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(Circulation Research. 2000;86:795.)
© 2000 American Heart Association, Inc.

Integrative Physiology

Enhanced Cardiac Function in Transgenic Mice Expressing a Ca²⁺-Stimulated Adenylyl Cyclase

Larissa Lipskaia, Nicole Defer, Giovanni Esposito, Iman Hajar, Marie-Claude Garel, Howard A. Rockman, Jacques Hanoune

Correspondence to Jacques Hanoune, Unite de Recherches, INSERM U-99, Hôpital Henri Mondor, F-94010 Créteil, France. E-mail hanoune{at}im3.inserm.fr

	Abstract

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Abstract—The predominant functional adenylyl cyclases normally expressed in cardiac tissue and coupled to ß-adrenergic receptors are inhibited by micromolar Ca²⁺ concentration. To modify the overall balance of activities, we have generated transgenic mice expressing the Ca²⁺-stimulatable adenylyl cyclase type 8 (AC8) specifically in the heart. AC activity is increased by at least 7-fold in heart membranes from transgenic animals and is stimulated by Ca²⁺ in the same range of concentration that inhibits the endogenous activity. Moreover, the in vivo basal protein kinase A activity was augmented 4-fold. Overexpression of AC8 in the heart has no detrimental consequences on global cardiac function. Basal heart rate and contractile function, measured by noninvasive echocardiography, were unchanged. In contrast, on release of parasympathetic tone, the intrinsic contractility is heightened and unresponsive to further ß-adrenergic receptor stimulation. AC8 transgenic mice thus represent an original model to investigate the relative influence of Ca²⁺ and cAMP on cardiac function within a phenotype of enhanced cardiac contractility and relaxation.

Key Words: adenylyl cyclase • transgenesis • cardiac function

	Introduction

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In the heart, the force of contraction is dependent on the influx of Ca²⁺ ions through voltage-dependent channels.^{1 2} ß-Adrenoceptor stimulation augments the amplitude of the L-type Ca²⁺ current and the force of contraction³ through binding to ß-adrenergic receptors (ß-ARs), stimulation of adenylyl cyclase (AC), and increase in the concentration of cAMP. To understand the physiological and pathological consequences of this cascade, murine models have been created with an enhanced efficacy of the ß-AR–Gs-AC signaling pathway.^{4 5 6} Overexpression of the ß₂-AR resulted in a maximal activation of the ß-AR signaling pathway, even in the absence of the agonist.^{4 7} Gs overexpression resulted in cardiomyopathy and substantial cardiac histological abnormalities.⁸

Another approach to enhance ß-AR–Gs-AC signaling would be to bypass the potential deleterious consequences of the receptor or G protein and directly to increase the expression of the effector, AC. At least 9 isoforms of AC are known.⁹ There is a significant heterogeneity in the distribution and biochemical properties of the different isoforms, and each tissue or cell type possesses a unique combination of these isoforms. In the heart, the Ca²⁺-inhibitable isoforms AC5 and AC6 are the most abundant.^{10 11} Elevation of Ca²⁺ concentration might inhibit cAMP synthesis and thereby provide a sensitive negative feedback.¹² In contrast, AC1 and AC8, which are essentially expressed in the central nervous system, are activated by Ca²⁺ through the Ca²⁺/calmodulin complex.^{13 14 15}

In this study, we describe transgenic mice overexpressing the Ca²⁺/calmodulin-activatable isoform AC8 specifically targeted to cardiomyocytes. Surprisingly, we observed that AC8 overexpression has no effect on the viability of the animals but leads to a higher basal intrinsic contractility that is unresponsive to further ß-AR stimulation.

	Materials and Methods

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Generation of Transgenic Mice
For the construction of transgenic mice, the murine -myosin heavy chain (MHC) promoter¹⁶ was ligated to the cDNA coding for human AC8.¹⁵ Mice were screened for the presence of the transgene by Southern blot performed on tail genomic DNA. Two founders were identified and propagated by crossbreeding with C57BL/6 wild-type mice. Number of transgene copies was determined by slot-blot analysis. The care and use of animals were in accordance with institutional guidelines.

Echocardiography
Echocardiography was performed in anesthetized mice (Avertin [tribromoethanol] 2.5%, 14 µL/g IP) using an ATL HDI 5000 (ATL Ultrasound, Bothell, Wash) echocardiograph as previously described.¹⁷ The following parameters were measured: left ventricular (LV) end-diastolic dimension (LVEDD), LV end-systolic dimension (LVESD), posterior and septal wall thickness, heart rate, percentage of fractional shortening (%FS) (calculated as [LVEDD–LVESD]/LVEDDx100), and mean velocity of circumferential fiber shortening (mean Vcfc).

Hemodynamic Evaluation
Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (2.5 mg/kg) and analyzed as previously described.^{6 18} Briefly, after endotracheal intubation, mice were connected to a rodent ventilator. After bilateral vagotomy, a 1.4F high-fidelity micromanometer catheter (Millar Instruments) was inserted into the right carotid artery and retrograde across the aortic valve into the LV. Hemodynamic measurements were recorded at baseline and 45 to 60 seconds after injection of incremental doses of isoproterenol (ISO). Doses of ISO were specifically chosen to maximize the contractile response but limit the increase in heart rate. Ten sequential beats were averaged for each measurement.

RNA Preparations and Northern Blotting
Total RNA was extracted,¹⁹ and Northern blots were carried out as described.²⁰ RNAs were hybridized with [-³²P]dCTP-labeled AC8, AC5, or AC6 cDNA probes.²⁰ A rat GAPDH cDNA was used to control the equal RNA loading.

AC Assays
AC activity was measured as described²⁰ on purified cardiac membranes. Hearts were homogenized in 10 volumes of ice-cold lysis buffer (in mmol/L, Tris-HCl [pH 7.6] 10, EDTA 0.1, DTT 0.5, and PMSF 0.5) and centrifuged at 500g for 5 minutes at 4°C. The supernatant was centrifuged at 15 000g for 30 minutes and the pellet washed 3 times in the same buffer. For analysis of Ca²⁺ effect, the membranes were previously washed twice with 1 mmol/L EGTA. Free Ca²⁺ concentrations were calculated as described.²¹

Protein Kinase A (PKA) Assay
PKA activity was measured on crude myocardial extracts using the Signa TECT PKA Assay System (Promega). Assays were performed with or without exogenous cAMP (5 µmol/L). Addition of the PKA peptide inhibitor completely abolished the enzyme activity.

ß-AR Binding
ß-ARs were estimated by saturation binding of [¹²⁵I]iodocyanopindolol (¹²⁵I-CYP) as described.^{6 22}

Western Blotting
Crude cardiac homogenates were prepared as described (Upstate Biotechnology). Proteins (25 to 100 µg/lane) were separated on 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with anti-calmodulin antibody as recommended by the manufacturer, and antigen was visualized using the enhanced chemiluminescence system from Amersham Pharmacia Biotech.

Statistical Analysis
All results are expressed as mean±SEM of at least 3 determinations. To examine the effect of ISO on changes in hemodynamic parameters between control and transgenic animals, a repeated-measures ANOVA was used. For echocardiographic data, a 1-factor ANOVA was used. Post hoc analysis with regard to differences in mean values between groups was conducted with a Scheffé test. P<0.05 was considered significant.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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Generation and Genetic Characterization of the Transgenic Mice
The -MHC-AC8-SV40 intron/pA transgene (Figure 1A) was microinjected into the pronuclei of fertilized mouse eggs. Two founders were obtained (L7 and L8). The transmission of the transgene was demonstrated by Southern blotting of the offspring (Figure 1B). Slot-blot analyses of genomic DNA from tail biopsies showed 7 to 8 copies of the transgene per genome (not shown). Both strains expressed AC8 mRNA and activity at a high level in the heart. For further studies, only the offspring of the L7 founder were used.

View larger version (30K): [in this window] [in a new window]   Figure 1. Genetic characterization of the transgenic mice. A, DNA construct used for generation of transgenic mice overexpressing human AC8 in the heart. B, Southern blot analysis of HpaI-digested genomic DNA from tail biopsies. Blots were hybridized with the -32P–labeled cDNA probe for AC8, washed (0.5x SSC, 0.1% SDS, at 65°C for 15 minutes), and exposed for 24 hours. Lanes 1 through 8 represent DNA from 13 littermates after the crossing (AC8TMxC57BL/6). N indicates C57BL/6 control animal; P-AC8TM, transgenic parent (L7 founder). C, Cardiac-specific mRNA expression of the AC8 transgene. Shown are total RNAs (30 µg) from heart (H), kidney (K), brain (B), and skeletal muscle (M) of 1 AC8TM or of 1 control mouse. Autoradiograms were obtained after 2 hours (GAPDH) or 12 hours (AC8, AC5, and AC6) of exposure.

Northern blot analysis revealed a high, cardiac-specific expression of the AC8 transgene in these animals (Figure 1C), which is consistent with the previously documented pattern of expression achieved with the murine -MHC promoter.^{4 6 23 24} In contrast, no variation in the mRNA expression of the 2 major cardiac cyclases, AC5 and AC6, was detected.

Anatomical examination of 2-month-old animals showed no fibrosis or any obvious differences between hearts of AC8 transgenic mice (AC8TM; n=6) and control littermates (n=6) with respect to gross morphology or myocyte appearance (not shown). Body weight (control, 34±0.4 g; transgenic, 33±0.7 g), heart weight (control, 153.9±2.8 mg; transgenic, 156.9±7.8 mg), tibia length (control, 19±0.1 g; transgenic, 18.6±0.1 g), and LV wet weight (control, 113.4±2.16 g; transgenic, 117.8±6.6 g) were unchanged by transgene expression (control mice [CM], n=14; transgenic mice, n=12). No differences in behavior or exterior aspect were observed. Neonatal mortality was not different between transgenic and nontransgenic animals.

AC Activity in Cardiac Membranes From AC8TM
AC activity was assayed in cardiac membranes prepared from pools of 10 transgenic or 10 CM. Basal AC activity was increased at least 7-fold in AC8TM as compared with their littermates (156.26±5.5 pmol cAMP/min mg^–1 proteins versus 21.0±3.4 for control hearts [n=5; P=0.001]; Figures 2A and 2B). In the presence of NaF (10 mmol/L), AC activity was increased by 3-fold in AC8TM and by 8-fold in CM (470±18.59 versus 178.43±6.75 pmol cAMP/min mg^–1 proteins in cardiac membranes from AC8TM and CM, respectively).

View larger version (18K): [in this window] [in a new window]   Figure 2. Effects of Ca2+ on basal and on FSK- and calmodulin-stimulated AC activities in cardiac membranes from AC8TM and CM. Membranes (25 µg of proteins) prepared from hearts of CM (A) or transgenic AC8 littermates (B) were incubated with increasing amounts of Ca2+ in the presence ( and ) or in the absence ( and ) of 1 µmol/L calmodulin. • indicates W7 (100 µmol/L) added to the incubation medium. C, Effects of Ca2+ on FSK-stimulated AC activity. AC activity measured in the presence of 10 µmol/L FSK and 0.08 µmol/L free Ca2+ was taken as 100% (CM, 589.92±11.99 pmol cAMP/min mg–1 protein; AC8TM, 2054.74±186.43 pmol cAMP/min mg–1 protein).

To document the AC activity in cardiac membranes from AC8TM heart, the enzyme activity was assayed in the presence of increasing concentrations of Ca²⁺. As expected, micromolar concentrations of Ca²⁺ inhibited the FSK-stimulated AC activity in normal nontransgenic animals by 25% (Figure 2C). In membranes from AC8TM, forskolin (FSK)-stimulated AC activity was higher by 3- to 4-fold than in CM hearts; it was only slightly inhibited by Ca²⁺. Addition of calmodulin (1 µmol/L) had no effect on control membranes (Figure 2A) but evoked a 3-fold stimulation of AC activity in transgenic membranes (Figure 2B). This stimulation was completely abolished by the addition of the calmodulin inhibitor, W7 (100 µmol/L). Because the basal activity increased from 21.0±3.4 to 156.26±5.5 pmol cAMP/min mg^–1 proteins in membranes from control and transgenic hearts, respectively, and to 450.47±39.0 pmol cAMP/min mg^–1 proteins in transgenic heart membranes under calmodulin stimulation, these results demonstrate that, in hearts from transgenic mice, AC8 represents the major part of the AC activity.

In mammalian cardiomyocytes, calmodulin plays an important role as a regulator of cell proliferation and function.^{25 26 27 28} Although its concentration decreases after birth in the heart, it remains high in the adult.^{28 29} We did not find any modification in the calmodulin expression in the heart of AC8TM as compared with their littermates (Figure 3).

View larger version (55K): [in this window] [in a new window]   Figure 3. Western blot analysis of calmodulin (CaM) expression. A, Expression in mouse brain and heart (50 µg protein/lane). Various concentrations of pure calmodulin were used as control as well as a positive control proposed by the manufacturer. B, Expression in mouse heart from 3 control and transgenic animals (50 µg/lane). C, Increasing amounts of cardiac extract from control and transgenic mouse hearts.

To determine whether the increase in the AC activity observed in vitro in the heart membranes of transgenic mice corresponds to an increase in the AC activity in vivo, we measured the cAMP-dependent PKA activity on crude heart extracts from AC8TM and CM. In transgenic mice, PKA activity was found to be higher by 4-fold than that of control animals (2.14±0.06 [n=3] versus 0.59±0.04 pmol ATP/min µg^–1 protein [n=3]; P=0.0004), whereas the total PKA activity measured in the presence of an excess of cAMP was unchanged (transgenic, 7.52±0.49 pmol ATP/min µg^–1 protein, n=3; control, 7.76±0.43 pmol ATP/min µg^–1 protein, n=3; P=NS). This indicates that cAMP level in hearts of AC8TM was considerably increased as compared with CM, suggesting that AC8 was functionally active in vivo.

Echocardiography and In Vivo Assessment of Cardiac Function in AC8-Overexpressing Mice
To determine whether the marked overexpression of AC8 would affect the physiological phenotype, transthoracic echocardiography was performed. Despite the increase in cardiac AC expression, basal heart rate and contractile functions were unchanged (Table); LV end-diastolic and end-systolic dimensions, heart rate, %FS, and mean Vcfc were similar between the 2 groups.

View this table: [in this window] [in a new window]   Table 1. Echocardiography Parameters in Control and AC8 Transgenic Mice

We further assessed the in vivo cardiac function by cardiac catheterization in intact anesthetized control and transgenic mice after bilateral vagotomy. The following parameters were recorded: heart rate, LV systolic pressure, and the 2 derivatives of LV systolic pressure (LV dP/dt_max and LV dP/dt_min) (Figure 4). Under basal conditions, LV dP/dt_max in AC8TM was twice that of wild-type littermates (15 651±1805 versus 8023±1705 mm Hg/s for AC8TM [n=14] and CM [n=12], respectively; P<0.00001) and unresponsive to further ß-adrenergic stimulation (16 736±2390 mm Hg/s; P=NS). Heart rate was significantly increased in AC8TM as compared with wild type (485±20 versus 377±16 bpm for AC8TM [n=14] and CM [n=12], respectively; P<0.0005). These results indicate that with overexpression of AC8, cardiac contractility is markedly increased and unresponsive to further ß-adrenergic stimulation.

View larger version (13K): [in this window] [in a new window]   Figure 4. In vivo assessment of LV contractile function in response to ß-agonist stimulation. Parameters measured were LV end-systolic and end-diastolic pressure, LV dP/dtmax and LV dP/dtmin, and heart rate. Four measured parameters are shown at baseline and after progressive doses of ISO in wild-type mice (, n=14) and AC8TM (•; n=12). A, Heart rate. B, LV systolic pressure. C, LV dP/dtmax. D, LV dP/dtmin. Data were analyzed with a repeated-measures ANOVA. *P<0.0001, P<0.05 vs AC8TM.

Characterization of the AC Signaling in Transgenic Heart
To document whether cardiac myocytes overexpressing AC8 were responsive to ß-adrenergic stimulation in vitro, AC assays were performed in the presence of ISO and in the presence or absence of calmodulin (Figure 5). In control heart, ß-agonist stimulation increased AC activity by 2-fold (Figure 5A). Addition of calmodulin (1 µmol/L) had no effect on the ISO-stimulated AC activity. In membranes from AC8TM, the AC activity was only poorly stimulated by 10 mmol/L ISO, from 219.6±2.99 to 262.25±5.94 pmol cAMP/min mg^–1 protein in the absence of calmodulin, and increased from 600.0±11.35 to 685.0±14.58 pmol cAMP/min mg^–1 protein in the presence of 1 µmol/L calmodulin (Figure 5B). The apparent affinity toward ISO was not different in control and transgenic mice. Thus, ISO stimulation does not appear to affect AC8 activity directly, and it is very likely that in AC8TM, the ISO-stimulated AC activity essentially corresponds to the effect of ISO on the endogenous isoforms. Furthermore, radioligand binding assays indicated a similar ß-AR number in control (57.95±3.10 fmol ¹²⁵I-CYP bound/mg protein) and transgenic (59.39±1.96 fmol ¹²⁵I-CYP bound/mg protein) animals, with no difference in the apparent affinity.

View larger version (18K): [in this window] [in a new window]   Figure 5. Effect of ISO or GTPS on basal or Ca2+/calmodulin-stimulated AC activities. AC activity was measured in the presence of indicated concentrations of ISO and 10 µmol/L GTP or in the presence of GTPS (C). Effect of ISO and GTPS on Ca2+/calmodulin-stimulated AC activity was assayed in the presence of GTP (10 µmol/L), calmodulin (1 µmol/L), and 1.7 µmol/L free Ca2+. , Control (basal); , control (1 µmol/L calmodulin); , AC8TM (basal); , AC8TM (1 µmol/L calmodulin).

The GTPS dose-response curve is shown in Figure 5C. The activity assayed on membranes from CM increased from 13.75±3.25 pmol cAMP/min mg^–1 protein in the absence of GTPS to 126.67±3.48 in the presence of 10 µmol/L GTPS. Addition of calmodulin does not affect this activity. In cardiac membranes from transgenic animals, the activity increased from 150.33±4.63 (GTPS=0) to 320.67±41.09 (GTPS=10 µmol/L) pmol cAMP/min mg^–1 protein in the absence of calmodulin and from 313.0±7.21 (GTPS=0) to 641.0±38.8 (GTPS=10 µmol/L) pmol cAMP/min mg^–1 protein in the presence of 1 µmol/L calmodulin. Thus, the increased AC activity in transgenic mice did not affect either the number of ß-ARs or the GTPS responsiveness of AC activity.

Figure 6 shows the effect of increasing Ca²⁺ concentration on ISO-stimulated AC activity. As expected, Ca²⁺ inhibited the ISO-stimulated endogenous activity by 30% in heart membranes from CM and to a lesser extent in heart membranes from AC8TM. Whereas calmodulin had no effect on inhibition of ISO-stimulated AC activity in CM, in the AC8TM, Ca²⁺ and calmodulin increased the ISO-stimulated activity from 265.0±5.34 to 463.0±32.25 pmol cAMP/min mg^–1 protein.

View larger version (20K): [in this window] [in a new window]   Figure 6. Effect of Ca2+ and calmodulin on ISO-stimulated AC activities. AC activity was measured in the presence of ISO (5 µmol/L), GTP (10 µmol/L), and indicated concentrations of free Ca2+, with or without addition of calmodulin (1 µmol/L). , Control (basal); , control (1 µmol/L calmodulin); , AC8TM (basal); , AC8TM (1 µmol/L calmodulin).

	Discussion

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In the heart, the inotropic effect of ß-adrenergic agonists is mediated by the stimulation of AC activity and the subsequent phosphorylation of specific proteins by cAMP-dependent protein kinase. The prevalence of AC5 and AC6 in the cardiomyocytes hints at a crucial role for the susceptibility of these ACs to Ca²⁺ inhibition in the regulation of cardiac function. It has been proposed that elevated [Ca²⁺]_i could inhibit cAMP synthesis by AC5 and AC6 and thereby provide sensitive negative feedback.^{12 30 31 32} We have developed an in vivo model of transgenic mice overexpressing the Ca²⁺-stimulatable isoform AC8 specifically in cardiomyocytes. Because of the presence of calmodulin in the heart,^{28 29} the activity of this isoform should be activated by Ca²⁺ when the activity of the endogenous isoforms, AC5 and AC6, are inhibited.

Two transgenic lines have been obtained, both expressing AC8 at high levels in cardiomyocytes. For both, AC activity was increased 7-fold and was strongly activated by Ca²⁺/calmodulin, with AC8 representing at least 80% of the total activity in the cardiomyocyte membranes. ISO does not stimulate directly AC8 activity in heart membranes from transgenic mice. The inability of AC8 to respond to ISO by increased cAMP accumulation has already been described in AC8-transfected HEK293 cells.¹⁴ Furthermore, Fagan et al³³ demonstrated that HEK 293 cells possess the capability to localize transfected AC appropriately, suggesting that the targeting information is encoded within the protein sequence. In this context, AC8 would appear to function as a "pure Ca²⁺ " detector.^{34 35} Baker et al³⁶ have demonstrated that the Gs-coupled receptor, 5-HT7 receptor, stimulates AC8 activity in vivo, by increasing [Ca²⁺]_i concentration. We cannot exclude a similar mode of action for the ß-AR to account for some effect on AC8 activity in vivo.

Despite the high AC and PKA activities, the basal cardiac function of the transgene-positive animals, as measured by echocardiography, was not affected. In contrast, when the phenotype was evaluated by invasive hemodynamics, LV dP/dt_max (an index of contractility) was increased and found to be unresponsive to further ß-AR stimulation. Our physiological data demonstrate that overexpression of AC8 does not have deleterious consequences on global cardiac function, because chamber size and fractional shortening are normal. Furthermore, heart rate is not affected as long as the autonomic nervous system is intact. However, release of parasympathetic tone shows that the intrinsic contractility is heightened, in part related to the higher heart rate, with loss of normal ß-AR function as shown by the lack of responsiveness to catecholamine stimulation. Because echocardiography is most sensitive for the determination of chamber dimension and not contractile function, it is not surprising that echo parameters of %FS and Vcfc are the same. For instance, overexpression of the ß₂-AR results in a marked increase in dP/dt max⁴ but has no effect on %FS or Vcfc.³⁷ Importantly, under certain conditions, these mice lose normal regulation of ß-AR coupling. Whether this will have an impact in the conscious animal will require further study.

Cardiac overexpression of the ß-AR, Gs, or ß-AR kinase inhibitor only slightly increases the basal AC activity and the ß-AR signaling.^{4 5 6 38} On the other hand, unlike our AC8 mice, AC6 overexpression in mice resulted in a strong amplification of the ß-AR signaling,³⁹ as evidenced by cAMP accumulation in isolated cardiomyocytes and physiological assessment of cardiac function, although the echocardiographic parameters were unchanged. The overall published literature points to a very complex relationship between ß₁- and ß₂-ARs, Ca²⁺, and cardiac contraction, which could explain the differences observed in the various models of transgenic animals. At present, the relationship between Ca²⁺, cAMP, and the various parameters of cardiac function still needs to be clarified. From this point of view, AC8TM represent an original model in which the AC activity is stimulatable by Ca²⁺. These mice can be used to investigate in more detail the relative influence of Ca²⁺ and cAMP on cardiac function within a phenotype of enhanced contractility and relaxation.

	Acknowledgments

 
This work was supported by the INSERM, the Université Paris XII, and the NIH Grant HL 56 687 (to H.A.R.). L.L. was a recipient of a fellowship from the Fondation pour la Recherche Médicale. We thank Dr Jeffrey Robbins (Children’s Hospital Medical Center, Cincinnati, Ohio) for providing the -myosin heavy chain promoter and Dr J.-F. Authier (Hôpital Henri Mondor, Créteil, France) for histology of the heart. We are grateful to F. Pecker, R. Fischmeister, and M. BestBelpomme for their helpful discussions.

	Footnotes

 
Unite de Recherches, INSERM U-99 (L.L., N.D., J.H.) and INSERM U-474 (I.H., M.-C.G.), Hôpital Henri Mondor, Créteil, France, and University of North Carolina at Chapel Hill (G.E., H.A.R.), Chapel Hill, NC. Present affiliation of G.E. and H.A.R. is Duke University Medical Center, Durham, NC.

Received July 13, 1999; accepted January 11, 2000.

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