The characteristics of the sodium currents across EGTA-modified calcium channels in the membrane of cultured rat cardiomyocytes]

In isolated, cultured neonatal rat ventricular myocytes sodium currents through calcium channels induced by lowering of extracellular calcium concentration 100 nmol/l have been investigated by whole-cell patch clamp technique. Such Na(+)-carried currents are modulated by classic Ca2+ agonists and antagonists. The potential-dependent characteristics of Na+ current are shifted at 20 mV in hyperpolarizing direction as compared to initial Ca(2+)-carried current. The inactivation decay of Na+ current through Ca2+ channels has the monoexponential behaviour. The possible action of extracellular Ca2+ lowering on Ca2+ channel selective filter and gating mechanisms is suggested.     

Characterization of nifedipine-resistant calcium current in neonatal rat ventricular cardiomyocytes

Calcium current was recorded from ventricular cardiomyocytes of rats at various stages of postnatal development using the whole cell patch-clamp technique. In cultured 3-day-old neonatal cells, the current carried by Ca(2+) or Ba(2+) (5 mM) was not completely inhibited by 2 microM nifedipine. A residual current was activated in the same voltage range as the L-type, nifedipine-sensitive Ca(2+) current, but its steady-state inactivation was negatively shifted by 16 mV. This nifedipine-resistant calcium current was not further inhibited by other organic calcium current antagonists such as PN200-110, verapamil, and diltiazem nor by nickel, omega-conotoxin, or tetrodotoxin. It was completely blocked by cadmium and increased by isoproterenol and forskolin. This current was >20% of total calcium current in ventricular myocytes freshly isolated from neonatal rats, and it decreased during postnatal maturation, disappearing at the adult stage. This suggests that this current could be caused by an isoform of the L-type calcium channel expressed in a way that reflects the developmental stage of the rat heart.     

L-type Ca2+ currents in ventricular myocytes from neonatal and adult rats

Postnatal changes in the slow Ca2+ current (I(Ca)(L)) were investigated in freshly isolated ventricular myocytes from neonatal (1-7 days old) and adult (2-4 months old) rats, using whole-cell voltage clamp and single-channel recordings. The membrane capacitance (mean+/-SEM) averaged 23.2+/-0.5 pF in neonates (n = 163) and 140+/-4.1 pF in adults (n = 143). I(Ca)(L) was measured as the peak inward current at a test potential of +10 mV (or +20 mV) by applying a 300-ms pulse from a holding potential of -40 mV; 1.8 mM Ca2+ was used as charge carrier. The basal ICa(L) density was 6.7+/-0.2 pA/pF in neonatal and 7.8+/-0.2 pA/pF in adult cells (p < 0.05). The time course of inactivation of the fast component (at +10 ms) was significantly longer in the neonatal (10.7+/-1.4 ms) than in the adult (6.6+/-0.4 ms) cells (p < 0.05). Ryanodine (10+/-M) significantly increased this value to 18.0+/-1.9 in neonate (n = 8) and to 17.7+/-2.0 in adult (n = 9). For steady-state inactivation, the half-inactivation potential (Vh) was not changed in either group. For steady-state activation, Vh was 5.1 mV in the neonatal (n = 6) and -7.9 mV in the adult cells (n = 7). Single-channel recordings revealed that long openings (mode-2 behavior) were occasionally observed in the neonatal cells (11 events from 1080 traces/11 cells), but not in the adult cells (400 traces/4 cells). Slope conductance was 24 pS in both the neonatal and adult cells. Results in rat ventricular myocytes suggest the following: (i) the peak Ca2+ current density is already well developed in the neonatal period (being about 85% of the adult value); (ii) the fast component of inactivation is slower in neonates than in adults; and (iii) naturally occurring long openings are occasionally observed in the neonatal stage but not in the adult. Thus, the L-type Ca2+ channels of the neonate were slightly lower in density, were inactivated more slowly, and occasionally exhibited mode-2 behavior as compared with those of the adult.     

Angiotensin II stimulates PGF(2 alpha)release in cultured neonatal rat ventricular myocytes via L-type calcium channels.

Angiotensin II (Ang II) has been shown to cause Prostaglandin F(2 alpha)(PGF(2 alpha)) release in neonatal rat ventricular myocytes and smooth muscle cells. In these cells, Ang II has also been shown to regulate growth. We used neonatal rat ventricular myocytes to investigate the role of calcium in maintenance of Ang II-induced PGF(2 alpha)release. The amount of PGF(2 alpha)produced was determined by radioimmunoassay. Ang II-induced PGF(2 alpha)release. Pretreatment of neonatal rat ventricular myocytes with different doses (10(-8)M, 10(-7)M, 10(-6)M and 10(-5)M) of diltiazm (voltage-sensitive L-type calcium channel blocker) produced significant inhibition in Ang II-induced PGF(2 alpha)release. Inhibition was first noted at 10(-8)M and was complete at 10(-6)M. Conversely, pretreatment of neonatal rat ventricular myocytes with different doses (10(-8)M, 10(-7)M, 10(-6)M and 10(-5)M) of calcium channel blockers (conotoxin; voltage-sensitive N-type calcium channel blocker or thapsigargin; intracellular calcium channel blocker) produced no changes in Ang II-induced PGF(2 alpha)release. These results strongly suggest that Ang II-induced PGF(2 alpha)release in neonatal rat ventricular myocytes is maintained, at least in part, via increase in extracellular calcium influx.     

慢性低氧对豚鼠右室心肌细胞钙、钾电流的影响

采用全细胞膜片箝技术,分别记录并比较正常对照组与慢性低氧组豚鼠单个右室心肌细胞的膜电容、L型钙电流和延迟整流钾电流峰值和电流-电压关系曲线,以探讨慢性低氧对豚鼠右室心肌细胞L型钙电流和延迟整流钾电流的影响。结果表明,上述两组细胞膜电容分别为（155±13．2）pF、（179±14,8）pF,低氧组显著大于正常对照组（P＜0．01）;L型钙电流峰值分别为（1．07±0．21）nA和（0．99±0．17）nA,两组之间无显著差异;在-20mV至＋20mV,慢性低氧组L型钙电流密度较正常对照组显著下降（P＜0．05）。在＋月mV至＋60mV之间,慢性低氧组豚鼠右室心肌细胞延迟整流钾电流幅度均小于正常对照组;在-20mV至＋60mV之间,慢性低氧组豚鼠右室心肌细胞延迟整流钾电流密度明显低于正常对照组。可见慢性低氧能使豚鼠右室心肌细胞膜电容增加,L型钙电流幅度不变,但L型钙电流密度下降;同时慢性低氧降低豚鼠右室心肌细胞延迟整流钾电流幅度和密度。     

Gating of mammalian cardiac gap junction channels by transjunctional voltage.

Numerous two-cell voltage-clamp studies have concluded that the electrical conductance of mammalian cardiac gap junctions is not modulated by the transjunctional voltage (Vj) profile, although gap junction channels between low conductance pairs of neonatal rat ventricular myocytes are reported to exhibit Vj-dependent behavior. In this study, the dependence of macroscopic gap junctional conductance (gj) on transjunctional voltage was quantitatively examined in paired 3-d neonatal hamster ventricular myocytes using the double whole-cell patch-clamp technique. Immunolocalization with a site-specific antiserum directed against amino acids 252-271 of rat connexin43, a 43-kD gap junction protein as predicted from its cDNA sequence, specifically stained zones of contact between cultured myocytes. Instantaneous current-voltage (Ij-Vj) relationships of neonatal hamster myocyte pairs were linear over the entire voltage range examined (0 less than or equal to Vj less than or equal to +/- 100 mV). However, the steady-state Ij-Vj relationship was nonlinear for Vj greater than +/- 50 mV. Both inactivation and recovery processes followed single exponential time courses (tau inactivation = 100-1,000 ms, tau recovery approximately equal to 300 ms). However, Ij recovered rapidly upon polarity reversal. The normalized steady-state junctional conductance-voltage relationship (Gss-Vj) was a bell-shaped curve that could be adequately described by a two-state Boltzmann equation with a minimum Gj of 0.32-0.34, a half-inactivation voltage of -69 and +61 mV and an effective valence of 2.4-2.8. Recordings of gap junction channel currents (ij) yielded linear ij-Vj relationships with slope conductances of approximately 20-30 and 45-50 pS. A kinetic model, based on the Boltzmann relationship and the polarity reversal data, suggests that the opening (alpha) and closing (beta) rate constants have nearly identical voltage sensitivities with a Vo of +/- 62 mV. The data presented in this study are not consistent with the contingent gating scheme (for two identical gates in series) proposed for other more Vj-dependent gap junctions and alternatively suggest that each gate responds to the applied Vj independently of the state (open or closed) of the other gate.     

Calcium signaling mechanisms in dedifferentiated cardiac myocytes: comparison with neonatal andadult cardiomyocytes

Our studies focused on calcium sparking and calcium transients in cultured adult rat cardiomyocytes and compared these findings to those in cultured neonatal and freshly isolated adult cardiomyocytes. Using deconvolution fluorescence microscopy and spec trophotometric image capture, sequence acquisitions were examined for calcium spark intensities, calcium concentrations and whether sparks gave rise to cell contraction events. Observations showed that the preparation of dedifferentiated cardiomyocytes resulted in stellate, neonatal-like cells that exhibited some aspects of calcium transient origination and proliferation similar to events seen in both neonatal and adult myocytes. Ryanodine treatment in freshly isolated adult myocytes blocked the calcium waves, indicating that calcium release at the level of the sarcoplasmic reticulum and t-tubule complex was the initiating factor, and this effect of ryanodine treatment was also seen in cultured-dedifferentiated adult myocytes. However, experiments revealed that in both neonatal and cultured adult myocytes, the inositol triphosphate pathway (IP3) was a major mechanism in the control of intracellular calcium concentrations. In neonatal myocytes, the nucleus and regions adjacent to the plasma membrane we re major sites of calcium release and flux. We conclude: (1) culturing of adult cardiomyocytes leads them to develop mechanisms of calcium homeostasis similar in some aspects to those seen in neonatal cardiomyocytes; (2) neonatal myocytes rely on both extracellular and nuclear calcium for contractile function; and (3) freshly isolated adult myocytes use sarcoplasmic reticulum calcium stores for the initiation of contractile function.     

Modulation of membrane potential by an acetylcholine-activated potassium current in trout atrial myocytes

Application of the current-clamp technique in rainbow trout atrial myocytes has yielded resting membrane potentials that are incompatible with normal atrial function. To investigate this paradox, we recorded the whole membrane current (I(m)) and compared membrane potentials recorded in isolated cardiac myocytes and multicellular preparations. Atrial tissue and ventricular myocytes had stable resting potentials of -87 +/- 2 mV and -83.9 +/- 0.4 mV, respectively. In contrast, 50 out of 59 atrial myocytes had unstable depolarized membrane potentials that were sensitive to the holding current. We hypothesized that this is at least partly due to a small slope conductance of I(m) around the resting membrane potential in atrial myocytes. In accordance with this hypothesis, the slope conductance of I(m) was about sevenfold smaller in atrial than in ventricular myocytes. Interestingly, ACh increased I(m) at -120 mV from 4.3 pA/pF to 27 pA/pF with an EC(50) of 45 nM in atrial myocytes. Moreover, 3 nM ACh increased the slope conductance of I(m) fourfold, shifted its reversal potential from -78 +/- 3 to -84 +/- 3 mV, and stabilized the resting membrane potential at -92 +/- 4 mV. ACh also shortened the action potential in both atrial myocytes and tissue, and this effect was antagonized by atropine. When applied alone, atropine prolonged the action potential in atrial tissue but had no effect on membrane potential, action potential, or I(m) in isolated atrial myocytes. This suggests that ACh-mediated activation of an inwardly rectifying K(+) current can modulate the membrane potential in the trout atrial myocytes and stabilize the resting membrane potential.     

Voltage-dependence of contraction in streptozotocin-induced diabetic myocytes

Cardiac contractile dysfunction is frequently reported in human patients and experimental animals with type-1 diabetes mellitus. The aim of this study was to investigate the voltage-dependence of contraction in ventricular myocytes from the streptozotocin (STZ)-induced diabetic rat. STZ-induced diabetes was characterised by hyperglycaemia and hypoinsulinaemia. Other characteristics included reduced body and heart weight and raised blood osmolarity. Isolated ventricular myocytes were patched in whole cell, voltage-clamp mode after correcting for membrane capacitance and series resistance. From a holding membrane potential of -40 mV, test pulses were applied at potentials between -30 and +50 mV in 10 mV increments. L-type Ca2+ current (I Ca,L) density and contraction were measured simultaneously using a video-edge detection system. Membrane capacitance was not significantly altered between control and STZ-induced diabetic myocytes. The I Ca,L density was significantly (p < 0.05) reduced throughout voltage ranges (-10 mV to +10 mV) in myocytes from STZ-treated rats compared to age-matched controls. Moreover, the amplitude of contraction was significantly reduced (p < 0.05) in myocytes from STZ-treated rats at all test potentials between -20 mV and +30 mV. However, in electrically field-stimulated (1 Hz) myocytes, the amplitude of contraction was not altered by STZ-treatment. It is suggested that in field-stimulated myocytes taken from STZ-induced diabetic hearts, prolonged action potential duration may promote increased Ca2+ influx via the sodium-calcium exchanger (NCX), which may compensate for a reduction in Ca2+ trigger through L-type-Ca2+-channels and lead to normalised contraction.     

Morphine and (D-Ala2, NMe-Phe4, Gly-ol)-enkephalin increase the intracellular free calcium in isolated rat myocytes--effect of naloxone or pretreatment with morphine.

The purpose of the present study was firstly to determine whether morphine and (D-Ala2, NMe-Phe4, Gly-ol)-enkephalin (DAGO), a highly selective mu-agonist, increased intracellular free calcium of rat myocytes and secondly to determine whether opioid receptors were involved. Two series of experiments were performed. In the first, the effect of morphine and DAGO on intracellular free calcium (Cai) of cultured isolated myocytes was studied with a spectrophotometric method using fura2-AM as the fluorescent Ca2+ indicator. In the second, the effect of morphine on Cai of isolated ventricular myocytes from rats which had received chronic daily injection of morphine for two weeks or myocytes which had been incubated in a solution with morphine for 12 hr was studied. It was found that both morphine at 100-250 microM and DAGO at 23-75 microM increased Cai dose-dependently and that the effect was significantly antagonized by naloxone at a concentration of 50 microM, which itself did not cause any significant alteration in Cai. Pretreatment with morphine also abolished the morphine-induced increase in Cai of isolated myocytes. The results suggest that morphine increases Cai by directly activating the cardiac receptors (most likely micro-receptors) on the membrane of ventricular myocytes.     

Developmental differences in delayed rectifying outward current in feline ventricular myocytes

In the present work, we found that the delayed rectifying outward potassium current (I(K)) in adult and neonatal cat ventricular myocytes consists of both rapid and slow components, I(Kr) and I(Ks), respectively, which can be isolated pharmacologically. Thus after complete blockade of I(Kr) with dofetilide, the remaining I(Ks) current is homogeneous, as shown by an envelope of tails test. I(Kr) maximum tail current density, measured at -40 mV, was similar in adult and neonatal myocytes. I(Ks) maximum tail current density in neonatal myocytes, measured at -40 mV, was significantly smaller than in adult myocytes. Activation kinetics of I(Kr) and I(Ks) was similar in both age groups. However, the I(Kr) deactivation time course was significantly faster in neonatal than in adult myocytes. Developmental differences in the subunit composition of I(Kr) that display distinctly different deactivation kinetics are suggested.     

Human cord blood CD34+ progenitor cells acquire functional cardiac properties through a cell fusion process

The efficacy of cardiac repair by stem cell administration relies on a successful functional integration of injected cells into the host myocardium. Safety concerns have been raised about the possibility that stem cells may induce foci of arrhythmia in the ischemic myocardium. In a previous work (36), we showed that human cord blood CD34(+) cells, when cocultured on neonatal mouse cardiomyocytes, exhibit excitation-contraction coupling features similar to those of cardiomyocytes, even though no human genes were upregulated. The aims of the present work are to investigate whether human CD34(+) cells, isolated after 1 wk of coculture with neonatal ventricular myocytes, possess molecular and functional properties of cardiomyocytes and to discriminate, using a reporter gene system, whether cardiac differentiation derives from a (trans)differentiation or a cell fusion process. Umbilical cord blood CD34(+) cells were isolated by a magnetic cell sorting method, transduced with a lentiviral vector carrying the enhanced green fluorescent protein (EGFP) gene, and seeded onto primary cultures of spontaneously beating rat neonatal cardiomyocytes. Cocultured EGFP(+)/CD34(+)-derived cells were analyzed for their electrophysiological features at different time points. After 1 wk in coculture, EGFP(+) cells, in contact with cardiomyocytes, were spontaneously contracting and had a maximum diastolic potential (MDP) of -53.1 mV, while those that remained isolated from the surrounding myocytes did not contract and had a depolarized resting potential of -11.4 mV. Cells were then resuspended and cultured at low density to identify EGFP(+) progenitor cell derivatives. Under these conditions, we observed single EGFP(+) beating cells that had acquired an hyperpolarization-activated current typical of neonatal cardiomyocytes (EGFP(+) cells, -2.24 ± 0.89 pA/pF; myocytes, -1.99 ± 0.63 pA/pF, at -125 mV). To discriminate between cell autonomous differentiation and fusion, EGFP(+)/CD34(+) cells were cocultured with cardiac myocytes infected with a red fluorescence protein-lentiviral vector; under these conditions we found that 100% of EGFP(+) cells were also red fluorescent protein positive, suggesting cell fusion as the mechanism by which cardiac functional features are acquired.     

Effects of allapinin on sodium currents in isolated neurons of the trigeminal ganglia and cardiomyocytes of rats

Block of sodium currents by allapinin (diterpene alkaloid with strong antiarrhythmic properties) was investigated in isolated, voltage clamped rat trigeminal neurons and cultured neonatal rat single ventricular myocytes. Allapinin produces a decrease in sodium current amplitude without any changes in voltage dependent properties. Possible differences between the mechanisms of antiarrhythmic effect of diterpene alkaloids and classic antiarrhythmic agents have been analysed.     

Comparison of Na(+)/Ca(2+) exchanger current and of its response to isoproterenol between acutely isolated and short-term cultured adult ventricular myocytes

The Na(+)/Ca(2+) exchanger protein is present in the cell membrane of many tissue types and plays key roles in Ca(2+) homeostasis, excitation-contraction coupling, and generation of electrical activity in the heart. The use of adult ventricular myocyte cell culture is important to molecular biological approaches to study the roles and modulation of the cardiac Na(+)/Ca(2+) exchanger. Therefore, we characterised the functional expression of the exchanger in adult guinea-pig ventricular myocytes maintained in short-term culture (for 4 days) and compared the response of ionic current (I(NaCa)) carried by the exchanger from acutely isolated and Day 4 cells to beta-adrenoceptor activation with isoproterenol (ISO). Functional activity of the exchanger was assessed by measuring I(NaCa) using whole cell patch clamp, under selective recording conditions. I(NaCa) amplitude measured at both +60 and -100mV declined significantly by Day 1 of cell culture, showing a further small decline by Day 4. However, cell surface area (assessed by measuring membrane capacitance) also declined over this time-frame. I(NaCa) normalised to membrane capacitance (I(NaCa) density) did not differ significantly between acutely isolated and cells cultured for 4 days. However, although ISO (1 microM) increased I(NaCa) in acutely isolated myocytes, it exerted no significant effect on I(NaCa) from Day 4 cells. This was not due to an inherent inability of these cells to respond to ISO, as L-type calcium current amplitude from Day 4 cells was increased by ISO to a similar extent as that from acutely isolated cells. Our data suggest that the functional expression of the Na/Ca exchanger is well maintained during short-term culture of adult ventricular myocytes. The lack of response to ISO of I(NaCa) from Day 4 cells suggests: (a) that, despite a well-maintained I(NaCa) density, cultured adult myocytes may not necessarily be suitable for studies of exchanger modulation by some agonists and (b) that there may exist subtle differences between beta-adrenergic regulation of the exchanger protein and of L-type Ca channels.     

Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat

TRPV4 protein forms a Ca²⁺-permeable channel that is sensitive to osmotic and mechanical stimuli and responds to warm temperatures, and expresses widely in various kinds of tissues. As for cardiac myocytes, TRPV4 has been detected only at the mRNA level and there were few reports about subcel-lular localization of the protein. The purpose of the present study was to investigate the expression profile of TRPV4 protein in cultured neonatal rat ventricular myocytes. Using Western blots, immunofluorescence, confocal microscopy and immuno-electron microscopy, we have shown that TRPV4 protein was predominantly located in the nucleus of cultured neonatal myocytes. Furthermore, cardiac myocytes responded to hypotonic stimulation by translocating TRPV4 protein out of the nucleus. The significance and mechanism concerning the unusual distribution and translocation of TRPV4 protein in cardiac myocytes remain to be clarified.Key words: TRPV4, nucleus, hypotonicity, translocation, ventricular myocytes, neonatal rat.     

Ventricular myocytes from neonatal rats are more responsive to dexamethasone than atrial myocytes in synthesis of atrial natriuretic peptide

Using the primary culture of neonatal rat ventricular myocytes, synthesis and secretion of rat atrial natriuretic peptide (rANP) were studied. Ventricular myocytes in culture, although contained less amounts of cellular immunoreactive (IR)-rANP, secreted substantial amounts of IR-rANP at a rate comparable to that of atrial myocytes. Dexamethasone markedly stimulated synthesis and secretion of IR-rANP by cultured ventricular myocytes in a dose-dependent manner (10(-10)-10(-6) M), of which effect was far more potent than that in atrial myocytes. Testosterone and triiodothyronine also stimulated synthesis and secretion of ventricular IR-rANP to the extent comparable to that of atrial IR-rANP. The present study suggests that tissue-dependent difference in glucocorticoids sensitivity plays an important role in the regulation of developmental ANP gene expression in mammalian heart.     

Ultrastructural morphometric analysis of cultured neonatal and adult rat ventricular cardiac muscle cells

Neonatal and adult rat ventricular cardiac muscle cells cultured on laminin differed from similar myocytes grown on plastic in the amount and distribution of their mitochondria and transverse tubules. Point-count morphometry was used at the electron microscopic level to quantify these differences. Adult myocytes grown on laminin contained more mitochondria per unit volume than adult myocytes grown on plastic. No significant differences were observed in the volume percent of myofibrils in either adult or neonatal ventricular myocytes when grown on laminin and compared to those grown on plastic. The transverse tubule system in neonatal and adult myocytes was reduced significantly when both groups were cultured on laminin. Furthermore, neonatal and adult myocytes cultured on laminin were flatter than those cultured on plastic. This may indicate a relationship between the surface/volume ratio and transverse tubule development in cultured myocytes. These studies establish that point-count morphometry can be used to quantify changes in the organelle volume densities of cultured cardiac muscle cells.     

Transient outward current carried by inwardly rectifying K+ channels in guinea pig ventricular myocytes dialyzed with low-K+ solution

There have been periodic reports of nonclassic (4-aminopyridine insensitive) transient outward K+ current in guinea pig ventricular myocytes, with the most recent one describing a novel voltage-gated inwardly rectifying type. In the present study, we have investigated a transient outward current that overlaps inward Ca2+ current (I(Ca,L)) in myocytes dialyzed with 10 mM K+ solution and superfused with Tyrode's solution. Although depolarizations from holding potential (Vhp) -40 to 0 mV elicited relatively small inward I(Ca,L) in these myocytes, removal of external K+ or addition of 0.2 mM Ba2+ more than doubled the amplitude of the current. The basis of the enhancement of I(Ca,L) was the suppression of a large transient outward K+ current. Similar enhancement was observed when Vhp was moved to -80 mV and test depolarizations were preceded by short prepulses to -40 mV. Investigation of the time and voltage properties of the outward K+ transient indicated that it was inwardly rectifying and unlikely to be carried by voltage-gated channels. The outward transient was attenuated in myocytes dialyzed with high-Mg2+ solution, accelerated in myocytes dialyzed with 100 microM spermine solution, and abolished with time in myocytes dialyzed with ATP-free solution. These and other findings suggest that the outward transient is a component of classic "time-independent" inwardly rectifying K+ current.     

The effects of CGRP on calcium transients of dedifferentiating cultured adult rat cardiomyocytes compared to non-cultured adult cardiomyocytes: possible protective and deleterious results in cardiac function

CGRP has potent cardiovascular effects but its role in heart failure is unclear. Effects of CGRP on calcium concentrations in fresh adult rat cardiomyocytes, cultured adult cardiomyocytes and neonatal cardiomyocytes were determined by real time fluorescence spectrophotometry. Treatment of cultured adult cardiomyocytes with CGRP resulted in a rapid cessation of beating and a reduction in intracellular calcium. Similar results were obtained in cultured neonatal myocytes. However, rod-shaped adult cardiomyocytes revealed a number of responses; (a) non-beating cells began to beat with increased intracellular calcium; (b) spontaneously beating cells exhibited increased intracellular calcium content and a faster beating rate or (c), myocytes increased their beating rate and became arrhythmic, suggesting that CGRP action on cultured dedifferentiated adult and neonatal myocytes depletes intracellular calcium, whereas in the rod-shaped mature myocytes calcium is retained, pointing to a different mode of action for CGRP on developing and dedifferentiating cardiomyocytes, compared to fully developed cardiomyocytes.     

Two types of single inward rectifying potassium channels in rat myocardial cells

The patch-clamp method was used to examine inward rectifying potassium channels in the membrane of rat ventricular myocytes. Two types of inward rectifying channels strongly selective for K+ ions and with different conductance and kinetics coexist in rat myocardial cells. When the concentration of K+ was 140 mmol/l on the extracellular side of the patch, the conductance was 38.9 pS for type I channels and 25.7 pS for the type II. The type II channels had a detectable conductance (4 pS) at potentials positive than the potassium equilibrium potential. The mean open time was 18 ms at -60 mV patch membrane potential and 12 ms at -100 mV for type I channels, and 1.3 s at -60 mV and 0.94 s at -105 mV for type II channels, respectively. The opening probability of type II channels decreased with hyperpolarization. The type II channels can adopt several (about 10 or more) conductance states, which can occur either within one opening or as individual events.