characterization of intrarenal arterial adrenergic ... · tension was induced by the goldblatt...

851

Characterization of IntrarenalArterial Adrenergic Receptors in

Renovascular HypertensionNeil D. McElroy and Ben G. Zimmerman

a-Adrenergic receptor subtypes were investigated using [3H]prazosin, an a, selective antagonist,and the a2 selective antagonist [3H]rauwolscine in a smooth muscle plasma membrane enrichedmicrosomal fraction prepared from rabbit intrarenal arterial vasculature. Both radioligandsdisplayed single components on Scatchard analysis. The specific binding of [3H]prazosin was ofhigh affinity (0.54±0.04 nM) with a maximum binding capacity (Bmax) of 212±15 fmol/mgprotein. The maximum number of [3H]rauwolscine binding sites was 64±4 fmol/mg of proteinwith a dissociation constant (Kd) of 5.60±0.27 nM. Binding of both radioligands was rapid,saturable, and specific, a,- and a2-adrenergic receptors in the intrarenal arterial membranepreparation were also characterized at 2-, 4-6- , and 10-12-week intervals during the course ofdevelopment and maintenance of chronic two-kidney, one clip (2K1C) Goldblatt hypertensionand in age-matched sham-operated normotensive control rabbits. The a,-adrenergic receptoraffinity for [3H]prazosin binding in hypertensive rabbits was significantly increased in thestenotic, but not contralateral, kidney at 2 weeks; however, at 6 weeks the receptor affinity ofboth kidneys was significantly increased compared with those of the normotensive controlgroup. No difference in a,-adrenergic receptor affinity was seen at 12 weeks, and there were nochanges in Bmax at any of the weekly intervals. Neither the KA, nor Bma, for [3H]rauwolscine ineither kidney showed a significant difference between hypertensive rabbits and normotensivecontrol rabbits. These studies demonstrate the existence in the rabbit intrarenal arterialvasculature of binding sites with a,- and a2-adrenergic receptor specificity. Further, intrarenalarterial a,-adrenergic receptor affinity is significantly increased early (to at least 6 weeks) butnot later (at most 12 weeks) during 2K1C Goldblatt hypertension. (Hypertension 1989;13:851-858)

Enhanced reactivity of regional vascular bedsto various vasoconstrictor stimuli is a com-mon feature in several models of experi-

mental and human hypertension.12 Such increasedvascular reactivity may involve, in part, adrenergicreceptor changes,34 which have been shown toprecede the elevation of blood pressure.5 Adrener-gic receptors located on the postjunctional mem-brane mediate catecholamine-induced vasoconstric-tion and therefore directly influence vascularresistance. Increased vascular reactivity may resultfrom differences in a number of factors, includingcell membrane properties, level of membrane poten-tial, and postreceptor phenomena. The sensitivityof blood vessels to catecholamines has also been

From the Department of Pharmacology, University of Minne-sota Medical School, Minneapolis, Minnesota.

Supported by a Minnesota Medical Foundation grant andgrants HL-17871 and R01 HL-39698 from the National Institutesof Health.

Address for correspondence: Neil D. McElroy, Department ofPharmacology, 3-249 Millard Hall, 435 Delaware Street, S.E.,University of Minnesota, Minneapolis, MN 55455.

shown to be influenced by a-receptor number andaffinity6 and may be modulated by a variety ofphysiological and pathological conditions.78

Several investigations have concerned changes invascular reactivity of the kidney in experimentalhypertension. Because of the pivotal role of thekidney in maintaining elevated arterial pressure,alterations in renal vascular reactivity could lead tochanges in renal hemodynamics that importantlyinfluence the development and maintenance of hyper-tension. Enhanced renal vascular tone may be dueto vasoconstrictor hyperresponsiveness9 or toincreased activity of the renal sympatheticinnervation.10 However, observations of increasedsympathetic nerve activity in experimental hyper-tension differ,10" and to our knowledge there is nofirm evidence of increased renal nerve activity inrenovascular hypertension.

Information on adrenergic functional alterationsin renovascular hypertension have come largelyfrom in vivo experiments, studies on isolated bloodvessels and perfused kidney, or from whole kidney

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852 Hypertension Vol 13, No 6, Part 2, June 1989

homogenates. Thus, previous investigations havenot provided a means of determining specificallyalterations in renal vascular adrenergic receptors inhypertension, nor have the subtypes of these recep-tors in renal vessels been defined. The presentinvestigation was conducted on a novel intrarenalarterial vascular preparation to characterize thesereceptors during various stages in the developmentand maintenance of the hypertensive process.

Materials and MethodsAnimal Protocol

Experiments were initiated with male NewZealand White rabbits having body weights of lessthan 1 kg. Rabbits were anesthetized with 10-15 mg/kg pentobarbital via the marginal ear vein. Hyper-tension was induced by the Goldblatt method, withplacement of a silver clip (0.22-mm slit) on the leftrenal artery in close proximity to the aorta.12 Youngrabbits were used to facilitate hypertension devel-opment by allowing the renal artery to grow into theclip. Rabbits in the normotensive group underwenta sham operation with an identical silver clip placednear the renal artery. All rabbits were maintainedon standard laboratory chow and tap water adlibitum.

Arterial pressures were determined in the con-scious state by means of a permanent catheterplaced in the carotid, auricular, or lower abdominalaorta (via the femoral artery). The arterial line wasalso used to obtain blood samples for determinationof biweekly plasma renin activity (PRA).13 Animalswere studied after 2, 6, and 12 weeks and 2, 4, and10 weeks of sustained hypertension for ar anda2-adrenergic receptor analysis, respectively.

Membrane PreparationRabbits were killed by rapid pentobarbital over-

dose, and the kidneys, abdominal aorta, and tho-racic aorta were immediately excised. Kidneys werebisected and immersed in ice-cold modified Tris-HC1 buffer (50 mM Tris-HCl, 10 raM MgCl2, pH7.4). Plasma membrane vesicles were prepared bymodifications of methods from Kwan et al.14 Briefly,medullary extravascular tissues were removed.Gross dissection of the remaining tissue revealedarcuate and interlobular arteries and branches ofthe interlobar arteries. This partially isolated vas-cular network was subjected to gentle agitation withborosilicate glass beads and ice-cold buffer for 2hours to facilitate removal of the remaining corticaltissue (e.g., adhering glomeruli and microvascularand tubular structures). Microscopic examinationof the resulting vascular network was used to verifyisolation of the arcuate, interlobular arterial, andafferent arteriolar network to the exclusion of othertissues. Cleaned arterial network was subjected tohomogenization with a Brinkman Polytron, setting8, for three 30-second bursts followed by stepwisedifferential centrifugation at 4° C, first at 900g for 10

minutes and then at 9,000g for 60 minutes. Theresulting supernatant was then centrifuged at100,000g for 60 minutes to sediment a microsomalfraction. This microsomal pellet was suspended inmodified sucrose-Tris-HCl buffer (0.25 M sucroseand 50 mM Tris, pH 7.4) and centrifuged again at9,000g for 10 minutes. The final supernatant waslayered gently on top of the differential sucrose-density gradient composed of 3 ml each 26.5% and38.5% sucrose and recentrifuged at 100,000g for 120minutes. The uppermost band at the 8-26.5% inter-face (F2) has been found to contain highly enrichedplasma membrane vesicles.14 The F2 band wasdiluted with modified Tris-HCl buffer (pH 7.4) andrecentrifuged at 100,000# for 30 minutes. The sedi-mented smooth muscle plasma membrane micro-somes were suspended in buffer to a final proteinconcentration of 1 mg/ml. An aortic smooth musclemicrosomal fraction was also prepared.

Binding Assays[3H]Prazosin and [3H]rauwolscine were used to

assess a r and a2-adrenergic receptors in the plasmamembrane fraction prepared from intrarenal arterialand aortic smooth muscle over the course of theinduction and maintenance of two-kidney, one clip(2K1C) Goldblatt hypertension. Experiments wereperformed on fresh or frozen (in modified Tris-HClbuffer, pH 7.4, at -60° C) plasma membrane-enriched F2 fractions. Previous experiments ofradioligand-receptor binding have shown that mem-branes stored frozen up to 1 month give resultscomparable with those from freshly preparedmembranes.15

In all radioligand binding assays, a modified Trisbuffer (50 mM Tris-HCl and 10 nM MgCl2, pH 7.2)was used in the incubation media. Subdued filamentroom lighting was used to prevent photolysis of[3H]rauwolscine radioligand. No degradation ofeither radioligand was observed in the presence ofmembrane during incubation under these condi-tions. The reactions were terminated by adding 3.0ml ice-cold buffer (4° C) to the incubation mixtures.Membranes were collected on 0.1% polyethylene-amine pretreated Whatman GF/C microfibre glassfilters by rapid vacuum filtration followed by threewashes with 5.0 ml ice-cold buffer. The filters wereair dried and placed in 5.0 ml scintillation solutioncontaining pseudocumene-1,2,4 trismethylbenzene,Biosolve-emulsifier, PPO primary fluor, and bis-o-methyl styrylbenzene secondary fluor. Filters wereleft to equilibrate in scintillation cocktail at roomtemperature for 12 hours and counted on a Beck-man Model LS5801 liquid scintillation counter.Quench correction in the counting of tritium wasmade using the external standard ratio method.Counting efliciency was 43%. Blanks were run forall the concentrations of radioligands as for bindingwith membranes except that in blank tubes therewas no membrane, but they contained the samevolume of modified Tris buffer.



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McElroy and Zimmerman Intrarenal Arterial a-Receptors 853

Specific binding for [3H]prazosin and [3H]rauwol-scine was defined as the radioactivity displaceableby 10 /xM phentolamine and comprised 80- 95% oftotal bound counts at a concentration of theseradioligands near their dissociation constant (Kd)values. Justification for the use of 10 (iM phentola-mine to define specific binding was based on dis-placement curves between various concentrationsof phentolamine and the radioligands in which max-imum displacement was reached at a concentrationnot greater than 5xlO"6 M phentolamine.

Protein content of the membrane was determinedby the method of Bradford,16 using crystalline serumbovine albumin as the standard. Data in all figures,text, and tables refer to specific binding and have beennormalized in terms of the protein concentration.

[3H]Prazosin (0.02-5.0 nM) and [3H]rauwolscine(0.1-40.0 nM) were used for construction of Scatch-ard plots in duplicate for each assay. The incubationmedia contained 250 /il buffer with or withoutunlabeled drug and 50 fi\ diluted radioligand. Thebinding interactions were started by adding 100 /xlmembrane suspension (1 mg/ml) to make a finalvolume of 400 /A. Incubations were carried out in acovered rotary shaking water bath at 25° C for 45minutes, at which time equilibrium had been reachedfor all concentrations of each radioligand used inthe study. Inhibition experiments were similar tosaturation studies except that in these assays ali-quots of the tissue homogenates were incubatedwith [3H]prazosin or [3H]rauwolscine for 45 minutesat 25° C in the presence of at least 12 increasingconcentrations of the various antagonists and (-)epinephrine.

The reaction kinetics of the radioligands[3H]prazosin and [3H]rauwolscine were studied usingone concentration of the radioligand under standardassay conditions but at different incubation times.For dissociation studies, binding was allowed toproceed for 45 minutes before 100 /uM phentol-amine was added at specified intervals to the assaymixture. Kinetic rate constants were determined asdetailed in Figure 3 and by the method described byWilliams and Lefkowitz.17

Data AnalysisSaturation radioligand binding curves were ana-

lyzed by the method of Scatchard18 using the com-puterized weighted least-squares curve-fitting algo-rithm approach for determination of Kd andmaximum binding capacity (Bmax) radioligand bind-ing parameters according to Munson and Rodbard.19

The apparent dissociation constant (K^ values forthe competitive unlabeled antagonist were calcu-lated by the method of Cheng and Prusoff.2° The K{for each agonist and antagonist was calculated fromthe equation: Ki=lC5Ql{] + *lJKd), where IC50 is theconcentration of an unlabeled antagonist giving 50%inhibition of radioligand binding, *L and Kd are theconcentration of free tritiated ligand in the assaysystem and the equilibrium dissociation constant of

the radioligand as determined from saturation exper-iments, respectively. To check whether ligand-receptor interaction studies" have followed the lawof mass action in saturation studies or displacementby the unlabeled compounds, the data have beentransformed into the Hill equation: logB/(Bmax-B)=nlog[*L]-log Kd: Log B/(Bmax-B) was plottedagainst log[*L] to obtain the value of the Hill slope(nH) and the Hill binding dissociation constantCKd).

21 Differences between mean values of Kd andBmax were tested for significance using a grouped ttest with a Bonferroni transformation.22 The dif-ference was taken to be significant when /?<0.05.Slopes of linear regression lines were fitted by theleast-squares method.

DrugsRadioligands [3H]prazosin (specific activity, 26.0

Ci/mmol) and [3H]rauwolscine (specific activity,73.7 Ci/mmol) were purchased from New EnglandNuclear, Boston, Massachusetts. Other drugs wereobtained as follows: rauwolscine (Regitine) andphentolamine (CIBA-GEIGY Corp., Summit, NewJersey), prazosin HC1 and (-)epinephrine (SigmaChemical Co., St. Louis, Missouri), idazoxan (giftfrom Reckitt & Colman, Kingston-Upon-Hull, UK).

Results[3H]Prazosin Binding

Specific saturation binding data disclosed a singleclass of sites in the intrarenal arterial preparationover the concentration range of [3H]prazosin stud-ied (Figure 1A). Kd and Bmax were 0.54+0.04 nMand 212±15 fmol/mg protein, respectively.

Specific binding of [3H]prazosin to this fractionwas rapid, with a half-time of approximately 3minutes. In the presence of 100 iM phentolamine,[3H]prazosin dissociated from the membranes witha half-time of approximately 2 minutes. Accordingto the pseudo first-order rate equations, the appar-ent rate constant for the pseudo first-order reactionof association (Kap) of [3H]prazosin binding at 0.5nM concentration was 0.253/min (Figure 2A, insert),and the second-order rate constant (Kt) was 2.04x 108

M"Vmin. The second-order dissociation rate con-stant (£_,) was 0.151/min (Figure 2B). The ratio ofthe K-XIK\ yielded a value of 0.74 nM, whichcompares well with the Kj values obtained by theequilibrium binding experiments (Figure 1A).

[3H]Rauwolscine BindingThe specific binding of increasing concentrations

of [3H]rauwolscine in the F2 fraction of the intrare-nal arterial preparation was saturable, and the lin-earity of the Scatchard analysis indicated a singleclass of binding sites over the concentration rangestudied (Figure IB). The Kd and Bmax were 5.6±0.27nM and 64±4 fmol/mg protein, respectively. Hillplots of the data were linear with an average Hillcoefficient of 0.99±0.02.



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The specific binding of [3H]rauwolscine (7.2 nM)to the membranes of the intrarenal arteries was alsotime dependent with a half-time of about 3 minutes(Figure 2C). Assuming pseudo first-order kinetics,the association reaction reached apparent equilib-rium with the Kip value of 0.209/min. In the disso-ciation experiments, AL, was 0.091/min (Figure2D). From the tf_, and the K3p, a Kx value of1.53X107 M/min was calculated. The dissociationconstant calculated from the ratio of K.XIKX was 5.9nM, which is in close agreement with trie KA calcu-lated from the Scatchard analysis.

Specificity of I3H]Prazosin and[3H]Rauwolscine Binding

Data for these competition studies are shown inTable 1. Investigation of competition between unla-beled antagonists versus [3H]prazosin and[3H]rauwolscine disclosed that [3H]prazosin and[3H]rauwolscine were labeling distinct sites. Theunlabeled prazosin was about 7,300-fold more potentin competing at the [3H]prazosin recognition sitesthan at the [3H]rauwolscine sites. In contrast, unla-beled rauwolscine displayed the reverse relation;rauwolscine was about 200-fold more potent at the[3H]rauwolscine sites than at the [3H]prazosin sites.Unlabeled a-adrenergic antagonists competed for[3H]prazosin binding sites with an order of potencyof: prazosin>phentolamine>rauwolscine indicativeof an a,-adrenergic receptor subtype. Competitioncurves with unlabeled antagonists against [3H]rauwol-scine revealed the reverse order of potency expectedof an a2-adrenergic receptor subtype. The a agonist(-)epinephrine competed equally for the [3H]prazosinor [3H]rauwolscine binding sites.

Effects of Two-Kidney, One ClipGoldblatt Hypertension

The time course of changes of mean arterial bloodpressure and PRA in sham-operated and hyperten-sive rabbits is summarized in Table 2. 2K1C Gold-blatt hypertensive rabbits in both the a r and a2-adrenergic receptor studies maintained an increasein mean arterial blood pressure of at least 20 mm Hgover the sham-operated controls. Plasma renin activ-ity in 2K1C hypertensive rabbits was increasedapproximately fourfold or greater to at least 4-6weeks in both groups.

The binding affinity of [3H]prazosin to aradrenergic receptors was significantly higher in theintrarenal arterial preparation derived from thestenotic, but not the contralateral, kidney of hyper-tensive rabbits at 2 weeks; at 6 weeks the receptoraffinity of both kidneys was significantly increasedcompared with that in sham-operated normotensivecontrol rabbits (Figure 3). However, at 12 weeks nodifference was observed in the affinity of a,-adrenergic receptor binding. B ^ did not differbetween sham-operated and hypertensive rabbitsover the intervals studied. Both the KA and B ^ inmicrosomes derived from aortic smooth muscle of

Alpha-1 Saturation Binding of[3H] Prazosin

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0 10 20 30 40 50[3H]Rauwolscine (nM)

FIGURE 1. Saturation curves for [3H]prazosin (Panel A)and [3H]rauwolscine (Panel B) specific binding to the F2

fraction of the rabbit intrarenal arterial preparation.Each point is the mean of duplicates. Insets, Scatchardanalysis of these same data. Dissociation constant (Kd)was determined from the negative reciprocal of the slopeand maximum binding capacity (Bmax) as the abscissaintercept. Data shown are representative of those obtainedin three separate experiments performed in duplicate.

these rabbits did not differ over these time intervals(data not shown).

The results of [3H]rauwolscine binding in theintrarenal arterial preparation are shown in Figure 4at 2-, 4-, and 10-week intervals. Neither the a2-adrenergic receptor affinity nor the total number ofbinding sites in either kidney showed a significantdifference between the hypertensive rabbits andtheir normotensive sham-operated controls.

DiscussionThis study identified and characterized pharma-

cologically for the first time a,- and a2-adrenergicreceptors in the intrarenal arterial vasculature. The



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Association Kinetic Analysis of[3H]-Prazosin Specific Binding


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FIGURE 2. Kinetic analysis of [3H]prazosin and [3H]rauwolscine binding to the F2 fraction of the rabbit intrarenalarterial preparation. Results shown are means of two determinations performed in duplicate. Panel A, time course ofassociation for specific [3H]prazosin binding at the radioligand concentration of 0.5 nM. Inset, determination of thesecond-order rate constant for [3H]prazosin binding. Beq is the amount of[3H]prazosin bound at equilibrium, and B, is theamount bound at each time, t. The slope of the line relating ln(Beq/Beq—B,) and time, determined by linear regressionanalysis is equal to the apparent rate constant for the pseudo first-order reaction of association (Kap). Second-order rateconstant (Kt) is calculated according to the equation K;=Kop—K_,//*L7, where K_; is the dissociation rate constant and[*L] is the concentration of[3H]prazosin used in the assay. These results indicated K.ap=0.253/min and Kt=2.04 x 10s M~'lmin with a linear regression correlation coefficient (f)=0.98. Panel B, time course for dissociation of [3H]prazosindetermined by the addition of phentolamine to a final concentration of 10 \iM. Inset, first-order rate plot of [3H]prazosindissociation. Dissociation rate constant (K_,,) is equal to the slope of the line relating In (BJB0) and time, where B, refersto the specific binding at each time, t, and Bo represents the binding at equilibrium before addition of phentolamine.K-i=0.151/min with r=0.96. Panel C, time course of association for specific [3Hjrauwolscine binding at the radioligandconcentration of 7.2 nM. Inset, determination of the second-order rate constant for [3H]rauwolscine binding. Theseresults indicated Kap=0.201lmin and K,=I.58xlO7 M~'lmin with x-0.99. Panel D, time course of dissociation ofI3H]rauwolscine on addition of phentolamine to a final concentration of 10 fiM. Inset, first-order rate plot of[3H]rauwolscine dissociation. K-j=0.091/min with r=0.99.

KA and B ^ for the aradrenergic receptors in theintrarenal arterial vessels and aorta obtained in thisstudy are within the range of previously reportedvalues for vascular smooth muscle. arAdrenergicreceptor number in the intrarenal vessels was approx-

imately fourfold greater than that of the a2. In 2K1CGoldblatt hypertension, the a,-adrenergic receptorsexhibited a significant affinity increase early in thecourse of development of the hypertension. Thisincrease was, however, not sustained during the



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TABLE 1. Inhibition Constants and Hill Coefficients for Competition of [3H]Prazosin and [3H]Rauwolscine BindingSites in Rabbit Intrarenal Arterial Smooth Muscle Microsomes

Drug Competitor

RauwolscineIdazoxanPhentolamine

Prazosin(-)Epinephrine

[3H]Rauwolscine

Kj(nM)

1.45±0.214.02+0.3828.6±4.1528±68

1,480± 120

nH

1.06+0.050.93 ±0.031.05±0.040.94+0.030.99±0.06

[3H]Prazosin

Ki(nM)

286±33N.D.

23.3+2.70.072±0.015l,730±280

nH

0.95±0.2N.D.

0.98±0.021.03 ±0.051.09±0.1

Inhibition constant (Kj) values calculated according to Cheng and Prusoff20; values are mean±SEM of duplicatedeterminations from two separate experiments in membrane preparations of six rabbits per group. Concentrationsof [3H]prazosin and [3H]rauwolscine used in these experiments were 0.2-0.6 nM and 4.0-6.0 nM, respectively. nH,Hill coefficient; N.D., not determined.

12-week interval when PRA was normal. An increasein a,-adrenergic affinity may relate to altered vas-cular reactivity in the hypertensive rabbit. Becauseno changes were seen in the vascular a2-adrenergicreceptors, they do not appear to be involved in thehypertension.

A relation between affinity of the aradrenergicreceptor in vascular smooth muscle and the sensi-tivity of the arterial contractile response has beendemonstrated in a series of 12 rabbit arteries.23

Accentuated adrenergic receptor-mediated responseshave also been demonstrated in renovascular hyper-tension both in vitro and in vivo. This was mani-fested by increased a-adrenergic receptor affinity inisolated aortic smooth muscle from Grollman hyper-tensive rats24 and by a decreased ED50 of norepi-nephrine and phenylephrine for renal vasoconstric-tor responses in the 2K1C Goldblatt hypertensivedog.25 Vascular wall hypertrophy did not appearresponsible for the altered reactivity in the latterstudy since there was a temporal difference in thereactivity change between norepinephrine and athromboxane analogue. Studies in the artificiallyperfused renal vascular preparation26 or in isolatedarterial strips27 have shown that alterations in reac-tivity persist in preparations devoid of humoral andneurogenic control. Therefore, it may be concludedthat an intrinsic change occurs in the vascular

smooth muscle resulting in enhanced sensitivity toexternal stimuli.

Regional differences in affinity of the «,-adrenergic receptors of arterial vascular smoothmuscle cells have been demonstrated.23 This maybe an important determinant of differential reactiv-ity of rabbit vascular smooth muscle. In the presentstudy, an increase in affinity was seen in one region,the intrarenal vasculature, but not in the aorta of2K1C Goldblatt hypertensive rabbits. This regionaldifference would seem to support the potential forregional variation in receptor plasticity. The regionaldifference, and the temporal variation in affinity,may explain, in part, failure of previous investiga-tions of a-adrenergic receptors in hypertension todemonstrate an affinity alteration. The present studyis consistent with the previous findings of Niels andBevan28 where arteries from spontaneously hyper-tensive rats were shown to be more sensitive tonorepinephrine than arteries from Wistar-Kyotorats. This increased sensitivity was associated withan increased norepinephrine Kd for the a-adrenergicreceptor but not with differences in receptor reserveor relative receptor occupation.

It is possible that the increased intrarenal arteriala,-adrenergic receptor affinity seen in the presentstudy was related to the increase in plasma angio-tensin II due to the early rise in PRA. At the 2-week

TABLE 2. Mean Arterial Blood Pressure and Plasma Renin Activity in Goldblatt Hypertensive and Sham-OperatedNormotensive Control Rabbits

Weeks afterprocedure

Group 126

12

Group 224

10

Sham

79+493±691+6

65+376±385±2

MAP (mm Hg)

Hypertensive

108±7*114±4*109±6*

98±4*114±6*107±6*

Sham

2.5±0.93.7±0.82.7±1.2

2.4+1.93.2±l.O3.4±1.4

PRA (ng/ml/hr)

Hypertensive

17.0+0.7*13.8+0.9*2.1±1.4

14.7+3.7*15.4±3.3*4.0±0.9

Group I, n=5; Group 2, n=6 for hypertensive and sham-operated rabbits in each time period. Group 1 and Group2 correspond to ar and a2-adrenergic receptor experiments, respectively. MAP, mean arterial pressure; PRA,plasma renin activity.

*p<0.05 by grouped (test when compared with sham-operated rabbits.



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Intrarenal Arterial Alpha-1Adrcnoceptor Dissociation Constants

Intrarenal Arterial Alpha-2Adrenoceptor Dissociation Constants

Weeks After Goldblattor Sham Operation

Intrarenal Arterial Alpha-1Adrenoceptor Binding Capacity

Left Kidney ShamLeft Kidney Hypert.Right Kidney ShamRight Kidney

Hypertensiven«5 for sham and hypertensive

in each time period


FIGURE 3. [3H]Prazosin binding to a,-adrenergic recep-tors. Time course of changes in the a,-adrenergic recep-tor dissociation constant (Kd) (Panel A) and maximumbinding capacity (Bmax) (Panel B) in the intrarenal arterialpreparation from left stenotic and right contralateralkidney of sham-operated and Goldblatt two-kidney, oneclip hypertensive rabbits. Each vertical bar represents themean±SEM of five separate experiments conducted induplicate. *p<0.002 versus sham value for all compari-sons. See Table 1 and text for details.

postclipping interval, PRA had maximally increasedat which time aradrenergic receptor affinity of thestenotic kidney was significantly enhanced. PRAremained elevated at the 6-week interval when a,affinity of both the stenotic and contralateral kid-ney was significantly increased. At 12 weeks bothPRA and affinity had returned to the control level.During this stage in the hypertension when recep-tor affinity normalized, other factors (e.g., struc-tural) may play a more important role in increasedvascular reactivity.

The present study revealed an early increase inaffinity of the intrarenal arterial but not in aorticaradrenergic receptors in 2K1C Goldblatt hyper-tension. This receptor affinity change is unlikely torepresent the sole basis of the hypertension, but itmay be important in regard to several factors (e.g.,structural remodeling, altered sodium balance, orincreased sympathetic tone) that eventually sustainthe elevated blood pressure.


Intrarenal Arterial Alpha-2Adrenoceptor Binding Capacity

Left Kidney ShamLeft Kidney

Hypertensive0 Right Kidney Sham• Right Kidney

Hypertensive••6 for sham and hypentnsivc

in each lime perod

2 4Weeks After Goldblatt

or Sham Operation

FIGURE 4. [3H]Rauwolscine binding to a2-adrenergicreceptors. Panel A, time course of changes in the a2-adrenergic receptor dissociation constant (Kd). Panel B,maximum binding capacity in the intrarenal arterialpreparation from left and right kidneys of sham-operatedand 2K1C Goldblatt hypertensive rabbits. Each verticalbar represents the mean±SEM of six separate experi-ments conducted in duplicate.

AcknowledgmentsWe thank Dr. Jack Miller for his support and

Brenda Zimmerman for doing the radioimmunoassays.

References1. Holloway ET, Bohr DF: Reactivity of vascular smooth

muscle in hypertensive rats. Ore Res 1973;33:687-6952. Brody MJ, Zimmerman BG: Peripheral circulation in arterial

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KEY WORDS • vascular smooth muscle • intrarenal arteries •a-adrenergic receptors • kidney



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N D McElroy and B G ZimmermanCharacterization of intrarenal arterial adrenergic receptors in renovascular hypertension.

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 1989 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Hypertension doi: 10.1161/01.HYP.13.6.851

1989;13:851-858Hypertension.

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