Biomedical subjects

D A Terrar

Publications and source records attributed to D A Terrar.

At least 19 recordsLinked to original sources

PubMed2001

Nitric oxide does not modulate the hyperpolarization-activated current, I(f), in ventricular myocytes from spontaneously hypertensive rats.

OBJECTIVE: : In sinoatrial (SA) node cells, nitric oxide (NO) exerts a dual effect on the hyperpolarization-activated current, I(f), i.e. in basal conditions NO enhances I(f) whereas in the presence of beta-adrenergic stimulation it decreases it. Recent studies have shown that I(f) is present in ventricular myocytes from hypertrophied or failing hearts where it may promote abnormal automaticity. Since these pathological conditions are associated with increased sympathetic tone and upregulation of myocardial NO production, we set out to investigate whether I(f) is similarly modulated by NO in hypertrophied ventricular myocytes. METHODS: Left ventricular myocytes were isolated from 18-20-month-old spontaneously hypertensive rats (SHRs). Membrane current was measured under whole-cell or amphotericin-perforated patch-clamp conditions, at 35 degrees C. RESULTS: Application of diethylamine-NO (DEA-NO, 1-100 microM) did not alter the amplitude or voltage dependence of activation of I(f) under basal conditions (half-activation voltage, V(h): control -82.9+/-2.6, DEA-NO -84.0+/-2.6 mV). Similarly, I(f) was not affected by the inhibition of endogenous NO production (L-NMMA, 500 microM) or guanylate cyclase (ODQ, 10 microM). Forskolin (10 microM) or isoprenaline (100 nM) elicited a positive shift in V(h) but subsequent application of DEA-NO did not further affect the properties of I(f). CONCLUSIONS: Our results show that, unlike in SA node cells, in SHR ventricular myocytes basal and adrenergically stimulated I(f) is not modulated by exogenous NO or by constitutive NO or cGMP production.

Adenylyl Cyclases↗

PubMed2001

NO-cGMP pathway increases the hyperpolarisation-activated current, I(f), and heart rate during adrenergic stimulation.

OBJECTIVES: The role of the nitric oxide (NO)-cGMP pathway in the autonomic modulation of cardiac pacemaking is controversial and may involve an interplay between the L-type calcium current, I(CaL), and the hyperpolarisation activated current, I(f). We tested the hypothesis that following adrenergic stimulation, the NO-cGMP pathway stimulates phosphodiesterase 2 (PDE2) to reduce cAMP dependent stimulation of I(f) and heart rate (HR). METHODS: In the presence of norepinephrine (NE, 1 microM), the effects of the NO donor sodium nitroprusside (SNP) were evaluated in sinoatrial node (SAN)/atria preparations and isolated SAN cells from adult guinea pigs. RESULTS: Contrary to our hypothesis, SNP (10 and 100 microM, n=5) or the membrane permeable cGMP analogue, 8Br-cGMP (0.5 mM, n=6) transiently increased HR by 5+/-1, 12+/-1 and 12+/-2 beats/min, respectively. The guanylyl cyclase inhibitor 1H-(1,2,4)-oxadiazolo-(4,3-a)-quinoxalin-1-one (ODQ, 10 microM, n=5) abolished the increase in HR to SNP (100 microM) as did the I(f) blockers caesium chloride (2 mM, n=7) and 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)-pyrimidinium chloride (ZD7288, 1 microM, n=7). Addition of SNP (10 microM) also transiently increased I(f) in SAN cells (n=5). After inhibition of PDE2 with erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA, 10 microM, n=5), the increase in HR to SNP in the presence of NE was significantly augmented and maintained. RT-PCR analysis confirmed the presence of PDE2 in addition to cGMP inhibited PDE3 mRNA in central SAN tissue. CONCLUSIONS: These results suggest that during adrenergic stimulation, activation of the NO-cGMP pathway does not decrease HR, but has a transient stimulatory effect that is I(f) dependent, and is limited in magnitude and duration by stimulation of PDE2.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2001

Inotropic actions of protein kinase C activation by phorbol dibutyrate in guinea-pig isolated ventricular myocytes.

The mechanisms which underlie the inotropic actions of phorbol dibutyrate (PDBu), a synthetic compound which can directly activate protein kinase C (PKC), were investigated in guinea-pig isolated ventricular myocytes. Exposure of cells to PDBu (10(-7) M) reduced myocyte contraction amplitude to 46 +/- 3% of control (n = 8; P < 0.05) with an associated shortening in action potential duration (action potential duration at 90% repolarisation (APD90) was reduced to 83 +/- 1% of control; P < 0.05). The negative inotropic actions of PDBu and the shortening in action potential duration were abolished in the presence of a selective PKC inhibitor, Ro31-8220. Calcium transients (constructed from calcium-activated tail currents following interruption of action potentials by voltage clamp to -70 mV) were reduced following exposure to 10(-7) M PDBu by 38 +/- 2% (n = 9, P < 0.05). L-type calcium currents were not significantly altered following exposure to 10(-7) M PDBu (98 +/- 2% of control; P > 0.05). In contrast, delayed rectifier potassium currents (I(K)) were enhanced to 154 +/- 8% of control (n = 7; P < 0.05) by 10(-7) M PDBu. This enhancement of I(K) may contribute to the observed shortening in action potential duration observed following exposure to PDBu under the conditions of our experiments. When the action potential configuration was maintained throughout the experiment by applying a voltage-clamp waveform, 10(-7) M PDBu still reduced contraction amplitude to 57 +/- 3% of control (P < 0.05). Exposure to 10(-7) M PDBu also suppressed spontaneous activity (both spontaneous potential fluctuations induced by the beta-adrenergic agonist isoprenaline (40 nM), and transient inward currents induced by the cardiac glycoside ouabain (1 microM) under voltage clamp). It therefore appears that the reduction in myocyte contraction amplitude induced by exposure to PDBu may result in part through mechanisms independent of action potential shortening, which may include direct actions of protein kinase C on the function of the sarcoplasmic reticulum (SR) calcium store and/or on contractile proteins (though action potential shortening would be expected to cause a further decrease as a consequence of reduced calcium loading of the SR). The reduction of spontaneous activity caused by PDBu may also result from changes in the function of the SR store mediated by protein kinase C.

Action Potentials↗

PubMed2001

Actions of arachidonic acid on contractions and associated electrical activity in guinea-pig isolated ventricular myocytes.

The actions of arachidonic acid (AA) were investigated in guinea-pig isolated ventricular myocytes. Exposure of myocytes to 10 microM AA reduced the amplitude of contractions and calcium transients accompanying action potentials at a frequency of 1 Hz. AA (10 microM) also reduced the amplitude of calcium currents recorded under voltage-clamp conditions. The suppression of contraction by AA was not prevented by either 10 microM trihydroindomethicin (to inhibit cyclo-oxygenase) or 10 microM ETYA (5,8,11,14-eicosatetraynoic acid, to inhibit AA metabolising enzymes), showing that the actions of AA appeared not to be mediated by these metabolites. The reduction of contraction by 10 microM AA was also not prevented by the protein kinase C inhibitor, Ro31-8220 (1 microM), showing that this pathway appeared not to be required for the observed effect. Direct effects of AA may be involved. A further action of 10 microM AA was to suppress spontaneous electrical activity induced by either the beta-adrenergic agonist isoprenaline or the Na(+) pump inhibitor, ouabain. This effect of AA on spontaneous activity might be associated with the observed reduction of calcium entry through L-type calcium channels, although additional effects of AA on calcium release from the sarcoplasmic reticulum might also be involved. Experimental Physiology (2001) 86.4, 437-449.

5,8,11,14-Eicosatetraynoic Acid↗

PubMed2000

Protein kinase C enhances the rapidly activating delayed rectifier potassium current, IKr, through a reduction in C-type inactivation in guinea-pig ventricular myocytes.

1. The rapidly activating delayed rectifier potassium current, IKr, was studied in guinea-pig ventricular myocytes in the presence of thiopentone, which blocks the more slowly activating component of the delayed rectifier potassium current, IKs, and using whole cell perforated patch clamp or switched voltage clamp with sharp electrodes to minimise intracellular dialysis. 2. Activation of protein kinase A (PKA) by isoprenaline or forskolin caused an increase in IKr tail currents. Following a 300 ms depolarising step to +20 mV, mean tail current amplitude was increased 47 +/- 12% by isoprenaline, and 73 +/- 13% by forskolin. No increase in IKr was observed when IKr was studied using whole cell ruptured patch clamp and there was no change in the reversal potential of IKr in the presence of isoprenaline. 3. The rectification of the current sensitive to E4031, a selective IKr blocker, was markedly reduced in the presence of isoprenaline and the region of negative slope was absent. This is consistent with a reduction in the inactivation of IKr and was supported by the finding that IKr, in the presence of isoprenaline, was somewhat less sensitive to block. E4031 (5 microM) blocked only 81 +/- 5% of IKr in the presence of isoprenaline compared to 100 +/- 0% in control. 4. The forskolin- and isoprenaline-induced increases in IKr were inhibited by staurosporine and by the selective protein kinase C (PKC) inhibitor bisindolymaleimide I. Direct activation of PKC by phorbol dibutyrate increased IKr tail currents by 24 +/- 5%. Both the isoprenaline- and forskolin-induced increases in IKr were inhibited when calcium entry was reduced by block of ICa with nifedipine or when myocytes were pre-incubated in BAPTA-AM. 5. The selective PKA inhibitor KT5720 prevented the isoprenaline-induced increase in IKr only when the increase in ICa was also suppressed. 6. These data show a novel mechanism of regulation of IKr by PKC and this kinase was activated by beta-adrenoceptor stimulation. IKr seems to be enhanced through a reduction in the C-type inactivation which underlies the rectification of the channel and such a mechanism may occur in other channels with this type of inactivation.

Adrenergic beta-Agonists↗

PubMed2000

Role of cGMP-inhibited phosphodiesterase and sarcoplasmic calcium in mediating the increase in basal heart rate with nitric oxide donors.

Nitric oxide (NO) donors increase heart rate (HR) through a guanylyl cyclase-dependent stimulation of the pacemaker current I(f), without affecting basal I(Ca-L). The activity of I(f)is known to be enhanced by cyclic nucleotides and by an increase in cytosolic Ca(2+). We examined the role of cGMP-dependent signaling pathways and intracellular Ca(2+)stores in mediating the positive chronotropic effect of NO donors. In isolated guinea pig atria, the increase in HR in response to 1-100 micromol/l 3-morpholino-sydnonimine (SIN-1; with superoxide dismutase, n=6) or diethylamine-NO (DEA-NO, n=8) was significantly attenuated by blockers of the cGMP-inhibited phosphodiesterase (PDE3; trequinsin, milrinone or Ro-13-6438, n=22). In addition, the rate response to DEA-NO or sodium nitroprusside (SNP) was significantly reduced following inhibition of PKA (KT5720 or H-89, n=15) but not PKG (KT5728 or Rp-8-pCPT-cGMPs, n=16). Suppression of sarcoplasmic (SR) Ca(2+)release by pretreatment of isolated atria with ryanodine or cyclopiazonic acid (2 micromol/l and 60 micromol/l, n=16) significantly reduced the chronotropic response to 1-100 micromol/l SIN-1 or DEA-NO. Moreover, in isolated guinea pig sinoatrial node cells 5 micromol/l SNP significantly increased diastolic and peak Ca(2+)fluorescence (+13+/-1% and +28+/-1%, n=6, P<0.05). Our findings are consistent with a functionally significant role of cAMP/PKA signaling (via cGMP inhibition of PDE3) and SR Ca(2+)in mediating the positive chronotropic effect of NO donors.

Animals↗

PubMed2000

Localisation and functional significance of ryanodine receptors during beta-adrenoceptor stimulation in the guinea-pig sino-atrial node.

OBJECTIVE: Recent evidence shows that calcium released from the sarcoplasmic reticulum (SR) plays an important role in the regulation of heart rate. The aim of this study was to investigate the subcellular distribution of ryanodine receptors in the guinea-pig sino-atrial (SA) node and to determine their functional role in the regulation of pacemaker frequency in response to beta-adrenoceptor stimulation. METHODS: Monoclonal antibodies raised against the cardiac ryanodine receptor were used with confocal microscopy to investigate ryanodine receptor distribution in single guinea-pig SA node cells. The functional role of ryanodine receptors was investigated in both multicellular SA node/atrial preparations and in single SA node cells. RESULTS: Ryanodine receptor labelling was observed in all SA node cells studied and showed both subsarcolemmal and intracellular staining. In the latter, labelling appeared as transverse bands with a regular periodicity of approximately 2 microm. This interval resembled that of the expected sarcomere spacing but did not, however, depend on the presence of transverse tubules. The bands of ryanodine receptors appeared to be located in the region of the Z lines, based on co-distribution studies with antibodies to alpha-actinin, myomesin and binding sites for phalloidin. Functional studies on single SA node cells showed that application of ryanodine (2 micromol/l) reduced the rate of firing of spontaneous action potentials (measured using the perforated patch clamp technique) and this was associated with changes in action potential characteristics. Ryanodine also significantly decreased the positive chronotropic actions of isoprenaline in both multicellular and single cell preparations. In single cells exposed to 100 nmol/l isoprenaline, ryanodine caused a decrease in the rate of firing and this was associated with a decrease in the amplitude of the measured calcium transients. CONCLUSIONS: These findings are the first to show immunocytochemical evidence for the presence and organisation of ryanodine receptor calcium release channels in mammalian SA node cells. This study also provides evidence of a role for ryanodine sensitive sites in the beta-adrenergic modulation of heart rate in this species.

Action Potentials↗

PubMed2000

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.

Adrenergic beta-Agonists↗

PubMed1999

Effects of photoreleased cADP-ribose on calcium transients and calcium sparks in myocytes isolated from guinea-pig and rat ventricle.

Actions of photoreleased cADP-ribose (cADPR), a novel regulator of calcium-induced calcium release (CICR) from ryanodine-sensitive stores, were investigated in cardiac myocytes. Photoreleased cADPR caused an increase in the magnitude of whole-cell calcium transients studied in mammalian cardiac ventricular myocytes (both guinea-pig and rat) using confocal microscopy). Approx. 15 s was required following photorelease of cADPR for the development of its maximal effect. Photoreleased cADPR also increased the frequency of calcium 'sparks', which are thought to be elementary events which make up the whole-cell calcium transient, and were studied in rat myocytes, but had little or no effect on spark characteristics (amplitude, rise time, decay time and distance to half amplitude). The potentiating effects of photoreleased cADPR on both whole-cell transients and the frequency of calcium sparks were prevented by cytosolic application of the antagonist 8-amino-cADPR (5 microM). These experiments, therefore, provide the first evidence in any cell type for an effect of cADPR on calcium sparks, and are the first to show the actions of photoreleased cADPR on whole-cell calcium transients in mammalian cells. The observations are consistent with the effects of cADPR in enhancing the calcium sensitivity of CICR from the sarcoplasmic reticulum in cardiac ventricular myocytes, leading to an increase in the probability of occurrence of calcium sparks and to an increase in whole-cell calcium transients. The slow time-course for development of the full effect on whole-cell calcium transients might be taken to indicate that the influence of cADPR on CICR may involve complex molecular interactions rather than a simple direct action of cADPR on the ryanodine-receptor channels.

Adenosine Diphosphate Ribose↗

PubMed1999

An antagonist of cADP-ribose inhibits arrhythmogenic oscillations of intracellular Ca2+ in heart cells.

Oscillations of Ca2+ in heart cells are a major underlying cause of important cardiac arrhythmias, and it is known that Ca2+-induced release of Ca2+ from intracellular stores (the sarcoplasmic reticulum) is fundamental to the generation of such oscillations. There is now evidence that cADP-ribose may be an endogenous regulator of the Ca2+ release channel of the sarcoplasmic reticulum (the ryanodine receptor), raising the possibility that cADP-ribose may influence arrhythmogenic mechanisms in the heart. 8-Amino-cADP-ribose, an antagonist of cADP-ribose, suppressed oscillatory activity associated with overloading of intracellular Ca2+ stores in cardiac myocytes exposed to high doses of the beta-adrenoreceptor agonist isoproterenol or the Na+/K+-ATPase inhibitor ouabain. The oscillations suppressed by 8-amino-cADP-ribose included intracellular Ca2+ waves, spontaneous action potentials, after-depolarizations, and transient inward currents. Another antagonist of cADP-ribose, 8-bromo-cADP-ribose, was also effective in suppressing isoproterenol-induced oscillatory activity. Furthermore, in the presence of ouabain under conditions in which there was no arrhythmogenesis, exogenous cADP-ribose was found to be capable of triggering spontaneous contractile and electrical activity. Because enzymatic machinery for regulating the cytosolic cADP-ribose concentration is present within the cell, we propose that 8-amino-cADP-ribose and 8-bromo-cADP-ribose suppress cytosolic Ca2+ oscillations by antagonism of endogenous cADP-ribose, which sensitizes the Ca2+ release channels of the sarcoplasmic reticulum to Ca2+.

Action Potentials↗

PubMed1997

Block by propofol and thiopentone of the min K current (IsK) expressed in Xenopus oocytes.

The slowly activating component of the delayed rectifier potassium current (I(Ks)) in the heart is important during the repolarization of the cardiac action potential. Injection into Xenopus oocytes of mRNA coding for the min K protein induces a similar current (IsK) and recent observations support the hypothesis that functional channels result from the association of the min K protein with an endogenous K+ channel similar to the recently cloned KvLQT1. The general anaesthetics propofol and thiopentone have been shown to suppress cardiac I(Ks) with no effect on the rapidly activating component of I(K) (Takahashi and Terrar 1995). It was therefore of interest to test whether IsK was also inhibited by propofol and thiopentone. IsK was induced following injection into oocytes of min K mRNA which was transcribed in vitro from a synthetic gene (Hausdorff et al. 1991). IsK was activated by step depolarizations to a series of potentials from a holding potential of -40 mV and measured as the deactivating tail current on repolarization to the holding potential. Following a 2 s depolarization to +45 mV, propofol and thiopentone caused concentration-dependent reductions in IsK. The estimated IC50 value for the block of IsK by propofol was 250 microM and by thiopentone was 56 microM. Block of IsK by both propofol and thiopentone was not dependent on voltage or time. The reductions in IsK caused by propofol and thiopentone are consistent with the previously reported effects of these anaesthetics on I(Ks) in the heart and support the hypothesis that the min K protein contributes to the molecular basis of the cardiac I(Ks) channel.

Anesthetics, Intravenous↗

PubMed1997

Actions of cADP-ribose and its antagonists on contraction in guinea pig isolated ventricular myocytes. Influence of temperature.

Although it is becoming widely accepted that cADP-ribose (cADPR) can regulate calcium release from the endoplasmic reticulum in sea urchin eggs and in a variety of mammalian cell types, it remains controversial whether this substance might influence calcium release during excitation-contraction coupling in cardiac muscle. We have investigated possible actions of cADPR in intact cells isolated from guinea pig ventricle, paying particular attention to the possible influence of temperature. At 36 degrees C, myocyte contraction was influenced by cytosolic application of cADPR in a concentration-dependent manner (showing an approximately 30% increase in contraction with 5 mumol/L cADPR applied via a patch pipette in myocytes stimulated to fire action potentials at 1 Hz). Calcium transients measured with fura 2 were also increased by 5 mumol/L cADPR. Antagonists of cADPR reduced contraction at 36 degrees C (by approximately 35% with either 50 mumol/L 8-Br-cADPR or 5 mumol/L 8-amino-cADPR applied via the patch pipette). At room temperature (approximately 20 degrees C to 24 degrees C), no significant effects on contraction were detected with either cADPR or its antagonists. At 36 degrees C, treatment of the cells with a mixture of 2 mumol/L ryanodine and 1 mumol/L thapsigargin to suppress function of the sarcoplasmic reticulum stores of calcium prevented the action of 5 mumol/L cADPR applied via a patch pipette. These observations are consistent with an action of cytosolic cADPR to enhance calcium-induced calcium release from the sarcoplasmic reticulum in guinea pig ventricular myocytes at 36 degrees C. The observed influence of temperature under the conditions of our experiments is one factor that might help to account for failure to detect actions of cADPR and its analogues in some previous studies.

Adenosine Diphosphate Ribose↗

PubMed1996

A specific cyclic ADP-ribose antagonist inhibits cardiac excitation-contraction coupling.

BACKGROUND: Cyclic ADP-ribose (cADPR) has been shown to act as a potent cytosolic mediator in a variety of tissues, regulating the release of Ca2+ from intracellular stores by a mechanism that involves ryanodine receptors. There is controversy over the effects of cADPR in cardiac muscle, although one possibility is that endogenous cADPR increases the Ca2+ sensitivity of Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum. We investigated this possibility using 8-amino-cADPR, which has been found to antagonize the Ca2+-releasing effects of cADPR on sea urchin egg microsomes and in mammalian cells (Purkinje neurons, Jurkat T cells, smooth muscle and PC12 cells). RESULTS: In intact cardiac myocytes isolated from guinea-pig ventricle, cytosolic injection of 8-amino-cADPR substantially reduced contractions and Ca2+ transients accompanying action potentials (stimulated at 1Hertz). These reductions were not seen with injection of HEPES buffer, with heat-inactivated 8-amino-cADPR, or in cells pretreated with ryanodine (2 microM) to suppress sarcoplasmic reticulum function before injection of the 8-amino-cADPR. L-type Ca2+ currents and the extent of Ca2+ loading of the sarcoplasmic reticulum were not reduced by 8-amino-cADPR. CONCLUSIONS: These observations are consistent with the hypothesis that endogenous cADPR plays an important role during normal contraction of cardiac myocytes. One possibility is that cADPR sensitizes the CICR mechanism to Ca2+, an action antagonized by 8-amino-cADPR (leading to reduced Ca2+ transients and contractions). A direct effect of 8-amino-cADPR on CICR cannot be excluded, but observations with caffeine are not consistent with a non-selective block of release channels.

Adenosine Diphosphate Ribose↗

PubMed1996

Separation of the components of the delayed rectifier potassium current using selective blockers of IKr and IKs in guinea-pig isolated ventricular myocytes.

Delayed rectifier potassium current (IK) was investigated in guinea-pig isolated ventricular myocytes under voltage-clamp conditions ('switched' single electrode clamp), using selective blockers and/or different activation protocols to separate its rapid (IKr) and slow (IKs) components. The class III antiarrhythmic compound E4031 (5 microM) was used to block IKr and the anaesthetic drugs propofol (100 microM) or thiopentone (100 microM) to block IKs. In all experiments L-type calcium currents were blocked with nifedipine (2 microM). Complementary effects of E4031 and the anaesthetic drugs on the components of IK were observed. The E4031-sensitive current (IKr) resembled the current remaining in the presence of the anaesthetics and, likewise, the anaesthetic-sensitive current (IKs) resembled the current remaining in the presence of E4031. Under the conditions of these experiments, the relative contribution of the two components to total IK tail current was found to be approximately equal after a 400 ms depolarization to +40 mV. For example, IKr was 58 +/- 10% of total IK tail current when measured as the E4031-sensitive current, 41 +/- 6% as the propofol-insensitive current and 43 +/- 7% as the thiopentone-insensitive current. In the presence of both E4031 and propofol or thiopentone the IK tail current deactivating at -40 mV was completely eliminated, leaving a residual current during the pulse which reversed at -46 +/- 1 mV. To avoid complication of the 'envelope of tails' test with this residual current, the tail:pulse ratio was calculated for the anaesthetic-sensitive component and this was constant, consistent with block of a single component of IK. Forskolin (1 microM) enhanced the current most consistent with IKs. Propofol (300 microM) caused a 64 +/- 3% increase in action potential duration in the presence of both E4031 (5 microM) and nifedipine (2 microM), consistent with an important role for IKs in the repolarization of the action potential in the guinea-pig heart. The observations therefore provide further support for separate components of IK with different characteristics in the guinea-pig heart; it appears that E4031 and propofol or thiopentone are useful complementary tools for their separation.

Action Potentials↗

PubMed1996

The deactivation kinetics of the delayed rectifier components IKr and IKs in guinea-pig isolated ventricular myocytes.

The deactivation kinetics of the delayed rectifier potassium current (IK) were studied in guinea-pig isolated ventricular myocytes at 35-36 degrees C using 5 microM E4031 to selectively block the rapidly activating component (IKr) and 300 microM thiopentone to selectively suppress the slowly activating component (IKs). IK was activated by depolarization of 400 or 1000 ms duration to +40 mV, and tail currents were analysed on repolarization to potentials between -30 and -60 mV. Before exposure to drugs, the deactivation of total IK was fitted by two exponential functions at all potentials and, at -40 mV, the time constants were 188 +/- 12 and 2510 +/- 185 ms (n = 25); increasing the pulse duration from 400 to 1000 ms (expected to increase IKs rather than IKr under these conditions) caused a significant increase in the amplitude of only the fast component of deactivation at -40 mV (from 372 +/- 32 to 479 +/- 46 pA). The decay of IKr was biexponential at all potentials when detected as the E4031-sensitive current and at -30 to -50 mV when detected as the thiopentone-resistant current and was accelerated as the membrane potential was made more negative. The amplitudes of the two components of decay of IKr (as either E4031-sensitive or thiopentone-resistant current) were similar and neither were significantly increased when pulse duration was increased from 400 to 1000 ms. The decay of IKs detected as the E4031-resistant current was biexponential at -30 mV and, in some cells, at -40 mV, but decayed with predominately a single, fast component of deactivation at -50 and -60 mV, which was accelerated at the more negative potentials. A slow component of decay of IKs was detected in more cells when thiopentone was used to isolate IKs, although it was of much smaller amplitude than the fast component of decay. The presence of a slow component of decay of the thiopentone-sensitive IKs may result from a slight block of IKr by thiopentone. The amplitude of only the fast decay phase of IKs (detected using either drug) was increased when pulse duration was lengthened from 400 to 1000 ms. It seems likely that the relative importance of IKr and IKs in rate-dependent shortening of action potential duration may need to be re-evaluated in view of the relatively rapid deactivation of IKs, together with the prominence of the slow component of deactivation of IKr compared with that of IKs.

Action Potentials↗

PubMed1996

Possible role of calcium release from the sarcoplasmic reticulum in pacemaking in guinea-pig sino-atrial node.

Ryanodine and cyclopiazonic acid were used to alter the ability of the sarcoplasmic reticulum (SR) to store calcium. Interventions of this kind significantly reduced the rate of spontaneous beating of guinea-pig sino-atrial node preparations. In addition, both of these drugs decreased the rate of rise and modified other characteristics of the action potentials recorded from this region. It is proposed that calcium released from the SR plays an important, previously unrecognized, role in pacemaking in the sino-atrial node, possibly through regulation of sarcolemmal ionic currents.

Acetylcholine↗

PubMed1995

Effects of caffeine on background potassium current in isolated guinea pig ventricular myocytes.

We investigated effects of caffeine on inward rectifier potassium current (Ik1) in voltage-clamped ventricular cells by slow ramp depolarization (15 mV/s). Caffeine 10 mM applied in the solution bathing the cells consistently reduced the slope of the current-voltage (I-V) relation over the range of -80 to -40 mV. This effect of caffeine was not prevented by loading cells with BAPTA (1,2-bis(2-aminophenoxy) ethane N,N,N',N'-tetra-acetic acid) to suppress contraction. In the absence of caffeine, reducing extracellular potassium from 5.4 to 2.7 mM caused the expected shift of the reversal potential for current in the negative direction and increased rectification. In low potassium, 10 mM caffeine continued to reduce the slope of the I-V relation. When 2 mM barium was applied to suppress Ik1, any effects of 10 mM caffeine were slight or absent. The observations are consistent with a blocking action of caffeine on Ik1 in guinea pig ventricular myocytes.

Action Potentials↗

PubMed1994

Effects of etomidate on whole-cell and single L-type calcium channel currents in guineapig isolated ventricular myocytes.

We have investigated the effects of etomidate 4.4 mumol litre-1, 1.0 mg litre-1 and 27.4 mumol litre-1, 6.25 mg litre-1 on whole-cell and single L-type calcium channel currents in myocytes from guineapig ventricles. For whole-cell recordings, the cells were voltage-clamped and step depolarizations were applied from holding potential of -40 mV to various potentials to elicit L-type calcium currents. Peak calcium currents were decreased significantly by both low and high concentrations of etomidate. Ethanol in the same concentration with the highest etomidate solution (1.1 mmol litre-1) had no significant effect on calcium currents. When single calcium channel activity was investigated, the high concentration of etomidate significantly decreased the open probability of the channel with little or no effect on channel conductance. Mean closed time was increased significantly, caused apparently by prolongation of the slower of two exponential components fitted to histograms of the closed times. The mean open time was virtually unaffected. The low concentration of etomidate did not significantly affect single channel kinetics. These results showed that etomidate decreased L-type calcium current by altering the kinetics of the channel to favour the closed state without any significant change in conductance. However, compared with other anaesthetics which were investigated previously in our laboratory, the overall effect on calcium current appeared to be small.

Animals↗