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Biomedical subjects

D DiFrancesco

Publications and source records attributed to D DiFrancesco.

At least 55 records · Page 3Linked to original sources
PubMed1994

Some properties of the UL-FS 49 block of the hyperpolarization-activated current (i(f)) in sino-atrial node myocytes.

Block of the hyperpolarization-activated "pacemaker" current (i(f)) by the bradycardic agent UL-FS 49 was studied in isolated sino-atrial (SA) node myocytes. Using repetitive activation/deactivation protocols, micromolar concentrations of UL-FS 49 blocked i(f) in a dose-dependent fashion. Block development was slow, with time constants decreasing with drug concentration and ranging from 25.8 s at 10 microM to 75.5 s at 1 microM UL-FS 49. Block did not develop in cells held at -35 mV, at which voltage i(f) channels are closed, indicating that channels must open before blocking occurs. Apparently in contrast with the requirement of negative voltages for block development, block was relieved by hyperpolarization with a time course slower than current kinetics. Due to the hyperpolarization-induced block relief, current/voltage (I/V) relations in the presence of UL-FS 49 displayed inward-going rectification. Experimental data fitted the hypothesis that UL-FS 49 behaves as an "open channel" blocker of "single-ion" i(f) channels. Block occurs within the pore, at a distance of about 39% of the membrane thickness from its internal side.

Animals↗
PubMed1993

Properties of the hyperpolarization-activated current in rat hippocampal CA1 pyramidal cells.

1. Voltage and current clamp recordings were performed on CA1 rat hippocampal pyramidal cells using the patch clamp technique on "in vitro" slice preparations. 2. Hyperpolarizations from a holding potential of -35 mV elicited activation of the hyperpolarization-activated current (Ih) starting at voltages near -50 mV. 3. Ih recorded in voltage clamp conditions was blocked by external caesium (5 mM). 4. Raising the external K concentration from 4.35 to 24.35 mM sensibly increased the slope of the current-voltage (I/V) curve. Decreasing the external Na concentration from 133.5 to 33.5 mM depressed Ih without grossly altering the I/V slope. 5. The Ih fully activated I/V relation measured in the range -140 to -45 mV was linear with an extrapolated reversal at -17.0 +/- -1.6 (SE) mV. The current activation curve comprised the range between about -50 and -140 mV with a half-maximal activation at about -98 mV. 6. Perfusion of unclamped neurons with Cs (2 mM) hyperpolarized their resting potential by 3.8 +/- 0.2 mV and decreased the membrane conductance, as expected if Ih were activated at rest. Firing caused by depolarizing current steps was prevented by Cs-induced hyperpolarization, and could be restored by returning the membrane voltage to resting level by constant current injection. 7. The Cd-insensitive (medium-duration) afterhyperpolarization (AHP) elicited by a train of action potentials at -60 mV had an amplitude of 3.9 +/- 0.3 mV and was nearly fully abolished by 2 mM Cs (82.7 +/- 7.4%). Cs removed the depolarizing part of the afterhyperpolarization as expected if Ih activation was responsible for this phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1992

Reciprocal role of the inward currents ib, Na and i(f) in controlling and stabilizing pacemaker frequency of rabbit sino-atrial node cells.

Experiments and computations were done to clarify the role of the various inward currents in generating and modulating pacemaker frequency. Ionic currents in rabbit single isolated sino-atrial (SA) node cells were measured using the nystatin-permeabilized patch-clamp technique. The results were used to refine the Noble-DiFrancesco-Denyer model of spontaneous pacemaker activity of the SA node. This model was then used to show that the pacemaker frequency is relatively insensitive to the magnitude of the sodium-dependent inward background current ib, Na. This is because reducing ib, Na hyperpolarizes the cell and so activates more hyperpolarizing-activated current, i(f), whereas the converse occurs when ib, Na is increased. The result is that i(f) and ib, Na replace one another and so stabilize nodal pacemaker frequency.

Animals↗
PubMed1992

Expression and characterization of a canine hippocampal inwardly rectifying K+ current in Xenopus oocytes.

1. An inwardly rectifying potassium current expressed in Xenopus laevis oocytes injected with canine hippocampal poly(A)+ RNA was investigated with the two-microelectrode voltage clamp technique. 2. Xenopus oocytes injected with canine hippocampal poly(A)+ RNA expressed a current activated by hyperpolarization. This current contained an instantaneous and a time-dependent component. Both components were inwardly rectifying and could be blocked by extracellular Cs+ or Ba2+. 3. The expressed current was carried mainly by K+. Its reversal potential measured in different [K+]os could be fitted by the Nernst equation with a slope of -50.7 per tenfold change in [K+]o. Extracellular Cl- and Na+ made minimal contributions to the current. 4. The activation of the expressed current depended on both voltage and [K+]o. Activation started near EK and the activation curve shifted along the voltage axis in parallel with EK when [K+]o was altered. 5. The activation time constants of the expressed current also depended on both voltage and [K+]o. The voltage dependence of the time constants was bell-shaped and the peak value was at a potential 30-50 mV more negative than EK. The voltage dependence of the time constants shifted along the voltage axis when EK was changed. 6. The poly(A)+ RNA extracted from canine hippocampus was fractionated in a 10-31% linear sucrose gradient. The size of the mRNA required to express the inwardly rectifying current was estimated to be around 4 kb. 7. In conclusion, the expressed current is an inwardly rectifying potassium current. The canine hippocampal mRNA should be an excellent source for expression-cloning of the inward rectifier channel.

Animals↗
PubMed1991

The effects of manganese and barium on the cardiac pacemaker current, if, in rabbit sino-atrial node myocytes.

The isolation of ionic fluxes contributing to electric currents through cell membranes often requires block of other undesired components which can be achieved, among others, by divalent cations. Mn2+ and Ba2+ are often used, for example, to block Ca and K currents. Here we have investigated the effects of these two cations on the properties of the hyperpolarization-activated pacemaker current if, in rabbit sino-atrial node myocytes, as obtained by voltage clamp analysis. We find that 2 mM Mn2+ shifts the if activation curve by 3.2 +/- 0.3 mV towards more positive values. However, when 1 mM Ba2+ is also added, the positive shift is more than halved (1.3 +/- 0.2 mV). We find, too, that in the absence of blocking cations the ACh-induced if inhibition is slightly higher than in their presence. These results indicate that the alteration of if kinetic properties by Ba2+ plus Mn(2+)-containing solutions is minimal.

Acetylcholine↗
PubMed1991

Direct activation of cardiac pacemaker channels by intracellular cyclic AMP.

Cyclic AMP acts as a second messenger in the modulation of several ion channels that are typically controlled by a phosphorylation process. In cardiac pacemaker cells, adrenaline and acetylcholine regulate the hyperpolarization-activated current (if), but in opposite ways; this current is involved in the generation and modulation of pacemaker activity. These actions are mediated by cAMP and underlie control of spontaneous rate by neurotransmitters. Whether the cAMP modulation of if is mediated by channel phosphorylation is, however, still unknown. Here we investigate the action of cAMP on if in excised patches of cardiac pacemaker cells and find that cAMP activates if by a mechanism independent of phosphorylation, involving a direct interaction with the channels at their cytoplasmic side. Cyclic AMP activates if by shifting its activation curve to more positive voltages, in agreement with whole-cell results. This is the first evidence of an ion channel whose gating is dually regulated by voltage and direct cAMP binding.

Biological Clocks↗
PubMed1991

Intracellular calcium does not directly modulate cardiac pacemaker (if) channels.

A study on the cardiac pacemaker current if in inside-out macro-patches of sino-atrial (SA) node cells has recently demonstrated that if channels are directly activated by intracellular cAMP. Using the same preparation, here we investigate the possibility that internal Ca2+ ions play a role in the modulation of if channels. Our results indicate that Ca2+ ions do not have a direct activating effect on if.

Animals↗
PubMed1991

Block of the cardiac pacemaker current (If) in the rabbit sino-atrial node and in canine Purkinje fibres by 9-amino-1,2,3,4-tetrahydroacridine.

We have investigated the action of 9-amino-1, 2, 3, 4-tetrahydroacridine (THA) on the pacemaker current If in rabbit sino-atrial node myocytes and in canine Purkinje fibers. THA at concentrations in the range 3-300 microM blocked If in a voltage-independent manner, as revealed by measurements on the fully activated I/V relation for If. The dose/response relationship of the If maximal slope conductance (Gf) can be fitted by assuming a cooperative binding reaction where two THA molecules are required to block one If channel. Half-maximal block occurred at 18.2 microM in the sino-atrial node and 36.6 microM in Purkinje fibers. THA also affected the If kinetic properties. This was examined in the sino-atrial node where the current activation curve was shifted in the negative direction on the voltage axis (- 21 mV at 30 microM THA). The delayed rectifier current, IK, was also reduced by THA in sino-atrial node myocytes: at - 40 mV the IK fully activated value was decreased to 37% of its control value by 30 microM THA, with only a minor modification of the position of the activation curve at the same potential. Thus, although THA blocks If at a lower concentration than other known If-channel blockers [DiFrancesco (1982) J Physiol (Lond) 329:485-507], its action on the pacemaker current is not specific.

Animals↗
PubMed1991

The contribution of the 'pacemaker' current (if) to generation of spontaneous activity in rabbit sino-atrial node myocytes.

1. The contribution to the diastolic depolarization of the hyperpolarization-activated current, if, relative to other components was investigated in isolated rabbit sino-atrial (SA) node myocytes. 2. During the diastolic phase the membrane potential depolarized by 0.1096 +/- 0.014 V/s, which requires only about 3 pA of inward current in a cell with an average capacity of 30 pF. The problem of which ionic component is responsible for initiating the diastolic depolarization was investigated by analysing the composition and the properties of the net inward current in the diastolic range of voltages. 3. The measured instantaneous 'background' current activated during voltage clamp steps from a holding potential of -35 mV was outward positive to approximately -61 mV, and had a region of negative slope conductance from -45 to -35 mV. 4. The instantaneous component lost its rectifying behaviour in the presence of Ni2+ (100 microM) and nitrendipine (10 microM). These blockers of Ca(2+)-dependent currents modified the instantaneous I-V relation at voltages positive to -45 to -50 mV, thus implying that Ca2+ currents become important at less negative potentials than -50 mV, towards the very end of diastolic depolarization. 5. Possible errors introduced by voltage clamp analysis with the whole-cell method on the instantaneous current and on if measurement were evaluated. Leakage through the seal resistance caused the instantaneous I-V relation to be displaced in the inward direction at negative voltages. Correction for the seal leakage moved the reversal potential for the instantaneous current toward the negative direction from -61 to approximately -66 mV. Thus, no depolarization can be driven by the background current beyond -66 mV. 6. During voltage clamp analysis, lack of series-resistance compensation led to lack of intracellular voltage control, as was apparent using a second pipette on the same cell. This slowed activation of if and led to a 1.5- to 2-fold reduction of if size in the range -55 to -115 mV. Thus, uncorrected measurements of the instantaneous component and of if may concur to underestimate the role of if in pacemaking. 7. These results lead to the conclusion that in the SA node cells analysed, pacemaker activity is generated with the essential contribution of the hyperpolarization-activated current, if. Numerical computation of SA node cell activity using an extension of the DiFrancesco-Noble model shows that the if-activation hypothesis can account for the presence of spontaneous action potentials and their sensitivity to if changes.

Action Potentials↗
PubMed1991

Effects of protein kinase inhibitors on canine Purkinje fibre pacemaker depolarization and the pacemaker current i(f).

1. The effects of the protein kinase inhibitors H-7 and H-8 were investigated on diastolic depolarization of the action potential with microelectrodes and on the pacemaker current if with the two-microelectrode voltage clamp in canine cardiac Purkinje fibres. 2. Both 200 microM-H-7 and 100 microM-H-8 had no significant effect on the slope of diastolic depolarization but eliminated the actions of isoprenaline (1 microM). 3. We examined the actions of H-7 and H-8 on if in the presence and absence of isoprenaline. H-7 (200 microM) shifted the pacemaker current if in the negative direction on the voltage axis, whereas 100 microM-H-8 had no significant effect by itself. Both 200 microM-H-7 and 100 microM-H-8 can reverse or prevent the actions of isoprenaline (1-5 microM) on if. 4. We applied activators of the cyclic AMP cascade down-stream to the beta-receptor, to further evaluate where H-7 and H-8 might be exerting their effects. When exposing Purkinje fibres to an adenylyl cyclase activator (forskolin, 10-50 microM), a phosphodiesterase inhibitor (IBMX, 100 microM) and a permeable cyclic AMP analogue (8-chlorophenylthio-cyclic AMP, 200 microM-1 mM), the amplitude of if was increased. H-7 and H-8 at 100-200 microM eliminated each of these actions. 5. These results suggest that a phosphorylation process is involved in the modulation of the pacemaker current, if, in Purkinje fibres. The different actions of H-7 and H-8 on basal if suggest the hypothesis that other protein kinases, possibly protein kinase C, might also be involved in regulating basal phosphorylation of if in Purkinje fibres.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Acetylcholine reverses effects of beta-agonists on pacemaker current in canine cardiac Purkinje fibers but has no direct action. A difference between primary and secondary pacemakers.

We have investigated the actions of acetylcholine in the absence and presence of the beta-agonist isoproterenol in cardiac Purkinje fibers. beta-Agonists, like isoproterenol, increase the magnitude of the pacemaker current (If) in cardiac myocytes by shifting its activation voltage more positive on the voltage axis. We find that acetylcholine has no effect on If in the absence of isoproterenol. However, if If is first increased by beta-agonist stimulation, acetylcholine can then return If to control levels. This effect on If is exerted through muscarinic receptors since atropine prevents this action of acetylcholine. Functionally, this action of acetylcholine can guarantee the maintenance of ventricular pacemakers when there is high parasympathetic tone but can also prevent extra ventricular beats when sympathetic and parasympathetic tone are both high.

Acetylcholine↗
PubMed1989

Muscarinic modulation of cardiac rate at low acetylcholine concentrations.

Slowing of cardiac pacemaking induced by cholinergic input is thought to arise from the opening of potassium channels caused by muscarinic receptor stimulation. In mammalian sinoatrial node cells, however, muscarinic stimulation also inhibits the hyperpolarization-activated current (If), which is involved in the generation of pacemaker activity and its acceleration by catecholamines. Acetylcholine at nanomolar concentrations inhibits If and slows spontaneous rate, whereas 20 times higher concentrations are required to activate the acetylcholine-dependent potassium current (IK,ACh). Thus, modulation of If, rather than IK,ACh, is the mechanism underlying the muscarinic control of cardiac pacing at low (nanomolar) acetylcholine concentrations.

Acetylcholine↗
PubMed1989

Intracellular Ca modulates K-inward rectification in cardiac myocytes.

In cardiac myocytes, instantaneous inward rectification of the K-rectifying channel is abolished by removal of divalent cations from the intracellular environment and can be restored by addition of Mg ions at submillimolar concentrations, which has led to the proposal that Mg ions regulate inward rectification in these cells (Matsuda et al., 1987; Vandenberg, 1987; Matsuda, 1988). Here we report that Ca, too, reduces outward current flow through single inward rectifier channels in cell-free inside-out patches at much lower (submicromolar) concentrations. Intracellular Ca induces rectification by decreasing the probability of the main open channel state and by favouring the opening of channel substrates. Ca concentrations generating rectification are in the range of the Ca transient during activity, suggesting that Ca ions can contribute to K-rectification during cardiac muscle contraction.

Animals↗
PubMed1989

Internal and external K+ help gate the inward rectifier.

Recent investigations have demonstrated substantial reductions in internal [K+] in cardiac Purkinje fibers during myocardial ischemia (Dresdner, K.P., R.P. Kline, and A.L. Wit. 1987, Circ. Res. 60: 122-132). We investigated the possible role these changes in internal K+ might play in abnormal electrical activity by studying the effects of both internal and external [K+] on the gating of the inward rectifier iK1 in isolated Purkinje myocytes with the whole-cell patch-clamp technique. Increasing external [K+] had similar effects on the inward rectifier in the Purkinje myocyte as it does in other preparations: increasing peak conductance and shifting the activation curve in parallel with the potassium reversal potential. A reduction in pipette [K+] from 145 to 25 mM, however, had several dramatic previously unreported effects. It decreased the rate of activation of iK1 at a given voltage by several-fold, reversed the voltage dependence of recovery from deactivation, so that the deactivation rate decreased with depolarization, and caused a positive shift in the midpoint of the activation curve of iK1 that was severalfold smaller than the associated shift of reversal potential. These changes suggest an important role of internal K+ in gating iK1 and may contribute to changes in the electrical properties of the myocardium that occur during ischemia.

Animals↗
PubMed1988

Inhibition of the hyperpolarization-activated current (if) induced by acetylcholine in rabbit sino-atrial node myocytes.

1. The action of acetylcholine (ACh) on the hyperpolarization-activated ('pacemaker') current if was studied in single myocytes from the sino-atrial (SA) node region of the rabbit heart, where low doses of ACh slow spontaneous activity by prolonging the diastolic depolarization phase. 2. Besides activating an outward component at voltages positive to the K+ equilibrium potential (iK,ACh), ACh depressed the current if activated on hyperpolarization at concentrations in the range 0.03-1 microM. 3. The ACh-dependent if depression was dissected from modifications of iK,ACh by blocking iK,ACh with barium and was studied under conditions that minimized the interference of other current changes caused by ACh. 4. The study of if modification by ACh with three-pulse protocols and the measurement of fully activated I-V relations of if with and without ACh revealed that ACh acted on if by shifting the current activation range to more negative voltages, with no obvious alteration of the fully activated current amplitude. 5. The action of ACh on if was opposite to that caused by catecholamines. The presence of isoprenaline (IP) did not prevent ACh inhibition of if, nor did the presence of ACh prevent the if stimulation caused by IP. The effects of IP and ACh on if were additive. 6. The ACh-induced inhibition of if was reversed by addition of atropine and could be mimicked by muscarine, indicating that muscarinic receptors mediate it. The implications of these findings on the regulation of pacemaker activity by ACh is discussed.

Acetylcholine↗