HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 551/1/239    most recent jphysiol.2003.045211v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jurevicius, J. Articles by Fischmeister, R. Search for Related Content PubMed PubMed Citation Articles by Jurevicius, J. Articles by Fischmeister, R.

J Physiol (2003), 551.1, pp. 239-252
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.045211

Role of cyclic nucleotide phosphodiesterase isoforms in cAMP compartmentation following ₂-adrenergic stimulation of I_Ca,L in frog ventricular myocytes

Jonas Jurevicius*, V. Arvydas Skeberdis† and Rodolphe Fischmeister‡

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

The role of cyclic nucleotide phosphodiesterase (PDE) isoforms in the ₂-adrenergic stimulation of the L-type Ca²⁺ current (I_Ca,L) was investigated in frog ventricular myocytes using double patch-clamp and double-barrelled microperfusion techniques. Isoprenaline (ISO, 1 nM to 10 µM) was applied on one half of the cell, either alone or in the presence of PDE inhibitors, and the local and distant responses of I_Ca,L were used to determine the gradient of local vs. distant cAMP concentration (). IBMX (100 µM), a non-selective PDE inhibitor, reduced from 40 to 4.4 indicating a 9-fold reduction in intracellular cAMP compartmentation when all PDE activity was blocked. While PDE1 and PDE2 inhibition had no effect, PDE3 inhibition by milrinone (3 µM) or PDE4 inhibition by Ro 20-1724 (3 µM) reduced by 6- and 4-fold, respectively. A simultaneous application of milrinone and Ro 20-1724 produced a similar effect to IBMX, showing that PDE3 and PDE4 were the major PDEs accounting for cAMP compartmentation. Okadaic acid (3 µM), a non-selective phosphatase inhibitor, or H89 (1 µM), an inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the distant response of I_Ca,L to ISO indicating that PDE activation by PKA played a minor role in cAMP compartmentation. Our results demonstrate that PDE activity determines the degree of cAMP compartmentation in frog ventricular cells upon ₂-adrenergic stimulation. PDE3 and PDE4 subtypes play a major role in this process, and contribute equally to ensure a functional coupling of ₂-adrenergic receptors with nearby Ca²⁺ channels via local elevations of cAMP.

(Resubmitted 14 April 2003; accepted after revision 2 June 2003; first published online 18 June 2003)
Corresponding author R. Fischmeister: INSERM U-446, Université Paris-Sud, Faculté de Pharmacie, F-92296 Châtenay-Malabry Cedex, France. Email: fisch{at}vjf.inserm.fr

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

Sympathomimetic amines, such as isoprenaline (ISO), exert a positive inotropic effect on the heart that is mediated by activation of adenylyl cyclase and phosphorylation by cAMP-dependent protein kinase (PKA) of a number of target proteins, including the L-type Ca²⁺ channels (Rapundalo, 1998; Kamp & Hell, 2000). In the mammalian heart, ISO activates ₁- and ₂-adrenergic receptors and both receptors contribute to cAMP synthesis and PKA-mediated stimulation of the macroscopic L-type Ca²⁺ current (I_Ca,L) (Hartzell et al. 1991; Skeberdis et al. 1997b; Zhou et al. 1997) and amplitude of contraction (Molenaar et al. 2000; for a review, see Steinberg, 1999). However, although both receptors share a common signalling pathway, the positive inotropic effect of ₂-adrenergic agonists differs from that of ₁-agonists in that it is not accompanied by a measurable production of intracellular cAMP (Xiao et al. 1994), by an accelerated relaxation of contractility (Xiao et al. 1993) or by cAMP-dependent phosphorylation of phospholamban (Xiao et al. 1994; but see Molenaar et al. 2000). This observation was unexpected, though, considering that ₂-receptors were shown to be more tightly coupled to adenylyl cyclase than ₁-receptors, both in native cardiac tissues (Waelbroeck et al. 1983; Bristow et al. 1989) and in reconstitution systems (Green et al. 1992; Levy et al. 1993). A possible explanation for the difference between cardiac ₁- and ₂-adrenergic responses came from studies performed in rat ventricular myocytes which indicated that the cAMP signal might be more localised upon ₂- than ₁-receptor activation (Zhou et al. 1997; see Steinberg & Brunton 2001 for a review). Such a hypothesis was further supported by additional experiments in transgenic mice overexpressing the ₂-adrenergic receptor in the heart (Milano et al. 1994). In these animals, cardiac force of contraction (Milano et al. 1994) and I_Ca,L (An et al. 1999; but see Zhou et al. 1999; Heubach et al. 1999) were found to be strongly enhanced due to an enhanced PKA activity resulting from a large constitutive activity of overexpressed ₂-adrenergic receptors (Bond et al. 1995). However, I_K,S, a slow component of the delayed rectifier K⁺ current also normally activated by PKA (Roden et al. 2002), was unchanged in these transgenic animals (An et al. 1999) indicating that cAMP may not rise uniformly inside the cell when its synthesis is triggered by ₂-receptors. A non-uniform distribution of intracellular cAMP was initially proposed by Brunton and his colleagues over 20 years ago in a series of elegant biochemical studies performed on the rat heart (Brunton et al. 1979) or isolated ventricular myocytes (Buxton & Brunton, 1983). When comparing the effects of ISO and prostaglandin E₁ (PGE₁) on the degree of cAMP accumulation and activation of PKA in particulate and soluble fractions, they found that ISO increased cAMP and PKA in both fractions, but PGE₁ increased cAMP and PKA in the soluble fraction only (Brunton et al. 1979; Buxton & Brunton, 1983). However, what causes cAMP to remain localised in one case and to diffuse more readily in the other case remains an open question (Bers & Ziolo, 2001; Steinberg & Brunton, 2001).

In the frog heart, we found that a single population of receptors is involved in the -adrenergic regulation of I_Ca,L (Skeberdis et al. 1997a). The pharmacological pattern of the response of I_Ca,L to different -adrenergic agonists and antagonists allowed us to conclude that these receptors are of the ₂-subtype (Skeberdis et al. 1997a). ISO activation of I_Ca,L in frog ventricular myocytes is thus entirely mediated by ₂-adrenergic receptors (Skeberdis et al. 1997a) and cAMP-dependent phosphorylation (Hartzell et al. 1991). The elongated shape of frog ventricular myocytes allows a direct investigation of the intracellular cAMP compartmentation in these cells. Indeed, using a double-barrelled microperfusion technique, it is possible to expose only half of a cell to a drug and record independently the 'local' and 'distant' effects of the drug on I_Ca,L (Jurevicius & Fischmeister, 1996; Dittrich et al. 2001).

As shown in our earlier study, the local effect of ISO or forskolin on I_Ca,L in frog ventricular myocytes was due to adenylyl cyclase activation in the part of the cell exposed to these drugs, while the distant effect was due to passive diffusion of cAMP from the exposed part to the remote part of the cell (Jurevicius & Fischmeister, 1996). Under control conditions, a small amount (~3 %) of cAMP was found to diffuse to the remote part of the cell when ISO was applied on one half of the cell only, indicating that the cAMP signal is indeed highly localised upon ₂-adrenergic receptor activation. Almost three decades ago, a somewhat similar finding was obtained by Venter and colleagues on a multicellular cardiac preparation. In an elegant series of studies, a local application of ISO immobilised on glass beads was shown to achieve a strong increase in contraction of cat papillary muscle without any change in whole-tissue cAMP concentration (Venter et al. 1975; Hu & Venter, 1977). We hypothesised that cyclic nucleotide phosphodiesterases (PDE) might play a role in limiting the amount of cAMP that diffuses from the membrane to the cytosol, since in the presence of 3-isobutyl-1-methylxanthine (IBMX), a broad-spectrum PDE inhibitor, cAMP appeared to diffuse more readily (Jurevicius & Fischmeister, 1996). In the present study, our aim was to examine this latter hypothesis further, in particular by investigating the role of two major PDE subtypes which have been shown earlier to regulate I_Ca,L in frog ventricular myocytes (Fischmeister & Hartzell, 1990): PDE3, the cGMP-inhibited PDE, and PDE4, the cGMP-independent cAMP-selective PDE.

A preliminary account of this work has been presented elsewhere (Jurevicius et al. 1997).

	METHODS

Top Abstract Introduction Methods Results Discussion References

The investigation conforms with the European Community guiding principles in the care and use of animals (86/609/CEE, CE Off J no. L358, 18 December 1986), the local (CREEA Ile-de-France Sud) ethics committee guidelines and the French decree no. 87/748 of October 19, 1987 (J Off République Française, 20 October 1987, pp. 12245-12248). Authorisations to perform animal experiments according to this decree were obtained from the French Ministère de l'Agriculture et de la Forêt (no. 04226, April 12, 1991).

Cell isolation

Ventricular cells were enzymatically dispersed from frog (Rana esculenta) heart, by a combination of collagenase and trypsin as described (Fischmeister & Hartzell, 1987). Frogs were killed by decapitation, the spinal cord was destroyed with a steel rod and the heart was then excised. The isolated cells were stored in storage Ringer solution, and kept at 4 °C until use (2-48 h following dissociation). This study is based on myocytes isolated from a total of 21 frogs.

Electrophysiology

Rod-shaped Ca²⁺-tolerant and single mononucleated frog ventricular myocytes 250-400 µm in length were sealed at both ends with two patch-clamp pipettes (1.0-1.5 M) as described previously (Jurevicius & Fischmeister, 1996). Cell membrane capacitance was 76.0 ± 1.1 pF (n = 19). Whole-cell recording conditions were established for both electrodes using two independent patch-clamp amplifiers (RK400, Bio-Logic, Claix, France). One electrode (EL1) was under voltage-clamp conditions and was used to depolarise the cell every 8 s to 0 mV for 200 ms from a holding potential of -80 mV. The other electrode (EL2) was under current-clamp conditions (at zero current) to allow the measurement of membrane potential at the most remote part of the cell. Voltage-clamp protocols applied to EL1 were generated by a challenger/09-VM programmable function generator (Kinetic Software, Atlanta, GA, USA). K⁺ currents were blocked by replacing all K⁺ ions with intracellular and extracellular Cs⁺ (Fischmeister & Hartzell, 1987) and the fast Na⁺ current was blocked by tetrodotoxin. All experiments were done at room temperature (19-26 °C), and the temperature did not vary by more than 1 °C in a given experiment.

Microperfusion of single cardiac myocytes

After breaking of the patch membrane at the tips of both electrodes and establishment of whole-cell recording conditions, EL1 and EL2 were moved separately so that the cell was positioned transversely at the mouth of two adjacent capillaries (square section: 400 µm 400 µm) made out of Plexiglas and separated by an intermediate wall ~5 µm-thick (Jurevicius & Fischmeister, 1996). The cell was thus exposed to two different solutions, which are indicated as S1 and S2 in subsequent figures. In all experiments, S1 was a regular Cs⁺-Ringer Ca²⁺-containing solution complemented with 1 µM propranolol to completely avoid any activation of -adrenergic receptors in the part of the cell not exposed to ISO. S2 varied during the course of the experiments from a zero-Ca²⁺ to a Ca²⁺-containing Cs⁺-Ringer solution, and all drugs tested here were added to S2. Mixing of the solutions at the cell membrane was minimised by (1) pressing the cell against the wall, (2) positioning the tips of EL1 and EL2 inside the mouth of the capillaries, and (3) pressure ejection of the solutions out of the capillaries at a linear velocity of 2 cm s^-1 (Jurevicius & Fischmeister, 1996).

Data analysis

Currents were sampled at a frequency of 10 kHz using a 16-bit analog-to-digital converter (PCL816, Advantech France, Levallois Perret, France) connected to a PC-compatible micro computer. Currents were not compensated for capacitive and leak currents. On-line analysis of the recordings was made possible by programming a PC-compatible microcomputer in Assembling language (Borland, USA) to determine, for each membrane depolarisation, peak and steady-state current values (Fischmeister & Hartzell, 1987).

I_Ca,L amplitude was measured as the difference between peak inward current and the current at the end of the 200 ms pulse (Fischmeister & Hartzell, 1987). When S1 and S2 are both identical to the control external Cs⁺-Ringer solution, I_Ca,L amplitude reflects the basal activity of L-type Ca²⁺ channels present in the whole myocyte. When S2 is switched to a zero-Ca²⁺ solution, the L-type Ca²⁺ channels exposed to S2 become silent, and I_Ca,L amplitude reflects only the activity of the channels exposed to S1 (S1 - I_Ca,L). The reduction in I_Ca,L amplitude upon switching S2 to zero-Ca²⁺, or the increment in I_Ca,L upon reintroduction of Ca²⁺ in S2, reflects the activity of the Ca²⁺ channels exposed to S2 (S2 - I_Ca,L). These manoeuvres allow separate measurement of the local and distant responses of I_Ca,L to drugs added to S2. Cumulative dose-response curves were obtained by testing four or five successively increasing concentrations of ISO on local and distant I_Ca,L in the presence or absence of a PDE inhibitor. For each condition, the distant and local responses of I_Ca,L to ISO are calculated as a percentage increase in distant (S1 - I_Ca,L) and local (S2 - I_Ca,L) I_Ca,L amplitudes with respect to their basal values in the absence of agonist. Thus: (%distant I_Ca,L response) = 100[(S1 - I_Ca,L with agonist) - (basal S1 - I_Ca,L)]/(basal S1 - I_Ca,L) and (%local I_Ca,L response) = 100[(S2 - I_Ca,L with agonist) - (basal S2 - I_Ca,L)]/(basal S2 - I_Ca,L).

Error bars are given as standard errors to the mean (S.E.M.). The number of cells is denoted by n. For statistical evaluation Student's paired or unpaired t test was used, and a difference was considered statistically significant when P was < 0.05.

Solutions and drugs

For the preparation of frog ventricular myocytes, the ionic composition of Ca²⁺-free Ringer solution was (mM): 88.4 NaCl; 2.5 KCl; 23.8 NaHCO₃; 0.6 NaH₂PO₄; 1.8 MgCl₂; 5 creatine; 10 D-glucose; 1 mg ml^-1 fatty acid-free bovine serum albumin; 50 i.u. ml^-1 penicillin; 50 µg ml^-1 streptomycin; pH 7.4 maintained with 95 % O₂-5 % CO₂. Storage Ringer solution was Ca²⁺-free Ringer solution to which was added 0.9 mM CaCl₂ and 10 µl ml^-1 non-essential and essential amino acid and vitamin solution (MEM 100 ). Dissociation medium was composed of Ca²⁺-free Ringer solution to which was added 0.2 mg ml^-1 trypsin type XIII (Sigma, St Louis, MO, USA), 0.14 mg ml^-1 collagenase (Yakult, Tokyo, Japan), and 10 µl ml^-1 M199 medium (Sigma).

For electrophysiology, the control external Cs⁺-Ringer solution contained (mM): 107 NaCl; 10 Hepes; 20 CsCl; 4 NaHCO₃; 0.8 NaH₂ PO₄; 1.8 MgCl₂; 1.8 CaCl₂; 5 D-glucose; 5 sodium pyruvate; 3 10^-4 tetrodotoxin; pH 7.4 adjusted with NaOH. Zero-Ca²⁺ external solutions were obtained by simply omitting Ca²⁺ ions from the control Cs⁺-Ringer solution. Patch electrodes were filled with control internal solution which contained (mM): 119.8 CsCl; 5 EGTA (acid form); 4 MgCl₂; 5 creatine phosphate disodium salt; 3.1 Na₂ATP; 0.42 Na₂GTP; 0.062 CaCl₂ (pCa 8.5); 10 Hepes; pH 7.1 adjusted with CsOH.

Tetrodotoxin was from Latoxan (Rosans, France). Milrinone was a gift of Sterling-Winthrop (USA) and Ro 20-1724 was kindly provided by Hoffman-La-Roche (Switzerland). All other drugs were from Sigma. All compounds were dissolved in distilled water. Each day, fresh 1-10 mM stock solutions were prepared and stored at 4 °C. Immediately before being applied to the cell, the drug was dissolved at the desired final concentration in external solutions, i.e. only fresh solutions were tested.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Milrinone increases the distant response of I_Ca,L to isoprenaline

I_Ca,L was measured in isolated frog ventricular myocytes using the whole-cell patch-clamp technique (Hamill et al. 1981). Single myocytes were attached to two patch pipettes and positioned transversely at the mouth of two adjacent capillaries, allowing two different solutions (S1 and S2) to superfuse the two halves of the cell (Jurevicius & Fischmeister, 1996). When S1 and S2 were both identical to the control external Cs⁺-Ringer solution, basal I_Ca,L amplitude was on average 122.1 ± 12.5 pA (mean ± S.E.M., n = 46) at 0 mV membrane potential, and mean I_Ca,L density, which represents the ratio of I_Ca,L amplitude to membrane capacitance, was 1.64 ± 0.19 pA pF^-1 (n = 46). In the experiment shown in Fig. 1, basal I_Ca,L amplitude was 66 pA. After Ca²⁺ withdrawal from S2, the current was reduced to 31 pA. This amplitude represents the 'distant' I_Ca,L, i.e. the Ca²⁺ current carried by the channels located in the cell membrane facing S1 where Ca²⁺ is still present. The 'local' I_Ca,L corresponds to the amount of current that disappeared after withdrawing Ca²⁺ from S2 (in this case 35 pA). Since L-type Ca²⁺ channels are uniformly distributed along the cell length (Jurevicius & Fischmeister, 1997), distant and local I_Ca,L amplitudes give an indication of how much cell membrane is exposed to each solution. In all experiments, the cells were positioned in such a way that distant and local I_Ca,L did not differ by more than 15 %, so that it could be assumed that a similar amount of cell membrane was exposed to each solution.

		View larger version [in this window] [in a new window]
Figure 1. Effect of PDE3 inhibition on the distant and local response of ICa,L to ISO A frog ventricular myocyte was sealed at both ends with two electrodes and positioned approximately half-way in front of channels S1 and S2 of a double-barrelled perfusion system (see Methods). The schematic drawing illustrates the experimental set-up and the solutions applied to S1 and S2 are listed below and above the dotted line, respectively. The bottom graph indicates the time course of the changes of absolute ICa,L amplitude () upon application of different solutions to S2. At first, S2 contained a Cs+-Ringer Ca2+-containing solution. Ca2+ ions were then omitted from S2 and ICa,L decreased by ~50 %. ISO (0.1 µM) was then added to S2 (in 0 Ca2+) and this produced a small distant effect on ICa,L (first bottom arrow). When a steady state was achieved, Ca2+ ions (1.8 mM) were briefly reintroduced in S2, together with ISO, to evaluate the local effect of ISO on ICa,L (first top arrow). Milrinone (3 µM) or IBMX (100 µM) was then added to S2 in the presence of ISO for the periods indicated, and the same 0 Ca2+/1.8 mM Ca2+ protocol was applied to test for the effects of these drugs on the local and distant response of ICa,L to ISO, as indicated by the top and bottom arrows, respectively. The individual current traces shown above the graph were obtained in each experimental condition at the times indicated by the corresponding letters in the bottom graph.

As shown in our previous study (Jurevicius & Fischmeister, 1996), addition of ISO to a zero-Ca²⁺ S2 solution allows estimation of the distant response of I_Ca,L to the -adrenergic agonist. Figure 1 shows that 0.1 µM ISO added to S2 induced a small (61 %) increase in distant I_Ca,L. However, at this concentration, ISO induced a much larger (~5-fold) increase in local I_Ca,L, as seen by the increment in current observed upon a transient reintroduction of Ca²⁺ in S2 (Fig. 1). As shown earlier, the difference between the local and distant responses of I_Ca,L to ISO is evidence for a strong cAMP compartmentation following -adrenoceptor activation (Jurevicius & Fischmeister, 1996). To examine whether PDE3 plays a role in this process, the PDE3 inhibitor milrinone was added to S2 in the continuing presence of ISO. Milrinone was used at 3 µM, a concentration shown earlier to produce a maximal stimulation of cAMP-elevated I_Ca,L (Fischmeister & Hartzell, 1990), while retaining a high selectivity towards PDE3 (Stoclet et al. 1995). Figure 1 shows that addition of milrinone increased distant I_Ca,L by 62 % while it had a smaller (28 %) effect on local I_Ca,L. Thus, the ratio of local vs. distant response of I_Ca,L to ISO was reduced by milrinone approximately from 8.3 to 4.2, indicating that PDE3 inhibition attenuated the gradient of local vs. distant intracellular cAMP concentration. After the effect of milrinone reached steady state, the drug was replaced by IBMX to examine the effect of a complete block of PDE activity in this cell. IBMX was used at 100 µM, a concentration shown earlier to produce a maximal stimulation of cAMP-elevated I_Ca,L (Fischmeister & Hartzell, 1990). Addition of IBMX to S2 increased distant I_Ca,L 1.8-fold compared to its level in milrinone, so that the distant response of I_Ca,L in ISO + IBMX was about three times larger than in ISO alone. However, IBMX increased the local response only by 30 % compared to its level in milrinone, and by 67 % compared to its level in ISO alone. Hence, the ratio of local vs. distant response of I_Ca,L to ISO was reduced from 8.3 to 2.4 by IBMX, indicating that complete PDE inhibition reduced the gradient of local vs. distant intracellular cAMP concentration to a larger extent than inhibition of PDE3 alone. At the end of the experiments, all drugs were washed out of S2 and local and distant I_Ca,L returned to their basal amplitudes.

Ro 20-1724 increases the distant response of I_{Ca, L} to isoprenaline

Similar experiments were performed to examine the contribution of PDE4 to the intracellular cAMP compartmentation following ISO application. To do this, the PDE4 inhibitor Ro 20-1724 was added to S2 in the continuing presence of ISO. Ro 20-1724 was used at 3 µM, a concentration shown earlier to produce a maximal stimulation of cAMP-elevated I_Ca,L (Fischmeister & Hartzell, 1990; Verde et al. 1999), while retaining a high selectivity towards PDE4 (Stoclet et al. 1995). In the experiment shown in Fig. 2, addition of Ro 20-1724 (3 µM) to S2 in the presence of 0.1 µM ISO increased the distant response of I_Ca,L 3.2-fold from 56 % to 177 % stimulation over basal. However, during the same protocol, the local response increased by only 49 %. Hence, the ratio of local vs. distant response of I_Ca,L to ISO was reduced by Ro 20-1724 approximately from 7 to 3.3, indicating that PDE4 inhibition attenuated the gradient of local vs. distant intracellular cAMP concentration. After the effect of Ro 20-1724 reached steady state, the drug was replaced by a saturating concentration of IBMX (100 µM) to compare the effect of PDE4 inhibition with a complete block of PDE activity. Addition of IBMX to S2 increased distant I_Ca,L by 47 % compared to its level in Ro 20-1724, so that the distant response of I_Ca,L in ISO + IBMX was 5.5 times larger than in ISO alone. However, during the same protocol, IBMX had no effect (6 %) on the local response. Hence, the ratio of local vs. distant response of I_Ca,L to ISO was reduced from 7 to 2 by IBMX, indicating that complete PDE inhibition reduced the gradient of local vs. distant intracellular cAMP concentration to a larger extent than inhibition of PDE4 alone. At the end of the experiments, all drugs were washed out of S2 and local and distant I_Ca,L returned to their basal amplitudes.

		View larger version [in this window] [in a new window]
Figure 2. Effect of PDE4 inhibition on the distant and local response of ICa,L to ISO Same protocol as detailed in Fig. 1. Ro 20-1724 (3 µM) or IBMX (100 µM) was added to S2 in the presence of ISO (0.1 µM) for the periods indicated, and 0 Ca2+/1.8 mM Ca2+ protocols were successively applied to test for the effects of these drugs on the local and distant response of ICa,L to ISO, as indicated by the top and bottom arrows, respectively. The individual current traces shown above the graph were obtained in each experimental condition at the times indicated by the corresponding letters in the bottom graph.

Relative contribution of PDE3 and PDE4 to the distant response of I_Ca,L to isoprenaline

From the above experiments, it appears that both PDE3 and PDE4 contribute to limit the distant response of I_Ca,L to ISO. However, inhibition of a single PDE subtype was not sufficient to reach the degree of response obtained with IBMX, i.e. when all PDE activity was blocked. Therefore, we next examined whether a simultaneous inhibition of PDE3 and PDE4 produced comparable effects to IBMX. In the experiment shown in Fig. 3, addition of 3 µM milrinone and 3 µM Ro 20-1724 to S2 in the presence of 0.1 µM ISO increased the distant response of I_Ca,L ~5-fold from 111 % to 517 % stimulation over basal. However, during the same protocol, the local response increased only by 65 %. Hence, the ratio of local vs. distant response of I_Ca,L to ISO was reduced by milrinone + Ro 20-1724 approximately from 7 to 2.5. After steady state, the drugs were replaced by 100 µM IBMX to compare the effect of PDE3 and PDE4 inhibition with a complete block of PDE activity. As shown, IBMX produced an identical effect to milrinone + Ro 20-1724, since neither distant nor local I_Ca,L was significantly modified by the drug. This was not due to some irreversible effect of the PDE inhibitors used since, at the end of the experiments, all drugs were washed out of S2 and local and distant I_Ca,L returned to their basal amplitudes.

		View larger version [in this window] [in a new window]
Figure 3. Effect of a dual PDE3 and PDE4 inhibition on the distant and local response of ICa,L to ISO Same protocol as detailed in Fig. 1. A combination of milrinone (Mil, 3 µM) and Ro 20-1724 (Ro, 3 µM) or IBMX (100 µM) was added to S2 in the presence of ISO (0.1 µM) for the periods indicated, and 0 Ca2+/1.8 mM Ca2+ protocols were successively applied to test for the effects of these drugs on the local and distant response of ICa,L to ISO, as indicated by the top and bottom arrows, respectively. The individual current traces shown above the graph were obtained in each experimental condition at the times indicated by the corresponding letters in the bottom graph.

Similar experiments to the ones shown in Figs 1-3 were performed and the results are summarised in Fig. 4. ISO (0.1 µM) was applied either alone, or in the presence of milrinone (3 µM), Ro 20-1724 (3 µM), milrinone + Ro 20-1724, or IBMX (100 µM), and the drugs were applied to S2 only. The results are expressed as percentage increase in local (open bars) and distant I_Ca,L (filled bars) with respect to their respective basal values in the absence of drugs (see Methods). When ISO was used alone, local I_Ca,L increased more than 6-fold on average, but distant I_Ca,L increased only by 62 %. This 10-fold difference between local and distant responses of I_Ca,L to ISO was strongly attenuated when PDE inhibitors were applied together with ISO. The difference was reduced to ~4 when either PDE3 or PDE4 was blocked, and to 2.8 when both PDEs were blocked simultaneously. A similar response was obtained when all PDE activity was blocked by IBMX (2.6-fold), indicating that PDE3 and PDE4 account for all PDE activity under our experimental conditions.

		View larger version [in this window] [in a new window]
Figure 4. Summary of the effects of PDE inhibitors on the distant and local response of ICa,L to ISO The results of similar experiments to those in Figs 1-3 are summarised. The effects of ISO (0.1 µM) alone, ISO + milrinone (Mil, 3 µM), ISO + Ro 20-1724 (Ro, 3 µM), ISO + Mil + Ro, or ISO + IBMX (100 µM) were tested by applying the drugs to only one half of the cell. The local effect of the drugs (open bars) corresponds to the response of ICa,L in the part of the cell exposed to the drugs. The distant effect of the drugs (filled bars) corresponds to the response of ICa,L in the remote part of the cell not exposed to the drugs. ICa,L amplitude is expressed as percentage increase over its control value, i.e. in the absence of drug. The bars indicate the means and the lines the S.E.M. of the number of cells indicated near the bars. Statistically significant differences relative to ISO alone condition are indicated as: * P < 0.05, ** P < 0.01, *** P < 0.001.

Role of PDE3 and PDE4 in the compartmentation of cAMP

The above experiments demonstrate that PDE3 and PDE4 play an important role during -adrenergic stimulation in limiting the amount of cAMP that diffuses away from where it is being synthesised. However, the contribution of each PDE subtype to the compartmentation of cAMP is likely to vary depending on the amount of cAMP produced. Therefore, we have examined the influence of PDE3 and PDE4 inhibition at different ISO concentrations, i.e. at different levels of adenylyl cyclase activity, in order to get a more quantitative estimate of the gradient of local vs. distant intracellular cAMP concentration in the various experimental conditions tested.

Figure 5 shows two typical experiments in which the local and distant responses of I_Ca,L to increasing concentrations of ISO were measured in the presence of either milrinone (3 µM, Fig. 5A) or Ro 20-1724 (3 µM, Fig. 5B). In both experiments, local and distant I_Ca,L were measured during a cumulative dose-response curve to ISO (0.01, 0.1, 1 and 10 µM). Unlike what was obtained in the presence of ISO alone (Jurevicius & Fischmeister, 1996), application of ISO in the presence of milrinone (Fig. 5A) or Ro 20-1724 (Fig. 5B) induced a substantial distant response of I_Ca,L at each concentration of ISO tested. The results of similar experiments are summarised in Fig. 6. The dose-response curves for the effects of ISO in the presence of milrinone (3 µM, Fig. 6A) or Ro 20-1724 (3 µM, Fig. 6B) show that the distant and local responses mainly differ by a 6- to 10-fold reduction in the sensitivity to the agonist. A simple theoretical analysis of the dose-response curves allowed estimation of the degree of cAMP compartmentation achieved by ISO when either PDE3 or PDE4 was blocked. The analysis leads to the hypothesis that I_Ca,L is an index of the concentration of cAMP near Ca²⁺ channels. Indeed, when exogenous cAMP is perfused inside a frog ventricular myocyte, a relationship is obtained between I_Ca,L and cAMP concentration which is well fitted by the Michaelis equation (Fischmeister & Hartzell, 1987). We thus assumed that local (E_l) and distant (E_d) responses of I_Ca,L to ISO, like those plotted in Fig. 6, reflect the changes in local ([cAMP]_l) and distant ([cAMP]_d) cAMP concentrations, respectively. When normalised to the maximal increase in I_Ca,L, a simple relationship between E_l and E_d can be found:

		View larger version [in this window] [in a new window]
Figure 5. Dose-response for the local and distant response of ICa,L to ISO in the presence of PDE inhibitors Same protocol as detailed in Fig. 1. Four increasing concentrations of ISO (0.01, 0.1, 1 and 10 µM) were successively added to S2 in the presence of either A, milrinone (3 µM), or B, Ro 20-1724 (3 µM), for the periods indicated, and 0 Ca2+/1.8 mM Ca2+ protocols were successively applied to test for the local and distant response of ICa,L, as indicated by the top and bottom arrows, respectively.

		View larger version [in this window] [in a new window]
Figure 6. Dose-response curve for the local and distant response of ICa,L to ISO in the presence of PDE inhibitors The results of similar experiments to those in Fig. 5 are summarised. Five different concentrations of ISO (0.001, 0.01, 0.1, 1 and 10 µM) were tested by applying the drug to only one half of the cell. The same half-cell was additionally exposed to either A, milrinone (3 µM), or B, Ro 20-1724 (3 µM). The local effect of ISO () corresponds to the response of ICa,L in the part of the cell exposed to the drug. The distant effect of ISO () corresponds to the response of ICa,L in the remote part of the cell not exposed to the drug. ICa,L amplitude is expressed as percentage increase over its control value, i.e. in the absence of drug. Each symbol indicates the mean and the lines the S.E.M. of the number of cells indicated near the symbols. Statistically significant differences between local and distant responses are indicated as: * P < 0.05, ** P < 0.01, *** P < 0.001.

E_d = E_l/( + (1 - )E_l). (1)

In this equation, the coefficient = [cAMP]_l/[cAMP]_d represents the gradient of local vs. distant cAMP concentration (for further details, see Jurevicius & Fischmeister, 1996). Equation (1) was applied to the experimental data of Fig. 6 for the effects of ISO on I_Ca,L in the presence of milrinone and Ro 20-1724. Each set of two curves in Fig. 6A and B, which gives E_l () and E_d () as a function of ISO concentration, was used to obtain a relationship of E_l as a function of E_d (not shown) which was then fitted to eqn (1). Calculated mean values derived from these fits were, respectively, 6.1 for ISO in the presence of milrinone and 10.2 for ISO in the presence of Ro 20-1724. Data from additional experiments performed in the presence of 100 µM IBMX were also fitted to eqn (1) and led to an value of 4.4. These values are to be compared with the value of 40 obtained when ISO was used alone (Jurevicius & Fischmeister, 1996). Thus, while under control conditions cAMP concentration is 40-fold higher in the part of the cell exposed to ISO than in the rest of the cell, inhibition of either PDE3 or PDE4 reduced this concentration gradient by a factor of 4-6 and a complete inhibition of all PDE activity reduced the gradient by a factor of ~9.

Role of cAMP-dependent phosphorylation in the compartmentation of cAMP

Several subtypes of cyclic nucleotide phosphodiesterases have been shown to be regulated by cAMP-dependent phosphorylation. In particular, PDE3 and PDE4 are stimulated in vitro by PKA (see e.g. Sette & Conti, 1996; Murthy et al. 2002). Thus, we tested the hypothesis that activation of PKA following ISO application leads to a stimulation of PDE activity in frog ventricular myocytes. To examine this hypothesis, we investigated the local and distant responses of I_Ca,L to ISO in the presence of either okadaic acid, a non-selective phosphatase inhibitor (Takai et al. 1987), or H89, a potent inhibitor of PKA (Chijiwa et al. 1990).

First, dose-response curves for the effects of ISO on I_Ca,L in the absence or presence of 3 µM okadaic acid were obtained (Fig. 7A). At this concentration, okadaic acid increased basal I_Ca,L about 4-fold and strongly amplified the stimulation of the current by a threshold concentration of ISO, an effect compatible with the expected inhibitory action of okadaic acid on phosphatase activity. However, okadaic acid did not modify the maximal response of I_Ca,L to ISO. Then, we tested the effect of 3 µM okadaic acid on the distant response of I_Ca,L to ISO. Figure 7B shows a typical experiment. ISO was used at 0.1 µM and the local response of I_Ca,L was 4-fold larger than the distant response (Fig. 7B). As expected from the dose-response curves shown in Fig. 7A, okadaic acid exerted little stimulation on the local response of I_Ca,L to ISO, because that response was near maximum with ISO alone. However, okadaic acid also induced a negligible effect on the distant response of I_Ca,L to ISO. On average, okadaic acid (3 µM) induced no significant change in the distant response of I_Ca,L to 0.1 µM ISO (12.1 ± 4.2 %, n = 4).

		View larger version [in this window] [in a new window]
Figure 7. Role of protein phosphatase in the local and distant response of ICa,L to ISO A, dose-response curves for the response of ICa,L to ISO in the absence () and presence () of 3 µM okadaic acid. ICa,L amplitude is expressed as current density (pA pF-1). Each symbol indicates the mean and the lines the S.E.M. of the number of cells indicated near the symbols. Statistically significant differences between control and okadaic acid responses are indicated as: ** P < 0.005, *** P < 0.001. B, same protocol as detailed in Fig. 1. Okadaic acid (3 µM) was added to S2 in the presence of ISO (0.1 µM) for the periods indicated, and 0 Ca2+/1.8 mM Ca2+ protocols were successively applied to test for the effects of these drugs on the local and distant response of ICa,L to ISO, as indicated by the top and bottom arrows, respectively.

As inhibition of phosphatase activity was without effect, we tried to inhibit PKA activity using H89. Figure 8A shows the effect of three increasing concentrations of H89 on the response of whole-cell I_Ca,L to 1 µM ISO. As expected from the PKA dependence of ISO response (Hartzell et al. 1991), H89 strongly antagonised the stimulation of I_Ca,L by ISO. At a concentration of 10 µM, H89 completely blocked the ISO response and reduced I_Ca,L to about 50 % below basal level (Fig. 8A). Since at this concentration H89 was shown to also block seven other protein kinases in addition to PKA by 80-100 % (Davies et al. 2000), we decided to use H89 at a 10-fold lower concentration in subsequent experiments. Figure 8B shows a typical experiment in which H89 (1 µM) was tested for its effect on the distant response of I_Ca,L to ISO. ISO was used at 1 µM and the local response of I_Ca,L was 3-fold larger than the distant response (Fig. 8B). As expected from the dose-response curves shown in Fig. 8A, 1 µM H89 reduced the local response of I_Ca,L to ISO by about 50 %. However, H89 induced negligible effect on the distant response of I_Ca,L to ISO. On average, H89 (1 µM) induced no significant change in the distant response of I_Ca,L to 1 µM ISO (-8.9 ± 25.3 % change, n = 3). Altogether, these experiments demonstrate that cAMP-dependent phosphorylation plays a minor role in the cAMP compartmentation observed in frog ventricular cells during -adrenergic stimulation.

		View larger version [in this window] [in a new window]
Figure 8. Role of PKA in the local and distant response of ICa,L to ISO A, inhibitory effect of H89 on ICa,L stimulated by 1 µM ISO. ICa,L amplitude is expressed as current density (pA pF-1). The open bars show the control ICa,L density and the stimulation obtained after application of 1 µM ISO. The filled bars indicate the ICa,L density in the presence of three increasing concentrations of H89 (0.1, 1 and 10 µM). Each bar indicates the mean and the lines the S.E.M. of the number of cells indicated near the bars. Statistically significant differences between ISO alone and ISO + H89 are indicated as: ** P < 0.01, *** P < 0.001. B, same protocol as detailed in Fig. 1. H89 (1 µM) was added to S2 in the presence of ISO (1 µM) for the periods indicated, and 0 Ca2+/1.8 mM Ca2+ protocols were successively applied to test for the effects of these drugs on the local and distant response of ICa,L to ISO, as indicated by the top and bottom arrows, respectively.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

In the present study, we investigated the modulation of I_Ca,L by isoprenaline and PDE inhibitors in isolated frog ventricular myocytes using double patch-clamp and double-barrelled microperfusion techniques for the recording of I_Ca,L and application of agonists at the local side. Since the amplitude of I_Ca,L can be used as a measure of cAMP levels (Hartzell & Fischmeister, 1990; Hartzell et al. 1991; Goaillard et al. 2001), we used this approach to study the localisation of the cAMP signal in relation to the activity of the L-type Ca²⁺ channels. Our main results demonstrate that PDE3 and PDE4 activities determine the degree of cAMP compartmentation in frog ventricular cells during -adrenergic stimulation. As -adrenergic stimulation of I_Ca,L is entirely due to activation of ₂-receptors in frog ventricular myocytes (Skeberdis et al. 1997a), we conclude that PDE3 and PDE4 subtypes contribute equally to ensure a functional coupling of ₂-adrenergic receptors with nearby Ca²⁺ channels via local elevations of cAMP.

Using unilateral drug application, we have demonstrated earlier in frog ventricular myocytes that a local application of ISO preferentially stimulates L-type Ca²⁺ channels near the locus of activated -receptors when PDEs were locally active. However, Ca²⁺ channel activity was more uniformly and spatially enhanced when PDEs were locally inhibited by a maximal concentration of IBMX (Jurevicius & Fischmeister, 1996). While our study was the first to demonstrate in an intact cell that PDE activity plays a critical role in limiting the amount of cAMP diffusing from membrane to cytosol, it did not provide any clue to what type(s) of PDE might be responsible for this effect. Thus, the present study was designed to specifically address that question by using selective inhibitors of different PDE isoforms.

PDEs constitute a large multigenic family, four members of which have been found and characterised in the heart: PDE1, which is activated by Ca²⁺-calmodulin; PDE2, which is stimulated by cGMP; PDE3, which is inhibited by cGMP; and PDE4, which is insensitive to cGMP. While PDE3 and PDE4 hydrolyse cAMP, PDE1 hydrolyses cAMP and cGMP equally and PDE2 hydrolyses both with a lower apparent K_m for cGMP than for cAMP (Beavo, 1995; Stoclet et al. 1995; Conti & Jin, 2000). All four isoforms have been found in the frog heart, with some differences in their distribution between membrane and cytosolic fractions. PDE1 is mainly expressed in the cytosolic fraction, and its activity overcomes any other PDE isoform upon activation of Ca²⁺-calmodulin (Lugnier et al. 1992). PDE2 is mainly expressed in the membrane fraction, and its activity dominates over any other PDE isoform when cGMP is elevated (Simmons & Hartzell, 1988; Lugnier et al. 1992). PDE3 and PDE4 are expressed in both cytosolic (Lugnier et al. 1992) and membrane fractions of frog ventricles (Méry et al. 1990; Brechler et al. 1992; Lugnier et al. 1992). However, while PDE4 is dominant over PDE3 in the cytosol, both PDE3 and PDE4 are equally active in the membrane fraction (Lugnier et al. 1992). This may explain why we found in this study that PDE3 and PDE4 inhibition produced identical effects on cAMP compartmentation in frog myocytes. The biochemical evidence for the presence of PDE2, 3 and 4 isoforms in frog heart and their respective contribution to total PDE activity have been confirmed by functional experiments, in which selective PDE inhibitors were tested for their effects on I_Ca,L under various conditions. For instance, PDE3 and PDE4 inhibitors have no effect on basal I_Ca,L but strongly increase the current after a previous stimulation by ISO or cAMP (Fischmeister & Hartzell, 1990; Méry et al. 1993; this study). Similarly, the PDE2 inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) has no effect on basal or ISO-stimulated I_Ca,L but strongly increases the current after an elevation of intracellular cGMP (Méry et al. 1995; Dittrich et al. 2001). To test for the participation of PDE1 is more difficult, because this isoform lacks a convenient selective inhibitor. However, nimodipine was shown earlier to block PDE1 with a high degree of selectivity among other PDE isoforms (Matsushima et al. 1987; Schachtele et al. 1987; Stoclet et al. 1995). Since nimodipine is also a potent antagonist of L-type Ca²⁺ channels, it cannot be used to evaluate the role of PDE1 while measuring I_Ca,L. However, we were able to examine the contribution of PDE1 to the compartmentation of cAMP, by performing a series of experiments in which we applied nimodipine on top of ISO on one half of the cell only. We found that nimodipine (1 µM) had no significant effect on the distant response of I_Ca,L to 1 µM ISO (-11.0 ± 7.7 % change, n = 4, data not shown), thus excluding a substantial contribution of PDE1 to the maintenance of a cAMP gradient within the cell. The contribution of PDE2 in this process was evaluated earlier using EHNA, a potent PDE2 inhibitor (Méry et al. 1995; Dittrich et al. 2001). In these studies, EHNA (30 µM) had no effect on basal or ISO-stimulated I_Ca,L, whether the drug was applied on the whole cell (Méry et al. 1995) or locally on top of a local application of ISO (Dittrich et al. 2001). Thus, PDE2 does not seem to make a major contribution to the gradient of local vs. distant cAMP concentration, as long as the cGMP cascade is not activated.

Unlike with PDE1 or PDE2, inhibition of PDE3 or PDE4 by milrinone or Ro 20-1724, respectively, at concentrations at which these drugs produce a maximal stimulation of cAMP-elevated I_Ca,L (Fischmeister & Hartzell, 1990) while retaining a high selectivity towards these PDE isoforms (Stoclet et al. 1995), reduced the gradient of local vs. distant cAMP concentration by 4- to 6-fold upon local ISO stimulation. However, inhibition of PDE3 or PDE4 alone produced only half the effect of IBMX, suggesting that each PDE isoform accounted for about half the hydrolytic activity necessary to maintain the large cAMP gradient. Indeed, when both PDEs were inhibited using a combination of milrinone and Ro 20-1724, the gradient of local vs. distant cAMP concentration was reduced to the same extent as with IBMX. Thus, PDE3 and PDE4, but neither PDE1 nor PDE2, contribute equally to the degradation of cAMP in the frog ventricle, hence limiting the amount of cAMP diffusing from the membrane to the cytosol. Surprisingly, okadaic acid, an inhibitor of protein phosphatase (Takai et al. 1987), or H89, an inhibitor of PKA (Chijiwa et al. 1990; Davies et al. 2000), had no apparent effect on the cAMP compartmentation when applied locally together with ISO, although the latter clearly inhibited local PKA-mediated ISO stimulation of I_Ca,L. This suggests that neither PDE3 nor PDE4 activity is regulated by PKA in frog ventricular myocytes, unlike what was found in vitro in mammalian preparations (see e.g. Sette & Conti, 1996; Murthy et al. 2002). Although we do not have an explanation for this observation, one possibility is that the frog heart lacks the upstream conserved region present in the long PDE4 isoforms, which is required for PKA regulation (MacKenzie et al. 2002).

Our demonstration that PDEs may contribute to the compartmentation of the cAMP signalling pathway is supported by several earlier studies (see e.g. Hohl & Li, 1991; Kuschel et al. 1999; Steinberg & Brunton, 2001 for a review). For instance, in mammalian heart, PDE3 and PDE4 were found to be associated to different subcellular structures: PDE4 is associated with the sarcolemma (Okruhlicova et al. 1996, 1998) and nuclear envelope (Lugnier et al. 1999), whereas PDE3 is mainly associated with the sarcoplasmic reticulum (Lugnier et al. 1993). Thus, PDE3 and PDE4 might locally regulate the cAMP level in the vicinity of functional proteins regulated by PKA (Eckly-Michel et al. 1997). Moreover, in guinea-pig ventricle, although rolipram, a selective PDE4 inhibitor, increased cAMP level, it was unable to affect the phosphorylation state of phospholamban and the inhibitory subunit of troponin, supporting the role of PDE4 in cAMP compartmentation (Boknik et al. 1997). More recently, Rich et al. (2000, 2001a,b), in an elegant series of studies using cyclic nucleotide-gated channels as cAMP sensors expressed in two different non-cardiac cell lines (excitable GH4C1 rat pituitary cells or non excitable HEK-293 human embryonic kidney cells), demonstrated (1) the presence of cAMP microdomains near the sarcolemmal membrane with restricted diffusional access of cAMP to the bulk cytosol (Rich et al. 2000, 2001a) and (2) the contribution of PDEs, and more specifically PDE4, to this cAMP compartmentation (Rich et al. 2001b). Many proteins may be involved in this spatial cAMP signal localisation. For instance, A-kinase anchoring proteins (AKAP), which have been shown to localise PKA near its target proteins during -adrenergic regulation of cardiac contractility (Fink et al. 2001; Zaccolo et al. 2002), may also serve to anchor PDE4 isoforms at specific organelles (Dodge et al. 2001; Verde et al. 2001). Finally, recent studies have emphasised the role of caveolae in the spatial organisation of various signalling complexes, which might facilitate the interaction between molecules and contribute to the localisation of the cAMP signal in a restrictive place (Rybin et al. 2000; for reviews see Shaul & Anderson, 1998; Steinberg & Brunton, 2001).

In conclusion, our study demonstrates that PDEs play a dynamic role in shaping the spatial profile of intracellular cAMP concentration in frog cardiac myocytes. Although cAMP is a relatively small molecule which should readily diffuse throughout the cell, we demonstrate that its concentration may be different in different cellular compartments due to the activity of PDE3 and PDE4. PDE activity close to the membrane may limit the amount of cAMP that distributes inside the cell upon activation of ₂-adrenergic receptors, hence affecting mainly PKA substrates that reside in an immediate vicinity of these receptors, such as L-type Ca²⁺ channels. Some support for this hypothesis, as well as a possible molecular rationale to our findings, was recently provided by Perry et al. (2002). They showed that PDE4 binds to -arrestins, and is recruited at the membrane of HEK-293 cells during the process of ₂-adrenergic receptor desensitisation. This mechanism was recently confirmed in neonatal rat ventricular myocytes (Baillie et al. 2003). It is tempting to speculate that such a spatial distribution of PDE activity may vary depending on which membrane receptor is activated. This would provide an elegant mechanism by which a cell could adjust its functional response to different external stimuli acting on the same cAMP signalling cascade.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

An RH, Heath BM, Higgins JP, Koch WJ, Lefkowitz RJ & Kass RS (1999). 2-Adrenergic receptor overexpression in the developing mouse heart: evidence for targeted modulation of ion channels. J Physiol 516, 19-30	[Abstract/Full Text]
Baillie GS, Sood A, McPhee I, Gall I, Perry SJ, Lefkowitz RJ & Houslay MD (2003). -Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates -adrenoceptor switching from Gs to Gi. Proc Natl Acad Sci U S A 100, 941-945
Beavo JA, (1995). Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiol Rev 75, 725-748	[Abstract]
Bers DM , & Ziolo MT (2001). When is cAMP not cAMP? Effects of compartmentalization. Circ Res 89, 373-375	[Full Text]
Boknik P, Neumann J, Schmitz W, Scholz H & Wenzlaff H (1997). Characterization of biochemical effects of CGS 21680C, an A2-adenosine receptor agonist, in the mammalian ventricle. J Cardiovasc Pharmacol 30, 750-758	[Medline]
Bond RA, Leff P, Johnson TD, Milano CA, Rockman HA, McMinn TR, Apparsundaram S, Hyek MF, Kenakin TP, Allen LF & Lefkowitz RJ (1995). Physiological effects of inverse agonists in transgenic mice with myocardial overexpression of the 2-adrenoceptor. Nature 374, 272-276	[Medline]
Brechler V, Pavoine C, Hanf R, Garbarz E, Fischmeister R & Pecker F (1992). Inhibition by glucagon of the cGMP-inhibited low-Km cAMP phosphodiesterase in heart is mediated by a pertussis toxin-sensitive G-protein. J Biol Chem 267, 15496-15501	[Abstract]
Bristow MR, Hershberger RE, Port JD, Minobe W & Rasmussen R (1989). 1-adrenergic and 2-adrenergic receptor-mediated adenylate cyclase stimulation in nonfailing and failing human ventricular myocardium. Mol Pharmacol 35, 295-303	[Abstract]
Brunton LL, Hayes JS & Mayer SE (1979). Hormonally specific phosphorylation of cardiac troponin I and activation of glycogen phosphorylase. Nature 280, 78-80
Buxton ILO , & Brunton LL (1983). Compartments of cyclic AMP and protein kinase in mammalian cardiomyocytes. J Biol Chem 258, 10233-10239	[Abstract]
Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T & Hidaka H (1990). Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5- isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265, 5267-5272	[Abstract]
Conti M , & Jin SLC (2000). The molecular biology of cyclic nucleotide phosphodiesterases. Prog Nucleic Acid Res Mol Biol 63, 2-33
Davies SP, Reddy H, Caivano M & Cohen P (2000). Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351, 95-105	[Medline]
Dittrich M, Jurevicius J, Georget M, Rochais F, Fleischmann BK, Hescheler J & Fischmeister R (2001). Local response of L-type Ca2+ current to nitric oxide in frog ventricular myocytes. J Physiol 534, 109-121	[Abstract/Full Text]
Dodge KL, Khouangsathiene S, Kapiloff MS, Mouton R, Hill EV, Houslay MD, Langeberg LK & Scott JD (2001). mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module. EMBO J 20, 1921-1930	[Abstract/Full Text]
Eckly-Michel A, Martin V & Lugnier C (1997). Involvement of cyclic nucleotide-dependent protein kinases in cyclic AMP-mediated vasorelaxation. Br J Pharmacol 122, 158-164	[Abstract]
Fink MA, Zakhary DR, Mackey JA, Desnoyer RW, Apperson-Hansen C, Damron DS & Bond M (2001). AKAP-mediated targeting of protein kinase A regulates contractility in cardiac myocytes. Circ Res 88, 291-297	[Abstract/Full Text]
Fischmeister R , & Hartzell HC (1987). Cyclic guanosine 3',5'-monophosphate regulates the calcium current in single cells from frog ventricle. J Physiol 387, 453-472	[Abstract]
Fischmeister R , & Hartzell C (1990). Regulation of calcium current by low-Km cyclic AMP phosphodiesterases in cardiac cells. Mol Pharmacol 38, 426-433	[Abstract]
Goaillard J-M, Vincent P & Fischmeister R (2001). Simultaneous measurements of intracellular cAMP and L-type Ca2+ current in single frog ventricular myocytes. J Physiol 530, 79-91	[Abstract/Full Text]
Green SA, Holt BD & Liggett SB (1992). 1- and 2-adrenergic receptors display subtype-selective coupling to Gs. Mol Pharmacol 41, 889-893	[Abstract]
Hamill OP, Marty A, Neher E, Sakmann B & Sigworth FJ (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391, 85-100	[Medline]
Hartzell HC, Méry P-F, Fischmeister R & Szabo G (1991). Sympathetic regulation of cardiac calcium current is due exclusively to cAMP-dependent phosphorylation. Nature 351, 573-576	[Medline]
Heubach JF, Trebess I, Wettwer E, Himmel HM, Michel MC, Kaumann AJ, Koch WJ, Harding SE & Ravens U (1999). L-type calcium current and contractility in ventricular myocytes from mice overexpressing the cardiac 2-adrenoceptor. Cardiovasc Res 42, 173-182	[Medline]
Hohl CM , & Li Q (1991). Compartmentation of cAMP in adult canine ventricular myocytes - relation to single-cell free Ca2+ transients. Circ Res 69, 1369-1379	[Abstract]
Hu EH , & Venter JC (1977). Adenosine cyclic 3',5'-monophosphate concentrations during the positive inotropic response of cat cardiac muscle to polymeric immobilized isoproterenol. Mol Pharmacol 14, 237-245	[Abstract]
Jurevicius J , & Fischmeister R (1996). cAMP compartmentation is responsible for a local activation of cardiac Ca2+ channels by beta-adrenergic agonists. Proc Natl Acad Sci U S A 93, 295-299	[Abstract]
Jurevicius J , & Fischmeister R (1997). Longitudinal distribution of Na+ and Ca2+ channels and -adrenoceptors on the sarcolemmal membrane of frog cardiomyocytes. J Physiol 503, 471-477	[Abstract]
Jurevicius J, Skeberdis VA, Fischmeister R (1997). Characterization of the phosphodiesterase isoforms involved in the compartmentation of cAMP following -adrenoceptor activation in frog cardiac myocytes. Biophys J 72, A23
Kamp TJ , & Hell JW (2000). Regulation of cardiac L-type calcium channels by protein kinase A and protein kinase C. Circ Res 87, 1095-1102	[Abstract/Full Text]
Kuschel M, Zhou YY, Cheng H, Zhang SJ, Chen Y, Lakatta EG & Xiao RP (1999). Gi protein-mediated functional compartmentalization of cardiac 2-adrenergic signaling. J Biol Chem 274, 22048-22052	[Abstract/Full Text]
Levy FO, Zhu X, Kaumann AJ & Birnbaumer L (1993). Efficacy of 1-adrenergic receptors is lower than that of 2-adrenergic receptors. Proc Natl Acad Sci U S A 90, 10798-10802	[Abstract]
Lugnier C, Gauthier C, Lebec A & Soustre H (1992). Cyclic nucleotide phosphodiesterases from frog atrial fibers - isolation and drug sensitivities. Am J Physiol 262, H654-660	[Medline]
Lugnier C, Keravis T, Le Bec A, Pauvert O, Proteau S & Rousseau E (1999). Characterization of cyclic nucleotide phosphodiesterase isoforms associated to isolated cardiac nuclei. Biochim Biophys Acta 1472, 431-446	[Medline]
Lugnier C, Muller B, Lebec A, Beaudry C & Rousseau E (1993). Characterization of indolidan-sensitive and rolipram-sensitive cyclic nucleotide phosphodiesterases in canine and human cardiac microsomal fractions. J Pharmacol Exp Ther 265, 1142-1151	[Abstract]
MacKenzie SJ, Baillie GS, McPhee I, MacKenzie C, Seamons R, McSorley T, Millen J, Beard MB, Vanheeke G & Houslay MD (2002). Long PDE4 cAMP specific phosphodiesterases are activated by protein kinase A-mediated phosphorylation of a single serine residue in Upstream Conserved Region 1 (UCR1). Br J Pharmacol 136, 421-433	[Abstract/Full Text]
Matsushima S, Tanaka T, Saitoh M, Watanabe M & Hidaka H (1987). Different sensitivities of Ca2+, calmodulin-dependent cyclic nucleotide phosphodiesterases from rabbit aorta and brain to dihydropyridine calcium channel blockers. Biochem Biophys Res Comm 148, 1468-1474	[Medline]
Méry P-F, Brechler V, Pavoine C, Pecker F & Fischmeister R (1990). Glucagon stimulates the cardiac Ca2+ current by activation of adenylyl cyclase and inhibition of phosphodiesterase. Nature 345, 158-161	[Medline]
Méry P-F, Pavoine C, Belhassen L, Pecker F & Fischmeister R (1993). Nitric oxide regulates cardiac Ca2+ current - involvement of cGMP-inhibited and cGMP-stimulated phosphodiesterases through guanylyl cyclase activation. J Biol Chem 268, 26286-26295	[Abstract]
Méry P-F, Pavoine C, Pecker F & Fischmeister R (1995). Erythro-9-(2-hydroxy-3-nonyl)adenine inhibits cyclic GMP-stimulated phosphodiesterases in isolated cardiac myocytes. Mol Pharmacol 48, 121-130	[Abstract]
Milano CA, Allen LF, Rockman HA, Dolber PC, McMinn TR, Chien KR, Johnson TD, Bond RA & Lefkowitz RJ (1994). Enhanced myocardial function in transgenic mice overexpressing the 2-adrenergic receptor. Science 264, 582-586
Molenaar P, Bartel S, Cochrane A, Vetter D, Jalali H, Pohlner P, Burrell K, Karczewski P, Krause EG & Kaumann A (2000). Both 2-and 1-adrenergic receptors mediate hastened relaxation and phosphorylation of phospholamban and troponin I in ventricular myocardium of fallot infants, consistent with selective coupling of 2-adrenergic receptors to Gs-protein. Circulation 102, 1814-1821	[Abstract/Full Text]
Murthy KS, Zhou HP & Makhlouf GM (2002). PKA-dependent activation of PDE3A and PDE4 and inhibition of adenylyl cyclase V/VI in smooth muscle. Am J Physiol 282, C508-517
Okruhlicova L, Tribulova N, Eckly A & Lugnier C (1996). Cytochemical distribution of cyclic AMP-dependent 3-,5'-nucleotide phosphodiesterase in the rat myocardium. Histochem J 28, 165-172	[Medline]
Okruhlicova L, Vrbjar N & Lugnier C (1998). Characterization of type 4 cyclic nucleotide phosphodiesterase (PDE4) in cardiac sarcolemma. Exp Clin Cardiol 3, 188-192
Perry SJ, Baillie GS, Kohout TA, McPhee I, Magiera MM, Ang KL, Miller WE, McLean AJ, Conti M, Houslay MD & Lefkowitz RJ (2002). Targeting of cyclic AMP degradation to 2-adrenergic receptors by -arrestins. Science 298, 834-836
Rapundalo ST, (1998). Cardiac protein phosphorylation: functional and pathophysiological correlates. Cardiovasc Res 38, 559-588	[Medline]
Rich TC, Fagan KA, Nakata H, Schaack J, Cooper DM & Karpen JW (2000). Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion. J Gen Physiol 116, 147-161	[Abstract/Full Text]
Rich TC, Fagan KA, Tse TE, Schaack J, Cooper DM & Karpen JW (2001a). A uniform extracellular stimulus triggers distinct cAMP signals in different compartments of a simple cell. Proc Natl Acad Sci U S A 98, 13049-13054	[Abstract/Full Text]
Rich TC, Tse TE, Rohan JG, Schaack J & Karpen JW (2001b). In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors. J Gen Physiol 118, 63-77	[Abstract/Full Text]
Roden DM, Balser JR, George AL & Anderson ME (2002). Cardiac ion channels. Annu Rev Physiol 64, 431-475	[Medline]
Rybin VO, Xu X, Lisanti MP & Steinberg SF (2000). Differential targeting of -adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae: a mechanism to functionally regulate the cAMP signaling pathway. J Biol Chem 275, 41447-41457	[Abstract/Full Text]
Schachtele C, Wagner B & Marme D (1987). Stereoselective inhibition of calmodulin-dependent cAMP phosphodiesterase from bovine heart by (+)- and (-)-nimodipine. Naunyn-Schmied Arch Pharmacol 335, 340-343
Sette C & Conti M (1996). Phosphorylation and activation of a cAMP-specific phosphodiesterase by the cAMP-dependent protein kinase - involvement of serine 54 in the enzyme activation. J Biol Chem 271, 16526-16534	[Abstract/Full Text]
Shaul PW & Anderson RGW (1998). Role of plasmalemmal caveolae in signal transduction. Am J Physiol 19, L843-L851
Simmons MA & Hartzell HC (1988). Role of phosphodiesterase in regulation of calcium current in isolated cardiac myocytes. Mol Pharmacol 33, 664-671	[Abstract]
Skeberdis VA, Jurevicius J & Fischmeister R (1997a). Pharmacological characterization of the receptors involved in the -adrenoceptor-mediated stimulation of the L-type Ca2+ current in frog ventricular myocytes. Br J Pharmacol 121, 1277-1286	[Abstract]
Skeberdis VA, Jurevicius J & Fischmeister R (1997b). 2 adrenergic activation of L-type Ca ++ current in cardiac myocytes. J Pharmacol Exp Ther 283, 452-461	[Abstract/Full Text]
Steinberg SF, (1999). The molecular basis for distinct -adrenergic receptor subtype actions in cardiomyocytes. Circ Res 85, 1101-1111	[Full Text]
Steinberg SF & Brunton LL (2001). Compartmentation of G protein-coupled signaling pathways in cardiac myocytes. Annu Rev Pharmacol Toxicol 41, 751-773	[Medline]
Stoclet JC, Keravis T, Komas N & Lugnier C (1995). Cyclic nucleotide phosphodiesterases as therapeutic targets in cardiovasculasr diseases. Exp Op Invest Drugs 4, 1081-1100
Takai A, Bialojan C, Troschka M & Ruegg JC (1987). Smooth muscle myosin phosphatase inhibition and force enhancement by black sponge toxin. FEBS Lett 217, 81-84	[Medline]
Venter JC, Ross JJ & Kaplan NO (1975). Lack of detectable change in cyclic AMP during the cardiac inotropic response to isoproterenol immobilized on glass beads. Proc Natl Acad Sci U S A 72, 824-828	[Medline]
Verde I, Pahlke G, Salanova M, Zhang G, Wang S, Coletti D, Onuffer J, Jin SlC & Conti M (2001). Myomegalin is a novel protein of the Golgi/centrosome that interacts with a cyclic nucleotide phosphodiesterase. J Biol Chem 276, 11189-11198	[Abstract/Full Text]
Verde I, Vandecasteele G, Lezoualc'h F & Fischmeister R (1999). Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca2+ current in rat ventricular myocytes. Br J Pharmacol 127, 65-74	[Abstract/Full Text]
Waelbroeck M, Taton G, Delhaye M, Chatelain P, Camus JC, Pochet R, Leclerc JL, De Smet JM, Robberecht P & Christophe J (1983). The human heart beta-adrenergic receptors. II. Coupling of beta2-adrenergic receptors with the adenylate cyclase system. Mol Pharmacol 24, 174-182	[Abstract]
Xiao RP, Hohl C, Altschuld R, Jones L, Livingston B, Ziman B, Tantini B & Lakatta EG (1994). 2-adrenergic receptor-stimulated increase in cAMP in rat heart cells is not coupled to changes in Ca2+ dynamics, contractility, or phospholamban phosphorylation. J Biol Chem 269, 19151-19156	[Abstract]
Xiao RP & Lakatta EG (1993). 1-Adrenoceptor stimulation and 2-adrenoceptor stimulation differ in their effects on contraction, cytosolic Ca2+, and Ca2+ current in single rat ventricular cells. Circ Res 73, 286-300	[Abstract]
Zaccolo M & Pozzan T (2002). Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 295, 1711-1715
Zhou YY, Cheng HP, Bogdanov KY, Hohl C, Altschuld R, Lakatta EG & Xiao RP (1997). Localized cAMP-dependent signaling mediates 2-adrenergic modulation of cardiac excitation-contraction coupling. Am J Physiol 42, H1611-1618
Zhou YY, Cheng HP, Song LS, Wang DJ, Lakatta EG & Xiao RP (1999). Spontaneous 2-adrenergic signaling fails to modulate L-type Ca2+ current in mouse ventricular myocytes. Mol Pharmacol 56, 485-493	[Abstract/Full Text]

Acknowledgements

We thank Patrick Lechêne for excellent technical help, and Florence Lefebvre and Isabelle Paic for preparation of the myocytes. This work was supported by an East-West cooperation grant from INSERM, France.

This Article Abstract Full Text (PDF) All Versions of this Article: 551/1/239    most recent jphysiol.2003.045211v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles