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L Hove-Madsen

Publications and source records attributed to L Hove-Madsen.

At least 19 recordsLinked to original sources
PubMed2005

Identification of cardiomyogenic lineage markers in untreated human bone marrow-derived mesenchymal stem cells.

BACKGROUND: Recent reports refute the classic paradigm by which human heart is unable to repair itself following disease or injury. Cardiac and noncardiac stem cells with cardiac regeneration potential have been documented. We studied whether untreated mesenchymal stem cells express markers of cardiomyogenic lineage in vitro. METHODS: Mesenchymal stem cells were obtained from human iliac crest marrow aspirates. Cells were isolated and characterized using flow cytometry by surface expression of CD105, CD166, CD29, CD44, CD14, and CD34. To evaluate their cardiomyogenic potential, presence of cardiac proteins (cardiac troponin I, sarcomeric alpha-actinin, beta myosin heavy chain (beta-MyHC), connexin-43, and SERCA-2), and transcription factors (GATA-4) were assessed. RESULTS: Mesenchymal stem cells expressed CD105 (4.25 +/- 0.35), CD166 (27.83 +/- 1.89), and CD29 (9.4 +/- 0.57) and were negative for CD34, CD14, and CD45. In absence of additional stimuli in the culture media, these cells expressed connexin-43, alpha-actinin, and GATA-4, and were negative for SERCA-2, cardiac troponin I, and beta-MyHC. CONCLUSIONS: Human adult mesenchymal stem cells spontaneously exhibit markers of cardiac phenotype in vitro. In the appropiate myocardial environment, these cells may transdifferentiate into mature cardiomyocytes.

Adult↗
PubMed2004

Otilonium bromide inhibits muscle contractions via L-type calcium channels in the rat colon.

The aim of this study is to evaluate in vitro the effect of otilonium bromide (OB) on the mechanical and electrical activities of the rat colonic smooth muscle using muscle bath, microelectrodes and patch-clamp techniques. Otilonium bromide dose dependently inhibited the spontaneous activity (logIC(50) +/- SE: -5.31 +/- 0.05). This effect was not modified by TTX (10(-6) mol L(-1)). Cyclic depolarizations were abolished by OB (10(-4) mol L(-1)). Electrical field stimulation induced inhibitory junction potentials (IJPs) followed by a depolarization with superimposed spikes causing a contraction. In the presence of OB (10(-4) mol L(-1)) IJPs were recorded, but spikes and contractions were abolished. Otilonium bromide (3 x 10(-6) mol L(-1)) inhibited inward current obtained in isolated cells (amphotericin perforated patch technique). The otilonium-sensitive current amplitude was maximal (75pA) around 0 mV. The effect of different doses of OB was tested by depolarizing cells from -70 mV to 0 mV. OB dose dependently inhibited the inward current with an EC(50) of 885 nmol L(-1). Abolishment of the otilonium-sensitive current by 3 x 10(-6) mol L(-1) nifedipine confirmed that it was an L-type Ca(2+) current. Our results show that OB inhibits the spontaneous and triggered muscular contractions. This effect is produced by the inhibition of muscular action potentials carried by L-type calcium current, confirming the spasmolytic properties of OB.

Action Potentials↗
PubMed2001

Characterization of the relationship between Na+ -Ca2+ exchange rate and cytosolic calcium in trout cardiac myocytes.

The whole-cell patch-clamp technique combined with rapid caffeine (CAF) applications was used to measure Na+-Ca2+ exchange (NCX) currents (I(NCX)). The rate of Ca2+ extrusion and the amount of Ca2+ extruded from the cell upon a rapid CAF exposure were obtained from I(NCX) and its time integral, respectively. This gave a maximal NCX rate (V(NCX)) of 151 amol pF(-1) s(-1) or 2.3 mM s(-1) and a half-maximal V(NCX) (K0.5) at a total cellular [Ca2+] ([Ca2+]tot) of 15.4 amol pF(-1). Using the same approach for the tail current induced by repolarization to -80 mV gave a K0.5 of 7.0 amol pF(-1) corresponding to 108 microM total or 2-4 microM free Ca2+. The relationship between [Ca2+]tot and V(NCX) was linear in the physiological range. Inhibition of the SR function with cyclopiazonic acid plus ryanodine reduced the slope significantly from 23.2+/-1.4 to 17.6+/-1.6 s(-1), while ryanodine alone had no effect. The relationship between [Ca2+]tot and V(NCX) was steeper at more negative membrane potentials, and with identical SR Ca2+ loads the maximal VNCX at -10 mV was reduced to 39.7+/-2.7% of the value at -90 mV. Long depolarizations caused SR Ca2+ loading through reverse-mode NCX. Between -30 and +10 mV reverse mode V(NCX)=Vm.0.047 amol pF(-1) s(-1) mV(-1)+2.51 amol pF(-1) s(-1), giving a reversal potential of -54 mV. In conclusion, the relationship between V(NCX) and [Ca2+]tot shows that the NCX is capable of removing a total Ca2+ transient of 60 microM at physiological heart rates, while reverse-mode NCX reloads the sarcoplasmic reticulum (SR) during depolarization. Furthermore, small alterations in the action potential configuration are predicted to change significantly the relative importance of the NCX in the regulation of cytosolic [Ca2+] and SR Ca2+ loading.

Animals↗
PubMed2001

The function of the sarcoplasmic reticulum is not inhibited by low temperatures in trout atrial myocytes.

The effect of temperature on sarcoplasmic reticulum (SR) Ca(2+) uptake and release was measured in trout atrial myocytes using the perforated patch-clamp technique. Depolarization of the myocyte for 10 s to different membrane potentials (V(m)) induced SR Ca(2+) uptake. The relationship between V(m) and SR Ca(2+) uptake was not significantly changed by lowering the experimental temperature from 21 to 7 degrees C, and the relationship between total cytosolic Ca(2+) and SR Ca(2+) uptake was similar at the two temperatures with a pooled V(max) = 66 amol/pF and K(0.5) = 4 amol/pF. Quantification of the Ca(2+) release from the SR elicited by 10-ms depolarizations to different V(m) showed an increasing SR Ca(2+) release at more positive V(m) between -50 and +10 mV, whereas SR Ca(2+) release stagnated between +10 and +50 mV. Lowering of the temperature did not affect this relationship significantly, giving an SR Ca(2+) release of 1.71 and 1.54 amol/pF at 21 and 7 degrees C, respectively. Furthermore, clearance of the SR Ca(2+) content slowed down inactivation of the L-type Ca(2+) current at both temperatures (the fast time constant increased significantly from 10.4 +/- 1.9 to 15.0 +/- 2.0 ms at 21 degrees C and from 38 +/- 15 to 73 +/- 24 ms at 7 degrees C). Thus the SR has the capacity to remove the entire Ca(2+) transient at physiologically relevant stimulation frequencies at both 21 and 7 degrees C, although it is estimated that ~40% of the total Ca(2+) transient is liberated from and reuptaken by the SR with continuous stimulation at 0.5 Hz independently of the experimental temperature.

Animals↗
PubMed2000

Na(+)/Ca(2+)-exchange activity regulates contraction and SR Ca(2+) content in rainbow trout atrial myocytes.

We have used the whole cell configuration of the patch-clamp technique to measure sarcolemmal Ca(2+) transport by the Na(+)/Ca(2+) exchanger (NCX) and its contribution to the activation and relaxation of contraction in trout atrial myocytes. In contrast to mammals, cell shortening continued, increasing at membrane potentials above 0 mV in trout atrial myocytes. Furthermore, 5 microM nifedipine abolished L-type Ca(2+) current (I(Ca)) but only reduced cell shortening and the Ca(2+) carried by the tail current to 66 +/- 5 and 67 +/- 6% of the control value. Lowering of the pipette Na(+) concentration from 16 to 10 or 0 mM reduced Ca(2+) extrusion from the cell from 2.5 +/- 0.2 to 1.0 +/- 0.2 and 0.5 +/- 0.06 amol/pF. With 20 microM exchanger inhibitory peptide (XIP) in the patch pipette Ca(2+) extrusion 20 min after patch break was 39 +/- 8% of its initial value. With 16, 10, and 0 mM Na(+) in the pipette, the sarcoplasmic reticulum (SR) Ca(2+) content was 47 +/- 4, 29 +/- 6, and 10 +/- 3 amol/pF, respectively. Removal of Na(+) from or inclusion of 20 microM XIP in the pipette gradually eliminated the SR Ca(2+) content. Whereas I(Ca) was the same at -10 or +10 mV, Ca(2+) extrusion from the cell and the SR Ca(2+) content at -10 mV were 65 +/- 7 and 80 +/- 4% of that at +10 mV. The relative amount of Ca(2+) extruded by the NCX (about 55%) and taken up by the SR (about 45%) was, however, similar with depolarizations to -10 and +10 mV. We conclude that modulation of the NCX activity critically determines Ca(2+) entry and cell shortening in trout atrial myocytes. This is due to both an alteration of the transsarcolemmal Ca(2+) transport and a modulation of the SR Ca(2+) content.

Animals↗
PubMed1999

Quantification of calcium release from the sarcoplasmic reticulum in rainbow trout atrial myocytes.

Using the whole-cell configuration of the patch-clamp technique, we quantified calcium release from the sarcoplasmic reticulum (SR) elicited by short depolarization pulses before and after clearance of the SR Ca2+ content with 10 mM caffeine (CAF). With a loaded SR, the first detectable contraction occurred with a pulse duration of 5 ms. CAF exposure increased this pulse duration to 85 ms and slowed the inactivation of the Ca2+ current (ICa). Repolarization of the cell to -80 mV after a short depolarization elicited a tail current that was attenuated markedly after CAF exposure. The difference between the charge carried by the tail currents obtained before and after CAF exposure was taken as a measure of the Ca2+ released from the SR. SR Ca2+ release increased with increasing SR Ca2+ load over the range of loads examined. In contrast, SR Ca2+ release reached a maximum when the duration of the preceding depolarization exceeded 10 ms. Maximal Ca2+ release was 1.64 amol/pF or 62 microM and elicited a contraction that was 40 +/- 6% of the amplitude of a normal contraction. This release could account for 48 +/- 10% of the total Ca2+ required to activate contraction but only a few percent of the CAF-releasable Ca2+. Thus, contrary to the general view of excitation-contraction coupling in lower vertebrates, SR Ca2+ release in trout atrial myocytes may account for up to 50% of the total Ca2+ transient at room temperature.

Animals↗
PubMed1999

Ca(2+) uptake in the sarcoplasmic reticulum from the systemic heart of octopod cephalopods.

We have measured Ca(2+) uptake in crude homogenates of heart tissue, as well as cell shortening and ionic currents in isolated myocytes exposed to caffeine, to characterize Ca(2+) uptake in the sarcoplasmic reticulum (SR) of the systemic heart of octopus. The maximal rate of SR Ca(2+) uptake in crude homogenates of octopus heart was 43+/-4 (mean +/- s.e.m., N=7), compared with 28+/-2 nmol min(-)(1 )mg(-)(1) protein (N=4) in homogenates of rat heart. The Ca(2+)-dependency of SR Ca(2+) uptake was similar for the two species, with a Ca(2+) activity at half-maximal uptake rate (pCa(50)) of 6.04+/-0.02 for octopus and 6.02+/-0.05 for rat. Exposure of isolated myocytes to 10 mmol l(-)(1) caffeine resulted in cell shortening to 53+/-2 % of the resting cell length and an inward trans-sarcolemmal ionic current. The charge carried by this current was 3.28+/-0.70 pC pF(-)(1) (mean +/- s.e.m., N=5) corresponding to extrusion of 34.0+/-0.7 amol Ca(2+ )pF(-)(1) from the cell by Na(+)/Ca(2+) exchange. This is approximately 50 times more than the Ca(2+) carried by the Ca(2+) current elicited by a 200 ms depolarization from -80 to 0 mV and corresponds to an increase in the total intracellular [Ca(2+)] of 404+/-86 (&mgr;)mol l(-)(1) non-mitochondrial volume due to Ca(2+) release from the SR. Thus, we find that at 20 degrees C in the SR both Ca(2+) content and Ca(2+) uptake rate in the systemic heart of octopus are comparable with or larger than the corresponding values obtained in the rat heart. These results support the argument that the SR may play an important role in the regulation of contraction in the systemic heart of cephalopods.

Journal Article↗
PubMed1998

L-type Ca2+ current and excitation-contraction coupling in single atrial myocytes from rainbow trout.

We have examined the contribution of L-type Ca2+ current (ICa) to the activation of contraction in trout atrial myocytes under basal and phosphorylating conditions. The average myocyte length was 197 +/- 14 micrometer, width was 5.5 +/- 0.2 micrometer, and cell capacitance was 36.2 +/- 2.2 pF. With 25 microM EGTA in the patch pipette and a stimulation frequency of 0.125 Hz, ICa was 2.6 +/- 0.4 pA/pF and it carried a total charge of 0.10 +/- 0.01 pC/pF, giving rise to a contraction of 15.2 +/- 2.8% of the resting cell length. With a cell volume of 2.4 +/- 0.3 pl, the charge carried by ICa corresponded to 14.7 +/- 2.2 micromol Ca2+/l nonmitochondrial cell volume (microM). This can account for only 30-40% of the Ca2+ binding to the myofilaments during a contraction. Increasing the stimulation frequency from 0.25 to 2 Hz decreased ICa amplitude and charge by 66 +/- 5 and 80 +/- 3%, respectively. Elevating the pipette EGTA concentration from 25 microM to 5 mM increased ICa amplitude and charge by approximately 290%. Both isoproterenol and cAMP increased ICa by approximately 230%. The total charge carried by the isoproterenol- or cAMP-stimulated current was increased by 170%. We conclude that the use of high-EGTA concentration may overestimate the total Ca2+ carried by ICa under physiological conditions. Furthermore, the results suggest that, in contrast to previous reports from other lower vertebrates, Ca2+ flux through L-type Ca2+ channels alone is not sufficient to fully activate contraction in trout atrial myocytes at room temperature.

Adrenergic beta-Agonists↗
PubMed1998

Quantification of Ca2+ uptake in the sarcoplasmic reticulum of trout ventricular myocytes.

We measured Ca2+ uptake by the sarcoplasmic reticulum (SR) in trout ventricular myocytes, measuring indo 1 fluorescence in permeabilized cells or ionic currents in single myocytes subjected to voltage clamp. Titration of the SR Ca2+ pumps with thapsigargin gave a pump site density of 454 pmol/mg cell protein. Lowering the temperature from 20 degreesC to 10 or 5 degreesC reduced the SR Ca2+ uptake rate in permeabilized myocytes by 50 and 63%, respectively. Surprisingly, Ca2+ leak from the SR also decreased with decreasing temperatures. Exposure of single myocytes to 10 mM caffeine (Caf) induced a cell contracture and an inward ionic current. Neither contracture nor current decreased significantly after rest periods of 120 and 320 s. The inward current was due to Ca2+ extrusion by the Na+/Ca2+ exchanger (NCX), and the time integral of the exchange current (INCX) was used to calculate the SR Ca2+ content. This gave a steady-state SR Ca2+ content of 22.5 +/- 2.8 amol Ca2+/pF or 750 microM. When the SR was loaded by depolarizing the cell to +50 mV, the Ca2+ content increased with increasing length of the depolarization, reaching a maximum of 52.0 +/- 5.9 amol Ca2+/pF. When the cell was depolarized to different voltages for 3 s, a subsequent Caf-induced INCX increased with increasing voltage. At +100 mV, the Ca2+ content was 36.6 +/- 3.8 amol/pF, giving a maximal SR Ca2+ uptake rate of 12.2 +/- 1.2 amol Ca2+. pF-1. s-1 or 417 microM/s. We conclude that maximal SR Ca2+ content and Ca2+ uptake rates can be estimated using specific SR Ca2+ loading protocols. Contrary to the general assumption that contraction in lower vertebrates depends largely on transsarcolemmal Ca2+ fluxes, we found that although the L-type Ca2+ current is insufficient to fully activate contraction, the SR is capable of participating in the regulation of the cytosolic Ca2+ during the excitation-contraction coupling in trout ventricular myocytes.

Animals↗
PubMed1997

Sympathetic modulation of the effect of nifedipine on myocardial contraction and Ca current in the rat.

The regulation of cardiac L-type Ca2- current (Ica) and contraction by dihydropyridine antagonists and beta-adrenergic receptor agonists has been the subject of numerous studies over the last decade. However, little is known on the crosstalk between these two regulatory pathways. For instance, a fundamental question that remains unanswered is: does activation of the beta-adrenergic receptors modify the sensitivity of the myocardium to dihydropyridine agonists? To answer this question, we examined in the present study how activation of the beta-adrenergic receptors modifies the effects of nifedipine on the mechanical and energetic parameters of the isolated perfused rat heart. Activation of the beta-adrenergic receptors was achieved by perfusing the hearts with isoprenaline, a non-selective beta-adrenergic receptor agonist, and could be reduced by atenolol, a beta-adrenergic receptor antagonist. To examine possible alterations during hypertension in the sensitivity of the hearts to the drug, tested, the study was performed in both normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive animals (SHR). While 0.1 microM nifedipine reduced left ventricular pressure (LVP) by 36% and 34% in WKY and SHR rats, respectively, under basal conditions, its effects became negligible in both groups of rats after stimulation of the hearts with 0.1 microM isoprenaline. Addition of 1 microM atenolol in the presence of isoprenaline restored the inhibitory effect of nifedipine to control values in both WKY and SHR rats. Additional experiments were performed in isolated ventricular myocytes from WKY rats using the whole-cell patch-clamp technique. The inhibitory effects of 0.1 to 1 microM nifedipine were significantly larger on basal Ica than after the current had been previously elevated by 0.1 microM isoprenaline. Addition of 1 microM atenolol in the presence of isoprenaline partially restored the inhibitory effect of nifedipine on Ica. Our results demonstrate a reduced sensitivity of the heart muscle to nifedipine during activation of beta 1-adrenergic receptors. This effect is partly explained by a reduced inhibitory effect of nifedipine on Ic during activation of cAMP-dependent phosphorylation.

Adrenergic beta-Agonists↗
PubMed1997

A comparative study of the effects of three guanylyl cyclase inhibitors on the L-type Ca2+ and muscarinic K+ currents in frog cardiac myocytes.

1. To investigate the participation of guanylyl cyclase in the muscarinic regulation of the cardiac L-type calcium current (ICa), we examined the effects of three guanylyl cyclase inhibitors, 1H-[1,2,4]oxidiazo-lo[4,3-a]quinoxaline-1-one (ODQ), 6-anilino-5,8-quinolinedione (LY 83583), and methylene blue (MBlue), on the beta-adrenoceptor; muscarinic receptor and nitric oxide (NO) regulation of ICa and on the muscarinic activated potassium current I(K,ACh), in frog atrial and ventricular myocytes. 2. ODQ (10 microM) and LY 83583 (30 microM) antagonized the inhibitory effect of an NO-donor (S-nitroso-N-acetylpenicillamine, SNAP, 1 microM) on the isoprenaline (Iso)-stimulated ICa which was consistent with their inhibitory action on guanylyl cyclase. However, MBlue (30 microM) had no effect under similar conditions. 3. In the absence of SNAP, LY 83583 (30 microM) potentiated the stimulations of ICa by either Iso (20 nM), forskolin (0.2 microM) or intracellular cyclic AMP (5-10 microM). ODQ (10 microM) had no effect under these conditions, while MBlue (30 microM) inhibited the Iso-stimulated ICa. 4. LY 83583 and MBlue, but not ODQ, reduced the inhibitory effect of up to 10 microM acetylcholine (ACh) on ICa. 5. MBlue, but not LY 83583 and ODQ, antagonized the activation of I(K,ACh) by ACh in the presence of intracellular GTP, and this inhibition was weakened when I(K,ACh) was activated by intracellular GTPgammaS. 6. The potentiating effect of LY 83583 on Iso-stimulated ICa was absent in the presence of either DL-dithiothreitol (DTT, 100 microM) or a combination of superoxide dismutase (150 u ml(-1)) and catalase (100 u ml(-1)). 7. All together, our data demonstrate that, among the three compounds tested, only ODQ acts in a manner which is consistent with its inhibitory action on the NO-sensitive guanylyl cyclase. The two other compounds produced severe side effects which may involve superoxide anion generation in the case of LY 83583 and alteration of beta-adrenoceptor and muscarinic receptor-coupling mechanisms in the case of M Blue.

Aminoquinolines↗
PubMed1997

Methylene blue is a muscarinic antagonist in cardiac myocytes.

We studied the mechanism of action of methylene blue (Mblue), a putative guanylyl cyclase inhibitor, on the L-type calcium current (ICa) and the muscarinic activated K+ current (IK,ACh) in rat ventricular and atrial myocytes, respectively, and on the binding of [3H]quinuclidinyl benzylate in rat ventricular membranes. Superfusion, but not internal dialysis, with 30 microM Mblue antagonized the inhibitory effect of acetylcholine (ACh, 1 microM) on beta-adrenergic stimulation of ICa with isoprenaline (Iso, 10 nM or 1 microM). However, Mblue had no effect on the basal ICa or on the stimulation of ICa by Iso in the absence of ACh. The activation of IK,ACh by 3 microM ACh was also antagonized by Mblue in a dose-dependent manner. In contrast, Mblue had no effect on the activation of IK,ACh by either guanosine-5'-O-(3-thio)triphosphate or guanosine-5'-(beta,gamma-imido)triphosphate. Chlorpromazine (CPZ), a piperazine derivative like Mblue, also inhibited the muscarinic activation of IK,ACh in a dose-dependent manner. The specific binding of [3H]QNB, a muscarinic ligand, to rat ventricular membranes was displaced in a dose-dependent manner by Mblue and CPZ. The piperazine derivatives behaved like competitive antagonists of [3H]QNB binding, exhibiting equilibrium dissociation constant (Ki) values of 187 nM for Mblue and 366 nM for CPZ. In conclusion, Mblue exerts antimuscarinic effects on ICa and IK,ACh in rat cardiac myocytes that are best explained by the binding of Mblue to the M2 subtype of muscarinic receptors. This property probably contributes to the antimuscarinic effect of the putative guanylyl cyclase inhibitor reported in previous studies.

Acetylcholine↗
PubMed1996

Binding constants determined from Ca2+ current responses to rapid applications and washouts of nifedipine in frog cardiac myocytes.

1. A fast perfusion system was used to analyse the kinetics of the response of L-type calcium current (ICa) to rapid applications and washouts of the dihydropyridine antagonist nifedipine in whole-cell patch-clamped frog ventricular myocytes. 2. Both the inhibition of ICa induced by nifedipine and the recovery from inhibition upon washout of the drug behaved as mono-exponential functions of time. 3. During application or washout of 100 nM nifedipine, only the peak amplitude of ICa varied but not its time course of activation or inactivation. 4. The rate constant of the onset of ICa inhibition increased with the concentration of nifedipine. However, the time course of the recovery from inhibition was independent of drug concentration. 5. Both rate constants were strongly sensitive to the holding potential but insensitive to the test potential. 6. Using simple rate equations and a one-binding-site analysis it was possible to determine the rate constants for association (k1) and dissociation (k-1) and the equilibrium dissociation constant (KD) of the reaction between nifedipine and Ca2+ channels. KD values for nifedipine were identical to IC50 values obtained from classical steady-state experiments. 7. With depolarized holding potentials, KD decreased strongly due to a large reduction in k-1 and a modest increase in k1. Assuming that these changes result from the distribution of Ca2+ channels between resting and inactivated states, a low-affinity binding to the resting state (R) and a high-affinity binding to the inactivated state (I) were obtained with the binding constants: k1R = 1.0 x 10(6) M-1 S-1, k-1R = 0.077 S-1, and KDR = 77 nM for the resting state; k1I = 4.47 x 10(6) M-1 S-1, k-1I = 7.7 x 10(-4) S-1, and KDI = 0.17 nM for the inactivated state. 8. Rapid application/washout experiments provide a unique way to determine, in an intact cell and in a relatively short period (2-4 min), the binding rate constants and the KD value of the reaction between a dihydropyridine antagonist and the Ca2+ channels.

Animals↗
PubMed1996

Regulation of myocardial calcium channels by cyclic AMP metabolism.

Hormonal regulation of cardiac inotropism is often correlated with modification of the L-type Ca-channel current. Among several regulatory pathways that control Ca-channel activity, the best described one is the cAMP cascade. Cyclic AMP-dependent phosphorylation of the Ca-channel results in an increase of the mean open probability of the individual Ca-channels and, thus, of the macroscopic Ca current. Modulation of cAMP concentration can take place at the level of adenylyl cyclases or cAMP phosphodiesterases. Of major interest is the fact that the activity of two different forms of phosphodiesterases is controlled by the level of intracellular cGMP. Thus, cAMP metabolism is intimately associated with cGMP metabolism, and both determine the degree of cAMP-dependent phosphorylation of cardiac Ca-channels. This brief discussion will focus on these two levels of control and their relative importance in the cAMP-dependent regulation of myocardial Ca-channels.

Adenylyl Cyclases↗
PubMed1996

Nitric oxide synthase does not participate in negative inotropic effect of acetylcholine in frog heart.

In the heart, the parasympathetic neurotransmitter acetylcholine (ACh) reduces the force of contraction. Although the effect of ACh can be partly explained by an inhibition of adenylyl cyclase, some of the effects of ACh may also be mediated via stimulation of nitric oxide synthase (NOS) and production of guanosine 3', 5'-cycle monophosphate (cGMP). NOS inhibitors can prevent the negative chronotropic effect of ACh on spontaneously beating cardiomyocytes and suppress the inhibition of the L-type calcium current (ICa) by ACh in sinoatrial myocytes. This pathway may be relevant not only to the chronotropic effect of ACh but also to its inotropic effect, because ACh, NO, and cGMP regulate the force of contraction and ICa in the cardiac ventricle. Here we report the effects of L-arginine (L-Arg), the substrate of NOS, and NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NNA), two NOS inhibitors, on muscarinic effects in the cardiac ventricle. We found that L-Arg, L-NMMA, and L-NNA have no effect on the muscarinic inhibition of ICa in isolated frog myocytes. In addition, these compounds have no significant effects on basal ICa or beta-adrenergic stimulation of ICa. L-Arg and its analogues did not change the negative inotropic effect of ACh in frog ventricular fibers. Basal active tension and the positive inotropic effect of isoproterenol, a beta-adrenergic agonist, also were unaffected. We conclude that NOS in not involved in muscarinic inhibition of ICa in isolated from ventricular myocytes or the negative inotropic effect of ACh in the frog ventricle.

Acetylcholine↗
PubMed1994

Protein kinase inhibitors reduce SR Ca transport in permeabilized cardiac myocytes.

Phosphorylation of the sarcoplasmic reticulum (SR) protein phospholamban by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) and Ca-calmodulin-dependent protein kinase (CaM-KII) stimulates Ca-adenosinetriphosphatase (ATPase) activity and SR Ca transport, but the role of CaM-KII-dependent phosphorylation is not well defined. We studied the PKA- and CaM-KII-dependent regulation of SR Ca transport in digitonin-permeabilized rabbit ventricular myocytes. SR Ca uptake and free Ca concentration were measured on line with indo 1 and Ca electrodes in the presence of 20 microM ruthenium red and 10 mM oxalate. neither N5,2'-w-dibutyryl-cAMP (up to 500 microM) nor the nonhydrolyzable cAMP agonist adenosine 3'5'-cyclic monophosphorothioate sodium salt (Sp-cAMP[S]; up to 275 microM) affected the maximum uptake rate (Vmax) or the dissociation constant (Kd) for Ca uptake. However, the PKA inhibitor H-89 significantly increased Kd (e.g., from 307 +/- 67 to 826 +/- 62 nM Ca at 40-65 microM H-89) without significantly affecting Vmax. Both CaM-KII inhibitors, KN-62 (60 microM) and a CaM-KII inhibitory peptide (10 microM), significantly decreased Vmax from 11.95 +/- 0.5 to 9.48 +/- 0.6 nmol.mg-1.min-1 and from 10.95 +/- 1.72 to 7.37 +/- 0.94 nmol.mg-1.min-1, respectively, without consistently changing Kd. The effects of H-89 on Kd and of KN-62 on Vmax were prevented by a monoclonal antibody to phospholamban 2D12 (consistent with the antibody removing the inhibitory effect of phospholamban on the SR Ca-ATPase).(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

Passive Ca buffering and SR Ca uptake in permeabilized rabbit ventricular myocytes.

Passive Ca binding was measured with a Ca-selective minielectrode in suspensions of permeabilized rabbit ventricular myocytes equilibrated with 5 microM thapsigargin and 30 microM ruthenium red to prevent sarcoplasmic reticulum (SR) or mitochondrial Ca uptake. Passive Ca binding was obtained by titration of the myocytes with Ca and subtraction of Ca binding in a blank titration without myocytes. Passive Ca binding could be described by a Michaelis binding curve with two sites: K1 = 0.42 microM n1 = 1.27 nmol/mg cell protein and K2 = 79 microM, n2 = 4.13 nmol/mg cell protein. The passive Ca buffering over the physiological Ca concentration was approximately twice the value expected from the values compiled by Fabiato [A. Fabiato. Am. J. Physiol. 245 (Cell Physiol. 14): C1-C14, 1983]. The maximal SR Ca uptake in the presence of 30 microM ruthenium red was fit by an uptake curve with a maximal uptake of 5.16 nmol/mg cell protein and a K 1/2 of 1.0 microM. In the presence of 5 microM thapsigargin and no ruthenium red, a significant Ca uptake attributed to mitochondria was measured between 10 and 100 microM free Ca. Rapid changes in free Ca concentration ([Ca]) measured with a Ca electrode were slower than simultaneous measurements of free [Ca] with indo-1 in permeabilized myocytes. However, oxalate, which buffers Ca and maximizes SR Ca uptake, increased the uptake rate and eliminated the difference in free [Ca] measured with Ca electrode and indo-1. This suggests that spatial gradients of [Ca] exist in permeabilized myocytes without Ca buffering. The new estimates of the buffering of intracellular Ca in cardiac myocytes should be valuable in developing quantitative insights into cardiac Ca regulation.

Animals↗