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Key points The mucosa may have neuron-like functions as urinary bladder mucosa releases bioactive substances that modulate sensory nerve activity as well as detrusor muscle contractility. However, such mucosal function in other visceral organs remains to be established., The role of mucosa in generating spontaneous contractions in seminal vesicles (SVs), a paired organ in the male reproductive tract, was investigated., The intact mucosa is essential for the generation of spontaneous phasic contractions of SV smooth muscle arising from electrical slow waves and corresponding increases in intracellular Ca2+. These spontaneous events primarily depend on Ca2+ handling by sarco-endoplasmic reticulum Ca2+ stores., A population of mucosal cells developed spontaneous rises in intracellular Ca2+ relying on sarco-endoplasmic reticulum Ca2+ handling., The spontaneously active cells in the SV mucosa appear to drive spontaneous activity in smooth muscle either by sending depolarizing signals and/or by releasing humoral substances., Abstract The role of the mucosa in generating the spontaneous activity of guinea-pig seminal vesicle (SV) was explored. Changes in contractility, membrane potential and intracellular Ca2+ dynamics of SV smooth muscle cells (SMCs) were recorded using isometric tension recording, intracellular microelectrode recording and epi-fluorescence Ca2+ imaging, respectively. Mucosa-intact but not mucosa-denuded SV preparations generated TTX- (1 μ m) resistant spontaneous phasic contractions that were abolished by nifedipine (3 μ m). Consistently, SMCs developed mucosa-dependent slow waves (SWs) that triggered action potentials and corresponding Ca2+ flashes. Nifedipine (10 μ m) abolished the action potentials and spontaneous contractions, while suppressing the SWs and Ca2+ flashes. Both the residual SWs and spontaneous Ca2+ transients were abolished by cyclopiazonic acid (CPA, 10 μ m), a sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor. DIDS (300 μ m) and niflumic acid (100 μ m), blockers for Ca2+-activated Cl− channels (CACCs), or low Cl− solution also slowed or prevented the generation of SWs. In SV mucosal preparations detached from the muscle layer, a population of mucosal cells generated spontaneous Ca2+ transients that were blocked by CPA but not nifedipine. These results suggested that spontaneous contractions and corresponding Ca2+ flashes in SV SMCs arise from action potential generation due to the opening of L-type voltage-dependent Ca2+ channels. Spontaneous Ca2+ transients appear to primarily result from Ca2+ release from sarco-endoplasmic reticulum Ca2+ stores to activate CACCs to develop SWs. The mucosal cells firing spontaneous Ca2+ transients may play a critical role in driving spontaneous activity of SV smooth muscle either by sending depolarizing signals or by releasing humoral substances. [ABSTRACT FROM AUTHOR]

We provide evidence for location and function of a small conductance, Ca 2+ -activated K + (SK Ca ) channel isoform 3 (SK3) in mitochondria (m) of guinea pig, rat and human ventricular myocytes. SK Ca agonists protected isolated hearts and mitochondria against ischemia/reperfusion (IR) injury; SK Ca antagonists worsened IR injury. Intravenous infusion of a SK Ca channel agonist/antagonist, respectively, in intact rats was effective in reducing/enhancing regional infarct size induced by coronary artery occlusion. Localization of SK3 in mitochondria was evidenced by Western blot of inner mitochondrial membrane, immunocytochemical staining of cardiomyocytes, and immunogold labeling of isolated mitochondria. We identified a SK3 splice variant in guinea pig (SK3.1, aka SK3a) and human ventricular cells (SK3.2) by amplifying mRNA, and show mitochondrial expression in mouse atrial tumor cells (HL-1) by transfection with full length and truncated SK3.1 protein. We found that the N-terminus is not required for mitochondrial trafficking but the C-terminus beyond the Ca 2+ calmodulin binding domain is required for Ca 2+ sensing to induce mK + influx and/or promote mitochondrial localization. In isolated guinea pig mitochondria and in SK3 overexpressed HL-1 cells, mK + influx was driven by adding CaCl 2 . Moreover, there was a greater fall in membrane potential (ΔΨ m ), and enhanced cell death with simulated cell injury after silencing SK3.1 with siRNA. Although SK Ca channel opening protects the heart and mitochondria against IR injury, the mechanism for favorable bioenergetics effects resulting from SK Ca channel opening remains unclear. SK Ca channels could play an essential role in restraining cardiac mitochondria from inducing oxidative stress-induced injury resulting from mCa 2+ overload. [ABSTRACT FROM AUTHOR]

Cardiac ion channels and their respective accessory subunits are critical in maintaining proper electrical activity of the heart. Studies have indicated that the K+ channel interacting protein 2 (KChIP2), originally identified as an auxiliary subunit for the channel Kv4, a component of the transient outward K+ channel (Ito), is a Ca2+ binding protein whose regulatory function does not appear restricted to Kv4 modulation. Indeed, the guinea pig myocardium does not express Kv4, yet we show that it still maintains expression of KChIP2, suggesting roles for KChIP2 beyond this canonical auxiliary interaction with Kv4 to modulate Ito. In this study, we capitalize on the guinea pig as a system for investigating how KChIP2 influences the cardiac action potential, independent of effects otherwise attributed to Ito, given the endogenous absence of the current in this species. By performing whole cell patch clamp recordings on isolated adult guinea pig myocytes, we observe that knock down of KChIP2 significantly prolongs the cardiac action potential. This prolongation was not attributed to compromised repolarizing currents, as IKr and IKs were unchanged, but was the result of enhanced L-type Ca2+ current due to an increase in Cav1.2 protein. In addition, cells with reduced KChIP2 also displayed lowered INa from reduced Nav1.5 protein. Historically, rodent models have been used to investigate the role of KChIP2, where dramatic changes to the primary repolarizing current Ito may mask more subtle effects of KChIP2. Evaluation in the guinea pig where Ito is absent, has unveiled additional functions for KChIP2 beyond its canonical regulation of Ito, which defines KChIP2 as a master regulator of cardiac repolarization and depolarization. [ABSTRACT FROM AUTHOR]

Astragaloside IV (AS-IV) is one of the main active ingredients of Astragalus membranaceus. This study is aimed to investigate AS-IV׳s effects on Ca 2+ channel activity of single cardiomyocytes and single Ca 2+ channels. Whole-cell Ca 2+ currents in freshly dissociated cardiomyocytes were measured using the whole-cell patch-clamp technique. Single Ca 2+ channel currents were examined in cell-attached patches and inside-out patches. In the whole-cell recording, AS-IV reduced the amplitude of l -type Ca 2+ currents ( I CaL ) in a concentration-dependent manner. Although AS-IV did not alter the steady-state activation curves, the voltage dependence of the current inactivation curves was negatively shifted by AS-IV in a concentration dependent manner. Consistent with the results of the whole-cell recording, in the inside-out configuration the ensemble average of single Ba 2+ current via l -type Ca 2+ channel was dose-dependently reduced by AS-IV. The reduction of unitary Ba 2+ current at 0.1 or 1 µM AS-IV was accounted for a decrease in the channel activity ( NPo ). In addition to the decrease in NPo , there was a reduction of Po without a change in channel number or an apparent change in single channel current. Furthermore, we found that the open-closed kinetics of the channel were affected by AS-IV. AS-IV induced the shift of l -type Ca 2+ channels from either brief openings (mode 1) or long-lasting openings (mode 2) to no active opening (mode 0). Our results suggest that AS-IV blocks the currents through Ca 2+ channels in guinea-pig ventricular myocytes by affecting the open-closed kinetics of l -type Ca 2+ channels to inhibit the channel activities. This study could provide theoretical basis for the drug exploiting of the monomer of Astragalus membranaceus . [ABSTRACT FROM AUTHOR]

Background and Purpose Subtypes of the hyperpolarization-activated cyclic nucleotide-gated ( HCN) family of cation channels are widely expressed on nerves and smooth muscle cells in many organ systems, where they serve to regulate membrane excitability. Here we have assessed whether HCN channel inhibitors alter the function of airway smooth muscle or the neurons that regulate airway smooth muscle tone. Experimental Approach The effects of the HCN channel inhibitors ZD7288, zatebradine and Cs+ were assessed on agonist and nerve stimulation-evoked changes in guinea pig airway smooth muscle tone using tracheal strips in vitro, an innervated tracheal tube preparation ex vivo or in anaesthetized mechanically ventilated guinea pigs in vivo. HCN channel expression in airway nerves was assessed using immunohistochemistry, PCR and in situ hybridization. Key Results HCN channel inhibition did not alter airway smooth muscle reactivity in vitro to exogenously administered smooth muscle spasmogens, but significantly potentiated smooth muscle contraction evoked by the sensory nerve stimulant capsaicin and electrical field stimulation of parasympathetic cholinergic postganglionic neurons. Sensory nerve hyperresponsiveness was also evident in in vivo following HCN channel blockade. Cs+, but not ZD7288, potentiated preganglionic nerve-dependent airway contractions and over time induced autorhythmic preganglionic nerve activity, which was not mimicked by inhibitors of potassium channels. HCN channel expression was most evident in vagal sensory ganglia and airway nerve fibres. Conclusions and Implications HCN channel inhibitors had a previously unrecognized effect on the neural regulation of airway smooth muscle tone, which may have implications for some patients receiving HCN channel inhibitors for therapeutic purposes. [ABSTRACT FROM AUTHOR]

The rapidly activating delayed rectifier potassium current (IKr) plays a critical role in cardiac repolarization. Although IKr is known to be regulated by both α1- and β1-adrenergic receptors (ARs), the cross-talk and feedback mechanisms that dictate its response to α1- and β1-AR activation are not known. In the present study, IKr was recorded using the whole-cell patch-clamp technique. IKr amplitude was measured before and after the sequential application of selective adrenergic agonists targeting α1- and β1-ARs. Stimulation of either receptor alone (α1-ARs using 1 μM phenylephrine (PE) or β1-ARs using 10 μM xamoterol (Xamo)) reduced IKr by 0.22 ® 0.03 and 0.28 α 0.01, respectively. The voltage-dependent activation curve of IKr shifted in the negative direction. The half-maximal activation voltage (V0.5) was altered by -6.35 ® 1.53 and -1.95 ® 2.22 mV, respectively, with no major change in the slope factor (k). When myocytes were pretreated with Xamo, PE-induced reduction in IKr was markedly blunted and the corresponding change in V05 was significantly altered. Similarly, when cells were pretreated with PE, Xamo-induced reduction of IKr was significantly attenuated. The present results demonstrate that functional cross-talk between α1- and β1-AR signaling regulates IKr. Such non-linear regulation may form a protective mechanism under excessive adrenergic stimulation. [ABSTRACT FROM AUTHOR]

Human ether-à-go-go-related gene (hERG) potassium channels conduct the rapid component of the delayed rectifier potassium current (IKr), which is crucial for repolarization of cardiac action potential. Patients with hERG-associated long QT syndrome usually develop tachyarrhythmias during physical and/or emotional stress, both known to stimulate adrenergic receptors. The present study aimed to investigate a putative functional link between β1-adrenergic stimulation and IKr in guinea-pig left ventricular myocytes and to analyze how IKr is regulated following activation of the β1-adrenergic signaling pathway. The IKr current was measured using a whole-cell patch-clamp technique. A selective β1-adrenergic receptor agonist, xamoterol, at concentrations of 0.01-100 μM decreased IKr in a concentration-dependent manner. The 10 μM xamoterol-induced inhibition of IKr was attenuated by the protein kinase A (PKA) inhibitor KT5720, the protein kinase C (PKC) inhibitor chelerythrine, and the phospholipase (PLC) inhibitor U73122, indicating involvement of PKA, PKC and PLC in β1-adrenergic inhibition of IKr The results of the present study indicate an association between IKr and the β1-adrenergic receptor in arrhythmogenesis, involving the activation of PKA, PKC and PLC. [ABSTRACT FROM AUTHOR]

The article looks at a model which mechanistically develops a detailed description of excitation-contraction complex (ECC) in the guinea pig cardiac myocyte with the combination of modules describing energetics and isometric forces. It explores how the incorporation of mitochondrial energetics has allowed the model to qualitatively reproduce changes in NADH in response to changes in cardiac workload. The development of computational model of the cardiac myocyte action potential is discussed.

Aims The aims of the study were to determine the effects of anisosmotic bathing solution on selected properties of IKs, the slowly activating delayed-rectifier K+ current important for repolarization of the action potential in cardiac cells. Methods and results Guinea-pig ventricular myocytes were voltage-clamped using either the ruptured-patch or perforated-patch technique, and the amplitude, time course, and voltage dependence of IKs were determined before [isosmotic (1T)] and during superfusion of hyposmotic (<1T) or hyperosmotic (>1T) bathing solution. Hyposmotic solution increased the amplitude of IKs, and hyperosmotic solution decreased it. Anisosmotic-induced changes in IKs amplitude were complete in 2–5 min, well-maintained, reversible, and not accompanied by significant changes in IKs time course and voltage dependence. There was little difference in the results obtained with the ruptured-patch technique and those obtained with the perforated-patch technique. The amplitude of IKs was sensitive to small (±10%) changes in osmolarity, maximally increased by hyposmotic solution with T < 0.7, and strongly decreased by hyperosmotic solution with T > 1.5. Experimental data on a plot of relative (1T = 1.0) IKs amplitude vs. the reciprocal of relative osmolarity are well-described by a Hill equation that has a lower asymptote of 0.0, an upper asymptote of 2.0, and a slope factor of 1.87 ± 0.07. Conclusion Modulation of IKs amplitude by anisosmotic solution is independent of patch configuration, unaccompanied by changes in current gating, and well-described by a Hill dose–response relation that predicts relatively strong responses of IKs to small perturbations in external osmolarity. [ABSTRACT FROM PUBLISHER]

This paper briefly recapitulates the Frank-Starling law of the heart, reviews approaches to establishing diastolic and systolic force-length behaviour in intact isolated cardiomyocytes, and introduces a dimensionless index called 'Frank-Starling Gain', calculated as the ratio of slopes of end-systolic and end-diastolic force-length relations. The benefits and limitations of this index are illustrated on the example of regional differences in Guinea pig intact ventricular cardiomyocyte mechanics. Potential applicability of the Frank-Starling Gain for the comparison of cell contractility changes upon stretch will be discussed in the context of intra- and inter-individual variability of cardiomyocyte properties. [ABSTRACT FROM AUTHOR]

The objective of the present study was to identify kit-positive interstitial cells (ICs) in guinea pig corpus cavernosum and examine their relationships with adjacent smooth muscle cells (SMCs) and intramural nerves. In addition, we investigated the possible involvement of ICs in nitric oxide (NO)-mediated relaxation of corpus cavernosum smooth muscle (CCSM). ICs were identified by their immunoreactivity to the kit receptor, a cell surface marker encoded by c-kit proto-oncogene and specific for interstitial cells of Cajal. ICs were abundantly distributed in guinea pig corporal tissues. Ultrastructural investigation by conventional transmission electron microscopy revealed the ultrastructural features of ICs and gap junctions located between ICs and adjacent SMCs, furthermore, a close contact between ICs and intramural nerves for the first time. Western blot analysis of purified ICs by fluorescence-activated cell sorting revealed coexpression of soluble guanylate cyclase (sGC)α1, sGCβ1 and kit receptor tyrosine kinase protein in them. These observations imply that ICs express the NO-sensitive sGC molecule and may be involved in the NO-mediated relaxation of CCSM in the guinea pig corpus cavernosum. [ABSTRACT FROM AUTHOR]

It has been hypothesized that an interaction among adenosine A1 receptors, protein kinase C (PKC) activation, and ATP-sensitive potassium channels (KATP) mediates ischemic preconditioning in experiments on different animal species. The purpose of this study was to determine if activation of KATP is functionally coupled to A1 receptors and (or) PKC activation during metabolic inhibition (MI) in guinea pig ventricular myocytes. Perforated-patch using nystatin and conventional whole-cell recording methods were used to observe the effects of adenosine and adenosine-receptor antagonists on the activation of KATP currents during MI induced by application of 2,4-dinitrophenol (DNP) and 2-deoxyglucose (2DG) without glucose, in the presence or absence of a PKC activator, phorbol 12-myristate 13-acetate (PMA). Adenosine accelerated the time course activation of KATP currents during MI under the intact intracellular condition or dialyzed condition with l mmol/L ATP in the pipette solution. The accelerated effect of adenosine activation of KATP under MI was not reversed by a nonselective Al adenosine receptor antagonist, 8-(p-sulfophenyl)theophylline (SPT), or a specific Al adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). However, the adenosine A2 receptor antagonist alloxazine reversed the time course activation of the KATP current under MI. An adenylate cyclase activator, forskolin, did not further abbreviate the time course activation of KATP with or without adenosine. Application of a PKC blocker, chelerythrine, reversed the time course activation of KATP by adenosine under MI. In addition, pretreatment with a PKC activator, PMA, had similar effects to adenosine, while adenosine did not further shorten the time required for activation of KATP currents during MI with PMA pretreatment. There is no direct evidence of activation of KATP currents by adenosine A1 receptor during metabolic inhibition under our experimental condition. However, adenosine A2 receptor activation is involved in the KATP channel activation in the guinea pig ventricular myocytes, of which effect is not mediated through the increase in intracellular cAMP. Adenosine seems to interact with PKC activation to open KATP during MI, but a possible link between the adenosine A2 receptor and PKC activation in this process needs further elucidation. Une interaction entre les récepteurs A1 de l'adénosine, l'activation de la protéine kinase C (PKC) et les canaux potassiques sensibles à l'ATP (KATP) participerait à la médiation du préconditionnement ischémique selon des études expérimentales sur différentes espèces animales. La présente étude a eu pour but de déterminer si l'activation des canaux KATP est fonctionnellement couplée aux récepteurs A1 et (ou) à l'activation de la PKC durant l'inhibition métabolique (IM) dans les myocytes ventriculaires de cobayes. Nous avons utilisé les techniques d'enregistrement en patch clamp configurations cellule entière et patch perforé avec nystatine pour examiner les effets de l'adénosine et d'antagonistes des récepteurs de l'adénosine sur l'activation des courants KATP durant l'IM induite par l'application de 2,4-dinitrophénol (DNP) et de 2-désoxyglucose (2DG), sans glucose, en présence et en l'absence de l'activateur de la PKC, phorbol 12-myristate 13-acétate (PMA). L'adénosine a accéléré le décours temporel de l'activation des courants KATP durant l'IM en condition intracellulaire intact ou en condition dialysée avec insertion de 1 mmol/L d'ATP dans la pipette. L'effet d'accélération de l'activation des courants KATP durant l'IM n'a pas été renversé par l'antagoniste non sélectif des récepteurs A1 de l'adénosine, 8-(p-sulfophényl)théophylline (STP), ou l'antagoniste spécifique des récepteurs A1 de l'adénosine, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Toutefois, l'antagoniste des récepteurs A2 de l'adénosine, alloxazine, a renversé le décours temporel de l'activation des courants KATP durant l'IM. L'activateur de l'adénylate cyclase, forskoline, n'a pas atténué davantage le décours temporel de l'activation de la KATP, en présence ou non d'adénosine. L'application du bloqueur de la PKC, chélérythrine, a renversé le décours temporel de l'activation des courants KATP par l'adénosine durant l'IM. Enfin, le prétraitement avec l'activateur de la PKC, PMA, a eu des effets similaires à ceux l'adénosine, qui n'a pas raccourci davantage le temps nécessaire à l'activation des courants KATP durant l'IM. Nos conditions expérimentales n'ont pas permis une observation directe de l'activation des courants KATP par les récepteurs A1 de l'adénosine durant l'inhibition métabolique. Cependant, les récepteurs A2 participent à l'activation des canaux KATP dans les myocytes ventriculaires du cobaye, effet qui n'est pas véhiculé par l'augmentation de l'AMPc intracellulaire. L'adénosine semble interagir avec l'activation de la PKC pour ouvrir les canaux KATP durant l'IM, mais l'existence d'un lien possible entre les récepteurs A2 de l'adénosine et l'activation de la PKC dans ce processus devra faire l'objet d'une autre étude. [ABSTRACT FROM AUTHOR]

2-Aminoethoxydiphenyl borate (2-APB) analogs are potentially better vascular gap junction blockers than others widely used, but they remain to be characterized. Using whole cell and intracellular recording techniques, we studied the actions of 2-APB and its potent analog diphenylborinic anhydride (DPBA) on vascular smooth muscle cells (VSMCs) and endothelial cells in situ of or dissociated from arteriolar segments of the cochlear spiral modiolar artery, brain artery, and mesenteric artery. We found that both 2-APB and DPBA reversibly suppressed the input conductance (Ginput) of in situ VSMCs (IC50 ≈ 4-8 μM). Complete electrical isolation of the recorded VSMC was achieved at 100 μM. A similar gap junction blockade was observed in endothelial cell tubules of the spiral modiolar artery. Similar to the action of 18β-glycyrrhetinic acid (18β-GA), 2-APB and DPBA depolarized VSMCs. In dissociated VSMCs, 2-APB and DPBA inhibited the delayed rectifier K+ current (IK) with an IC50 of ∼120 μM in the three vessels but with no significant effect on Ginput or the current-voltage relation between -140 and -40 mV. 2-APB inhibition of IK was more pronounced at potentials of ≤20 mV than at +40 mV and more marked on the fast component than on the slow component, which was mimicked by 4-aminopyridine but not by tetraethylammonium, nitrendipine, or charybdotoxin. In contrast, 18β-GA caused a linear inhibition of IK between 0 to +40 mV, which was similar to the action of tetraethylammonium or charybdotoxin. Finally, the 2-APB-induced inhibition of electrical coupling and IK was not affected by the inositol 1,4,5-trisphosphate receptor antagonist xestospongin C. We conclude that 2-APB analogs are a class of potent and reversible vascular gap junction blockers with a weak side effect of voltage-gated K+ channel inhibition. They could be gap junction blockers superior to 18β-GA only when Ca2+-actived K+ channel inhibition by the latter is a concern but inositol 1,4,5-trisphosphate receptor and voltage-gated K+ channel inhibitions are not. [ABSTRACT FROM AUTHOR]

Electroporation induced by high-strength electrical fields has long been used to investigate membrane properties and facilitate transmembrane delivery of molecules and genes for research and clinical purposes. In the heart, electric field-induced passage of ions through electropores is a factor in defibrillation and postshock dysfunction. Voltage-clamp pulses can also induce electroporation, as exemplified by findings in earlier studies on rabbit ventricular myocytes: Long hyperpolarizations to ≤-110 mV induced influx of marker ethidium and irregular inward currents that were as large with external NMDG(+) as Na(+). In the present study, guinea pig ventricular myocytes were bathed with NMDG(+), Na(+) or NMDG(+) + La(3+) solution (36°C) and treated with five channel blockers. Hyperpolarization of myocytes in NMDG(+) solution elicited an irregular inward current (I (ep)) that reversed at -21.5 ± 1.5 mV. In myocytes hyperpolarized with 200-ms steps every 30 s, I (ep) occurred in "episodes" that lasted for one to four steps. Boltzmann fits to data on the incidence of I (ep) per experiment indicate 50% incidence at -129.7 ± 1.4 mV (Na(+)) and -146.3 ± 1.6 mV (NMDG(+)) (slopes ≈-7.5 mV). I (ep) amplitude increased with negative voltage and was larger with Na(+) than NMDG(+) (e.g., -2.83 ± 0.34 vs. -1.40 ± 0.22 nA at -190 mV). La(3+) (0.2 mM) shortened episodes, shifted 50% incidence by -35 mV and decreased amplitude, suggesting that it inhibits opening/promotes closing of electropores. We compare our findings with earlier ones, especially in regard to electropore selectivity. In the Appendix, relative permeabilities and modified excluded-area theory are used to derive estimates of electropore diameters consistent with reversal potential -21.5 mV. [ABSTRACT FROM AUTHOR]

Diastolic depolarization (DD) of atrial myocytes can lead to spontaneous action potentials (APs) and, potentially, atrial tachyarrhythmias. This study examined the hypotheses that 1) a slowly inactivating component of the Na+ current (referred to as late INa) may contribute to DD and initiate AP firing and that 2) blocking lateINa will reduce spontaneous and induced firing of APs by atrial myocytes. Guinea pig atrial myocytes without or with DD and spontaneous AP firing were studied using the whole cell patch-clamp technique. In experiments using cells with a stable resting membrane potential (no spontaneous DD or firing), hydrogen peroxide (H2O2, 50 μmol/I) caused DD and AP firing. The H2O2-induced activity was suppressed by the late INa inhibitors tetrodotoxin (TFX, 1 μmol/l) and ranolazine (5 μmol/l). In cells with DD but no spontaneous APs, the late INa enhancer anemone toxin II (ATX-II, 10 nmol/l) accelerated DD and induced APs. In cells with DD and spontaneous AP firing, TTX and ranolazine (both, 1 μmol/l) significantly reduced the slope of DD by 81 ± 12% and 75 ± 11% and the frequency of spontaneous firing by 70 ± 15% and 74 ± 9%, respectively. Ramp voltage-clamp simulating DD elicited a slow inward current. TTX at 1, 3, and 10 p.mol/l inhibited this current by 41 ± 4%, 73 ± 2%, and 91 ± 1%, respectively, suggesting that a slowly inactivating `Na underlies the DD. ATX-II and H2O2 increased the amplitude of this current, and the effects of ATX-II and H2O2 were attenuated by ranolazine or TTX. In conclusion, late INa can contribute to the DD of atrial myocytes and the inhibition of this current suppresses atrial DD and spontaneous APs. [ABSTRACT FROM AUTHOR]

Abstract: Sarcoplasmic reticulum contains the internal Ca2+ store in smooth muscle cells and its lumen appears to be a continuum that lacks diffusion barriers. Accordingly, the free luminal Ca2+ level is the same all throughout the SR; however, whether the Ca2+ buffer capacity is the same in all the SR is unknown. We have estimated indirectly the luminal Ca2+ buffer capacity of the SR by comparing the reduction in SR Ca2+ levels with the corresponding increase in [Ca2+] i during activation of either IP3Rs with carbachol or RyRs with caffeine, in smooth muscle cells from guinea pig urinary bladder. We have determined that carbachol-sensitive SR has a 2.4 times larger Ca2+ buffer capacity than caffeine-sensitive SR. Rapid inhibition of SERCA pumps with thapsigargin revealed that this pump activity accounts for 80% and 60% of the Ca2+ buffer capacities of carbachol- and caffeine-sensitive SR, respectively. Moreover, the Ca2+ buffer capacity of carbachol-sensitive SR was similar to caffeine-sensitive SR when SERCA pumps were inhibited. Similar rates of Ca2+ replenishments suggest similar levels of SERCA pump activities for either carbachol- or caffeine-sensitive SR. Paired pulses of caffeine, in conditions of low Ca2+ influx, indicate the relevance of luminal SR Ca2+ buffer capacity in the [Ca2+] i response. To further study the importance of luminal SR Ca2+ buffer capacity in the release process we used low levels of heparin to partially inhibit IP3Rs. This condition revealed carbachol-induced transient increase of luminal SR Ca2+ levels provided that SERCA pumps were active. It thus appears that SERCA pump activity keeps the luminal SR Ca2+-binding proteins in the high-capacity, low-affinity conformation, particularly for IP3R-mediated Ca2+ release. [Copyright &y& Elsevier]

Background and purpose: Changes in smooth muscle tone of the prostate gland are involved in aetiology of symptomatic prostatic hyperplasia, however the control mechanisms of prostatic smooth muscle are not well understood. Here, we have examined the role of internal Ca2+ compartments in regulating slow wave activity in the guinea pig prostate. Experimental approach: Standard intracellular membrane potential recording techniques were used. Key results: The majority (89%) of impaled cells displayed ‘slow wave’ activity. Cyclopiazonic acid (10 µmol·L−1) transiently depolarized (3–9 mV) the membrane potential of the prostatic stroma and transiently increased slow wave frequency. Thereafter, slow wave frequency slowly decreased over 20–30 min. Ryanodine transiently increased slow wave frequency, although after 30 min exposure slow wave frequency and time course returned to near control values. Caffeine (1 mmol·L−1) reduced slow wave frequency, accompanied by membrane depolarization of about 8 mV. Blockade of inositol trisphosphate receptor (IP3R)-mediated Ca2+ release with 2-aminoethoxy-diphenylborate (60 µmol·L−1) or Xestospongin C (3 µmol·L−1) or inhibiting phospholipase C and IP3 formation using U73122 (5 µmol·L−1) or neomycin (1 and 4 mmol·L−1) reduced slow wave frequency, amplitude and duration. The mitochondrial uncouplers, p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (1–10 µmol·L−1), carbonyl cyanide m-chlorophenylhydrazone (1–3 µmol·L−1) or rotenone (10 µmol·L−1), depolarized the membrane (8–10 mV) before abolishing electrical activity. Conclusion and implications: These results suggest that slow wave activity was dependent on the cyclical release of Ca2+ from IP3-controlled internal stores and mitochondria. This implies that intracellular compartments were essential in the initiation and/or maintenance of the regenerative contractile activity in the guinea pig prostate gland. [ABSTRACT FROM AUTHOR]

Gallbladder disease is prevalent during pregnancy. It has been suggested that this complication of pregnancy is attributable to increased bile cholesterol (Ch) induced by estrogens and to gallbladder hypomotility caused by increasing levels of progesterone (P4). Studies on nonpregnant gallbladders have shown that increased levels of bile Ch contribute to both gallstone formation and bile stasis. These studies investigated the effects of high levels of plasma membrane Ch on P4 on gallbladder muscle cells from human and guinea pigs. Contraction was studied in intact and permeabilized muscle cells. G proteins were determined by Western blot, and 3H-P4 incorporation by muscle cells was measured in the β-scintillation counter. High levels of caveolar Ch blocked the effects induced by P4 treatment for 6 h. They suppressed the expected P4 inhibition of GTP-γS (a G protein activator)-induced contraction and changes in G proteins by downregulating Gi3 and upregulating Os protein levels. Ch inhibited these P4 actions at the caveolar 3 (CAV-3) level, since the P4 effects were antagonized by treatment with CAV-3 antibody, by reducing CAV-3 expression through CAV-3 siRNA. CAV-3 antibody and siRNA reduced caveolar Ch levels. High caveolar levels of Ch and CAV-3 antibody blocked the incorporation of 3H-P4 into caveolae. Treatment with GDP-βS (a G protein antagonist) had no effect on P4 actions. High caveolar Ch levels blocked the P4 effects on muscle contraction and G protein changes probably because both Ch and P4 require CAV-3 proteins for their transport across the plasma membrane. [ABSTRACT FROM AUTHOR]

Abstract: Ca2+ channel antagonists are useful to reduce the abnormal motility in patients with irritable bowel syndrome. We have therefore examined the effects of a newly synthesized antagonist AJG049, on voltage-dependent L-type Ca2+ channels in gastric antrum. Intracellular recordings were made from sheets of the circular muscle layer of guinea-pig gastric antrum, with simultaneous measurement of spontaneous contraction activity, and the effects of AJG049 were studied. The effects of AJG049 on voltage-dependent Ba2+ currents (I Ba) and the basal membrane currents at −70 mV in dispersed smooth muscle cells were also investigated by the use of conventional whole-cell patch-clamp techniques. Although AJG049 (100 nM) enhanced the peak amplitude of spontaneous contractions, high concentrations (≥10 µM) had inhibitory effects. In whole-cell configuration, AJG049 (10 nM) reversibly enhanced the peak amplitude of I Ba in a voltage-dependent manner whilst high concentrations (≥100 nM) suppressed the peak amplitude in a concentration- and voltage-dependent manner. AJG049 (300 nM) caused little shift in the activation curve at a holding potential of −70 mV although the voltage dependence of the steady-state inactivation was shifted to more negative potentials by 5 mV in its presence. AJG049 caused a delay of the recovery from the inactivated state of I Ba. Furthermore, AJG049 reduced the amplitude of the basal membrane currents at −70 mV in a concentration-dependent manner. These results suggest that AJG049 possesses a dual action on voltage-dependent Ca2+ channels in circular layer of guinea-pig antrum. [Copyright &y& Elsevier]

It is well known that choline has protective effects on ischemic arrhythmias. We designed the present study to evaluate the antiarrhythmic effects of choline and to detect its related mechanisms in aconitine-induced rat and ouabain-induced guinea pig models of arrhythmia. Laser scanning confocal microscopy and patch-clamp technique were utilized to study the action of choline on intracellular calcium concentration and L-type calcium current (ICa-L) of cardiac myocytes. M3 receptor antagonist 4-DAMP (4-diphenylacetoxy-N-methylpiperidine-methiodide) was applied preliminarily to evaluate the role of the M3 receptor. Choline significantly increased the survival time of arrhythmic rats and guinea pigs, delayed the onset of arrhythmias and ventricular tachycardia, and decreased the arrhythmia score. The overload of intracellular Ca2+ induced by aconitine or ouabain was reduced in isolated myocytes pretreated with choline. Choline reduced the increased density of ICa-L induced by aconitine or ouabain. Moreover, the beneficial effects of choline were reversed by 4-DAMP. Choline produced antiarrhythmic actions on arrhythmia models by stimulating the cardiac M3 receptor. The mechanism may be related to the improvement of Ca2+ handling. Il est bien connu que la choline a des effets protecteurs sur les arythmies ischémiques. Pour examiner si l’effet antiarythmique de la choline sur le myocarde ischémique pourrait être reproduits dans d’autres modèles animaux arythmogènes, nous avons conçu la présente étude pour évaluer les effets antiarythmiques de la choline et identifier ses mécanismes. Nous avons observés les effets de la choline sur des modèles de rats présentant une arythmie induite par la choline et de cobayes présentant une arythmie induite par l’ouabaïne. Nous avons utilisé la microscopie confocale à balayage laser et la technique du patch clamp pour examiner l’action de la choline sur la concentration de calcium intracellulaire et le courant calcique de type L (ICa-L) des myocytes cardiaques. Nous avons d’abord appliqué l’antagoniste du récepteur M3, 4-DAMP (4-diphénylacétoxy-N-méthylpyridine-méthiodide), pour évaluer le rôle du récepteur M3. La choline a augmenté significativement le temps de survie des rats et des cobayes présentant une arythmie, retardé le déclenchement des arythmies et de la tachycardie ventriculaire, et diminué le score d’arythmie. La surcharge de Ca2+ intracellulaire induite par l’aconitine ou l’ouabaïne a été réduite dans les myocytes isolés prétraités à la choline. La choline a diminué l’augmentation de la densité de l’ICa-L induite par l’aconitine ou l’ouabaïne. Le 4-DAMP a renversé les effets bénéfiques de la choline. La choline a eu des actions antiarythmiques sur les modèles d’arythmie en stimulant le récepteur M3 cardiaque. Le mécanisme pourrait être lié à l’amélioration du métabolisme du Ca2+. [ABSTRACT FROM AUTHOR]

Abstract: Emodin is known to be used in the treatment of cholesterol stones and cholecystitis. This study sought to investigate the effects of emodin on the contraction of gallbladder smooth muscle (GBSM), intracellular Ca2+ concentration and L-type calcium current in GBSM cells. Gallbladder muscle strips were obtained from adult guinea pigs and the resting tension was recorded. Gallbladder smooth muscle cells were isolated by enzymatic digestion. Cells were loaded with fluo-3/AM and [Ca2+]i was determined by a laser confocal microscope. Calcium current was recorded by the whole-cell patch clamp method. Emodin increased the resting tension of GBSM strips in a dose-dependent manner. Emodin elevated [Ca2+]i in GBSM cells, and this effect was attenuated by pretreatment with nifedipine. In addition, Emodin increased L-type calcium current at concentrations of 1 to 30 µM (at +10 mV, 10 µM, 45.1±5.2% compared to control, EC50 =3.11 µM). In the presence of protein kinase C (PKC) inhibitor, Staurosporine, emodin did not significantly affect the calcium current. However, phorbol 12, 13-dibutyrate mimicked emodin in enhancement of the calcium current. These results suggest that emodin promotes gallbladder contraction by increasing Ca2+ influx through L-type calcium channel via PKC pathway. [Copyright &y& Elsevier]

Components of excitation- contraction (EC)-coupling were compared at 37°C and 22°C to determine whether hypothermia altered the gain of EC coupling in guinea pig ventricular myocytes. Ca2+ concentration (fura-2) and cell shortening (edge detector) were measured simultaneously. Hypothermia increased fractional shortening (8.3 ± 1.7 vs. 2.6 ± 0.3% at 37°C), Ca2+ transients (157 ± 33 vs. 35 ± 5 nM at 37°C), and diastolic Ca2+ (100 ± 9 vs. 60 ± 6 nM at 37°C) in field-stimulated myocytes (2 Hz). In experiments with high-resistance microelectrodes, the increase in contractions and Ca2+ transients was accompanied by a twofold increase in action potential duration (APD). When voltage-clamp steps eliminated changes in APD, cooling still increased contractions and Ca2+ transients. Hypothermia increased sarcoplasmic reticulum (SR) Ca2+ stores (83 ± 17 at 37°C to 212 ± 50 nM, assessed with caffeine) and increased fractional SR Ca2+ release twofold. In contrast, peak Ca2+ current was much smaller at 22°C than at 37°C (1.3 ± 0.4 and 3.5 ± 0.7 pA/pF, respectively). In cells dialyzed with sodium-free pipette solutions to inhibit Ca2+ influx via reverse-mode Na+/Ca2+ exchange, hypothermia still increased contractions, Ca2+ transients, SR stores, and fractional release but decreased the amplitude of Ca2+ current. The rate of SR Ca2+ release per unit Ca2+ current, a measure of EC-coupling gain, was increased sixfold by hypothermia. This increase in gain occurred regardless of whether cells were dialyzed with sodium-free solutions. Thus an increase in EC-coupling gain contributes importantly to positive inotropic effects of hypothermia in the heart. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the effects of γ-radiation on cells isolated from the longitudinal smooth muscle layer of the guinea pig ileum, a relatively radioresistant tissue. Single doses (up to 50 Gy) reduced the amount of sarcoplasmatic reticulum and condensed the myofibrils, as shown by electron microscopy 3 days post-irradiation. After that, contractility of smooth muscle strips was reduced. Ca2+ handling was altered after irradiation, as shown in fura-2 loaded cells, with elevated basal intracellular Ca2+, reduced amount of intrareticular Ca2+, and reduced capacitive Ca2+ entry. Radiation also induced apoptosis, judged from flow cytometry of cells loaded with proprium iodide. Electron microscopy showed that radiation caused condensation of chromatin in dense masses around the nuclear envelope, the presence of apoptotic bodies, fragmentation of the nucleus, detachment of cells from their neighbors, and reductions in cell volume. Radiation also caused activation of caspase 12. Apoptosis was reduced by the administration of the caspase inhibitor Z-Val-Ala-Asp-fluoromethyl-ketone methyl ester (Z-VAD-FMK) during the 3 day period after irradiation, and by the chelator of intracellular Ca2+, 1,2-bis(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA), from 1 h before until 2 h after irradiation. BAPTA also reduced the effects of radiation on contractility, basal intracellular Ca2+, amount of intrareticular Ca2+, capacitative Ca2+ entry, and apoptosis. In conclusion, the effects of gamma radiation on contractility, Ca2+ handling, and apoptosis appear due to a toxic action of intracellular Ca2+. Ca2+-induced damage to the sarcoplasmatic reticulum seems a key event in impaired Ca2+ handling and apoptosis induced by γ-radiation. [Copyright &y& Elsevier]

Early afterdepolarizations (EADs) induced by suppression of cardiac delayed rectifier I (Kr) and/or I (Ks) channels cause fatal ventricular tachyarrhythmias. In guinea pig ventricular myocytes, partial block of one of the channels with complete block of the other reproducibly induced EADs. Complete block of both I (Kr) and I (Ks) channels depolarized the take-off potential and reduced the amplitude of EADs, which in some cases were not clearly separated from the preceding action potentials. A selective L-type Ca(2+) (I (Ca,L)) channel blocker, nifedipine, effectively suppressed EADs at submicromolar concentrations. As examined with the action potential-clamp method, I (Ca,L) channels mediated inward currents with a spike and dome shape during action potentials. I (Ca,L) currents decayed mainly due to inactivation in phase 2 and deactivation in phase 3 repolarization. When EADs were induced by complete block of I (Kr) channels with partial block of I (Ks) channels, repolarization of the action potential prior to EAD take-off failed to increase I (K1) currents and thus failed to completely deactivate I (Ca,L) channels, which reactivated and mediated inward currents during EADs. When both I (Kr) and I (Ks) channels were completely blocked, I (Ca,L) channels were not deactivated and mediated sustained inward currents until the end of EADs. Under this condition, the recovery and reactivation of I (Ca,L) channels were absent before EADs. Therefore, an essential mechanism underlying EADs caused by suppression of the delayed rectifiers is the failure to completely deactivate I (Ca,L) channels. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the effects of aging in Ca2+ extrusion mechanisms in smooth muscle bladder cells from 4 and 20–24-month-old guinea pigs using fluorescence microscopy and fura-2. Cells were challenged with a pulse of KCl immediately before perfusion with a Ca2+ free solution containing no inhibitors (control, untreated cells) or inhibitors of plasma membrane Ca2+ pump (PMCA, 1mM La3+), Na+/Ca2+ exchanger (NCX, 1μM SEA0400) or the sarcoendoplasmic Ca2+ pump (SERCA, 1μM thapsigargin). Treatment of young adult cells with the inhibitors allowed estimating a relative contribution of 55% for NCX, 27% for PMCA and 31% for SERCA. Combination of two inhibitors at the same time showed the presence of interaction between extrusion mechanisms. In aged cells the [Ca2+] i extrusion was impaired due to decrease of PMCA activity, as revealed by the loss of effect of La3+, and to inhibitory interactions between NCX and SERCA activities, indicated by acceleration of decay in response to their respective inhibitors. In conclusion, in smooth muscle cells aging decreases the overall Ca2+ extrusion activity and modifies the interactions between the activities of the main Ca2+ removing mechanisms. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

The motility of the gut depends on the chemicals contained in the lumen, but the stimuli that modify motility and their relationship to enteric neural pathways are unclear. This study examined local inhibitory reflexes activated by various chemical stimulants applied to the mucosa to characterize effective physiological stimuli and the pathways they excite. Segments of the jejunum were dissected to allow access to the circular muscle on one-half of the preparation while leaving the mucosa intact on the circumferentially adjacent half. Chemicals were transiently applied to the mucosa, and responses were recorded intracellularly in nearby circular muscle cells. The amino acids L-phenylalanine, L-alanine, or L-tryptophan (all 1 mM) evoked inhibitory junction potentials (IJPs; latency 150–300 ms, amplitude 3–8 mV, each n > 6) that were blocked by TTX and partially blocked by antagonists of P2X receptors and/or a combination of antagonists at 5-HT3 and 5-HT4 receptors. The putative mediators 5-HT (10 μM), ATP (1 mM), and CCK-8 (1–10 μM) elicited IJPs mediated via 5-HT3, P2X, and CCK-B receptors, respectively. Responses were only partially reduced by the effective antagonists. IJPs evoked by electrically stimulating the mucosa were unaffected by antagonists that reduced chemically evoked responses. Both chemically and electrically evoked IJPs were resistant to nicotinic, NK1, NK3, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, N-methyl-D-aspartate, or CGRP receptor blockade. We conclude that mucosal stimulation by amino acids activates local neural pathways whose pharmacology depends on the nature of the stimulus. Transmitters involved at some synapses in these pathways remain to be identified. [ABSTRACT FROM AUTHOR]

Na+/K+ pump is one of key mechanisms to maintain cell volume. When it is inhibited, cells are at risk of swelling. However, in guinea pig ventricular myocytes, the cell area as an index of cell volume was almost constant during 90 min Na+/K+ pump blockade with 40 μM ouabain despite the marked membrane depolarization. In this study, involvements of Ca2+ transporters and channels in the cardiac cell volume regulation were proposed by conducting the computer simulation in parallel with the experimental validation. [ABSTRACT FROM AUTHOR]

We examined the effect of SN-6 on the Na+/Ca2+ exchanger (NCX) current (INCX) and other membrane currents in isolated guinea pig ventricular myocytes using the whole-cell voltage clamp technique. SN-6 suppressed the bidirectional INCX in a concentration-dependent manner. The IC50 values of SN-6 were 2.3 μM and 1.9 μM for the outward and inward components of the bidirectional INCX, respectively. On the other hand, SN-6 suppressed the unidirectional outward INCX more potently than the inward INCX, with an IC50 value of 0.6 μM. SN-6 at 10 μM inhibited the unidirectional inward INCX by only 22.4 ± 3.1%. SN-6 suppressed INCX more potentially when intracellular Na+ concentration became higher. SN-6 inhibited INa, ICa, IKr, IKs, and IK1 by about 13%, 34%, 33%, 18%, and 13%, respectively. SN-6 shortened the action potential duration (APD) by about 34% and 25% at APD50 and APD90, respectively. These results indicate that SN-6 inhibits NCX in a similar manner to that of KB-R7943. SN-6 and KB-R7943 inhibit the unidirectional outward INCX more potently than the unidirectional inward INCX. Both drugs inhibit NCX in an intracellular Na+ concentration-dependent manner. However, SN-6 affected other membrane currents less potently than KB-R7943. [ABSTRACT FROM AUTHOR]

Genetic and drug-induced abnormalities of cardiac repolarization have been linked to fatal arrhythmias. These arrhythmias result from a complex interaction of the remaining currents during excitation and repolarization. In this review, we examine recent advancement in investigations of genetic heart diseases and mechanisms of arrhythmia generation. We also present our simulation of repolarization during rapid pacing for different levels of block of the rapid delayed rectifier current, IKr, and pharmacological interventions using the Luo–Rudy model. Control simulations showed the development of alternans at a basic cycle length (BCL) of 131 ms. Two levels of IKr block were simulated corresponding to type 2 of familial long QT syndrome, LQT2. At 100% IKr block, the threshold BCL for the appearance of alternans increased to 145 ms and for shorter cycle lengths showed increasingly complex patterns of periodic and chaotic behavior. We examined the potential of other currents to correct this complex behavior. Improvement of the threshold for bifurcation as a function of BCL was achieved by: (1) 100% block of a nonspecific Ca2+-activated current; (2) 15% block of L-type Ca2+ current; (3) 20% increase of Na+/K+ pump current; (4) 50% increase of SERCA2 pump activity. Conversely, increased L-type Ca2+ current, decreased Na+/K+ pump current, or decreased SERCA2 pump activity increased the threshold BCL. Modification of several other currents had little effect. Alternans and chaotic activity develop at fast pacing rates in model guinea pig ventricular myocytes through a sequence of bifurcations. We elucidated mechanisms that modify the development of alternans which may provide novel targets for treatment of patients with LQT2. [ABSTRACT FROM AUTHOR]

1. 5-Hydroxytryptamine (5-HT) has an important role in the pathogenesis of irritable bowel syndrome. To investigate the effects of 5-HT on the contractile activity of myocytes of the guinea-pig proximal colon, cell imaging before and after contraction was undertaken and images were analysed using image-analysis software. Ion currents and membrane potentials were measured. Cytoplasmic free Ca2+ was recorded using a confocal microscope followoing loading of the cells with the fluorescent probe Fura-2AM. 2. 5-Hydroxytryptamine reduced cell length in a dose-dependent manner (EC50 = 0.189 µmol/L). Under current clamp, 10 µmol/L 5-HT reduced action potential amplitude (measured as peak height) and decreased action potential duration, as well as depolarizing the resting potential from −68.4 ± 3.6 to −22.96 ± 4.65 mV. Iberiotoxin (1 µmol/L) blocked the effects of 5-HT in reducing the time to repolarization (T90) and nicardipine (5 µmol/L) blocked the effects of 5-HT in reducing action potential amplitude. 3. In the whole-cell mode, 5-HT enhanced L-type Ca2+ currents, large conductance K+ channel (BKCa) currents and spontaneous transient outward currents (STOC). In addition, 5-HT increased intracellular Ca2+ levels. Ondansetron (10 µmol/L) blocked the effects of 5-HT in enhancing L-type Ca2+ currents, BKCa currents and STOC. 4. In conclusion, 5-HT induces contraction of colonic myocytes, mostly as a result of Ca2+ release from the sarcoplasmic reticulum (SR) following activation of 5-HT3 receptors and the inositol 1,4,5-trisphosphate pathway. In addition, the effect of 5-HT in decreasing action potential amplitude is mediated by the release of Ca2+ from the SR, as well as by enhanced L-type Ca2+ current. 5-Hydroxytryptamine decreased action potential duration by enhancing BKCa current. [ABSTRACT FROM AUTHOR]

Although tile Na+/K+ pump is one of tile key mechanisms responsible for maintaining cell volume, we have observed experimentally that cell volume remained almost constant during 90 rain exposure of guinea pig ventricular myocytes to ouabain. Simulation of this finding using a comprehensive cardiac cell model (Kyoto model incorporating CI- and water fluxes) predicted roles for the plasma membrane Ca+-ATPase (PMCA) and Na+/Ca2+ exchanger, in addition to low membrane permeabilities for Na+ and CI-, in maintaining cell volume. PMCA might help maintain the [Ca2+] gradient across the membrane though compromised, and thereby promote reverse Na+/Ca2+ exchange stimulated by the increased [Na+]i its well as the membrane depolarization. Na+ extrusion via Na+/Ca2+ exchange delayed cell swelling during Na+/K+ pump block. Supporting these model predictions, we observed ventricular cell swelling after blocking Na+/Ca2+ exchange with KB-R7943 or SEA0400 in the presence of ouabain. When Cl- conductance via the cystic fibrosis transmembrane conductance regulator (CFTR) was activated with iso-proterenol during the ouabain treatment, cells showed an initial shrinkage to 94.2 ± 0.5%, followed by a marked swelling 52.0 ± 4.9 rain after drug application. Concomitantly with the onset of swelling, a rapid jump of membrane potential was observed. These experimental observations could be reproduced well by the model simulations. Namely, the Cl- efflux via CFTR accompanied by a concomitant cation efflux caused the initial volume decrease. Then, the gradual membrane depolarization induced by the Na+/K+ pump block activated the window current of the L-type Ca2+ current, which increased [Ca2+]i. Finally, the activation of Ca2+-dependent cation conductance induced the jump of membrane potential, and the rapid accumulation of intracellular Na+ accompanied by the Cl- influx via CFTR, resulting in the cell swelling. The pivotal role of L-type Ca2+ channels predicted in the simulation was demonstrated in experiments, where blocking Ca2+ channels resulted in a much delayed cell swelling. [ABSTRACT FROM AUTHOR]

The stomach generates a characteristic pattern of coordinated activity whereby rings of contraction regularly start in the corpus and migrate slowly down the stomach to the duodenum. This behaviour persists after isolating the stomach and after blocking nervous activity; hence the response is myogenic, resulting from organized contractions of smooth muscle cells lying in the stomach wall. Each ring of contraction is triggered by a long lasting wave of depolarization, termed a slow wave. Slow waves are now known to be generated by sets of interstitial cells of Cajal (ICC), which intermingle with gastric smooth muscle cells. This article describes some studies which identify the roles played by ICC in the on-going generation of coordinated gastric movements. Intramuscular ICC in the corpus generate slow waves and these provide the dominant pacemaker frequency in the stomach. Corporal slow waves, in turn, activate a network of myenteric ICC, which starts in the antrum and slowly conducts waves of depolarization down the stomach. As these waves pass over bundles of circularly orientated muscle cells, they activate a set of intramuscular ICC which lie in the circular muscle layer: these generate slow waves that rapidly spread radially, so triggering each ring of contraction. [ABSTRACT FROM AUTHOR]

Increases in contraction amplitude following rest or in elevated extracellular Ca2+ concentration ([Ca2+]) have been attributed to increased sarcoplasmic reticulum (SR) Ca2+ stores and/or increased trigger Ca2+. However, either manipulation also may elevate diastolic [Ca2+]. The objective of this study was to determine whether elevation of diastolic [Ca2+] could contribute to positive inotropy in isolated ventricular myocytes. Voltage-clamp experiments were conducted with high-resistance microelectrodes in isolated myocytes at 37°C. Intracellular free [Ca2+I was measured with fura-2, and cell shortening was measured with an edge detector. SR Ca2+ stores were assessed with 10 mM caffeine (0 mM Na+, 0 mM Ca2+). Following a period of rest, cells were activated with trains of pulses, which generated contractions of increasing amplitude, called positive staircases. Positive staircases were accompanied by increasing diastolic [Ca2+] but no change in Ca2+ transient amplitudes. When extracellular [Ca2+] was elevated from 2.0 to 5.0 mM, resting intracellular [Ca2+] increased and resting cell length decreased. Amplitudes of contractions and L-type Ca2+ current increased in elevated extracellular [Ca2+], although SR Ca2+ stores, assessed by rapid application of caffeine, did not increase. Although Ca2+ transient amplitude did not increase in 5.0 mM extracellular [Ca2+], diastolic [Ca2+] continued to increase with increasing extracellular [Ca2+]. These data suggest that increased diastolic [Ca2+] contributes to positive inotropy following rest or with increasing extracellular [Ca2+I in guinea pig ventricular myocytes. [ABSTRACT FROM AUTHOR]

Abstract: In cardiac muscle the amplitude of Ca2+ transients can be increased by enhancing Ca2+ influx. Among the processes leading to increased Ca2+ influx, agonists of the L-type Ca2+-channel can play an important role. Known pharmacological Ca2+-channel agonists act on different binding sites on the channel protein, which may lead not only to enhanced peak currents, but also to distinct changes in other biophysical characteristics of the current. In this study, membrane currents were recorded with the patch–clamp technique in the whole-cell configuration in guinea pig isolated ventricular myocytes in combination with confocal fluorescence Ca2+ imaging techniques and a variety of pharmacological tools. Testing a new positive inotropic steroid-like compound, we found that it increased the L-type Ca2+-current by 2.5-fold by shifting the voltage-dependence of activation by 20.2mV towards negative potentials. The dose–response relationship revealed two vastly different affinities (EC50(high-affinity) =4.5±1.7nM, EC50(low-affinity) =8.0±1.1μM) exhibiting differential pharmacological interactions with three classes of Ca2+-current antagonists, suggesting more than one binding site on the channel protein. Therefore, we identified and characterized a novel positive inotropic compound (F90927) as a member of a new class of Ca2+-channel agonists exhibiting unique features, which set it apart from other presently known L-type Ca2+-channel agonists. [Copyright &y& Elsevier]

Activators of the slow delayed rectifier K+ current (IKs) are promising tools to suppress ventricular arrhythmias originating from prolongation of action potentials. A recently synthesized compound, L-364,373, was shown to activate IKs in ventricular cells isolated from guinea pigs and rabbits. Due to the interspecies differences known to exist in the properties of the delayed rectifier K+ currents, the effect of L-364,373 on IKs was studied and compared with that of another IKs activator mefenamic acid in canine ventricular myocytes. Mefenamic acid (100 μM) significantly increased the amplitude of the fully activated IKs current, as well as the IKs current tails, by shifting the voltage dependence of its activation towards negative voltages and increased the time constant for deactivation. In contrast, L-364,373, up to concentrations of 3 μM, failed to augment IKs at any membrane potential studied, but slightly increased the time constant of deactivation. It is concluded that human studies are required to evaluate the therapeutically beneficial effects of IKs activators. Rodent cardiac tissues are not suitable for this purpose. [ABSTRACT FROM AUTHOR]

Abstract: We measured mitochondrial NADH autofluorescence or Ca2+ using Rhod-2, simultaneously with cell shortening in isolated guinea-pig ventricular myocytes. When both frequency and amplitude of twitch shortening (work intensity) were increased by raising stimulus frequency in incremental steps from 0.1 to 3.3 Hz, the steady level of NADH signal increased in a frequency-dependent manner. Mitochondrial Ca2+ also increased with increasing work intensity. Applying Ru360, an inhibitor of mitochondrial Ca2+ uniporter, largely attenuated the response of both NADH fluorescence and mitochondrial Ca2+. The increase in mitochondrial Ca2+ was slow with t1/2 =~12 s and no obvious cyclic changes were observed in the NADH signal. When a step change from 0.1 to 3.3 Hz stimulation was applied, the NADH signal first decreased to 83% and then increased to 155% of the control level. Upon returning to 0.1 Hz, the NADH signal showed an overshoot before declining to the control level. The biphasic onset time course was well explained by the delayed Ca2+ activation of the substrate dehydrogenation superimposed on the feedback control of the ATP synthesis, while the offset time course with a delayed deactivation of dehydrogenation. A computer simulation using an oxidative phosphorylation linked to the cardiac excitation contraction model well reconstructed the response of NADH. This model simulation predicts that the activation of substrate dehydrogenation provides ~23% of driving force of the ATP synthesis to meet the increased workload induced by the jump of stimulus from 0.1 to 3.3 Hz, and remaining ~77% is supplied by the feedback control. [Copyright &y& Elsevier]

We used Fluo-3/AM to examine the effect of platelet-activating factor on the intracellular Ca2+([Ca2+]i) levels in isolated myocytes of guinea pig ventricle. Myocytes were isolated with Langendorff perfusion technique and were challenged with platelet-activating factor. Addition of platelet-activating factor (1 pM to 10 nM) significantly increased the [Ca2+]i in the presence and absence of extracellular Ca2+. The notion that increases in intracellular Ca2+ induced by platelet-activating factor is the result of stimulation of intracellular Ca2+ pool rather than increasing Ca2+ influx was further supported by the whole cell patch-clamp experiments in which the platelet-activating factor did not alter the activity of L-type of Ca2+ channels ( ICa-L). Treatment of myocytes with ryanodine failed to abolish the stimulatory effect of platelet-activating factor on [Ca2+]i. In contrast, inhibition of IP3-sensitive Ca2+ release pool with 2-aminoethoxydiphenyl borate (2-APB) blocked the effect of platelet-activating factor. We conclude that the platelet-activating factor-induced increase in intracellular Ca2+ is mediated by stimulation of IP3 receptor but not by stimulation of ICa-L and ryanodine-sensitive receptor. [ABSTRACT FROM AUTHOR]

1. Sipatrigine (BW 619C89), a blocker of neuronal Na+ and Ca2+ channels that is structurally related to lamotrigine, has been shown to be neuroprotective in models of cortical ischaemia. Although associated with cardiovascular effects in animal models in vivo, there is no published information concerning the effects of sipatrigine on cardiac ion currents and action potentials (AP). 2. The aim of the present study was to examine the effects of sipatrigine on the delayed rectifier currents (IKr and IKs), the inward rectifier current (IK1), the L-type Ca2+ current (ICa,L) and the fast Na+ current (INa), as well as on AP duration at 30% (APD30) and 90% (APD90) repolarization, in guinea-pig isolated ventricular myocytes. 3. Each of the currents was inhibited by sipatrigine, demonstrating the drug to be a relatively broad-spectrum blocker of cation channels in the heart. However, sipatrigine was a comparatively more potent inhibitor of IKr (IC50 = 0.85 µmol/L) and IKs (IC50 = 0.92 µmol/L) than of IK1 (IC50 = 5.3 µmol/L), ICa,L (IC50 = 6.0 µmol/L) and INa (IC50 = 25.5 µmol/L). 4. Consistent with block of IKr, IKs and IK1, sipatrigine (1–30 µmol/L) produced a concentration-dependent prolongation of APD90. Although lower concentrations of sipatrigine (≤ 3 µmol/L) caused APD30 prolongation, higher concentrations (≥ 10 µmol/L) shortened APD30, consistent with an involvement of ICa,L blockade. The contrasting effects of sipatrigine on APD30 and APD90 at higher concentrations resulted in a marked concentration-dependent triangulation of the AP. 5. The results of the present study demonstrate that sipatrigine, at concentrations previously shown to be neuroprotective in vitro, modulates cardiac K+, Ca2+ and Na+ currents and repolarization of the cardiac ventricular action potential. [ABSTRACT FROM AUTHOR]

Abstract: Objective: It was our aim to investigate effects of human LDL, copper-, or AAPH-oxidized over different periods of time to different degrees (ox-LDL), on viability and electrophysiological parameters of isolated ventricular myocytes of guinea pigs. Methods: Guinea pig ventricular myocytes were incubated with ox-LDL or native LDL (at 0.5 mg/ml) for 12 h, and afterwards myocyte damage, action potentials, and transmembrane ion currents were studied (at 37 °C). Results: Ox-LDL was found to induce severe myocyte damage, whereas native LDL had no effect. Myocyte damage was dependent on the content of total lipid hydroperoxides in both copper-oxidized and AAPH-oxidized LDL. Incubation with ox-LDL led to intense contractile and electrophysiological effects including prolongation of action potential duration, depolarization of resting membrane potential, spontaneous activity, generation of afterdepolarizations, and modification of transmembrane ion currents (e.g. inward rectifier, calcium, and background currents). Conclusions: Ox-LDL induced cell damage and irregular electrical activity in ventricular myocytes. These effects were dependent on the lipid hydroperoxide content of ox-LDL and were similar to oxidative stress (OS) induced by various OS-generating systems. The observed effects may play a role for functional cardiac abnormalities in patients with increased ox-LDL levels. [Copyright &y& Elsevier]

Ca+ waves have been implicated in Ca2+ overload-induced cardiac arrhythmias. To deepen understanding of the behavior of Ca2+ waves in a multicellular system, consecutive two-dimensional Ca2+ images were obtained with a confocal microscope from surface cells of guinea pig ventricular papillary muscles loaded with fluo 3 or rhod 2. In intact muscles, no Ca2+ waves were detected under the resting condition, whereas they were frequently observed during the rest immediately after high-frequency stimulations where cytoplasmic Ca2+ concentration and Ca2+ stored in the sarcoplasmic reticulum (SR) were gradually decreasing. The intervals of Ca2+ waves increased as they occurred later, their amplitudes and velocities remaining unchanged. A SERCA inhibitor reversibly prolonged the wave intervals. In Na+-free/Ca2+- free medium where neither Ca2+ influx nor Na+/Ca2+exchange took place, recurrent Ca2+ waves emerged at constant intervals in each cell. These results are consistent with the conclusion that the loading level of the SR is critical for induction of Ca2+ waves. Each cell independently exhibited its own regular rhythm of Ca2+ wave with a distinct interval. These waves propagated in either direction along the longitudinal axis within a muscle cell, but seldom beyond the cell boundary. In contrast, in partially damaged muscles that showed spontaneous Ca2+ waves at rest in normal Krebs solution, their propagation often was unidirectional, decreasing in frequency. In these cases, however, Ca2+ waves rarely moved beyond the cellular boundary. The gradient of the cytoplasmic Ca2+ concentration was suggested to be the cause of the one-way propagation. [ABSTRACT FROM AUTHOR]

1. The aim of the present study was to analyse the mechanism that underlies the force development induced by ouabain (ED100 = 100 µmol/L) in guinea-pig tracheal rings.2. The dose–response curve showed that concentrations of ouabain above 100 µmol/L evoked smaller contractions.3. Ouabain, at 100 µmol/L, produced two long-lasting consecutive transient contractions. The peak of the first contraction was 750 ± 75 mg, whereas the peak of the second contraction was 280 ± 46 mg.4. Both contractions induced by ouabain were dependent on extracellular Ca2+. Consistent with this, verapamil (10 µmol/L) inhibited the first and second contractions by 77 and 59%, respectively.5. 3,4-Dichlorobenzamil (20 µmol/L) inhibited the first and second contractions by 68 and 97%, respectively.6. Simultaneous exposure to 15 mmol/L sodium solution and 100 µmol/L ouabain evoked only one transient contraction, larger (987 ± 135 mg) than either of the ouabain-induced contractions.7. Inhibition of the sarcoendoplasmic reticulum Ca-ATPase with cyclopiazonic acid potentiated the first and second ouabain-induced contractions by 47 and 300%, respectively.8. Atropine (1 µmol/L) inhibited the first and second contractions by 44 and 76%, respectively.9. In conclusion, the results of the present study are relevant to the understanding of the mechanisms by which ouabain (100 µmol/L) contracts guinea-pig tracheal rings. At the muscular level, oubain induces Ca2+ influx through L-type Ca2+ channels and the reverse mode of the sodium–calcium exchanger. At the nerve terminals, ouabain promotes the release of acetylcholine secondary to the increase in Ca2+ influx mediated by the reverse mode of the sodium–calcium exchanger. [ABSTRACT FROM AUTHOR]

Abstract The properties of cationic channels of an average unitary conductance were studied in guinea-pig ileum smooth muscle cells using a patch-clamp technique in the outside-out configuration. Cationic channels were activated by addition of 200 M GTP?S to an intrapipette solution, which resulted in stable activation of G proteins. The replacement of external cesium-containing (in the absence of Ca2+ and Mg2+ ions) solution with a more physiological sodium solution containing 2.5 mM Ca2+ and 2.0 mM Mg2+ led to smaller values of both the amplitude of currents through the unitary cationic channels under study and the probability of the stay of the channel in the open state (Po). The drop in current amplitude was related mostly to the blocking effect of bivalent cations, while a decrease in the Po resulted from the replacement of Cs+ with Na+. Just a drop in the Po, which was responsible for approximately 85% of the inhibitory effect, played a crucial role in the suppression of the integral transmembrane current. [ABSTRACT FROM AUTHOR]

This study in- vestigated the interaction between L-type Ca[sup 2+] current (ICaL) and Ca[sup 2+] release from the sarcoplasmic reticulum (SRCR) in whole cell voltage-clamped guinea pig ventricular myocytes. Quasiphysiological cation solutions (Na[sup +, sub o]:K[sup +, sub I]) were used for most experiments. In control conditions, there was no obvious interaction between ICaL and SRCR. In isoproterenol, activation of ICaL from voltages between -70 and -50 mV reduced the amplitude and accelerated the decay of the current. Short (50 ms), small-amplitude voltage steps applied 60 or 510 ms before stimulating ICaL inhibited and facilitated the current, respectively. These changes were blocked by ryanodine. Low-voltage activated currents such as T-type Ca[sup 2+] current, TTX-sensitive ICa (ICaTTX), or "slip mode" Ca[sup 2+] conductance via INa+ were not responsible for low-voltage SRCR. However, L-type Ca[sup 2+] currents could be distinguished at voltages as negative as -45 mV. It is concluded that in the presence of isoproterenol, Ca[sup 2+] release from the SR at negative potentials is due to activation of L-type Ca[sup 2+] channels. [ABSTRACT FROM AUTHOR]

1The objective of this study was to determine the concentration-dependent effects of nisoldipine. a dihydropyridine Ca[sup2+]channel blocker, on K[sup+] currents in guinea-pig ventricular myocytes. 2 Myocytes in the conventional whole-cell configuration were bathed in normal Tyrode's solution or K[sup+]-free Tyrode's solution for the measurement of the effects of 0.01 A—l00μM nisoldipine on rapidly activating delayed-rectifier K[sup+] current (I[subKr]), slowly activating delayed-rectifier K[sup+] current (I[subKs]), inwardly rectifying K[sup+] current(I[subKt]), and reference L-type Ca[sup2+] current (I[subCa 1)]. 3 Nisoldipine inhibited I[sub Kr] with an IC[sub50] of 23μM. and I[subKs] with an IC[sub50] of 40μM.. The drug also had weak inhibitory effects on inward- and outward-directed I [subKr]; the IC[sub30] determined for outward-directed current was 80μM. 4 Investigation of nisoldipine action on I[subKs] showed that inhibition occurred in the absence of previous pulsing, and with little change in the time courses of activation and deactivation. However, the drug-induced inhibition was significantly weaker at ≥ +30mV than at + 10mV.5 We estimate that nisoldipine is about 30 times less selective for delayed-rectifier K[sup+] channels than for L-type Ca[sup2+] channels in fully polarised guinea-pig ventricular myocytes, and several orders less selective in partially depolarised myocytes. [ABSTRACT FROM AUTHOR]

While cardiac pacemakers are frequently used for the treatment of bradydysrhythmias (from diseases of the cardiac conduction system), their use is still limited by complications that can be life-threatening and expensive. Genetic engineering approaches offer an opportunity to modulate cellular automaticity in a manner that could have significant therapeutic potential. It is well known that ventricular myocytes exhibit a more negative diastolic potential than do pacemaker cells, in large part because of the inward rectifying potassium current/[subK1] (which pacemaker cells lack). Taking advantage of these intrinsic electrophysiological differences, a biological pacemaker has recently been developed by Miake et al (Nature 2002; 419:132-133) using adenoviral gene transfer approaches. By isolating the gene responsible for/[subK1] (the Kir2. 1 gene), mutating it to make it a dysfunctional channel (a dominant-negative), inserting the mutated gene into an adenoviral vector, and delivering the virus to the hearts of guinea pigs, the investigators were able to successfully convert some ventricular myocytes to pacemaker cells. While issues of safety and long-term efficacy need to be further established, the results of these experiments provide proof of principle that gene transfer offers great promise for treatment of electrophysiological disorders including conduction system disease. [ABSTRACT FROM AUTHOR]

Inactivation of the L-type Ca[sup 2+] current (I[sub CaL]) was studied in isolated guinea pig ventricular myocytes with different ionic solutions. Under basal conditions, I[sub CaL] of 82% of cells infused with Cs[sup +]based intracellular solutions showed enhanced amplitude with multiphasic decay and diastolic depolarization-induced facilitation. The characteristics of I[sub CaL] in this population of cells were not due to contamination by other currents or an artifact. These phenomena were reduced by ryanodine, caffeine, cyclopiazonic acid, the protein kinase A inhibitor H-89, and the cAMP-dependent protein kinase inhibitor. Forskolin and isoproterenol increased I[sub CaL] by only ∼60% in these cells. Cells infused with either N-methyl-D-glucamine or K[sup +]-based intracellular solutions did not show multiphasic decay or facilitation under basal conditions. Isoproterenol increased I[sub CaL] by ∼200% in these cells. In conclusion, we show that multiphasic inactivation of I[sub CaL] is due to Ca[sup 2+]-dependent inactivation that is reversible on a time scale of tens of milliseconds. Cs[sup +] seems to activate the cAMP-dependent protein kinase pathway when used as a substitute for K[sup +] in the pipette solution. [ABSTRACT FROM AUTHOR]

1 The modulation of large conductance Ca2+-activated K+ (BKCa) channels by the nitric oxide (NO') donor, S-nitroso-L-cysteine (NOCys) and three sutfhydryl-specific methanethiosulfonate (MTS) reagents, positively charged 2-aminoethyl MTS hydrobromide (MTSEA C3H9NO2S2HBr) and [2-(trimethylammonium) ethyl MTS bromide (MTSET C6H16NO2S2HBr) and negatively charged sodium (2-sulfonatoethyl) MTS (MTSES C3H7O5S3Na) were compared in excised inside-out membrane patches of the guinea-pig taenia caeca. 2 In membrane patches bathed in a low Ca2+ (15 riM) high K F physiological salt solution. l-3 BK~, channels opened with a low probability (~V.P,) of 0.019±0.011 at OmV. N.P~ readily increased with membrane depolarization, raised Ca2 ~ concentration or upon the addition of NOCys (l0~iM for 2- 5mm) such that 5-15 open BK~. channels were evident. 3 MTSEA (2.5 nms) decreased, while MTSES (2.5 mM) increased NP0 (at 0 mV) and the number of open ~ channels at positive potentials. These changes in channel activity remained after a prolonged washout of these two MTS reagents. 4 MTSET 2.5mM) increased N.P~ (at OmV) and voltage-dependently decreased BK~. current amplitudes in a manner readily reversed upon washout. 5 Pre-exposure of excised membrane patches to MTSES or N-ethylmaleimide (NEM 1 mM), a specific alkylating agent of cysteine sulfliydryls, but not MTSEA or MTSET, prevented the excitatory actions of NOCys (10pM). 6 It was concluded that NOCys-evoked increases in BK~, channel activity occur via the S- nitrosylation of cysteine residues within basic regions of the channel ~ subunit that have an accessible water interface. [ABSTRACT FROM AUTHOR]

1 The aim of this study was to investigate the effect of nicotinamide-adenine dinucleotide (NADH) supplementation on the metabolic condition of isolated guinea-pig ventricular cardiomyocytes. The pinacidil-primed ATP-dependent potassium current 1K(ATP) was used as an indicator of subsarco-lemmal ATP concentration and intracellular adenine nucleotide contents were measured. 2 Membrane currents were studied using the patch-clamp technique in the whole-cell recording mode at 36- 37°C. Adenine nucleotides were determined by HPLC. 3 Under physiological conditions (4.3 mM ATP in the pipette solution, ATP,) 1{subK(ATP)] did not contribute to basal electrical activity. 4 The ATP-dependent potassium (K(ATP) channel opener pinacidil activated 1K(ATP) dependent on [ATP], showing a significantly more pronounced activation at lower (1 mM) [ATP]. 5 Supplementation of cardiomyocytes with 300/μgml NADH (4-6h) resulted in a significantly reduced 1K(ATP) activation by pinacidil compared to control cells. The current density was 13.±3.78 (n= 6) versus 28.9± 3.38 pA pF (n= 19; P < 0.05).6 Equimolar amounts of the related compounds nicotinamide and NAD did not achieve a similar effect like NADH. 7 Measurement of adenine nucleotides by HPLC revealed a significant increase in intracellular ATP (NADH supplementation: 45.6±1.88 nmolmg1 protein versus control: 35.4±2.57 nmol mg protein. P < 0.000005), 8 These data show that supplementation of guinea-pig ventricular cardiomyocytes with NADH results in a decreased activation of 1K(ATP) by pinacidil compared to control myocytes, indicating a higher subsarcolemmal ATP concentration. 9 Analysis of intracellular adenine nucleotides by HPLC confirmed the significant increase in ATP. [ABSTRACT FROM AUTHOR]

The mean sarcomere length (SL) of guinea-pig cardiac myocytes was recorded simultaneously with the whole-cell current under voltage-clamp conditions. After blocking both sarcoplasmic reticulum (SR) and L-type Ca2+ channels with ryanodine, cyclopiazonic acid and nicardipine, strong depolarizing pulses induced only the tonic component of SL shortening through the reverse mode of Na+/Ca2+ exchange (NCX). A positive staircase of SL shortening was observed on applying a train pulses to +60~+100 mV at 2 Hz and trans-membrane Ca2+ flux was calculated from the time integral of the Na+/Ca2+ exchange current (INCX). Changes in cytosolic [Ca2+] ([Ca2+]i) were determined indirectly using the experimental [Ca2+]i/SL relationship. Cellular Ca2+ buffering was characterized by a lumped single-component system with a maximum binding capacity of 200 µM and a dissociation constant of 613 nM. Despite the decrease in driving force, the amplitude of the outwards INCX at +60 mV gradually increased along with the positive staircase. The model simulation suggested that this increase of outwards INCX is caused by a dramatic increase in Ca2+-mediated activation of NCX. [ABSTRACT FROM AUTHOR]