The antiestrogen tamoxifen blocks the delayed rectifier potassium current, IKr, in rabbit ventricular myocytes

Recently, two reports [R. E. Davis et al. (1997) Proc. Natl. Acad. Sci. USA 94, 4564-4569 and E. Fahy et al. (1997) Nucleic Acids Res. 25, 3102-3109] described a series of heteroplasmic mitochondrial DNA (mtDNA) mutations in the genes encoding two cytochrome c oxidase subunits (CO1 and CO2) which segregated in higher abundance with Alzheimer's disease subjects than controls. Using mtDNA-depleted NT2 cells, we provide further evidence that these two reports are erroneously based on a PCR artifact arising from the amplification of nuclear DNA encoded mtDNA pseudogenes (mtDNA psi s). Our findings are similar, but not identical, to other recent studies of these putative mtDNA psi sequences. This sequence variability may indicate that multiple mtDNA psi s, all of comparatively recent evolutionary origin are involved. While such pseudogenes are interesting in that they provide a molecular evolutionary "snapshot" of human ancestral mtDNA, it is unlikely that they play any role in the etiology of Alzheimer's disease.     

Gadolinium blocks the delayed rectifier potassium current in isolated guinea-pig ventricular myocytes

The effect of Gd3+ on the delayed rectifier potassium current (IK) in single guinea-pig ventricular myocytes was tested using whole-cell patch-clamp techniques. It was found that Gd3+ blocked 70% of the IK tail current at a concentration of 100 microM. The EC50 was 24 microM. Action potential durations were, however, reduced, consistent with a predominant effect on depolarizing L-type Ca2+ current (Ica.L). In the presence of 5 microM nifedipine Gd3+ prolonged the action potential. Using carbon fibres to stretch cells we observed that 10 microM Gd3+ was not effective in reducing a large stretch-activated increase in resting calcium. Modelling studies using the OXSOFT HEART program suggest that this lack of response is influenced by blockade of repolarizing current but is best reproduced by additional blockade of Ca2+ extrusion via the Na(+)-Ca2+ exchanger. When Gd3+ is used as a blocker of stretch-activated channels its actions upon both Ica.L and IK must therefore be accounted for.     

Reduced inward rectifying and increased E-4031-sensitive K+ current density in arrhythmogenic subendocardial purkinje myocytes from the infarcted heart

Astrocytes in primary culture from rat cerebral cortex were probed concerning the expression of delta-opioid receptors and their coupling to changes in intracellular free calcium concentrations ([Ca2+]i). Fluo-3 or fura-2 based microspectrofluorometry was used for [Ca2+]i measurements on single astrocytes in a mixed astroglial-neuronal culture. Application of the selective delta-opioid receptor agonist, [D-Pen2, D-Pen5]-enkephalin (DPDPE), at concentrations ranging from 10 nM to 100 microM, induced concentration-dependent increases in [Ca2+]i (EC50 = 114 nM). The responses could be divided into two phases, with an initial spike in [Ca2+]i followed by either oscillations or a sustained elevation of [Ca2+]i. These effects were blocked by the selective delta-opioid receptor antagonist ICI 174864 (10 microM). The expression of delta-opioid receptors on astroglial cells was further verified immunohistochemically, using specific antibodies, and by Western blot analyses. Pre-treatment of the cells with pertussis toxin (100 ng/ml, 24 h) blocked the effects of delta-opioid receptor activation, consistent with a Gi- or Go-mediated response. The sustained elevation of [Ca2+]i was not observed in low extracellular Ca2+ and was partly blocked by nifedipine (1 microM), indicating the involvement of L-type Ca2+ channels. Stimulating neurons with DPDPE resulted in a decrease in [Ca2+]i, which may be consistent with the closure of the plasma membrane Ca2+ channels on these cells. The current results suggest a role for astrocytes in the response of the brain to delta-opioid peptides and that these opioid effects in part involve altered astrocytic intracellular Ca2+ homeostasis.     

Comparison of the effects of class I anti-arrhythmic drugs, cibenzoline, mexiletine and flecainide, on the delayed rectifier K+ current of guinea-pig ventricular myocytes

The effect of class I anti-arrhythmic drugs, cibenzoline, mexiletine and flecainide, on the delayed rectifier potassium current (IK) in guinea-pig ventricular myocytes was studied using whole cell voltage clamp techniques and under blockade of the L-type calcium current by 5 microM nitrendipine. IK consisted of two different current systems, as reported by Sanguinetti and Jurkiewicz (1990), i.e. an E4031-sensitive rapidly activating component (IKr) with a strong inward-going rectification property and an E4031-insensitive slowly activating component (IKs) with little rectification. Cibenzoline (30 microM) decreased both IKr and IKs while flecainide (10 and 30 microM) decreased the IKr exclusively. Mexiletine (30 microM), in contrast, affected neither IKr nor IKs. Since the inhibition of IK(r) and/or IKs prolongs duration of action potentials and refractory periods, class I drugs which also possess the class III effect may have additional effects in treating certain re-entrant arrhythmias.     

Concentration-dependent block of sodium current in guinea pig ventricular myocytes by a class III antiarrhythmic agent, MS-551

The 32P-post-labelling assay for DNA adduct quantification gives the opportunity to examine endogenous exposure to DNA reactive compounds. Most human biomonitoring studies applied white blood cells (WBC) or cells obtained by broncho-alveolar lavages (BAL) as source of DNA, but still it is not clear what cell type represents the most reliable indicator for exposure to cigarette smoke-associated genotoxins. At first, we examined DNA adduct levels by means of nuclease P1 (NP1) enriched 32P-post-labelling in separated WBC subpopulations after in vitro incubations for 18 h with 10 microM benzo[a]pyrene (B[a]P). DNA adduct levels were highest in monocytes (10.7 +/- 2.9 adducts/10(8) nucleotides, n = 8), followed by lymphocytes (5.9 +/- 1.7, n = 8), and granulocytes (0.5 +/- 0.2, n = 8). Secondly, aromatic-DNA adduct levels were determined in BAL cells and WBC-subsets from (non-)smoking volunteers. In smoking individuals, adduct levels were in the ranking order: BAL cells (3.7 +/- 1.0, n = 5) > monocytes (2.0 +/- 0.5, n = 8) > or = lymphocytes (1.6 +/- 0.4, n = 8) > granulocytes (0.8 +/- 0.2, n = 8) by NP1-enrichment and monocytes (9.0 +/- 3.2, n = 5) > or = lymphocytes (8.0 +/- 2.1, n = 6) > granulocytes (2.1 +/- 0.3, n = 7) by butanol-enriched 32P-post-labelling. Aromatic-DNA adduct levels were significantly higher in WBC-subsets of smokers as compared with non-smokers, except for DNA adducts in granulocytes using butanol enrichment. Thirdly, dose-response relationships were investigated in mononuclear white blood cells (MNC, i.e. monocytes plus lymphocytes) and BAL-cells of a larger group of smoking individuals (n = 78). Adduct levels in MNC were related to daily exposure to cigarette-tar (r = 0.31, P < 0.01). Adduct levels in BAL cells seemed to be correlated with pack-years, but after correction for age this relationship was lost. Butanol extraction resulted in 5-6-fold higher DNA adduct levels in MNC, whereas butanol extraction of BAL-DNA of the same individuals yielded only 2-fold higher adduct levels. The two enrichment procedures of 32P-post-labelling were correlated in BAL cells (r = 0.86, P < 0.001, n = 12). We conclude that particularly MNC are good surrogates for the detection of smoking-related DNA adducts.     

Comparative effects of glibenclamide, tedisamil, dofetilide, E-4031, and BRL-32872 on protein kinase A-activated chloride current in guinea pig ventricular myocytes

The modulation of the protein kinase A-activated chloride current (PKA-I[Cl]) may lead to modification of the cardiac action potential shape. The purpose of this study was to evaluate the effects of glibenclamide, tedisamil, dofetilide, E-4031, and BRL-32872 on the PKA-I(Cl). Experiments were conducted by using the patch-clamp technique in guinea pig ventricular myocytes. PKA-I(Cl) was activated by application of 1 microM isoproterenol and was inhibited by 1 microM propranolol, 10 microM acetylcholine, or 1 mM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS). The sulfonylurea receptor inhibitor, glibenclamide, inhibited PKA-I(Cl) at micromolar concentration. Among class III antiarrhythmic agents, tedisamil induced a dose-dependent inhibition of PKA-I(Cl) with a half effective concentration (EC50) of 7.15 microM (Hill coefficient, 0.54). This effect may contribute to action potential widening induced by tedisamil. In contrast, the selective inhibitors of the rapid component of the delayed rectifier K current (I[Kr]), dofetilide, and E-4031, as well as BRL-32872, that blocks I(Kr) and the L-type calcium current, did not significantly affect the amplitude of PKA-I(Cl), even at high concentrations (10-30 microM). These results demonstrate that compounds such as glibenclamide and tedisamil that are known to block the adenosine triphosphate (ATP)-sensitive K current also affect PKA-I(Cl). Furthermore it appears that blockade of PKA-I(Cl) is not a common feature for all class III antiarrhythmic agents.     

Effects of troglitazone and pioglitazone on the action potentials and membrane currents of rabbit ventricular myocytes

The effects of the antidiabetic thiazolidinediones troglitazone and pioglitazone on action potentials and membrane currents were studied in rabbit ventricular myocytes. Troglitazone (10 microM) reversibly reduced excitability of the myocytes and modified their action potential configuration. It significantly increased the stimulation threshold required to elicit action potentials and decreased action potential amplitude and the maximum upstroke velocity of the action potentials. The Inhibition of the maximum upstroke velocity by troglitazone was also significant at 1 microM. Voltage-clamp experiments revealed that troglitazone (10 microM) reversibly inhibited both the slow inward Ca2+ current and the steady-state K+ current. In contrast to troglitazone, pioglitazone (1-10 microM) had no significant effect on the excitability, action potential configuration, or membrane currents of myocytes. These results suggest that troglitazone, but not pioglitazone, modulates Na+, Ca2+ and K+ currents, leading to the changes in excitability and action potential configuration of ventricular myocytes.     

Comparison of direct negative chronotropic and positive inotropic effects of sematilide to those of E-4031 and MS-551 and the reverse frequency-dependent prolongation of cardiac refractoriness of sematilide

Direct cardiac effects of sematilide, a new class III antiarrhythmic drug, were compared with those of E-4031 and MS-551 in canine isolated blood-perfused heart preparations. Doses of sematilide, E-4031, and MS-551 causing a 10% decrease in the spontaneous sinoatrial beating rate were 58 +/- 15, 9 +/- 5, and 84 +/- 10 micrograms (n = 5); those causing a 10% increase in developed tension of the papillary muscle were 485 +/- 49, 17 +/- 2, and 267 +/- 50 micrograms (n = 6); and those causing a 10% prolongation of effective refractory period (ERP) of the atrioventricular node were 68 +/- 10, 11 +/- 2, and 53 +/- 15 micrograms (n = 5), respectively. There were few effects on atrio-His or His-ventricular intervals. Also, in in situ open-chest dog hearts, the percent increases in ERP of the atrioventricular conduction system caused by 1 mg/kg of sematilide were 21 +/- 3, 16 +/- 2 and 9 +/- 1% at cycle lengths of 800, 600, and 400 ms, respectively (p < 0.01; n = 8). These results indicate that (a) sematilide, as well as E-4031 and MS-551, has direct negative chronotropic and positive inotropic effects and prolongs cardiac refractoriness without affecting conduction velocities; (b) quantitatively, the cardiac effects of sematilide were almost identical to those of MS-551 and five to ten times less potent than those of E-4031; (c) and prolongation of ERP of the atrioventricular conduction system by sematilide occurred in a reverse frequency-dependent manner.     

Effects of volatile anesthetic isoflurane on ATP-sensitive K+ channels in rabbit ventricular myocytes

It has been suggested that volatile anesthetic, isoflurane mediates cardioprotective effects via activation of the ATP-sensitive K+ (KATP) channels. However, no direct evidence has been provided to define whether isoflurane activates cardiac KATP channels using patch-clamp technique. We examined the effects of isoflurane on the KATP channels in rabbit ventricular myocytes by use of patch-clamp technique. Contrary to the results of the in vivo experiments, isoflurane inhibited the channel activity without a change in the single-channel conductance. Isoflurane decreased the channel activity by a decrease in burst duration and an increase in the inter-burst duration. On the other hand, isoflurane diminished the ATP sensitivity of KATP channels, indicating an increased probability of KATP channel opening for a given concentration of ATP after isoflurane anesthesia. The result supports, at least in part, the hypothesis that isoflurane mediates cardioprotective effects via KATP channel activation.     

The new antiarrhythmic substance AWD 23-111 inhibits the delayed rectifier potassium current (IK) in guinea pig ventricular myocytes

In a family with three siblings, one developed classical late infantile metachromatic leukodystrophy (MLD), fatal at age 5 years, with deficient arylsulfatase A (ARSA) activity and increased galactosylsulfatide (GS) excretion. The two other siblings, apparently healthy at 12(1/2) and 15 years, respectively, and their father, apparently healthy as well, presented ARSA and GS values within the range of MLD patients. Mutation screening and sequence analysis disclosed the involvement of three different ARSA mutations being the molecular basis of intrafamilial phenotypic heterogeneity. The late infantile patient inherited from his mother the frequent 0-type mutation 459+1G>A, and from his father a novel, single basepair microdeletion of guanine at nucleotide 7 in exon 1 (7delG). The two clinically unaffected siblings carried the maternal mutation 459+1G>A and, on their paternal allele, a novel cytosine to thymidine transition at nucleotide 2435 in exon 8, resulting in substitution of alanine 464 by valine (A464V). The fathers genotype thus was 7delG/A464V. Mutation A464V was not found in 18 unrelated MLD patients and 50 controls. A464V, although clearly modifying ARSA and GS levels, apparently bears little significance for clinical manifestation of MLD, mimicking the frequent ARSA pseudodeficiency allele. Our results demonstrate that in certain genetic conditions MLD-like ARSA and GS values need not be paralleled by clinical disease, a finding with serious diagnostic and prognostic implications. Moreover, further ARSA alleles functionally similar to A464V might exist which, together with 0-type mutations, may cause pathological ARSA and GS levels, but not clinical outbreak of the disease.     

Inhibition of the delayed rectifier K current in guinea-pig cardiomyocytes by thiamine tetrahydrofurfuryl disulfide

In this study, we attempted to determine which method was the best for reinnervating the laryngeal adductor muscles by comparing nerve suture, nerve implantation, and nerve-muscular pedicle (NMP) transfer, as well as the length of time that could elapse after denervation and still allow for successful reinnervation with the ansa cervicalis. Reinnervation was performed in 36 dogs, at 6-, 8-, 10-, 12- and 18-month intervals after denervation via the three methods of muscle reinnervation described above. We noted some return of adduction in the cases using nerve suture before a 10-month interval after denervation, and with nerve implantation and NMP transfer before the 8-month intervals. The variable adduction was caused by reinnervation of the adductor muscles from the ansa cervicalis, as demonstrated by laryngeal spontaneous and evoked electromyography, the strength of muscle contraction, and histologic findings. Adduction was not observed in the cases after the above-mentioned intervals but partial improvement of the bulk and strength of the reinnervated vocal cord was still achieved. An analysis of the experimental results showed that nerve suture was superior to nerve implantation and the NMP technique. Little difference was noted between nerve implantation and the NMP technique.     

AMPA/kainate receptor activation inhibits neuronal delayed rectifier K+ current via Na+ entry in rat cortical neurons

In the present study we evaluated the modulation of neuronal delayed rectifier K+ current (IK) by activation of ionotropic glutamate receptors. In whole-cell voltage-clamp experiments, an external application of 10-100 microM kainate suppressed the amplitude of IK following an inward shift of holding current. The effect of kainate on IK was eliminated by CN QX, an AMPA/kainate receptor antagonist, indicating that the receptor-mediated cation entry caused IK suppression. When external Na+ was completely replaced by equimolar choline+ or N-methyl-D-glucamine, kainate-induced IK suppression was abolished. Our results suggest that in cultured rat cortical neurons, AMPA/kainate receptor activation leads to an intracellular Na+ increase which blocks delayed rectifier K+ channels. This contributes to feed-forward excitation of neuronal cells in glutaminergic responses.     

Differential effects of fentanyl and morphine on intracellular Ca2+ transients and contraction in rat ventricular myocytes

BACKGROUND: Our objective was to elucidate the direct effects of fentanyl and morphine on cardiac excitation-contraction coupling using individual, field-stimulated rat ventricular myocytes. METHODS: Freshly isolated myocytes were loaded with fura-2 and field stimulated (0.3 Hz) at 28 degrees C. Amplitude and timing of intracellular Ca2+ concentration (at a 340:380 ratio) and myocyte shortening (video edge detection) were monitored simultaneously in individual cells. Real time Ca2+ uptake into isolated sarcoplasmic reticulum vesicles was measured using fura-2 free acid in the extravesicular compartment. RESULTS: The authors studied 120 cells from 30 rat hearts. Fentanyl (30-1,000 nM) caused dose-dependent decreases in peak intracellular Ca2+ concentration and shortening, whereas morphine (3-100 microM) decreased shortening without a concomitant decrease in the Ca2+ transient. Fentanyl prolonged the time to peak and to 50% recovery for shortening and the Ca2+ transient, whereas morphine only prolonged the timing parameters for shortening. Morphine (100 microM), but not fentanyl (1 microM), decreased the amount of Ca2+ released from intracellular stores in response to caffeine in intact cells, and it inhibited the rate of Ca2+ uptake in isolated sarcoplasmic reticulum vesicles. Fentanyl and morphine both caused a downward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on the Ca2+ transient. CONCLUSIONS: Fentanyl and morphine directly depress cardiac excitation-contraction coupling at the cellular level. Fentanyl depresses myocardial contractility by decreasing the availability of intracellular Ca2+ and myofilament Ca2+ sensitivity. In contrast, morphine depresses myocardial contractility primarily by decreasing myofilament Ca2+ sensitivity.     

Ionic mechanisms mediating the differential effects of methohexital and thiopental on action potential duration in guinea pig and rabbit isolated ventricular myocytes

BACKGROUND: The stress response to injury concept has been proposed as a mechanism of chronic rejection. This hypothesis has been tested with a rat cardiac allograft model in recipients pretreated with donor bone marrow (BM) cells. Chronic rejection is manifested in this BM group by obliterative arteriopathy and the epicardium and endocardium contains lymphocytic infiltrates resembling Quilty lesions. Pretreatment with a liver allograft (the orthotopic liver transplant [OLTx] group) is associated with an absence of chronic rejection in the transplanted heart. METHODS AND RESULTS:. Stress responses in the allograft were assessed by determining heat shock protein (hsp) expression by immunohistology of graft tissues and immunoblot analysis of stromal tissue lysates with monoclonal antibodies (mAb) to mammalian hsp60, the inducible hsp72, the constitutively expressed hsc73, and the grp78 C-terminal sequence KSEKDEL (grp78seq). Immunostaining showed clusters of grp78seq-positive cells in the inflammatory infiltrates of obliterated blood vessels and Quilty lesions in the BM group of cardiac allografts. Such grp78seq-positive cells were not seen in the OLTx group of heart allografts nor in syngrafts. Neither group showed significantly different graft myocyte staining of grp78 or hsp72, whereas hsp60 and hsc73 showed higher expression in the BM group and, to a lesser extent, the OLTx group. The increased expression of hsc73 was seen especially in the obliterated arteries and in myocytes nearby cellular infiltrates. Immunoblot analysis of graft stromal tissue lysates showed additional bands with mAb to hsp60 and hsc73 for the OLTx and especially the BM group. No significant bands were seen for hsp72 and grp78. Lymphocytes isolated from chronically rejecting allografts reacted with irradiated autologous spleen cells in the presence of mycobacterial hsp65 and interleukin-2. Culturing of graft-infiltrating cells with mycobacterial hsp71 and interleukin-2 yielded lymphocyte clones without alloreactivity, but with strong proliferative responsiveness to self-antigen-presenting cells and, only in the presence of mycobacterial hsp71 or murine grp78. This T-cell reactivity seemed to require intact hsp molecules because treatment of hsp71 with proteolytic enzymes, polymyxin, or ATP abrogated this induction of the stimulatory effect of self-antigen-presenting cells. These T cells are similar to the hsp-dependent, autoreactive lymphocytes cloned from acutely rejecting rat allografts. CONCLUSIONS: These findings support the concept that the pathogenesis of chronic rejection involves a stress response and the participation of graft-infiltrating autoreactive T cells that operate under hsp-dependent mechanisms.     

Activation of Ca2+-sensitive Cl- current by reverse mode Na+/Ca2+ exchange in rabbit ventricular myocytes

We investigated how Ca2+-sensitive transient outward current, Ito(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na+ (Na+i) using the whole-cell patch-clamp technique at 36 degreesC. In cells dialysed with Na+-free solutions, the application of nicardipine (5 microM) to block L-type Ca2+ current (ICa) completely inhibited Ito(Ca). In cells dialysed with a [Na+]i>/=5 mM, however, Ito(Ca) could be observed after blockade of ICa, indicating the activity of an ICa-independent component. The amplitude of ICa-independent Ito(Ca) increased with voltage in a [Na+]i-dependent manner. The block of Ca2+ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked ICa-independent Ito(Ca). In Ca2+-free bath solution Ito(Ca) was completely abolished. The application of 2 mM Ni2+ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na+/Ca2+ exchange, or perfusion with pipette solution containing XIP (10 microM), a selective blocker of the exchanger, blocked ICa-independent Ito(Ca). From these results we conclude that, in the presence of Na+i, Ito(Ca) can be activated via Ca2+-induced Ca2+ release triggered by Na+/Ca2+ exchange operating in the reverse mode after blockade of ICa.     

Influence of postnatal changes in action potential duration on Na-Ca exchange in rabbit ventricular myocytes

Cardiac Na-Ca exchanger (NCX) expression and current density are significantly greater in newborn rabbit hearts compared with adults. However, the relatively short action potential (AP) at birth may limit the impact of increased NCX expression by diminishing Ca2+ entry via Na-Ca exchange current (INaCa). To address the interdependence of AP duration and NCX activity, we voltage-clamped newborn (NB, 1-5 day), juvenile (JV, 10-14 day) and adult (AD) rabbit myocytes with a series of APs of progressively increasing duration (APD90: 108-378 ms) under nominally chloride-free conditions. In each age group we quantified an increase in outward (QExout) and inward (QExin) Ni2+-sensitive charge movement in response to AP prolongation. QExout and QExin measured during age-appropriate APs declined postnatally [QEXout: NB (2 day) 0.19 +/- 0.02, JV (10 day) 0.10 +/- 0.01, AD 0.04 +/- 0.002; QEXin: NB -0. 2 +/- 0.01, JV -0.11 +/- 0.02; AD -0.04 +/- 0.003 pC/pF] despite the significantly shorter APD90 of newborn myocytes (NB 122 +/- 10; AD 268 +/- 22 ms). When Ca2+ fluxes by other transport pathways were blocked with nifedipine, ryanodine and thapsigargin, age-appropriate APs elicited contractions in NB and JV but not AD myocytes (NB 4.8 +/- 0.5, JV 1.2 +/- 0.3% resting length). These data demonstrate that a shorter AP does not negate the impact of increased NCX expression at birth.     

Effect of pregnenolone sulfate on delayed rectifier K+ channel in pyramidial neuron of cerebral cortex

The possible preventive effect of Kampo medicine Hange-shashin-to (TJ-14) on chronic diarrheal symptoms induced by the administration of the anticancer agent irinotecan hydrochloride (CPT-11) was investigated in the rat. Repeated oral administrations of TJ-14 at 125 and 500 mg/kg significantly prevented the reduction in body weight and the onset of chronic diarrheal symptoms due to CPT-11 in a dose-dependent manner, even though it failed to show a definite effect on acute diarrheal symptoms. In addition, treatment with TJ-14 accelerated the healing of the intestinal tract injured by repeated dosing of CPT-11 and inhibited significantly the increase of colonic prostaglandin E2 (PGE2) which is closely related to the onset of diarrhea. TJ-14 also improved colonic water absorption impaired by repeated dosing of CPT-11 in rats. These results demonstrate that TJ-14 is an effective medicine for the prevention and/or treatment of CPT-11-induced chronic diarrheal symptoms.     

Cholinergic short-term conditioning and activation of ATP-sensitive K+ current in cat atrial myocytes

In atrial myocytes, an initial exposure to acetylcholine (ACh1) exerts a short-term conditioning effect such that a second ACh exposure (ACh2) activates ATP-sensitive K+ current (IK,ATP). The purpose of the present study was to determine the mechanism underlying the short-term conditioning induced by ACh that results in subsequent ACh-induced activation of IK.ATP. Cat atrial myocytes were studied using a nystatin-perforated patch whole cell recording method. Changes in L-type Ca2+ current (ICa,L) amplitude were used as an index of relative changes in cyclic AMP (cAMP). The results show that when atrial myocytes are treated with two consecutive exposures to 10 microM ACh separated by a recovery interval, ACh2 activates a larger increase in potassium conductance (gK+) than ACh1. The additional ACh2-induced increase in gK+ is selectively blocked by 10 microM glibenclamide, identifying the current as IK,ATP. Moreover, ICa,L activated immediately after the withdrawal of ACh1 exhibited a transient increase in amplitude above control (+ 76%), consistent with rebound stimulation of cAMP. Rp-cAMPs (50 microM), a selective antagonist of cAMP-dependent protein kinase A, blocked the rebound stimulation of ICa,L and abolished ACh2-induced activation of IK,ATP. Thapsigargin (5 microM), an inhibitor of Ca2+ ATPase in the sarcoplasmic reticulum (SR), abolished ACh2-induced activation of IK,ATP without decreasing rebound stimulation of ICa,L. Rebound stimulation of ICa,L and ACh2-induced activation of IK,ATP both varied as a function of ACh1 duration. We conclude that withdrawal of an initial ACh exposure elicits a rebound cAMP-mediated stimulation of SR Ca2+ uptake. This mechanism induces a short-term conditioning in atrial myocytes such that a subsequent ACh exposure activates IK,ATP. The present results demonstrate novel cholinergic signaling mechanisms in the regulation of IK,ATP.     

Effects of dopamine on L-type Ca2+ current in single atrial and ventricular myocytes of the rat

1. The effects of dopamine on the L-type Ca2+ current (ICa,L) of both atrial and ventricular single myocytes and on the force of contraction of atrial trabeculae in rat heart were investigated. 2. Dopamine increased atrial ICa,L at concentrations higher than 1 microM, but had little or no effect on ICa,L at lower concentrations. The increase in ICa,L at high concentrations was reversed by propranolol and acetylcholine, but not by phentolamine. Activation and inactivation kinetics of ICa,L were not altered by dopamine. 3. In rat ventricular myocytes in which the D4 receptor mRNA does not express, dopamine (20-100 microM) also increased the ICa,L amplitude and propranolol reversed this effect. 4. Clozapine, a potent D4 receptor antagonist, blocked the augmenting effect of dopamine on ICa,L. However, this effect could be explained by beta-antagonism, since clozapine also inhibited the isoprenaline effect. 5. In the atrial trabeculae, the increase in contraction by dopamine (1 to 30 microM) was reversed by 1 microM propranolol, but not by 2 microM phentolamine. Low doses of dopamine (0.01 to 0.3 microM) did not affect the contraction in the controls or during a modest stimulation of the beta-adrenoceptor with 0.01 microM isoprenaline. 6. These results indicate that the positive inotropic action of dopamine is mediated through direct stimulation of the beta-adrenoceptor in both atrial and ventricular myocytes. Involvement of D4 receptor appears unlikely in the regulation of the atrial contraction.