Pierre-François Méry

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.

Muscarinic regulation of the L-type calcium current in isolated cardiac myocytes

Muscarinic agonists regulate the L-type calcium current in isolated cardiac myocytes. The second messengers pathways involved in this regulation are discussed briefly, with particular emphasis on the involvement of cAMP and cGMP pathways.

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

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]oxidiazolo[4,3‐a]quinoxaline‐1‐one (ODQ), 6‐anilino‐5,8‐quinolinedione (LY 83583), and methylene blue (MBlue), on the β‐adrenoceptor; muscarinic receptor and nitric oxide (NO) regulation of ICa and on the muscarinic activated potassium current IK,ACh, in frog atrial and ventricular myocytes. ODQ (10 μM) and LY 83583 (30 μM) antagonized the inhibitory effect of an NO‐donor (S‐nitroso‐N‐acetylpenicillamine, SNAP, 1 μM) on the isoprenaline (Iso)‐stimulated ICa which was consistent with their inhibitory action on guanylyl cyclase. However, MBlue (30 μM) had no effect under similar conditions. In the absence of SNAP, LY 83583 (30 μM) potentiated the stimulations of ICa by either Iso (20 nM), forskolin (0.2 μM) or intracellular cyclic AMP (5–10 μM). ODQ (10 μM) had no effect under these conditions, while MBlue (30 μM) inhibited the Iso‐stimulated ICa. LY 83583 and MBlue, but not ODQ, reduced the inhibitory effect of up to 10 μM acetylcholine (ACh) on ICa. MBlue, but not LY 83583 and ODQ, antagonized the activation of IK,ACh by ACh in the presence of intracellular GTP, and this inhibition was weakened when IK,ACh was activated by intracellular GTPγS. The potentiating effect of LY 83583 on Iso‐stimulated ICa was absent in the presence of either DL‐dithiothreitol (DTT, 100 μM) or a combination of superoxide dismutase (150 u ml−1) and catalase (100 u ml−1). 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 β‐adrenoceptor and muscarinic receptor‐coupling mechanisms in the case of MBlue.

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.

Regulation of cardiac Ca2+ channels by cGMP and NO

There is ample evidence that cAMP and cGMP lead to opposite contractile and electrical responses in the heart [1–11]. Since regulation of the L-type calcium channel current (ICa) is often correlated with the regulation of cardiac contraction, it was conceivable that both nucleotides exert opposite actions on ICa. The stimulatory effect of cAMP on ICa has been well documented. Cyclic AMP activation of cAMP-dependent protein kinase (cAMP-PK) leads to phosphorylation of L-type Ca2+ channels (or a closely associated protein), resulting in an increase in the mean probability of channel opening and stimulation of macroscopic ICa [11–13], By symmetry, it has been proposed that the negative inotropic effect of cGMP was mediated by a decrease in ICa. Among the evidence supporting this hypothesis are the findings that cGMP reduces 45Ca flux [3], shortens action potential duration [14], and inhibits Ca2+ -dependent action potentials [15–18].

A loudspeaker-driven system for rapid and multiple solution exchanges in patch-clamp experiments

A new and inexpensive system allowing rapid and synchronized changes of solutions around a membrane patch or a cell under voltage-clamp conditions is described. Four plastic capillary tubings (OD 640 μm; ID 430 μm) were glued together horizontally and attached to a coil of a commercially available loudspeaker. Servo-control of the position of the coil allowed the mouth of any of the capillaries to be positioned near the pipette tip within 6 ms. A high flow speed of the test solution was crucial to achieve rapid solution exchange. At a flow speed of 5 cm/s, complete exchange of the external environment of a frog ventricular cell was achieved within 20–30 ms. The time course of solution change was found to be 3–5 times faster at the tip of an open patch pipette. To preserve the physical integrity of the cell, the cell was usually perfused by a control capillary at a slow velocity (0.2 –0.4 cm/s) and test solutions flowing out of adjacent capillaries at high velocity (4–5 cm/s) were applied to the cell only for short periods. Determination of the three-dimensional contamination profile around the mouth of the control capillary allowed the optimal conditions for the use of the system to be established and possible sources of contamination to be avoided between adjacent capillaries with unmatched flow speeds. Successive and multiple changes in external solutions could be easily synchronized with voltage-clamp depolarizations to examine the time course of the effect of drugs on voltage-operated ion channels. An example of this application is given with rapid applications of the dihydropyridine agonist (-)BayK 8644 to the L-type Ca2+ channel current in frog ventricular myocytes.

Hormonal and Non-Hormonal Regulation of Ca2+ Current and Adenylate Cyclase in Cardiac Cells

The influx of Ca2+ ions through transmembrane Ca2+ channels is fundamental in many aspects of cardiac function. Regulation of the heart beat by noradrenaline and acetylcholine (ACh) is in part mediated by the effects of these neurotransmitters on calcium current, ICa (1). β-adrenergic stimulation of ICa is mediated by a guanine-nucleotide binding protein, Gs (2), which triggers the activation of adenylate cyclase (AC) and in turn stimulates cAMP-dependent phosphorylation of Ca2+ channels (1,3). Gs has also been shown to directly activate Ca2+ channels (4). This latter mechanism, however, may play only a minor role in the physiological response to noradrenaline since the effects of β-adrenergic agonists on ICa were mimicked by external application of cAMP, its analogues or phosphodiesterase inhibitors (5), forskolin (6) [a direct activator of AC (7)], and by intracellular application of cAMP (8,9) or the catalytic subunit of cAMP-dependent protein kinase (PKA; ref. 8).