Inotropy and L-type Ca2+ current,activated by β1- and β2-adrenoceptors,are differently controlled by phosphodiesterases 3 and 4 in rat heart

Background and purpose

β₁- and β₂-adrenoceptors coexist in rat heart but β₂-adrenoceptor-mediated inotropic effects are hardly detectable, possibly due to phosphodiesterase (PDE) activity. We investigated the influence of the PDE3 inhibitor cilostamide (300 nmol·L⁻¹) and the PDE4 inhibitor rolipram (1 µmol·L⁻¹) on the effects of (−)-catecholamines.

Experimental approach

Cardiostimulation evoked by (−)-noradrenaline (ICI118551 present) and (−)-adrenaline (CGP20712A present) through β₁- and β₂-adrenoceptors, respectively, was compared on sinoatrial beating rate, left atrial and ventricular contractile force in isolated tissues from Wistar rats. L-type Ca²⁺-current (I_Ca-L) was assessed with whole-cell patch clamp.

Key results

Rolipram caused sinoatrial tachycardia. Cilostamide and rolipram did not enhance chronotropic potencies of (−)-noradrenaline and (−)-adrenaline. Rolipram but not cilostamide potentiated atrial and ventricular inotropic effects of (−)-noradrenaline. Cilostamide potentiated the ventricular effects of (−)-adrenaline but not of (−)-noradrenaline. Concurrent cilostamide + rolipram uncovered left atrial effects of (−)-adrenaline. Both rolipram and cilostamide augmented the (−)-noradrenaline (1 µmol·L⁻¹) evoked increase in I_Ca-L. (−)-Adrenaline (10 µmol·L⁻¹) increased I_Ca-L only in the presence of cilostamide but not rolipram.

Conclusions and implications

PDE4 blunts the β₁-adrenoceptor-mediated inotropic effects. PDE4 reduces basal sinoatrial rate in a compartment distinct from compartments controlled by β₁- and β₂-adrenoceptors. PDE3 and PDE4 jointly prevent left atrial β₂-adrenoceptor-mediated inotropy. Both PDE3 and PDE4 reduce I_Ca-L responses through β₁-adrenoceptors but the PDE3 component is unrelated to inotropy. PDE3 blunts both ventricular inotropic and I_Ca-L responses through β₂-adrenoceptors.     

Differential regulation of β2 -adrenoceptor-mediated inotropic and lusitropic response by PDE3 and PDE4 in failing and non-failing rat cardiac ventricle

BACKGROUND AND PURPOSE

β-Adrenoceptors play a major role in regulating myocardial function through cAMP-dependent pathways. Different phosphodiesterases (PDEs) regulate intracellular cAMP-pools and thereby contribute to the compartmentalization of cAMP-dependent effects. We explored the involvement of PDEs in limiting the β₂ adrenoceptor-mediated positive inotropic (PIR) and lusitropic (LR) responses in sham-operated (Sham) and failing rat hearts.

EXPERIMENTAL APPROACH

Extensive myocardial infarctions were induced by coronary artery ligation in Wistar rats. Rats developing heart failure were studied 6 weeks after surgery. Contractility was measured in left ventricular strips from failing and Sham hearts. cAMP was quantified by RIA.

KEY RESULTS

In ventricular strips, stimulation of β₂-adrenoceptors with (-)-adrenaline (300 nM CGP20712A present) exerted a small PIR and LR. In Sham hearts, β₂-adrenoceptor-mediated as well as β₁-adrenoceptor-mediated PIR and LR were increased by selective inhibition of either PDE3 (1 µM cilostamide) or PDE4 (10 µM rolipram). In failing rat hearts, PDE3 inhibition enhanced PIR and LR to both β₁- and β₂-adrenoceptor stimulation while PDE4 inhibition had no effect on these responses despite a significant increase in cAMP levels. Combined PDE3/4 inhibition further enhanced the PIR and LR of β₂- and β₁-adrenoceptor activation both in Sham and failing hearts, compared with PDE3 inhibition alone. PDE4 enzyme activity was reduced in failing hearts.

CONCLUSIONS AND IMPLICATIONS

Both PDE3 and PDE4 attenuated β₂- and β₁-adrenoceptor-mediated contractile responses in Sham hearts. In failing hearts, these responses are attenuated solely by PDE3 and thus even selective PDE3 inhibitors may provide a profound enhancement of β-adrenoceptor-mediated responses in heart failure.     

Phosphodiesterases do not limit β1-adrenoceptor-mediated sinoatrial tachycardia: evidence with PDE3 and PDE4 in rabbits and PDE1–5 in rats

The mammalian heart expresses at least five phosphodiesterases (PDE1–5). Catecholamines produce surges of inotropically relevant cAMP through β₁-adrenoceptor stimulation. cAMP is mainly hydrolysed by PDE3 and/or PDE4 thereby blunting contractility. Basal sinoatrial beating rate in mouse, rat, piglet and rabbit sinoatrial cells is reduced by PDE3 and/or PDE4 through hydrolysis of cAMP. However, in rodents, the tachycardia elicited by catecholamines through production of cAMP by β-adrenoceptor activation is not controlled by PDE3 and PDE4, despite a blunting effect of PDE3 or/and PDE4 on basal sinoatrial beating, but it is unknown whether PDE3 limits catecholamine-evoked tachycardia in the rabbit. Since rabbit sinoatrial cells are an important model for pacemaker research, we investigated whether the positive chronotropic effects of (?)-noradrenaline on spontaneously beating right atria of the rabbit are potentiated by inhibition of PDE3 with cilostamide (300 nM). We also studied the sinoatrial effects of the PDE4 inhibitor rolipram (10 μM) and its influence on the responses to (?)-noradrenaline. For comparison, we investigated the influence of cilostamide and rolipram on the positive inotropic responses to (?)-noradrenaline on rabbit left atria and right ventricular papillary muscles. Cilostamide and concurrent cilostamide?+?rolipram, but not rolipram alone, increased sinoatrial rate by 15% and 31% of the effect of (?)-isoprenaline (200?µM) but the PDE inhibitors did not significantly change the chronotropic potency of (?)-noradrenaline. In contrast in papillary muscle, the positive inotropic effects of (?)-noradrenaline were potentiated 2.4-, 2.6- and 44-fold by cilostamide, rolipram and concurrent cilostamide?+?rolipram, respectively. In left atrium, the positive inotropic effects of (?)-noradrenaline were marginally potentiated by cilostamide, as well as potentiated 2.7- and 32-fold by rolipram and by concurrent cilostamide and rolipram respectively. To compare the influence of PDE1–5 on basal sinoatrial rate and (?)-noradrenaline-evoked tachycardia, we investigated on rat right atria the effects of selective inhibitors. The PDE4 inhibitor rolipram and non-selective inhibitor isobutyl-methylxanthine caused tachycardia with –logEC₅₀s of 7.2 and 5.0 and E _max of 18% and 102% of (?)-isoprenaline, respectively. Rolipram did not change the chronotropic potency of (?)-noradrenaline. At high concentrations (10–30?µM), the PDE1, PDE3 and PDE5 inhibitors 8-methoxymethyl-3-isobutyl-1-methylxanthine, cilostamide and sildenafil, respectively, caused marginal tachycardia but did not significantly change the chronotropic potency of (?)-noradrenaline. The PDE2-selective inhibitor erythro-9-[2-hydroxy-3-nonyl]adenine caused marginal bradycardia at 30?µM and tended to reduce the chronotropic potency of (?)-noradrenaline. Rabbit PDE3 reduces basal sinoatrial rate. Although PDE4 only marginally reduces rate, under conditions of PDE3 inhibition, it further reduces sinoatrial rate. Both PDE3 and PDE4 control atrial and ventricular positive inotropic effects of (?)-noradrenaline. In contrast, neither PDE3 nor PDE4 limit the sinoatrial tachycardia induced by (?)-noradrenaline. In the rat, only PDE4, but not PDE1, PDE2, PDE3 and PDE5, reduces basal sinoatrial rate. None of the five rat PDEs limits the (?)-noradrenaline-evoked tachycardia. Taken together, these results confirm and expand evidence for our proposal that the cAMP-compartment modulating basal sinoatrial rate, controlled by PDE3 and/or PDE4, is different from the PDE-resistant cAMP compartment involved in β₁-adrenoceptor-mediated sinoatrial tachycardia.     

Phosphodiesterases PDE3 and PDE4 jointly control the inotropic effects but not chronotropic effects of (−)-CGP12177 despite PDE4-evoked sinoatrial bradycardia in rat atrium

Acting through a low-affinity site of the β₁-adrenoceptor (β_1LAR), CGP12177 causes sinoatrial tachycardia and positive inotropic effects in left atrium but not in the ventricle of the rat. However, inhibition of either PDE3 or PDE4 also uncovers positive inotropic effects of CGP12177 in ventricle, but whether these phosphodiesterases also control the atrial agonist effects of CGP12177 was unknown. We, therefore, investigated the effects of the PDE3-selective inhibitor cilostamide (300 nM) and PDE4 inhibitor rolipram (1 μM) on the (−)-CGP12177-evoked increases of sinoatrial beating rate and force of paced left atria of the rat. Rolipram (n = 8) increased basal sinoatrial rate by 27 ± 5 bpm but cilostamide (n = 8) had no effect. The chronotropic potency of (−)-CGP12177 (−logEC₅₀M = 7.5) was not changed by rolipram and cilostamide or their combination. (-)-CGP12177 increased left atrial force with intrinsic activity 0.25 compared to (-)-isoprenaline. Rolipram (n = 8) and cilostamide (n = 8) did not change basal force of left atria but concurrent rolipram + cilostamide (n = 8) increased force by 52 ± 9% of the effect of 200 μM (−)-isoprenaline. Neither rolipram nor cilostamide affected the inotropic potency of (−)-CGP12177 (−logEC₅₀M = 7.4) but concurrent rolipram + cilostamide caused potentiation (−logEC₅₀M = 8.2) and converted (-)-CGP12177 into a full agonist compared to (-)-isoprenaline. Cyclic AMP appears to maintain sinoatrial rate and PDE4 elicits bradycardia through hydrolysis of cAMP in a compartment distinct from the β_1LAR-induced cAMP compartment through which (−)-CGP12177 causes tachycardia. In contrast to the (−)-CGP12177-evoked tachycardia, not controlled by PDE3 and PDE4, these isoenzymes jointly reduce (−)-CGP12177-evoked increases of left atrial contractility through β_1LAR.     

Phosphodiesterase PDE3 blunts the positive inotropic and cyclic AMP enhancing effects of CGP12177 but not of noradrenaline in rat ventricle

1.--The cardiostimulant effects of CGP12177, mediated through a beta(1)-adrenoceptor site with low affinity for (-)-propranolol, are potentiated by the nonselective PDE inhibitor IBMX but the role of PDE isoenzymes is unknown. We studied the effects of the PDE3-selective inhibitor cilostamide (300 nM) and PDE4-selective inhibitor rolipram (1 microM) on the positive inotropic and cyclic AMP-enhancing effects of CGP12177 and noradrenaline in right ventricular strips of rat. 2.--CGP12177 (under (-)-propranolol 200 nM) only increased contractile force in the presence of either cilostamide or rolipram with -logEC(50)M 6.7 (E(max)=23% over basal) and 7.1 (E(max)=50%) respectively. The combination of cilostamide and rolipram caused CGP12177 to enhance contractile force with -logEC(50)M=7.7 and E(max)=178%. 3.--The positive inotropic effects of noradrenaline (-logEC(50)M=6.9) were potentiated by rolipram (-logEC(50)M=7.4) but not by cilostamide (-logEC(50)M=7.0). 4.--In the presence of rolipram and (-)-propranolol, noradrenaline (2 microM) and CGP12177 (10 microM) produced matching inotropic effects but failed to increase cyclic AMP levels. 20 microM (-)-noradrenaline increased cyclic AMP levels, a response further enhanced by rolipram. 5.--Both PDE3 and PDE4 of rat ventricle appear to hydrolyse cyclic AMP generated through the low-affinity beta(1)-adrenoceptor site, thereby preventing inotropic responses of CGP12177. When (-)-noradrenaline interacts with the beta(1)-adrenoceptor, the generated cyclic AMP is hydrolysed only by PDE4, thereby reducing cardiostimulation.     

PDE3, but not PDE4, reduces β1- and β2-adrenoceptor-mediated inotropic and lusitropic effects in failing ventricle from metoprolol-treated patients

Background and Purpose

PDE3 and/or PDE4 control ventricular effects of catecholamines in several species but their relative effects in failing human ventricle are unknown. We investigated whether the PDE3-selective inhibitor cilostamide (0.3–1 μM) or PDE4 inhibitor rolipram (1–10 μM) modified the positive inotropic and lusitropic effects of catecholamines in human failing myocardium.

Experimental Approach

Right and left ventricular trabeculae from freshly explanted hearts of 5 non-β-blocker-treated and 15 metoprolol-treated patients with terminal heart failure were paced to contract at 1 Hz. The effects of (-)-noradrenaline, mediated through β₁ adrenoceptors (β₂ adrenoceptors blocked with ICI118551), and (-)-adrenaline, mediated through β₂ adrenoceptors (β₁ adrenoceptors blocked with CGP20712A), were assessed in the absence and presence of PDE inhibitors. Catecholamine potencies were estimated from –logEC₅₀s.

Key Results

Cilostamide did not significantly potentiate the inotropic effects of the catecholamines in non-β-blocker-treated patients. Cilostamide caused greater potentiation (P = 0.037) of the positive inotropic effects of (-)-adrenaline (0.78 ± 0.12 log units) than (-)-noradrenaline (0.47 ± 0.12 log units) in metoprolol-treated patients. Lusitropic effects of the catecholamines were also potentiated by cilostamide. Rolipram did not affect the inotropic and lusitropic potencies of (-)-noradrenaline or (-)-adrenaline on right and left ventricular trabeculae from metoprolol-treated patients.

Conclusions and Implications

Metoprolol induces a control by PDE3 of ventricular effects mediated through both β₁ and β₂ adrenoceptors, thereby further reducing sympathetic cardiostimulation in patients with terminal heart failure. Concurrent therapy with a PDE3 blocker and metoprolol could conceivably facilitate cardiostimulation evoked by adrenaline through β₂ adrenoceptors. PDE4 does not appear to reduce inotropic and lusitropic effects of catecholamines in failing human ventricle.

Linked Article

This article is commented on by Eschenhagen, pp 524–527 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12168     

Ontogenic changes of the control by phosphodiesterase-3 and -4 of 5-HT responses in porcine heart and relevance to human atrial 5-HT4 receptors

Background and purpose:

Atrial inotropic responses to 5-HT mediated through 5-HT₄ receptors fade, presumably through phosphodiesterase (PDE) activity. We investigated the influence of a selective inhibitor of PDE3 (cilostamide) or of PDE4 (rolipram) on the fade of 5-HT responses in atrial muscle.

Experimental approach:

5-HT responses were compared, ex vivo, on sinoatrial beating rate of newborn piglets, porcine atrial and ventricular force, and human atrial force. cAMP levels were assessed in piglet atrium.

Key results:

5-HT-evoked sinoatrial tachycardia did not fade and was not potentiated by cilostamide (300 nmol·L⁻¹) or rolipram (1 µmol·L⁻¹). Inotropic responses to 5-HT faded in atria from piglets, adolescent pigs and humans. Cilostamide reduced atrial fade of 5-HT responses in adolescent pigs and humans but not in newborn piglets. Cilostamide disclosed 5-HT ventricular responses in newborn, but not adolescent pigs. Rolipram reduced fade of atrial 5-HT responses in newborn and adolescent pigs but not in humans. Concurrent cilostamide + rolipram abolished fade of 5-HT responses in porcine left atria and facilitated ventricular 5-HT responses, but did not reduce residual fade in human atrium in the presence of cilostamide. 5-HT-evoked increases in cAMP faded; fade was abolished by concurrent cilostamide + rolipram.

Conclusions and implications:

PDE3-induced control of porcine 5-HT responses differed in atrium and ventricle and changed with age. PDE3 and PDE4 jointly prevented fade of inotropic and cAMP responses to 5-HT in porcine atrium. Unlike porcine atria, only PDE3 induced fade of 5-HT responses in human atria.     

Carvedilol induces greater control of β2- than β1-adrenoceptor-mediated inotropic and lusitropic effects by PDE3, while PDE4 has no effect in human failing myocardium

The β-blockers carvedilol and metoprolol provide important therapeutic strategies for heart failure treatment. Therapy with metoprolol facilitates the control by phosphodiesterase PDE3, but not PDE4, of inotropic effects of catecholamines in human failing ventricle. However, it is not known whether carvedilol has the same effect. We investigated whether the PDE3-selective inhibitor cilostamide (0.3 μM) or PDE4-selective inhibitor rolipram (1 μM) modified the positive inotropic and lusitropic effects of catecholamines in ventricular myocardium of heart failure patients treated with carvedilol. Right ventricular trabeculae from explanted hearts of nine carvedilol-treated patients with terminal heart failure were paced to contract at 1 Hz. The effects of (-)-noradrenaline, mediated through β₁-adrenoceptors (β₂-adrenoceptors blocked with ICI118551), and (-)-adrenaline, mediated through β₂-adrenoceptors (β₁-adrenoceptors blocked with CGP20712A), were assessed in the absence and presence of the PDE inhibitors. The inotropic potency, estimated from –logEC₅₀s, was unchanged for (-)-noradrenaline but decreased 16-fold for (-)-adrenaline in carvedilol-treated compared to non-β-blocker-treated patients, consistent with the previously reported β₂-adrenoceptor-selectivity of carvedilol. Cilostamide caused 2- to 3-fold and 10- to 35-fold potentiations of the inotropic and lusitropic effects of (-)-noradrenaline and (-)-adrenaline, respectively, in trabeculae from carvedilol-treated patients. Rolipram did not affect the inotropic and lusitropic potencies of (-)-noradrenaline or (-)-adrenaline. Treatment of heart failure patients with carvedilol induces PDE3 to selectively control the positive inotropic and lusitropic effects mediated through ventricular β₂-adrenoceptors compared to β₁-adrenoceptors. The β₂-adrenoceptor-selectivity of carvedilol may provide protection against β₂-adrenoceptor-mediated ventricular overstimulation in PDE3 inhibitor-treated patients. PDE4 does not control β₁- and β₂-adrenoceptor-mediated inotropic and lusitropic effects in carvedilol-treated patients.     

Physiological antagonism between ventricular beta 1-adrenoceptors and alpha 1-adrenoceptors but no evidence for beta 2- and beta 3-adrenoceptor function in murine heart

1. Murine left atrium lacks inotropic beta(2)-adrenoceptor function. We investigated whether beta(2)-adrenoceptors are involved in the cardiostimulant effects of (-)-adrenaline on spontaneously beating right atria and paced right ventricular myocardium of C57BL6 mice. We also studied a negative inotropic effect of (-)-adrenaline. 2. Sinoatrial tachycardia, evoked by (-)-adrenaline was resistant to blockade by beta(2)-selective ICI 118,551 (50 nM) but antagonized by beta(1)-selective CGP 20712A (300 nM). This pattern was unaffected by pretreatment with pertussis toxin (PTX, 600 microg kg(-1) i.p. 24 h) which reversed carbachol-evoked bradycardia to tachycardia. 3. Increases of ventricular force by (-)-adrenaline and (-)-noradrenaline were not blocked by ICI 118,551 but antagonized by CGP 20712A. 4. Under blockade of beta-adrenoceptors, (-)-adrenaline and (-)-noradrenaline depressed ventricular force (-logIC(50)M=7.7 and 6.9). The cardiodepressant effects of (-)-adrenaline were antagonized by phentolamine (1 microM) and prazosin (1 microM) but not by (-)-bupranolol (1 microM). Prazosin potentiated the positive inotropic effects of (-)-adrenaline (in the absence of beta-blockers) from -logEC(50)M=6.2 - 6.8. 5. PTX-treatment reduced carbachol-evoked depression of ventricular force in the presence of high catecholamine concentrations. Inhibition of ventricular function of G(i) protein was verified by 82% reduction of in vitro ADP-ribosylation. PTX-treatment tended to increase the positive inotropic potency of (-)-adrenaline under all conditions investigated, including the presence of ICI 118,551. 6. (-)-Adrenaline causes murine cardiostimulation through beta(1)-adrenoceptors but not through beta(2)-adrenoceptors. The negative inotropic effects of (-)-adrenaline are mediated through ventricular alpha(1)-adrenoceptors but not through beta(3)-adrenoceptors. Both G(i) protein and alpha(1)-adrenoceptors restrain (-)-adrenaline-evoked increases in right ventricular force mediated through beta(1)-adrenoceptors.     

The effects of both noradrenaline and CGP12177, mediated through human β1-adrenoceptors, are reduced by PDE3 in human atrium but PDE4 in CHO cells

(-)-Noradrenaline and (-)-CGP12177 activate β₁-adrenoceptors through a high (H)- and low-affinity (L) site, respectively. The positive inotropic effects of (-)-noradrenaline are blunted by phosphodiesterase4 (PDE4) but not PDE3, while both PDE isoenzymes, acting in concert, prevent the effects of (-)-CGP12177 through β₁-adrenoceptors in rat ventricle. We sought to unravel the role of PDE3 and PDE4 on signals through the H and L sites in human myocardium. The kinetics of matching positive inotropic effects of (-)-noradrenaline (20 nM) and (-)-CGP12177 (100 nM) were investigated on human atrial trabeculae in the absence and presence of the PDE3 inhibitor cilostamide (300 nM), PDE4 inhibitor rolipram (1 μM) or both. The influence of cilostamide and rolipram on agonist-evoked cyclic adenosine monophosphate (cAMP) increases were also compared in Chinese hamster ovary (CHO) cells expressing recombinant human β₁-adrenoceptors. (-)-Noradrenaline and (-)-CGP12177 caused matching inotropic responses that faded during a 60-min time course. Cilostamide, but not rolipram, increased the positive inotropic effects and abolished the time dependent fade of both agonists. In CHO cells, rolipram, but not cilostamide, enhanced the cAMP signals caused by both (-)-noradrenaline and (-)-CGP12177. PDE3, but not PDE4, blunts the positive inotropic effects of both (-)-noradrenaline and (-)-CGP12177 through H and L sites, respectively, of human atrial β₁-adrenoceptors. However, in CHO cells, PDE4 blunts the cAMP signals of both (-)-noradrenaline and (-)-CGP12177. Neither CHO cells nor the rat ventricle are appropriate models for the β₁-adrenoceptor-evoked signalling to PDE3 observed in human atrium.     

Regional differences of β1- and β2-adrenoceptor-mediated functions in feline heart

The effects of (-)-adrenaline and (-)-noradrenaline were studied on isolated preparations of kitten heart. To define the contribution of beta 1-adrenoceptors (beta 1AR) and beta 2-adrenoceptors (beta 2AR) we used as tools the highly beta 1AR-selective antagonist CGP 20,712 A and non-linear analysis of antagonism. The beta 2AR-mediated responses to the catecholamines, disclosed by CGP 20,712 A, were verified by blockade with the beta 2AR-selective ICI 118,551. The relative density and contribution of beta 1AR and beta 2AR to (-)-adrenaline- and (-)-noradrenaline-induced adenylyl cyclase stimulation was also estimated in right ventricular membranes. 1. In the sinoatrial pacemaker (-)-adrenaline caused positive chronotropic effects through both beta 1AR and beta 2AR while (-)-noradrenaline does so predominantly through beta 1AR. During beta 1AR blockade (-)-adrenaline did cause the same maximum effects through beta 2AR as (-)-noradrenaline did through beta 1AR. 2. In left atria (-)-adrenaline caused positive inotropic effects predominantly through beta 1AR. CGP 20,712 A also uncovered a beta 2AR component at high (-)-adrenaline concentrations comprising one third of the maximum beta 1AR-mediated response. 3. Receptor binding assays revealed that 80% of right ventricular beta AR were beta 1AR and 20% beta 2AR. Consistent with this finding, around 80% of the adenylyl cyclase stimulation by both (-)-noradrenaline and (-)-adrenaline was mediated through beta 1AR, around 20% through beta 2AR. The positive inotropic effects of (-)-noradrenaline appeared to be nearly exclusively mediated through beta 1AR in right ventricular papillary muscles. 4. The positive inotropic effects of (-)-adrenaline were quite variable with regard to beta 1AR and beta 2AR in right ventricular papillary muscles. Although beta 1AR-mediated effects are predominant in many muscles with only a small contribution of beta 2AR, in some muscles beta 2AR mediated around 50% of the maximum effect elicited through beta 1AR. In 3 out of 17 muscles beta 2AR mediated the same maximum effect of (-)-adrenaline as beta 1AR. 5. On occasion, we found marked beta AR heterogeneity amongst two muscles from the same right ventricle. One muscle only exhibited beta 1AR-mediated effects of (-)-adrenaline whereas in the other muscle maximal effects could be elicited through beta 2AR.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mode of action of (-)-pindolol on feline and human myocardium.

(-)-Pindolol antagonized competitively and to a similar extent the positive inotropic effects of both (-)-noradrenaline and (-)-adrenaline in human ventricular preparations. An equilibrium dissociation constant KD (-log mol 1(-1) = pKD) of 9.2-9.3 was estimated regardless of disease present or agonist used. (-)-Pindolol antagonized competitively the positive inotropic effects of (-)-adrenaline more than those of (-)-noradrenaline in human atrial preparations. pKD values of (-)-pindolol were 9.6 against (-)-adrenaline and 9.1 against (-)-noradrenaline. The results are consistent with a moderate selectivity of (-)-pindolol for beta 2-compared to beta 1-adrenoceptors in human atrium. (-)-Pindolol competed with [3H]-(-)-bupranolol with a pKD of 9.4 for beta-adrenoceptors of human ventricle. Positive inotropic effects of (-)-pindolol were not detected on human atrium or ventricle in a concentration range of 1-1000 nmol 1(-1). The affinity of (-)-pindolol estimated for human myocardial beta-adrenoceptors, its moderate beta 2-selectivity and its lack of intrinsic activity for contractile force agreed with similar characteristics in other species. (-)-Pindolol caused marked positive chronotropic effects in kitten right atria with an intrinsic activity of 0.5 with respect to catecholamines. On kitten left atria it caused only weak positive inotropic effects with an intrinsic activity of 0.1. (-)-Pindolol (0.6-6000 nmol-1) did not cause positive inotropic effects in kitten papillary muscle. The concentration-effect curve for (-)-pindolol on kitten right and left atria was biphasic. Its positive chronotropic and inotropic effects were not blocked by methysergide, suggesting that 5-hydroxytryptamine (5-HT)-receptors were not involved. Low concentrations of antagonists selective for beta 1- and beta 2-adrenoceptors blocked the high sensitivity component but not the low sensitivity component of the positive chronotropic and inotropic effects. The biphasic nature of the positive chronotropic effects of (-)-pindolol in kitten agreed with previous observations made on guinea-pig right atria and support the concept that 3 receptors in the sinoatrial pacemaker contribute to these chronotropic effects: beta 1, beta 2 and a low-affinity receptor for (-)-pindolol which is neither beta 1 nor beta 2. The partial agonistic activity of (-)-pindolol in the heart appears to be mainly (kitten) or completely (man) restricted to the sinoatrial pacemaker.     

Pertussis toxin suppresses carbachol-evoked cardiodepression but does not modify cardiostimulation mediated through β1- and putative β4-adrenoceptors in mouse left atria: no evidence for β2- and β3-adrenoceptor function

Activation of beta1-, beta2-, beta 3- and putative beta4-adrenoceptors modifies cardiac function. These receptors are usually coupled to Gs protein, but beta2- and beta3-adrenoceptors could also couple to Gi/o proteins. The mouse heart is used increasingly for studies of genetically disrupted or overexpressed proteins, including beta-adrenoceptor subtypes. We therefore investigated in contracting mouse left atria (2 Hz, 37 degrees C) if inactivation of Gi/o proteins with pertussis toxin modifies or uncovers effects mediated through beta-adrenoceptor subtypes. The negative inotropic effects of carbachol in atria exposed to catecholamine or high calcium (6.8 mmol/l) were assumed to be mediated through activation of muscarinic receptors coupled to Gi/o. We report conditions under which incubation of left atria with 200 ng/ml pertussis toxin for 24 h nearly abolished the carbachol responses. Although it has been reported that muscarinic receptor-mediated cardiodepression has an obligatory contribution of nitric oxide, the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (0.1-1 mmol/l) did not modify the negative inotropic effects of carbachol, inconsistent with an involvement of nitric oxide. The positive inotropic effects of (-)-noradrenaline and (-)-adrenaline, mediated through beta1-adrenoceptors, were not affected by pertussis toxin. (-)-Adrenaline did not cause positive inotropic effects attributable to beta2-adrenoceptor-mediation, in the presence of CGP 20712A (300 nmol/l) to block beta1-adrenoceptors, in control atria or atria pretreated with pertussis toxin. The positive inotropic effects of (-)-CGP 12177 (1 micromol/l), a compound with agonist activity at the putative beta4-adrenoceptor, were unaffected by pertussis toxin. The beta3-adrenoceptor-selective agonist BRL 37344 (1 micromol/l), in the presence of (-)-propranolol (200 nmol/l), did not cause positive or negative inotropic effects in control and pertussis toxin-treated atria. In left atria obtained from mice injected with 150 microg/kg i.p. pertussis toxin which abolished carbachol-evoked cardiode-pression, the positive inotropic effects of (-)-adrenaline were antagonised by CGP 20712A. The beta2-adrenoceptor-selective antagonist ICI 118551 (50 nmol/l) did not cause additional blockade of the effects of high (-)-adrenaline concentrations in the presence of CGP 20712A, ruling out the involvement of beta2-adrenoceptors. The results with intraparenteral PTX validate our in vitro PTX method. We conclude that inhibition of murine Gi/o proteins does not alter atrial positive inotropic effects mediated through beta1- and putative beta4-adrenoceptors and does not reveal functional beta2- and beta3-adrenoceptors.     

Functional antagonism of β-adrenoceptor subtypes in the catecholamine-induced automatism in rat myocardium

BACKGROUND AND PURPOSE

Myocardial automatism and arrhythmias may ensue during strong sympathetic stimulation. We sought to investigate the relevant types of adrenoceptor, as well as the role of phosphodiesterase (PDE) activity, in the production of catecholaminergic automatism in atrial and ventricular rat myocardium.

EXPERIMENTAL APPROACH

The effects of adrenoceptor agonists on the rate of spontaneous contractions (automatic response) and the amplitude of electrically evoked contractions (inotropic response) were determined in left atria and ventricular myocytes isolated from Wistar rats.

KEY RESULTS

Catecholaminergic automatism was Ca²⁺-dependent, as it required a functional sarcoplasmic reticulum to be exhibited. Although both α- and β-adrenoceptor activation caused inotropic stimulation, only β₁-adrenoceptors seemed to mediate the induction of spontaneous activity. Catecholaminergic automatism was enhanced and suppressed by β₂-adrenoceptor blockade and stimulation respectively. Inhibition of either PDE3 or PDE4 (by milrinone and rolipram, respectively) potentiated the automatic response of myocytes to catecholamines. However, only rolipram abolished the attenuation of automatism produced by β₂-adrenoceptor stimulation.

CONCLUSIONS AND IMPLICATIONS

α- and β₂-adrenoceptors do not seem to be involved in the mediation of catecholaminergic stimulation of spontaneous activity in atrial and ventricular myocardium. However, a functional antagonism of β₁- and β₂-adrenoceptor activation was identified, the former mediating catecholaminergic myocardial automatism and the latter attenuating this effect. Results suggest that hydrolysis of cAMP by PDE4 is involved in the protective effect mediated by β₂-adrenoceptor stimulation.     

Cardiostimulant and cardiodepressant effects through overexpressed human β<Subscript>2</Subscript>-adrenoceptors in murine heart: regional differences and functional role of β<Subscript>1</Subscript>-adrenoceptors

(-)-Isoprenaline enhances cardiac contractility through beta-adrenoceptors. However, in cardiac tissue from transgenic mice with a 200-400-fold cardiac overexpression of the human beta(2)-adrenoceptor (TG4) we observed a pronounced cardiodepression at high (-)-isoprenaline concentrations. Here, we investigated the functional role of the coexisting beta(1)-, beta(2)-, and beta(3)-adrenoceptor subtypes in several regions of the TG4 heart, and in particular their contribution to the negative inotropic effect. In paced TG4 left atria, (-)-isoprenaline produced bell-shaped concentration-effect curves increasing (-logEC(50)M=9.0) and decreasing (-logIC(50)M=6.4) contractile force. These effects were unaffected by the beta(1)-selective CGP 20712A (300 nM). The beta(2)-selective inverse agonist ICI 118,551 (30-1,000 nM) antagonised in surmountable manner both the positive and negative inotropic effects of (-)-isoprenaline with similar concentration-dependence, consistent with an exclusive mediation through beta(2)-adrenoceptors. The beta(3)-adrenoceptor-selective agonist BRL37344 (1 nM-10 microM) failed to produce significant inotropic effects in TG4 left atria. Subsequently, we measured left atrial action potentials accompanying the inotropic changes induced by (-)-isoprenaline. Action potentials tended to have shorter duration in left atria from TG4 mice than from non-transgenic littermate mice. However, (-)-isoprenaline prolonged the duration of 30% repolarisation in atria from non-transgenic littermate but not from TG4 mice, while 90% repolarisation was abbreviated in both groups of atria. Negative inotropic effects of (-)-isoprenaline were also observed in right ventricular preparations. Pertussis toxin-treatment of the mice abolished the negative inotropic effects in left atria and reduced cardiodepression in right ventricle, indicating an involvement of beta(2)-adrenoceptor coupling to PTX-sensitive G-proteins. In additional experiments, designed to study the native murine beta(1)-adrenoceptor function, we used the physiological beta(1)-adrenoceptor agonist (-)-noradrenaline. In the presence of 600 nM ICI 118,551 we failed to find a functional role of the beta(1)-adrenoceptors in left atria, and detected only a marginal contribution to the positive chronotropic effect in right atria. We also investigated the effects of the non-conventional partial agonist (-)-CGP 12177 (0.2 nM-6 microM), which in wild-type mice causes tachycardia through beta(1)-adrenoceptors. In TG4 right atria, however, (-)-CGP 12177-evoked tachycardia was resistant to blockade by CGP 20712A but antagonised by ICI 118,551, consistent with mediation through human beta(2)-adrenoceptors.The results from TG4 mice suggest that the positive and negative inotropic effects of (-)-isoprenaline are mediated through human overexpressed beta(2)-adrenoceptors coupled to G(s) protein and G(i) protein, respectively. The (-)-isoprenaline-evoked shortening of the atrial action potential combined with reduced responses of L-type Ca(2+) current may contribute to the negative inotropic effects. The function of murine cardiac beta(1)-adrenoceptors is suppressed by overexpressed human beta(2)-adrenoceptors.     

Cilostamide potentiates more the positive inotropic effects of (−)-adrenaline through β2-adrenoceptors than the effects of (−)-noradrenaline through β1-adrenoceptors in human atrial myocardium

Activation of both β₁- and β₂-adrenoceptors increases the contractility of human atrial myocardium through cyclic AMP-dependent pathways. Cyclic AMP is hydrolised by phosphodiesterases, but little is known about which isoenzymes catalyse inotropically relevant cyclic AMP accumulated upon stimulation of β-adrenoceptor subtypes. We have compared the positive inotropic effects of (−)-noradrenaline and (−)-adrenaline, mediated through β₁- and β₂-adrenoceptors, respectively, in the absence and presence of the PDE3 inhibitor cilostamide (300 nM) or PDE4 inhibitor rolipram (1 μM) on human atrial trabeculae from non-failing hearts. Cilostamide, but not rolipram, potentiated the effects of both (−)-noradrenaline and (−)-adrenaline. Cilostamide increased the −logEC₅₀M of (−)-adrenaline more than of (−)-noradrenaline (P < 0.05), regardless of whether or not the patients had been chronically treated with β-blockers. The results are consistent with a greater PDE3-catalysed hydrolysis of inotropically relevant cyclic AMP produced through β₂-adrenoceptors than β₁-adrenoceptors in human atrium.     

The β2-adrenoceptor antagonist CGP 20712 A unmasks β2-adrenoceptors activated by (−)-adrenaline in rat sinoatrial node

Summary The role of sinoatrial ₁- and ₂-adrenoceptors mediating positive chronotropic effects of (–)-adrenaline and (–)-noradrenaline was investigated in rat right atria. Concentration effect curves for (–)-adrenaline, but not for (–)-noradrenaline, became biphasic in the presence of the ₁-adrenoceptors antagonist CGP 20712 A. The curves for (–)-adrenaline in the presence of 300 nmol/l CGP 20712 A (equivalent to 1,000 times its K _B, K _B=0.3 nmol/l for ₁-adrenoceptors) comprise a high-sensitivity component that saturates at 1/4 of maximum effect, and a low sensitivity component. The high-sensitivity component is blocked by the ₂-adrenoceptor-selective antagonist ICI 118,551. These results are consistent with an involvement in the rat of both ₁-adrenoceptors (to a major extent) and ₂-adrenoceptors [only at high (–)-adrenaline concentrations] in the positive chronotropic effects of (–)-adrenaline. (–)-Noradrenaline appears to activate mostly rat sinoatrial ₁-adrenoceptors.     

Differences between the third cardiac beta-adrenoceptor and the colonic beta 3-adrenoceptor in the rat.

1. The heart of several species including man contains atypical beta-adrenoceptors, in addition to coexisting beta 1- and beta 2-adrenoceptors. We now asked the question whether or not the third cardiac beta-adrenoceptor is identical to the putative beta 3-adrenoceptor. We compared the properties of the third cardiac beta-adrenoceptor with those of beta 3-adrenoceptors in isolated tissues of the rat. To study the third cardiac beta-adrenoceptor we used spontaneously beating right atria, paced left atria and paced left ventricular papillary muscles. As a likely model for putative beta 3-adrenoceptors we studied atypical beta-adrenoceptors of the colonic longitudinal muscle precontracted with 30 mM KCl. We used beta 3-adrenoceptor-selective agonists, antagonists and non-conventional partial agonists (ie high-affinity blockers of both beta 1- and beta 2-adrenoceptors know to exert also stimulant effects through beta 3-adrenoceptors). 2. The non-conventional partial agonist (-)-CGP 12177 caused positive chronotropic effects in right atria (pD2 = 7.3) and positive inotropic effects in left atria (pD2 = 7.5). The stimulant effects of (-)-CGP 12177 were resistant to blockade by 200 nM-2 microM (-)-propranolol and 3 microM ICI 118551 (a beta 2-selective antagonist) but antagonized by 1 microM (-)-bupranolol (pKB = 6.4-6.8), 3 microM CGP 20712A (a beta 1-selective antagonist) (pKB = 6.3-6.4) and 6.6 microM SR 59230A (a beta 3-selective antagonist, pKB = 5.1-5.4). 3. The non-conventional partial agonist cyanopindolol caused positive chronotropic effects in right atria (pD2 = 7.7) and positive inotropic effects in left atria (pD2 = 7.1). The stimulant effects of cyanopindolol were resistant to blockade by 200 nM (-)-propranolol but antagonized by 1 microM (-)-bupranolol (pKB = 6.8-7.1). 4. Neither (-)-CGP 12177 nor cyanopindolol caused stimulant effects in papillary muscles at concentrations between 0.2 nM and 20 microM. 5. In the presence of 200 nM (-)-propranolol the beta 3-adrenoceptor-selective agonists BRL 37344 (6 microM), SR 58611A (6 microM), ZD 2079 (60 microM) and CL 316243 (60 microM) did not cause stimulant effects or modify the potency and efficacy of the effects of (-)-CGP 12177 in right and left atria. The combination of 2 microM (-)-propranolol and 2 microM (-)-noradrenaline did not modify the chronotropic potency and efficacy of (-)-CGP 12177 compared to the potency and efficacy in the presence of 2 microM (-)-propranolol alone. 6. (-)-CGP 12177 relaxed the colon with a pD2 of 6.9 and a maximum effect of 55% compared to (-)-isoprenaline. The relaxant effects of (-)-CGP 12177 were resistant to blockade by 200 nM (-)-propranolol, 3 microM CGP 20712A, 3 microM ICI 118551 but blocked by 2 microM (-)-propranolol (pKB = 6.0), 1 microM (-)-bupranolol (pKB = 6.4) and 3 microM SR 59230A (pKB = 6.3). In the presence of 200 nM (-)-propranolol, (-)-CGP 12177 (20 microM) antagonized surmountably the relaxant effects of BRL 37344 (pKP = 7.3) (-)-noradrenaline (pKP = 7.0); and CL 316243 (pKP = 7.0). 7. Cyanopindolol in the presence of 200 nM (-)-propranolol relaxed the colon with a pD2 of 7.0 and a maximum effect of 40% compared to (-)-isoprenaline. As expected from a partial agonist, cyanopindolol antagonized the relaxant effects of both BRL 37344 and CL 316243 with a pKP = 7.6 and (-)-noradrenaline with a pKP = 7.4. 8. The following beta 3-adrenoceptor-selective agonists were potent colonic relaxants (pD2 values between parentheses): BRL 37344 (9.1), ZD 2079 (7.0), CL 316243 (9.0) and SR 58611A (8.2). The relaxant effects of these agonists were only marginally affected by 200 nM (-)-propranolol, not blocked by 3 microM CGP 20712A or 3 microM ICI 118551, and blocked by SR 59230A 3 microM (pKB = 6.9-7.5), 1 microM (-)-bupranolol (pKB = 6.2-6.4) and 2 microM (-)-propranolol (pKB = 6.3-6.5). 9...     

Direct labelling of myocardial β 1-adrenoceptors

A radioligand that selectively labels beta 1-adrenoceptors, 3H-(-)-bisoprolol (3H-BIS), is introduced. The binding properties of 3H-BIS to membrane particles of kitten heart are compared with the blocking properties of (-)-bisoprolol against stimulant effects of (-)-adrenaline and (-)-noradrenaline in heart preparations of kitten and guinea pig. 1. On kitten heart tissues (-)-bisoprolol antagonized the positive chronotropic and inotropic effects of catecholamines competitively. The effects of (-)-adrenaline were antagonized considerably less by (-)-bisoprolol than the corresponding effects of (-)-noradrenaline on sinoatrial pacemakers. The antagonism was nearly the same against both (-)-adrenaline and (-)-noradrenaline in left atria and papillary muscles. The data were analyzed with a model for 2-receptor subtypes by non-linear regression. Equilibrium dissociation constants KB (mol/l; -log KB = pKB) for a high-affinity beta 1-adrenoceptor of 8.8 and for a low-affinity beta 2-adrenoceptor of 7.0 were estimated in the three classes of tissues. In kitten sinoatrial pacemaker beta 1-adrenoceptors contribute 76% to the stimulus induced by (-)-adrenaline and 97% to the stimulus induced by (-)-noradrenaline. In ventricle and left atrium beta 1-adrenoceptors contribute 97-99% and 100% to the stimulus caused by (-)-adrenaline and (-)-noradrenaline, respectively. 2. Both 3H-BIS and unlabelled (-)-bisoprolol caused competitive blockade of the positive chronotropic effects of (-)-noradrenaline in guinea-pig right atria. pKB-values of 8.7 were estimated for both unlabelled and tritiated (-)-bisoprolol. The positive chronotropic effects of (-)-adrenaline were antagonized considerably less by (-)-bisoprolol than those of (-)-noradrenaline in guinea-pig atria. In the presence of low concentrations of beta 2-selective ICI 118,551, which did not antagonize beta 1-adrenoceptor mediated effects, (-)-bisoprolol antagonized positive chronotropic effects of (-)-adrenaline to the same extent as those of (-)-noradrenaline. The results are consistent with the concept of a significant role of sinoatrial beta 2-adrenoceptors of guinea pig for the effects of (-)-adrenaline but not for those of (-)-noradrenaline. 3. 3H-BIS associated and dissociated quickly with and from ventricular beta 1-adrenoceptors. A koff of 1.0 min-1 was estimated. An equilibrium dissociation constant pKL* of 8.2 for 3H-BIS was estimated from saturation binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

5-HT4-elicited positive inotropic response is mediated by cAMP and regulated by PDE3 in failing rat and human cardiac ventricles

Background and purpose:

The left ventricle in failing hearts becomes sensitive to 5-HT parallelled by appearance of functional G_s-coupled 5-HT₄ receptors. Here, we have explored the regulatory functions of phosphodiesterases in the 5-HT₄ receptor-mediated functional effects in ventricular muscle from failing rat and human heart.

Experimental approach:

Extensive myocardial infarctions were induced by coronary artery ligation in Wistar rats. Contractility was measured in left ventricular papillary muscles of rat, 6 weeks after surgery and in left ventricular trabeculae from explanted human hearts. cAMP was quantified by RIA.

Key results:

In papillary muscles from postinfarction rat hearts, 5-HT₄ stimulation exerted positive inotropic and lusitropic effects and increased cAMP. The inotropic effect was increased by non-selective PDE inhibition (IBMX, 10 μM) and selective inhibition of PDE3 (cilostamide, 1 μM), but not of PDE2 (EHNA, 10 μM) or PDE4 (rolipram, 10 μM). Combined PDE3 and PDE4 inhibition enhanced inotropic responses beyond the effect of PDE3 inhibition alone, increased the sensitivity to 5-HT, and also revealed an inotropic response in control (sham-operated) rat ventricle. Lusitropic effects were increased only during combined PDE inhibition. In failing human ventricle, the 5-HT₄ receptor-mediated positive inotropic response was regulated by PDEs in a manner similar to that in postinfarction rat hearts.

Conclusions and implications:

5-HT₄ receptor-mediated positive inotropic responses in failing rat ventricle were cAMP-dependent. PDE3 was the main PDE regulating this response and involvement of PDE4 was disclosed by concomitant inhibition of PDE3 in both postinfarction rat and failing human hearts. 5-HT, PDE3 and PDE4 may have pathophysiological functions in heart failure.