Myocardial P-adrenoceptors in pacing-induced heart failure: regulation by enalapril?

C H ~ S T I N B ~ ~ W S T E W , ' GEORGE 0 .A. NAIK, AND GHULHA EAROSA Division of Cardiology, Department of Medicine, and Dgai-tmene sf P h a m ~ o k o g y ~ University of Toronto,

Ste Michael's Hospital, 30 B o d St. , Torunto, ON M5B 4 W8, Canada

Received November 17, 1993

FORSTER, C., NAIK, G.Q A., and LARBSA, G. 1994. Myocardial 0-adrenoceptors in pacing-induced heart failure: regulation by enalapril? Can. J. Physiol. Phamcol . 72: 667-672.

In heart failure, both the sympathetic nervous system and the renin angiotensin system play important pathophysiological roles, and the two systems m y interact with each other, e.g., angiotensin fl facilitating noradrenaline release. An abnomdlity in 8-adrenoceptor density (i-e., a decrease) occurs in clinical and pacing-induced heart failure- This observation together with the therapeutic effectiveness of converting-enzyme inhibitors in the management of patients with heaH% failure led to the current investigation. The aim was to explore the impact of chronic enalapril treatment on the status of myocardial /3-adrens- ceptors in dogs paced (250 h a t s . rnin-l) to end-stage heart failure. Placebo or enalapril treatment (5 rng b.i.d,) commenced 1 week after the onset of ventricular pacing and continued until end-stage heart failure was reached. Myocardial @-adreno- ceptor density and affinity were assessed by radioligand binding with ['""Iiodocyanopinddol. Left ventricular angiotensin II formation and noradrenaline concentration were measured. In addition, plasma renin activity and plasma noradrenaline levels were determind. The results showed that there was a significant increase in 8-adrenoceptor density following enalapril treatment compared with placebo in the heart-failure group. Enalapril did not change the fl-adrenoceptor density in the control animals. However, in both heart failure and control animals, enalapril caused an unexpected increase in Kd. Furthermore, in heart failure, enalapril caused a significant increase in myocardial angiotensin I.l fomation. We conclude that enalapril prevents or reverses the myocardial 8-adrenoceptor abnormality seen in heart failure and promotes angistensin EH formation.

Key words: congestive heart failure, emlapril, cardiac fl-adrenweptors, angiotensin HI formation.

FORSTER, C., NAHK, G.O.A., et E A ~ S A , G. 1994. Myocardial 6-adrenoceptors in pacing-induced heart failure: regulation by enalaprilKan. J. Physiol. Phamcol . 72 : 667-672.

Dans l'insuffisance cardiaque, le systkrne nerveux syrnpathique et le systhme renine-angioitensine ont des rales patho- physiolsgiques importants et peuvent s'imflueaacer mutuellernent, par exemple, I'angiotensine 11 faciliter la liberation de noradrCndine. II se prduit une altkration dams la densitd des 8-adrtnocepteurs (c.-8-d. une diminution) dans l'insuffisance cardiaque induite par stimulation de mCme que dans l'insuffisance cardiaque climique. Cette observation, ainsi que I'efficacitd thkrapeutique des inhibiteurs des enzymes de conversion dans le traitement des patients ssuffrant d'insuffisance cardiaque, ese B l'origine de la prksente ttude. E'objectif a CtC d'examiner l'effet d'un traitement chronique 8 l7Cna1april sur l'ktat des 8-adr6nocepteurs chez des chiens stimulks (250 battements min-') 8 un stade d'insuffisance cardiaque terminale. Uaa traite- ment (5 mg b i d . ) avm de lqCnalapril ou un placebo, initiC 1 semine aprhs le debut de la stimulation cardiaque, a Ctt pour- suivi jusqu98 ce que le sbde d'insuffnsamce cardiaque terninale soit atteint. L'affinitC et la densit6 de fl-adrCmcepteurs myocardiques ont tt6 6valuCes par fixation de radisligands avec de l'['~f]iodocyanopisadol. La concentration de nsradrknaline et la fomation d'angiotensine II dans le ventricule gauche, ainsi que l'activitt rCnine plamatique et les taux de nsradrCmline plasmtique ont C d dCkminCs. Ees rbsultzits snt montrt une augmentation significative de la densit6 des 8-adrtncxepteurs aprks le traitement i 19Cnalapril comparativement au traitement avec placebo chez le g roup souffrant d9insu%sance cardiaque. E'Cndapril n'a pas modifit la densitt des fl-adrCnwepteurs chez les animaux dmoins; toutefois, il a provoqut une augmentation imprCvue de Kd hnt chez les animaux ttmoins que chez les animux souffrant d'insuffisance cardiaque. De plus, l'tnalapril a causC une augmentation significative de la formation d'angiotensine I1 mysmrdique chez les animaux souffrant d9insuffisamce cardiaque. Ainsi, 19Cnalapril prtvient ou renverse l'dtkration des b-adrCncxepteurs obsemCe dans l'insuffisance cardiaque et favorise la fomation d'angiotensine II.

Mots c&&s : insuffisance cardiaque, Cnalapril, 0-adrtnocepteurs cardiaques, fomation d 'angiotemine II. [Traduit par la r&action]

$ntroduc&iow However, the reason for ,heir success is unclear, but may be

Activation sf the sympathetic nervous system and the renin - angiotensin system plays important roles in the pathophysisl- ogy of congestive heart failure (kvine et al. 1982; Thomas and Marks 1982; Francis et d . 1984; Packer 1988). It is proposed that the increased sympathetic activity initially helps maintain pafusion pressure but ultimately contributes to cardiac deterioration, possibly as a result of reduced P-adrenoceptor density (Bristow et d. 1982, 1 986). The remain - amagiotensin system may modulate sympathetic nervous system activity by facilitating noradrendine release. Converting-enzyme inhibi- tors have hemodynamic and clinical benefits in the treatment of congestive heart failure (Lipkin and Pwle-Wilson 1985; Nicho11s et al. 1982; The Consensus Trial Study Group 1987).

related to the inhibition of the facilitatory action of angiotehsin II on noradrendine release (Axatonaccis and Kerwin 198 1). Therefore increased angiotensin II levels associated with con- gestive heart failure may contribute to the downregulation of mymu&d @-adrenoceptors by enhmcing norackendine release. Thus, the beneficid effects sf converting-enzyme inhibitors in heart failure may be due to the resensitisation of the @-adreno- ceptors as a result of blockade of noradremadine release (Maisel et d. 1989).

Rapid ventricular pacing in the dog has been shown to produce congestive heart failure (Riegger and Liebau 1982; Armstrong et a!. 1986). This mdel closely resembles dilated cardiomyopathy in humans (Shannon et al. 199 1 ; Wilson et d . 1987). Furthermore, the model is advantageous over other

'Author for correspondence.

Printed in Canada 1 Imprid au Canada

heart-failure models because it allows sequential analysis of neurohumord indices and hemodynamic parameters, as well

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668 CAW. J. PMYSIOL. PMARMACOL. VOL. 72. 1994

as in vitro assessment of vascular responsiveness (Armstrong et al. 1986; Forster and Armstrong 1990; Main et al. 1991; Forster et aH. 1992a). Moreover, the model has recently been used 8 s compare the status of rnyocudid fl-adrenoeeptors at various stages of heart-failure development as well as during recovery from heart failure (Larosa et al. 1993). This study demonstrated a dynamic nature of rnyocu&d @-adrenoceptors in evolving heart failure and indicated that these changes could be reversed by cessation of pacing. Other reports from our laboratory also demonstrated that the hemdynamic and news- humsrd parameters could d so be reversed after cessation of pacing (Howard et d. 1988). Vascular abnormdities, how- ever, do not return to control state (Forster et d. 1 9 9 2 ~ ~ ; Larosa et d. 1991).

The objective of the present study was to explore the impact sf chronic endapril or placebo treatment on the status of myo- cardid @-adrenoceptars in dogs paced to end-stage heart failure. A secondary objective was to explore the effect of endapril and heart failure on the rate of left ventricular angis- tensin I1 formation.

Materials and methods Adult male mongrel dogs (268 -25 kg) were preconditioned to the

study environment 2 - 3 weeks prior to the study. Twenty dogs were used: 11 dogs had pacemaker-induced congestive heart failure (and were to receive either enalapril (n = 6; weight 21.6 k O.$ kg; desig- nated enalapril-CHF) or placebo (rz = 5; weight 21.4 f 0.8 kg; designated placebo-CHF) and 9 dogs acted as control nonpaced a n i d s (4 received enalapril (weight 25.3 f 1 .6 kg; designated enalapril-control) and 5 (weight 23.0 + 0.9 kg; designated control) did not).

Bmp&antati~n of pacetmker Animals were anesthetised with sodium thiopental (intravenous,

25 mg - kg-', and pacemakers were implanted according to methods previously described (Amstrong et al. 8986). Briefly, under general anesthesia, a pacemaker generator (Medtronics Canada, Mississauga, Ont.) was inserted into a cervical pocket, and a unipolar pacemaker lead was positioned transvenously into the right ventricle. At least 1 week postoperative recovery was allowed, after which time the pace- makers were programed to deliver 250 beats . min-' asynchron- ously. Pacing continued until end-stage heart failure was manifested to the same degree in both enalapril-CHF and placebo-CHF. The criteria for end-stage heart failure were a 25% increase in heart size as measured by planimetry with the presence of pulmonary eedem (as evidenced by radiography) and (or) a 10% increase in body weight- When these criteria were met, animals were sacrificed with an over- dose s f sodium thiopental.

Protocol for enalapril admkni~tmti~n One week following the onset of pacing, animals were assigned to

receive either enalapril (oral administration of 5 mg two times per day) or placebo according to Forster et al. (1992bB. This dosage regi- men represents a human dose and was based on previously published canine research (Leddy et al. 1983). Weekly venous blood samples were taken for determination of plasma noradrenaline concentration and plasm renin activity. For noradrenaline, approximately 4 mL sf blood was collected into tubes containing ethylene glycol tetraacetic acid (90 mg a91L-I) and reduced glutathione (60 mg - mL-I) and centrihgd and assayed by high performance liquid chromatography (BPLG) as described by Forster et al. (1994). Plasma renin activity was determined from blood (6 mE) collected in tubes containing EDTA and was measured by radisimunsassay (Incstar, Stillwater, Minn.). This assay has a sensitivity of 3 pg angiotensin I and 7.8 and 6.9% precision, respectively, within and between samples. Weekly chest x-rays were taken, and heart size was quantified by planimetry of each cardiac silhouette and the results were expressed in square

centimetres. Briefly, the radiographs were obtained from dogs in the right lateral recumbent position, and care was taken to ensure identi- cal positioning, milliampere and kilovolt settings, and central beam distance and orientation during sequential examinations. Repro- ducibility was further ensured by marking exterior skin p in t s to indi- cate positioning of the central beam. Animals continued to be paced and were examined biweekly for signs of dyspnea, pulmonary edema, and ascites as described previously (Amstrong et al. 1986). The enalapril-control group received the drug in a similar dosage regi- men as the enalapril -CHF group (i.e., treatyent for a total of 26 days) and were sacrificed. The control animals were sacrificed acutely. All animal procedures were approved by the animal care committee of St. Michael's Hospital in accordance with the guide- lines of the Canadian Council on Animal Care and The Animals of Research Act.

Membrane prepamtion The heart was excised, and three adjacent segments (approximately

1 g each) of lee ventricular free wall were obtained. One piece was used for the subsequent determination of tissue noradrenaline con- tent, as described previously by Amstrong and col%eagues (1986), and another piece was used for the determination of angiotensin 11 formation (see below). The remaining piece was weighed and placed in 10 mL ice-cold buffer (10 mM Tris, 1 mM EGTA, pH 8.0) per gram tissue. Samples were homogenized, and 0.1-mL diquots were stored at - 80°C until ready for use. Myocardial P-adrenoceptor density was determined by the specific binding of ['251]iodocyano- pindolol (ICYP, 2208 Ci - m o l - I (1 Ci = 37 GBq); (New England Nuclear, Boston, Mass.). Briefly, after extraction of contractile pro- teins with 0.5 M KCI, the homogemte was serially centrihged as outlined previously (Larosa et al. 1993). The final pellet was resuspended in assay buffer (150 mM NaCl, 20 Tris, 1 mM ascorbic acid, pH 7.5) to give a final tissue concentration of 10 mg - mL-'. Approximately 3 mg of tissue was incubated with increasing conceitrations of ICYB (5-150 pmol - L-') in the absence and presence of 1-propranolol (5 yM) at 37°C for 2 h in a final volume of 450 pL. Following incubation, each assay was vacuum filtered through Whatman GF/B filters (Gelman Science, Ann Arbor, Mich.) and washed with 30 m% assay buffer at a flow rate of 2 mL - s-'. Filters were placed in a gamma counter (Beck- man Gam 5500, B e c h a n Instruments, Toronto, Ont.) and read. All assay points were performed in triplicate.

Specific binding was defined as the difference between total binding and nonspecific binding. Free ligand was determined by subtracting counts in the absence s f propranolol from counts of added ligand at each concentration of ICYP. Total ventricular @-adrenoceptor density (B,,,) and dissociation constant (Kd) were determined by Scatchard analysis (Scatchard 1949). The %-intercept of the plot represented B,,, (fmol . g-I wet weight), whereas the negative reciprocal of the slope, corrected for total assay vslume, determined the Kd (pmol L- I ) .

Rate of angistensin 11 f o m t i s n Briefly, angiotensin 11 formation was assayed with certain modifi-

cations according to Urata and colleagues (1991). The section of left ventricle was homogenized and incubated with 100 fmol 1125-Eabelled angiotensin I with 18 mM NaH2P0, containing 100 mM NaCl in a total incubation volume of 150 p&. Membranes were incubated for 20 min at 37°C. The precipitated proteins were removed by centrifuga- tion at 128 x g, and the supernatant was evaporated to dryness. The residues were resuspended in 25 mM triethylammonium phosphate and applied to a C,, Nova Pak HPLC column. Radioactivity was detected as a continuous-flow radioactive detector (model LB 507 A, Berthold, Germany). Angiotensin HI formation by the tissue samples was expressed as fmol ['251]AII fom& . mg - ' - min- '. Data aanlysisis

Results are expressed as mean f SEM or mean (95% confidence intervals). For comparisons between two groups, Student's t test was

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FORSTER ET AL. 669

TABLE 1. Left ventricular @-adrenoceptor density (B,,, fmol - g - I ) and dissociation constant (Kd, pmol - L-') from contrd, enalapril- contreB1, end-

april - CHF, and placebo - CHF animals

Bm, Kd

Control 2784f 12%" 21(5-66) Enalapril -control 3390 + 380a 65(37 - 1 1 7)'pb Enalapril - CHF 49 19 + 170a,V7BB(56 - $9)"~" Placebo - CHF 4 180 f lWb 12(3 - 34)

NOTE: B,, values are means f SBM; Kd values are geometric maws, with 95% confidence limits in pren- theses.

"Significantly different at p < 0.05 versus placebo-CBE bSignificantly different at p < 8.85 versus control.

TABLE 2. Left ventricular angiotensin 11 formation (LVAII, h o l mg-' . mian-'), noradrenaline concentration (LVNA, pmol g- I ) ,

plasm renin activity ( P M , ng . mL-I - Lsl), and plasma noradren- dine concentration (PNA, nmol - L-I) from control, enalapril -

control, enalaprd - CHF, and placebo - CHF dogs

LVAII LVNA P M PNA

Control 4.3f8.6 4.2f 0.3" 1.0f0.3 1.7f0.6" Enalapril-copnBml4.1f8.5 5.1f0.4" 1.2fgB.2 B.7f0.Ha Enalapril-CHF 9.8+B.5"~b 1.4f 0.4"1.1 f 2.5"."6.f 1.1" Placebo-CHF 5.2-1.6 1.9f 8.3b 2.9f 1.0 5.9f 1.3"

NOTE: Each value is mean k SSEM for at least PI = 4. "Significantly different at p < 8.85 versus placebo - CHF. bSigwificantly different at p < 0.05 versus confro1.

employed. For comparisons between more than two groups, a one- way analysis of variance was used. If an overall sigmificance was found, then the level of significance was determined by the Bon- ferroni multiple-comparison test. The Mann-Whitney test was used to assess significance between the Kd values. A g value of <0.05 was considered statistically significant.

Results The hernodynamic profiles of these groups of animals have

been described previously (Moe et al. 1993). Briefly, in the control dogs, there was no change in the hemodynmic vari- ables between baseline and repeat evaluation. In the paced dogs, at severe heart failure, there was a reduction in cardiac output and an elevation in cardiac filling pressures in both placebo- and endapril-treated groups compared with control.

With developing heart failure, @-adrenoceptor binding was reduced at end-stage heart failure (Fig. 1) compared with con- trol. The impact of endapril treatment on 6-adrenoceptor density of control animals, i.e., enalapril-control, was not signifi- cants When endapril was given to animals that were paced to end-stage heart failure, there was an apparent, significant increme in binding profile compared with the placebo - CHF group. While the binding isotherm approached that of control, the B,,, was still significantly lower than that of the control group. The actual B,, values and dissociation constants are given in Table 1 . In contrast to previous observations from this laboratory (Laosa et d. 1993), in which changes only in B,,, were observed, Kd was increased in both the enalapril- control and the endapril - CHF group.

FIG. 1. Saturation curves of ["551]iodocyanopindolol binding to mymardial membranes prepared from left ventricles of control (A), enalapril -control (B), enalapril - CHF (g), and placebo - CHF (O). ['251]idwyanopindoIoB bound to &adrenoceptors is given on the ordinate in h o l - g-I wet weight. The curves represent the best fit as determined by nonlinear regression. Data points represent mean f SEM. *Significantly different ( p < 0.05) versus pBacebo - CHF animals.

The left ventricular angiotensin II formation, left ventricular noradrenaline concentration, plasma renin activity, and plasma noradrenaline concentration for the four groups of dogs are presented in Table 2. The rate of angiotensin II formation did not change with developing heart failure (i .e., 4.3 1 0.6 for the control group versus 5.2 f 1.6 for the placebo -CHF group). Likewise, here was no effect on angiotensin II forma- tion in the endapd - control group. However, in the edapri l - CHF group, there was a significant increase in angiotensin II formation. As previously demonstrated, the left ventricular noradrenaline concentration significantly declined at end-stage heart failure (Amstrong et d. 1986). Left ventricular noradren- dine concentration was not dtered with the impact of chronic endapril treatment in the control or the CHF groups. The enalapril -CHF group had a significantly higher plasma renin activity compared with the other three groups. Neither endapril (endapril -control) nor the impact of heart failure (placebo - CHF) produced any change in plasma renin activity. Plasma noradrendine was significantly elevated in both heart failure groups compared with control.

The relationship of all the above parameters to the p-adreno- cepor density is shown in Fig. 2. With respect to the relation- ship with angiotensin 11 formation, group by group regression analysis revealed a significant correlation in only the placebo - CHF group (r = -0.9, p < 0.05). When all the data points were andysed, the r value decreased to -0.4 and the p value became not significant ( p < 0.1). Similarly a significant rela- tionship with left ventricular noradremline was found in the placebo - CHF group (r = 0.8, p < 0.05), and when all the points were pooled, the r value became 0.6 with a p value of 0.05. No significant correlations were found when the relation- ship between plasma renin activity and @-adrenoceptor density was analysed. Finally, the relationship between P-adrenocep- tor density and plasma noradrenaline was examined. The only group to show a significant correlation was in the endapril- CHF group (r = 0.9, p < 8.05). When d l the data points were considered, a significant inverse relationship was found (r = -0.5, p < 0.05).

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670 CAN. J. PHYSIOL. PBAMACOL. VOL. 72, 1994

FIG. 2. Plots of angiotensia I1 (AII) formation, left ventricular noradrenaline (NA), plasma renia activity (PEW), and plasma noradrenaline, versus P-adremoceptor density (B,,) for cesmtml (A) , enalapril -control (H) , emdapril - CHF (@) , and placebo - CHF (@) . The r values are cited in the Results and the only significant correlations were between B,, and left ventricular noradrenaline (r = 0.6, g < 0.05) and between B,, and plasma noradrenaline (r = -0.05, p < 8.05).

Discussion The principal Ending of the current investigation was that

chronic enalapril treatment caused a significant increase in ~-adrenoceptor density compued with &at of placebo-treatment following the development of pacing-induced heart failure. A subsidiary observation was that enalapril in both control and CHF animals caused an unexpected increase in Kd. Enalapril when given to paced dogs produced a paradoxical increase in the rate of myocardial angistensin I% formation.

In rapid ventricular paced dogs, there was a significant increase in left ventricular P-adrenoceptor density in response to chronic endapril treatment compared with placebo. The 6-adrenmeptor density in the placebo -GMF group was simi- lar to that reported previously by this laboratory (Larosa et al. 1993). We had dso previously demonstrated that the @-adreno- ceptor density returned to control values following a 4-week period of recovery (Larosa et d. 1993). In this regard, it was noted by Packer et al. (1986) that there was a recovery in left ventricular systolic function in patients with chronic rapid supraventricular tachycardia. In the enalapril- CHP group, there was a significant increase in P-adrenoceptor density. This increase was intermediate between the placebo-CHP md the control group, suggesting that enalapril partidly reverses this parameter. It is, perhaps, of greater importance to note that in dogs that were paced for only 1 week, /3-adrenoceptor densities were similar to those in the enalapril-CMF group (Larosa et d . 1993). This raises the question whether the onset

of enalapril treatment (e.g . , 1 week following the onset of pac- ing) merely prevents the downregulation of receptors. This appears unlikely since it has been reported that &-adrenoceptor density increases, decreases, or remains undtered in different models of experimental heart failure and, therefore, altera- tions of this aspect seem to depend on the type of heart failure rather than the stage of heart failure (Dixon and Dhdla 1991).

With respect to the P-adrenoceptor affinity, enalapril in both the endapril-control and the enalapril-CHH group caused an increase in Kd. In other binding studies, no change in Kd was seen (Bristow et d. 1986; Larosa et d. 1993). That the Kd was increased in both the endapril-treated groups to a similar extent implies that the decrease in affinity was a result of the enalapril per se rather than the heart-failure state. The mechanism for the decrease in affinity is unclear. Angistensin II has been shown to have a facilitatory role on noradrenaline release (Zimmerman et al. 19'92; Rother 1972), and this may contribute to the downregulation. Moreover, it might be expected that endapril inhibits the facilitation and hence reverses the ,B-adrenoceptor abnormality; this does not explain the effect sf endapril on ,6-adrenoceptor affinity. It is possible that different ,@-adrenoceptor subtypes may be involved. In this regard, several laboratories have published data on the relative distribution of bI- and &-adrenoceptors in pacing- induced heart failure. A selective downregulation of ,Bl-adreno- ceptors was seen in early and end-stage heart failure by Kiuchi et al. (1991) and Marzo et $1- (1991). Similarly this selectivity

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FOSTER ET AL. 67 1

has d s s been seen in human failed ventricles (Bristow et ale 1986). Nevertheless, controversial reprts have appeared in the literature regarding the status of PI- and P2-adrenoceptors in heart failure (Bristow et d. 11986; Karliner et al. 19863). The general consensus is that there is a downregulation of only the /31-adrenoceptor in heart failure (Bristow et d. 1986; Vatner et d. 1985). Thus the differentid effects of enalapril treatment on 6-adrenoceptor affinity and density support an effect (inde- pendent of heart-failure state) on &-adrenoceptors md an effect (dependent on heart-failure state) on @l-adrenoceptors. To establish these differential effects, competition binding studies using selective agents for each receptor should be performed; this was beyond the scope of the current study. However, it is interesting to note that competition studies per- formed in other laboratories have revealed that neither cap- topril nor lisinopril displaced iodocyanopindolcbl from human cardiac or lymphocyte membranes (Maisel et al. 1989; Horne et al. 1988).

Changes in pastreceptor mechanisms may also be involved, and changes have been reported in rapid ventricular pacing (Juneau et ale 1992). Adenylyl cyclase activity might be expected to complement fie @-adrenoceptor abnormality. Inded the cyclase activity was found to be defective in membranes from failing hearts (Marzo et d. 199%), which was suggested to be caused by an impairment in the ability of the cyclase to couple to the G protein (Gs). The implications of these findings to the current study and cardiac function in heart failure are unclear and warrant further investigation.

We discovered that the rate of angiotensiar II formation in the left ventricle was increased paradoxically in the endapril- CHF group. This observation raises many questions with respect to the degree of enzyme inhibition with the dosage regimen. Measuring converting enzyme per se would provide a better understanding of the relative effects of converting enzyme on tissue versus plasma renin - angiotensin systems. First, it seems highly unlikely that a higher dose of enalapril would have resulted in different findings. Plasma renin activity was greatly enhanced in the endapril -CHF group. In addi- tion, we have confirmed the effect of this dose on coronary vascular reactivity in which angiotensin receptors become supersensitive (Forster et d. 1992b). This study provided our best evidence that converting emyme was satisfactorily inhibited (albeit in another tissue). Moreover, 5 mg (b id . ) has been established as an effective dose in dogs as well as humans ( M d y et d. 1983). Measurement of converting enzyme may have been a superior approach to these experiments and would provide circumferential evidence. The methodology for measuring converting enzyme is controversial, and different assay technologies render different results. There are several examples in the literature that allude to the problems encoun- tered. Several different methods have been employed with conflicting results from different laboratories (Alhenc-Gelas et d. 1991). There are age-related variations in tissue and plmma-converting enzyme (Grima et d . 1990). In the absence of these measurements, in heart failure, we propose that the increase in angiotensin I% formation in the setting of adequate converting-enzyme inhibition was due to an overcompensatory production through an alternate pathway. This pathway may compensate following inhibition of converting enzyme, allow- ing diffusion of precursors into the tissue.

Finally, considering the relationship of the parameters with 8-adrenoceptor density, because of the small n values, it was felt that the correlations in the individual groups should be con-

sidered cautiously. Notwithstanding, there were some individual groups with significant correlations. For /3-adrenoceptor density versus plasma norad~naline, the overall relationship was of inverse nature and consistent with our previous research (Larosa et d . 1993), but in the enalapril- CHF group, the situation was reversed and a positive correlation was seen. This was unexpected, and the explanation remains unclear. In the placebo-CHF group an inverse relationship was found between P-adrenoceptor density and angiotensin H% formation, but the relationship was not significant when all data paints were considered.

In conclusion, endapril therapy prevents or reverses the 0-adrenoceptor abnormdity seen in heart failure, it produces a decrease in 6-adrenoceptor affinity independent of the heart- failure state, a new finding that adds to our howledge. Fur- ther studies are warranted to examine the relative contribution of distinct fl-adrenoceptor populations, as well as to evaluate novel angiotensin-formirag pathways.

This study was supported through a grant-in-aid from the Heart and Stroke Foundation of Ontario. The authors thank Medtronics Canada for their generous supply of pacemakers and Merck Frosst Canada for supplying enalapril.

Alhenc-Gelas, F., Richard, .Ia9 Courbon, D., Warnet, J.M., and Cowol, P. 1991. Distribution sf plasma angiotensin I-converting enzyme levels in healthy mew. Relationship to environmenbl and hormonal parameters. J . Lab- Cliplo Med. 117: 33 -39.

Antonaccis, M.S., and Kerwin, %. 198 1 . Pre- and postjunctional inhibition of vascular sympathetic hnctisn by captopria in SHR. Hypertension (Dallas), 36Suppl. 1) : 54 - 62.

Armstrong, B.W., Stopps, T.B., Ford, S.E., and de Bold, A.B. 1986. Rapid ventricular pacing in the dog: pathophysiologic studies of heart failure. Circulatisn, 94: 1075 - 1084.

Brbtow, M.R., Ginsburg, R., Minobe, W., Cubiccioti, R.S., Sagernan, W.S., Lurie, K., Billingham, M.E., Harrison, D.C., and Stinson, E.B. 1982. Decreased catwholamine sensitivity and 8-adrenergic receptor density in failing human hearts. New Engl. J . Med. 307: 205-211.

Bristow, M.R., Ginsburg, R., Umans, V., Howler, M., Minobe, W., Rasmussen, W., %era, P., Medove, R., Shah, P., Jarnieson, S. , and Stinson, E.B. 1986. Dl- and 6,-Adrenergic receptor sub- populations in wonfailing and failing human ventricular myo- cardium: coupling of both receptor subtypes to muscle contraction and selective 8,-down-regulation in heart failure. Circ. Res. 59: 297 - 309.

Dixon, I.M.C., and Dhalla, N.S. 1991. Alterations in cardiac adrenoceptors in congestive heart failure secondary to myocardial infarction. Coron. Artery Dis. 2: 805 -814.

Forster, C, , and Armstrong, P. W. 1990, Pacing-induced heart failure in the dog: evaluation of peripheral vascular a-adrenoceptsr sub- types. 3. Cardiovasc. Pharmacol. 16: 708 - 7 18.

Forseer, C . , Campbell, P. M . , and Armstrong, P. W. 1992~. Temporal alterations in peripheral vascular responsiveness daring both the development and recovery from pacing-induced heart failure. I. Cardiovasc. Phamcol . 20: 206 - 2 15.

Forster, C., Larosa, G . , and Armstrong, B.W. 19926. Impact of enalapril therapy on in vitro coronary artery responsiveness in pacing-induced heart failure. Can. J. Physiol. Pharrnacol. '$0: 1417- $422.

Forster, C., Naik, G . , and Amstrong, P.W. B?XM. Noradrem1ine biosynthesis and metabolism during development and recovery from pacing-induced heart failure in the dog. Can. J. Physiol. Phamacol. 72: 45 - 49.

Francis, G.S. , Goldsmith, S.R., Levine, T.B., Qlivari, M.R., and

Can

. J. P

hysi

ol. P

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. Dow

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16/1

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672 CAM. J. PHYSIOL. PHAWACOL. VOL. 72, 19%

Cohn, J.N. 1984. The neurohumoral axis in congestive heart failure. Awn. Intern. Med. 181: 3'50-377.

G r h a , M., Welsch, C., Giesen-Crouse, E.M., Coquad, C., BartheEmebs, M., and Embs, J.L. 1990. Age-related variations in tissue angiotensin-convefiing enzyme activities: comparison between spontaneously hypertensive and Wistar Kyoto rats. J. Hypertens. 8: 697-702.

Horn, E.M., Conwin, S.I., Steinberg, S.F., Chow, Y.K., Neuberg, G.W., Cannon, P.J., Powers, E.R., and Bilezikan, B.P. 1988. Reducd lymphocyte stimulatory guanine nucleotide regulatory protein and 8-adrenergic receptors in congestive h a r t failure and reversal with angiotensin converting e q m e inhibitor therapy. Circulation, 78: 1373 - 1379.

Howard, R.J., Stopps, T.P., Moe, @.We, Gotlieb, A., and Amstrong, P.W. 1988. Recovery from heart failure: structural and functional analysis in a canine model. Can. J . Physiol. Phar- mcol . 46: 1505-1512.

Juneau, C., Calderone, A., and RouBeau, J.E. 1992. Myocardial @-adrenergic and mechanical properties in pacing-induced heart failure in dogs. Am. J. Physiol. 262: H1458 -H1464.

Karliner, J.S., Barnes, B., Brown, M., and Dollery, C. 1980. Chronic heart failure in the guinea pig increases cardiac a,- and 8-adrenoceptors. Eur . I. Phamacol. 67: 1 15 - 1 1 8.

Kiuchi, K., Komamura, K., Shannon, R.P., Hittiinger, E., Homcy, C.I., Vatner, S.F., and Vatner, D.E. 1991. Initial changes in B-adrenergic receptor hnction in pacing induced cardiomyogathy . Circulation, WCSuppl. 11) : 11 - 394.

Larosa, G. , Amstrong, P.W., and Forster, C. 1991. Endothelial dependent relaxations modify contractile function of a-agonists in pacing-induced heart failure. Can. J. Cardiol. 7(SuppB. A) : 1 10A.

Larosa, G., Amstrong, P.W., Seeman, P., and Forster, C. 1993. @ adrenoceptor recovery after heart failure in the dog. Cardiovasc. Res. 2'8: 489-493.

Leddy , C.L., Wilen, M., and Franciosa, J.A. 1983. Effects of a new angiotensin converting enzyme inhibitor, enalagril, in acute and chronic left ventricular failure in dogs. J. Clin. Phrmacol. 23: 189- 198.

Levine, T.B., Francis, G.S., Goldsmith, S.W., Simon, A.B., and Cohn, J.N. 1982. Activity of the sympathetic nervous system assessed by plasma hormone levels a d heir relation to hemo- dynamic abnormalities in congestive heart failure. Am. J. Cardiol. 49: 1659-1667.

Lipkin, D.P., and Poole-Wilson, P. A. 1985. Treatment of chronic heart failure: a review of recent drug trials. Br. Med. I. 291: 993 - 996.

Main, I.%. , Foaster, C., and Amstrong, P.W. 1991. Inhibitory role of the coronary arterial endothelium to a-adrenergic stimulation in experimental h a r t failure. Circ. Res. 68: 940-946.

Maisel, A.S., Phillips, C., Michel, M.C., Ziegler, M.G., and Carter, S . M . B 989. Regulation of cardiac 8-adrenergic receptors by captopril. Implications for congestive heart failure. Circulation, 80: 666-695.

Marzo, K.P., Prey, M.B., Wison, J.W., Liang, B.T., Manning, D.R., Lanoce, \I., and Molinoff, P.B. 199 1. /3-Adrenergic receptor - 6 protein - adenyIate cyclase complex in experimental canine con- gestive heart failure produced by rapid ventricular pacing. Circ. Res. 69: 1546-1556.

Moe, G.W., Montgomery, C., Howad, R.J., Grima, E., and A m - strong, P. W. 1993. Left ventricular myocardial blood flow, metabolism and effects of early treatment with endapril in canine experimental heart failure. J. Lab. Clin. Invest. 121 : 294 - 30 1.

NichoBEs, M.G., Ikam, H., Kepsinger, E., Muslowski, A., Scmdrett, M.S., and Penman, T. 1982. Hemodynamic and hor- monal responsiveness during captopril therapy for heart failure: acute, chronic and withdrawal studies. Am. J. Cardiol- 49: 1497- 1501.

Packer, D.M., Bardy, G.H., Worley, S.J., Smith, M.S., Cobb, FA., Coleman, R.E., Gallagher, J.I., and German, L.D. 1986. Tachycardia-induced cardismyopathy: a reversible f o m of left ventricular dysfunction. Am. J, Cardiol. 57: 563-570.

Packer, M. 1988. Neurohormonal interactions and adaptations in congestive heart failure. Circulation, 77: 42 1 - 730.

Riegger, A. J.G., and Liebau, G. 1982. The renin -angiotensin - aldosterone system, antidiuretic hormone, and sympathetic nerve activity in an experimental model of congestive heart failure in the dog. CIin. Sci. 62: 465 -469.

Roh, W .H. 1972. Action s f angiotensin on adrenergic nerve endings: enhancement of norepinephrine biosy nthesis. Fed. Proc . 31 : 1358- 1364.

Scatchad, G. 1949. The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51: 660-672.

Shannon, W.P., Komamura, K., Stambler, B.S., Bigaud, M., Mandeas, W.T., and Vatner, S. H. 199 1. Alterations in myocardial contractility in conscious dogs with dilated eardiomyopathy. Ana. I . Physiol. 260: HI903 -H191 1.

The Consensus Trial Study Group. 1987. Effects of enalapril on mor- tality in severe congestive heart failure. New Engl. J . Med. 316: 1429 - 1435.

Thomas, J.A., and Marks, B.W. 1982. Plasma norepinephrine in congestive heart failure. Am. J . Cardiol. 41: 233 - 243.

Urata, H., Healy, B., Stewart, R.W., Merlin Bumpus, F., and Husain, A. 1991. Angiotensin 18-forming pathways in normal and failing human hearts. Circ. Wes. 66: 883 - 898.

Vatner, D.E., Vatner, S.F., Fujii, A.M., and Homcy, C.J. 1985. Loss of high affinity beta-adreslergic receptors in dogs with heart failure. J . Clin. Invest. '86: 2259 - 2264.

Wilson, J.R., Douglas, P., Hickey, W.F., Laore, V,, Ferrars, N., Muhammad, A., and Weichek, N. 198'5. ExperimentaE congestive heart failure produced by rapid ventricular pacing in the dog: cardac effects. Circulation, 75: 857 - 867.

Z i m e m a n , B.G., Gornes, S.K., and kiano, J.C. 1972. Action of angistensin on vascular nerve adrenergic endings: facilitation of norepinephrine release. Fed. ROC. 31: 1344 - 1350.

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