Normal Tyrode Solution Research Articles

This Month in ANESTHESIOLOGY

This Month in ANESTHESIOLOGY

Direct myocardial depressant effect of methylmethacrylate monomer: mechanical and electrophysiologic actions in vitro.

The present study explored the mechanism of direct myocardial depression by methylmethacrylate monomer (MMA). Isometric contraction of isolated guinea pig right ventricular papillary muscle was measured in modified normal and 26 mm K+ Tyrode solutions at various stimulation rates. Normal and slow action potentials were evaluated by conventional microelectrode technique. MMA effects on various aspects of sarcoplasmic reticulum function were evaluated by its effect on rapid-cooling contractures, rested-state contraction in rat papillary muscle in modified normal Tyrode solution, and in guinea pig papillary muscle under low Na+ (25 mm) Tyrode solution. Whole cell patch clamp techniques were applied to measure the inward Ca2+ currents (I(Ca)). MMA (0.5, 1.5, and 4.7 mm) caused concentration-dependent depression of peak force and maximal rate of force development to approximately 70, 50, and 20% of baseline from rested state to 3 Hz stimulation rates, respectively. Depression of peak force and maximal rate of force development by MMA was dependent on stimulation frequency, with less depression at higher stimulation rates. In low Na+ Tyrode solution, 1.5 mm MMA depressed peak force of rat and guinea pig myocardium by 20-30%. In 26 mm K+ Tyrode solution, 0.5 and 1.5 mm MMA caused selective and marked concentration-dependent depression of late force development (0.5 mm: approximately 60% of baseline, 1.5 mm: approximately 30% of baseline) with no alteration in early force development. MMA (1.5 mm) depressed rapid-cooling contracture to 53 +/- 10% of baseline, accompanied by approximately 63% prolongation of time to peak contracture. In patch clamp studies, MMA reduced I(Ca) in a concentration-dependent manner. The direct myocardial depressant effect of MMA seems to be caused in part by depression of Ca2+ influx through cardiac membrane, while depolarization-activated sarcoplasmic reticulum Ca2+ release appears modestly depressed.

Effects of Reactive Oxygen Species on ATP-induced intracellular Ca2+ Activity in Osteoblasts

Background: The physiological activity of osteoblsts is known to be closely related to increased intracellular activity ([]i) in osteoblasts. The cellular regulation of ([]i) in osteoblasts is mediated by movements associated with release from intracellular stores, and transmembrane influx via exchanger, and ATPase. Reactive oxygen species, such as , play an important role in the regulation of cellular functions, and act as signaling molecules or as toxins in cells. Methods: Osteoblasts were isolated from the femurs and tibias of neonatal Sprague-Dawley rats, and cultured for 7 days. The cultured osteoblasts were loaded with a -sensitive fluorescent dye, Fura-2 AM ester, and fluorescence images were monitored using a cooled CCD camera. Ca-spike changes upon ATP application were checked for (1) osteoblasts in -free and 2.5 mM normal Tyrode solution, (2) osteoblasts in which the of the endoplastic reticulumin had been depleted with ryanodine, thapsigargin ord caffein, and (3) osteoblasts pretreated with , in which the expression of IP3 receptor was checked by Western blotting. Results: ATP increased intracellular free regardless of extracellular concentration. When the intracellular store was depleted, the level of increased activity by ATP was suppressed. sustained the increase induced by ATP. The expression of IP3 receptor was enhanced by . Conclusions: modulates intracellular activity in osteoblasts by increasing release from the intracellular stores.

Direct Myocardial Depressant Effect of Desflurane: Mechanical and Electrophysiological Actions in Vitro

Background: Desflurane actions on myocardial contractility and cellular electrophysiologic behavior were studied in isolated guinea pig and rat right ventricular papillary muscles. Methods: The isometric force of isolated guinea pig ventricular papillary muscles was studied in normal and 26 mM Tyrode's solution at various stimulation rates. Experiments using rat papillary muscles under normal Tyrode's solution at the rested-state (RS) and using guinea pig papillary muscles under low Tyrode's solution (25 mM) were performed to evaluate the effect of release from the sarcoplasmic reticulum (SR). Effects of desflurane on SR function in situ were examined by its effect on rapid cooling contractures (RCCs). Normal and slow action potentials (APs) were evaluated by using a conventional microelectrode technique. Finally, currents of isolated guinea pig ventricular myocytes were examined using the whole cell patch clamp technique. Results: 1 MAC (minimum alveolar concentration: 6%) and 2 MAC desflurane were applied. 1 MAC and 2 MAC desflurane depressed guinea pig myocardial contractions by 30% and 60%, respectively, from RS to 3 Hz stimulation rates. 1 MAC (1.15%) and 2 MAC isoflurane depressed peak force by 25% and 45%, respectively. Contractile force after rest in rat and guinea pig myocardium under low Tyrode's solution showed modest depression. In the partially depolarized, adrenergically stimulated myocardium, 1 MAC and 2 MAC desflurane caused a marked depression of the late peak force (1 MAC:60%, 2 MAC:80%) with moderate changes of the early peak force (1 MAC: 20%, 2 MAC: 40%). RCCs were abolished at 1 MAC desflurane. Desflurane did not alter the peak amplitude or maximum depolarization rate of normal and slow APs, however, AP duration was significantly prolonged. In isolated guinea pig myocytes at room temperature, 1 MAC and 2 MAC desflurane caused a 28% and 55% decrease in currents, respectively. Conclusions: These results indicate that desflurane causes a dose-dependent depression of contractile force in isolated myocardium, which is comparable to that of isoflurane. The depression seems to be related, at least in part, to its ability to reduce inward currents through the cardiac membrane. Therefore, it is likely that various methods employed to enhance inward current may improve the hemodynamic depression induced by desflurane.

Ischemia alters the electrical activity of pacemaker cells isolated from the rabbit sinoatrial node

The purpose of this study was to investigate the mechanisms responsible for ischemia-induced changes in spontaneous electrical activity. An ischemic-like Tyrode solution (pH 6.6) reversibly depolarized the maximum diastolic potential (MDP) and reduced the action potential (AP) overshoot (OS). We used SNARF-1, which is an indicator of intracellular pH (pH(i)), and perforated-patch techniques to test the hypothesis that acidosis caused these effects. Acidic but otherwise normal Tyrode solution (pH 6.8) produced similar effects. Basic Tyrode solution (pH 8.5) hyperpolarized the MDP, shortened the AP, and slowed the firing rate. In the presence of "ischemic" Tyrode solution, hyperpolarizing current restored the MDP and OS to control values. HOE-642, an inhibitor of Na/H exchange, did not alter pH(i) or electrical activity and did not prevent the effects of ischemic Tyrode solution or recovery after washout. Time-independent net inward current but not hyperpolarization-activated inward current was enhanced by ischemic Tyrode solution or by 30 microM BaCl(2), a selective blocker of inward-rectifying K currents at this concentration. The results suggest that 1) acidosis was responsible for the ischemia-induced effects but Na/H exchange was not involved, 2) the OS was reduced because of depolarization-induced inactivation of inward currents that generate the AP upstroke, and 3) reduction of an inward-rectifying outward K current contributed to the depolarization.

Intracellular Calcium Level Required for Calpain Activation in a Single Myocardial Cell

We have hypothesized that calpain mediates myocardial injury induced by Ca2+overload. However, in vitro study demonstrated that the calcium requirement for calpain activation is around 10 μ m, which is difficult to reach without the cell collapsing. Furthermore, because calpastatin is abundant in the myocardial cell, calpain may not be activated in physiological conditions. To elucidate whether calpain is activated by the calcium concentration reachable in myocardial living cells, we measured the calpain activity and the calcium concentration simultaneously in isolated guinea-pig cardiomyocytes. t-Butoxycarbonyl-Leu-Met-7-amino-4chlorimethylcoumarin (Boc-Leu-Met-CMAC), a fluorescent substrate of calpain, and/or fura red, a calcium indicator, were loaded into isolated cardiomyocytes together, and their fluorescence were measured separately. Intracellular Ca overload was induced by changing the superfusate from normal Tyrode solution to a sodium-free one. After changing the solution, fluorescence intensity of fura red and Boc-Leu-Met-CMAC did not change for a while, then fluorescence intensity of fura red began to rise. This was followed by the fluorescence intensity of Boc-Leu-Met-CMAC starting to rise 160±45 s after [Ca2+]iincrease. The relative fluorescence intensity of fura red increased to 1.37±0.32 folds of the control at the point that calpain became active. The calcium concentration at this point was estimated as 451 n m. These results indicate that calpain is activated by the slight rise of Ca concentration in intact cardiomyocytes.

Inhibition of oxotremorine-induced desensitization of guinea-pig ileal longitudinal muscle in Ca2+-free conditions.

The aim of this study was to investigate the differences between oxotremorine-induced and acetylcholine (ACh)-induced desensitization, particularly under Ca2+-free conditions, in guinea-pig ileal longitudinal muscle, and to elucidate the different mechanisms of desensitization that might exist between these two muscarinic agonists. Pretreatment of the tissue with 10(-7)-10(-5) M oxotremorine (desensitizing treatment) in normal Tyrode solution caused desensitization of the responses to ACh, as did the desensitizing treatment with ACh. However, Ca2+-free conditions significantly reduced oxotremorine-induced desensitization, contrary to the previous findings that Ca2+-free conditions enhanced ACh-induced desensitization. The desensitizing treatment with oxotremorine caused suppression of the responses to high K+ (tonic phase), as did the ACh treatment. Ca2+-free conditions removed this suppression, whereasthis condition enhanced ACh-induced suppression of the K+ response. A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (10(-4) M) had no effect on oxotremorine-induced desensitization of the ACh response. The results suggest that a voltage-gated Ca2+ channel was involved in oxotremorine-induced desensitization, as in ACh-induced desensitization, but that the process of inactivation of Ca2+ channels was different between oxotremorine and ACh, and that oxotremorine-induced desensitization was due not only to Ca2+ channel, but also to other unknown factors. Protein kinase C did not participate in oxotremorine-induced desensitization.

Vacuole formation in fatigued skeletal muscle fibres from frog and mouse: effects of extracellular lactate.

Isolated, living muscle fibres from either Xenopus or mouse were observed in a confocal microscope and t-tubules were visualized with sulforhodamine B. Observations were made before and after fatiguing stimulation. In addition, experiments were performed on fibres observed in an ordinary light microscope with dark-field illumination. In Xenopus fibres, recovering after fatigue, t-tubules started to show dilatations 2-5 min post-fatigue. These swellings increased in size over the next 10-20 min to form vacuoles. After 2-3 h of recovery the appearance of the fibres was again normal and force production, which had been markedly depressed 10-40 min post-fatigue, was close to control. Vacuoles were not observed in mouse fibres, fatigued with the same protocol and allowed to recover. In Xenopus fibres, fatigued in normal Ringer solution and allowed to recover in Ringer solution with 30-50 mM L-lactate substituting for chloride (lactate-Ringer), the number and size of vacuoles were markedly reduced. Also, force recovery was significantly faster. Replacement of chloride by methyl sulphate or glucuronate had no effect on vacuolation. Resting Xenopus fibres exposed to 50 mM lactate-Ringer and transferred to normal Ringer solution displayed vacuoles within 5-10 min, but to a smaller extent than after fatigue. Vacuolation was not associated with marked force reduction. Mouse fibres, fatigued in 50 mM lactate-Tyrode (L-lactate substituting for chloride in Tyrode solution) and recovering in normal Tyrode solution, displayed vacuoles for a limited period post-fatigue. Vacuolation had no effect on force production. The results are consistent with the view that lactate, formed during fatigue, is transported into the t-tubules where it attracts water and causes t-tubule swelling and vacuolation. This vacuolation may be counteracted in vivo due to a gradual extracellular accumulation of lactate during fatigue.

The mechanism of actions of 3-(5′-(hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1) on Ca 2+-activated K + currents in GH 3 lactotrophs

The effects of 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1), an activator of soluble guanylyl cyclase, on ionic currents have been assessed in rat pituitary GH 3 lactotrophs. In GH 3 cells bathed in normal Tyrode's solution, YC-1 (1 μM) reversibly suppressed the amplitude of the Ca 2+-activated K + current ( I K(Ca)). YC-1 at a concentration above 10 μM produced a biphasic response in the amplitude of I K(Ca), i.e., an initial decrease followed by a sustained increase. When the pipette solutions were filled with high EGTA (10 mM), the YC-1-induced stimulatory effect on I K(Ca) was abolished. Over a similar concentration range, YC-1 also effectively inhibited the voltage-dependent K + current ( I K(V)) in GH 3 cells. The IC 50 value required for the inhibition of I K(V) by YC-1 was 1 μM. Unlike YC-1, 8-bromo cGMP did not inhibit I K(Ca). However, YC-1 (10 μM) did not affect the amplitude of L-type Ca 2+ current. In the cell-attached configuration, application of YC-1 (10 μM) to the bath did not change the single-channel conductance of the large-conductance Ca 2+-activated K + (BK Ca) channels; however, it did increase the opening probability of BK Ca channels. In contrast, in the outside-out configuration, YC-1 (10 μM) significantly suppressed the opening probability of BK Ca channels. The present study shows dual effects of YC-1 on I K(Ca) in GH 3 cells. The YC-1-mediated stimulation of I K(Ca) may result from elevated cytosolic Ca 2+, whereas the inhibition of I K(Ca) and I K(V) by YC-1 appears to be direct and independent of the activation of soluble guanylyl cyclase. Caution thus needs to be used in attributing the YC-1-mediated response to the activation of soluble guanylyl cyclase.

M cells and transmural heterogeneity of action potential configuration in myocytes from the left ventricular wall of the pig heart

Heterogeneity of action potential configuration in the left ventricle (LV), and the contribution of M cells to it, has been observed in the human heart and is important for arrhythmogenesis. Whether the pig heart has similar properties remains a controversial but important issue as the pig heart is currently under study for use in xenotransplantation. Single myocytes were enzymatically isolated from the epicardium (EPI, ncells = 29), midmyocardium (MID, ncells = 38), and endocardium (ENDO, ncells = 13) of the free LV wall (npigs = 26, 14-22 weeks old, 55-80 kg), and studied at different stimulation rates during whole-cell recording (normal Tyrode's solution, K(+)-aspartate-based pipette solution, 50 microM K5fluo-3 as [Ca2+]i indicator, 37 degrees C). Standard six-lead ECGs were recorded from anesthetized pigs. The action potential duration (APD) was not significantly different at 0.25 Hz vs. 2 Hz for the majority of cells in all three layers. However, a subpopulation of cells behaved like M cells and had a very steep frequency response (APD90 at 0.25 Hz 538 +/- 30 ms, vs. 337 +/- 9 ms at 2 Hz, P < 0.05, n = 22). These cells were found predominantly in the MID layer (34% of cells), but also (24%) in EPI. M cells had a more pronounced spike-and-dome configuration, with a significantly larger phase 1 magnitude and plateau voltage. The frequency response of these parameters was different from the other cell types. [Ca2+]i transients tended to be larger in M cells. For the in vivo ECG of anesthetized pigs, the QT time was close to the APD90 of M cells, and J waves were seen in 7/12 recordings. In young adult pigs, M cells can be identified by a steep frequency response of the APD and by a spike-and-dome configuration. These cells are mostly, but not exclusively, found in the midmyocardium, and could contribute to the ECG characteristics. Their properties may however be different from those of other species, including humans.

Stretch-activated whole cell currents in adult rat cardiac myocytes.

Mechanoelectric transduction can initiate cardiac arrhythmias. To examine the origins of this effect at the cellular level, we made whole cell voltage-clamp recordings from acutely isolated rat ventricular myocytes under controlled strain. Longitudinal stretch elicited noninactivating inward cationic currents that increased the action potential duration. These stretch-activated currents could be blocked by 100 microM Gd(3+) but not by octanol. The current-voltage relationship was nearly linear, with a reversal potential of approximately -6 mV in normal Tyrode solution. Current density varied with sarcomere length (SL) according to I (pA/pF) = 8.3 - 5.0 SL (microm). Repeated attempts to record single channel currents from stretch-activated ion channels failed, in accord with the absence of such data from the literature. The inability to record single channel currents may be a result of channels being located on internal membranes such as the T tubules or, possibly, inactivation of the channels by the mechanics of patch formation.

Long-term treatment of spontaneously hypertensive rats with losartan and electrophysiological remodeling of cardiac myocytes

Cardiac hypertrophy due to pressure overload is associated with several cellular electrophysiological alterations such as prolongation of action potential duration (APD), decrease in transient outward current (Ito) and occurrence of the pacemaker current I(f). These alterations may play a role in sudden arrhythmic death, which is a major risk factor in myocardial hypertrophy and failure. Since angiotensin II is a key signal for myocyte hypertrophy, we tested if an 8-week treatment of old spontaneously hypertensive rats (SHR) with the antagonist of type-1 angiotensin II receptor (AT1), losartan (10 mg/kg/day), was able to influence the cellular electrophysiologic remodeling associated with cardiac hypertrophy. Left ventricular myocytes were isolated from control (CTR) or losartan-treated (LOS) 18-month old SHR. Patch-clamped LVM were superfused with a normal Tyrode's solution (to measure action potential) or appropriately modified Tyrode's solution (to measure Ito and I(f)). Heart weight to body weight ratio (HW/BW) was significantly smaller in LOS (5.69 +/- 0.25 mg/g) than in CTR rats (6.67 +/- 0.37 mg/g; P < 0.05). Membrane capacitance, an index of cell size, was significantly reduced in LOS (342 +/- 12, n = 92) vs. CTR (422 +/- 14 pF, n = 96, P < 0.001). APD was significantly shorter in LOS than in CTR (at -60 mV: 197 +/- 23 vs. 277 +/- 19 ms, n = 28, P < 0.001); this effect was paralleled by a larger maximum Ito density in the LOS group (LOS: 15.1 +/- 1.4 pA/pF, CTR: 10.0 +/- 0.8 pA/pF) (n = 27, P < 0.02). I(f), elicited by hyperpolarizing steps (range: -60 to -130 mV), was consistently recorded in SHR cells; however, its maximal specific conductance was significantly lower in LOS than in CTR rats (28.6 +/- 3.6 vs. 54.2 +/- 8.0 pS/pF, n = 55, P < 0.001). Voltage of half-maximal activation (V1/2) of both Ito and I(f) was unchanged by the treatment. AT1 receptor blockade with losartan prevents the development of myocyte hypertrophy and associated electrophysiological alterations in old SHR.

Enhancement of acetylcholine-induced desensitization of guinea-pig ileal longitudinal muscle in Ca2+-free conditions.

1. To determine the role of cellular Ca2+ in desensitization, acetylcholine(ACh)-induced desensitization was studied under Ca2+-free condition in guinea-pig ileal longitudinal muscle. 2. Pretreatment of the tissue with 10(-4) M ACh (desensitizing treatment) in normal Tyrode solution caused desensitization of the responses both to ACh and histamine. The desensitizing treatment performed in Ca2+-free solution enhanced desensitization of the responses to ACh and histamine significantly. 3. The desensitizing treatment with ACh caused suppression of the responses to high K+ (tonic component) and Bay K 8644. The desensitizing treatment performed in Ca2+-free solution potentiated the suppression of the responses to high K+ and Bay K 8644 significantly. 4. ACh-induced desensitization was enhanced significantly in the presence of a protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine(H-7, 10(-4) M) to a similar extent as desensitization obtained under Ca2+-free condition, but not in the presence of a non-specific and less potent kinase inhibitor, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004, 10(-4) M). 5. These results suggested that voltage-gated Ca2+ channels were involved in ACh-induced desensitization and that intracellular Ca2+, which was increased during the stimulation with ACh, inhibited desensitization through the activation of protein kinase C. This kinase could have activated or protected Ca2+ channels during the desensitization process to reduce desensitization.

Influence of Ischemic-Simulation on the Action Potential Characteristics in Rat Atrial Fibers

Background:To investigate the mechanisms of myocardial ischemia induced changes of electrophysiological properties, influences of various ischemic-simulated Tyrode's solutions on the changes of action potential characteristics were examined. Method:Action potential characteristics were measured during superfusion with various ischemic-simulated solutions (modified physiologic salt solution;MPSS by the method of conventional microelectrode technique in rat atrial fibers. Results:Hypoxic-, hyperkalemic-, and mixed-MPSS decreased ' maximum diastolic potential' (MDP and ' action potential amplitude' (APA, however, no significant changes of MDP and APA were observed by acidic- and glucose-free-MPSS.' Maximum velocity of phase 0 depolariza- tion' (dV/dtmax and ' time for 90% repolarization' (APD90 significantly decreased during hypoxic- and mixed- MPSS superfusion, and hyperkalemic-MPSS also decreased the dV/dtmax and APD90. However, no significant changes in dV/dtmax and APD90 were observed by acidic- and glucose-free-MPSS. The decreasing effects of dV/ dt max and APD 90 by the MPSSes were attenuated when the MPSSes were replaced with normal Tyrode's solution. DPCPX (2×10

Evaluation of intrinsic modulation of synaptic transmission by ATP in mouse fast twitch muscle.

This study aims to evaluate whether endogenous ATP or adenosine modulates the neurotransmission and contractile function of mouse phrenic nerve-diaphragm. Bath application of ATP (1 mM) and alpha, beta-methylene ATP (m-ATP, 0.1 mM) elevated muscle tones, depressed contractions (approximately 12%), and depolarized muscle membranes (approximately 20 mV). Adenosine (1 mM) or low concentrations of ATP (0.1 mM) had no effect. In a low Ca2+ media, ATP caused prolonged inhibitions of endplate potentials (EPPs), whereas m-ATP augmented EPPs while both agents produced slight effects in normal Tyrode solution. When applied by puff ejection, ATP and m-ATP additionally elicited fast transient suppressions of EPPs in association with inhibitions of high K+-evoked releases of miniature EPPs. Blockades of P2 purinoceptors with suramin antagonized all the effects of ATP and m-ATP except the prolonged inhibitions of EPPs induced by ATP, which were antagonized instead by 8-cyclopentyl-1,3-dipropylxanthine (CPDPX), an A1 adenosine receptor antagonist. Suramin and CPDPX did not change contractions nor alter EPPs evoked by a low- or high-frequency nerve stimulation. The results indicate that exogenously applied ATP and m-ATP, via activations of distinct pre- and postsynaptic purinoceptors, exert inhibitory and facilitatory pharmacological modulations on the mature neuromuscular junction. However, because of intrinsic high efficiency of the synaptic transmission under physiological conditions, endogenously released ATP and its degradation product-adenosine-do not build up to concentrations high enough to alter motor functions.

Inhibition of the rapid component of the delayed-rectifier K+ current by therapeutic concentrations of the antispasmodic agent terodiline.

Prolongation of the QT interval and malignant ventricular arrhythmia have been observed in patients administered terodiline for urinary incontinence. Since this adverse reaction might be caused by inhibition of delayed-rectifier K+ current (IK), we investigated whether clinically relevant (< or = 10 microM) concentrations of the drug modify IK in guinea-pig ventricular myocytes. Myocytes superfused with normal Tyrode's solution were pulsed from -40 mV to more positive test potentials (V) for 0.2 - 1 s to elicit tail IK on repolarization and measure tail IK-V relationships. IKr was distinguished from IKs by its sensitivity to the selective blocker E4031. Inhibition of IKr by 5 microM E4031 was completely occluded by pretreatment with 3 microM terodiline. In addition, action potential lengthening by E4031 in guinea-pig papillary muscles (29+/-3%) was abolished (3+/-2%) (P<0.001) by terodiline pretreatment. Inhibition of IKr by terodiline appeared to be voltage-independent, and the parameters of the Hill equation describing the inhibition were IC50 = 0.7 microM and nH = 1.6. High concentrations of the drug also affect IKs; in experiments with K+-free Tyrode's, 10 microM terodiline inhibited tail IKs by 27+/-3% (n=5) (P< 0.001). These data suggest that QT lengthening at therapeutic concentrations of the drug (approximately equal to 1.5 microM) is primarily due to inhibition of IKr. Inhibition of other K+ currents such as IKs is likely to be important at higher concentrations.

Chloride-sensitive nature of the histamine-induced Ca2+ entry in cultured human aortic endothelial cells.

1. Whole-cell currents and intracellular Ca2+ concentration ([Ca2+]i) were recorded in cultured human aortic endothelial cells (HAECs) to study the mechanisms underlying Cl--sensitive Ca2+ entry. 2. In the absence of histamine the membrane potential ranged between -90 and +5 mV and showed bimodal distribution with peaks at -17.8 and -67.5 mV. 3. Histamine (1-100 microM) activated an outward current, followed by a sustained inward current at -50 mV. The reversal potential (Vrev) was more negative than -60 mV for the initial outward current, and approximately -30 mV for the sustained inward current with normal Tyrode solution and internal solution containing 30 mM Cl-. 4. Vrev of the sustained inward current was hardly affected by varying the external concentrations of K+, Na+ and Ca2+, but was greatly changed by varying the external Cl- concentration ([Cl-]o). The relationship between Vrev and log[Cl-]o showed a slope of -44.8 mV per tenfold increase of [Cl-]o. 5. The Cl- channel blockers 9-anthracene carboxylic acid (1 mM), N-phenylanthranilic acid (0.1 mM) and niflumic acid (0.1 mM) all depressed the histamine-induced inward current. The non-selective cation channel blocker Gd3+ (10 microM) was without effect on the current. 6. In the absence of histamine, [Ca2+]i was not affected by varying the membrane potential. During the continuous presence of histamine, however, hyperpolarization increased and depolarization decreased [Ca2+]i, indicating that Ca2+ entry through the plasma membrane was activated by histamine. 7. Vrev of the histamine-induced Cl- current, measured by the gramicidin-perforated patch clamp method, was -28.4 +/- 6.6 mV (n = 8), which gave an intracellular Cl- concentration of approximately 34 mM. Under the current clamp condition, the membrane potential varied from cell to cell in the control, but application of histamine induced either depolarization or hyperpolarization, depending on the membrane potential before histamine application, and the membrane potential became stable near the equilibrium potential for Cl-. 8. We conclude that the histamine-induced inward current is carried mainly by Cl-. Although Ca2+ entry was also activated, we consider that its amplitude was too small to be resolved by the patch clamp method. The Cl- current may play a functional role in the sustained [Ca2+]i elevation by providing a constant driving force for Ca2+ entry in the presence of histamine.

Inhibition of the Fast Na + Current by Taurine in Guinea Pig Ventricular Myocytes

1. Effects of taurine on the fast Na + current ( I Na ) in isolated guinea pig ventricular cardiomyocytes were examined by using the whole-cell voltage-clamp method. Experiments were performed at room temperature (22°C). 2. Test pulses were applied from −60 to +40 mV from a holding potential of −90 mV. Addition of taurine to the bath solution markedly inhibited the I Na in a concentration-dependent manner; at −30 mV, by 39.0±4.1% ( n=8, P<0.01) at 10 mM and by 56.1±4.7% ( n=9, P<0.001) at 20 mM. 3. Simultaneously, the time constant of inactivation phase for I Na decreased to 0.95±0.4 ms ( n=8, P<0.05) at 10 mM and to 1.02±0.3 ms ( n=9, P<0.05) at 20 mM, from 1.29±0.3 ms in normal Tyrode solution. 4. These results indicate that taurine inhibits the I Na current, which would play an important role in the cell functions.

Active and passive compliance of the fetal bovine bladder.

Others have shown that the fetal bovine bladder is relatively noncompliant. Previous studies on compliance of fetal bovine bladders have demonstrated that the youngest fetal bladders had lowest and the oldest fetal bladders (near full-term) had greatest compliance. Our study was designed to determine the level of participation of active tension in the compliance of fetal bladders during gestation. Fetal bovine bladders were obtained immediately after maternal harvest and crown-to-rump length was measured to determine gestational age. The fetus was inspected for genitourinary anomalies and the bladder was immediately placed in chilled M199 media. Strips (1 x 0.5 cm.) were excised from the anterior sagittal plane of the bladder and subjected to length-tension analysis in oxygenated Tyrode's buffer at 37C. Tension was measured using a force transducer and length was increased using a micropositioner. Compliance refers to the length-tension studies performed in normal Tyrode's solution and consists of a combination of active (smooth muscle tone) and passive properties. Passive compliance refers to length-tension studies performed after inactivation of bladder smooth muscle tone. Compliance with muscle tone intact was determined by incrementally stretching the strips to twice resting length in physiological buffer and then permitting them to return to resting length. Passive compliance with muscle tone ablated was determined in the same fashion after overnight incubation in calcium-free Tyrode's buffer in the presence of 5 mM. egtazic acid and 10 mM. sodium azide. An exponential function was fit to the normalized length-tension curves, where the exponential coefficient (EC) is numerically inversely proportional to compliance. Passive compliance was greatest in the youngest bladders (EC = 0.5 in the first trimester) and gradually decreased with increasing fetal age (EC = 1.2 in the third trimester). Active compliance demonstrated the opposite pattern, since the younger bladders were more stiff (EC = 2.1 in the first and 1.6 in the third trimesters). These studies demonstrate that passive compliance is greatest in the youngest bladders and progressively decreases with gestation. However, active smooth muscle tone is greatest in the youngest bladders and decreases with gestation. Thus, high active smooth muscle tone in the youngest fetal bladders results in relatively poor compliance of the early stage fetal bladder.

Amiodarone blocks L-type calcium current in single myocytes isolated from the rabbit atrioventricular node

1. 1. Single myocytes were isolated from the rabbit atrioventricular node (AVN) and whole cell patch clamped, using a Cs +-based internal dialysis solution. Depolarizing voltage clamps from a holding potential of −40 mV were applied to investigate the effects of the antiarrhythmic agent amiodarone on L-type calcium current (I Ca,L). 2. 2. The current-voltage (I–V) relation for I Ca,L was bell shaped in normal Tyrode's solution, with a peak at +10 mV. After a 1-min exposure to 10 μM amiodarone, I Ca,L at all potentials between −30 mV and +60 mV was largely and reversibly blocked ( n=4). In 13 cells, peak I Ca,L was blocked by 85% after amiodarone application. 3. 3. The time course of the blocking effect was monitored during continuous pulsing (at 0.33 Hz) from −40 mV to + 10 mV to observe the time course of I Ca,L blockade. This could be described by a single exponential function with a rate constant of 0.21 per second. In other cells, amiodarone was applied for 30 sec without any stimulation; and, when stimulation was resumed, the I Ca,L amplitude with the first pulse was 22% of the control amplitude ( n=4), indicating that a substantial portion of the blockade of I Ca,L by amiodarone was tonic in nature. 4. 4. In addition to the effect of amiodarone on the I Ca,L channel, when the β adrenergic agonist isoprenaline (1 μM) was applied to cells that had been pretreated with amiodarone, I Ca,L amplitude was increased by 44.5±10.8% ( n=4). In cells that had received no such pretreatment with amiodarone, isoprenaline increased the I Ca,L amplitude by 101.5±5.9% ( n=4). Thus, isoprenaline produced a larger increase in I Ca,L in the absence of amiodarone than in its presence ( P<0.001). 5. 5. It is concluded that, in single AVN cells, amiodarone exerts a direct I Ca,L blocking action. In addition, the attenuated increase in I Ca,L with isoprenaline in amiodarone pretreated cells appears consistent with an antagonism of β adrenergic stimulation of I Ca,L.