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169

Ontogenesis of Sympathetic Responsiveness inSpontaneously Hypertensive Rats

I. Renal a,-, as-, and /3-Adrenergic Receptorsand Their Signaling

Martin C. Michel, Frank Siepmann, Rainer Biischer, Thomas Philipp, Otto-Erich Brodde

We studied the ontogenetic development of renal t v , o^-, and /3-adrenergic receptors and their couplingto inositol phosphate and cyclic AMP formation in spontaneously hypertensive and normotensiveWistar-Kyoto rats, a,-, <%-, and /3-Adrenergic receptor number was significantly increased in hypertensivecompared with normotensive rats, but the increase did not precede blood pressure elevation. Despiteincreased a,-adrenergic receptors, basal and norepinephrine-stimulated inositol phosphate formationremained unchanged in all age groups. Rat kidney contains a,A- and «,B-adrenergic receptors coupling toinositol phosphate formation by different mechanisms, but the relative contribution of «,A- andff,B-adrenergic receptors to norepinephrine-stimulated inositol phosphate formation was similar innormotensive and hypertensive rats. Despite increased /3-adrenergic receptors, basal, isoproterenol-, andforskolin-stimulated cyclic AMP accumulation was similar in normotensive and hypertensive rats. Weconclude that the number but not the functional responsiveness of renal adrenergic receptors increases inspontaneously hypertensive rats. Thus, the additional receptors are unlikely to contribute to thepathophysiology of elevated blood pressure in this model. (Hypertension 1993^22:169-177)

KEY WORDS • receptors, adrenergic • inositol phosphates • adenosine cyclic monophosphate •kidney • rats, inbred SHR

The kidney is the tissue with the greatest long-term effect on blood pressure control.1 Cross-transplantation studies have demonstrated that

transplantation of the kidney of a genetically hyperten-sive rat can elevate blood pressure in immunotolerantrecipients, whereas transplantation of the kidney of anormotensive animal can lower blood pressure in im-munotolerant borderline hypertensive rats.2 The adren-ergic system is a major mechanism for the neural andhormonal control of renal function. This occurs ex-trarenally by the well-known alterations of cardiac andvascular function that affect efferent renal nerves viabaroreceptor and volume reflexes.3 Perhaps even moreimportant are intrarenal effects of the sympathoadrenalsystem, which can occur by alterations of renin release,4

renal blood flow,56 glomerular filtration rate,7 and tu-bular function.8 Thus, the kidney is richly endowed withadrenergic receptors. In the rat, renal aradrenergicreceptors are located mainly postsynaptically, but somemay also exist presynaptically.9'10 Rat renal a2-adrener-gic receptors have been demonstrated presynaptically,but extrajunctional a2-adrenergic receptors have alsobeen found.1011 Some renal /3-adrenergic receptors arelocated presynaptically,12 but many are found on tubularcells.8

Received October 5, 1992; accepted in revised form March 29,1993.

From the Department of Medicine, University of Essen(Germany).

Correspondence to Nephrology Laboratory IG 1, Klinikum,Hufelandstr. 55, D-45122 Essen 1, FRG (Dr Michel).

Based on these considerations, many studies haveinvestigated possible alterations of renal adrenergicreceptors in states of elevated blood pressure and theirpossible pathophysiological role in this disease state.Thus, it has been found repeatedly that the number ofrenal a2-adrenergic receptors is increased in geneticallyhypertensive rats of the Kyoto spontaneously hyperten-sive rat (SHR) strain but also of some other strains andthat this increase is specific for the genetic models ofhypertension.13'14 Studies on renal a2-adrenergic recep-tors in SHR have been performed using [3H]yohimbineor [3H]rauwolscine as the ligand that labels homoge-neous populations of a^-adrenergic receptors15 andfails to detect the minor population of a^-adrenergicreceptors that is also present in rat kidney.16 Elevationsof renal aradrenergic receptors have also been foundspecifically in SHR, but this finding was somewhat lessconsistent.13'14 Such studies were performed usingpHJprazosin or [125I]BE-2254 as the ligand that labelstotal aradrenergic receptors; rat kidney contains botha1A- and a1B-adrenergic receptors, but two recent stud-ies have demonstrated that the ratio of the two subtypesremains unchanged in SHR when compared with nor-motensive Wistar-Kyoto (WKY) rats.1718 Elevations ofboth a r and a2-adrenergic receptor numbers have beenreported to precede the development of hyperten-sion.19'20 Based on these findings, we have previouslyhypothesized that genetically determined alterations ofrenal a-adrenergic receptors might play an importantpathophysiological role in the development of hyperten-sion in the SHR.21 More recent studies in our labora-

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170 Hypertension Vol 22, No 2 August 1993

tory, however, have argued against a role for geneticallydetermined alterations for a1A-, a1B-, or a^-adrenergicreceptor number in the development of hypertension inthe SHR.17 Elevations of /3-adrenergic receptors havealso been described frequently in SHR,13 but they donot precede blood pressure elevations22 and are notspecific for genetic models of hypertension.23

Thus, alterations in the number of renal adrenergicreceptors (whether they are specific for genetic hyper-tension or not) do not appear to play a dominant role inthe development of hypertension. Adrenergic receptornumber, however, is a poor predictor of adrenergicreceptor responsiveness, and only a few studies haveinvestigated renal adrenergic receptor responsiveness inhypertensive animals, all of which were performed inthe established phase of hypertension. Therefore, wehave studied a- and /3-adrenergic receptor signaling inrelation to the ontogenetic development of hyperten-sion in WKY and SHR kidneys. Our data demonstratethat increased renal a,- and ^-adrenergic receptors arenot associated with enhanced signaling and thus providefurther evidence against an important pathophysiologi-cal role of alterations in renal adrenergic receptors inthe SHR model of hypertension.

MethodsAnimals

WKY rats and SHR were obtained from Mollegard,Skensved, Denmark. Male and female rats of eachstrain were interbred to generate male progeny thatwere killed at the ages of 3, 6, and 8 weeks by cervicaldislocation without prior anesthesia to avoid anesthesia-induced activation of the sympathetic nervous system.After breeding was complete, male breeders were killedat the age of 28 weeks. One day before death, systolicblood pressure was determined by a tail plethysmo-graphic method; blood pressure measurements in3-week-old rats were not possible for technical reasons.At the time of death, body and kidney weights weredetermined to assess the development of possible renalhypertrophy.

Radioligand BindingRadioligand binding to a- and /3-adrenergic receptors

was performed as previously described.17-22 Briefly, renalmembranes were prepared in a glass homogenizer with amotor-driven PTFE (Teflon) pestle (1500 rpm, 10 strokes)followed by centrifugation at 50 OOOg for 20 minutes at4°C. This method yields a low receptor density whenexpressed in femtomoles per milligram of protein but hasthe advantage of making all receptors present in the tissueavailable to the ligand; more refined membrane prepara-tions usually suffer from considerable loss in yield. Thepellet was resuspended in binding buffer (50 mmol/LTris-HQ and 0.5 mmol/L EDTA, pH 7.5, for o-adrenergicreceptors; 154 mmol/L NaCl and 10 mmol/L Tris-HCl, pH7.4, for /3-adrenergic receptors). Experiments were per-formed in a total volume of 1000 /JLL (aradrenergicreceptors) or 250 JAL (a2- and /3-adrenergic receptors)containing approximately 200 (a,), 100 (a2), or 25 (/3) jigof protein per assay. arAdrenergic receptors were labeledwith [3H]prazosin, a2-adrenergic receptors with pHjrauwol-scine, and /3-adrenergic receptors with [125I]iodocyanopin-dolol. The mixtures were incubated at 25°C (oadrenergic

receptors) or 37°C (^-adrenergic receptors) for 45(aj-adrenergic receptors), 60 (a2-adrenergic receptors),or 90 (/3-adrenergic receptors) minutes. The incubationswere stopped by rapid vacuum filtration over WhatmanGF/C filters and two subsequent washes with 10 mLeach of binding buffer. Nonspecific binding was definedas binding in the presence of 10 /xmol/L phentolamine(a-adrenergic receptors) or 1 ̂ tmol/L of the hydrophilicantagonist (±)-CGP 12177 (/3-adrenergic receptors).The affinity (K^) and number (£„,„) of binding siteswere determined from the experimental data with plotsaccording to Scatchard.24

Inositol Phosphate MeasurementsInositol phosphate accumulation was determined in

renal cortical slices (350x350 y.m) as described previ-ously.25 Briefly, the slices were resuspended in Krebs-Henseleit buffer of the following composition (mmol/L): NaCl, 118; KC1, 4.7; CaCl2, 1.3; MgSO4, 1.2;KH2PO4, 1.2; NaHCO3, 24.9; glucose, 11; and EDTA,0.001. The buffer was supplemented with 10 mmol/LUCl to block inositol phosphate degradation, 2 U/mLadenosine deaminase to remove adenosine liberatedduring tissue chopping from the assay, and 20 /imol/Lcocaine to block neuronal catecholamine uptake. Aftertwo washes with fresh buffer during a 30-minute incu-bation at 37°C, the slices were labeled for 60 minuteswith 100 /iCi of [3H]myoinositol per 11 mL of suspen-sion containing 150 to 300 mg of slices. Then, 300-/xLaliquots of the suspension were pipetted into flat-bottomed polystyrene tubes under gentle swirling, andagonists and antagonists were added to yield a finalvolume of 330 /iL. After 45 minutes in the presence ofagonists and antagonists, the incubation was stopped byaddition of 330 fiL methanol and 660 /xL chloroform.After vigorous mixing, the phases were separated bycentrifugation at 820g for 10 minutes at 4°C, andaliquots (450 ylS) of the upper phase were placed onDowex AG 1-X8 columns (200 mg per column). Afterelution of free inositol with twice 5 mL H2O and twice 5mL of 60 mmol/L ammonium formate, total inositolphosphates were eluted by addition of twice 1 mL of 1mol/L ammonium formate dissolved in 100 mmol/Lformic acid. The recovered radioactivity was deter-mined in a scintillation counter at 42% efficiency.

[3H]Inositol incorporation was assessed by countingthe radioactivity in aliquots of the inositol-loaded slicesuspension after three washes with buffer. [3H]Inositolphosphate formation was expressed as the percentage ofincorporated [3H]inositol.

Cyclic AMP MeasurementsRenal cortical slices of 350x350 /im were prepared with

a tissue chopper (Bachofer, Reutlingen, Germany). Sliceswere washed four times with fresh Krebs-Henseleit buffer(supplemented with 2 U/mL adenosine deaminase toinactivate endogenous adenosine) during a 1-hour incuba-tion at 37°C. Slices were aliquoted (approximately 15 to 30mg wet weight per assay) and incubated in a total volumeof 500 /xL containing 100 /xmol/L isobutylmethylxanthineto inhibit phosphodiesterases for 10 minutes at 37°C;additionally, some tubes contained 100 /xmol/L isopro-terenol or 10 ^mol/L forskolin. The incubation wasstopped by addition of 500 /xL stopping buffer (2% sodiumdodecyl sulfate and 50 mmol/L Tris, pH 7.5) and boiling



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Michel et al Renal Adrenergic Receptors in Hypertension 171

TABLE 1. Development of Systolic Blood Pressure, Body Weight, and Renal Weight

Parameter

Systolic blood pressure (mm Hg)

Body weight (g)

Kidney weight (mg)

Kidney/body weight (g/kg)

3

WKY

n.d.

36±1

(43)

191±7

(43)

5.3±0.1

(43)

Weeks

SHR

n.d.

28±1'

(85)

170±5t(85)

6.2±0.1'

(85)

6 Weeks

WKY

82±3

(6)119±4

(28)

523±16

(28)

4.4±0.1

(28)

SHR

122±2*

(6)

m±4(30)

49O±16

(30)

4.5±0.1

(30)

8 Weeks

WKY

96±1

(6)195±4

(23)

785 ±15

(23)

4.0±0.1

(23)

SHR

163±T

(6)195±4

(26)

767±16

(26)

3.9±0.1

(26)

28

WKY

111±4

(6)366±4

(15)

1227 ±24

(15)

3.4±0.1

(15)

Weeks

SHR

190±4*

(6)357±5

(15)

1212 + 17

(15)

3.4±0.0

(15)

WKY, Wistar-Kyoto rats; SHR, spontaneously hypertensive rats; n.d., not determined for technical reasons. Data are mean±SEM of thenumber of animals indicated in parentheses.

*/><.001, tP<-05 compared with age-matched WKY rats.

for 5 minutes. The boiled samples were centrifuged at13 OOOg for 5 minutes, and the supernatants were placedon neutral alumina columns. The columns were elutedwith 1 mL of 100 mmol/L Tris at pH 7.5, and the cyclicAMP (cAMP) content in the eluate was determined witha commercially available radioimmunoassay (NEN, Drei-eich, Germany). Recovery from the columns was typically80%. The amount of cAMP accumulated in each samplewas calculated and normalized for wet weight.

ChemicalspH]Prazosin (specific activity, approximately 80 Ci/

mmol), [3H]rauwolscine (specific activity, approximately80 Ci/mmol), and [125I]iodocyanopindolol (specific activity,2200 Ci/mmol) were obtained from NEN; [3H]m>>o-inositol(specific activity, 80 to 120 Ci/mmol, prepurified withPT6-271) was from Amersham, Braunschweig, Germany.Phentolamine and (±)-CGP 12177 (l-[2-(3-carbamoyl-4-hydroxy)phenoxyethylamino]-3-[4-(l-methyl-4-trifluoro-methyl-2-imidazolyl)phenoxy]-2-propanol methanesul-fonate) were kind gifts of CIBA-GEIGY, Basel, Swit-zerland, and (+)-niguldipine of BYK-Gulden, Kon-stanz, Germany.

Data EvaluationAll data are mean±SEM of n experiments. Statistical

significance of differences between groups was assessedby two-tailed unpaired t tests when two groups werecompared and by one-way analysis of variance whenmultiple groups were compared. If one-way analysis ofvariance demonstrated that the variance betweengroups was significantly greater than that within groups,individual groups were compared by t tests with Bonfer-roni corrections for multiple comparisons. All statisticalcalculations were performed with the INSTAT program(GraphPAD Software, San Diego, Calif). A value ofP<.05 was considered significant.

ResultsPhysiological Parameters

As expected, systolic blood pressure increased onlyslightly during development in WKY rats but increasedmarkedly in SHR (Table 1). Thus, starting at 6 weeks ofage, systolic blood pressure was significantly elevated inSHR compared with age-matched WKY rats. For tech-nical reasons, we were not able to determine systolicblood pressure in 3-week-old rats, but it is well docu-

mented that 3-week-old SHR have only marginal if anyblood pressure elevations compared with WKY rats2026;thus, we refer to the 3-week-old age group as prehyper-tensive in this article. Body and renal weights alsoincreased with age but did not differ between strains,with the exception of the 3-week-old age group, inwhich both were significantly reduced (Table 1). Be-cause the delay in body weight gain exceeded that inrenal weight gain, a slight but significant renal hyper-trophy (assessed as renal weight-body weight ratio) wasdetected in 3-week-old SHR (Table 1).

oti-Adrenergic ReceptorsThe affinity of [3H]prazosin for rat a,-adrenergic

receptors ranged between 350 and 600 pmol/L and didnot differ significantly between age groups or strains(data not shown). In our preparation, renal a,-adrener-gic receptor density ranged between 20 and 40 fmol/mgprotein (Fig 1). In both strains, it tended to be lowest inthe 6- and 8-week-old groups, but no consistent agedependency was detectable in either strain. A compar-ison between strains demonstrated that Oi-adrenergicreceptor number was similar in both strains in theprehypertensive phase but was elevated in SHR of allother age groups, although this did not reach statisticalsignificance in the 8-week-old group (Fig 1).

To test the functional relevance of the elevated renala,-adrenergic receptor number in hypertensive SHR,

28weeks

FIG 1. Line graph shows development of aradrenergicreceptor density determined in renal membranes of Wistar-Kyoto (WKY) rats (o) and spontaneously hypertensive rats(SHR) (•) from [3H]prazosin saturation binding experi-ments. Data are mean±SEM of eight rats per age group.*P<.05 vs age-matched WKY rats.



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0 3 6 9 28ige, wetks

FIG 2. Line graphs show development of norepinephrine(noradrenaline) -stimulated inositol phosphate formation inWistar-Kyoto (WKY) rat and spontaneously hypertensive rat(SHR) kidneys. Inositol phosphate generation stimulated by 1yjnol/L (top panel) and 100 fimol/L (bottom panel) norepi-nephrine in renal slices from WKY rats (o) and SHR (•) wasdetermined during a 45-minute incubation in the presence of10 mmol/L LACL Data are expressed as percentage of inositolphosphates formed above basal, with basal values beingsimilar in WKY rats and SHR (data not shown). Data aremean±SEM of 8 to 10 experiments in each age group; noneof the observed differences between WKY rats and SHR wasstatistically significant.

we performed experiments on norepinephrine-stimu-lated inositol phosphate formation in renal corticalslices. Incorporation of [3H]m>w-inositoI was similar inall age groups of both strains and typically rangedbetween 6000 and 20 000 cpm/mg wet wt (data notshown). Basal inositol phosphate formation was lowestat 6 weeks (approximately 0.6% of incorporated radio-activity) and highest at 28 weeks (approximately 2.2% ofincorporated radioactivity, P<.001 vs 6 weeks), withintermediate values in the other two groups. However,we did not detect significant differences between strainsin any age group (data not shown). Norepinephrine-stimulated inositol phosphate formation (expressed aspercent over basal) was tested at agonist concentrationsof 1 and 100 uM to detect possible alterations in thepotency as well as maximal effects of the agonist.Norepinephrine-stimulated inositol phosphate forma-tion was a mirror image of basal formation, ie, smallestat 28 weeks of age and greatest at 6 weeks of age, butwas not significantly different between strains at any ageregardless of which norepinephrine concentration wasused (Fig 2). To estimate the possibility of a type IIerror regarding the lack of differences between WKYrats and SHR, we performed power calculations. Thesedemonstrated that with the given sample size andvariance our study would have detected alterations ofinositol phosphate formation by 30% to 50% as beingsignificant in individual age groups. When the data of all

3 weeks 28 weeks

FIG 3. Bar graph shows contribution of aM- and aIB-adrenergic receptors to norepinephrine-stimulated inositolphosphate formation in Wistar-Kyoto (WKY) rat and spon-taneously hypertensive rat (SHR) kidneys. Inositol phosphategeneration stimulated by 100 fimol/L norepinephrine wasdetermined in renal slices from WKY rats (open bars) andSHR (hatched bars) during a 45-minute incubation in thepresence of 10 mmol/L LiCl in the absence and presence of100 nmol/L (+)-niguldipine. Data are shown as percentinhibition by (+)-niguldipine and reflect the relative contri-bution of ait-adrenergic receptors to the inositol phosphateresponse. Data are mean±SEM of 8 to 10 experiments ineach age group.

hypertensive age groups were pooled, an alteration of atleast 15% would have been detected as statisticallysignificant, with an a of 0.05 and /3 of 0.05.

Although the ratio of renal a1A- to a1B-adrenergicreceptor number remains unchanged in SHR comparedwith WKY rats,1718 the different pathways used by bothreceptor subtypes to couple to inositol phosphate for-mation in rat kidney27 opened the possibility that thecontribution of both subtypes to norepinephrine-stimu-lated inositol phosphate formation might be altered inSHR. To test whether differential regulations of alA-and a,B-adrenergic responsiveness might offset eachother, we determined the inhibition of norepinephrine-stimulated inositol phosphate formation by 100 nmol/Lof the highly a,A-selective antagonist (-l-)-niguldipine28

in 3- and 28-week-old WKY rats and SHR. This con-centration of (+)-niguldipine was chosen because itoccupies more than 92% of a1A- and less than 2% ofa1B-adrenergic receptors as calculated based on itsaffinities1718 after correction for the presence of 100jtmol/L norepinephrine based on its potency in thissystem (EQo, 3.1 /xM25). (+)-Niguldipine inhibitednorepinephrine-stimulated inositol phosphate forma-tion to a similar extent in 3- and 28-week-old WKY ratsand SHR (Fig 3). Moreover, there was no significantdifference between WKY rats and SHR with regard tostimulation of inositol phosphate formation by thesomewhat a1A-selective agonist methoxamine in 6- and8-week-old rats (data not shown).

arAdrenergic ReceptorsThe affinity of [3H]rauwolscine for rat renal a2-

adrenergic receptors ranged between 3.3 and 4.3nmol/L and did not vary significantly between strains orage groups (data not shown). The density of a2-adren-ergic receptors in young and adolescent WKY ratsranged between 42 and 49 fmol/mg protein and signif-icantly increased in adult WKY rats (72±5 fmol/mgprotein, P<.05, Fig 4). In contrast, renal a2-adrenergic



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3 6 9 28age. weeks

FIG 4. Line graph shows development of aradrenergicreceptor density determined in renal membranes of Wistar-Kyoto (WKY) rats (o) and spontaneously hypertensive rats(SHR) (•) from [3H]rauwolseine saturation binding experi-ments. Data are mean±SEM of eight rats per age group.*?<.O5, **?<.O1 vs age-matched WKY rats.

receptor density gradually increased in SHR from 55 ±8fmol/mg protein at 3 weeks to 90±5 fmol/mg protein at28 weeks (P<.05 for 28 vs 3 or 6 weeks, Fig 4). In allhypertensive age groups, the renal a2-adrenergic recep-tor density was significantly greater in SHR than inage-matched WKY rats, whereas we did not detect asignificant difference for prehypertensive SHR (Fig 4).

To determine the functional relevance of increaseda2-adrenergic receptors in SHR kidney, we tried tomeasure inhibition of cAMP accumulation in renalcortical slices from WKY rats and SHR. Regardless ofwhether forskolin (10 /xmol/L) or parathyroid hormone(100 nmol/L) was used to stimulate cAMP formation,we were unable to obtain consistent inhibition of cAMPformation by the a2-adrenergic agonists epinephrine,a-methyl-norepinephrine, bromoxidine, moxonidine, orazepexole (all in the presence of 10 ^.mol/L [±]-pro-pranolol to block /3-adrenergic receptors, data notshown). In contrast, epinephrine (100 /xmol/L in thepresence of 10 /xmol/L [±]-propranolol) consistentlyenhanced cAMP formation in the presence of 10/xmol/L forskolin, which might indicate the presence ofatypical (propranolol-insensitive) "/33-like" adrenergicreceptors in rat kidney. This stimulatory effect of epi-nephrine was similar in WKY rats and SHR at 6 and 8weeks of age (data not shown) and was not furtherinvestigated in the present study.

^-Adrenergic Receptors[123I]Iodocyanopindolol bound to rat renal /3-adrener-

gic receptors with affinities ranging between 10 and 40pmol/L that did not vary significantly between strains orage groups (data not shown). /3-Adrenergic receptordensity ranged from 8 to 13 fmol/mg protein in WKYkidneys and from 12 to 16 fmol/mg protein in SHRkidneys (Fig 5). In all hypertensive age groups, it washigher in SHR than in WKY rats, although this differ-ence did not reach statistical significance in the 8-week-old group (Fig 5).

To test the functional relevance of /3-adrenergic re-ceptor elevations in hypertensive SHR, we investigatedcAMP accumulation in renal cortical slices. BasalcAMP accumulation was approximately 20 pmol per 10minutes per 100 mg wet weight. This value was similar inall age groups and did not significantly differ between

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rote

:

s

15-

10-

5.

0-

i

o WKY• SHR

n = 6

tX

. - " " * • • • - -.1

I

*

A

0 3 6 9 28age, weeks

FlG 5. Line graph shows development of ^-adrenergic re-ceptor density determined in renal membranes of Wistar-Kyoto (WKY) rats (o) and spontaneously hypertensive rats(SHR) (•) from [125I]iodocyanopindolol saturation bindingexperiments. Data are mean±SEM of eight rats per agegroup. *P<.05 vs age-matched WKY rats.

strains (Fig 6). Isoproterenol (100 ^mol/L) increasedcAMP accumulation by approximately 40%; this en-hancement was similar in all age groups and in WKYrats and SHR (Fig 6). Power calculations demonstratedthat with the given sample size and variance our studywould have detected alterations of 25% to 50% inisoproterenol-stimulated cAMP accumulation as statis-tically significant when individual age groups were ana-lyzed. When pooled data from all hypertensive agegroups were analyzed, our study would have detected a25% alteration as statistically significant, with an a of0.05 and /3 of 0.2.

Parathyroid hormone (100 nmol/L) increased cAMPformation in 3- and 28-week-old WKY rats and in3-week-old SHR by approximately 20%; in 28-week-oldSHR, however, parathyroid hormone failed to signifi-cantly stimulate cAMP formation (Table 2). Forskolin(10 /xmol/L) increased cAMP accumulation much moreefficiently (by approximately 350%) than the hormonereceptor agonists, but forskolin-stimulated cAMP for-mation did not differ significantly among age groups orstrains (Fig 6).

DiscussionA rich sympathetic innervation, a large complement

of adrenergic receptors, and a huge body of biochemicaland physiological data demonstrate the important roleof the sympathoadrenal system in the regulation ofrenal function. Both the sympathetic nervous systemand the kidney have frequently been implied in theoriesregarding the development of elevated blood pressurein SHR or hypertensive patients,'•21-29-30 so many labo-ratories have studied possible alterations of renal a- and/3-adrenergic receptors in SHR and other rat models ofhypertension.1314 The evolving consensus of such stud-ies is that renal a,-, a2-, and /3-adrenergic receptornumbers increase in SHR with established hyperten-sion. Elevated a2- and ^-adrenergic receptors werefound in almost all studies; the increase in renal ar

adrenergic receptors is somewhat less consistent, butreduced renal a,-adrenergic receptors were not re-ported. The elevations of renal ar and a2-adrenergicreceptors are specific for SHR and possibly some othermodels of genetic hypertension, because unchanged oreven reduced a,- and a2-adrenergic receptors are found



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ban1

3 6 9age, weeks

FIG 6. Line graphs show development of basal and isopro-terenol- and forskolin-stimulated cyclic AMP (cAMP) forma-tion in Wistar-Kyoto (WKY) rat and spontaneously hyperten-sive rat (SHR) kidney. Cyclic AMP accumulation wasdetermined in renal slices of WKY rats (O) and SHR (•)during a 10-minute incubation in the absence of agonists (toppanel) or in the presence of either 100 fimol/L isoproterenol(isoprenaline, middle panel) or 20 pmol/L forskolin (bottompanel). Data are mean±SEM of 8 to 10 rats per age group.

in rat models with acquired hypertension.1314 Despitethis specificity, increased renal a,-, a2-adrenergic recep-tors, or both do not appear to be directly involved in thepathogenesis of blood pressure elevations in the SHR.17

Renal /3-adrenergic receptors are elevated in variousforms of genetic and acquired hypertension, and themagnitude of the receptor increase appears to correlate

TABLE 2. ParathyroidAMP Accumulation

Age

3 Weeks (n=8)28 Weeks (n = 10)

Hormone-Stimulated Cyclic

WKY

28±1325±5

SHR

20+13-10±6'

WKY, Wistar-Kyoto rats; SHR, spontaneously hypertensiverats. Data are mean±SEM of 100 nmol/L parathyroid hormone-stimulated cyclic AMP accumulation expressed as percent overbasal (shown in Fig 6).

*P<M vs age-matched WKY rats.

with that of the blood pressure elevation.23 Thus, theregulation of renal /3-adrenergic receptors appears to beclosely linked to that of blood pressure but does notappear to be specific for genetic hypertension.

It is now clear that the three major classes of adren-ergic receptors, ie, au a2, and /3, all exist in multiplesubtypes, and the possibility exists that the subtypesundergo differential regulation in hypertension. Ratkidney expresses both alA- and aiB-adrenergic recep-tors, but their ratio remains unchanged in SHR.17'18

Ligands such as [3H]rauwolscine or [3H]yohimbine de-tect only a2B-adrenergic receptors in rat kidney,15 butNorthern blot analysis31 and binding with pH]RX821002 as the ligand16 suggest that a minor populationof a2A-adrenergic receptors also exists; studies on renala2-adrenergic receptors in hypertension, however, havealways used rauwolscine oryohimbine as the ligand. Ratkidney expresses /3 r and ^-adrenergic receptors, buttheir ratio remains constant during the development ofSHR22 and in various other models of hypertension.23

Thus, it can be concluded on the basis of previouslypublished data that alterations in the number of renal«IA-, "IB", «2B-, /3 r , or ft-adrenergic receptors in estab-lished hypertension are unlikely to have a causativeeffect on blood pressure. Therefore, the present studyhas investigated two questions regarding a possible roleof adrenergic receptor alterations for the pathophysiol-ogy of SHR: What is the relation between increasedadrenergic receptors and blood pressure over time?Does the increased adrenergic receptor number trans-late into altered receptor responsiveness?

Few previous studies have investigated renal adren-ergic receptors in very young SHR. Sanchez et al20 havecompared the ontogenesis of renal ar and a2-adrener-gic receptors in WKY rats and SHR and detectedsignificant increases in both receptors in SHR at 5 and8 weeks and in all older age groups but not at 1, 4, 6, or7 weeks of age. Although systolic blood pressure washigher in SHR than in WKY rats at all age groups inthat study, this difference reached statistical significanceonly starting at 8 weeks with the given sample size.Fukuda et al19 detected significantly elevated renala2-adrenergic receptors in 4-week-old SHR but did notreport blood pressure values for these animals. Thepresent study failed to detect significant alterations of«]- or a2-adrenergic receptors in 3-week-old SHR. Wehave previously reported significant elevations (29%) ofrenal /3-adrenergic receptors in 5- to 6-week-old SHR22;although the present study confirmed this finding in6-week-old SHR, it did not detect such increases in3-week-old SHR. Thus, increased renal adrenergic re-ceptors are not consistently found in very young SHR.Because increases in peripheral resistance have beendetected even in very early ages of the SHR with the useof appropriately sensitive techniques and sample sizes,we conclude that increased renal adrenergic receptorsare unlikely to precede blood pressure elevations inSHR; if anything, they accompany such elevations andmay be part of the response to a small but physiologi-cally important blood pressure increase, sodium reten-tion, or both.

Because receptor number and responsiveness do notalways correlate, a more important question is whetherincreased adrenergic receptor numbers translate intoincreased functional responsiveness in SHR kidney. The



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first step in renal a,-adrenergic receptor signaling is theformation of inositol phosphates.32 Despite significantlyincreased a,-adrenergic receptor number, we failed todetect enhanced norepinephrine-stimulated inositolphosphate formation. It could be argued that the max-imal inositol phosphate formation was unchanged de-spite receptor increases, because something other thanreceptor number is limiting inositol phosphate forma-tion. This is unlikely to be the case, because the inositolphosphate response to submaximally effective norepi-nephrine concentrations was also unchanged in ourstudy. Moreover, we have previously demonstrated inkidneys from adult SHR that the ECJO for norepineph-rine-stimulated inositol phosphate formation was notaltered, whereas the maximal stimulatory effect wasreduced.33 Reduced inositol phosphate formation ac-companied by unchanged apadrenergic receptor num-bers has also been found by other investigators.34

Whereas these three studies differ with regard to thepresence of increased apadrenergic receptor numberand reduced inositol phosphate formation, they alldemonstrate that the amount of inositol phosphatesformed per a,-adrenergic receptor is reduced in hyper-tensive SHR; thus, they agree that renal a,-adrenergicreceptor function is not enhanced in SHR.

Because both atA- and a1B-adrenergic receptors cou-ple to inositol phosphate formation in rat kidney25-35 butuse different pathways to do so,27 it could be possiblethat opposite changes in the responsiveness of bothsubtypes offset one another. We have previously dem-onstrated that the ratio of renal a1A- to a,B-adrenergicreceptors remains unchanged in SHR.1718 The presentstudy demonstrates that the same is true for the contri-bution of both subtypes to norepinephrine-stimulatedinositol phosphate formation in 3- and 28-week-oldSHR. Moreover, the stimulatory effects of the a,A-selective agonist methoxamine36 were also unchanged in6- and 8-week-old SHR (data not shown).

Thus, increased or unchanged renal a,-adrenergicreceptors in SHR translate into unchanged or evendecreased signaling of these receptors. These data givefurther evidence against the previous hypothesis21 thatalterations in number or coupling efficiency of renala,-adrenergic receptors are important for the develop-ment of hypertension in the SHR. This idea is furthersupported by recent data demonstrating that the reduc-tions in urine formation and fractional and absolutesodium excretion caused by renal nerve stimulation andmediated by a,-adrenergic receptors9 are similar inextent in WKY rats and SHR.37 Moreover, continuoustreatment with the a,-adrenergic antagonist terazosinhas been demonstrated not to prevent the developmentof hypertension in SHR.38 On the other hand, increasedafferent and efferent arteriolar vasoconstrictive respon-siveness to a,-adrenergic stimulation has recently beenreported,6 but in light of the above findings, we suggestthat this is related to structural vessel alterations (seebelow) rather than to a specific alteration in a^-adren-ergic responsiveness.

Studies in SHR aorta have suggested that the basalactivity of one or more phospholipase C isozymes maybe increased in SHR.14 Studies in resistance vessels39

and the present and previous data33-34 in kidney do notconfirm an enhanced basal inositol phosphate formationand thus indicate a tissue-specific regulation of phos-

pholipase C activity in SHR. Multiple allelic polymor-phisms of phospholipase C isozyme genes have beendetected in SHR compared with WKY rats,4041 but onlyone polymorphism in the phospholipase C-S gene ap-pears to be specific for genetic forms of hypertension.Although this polymorphism encodes a point mutationin the catalytic domain of the enzyme,41 it is not clearwhether this yields altered enzymatic activity. In fact, arecent breeding study demonstrated that the SHR alleleof phospholipase C-5 cosegregates with low rather thanwith high blood pressure in an F2 population ofWKY x SHR hybrid rats.42 Because increased wallstress, eg, caused by enhanced pressure or by osmoticfactors, can also activate phospholipase C,43 we there-fore speculate that such effects rather than geneticfactors could explain the enhanced basal inositol phos-phate formation sometimes seen in some SHR vessels.

An elevation of renal a2-adrenergic receptors in SHRis one of the most consistent findings in studies of thesympathoadrenal system in hypertension.1314 This ap-pears to be important because a2-adrenergic receptorsoutnumber all other forms of adrenergic receptors in ratkidney. However, surprisingly little is known regardingthe functional role of renal a2-adrenergic receptors inthe rat. They have been demonstrated to inhibit reninrelease,4445 to functionally antagonke the actions ofvasopressin in collecting ducts,4* and to promote intra-renal vasoconstriction in conscious rats,5 but the major-ity of rat renal a2-adrenergic receptors are located inproximal tubules.47 Tubular a2-adrenergic receptorscouple to enhanced sodium uptake4849 and oxygenconsumption,50 but it remains unclear how this relatesto overall renal sodium excretion. Because ^-adrener-gic receptors couple to inhibition of cAMP formation inall known model systems,51 we tried to study theirfunction at this level of the signaling pathway. Previousstudies have detected inhibition of cAMP formation bya2-adrenergic agonists in some renal preparations butnot in others.44 Unfortunately, we were not able todetect inhibition of cAMP formation in renal corticalslices in normotensive or hypertensive rats despite theuse of various chemically distinct a2-adrenergic agonists.Because no other studies have reported on a2-adrener-gic function in SHR kidney, it remains unclear whetherincreased a2-adrenergic receptor numbers are accompa-nied by enhanced responsiveness despite the huge num-ber of rat renal a2-adrenergic receptors.

Finally, we have investigated the coupling of renal/3-adrenergic receptors to cAMP formation in the SHR.Previous data have reported enhanced,52 unchanged,53

and reduced54 cAMP formation in kidneys of hyperten-sive rats after stimulation by isoproterenol; our studydid not detect altered cAMP formation. An unalteredcAMP formation despite elevated renal /J-adrenergicreceptors in this and various previous studies13 indicatesthe existence of a disturbed effector coupling of /3-ad-renergic receptors and perhaps other G,-like hormonereceptors. This idea is supported by the desensitizedcAMP formation in response to parathyroid hormone inhypertensive SHR in this and a previous study,53 al-though interpretation of the parathyroid hormone datais hampered by lack of data regarding a possible alter-ation in receptors for this hormone in SHR kidney andby lack of knowledge regarding a possible colocalizationof /3-adrenergic receptors and parathyroid hormone



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receptors on the same renal cells. On the other hand,cAMP formation in response to the direct adenylatecyclase stimulator forskolin55 remained unchanged inour study, indicating that the defect is located some-where between the /3-adrenergic receptors and adenyl-ate cyclase, possibly at the level of G proteins. However,an alternative hypothesis would be that the increasedrenal /3-adrenergic receptor number translates into en-hanced function via increased sensitivity to submaxi-mally effective agonist concentrations. The presentstudy cannot rule out this possibility, and further studieswill be necessary to test this hypothesis directly.

Taken together, the data of our study do not supportthe idea that increased renal adrenergic receptor num-bers are accompanied by enhanced functional respon-siveness. It should be noted, however, that functionalresponsiveness could theoretically occur via signalingpathways not investigated here, especially for a2-adren-ergic receptors, but because such pathways have notbeen described in rat kidney, this alternate hypothesisremains purely speculative. Therefore, quantitative dataon the effects of adrenergic agonists on renal vascular ortubular function or both are required to settle thequestion of whether renal adrenergic receptor respon-siveness is altered in SHR and what pathophysiologicalimportance that might have.

More generally, the question remains of why thenumber of ar and /3-adrenergic receptors increases inSHR kidney whereas the function apparently does not.Three explanations are possible: Function could declineto compensate for nonphysiological increases in num-ber; number could increase to compensate for impairedfunction; or the two processes could be unrelated.Moreover, different explanations might exist for ar and/3-adrenergic receptors, because the former increasespecifically in SHR, whereas the latter increase in manymodels of hypertension. Because reduced aradrenergicfunction was detected in SHR, in which receptor num-ber remained unchanged,34 a compensatory reductionin a,-adrenergic responsiveness is unlikely. A direct testof the other possibilities awaits further experimentaldata, specifically, quantitation of the G protein subunitsand effector enzymes.

In summary, we have demonstrated that a,-, a2-, and/3-adrenergic receptor numbers are increased in SHRkidney and that these increases do not precede bloodpressure elevations. Moreover, increased receptors arenot accompanied by enhanced functional responsive-ness or by an altered relative involvement of a1A- anda,B-adrenergic receptors in norepinephrine-stimulatedinositol phosphate formation. Thus, our data providefurther evidence that increased renal adrenergic recep-tor numbers are unlikely to contribute to the pathophys-iology of blood pressure elevations in SHR.

AcknowledgmentsThis work was supported by the Deutsche Forschungsge-

meinschaft (Ph 43/1-2). The help of Dr Dietmar Buttner withthe animal breeding is gratefully acknowledged.

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M C Michel, F Siepmann, R Büscher, T Philipp and O E Broddealpha 1-, alpha 2-, and beta-adrenergic receptors and their signaling.

Ontogenesis of sympathetic responsiveness in spontaneously hypertensive rats. I. Renal

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