₁-Adrenergic Stimulation of Sarcolemmal Na⁺-H⁺ Exchanger Activity in Rat Ventricular Myocytes

Evidence for Selective Mediation by the _1A-Adrenoceptor Subtype

From Cardiovascular Research, The Rayne Institute, St Thomas' Hospital, London, UK.

Correspondence to Dr Metin Avkiran, Cardiovascular Research, The Rayne Institute, St Thomas' Hospital, Lambeth Palace Rd, London SE1 7EH, UK. E-mail m.avkiran{at}umds.ac.uk

	Abstract

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Abstract—₁-Adrenoceptor (₁-AR) stimulation increases sarcolemmal Na⁺-H⁺ exchanger (NHE) activity. The present study was designed to determine the role(s) of ₁-AR subtype(s) in mediating this response. As an index of NHE activity, acid efflux rates (J_Hs) were determined in single rat ventricular myocytes loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 after 2 consecutive intracellular acid pulses in bicarbonate-free medium. J_H at pH_i 6.90 did not change significantly during the second pulse relative to the first in control cells but increased in a dose-dependent manner when the second pulse occurred in the presence of phenylephrine (nonselective ₁-AR agonist) or A61603 (_1A-AR–selective agonist), with EC₅₀ values of 1.24 µmol/L and 3.6 nmol/L, respectively (both agonists given together with 1 µmol/L atenolol). Stimulation of NHE activity by 10 µmol/L phenylephrine was inhibited in a dose-dependent manner by the competitive antagonists prazosin, WB4101, and 5-methylurapidil, with IC₅₀ values of 12, 32, and 149 nmol/L, respectively. Analyses of the relative EC₅₀ and IC₅₀ values obtained (and K_i values estimated from the antagonist IC₅₀s) in relation to the relative potencies of these agents at native rat ₁-AR subtypes and their relative affinities for recombinant rat ₁-ARs suggest that ₁-adrenergic stimulation of sarcolemmal NHE activity is likely to be mediated selectively by the _1A-AR.

Key Words: Na⁺-H⁺ exchanger • ₁-adrenoceptor subtype • myocyte • receptor selectivity

	Introduction

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The sarcolemmal NHE is a primary acid extrusion mechanism in cardiac myocardium^{1 2} whose activity is subject to modulation by neurohormonal stimuli.³ In this regard, there is now convincing evidence that ₁-adrenergic stimulation increases sarcolemmal NHE activity in ventricular myocytes.^{4 5 6} However, the question of whether this response is mediated by a specific ₁-AR subtype(s) has not been addressed.

In recent years, there has been considerable confusion regarding the classification of ₁-AR subtypes, particularly over the relationship between pharmacologically identified native ₁-ARs and the cloned ₁-ARs. However, much of this confusion has now been resolved (see Ford et al⁷ and Graham et al⁸), such that the classification currently recommended by the International Union of Pharmacology⁹ identifies ₁-AR subtypes as _1A-, _1B-, and _1D-ARs, which correspond respectively to the recombinant subtypes previously referred to as _1c-, _1b-, and _1d-ARs (the last of which has also been referred to as the _1a- or _1a/d-AR). Within the context of the present study, it is important to note that _1A- and _1B-AR transcripts represent the dominant subtypes that are expressed in adult rat myocardium (81% of total in isolated myocytes¹⁰ and 97% of total in whole hearts¹¹). A similar pattern of expression may exist at the protein level also, on the basis of radioligand binding studies in adult rat myocardium that have shown (1) the presence of 2 high-affinity binding sites with the characteristics of _1A- and _1B-ARs^{12 13} and (2) the absence of a high-affinity binding site for BMY7378, an _1D-AR–selective antagonist.¹⁴

Determination of the role of _1A-AR versus _1B-AR subtypes in mediating physiological responses to ₁-adrenergic stimulation in myocardium (as in other tissues) is complicated by the paucity of highly selective ligands (ie, ligands that possess at least a 100- to 1000-fold higher affinity for one subtype relative to the other).⁸ Studies with recombinant ₁-ARs have confirmed that endogenous catecholamines and the synthetic agonist phenylephrine do not discriminate between _1A- and _1B-ARs.^{15 16} In contrast, the recently described potent ₁-adrenergic agonist A61603 appears to exhibit considerable selectivity for the _1A-AR.¹⁷ The alkylating agent CEC inactivates primarily the _1B-AR but can produce partial inactivation of the other subtypes also, particularly with prolonged exposure at high concentration.^{15 16} Of available competitive antagonists, prazosin is nonselective, but WB4101 and 5-methylurapidil exhibit 25-fold greater affinity for _1A- versus _1B-ARs (although WB4101 exhibits high affinity also for _1D-ARs).^{15 16} From the above, it is clear that characterization of ₁-AR subtype–mediated responses in intact myocardium or isolated myocytes may be achieved only through the methodical application of multiple agents.

The objective of the present study was to determine the role(s) of ₁-AR subtype(s) in mediating ₁-adrenergic stimulation of sarcolemmal NHE activity. Toward this aim, we have used isolated ventricular myocytes from the adult rat heart in conjunction with a microepifluorescence-based assay for sarcolemmal NHE activity. In preliminary experiments, we studied the relative effects of CEC versus WB4101 on phenylephrine-induced stimulation of NHE activity to determine the likelihood of a receptor subtype–selective response. The results of the preliminary experiments led us to formulate the hypothesis that ₁-adrenergic stimulation of sarcolemmal NHE activity is mediated selectively by the _1A-AR subtype. In order to test this hypothesis rigorously, we then carried out extensive dose-response studies with 2 agonists (phenylephrine and A61603) and 3 antagonists (prazosin, WB4101, and 5-methylurapidil; all in conjunction with phenylephrine) and compared the relative EC₅₀ and IC₅₀ values obtained (and the K_i values estimated from the antagonist IC₅₀s) with the relative potencies and affinities of these agents at native and recombinant rat ₁-AR subtypes.

	Materials and Methods

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This investigation was performed in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, published by Her Majesty's Stationery Office, London, UK.

Determination of Sarcolemmal NHE Activity in Isolated Ventricular Myocytes
Sarcolemmal NHE activity was determined in single ventricular myocytes from the rat heart using a microepifluorescence-based approach that we have used in previous studies.^{18 19 20} In brief, adult male Wistar rats (200 to 250 g body weight) were anesthetized by inhalation of diethyl ether, and hearts were excised and perfused (37°C) in the Langendorff mode for four sequential periods as follows: (1) with Tyrode's solution (mmol/L: NaCl 137, KCl 5.4, CaCl₂ 1.8, MgCl₂ 0.5, HEPES 10, and glucose 10, adjusted to pH 7.4 at 34°C with NaOH) for 5 minutes, (2) with nominally Ca²⁺-free Tyrode's solution (mmol/L: NaCl 135, KCl 5.4, NaH₂PO₄ 0.33, MgCl₂ 1.0, HEPES 10, and glucose 10, adjusted to pH 7.2 at 34°C with NaOH) for 5.5 minutes, (3) with nominally Ca²⁺-free Tyrode's solution containing collagenase (Worthington type 1, 94 U/mL) for 8.5 minutes, and (4) with storage buffer (mmol/L: KOH 78, KCl 30, KH₂PO₄ 30, MgSO₄ 3, EGTA 0.5, HEPES 10, glutamic acid 50, taurine 20, and glucose 10, adjusted to pH 7.2 at 34°C with KOH) for 5 minutes. All solutions were gassed with 100% O₂. After the perfusion procedure, the ventricles were removed and chopped into several pieces in storage buffer. The tissue fragments were then gently agitated to facilitate cell dispersion, and the cell suspension (>80% rod-shaped cells¹⁸) was maintained in storage buffer at 25°C for at least 1 hour before use in the microepifluorescence studies.

pH_i was monitored in single ventricular myocytes using the pH-sensitive fluorescent dye C-SNARF-1. Cells loaded with C-SNARF-1 were placed on a glass coverslip in a 100 µL chamber and continuously superfused (3.5 mL/min) with Tyrode's solution (34°C) of the composition described above. Since cells were maintained in bicarbonate-free medium throughout the experimental protocol, J_H calculated during recovery from intracellular acidosis (see below) could be used as an indicator of sarcolemmal NHE activity.^{18 19 20}

Experimental Protocols
Experiments were performed according to a protocol involving 2 consecutive acid pulses, as we have described previously.¹⁸ After 5 to 10 minutes of superfusion with normal Tyrode's solution (pH 7.4), cells (n=7 to 10 per group) were subjected to intracellular acidosis by transient (3-minute) exposure to 20 mmol/L NH₄Cl (first acid pulse). After a 6-minute period of NH₄Cl washout, cells were superfused with Tyrode's solution for an additional 6 minutes before a second transient exposure to NH₄Cl (second acid pulse). In control cells, both acid pulses occurred under identical conditions. When studying the effects of phenylephrine (0.1 to 100 µmol/L) or A61603 (0.1 to 300 nmol/L), the ₁-AR agonist was present throughout the second pulse (ie, during exposure to and washout of NH₄Cl) together with 1 µmol/L atenolol (to preclude ß₁-AR–mediated effects). When studying the effects of phenylephrine (100 µmol/L) in the presence of the alkylating agent CEC (3 µmol/L) or the competitive ₁-AR antagonist WB4101 (3 µmol/L) in our preliminary studies, these agents were included in all solutions from 6 minutes before the second pulse to the end of the experiment. When the inhibition curves were constructed for prazosin (0.1 to 300 nmol/L), WB4101 (1 to 1000 nmol/L), and 5-methylurapidil (3 to 3000 nmol/L) versus phenylephrine (10 µmol/L), the antagonists were included in all solutions from 3 minutes before the second pulse. J_H was calculated at pH_i intervals of 0.05 during the recovery phases after both acid pulses.¹⁸ Each cell received only a single drug intervention.

Drugs
Drugs were purchased from Sigma Chemical Co and were dissolved directly in Tyrode's solution, unless stated otherwise. Prazosin, WB4101, and 5-methylurapidil were purchased from Research Biochemicals International (via Semat Technical) and were dissolved in deionized water, ethanol, and dimethyl sulfoxide, respectively. A61603 was a gift from Abbott Laboratories (Abbott Park, Ill) and was dissolved in 0.3 mmol/L ascorbate. All stock solutions were diluted (1:1000) in Tyrode's solution to obtain the appropriate concentrations shortly before the beginning of experiments (concomitant controls received the appropriate vehicle).

Data Analysis
Experiments within each study subsection were carried out in a randomized manner. Data are expressed as mean±SEM. Within individual groups, the paired t test was used to assess changes in J_H at identical pH_i levels between the first and second acid pulses (P<0.05 was considered significant). Agonist dose-response and antagonist inhibition curves were constructed by measuring as the agonist response the change in J_H6.9 during the second pulse (ie, in the presence of agonist) relative to the first (ie, in the absence of agonist). The curves were fitted to a variable slope logistic equation, and EC₅₀ and IC₅₀ values were determined using Prism 2.0 for Macintosh software (GraphPad).

	Results

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Effects of CEC and WB4101 on Phenylephrine-Induced Stimulation of Sarcolemmal NHE Activity
The objective of this preliminary study was to determine whether the NHE-stimulatory effect of 100 µmol/L phenylephrine (a concentration shown previously to significantly increase sarcolemmal exchanger activity in ventricular myocytes from both guinea pig⁵ and rat⁶) could be inhibited by pretreatment with either CEC or WB4101. Figure 1 showsrepresentative recordings of pH_i from single myocytes, as well as quantitative data in the form of J_H-versus-pH_i curves, in 4 study groups. In control cells, in which the consecutive acid pulses occurred under identical conditions, the profiles of pH_i recovery from acidosis and the J_H-versus-pH_i curves were similar during both acid pulses (Figure 1A), indicating that temporal changes in NHE activity do not occur in the absence of agonist stimulation. In contrast, exposure to 100 µmol/L phenylephrine plus 1 µmol/L atenolol during the second pulse accelerated recovery from intracellular acidosis and produced an upward shift of the J_H-versus-pH_i curve (Figure 1B). At pH_i 7.15, J_H was significantly greater in the presence of 100 µmol/L phenylephrine, indicating increased sarcolemmal NHE activity in response ₁-adrenergic stimulation (in agreement with earlier reports^4–6). When cells were pretreated with 3 µmol/L CEC before exposure to 100 µmol/L phenylephrine, the NHE-stimulatory response to the ₁-AR agonist was retained (Figure 1C); in contrast, when cells were pretreated with 3 µmol/L WB4101, the response to phenylephrine was abolished (Figure 1D). This finding suggests that the _1B-AR may not be the primary mediator of the NHE-stimulatory action of phenylephrine and implicates a role for the _1A-AR.

View larger version (32K): [in this window] [in a new window]   Figure 1. Representative pHi recordings (left) and JH-vs-pHi curves (right) obtained during 2 consecutive acid pulses in control cells (A) and in cells that were exposed to 100 µmol/L phenylephrine throughout the second pulse in the absence (B) or presence of 3 µmol/L CEC (C) or 3 µmol/L WB4101 (D). In all cases, phenylephrine was administered together with 1 µmol/L atenolol. *P<0.05 vs first pulse (n=7 cells per group).

Relative Potencies of Phenylephrine and A61603 in Stimulating Sarcolemmal NHE Activity
As a first step in testing rigorously the hypothesis that ₁-adrenergic stimulation of sarcolemmal NHE activity is mediated selectively by the _1A-AR subtype, we determined and compared the dose-response characteristics of phenylephrine and A61603. Figure 2A shows the effects of the 2 agonists on J_H6.9 during the second acid pulse; as can be seen, both phenylephrine and A61603 increased J_H6.9 in a dose-dependent manner. The agonist dose-response curves derived from these data (Figure 2B) revealed EC₅₀ values of 1.24 µmol/L and 3.6 nmol/L for phenylephrine and A61603, respectively, indicating a 340-fold greater potency for the latter agonist.

View larger version (19K): [in this window] [in a new window]   Figure 2. Panel A shows JH6.9 during the second acid pulse in the presence of various concentrations of phenylephrine or A61603 (n=7 to 10 cells per concentration). Solid symbols indicate the mean JH6.9 during the first acid pulse, in the absence of agonist, in the same cells. In all cases, phenylephrine or A61603 was administered together with 1 µmol/L atenolol. Panel B shows the agonist dose-response curves, which revealed EC50 values of 1.24 µmol/L for phenylephrine and 3.6 nmol/L for A61603.

Relative Inhibitory Potencies of Prazosin, WB4101, and 5-Methylurapidil Against Phenylephrine-Induced Stimulation of Sarcolemmal NHE Activity
To further examine the role of the _1A-AR in stimulating sarcolemmal NHE activity, we also determined the inhibitory potencies of the competitive ₁-AR antagonists prazosin, WB4101, and 5-methylurapidil against the stimulatory effect induced by phenylephrine at the 10 µmol/L concentration. In concomitant experiments with phenylephrine alone (n=27 cells), J_H6.9 was 3.80±0.45 mmol/L per minute during the first pulse and was increased by 98% to 7.51±0.52 mmol/L per minute during the second pulse. Figure 3 shows the effects of the agonist on J_H6.9 during the second pulse, in the presence of various concentrations of prazosin (Figure 3A), WB4101 (Figure 3B), or 5-methylurapidil (Figure 3C). As illustrated, the NHE-stimulatory response to phenylephrine was inhibited in a dose-dependent manner by all 3 antagonists. The antagonist inhibition curves derived from these data (Figure 3D) revealed IC₅₀ values of 12, 32, and 149 nmol/L for prazosin, WB4101, and 5-methylurapidil, respectively. These IC₅₀ values and the phenylephrine EC₅₀ (1.24 µmol/L) were then entered into a functional equivalent of the Cheng-Prusoff equation (Craig²¹) to obtain estimates of antagonist K_i values for comparison with published K_i values (from radioligand binding studies) at recombinant rat ₁-AR subtypes (Table 1).

View larger version (28K): [in this window] [in a new window]   Figure 3. Panels A through C show JH6.9 during the second acid pulse in the presence of 10 µmol/L phenylephrine in cells that received various concentrations of prazosin (A), WB4101 (B), or 5-methylurapidil (C) (n=7 to 9 cells per concentration). Solid symbols indicate the mean JH6.9 during the first acid pulse, in the absence of agonist or antagonist, in the same cells. In all cases, phenylephrine was administered together with 1 µmol/L atenolol. Panel D shows the antagonist inhibition curves, which revealed IC50 values of 12 nmol/L for prazosin, 32 nmol/L for WB4101, and 149 nmol/L for 5-methylurapidil.

	Discussion

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₁-AR Subtype(s) Mediating ₁-Adrenergic Stimulation of Sarcolemmal NHE Activity
In previous studies investigating adrenergic modulation of sarcolemmal NHE activity, a stimulatory role for the ₁-AR was concluded on the basis of (1) the ability of ₁-AR agonists, such as phenylephrine^{5 6} or 6-fluoronorepinephrine⁴ (usually in the presence of a ß₁-AR antagonist), to increase NHE activity and (2) the ability of ₁-AR antagonists, such as prazosin,^{4 5} to inhibit such NHE-stimulatory effects. However, because of the lack of selectivity of the agents used, it is not possible to draw any conclusions from these studies regarding the identity of the ₁-AR subtype(s) involved in the NHE-stimulatory response.

In the present study, the novel _1A-AR-selective agonist A61603 exhibited a 340-fold greater potency than phenylephrine in stimulating sarcolemmal NHE activity. Relative to phenylephrine, A61603 has been shown to possess 330-fold greater potency in inducing contraction of the rat vas deferens (an _1A-AR–mediated response), only 40-fold greater potency in inducing contraction of the rat spleen (an _1B-AR–mediated response), and 33-fold less potency in inducing contraction of rat aortic rings (an _1D-AR–mediated response).¹⁷ The close agreement between the relative potencies of A61603 and phenylephrine in inducing contraction of the rat vas deferens (as reported by Knepper et al¹⁷) and stimulating sarcolemmal NHE activity (as shown in the present study) is supportive of our hypothesis that ₁-adrenergic stimulation of sarcolemmal NHE activity is mediated by the _1A-AR subtype. Furthermore, the absolute EC₅₀ values for A61603 and phenylephrine obtained in the present study (3.6 nmol/L and 1.24 µmol/L, respectively) are comparable to those reported by Knepper et al¹⁷ for _1A-AR–mediated contraction of the rat vas deferens (6.2 nmol/L and 2.05 µmol/L, respectively) but not for _1B-AR–mediated contraction of the rat spleen (380 nmol/L and 15.70 µmol/L, respectively) or _1D-AR–mediated contraction of rat aortic rings (6.55 µmol/L and 198 nmol/L, respectively).

In interpreting our data with prazosin, WB4101, and 5-methylurapidil, the relative affinities of these antagonists for recombinant ₁-AR subtypes, expressed in ₁-AR–deficient cells, need to be taken into consideration. Such consideration is hindered, however, by the variability in the K_i values reported for these antagonists at recombinant receptors expressed in cell lines such as COS-7 and Rat-1 (Table 2), which may arise (at least in part) from differences in the species of origin of the receptors (eg, see Shibata et al²²). In this regard, only Laz et al¹⁵ have reported K_i values for prazosin, WB4101, and 5-methylurapidil (the 3 antagonists used in the present study) at recombinant ₁-AR subtypes exclusively of rat origin. Therefore, in view of the commonality in species, we have chosen to use the these values for comparison with those estimated in the present study. As shown in Table 1, both the absolute and the relative K_i values for prazosin, WB4101, and 5-methylurapidil estimated in the present study are in good agreement with those reported for these antagonists at recombinant _1A-ARs but not _1B- or _1D-ARs of rat origin. This observation provides additional support for our hypothesis that ₁-adrenergic stimulation of sarcolemmal NHE activity is mediated selectively by the _1A-AR subtype.

View this table: [in this window] [in a new window]   Table 2. Antagonist Ki Values at Recombinant 1-AR Subtypes

Potential Physiological/Pathophysiological Significance of Findings
Myocardial ₁-ARs have been implicated as mediators of a variety of physiological and pathophysiological responses to adrenergic stimulation (for reviews, see Fedida et al²³ and Terzic et al²⁴). _1A-AR–mediated stimulation of sarcolemmal NHE activity is likely to contribute to at least some of these responses, such as enhanced contractility (see Capogrossi²⁵ versus Pucéat²⁶) and increased susceptibility to ischemia- and reperfusion-induced dysfunction,²⁷ with the latter most probably resulting from the exacerbation of intracellular Na⁺ and Ca²⁺ accumulation.²⁸ With regard to ischemia and reperfusion, ₁-AR stimulation has also been implicated in the induction of ischemic preconditioning in rat myocardium.²⁹ Since ischemic preconditioning is associated with reduced intracellular acidosis during the prolonged ischemia,³⁰ it is possible that _1A-AR–mediated stimulation of sarcolemmal NHE activity may contribute to this phenomenon. However, Gabel et al³¹ have shown recently that H⁺ efflux during prolonged ischemia is not increased in preconditioned hearts. Furthermore, any stimulation of sarcolemmal NHE activity is unlikely to contribute to the cardioprotective mechanism(s) of ischemic preconditioning, since we have shown such protection to be retained (and indeed enhanced) in the presence of NHE inhibition.²⁰

_1A-AR–mediated stimulation of exchanger activity may be involved in the induction of hypertrophy by ₁-adrenergic agonists, since the _1A-AR subtype has been implicated as the mediator of this response in cultured myocytes³² and since other stimuli that can induce such a response (eg, thrombin³³ and endothelin³⁴ ) also share the ability to increase sarcolemmal NHE activity.^{18 35} On a related note, Rokosh et al³⁶ have shown recently that hypertrophy of rat myocardium is associated with transcriptional induction of the _1A-AR subtype, suggesting that _1A-AR–mediated responses (such as stimulation of sarcolemmal NHE activity) may assume greater significance in hypertrophied myocardium. Finally, our findings may have particular relevance to cardiac ₁-adrenergic (patho)physiology in humans, since the _1A-AR appears to be the predominant subtype expressed in human ventricular myocardium.³⁷

Concluding Comments
By the application of an established microepifluorescence-based assay for sarcolemmal NHE activity and a variety of pharmacological tools to stimulate or block ₁-AR subtypes, the present study has shown that ₁-adrenergic stimulation of the exchanger is likely to be mediated selectively by the _1A-AR subtype. Since this pathway may be of significance in mediating a variety of physiological and pathophysiological responses to ₁-adrenergic stimuli, definitive confirmation of the role of the _1A-AR in regulating sarcolemmal NHE activity (perhaps through targeted disruption of _1A-AR expression) appears desirable.

	Selected Abbreviations and Acronyms

 

AR = adrenoceptor C-SNARF-1 = carboxy-seminaphthorhodafluor-1 CEC = chloroethylclonidine JH = rate of acid efflux JH6.9 = JH at pHi 6.9 NHE = Na+-H+ exchanger

	Acknowledgments

 
This study was supported by a Basic Science Senior Lectureship Award from the British Heart Foundation (BS/93002) and equipment grants from the Special Trustees for St Thomas' Hospital (523) and The Wellcome Trust (048021/Z/96/Z) to Dr Avkiran. Drs Yokoyama and Yasutake were Visiting Research Fellows from the Nippon Medical School, Tokyo, whose visits were supported in part by the Daiwa Anglo-Japanese Foundation.

Received September 26, 1997; accepted March 16, 1998.

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