Inotropic Response to Norepinephrine Is AugmentedEarly …circres.ahajournals.org/content/circresaha/68/2/543.full.pdf · Inotropic Response to Norepinephrine Is AugmentedEarly andMaintained

543

Inotropic Response to NorepinephrineIs Augmented Early and Maintained

Late in Conscious Dogs WithPerinephritic Hypertension

Richard P. Shannon, Ricardo J. Gelpi, Luc Hittinger, Dorothy E. Vatner, Charles J. Homcy,

Robert M. Graham, and Stephen F. Vatner

We studied the inotropic responses to intravenous infusions of norepinephrine in nineconscious chronically instrumented dogs before and early (2-4 weeks) in the development ofperinephritic hypertension; seven conscious dogs were studied later (- 14 weeks), during a morestable phase of hypertension. Perinephritic hypertension was associated with a 24% increase inleft ventricular (LV) mass during developing hypertension; no further increase was seen duringthe stable hypertension phase. LV end-systolic stress was increased early (p<0.01) but wasnormalized later. The LV end-systolic stress-volume relation demonstrated an enhancedcontractile response to norepinephrine during developing hypertension, which returned towardcontrol later in the course of stable hypertension. The LV dP/dt responses to norepinephrine(0.4 ,ug/kg/min) were significantly greater during developing hypertension (7,509±337 mmHg/sec, p<0.05) compared with the control period (4,737±286 mm Hg/sec) and returnedtoward the control value during stable hypertension (5,168±465 mm Hg/sec). The enhancedinotropic responses to norepinephrine in developing hypertension were preserved in thepresence of ganglionic blockade, suggesting that the augmentation was not mediated via reflexmechanisms. These physiological responses were associated with an increase in 3-adrenergicreceptor density, but no significant change in basal or maximal adenylate cyclase stimulationoccurred during developing hypertension. Thus, in contrast to prior studies in anesthetizedanimals, the inotropic response to fg-adrenergic stimulation is not depressed in conscious dogsbut is enhanced selectively during the development of hypertension and maintained duringstable hypertension. (Circulation Research 1991;68:543-554)

M yocardial hypertrophy is a complex patho-physiological response to excessive cardiac

v load, designed to normalize ventricularwall stress and maintain normal systolic function.However, the hypertrophic process is heterogeneousin terms of its morphological, biochemical, and func-tional consequences and differs in systemic hyperten-sion from other causes of cardiac hypertrophy.1'2Recent evidence from our laboratory3 revealed that

From the Departments of Medicine, Harvard Medical School,Beth Israel Hospital, Brigham and Women's Hospital, Massachu-setts General Hospital, Boston, and the New England RegionalPrimate Research Center, Southborough, Mass.

Supported in part by National Institutes of Health grantsHL-38070, HL-33107, HL-37404, and RR-00168. D.E.V. is therecipient of US Public Health Service RCDA grant HL-01909.Address for correspondence: Stephen F. Vatner, MD, New

England Regional Primate Research Center, 1 Pine Hill Drive,Southborough, MA 01772.

Received January 9, 1990; accepted October 9, 1990.

left ventricular (LV) systolic function is enhanced atbaseline early (2-4 weeks) in the development ofcardiac hypertrophy, secondary to perinephritic hy-pertension, in conscious dogs and that the enhancedinotropic state at baseline was independent of al-tered loading conditions but was dependent on theintegrity of the sympathetic nervous system. Eitherganglionic or 83-adrenergic blockade abolished theaugmented response at baseline.3 These data raisethe question as to whether adrenergic responsivenessis enhanced during the development of cardiac hy-pertrophy secondary to systemic hypertension andwhether adrenergic responsiveness is maintained oraltered later in the course of hypertension whencompensated LV hypertrophy is established. Priorstudies using genetic or renovascular models of hy-pertension in the rat have suggested a reducedmyocardial response to adrenergic stimulation4-11associated with decreases in (-receptor densityand/or affinity.6'10-15 However, these studies are con-

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544 Circulation Research Vol 68, No 2, February 1991

founded by methodologies using either isolatedhearts or papillary muscle preparations or are miti-gated by the use of anesthesia in intact animalpreparations. In addition, there are important spe-cies differences: the rat exhibits more prominenta-adrenergic receptor control of LV contractili-ty,7,13,16,17 as well as alterations in myocardial isoen-zyme forms that occur during hypertrophy.18

Thus, we first sought to establish whether ,3-adren-ergic responsiveness to the endogenous sympatheticneurotransmitter, norepinephrine, is altered both early,during the development of perinephritic hypertension,and later, during stable hypertension. A second aimwas to determine if the alterations in response tonorepinephrine could be attributed to alterations inautonomic reflex buffering. This was addressed byreexamining responses to norepinephrine in the pres-ence and absence of ganglionic blockade. Finally, thephysiological responses were correlated with changes inmyocardial /3-adrenergic receptor density.

Materials and MethodsInstrumentation

Twenty-four mongrel dogs of either sex, weighingbetween 20 and 31 kg, were sedated with xylazine (2mg/kg i.m.) and anesthetized using halothane anes-thesia (1 vol%). With the use of sterile technique andthrough an incision in the left fifth intercostal space,Tygon catheters (Norton Plastics and Synthetic Divi-sion, Akron, Ohio) were implanted in the descendingthoracic aorta and left atrium. In all dogs, piezoelec-tric ultrasonic dimension crystals were implanted onopposing anterior and posterior endocardial surfacesof the LV to measure the internal short axis and onopposing endocardial and epicardial surfaces in thesame equatorial plane as the internal short axisdiameter crystals to measure wall thickness. Theendocardial wall-thickness crystals were implantedobliquely to avoid damage to the myocardium be-tween the two wall-thickness crystals. Ultrasonictransducers were also implanted at the basal epicar-dial surface and apical endocardial surface to mea-sure LV long axis. A solid-state miniature pressuretransducer (model P22, Konigsberg Instruments,Inc., Pasadena, Calif.) was implanted in the apex tomeasure LV pressure in all dogs (Figure 1). Thethoracotomy was closed, and the dogs were allowedto recover for 2-3 weeks. The dogs used in this studywere maintained in accordance with the guidelines,Guide for the Care and Use ofLaboratory Animals, ofthe Institute of Laboratory Animal Resources, Na-tional Council (Department of Health and HumanServices publication No. [NIH] 85-23, revised 1985),and the Standing Committee on Animal Care ofHarvard Medical School.

Aortic and left atrial pressures were sampled fromchronically implanted catheters and measured withstrain-gauge manometers (model P23 ID, StathamInstruments, Oxnard, Calif.), which were calibratedwith a mercury manometer. LV pressure was mea-

sured using the solid-state miniature pressure gaugecalibrated in vitro with a mercury manometer and invivo using the left atrial and aortic catheters andStatham strain-gauge manometers.19,20 An ultrasonicdimension gauge was used to measure LV dimen-sions.21 The dimension gauge generates a voltagelinearly proportional to the transit time of ultrasonicimpulses traveling at the velocity of 1.58x 106 m/secbetween the crystals. At constant room temperature,the thermal drift of the instrument is minimal, that is,less than 0.02 mm in 6 hours. Any drift in themeasurement system was eliminated during the ex-periment by periodic calibration accomplished bysubstituting impulses of known duration from a crys-tal-controlled pulse generator having a stability of0.001%. The position of all transducers was con-firmed at autopsy.

Model of Perinephritic HypertensionAfter completion of control studies in the nor-

motensive state, perinephritic hypertension was in-duced in nine mongrel dogs according to the methodintroduced by Page22 and used previously in ourlaboratory.3 After removal of perinephric fat, the leftkidney was wrapped loosely in a silk pouch through aleft flank incision. Care was taken to avoid inadvert-ent stenosis of the renal artery. One week later, acontralateral nephrectomy was performed through aright flank incision. Technical limitations and long-term morbidity precluded the study of these samedogs during a more chronic phase of hypertension.Accordingly, a separate group of seven dogs wasmade hypertensive initially by performing a simulta-neous bilateral renal wrap, as described above,through a midline laparotomy. Subsequently, thesedogs underwent chronic instrumentation at 10-14weeks after developing hypertension, thereby avoid-ing the morbidity associated with chronic instrumen-tation and renal insufficiency over the prolongedperiod of hypertension. All surgical procedures wereperformed using sterile surgical techniques, and gen-eral anesthesia was induced by halothane (1 vol%).An additional five dogs were prepared as shamhypertensive controls; that is, the left kidney wasdissected but not wrapped, and the right kidney wasremoved 1 week later. An additional three dogs wereinstrumented fully for a period of 8 weeks and wereincluded as sham-operated controls. Together, theseeight dogs were used as a separate comparison groupfor the stable hypertensive dogs and for pathologicaland biochemical measurements.

ProtocolsNine dogs were studied as controls in a normoten-

sive state beginning 2-3 weeks after instrumentation,when they were healthy and had completely recov-ered from the effects of surgery. The same nine dogswere studied 2-4 weeks after induction of perine-phritic hypertension (renal wrap and contralateralnephrectomy). An additional seven dogs were stud-ied during stable hypertension, that is, approximately

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14 weeks after induction of hypertension. At no timewere significant electrolyte disturbances or renalinsufficiency noted.During the control period and during both devel-

oping and stable hypertension, each dog received agraded infusion of norepinephrine (0.05, 0.1, 0.2, and0.4 ,jug/kg/min). On a separate occasion, the re-sponses to norepinephrine were determined in thepresence of ganglionic blockade effected by hexa-methonium (20 mg/kg) and atropine methylbromide(0.1 mg/kg). The efficacy of ganglionic blockade wasestablished by demonstrating the absence of a heartrate response to the hypotensive effects of bolusadministration of nitroglycerin (120 ,ug). Finally, on aseparate occasion, each dog received a graded intra-venous infusion of phenylephrine (1, 2, and 5 ,ug/kg/min) to study the hemodynamic and inotropic re-sponses to a-adrenergic stimulation. These studieswere designed to investigate the potential role thata-adrenergic stimulation might play in the observedresponses to norepinephrine. Similar protocols wereused in the eight sham-operated dogs, which wereused as comparisons for the responses in the stablehypertensive dogs.

Biochemical PreparationThe left ventricle and septum were weighed,

trimmed of fat and connective tissue, minced, andhomogenized in 4 vol buffer I (0.75 M NaCl and 10mM histidine, pH 7.5) with a Polytron (model PT-20S, Brinkmann Instruments, Inc., Westbury, N.Y.)for 5 seconds at half speed. The homogenate wascentrifuged at 14,000g for 20 minutes. The pellet wasresuspended in buffer L, homogenized for 5 secondsat half speed, and centrifuged at 14,000g for 20minutes. The pellet was homogenized and centri-fuged as before. The pellet was resuspended in buffer11 (10 mM NaHCO3 and 5 mM histidine), homoge-nized for 30 seconds three times at half speed, andcentrifuged at 14,000g for 20 minutes. The pellet wasfiltered through one layer of Japanese silk screen,size 12, and saved as the crude membranes.23

All studies were performed in triplicate in thepresence of Tris buffer (100 mM Tris, 1 mM EGTA,and 5 mM MgCl2, pH 7.2). 83-Adrenergic receptorantagonist binding studies were performed usingeight concentrations of 25 ,Ll [1251]cyanopindololranging from 0.02 to 1.0 nM, 25 ,ul isoproterenol (0.1mM) or buffer, and 100 gl crude membrane protein(10 ,ug/assay). The antagonist binding data wereanalyzed by the LIGAND program.24 A linear regres-sion was performed on the amount bound versusbound/free ligand. An r2 value of 0.7 was the criterionused for acceptability of the data.

Adenylate cyclase activity was assayed according tothe method of Salomon et al.25 Dose-response curveswere performed for each stimulator of adenylatecyclase activity to determine that maximally stimu-lated activity would be measured. The concentrationsof the following compounds were found to producemaximally stimulated adenylate cyclase activity

(mM): GTP 0.1, GTP 0.1 plus isoproterenol 0.1,Gpp(NH)p 0.1, NaF 10, and forskolin 0.1.

Tissue and plasma catecholamines were measuredby the radioenzymatic assay of Peuler and Johnson.26Na+,K+-ATPase activity was determined by themethod of Jones and Besch.27

Data AnalysisMeasurements were made at each dose of norepi-

nephrine and phenylephrine, 4-5 minutes after be-ginning the infusion when hemodynamics had stabi-lized. The data were recorded on a multichannel taperecorder (model 101, Honeywell, Denver) and on adirect-writing oscillograph (Mark 200, Gould-Brush,Cleveland, Ohio). A cardiotachometer (model9857B, Beckman Instruments, Inc., Fullerton, Calif.),triggered by the LV pressure pulse, provided acontinuous recording of heart rate. Continuous re-cordings of LV dP/dt and LV dD/dt were derivedfrom LV pressure signals and from the short axisdimension signals, respectively, using Philbrick oper-ational amplifiers (Teledyne Philbrick, Dedham,Mass.), which were operated as differentiators andhad a frequency response of 700 Hz. A triangularwave signal was substituted for the pressure anddimension signals to calibrate the differentiator di-rectly. LV end-diastolic dimensions were measuredimmediately before the onset of ventricular contrac-tion. LV end-systolic dimensions were measured atthe time of maximum negative dP/dt. Ejection timewas taken as the interval between maximum andminimum LV dP/dt.

Cavity volume was calculated using an ellipsoidalmodel28:

EDV= (wr/6)(EDD)2(EDL-0.55 x EDW)/1,000

ESV= (w/6)(ESD)2(ESL- 0.55 x ESW)/1,000

where EDV is end-diastolic volume, ESV is end-systolic volume, EDD is the end-diastolic short axis,ESD is the end-systolic short axis, EDL is the end-diastolic long axis, ESL is the end-systolic long axis,EDW is end-diastolic wall thickness, and ESW isend-systolic wall thickness. Notice that a wall thick-ness factor is subtracted from the measured long axisbecause one of the long axis crystals is endocardialand the second is epicardial.

End-systolic wall stress (Es oa) was calculated byusing a cylindrical model:

Esu = 1.36 x [(APes x SAXes)/(2 xWTHes)]where APes is the aortic pressure at end systole,SAXes is the short axis diameter at end systole, andWTHes is wall thickness at end systole.The individual end-systolic stress-volume relations

for each dog were generated from playback of on-lineacquired data during both phenylephrine and norepi-nephrine infusions. End systole was defined as themaximum stress-volume point. The end-systolicstress-volume points were averaged, and the relation

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Shannon et al Enhanced Inotropic Response to Norepinephrine in Hypertension 547

TABLE 1. Pathology

Developing StableSham HTN HTN(n=8) (n=9) (n=7)

Body wt (kg) 26+1 25+--1 23±1LVfreewallwt (g) 92+7 114+6* 111+3*LV wt/body wt (g/kg) 3.6+0.2 4.5±+0.2* 4.7+0.2*LV+septum/body wt (g/kg) 4.8±0.3 6.1±0.2* 5.9±0.2*RVwt (g) 45±3 48+3 45±3RV wt/body wt (g/kg) 1.8±0.1 1.9±0.2 1.9±0.1

Values are mean±SEM. Sham, eight sham-operated controldogs; developing HTN, nine dogs studied early (2-4 weeks) in thedevelopment of perinephritic hypertension; stable HTN, dogsstudied later (-14 weeks) in the development of perinephritichypertension; LV, left ventricular; RV, right ventricular.

*p<0.05 compared with sham-operated control values.

was established for each group at control and duringdeveloping and stable hypertension. The slope of theindividual relations was determined as the end-sys-tolic elastance (Ees).29,30

Statistical AnalysesAll analyses31 were performed using BMDP bio-

medical computer programs (BMDP, Los Angeles).The group of sham dogs and the group of dogsstudied at control were compared with Hotelling's T2test. This test showed no significant difference be-tween these two groups atp<O.05. The comparisonsperformed to study the differences between effects inthe different groups were as follows: first, the com-parison of the paired data (normotensive controlperiod versus period of developing hypertension);then, the comparison between the stable hyperten-sion group and the sham group (used as the nor-

motensive control for the stable hypertension group);and finally, the comparison between developing andstable hypertension groups. Significance was set atp<O.05. The method of statistical analysis was therepeated-measures analysis of variance. This wasfollowed, when required, by an analysis of linearcontrasts to compare the baseline with the drugresponses and, when required, by Student's t test tocompare two groups at each drug response level. Theindividual slopes (Ees) of the end-systolic stress-volume relation were compared in the same dogs atcontrol and during developing hypertension using a

paired Student's t test corrected, when necessary, formultiple comparisons.

ResultsPathology

Table 1 reveals the postmortem heart weights ineach of the three study groups. There were nodifferences in body weight, but there was a 24%increase in LV free wall mass in developing hyper-tension (p<0.05) and a similar significant increase instable hypertension (p<0.05). Both the LV free walland free wall plus septum to body weight ratios weresignificantly greater in the two hypertensive groupscompared with sham-operated controls (p<0.05).There were no differences in the right ventricularweights nor the right ventricular weight/body weightratios among the three groups.

Baseline HemodynamicsTable 2 lists the hemodynamics and LV contractile

indexes at baseline in the groups. Baseline LV peaksystolic, end-diastolic, and mean arterial pressureswere significantly greater (p<O.O1) in dogs that were

TABLE 2. Effects of Hypertension on Baseline Hemodynamics and Left Ventricular Function

Developing Sham StableControl HTN HTN HTN(n=9) (n=9) (n=8) (n=7)

LV systolic pressure (mm Hg) 124+3 160±5* 121±3 161±6tLV end-diastolic pressure (mm Hg) 8±1 17±2* 8±1 11±2Mean arterial pressure (mm Hg) 96±3 128±3* 97±3 131±7*Heart rate (beats/min) 94±3 83±5 96±5 105±2LV end-diastolic diameter (mm)

Short axis 40.0±1.4 42.8±1.2 39.8±0.7 39.8±1.5Long axis 63.7±2.7 65.4±3.1 65.1±3.1 67.1±2.0

LV end-systolic diameter (mm)Short axis 29.8±1.4 30.8±1.4 30.4±0.6 29.4±1.7Long axis 60.6±2.7 62.7+2.8 62.3±2.9 64.4±1.9

LV end-diastolic wall thickness (mm) 12.3±0.5 12.5±0.4 12.2±0.9 13.7±0.5LV end-systolic wall thickness (mm) 14.2±0.7 15.5±0.8 14.9±1.0 17.4±0.4LV end-systolic stress (g/cm2) 152±11 203±18* 158±9 162±17LV +dP/dt (mm Hg/sec) 3,011+112 4,068±213* 3,187±92 3,202±238

Values are mean±SEM. Control, dogs studied before induction of perinephritic hypertension; developing HTN,dogs from control group studied early (2-4 weeks) in the development of perinephritic hypertension; sham, separategroup of sham-operated control dogs; stable HTN, separate group of dogs studied later (-14 weeks) in thedevelopment of perinephritic hypertension.

*p<0.01 developing hypertension compared with control values.tp<0.01 stable hypertension compared with sham values.

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studied during both developing and stable perine-phritic hypertension. There were no differences inheart rate among the groups. During developinghypertension, there was a significant increase in LVend-systolic wall stress compared with the controlvalue (p<O0.01). End-systolic wall stress was normal-ized later during the stable phase of perinephritichypertension, as a consequence of changes in LVgeometry, despite similar increases in systolic pres-sure. During developing hypertension, there wereincreases of a similar magnitude in both wall thick-ness and LV cavity diameter, such that the ratio ofwall thickness to cavity diameter was not differentcompared with the control value. During stable hy-pertension, LV cavity diameter returned to controllevels, whereas there were further increases in wallthickness, both at end diastole and end systole, suchthat the ratio of cavity diameter to wall thicknessdeclined significantly (p<O.OS). Thus, during stablehypertension, despite the persistently elevated LVpressures, end-systolic wall stress was normalized tocontrol levels through a proportionally greater in-crease in LV wall thickness compared with LV shortaxis diameter. There were no significant alterationsin long axis diameter.At baseline, the isovolumic index of contractility,

LV dP/dt, was significantly greater (p<O.O1) duringdeveloping hypertension compared with the controlperiod. Furthermore, there was no depression inbaseline LV dP/dt between sham-operated dogs anddogs studied late in the course of stable hypertension.

Response to NorepinephrineBecause important alterations in loading conditions

occurred during the course of hypertension, the end-systolic stress-volume relations, a relatively load-inde-pendent index ofLV contractility,29,30 were constructed

FIGURE 2. Graphs showing the average

0o/ end-systolic stress-volume relation foreach group of dogs at baseline (left pan-els) and in response to inotropic stimula-

0 t tion with norepinephrine (right panels).Control, dogs studied before induction ofperinephritic hypertension; devel HTN,

o dogsfrom control group studied early (2-4weeks) in the development ofperinephritic

o hypertension; stable HTN, separate groupof dogs studied later (-14 weeks) in thedevelopment ofperinephritic hypertension.

o *// .In response to norepinephrine, the relationwas shifted further upward in developinghypertension compared with the nor-

0 27.motensive control period, which is consis-tent with an enhanced inotropic responseto adrenergic stimulation.

in response to norepinephrine. Figure 2 depicts theaverage relation for control dogs in the normotensivestate and for the same dogs studied during developingperinephritic hypertension. The relation is also de-picted for the dogs with stable hypertension comparedwith controls. At baseline, the relations were estab-lished based on changes in stress-volume loops con-structed during an infusion of phenylephrine. At base-line, there was an increase in Ees during the period ofdeveloping hypertension (27.8±0.5 g/cm2/ml) com-pared with the control period (16.9±+2.5 g/cm2/ml),although the difference was not significant. These datasuggest an enhanced contractile state at baseline indeveloping hypertension compared with the controlperiod, although of a lesser magnitude than noted inour prior findings.3 Later, in the course of stablehypertension, the end-systolic stress-volume relations(Ees= 17.2+1.6 g/cm2/ml) returned toward the controlvalue. In contrast, in the course of developing hyper-tension, Ees was further increased (44.6+10.6 g/cm2/ml) in response to the positive inotropic effect ofnorepinephrine compared with the response in nor-motensive control studies (17.9+5.1 g/cm2/ml;p<0.05).The relation returned to control levels during the later,more stable phase of hypertension (26.3+±3.2 g/cm2/ml). Thus, the developing course of perinephritic hy-pertension is associated with a shift in the averageend-systolic stress-volume relation in response to nor-epinephrine, reflecting an enhanced inotropic respon-siveness to adrenergic stimulation. The inotropic stateis maintained at or above control levels later in hyper-tension. In either case, there is no evidence of de-pressed LV systolic function over the nearly 4-monthperiod of perinephritic hypertension, when using anindex that is relatively independent of load.

Table 3 depicts the hemodynamic responses toincreasing doses of intravenous norepinephrine.

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Shannon et al Enhanced Inotropic Response to Norepinephrine in Hypertension 549

TABLE 3. Hemodynamic Responses to Norepinephrine Infusions Before and After the Development of Perinephritic Hypertension

Dose of norepinephrine (,ug/kg/min)

Baseline 0.05 0.1 0.2 0.4

LV systolic pressure (mm Hg)Control 124±3 134+3* 142±4* 165±6* 193±9*Developing HTN 160±5t 181±7*t 207+8*t 239±10*t 268±11*tStable HTN 161±6t 169±5*t 189±9*t 238±12*t 269+12*tSham HTN 121±3 126±2* 141±4* 170±5* 197±6*

LV end-diastolic pressure (mm Hg)Control 8±1 9±1* 11±1* 14±2* 18±2*Developing HTN 17±2t 19±2t 22±2*t 24±2*t 30±2*tStable HTN 11±2 12±2 16±2* 22±3* 28±2*Sham HTN 8±1 9±1 12±1* 16±1* 21±1*

Mean arterial pressure (mm Hg)Control 96±3 102±3* 109±3* 128±5* 147±7*Developing HTN 128±3t 144+4*t 161±5*t 182±6*t 201±8*tStable HTN 131+7t 136-7t 154±10*t 187±10*t 205+9*tSham HTN 97±3 102±3 113±3* 139±5* 151±5*

Heart rate (beats/min)Control 94+3 93±3 86±5 84±4 84±4Developing HTN 83±5 94±5 85±8 89±5 93±11Stable HTN 105±3 99±6 96±6 92±8 91±7Sham HTN 96±5 92±6 82±3* 89±4 86±6

LV end-systolic stress (g/cm2)Control 152±11 171±10* 188±10* 216±10* 236±14*Developing HTN 203±18t 235±19*t 265±22*t 295±21*t 320+21*tStable HTN 162±17 178±14* 209±19* 268±28* 284±24*Sham HTN 158±9 171±8* 192±9* 239±18* 244±17*

LV dP/dt (mm Hg/sec)Control 3,011+112 3,150±115* 3,255 ± 105* 3,831±177* 4,737+286*Developing HTN 4,068±213t 4,312±222*t 4,738±229*t 5,752±326*t 7,509+337*tStable HTN 3,202±238 3,250±229 3,459±248* 4,283±267* 5,168±465*Sham HTN 3,187+92 3,220±115 3,381+103* 4,134±147* 4,953±212*

Values are mean±SEM. LV, left ventricular; control, dogs studied before induction of perinephritic hypertension; developing HTN, dogsfrom control group studied early (2-4 weeks) in the development of perinephritic hypertension; stable HTN, separate group of dogs studiedlater (-14 weeks) in the development of perinephritic hypertension; sham HTN, separate group of sham-operated control dogs.

*p<0.05 compared with baseline values.tp<0.05 compared with control values.4p<0.05 compared with sham HTN values.

There were dose-related increases in LV systolic,end-diastolic, and mean arterial pressures and LVend-systolic wall stress in each group. Of note is thefinding that the magnitude of the pressor response tonorepinephrine was significantly greater (p<0.05) instudies during developing hypertension comparedwith control studies. The increases in LV dP/dt inresponse to intravenous norepinephrine were signif-icantly greater during developing hypertension com-pared with the normotensive control period, whereasresponses to intravenous norepinephrine were main-tained later during the stable hypertensive phase,suggesting again that there was no depression inadrenergic responsiveness over the period studied(Table 3). Figure 3 depicts the response of theisovolumic index, LV dP/dt, to intravenous norepi-nephrine, expressed as the change from baseline.There remained a significantly enhanced response

during developing hypertension compared with thecontrol period, with preservation of adrenergic re-sponsiveness to norepinephrine seen later in hyper-tension. Similar significant differences were notedwhen the data were expressed as percent changefrom baseline, suggesting that the enhanced respon-siveness to norepinephrine seen in developing hyper-tension was not attributable to the augmented con-tractile state noted at baseline.To establish whether the enhanced responsiveness

to norepinephrine during developing hypertensionwas due to direct myocardial effects or mediated viaautonomic reflex mechanisms, the dose-response re-lation to norepinephrine was examined under gangli-onic blockade produced by hexamethonium and at-ropine methylbromide. Expressed as a change frombaseline (Figure 4), the LV dP/dt response to norepi-nephrine (0.4 ,ug/kg/min) in developing hypertension

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0.05 0.1 0.2 0.4

DOSE OF NOREPINEPHRINE (gl/kg/min)

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DOSE OF NOREPINEPHRINE (jsg/kg/min)

FIGURE 3. Graphs showing the dose-response relation be-tween norepinephrine and left ventricular (LV) dP/dt, ex-

pressed as the change from baseline. Control, dogs studiedbefore induction of perinephritic hypertension; developingHTN, dogs from control group studied early (2-4 weeks) inthe development ofperinephritic hypertension; sham, separategroup of sham-operated control dogs; stable HTN, separategroup of dogs studied later (-14 weeks) in the development ofperinephritic hypertension. There were significant increases inresponse to norepinephrine in developing hypertension,whereas the response was neither enhanced nor depressed laterin stable hypertension (*p<O.O5).

0-0 Control

0-0 Developing HTN

*

-lUU i-0.05 0.1 0.2 0.4

DOSE OF NOREPINEPHRINE (Mg/kg/min)

A-A Shom,-A Stable HTN

TA

0.05 0.1 0.2 0.4

DOSE OF NOREPINEPHRINE (jg/kg/min)

FIGURE 4. Graphs showing the dose-response relation be-tween norepinephrine and left ventricular (LV) dP/dt. Control,dogs studied before induction of perinephritic hypertension;developing HTN, dogs from control group studied early (2-4weeks) in the development ofperinephritic hypertension; sham,separate group of sham-operated control dogs; stable HTN,separate group of dogs studied later (-14 weeks) in thedevelopment ofperinephritic hypertension. The dose-responserelation remained enhanced during developing hypertensionand was maintained during stable hypertension compared withthe nonnotensive control period in the presence ofganglionicblockade (*p <O. 05).

remained enhanced (+7,423±777 mm Hg/sec) underganglionic blockade compared with the normotensivecontrol period (+4,938+662 mm Hg/sec) and wasmaintained later in the course of perinephritic hyper-tension (+4,937+290 mm Hg/sec). Thus, the en-hanced responsiveness to norepinephrine that wasseen during developing hypertension was not abol-ished by ganglionic blockade, suggesting that the aug-mentation was due in part to direct myocardial effectsof norepinephrine. Interestingly, in the presence ofganglionic blockade, heart rate increases were greaterduring developing hypertension (+57+10 beats/min)compared with the control period (+27±+6 beats/min;p<O.OS), suggesting an enhanced chronotropic re-sponsiveness to norepinephrine, consistent with theenhanced inotropic responsiveness.

Response to PhenylephrineTo establish the potential contribution of a-adren-

ergic stimulation to the observed responses to nor-

epinephrine, we assessed the inotropic and pressorresponses to intravenous phenylephrine. Figure 5reveals that there was no significant increase in LVdP/dt in response to increasing doses of phenyleph-rine. However, a dose-related increase in mean arte-rial pressure was observed in each group, consistentwith the anticipated vasopressor response to a-ad-renergic stimulation. As has been reported previous-ly,3 the baseline levels of LV dP/dt were significantlygreater during developing hypertension comparedwith the control period. Thus, although there was theexpected vasopressor responses to the a-agonist,phenylephrine, there were no detectable inotropiceffects attributable to a-adrenergic stimulation.

/-Adrenergic Receptor BiochemistryDuring developing hypertension, there was a signif-

icant (41%) increase in ,-adrenergic receptor density(80±+6 fmol/mg protein) compared with the controlperiod (57+±5 fmol/mg protein, p<0.05). During thestable hypertensive phase, 3-adrenergic receptor den-

Intact

o-o Control

*-* Developing HTN

*4000-

1-z

2000

3000.Z >

-Sw EC 200

ow 1 000-

3

*

Gonglionic Blockade

9000-

, 7000-

Z0 3000-

a.

4000-

0

: 3000-

-z0 E 2000

[L 1000-

0O

-1000

-1iWLt I

-100 UU I

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Shannon et al Enhanced Inotropic Response to Norepinephrine in Hypertension 551

50001

_

s. 4000

E

X 3000-

i 2000-

0-0 Control*-* Developing HTN

A-A Stable HTN

I =iT

Baseline 1 2 5 (Ig/kg/min)

DOSE OF PHENYLEPHRINE

, 250-

E-E 200-Wcc

v) 150-V)

[Li_i 100-

I-< 50-z

0

T C

AL

Baseline 1 2 5 (ig/kg/min)

DOSE OF PHENYLEPHRINE

FIGURE 5. Graphs showing the inotropic and pressor re-

sponses to phenylephrine. Control, dogs studied before induc-tion ofperinephritic hypertension; developing HTN, dogs fromcontrol group studied early (2-4 weeks) in the development ofperinephritic hypertension; stable HTN, separate group ofdogsstudied later (-14 weeks) in the development ofperinephritichypertension. There were dose-related increases in mean arte-rialpressure, but there was no increase in left ventricular (LV)dPldt. The enhanced inotropic state in developing hyperten-sion is reflected in the greater baseline measures ofLV dPldt.*p<O.OS compared with control.

sity returned toward the control value (65±7 fmol/mgprotein). The dissociation constants were unchangedduring both developing and stable hypertension com-pared with the control period, suggesting that therewere no significant alterations in receptor affinity. Toconfirm that the differences in P-receptor density werenot due to differences in the purity of the sarcolemmalmembrane preparation, we measured Na+,K+-ATPaseactivity from the same samples. There were no differ-ences between the control period (2.7+0.4 ,umol Pi/hr/mg), developing hypertension (2.4+0.2,mol Pihr/mg),or stable hypertension (2.6±0.4 ,umol Pihr/mg). Inaddition, basal levels of myocardial adenylate cyclasewere not different for the normotensive control period(96±7 pmol cyclic AMP [cAMP]/min/mg protein), de-veloping hypertension (79± 6 pmol cAMP/min/mg pro-tein), or stable hypertension (85_± 11 pmol cAMP/min/mg protein). Similarly, maximally stimulatedadenylate cyclase activity was not significantly differentamong the three groups. For example, the adenylate

cyclase activity in response to NaF (10 mM) was similarfor the control period (359+±21 pmol cAMP/min/mgprotein), developing hypertension (323± 16 pmolcAMP/min/mg protein), and stable hypertension(333+±28 pmol cAMP/min/mg protein), as was theresponse to forskolin (0.1 mM) (control, 891±115 pmolcAMP/min/mg protein; developing hypertension,770±+72 pmol cAMP/min/mg; stable hypertension,843+±120 pmol cAMP/min/mg).

In a subset of dogs (n =5), plasma catecholamineswere measured before and after the development ofhypertension. There were no differences in plasmanorepinephrine (control, 215±45 pg/ml; developinghypertension, 226±28 pg/ml; stable hypertension,205±18 pg/ml) or in plasma epinephrine (control,125 ± 32 pg/ml; developing hypertension, 113±23 pg/ml; stable hypertension, 104±14 pg/ml). Myocardialcatecholamine content was also unchanged.

DiscussionIn the present study, we found significant changes

in LV hemodynamics during the course of perine-phritic hypertension. In developing hypertension(2-4 weeks), there were significant increases in end-systolic wall stress, which was normalized over time,through an increase in LV wall thickness. The in-crease in LV short axis diameter observed duringdeveloping hypertension returned toward the controlvalue later during stable hypertension. Thus, al-though neither LV mass nor LV systolic pressure wasdifferent during developing and stable hypertension,there were important changes in LV geometry, spe-cifically, a decrease in the ratio of cavity size to wallthickness, with the resultant normalization of end-systolic stress in the stable hypertensive phase. Inaddition, at baseline, we found that LV systolicfunction was augmented during the course of devel-oping hypertension and was maintained at controllevels later, during the course of stable perinephritichypertension, but importantly, there was no evidenceof depressed LV systolic performance.We also observed important increases in respon-

siveness to intravenous norepinephrine during thedevelopment of perinephritic hypertension. Giventhe dynamic changes in loading conditions accompa-nying the evolving hypertensive process, we used therelatively load-independent index, the end-systolicstress-volume relation,29,30 to assess the LV contrac-tile response to norepinephrine. These analyses indi-cated that the LV inotropic state was enhanced atbaseline in developing hypertension and was main-tained at control levels later, in the stable phase ofhypertension. Subsequently, in response to norepi-nephrine, there were further shifts in the end-systolicstress-volume relation in developing hypertensioncompared with the normotensive control period,consistent with an enhanced inotropic response tothis adrenergic agonist. Although the sensitivity andlinearity of this relation and the significance ofchanges in the volume intercept have been ques-tioned,32 the slope of the end-systolic stress-volume

1 for)

,i

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552 Circulation Research Vol 68, No 2, February 1991

relation (Ees) was clearly greater during developinghypertension compared with the normotensive con-trol period in response to adrenergic stimulation withnorepinephrine.

Given the relative insensitivity of this index toalterations in contractility,30,32 the significant in-creases in Ees seen in response to norepinephrine indeveloping hypertension were a strong endorsementof the findings of enhanced adrenergic responsive-ness, independent of changes in loading conditions.To further support these findings, we observed en-hanced responsiveness of the isovolumic index, LVdP/dt, to norepinephrine in developing hypertensionand maintenance of the response later, during thestable phase of hypertension. Thus, by using load-dependent and load-independent indexes of LV con-tractility, the inotropic responses to norepinephrinewere enhanced early, during developing hyperten-sion, and maintained late in the course of perine-phritic hypertension. At no time did we observe animpaired responsiveness to this adrenergic agonist.These findings differ from the majority of previous

reports,4 6,8 111 which have suggested an impair-ment in /8-adrenergic LV contractile responsivenessin hypertension. Likely explanations for these differ-ences stem from the use of conscious, chronicallyinstrumented dogs in the present study and a com-plete characterization of the inotropic state, using anisovolumic index of contractility and the end-systolicstress-volume relation. In addition, we characterizedthe inotropic responses at two discrete stages in thehypertensive process and used the endogenous neu-rotransmitter, norepinephrine, as the adrenergic ago-nist. Prior studies5,69-11 in rats have used differenthypertensive models and have used isolated papillarymuscle or whole-heart preparations devoid of sym-pathetic innervation. Saragoca and Tarazis demon-strated an impaired contractile response to the intra-venous administration of isoproterenol in a group ofspontaneously hypertensive rats compared with con-trols. Gende et al"l demonstrated marked cardiacimpairment to isoproterenol infusions in rats withtwo-kidney, one-clip Goldblatt hypertension, where-as Ayobe and Tarazi10 have noted that the impair-ment in f3-adrenergic responsiveness is reversiblewith regression of hypertrophy. Tarazi2 has offeredimportant caveats about the heterogeneity of thealteration in 18-adrenergic physiology depending onwhether genetic or acquired models of hypertensionare used. Major species differences and model de-pendency make it difficult to reach a consensus onalterations in LV contractile responses to catechola-mines during the development of hypertension in therat. In addition to differences in the proportion ofmyocardial a- and /3-receptors,16,17 there are impor-tant species differences in changes in myosin isoen-zyme forms to the V3 subtype during hypertension,'8which result in slower crossbridge formation and maypredominate over changes in sarcolemmal membranereceptor responses.

There have been fewer prior studies investigatingalterations in adrenergic responsivensss in hyperten-sive canine models. Davidson et a133 suggested that,B-adrenergic stimulation was maintained early (2weeks) in the development of renovascular hyperten-sion in dogs. Although there were some methodolog-ical differences (e.g., they conducted their studies inresponse to bolus infusions of isoproterenol, theanimals were sedated, and changes in loading condi-tions were not considered), their data might really beconsistent with those in the current investigation. It isimportant to consider that the net inotropic effect ofisoproterenol is the sum of direct myocardial f-ad-renergic stimulation plus reflex mediated effects.Since Davidson et a133 demonstrated depressed reflexresponses, the portion of the inotropic and chrono-tropic responses to isoproterenol that is reflexlymediated was lost. In the absence of the reflex-mediated component, the observation that therewere no differences in the inotropic response toisoproterenol implies that the direct myocardial stim-ulation must have been enhanced. If reflex controlhad been intact in these hypertensive dogs, or hadthe dogs been studied under ganglionic blockade,enhanced inotropic and chronotropic responses toisoproterenol might have been observed.

Thus, there are major methodological differencesbetween prior reports in either isolated or anesthe-tized animal preparations and the current observa-tions conducted in chronically instrumented dogswith perinephritic hypertension. Recent studies fromour laboratory3 have underscored the importance ofintact autonomic nervous system function in theenhanced LV contractile response seen at baseline indeveloping perinephritic hypertension. The currentdata extend these observations to include augmentedadrenergic responsiveness to norepinephrine in de-veloping hypertension and maintenance of a normalinotropic response to this adrenergic agonist later,when the hypertensive process has stabilized.

It is conceivable that alterations in reflex controlmay contribute to altered inotropic responsiveness inthe intact, conscious animal.3435 Accordingly, weinvestigated the responses to norepinephrine underganglionic blockade and observed that the enhancedresponses to norepinephrine persisted in developinghypertension compared with the control period.Thus, the enhanced inotropic responsiveness to nor-epinephrine was not abolished by ganglionic block-ade, suggesting that the effects were not mediated viareflex mechanisms. The enhanced chronotropic re-sponses to norepinephrine under ganglionic blockadeare consistent with the augmented direct myocardialeffects of this adrenergic agonist.Another difference between the current investiga-

tion and most prior studies is the use of the endog-enous neurotransmitter, norepinephrine, to induceadrenergic stimulation, as opposed to a specific ,l-ad-renergic agonist.4-6,9-11,32 Thus, it is conceivable thatthe observed differences may be attributed to theassociated a-adrenergic properties of norepineph-

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Shannon et al Enhanced Inotropic Response to Norepinephrine in Hypertension 553

rine. This explanation is unlikely in view of the virtualinability of norepinephrine to increase LV dP/dt inthe presence of ,B-adrenergic blockade in the caninemodel.16 Similarly, we observed that there was noinotropic response to a-adrenergic stimulation in anygroup, suggesting that the increased inotropic re-sponses to norepinephrine were mediated via itsP-adrenergic properties.There are several mechanisms that may account for

the enhanced responses that were observed. Of obvi-ous importance are alterations in f-adrenergic recep-tor biochemistry. We observed significant increases inmyocardial 13-adrenergic receptor density during de-veloping hypertension, which may account for theselective increase in adrenergic myocardial respon-siveness to norepinephrine. There were no significantdifferences in plasma or myocardial catecholaminelevels for the three study periods, indicating that thechanges in /3-adrenergic receptor density were not dueto altered levels of measurable catecholamines.The finding of increased /3-receptor density but

similar maximal adenylate cyclase activity in develop-ing hypertension provides an important and recog-nized method of amplification within the ,3-adrener-gic receptor system36 by allowing for an increase inreceptor occupancy at any given agonist concentra-tion. A similar pattern was observed in a recentstudy37 examining mechanisms of denervation super-sensitivity, which was characterized by increased/3-adrenergic receptor density and no change in max-imal adenylate cyclase stimulation. However, this isthe first report that such mechanisms may be opera-tive during developing hypertension, when wall stressis increased, thereby allowing for enhanced contrac-tile responses to ,3-adrenergic stimulation.The findings of increased /8-adrenergic receptor den-

sity in developing hypertension are in contrast to priorreports in rats.2,12-15 Ayobe and Tarazi6 demonstrateddecreases in myocardial /8-receptor number in rats withrenovascular hypertension and LV hypertrophy. How-ever, Sen and Tarazil have also noted that there wasmarked variability in myocardial catecholamine contentdepending on whether genetic or acquired models ofhypertension were used, even when the degree ofhypertrophy was similar. Such variations likely accountfor the differences in findings that have been reported.For example, using a similar model, Gende et al"lnoted depressed adrenergic responsiveness to isopro-terenol but no change in /3-receptor density or affinity.In their canine model of perinephritic hypertension,Davidson et a133 found no perturbation in the myocar-dial /3-receptor-cyclase system but also found no evi-dence of hypertrophy after a brief period of hyperten-sion. Thus, important differences in myocardialadrenergic receptor pharmacology among species, theduration of the hypertensive lesion, and the degree ofassociated hypertrophy may all account for the signifi-cant differences between our findings and others.Other potential mechanisms that may help to

reconcile the differences between our findings and

include abnormalities in coronary flow reserve asso-

ciated with hypertensive hypertrophy. Alfaro et a138noted impaired coronary perfusion in rats with reno-

vascular hypertension and associated this with im-pairments in contractile function. Marcus et a139showed a modest decrease in coronary vasodilatorreserve at 6 weeks after the development of renovas-

cular hypertension in dogs but did not study contrac-tile function. Furthermore, they found no furtherdecrease in vasodilator reserve when the hyperten-sive lesion was allowed to persist for 6 months. Asreported elsewhere,40 we found modest reductions incoronary vasodilator reserve in dogs with eitherdeveloping or stable hypertension. However, despitethis modest impairment in coronary vasodilator re-

serve, we observed enhanced inotropic responsive-ness to norepinephrine in developing hypertension,the magnitude of which may have been even greater,if coronary vasodilator reserve was not affected.Another potential explanation for the differences

between our findings and those of others pertains toalterations in myocardial structure. Another investiga-tor41 has observed an increase in fibrous connectivetissue in hypertensive rats and has suggested thatthese changes may contribute to systolic impairmentin these hypertensive models. Weber et a142 noted an

increase in the volume and a shift in the type ofcollagen in evolving hypertension (4 weeks) in a

nonhuman primate model of perinephritic hyperten-sion. These changes were associated with diminishedsystolic performance both in vivo and in vitro. Incontrast, we found no significant increase in collagencontent in the LV of dogs with stable hypertension,40suggesting that this mechanism is not playing a role inmodulating the inotropic responsiveness to catechol-amines in this model. Potentially with more chronichypertension and more severe hypertrophy, where LVfibrosis is increased substantially, this mechanismmight act to decrease catecholamine responsivenessand might have been a factor in prior studies reportingdepressed responsiveness to /3-adrenergic stimulation.

Finally, the findings of enhanced inotropic respon-siveness to norepinephrine during developing hy-pertension and maintenance late in the course ofhypertension have important implications in thepathogenesis of the hypertensive state. Previous stud-ies43-45 have suggested that the heart may play an

important role in the development of hypertension.However, prior studies in rats have been unable toconclusively demonstrate such a role. These data dem-onstrate for the first time that the myocardial substrateis poised to respond to the sympathetic neurotrans-mitter, norepinephrine, with enhanced responsivenessearly in the hypertensive state and that diminishedresponses to norepinephrine were not observed even

during a more chronic phase of stable hypertension.

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554 Circulation Research Vol 68, No 2, February 1991

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22. Page IH: The production of persistent arterial hypertension bycellophane perinephritis. JAMA 1939;113:2046-2048

23. Vatner DE, Vatner SF, Nejima J, Uemura N, Susanni EE,Hintze TH, Homcy CJ: Chronic norepinephrine elicits desen-

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24. Munson PJ, Rodbard D: LIGAND: A versatile computerizedapproach for characterization of ligand binding systems. AnalBiochem 1980;107:220-239

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26. Peuler JD, Johnson GA: Simultaneous single isotope radioen-zymatic assay of plasma norepinephrine, epinephrine, anddopamine. Life Sci 1977;21:625 -636

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29. Suga H, Sagawa K, Shoukas AA: Load independence of theinstantaneous pressure volume ratio of the canine left ventri-cle and the effects of epinephrine and heart rate on the ratio.Circ Res 1973;32:314-322

30. Kass DA, Maughan WL: From 'Emax' to pressure-volumerelations: A broader view. Circulation 1988;77:1203-1212

31. Snedecor GW, Cochran WG: Statistical Methods, ed 7. Ames,Iowa, Iowa State University Press, 1982, pp 144-145

32. Crottogini AJ, Willshaw P, Barra JG, Armentano R, CabreraFischer El, Pichel RH: Inconsistency of the slope and volumeintercept of the end-systolic pressure-volume relationship asindividual indexes of inotropic state in conscious dogs: Pre-sentation of an index combining both variables. Circulation1987;76:1115-1126

33. Davidson WR, Kawashima S, Banerjee SP, Liang C: Preservedcardiac ,3-adrenergic sensitivity in early renovascular hyper-tension. Hypertension 1987;9:467-472

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35. Vatner SF, Rutherford JD, Ochs HR: Baroreflex and vagalmechanisms modulating left ventricular contractile responsesto sympathomimetic amines in conscious dogs. Circ Res 1979;44:195-207

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KEY WoRDs * systemic hypertension * left ventricular hypertrophy* contractility ,3-adrenergic receptors * adenylate cyclase

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R P Shannon, R J Gelpi, L Hittinger, D E Vatner, C J Homcy, R M Graham and S F Vatnerdogs with perinephritic hypertension.

Inotropic response to norepinephrine is augmented early and maintained late in conscious

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1991 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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