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J. Biol. Chem., Vol. 276, Issue 44, 40811-40816, November 2, 2001

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Different Subtypes of ₁-Adrenoceptor Modulate Different K⁺ Currents via Different Signaling Pathways in Canine Ventricular Myocytes^*

Huizhen Wang^§¶, Baofeng Yang^¶, Yiqiang Zhang, Hong Han^**, Jingxiong Wang, Hong Shi, and Zhiguo Wang^§§

From the  Research Center, Montreal Heart Institute, Montreal, Quebec H1T 1C8, Canada, the  Department of Medicine, University of Montreal, Montreal, Quebec H3C 3J7, Canada, and the  Department of Pharmacology, Harbin Medical University, Harbin, Heilongjiang 150086, China

Received for publication, June 18, 2001, and in revised form, August 2, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Multiple subtypes (_1A, _1B, and _1D) of ₁-adrenoreceptors (₁ARs) co-exist in the heart and mediate a variety of cellular functions. We studied ₁AR modulation of inward rectifier (I_K1) and transient outward (I_to) K⁺ currents in canine ventricular myocytes. Phenylephrine at 10 µM depressed only I_to without affecting I_K1 and at 100 µM inhibited both I_to and I_K1. The effect of phenylephrine on I_to was abolished by (+)niguldipine (10 nM) to inhibit _1AARs but not by chloroethyclonidine (10 µM) to inactivate _1BARs nor by BMY-7378 to antagonize _1DARs. In contrast, phenylephrine inhibition of I_K1 was reversed only by BMY-7378 (1 nM). PDD (100 nM, phorbol ester activator of protein kinase C (PKC)) simulates and bisindolylmaleimide (50 nM, PKC inhibitor) weakens phenylephrine modulation of I_to but not I_K1. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 and inhibitor peptides abolished the effects of phenylephrine on I_K1. Enhancement of PKC or CaMKII activities was seen in _1aAR- or _1dAR-transfected HEK293 cells and in myocytes pretreated with 10 or 100 µM phenylephrine, respectively. Our data suggest that different subtypes of ₁ARs selectively modulate different cardiac K⁺ currents via different signal transduction mechanisms; _1AARs mediate I_to regulation via PKC, and _1DARs mediate I_K1 regulation via CaMKII.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Over the past decade, evidence from pharmacological studies and molecular cloning has been accumulating indicating that ₁-adrenoreceptors (₁ARs)¹ are actually a heterogeneous group of distinct but related protein subsets. Many cellular responses to ₁ARs are mediated by multiple subtypes (_1A, _1B, and _1D) (1-4). In the heart, whereas the _1A and _1B subtypes have been well characterized, the presence of _1DAdR was indicated only recently (5-7). Moreover, although the pathophysiological roles of _1A and _1B receptors have been well appreciated, those of _1D subtype in the heart remain to be determined.

Enhanced ₁AR activity has been implicated in various types of arrhythmias, particularly those in the pathogenesis of myocardial ischemia, ischemia-reperfusion and preconditioning, cardiac hypertrophy, etc. (1, 3). Drug intervention with ₁ARs has thus become an attractive issue for developing new compounds for potential therapy. A significant mechanism underlying ₁AR-induced alteration of cardiac electrical activity is attributable to the ability of ₁ARs to modulate ion channels. To date, no less than seven cardiac ionic currents are on the list of ₁AR modulation, including inward rectifier K⁺ current (I_K1), transient outward K⁺ current (I_to), delayed rectifier K⁺ current (I_K), ultrarapid delayed rectifier K⁺ current (I_Kur), acetylcholine-induced K⁺ current (I_KACh), calcium current (I_Ca), and chloride current (1, 3, 8-11). However, it is not known whether the effects are the results from participation of all three different subtypes of ₁ARs or of a particular individual subtype, although evidence is accumulating that different subtypes may have different roles in regulating cardiac contraction and electrical activities (12-16). Moreover, recent studies also demonstrated subtype differences in the signal transduction (17-19). In light of these studies, we speculated that different subtypes of ₁ARs may have distinct effects on ion channels. Understanding subtype specificity of ₁ARs in ion channel regulation is of theoretical and practical importance.

K⁺ currents play critical roles in determining cardiac electrical activities. Besides stabilizing resting potential, I_K1 in cardiac cells also plays an important role in modulating cellular excitability and regulating membrane repolarization, therefore an important determinant of action potential initiation. Another important cardiac K⁺ current is transient outward K⁺ current (I_to), which is known to be critical for initiating cardiac repolarization in the early phase of action potentials. Both of these currents have been implicated in the pathology of cardiac electrophysiological disorders and heart failure (20). For these reasons, we explored the potential subtype selectivity and signal transduction mechanisms of ₁ARs in regulating I_to and I_K1 in isolated canine ventricular myocytes using whole cell patch clamp techniques.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Myocyte Isolation-- Single canine myocytes were isolated from the epicardium of left ventricles with techniques as described previously (21). For electrophysiological experiments, the dispersed cells were stored in KB medium (20 mM KCl, 10 mM KH₂PO₄, 25 mM glucose, 70 mM potassium glutamate, 10 mM -hydroxybutyric acid, 20 mM taurine, 10 mM EGTA, 0.1% albumin, and 40 mM mannitol, pH 7.4).

Whole Cell Patch Clamp Recording-- Patch clamp recording techniques used have been described in detail elsewhere (22-24). Borosilicate glass electrodes (outer diameter, 1 mm) had tip resistances of 1-3 M when filled with pipette solution. Junction potentials were zeroed before formation of the membrane-pipette seal in Tyrode's solution. The capacitance and series resistance was electrically compensated to minimize the duration of the capacitive surge on the current recording and the voltage drop across the clamped cell membrane. I_to was defined as the peak current amplitude, and I_K1 was measured as the amplitude at the end of 400-ms pulses. The experiments were conducted at 36 °C.

Solutions and Drugs-- The bath solution for whole cell patch clamp recording had the following composition 136 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 0.33 mM NaH₂PO₄, 5 mM HEPES, 10 mM glucose, and 1 mM CaCl₂; pH was adjusted to 7.4 with NaOH. Unless otherwise specified, the pipette solution contained 0.1 mM GTP, 110 mM potassium aspartate, 20 mM KCl, 1 mM MgCl₂, 5 mM Mg-ATP, 10 mM HEPES, and 5 mM phosphocreatine, pH 7.3. Sodium current was prevented by holding the cells at 20 or 50 mV, and calcium current was blocked by inclusion of Cd²⁺ (200 µM) in the bathing solution. All chemicals were purchased from Sigma.

Phenylephrine (Phen; used as a non-subtype-selective ₁AR agonist), prazosin (a non-subtype-selective ₁AR antagonist), (+)niguldipine (Nig; a specific inhibitor of _1AARs), chloroethyclonidine (CEC; an alkylating agent for _1BARs), BMY-7378 (a specific antagonist of _1DARs), and propranolol were from Sigma (Oakville, Canada). Phen, Nig, CEC, and BMY-7378 were prepared as 100 mM, 10 µM, 10 mM, and 1 µM stock solutions in distilled water, respectively. Phen was always administered along with 1 µM propranolol to prevent any collateral effects mediated by -adrenergic receptor stimulation. Prazosin was dissolved in Me₂SO. The protein kinase C (PKC)-stimulating phorbol ester 12,13-didecanoate (PDD) and its inactive congener 4-PDD were obtained from Calbiochem-Novobiochem International (La Jolla, CA) and prepared as a 10 mM stock solution in Me₂SO. Bisindolylmaleimide (Bis; PKC inhibitor; Sigma) was dissolved as a 50 µM stock solution in Me₂SO. KN-93 (a specific inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII)), KN-92 (inactive analog of KN-93), synthetic CaMKII inhibitor peptides (peptide 281-309 (P281-309), a potent calmodulin antagonist containing the calmodulin-binding site amino acids 290-309 and the autophosphorylation site Thr²⁸⁶ of CaMKII)), and autocamtide-2-related peptide (ACP) were all purchased from Calbiochem (San Diego, CA). Calmidazolium, a calmodulin inhibitor, was purchased from Sigma. KN-93 and KN-92 were dissolved in Me₂SO and added to the bathing solution to the desired concentrations. P281-309 and ACP were dissolved in the pipette solution.

PKC Activity Assay and Immunoblotting Analysis of CaMKII Activity-- The HEK293 cells stably transfected with _1aARs and _1dARs were a kind gift from Dr. Kenneth P. Minneman (Emory University, Atlanta, GA). The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. The cells were passaged regularly and subcultured to ~90% confluence before experimental procedures. For drug treatment, the cells were incubated with Phen at 10 or 100 µM or vehicle for 20 min before being collected for protein preparation. Similarly, isolated canine ventricular myocytes were also treated with Phen or vehicle in KB solution for 20 min. The proteins were prepared following the same procedures as described previously (25, 26). The protein content was determined with Bio-Rad protein assay kit using bovine serum albumin as a standard.

PKC activity was assayed with the PKC enzyme assay system from Amersham Pharmacia Biotech, and the assay procedures were performed according to the system instructions. The Anti-ACTIVE^® CaM KII polyclonal antibody system from Promega was used to assess CaMKII activity, containing the rabbit polyclonal antibody, which recognizes autophosphorylation of Thr²⁸⁶ of all isoforms of CaMKII. Experiments were performed according to the manufacturers' protocol. Bound antibodies were detected with Western blot Chemiluminescence Reagent Plus (PerkinElmer Life Sciences) and quantified by densitometry, as detailed previously (25-26). Coomassie staining was performed to verify the amount of protein inputs, as described previously (25-26).

Statistical Analysis-- The group data are expressed as the means ± S.E. Statistical comparisons were made with Student's nonpaired t test. All enzyme activity and Western blot determinations were performed in parallel with cells from each group for each experiment to minimize contamination by inter-day and inter-lot reagent variation, and the results for each experiment are expressed as the values normalized to control group determinations.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

₁AR Modulation of I_to and I_K1-- Application of depolarizing or hyperpolarizing voltage steps (voltage protocols shown in the non-graphical parts of Fig. 1) elicited outward I_to with transient properties (rapid activation and inactivation) or I_K1 with strongly inward rectification. Bath application of Phen at 10 µM produced marked reduction of I_to amplitude without apparent alterations in its activation and inactivation kinetics but did not affect I_K1 (Fig. 1). When Phen concentration was elevated to 100 µM, I_K1 was significantly decreased, I_to was further reduced, and the inactivation kinetics was slightly decelerated. Co-application with prazosin (1 µM) completely converted the depressed I_to and I_K1 caused by Phen back to predrug base-line values, indicating the role of ₁ARs in mediating the effects of Phen.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   1AR modulation of transient outward K+ current (Ito) and inward rectifier K+ current (IK1) in canine ventricular myocytes. A and B, raw traces recorded from representative cells showing the inhibition of Ito and IK1 produced by Phen at concentrations of 10 and 100 µM, respectively. Voltage protocols for current recordings are shown in the insets. C and D, current-voltage (I-V) relationships of Ito (n = 19 cells) and IK1 (n = 22 cells). When Phen concentration was 10 µM, statistically significant inhibition (p < 0.05) of Ito was seen at potentials positive to 0 mV, and the effect on IK1 was not statistically significant (p > 0.05). When Phen concentration was raised to 100 µM, Ito blockade was statistically significant (p < 0.05) at potentials positive to 20 mV, and IK1 blockade was statistically significant (p < 0.05) at potentials more negative than 80 mV.

Subtype Specificity of ₁AR Modulation of I_to and I_K1-- To determine which receptor subtypes, _1A, _1B, or _1D, mediate the effect of ₁AR modulation of I_to and I_K1, we performed experiments using subtype-selective antagonists. Current recordings made under control conditions were repeated 10 min after exposure of the cells to Phen (10 µM). Subsequently, Nig (10 nM) was concurrently applied with Phen to the bath solution. As illustrated in Fig. 2, Nig reversed Phen-induced I_to depression. Nig also reduced the further reduction of I_to caused by raising Phen concentration to 100 µM. On the other hand, Nig failed to change the depressed I_K1 caused by Phen (100 µM).

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Effects of subtype-selective antagonists on 1AR modulation of Ito (A) and IK1 (B). Analog data shown were elicited by the voltage protocols shown in the insets. Nig (10 nM), CEC (10 µM), and BMY-7378 (BMY, 1 nM). Nig or BMY-7378 was co-applied with Phen to the bathing solution. For CEC experiments, the cells after control current recordings were superfused with CEC for 20 min, following a 30-min wash-out period (After CEC), and then Phen was applied. The averaged values were the data obtained at a potential of +30 mV for Ito and 120 mV for IK1. The values in parentheses indicate the number of cells studied. *, p < 0.05, Student t test, comparison versus control; +, p < 0.05, comparison versus Phen or After CEC.

We then turned to study the effects of CEC (10 µM), an alkylating agent selective toward _1BARs over other subtypes. Following a 30-min incubation with CEC to inactivate the _1BARs, the cells were superfused with drug-free Tyrode's solution for 20 min, and then measurements of I_to and I_K1 were made as base-line values. Then Phen was added to the superfusate. The same degrees of I_to and I_K1 diminishment as in cells without pretreatment with CEC were consistently seen in a total of six cells (Fig. 2).

BMY-7378 (1 nM), a specific antagonist of _1DARs (3, 4, 27), when co-applied with Phen, nearly fully reversed the inhibitory effects of Phen (100 µM) on I_K1 but not I_to (Fig. 3). BMY-7378 alone did not exert any detectable effects on I_K1 (n = 5).

View larger version (47K): [in this window] [in a new window]   Fig. 3.   Roles of PKC in 1AR modulation of Ito (A) and IK1 (B). For PDD (or 4-PDD) experiments, the data were acquired in control conditions, 10 min after bath application with Phen (10 µM for Ito and 100 µM for IK1), 15 min after wash-out of Phen (After Phen), and 10 min after superfusion with PDD (100 nM, or 4-PDD 100 nM). Only current traces recorded in control and after PDD were shown for the sake of clarity. For Bis experiments, current recordings made in control conditions and 10 min after Phen were repeated 15 min after co-application of Phen and Bis (50 nM) and 10 min after Bis and PDD. The averaged values were the data obtained at a potential of +30 mV for Ito and 120 mV for IK1. The values in parentheses indicate the number of cells studied. *, p < 0.05, Student t test, comparison versus control; +, p < 0.05, comparison versus Phen or After Phen. The order of the bar data follows the same order of experimental procedures.

Signal Transduction Mechanisms of ₁AR Modulation of I_to and I_K1-- ₁ARs are known to stimulate activation of PKC (17, 19, 28), which in turn can phosphorylate the channel proteins. To investigate whether PKC can account for the ₁AR modulation of I_to and I_K1, the effects of PKC inhibitor Bis and activator PDD were assessed (10-11). 10 min after superfusion with Phen (10 µM) to verify the effect on I_to, Bis (50 nM) was co-applied. As illustrated in Fig. 3, Bis nearly abolished the depressing effects of Phen on I_to. By comparison, Bis failed to affect the reduction of I_K1 by 100 µM Phen, even with elevated Bis concentrations up to 200 nM. External application of PDD (100 nM), mimicking the effect of Phen on I_to, caused marked suppression of I_to amplitude. Moreover, no significant effect of 4-PDD (100 nM, the inactive stereoisomer of PDD) on I_to was seen, and when in the presence of Bis, PDD had little effect on I_to. Slight reduction of canine I_K1 (about 7%) was observed when treated with PDD (100 nM).

CaMKII has recently been reported to be involved in regulating ion currents (29-31). Accordingly, we also studied the potential participation of CaMKII in ₁ARs or Phen modulation of I_to and I_K1. As illustrated in Fig. 4, Phen at 10 µM produced the similar effects on I_to with and without KN-93 (3 µM, a potent CaMKII inhibitor) present in the bath solution. However, with 100 µM Phen, I_to reduction was slightly smaller in the presence of KN-93 than in the absence of the compound (Fig. 4B). The CaMKII inhibitor peptides P281-309 or ACP dialyzed into the cells did not affect I_to neither the ability of Phen to inhibit I_to (Fig. 4, A and C). By comparison, the reduction of I_K1 caused by Phen (100 µM) alone was completely prevented by KN-93 (Fig. 5). We then examined whether the effects of KN-93 on I_K1 were related to the inhibition of CaMKII activity or to a direct effect on K⁺ channels. KN-92 (10 µM), the inactive analog of KN-93, failed to affect I_K1 reduction caused by Phen (Fig. 5B). Further evidence that I_K1 is regulated by CaMKII was obtained by dialyzing cells with the inhibitor peptides P281-309 or ACP. The cells were bathed in the solution containing Phen (100 µM) for at least 10 min before the formation of whole cell configuration. The currents recorded immediately after membrane rupture with minimal dialysis were considered the base-line data, and the data acquired 15 min after membrane rupture with complete dialysis were taken as the effects of CaMKII inhibition by ACP or P281-309 (Fig. 5). In addition, addition of EGTA (10 mM) to the pipette solution also substantially weakened the ability of Phen to suppress I_K1 (data not shown). The effects of KN-93 on Phen-induced decrease in I_K1 were also assessed in myocytes pretreated with the calmodulin inhibitor calmidazolium. Similar to KN-93, calmidazolium converted the depressed I_K1 to the base-line amplitude (Fig. 5B). External application of KN-93 (3 µM) when the steady-state effect of calmidazolium had been achieved failed to cause further changes on I_K1 (data not shown).

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Roles of CaMKII in 1AR modulation of Ito. For experiments involving KN-93, the currents were measured in control conditions, 10 min after Phen (10 µM), and 15 min after co-administration of Phen and KN-93 (3 µM). For experiments with inhibitor peptides (P281-309 (60 µM) and ACP (50 µM)) on Ito, the currents recorded right after membrane rupture were taken as control base-line data, followed by 15 min of dialysis of the peptides into the cells through the recording pipette, and then Phen (10 µM) was superfused for 10 min. The averaged values were the data obtained at a potential of +30 mV. The values in parentheses indicate the number of cells studied. *, p < 0.05, Student t test, comparison versus control. The order of the bar data follows the same order of experimental procedures.

View larger version (43K): [in this window] [in a new window]   Fig. 5.   Roles of CaMKII in 1AR modulation of IK1. For experiments involving KN-93 or calmidazolium, the currents were measured in control conditions, 10 min after Phen (100 µM), and 15 min after co-administration of Phen and KN-93 (3 µM) or KN-92 (10 µM) or calmidazolium (50 µM). For experiments involving inhibitor peptides on IK1, the cells were superfused with the solution containing Phen (100 µM) throughout the experiments. The currents recorded right after patch formation and 15 min after rupture were taken as base-line and inhibitor peptide data, respectively. As a negative control, experiments were also conducted in the absence of Phen. The averaged values were the data obtained at a potential of 120 mV. The values in parentheses indicate the number of cells studied. *, p < 0.05, Student t test, comparison versus control; +, p < 0.05, comparison versus Phen.

PKC Activity Assay and Immunoblotting Analysis of CaMKII Activity-- Based on the results from the above functional studies, we believed that suppression of I_to by Phen at 10 µM is primarily mediated by PKC activation as a result of _1AAR activation, whereas an increase in CaMKII activity caused by _1DARs stimulation by 100 µM Phen leads to I_K1 inhibition. To test this point, we performed analyses for PKC and CaMKII activities. Fig. 6 shows the results from PKC activity assay. A pronounced increase in PKC activity was seen in HEK293 cells stably transfected with _1aARs and pretreated with Phen, relative to nontransfected and untreated cells. Only a minor increase in PKC activation was observed in _1dAR-transfected cells, even with elevated Phen concentration to 100 µM. Coincidentally, canine ventricular myocytes pretreated with 10 µM (or higher) Phen also resulted in a significant increase in PKC activity relative to untreated cells.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   PKC activities stimulated by phenylephrine and effects of 1AR subtype selective inhibitors. A, PKC activity assayed with protein samples from 1aAR- or 1dAR-transfected HEK293 cells. 10 nM Nig and 1 nM BMY-7378 (BMY) were used. B, PKC activity assayed with protein samples from nontransfected HEK293 cells as a control. PZ, prazosin (2 µM). C, PKC activity assayed with protein samples from canine ventricular myocytes. The values in parentheses indicate the number of independent samples tested for each group. *, p < 0.05, unpaired Student t test, comparison versus control; +, p < 0.05, comparison versus Phen (100 µM).

The antibody to the autophosphorylation site Thr²⁸⁶ of CaMKII recognized a band of 51 kDa in both HEK and a band of 48 kDa in canine ventricular cells, which is in agreement with the size of CaMKII reported by other laboratories (29-31). As shown in Fig. 7, a more prominent band was seen only in _1dAR-transfected cells pretreated with 100 µM Phen. There are slight increases in the phosphorylated CaMKII in _1aAR-transfected cells or in _1dAR-transfected cells treated with 10 µM Phen. For canine ventricular myocytes, only samples from cells pretreated with 100 µM Phen showed an enhanced phosphorylation of CaMKII, and the immunoreactive band was nearly invisible in control and 10 µM Phen-treated cells.

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Western blot analysis of the activity of CaMKII. The antibody raised against the phosphorylated form of CaMKII recognized a 51-kDa band in the protein samples from 1AR-transfected (A) or nontransfected (B) HEK293 cells and a 48-kDa band in the samples from canine ventricular cells (C), which were abolished when the antibody was pretreated with the antigenic peptide (the lanes labeled with Antigen) from which the antibody was raised. The values in parentheses indicate the number of independent samples tested for each group. *, p < 0.05, unpaired Student t test, comparison versus control; +, p < 0.05, comparison versus Phen (100 µM).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have shown in this study that ₁AR stimulation by phenylephrine produces important suppression of I_to and I_K1 in dog ventricular myocytes. The effects on I_to and I_K1 can be separated by the strength of ₁AR stimulation (Phen concentrations), and the use of subtype-selective antagonists suggests a possibility of differential modulation of I_to and I_K1 by activation of different subtypes of ₁ARs: _1AAR for I_to and _1DAR for I_K1. Moreover, our data also suggest that the differential modulation of I_to and I_K1 by ₁ARs is mediated by different signaling systems: I_to primarily by PKC and I_K1 mainly by CaMKII. Our data therefore provide evidence that different subtypes of ₁ARs modulate different cardiac K⁺ currents via different signal transduction mechanisms. Our results also represent, to our knowledge, the first to define the functional role of _1DARs in modulating cardiac ion channels and the role of CaMKII in modulating inward rectifier K⁺ current.

With the development of pharmacological tools and molecular cloning, it is now possible to study ₁AR subtypes separately. The _1AARs show a high affinity to (+)niguldipine that has been demonstrated to be 100~4000-fold more selective toward _1AAR over the other two subtypes (4, 32). The _1BARs are readily and irreversibly inactivated by the alkylating agent CEC (4, 33). BMY-7378 distinguishes _1DAR from the other two subtypes (_1A and _1B) with a binding affinity at least 2 orders of magnitude higher for the former over the latter (4, 7, 27). We show here that neither Nig nor CEC produced any appreciable influences on the inhibitory effects of Phen on I_K1. In sharp contrast, BMY-7378, at a concentration of as low as 1 nM, readily reversed Phen actions on I_K1. These data strongly suggest that _1DARs mediate ₁AR/Phen modulation of I_K1 in canine ventricular myocytes. ₁AR modulation of I_K1 in rabbit (34) and human (9) atria has also been previously studied, and consistent with the present study ₁AR stimulation was found to decrease atrial I_K1. However, the involvement of particular subtypes of ₁ARs was not investigated in these studies. Although our study probably is the first to establish the functions of _1DARs in the heart, its roles may not be restricted to the modulation of I_K1.

The present study appears to favor the notion that modulation of I_to in canine ventricular cells by ₁AR stimulation is primarily mediated by a subclass of ₁ARs, that is, _1AARs. A similar decrease in I_to upon ₁AR stimulation was also previously documented in other species such as rabbits (36) and rats (37, 38), but no information regarding involvement of particular subtypes of ₁ARs was provided. In another study reported by Wang et al. (39) in rat ventricular cells, phenylephrine at 30 µM was shown to reduce peak I_to, and both of the _1AAR-selective antagonists 5-methylurapidil and (+)niguldipine (0.1 µM each) and the irreversible _1BAR-subtype antagonist CEC (100 µM) blocked the phenylephrine effect on I_to. They concluded that stimulation of both ₁AR subtypes contributes to the phenylephrine-induced reduction in I_to of rat myocytes. However, it should be noted that CEC concentration used in this study was 100 µM, high enough to inactivate subtypes (e.g. _1A and _1D) other than _1BARs.

For the signal transduction mechanisms underlying ₁AR modulation of I_to, studies on cloned channels that generate I_to-like K⁺ currents in heterologous expression systems demonstrated that activation of PKC reduces Kv4.2 and Kv4.3 (40). This result is in good agreement with ours, which points to an important role of PKC activation mediated by ₁AR stimulation in regulating I_to, particularly when considering that Kv4.3 is the major molecular component of native I_to in dogs (41). The results from previous studies on PKC modulation of inward rectifier K⁺ channels (Kir) have been controversial. For the cloned Kirs, the study from Henry et al. (42) convincingly demonstrated that PKC activation by phorbol 12-myristate 13-acetate or phorbol 12,13-dibytyrate significantly inhibited Kir2.3 but did not alter Kir1.1 and Kir2.1. In light of our previous finding that Kir2.1 is the most abundantly expressed Kir subunit in human hearts (22), our present data are in line with the results from the study by Henry et al. Yet Fakler et al. (43) reported that stimulation of PKC by 12-O-tetradecanoylphorbol 13-acetate suppressed Kir2.1. It is unclear whether this is due to the use of different PKC activators by the two laboratories. Similarly, in native cells no consistent data have been reported. In the study performed by Braun et al. (34) it was shown that ₁AR inhibition of I_K1 in rabbit atrial cells did not depend on PKC activation, and direct PKC activation or inhibition did not affect I_K1 either. In contrast, one study conducted in human atrial myocytes suggested that ₁AR inhibition of I_K1 is mediated by PKC activation (9). One possible explanation for the discrepancy is that different species might have distinct molecular compositions of I_K1 because to date no less than 10 different Kir cDNAs have been cloned (44).

Several lines of evidence from the present study suggest that suppression of I_K1 by ₁AR stimulation in canine ventricular myocytes is mediated by CaMKII activation. To date, no other studies have published regarding CaMKII modulation of I_K1. However, CaMKII modulation of I_to or the cloned channels expressing I_to-like currents has been documented in several studies. Consistent in all these studies is the reduction of current amplitude. In the present study, we show that Phen at a concentration of 10 µM mainly activates PKC pathway, as indicated by inhibitor experiments, PKC assay, and CaMKII immunoblotting analysis. We therefore speculate that I_to modulation by 10 µM Phen is mainly mediated by PKC phosphorylation, whereas I_to reduction by 100 µM Phen might be the consequence of combined PKC and CaMKII activation. This is supported by our data showing that inhibition of CaMKII partially reversed I_to reduction caused by Phen at 100 µM but not at 10 µM. In addition, 100 µM Phen slightly slowed the inactivation kinetics of canine I_to (Fig. 1A), which was not seen with 10 µM Phen.

The _1AAR is generally far more efficient in stimulating PKC activation than the _1DAR (19). For example, Taguchi et al. (17) showed that Phen significantly stimulated PKC in rat-1 fibroblasts stably expressing _1AARs and _1BARs but not _1DARs. Our data are consistent with this notion. Intriguingly, a study performed in a vascular smooth muscle cell line (AC01) (18) demonstrated that the _1DARs, although representing the minor population compared with the _1BARs in this cell, are the main mediators of phosphoinositide/Ca²⁺ signaling. Moreover, based on their experimental data from isolated hepatocytes, Butta et al. (45) concluded that there are at least two major ₁AR signaling pathways; one is PKC-dependent and independent of variations in free cytosolic Ca²⁺, and the other one is dependent on variations in free cytosolic Ca²⁺ but is PKC-independent. Actually, the ability of ₁AR to activate CaMK has been previously realized. The study reported by Guo et al. (46) found that CaMK contributed to the ₁AR-mediated decrease in Kv1.5 K⁺ channel expression in cultured newborn rat ventricular cells. However, it was not characterized which subtype of ₁ARs is responsible for the effect in these studies. Our data suggest that _1AARs are mainly associated with PKC activation, whereas _1DARs are primarily coupled to CaMKII activation. Yet it should be noted that our data do not allow us to reach a conclusion on how _1AARs are coupled to PKC and how _1DARs are coupled to CaMKII. More detailed studies are necessary for verifying this notion and for delineating the subtype-specific signaling coupling mechanisms.

	ACKNOWLEDGEMENT

We thank XiaoFan Yang for excellent technical support.

	FOOTNOTES

^* This work was supported in part by funds from the Canadian Institute of Health Research, the Heart and Stroke Foundation of Quebec, and the Fonds de la Recherche de l'Institut de Cardiologie de Montreal (to Z. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Research fellow of the Canadian Institute of Health Research.

^¶ These authors contributed equally to this work.

^** Research fellow of the Heart and Stroke Foundation of Canada.

^§§ Research scholar of the Heart and Stroke Foundation of Canada. To whom correspondence should be addressed: Research Center, Montreal Heart Inst., 5000 Belanger East, Montreal, PQ H1T 1C8, Canada. Tel.: 514-376-3330; Fax: 514-376-1355; E-mail: wangz@icm.umontreal.ca.

Published, JBC Papers in Press, August 27, 2001, DOI 10.1074/jbc.M105572200

	ABBREVIATIONS

The abbreviations used are: AR, adrenoreceptor; Phen, phenylephrine; Nig, (+)niguldipine; CEC, chloroethyclonidine; BMY-7378, 8-[2-[4-(2-Methoxyphenyl)-l-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride; PKC, protein kinase C; PDD, phorbol ester 12,13-didecanoate; Bis, bisindolylmaleimide; CaMKII, Ca²⁺/calmodulin-dependent protein kinase II; P281-309, peptide 281-309; ACP, autocamtide-2-related peptide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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