Biomedical subjects

W R Giles

Publications and source records attributed to W R Giles.

At least 91 records · Page 5Linked to original sources

PubMed1990

Alpha-adrenergic modulation of the transient outward current in rabbit atrial myocytes.

1. A whole-cell voltage-clamp technique has been used to study the alpha-effects of the adrenergic agonists noradrenaline, methoxamine and phenylephrine on the action potentials and membrane currents of rabbit atrial myocytes. Experiments were carried out at 22-23 degrees C. 2. In the presence of 10(-6) M-propranolol, all three agents prolonged action potential duration. This change could be ascribed principally to changes in membrane current early during the plateau phase of the action potential. In the presence of 10(-3) M-4-aminopyridine, no changes in calcium current (ICa) were observed on exposure to alpha-agonists. No significant shift in the voltage dependence or change in the amplitude of the calcium current-voltage relation was observed. 3. Exposure to 3 x 10(-4) M-CdCl2 to block ICa reduced the action potential prolongation caused by alpha-adrenergic agonists. Measurement of unloaded cell shortening revealed that action potential prolongation caused by alpha-agonists, especially at low stimulus rates, could contribute significantly to the positive inotropic effect of alpha-adrenoceptor stimulation. 4. The voltage-activated transient outward current (It) was markedly reduced during exposure to alpha-adrenergic agonists in a dose-dependent manner in the presence of CdCl2 (3 x 10(-4) M) and propranolol in sufficient concentration to prevent beta-adrenoceptor activation. Noradrenaline exhibited a higher potency for this effect than either methoxamine or phenylephrine. The noradrenaline concentration required to give 50% of the maximal effect was 6 x 10(-6) M compared with 2.3 x 10(-4) M for methoxamine. Noradrenaline reduced It by only about 60% of the maximum reduction produced by methoxamine suggesting that it could be classified as a partial agonist for this effect. 5. The reduction of It during exposure to alpha-adrenergic agonists was rate dependent in that larger current reductions were observed at very low rates of stimulation (less than 0.1 Hz). 6. The magnitudes of current-voltage relations for It were reduced over the entire voltage range studied during exposure to alpha-adrenergic agonists and reductions were dose dependent. No shift of these relations along the voltage axis was observed. 7. The steady-state inactivation relations for It were studied using two voltage clamp protocols. A two-step method resulted in a relatively steep sigmoid 'quasi-steady-state' relation. The half-inactivation potential of -27 mV was unaffected by alpha-adrenergic agonists.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1990

Electrophysiology of parasympathetic neurones isolated from the interatrial septum of bull-frog heart.

1. Whole-cell voltage-clamp techniques were used to study the voltage-dependent membrane conductances in parasympathetic neurones enzymatically isolated from the interatrial septum of bull-frog heart and maintained in short-term (1-10 day) tissue culture. 2. The resting potential of the isolated neurones averaged -55.4 +/- 1.1 mV (+/- S.E.M., n = 11). Action potentials evoked in the isolated cells by brief (1-2 ms) current injections were similar to those recorded from neurones in the 'intact' septum. The amplitude of action potentials of isolated neurones averaged about 113 mV, with a peak depolarization of +32.8 +/- 2.8 mV and after-hyperpolarization of -80.0 +/- 2.8 mV. 3. The pattern of membrane currents recorded using voltage clamp with 'normal' external (containing 110 mM-Na+) and internal (110 mM-K+) solutions consisted of a rapidly activating and inactivating inward current followed by a slower, sustained outward current. 4. The inward components of current were isolated by using an internal solution in which Cs+ and TEA+ (tetrathylammonium) ions replaced K+. Depolarizations from holding potentials of -50 to -70 mV produced inward currents which had an initial transient phase followed by a maintained, or very slowly inactivating, component. The current-voltage relation for the initial transient phase reached a peak at membrane potentials near 0 mV, while the maintained phase, measured, for example, at the end of 50 ms voltage-clamp steps, had its peak near +10 mV. 5. The transient component of inward current was carried primarily by Na+ ions, as replacement of Na+ by TEA+ in the external solution abolished the transient. This current was thus identified as a voltage-dependent Na+ current, INa. The maintained component was greatly attenuated by removing 80-90% of the external Ca2+ ions, and it was abolished by divalent cations such as Cd2+ (0.2-0.4 mM), Ni2+ (0.5 mM) and La3+ (10-100 microM). This maintained component was thus a voltage-dependent calcium current, ICa. 6. About 80% of INa recorded in the presence of low (0.2-0.5 mM) external Ca2+ and 2 microM-LaCl3 was blocked by tetrodotoxin (TTX) with an apparent Kd of about 8 nM. The remaining 20% of INa was resistant to block by 2-10 microM-TTX. However, the 'TTX-resistant' component of INa was blocked by Cd2+ (0.2-0.4 mM). 7. The voltage-dependent calcium current, ICa, measured in saline in which Na+ was replaced by N-methyl-D-glucamine, activated near -40 mV and reached a peak near +10 to +15 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1990

Muscarinic modulation of calcium current in neurones from the interatrial septum of bull-frog heart.

1. The effects of activation of muscarinic receptors on the voltage-dependent calcium current, ICa, in parasympathetic neurones were examined. 2. Neurones were enzymatically isolated from the interatrial septum of bull-frog (Rana catesbeiana) heart, and were maintained in short-term (1-6 day) tissue culture. ICa was recorded from the cells using whole-cell patch-clamp methods (Clark, Tse & Giles, 1990). 3. External application of 2 nM to 10 microM acetylcholine (ACh) reduced the amplitude and slowed the time course of activation of ICa. These effects were dependent on membrane potential; they were most pronounced at potentials near the peak of the current-voltage relation for ICa (i.e. +10 to +15 mV), whereas at more-negative potentials (i.e. -15 to -25 mV) the effects on both amplitude and time course were relatively small. 4. Atropine (1 microM) completely blocked the action of 1 microM-ACh, indicating that the effects of ACh on ICa were mediated by activation of muscarinic receptors. 5. Other muscarinic agonists, such as carbamylcholine (0.1-10 microM), DL-muscarine (0.1-2.5 microM) and oxotremorine (5 microM), had similar effects on ICa to ACh. 6. A guanine nucleotide-binding protein (G-protein) is involved in this muscarinic inhibition of ICa. Inclusion of the non-hydrolysable guanosine triphosphate analogue guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S; 200 microM) in the intracellular solutions mimicked the effects of ACh, and application of external ACh in the presence of internal GTP-gamma-S produced smaller changes in ICa than in control conditions. Inclusion of another non-hydrolysable analogue, guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S; 0.5-5 mM), blocked the inhibitory effect of ACh on ICa. 7. The G-protein involved in the inhibition of ICa was sensitive to pertussis toxin (islet-activating protein; IAP). The inhibition of ICa by carbamylcholine (5 microM) was reduced by about 90% after incubating cells for 12-15 h in culture medium containing 200 ng/ml IAP. 8. The possible roles of cyclic AMP or cyclic GMP-dependent protein kinases, or protein kinase C, in the muscarinic inhibition of ICa were tested, but these enzymes appear not to be directly involved.

Acetylcholine↗

PubMed1990

Intracellular mechanisms for alpha 1-adrenergic regulation of the transient outward current in rabbit atrial myocytes.

1. The intracellular mechanism(s) underlying the decrease of a transient outward K+ current (It) induced by alpha 1-adrenergic agonists was studied in isolated adult rabbit atrial myocytes using whole-cell voltage clamp and cell-attached patch clamp techniques. Experiments were carried out at 22-23 degrees C. 2. Application of the specific alpha 1-adrenergic agonist, methoxamine, produced a decrease in It which was irreversible after the non-hydrolysable GTP analogues, GTP gamma S and Gpp(NH)p, had been introduced into cells via the recording micropipette. 3. Pre-treatment of cells with 0.1-0.15 microgram/ml pertussis toxin (PT) for 8-9 h at 30-34 degrees C did not prevent the alpha 1-induced decrease in It. Yet, this protocol, as measured by the PT-catalysed incorporation of [32P]ADP-ribose in membrane-associated 40 and 41 kDa proteins, effectively caused the ADP-ribosylation of approximately 70% of the PT-sensitive GTP-binding proteins (i.e. Gi) in these treated cells. After taking into account the proportion of non-viable cells (20-30%), the effectiveness of this treatment probably approaches 100% in the viable myocytes from which electrophysiological recordings were made. 4. Cell-attached patch recordings showed that bath application of methoxamine altered the single-channel events underlying It by decreasing their opening probability. Averaged currents from ensemble single-channel openings recorded in the presence of 0.2 mM-methoxamine outside the patch reproduced the features of alpha 1-adrenergic modulation of the macroscopic It observed during whole-cell voltage clamp measurements. This observation provides evidence for the involvement of a diffusible intracellular second messenger in the alpha 1-adrenergic modulation of It. 5. The protein kinase C (PKC) activators, 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl-2-acetylglycerol (OAG) increased It, when included in the bath perfusate, whereas the inactive analogues, 4 alpha-phorbol and 4 alpha-phorbol 12,13-didecanoate, had no effect on It. 6. Exposure of cells to the PKC inhibitors, staurosporine and H-7, either by bath superfusion or intracellularly, via the recording micropipette, did not block the decrease in It produced by methoxamine. 7. Prolonged stimulation of atrial myocytes for 7-9 h at 22 degrees C with 500 nM-PMA produced a 'down-regulation' of endogenous PKC activity, as well as a physical loss of the immunoreactive enzyme, as measured by an in vitro assay, and an anti-PKC monoclonal antibody, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1990

A mathematical model of a bullfrog cardiac pacemaker cell.

Previous models of cardiac cellular electrophysiology have been based largely on voltage-clamp measurements obtained from multicellular preparations and often combined data from different regions of the heart and a variety of species. We have developed a model of cardiac pacemaking based on a comprehensive set of voltage-clamp measurements obtained from single cells isolated from one specific tissue type, the bullfrog sinus venosus (SV). Consequently, sarcolemmal current densities and kinetics are not influenced by secondary phenomena associated with multicellular preparations, allowing us to realistically simulate processes thought to be important in pacemaking, including the Na(+)-K+ pump and Na(+)-Ca2+ exchanger. The membrane is surrounded extracellularly by a diffusion-limited space and intracellularly by a limited myoplasmic volume containing Ca2(+)-binding proteins (calmodulin, troponin). The model makes several predictions regarding mechanisms involved in pacing. 1) Primary pacemaking cannot be attributed to any single current but arises from both the lack of a background K+ current and a complex interaction between Ca2+, delayed-rectifier K+, and background leak currents. 2) Ca2+ current displays complex behavior and is important during repolarization. 3) Because of Ca2+ buffering by myoplasmic proteins, the Na(+)-Ca2+ exchanger current is small and has little influence on action potential repolarization but may modulate the maximum diastolic potential. 4) The Na(+)-K+ pump current does not play an active role in repolarization but is of sufficient size to modulate the rate of diastolic depolarization. 5) K+ accumulation and Ca2+ depletion may occur in the extracellular spaces but play no role in either the diastolic depolarization or repolarization of a single action potential. This model illustrates the importance of basing simulations on quantitative measurements of ionic currents in myocytes and of including both electrogenic transporter mechanisms and Ca2+ buffering by myoplasmic Ca2(+)-binding proteins.

Animals↗

PubMed1990

A mathematical model of electrophysiological activity in a bullfrog atrial cell.

We have developed a model of cardiac atrial electrical activity based on voltage-clamp measurements obtained from single cells isolated from the bullfrog atrium. These measurements have allowed us to simulate a number of processes thought to be important in action potential initiation, repolarization, and the excitation-contraction (EC) coupling process. In this atrial model, the cell membrane contains both channel-mediated (Na+, Ca2+, inward rectifier K+, delayed rectifier K+, linear background leak) and transporter-mediated (Na(+)-K+ pump, Na(+)-Ca2+ exchanger, Ca2+ pump) currents. The cell is surrounded extracellularly by a diffusion-limited space. The intracellular volume contains Ca2(+)-binding proteins (calmodulin, troponin). The model makes several important predictions. 1) Incomplete inactivation of the Ca2+ current provides an inward current the maintains the plateau of the action potential. 2) Activation of the delayed rectifier K+ current initiates repolarization. 3) Due to Ca2+ buffering by myoplasmic proteins the Na(+)-Ca2+ exchanger current is relatively small and has little influence on repolarization. 4) The Na(+)-K+ pump current does not play a major role in repolarization. 5) K+ accumulation and Ca2+ depletion may occur in the extracellular spaces. 6) Modulation of EC coupling is governed by interactions between the myoplasmic Ca2(+)-binding proteins; specifically, the inotropic "positive staircase effect" may be explained by interactions between Ca2+ and Mg2+ at a competitive binding site on troponin. When considered in conjunction with the results of our model of primary pacemaking in the sinus venosus [Rasmusson et al., Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H352-H369, 1990], this atrial model shows how the presence or absence of certain transmembrane currents can change action potential characteristics and consequently alter the relative influence of the various transporter-mediated and channel-mediated currents.

Action Potentials↗

PubMed1989

A novel effect of norepinephrine on cardiac cells is mediated by alpha 1-adrenoceptors.

In the heart, alpha-adrenergic agonists have long been known to produce a positive inotropic effect that is rate dependent and associated with action potential prolongation but is not accompanied by adenosine 3',5'-cyclic monophosphate (cAMP) elevation. The ionic mechanism of these effects is unknown. We report that a transient outward K+ current, a major determinant of plateau duration in rabbit and human atria, is strongly inhibited by norepinephrine and the alpha-adrenoceptor agonists methoxamine and phenylephrine. These effects of alpha-stimulation can be blocked by prazosin. The reduction in the transient outward current substantially slows action potential repolarization. These results can explain the regional and species-dependent positive inotropic effects of alpha-adrenergic stimulation in the heart and give important new insight into the autonomic regulation of cardiac function. In addition, reduction in this repolarizing current during the enhanced alpha-adrenergic responsiveness of myocardial ischemia may be a factor in the genesis of arrhythmias produced by catecholamines.

Adrenergic alpha-Agonists↗

PubMed1988

Ion transfer characteristics of the calcium current in bull-frog atrial myocytes.

1. Voltage clamp studies on single cells from bull-frog atrium have been carried out to study the ion transfer characteristics of the calcium current, ICa. In agreement with the preliminary results of Hume & Giles (1983), a TTX-resistant, 'second transient inward current' was recorded consistently. Its average peak size at 0 mV in 2.5 mM [Ca2+]o Ringer solution was approximately -200 pA, and it was blocked by Cd2+ and La3+ but not by tetrodotoxin (TTX, 3 x 10(-6) M). 2. The peak size of this current increases by approximately 4 times when [Ca2+]o is raised from 1.25 to 7.5 mM, indicating that Ca2+ is a major charge carrier. 3. A well-defined reversal potential, Erev, for ICa can be recorded in normal Ringer solution and also when Ba2+ or Sr2+ serve as the charge carriers. When [Ca2+]o is changed the shifts in Erev follow the predictions of a Nernstian Ca2+ electrode. However, all Erev values are well below those predicted from the thermodynamic Nernstian ECa values (see Campbell, Giles, Hume, Noble & Shibata, 1988a). 4. The Ca2+ current exhibits voltage-dependent inactivation, whether the direction of net current flow is inward or outward; however, the rate of inactivation is affected by the species of cation carrying the current. Inactivation is reduced substantially in Ba2+ Ringer solution. 5. Magnesium (5 mM) is not a significant carrier or blocker of ICa in normal [Ca2+]o Ringer solution; however, 5 mM [Mg2+]o can block the current carried by either Sr2+ or Ba2+. In the absence of Mg2+, equimolar substitutions of Sr2+ or Ba2+ for Ca2+ result in larger currents than those carried by Ca2+ in the normal Ringer solution. 6. Sodium appears not to be a significant charge carrier in the presence of normal [Ca2+]o. However, after free [Ca2+]o has been reduced to extremely low levels (less than 10(-6) M) Na+ can carry a significant fraction of 'ICa'. Thus, it appears that the high selectivity of ICa for Ca2+ ions depends upon the presence of Ca2+. 7. 'Slow tails' are frequently recorded after repolarizing clamp steps back to the holding potential. These 'slow tails' are prominent in normal [Na+]o, [Ca2+]o and [Sr2+]o Ringer solution; however, they are markedly reduced in [Ba2+]o, in Na+-free and Ca2+-free Ringer solutions. Experimental and theoretical work suggests these slow tails may be generated by an electrogenic Na+-Ca2+ exchanger (see Campbell, Giles, Robinson & Shibata, 1988b).(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1988

Reversal potential of the calcium current in bull-frog atrial myocytes.

1. Voltage clamp recordings of the calcium current (ICa) in single myocytes which were enzymatically isolated from bull-frog atrium show that a genuine reversal of the current flowing through Ca2+ channels can be recorded (ef. Reuter & Scholz, 1977; Lee & Tsien, 1982, 1984; Campbell, Giles & Shibata, 1988c). In normal 2.5 mM [Ca2+]0 Ringer solution this apparent reversal potential (Erev) is near +50 mV, a value well below the predicted thermodynamic Ca2+ equilibrium potential (ECa). 2. None the less, Erev shifts with variations in extracellular divalent ion concentrations (Ca2+, Sr2+ and Ba2+) according to the predictions of a Nernstian divalent cation electrode, i.e. approximately 29 mV per 10-fold change in the external concentration of divalent ion. 3. The existing theoretical analysis of this Erev has been extended in order to clarify its interpretation with regard to the selectivity characteristics of ICa. 4. The apparent reversal potential is analysed using a form of the constant field equation which has been modified to include (i) simultaneous monovalent and divalent cation movements and (ii) the presence of a surface potential (V'). This equation can be solved to yield an explicit expression for Erev. The effects of V' on apparent permeability ratios for the Ca2+ channel Erev are demonstrated. 5. In combination, our experimental results and calculations suggest that: (i) previous estimates of V' which were used to describe permeability (P) ratios of Ca2+ channels in various cardiac preparations may be in error, (ii) in normal [Ca2+]o the PNa/PCa ratio is very small, and (iii) PCa/PK must be greater than 1000. An analysis of the relative selectivity of the channel for divalent cations compared to K+ shows that PCa greater than PSr greater than PBa, assuming that PK remains the same after the divalent substitutions. 6. The Ca2+ channel in bull-frog atrial cells is thus much more selective for Ca2+ ions than had previously been estimated; in particular, inward flow of monovalent cations (e.g. Na+) through these channels does not contribute significantly to the observed ICa. The physiological implications of this high selectivity for Ca2+ ions are discussed.

Action Potentials↗

PubMed1988

Inactivation of calcium current in bull-frog atrial myocytes.

1. A single-microelectrode technique has been used to study the voltage dependence and the kinetics of inactivation and reactivation of a tetrodotoxin-resistant inward current (ICa) in single cells from bull-frog atrium. 2. In most cases the kinetics of both inactivation and reactivation can be well described as a single-exponential process. 3. Several different observations indicate that inactivation of ICa in these cells is controlled by both voltage-dependent and current-dependent processes, as has been demonstrated previously in heart (Kass & Sanguinetti, 1984; Lee, Marban & Tsien, 1985) and in other tissues (Hagiwara & Byerly, 1981; Tsien, 1983; Eckert & Chad, 1984). 4. Evidence in favour of a voltage-dependent inactivation mechanism included: (a) In paired-pulse measurements of steady-state inactivation ('f infinity') a 'conventional' steady-state f infinity vs. membrane potential (Vm) relationship was obtained in the range of membrane potentials from -60 to 0 mV. (b) Increasing [Ca2+]o from 2.5 to 7.5 mM, which resulted in a 2-3-fold increase in ICa, did not produce any significant increase in the amount of inactivation. (c) Using a 'gapped' double-pulse protocol non-monotonic U-shaped inactivation relationships were obtained, i.e. positive to approximately +20 mV some removal of inactivation occurred. However, f never approached a value near 1.00 at very depolarized potentials; it reached a maximum between 0.5 and 0.6. (d) In constant [Ca2+]o and at fixed Vm, the kinetics of ICa inactivation were independent of peak size of ICa. This was demonstrated by: (i) varying the holding potential (-90 to -30 mV), (ii) using paired-pulse 'recovery' protocols, and (iii) partial block by La3+ (1-10 microM) and Cd2+ (0.1 mM). (e) Influx of Ca2+ ions was not an obligatory prerequisite for development of inactivation. In all ionic conditions (Ca2+, Sr2+, Ba2+, Na+-free and Ca2+-free Ringer solutions) currents displayed inactivation phenomena, although the extent and kinetics of inactivation were dependent upon ionic conditions. Outward currents recorded above the reversal potential for ICa exhibited time- and voltage-dependent inactivation, and could be inactivated by brief depolarizing pre-pulses that produced no net inward current flow. Evidence against a possible role of the electrogenic Na+-Ca2+ exchanger in producing inactivation of these outward currents was obtained.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1988

Studies of the sodium-calcium exchanger in bull-frog atrial myocytes.

1. Experimental measurements and computer simulations have been used in attempts to identify an exchanger current (Iex) generated by an electrogenic Na+-Ca2+ exchanger in single cells from bull-frog atrium. 2. Voltage clamp measurements of an inward 'slow tail' current observed upon repolarization after depolarizing clamp pulses that elicit net inward Ca2+ currents (ICa) (see Campbell, Giles & Shibata, 1988c), show that these slow tails have a cationic dependence different from ICa. Slow tails are large and prominent in normal [Na+]o solutions containing either Ca2+ or Sr2+, but they are markedly reduced or absent in Ba2+, Ca2+-free, and Na+-free solutions. 3. Kinetic measurements on the slow tails show that they are not generated by deactivation of ICa, and suggest that they may be due to activation of Iex at negative potentials (-70 to -100 mV). 4. Computer simulations of the influx, buffering, and extrusion of Ca2+ provide further indirect evidence that the slow tails correspond to Iex. In addition, these calculations give insights into one plausible mechanism of Ca2+ homeostasis in frog atrium. When the Na+-Ca2+ exchanger formalism of Mullins (1979, 1981), as modified by DiFrancesco & Nobel (1985), is combined with equations for intracellular Ca2+ buffering by myoplasmic proteins (cf. Robertson, Johnson & Potter, 1981), slow inward tails are produced which are qualitatively similar to those recorded experimentally. 5. Comparisons of the size and time course of ICa with those of Iex suggest that Iex does not generate a physiologically significant current (or membrane potential change) during the plateau of the action potential. However, at potentials near the resting potential the inward current due to Iex may be significant. 6. Our theoretical results suggest that in the intact single atrial cell myoplasmic Ca2+-binding proteins (e.g. calmodulin and troponin) could be physiologically important modulators of the amplitude, polarity and kinetics of Iex. Hence, the specificity, capacity and kinetics of intracellular Ca2+ binding are essential components of any quantitative treatment of Iex in excitable tissue.

Action Potentials↗

PubMed1988

Comparison of potassium currents in rabbit atrial and ventricular cells.

1. In rabbit and human hearts there are significant differences in the action potential configuration in atrium and ventricle, and the action potential waveform exhibits marked frequency dependence in both tissues. To study the ionic mechanism(s) of these phenomena, the size and time course of the potassium (K+) currents responsible for repolarization have been recorded from single cells using a whole-cell microelectrode voltage clamp method. 2. At physiological heart rates, the action potential in atrial cells has a short plateau phase; however, the rapid early repolarization is strongly frequency dependent. Ventricular myocytes have a long plateau (400-700 ms at 23 degrees C), and the late repolarizing phase of the action potential is much faster in ventricle than in atrium. 3. In both cell types, four different outward currents can be recorded: (i) a large transient outward current, It; (ii) IK(Ca), a smaller Ca2+-dependent K+ current; (iii) IK, a small, maintained time- and voltage-dependent delayed rectifier K+ current; (iv) IK1, an inwardly rectifying K+ current. 4. It, which is responsible for early repolarization, is much larger in atrium than in ventricle. It has very rapid activation and inactivation kinetics but a very slow time course of recovery from inactivation (tau = 5.4 s at 23 degrees C). Our results show that the reactivation kinetics of It are responsible for the pronounced dependence of the shape of the atrial action potential on stimulus frequency. 5. IK(Ca) is variable from cell to cell and is larger in atrium than in ventricle. In both cell types, IK(Ca) is much smaller than It. 6. The delayed rectifier current, IK, is very small and turns on relatively slowly in both cell types. It is therefore not activated strongly during the relatively short plateau of the atrial action potential. Even in ventricle, it contributes only a small repolarizing current. 7. IK1, the inward rectifier K+ current, is much larger in ventricle than in atrium. The current-voltage relationship for IK1 in ventricle exhibits a negative slope conductance between -50 and 0 mV. IK1 is the outward current which generates the resting membrane potential and it modulates the final repolarization phase of the action potential in both cell types. 8. These data strongly suggest that the action potential configuration and its frequency dependence in rabbit atrial and ventricular cells are mainly due to the differences in sizes and kinetics of It and IK1.

4-Aminopyridine↗

PubMed1988

Properties of the transient outward current in rabbit atrial cells.

1. Whole-cell and patch clamp techniques have been used to study the steady-state voltage dependence and the kinetics of a transient outward current, It, in single cells from rabbit atrium. 2. The steady-state voltage dependence of both activation and inactivation of It are well described by Boltzmann functions. Inactivation is fully removed at potentials negative to -70 mV and it is complete near 0 mV. The threshold for activation of It is near -30 mV and it is fully activated at +30 mV. The region of overlap between the activation and inactivation curves indicates that a steady non-inactivating current will be recorded over a membrane potential range from approximately -30 to 0 mV. 3. In general, the time course of inactivation at potentials in the range 0 to +50 mV is best described as a sum of two exponential functions. The kinetic parameters controlling these processes exhibit only very weak voltage dependence. 4. Comparison of the time course of the development of inactivation in response to long depolarizing voltage clamp steps with the development of inactivation in response to trains of brief depolarizing pulses indicates that inactivation develops very quickly and decays relatively slowly at potentials near the resting potential (e.g. -70 mV). Thus, in response to (i) a train of voltage-clamp pulses or (ii) a series of action potentials, the magnitude of It decreases due to a progressive increase in the amount of inactivation. 5. A simple model of channel gating is presented: it can account for the major aspects of the voltage dependence and kinetics of It (cf. Aldrich, 1981). 6. Cell-attached patch clamp recordings have been used to identify the single-channel or unitary events underlying the current, It. In general, only one active channel is present per patch. The single-channel conductance in normal Tyrode solution is approximately 14 pS and the current-voltage relationship is approximately linear between +50 and +150 mV with respect to rest. This information, in combination with the fully activated current-voltage characteristics from the whole-cell data, can be used to estimate the number and density of It channels per cell: these are 1600 and one per 3-4 micron 2, respectively. 7. Ensemble averages obtained from patch recordings are very similar in time course to the macroscopic or whole-cell current itself: the ensemble current rises to a peak within approximately 5 ms and decays with a biexponential time course in response to depolarizations to approximately +50 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Ionic mechanisms of adenosine actions in pacemaker cells from rabbit heart.

1. Whole-cell and patch clamp techniques have been applied to cells isolated from the rabbit sino-atrial (S-A) node to study the ionic mechanism(s) of adenosine-induced slowing of cardiac pacemaker activity. 2. Viable spontaneously active cells were isolated from the central region of the S-A node of the rabbit heart by an enzymatic dispersion procedure similar to that reported by Giles & van Ginneken (1985) and van Ginneken & Giles (1988). In these spontaneously beating cells application of adenosine caused a dose-dependent slowing accompanied by a small hyperpolarization of the maximum diastolic potential. Relatively high doses of adenosine (greater than 20 microM) caused complete arrest, associated with a hyperpolarization of 12-15 mV. 3. In corresponding whole-cell voltage clamp experiments adenosine activated a time-independent potassium current, IK(ADO), which at -50 mV is approximately 30 pA in normal Tyrode solution and 50 pA in high [K+]o (20 mM) Tyrode solution. This current is similar to the one identified previously in guinea-pig atrium (Belardinelli & Isenberg, 1983a; Kurachi, Nakajima & Sugimoto, 1986). 4. Patch clamp recordings of the single-channel events underlying IK(ADO) showed that they have a conductance of approximately 25.0 +/- 1.9 pS. The whole-cell or macroscopic current, IK(ADO), and the adenosine-induced single-channel events exhibit strong inward-going rectification. 5. Adenosine in doses (10 microM) which significantly activate IK(ADO) failed to produce any measurable effect on the calcium current, ICa, in these isolated cardiac pacemaker cells. However, after ICa has been enhanced by the addition of isoprenaline, adenosine (1-10 microM) caused a significant inhibition: it reduced ICa back to approximately the control levels. 6. A similar 'indirect' effect of adenosine was observed on If, the slow time- and voltage-dependent inward current which is activated by hyperpolarizing these S-A node cells. Adenosine (10(-5) M) failed to influence the control or basal If; however, after If was enhanced by isoprenaline, adenosine markedly inhibited it. 7. These results provide explanations for both the direct and the indirect effects of adenosine in mammalian cardiac pacemaker tissue: activation of IK(ADO), and of a time-independent background potassium current and inhibition of ICa and If, respectively. Since it is known that there is significant adrenergic tone in the mammalian S-A node both the indirect and the direct effects of adenosine may be of physiological importance.

Adenosine↗

PubMed1987

Alinidine as a model of the mode of action of specific bradycardic agents on SA node activity.

Three different bradycardic agents, alinidine, AQ-A39 and UL-FS49 increase the intrinsic cycle length of the isolated SA node preparation of the rabbit. This increase is mainly caused by a decrease in rate of diastolic depolarization. One of these agents, alinidine, was used to study the underlying ionic mechanism of the decrease in the diastolic depolarization rate in isolated cells and small cell clusters of the rabbit SA node. In these preparations alinidine slowed down the rate of spontaneous activity at higher concentrations (80 microM). At lower concentrations (10 microM) the decrease in rate of spontaneous activity was variable, but injection of a hyperpolarizing current slowed the spontaneous rate more in the presence of alinidine, indicating an increase in membrane resistance. In voltage clamp experiments we found that the main effect of alinidine was a block of the hyperpolarization activated current if. The block was potential dependent and was maximal in the potential range in which diastolic depolarization occurs. These results are discussed in relation to previous findings of others.

Animals↗

PubMed1987

Quinidine-induced inhibition of transient outward current in cardiac muscle.

Quinidine is frequently used as a class I antiarrhythmic agent in the management of cardiac rhythm disturbances. It depresses the rapid initial depolarization of the action potential by blocking the sodium current, INa. In addition, quinidine increases the duration of the action potential and lengthens the refractory period. We have used a whole cell voltage-clamp technique to study the ionic mechanism underlying the lengthening of the action potential in single cells from the atrium and ventricle of the rabbit heart. Our data show that quinidine at therapeutic doses (3-10 microM) is a potent and selective inhibitor of a transient outward current, which controls the early repolarization of the action potential. In contrast, neither the calcium current, ICa, nor the time-independent background K+ current, IK1, is changed significantly by 10 microM quinidine. The reduction in the transient outward current can explain the lengthening of action potential and provides new insight into the mechanism of action of quinidine as an antiarrhythmic agent.

Action Potentials↗

PubMed1986

Comparison of steady-state electrophysiological properties of isolated cells from bullfrog atrium and sinus venosus.

Single electrode whole cell voltage-clamp experiments and frequency domain analyses have been used to study and compare the K+ currents in enzymatically dispersed single cells from the atrium and the sinus venosus (pacemaker region) of the bullfrog heart. Admittance measurements made near the "resting' or zero-current potential yield data from which the equivalent circuit of each cell type may be obtained. Data from both atrial and pacemaker cells are well-fitted by a model consisting only of parallel resistance-capacitative elements, as predicted from their micro-anatomy. Neither of these amphibian cardiac cells contain a transverse tubule system (TT) and both have very little sarcoplasmic reticulum (SR). These results complement and extend two earlier investigations: (i) Moore, Schmid and Isenberg (J. Membrane Biol. 81:29-40, 1984) have reported that in guinea pig ventricle cells (which do contain an internal membrane system consisting of transverse tubules and a substantial SR) the SR may be electrically coupled to the sarcolemma; (ii) Shibata and Giles (Biophys. J. 45:136a, 1984) have shown that although bullfrog atrial cells have an inwardly rectifying background K+ current, IK1, pacemaker cells from the immediately adjacent sinus venosus do not. Data from admittance measurements also provide evidence that a TTX-insensitive inward Ca2+ current is activated in the pacemaker range of potentials.

Action Potentials↗

PubMed1985

Ionic currents that generate the spontaneous diastolic depolarization in individual cardiac pacemaker cells.

An enzymatic dispersion procedure has been developed to obtain viable, spontaneously active single myocytes from cardiac pacemaker tissue: the bullfrog (Rana catesbeiana) sinus venosus. Recordings of time- and voltage-dependent Ca2+ and K+ currents have been made by using a single suction-microelectrode technique. The results show that two time- and voltage-dependent currents interact to modulate the slope of the pacemaker potential. These are: (i) the decay of a delayed rectifier K+ current and (ii) the activation of a Ca2+ current. In addition, the data strongly suggest that cardiac pacemaker tissue does not have an inwardly rectifying background K+ current.

Animals↗