290

Biochimica el Biol~'sica Acta. 1136(1992)290-296 © 1992F_lsevictSciencePulflishetsB.V.All fightsreserved0167-4889/92/$05.00

B3AMCR "3-238

Guanine nucleotide regulatory protein levels and function in spontaneously hypertensive rat vascular smith-muscle cells C a t h e r i n e J. Clark a, G r a e m e Milligan b, Alastair R. McLellan a a n d J o h n M . C Connell ~

s M~r

a MRC Blood PressureUnit, WesternInfnnm3; Gkttgow (UK) and Ftzarm~cologyGroup, Department of Biochemistr); Unirodly of Glazgo~; Glasgow(UK)

(Received12March1992)

Keywards: Adenylytcyclase:G protein;Spontaneouslyhypertensiverat;(Ralvascularsmooth-musclecell) We compared G-protein levelsand function in membranes from vascular srnooth-musclecells (VSMC) derived from mesenteric arteries from SHR, WKY and Wistar rats. Basal adenylylcyclaseactivilywas significantlyreduced in SHR membranescompared with W'tstar, but was similar to WKY. Isopmtereno| stinmlation(20-4 M) was signil-u:antb"lower in SHR membranescompared to WKY, but was similarto that in Wislar, whichwas also significantlylowerthan WKY. Forskolin(10-4 M} and NaF (10 -2 M), resulted in a higher stimulatoryresponse in SHR membranes. Biphasic effects of GTP on isoproterenol-stimulatedmembranes demonstrated unaltered G, function in SHR membranes. No significant differences were seen in the levels of G~a (44- and 42-kDa forms), G~2a and ~e 13-subunitin immunobIotlingstudies of the membranes.;unounts of Goa/GHa and Gi3a were also unchanged, in conclusion,there are differences in adenylylcyclaseresponses in SHR VSMC membraneswhich are not a consequenceof altered levels of G-proteins, but may reflect genetic difference~rather than effects of hypertension.

Introduction Guanine nucleotide regulatory proteins (G proteins) are ke~vcomponents in cellular signalling processes [1]; they transduce cellular signals between occupied receptors and second messenger generating systems, such as adenylyl cyclase [2-7] and ph~p~olipase C (PLC) [8]. Generation of ojclic AMP (cAMP) by adenylyl cyclase is under dual control from stimulatory (G~) and inh~itory (G~) G proteins, while generation of diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP 3) by PLC in response to receptor activation is mediated by a G protein(s) genetically termed Gp. Abnormalities cf adenyly! cyclase and PLC activa-

Correspondence to: IM.C ConnelL MRC Blood Pressure Unit, WesternInfirmar/~Glasgow,GI 1 6NT,UK. Abbreviations: Ab, antibod5 ACA, adenylylcyclaseactivity;,DG. diacytglycerot:G protein, guanine nucleotide regulatoryero~ein: HRP, horseradishpero.~dase:IgG. immunoglobulinG; IP~,inosdol 1,4~fi-tfisphosphatc;PLC, pho~hoiip~,seC; RMM, rat and mouse meintenancediet: SDS-PAGE.sodiumdodecylsulphaYepoiyacrsla.~ide gel clectrophoresis:S~E.,slandarderror of 1he mean;S'.-IR, .~pontaneouslyhypetlensiverat; ~'7"uS,Tris.bufferedsaline; VSMC, '~,ascularsnmoth-rnusc|ccells;WKY,Wislar-Kyoto.

tion have been reported in vascular (including vascular smooth-muscle of resistance vessels) [9-18] and nonvascular tissues [19-21] from the spontaneottsly hypertensive rat (SHR). in particular, reduced arterial relaxation in response to/3-adrenoceptor stimulation udng isolated femoral artery material from the SHR has been observed [22], which parallels reduced adenylyl cyclase activity (ACA) response to this and other s t ~ ulatory ligands. The mechanism underlying this reduced responsiveness has not been examined in detail. Similar changes in ACA have been noted in human non-insulin-dependent diabetes meilitus [23], and in animal models of diabetes which are insulin-resistant [24]. in these studies~ major changes in actMty and expression of both stimulatory and inlu'bitory G proteins have been reported which contribute to the observed changes in ACA. Human essential hypertension, and its animal model, the SHR, are both insulin-resistant states with hyperinsulinaemia, in this way, they resemble human diabetes and animal models of the ~ndition, and we wished to examine in this study whether abnormal ACA responses could be observed in cultured vascular tissue from the SHR and to test the hypothesis that changes in ACA might be a consequenc,~. of altered activity or expression of G s or G i spe.cies.

291 Materials and Methods

Materials All chemicals were supplied ny Sigma (UK), with the exception of gaanosine 5'-triphosphate (GTP), creatine kinase and creatine phosphate, which were supplied by Boehringer Mannheim UK (Diagnosti~ & Bioehemieals). All tissue culture products were supplied by Gibco-BRL with the exception of Lglutamine, which was supplied by ICN Flow Laboratories. The radioisotopes ([a-32P] adenosine 5'-ttiphosphate, [8-3H] adenosine Y:5'-cyclie monophosphate and anti-rabbit, whole Ab from donkey [t2Sl]) were supplied by Amersham International. Horseradish peroxidase (HRP)-iabelled anti-rabbit lgG (donkey polydonal) was supplied by the Scottish Antibody Production Unit, Law Hospital, Carluke.

Methods Rats. SHR, WK¥ and Wistar rats were supplied by Harlan Olac. Mesenterie arteries were excised from male rats that were 10 or 11 weeks old. Systolic blood pressures (tail cuff method [25]) and weights of the rats were measured before killing by a blow to the head: SHR 202 + 9 mmHg; WKY 130 + 6; Wistar 137 + 4 (P = 0.001) WKY vs. SHR and Wistar vs. SHR, SHR 205+5 g; WKY 212+8; Wistar 270+2 (P=0.001) SHR vs. Wistar and WKY vs. Wistar, respectively (significance assessed by t-test analysis). The rats were fed on rat and mouse maintenance diet (RMM), allowed free access to H20 and kept under constant light conditions. For each VSMC preparation, 6 rats of each strain were killed. Smooth.musde cell preparation. Rat mesenteries were excised under steri!e conditions, deaned and transferred to a mixture (10 ml) containing collagenase (1.25 mg/ml), elastase (0.25 mg/ml) and soybean trypsin inhibitor (0.05 mg/ml). After incubation at 3"PC for 5 rain any remaining fat, adventitia and endothelium were removed. The arteries were cut into small pieces (1-2 ram), transferred to fresh enzyme mixture and incubated until a single-cell suspension was obtained. The cells ~a,ere gro.~.~ in D~beeco's modified Eagle's medium containing 10% (v/v) foetal calf serum, 10% (v/v) horse serum, L-glutamine (2 mmol/l), penicillin (100 U/ml) and streptomycin (100 ~g/ml) at 3"PC in a humidii'i~d .,,o¢"CO2 to 95% zir atmosphere. Vascular smooth.muscle cells (VSMC) were used in these experiments at passage 4 or 5. Plasma membranepreparation. VSMC were h~mogenized in ice-cold 10-raM Tris/1 raM EDTA (pH 7.5) using a "Polytron' at setting '5' for 2 x 12 s. The homogenate was centrifuged at 2000 x g for 10 rain at 4"C. The resulting supernatant was centrifuged at 110000 x g for 1 h at 40C to obtain a crude plasmamembrane fraction which was resuspended in 10 mM

Tris-HCi (pH 7.5), aliquoted and stored at -70"(2. The protein concentration in the plasma-membrane preparation was measured colorimetrically using Peterson's modification [26] of Lowry's protocol [27] based on bovine serum albumin as standard. Adenylyl cyclase assay. G protein function in plasma-membranes was assessed by measuring ACA by the method of Salomon [28]. in this assay, the formation of [32p]cAMP from [a32p]ATP was measured in the presence of GTP and magnesium ions. The reaction mix (final volume of 50 ttl) was essentially that described by Sharma et al. [16] and included an ATPregenerating system. ACA was studied under basal conditions and in the presence of forskolin (10 '4 M), MnCt z (2 × 10-2 M), NaF (10 -2 M) and (-)-isoproterenol (10 -4 M). The concentrations of the various stimulatory agents were saturating with respect to their effects (from previous dose response curves (data not shown)). Dose-effect experiments for GTP on isoproterenol-stimulated ACA were also performed.

Sodium dodecyl sulphate polyacrylamide gel electrophorcsis (SDS-PAGE) and immunoblotting. The amount of plasma-membrane protein to be loaded to achieve optimal blots was determined from plots of protein concentration against cpm obtained by excision and counting of labelled bands constructed for each antibody used. Protein molecular-weight sta.ndards (molecular-weight range 14300-200000) were run in parallel with the samples under study. In addition, a 'standard' human platelet plasma-membrane prepara. tion was included on all blots to facilitate comparison of data frc,n different blots. The plasma-membrane protein was acid-precipitated with triehloroacetic acid (24% (w/v)), solubilized in Laemmli buffer 129] and then boiled for 3 rain [30]. The protein was resolved by SDS-PAGE on 10% acrylamide/0.27% N,N'-metbylenebisac 7¢lamide(v/v) gels. lmmunoblotting with specific antit'eptide antisera was then performed as described in detail previously [24,31]. Briefly, the resolved prote:n w:,s electrophoretically transferred (blotted) to nitlocel.~dose paper [32] which was then blocked for 2 h at 37°C in 5% (w/v) dried skimmed milk (Marvel) in Tri,:-buffered saline (TBS). After thorough washing with ,!istilled water, the blot was incubated at 37°(2 overnigi~t with the appropriate antipeptide antiserum. After at.propriate washing, the blot was incubated at 37°C for 2 h with horseradish peroxidase (HRP)-labelled anti-rabbit IgG (donkey polyclonal). The HRP-labelled bands were visualized by development with a 0.025% (w/v) solu'tion of ortho-dianisidine. After blotting, ~'he gel was retained and residual proteie, not transferred electropheretically, was visualized by staining with a solu+:'.,n of Coomassie brilliant blue G. The bands on the blots were quantified by incubation at 37°C for 2 h with

292 TABLE. ! Specificities of anti-G protein antisera Antiserum

Peptide employed

Co~¢spoading G-prutein

Antiserum identifies

sequence

CSI SG2 I3C BN3 CO2

RMIILRQY£LL KENLKDCGLF KNNLKECGLY MSELDOLRQE QLNLKEYNLV

G~a 385-394 Transd,.gin a 3~1-350 Gi3a 345-354 ~61(I-!0) Gqa 351-360 Gnu 350-359

t251-1abelled anti-rabbit lgG. The bands were subsequently cut out and counted using a NE 1612 gamma counter (Nuclear Enterprises, EMI, UK). Antisera. The antisera were generated in New Zealand White rabbits using a con.~ugate of the synthetic peptide and Keyhole Limpet haemo~.5'anin as detailed by Goldsmith et ai. [33]. The specificity of the antisera in recognising G~a and G~a species has been fully d , - ~ d elsewhere [34]. Gila and Gi2a have identica. C-terminal decapeptides and thus antiserum SG2 identifies each of these polypeptides eqtmlly (Table I). Trausducin a is also identified by antiserum SG2, but as this G protein is restricted in distribution to photoreceptor containing tissues, then the antiserum can be used as a probe for G~I or Gt2 in all other locations. Rat VSMC pla.qna-membranes express high levels of G~2, but do not express detectable levels of G~I (data not shown). Statistical a n , ~ . The adenylyl cyclase assay results were analyzed by the Mann-Whitney U test for noni:.arametric data. Results

G, Transducin. Gil Gi2 Gi3 Gq, G .

resulted in an inhibition of ACA in ~HR, WKY and Wisgar memb_~nes.A high concentration of Mn2+ ions uncouples adenylyl cyclase from its regulatory G proteins [35]. In these VSMC, there appears to be a much greater measurable input from G~ than G i, hence uncoupling these G proteins would probably tend towards inhibition of ACA. A similar trend in activities was seen when measured in the presence of forskolin 110 -4 M), which activates the catal~ic unit of adenylyi cyclase directly (Fig. 1). In view of these inherent differences in enzyme activity, the responses to the variow. agents studied were expressed as % change over basal ~measured in the presence of Mn2+ ions as this gives the closest approximation to the true basal activity [35]). Fig. 2 shows the % change in ACA over basal in SHR, Wistar and WKY membranes after the addition of forskolin 110 -4 M), NaF (10 -2 M) and (-)-isoproterenol 110 -4 M). Use of the tS-adrenergic agonist, isoproterenol, allowed measurement of receptor-dependent stimulation of ACA. there was significantly less stimulation in SHR and Wistar membranes compared with WKY ( P = 0.04 for both). However, t~.e stimulatory response caused by isoproterenol was simi-

Basal ACA (whether measured in presence/absence of Mn2+ ions) was similar in SHR and WKY, but both were significantly lower than Wistar membranes (P = 0.003, SHR vs. Wistar: P= 0.002, WKY vs. Wistar) (Table !! and Fig. 1). Thc addition of Mn2~ ions

I

TABLE !1 Basal adenylfl cydase actirilies in the presence and abseqce of Mn 2 + ions in SHR. BrL~tarand WKY VSMC merabranes

oE

Basal adenylyl q/clase acGcity(pmol cAMP/15 min per mg) was .,neasured in the presence and absence of 20 raM MnCI2. Data represent mean+S.IL for eight experiments using different membt.me preparatior'..s.

~g

SHR

WISTAR

WKY

1220+!21 *

2880_+454

J203_+56

1024±131*

19fb-+li9

998+_86

¢ o

10000

0 InoMn2÷ ions) Basal (+ Mn~+ ions)

• P < 0,005 (statistical analyfisof SHR ar,d Wietar results).

~

Basal Fig. 1. Basal a~n34yl ojctase activity(in the absence of Mn2+ ions) and after the addition of forskolin (10 -4 M) in SHIL Wistar and WKY VSMC membranes. Data are mean_+S.E, for eight experiments using different membrane preparations.

293 .2000.

p,,0.015

z~looo g

p~o.os

e~

F'-I

p=o.o4

I--'--)

0@

1 Fig. 2. ~: Ch.ange in ACA over baLSa!(in the presence of Mnz* ions) in SHR. Wistar and WKY VSMC membranes in the presence of forskolin (10 -'s ML NaF (10 -2 M)and ( - bisopro[erenol (10 -4 M). Data are mean±S.E, for eight experiments using different membrane preparations.

2

3

4

5

6

7

Fig. 4. Nitrocellulose membrane probed with G~a-specific Ab (CSI). 50 p.g of membrane protein was loaded in each lane. Te,~ forms of G,a are apparent (44 and 42 kDaL with the 44 kDa form being predominant in VSMC membranes. I and 2, Wistar membranes: 3 and 4. SHR membranes: 5 and 6, WKY membranes: and 7. human platelet membranes.

lar in SHR and Wistar membranes. To examine whether altered regulation of ACA in SHR was only apparent at the/3-adrenoceptor level o '~ the adenylyi cyclase system, or whether abnormalities were present at other levels of the signalling pathway, the response of ACA to forskolin and NaF was studied. There was a significantly greater stimulatory response to forskolin in SHR membranes compared with WKY (P = 0.015). However, although forskolin generated a greater stimnlatory response in SHR membranes compared with Wistar, the difference waz not significant. Another receptor-independent stimulatory agent, NaF, caused greater stimulation in SHR membranes compared with both Wistar and WKY, but the difference in stimulatory response was only significant with the former control membranes (P--0.05). When the data were

10D0.

~

•

sHn

1

T

expressed as % change from basal (no Mn z+ ions) a similar trend was observed. G, function in SHR, Wistar and WKY membranes was dem¢nstrated by the biphasic effects of GTP on isop~oterenol-stimulated ACA {Fig. 3). These GTP dose-effect experiments showed that activation of G s and Gi occurred over similar concentration ranges of GTP in SHR, WKY and Wistar membranes, indicating no difference in the function of G i in membranes from the three strains. However, although the GTP response in SHR and WKY membranes was similar, it was significantly reduced in comparison to the response measured in Wistar membranes.

._

Wh'lar

800.

Gi_2oc(4OkDa)

i! ° o

1 0

--41

NO GT'P

•

. ,

-8

-6'

.....

-4

'

-2

'

JoglTC--P](M}l

Fig. 3. Dose,.eff¢.clexperiment for GTP on isoproterenol-stimulated ACA in SHR, Wislar and WKY VSMC membranes. ACA was monitored in the pecscncc of (-)-isoproterenol (10 -'1 M). Data are mcaa +S.E. fcr ,'ire (SHR and Wistar) and four (WKY) experiments.

2

3

q

5

6

7

Fig. 5. Nitrocellulose membrane probed with transducin a, G i l a ,

Gi2a-spccifi~ Ab (SG2). Since VSMC membranes do not express delectable levels of Gil and transaucin distribution is restricted to photoreeeplor-containing tissues, the antiserum SG2 will only identify Gi2 in VSMC membrane~, fi~ gg of memhra.o protein was toaded in ca:h lane. I and 2, Wislar membranes: 3 and 4, SHR membranes: 5 and 6, WKY membranes: and 7, human platelet membranes.

294 pro!eins (Fig. 7). There was no significant difference in the level of Gqa/Gnapresent in SHR, Wistar and WKY membranes.

(55kDa)

1

2

3

14

5

6

7

,"~g. ~. NitroceUulosemembraae probed ~itb/3-subunit-specific Ab (BN3). 50 #g of m ~ protein was [oaded in each lane. i and 2, W-~tar mcmbrartes: 3 and 4. SHR membranes; 5 and 6. WKY membranes; and 7. human platelet membranes.

Preliminary immunoblotting experiments identified G~a (two fon~; 44 and 42 kDa), Gi2a and G~3a in VSMC plasma-membranes, whereas Goa and Gila were absenL The levels of these different G protein a-subunits and the level of the//-subunit were quantified in SHIL Wistar and WKY VSMC membranes using t251-1abelled anti*rabbit lgG (Figs. 4, 5 and 6). There were no significant differences in the levels of the different G protein a-subunits and the level of the /3-subunit in SHIL Wistar and WKY membranes. The adenylyl cyclase e:t~.yme is not the only effector system linked to cell surface :eceptors by G proteins. The enzyme PLC, which alters the intracellular concentration of the second messengers I P 3 and DG, is linked te cell surface receptors by a G protein named Gp. Recent evidence has indicated that this polypeptide is a member of the Gq/G. subfamily of G proteins [36]. We quantified the level of the a-subunit for G q / G n in SttIL Wistar and WKY n~embranes using an antiserum raised against a synthetic peptide which represents the C-terminal decapeptide of these two G

(~2kDal

1

2

3

k

5

6

7

Fig. 7. N~rocellulo~emembrane probed with Gqo/Gl:a-specific Ab (CQ2). 25 ~g of membrane protein was loaded in each lane. I and 2, Wistar membranes; 3 and 4. SHR membranes; 5 and 6. ~'KY membranes; and 7, human platelet membranes.

~ ~ We chose to use the outbred Wistar strain as a normotensive control group in addition to WKY rats in our experiments in view of evidence that the WKY rats obtainable from most commercial suppliers display marked genetic heterogeneity [37]. We have shown differences in ACA in membranes from cultured VSMC comparing SHR with WKY and Wistar control strains. However, differences in ACA exist between the Wistar and WKY strains of rat, raising the possibili',' that genetic heterogeneity between the WKY and Wistar strains may be the cause for our observed differences in ACA and that this may not be related to raised blood pressure in the SHR. We observed that basal activity in our SHR and WKY membranes was significantly lower (+ Mn 2+ ions) than in W'tstar membranes. We confirmed in our membra, e:~ the .3bservation that SHR membranes are less responsive to isoprotercnol stimulation than WKY membran~es, a finding which has been previously reported L-. m_,~ze._~te,ievascular smooth-muscle [9], aortic smooth-mu.~le [9-13] and in myocardium [9,13-17]; however, Wistar membranes behaved similarly to SHR membranes. There are several explanations for the difference in responsiveness to isoproterenol including changes in ~-adrenoceptor number, changes in amounts of Gs and/or Gi2 and their coupling to receptor and adenylyl cyelase catalylie unit, and changes in amount or activity of adenylyl cyclase catalytic unit itself. Although not studied here, other investigators ha~e failed to agree on observed changes in//-adrenoceplor number a.-.~ affinity in SHR tissues [15,17]. Our findin~ arc consistent with altered//-adrenoceptor number/function in SHIL as the observed trends in adenylyl cyclase ,-,ctivation were not only eliminated, but actually reversed when we studied the effects of forskolin end fluoride ions which act/':ate a~enyly! ~,clase (via G~ in the case of fluoride ions) independently of receptors. It is poss~le that changes at the level of the G proteins " " and Gi2) which regulate the activity of adenylyl cyclase might account for the differences in ba~! and stimulated enzyme activity noted. Alterations ef G proteins have previously been reported in platelet membranes [19] and myocardial membranes [38] from SHR. Coquil and Brunelle reported decreased G~ function in platelets from SHR, while Murakami and colleagues observed a reduction in the functional activit~ of G~ in SHR cardiac membranes. However, we found no changes in the levels of G~ (both forms) and

295 the Gi sub-types, as measured by immunoblotting, in the membranes ~'rom the three strains studied. Furthennore, in assessing Gs function by direct .,:timulation with NaF, we saw an increased adenylv] cyclase response in the S H ~ It appears, therefore, that the reduced response in the SHR with respect to isoproterenol is not a consequence of reduced G, a-subunit activity, and appears to reflect an alteration in the linkage of/]-adrenergic recepto~ to the G protein. We d,emonstrated G t function by showing biphasic effects of GTP on isoproterenol-stimulated ACA. In this instance, low concentrations of GTP promote activation of adenylyl cyclase by stimulating the coupling of tile ~-adrenoceptor to G~, whereas high concentrations cause ialu'bition due to activation of G~ [39,40]. We observed biphasic effects due to GTP in SHR, Wistar and WKY VSMC membranes showing that G~ function was not different among the strains. The GTP response in SHR membranes was greatly reduced compared to Wistar, but was similar to the response observed in WKY membranes. This suggests an alteration in the coupling of ~-adrenoceptors to G~ in SHR membranes compared to Wistar. Differences in the amount and/or activity of adenylyl cyclase cat~lytie unit may contn'bute to our observed differences in ACA. This possibility is supported by our observation of the effect of forskolin, which activates adenylyl cyclase directly. When expressed as absolute activity or as fold stimulation (% change), SHR was more responsive to forskolin than WKY, suggesting that in the former strain there is a greater level/ responsiveness of adenylyl cyclase catalytic unit. In a study of another animal model which displays insulin resistance (the streptozotocin-diabetic rat) Bushfieid et al. [31] reported increa~d levels of the catalytic subunit of adenylyl cyclase in hepatocyte and liver plasma-membranes compared with control, although basal ACA was actually decreased in hepatocyte plasma-membranes from diabetic animals. We were unable to quantify the level of the catalytic subunit of adenyly~ cyclase in the three sets of membranes as an appropriate antibody was not available to us. The reduced ACA response to isoproterenoi is consistent with the observed reduction in arterial relaxation in response to ,8-adrenoceptor stimulation in the SHR: studies by Asano and colleagues [22] using iso!2~.:~ .¢~moral artery material from the SHR suggested, on the basis of indirect evidence, that ttJs was a consequence of reduced G~ function. In contrast, while we confirm, that adenylyl cyclase response to/~-adrenoceptor stimulation is red,zced, we have good evidence that G, function is normal in these membranes. The SHR is a model of ilypertension that is insulinresistant and hyperinsulinaemic, and it is relevant to compare our data with experimental findings from other disease states which are related to hypertension

and which also show this phenomenon. In a genetic model of insulin-resistant diabetes, Strassheim and colleagues [4I] showed that reduced ACA in adipose tissue in response to E-receptor stimulation was probably a consequence of reduced receptor number. In the obese Zucker rat, which is severely insulin-resistant and hypertensive, complex changes in the expression of G~ and activity of Gt species were present in hepatic tissue [42]. Finally, in platelets from humans with noninsulin-dependent diabetes mellitus, which is an insulin-resistant state with close epidemiological links to essential hypertension, ACA was reduced in response to forskolin, and Gi2 and G,3 levels were reduced [23]. It appears, therefore, that there is a range of alterations of ACA in these hypertensive and/or insulin-resistant states, with most studies showing reduced responses in a number of tissues. However, the changes in G protein function are variable, and do not appear to account for the observed alterations in ACA. in summary, we have identified differences in ACA among the three strains studied. The increased response of SHR adenylyl cyclase to agents which can activate this enzyme directly may reflect increased catalytic subunit activity. In contrast, the decreased ACA response to isoproterenol in th~ SHR may reflect reduced /]-adrenoceptor number/coupling, and not changes in the levels of G., and Gi2. While these changes may reflect hypertension and its genetic antecedent, the possibility is raised, by inclusion of the Wistar control group, that the genetic heterogeneity reported in SHR and WKY strains ~43,37] may contribute substantially to the differences which we and others have observed in studies of ACA and raise the possibility that these changes are not directly linked to the development of increased blood pressure. Acknowledgements We thank Dr. Fiona Lyall for her expert assistance with culturing rat vascular smooth.muscle cells and many helpful discussions. This work was funded by the British Heart Foundation (Grant No. 1890357). References I Milligan,G. (1989)Curt. OpinionCell. Biol. I, 190-200. 2 Gilman,A.G.(1987)Anne. Rev.Biochem.56. 615-049, 3 Bimbaumer,L., Codina, J.. Mattera, R, Cerione, R.A, Hildebrandt. J.D., Sunya,T., Ruins.FJ., Canon,MJ., i.~fkowilz,RJ. and Iyengar,R. (1985~Mol.AspectsCell. Regul.4. 131-182. 4 Houslay,M.D.(198~ TrendsBi~hem.Sci.9, 39-40. 5 Levitzki,A. (1990)Mol.Pharmacol.Cell. Regul.1, 1-14. 6 Gierschik. P. and ~akobs, K.H. 0990) Mol. Pharmacol.Cell. Rrgul. 1, 67-82. 7 Spiegel.A.M.(17~0)Mol. PharmacoLCell. Regul.1, 15-30. 8 Cockcroft,S. and Gomperts,B.D.(1985)Nalure314. 534-536.

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