Involvement of tyrosine kinase in the hyposmotic stimulation of I Ks in guinea-pig ventricular myocytes

The objective of this study was to investigate the involvement of tyrosine phosphorylation in the hyposmotic stimulation of cardiac I _Ks, a slowly activating delayed-rectifier K⁺ current that promotes repolarization of the action potential. The current was recorded from whole-cell-configured guinea-pig ventricular myocytes before, during, and after their exposure to solution whose osmolarity was 0.75 times normal. Exposure to hyposmotic solution caused a near-doubling of the amplitude of I _Ks, with little change in the voltage dependence of current activation. Stable, hyposmotically stimulated I _Ks (I _Ks,Hypo) was decreased by broadspectrum tyrosine kinase (TK) inhibitors tyrphostin A23 (IC₅₀ ≈ 5 μM) and tyrphostin A25 (IC₅₀ 15.8 ± 1.6 μM) but not by TK-inactive tyrphostin analogs, suggesting that tyrosine phosphorylation is important for maintenance of the current. In agreement with that view, we found that the TK-inhibitor action on I _Ks,Hypo was strongly antagonized by vanadate compounds known to inhibit phosphotyrosyl phosphatase. When myocytes were pretreated with TK inhibitors, the stimulation of I _Ks was attenuated in a concentration-dependent manner. The attenuation was not due to concomitant attenuation of a stimulation of tyrosine phosphorylation because neither the stimulation of I _Ks nor its rate of decay following removal of hyposmotic solution was affected by pretreatment with vanadates. We suggest that the stimulation of I _Ks by hyposmotic solution is dependent on a basal tyrosine phosphorylation that modulates a swelling-induced I _Ks-stimulatory signal and/or the receptivity of Ks channels to that signal.     

Contribution of I Ks to ventricular repolarization in canine myocytes

The role of the slow delayed rectifier K⁺ current (I _Ks) in cardiac repolarization seems to be largely influenced by the experimental conditions including the species and tissue studied. The aim of this study was to determine the contribution of I _Ks to repolarization in canine ventricular myocytes by measuring the frequency dependent action potential lengthening effect of 10 μM chromanol 293B using sharp microelectrodes. Pretreatment with isoproterenol (2 nM), E-4031 (1 μM), and injection of inward current pulses were applied to modify action potential configuration. Chromanol alone caused moderate but statistically significant lengthening of action potentials at cycle lengths longer than 500 ms. The lengthening effect of chromanol, which was strongly enhanced in the presence of either isoproterenol or E-4031, was proportional to the amplitude of plateau, whereas poor correlation was found with action potential duration. Similar results were obtained when action potential configuration was modified by injection of depolarizing current pulses. Computer simulations revealed that activation of I _Ks is a sharp function of the plateau amplitude within the physiological range, while elongation of repolarization may enhance I _Ks only when it is excessive. It was concluded that the effect of I _Ks on ventricular repolarization critically depends on the level of action potential plateau; however, other factors, like action potential duration, cycle length, or suppression of other K⁺ currents can also influence its contribution.     

Sex differences in repolarization and slow delayed rectifier potassium current and their regulation by sympathetic stimulation in rabbits

Slow delayed rectifier potassium current (I_Ks) is important in action potential (AP) repolarization and repolarization reserve. We tested the hypothesis that there are sex-specific differences in I_Ks, AP, and their regulation by β-adrenergic receptors (β-AR’s) using whole-cell patch-clamp. AP duration (APD₉₀) was significantly longer in control female (F) than in control male (M) myocytes. Isoproterenol (ISO, 500 nM) shortened APD₉₀ comparably in M and F, and was largely reversed by β₁-AR blocker CGP 20712A (CGP, 300 nM). Inhibition of I_Ks with chromanol 293B (10 μM) resulted in less APD prolongation in F at baseline (3.0 vs 8.9 %, p?<?0.05 vs M) and even in the presence of ISO (5.4 vs 20.9 %, p?<?0.05). This suggests that much of the ISO-induced APD abbreviation in F is independent of I_Ks. In F, baseline I_Ks was 42 % less and was more weakly activated by ISO (19 vs 68 % in M, p?<?0.01). ISO enhancement of I_Ks was comparably attenuated by CGP in M and F. After ovariectomy, I_Ks in F had greater enhancement by ISO (72 %), now comparable to control M. After orchiectomy, I_Ks in M was only slightly enhanced by ISO (23 %), comparable to control F. Pretreatment with thapsigargin (to block SR Ca release) had bigger impact on ISO-induced APD shortening in F than that in M (p?<?0.01). In conclusion, we found that there are sex differences in I_Ks, AP, and their regulation by β-AR’s that are modulated by sex hormones, suggesting the potential for sex-specific antiarrhythmic therapy.     

Expression of angiotensin II and its receptors in the normal and hypoxic amoeboid microglial cells and murine BV-2 cells

Involvement of the local angiotensin receptor system in the central nervous system is well documented, yet its cellular localization and role in the glial cells have remained elusive. This study reports expression of angiotensin II and its receptors namely, angiotensin II receptor type 1 (AT₁) and angiotensin II receptor type 2 (AT₂) in the amoeboid microglial cells in the neonatal rat brain. In rats subjected to hypoxia, the amount of angiotensin II released in the corpus callosal tissue was reduced as revealed by enzyme immunoassay. Expression of AT₁ mRNA and protein was down-regulated after hypoxic exposure, but AT₂ was up-regulated. In BV-2 cells exposed to hypoxia for 4 h, expression of AT₁ mRNA was reduced but AT₂ was increased. These changes were further intensified respectively in LPS-stimulated microglia. Edaravone enhanced AT₁ expression but suppressed AT₂ expression significantly in lipopolysaccharide-stimulated cells. Neutralization of AT₂ with its antiserum significantly increased mRNA expression of tumor necrosis factor-α and interleukin-1β but decreased that of transforming growth factor-beta1. In conclusion, the present results suggest that AT₁ may be linked to regulation of vasodilation for increase of blood flow in hypoxic conditions, while up-regulated expression of AT₂ may reduce inflammatory responses through suppression of proinflammatory cytokines and elimination of free radicals.     

Atrial arrhythmogenesis in wild-type and Scn5a+/Δ murine hearts modelling LQT3 syndrome

Long QT(3) (LQT3) syndrome is associated with abnormal repolarisation kinetics, prolonged action potential durations (APD) and QT intervals and may lead to life-threatening ventricular arrhythmias. However, there have been few physiological studies of its effects on atrial electrophysiology. Programmed electrical stimulation and burst pacing induced atrial arrhythmic episodes in 16 out of 16 (16/16) wild-type (WT) and 7/16 genetically modified Scn5a+/Δ (KPQ) Langendorff-perfused murine hearts modelling LQT3 (P < 0.001 for both), and in 14/16 WT and 1/16 KPQ hearts (P < 0.001 for both; Fisher’s exact test), respectively. The arrhythmogenic WT hearts had significantly larger positive critical intervals (CI), given by the difference between atrial effective refractory periods (AERPs) and action potential durations at 90% recovery (APD₉₀), compared to KPQ hearts (8.1 and 3.2 ms, respectively, P < 0.001). Flecainide prevented atrial arrhythmias in all arrhythmogenic WT (P < 0.001) and KPQ hearts (P < 0.05). It prolonged the AERP to a larger extent than it did the APD₉₀ in both WT and KPQ groups, giving negative CIs. Quinidine similarly exerted anti-arrhythmic effects, prolonged AERP over corresponding APD₉₀ in both WT and KPQ groups. These findings, thus, demonstrate, for the first time, inhibitory effects of the KPQ mutation on atrial arrhythmogenesis and its modification by flecainide and quinidine. They attribute these findings to differences in the CI between WT and mutant hearts, in the presence or absence of these drugs. Thus, prolongation of APD₉₀ over AERP gave positive CI values and increased atrial arrhythmogenicity whereas lengthening of AERP over APD₉₀ reduced such CI values and produced the opposite effect.     

兔左室肥厚心肌跨室壁复极不均一性及其膜离子流变化的实验研究

目的:探讨兔左心室肥厚心肌心外膜下、中层、心内膜下3层心肌细胞动作电位及膜离子流变化的不均一性。方法: 心肌肥厚组以腹主动脉缩窄术复制兔压力超负荷心肌肥厚模型,并设正常对照组以作比较。胶原酶两步消化法分离获取兔单个心室肌细胞,其中用植皮刀分离左室游离壁内膜下、中层、外膜下3层心肌。以全细胞膜片钳记录单细胞跨膜动作电位和离子电流。结果: 肥厚组3层心肌细胞动作电位时程(APD₉₀)较对照组3层心肌细胞APD₉₀均分别有明显延长,而以中层心肌细胞APD₉₀延长最为明显(延长比例:中层26.0%±2.7%,外膜14.0%±1.6%,内膜10.0%±1.1%),使肥厚心肌跨室壁复极不均一性明显大于对照组。肥厚组各层心肌细胞瞬时外向钾电流(I_to)和缓慢激活的延迟整流钾电流(I_Ks)密度均低于对照组,且均以中层细胞下降的幅度最大。肥厚组各层心肌细胞L型钙电流(I_Ca,L)与快速激活的延迟整流钾电流(I_Kr) 密度与对照组均无明显差异。肥厚组各层心肌细胞内向整流钾电流(I_K1) 均明显低于对照组,但各层变低的幅度无明显差异。 结论:兔肥厚心肌跨室壁复极不均一性明显增大,I_to及I_Ks的跨室壁不均一性下降可能是其主要原因。     

Effects of Angiotensin II on sustained outward currents in rat ventricular myocytes

We investigated the effects of angiotensin II (Ang II) on the sustained outward current (I _sus) and action potential of rat ventricular myocytes using the whole-cell patch-clamp technique. Ang II at 30 nM~3 µM inhibited I _sus with an IC₅₀ of 240 nM, a Hill coefficient of 1.0 and maximum inhibition of 19.4%. Ang II-mediated inhibition of I _sus was voltage independent, was due to a decrease in the K⁺ current and was abolished by the Ang II type-I (AT₁) receptor blocker, valsartan. The protein kinase C (PKC) inhibitors PKC19–36 or calphostin C, abolished Ang II-mediated inhibition of I _sus. In contrast, pretreatment with the protein kinase A (PKA) inhibitor PKA6–22 (100 µM) significantly enhanced the suppression of I _sus by 1 µM Ang II: (33.7±5.1% vs. control 17.1±2.3%). These results indicate that Ang II inhibits I _sus via the AT₁ receptor and activation of PKC. Ang II significantly prolonged action potential duration (APD) when the control APD was lengthened by a Ca²⁺ channel activator, BAY K8644. In myocytes with a relatively long APD, Ang II may prolong APD by inhibiting I _sus.     

Redox reaction modulates transient and persistent sodium current during hypoxia in guinea pig ventricular myocytes

Whole-cell and cell-attached patch clamp techniques were applied on isolated guinea pig ventricular myocytes to study the possible regulatory mechanisms of redox agent on persistent and transient sodium current related to hypoxia. The results showed that hypoxia for 15 min increased persistent sodium current (I _Na.P) and decreased transient sodium current (I _Na.T) at the same time, while 1 mmol/l of reduced glutathione (GSH) could reverse the increased I _Na.P and the decreased I _Na.T simultaneously. Both persistent and transient sodium channel activities could be reversed concurrently again by application of 1 mmol/l oxidized glutathione (GSSG). Hypoxia for 15 min decreased the action potential amplitude (APA) and shortened action potential duration at 90% repolarization (APD₉₀) of ventricular papillary cells simultaneously, while 1 mmol/GSH could reverse the decreased APA and the shortened APD₉₀ at the same time; 1 mmol/l GSSG strengthened the decrease of APA induced by hypoxia and attenuated the decurtation of APD₉₀ induced by hypoxia compared with pure hypoxia. The correlation between I _Na.P and I _Na.T and the effects of GSH and GSSG on them suggested that during hypoxia, redox regulation played a tremendous part in sodium channel activity and that I _Na.P and I _Na.T might be charged by the same channel with different gating modes in guinea pig ventricular myocytes. Judging from their alterations during hypoxia and exposure to GSH and GSSG, we speculated that an interconversion might exist between I _Na.P and I _Na.T. That was when one of them was increased, the other was decreased, and vice versa.     

Niflumic acid reduces the hyperpolarization-activated current (Ih) in rod photoreceptor cells

We examined the effects of niflumic acid (NFA), a chloride channel blocker, on the hyperpolarization-activated current (I_h) in newt rod photoreceptors. At 100 μM, NFA delayed the activation of I_h induced by hyperpolarizing voltage pulses to −83 mV from a holding potential of −43 mV, and reduced the steady-state current. However, reduction by NFA was weakened when I_h was activated by hyperpolarizing steps to −123 mV, suggesting that these effects were voltage-dependent. The suppressive effects of NFA on I_h were accompanied by a negative shift in activation voltage. NFA also delayed the relaxation of I_h tail currents, showing that this drug also inhibited deactivation of the current. The reversal potential and the fully activated conductance were not affected. These observations suggest that NFA reduces I_h by modifying the gating kinetics of the underlying channels. The suppressive actions of NFA remained when intracellular Ca²⁺ was strongly chelated, and the failure of suppression by NFA in inside-out patches suggests that the agent may act on the I_h channel from the extracellular side. These results, obtained in rod photoreceptors, are consistent with similar effects of NFA on I_f in cardiac myocytes, suggesting that both currents share similar pharmacological properties.     

Genetic deletion and overexpression of angiotensin II receptors

Angiotensin II receptors are essential components of the renin-angiotensin system transducing angiotensin II mediated signals across the plasma membrane of many cell types in the cardiovascular system. To date, three subtypes of angiotensin II receptors have been identified by molecular cloning, termed angiotensin II type 1 (AT_1A, AT_1B) and type 2 (AT₂) receptors. This review focuses on recent transgenic animal models which have been generated to study the in vivo significance of angiotensin receptor diversity. AT_1A receptors are the major blood pressure regulators and have a potent growth-stimulatory effect on cardiac myocytes in vivo. The AT_1B receptor subtype may participate in the control of vascular tone if AT_1A receptors are absent. AT₂ receptors are abundantly expressed during embryonic development and may also play a role in blood pressure regulation by influencing vascular development and differentiation. Received: 16 February 1998 / Accepted: 10 August 1998     

Remodelling of human atrial K<Superscript>+</Superscript> currents but not ion channel expression by chronic β-blockade

Chronic β-adrenoceptor antagonist (β-blocker) treatment in patients is associated with a potentially anti-arrhythmic prolongation of the atrial action potential duration (APD), which may involve remodelling of repolarising K⁺ currents. The aim of this study was to investigate the effects of chronic β-blockade on transient outward, sustained and inward rectifier K⁺ currents (I_TO, I_KSUS and I_K1) in human atrial myocytes and on the expression of underlying ion channel subunits. Ion currents were recorded from human right atrial isolated myocytes using the whole-cell-patch clamp technique. Tissue mRNA and protein levels were measured using real time RT-PCR and Western blotting. Chronic β-blockade was associated with a 41% reduction in I_TO density: 9.3 ± 0.8 (30 myocytes, 15 patients) vs 15.7 ± 1.1 pA/pF (32, 14), p < 0.05; without affecting its voltage-, time- or rate dependence. I_K1 was reduced by 34% at −120 mV (p < 0.05). Neither I_KSUS, nor its increase by acute β-stimulation with isoprenaline, was affected by chronic β-blockade. Mathematical modelling suggested that the combination of I_TO- and I_K1-decrease could result in a 28% increase in APD₉₀. Chronic β-blockade did not alter mRNA or protein expression of the I_TO pore-forming subunit, Kv4.3, or mRNA expression of the accessory subunits KChIP2, KChAP, Kvβ1, Kvβ2 or frequenin. There was no reduction in mRNA expression of Kir2.1 or TWIK to account for the reduction in I_K1. A reduction in atrial I_TO and I_K1 associated with chronic β-blocker treatment in patients may contribute to the associated action potential prolongation, and this cannot be explained by a reduction in expression of associated ion channel subunits.     

Isolation and possible role of fast and slow potassium current components in hair cells dissociated from frog crista ampullaris

Potassium-current inactivation and recovery kinetics are pivotal in sustaining dynamic processing of time-varying sensory signals in hair cells. We report a detailed analysis of K⁺-currents in isolated hair cells from the frog crista ampullaris. The single components were dissected using a novel procedure based on their differential kinetic properties: The fast IA component exhibited two processes of inactivation removal; the persistent I _KD component (I _KV + I _KCa), unexpectedly displayed partial inactivation, removed by negative potentials with particularly slow, delayed kinetics. The physiological relevance of these observations was investigated by imposing sinusoidal membrane potential changes to mimic receptor response to hair bundle deflection. The excitatory phase elicited extra-currents (hysteresis) only if the off phase went sufficiently negative to remove IA inactivation. Native, resting hair cells are depolarised by receptor current; thus, voltage continuously modulates I_KD, whereas IA only transiently ensues when the receptor current vanishes (zero-current potential ≈−70 mV) and polarisation removes IA inactivation.     

Effect of vinpocetine on various types of high-threshold potassium currents in snail neurons

A high-threshold (−20 mV) K⁺ current was recorded from isolated edible snail neurons by a two-microelectrode voltage clamp technique. This current consisted of three components: fast-inactivating K⁺ currents (I_A), noninactivating K⁺ current (I_KD), and Ca²⁺-dependent K⁺ current (I_K(Ca)). Different cells had one to three components of K⁺ current. Vinpocetine increased I_A, moderately inhibited I_KD (by 30–50%) and strongly suppressed I_K(Ca) (by 60–90%). Inhibition of I_K(Ca) was not related to the effect of vinpocetine on the inward Ca²⁺ current. When K⁺ current consisted of all three components, the effect of vinpocetine on the ionic current amplitude was opposite at different potentials. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 10, pp. 408–411, October, 1998     

Stable transfection of rat angiotensin II receptor subtypes in mouse adrenocortical Y1 cells

Summary A procedure is described for stably transfecting mouse adrenocortical Y1 cells with AT_1a and AT_1b angiotensin II (AII) receptor subtypes using the lipofectin reagent, DOTAP. The transfected cells are screened by radioreceptor assay using¹²⁵I-[Sar¹,Ile⁸]AII to select those cells that express high levels of the AII receptors. The establishment of individual transfected Y1 cell lines expressing AT_1a and AT_1b receptors provides a valuable model system in which to compare the function and signal transduction mechanisms of these two highly homologous AII receptor subtypes.     

Does the threshold of transcutaneous partial pressure of carbon dioxide represent the respiratory compensation point or anaerobic threshold?

On reaching the respiratory compensation point (RCP) during rapidly increasing incremental exercise, the ratio of minute ventilation (V_E) to CO₂ output (VCO₂) rises, which coincides with changes of arterial partial pressure of carbon dioxide (P _aCO₂). Since P _aCO₂ changes can be monitored by transcutaneous partial pressure of carbon dioxide (PCO_2,tc) RCP may be estimated by PCO_2,tc measurement. Few available studies, however, have dealt with comparisons between PCO_2,tc threshold (T _AT) and lactic, ventilatory or gas exchange threshold (V _AT), and the results have been conflicting. This study was designed to examine whether this threshold represents RCP rather than V _AT. A group of 11 male athletes performed incremental excercise (25 W · min^–1) on a cycle ergometer. The PCO_2,tc at (44°C) was continuously measured. Gas exchange was computed breath-by-breath, and hyperaemized capillary blood for lactate concentration ([la^–]_b) and P _aCO₂ measurements was sampled each 2 min. The T _AT was determined at the deflection point of PCO_2,tc curve where PCO_2,tc began to decrease continuously. The V _AT and RCP were evaluated with VCO₂ compared with oxygen uptake (VO₂) and V_E compared with the VCO₂ method, respectively. The PCO_2,tc correlated with P _aCO₂ and end-tidal PCO₂. At T _AT, power output [P, 294 (SD 40) W], VO₂ [4.18 (SD 0.57)l · min^–1] and [la^–] [4.40 (SD 0.64) mmol · l^–1] were significantly higher than those at V _AT[P 242 (SD 26) W, VO₂ 3.56 (SD 0.53) l · min^–1 and [la^–]_b 3.52 (SD 0.75), mmol · l^–1 respectively], but close to those at RCP [P 289 (SD 37) W; VO₂ 3.97 (SD 0.43) l · min^– and [la^–]_b 4.19 (SD 0.62) mmol · l^–1, respectively]. Accordingly, linear correlation and regression analyses showed that P, VO₂ and [la^–]_b at T _AT were closer to those at RCP than at V _AT. In conclusion, the T _AT reflected the RCP rather than V _AT during rapidly increasing incremental exercise.     

Mechanism of hyperthyroidism-induced modulation of the L-type Ca2+ current in guinea pig ventricular myocytes

The positive inotropic effects of thyroid hormone in the heart, increased force and velocity of contraction have been mostly attributed to modulation of myosin ATPase isoenzymes (V₁, V₂ and V₃), and sarcoplasmic reticulum Ca²⁺ pumping activity. In addition, we have suggested that the effects on ventricular contraction result from a thyroid hormone-induced increase in L-type Ca²⁺ current (I _{Ca, L}). Due to the central role of I _{Ca, L} in excitation-contraction coupling, we studied mechanisms whereby thyroid hormone augments this current. Since thyroid hormone modulates adenylate cyclase activity in various tissues, we tested the hypothesis that the hormone activates adenylate cyclase, leading to increased cyclic adenosine monophosphate (cAMP) levels, protein kinase A activation, Ca²⁺ channel phosphorylation and increased I _{Ca, L}. We therefore stimulated or inhibited different sites along the adenylate cyclase cascade, and measured I _{Ca, L} and isometric twitch in ventricular myocytes and papillary muscles from euthyroid and hyperthyroid guinea pigs. Our major findings were as follows. In euthyroid myocytes, 0.1 M isoproterenol (Iso) increased I _{Ca, L} (at V _M=0 mV) from –7.04±0.72 to –22.26±1.88 pA/pF, P<0.05, while in hyperthyroid myocytes (I _{Ca, L}=-21.48±2.94 pA/pF), Iso was ineffective. In euthyroid myocytes, intracellular application of cAMP (50 M) was as potent as Iso, but ineffective in hyperthyroid myocytes. In hyperthyroid myocytes, a protein kinase A inhibitor (2 M) lowered I _{Ca, L} from –26.82±1.54 to -10.17±1.70 pApF (P<0.05), but had no effect in euthyroid myocytes. In hyperthyroid myocytes, acetylcholine (ACh) (1 M) decreased I _{Ca, L} from –26.86±1.49 to –18.33±1.25 pA/pF (P<0.05), while in euthyroid myocytes ACh decreased I_{Ca, L} from –6.80±0.61 to –6.00±0.39 pA/pF (NS). Accordingly, in hyperthyroid papillary muscles, ACh decreased twitch tension by 36.4±2.8%, but in euthyroid preparations only by 9.4±5.1% (P<0.05). These findings suggest that thyroid-hormone-induced increase in I _{Ca, L} contributing to positive inotropy, is mediated by activation of the adenylate cyclase cascade.     

The effects of over-expression of the FK506-binding protein FKBP12.6 on K+ currents in adult rabbit ventricular myocytes

This study examines the effects of the intracellular protein FKBP12.6 on action potential and associated K⁺ currents in isolated adult rabbit ventricular cardiomyocytes. FKBP12.6 was over-expressed by ~6 times using a recombinant adenovirus coding for human FKBP12.6. This over-expression caused prolongation of action potential duration (APD) by ~30%. The amplitude of the transient outward current (I _to) was unchanged, but rate of inactivation at potentials positive to +40 mV was increased. FKBP12.6 over-expression decreased the amplitude of the inward rectifier current (I _K1) by ~25% in the voltage range −70 to −30 mV, an effect prevented by FK506 or lowering intracellular [Ca²⁺] below 1 nM. Over-expression of an FKBP12.6 mutant, which cannot bind calcineurin, prolonged APD and affected I _to and I _K1 in a similar manner to wild-type protein. These data suggest that FKBP12.6 can modulate APD via changes in I _K1 independently of calcineurin binding, suggesting that FKBP12.6 may affect APD by direct interaction with I _K1.     

Accentuated antagonism by angiotensin II on guinea-pig cardiac L-type Ca-currents enhanced by β-adrenergic stimulation

 To examine mechanism(s) underlying the accentuated antagonism by angiotensin II (A-II) on twitch tension, we recorded L-type Ca²⁺ currents (I _Ca,L) using conventional patch-clamp techniques in single, guinea-pig, ventricular myocytes. I _Ca,L was recorded by a step-pulse protocol after eliminating K⁺ conductances (internal Cs⁺ plus tetraethylammonium chloride and K⁺-free extracellular solution). A-II (100 nM) did not affect basal I _Ca,L, but inhibited I _Ca,L that had been enhanced (approximately 200% of control) by (ISO, isoproterenol 100 nM). The inhibitory action of A-II was concentration dependent (concentration eliciting 50% inhibition 88±9 pM, n=41) and the ISO-enhanced component of I _Ca,L was completely blocked by A-II at concentrations above 10 nM. CV-11974 (500 nM), an A-II type-1 receptor (AT₁) antagonist, prevented the inhibitory action of A-II. Pre-incubation with pertussis toxin (PTX) abolished the inhibitory effect of A-II. A-II also inhibited the I _Ca,L enhanced by histamine (500 nM) and forskolin (1 μM), but failed to affect I _Ca,L enhanced by intracellular cyclic adenosine monophosphate (1 mM). The inhibitory action of A-II may therefore involve AT₁ receptors/PTX-sensitive, guanine nucleotide-binding (G) proteins (Gi)/adenylate cyclase and partially explains the A-II-dependent accentuated antagonism of inotropy.     

Receptor-mediated deactivation of Gk in cardiac myocytes

The muscarinic potassium current I_k(ACh) of atrial myocytes can be evoked in the absence of agonists by intracellular application of stable GTP analogs (GXP). This receptor-independent opening of K_ACh channels is a consequence of the direct activation of the guanyl nucleotide binding protein G_k that couples muscarinic receptors to K_ACh channels, and was previously thought to be unaffected by subsequent application of agonist. We report here that in the presence of GTP, application of a pulse of muscarinic agonist to atrial cells can abolish the GXP-induced I_K(ACh). The results imply that in intact cells the agonist-bound receptor can interact with G_k not only in its inactive, GDP-bound form, but also in its active, GXP-bound form in a process that promotes the release of guanine nucleotide from its binding site.     

Role of actin microfilament in osmotic stretch-induced increase of voltage-operated calcium channel current in guinea-pig gastric myocytes

 Using the whole-cell patch clamp technique, the role of actin microfilament in hyposmotic increase of voltage-operated calcium channel current (I _Ba) was studied in guinea-pig gastric myocytes. Hyposmotic superfusate (212 mOsm) increased peak I _Ba amplitude by 32.7 ± 6.5%; when cytochalasin-D (Cyt-D, 20 μM), an actin cytoskeleton disruptor, was used, an increase of only 9.7 ± 3.1% was seen. I _Baresponse to osmotic stress was potentiated (45.1 ± 4.1% increase) by 20 μM phalloidin, an actin microfilament stabilizer. However, colchicine (100 μM), an microtubule cytoskeleton disruptor, had no effect on either I _Ba or its response to hyposmotic solution. Phalloidin also induced a rightward shift of the I/V relationship of I _Ba, while Cyt-D itself had no effect. These results suggest that actin cytoskeleton may mediate hyposmotic stretch-induced I _Ba increase in gastric smooth muscle. Received: 26 March 1997 / Received after revision: 28 May 1997 / Accepted: 3 June 1997