undergo simple shifts along the voltage axis, changes in extracellular K concentration were also associated with changes in its slope. Again, a large variability was observed in the increase of I-V slope on raising the external K concentration. The current if was strongly depressed by Cs, and the block induced by 5 mM-Cs was markedly voltage dependent. Adrenaline (1-5 microM) and noradrenaline (1 microM) increased the current if around the half-activation voltage range and accelerated its activation at more negative voltages. Often, however, drug application failed to elicit any modification of if. Current run-down was observed in nearly all cells, although at a highly variable rate. It was accelerated by raising the extracellular K concentration but did not show a marked use dependence. Both the if activation curve and the fully activated I-V relation were affected by run-down, the former being shifted to more negative values along the voltage axis and the latter being depressed with no apparent change of the if reversal potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1985

Cadmium-induced blockade of the cardiac fast Na channels in calf Purkinje fibres.

The fast transient inward current elicited by depolarizations above about -60 mV in calf Purkinje fibres was found to be depressed by Cd in concentrations less than 1 mM. The Cd-sensitive current, which strongly depended on external Na, was recorded in the presence of 2 mM MnCl2 and was blocked by TTX, indicating that a contamination from slow Ca-dependent currents could be discounted. The current reduction caused by Cd was also observed in nominally Ca-free solutions. The Cd-induced depression of the fast Na current was not accompanied by changes in the current kinetic parameters, as revealed by comparing inactivation curves and peak current voltage relations at different Cd concentrations, and could be attributed to a voltage-independent channel blocking action. Half-blockade occurred at 0.182 +/- 0.06 mM (n = 4). Plots of peak current amplitude as a function of the Cd concentration showed that the cooperation of two Cd ions was required to block a single channel.

Animals↗
PubMed1984

Properties of the cardiac pacemaker (if) current.

The cardiac pacemaker if current, once thought to be a pure K+ current, is shown to be carried by both Na and K. It is inward and activating during hyperpolarizations negative to -50/-60 mV in normal conditions. Its kinetics are delayed during both activation and deactivation.

Animals↗
PubMed1984

Barium-induced blockade of the inward rectifier in calf Purkinje fibres.

The Ba-induced blockade of iK1 in calf Purkinje fibres was studied in the low concentration range (3-1,000 microM). The results showed that the blockade induced by hyperpolarizations was time-dependent, and that the rate of blockade increased with the Ba concentration and the negative pulse amplitude. At Ba concentrations higher than 1 mM the time course of iK1 blockade was not separable from the capacity transient. At these concentrations, and in a limited voltage range, the Ba-sensitive instantaneous current displayed a marked inward rectification and behaved with external K as expected for a pure K+ current, suggesting that under these conditions the only current affected by Ba was iK1. The Ba-sensitive current deceased with time during hyperpolarizations even when high external K was present, indicating that a voltage-dependent inactivation process could be at least in part responsible for the observed current decline.

Animals↗
PubMed1984

Characterization of the pace-maker current kinetics in calf Purkinje fibres.

Kinetics of the cardiac pace-maker current (if) were studied using high K+, low Na+ solutions under conditions where the current time course could be dissected from other components. Activation of if during relatively large negative pulses is S-shaped, and is approximated by an exponential function of time to the third power. Less-pronounced S-shaped activation occurs at potentials close to the middle of the activation curve (near -70/-80 mV). Here, allowing for the presence of a very slow component, the power required to fit the current activation approaches 1. The comparison between current activation and deactivation at the same potentials shows that although deactivation can be approximated by a single exponential, the two processes have a quite different time dependence, and this difference depends on the membrane potential. This behaviour is not compatible with Hodgkin-Huxley kinetics. While near the half-activation range the current decays with an apparently single exponential time course, at more positive potentials the current deactivation becomes sigmoidal. At least the third power of an exponential is required to fit its time course at potentials positive to about -40 mV. These data imply that both open and closed states correspond to several distinct channel configurations. The 'delay' in the current onset during a hyperpolarization is decreased by applying large, short hyperpolarizations before activation. Suitable pre-pulse durations and/or amplitudes can reduce the subsequent current activation to a single exponential. Records with and without a pre-pulse do not always superimpose. After the activation 'delay' has been removed by a suitable hyperpolarization preceding an activating pulse, the time course of its recovery can be studied by applying depolarizations of given amplitude and variable duration. The time course of the delay recovery does not seem to be linked to the time course of current deactivation recorded at the same voltage. Reduction of the activation 'delay' by conditioning pre-hyperpolarizations does not affect current decay during a subsequent depolarizing pulse. The current decay appears to depend only on the current amplitude reached before a deactivating pulse is applied. This, and the evidence in the preceding paragraph, suggest that the delay recovery and the current deactivation are independent processes. A reaction scheme is proposed, which has been developed on the basis of the experimentally determined kinetic properties of if. The channel model is composed of five gating subunits of three different types, not all independent in their movements.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Delayed activation of the cardiac pacemaker current and its dependence on conditioning pre-hyperpolarizations.

The activation time course of the pacemaker current if in Purkinje fibres during hyperpolarizations is s-shaped, which requires kinetics more complex than first order. Large, short hyperpolarizations preceding a fixed activating test pulse cause the current trace to shift to the left on the time-axis, as if a delay in if activation were removed, and eventually lead to a current trace which is a simple exponential and activates with no delay during a test pulse. Current traces before and after a relatively short and small prepulse will superimpose after a time translation. Further increasing prepulse amplitude and/or duration gives current traces that cannot be made to superimpose with the test current trace. The channel opening can be described by first order kinetics only if a delay in current activation is introduced.

Animals↗
PubMed1982

Block and activation of the pace-maker channel in calf purkinje fibres: effects of potassium, caesium and rubidium.

1. The effects of low concentrations of Cs(+) (0.01-3mM) on the fully activated I-V relation ī(f)(E) for the pace-maker current in calf Purkinje fibres have been investigated. The action of Cs(+) is two-fold: in the negative region of the I-V curve Cs(+) induces a channel blockade; on the other hand, at more positive potentials Cs(+) can produce the opposite effect, i.e. a current increase.2. Cs(+)-induced blockade is concentration- and voltage-dependent, as observed on other cation channels. Data in the far negative voltage range (about - 150 to - 50 mV) can be fitted by a simple block model (Woodhull, 1973), which gives a mean value of 0.71 for the fraction of membrane thickness (delta) crossed by Cs(+) ions before reaching the blocking site. The value of delta does not appear to be affected by either external Na or external K concentrations. Values for the dissociation constant of the blocking reaction at E = 0 mV (k(0)) are found in the range 0.5-3.7 mM. In the positive region of the ī(f)(E) relation the current depression caused by channel blockade vanishes. Unexpectedly, in this range the current can be observed to increase with Cs(+), and ī(f)(E) curves in different Cs(+) concentrations show cross-over.3. Changing external K(+) also produces similar cross-over phenomena. Investigation of this effect reveals that the increase in slope of the I-V curve on raising the external K(+) concentration follows Michaelis-Menten kinetics, and can be interpteted in terms of K(+)-induced channel activation. It is found that 44+/-6 mM-K(+) half-saturates the channel activating reaction.4. The Cs(+)-induced current increase is large in low-K(+) solutions and vanishes in high-K(+) solutions, suggesting a competition between Cs(+) and K(+) ions in their activating action. Increasing Na(+) also limits the Cs(+)-induced current increase.5. Rb(+) also blocks the i(f) channel, though less efficiently than Cs(+). The block caused by Rb(+) is, unlike that of Cs(+), nearly voltage-independent, and is explained by assuming that the blocking reaction occurs near the external mouth of the channel (mean value of delta is 0.05). The zero-voltage dissociation constant (k(0)) of the Rb(+)-blocking reaction ranges between 1.4 and 5.4 mM, and is lower in low-Na(+), high-K(+) solutions.6. A possible characterization of the i(f) channel which explains these results includes an inner ;blocking' site, to which external Cs(+) ions bind, blocking the channel, and a more external ;activatory' site, to which K(+), Cs(+), Rb(+) and possibly Na(+) ions bind. Binding of K(+) to this site induces a current increase either by modulating the channel, or actually by opening the channel itself. A similar mechanism can apply to Cs(+) and to Rb(+) binding.

Animals↗
PubMed1981

A new interpretation of the pace-maker current in calf Purkinje fibres.

1. The properties of the 'pace-maker' current iK2 of Purkinje fibres are investigated to verify whether its behaviour during voltage-clamp pulses is consistent with the view that iK2 is a K current deactivating on hyperpolarization in the range -50 to -100 mV. 2. During voltage-clamp pulses in the range positive to the apparent reversal potential (Erev), low concentrations of Cs depress the time-dependent current change without altering the time-independent current component. The total current becomes more outward in Cs, which on the assumption that Cs only affects the iK1 and iK2 channels implies that iK2 is inward in the voltage range analysed. 3. Ba strongly reduces the time-independent component due to iK1 and therefore limits the contribution of K depletion to the total current time course during hyperpolarizations. In the presence of barium a current reversal is not obtained even with large hyperpolarizations in normal Tyrode solution. If in Ba-containing solutions the external K concentration is raised from 3 up to 48 mM, iK2 greatly increases in the inward direction during hyperpolarizations in the range -51 to -101 mV, implying that it cannot be carried by K only. 4. In the presence of Ba, measurements of the membrane conductance due to iK2 indicate a channel-opening process during hyperpolarizations and a channel-closing process during depolarizations, in the K-concentration range analysed. 5. It is concluded that iK2 is not, as had been previously thought, a pure K current, but is rather an inward current activated during hyperpolarizations negative to about -50 mV. This provides further evidence for a possible identity between iK2 in Purkinje fibres and the current if in the SA node.

Animals↗
PubMed1981

A study of the ionic nature of the pace-maker current in calf Purkinje fibres.

1. Properties of the pace-maker current (if) in Purkinje fibres were studied in the presence of Ba, which by partially blocking the iK1 channel reduces K depletion during hyperpolarizing voltage-clamp pulses, and eliminates the main cause of distortion in the current time course. 2. On raising the external potassium concentration (Kb), the if fully activated current--voltage relation (if(E)) increases in the inward direction. In the range 3--36 mM--Kb and negative to -50 mV the current is inward, and no cross-over is observed. 3. In normal conditions, the reversal potential (Ef) for if lies in the voltage region positive to -50 mV, and can be observed on lowering the external sodium concentration (Nab). Ef shifts to the negative direction when Nab is decreased. Slopes ranging between 29 and 35 mV/decade are found for Nernst plots of Ef against Nab. Changing Nab in the range 140--4.4 mM causes the if(E) relation to undergo a simple shift along the voltage axis, without significant change in its slope. 4. Ef also depends on Kb, as can be observed in low Nab (35 mM), and shifts to the positive direction by about 26 mV for every 10-fold change in Kb. The fully activated slope conductance increases when Kb is increased. 5. It is concluded that Na and K both participate in carrying if. The slope of the fully activated if(E) relation increases with Kb, but is unchanged in different Nab, indicating that the channel conductance depends on Kb, but not appreciably on Nab.

Animals↗
PubMed1980

Separation of current induced by potassium accumulation from acetylcholine-induced relaxation current in the rabbit S-A node.

In a previous analysis on the rabbit S-A node the ACh-induced current was separated from the membrane current by subtracting the control from the current recorded in presence of ACh. In view of a possible interference of K accumulation processes, in the present paper the validity of the subtraction method was tested by studying the direct and indirect effects of ACh on the outward potassium current (iK). The following results were obtained. (1) The ACh-dependent channel activation and the iK-channel activation are different processes. (2) The activation curve of iK and the time constant of decay of iK current on return from a depolarizing clamp pulse were not affected by ACh. (3) In the majority of the experiments the presence of an accumulation component in the extra-current elicited by ACh could not be resolved. In a few cases the amplitude of the tail current was decreased in the presence of ACh. (4) In the case where iK was reduced, the fully-activated current-voltage relationship (i/K) was altered in the same way as that observed when the external K concentration was increased. In this case the difference between the control and the current recorded in the presence of ACh yielded a current component having a time constant similar to that of iK. We concluded that the decrease in the amplitude was due to an increase in K concentration in the clefts between the cells (K accumulation), associated with ACh application. No direct effect of ACh on the iK channel is apparent. (5) Because of the difference in the time constants of the relaxation current and the current change induced by accumulation the two processes could be clearly separated from each other.

Acetylcholine↗
PubMed1980

The contribution of potassium accumulation to outward currents in frog atrium.

1. Voltage-clamp experiments on frog atrial muscle were designed to distinguish effects due to K accumulation in extracellular spaces from those due to activation of K conductance mechanisms in the membrane. 2. The set of instantaneous current-voltage relations obtained at various external K concentrations following depolarization to about -10 mV for several seconds was found to be quite different from that obtained before the depolarization. Hence the process of increasing the extracellular K concentration cannot account for all the time-dependent changes in outward current during depolarization. 3. Although the instantaneous current-voltage relations obtained at different values of external K concentration before prolonged depolarization show the cross-over phenomenon (Noble, 1965), those obtained at the end of the depolarization did not show this feature. It is concluded that the current-voltage relations for the channels conducting the time-dependent K current do not show cross-over. 4. These results were used to construct a model involving both K activation and K accumulation. This model successfully reproduces the appearance of a very slow component in outward current decay tails which, when subtracted by semi-exponential curve-stripping leaves a component with the real time constant of conductance change. The model does not however reproduce the appearance of a fast decaying component without adding a second conductance mechanism, or assuming non-exponential decay of a single conductance mechanism. 5. It is therefore suggested that i chi, fast is not a perturbation of i chi, slow or of iK1 by the process of K accumulation. This conclusion is reinforced by the results of experiments showing that the relative magnitude of i chi, fast is not greatly changed by substantially increasing the external K concentration in order to reduce the proportionate effect of K accumulation on the K concentration.

Animals↗
PubMed1980

The time course of potassium current following potassium accumulation in frog atrium: analytical solutions using a linear approximation.

1. Regular perturbation theory was used to obtain analytical solutions for the time course of membrane current decay following voltage-clamp depolarizing pulses when both time-dependent K conductance mechanisms and the process of K accumulation in extracellular spaces are present. These solutions apply when the current and K concentration changes are small enough for linear relations to be assumed between current and K concentration. 2. In the case of a single Hodgkin-Huxley type conductance variable with time constant tau chi the presence of an accumulation process which, by itself, would produce a current decay with time constant tau alpha, induces the appearance of two infinite sets of components with decreasing time constants (1/(n+1/tau chi) and 1/(1/tau alpha + n/tau chi), where n is integer), and decreasing magnitudes. 3. The analytical solutions are used to investigate the range of conditions over which semi-exponential (curve-stripping) analysis of current decay tails may give useful information on the kinetics of current change. It is shown that, except at very large decay tail amplitudes, the method may give a good estimate of the true time constants of conductance decay even when the currents are assumed to be strongly dependent on external K concentration. 4. The method introduces error in current amplitude, but over the range in which curve-stripping gives useful results, the direct distortion of activation curves by variations in external K concentration is fairly small. However, as the current decay becomes grossly distorted in its time course by accumulation, so does the activation curve. The effects are very similar both to those obtained using numerical computation without linearization, and to those obtained experimentally. 5. Even with a large dependence of current on external K concentration the linear model does not reproduce i chi, fast as a perturbation of i chi, slow by K accumulation.

Animals↗
PubMed1980

Properties of the current if in the sino-atrial node of the rabbit compared with those of the current iK, in Purkinje fibres.

1. Properties of the 'pace-maker' current if in rabbit sino-atrial node have been investigated by voltage clamp of small preparations and compared with those of the iK2 current in the Purkinje fibre. Besides having a similar voltage range of activation and responding in a similar way to adrenaline, if resembles iK2 in other respects. 2. When external Na is reduced, if decreases proportionally. In 25% Na the time-dependent current change due to if disappears. 3. 20 mM-Cs completely abolishes it. 4. The time constant of if during a hyperpolarizing voltage-clamp pulse displays a relatively high temperature dependence. 5. In spite of the similarities between the two current systems, experiments in high K solutions (48 mM) rule out the possibility that the current change seen on a hyperpolarization reflects the decay of a pure K current. 6. From conductance measurements during onset of if it is deduced that if behaves as an inward current activated by hyperpolarizations.

Animals↗