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(Circulation. 1998;97:1062-1070.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Evidence for cAMP-Independent Mechanisms Mediating the Effects of Adrenomedullin, a New Inotropic Peptide

István Szokodi, MD; Pietari Kinnunen, MD; Pasi Tavi, MSc; Matti Weckström, MD, PhD; Miklós Tóth, MD, PhD; ; Heikki Ruskoaho, MD, PhD

From the Department of Cardiovascular Surgery and First Department of Internal Medicine, Semmelweis University Medical School, Budapest, Hungary (I.S., M.T.), and Departments of Pharmacology and Toxicology (P.K., H.R.) and Physiology (P.T., M.W.), Biocenter Oulu, University of Oulu, Finland.

Correspondence to Heikki Ruskoaho, MD, PhD, Department of Pharmacology and Toxicology, University of Oulu, Kajaanintie 52 D, FIN-90220 Oulu, Finland. E-mail heikki.ruskoaho{at}oulu.fi

	Abstract

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Background—Adrenomedullin (ADM), a new vasorelaxing and natriuretic peptide, may function as an endogenous regulator of cardiac function, because ADM and its binding sites have been found in the heart. We characterize herein the cardiac effects of ADM as well as the underlying signaling pathways in vitro.

Methods and Results—In isolated perfused, paced rat heart preparation, infusion of ADM at concentrations of 0.1 to 1 nmol/L for 30 minutes induced a dose-dependent, gradual increase in developed tension, whereas proadrenomedullin N-20 (PAMP; 10 to 100 nmol/L), a peptide derived from the same gene as ADM, had no effect. The ADM-induced positive inotropic effect was not altered by a calcitonin gene–related peptide (CGRP) receptor antagonist, CGRP_8–37, or H-89, a cAMP-dependent protein kinase inhibitor. ADM also failed to stimulate ventricular cAMP content of the perfused hearts. Ryanodine (3 nmol/L), a sarcoplasmic reticulum Ca²⁺ release channel opener, suppressed the overall ADM-induced positive inotropic effect. Pretreatment with thapsigargin (30 nmol/L), which inhibits sarcoplasmic reticulum Ca²⁺ ATPase and depletes intracellular Ca²⁺ stores, attenuated the early increase in developed tension produced by ADM. In addition, inhibition of protein kinase C by staurosporine (10 nmol/L) and blockade of L-type Ca²⁺ channels by diltiazem (1 µmol/L) significantly decreased the sustained phase of ADM-induced increase in developed tension. Superfusion of atrial myocytes with ADM (1 nmol/L) in isolated left atrial preparations resulted in a marked prolongation of action potential duration between 10 and -50 mV transmembrane voltage, consistent with an increase in L-type Ca²⁺ channel current during the plateau.

Conclusions—Our results show that ADM enhances cardiac contractility via cAMP-independent mechanisms including Ca²⁺ release from intracellular ryanodine- and thapsigargin-sensitive Ca²⁺ stores, activation of protein kinase C, and Ca²⁺ influx through L-type Ca²⁺ channels.

Key Words: adrenomedullin • contractility • calcium • peptides • signal transduction

	Introduction

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Adrenomedullin is a newly discovered, potent, vasorelaxing and natriuretic peptide that was originally isolated from human pheochromocytoma.¹ The peptide, consisting of 52 amino acids in humans and 50 amino acids in the rat, is classified in the CGRP family.^{2 3} ADM may function as a paracrine and/or autocrine factor in the regulation of cardiac function, because high mRNA expression,⁴ a considerable amount of ADM-like immunoreactivity,^{5 6 7} and a high level of ¹²⁵I-ADM binding⁸ have been found in the heart. In agreement with this hypothesis, ADM has been reported to increase cardiac output and left ventricular contractility in vivo^{9 10} and exert a direct inotropic effect in vitro.¹¹ Recently, the plasma concentration of circulating ADM has been shown to be increased in patients with congestive heart failure.^{5 12 13 14} Moreover, Jougasaki et al⁵ reported that immunohistochemical staining for ADM is significantly increased in the failing human ventricular myocardium compared with the normal human ventricle. These observations suggest that circulating or locally produced ADM may act within the heart to enhance myocardial contractility, in addition to its hypotensive and natriuretic effects. However, the signal transduction pathway of the cardiac effect of this new, possibly endogenous inotropic factor is unknown.

Originally, ADM was purified by monitoring its ability to elevate adenosine 3',5'-cAMP levels in rat platelets.¹ Later, the peptide was shown to induce cAMP accumulation in a variety of cultured cells, including smooth muscle cells,^{15 16} endothelial cells,¹⁷ and glomerular mesangial cells.¹⁸ Similarly, ADM has been reported to stimulate cAMP formation in isolated cardiac myocytes.^{19 20} These observations suggest that activation of the adenylate cyclase–cAMP system, which is one of the major pathways for the regulation of cardiac contractility in the mammalian heart,²¹ may also mediate the cardiac effects of ADM. However, recent findings that ADM inhibits adrenocorticotropin secretion in anterior pituitary cells²² and increases intracellular Ca²⁺ levels in aortic endothelial cells¹⁷ independently of its effect on cAMP suggest the involvement of other signaling pathways in mediating the effects of ADM, at least in some tissues.

The aim of the present study was to characterize the role of several signaling pathways involved in the positive inotropic effect of ADM by using isolated perfused rat heart and left atrial preparations. We examined the role of cAMP as a second messenger for ADM-induced increase in contractile force using H-89, a cAMP-dependent protein kinase inhibitor,²³ and by measuring cAMP production in ventricles in response to ADM infusion. Because these studies showed that cAMP does not appear to mediate the positive inotropic effect of ADM, we focused on other known mechanisms regulating excitation-contraction coupling, including Ca²⁺ release from SR, activation of protein kinase C, and Ca²⁺ influx through L-type Ca²⁺ channels.²¹ Finally, because a considerable amount of ADM-like immunoreactivity has been found in cardiac atria,⁶ the main storage site for ANP,²⁴ we hypothesized that ADM may influence ANP secretion. Therefore, we examined the direct effect of ADM on ANP secretion in perfused rat hearts.

	Methods

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Drugs
Drugs used were rat ADM_1–50 (Peninsula Laboratories Europe); rat PAMP (prodepin) and rat CGRP_8–37 (Phoenix Pharmaceuticals, Inc); N-[2-((p-bromo-cinnamyl)amino)ethyl]-5-isoquinoline-sulfonamide (H-89) (Seigaku Corp); ryanodine and thapsigargin (Calbiochem); staurosporine and isoproterenol hydrochloride (Sigma Chemical Co); diltiazem hydrochloride (Orion Pharmaceutical Ltd); and heparin (Leiras). ADM, PAMP, CGRP_8–37, ryanodine, isoproterenol, and diltiazem were dissolved in 0.9% saline; H-89, thapsigargin, and staurosporine were dissolved in DMSO. The final concentration of each solvent was <0.003%. The addition of an appropriate concentration of each solvent caused no significant change in hemodynamic variables.

Animals
Male Sprague-Dawley rats (weighing 210 to 270 and 290 to 400 g for isolated perfused rat heart preparation and atrial superfusion, respectively) from the Center for Experimental Animals at the University of Oulu were used. The rats were housed in plastic cages in a room with controlled 40% humidity and temperature of 22°C. A 12-hour light/dark cycle was maintained. The experimental design was approved by the Animal Experimentation Committee of the University of Oulu.

Isolated Perfused Rat Heart Preparation
The isolated perfused rat heart preparation used in the present study was similar to that described previously.²⁵ Briefly, 20 minutes after intraperitoneal injection of heparin (500 IU/kg body weight), rats were decapitated, and hearts were quickly removed and arranged for retrograde perfusion by the Langendorff technique. The hearts were perfused with a modified Krebs-Henseleit bicarbonate buffer, pH 7.40, equilibrated with 95% O₂-5% CO₂ at 37°C. The composition of the buffer was (in mmol/L) NaCl 113.8, NaHCO₃ 22.0, KCl 4.7, KH₂PO₄ 1.2, MgSO₄ 1.1, CaCl₂ 2.5, and glucose 11.0.

Contractile force (apicobasal displacement) was obtained by connecting a force-displacement transducer (Grass Instruments, model FT03) to the apex of the heart at an initial preload stretch of 2 g. Heart rate was counted from contractions by the Grass tachograph and was increased 15% to 20% above the spontaneous beating frequency by use of a Grass stimulator (model S88, 11 V, 0.5 ms). Perfusion pressure reflecting coronary vascular resistance was measured by a pressure transducer (Micron Instruments, model MP-15) situated on a side arm of the aortic cannula. All recordings were made with the use of a Grass 7DA polygraph. Each experiment was started by perfusing the hearts for 60 minutes (equilibration period) using a flow rate of 7 mL/min with a peristaltic pump (Minipuls 3, model 312). To exclude any secondary effects caused by the vasorelaxation of the coronary arteries induced by ADM,⁹ the vasculature was dilated by decreasing the perfusion rate to 5 mL/min before the initiation of the experimental protocol, as described previously.²⁵

Experimental Design
A 10-minute control period was followed by addition of vehicle, ADM, or various drugs in combination with vehicle or ADM into the aortic perfusion cannula as a continuous infusion via an infusion pump (Skyelectronics, model Secan PSA 55) at a rate of 0.5 mL/min for 30 minutes. All hearts were used only for one experiment, and the study was conducted in a controlled and randomized manner, ie, vehicle and drugs were run concomitantly and randomly. In an initial set of experiments, we determined the concentration-dependent inotropic effects of ADM (0.1 to 1 nmol/L) as well as PAMP (10 to 100 nmol/L), a peptide derived from the same gene as ADM.²⁶ CGRP_8–37, a CGRP receptor antagonist, was infused at a concentration of 100 nmol/L because this concentration significantly inhibited cAMP and NO generation stimulated by ADM in cytokine-activated cardiac myocytes.¹⁹

For signal transduction studies, ADM was infused at a concentration of 1 nmol/L, evoking maximal effect on contractility in the isolated perfused rat heart preparation. The concentration of H-89 (100 nmol/L) was chosen because this concentration has been shown to attenuate cAMP-dependent protein kinase activity in pheochromocytoma cells.²⁷ To validate the effect of H-89 as a protein kinase A inhibitor in the present study, in a separate series of experiments, 1 µmol/L isoproterenol was infused alone or in combination with H-89. Ryanodine, an SR Ca²⁺ release channel opener,^{28 29} was infused at a concentration of 3 nmol/L to avoid a marked effect on contractility. The concentrations of thapsigargin (30 nmol/L), staurosporine (10 nmol/L), and diltiazem (1 µmol/L) were chosen because these concentrations were shown to inhibit SR Ca²⁺-adenosinetriphosphatase (Ca²⁺-ATPase) in microsomes from hepatocytes³⁰ and COS cells,³¹ suppress protein kinase C activity,³² and block L-type Ca²⁺ channels³³ in the isolated rat heart preparation, respectively.

Atrial Superfusion, Electrophysiological Recordings, and Analysis
The left atrial appendage was prepared as described in detail previously.³⁴ Atrium, placed in a constant-temperature (37°C) organ bath, was superfused with a modified Krebs-Henseleit bicarbonate buffer (composition described above) at a flow rate of 3 mL/min with a peristaltic pump (Cole-Parmer Instrument, model 7553–85). Glass microelectrodes filled with a solution containing 2 mol/L K-acetate and 5 mmol/L KCl, pH 7.0, and having input resistance of 70 to 120 M were used for membrane potential recordings. The atrial appendage was quiescent unless stimulated electrically through bipolar Ag/AgCl electrodes placed in contact with the auricle. Electrical stimulation (steps of 1-ms duration, 50% over threshold voltage) was provided by a stimulator (Grass Instruments, model S44). All electrical signals were amplified with an intracellular amplifier (Dagan model 8100–1) and stored by a DAT recorder (Biologic DTR-1800). Data analysis was done with DT VEE (Data Translation Inc) and MATLAB (The Math Inc Natick) software programs. Sampling frequency was 3 kHz in all recordings.

Measurement of Tissue cAMP and Immunoreactive ANP in the Perfusate
For measurement of ventricular cAMP accumulation, the hearts were perfused with vehicle, ADM (1 nmol/L), or isoproterenol (1 µmol/L) for 2, 5, or 30 minutes. After the appropriate period of infusion, atrial tissue was removed, and the ventricles were immediately frozen with liquid nitrogen and stored at -70°C until assayed. Tissue samples were homogenized with 6% trichloroacetic acid at 4°C to give a 10% (wt/vol) homogenate, followed by centrifugation at 2000g for 15 minutes. Then supernatants were collected and washed with 5 vol of water-saturated diethyl ether four times. The extracts were lyophilized and processed for the measurement of cAMP content by use of a standard ¹²⁵I radioimmunoassay kit supplied by Amersham International.

For the ANP radioimmunoassay, the coronary venous effluents were collected at 2-minute intervals during a 10-minute control period and a 30-minute infusion period, placed immediately on dry ice, and stored at -20°C until assayed. Unextracted perfusate samples were incubated as duplicates of 100 µL with 100 µL of a specific rabbit ANP antiserum (final dilution of 1:25 000).³⁵ Synthetic rat ANP_99–126 ranging from 0 to 500 pg per tube was used to construct the standard curve. After incubation for 48 hours at 4°C, ¹²⁵I-labeled rat ANP_99–126 with normal rabbit serum was added. After incubation for another 24 hours at 4°C, the immunocomplexes were precipitated with anti-rabbit -globulin in the presence of 500 µL of polyethylene glycol 6000, pH 7, followed by centrifugation at 3000g for 30 minutes. The lowest amount of ANP yielding a binding different from the blanks at the 95% confidence level was 1 fmol per tube. The 50% intercept of the standard curve was 11 fmol per tube, and the intra-assay and interassay variations were <10% and <15%, respectively.

Statistical Analysis
Results are presented as mean±SEM. Data were analyzed with two- or one-way ANOVA followed by Bonferroni t test. The statistical significance of the differences between two groups was determined with Student's t test. Differences were considered statistically significant at the level of P<.05.

	Results

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Effect of ADM on Contractility in Isolated, Paced Rat Hearts
We¹¹ reported earlier that ADM at concentrations of 0.03 to 1 nmol/L induced a dose-dependent inotropic effect in the spontaneously beating perfused rat heart. To exclude any possible secondary effects caused by the changes in heart rate, we used a paced rat heart preparation (n=162) in the present study to examine the signaling pathways involved in the ADM-induced positive inotropic effect. The preparation was stable for the period of time used in these studies. When vehicle was infused for 30 minutes, the contractile force remained constant (Fig 1A, Table 1). Addition of ADM (0.1 to 1 nmol/L) into the perfusion fluid induced a dose-dependent positive inotropic effect (F=17.8, P<.001, ADM 0.1 nmol/L versus vehicle; F=29.9, P<.001, ADM 1 nmol/L versus vehicle; F=2.7, P<.01, ADM 0.1 nmol/L versus ADM 1 nmol/L; for drug and time interaction, two-way ANOVA, see Fig 1A, Table 1). The elevation of developed tension in response to ADM infusion was gradual. A significant increase in contractility was observed 5 minutes after the start of ADM infusion, and the maximal increase was seen at 30 minutes (Fig 1A).

View larger version (22K): [in this window] [in a new window]   Figure 1. A, Effect of ADM and PAMP on developed tension (DT) in isolated perfused, paced rat hearts. After a control period, as shown by the arrow, vehicle or peptides were added to the perfusion fluid for 30 minutes. Results are expressed as a percent change versus baseline values. Each point is the mean±SEM from four to seven separate experiments run on different isolated rat hearts. For numbers of experiments and baseline values of contractility in each group, see Table 1. B, Effect of ADM (1 nmol/L) alone or in combination with CGRP8–37 (100 nmol/L), a CGRP receptor antagonist, on DT in isolated perfused, paced rat hearts. After a control period, as shown by the arrow, vehicle or drugs were added to the perfusion fluid for 30 minutes. Results are expressed as a percent change versus baseline values. Each point is the mean±SEM from four to seven separate experiments run on different isolated rat hearts. For numbers of experiments and baseline values of contractility in each group, see Table 1.

View this table: [in this window] [in a new window]   Table 1. Effect of ADM on Developed Tension in the Perfused Rat Heart

To characterize the specificity of the action of ADM, we studied the inotropic effect of PAMP, a peptide derived from the same gene as ADM.²⁶ In contrast to ADM, PAMP (10 to 100 nmol/L) had no effect on developed tension (Fig 1A, Table 1). Previously, it has been shown that various responses to ADM can be blocked by a CGRP receptor antagonist, CGRP_8–37, suggesting that ADM interacts with CGRP receptors.^{15 19 36 37} CGRP_8–37 at a concentration of 100 nmol/L, which significantly inhibited cAMP and NO generation stimulated by ADM in cardiac myocytes,¹⁹ failed to attenuate the inotropic response to ADM (1 nmol/L) (F=0.3, P=NS, ADM plus CGRP_8–37 versus ADM alone; Fig 1B, Table 1).

cAMP and ADM-Induced Positive Inotropic Effect
Studies in many tissues have demonstrated that ADM induces a dose-dependent increase in cellular production of cAMP.^{15 16 17 18 19} To examine whether cAMP is a second messenger for the positive inotropic action of ADM, we studied first the effect of H-89 on the inotropic response to ADM. H-89 has been shown to be a potent inhibitor of protein kinase A with an inhibition constant in the nanomolar range.^{23 27} Vehicle or ADM alone or in combination with H-89 was added into the aortic perfusion cannula for 30 minutes. Infusion of H-89 at a concentration of 100 nmol/L alone had no influence on developed tension (F=0.5, P=NS, H-89 versus vehicle) and did not alter ADM-induced positive inotropic response (F=1.46, P=NS, ADM plus H-89 versus ADM alone; Fig 2A, Table 2). To validate the concentration of H-89 as an inhibitor of cAMP-dependent responses under these experimental conditions, we tested its effect on the positive inotropic effect of isoproterenol, known to increase cardiac cAMP levels and activate protein kinase A in the heart.³⁸ Infusion of isoproterenol at a concentration of 1 µmol/L caused a marked increase in resting tension (from 2.0±0.1 to 3.7±0.2 g, n=6; F=58.8, P<.001, isoproterenol versus vehicle), and this increase was significantly reduced in the presence of 100 nmol/L H-89 infusion (from 3.7±0.2 to 2.6±0.1 g, n=6; F=12.8, P<.001, isoproterenol plus H-89 versus isoproterenol alone). It was also of interest to note that maximal developed tension elevation produced by ADM (1 nmol/L) was 90% of that produced by isoproterenol (1 µmol/L) (44.3±4.2% versus 49.3±7.0%, P=NS).

View larger version (18K): [in this window] [in a new window]   Figure 2. A, Effect of ADM (1 nmol/L) alone or in combination with H-89 (100 nmol/L), a protein kinase A inhibitor, on developed tension (DT) in isolated perfused, paced rat hearts. After a control period, as shown by the arrow, vehicle or drugs were added to the perfusion fluid for 30 minutes. Results are expressed as a percent change versus baseline values. Each point is the mean±SEM from 6 to 10 separate experiments run on different isolated rat hearts. For numbers of experiments and baseline values of contractility in each group, see Table 2. B, Effect of ADM (1 nmol/L) and isoproterenol (ISO, 1 µmol/L) on cAMP levels of the perfused hearts. Hearts were perfused and freeze-clamped at various times, and assays for cAMP content were performed as described in "Methods." Values represent mean±SEM from 4 to 8 separate experiments as detailed in text. The basal content of cAMP was 362±4 pmol/g tissue (n=14). *P<.05 ISO vs vehicle; P<.01 ISO vs vehicle.

View this table: [in this window] [in a new window]   Table 2. Effect of Various Drugs on Developed Tension in the Perfused Rat Heart

Next, we measured cAMP production in the ventricles during infusion of ADM and isoproterenol. The basal values of cAMP were similar to previously reported values.³⁹ As shown in Fig 2B, isoproterenol (1 µmol/L) caused a significant increase in ventricular cAMP content by 62±13% (n=6, P<.05) and 79±16% (n=4; P<.01) at 2 and 5 minutes after the start of isoproterenol administration, respectively, which agrees with previous data in rat heart preparation.³⁹ During longer exposure (30 minutes) to isoproterenol, cAMP levels almost returned to baseline values (n=6, P=NS, isoproterenol versus vehicle). Infusion of ADM at the concentration of 1 nmol/L did not increase cAMP production in the ventricles of the perfused hearts either at 2 (n=6), 5 (n=4), or 30 minutes (n=8) (Fig 2B). These data suggest that ADM increases contractile force through a cAMP-independent process.

Ryanodine, Thapsigargin, Staurosporine, and ADM-Induced Positive Inotropic Effect
To examine the role of other potential signaling pathways in the inotropic effect of ADM, we evaluated the importance of two distinct intracellular Ca²⁺ cycles: the intracellular ryanodine-sensitive Ca²⁺ release channels, which allow Ca²⁺ to enter the cytosol from intracellular stores, and the energy-dependent Ca²⁺ pump of SR (Ca²⁺-ATPase), which removes Ca²⁺ from the cytosol.²¹ Infusion of ryanodine, an SR Ca²⁺ release channel opener,^{28 29} at a concentration of 3 nmol/L slightly decreased developed tension at the end of 30 minutes of infusion (F=3.97, P<.01, ryanodine versus vehicle; Fig 3A, Table 2). When ryanodine was administered in combination with ADM (1 nmol/L), it significantly reduced the positive inotropic effect of ADM after 8 minutes throughout the entire experimental period and maximally by 72% at the end of the infusion period (F=14.3, P<.001, ADM plus ryanodine versus ADM alone; Fig 3A).

View larger version (18K): [in this window] [in a new window]   Figure 3. Effects of (A) ryanodine (RYA, 3 nmol/L) and (B and C) thapsigargin (THAP, 30 nmol/L) on the ADM (1 nmol/L)–induced developed tension (DT) elevation. After control period, as shown by the arrows, vehicle or drugs were added to the perfusion fluid for 30 minutes (A, B) or a modified protocol was used when 10 minutes' pretreatment with thapsigargin (THAP PT) was followed by a control period and a 30-minute infusion of vehicle or ADM (C) as described in "Methods." Each point is the mean±SEM and depicts percentage of predrug values from 6 to 10 separate experiments run on different isolated rat hearts. For numbers of experiments and baseline values of contractility in each group, see Table 2. *P<.05 vs ADM; P<.01 vs ADM; P<.001 vs ADM; #P<.05 vs vehicle; §P<.01 vs vehicle.

As shown in Fig 3B, infusion of thapsigargin, a selective inhibitor of SR Ca²⁺-ATPase,^{30 31} at a concentration of 30 nmol/L had no effect on contractility up to 16 minutes of drug administration; however, afterward it progressively attenuated the developed tension (F=18.2, P<.001, thapsigargin versus vehicle; Table 2). When ADM was administered in the presence of thapsigargin, the increase in developed tension was augmented between 5 and 10 minutes (F=27.6, P<.001, ADM plus thapsigargin versus ADM alone; Fig 3B). Subsequently, the developed tension elevation was attenuated by thapsigargin and was significantly lower than that during ADM infusion from 20 minutes onward (F=6.94, P<.001).

To further analyze the role of the thapsigargin-sensitive Ca²⁺ pump of SR, a modified protocol was used in which hearts were pretreated with thapsigargin (30 nmol/L) for 10 minutes followed by a 10-minute control period and 30 minutes of infusion with either vehicle or ADM. Our aim was dual: on the one hand, we intended to deplete the Ca²⁺ content of intracellular stores before ADM administration; on the other hand, we wanted to prevent the depression of contractility observed during 30 minutes' exposure to thapsigargin (see Fig 3B). Because thapsigargin has been reported to act irreversibly on SR Ca²⁺-ATPase,³¹ we assumed that 10 minutes' pretreatment with thapsigargin would evoke the expected effect. Thapsigargin pretreatment significantly depressed the positive inotropic effect of ADM between 3 and 12 minutes (F=2.8, P<.01, thapsigargin pretreatment plus ADM versus ADM alone). Later, there was also a trend for lower developed tension; however, this change was not statistically significant (Fig 3C). Pretreatment with thapsigargin alone followed by a 10-minute control period resulted in a modest (16.7±2.2%) decrease in developed tension per se, and during vehicle infusion, a slight elevation in developed tension was observed (F=2.1, P<.05, thapsigargin pretreatment plus vehicle versus vehicle alone; Fig 3C).

An important mechanism for regulation of cellular processes in cardiac myocytes involves activation of phosphoinositide hydrolysis with subsequent production of IP₃ and DAG. The IP₃ released into the cytoplasm mobilizes Ca²⁺ from internal stores, whereas DAG activates protein kinase C.^{40 41} The latter system may be involved in the slow contractile response in cardiac myocytes.²¹ In the present study, we used a potent protein kinase C inhibitor, staurosporine,²³ to examine the possible involvement of the protein kinase C–dependent pathway in the positive inotropic action of ADM. As shown in Fig 4, the infusion of staurosporine (10 nmol/L) had no effect on contractile force (F=0.94, P=NS, staurosporine versus vehicle; Table 2). When given together with ADM, staurosporine significantly attenuated the ADM-induced positive inotropic effect from 18 minutes onward (F=10.7, P<.001, ADM plus staurosporine versus ADM alone; Fig 4), the maximal reduction being 62.5% at the end of 30 minutes' infusion time. Previously we have determined the concentration of staurosporine needed to inhibit protein kinase C–dependent responses under these experimental conditions. Coronary vasoconstrictor and ANP secretory responses induced by phorbol ester TPA (12-0-tetradecanoyl-phorbol-13-acetate), known to stimulate protein kinase C activity in the isolated perfused rat heart preparation,⁴² were completely abolished by staurosporine at concentrations from 10 to 100 nmol/L.^{32 43} Staurosporine also inhibited the cardiac and ANP secretory effects of a protein kinase C–activating peptide, endothelin-1.⁴³

View larger version (17K): [in this window] [in a new window]   Figure 4. Effect of staurosporine (STA, 10 nmol/L) on the ADM (1 nmol/L)–induced developed tension (DT) elevation. After a control period, as shown by the arrow, vehicle or drugs were added to the perfusion fluid for 30 minutes. Each point is the mean±SEM and depicts percentage of predrug values from 6 to 10 separate experiments run on different isolated rat hearts. For numbers of experiments and baseline values of contractility in each group, see Table 2. *P<.05 vs ADM; P<.01 vs ADM.

Ca²⁺ Channels and ADM-Induced Positive Inotropic Effect
The plasma membrane Ca²⁺ channels provide the major pathways for Ca²⁺ entry into myocardial cells. The most abundant of the plasma membrane Ca²⁺ channels in cardiac muscle cells are the L-type channels, which play a key role in the intracellular Ca²⁺ cycle by opening the intracellular Ca²⁺ release channels.²¹ To examine the requirement of extracellular Ca²⁺ entry for an ADM-induced positive inotropic effect, we administered ADM in the presence of diltiazem, an L-type Ca²⁺ channel blocker.³³ Infusion of diltiazem alone at a concentration of 1 µmol/L had no effect on developed tension in paced rat heart preparation (F=0.86, P=NS, diltiazem versus vehicle; Fig 5A, Table 2). When ADM was infused in the presence of diltiazem, the inotropic response was significantly attenuated from 22 minutes onward and maximally by 40% at the end of the 30-minute infusion period (F=2.5, P<.01, ADM plus diltiazem versus ADM alone; Fig 5A, Table 2).

View larger version (14K): [in this window] [in a new window]   Figure 5. A, Effect of diltiazem (DILT, 1 µmol/L) on the ADM (1 nmol/L)–induced developed tension (DT) elevation. After a control period, as shown by the arrow, vehicle or drugs were added to the perfusion fluid for 30 minutes. Each point is the mean±SEM and depicts percentage of predrug values from 6 to 10 separate experiments run on different isolated rat hearts. For numbers of experiments and baseline values of contractility in each group, see Table 2. *P<.05 vs ADM. B, Effect of ADM (1 nmol/L) on action potentials of rat atrium. Ten action potentials from each cell in the control group (n=17) and adrenomedullin group (n=6) were averaged.

If ADM has any effect on Ca²⁺ currents through sarcolemma, it should cause changes in cardiac action potentials. To test this hypothesis, we recorded intracellular action potentials from myocytes⁴⁴ by using an isolated left atrial preparation.³⁴ The isolated left atrium was chosen for electrophysiological experiments because of the absence of spontaneous activity and because the length of the action potential is considerably shorter than in the ventricles, making changes in Ca²⁺ currents more prominent. Criteria for acceptable recordings were a stable impalement, resting potential at least -70 mV, and an overshoot of action potential of at least 10 mV. When atria were superfused with buffer containing 1 nmol/L ADM (n=6), the shape of the action potentials was changed. The duration parameters were dramatically increased at 15%, 30%, 60%, and 90% repolarization levels (Table 3). As shown in Fig 5B, the most prominent increase of action potential duration occurred with membrane voltage between 10 and -50 mV. There was little or no change in resting potential, amplitude, overshoot, and rate of rise of action potentials (Table 3). The same findings, with a somewhat more prominent increase in action potential duration, were found with a larger concentration of ADM (10 nmol/L, data not shown), showing that even then the effect was fairly specific. The prolongation of the action potentials by ADM is consistent with an increase in L-type Ca²⁺-channel current during the plateau.

View this table: [in this window] [in a new window]   Table 3. Effect of ADM (1 nmol/L) on Action Potential Parameters in Rat Left Atrium

Effect of ADM on Hemodynamic Variables and Perfusate Immunoreactive ANP
Infusion of vehicle or ADM alone or in the presence of different substances did not affect resting tension (from 2.0±0.01 to 2.08±0.02 g, P=NS) or heart rate (from 307±1 to 307±1 beats/min, P=NS) in any of the groups. Overall, the changes in perfusion pressure were also small (from 29±0.3 to 31±0.4 mm Hg, P=NS), except that administration of thapsigargin at the dose of 30 nmol/L significantly increased perfusion pressure (from 29±2 to 42±3 mm Hg, F=14.1, P<.001, thapsigargin versus vehicle), as reported previously under these experimental conditions.⁴⁵ The concentration of 1 nmol/L of ADM reversed the vasoconstrictor effect of thapsigargin (34±3 versus 42±3 mm Hg, F=9.9, P<.001, ADM plus thapsigargin versus thapsigargin alone) consistent with the vascular smooth muscle–relaxant effect of ADM.^{2 3}

The basal concentration of ir-ANP in the perfusate after a 60-minute equilibration period was 337±43 pg/mL (n=16). A small decrease of perfusate ir-ANP concentration (from 398±56 to 252±45 pg/mL, n=7) was noted toward the end of the 30-minute infusion period during vehicle infusion, as reported previously.⁴⁵ Administration of ADM (1 nmol/L) failed to modulate the ir-ANP secretion, and the magnitude of decrease was similar to that observed with vehicle infusion (from 277±59 to 168±38 pg/mL, n=9; F=0.88, P=NS). Thus, we did not further evaluate the effects of various drugs alone or in combination with ADM on ANP levels of the coronary venous effluents.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Previous investigations suggest an intimate relation between ADM-induced biological effects and the increased cAMP levels in several cell types, including vascular smooth muscle cells,^{15 16} endothelial cells,¹⁷ and glomerular mesangial cells.¹⁸ In addition, ADM has been reported to augment NO synthesis under cytokine-stimulated conditions in isolated rat cardiac myocytes¹⁹ and to inhibit ANP gene expression,²⁰ at least in part via a cAMP-dependent pathway. Our results disagree with these previous findings and suggest that cAMP is not the major second messenger of the inotropic effect of ADM. First, H-89, a protein kinase A inhibitor, did not affect the inotropic response induced by ADM, although it significantly attenuated the cardiac effects of isoproterenol. Second, ADM failed to increase cAMP content of the ventricles of the perfused hearts, whereas marked production of cAMP was observed in isoproterenol-treated hearts. Third, the submaximal inotropic effect in response to a lower concentration of ADM (0.1 nmol/L) could not be enhanced in the presence of isobutyl methylxanthine, a phosphodiesterase inhibitor (data not shown). Finally, the development of the inotropic effect of ADM appeared to be slow compared with the observed rapid responses produced by agents acting through a cAMP-dependent process.⁴⁶

The lack of requirement for cAMP suggests that other signaling mechanisms regulating excitation-contraction coupling, such as Ca²⁺ release from intracellular stores, may be responsible for ADM-induced positive inotropic action. In agreement with this hypothesis, ryanodine markedly attenuated both the initial and the sustained phase and thapsigargin pretreatment attenuated the early phase of the inotropic effect of ADM. Although the inhibitors of SR function may not affect ADM activity directly, our results suggest that ADM may produce its positive inotropic effect via Ca²⁺ release from ryanodine- and thapsigargin-sensitive intracellular Ca²⁺ pools. IP₃ receptors are also present in cardiac tissue,⁴⁷ and IP₃-induced Ca²⁺ release from SR can serve as a mechanism for positive inotropy.⁴⁸ Recently, Shimekake et al¹⁷ reported that in bovine aortic endothelial cells, ADM induced Ca²⁺ mobilization from thapsigargin-sensitive intracellular Ca²⁺ pools through activation of phospholipase C. Our observation that staurosporine, a potent protein kinase C inhibitor,²³ markedly attenuated the sustained phase of the inotropic effect of ADM was consistent with the hypothesis that phospholipase C and DAG, which activates protein kinase C,⁴¹ may be involved in the ADM-induced increase in developed tension.

In cardiac muscle cells, Ca²⁺ influx via voltage-dependent L-type Ca²⁺ channels has been considered to be the main mechanism by which Ca²⁺-induced Ca²⁺ release from SR is triggered during depolarization.⁴⁹ Previously, ADM was reported to enhance Ca²⁺ entry through sarcolemmal receptor–operated Ca²⁺ channels in endothelial cells.¹⁷ In our experiments, diltiazem, an L-type Ca²⁺ channel blocker, suppressed the prolonged phase of the inotropic effect of ADM. Furthermore, in intracellular recordings of action potentials from rat atrial myocytes, ADM increased action potential duration between -50 and 10 mV, which is within the operating voltage of L-type Ca²⁺ channels.⁵⁰ These results suggest that ADM-induced positive inotropic action may involve enhanced Ca²⁺ influx through L-type Ca²⁺ channels, which could then enhance contractility further by increasing Ca²⁺ release from SR.⁴⁹

Recently, a cDNA clone has been identified as a functional ADM receptor. When expressed in COS-7 cells, the receptor mediates a cAMP response to ADM, suggesting that the receptor is coupled to adenylate cyclase.⁵¹ Although the receptor is also expressed in the heart,⁵¹ ADM failed to stimulate ventricular cAMP accumulation in the present study. The discrepancy may be explained by the finding that maximal cAMP accumulation occurred at a concentration of 100 nmol/L in COS-7 cells,⁵¹ whereas the concentration of ADM used in the present study (0.1 to 1 nmol/L) produced a moderate effect, if any. Thus, ADM may produce its positive inotropic effect via the cloned receptor but at concentrations lower than that of the stimulation threshold required for the activation of adenylate cyclase. Alternatively, our results suggest the existence of another ADM receptor subtype not coupled to adenylate cyclase in the heart. It is also possible that the discrepancy between our study and previous studies may be related to the different experimental models used (isolated cells versus perfused rat hearts).

ADM shares modest sequence homology with CGRP, in particular the presence of a six-residue intramolecular disulfide-linked ring structure.¹ In the rat heart, it has been demonstrated that ADM could inhibit ¹²⁵I-CGRP binding, suggesting that ADM may mediate its effects not only via specific receptors but also through CGRP receptors.⁸ Recently, ADM has been reported to augment NO synthesis in cardiac myocytes under cytokine-stimulated conditions, which effect can be blocked by a CGRP receptor antagonist, CGRP_8–37.¹⁹ Because CGRP_8–37 failed to attenuate the inotropic response to ADM, and CGRP itself, in agreement with previous reports,^{52 53} did not alter contractile force in our isolated perfused heart preparation (data not shown), the involvement of CGRP receptors in the ADM-induced positive inotropic effect is unlikely. To further characterize the specificity of the ADM effect, we compared the action of endogenous rat PAMP, an ADM-related peptide,²⁶ with that of ADM. Immunoreactive PAMP²⁶ and its specific binding sites have been found in considerable amounts in the rat heart.^{54 55} Because PAMP had no effect on contractility, it appears that ADM can bind and activate its own receptors in the heart fairly specifically, independently of the actions mediated via PAMP and CGRP binding sites.

Because several vasoactive substances are known to modulate ANP secretion²⁴ and cardiac atria contain a considerable amount of ADM-like immunoreactivity,^{6 7} it is of special interest whether ADM can influence ANP release. In patients with congestive heart failure, there was a positive correlation between plasma concentrations of ADM and ANP.¹⁴ Our present results show that ADM did not alter basal secretion of ANP in the isolated perfused rat heart. Previously it was reported that natriuretic peptides do not influence basal production of ADM in vascular smooth muscle cells.⁵⁶ These data suggest that the peptides are functioning independently, at least under nonstimulated conditions. It is widely accepted that substances producing a positive inotropic effect by increasing cytosolic Ca²⁺ levels via mobilization of intracellular stores and/or increase of extracellular Ca²⁺ influx simultaneously increase ANP secretion.²⁴ Our observation that the ADM-induced inotropic effect was not accompanied by an increased ANP secretion raises the possibility of the existence of different intracellular pools involved in the regulation of the inotropic and secretory responses.

Although the physiological significance of the present findings will require additional studies, several lines of evidence suggest that ADM may play an important role in the regulation of cardiac function. Previously, endothelin-1 has been considered to be the most potent positive inotropic factor.^{57 58} Because ADM was active in the subnanomolar range, with an EC₅₀ value of 50 pmol/L (similar to that of endothelin-1),¹¹ ADM appears to be one of the most potent endogenous positive inotropic substances yet identified. Furthermore, in isolated perfused, paced rat hearts, the ADM-induced increase in developed tension was comparable to the marked positive inotropic effect of isoproterenol (1 µmol/L), a ß-receptor agonist. However, the unique, slowly developing effect may suggest a different role for ADM in the modulation of myocardial contractility than that governed by ß-adrenoceptor stimulation.⁴⁶ Very recently, Ikenouchi et al⁵⁹ suggested that ADM exerts a negative inotropic effect, with a rapid onset mediated via the L-arginine–NO pathway in isolated adult rabbit ventricular myocytes. Using the isolated perfused rat heart preparation, we found that L-NAME, an inhibitor of NO synthase, at a concentration of 300 µmol/L significantly augmented the early phase of the positive inotropic effect of ADM (2 minutes, 5.6±2.4% versus -0.5±1.2%; 5 minutes, 20.5±3.1% versus 10.8±1.9%, ADM plus L-NAME versus ADM, n=5, P<.05), whereas the sustained phase of ADM-induced developed tension elevation was not modified. These results show that ADM may activate NO production in the isolated perfused rat hearts resulting in the attenuation of the initial phase of positive inotropic effect of ADM.

In summary, this is the first report showing that ADM enhances cardiac contractility independently of the cAMP-dependent signaling pathway. Our results suggest that the positive inotropic effect of ADM may involve Ca²⁺ release from intracellular ryanodine- and thapsigargin-sensitive Ca²⁺ stores, activation of protein kinase C, and Ca²⁺ influx via L-type Ca²⁺ channels. Taking into account the potent inotropic effect of ADM and the increased ventricular production of ADM in severely failing human myocardium,⁵ the present results are consistent with the hypothesis that ADM as an endogenous positive inotropic substance may play a role in the compensatory mechanisms against deterioration of cardiac performance by enhancing myocardial contractility in congestive heart failure.^{5 12 13 14}

	Selected Abbreviations and Acronyms

 

ADM = adrenomedullin ANP = atrial natriuretic peptide CGRP = calcitonin gene–related peptide DAG = diacylglycerol IP3 = inositol 1,4,5-trisphosphate ir-ANP = immunoreactive atrial natriuretic peptide L-NAME = NG-nitro-L-arginine methyl ester NO = nitric oxide PAMP = proadrenomedullin N-20 SR = sarcoplasmic reticulum

	Acknowledgments

 
This work was supported by the Medical Research Council, Academy of Finland, the Sigrid Juselius Foundation, and the Finnish Heart Research Foundation. We thank Marja-Leena Vainikka for expert technical assistance.

Received August 6, 1997; revision received October 15, 1997; accepted October 31, 1997.

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