Biomedical subjects

M R Boyett

Publications and source records attributed to M R Boyett.

At least 19 recordsLinked to original sources

PubMed2001

Heterogeneous expression of the delayed-rectifier K+ currents i(K,r) and i(K,s) in rabbit sinoatrial node cells.

1. The electrical activity of sinoatrial node cells is heterogeneous. To understand the reasons for this, the density of the delayed-rectifier K+ current and its two components, i(K,r) and i(K,s), as a function of the size (as measured by cell capacitance) of rabbit sinoatrial node cells was investigated using the whole-cell voltage-clamp technique at 35 degrees C. 2. i(K,r) and i(K,s) were isolated using E-4031 and 293B. Features of the E-4031-sensitive and 293B-insensitive currents corresponded well to those of i(K,r), while features of the E-4031-insensitive and 293B-sensitive currents corresponded well to those of i(K,s). 3. The densities of the outward current under control conditions and the drug-sensitive and -insensitive currents were significantly (P < 0.01) correlated with cell capacitance, with current densities being greater in larger cells. 4. The effects of partial blockade of i(K,r) by 0.1 microM E-4031 on spontaneous action potentials were greater in smaller cells. 5. It is concluded that there are cell size-dependent differences in the density of the i(K,r) and i(K,s) components, and these may be involved in the heterogeneity of the electrical activity of single sinoatrial node cells as well as that of the intact sinoatrial node.

Action Potentials↗

PubMed2001

Gradient model versus mosaic model of the sinoatrial node.

BACKGROUND: A radical reinterpretation (mosaic model) of the makeup of the sinoatrial (SA) node has been proposed to explain the characteristic regional differences in electrical activity between the periphery and center of the SA node. According to the mosaic model, the differences result from a change in the mix of atrial cells and uniform SA node cells from periphery to center, whereas according to the alternative gradient model, there are no atrial cells within the functional SA node, and the differences result from a change in the intrinsic properties of SA node cells from periphery to center. METHODS AND RESULTS: A mosaic model of peripheral and central tissue has been constructed computationally by use of a coupled ordinary differential equation network (CODE) in a 2D lattice (20x20), with each node of the lattice designated randomly as an atrial cell or SA node cell (in correct proportions for periphery and center). The mosaic model fails to predict the characteristic differences in action potential rate and shape between the periphery and center, whereas the existing gradient model can do so. CONCLUSIONS: The mosaic model of the SA node is untenable, and the SA node is adequately described by the gradient model.

Animals↗

PubMed2001

Control of the cardiac muscarinic K+ channel by beta-arrestin 2.

Control of the cardiac muscarinic K(+) current (i(K,ACh)) by beta-arrestin 2 has been studied. In Chinese hamster ovary cells transfected with m2 muscarinic receptor, muscarinic K(+) channel, receptor kinase (GRK2), and beta-arrestin 2, desensitization of i(K,ACh) during a 3-min application of 10 micrometer ACh was significantly increased as compared with that in cells transfected with receptor, channel, and GRK2 only (fade in current increased from 45 to 78%). The effect of beta-arrestin 2 was lost if cells were not co-transfected with GRK2. Resensitization (recovery from desensitization) of i(K,ACh) in cells transfected with beta-arrestin 2 was significantly slowed (time constant increased from 34 to 232 s). Activation and deactivation of i(K,ACh) on application and wash-off of ACh in cells transfected with beta-arrestin 2 were significantly slowed from 0.9 to 3.1 s (time to half peak i(K,ACh)) and from 6.2 to 13.8 s (time to half-deactivation), respectively. In cells transfected with a constitutively active beta-arrestin 2 mutant, desensitization occurred in the absence of agonist (peak current significantly decreased from 0.4 +/- 0.05 to 0.1 +/- 0.01 nA). We conclude that beta-arrestin 2 has the potential to play a major role in desensitization and other aspects of the functioning of the muscarinic K(+) channel.

Animals↗

PubMed2001

Engineering virtual cardiac tissue.

The kinetics of proteins involved in ion transfer, sequestration and binding in cardiac cells can be modelled to construct a model of the electrical activity of isolated cardiac cells as a system of ordinary differential equations. These cell models may be incorporated into tissue models, which, when combined with histology and anatomy, form virtual tissues. The effects of changes in specific protein expression, or changes in protein kinetics, produced by mutations or pharmacological agents, can be simulated using these tissue models and used to account for the whole organ effects of changes in specific ion-transport protein activity.

Action Potentials↗

PubMed2001

Pacemaker shift in the rabbit sinoatrial node in response to vagal nerve stimulation.

Effects of brief postganglionic vagal nerve stimulation on the activation sequence of the rabbit sinoatrial (SA) node were investigated. Activation sequences in a small area (7 mm x 7 mm) on the epicardial surface were measured in a beat-to-beat manner using an extracellular potential mapping system composed of 64 modified bipolar electrodes with high-gain and low-frequency band-pass filtering. The leading pacemaker site was recognised clearly from both the activation sequence and the characteristic morphology of the potentials. Vagal stimulation resulted in a short-lasting initial slowing of spontaneous rate followed by a long-lasting secondary slowing; a brief period of relative or absolute acceleration was interposed between the two slowing phases. During these changes of spontaneous rate, the leading pacemaker site shifted in a complex beat-to-beat manner by 1-6 mm alongside the crista terminalis in the superior or inferior direction. For the first spontaneous excitation following stimulation, the greater the slowing, the larger the distance of the pacemaker shift. There was no such linear relationship between the extent of slowing and the distance of pacemaker shift for the subsequent beats. These changes in the leading pacemaker site in response to vagal stimulation may be the result of the functional and morphological heterogeneity of the mammalian SA node in terms of innervation, receptor distribution and ion channel densities. Experimental Physiology (2001) 86.2, 177-184.

Animals↗

PubMed2001

Evidence of involvement of GIRK1/GIRK4 in long-term desensitization of cardiac muscarinic K+ channels.

The cardiac M2 muscarinic receptor/G protein/K+ channel system was studied in neonatal rat atrial cells cultured with and without 10 microM carbachol (CCh) for 24 h. Channel activity in CCh-pretreated cells was substantially reduced as a result of long-term desensitization regardless of whether the channel was activated by ACh in cell-attached patches or GTP in inside-out patches. Channel activity in CCh-pretreated cells was also low when the receptor was bypassed and the G protein and channel were directly activated by [gamma-S]GTP or both the receptor and G protein were bypassed and the channel was directly activated by trypsin. Finally, in CCh-pretreated cells, the whole cell K+ current was low when the channel was activated via the independent adenosine receptor. This suggests that the channel is involved in long-term desensitization. However, in CCh-pretreated cells, although the receptor was internalized, there was no internalization of the channel. We suggest that the function of the muscarinic K+ channel declines in long-term desensitization of the cardiac M2 muscarinic receptor/G protein/K+ channel system.

Acetylcholine↗

PubMed2001

Distribution of the muscarinic K+ channel proteins Kir3.1 and Kir3.4 in the ventricle, atrium, and sinoatrial node of heart.

The functionally important effects on the heart of ACh released from vagal nerves are principally mediated by the muscarinic K+ channel. The aim of this study was to determine the abundance and cellular location of the muscarinic K+ channel subunits Kir3.1 and Kir3.4 in different regions of heart. Western blotting showed a very low abundance of Kir3.1 in rat ventricle, although Kir3.1 was undetectable in guinea pig and ferret ventricle. Although immunofluorescence on tissue sections showed no labeling of Kir3.1 in rat, guinea pig, and ferret ventricle and Kir3.4 in rat ventricle, immunofluorescence on single ventricular cells from rat showed labeling in t-tubules of both Kir3.1 and Kir3.4. Kir3.1 was abundant in the atrium of the three species, as shown by Western blotting and immunofluorescence, and Kir3.4 was abundant in the atrium of rat, as shown by immunofluorescence. Immunofluorescence showed Kir3.1 expression in SA node from the three species and Kir3.4 expression in the SA node from rat. The muscarinic K+ channel is activated by ACh via the m2 muscarinic receptor and, in atrium and SA node from ferret, Kir3.1 labeling was co-localized with m2 muscarinic receptor labeling throughout the outer cell membrane.

Animals↗

PubMed2000

Residues and mechanisms for slow activation and Ba2+ block of the cardiac muscarinic K+ channel, Kir3.1/Kir3.4.

Mechanisms and residues responsible for slow activation and Ba(2+) block of the cardiac muscarinic K(+) channel, Kir3.1/Kir3.4, were investigated using site-directed mutagenesis. Mutagenesis of negatively charged residues located throughout the pore of the channel (in H5, M2, and proximal C terminus) reduced or abolished slow activation. The strongest effects resulted from mutagenesis of residues in H5 close to the selectivity filter; mutagenesis of residues in M2 and proximal C terminus equivalent to those identified as important determinants of the activation kinetics of Kir2.1 was less effective. In giant patches, slow activation was present in cell-attached patches, lost on excision of the patch, and restored on perfusion with polyamine. Mutagenesis of residues in H5 and M2 close to the selectivity filter also decreased Ba(2+) block of the channel. A critical residue for Ba(2+) block was identified in Kir3.4. Mutagenesis of the equivalent residue in Kir3.1 failed to have as pronounced an effect on Ba(2+) block, suggesting an asymmetry of the channel pore. It is concluded that slow activation is principally the result of unbinding of polyamines from negatively charged residues close to the selectivity filter of the channel and not an intrinsic gating mechanism. Ba(2+) block involves an interaction with the same residues.

Animals↗

PubMed2000

Inhibition of the K+ channel kv1.4 by acidosis: protonation of an extracellular histidine slows the recovery from N-type inactivation.

1. Acidosis alters the transient outward current, ito, in the heart. We have studied the mechanism underlying the effect of acidosis on one of the K+ channels, Kv1.4 (heterologously expressed in Xenopus laevis oocytes), known to underlie ito. 2. At pH 6.5, wild-type Kv1.4 current was inhibited during repetitive pulsing, in part as a result of a slowing of recovery from N-type inactivation. 3. Acidosis still caused slowing of recovery after deletion of just one (either the first or second) of the N-terminal inactivation ball domains. However, deletion of both the N-terminal inactivation ball domains greatly reduced the inhibition. 4. As well as the N-terminus, other parts of the channel are also required for the effect of acidosis, because, whereas the transfer of the N-terminus of Kv1.4 to Kv1.2 conferred N-type inactivation, it did not confer acidosis sensitivity. 5. Replacement of an extracellular histidine with a glutamine residue (H508Q) abolished the slowing of recovery by acidosis. Reduction of C-type inactivation by raising the bathing K+ concentration or by the mutation K532Y also abolished the slowing. 6. It is concluded that binding of protons to H508 enhances C-type inactivation and this causes a slowing of recovery from N-type inactivation and, thus, an inhibition of current during repetitive pulsing.

Amino Acid Substitution↗

PubMed2000

Cs+ block of the cardiac muscarinic K+ channel, GIRK1/GIRK4, is not dependent on the aspartate residue at position 173.

Cs+ block of GIRK1/GIRK4 expressed in Xenopus oocytes has been investigated. It has been reported that a negatively charged aspartate residue at position 172 in IRK1 is responsible for Cs+ block of the channel. IRK1, a homotetramer, has four aspartate residues at this position. GIRK1/GIRK4 is a heterotetramer and has two aspartate residues at the equivalent position (GIRK1-D173) and, consequently, it should be less sensitive to Cs+. Cs+ caused voltage-dependent block of GIRK1/GIRK4 current (measured with the two-microelectrode voltage-clamp technique). The apparent fraction of the electrical field through which Cs+ moves in order to reach its site of block (delta approximately equals 1.66) is comparable to that in IRK1, suggesting that Cs+ binds to a similar site in the two channels. GIRK1/GIRK4 was less sensitive than IRK1 to Cs+ -the Kd was 3.0-8.5 times greater and at potentials more negative than approximately or = to 130 mV there was voltage-dependent relief of block of GIRK1/GIRK4 (not the case with IRK1). However, the mutations GIRK1-D173A and GIRK1-D173Q increased the sensitivity of the channel to Cs+, while adding a negatively charged aspartate residue to GIRK4 at the equivalent position (GIRK4-N 79D) decreased Cs+ sensitivity. GIRK1-D173 cannot be the site of Cs+ block of GIRK1/GIRK4.

Amino Acid Sequence↗

PubMed2000

Characterisation of the transient outward K+ current in rabbit sinoatrial node cells.

OBJECTIVE: To (i) characterise the electrophysiological and pharmacological properties of the transient outward K+ current, I(to), (ii) determine the relationship between the density of I(to) and cell size, and (iii) determine the role of I(to) in electrical activity in rabbit sinoatrial node cells at 35 degrees C. METHODS: Rabbit sinoatrial node cells were studied using whole-cell voltage and current clamp techniques. RESULTS: I(to) showed half activation and inactivation at +11 and -49 mV, respectively. I(to) was blocked by 4-aminopyridine (4-AP) as well as the class I agents, quinidine and flecainide, with EC50 values of 326, 21 and 19 microM, respectively. The densities of the transient and sustained components of 4-AP-sensitive current were significantly correlated with cell capacitance, a measure of cell size, and were greater in cells with a larger capacitance. Block of I(to) by 4-AP affected both the action potential and pacemaker activity of sinoatrial node cells and the effects were greater in cells with a larger capacitance. CONCLUSIONS: I(to) in sinoatrial node cells shows similar electrophysiological and pharmacological properties to I(to) in atrial and ventricular cells. The expression of I(to) in sinoatrial node cells is heterogeneous and differs in large and small cells (likely to be from the periphery and centre of the sinoatrial node, respectively). I(to) plays an important role in action potential configuration and pacemaker activity in sinoatrial node cells, especially in larger cells.

4-Aminopyridine↗

PubMed2000

The sinoatrial node, a heterogeneous pacemaker structure.

This article focuses on the regional heterogeneity of the mammalian sinoatrial (SA) node in terms of cell morphology, pacemaker activity, action potential configuration and conduction, densities of ionic currents (i(Na), i(Ca,L), i(to), i(K,r), i(K,s) and i(f)), expression of gap junction proteins (Cx40, Cx43 and Cx45), autonomic regulation, and ageing. Experimental studies on the single SA node cell to the whole animal are reviewed. The heterogeneity is considered in terms of the gradient model of the SA node, in which there is gradual change in the intrinsic properties of SA node cells from periphery to centre, and the alternative mosaic model, in which there is a variable mix of atrial and SA node cells from periphery to centre. The heterogeneity is important for the dependable functioning of the SA node as the pacemaker for the heart, because (i) via multiple mechanisms, it allows the SA node to drive the surrounding atrial muscle without being suppressed electrotonically; (ii) via an action potential duration gradient and a conduction block zone, it promotes antegrade propagation of excitation from the SA node to the right atrium and prevents reentry of excitation; and (iii) via pacemaker shift, it allows pacemaking to continue under diverse pathophysiological circumstances.

Action Potentials↗

PubMed2000

Effects of halothane on the transient outward K(+) current in rat ventricular myocytes.

1. Halothane has been shown to affect several membrane currents in cardiac tissue including the L-type calcium current (I(Ca)), sodium current and a variety of potassium currents. However, little is known about the effects of halothane on the transient outward K(+) current (I(to)). 2. Single ventricular myocytes from rat hearts were voltage clamped using the whole cell patch configuration and an EGTA-containing pipette solution to record the Ca(2+)-independent, 4-aminopyridine sensitive component of I(to). 300 microM Cd(2+) or 10 microM nifedipine was used to block I(Ca). 3. At +80 mV, I(to) (peak current minus current at the end of the pulse) was 1.8+/-0.2 nA under control conditions which was reduced to 1.3+/-0.2 nA by 1 mM halothane (P:<0.001, mean+/-s.e.mean, n=9). The inhibition of I(to) by halothane was concentration-dependent (K(0.5), 1.1+/-0.2 mM). 4. One mM halothane led to a 16 mV shift in the steady-state inactivation curve towards negative membrane potentials (P:=0.005, n=8) but had no significant effect on the activation-voltage relationship (P:=0. 724). One mM halothane also increased the rate of inactivation of I(to); the dominant time constant of inactivation was reduced from 14+/-1 to 9+/-1 ms (P:=0.017, mean+/-s.e.mean, n=6). 5. These data show that halothane reduced I(to); 0.3 mM, close to the MAC(50) value for halothane, inhibited the current by 15% and as such, the inhibition of I(to) will be relevant to the clinical situation. Halothane induced a shift in the steady-state inactivation curve and accelerated the inactivation process of I(to) which could be responsible for its inhibitory effect. 6. Due to the differential transmural expression of I(to) in ventricular tissue, inhibition of I(to) would reduce the transmural dispersion of refractoriness which could contribute to the arrhythmogenic properties of halothane.

Action Potentials↗

PubMed2000

The concentration-dependent effects of propofol on rat ventricular myocytes.

UNLABELLED: Whether propofol contributes a direct negative inotropic effect is controversial. Our principal aim in this study was to determine whether negative inotropic effects of propofol occur at clinically relevant concentrations. We constructed the concentration-response relationship for the negative inotropic effects on intact, isolated, stimulated rat ventricular myocytes. Contraction was measured as cell shortening by using an optical system. Propofol was applied as dilutions of the commercial preparation in physiological saline solution. The drug vehicle had a minimal effect on myocyte contractility. Propofol produced a concentration-dependent reduction in evoked contraction at concentrations greater than 5 microM. The maximum effect was observed at >100 microM, with the K(0.5) calculated to be 34.5 microM (95% CI, 21.8-54.7 microM). In further experiments, we investigated the relationship between changes in contractility and changes in Ca(2+) transient (measured by using fura-2 fluorescence) after the application of propofol. By using the shift in the relationship of the cell length to fura-2 fluorescence ratio in the relaxation phase of a contraction as an index of Ca(2+) response of the myofilaments, we demonstrated that some of the negative inotropic effect of propofol may be caused by a reduction in myofilament Ca(2+) sensitivity. We confirmed this by comparing the reduction in contractility in the presence of propofol with that caused by reducing the extracellular Ca(2+) concentration. We observed that, for a decrease in the fura-2 fluorescence ratio of 21%, propofol caused a 12% (95% CI, 2% to 22%) greater reduction in contractility than predicted from reducing the extracellular Ca(2+) concentration. However, the K(0.5) for the negative inotropic effect of propofol we observed is more than 80 times the 50% effective concentration value for anesthesia. The potential relevance of these findings for clinical use of propofol in humans is discussed. IMPLICATIONS: By using intact, isolated rat heart ventricle cells, we investigated the mechanisms and concentration dependence of the depressant effect of propofol on contractility of the heart. We conclude that direct effects of propofol on the heart are unlikely to be of significance at the clinical dosage usually given.

Anesthetics, Intravenous↗

PubMed2000

Effects of isoflurane, sevoflurane, and halothane on myofilament Ca2+ sensitivity and sarcoplasmic reticulum Ca2+ release in rat ventricular myocytes.

BACKGROUND: The aim of this study was to describe and compare the effects of isoflurane, sevoflurane, and halothane at selected concentrations (i.e., concentrations that led to equivalent depression of the electrically evoked Ca2+ transient) on myofilament Ca2+ sensitivity, sarcoplasmic reticulum (SR) Ca2+ content, and the fraction of SR Ca2+ released during electrical stimulation (fractional release) in rat ventricular myocytes. METHODS: Single rat ventricular myocytes loaded with fura-2 were electrically stimulated at 1 Hz, and the Ca2+ transients and contractions were recorded optically. Cells were exposed to each anesthetic for 1 min. Changes in myofilament Ca2+ sensitivity were assessed by comparing the changes in the Ca2+ transient and contraction during exposure to anesthetic and low Ca2+. SR Ca2+ content was assessed by exposure to 20 mm caffeine. RESULTS: Isoflurane and halothane caused a depression of myofilament Ca2+ sensitivity, unlike sevoflurane, which had no effect on myofilament Ca2+ sensitivity. All three anesthetics decreased the electrically stimulated Ca2+ transient. SR Ca2+ content was reduced by both isoflurane and halothane but was unchanged by sevoflurane. Fractional release was reduced by both isoflurane and sevoflurane, but was unchanged by halothane. CONCLUSIONS: Depressed myofilament Ca2+ sensitivity contributes to the negative inotropic effects of isoflurane and halothane but not sevoflurane. The decrease in the Ca2+ transient is either responsible for or contributory to the negative inotropic effects of all three anesthetics and is either primarily the result of a decrease in fractional release (isoflurane and sevoflurane) or primarily the result of a decrease in SR Ca2+ content (halothane).

Actin Cytoskeleton↗

PubMed2000

Mathematical models of action potentials in the periphery and center of the rabbit sinoatrial node.

Mathematical models of the action potential in the periphery and center of the rabbit sinoatrial (SA) node have been developed on the basis of published experimental data. Simulated action potentials are consistent with those recorded experimentally: the model-generated peripheral action potential has a more negative takeoff potential, faster upstroke, more positive peak value, prominent phase 1 repolarization, greater amplitude, shorter duration, and more negative maximum diastolic potential than the model-generated central action potential. In addition, the model peripheral cell shows faster pacemaking. The models behave qualitatively the same as tissue from the periphery and center of the SA node in response to block of tetrodotoxin-sensitive Na(+) current, L- and T-type Ca(2+) currents, 4-aminopyridine-sensitive transient outward current, rapid and slow delayed rectifying K(+) currents, and hyperpolarization-activated current. A one-dimensional model of a string of SA node tissue, incorporating regional heterogeneity, coupled to a string of atrial tissue has been constructed to simulate the behavior of the intact SA node. In the one-dimensional model, the spontaneous action potential initiated in the center propagates to the periphery at approximately 0.06 m/s and then into the atrial muscle at 0.62 m/s.

Action Potentials↗

PubMed2000

Presence of the Kv1.5 K(+) channel in the sinoatrial node.

The aim of this study was to establish, using immunolabeling, whether the Kv1.5 K(+) channel is present in the pacemaker of the heart, the sinoatrial (SA) node. In the atrial muscle surrounding the SA node and in the SA node itself (from guinea pig and ferret), Western blotting analysis showed a major band of the expected molecular weight, approximately 64 kD. Confocal microscopy and immunofluorescence labeling showed Kv1.5 labeling clustered in atrial muscle but punctate in the SA node. In atrial muscle, Kv1.5 labeling was closely associated with labeling of Cx43 (gap junction protein) and DPI/II (desmosomal protein), whereas in SA node Kv1.5 labeling was closely associated with labeling of DPI/II but not labeling of Cx43 (absent in the SA node) or Cx45 (another gap junction protein present in the SA node). Electron microscopy and immunogold labeling showed that the Kv1.5 labeling in atrial muscle is preferentially associated with desmosomes rather than gap junctions.

Animals↗

PubMed2000

A novel background potassium channel in rat atrial cells.

A K+ channel activated by intracellular ATP has been observed in inside-out patches from rat atrial cells. The channel has a slope conductance of 130 +/- 5 pS in symmetrical 140 mM K+ solution, and is almost independent of voltage over the range from -80 to +80 mV. There is no detectable inactivation during application of ATP over a few minutes. In the presence of 3 mM intracellular ATP, channel openings occur as bursts with a mean open time of 1.7 ms, a mean closed time of 0.4 ms, a mean burst duration of 18 ms and a mean burst interval of 41 ms. Kinetic analysis suggests that ATP mainly affects the burst duration and the burst interval of the channel. Based on the properties above, the channel differs from other known K+ channels in cardiac cells and may contribute to background K+ current.

Adenosine Triphosphate↗