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Biomedical subjects

D DiFrancesco

Publications and source records attributed to D DiFrancesco.

At least 37 records · Page 2Linked to original sources
PubMed1997

Activation of f-channels by cAMP analogues in macropatches from rabbit sino-atrial node myocytes.

1. The action of the two diastereometric phosphorothioate derivatives of cAMP, Rp-cAMPs and Sp-cAMPs, was investigated on hyperpolarization-activated 'pacemaker' current (i(f)) recorded in inside-out macropatches from rabbit sino-atrial (SA) node myocytes. 2. When superfused on the intracellular side of f-channels at the concentration of 10 microM, both cAMP derivatives accelerated i(f) activation; their action was moderately less pronounced than that due to the same concentration of cAMP. 3. The measurement of the i(f) conductance-voltage relation by voltage ramp protocols indicated that both cAMP analogues shift the activation curve of i(f) to more positive voltages with no change in maximal (fully activated) conductance. 4. Dose-response relationships of the shift of the i(f) activation curve showed that both Rp-cAMPs and Sp-cAMPs act as agonists in the cAMP-dependent direct f-channel activation. Fitting data to the Hill equation resulted in maximal shifts of 9.6 and 9.5 mV, apparent dissociation constants of 0.82 and 5.4 microM, and Hill coefficients of 0.82 and 1.12 for Sp-cAMPs and Rp-cAMPs, respectively. 5. The activating action of Rp-cAMPs, a known antagonist of cAMP in the activation of cAMP-dependent protein kinase, confirms previously established evidence that f-channel activation does not involve phosphorylation. These results also suggest that the cAMP binding site of f-channels may be structurally similar to the cyclic nucleotide binding site of olfactory receptor channels.

Animals↗
PubMed1997

Differential control of the hyperpolarization-activated current (i(f)) by cAMP gating and phosphatase inhibition in rabbit sino-atrial node myocytes.

1. The actions of the phosphatase inhibitor calyculin A on the hyperpolarization-activated cardiac 'pacemaker' current (i(f)) were determined in single cells isolated from the sino-atrial (SA) node of the rabbit. 2. Cells were incubated for 8 min in Tyrode solution containing calyculin A (0.5 microM) and then superfused with normal Tyrode solution. The mean normalized i(f) measured in eight cells at mid-activation voltages during and after exposure to calyculin A increased maximally by 47% with a time constant of 466 s, a time much longer than that required for cAMP-mediated i(f) stimulation (about 8 s). 3. In two-pulse protocols, calyculin A treatment increased i(f) at full as well as at mid-activation voltages, indicating a higher i(f) conductance. 4. Measurement of the conductance-voltage (gf(V)) relation by voltage ramp protocols confirmed a conductance increase by calyculin A, with no significant change in the position of the activation curve on the voltage axis. Data pooled together from ramp and two-pulse protocols yielded a calyculin A-induced increase in fully activated i(f) conductance of 39.6 +/- 6.4% (n = 16 cells). 5. The positive and negative shift of i(f) voltage dependence in response to beta-adrenergic (1 microM isoprenaline) and muscarinic stimulation (1 microM acetylcholine), respectively, was preserved after the calyculin A-induced increase in conductance. The shift of the i(f) activation curve induced by 1 microM isoprenaline was significantly larger in calyculin A-treated cells (8.8 vs. 5.8 mV). 6. These data indicate that phosphatase inhibition increases i(f) in a manner distinct from the direct cAMP pathway and potentiates the beta-adrenergic-mediated i(f) modulation.

Acetylcholine↗
PubMed1997

Reduction of K+ uptake in glia prevents long-term depression maintenance and causes epileptiform activity.

Extracellular cesium causes synchronous, interictal-like bursting and prevents maintenance of long-term depression (LTD) in the CA1 hippocampal region. We have investigated the cellular mechanisms underlying cesium actions. Whole-cell recordings showed that brief (2 min) bath exposures to cesium caused pyramidal cell hyperpolarization associated with decreased membrane conductance attributable to blockade of an inward h-type current. After prolonged (>2 min) exposures, a late depolarizing response was observed; this effect was not associated with changes in cell membrane conductance. Recordings from interneurons revealed that Ih is expressed in a subpopulation of cells and that cesium effects on interneurons expressing Ih are comparable to those observed in pyramidal cells. Consistent with this effect, cesium decreased the early component of the IPSP recorded in pyramidal cells. Interneurons lacking Ih were not affected by cesium but developed a depolarizing response when drug applications were paired to orthodromic stimulation. We concluded that cesium actions on LTD and cesium-induced epileptiform activity were not attributable exclusively to its direct effects on neurons. Recordings from hippocampal slice astrocytes revealed that cesium interfered with glial electrical responses during LTD induction. Cesium blocked glial inwardly rectifying potassium channels and increased the amplitude and duration of stimulation-evoked [K+]out increases. Thus, the effects of cesium on CA1 synchronization and synaptic plasticity appear to be mediated predominantly by blockade of glial voltage-dependent potassium uptake.

Afferent Pathways↗
PubMed1997

The newborn rabbit sino-atrial node expresses a neuronal type I-like Na+ channel.

1. Newborn rabbit sino-atrial node (SAN) myocytes were recently found to express a tetrodotoxin (TTX)-sensitive Na+ current. We now report that the dose-response relation indicates that this SAN Na+ channel has unusually high TTX sensitivity, with half-maximal inhibition (26 +/- 5 nM) which is more typical of neuronal than cardiac tissue. 2. Additional characterization used mu-conotoxin GIIIA and Cd2+ as relatively selective blockers of the skeletal and cardiac isoforms, respectively. mu-Conotoxin GIIIA had no effect on the current recorded from SAN myocytes, but the Cd2+ sensitivity was unexpectedly high for a neuronal isoform (half-maximal inhibition = 185 +/- 8 microM). 3. Analysis of the time constant of inactivation did not reveal evidence of multiple inactivation processes, with the data well fitted by a single, relatively rapid exponential (inactivation time constant = 0.58 +/- 0.03 ms at 0 mV). 4. In situ hybridization with anti-sense cDNA probes was used to test for expression of neuronal type I, II and III Na+ channel isoforms. Myocardial cells in newborn SAN tissue exhibited clear hybridization to the type I, but not the type II or III probes. No hybridization was observed in adult SAN tissue with any of the three probes. 5. It is concluded that the newborn SAN expresses a neuronal type I-like Na+ channel isoform, and that this probably accounts for the unusual characteristic of high sensitivity to both TTX and Cd2+.

Animals↗
PubMed1997

Action of the hyperpolarization-activated current (Ih) blocker ZD 7288 in hippocampal CA1 neurons.

The effects of ZD 7288, a "bradycardic" agent, in young rat hippocampal slices in vitro were studied. ZD 7288 (1-1000 microM) reduced the hyperpolarization-activated current (Ih) in CA1 pyramidal neurons by a voltage-independent blocking mechanism. Under current-clamp conditions, the bradycardic agent (10 microM) caused membrane hyperpolarization (by 5.9 +/- 0.5 mV) and a reduction of membrane conductance (by 17.9 +/- 4.1%). These data are consistent with the block of an inward current which is active at rest. The drug-induced hyperpolarization depressed the cell's excitability by increasing the threshold current necessary to induce firing. When the drug-induced hyperpolarization was compensated for by injection of a tonic depolarizing current, ZD 7288 caused a reduction of the inhibitory post-synaptic potential (IPSP) in EPSP-IPSP sequences. Since Cs+, another known blocker of Ih, is able to reverse long-term depression (LTD) of the CA3-CA1 synapse in hippocampal slices, we tested the effect of ZD 7288 on synaptic transmission. We found that ZD 7288 did not significantly modify LTD, suggesting that Cs+-induced inhibition of LTD maintenance is not directly related to block of Ih.

Animals↗
PubMed1997

Properties and modulation of If in newborn versus adult cardiac SA node.

The hearts in newborn mammals have greater intrinsic beating rates, rates of diastolic depolarization, and sensitivity to autonomic stimulation than those in adults. The differences could be explained partly by altered properties of the hyperpolarization-activated current (If). To test this possibility, sinoatrial node myocytes from the hearts of newborn (9- to 10-day) and adult (>30-day) rabbits were isolated, and the If was examined with the perforated-patch-clamp technique. The fully activated current-voltage relationship yielded a larger slope conductance of If in newborn SA node myocytes (0.244 +/- 0.020 vs. 0.158 +/- 0.012 pS/pF), compatible with the more rapid diastolic depolarization. Activation curves of the If had similar midactivation voltages (newborn, -66.71 +/- 1.94 mV; adult, -66.33 +/- 2.60 mV), but the slope was significantly greater in newborns (inverse slope factor: newborn, -9.57 +/- 0.35 mV; adult, -11.34 +/- 0.54 mV). No differences in shifts of the If activation curve in response to maximal concentrations of acetylcholine (newborn, -9.70 +/- 1.8 mV; adult, -12.60 +/- 2.10 mV) and isoproterenol (newborn, 6.90 +/- 2.5 mV; adult, 5.3 +/- 1.5 mV) or in the total shift in response to these agonists (newborn, 16.60 +/- 3.30 mV; adult, 18.00 +/- 1.00 mV) were observed. The greater If density and steeper voltage dependence can contribute to both the greater heart rate and the greater sensitivity of the SA node to autonomic modulation in newborn animals.

Acetylcholine↗
PubMed1996

Modulation of the hyperpolarization-activated current (I(f)) by adenosine in rabbit sinoatrial myocytes.

BACKGROUND: Modulation of sinoatrial pacemaking by adenosine (Ado) in the absence of concomitant adrenergic stimulation (direct modulation) has been attributed to activation of a K+ conductance. In the present study, we evaluated the direct effects of Ado on the pacemaking current I(f) and tested their interaction with those of acetylcholine (ACh). METHODS AND RESULTS: Rabbit sinoatrial myocytes were patch-clamped at 35 degrees C in the presence of 1 mmol/L BaCl2 and 2 mmol/ L MnCl2, Ado (1 mumol/L) reversibly reduced I(f) by 33.1 +/- 5.7% of control (n = 5; P < .05). Ado (1 mumol/L) reversibly shifted I(f) midactivation potential by -6.63 +/- 1.18 mV (n = 4; P < .05). Fully activated I(f) conductance (0.262 +/- 0.037 versus 0.254 +/- 0.036 nS/ pF; n = 6, NS) and reversal potential (-17.35 +/- 0.99 versus -18.01 +/- 1.42 mV; n = 6, NS) were not changed by 10 mumol/L Ado. The Ado receptor antagonist 8-PST (10 mumol/L) reversed the effect of 0.3 mumol/L Ado by 64.9 +/- 4.2% (n = 6; P < .05). Ado maximally shifted the I(f) activation curve by -5.85 mV, with a half-maximal concentration of 0.0796 mumol/L (n = 93). The shifts in I(f) activation induced by Ado (0.3 mumol/L) and ACh (1 mumol/ L) separately were -4.89 +/- 0.05 and -8.84 +/- 0.51 mV, respectively; concomitant Ado and ACh superfusion shifted activation by -9.7 +/- 0.45 mV (NS versus ACh alone; n = 9). Threshold Ado concentrations dose-dependently reduced the rate of spontaneous pacemaker activity (eg, -18.8 +/- 3.4% at Ado 0.03 mumol/L). CONCLUSIONS: Submicromolar Ado directly inhibits I(f) and slows pacemaking in sinoatrial myocytes; the mode of I(f) inhibition is similar to that previously described for ACh. Thus, Ado may exert local modulation of sinus rate through signaling pathways similar to those used by ACh.

Acetylcholine↗
PubMed1996

A TTX-sensitive inward sodium current contributes to spontaneous activity in newborn rabbit sino-atrial node cells.

1. Single cells were isolated from the sinus node region of rabbits (2 days old to adult) to study the age-dependent contribution of the sodium current (iNa) to pacemaker activity. 2. Experiments were conducted in 50 mM Na(+)-Ca(2+)-free solution. All newborn cells (2-19 days) exhibited a TTX-sensitive, Mn(2+)-insensitive fast inward Na+ current (peak current density 115.5 +/- 11.9 pA pF-1 at 0 mV). Fifty per cent of young cells (20-40 days) possessed the current, but only one in ten adult cells. Current density decreased with development independently of cell capacitance. 3. Newborn cells exhibited a noticeable window current. With development, the position of the activation curve was shifted in the positive direction, while the inactivation was unaltered, resulting in reduced overlap of the two curves and hence less window current. 4. In newborn cells, 3 microM TTX significantly reduced all measured parameters of spontaneous action potentials, slowing rate by 63%. In contrast, there was no significant effect of TTX on rate or most of the same parameters in adult cells. 5. These results indicate that cells of the sinus node region exhibit a substantial TTX-sensitive current at birth. With development, both the density and frequency of occurrence of this current within the sinus node decrease, as does its contribution to automaticity.

Action Potentials↗
PubMed1996

Basal responses of the L-type Ca2+ and hyperpolarization-activated currents to autonomic agonists in the rabbit sino-atrial node.

1. The dose dependence of the cholinergic agonist acetylcholine (ACh) and the beta-adrenergic agonist isoprenaline (Iso) were determined for the hyperpolarization-activated current (If) and the L-type Ca2+ current (ICa,L) in single cells isolated from the rabbit sino-atrial (SA) node. 2. ACh inhibited If by a negative shift of its activation curve with a maximal effect of -9.9 mV; half-maximal effect was produced by 0.019 microM ACh. High ACh concentrations were required to inhibit ICa,L only partially (31% inhibition at 300 microM). 3. In contrast, If and ICa,L responded to Iso over a similar dose range, with concentrations for half-maximal enhancement of 0.0136 and 0.0070 microM, respectively. 4. The effects on spontaneous activity of ACh (range 0.001-0.03 microM) and Iso (range 0.001-1 microM) were investigated. ACh decreased the slope of diastolic depolarization at concentrations similar to those inhibiting If (> 50% at 0.03 microM). Iso enhanced diastolic depolarization at concentrations similar to those affecting both If and ICa,L (half-maximal effect at 0.027 microM). 5. In a ramp-clamp protocol simulating diastolic depolarization, the threshold for activation of inward nifedipine-sensitive current was -41.22 +/- 0.68 mV. Although enhancing ICa,L, Iso did not affect this threshold. 6. Half-maximal ACh concentrations for inhibition of automaticity and If are similar and are lower than the threshold concentrations for modulation of ICa,L; this argues against a role of ICa,L in direct muscarinic modulation of pacemaking. In contrast, modulation of If, ICa,L and automaticity occur at similar Iso concentrations. The difference between maximum diastolic potential (-61.95 +/- 0.93 mV) and the threshold for Iso-stimulated ICa,L (-39.54 +/- 1.03 mV) suggests that this current plays a role only at later stages of diastolic depolarization.

Acetylcholine↗
PubMed1996

Inhibition of the hyperpolarization-activated current (if) of rabbit SA node myocytes by niflumic acid.

The effects of the amphiphilic substance niflumic acid (NFA) were examined in myocytes isolated from the sino-atrial node of the rabbit heart. NFA (50 and 500 microM), for 30-60 s, produced a reversible negative chronotropic effect by reducing the rate of diastolic depolarization, suggesting an inhibitory effect on the hyperpolarization-activated pacemaker current (if). NFA (from 0.05 to 500 microM) inhibited if by modifying the current kinetics, without alteration of the conductance. This was shown by evidence indicating that: (1) NFA inhibited if during hyperpolarizing pulses to the mid-point of if activation but not at fully activating voltages; (2) the slope and reversal potential of the fully activated current/voltage (I/V) relation were not altered by NFA, indicating no change in slope conductance or ion selectivity; and (3) hyperpolarizing ramp protocols confirmed the lack of action of 50 microM NFA on the fully activated current and a shift of approximately -8 mV. Although similar to inhibition by acetylcholine (ACh), inhibition by NFA was only partly additive with the action of ACh and was not altered by atropine or pertussis toxin, both of which eliminated the action of ACh. The effect of NFA was present after stimulation of adenylate cyclase by forskolin and after inhibition of phosphodiesterase by isobutylmethylxanthine (IBMX). In cell-attached patch measurements, NFA applied externally did not affect if measured in the patch. Finally, application of NFA to the cytoplasmic side of excised patches did not alter the current in the absence or presence of adenosine 3',5'-cyclic monophosphate (cAMP). These results suggest an external, membrane-delimited action of NFA on if.

Animals↗
PubMed1996

Activation of the hyperpolarization-activated current (if) in sino-atrial node myocytes of the rabbit by vasoactive intestinal peptide.

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter found in extrinsic and intrinsic nerves of the heart. VIP can be released by vagal stimulation but, contrary to ACh, causes positive chronotropic effects as a result of binding to cardiac receptors which stimulate adenylate cyclase, and thus has been implicated in vagal tachycardias. Since the rate of diastolic depolarization of sinoatrial (SA) node myocytes depends on the hyperpolarization-activated current (if), which is directly activated by cytoplasmic cAMP, we studied the action of VIP on if in myocytes isolated from the SA node of the rabbit. VIP (0.65 microM) reversibly increased if at -65 mV but had no effect at -115 mV suggesting that its primary effect was to shift the activation curve to more positive voltages. Hyperpolarizing ramp and voltage compensation protocols indicated that VIP shifts the activation curve of if by approximately 5-6 mV in the positive direction with no change in maximal conductance. This shift may be the mechanism by which VIP produces its positive chronotropic effect and supports a negative feedback role for this peptide during elevated vagal activity.

Animals↗
PubMed1996

Cytoskeletal control of rectification and expression of four substates in cardiac inward rectifier K+ channels.

Cardiac inward rectifiers may have a three-barrel channel structure, based on evidence for three substates in single-channel recordings. However, some reports indicate four substates, a feature more compatible with the four-subunit structure for which there is evidence in cloned voltage-activated K+ channels. Here we show that although the fourth is easily missed, inward rectifier channels have four substates whose expression is controlled by intracellular Ca(2+) ions. Fourth substate openings also appear after rectification loss in intracellular divalent caution-free solution. We find that this process is accelerated by cytochalasin, a microfilament disrupter. Cytochalasin also abolishes Ca(2+), but not Mg(2+),-induced rectification by restoring fourth substate openings. Thus, cytoskeletal elements control Ca(2+)-dependent substate expression and rectification in native inwardly rectifying K+ channels.

Animals↗
PubMed1995

Cardiac pacemaker: 15 years of "new" interpretation.

After more than 15 years since the "new" interpretation of the Purkinje fibre's pacemaker current was proposed, much progress has been made in the understanding of the basic functional principles of cardiac pacemaking. We now know that, in both the SA node and Purkinje fibres, the diastolic depolarization is generated by the interplay of several ionic components, the key process being represented by the turning-on of the hyperpolarization-activated i(f) current towards the end of the action potential repolarization phase. The properties of i(f) are well suited not only to generate, but also to mediate the control of cardiac rate by autonomic transmitters. This control is exerted through modulation of adenylate-cyclase and of cAMP, and allows a fine and rapid adjustment of heart rate to the changing needs of our normal day-life. Still, several problems remain to be clarified : for example, it is not clear how the degree of involvement of i(f) and other components changes in different areas of the nodal region, and whether this process is under control of the autonomic nervous system; more importantly, it is still unknown if the pacemaking mechanisms are similar in the newborn and in the adult, or if developmental changes in the way pacemaker activity is generated and modulated exist.

Autonomic Nervous System↗
PubMed1994

Cesium prevents maintenance of long-term depression in rat hippocampal CA1 neurons.

Long-term depression of field excitatory postsynaptic potentials (EPSP) in the CA1 region of hippocampal slices was evoked by delivering a 15 min train of pulses at 1 Hz to the Schaffer-commissural-CA1 pathway, and prevented by adding an N-methyl-D-aspartate (NMDA) receptor antagonist (AP-5, 50 microM) to the perfusing medium. Superfusion of the slices with Cs (2 mM) during the 1 Hz stimulation period could both inhibit the maintenance phase of the depression itself and elicit spontaneous rhythmic activity. Cs had no effect on the postsynaptic response to the GABA-B agonist, baclofen. As a major effect of Cs is a block of the hyperpolarization-activated current (Ih), these results suggest the possible involvement of Ih in the maintenance of long-term depression.

2-Amino-5-phosphonovalerate↗
PubMed1994

Modulation of single hyperpolarization-activated channels (i(f)) by cAMP in the rabbit sino-atrial node.

1. The hyperpolarization-activated 'pacemaker' current (i(f)) was recorded in inside-out patches excised from rabbit sino-atrial (SA) node cell membranes. 2. Single-channel activity could be resolved in patches containing only a few channels; the voltage dependence of single-channel size and single-channel conductance (0.97 pS) were similar to those measured previously in cell-attached conditions. 3. Perfusion of the intracellular side of the patch membrane with 10 microM cAMP facilitated the opening of single i(f) channels on hyperpolarization. The cAMP-induced i(f) current activation occurred without modification of the single-channel conductance. 4. Modification by cAMP of the probability of channel opening was investigated with respect to the latency to first opening during hyperpolarization and in patches containing a large number of channels (macro-patches). First-latency histograms showed that cAMP shifts the probability curve of first openings to shorter times, in agreement with a cAMP-induced facilitation of channel opening. In macro-patches, measurement of the voltage dependence of the open probability by a slow voltage ramp protocol showed that cAMP shifts the probability curve to more positive voltages without modifying its shape. 5. In cell-free macro-patches the normalized open probability curve in control solutions was centred around -121.9 mV, a voltage some 30 mV more negative than in cell-attached macro-patches. Negative shifting of the curve after patch excision could only partly be explained by the removal of intracellular cAMP, and progressed with time during the ramp protocol, suggesting the presence of a run-down process independent from cAMP.

Animals↗