Claudin-1 overexpression in melanoma is regulated by PKC and contributes to melanoma cell motility.

Serial analysis of gene expression followed by pathway analysis implicated the tight junction protein claudin-1 (CLDN1) in melanoma progression. Tight junction proteins regulate the paracellular transport of molecules, but staining of a tissue microarray revealed that claudin-1 was overexpressed in melanoma, and aberrantly expressed in the cytoplasm of malignant cells, suggesting a role other than transport. Indeed, melanoma cells in culture demonstrate no tight junction function. It has been shown that protein kinase C (PKC) can affect expression of claudin-1 in rat choroid plexus cells, and we observed a correlation between levels of activated PKC and claudin expression in our melanoma cells. To determine if PKC could affect the expression of CLDN1 in human melanoma, cells lacking endogenous claudin-1 were treated with 200 nM phorbol myristic acid (PMA). PKC activation by PMA caused an increase in CLDN1 transcription in 30 min, and an increase in claudin-1 protein by 12 h. Inhibition of PKC signaling in cells with high claudin-1 expression resulted in decreased claudin-1 expression. CLDN1 appears to contribute to melanoma cell invasion, as transient transfection of melanoma cells with CLDN1 increased metalloproteinase 2 (MMP-2) secretion and activation, and subsequently, motility of melanoma cells as demonstrated by wound-healing assays. Conversely, knockdown of CLDN1 by siRNA resulted in the inhibition of motility, as well as decreases in MMP-2 secretion and activation. These data implicate claudin-1 in melanoma progression.

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.

Effects of catecholamines and potassium on cardiovascular performance in the rabbit.

Resting subjects risk cardiac arrest if plasma potassium ([K+]p) is raised rapidly to 7-9 mM, but brief bouts of exhaustive exercise in healthy subjects can give similar [K+]p without causing cardiac problems. We investigated the effects of [K+]p and catecholamines on systolic blood pressure (SBP) and mean aortic flow (MAF) in anesthetized rabbits and on maximum output pressure (MOP) in isolated working rabbit hearts. In six rabbits, hyperkalemia (11.4 +/- 0.4 mM) caused a fall in SBP from 116 +/- 6 to 49 +/- 6 mmHg and in MAF from 373 +/- 30 to 181 +/- 53 ml/min (P < 0.01). Raising [K+]p (11.6 +/- 0.3 mM) with norepinephrine (NE) (1.3 micrograms.kg-1.min-1 iv), however, increased SBP from 108 +/- 7 to 150 +/- 6 mmHg (P < 0.01) and MAF from 347 +/- 42 to 434 +/- 35 ml/min (P < 0.01). In 19 isolated working hearts, perfusion with 8 mM K+ Tyrode and then 12 mM K+ Tyrode reduced MOP from 87 +/- 3 (control 4 mM K+) to 67 +/- 3 (8 mM K+) and 51 +/- 2 cmH2O (12 mM K+) (P < 0.01); 12 mM K+ Tyrode with 0.08 microM NE or epinephrine, however, increased MOP from 67 +/- 6 (in 8 mM K+) to 85 +/- 6 cmH2O (NE) and from 58 +/- 2 to 76 +/- 5 cmH2O (epinephrine) (P < 0.01). Catecholamines may therefore play a key role in protecting the heart from exercise-induced hyperkalemia.

Inhibition by Compound II, a sotalol analogue, of delayed rectifier current (iK) in rabbit isolated sino-atrial node cells.

The effects of Compound II, a sotalol analogue, on spontaneous electrical activity and on three membrane currents (the delayed rectifier current, iK, the long-lasting inward calcium current, i(Ca,L) and hyperpolarization activated inward current, i(f)) were investigated in rabbit isolated sino-atrial node cells by whole cell clamp with amphotericin-permeabilised patches. A submaximal concentration of Compound II (50 nM) had a significant effect on the time and voltage dependent activation of iK and caused a positive shift of the iK activation curve. As well as blocking i(Kr), it caused some degree of block of i(Ks). Block of iK by Compound II was found to be concentration dependent with an IC50 of approximately 40 nM. 1 microM Compound II nearly completely blocked iK without significantly affecting the peak current or I/V relationships of i(Ca,L) or i(f). 50 nM Compound II caused a significant prolongation of APD100 and of cycle length. It also decreased diastolic depolarization rate without significantly affecting MDP and action potential amplitude. It is concluded that Compound II, a sotalol analogue, slows spontaneous activity of isolated rabbit SA node cells through a selective inhibition of iK.

Pacemaking in rabbit isolated sino-atrial node cells during Cs+ block of the hyperpolarization-activated current if.

1. Experiments have been carried out using the whole-cell patch clamp technique to investigate how the spontaneous pacemaker activity of rabbit isolated sino-atrial (SA) node cells is affected by the block of the hyperpolarization-activated current if by 1 and 2 mM-caesium. 2. Two millimolar caesium reduced the amount of if activated at -90 mV to less than 10% of the control value. In the pacemaking range of SA node cells, if was often completely blocked by this concentration of Cs+. 3. Two millimolar caesium slowed but did not arrest spontaneous pacemaking in isolated SA node cells. In freely beating non-patched cells, 2 mM-CsCl caused a 30% reduction in rate of beating, indicating that in all cells observed if was normally contributing to pacemaking. 4. No increase in instantaneous inward current was seen in response to hyperpolarizing voltage clamp pulses from a holding potential of -40 mV when 1 or 2 mM-CsCl was applied to SA node cells. These concentrations of Cs+ do not therefore induce an 'extra' inward current. 5. Neither the inward calcium current (iCa) nor the outward potassium current (iK) showed changes which could be attributed to Cs+ application. 6. Since spontaneous pacemaking continues during Cs+ block of if while other membrane currents show no Cs(+)-induced changes which could account for this, these experiments provide strong, though indirect, evidence for the presence in SA node of a time-independent background current, ib, which will contribute to a mode of pacemaking controlled by the decay of potassium conductance (gK). The nature of this inward current has yet to be clarified. 7. The results strongly suggest that the hyperpolarization-activated current if normally makes an important contribution to the pacemaker depolarization of all SA node cells.

Rabbit sino-atrial node cells: isolation and electrophysiological properties.

1. A method has been developed for isolating calcium-tolerant, single rabbit sinoatrial node cells which maintain their natural shape following isolation. The majority of viable, spontaneously active cells were elongated and measured about 100 microns in length. 2. Staining fixed cells with Haematoxylin-Eosin revealed that a 'cell' with projections was usually an aggregate of more than one cell. 3. Single, elongated, spontaneously active cells were current and voltage clamped using the whole-cell configuration of the patch-clamp recording technique. The spontaneous activity and time-dependent currents recorded were similar to those reported previously in multicellular nodal preparations and in single cells. 4. An assessment was made of the time course of L-type calcium current run-down: a stable period of between 10 and 20 min followed by a rapid run-down (over about 2 min) was typically observed. 5. In most cells, a fast, TTX-sensitive Na+ current component was seen. A few cells showed a transient outward K+ current (iA). 6. The activation range for the hyperpolarization-activated current, if, varied from cell to cell. In the majority of actively beating cells, the threshold for if was near the maximum diastolic potential (about -65 mV in most cells) but in other cells, no if could be recorded within the pacemaker range. 7. Millimolar concentrations of MnCl2 caused a marked increase in if, but only when the pipette solution did not contain EGTA. Inclusion of EGTA (to buffer Ca2+ to about pCa 8) significantly reduced the effect of Mn2+ which therefore probably occurs through inhibition of Na(+)-Ca2+ exchange and consequent rise in intracellular Ca2+ concentration.

Effect of raised extracellular calcium on characteristics of the guinea-pig ventricular action potential.

The whole-cell patch-clamp method was used to investigate the role of Na/Ca exchange current (INa,Ca) in the shortening action of raised extracellular calcium on the ventricular action potential. Experiments were performed either using BAPTA to buffer intracellular calcium, or by replacing extracellular Na+ with Li- to abolish INa,Ca. A blocker of Ik, compound II. was used to investigate whether changes in this current may also play a role. Raising extracellular calcium from 1.8 mM to 5.0 mM increased the amplitude of the action potential by 8% and decreased its duration (at 90% repolarization, APD90) by 23%. Compound II increased APD90 by 40% and resulted in the appearance of early afterdepolarizations but did not affect the response to raised extracellular calcium. Intracellular BAPTA (20 mM) did not prevent the calcium-induced shortening but did abolish the initial rapid phase of repolarization and increase the inactivation time constant of Icsl recorded under voltage clamp. Replacement of extracellular Na+ with Li+ dramatically shortened the action potential and under these conditions raising extracellular calcium lengthened the action potential. Using a voltage-clamp protocol to mimic an action potential, whole-cell current in the absence and in the presence of Li(+)-replacement was recorded in normal and raised extracellular calcium. The lithium-sensitive current was inward during the "plateau" and was reduced by raising extracellular calcium. The results do not support a role for Ik in mediating action potential shortening in raised extracellular calcium. It is suggested that a decrease in inward INa,Ca may be largely responsible, with a role for increased Icsl inactivation in the early part of the action potential.

Two components of the delayed rectifier potassium current, IK, in rabbit sino-atrial node cells.

The delayed rectifier current was studied in rabbit isolated sino-atrial (SA) node cells using the whole-cell voltage clamp technique with amphotericin-permeabilized patches. The envelope of tails test indicated that in SA node cells the decay of IK (IK.tall) comprises two distinct current components similar to the specific fast and slow components of IK (IKr and JKs) that have been found in atrial and ventricular myocytes. Dofetilide, a Class III antiarrhythmic agent and a selective blocker of IKr. separated the delayed rectifier current into drug-sensitive current (IKr) and drug-insensitive current (IKs). The dofetilide-sensitive current activated rapidly and it showed two components of deactivation, the larger of which was very slow, while the dofetilide-insensitive current activated more slowly and deactivated quickly. The effects of propofol, an IKs blocker, on the delayed rectifier current were also investigated. The results show that IKs contributes to IK.tall and that the more positive the membrane potential, the more the contribution of IKs. The ratio of IKs to IKr in tail currents at -40 mV after a 1 s clamp pulse to +40 mV was 0.3-0.4:1. Dofetilide slowed spontaneous activity, suggesting that IKr contributes to the pacemaker activity of the SA node cell.

Membrane currents underlying delayed rectification and pace-maker activity in frog atrial muscle.

1. A double sucrose gap method has been used to polarize and voltage clamp frog atrial muscle strips.2. In response to steady depolarizing currents, normally quiescent strips often show pace-maker activity, and long lasting depolarization occurs when the current is terminated.3. Voltage clamp experiments reveal the presence of two current components underlying delayed rectification.4. The first of these components has a time constant which varies with potential and is approximately 500 msec at -90 mV. Its reversal potential usually lies between -70 and -40 mV and has always been found to be positive to the resting potential of normally quiescent fibres.5. The time constant of the second component is extremely slow (tau [unk] 5 sec at -90 mV). Its reversal potential is much more positive than that of the faster component.6. The results confirm the presence of a component of inward current which is insensitive to tetrodotoxin (TTX), having an activation threshold about 20 mV positive to the sodium threshold. This current differs from the two components underlying delayed rectification both in its greater speed of activation and in showing inactivation. The inactivation of this TTX-insensitive current is also a fairly rapid process.7. It is suggested that pace-making in sino-atrial muscle may depend upon the deactivation of the faster component of delayed rectification and that the TTX-insensitive inward current is also involved.

A quantitative analysis of the slow component of delayed rectification in frog atrium.

1. The slow component of delayed rectification in the atrial muscle membrane of Rana ridibunda has been analysed quantitatively using a voltage-clamp technique.2. It is shown that the current is proportional to a variable which obeys first-order kinetics. At negative potentials the time constant of this process is very long (tau = 3 sec at -55 mV) but it becomes faster at positive potentials (tau = 1.85 sec at +25 mV).3. In the steady state the degree of activation is a sigmoid function of potential. The activation threshold varies substantially between preparations but is usually negative to -30 mV.4. The instantaneous current-voltage relation is nearly linear and its reversal potential most frequently lies between zero and -30 mV.5. The properties of this current system resemble those of the current system i(x2) found in mammalian Purkinje fibres.

Internal K ions modulate the action of external cations on hyperpolarization-activated inward current in rabbit isolated sinoatrial node cells.

We have investigated the effect of change in external Na+ concentration on the hyperpolarization-activated inward current (I(f)) in the presence of different internal cations. Rabbit single isolated sinoatrial node cells were studied using the whole-cell patch-clamp technique. With 140 mM K+ pipettes, lowering [Na+]o causes the fully activated I/V curve for I(f) to shift in a negative direction without a significant decrease of the slope conductance. The PNa/PK ratio, as defined by the Goldman-Hodgkin-Katz equation, is concentration-dependent: the lower the [Na+]o, the higher PNa/PK. The conductance/concentration relationship for I(f) shows saturation at low [Na+]o or [K+]o, indicating that the channel has a strong affinity for external cations. With 140 mM Cs+ pipettes, the I/V curve shows strong inward rectification and inward I(f) current decreases almost proportionally to the decrease in [Na+]o; the conductance/concentration relationship for I(f) shifts to the right suggesting that the binding affinity of the external binding site is reduced. These results suggest that the I(f) channel is a multi-ion channel with a high-affinity external binding site, the affinity of which is modulated by internal cations.

Identification of the pace-maker current in frog atrium.

1. The nature and interactions of the membrane currents underlying induced pace-maker activity in frog atrial muscle have been investigated using a double sucrose gap technique. 2. The membrane current which controls the speed of the atrial pacemaker depolarization (the pace-maker current, ip), is shown to be an outward current activated within the plateau potential range of a normal action potential. The subsequent deactivation of ip at more negative potentials unmasks the depolarizing action of time-independent inward membrane currents so that a pace-maker potential can result. 3. The deactivation of ip over a limited potential range (between about -30 and -60 mV) can be reliably recorded by switching on the voltage clamp during an induced pace-maker depolarization. 4. Investigation of the time and voltage-dependent behaviour of ip over a much wider potential range is less straightforward. How ip can be separated from other components of outward current present in the decay tails following square voltage clamp depolarizations is described. 5. The majority of such current decay tails contain three components of outward current. It appears that two of these components, one of which is ip, are true Hodgkin-Huxley conductance systems chiefly carrying potassium ions. 6. The nature of the third current, which decays very slowly at moderate membrane potentials (about -40 mV), is discussed and reasons are briefly given for considering it to result from the accumulation of potassium ions in extracellular spaces. Preliminary evidence that potassium depletion occurs at potentials negative to the resting potential of the trabeculum is also presented. 7. Because of the obvious complexities involved, a quantitative analysis of the atrial outward currents is not attempted here but forms the subject of a following paper (Brown, Clark & Noble, 1976a).

Analysis of pace-maker and repolarization currents in frog atrial muscle.

1. A quantitative analysis of the time-dependent component of outward membrane current in atrial wall trabeculae from Rana catesbeiana and Rana ridibunda has been carried out using a double sucrose gap technique. 2. Separation of the different components of delayed outward current was hampered by the sigmoid onset of one of the outward current systems ixfast and by the development of potassium ion accumulation which prevented current activation from reaching a steady state at positive membrane potentials. Semilogarithmic analysis of positive current decay tails recorded immediately following square voltage clamp depolarizations was therefore used to separate the two membrane conductance components ixfast and ixslow and the third component, attributable to potassium ion accumulation, which was almost invariably present in the tails. 3. It is shown that inaccuracies in this method of semilogarithmic separation of components caused by visual assessment of the i3 (accumulation) line are minor compared with the large changes in the time constants of ixfast and more especially of ixslow which would result from ignoring the potassium accumulation component. 4. Such semilogarithmic separation of the three components of outward current gave separate activation curves for each of the two membrane conductance components, ixfast and ixslow. 5. Measurement of 'total' activation curves in which all components of outward current were represented could be made more easily and fairly reliably. The position and shape of these activation curves on the voltage axis were found to closely resemble those obtained by three component separation. It is therefore suggested that such a simplified analysis reflects the properties of the individual currents sufficiently well for it to be of use in preliminary studies of, for example, drug action. 6. The kinetic properties of the atrial outward currents have been investigated over a wide potential range. Because of the presence of potassium ion accumulation, an indirect method of obtaining the average value of 1/gamma for outward current decay at negative potentials had to be employed. 7. It is shown that some degree of inward-going rectification is associated with the outward current systems of frog atrium. 8. The possible reasons for the differences between the analysis presented here and those presented earlier by us (Brown & Noble, 1969a, b) and by Ojeda & Rougier (1974) are discussed.

The influence of non-uniformity on the analysis of potassium currents in heart muscle.

1. A method is described for determining the space constant gamma of heart muscle strips using a sucrose gap technique. 2. The average value of gamma for frog atrial trabeculae was found to be nearly 700 mum. This value is nearly twice the length of the test gap (400 mum). Near the resting potential, the voltage non-uniformity should be about 10%. This was confirmed experimentally by comparing the membrane voltages recorded across the current-passing and voltage-recording sucrose gaps. 3. The non-uniformity during large depolarizations was calculated using a computer model. This model includes the inward-going rectification displayed by iK1 and the delayed rectification that occurs following depolarizations beyond -40mV. A single component of delayed rectification was included. 4. It is shown that even very large non-uniformities have relatively small effects on the shape of the activation curve and on the time course of onset or decay of current. 5. It is comcluded that the fast component of current decay described in a previous paper (Brown, Clark & Noble, 1976b) is not attributable to a non-uniformity artifact.

High selectivity of the i(f) channel to Na+ and K+ in rabbit isolated sinoatrial node cells.

The ionic selectivity of the hyperpolarization-activated inward current (i(f)) channel to monovalent cations was investigated in single isolated sinoatrial node cells of the rabbit using the whole-cell patch-clamp technique. With a 140 mM K+ pipette, replacement of 90% external Na+ by Li+ caused a -24.5 mV shift of the fully activated current/voltage I/V curve without a significant decrease of the slope conductance. With a 140 mM Cs+ pipette, the i(f) current decreased almost proportionally to the decrease in external [Na+]o as Li+ was substituted. These responses are practically the same as those observed with N-methyl glucamine (NMG+) substitution, suggesting that the relative permeability of Li+ compared with Na+ for the i(f) channel is as low as that of NMG+. When Cs+ or Rb+ was substituted for internal K+, the fully activated I/V relationship for i(f) showed strong inward rectification with a positive reversal potential, indicating low permeability of the i(f) channel for Cs+ and Rb+. These results show that the i(f) channel is highly selective for Na+ and K+ and will not pass the similar ions Li+ and Rb+. Such a high degree of selectivity is unique and may imply that the structure of the i(f) channel differs greatly from that of other Na+ and K+ conducting channels.

Modulation of delayed rectifier potassium current, iK, by isoprenaline in rabbit isolated pacemaker cells.

Permeabilized patch whole-cell voltage clamp methods were used to investigate the effects of isoprenaline (ISO) on total delayed rectifier potassium current, iK, in rabbit sino-atrial (SA) node pacemaker cells; total iK is composed of the rapidly activating iKr and the slowly activating iKs, but predominantly iKr in this species. ISO (20 nM) increased the amplitude of total iK and caused a negative shift of approximately 10 mV in the activation curve for iK, both in the absence and in the presence of 300 nM nisoldipine to block the L-type Ca2+ current, iCa,L. The same concentration (20 nM) of ISO increased the spontaneous pacemaker rate of SA node pacemaker cells by 16%. In addition to increasing the amplitude of iK, ISO (20-50 nM) also increased the rate of deactivation of this current. The stimulation of iK by ISO was reversed by 10 microM H-89, a selective protein kinase A inhibitor, but not by 200 nM bisindolymaleimide I, a selective protein kinase C inhibitor. It therefore appears that the mechanisms by which -adrenoceptor agonists increase pacemaking rate in sinoatrial node pacemaker cells include an increase in the rate of deactivation of iK in addition to the well-documented augmentation of iCa,L and the positive shift of the activation curve for the hyperpolarization-activated inward current, if. The observations are also consistent with a role for protein kinase A in the stimulation of iK by ISO in SA node cells.

Membrane currents underlying activity in frog sinus venosus.

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

Nitric oxide can increase heart rate by stimulating the hyperpolarization-activated inward current, I(f).

We investigated the chronotropic effect of increasing concentrations of sodium nitroprusside (SNP, n = 8) or 3-morpholinosydnonimine (SIN-1, n = 6) in isolated guinea pig spontaneously beating sinoatrial node/atrial preparations. Low concentrations of NO donors (nanomolar to micromolar) gradually increased the beating rate, whereas high (millimolar) concentrations decreased it. The increase in rate was (1) enhanced by superoxide dismutase (50 to 100 U/mL, n = 6), (2) prevented by the guanylyl cyclase inhibitors 6-anilino-5,8-quinolinedione (5 mumol/L, n = 6) or 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (10 mumol/L, n = 6), and (3) mimicked by 8-bromo-cGMP (n = 6) with no additional positive chronotropic effect of SIN-1 (n = 5). The response to 10 mumol/L SNP (n = 28) or 50 mumol/L SIN-1 (n = 16) was unaffected by IcaL antagonism with nifedipine (0.2 mumol/L) but was abolished after blockade of the hyperpolarization-activated inward current (I(f)) by Cs+ (2 mmol/L) or 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium chloride (1 mumol/L). The effect on I(f) was further evaluated in rabbit isolated patch-clamped sinoatrial node cells (n = 21), where we found that 5 mumol/L SNP or SIN-1 caused a reversible Cs(+)-sensitive increase in this current (+130% at -70 mV and +250% at -100 mV). In conclusion, NO donors can affect pacemaker activity in a concentration-dependent biphasic fashion. Our results indicate that the increase in beating rate is due to stimulation of I(f) via the NO-cGMP pathway. This may contribute to the sinus tachycardia in pathological conditions associated with an increase in myocardial production of NO.

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

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

Effects of high potassium and the bradycardic agents ZD7288 and cesium on heart rate of rabbits and guinea pigs.

The effects of 2 mM cesium (Cs+) and a novel selective bradycardic agent ZD7288 (0.64 microM) on sinoatrial Node (SAN) pacing rate were investigated in an isolated guinea pig SAN/atrial preparation, rabbit SAN preparation, and isolated working rabbit heart preparation. The effect of Cs+ and ZD7288 on the response of the preparations to increased extracellular potassium concentration ([K+]o) was also studied. Cs+ reduced beating frequency by 24% in isolated rabbit SAN (n = 16, p < 0.01) and by 21% in isolated working rabbit heart (n = 9, p < 0.01). ZD7288 decreased beating rate by 53% in guinea pig SAN (n = 7, p < 0.01) and by 38% in isolated working rabbit heart (n = 6, p < 0.01). In all three preparations, increased [K+]o significantly decreased the rate (p < 0.01) in normal Tyrode's solution but had no effect in the presence of Cs+ and caused tachycardia (p < 0.01) in the presence of ZD7288. We conclude that Cs+ and ZD7288 decrease pacing rate in rabbits and guinea pigs, possibly through modulation of the hyperpolarization-activated current (I(f)). ZD7288 is a more effective bradycardic agent than Cs+.