Enhanced Expression of Gi-Protein Precedes the Development of Blood Pressure in Spontaneously Hypertensive Rats

J Mol Cell Cardiol 29, 1009–1022 (1997)

Enhanced Expression of Gi-Protein Precedes the Development of Blood Pressure in Spontaneously Hypertensive Rats∗ Jose´e Marcil, Christelle Thibault and Madhu B. Anand-Srivastava† Department of Physiology, Groupe de Recherche sur le Syste`me Nerveux Autonome, Faculty of Medicine, University of Montreal, Montre´al, Que´bec, Canada, H3C 3J7 (Received 24 May 1996, accepted in revised form 25 November 1996) J. M, C. T  M. B. A-S. Enhanced Expression of Gi Protein Precedes the Development of Blood Pressure in Spontaneously Hypertensive Rats. Journal of Molecular and Cellular Cardiology (1997) 29, 1009–1022. In the present studies we have investigated if the increased expression of Gia proteins reported earlier in heart and aorta from SHR (spontaneously hypertensive rats) is the cause or effect of hypertension. The SHRs at various ages of the development of blood pressure (3–5 days, 2 weeks, 4 weeks and 8 weeks) and their age-matched WKY were used for these studies. The expression of Gia-2 and Gia-3 (inhibitory guanine nucleotide regulatory protein and Gsa (stimulatory guanine nucleotide regulatory protein) at protein and mRNA level was determined by immunoblotting and Northern blotting technique using specific antibodies and cDNA probes. The SHR at early ages up to 2 weeks did not show any increase in blood pressure, however it started to go up from 4 weeks. The levels of Gia2 and Gia3 at protein and mRNA in heart from SHR were not different in 3–5 days old SHR as compared to WKY (Wistar-Kyoto rats), however, the expression of Gia-2 and Gia-3 protein and mRNA was significantly increased in 2 weeks and older SHR. The mRNA level of the catalytic subunit type V enzyme was significantly decreased in SHR 2 weeks and later ages as compared to their age-matched WKY. On the other hand, the expression of Gsa was not different in SHR as compared to WKY at all the ages studied. In addition, the oxotremorine and C-ANF4–23 (a ring deleted analog of atrial natriuretic factor) mediated inhibitions of adenylyl cyclase in hearts and aorta were also significantly enhanced in 2 weeks and older SHRs as compared to WKY rats, whereas, at younger age of SHR (3–5 days old), no change in the percent inhibition of adenylyl cyclase by C-ANF4–23 was observed and oxotremorine was unable to inhibit adenylyl cyclase activity. Furthermore, the basal enzyme activity and the stimulatory responses of isoproterenol, NECA (N-ethylcarboxamideadenosine), glucagon and forskolin on adenylyl cyclase were significantly decreased at all ages of SHR as compared to WKY. These results suggest that the increased expression of genes for Gia-2 and Gia-3, decreased expression of type V enzyme mRNA and decreased cAMP levels precedes the development of blood pressure and may participate in the pathogenesis of hypertension.  1997 Academic Press Limited K W: Gi-proteins; Adenylyl cyclase; Heart; Aorta; Spontaneously hypertensive rats.

Introduction The elevation of blood pressure in essential hypertension is due to a general increase in the resistance

of peripheral vessels (Ferrario and Page, 1978; Smith and Hutchins, 1979). A part of this heightened peripheral resistance has been attributed to structural changes in the vessels (Folkow

Please address all correspondence to: J. Marcil, Department of Physiology, Groupe de Recherche sur le Syste`me Nerveux Autonome, Faculty of Medicine, University of Montreal, Montre´al, Que´bec, Canada, H3C 3J7. ∗ This work was supported by grants from Quebec Heart Foundation and the Medical Research Council of Canada. † M.B.A-S is a recipient of the Medical Research Council Scientist Award from the Medical Research Council of Canada during the course of these studies.

0022–2828/97/031009+14 $25.00/0

mc960343

 1997 Academic Press Limited

1010

J Marcil et al.

et al., 1973), abnormalities in Ca2+ movements (Daniel, 1981), and aberrations in cyclic nucleotide metabolism (Amer, 1981). Adenylyl cyclase/cAMP signal transduction system has been implicated in the regulation of various physiological functions such as vascular tone and reactivity, cardiac functions, platelet functions etc. (Triner et al., 1972). The adenylyl cyclase system is composed of three components: receptor, catalytic subunit and stimulatory (Gs) and inhibitory (Gi) guanine nucleotide regulatory proteins. The guanine nucleotide regulatory proteins act as a transducers and, in the presence of guanine nucleotide, transmit the signal from the hormone-occupied receptor to the catalytic subunit (Iismaa and Shine, 1992; Fleming et al., 1992; Neer, 1995). G-proteins are heterotrimeric proteins composed of a, b and c subunits, and the specificity of G-proteins is attributed to a-subunits. The stimulation and inhibition of adenylyl cyclase by hormones are mediated through Gs and Gi proteins (Gilman, 1984). Molecular cloning has revealed four different forms of Gsa-resulting from the differential splicing of one gene (Murakami and Yasuda, 1986; Robishaw et al., 1986; Bray et al., 1996) and three distinct forms of Gia; Gia-1, Gia2 and Gia-3 encoded by three distinct genes (Itoh et al., 1986, 1988; Jones and Reed, 1987). All three forms of Gia (Gia1–3) have been shown to be implicated in adenylyl cyclase inhibition (Wong et al., 1992). Both the Ga and Gbc mediate G-protein signalling (Neer, 1995). Of the eight types of adenylyl cyclase that have been cloned and expressed (De Vivo and Iyengar, 1994) only two types, types V and VI have been identified in heart, aorta and brain (Katsushika et al., 1992; Premont et al., 1992). Adenylyl cyclase type II and IV are activated by Gbc in the presence of Gsa, type I is inhibited by Gbc and type III, V and VI don’t appear to be directly regulated by Gbc (Toro et al., 1987; Taussig et al., 1993). Adenylyl cyclase activity and its responsiveness to various hormones has been shown to be altered in various models of hypertension (Anand-Srivastava, 1988, 1992, 1993; Bohm et al., 1992; AnandSrivastava et al., 1993). We have recently shown an increased expression of Gia-2 and Gia-3 at protein and mRNA levels and their relationship with hormonal inhibiton and stimulation of adenylyl cyclase in heart and aorta from 12 week-old spontaneously hypertensive rats as well as in DOCAsalt hypertensive rats (Anand-Srivastava, 1992; Anand-Srivastava et al., 1991, 1993) whereas an unaltered expression of Gia and Gsa proteins has also been reported in hearts from SHR (spontaneously hypertensive rats) and other models of

hypertension (McLellan et al., 1992, Michel et al., 1993). The present studies were undertaken to further investigate if the increased expression of Gia-2 and Gia-3 protein and mRNA level in SHR is the cause or effect of hypertension. We used SHRs and age-matched WKY (Wistar-Kyoto rats) at different ages of development of blood pressure and determined adenylyl cyclase activity and the levels of G-proteins in heart and aorta from these rats. We have shown for the first time that the enhanced expression of Gia-2 and Gia-3 in heart and aorta precedes the development of blood pressure and may participate in the pathogenesis of hypertension.

Materials and Methods Materials Plasmid containing rat cDNAs encoding Gia2, Gia3 and Gsa were kindly obtained from Dr Randall Reed from the Johns Hopkins University, and Dr Hiroshi Itoh from the University of Tokyo. The 32-mer oligonucleotide which recognizes a highly conserved region in the 28 S ribosomal RNA was kindly donated by Dr Yoshihiro Ishikawa from Lederle laboratory of New York. The cDNA for adenylyl cyclase type V enzyme was kindly obtained from Dr Yoshihiro Ishikawa, Columbia University, New York in collaboration with Lederle Laboratory. Adenosine triphosphate, Cyclic AMP, isoproterenol, oxotremorine, glucagon were purchased from Sigma (St-Louis, MO, USA). Creatine kinase, myokinase (EC 2.7.4.3), guanosine 5′- [3′-thio] triphosphate (GTPcS), guanosine triphosphate (GTP) and adenosine deaminase (EC 3.5.4.4) were from Boehringer-Mannheim (Montreal, Quebec, Canada). N-ethylcarboxamide adenosine (NECA) was from Research Biochemicals (Wayland, MA, USA). [a-32P] ATP and Western Blotting detection kit were from Amersham (Oakville, Ontario, Canada). AS/ 7, EC/2 and RM/1 antibodies were from Dupont (Mississauga, Ontario, Canada). cDNAs of G-proteins are a gift from Dr. Randall Reed (John Hopkins University) and Hiroshi Itoh (University of Tokyo).

Experimental animals Male SHR of different ages starting from newborn (3–5 days), 2 weeks, 4 weeks and 8 weeks and their age-matched normotensive Wistar-Kyoto (WKY) were purchased from Charles Rivers Canada

G Proteins and Hypertension

(St-Constant, Quebec, Canada). The blood pressure was measured by tail-cuff method without anaesthesia. The rats were sacrificed by decapitation and the heart and aorta were dissected out and frozen immediately in liquid nitrogen and stored at −80°C.

Preparation of heart particulate fraction Heart particulate fraction was prepared as described previously (Pandey and Anand-Srivastava, 1996). Before the assay, the frozen heart was pulverized to a fine powder, in a mortar cooled in liquid nitrogen. The heart powder was homogenized in a Teflon/glass homogenizer in a buffer containing 10 mmol/l Tris-HCl and 1 mmol/l EDTA pH 7.5. The homogenate was centrifuged twice at 1000×g for 10 min. The pellet was then resuspended and homogenized in Tris-EDTA buffer pH 7.5 and used for determination of adenylyl cyclase activity and immunoblotting studies.

Preparation of aorta washed particles Aorta washed particles were prepared as described previously (Anand-Srivastava, 1988). The dissected aortae were quickly frozen in liquid nitrogen and pulverized to a fine powder, in a mortar cooled in liquid nitrogen. The aorta powder was homogenized in a Teflon/glass homogenizer in a buffer containing 10 mmol/l Tris-HCl and 1 mmol/l EDTA pH 7.5. The homogenate was centrifuged at 16 000 g for 15 min at 4°C. The pellet was finally suspended and homogenized in Tris-EDTA buffer pH 7.5 and used for determination of adenylyl cyclase activity and immunoblotting studies.

Adenylyl cyclase activity determination Adenylyl cyclase activity was determined by measuring [32P]cAMP formation from [a32−P]ATP, as described previously (Anand-Srivastava, 1988, 1992). Typical assay medium contained 50 mmol/l glycylglycine pH 7.5, 0.5 mmol/l MgATP, 5 mmol/l MgCl2, 0.5 mmol/l cAMP, 5 U/ml ADA (or otherwise as indicated), 100 mmol/l NaCl, 1 mmol/l 3-isobutyl-1-methylxanthine (or otherwise as indicated), 0.1 mmol/l EGTA, 10 lmol/l GTP (or otherwise as indicated), [a-32P]ATP (1–1.5×106 CPM) and an ATP-regenerating system consisting of 2 mmol/l creatine phosphate, 0.1 mg/ml creatine kinase and 0.1 mg/ml myokinase in a final volume of 200 ll.

1011

Incubations were initiated by the addition of the reaction mixture to the membranes which has been thermally equilibrated for 2 min at 37°C. The reactions conducted in triplicate for 10 min at 37°C were terminated by the addition of 0.6 ml of 120 mmol/l zinc acetate containing 0.5 mmol/l unlabelled cAMP. cAMP was purified by coprecipitation of other nucleotides with ZnCO3 by the addition of 0.5 ml of 144 mmol/l Na2CO3 and subsequent chromatography by the double-column system as described by Salomon et al. (1974). The unlabelled cAMP served to monitor the recovery of the [32P]cAMP by measuring absorbance at 259 nm. Under the assay conditions used, adenylyl cyclase activity was linear with respect to protein concentration and time of incubation. Protein concentration was determined by Lowry et al. (1951) with crystalline bovine serum albumin (BSA) as standard. Immunoblotting Immunoblotting of G-proteins was performed as described earlier (Anand-Srivastava, 1992). After SDS-PAGE, the separated proteins were transferred to a nitrocellulose paper (Schleicher & Schuell) using a semidry transblot apparatus (Bio-Rad) at 15 V for 45 min. After transfer, the membranes were washed twice in phosphate-buffered saline (PBS) and were incubated in PBS containing 3% dehydrated milk at room temperature for 2 h. The blots were then incubated with antisera against G proteins in PBS containing 1.5% dehydrated milk and 0.1% Tween 20 at room temperature overnight. The antigen-antibody complexes were detected by incubating the blots with goat anti-rabbit IgG (BioRad) conjugated with horseradish peroxidase for 3 h at room temperature. The blots were washed three times with PBS before reacting with enhanced-chemiluminescence (ECL) Western blotting detection reagents from Amersham. The autoradiograms were quantified by densitometric scanning using an enhanced laser densitometer (LKB Ultroscan XL, Pharmacia, Quebec, Canada) and gel scan XL evolution software (version 2.1, Pharmacia). The scanning was one dimensional and scanned the entire area of protein bands in autoradiograms. Extraction of total RNA and Northern Blot analysis Total RNA was isolated as described previously (Thibault and Anand-Srivastava, 1992) by the guanidinium thiocyanate-phenol-chloroform method

1012

J Marcil et al.

described by Chomczynski and Sacchi (1987). Briefly, frozen heart ventricles were homogenized in a denaturating solution (solution D) containing 4 mol/l guanidinium thiocyanate, 25 mmol/l sodium citrate, pH 7.0. 0.5% sarkosyl and 0.1 mol/l 2mercaptoethanol. The homogenates were extracted once with 1 volume of phenol and 0.2 volume of chloroform-isoamylalcohol (49:1) in the presence of 0.2 mol/l sodium acetate, pH 4.0, and once with 1 volume of chloroform-isoamylalcohol (49:1). Total RNA was then precipitated with isopropanol. Following a second precipitation in solution D and isopropanol (v:v), total RNA was washed in 70% ethanol and resuspended in water.

al. (1989). RNA was directly quantified by densitometric scanning using an enhanced laser densitomer (LKB Ultroscan XL, Pharmacia, Quebec, Canada) and gel scan XL evolution software (version 2.1, Pharmacia). The scanning was 1-dimensional and scanned the entire area of protein in the autoradiograms.

Radiolabeling of the probes and Northern analysis

Results

DMSO/Glyoxal treated total RNAs (5 lg for 3–5 days and 2 weeks old WKY and SHR and 10 lg for 4 and 8 weeks old WKY and SHR) were resolved on 1% agarose gel electrophoresis and transferred to Hybond-N-filters (Amersham). The filters were prehybridized in a hybridyzation solution containing 600 mmol/l NaCl, 8 mmol/l EDTA, 120 mmol/l Tris, pH 7.4, 0.1% sodium pyrophosphate, 0.2% SDS and 500 U/ml heparin at 65°C for 6 h before the addition of cDNA probe. The probes were radiolabeled with (a-32P) dCTP (3000 Ci/mmol) by random priming essentially as described by Feinberg and Vogelstein (1983). Filters were hybridized at 65°C for 16 h in hybridization solution containing 10% of dextran sulfate and cDNA probe (1–3×106 ct/min/ml). Filters were then rinsed twice for 30 min at 65°C with a solution containing 300 mmol/l NaCl, 4 mmol/l EDTA, 60 mmol/l Tris, pH 7.4, and 0.2% SDS and one time for 30 min at 65°C with a solution containing 150 mmol/l NaCl, 2 mmol/l EDTA, 30 mmol/l Tris, pH 7.4 and 0.1% SDS. Autoradiography was performed with X-ray films at −70°C for 24 to 48 h. In order to assess the possibility of any variation in the amounts of total RNA in individual samples applied to the gel, each filter was hybridized with a 32-mer oligonucleotide recognizing a highly conserved region in the 28S ribosomal RNA. The blots which had been probed with the G-protein cDNA were de-hybridized by washing for 2 h at 65°C in 50% formamide, 300 mmol/l NaCl, 4 mmol/l EDTA and 60 mmol/l Tris, pH 7.4, and re-hybridized overnight at room temperature with the oligonucleotide. The 32-mer oligonucleotide recognizing the 28S rRNA was end-labelled with [c-32P]ATP using T4 polynucleotide kinase as described by Sambrook et

The physiological parameters of SHR and agematched WKY are shown in Table 1. The systolic blood pressure was not significantly different in 3 week-old SHR as compared to their age-matched WKY, whereas it was significantly increased in 4 and 8 week-old SHR as compared to their agematched WKY. The blood pressure of younger rats could not be measured because of the limitation of the method. The heart weight to body weight ratio was not significantly different in SHRs as compared to their age-matched WKY at all the ages studied.

Analysis of data Results are means ± ... and they were considered significant if P<0.05. Comparison between groups were made using either Student’s t-test or Analysis of Variance (ANOVA) where appropriate.

G-proteins levels G-proteins play an important role in the regulation of adenylyl cyclase activity. We have recently shown an altered expression of G-proteins in heart and aorta of SHR 12 weeks. To investigate if these changes occur before the onset of hypertension, we determined the levels of G-proteins in heart from SHR of different ages starting at 3–5 days up to 8 weeks by immunoblotting experiments using specific antibodies AS/7 against Gia-1 and Gia-2, EC/2 against Gia-3 and RM/1 against Gsa. Figure 1(a) shows that RM/1 antibody recognized three isoforms of Gsa, Gsa45, Gsa47 and Gsa52 in heart from both SHR and their age-matched control WKY, however the relative amount of immunodetectable Gsa as determined by densitometric scanning was not significantly different at all the ages studied (in arbitrary units, 3–5 days old; WKY, Gsa45 1.73±0.2; SHR, 1.75±0.2; Gsa47 WKY, 9.02±0.4; SHR, 10.11±0.9; 2 weeks old WKY, Gsa45 1.24±0.3; SHR, 1.27±0.3; Gsa47 WKY, 11.13±0.5; SHR, 11.99±1.4; 4 week-old WKY

1013

G Proteins and Hypertension

Table 1 Body weight, heart weight, body weight to heart weight ratio and blood pressure of SHR and WKY at various ages of development Strains

WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR

Age

Body weight (g)

Heart weight (mg)

Heart weight/ body weight (mg/g)

Systolic blood pressure (mmHg)

3–5 days 3–5 days 2 weeks 2 weeks 3 weeks 3 weeks 4 weeks 4 weeks 8 weeks 8 weeks

7.22±0.24 6.48±0.21 28.2±0.65 22.2±0.24 39.5±1.50 26.0±2.00 51.6±1.60 46.8±2.50 176±3.10 183±1.90

44.0±2.7 40.0±2.4 150.0±5.2 121.3±3.5 210.0±6.0 125.0±15.0 260.0±10.0 280.0±30.0 930.0±20.0 980.0±40.0

6.10±0.58 6.17±0.57 5.32±0.31 5.46±0.22 5.32±0.35 4.81±0.95 5.04±0.35 5.98±0.96 5.28±0.21 5.35±0.27

N.D. N.D. N.D. N.D. 124.7±13.7 120.3±1.3 124.6±3.0 159.2±4.9† 126.3±3.0 172.9±3.1†

WKY = Wistar-Kyoto; SHR = spontaneously hypertensive rat. Values are mean ± .. from 6 different rats of 2-weeks old WKY and their age-matched SHR and 10 different rats of 4- and 8-weeks-old WKY and their age-matched SHR. †P<0.001.

Heart Gs

(a) 3–5 days

2 weeks

4 weeks

8 weeks

WKY SHR WKY SHR WKY SHR WKY SHR

52 kDa 47 kDa 45 kDa

(b)

Gi 3–5 days

2

2 weeks

4 weeks

8 weeks

WKY SHR WKY SHR WKY SHR WKY SHR

40 kDa

(c)

Gi 3–5 days

2 weeks

3

4 weeks

8 weeks

WKY SHR WKY SHR WKY SHR WKY SHR

41 kDa

Figure 1 Quantification of G-proteins (Gsa, Gia-2 and Gia-3) in hearts from 3–5 day-, 2 week-, 4 week-, and 8 week-old WKY and age-matched SHR by immunoblotting. The heart proteins (50 lg) from WKY and SHR were resolved by SDS/PAGE and transferred to nitrocellulose, which was then immunoblotted with antibody RM/1 for Gsa (a), As/7 for Gia-2 (b), EC/2 for Gia-3 (c) as described in the Methods section. The detection of the different G-proteins was performed by using the chemiluminescence (ECL) Western blotting detection reagents from Amersham. The autoradiograms shown are representative of six or seven separate experiments.

Gsa45 1.45±0.2; SHR, 1.53±0.3; Gsa47 WKY 15.35±1.92; SHR, 17.02±2.0; 8 week-old WKY, Gsa45 2.07±0.47; SHR, 1.99±0.3; Gsa47 WKY, 12.40±1.8; SHR, 12.80±2.1 (n=4). On the other hand, AS/7 antibody recognized a single protein of 40 kDa referred to as Gia-2 (Gia-1 has been shown to be absent in heart (Jones and Reed, 1987) in both SHR and their age-matched control WKY [Fig. 1(b)] and the relative amount of immunodetectable Gia2 as determined by densitometric scanning (Table 2) was significantly increased by 118.3±23.6% (n=7) in 2 week-old SHR, 163.8±6.4% (n=7) in 4 week-old SHR and 119.6±8.3% (n=7) in 8 week-old SHR as compared to their age-matched WKY, whereas no alteration in the levels of Gia2 was observed in younger SHR as compared to WKY. Similarly, EC/2 antibody recognized a single protein of 41 kDa referred to as Gia-3 in heart in both SHR and their age-matched WKY, however, the relative amount of immunodetectable Gia-3 was also significantly increased by 82.4±7.8% (n=6) in 2 week-old SHR, 56.1±7.3% (n=6) in 4 week-old SHR and 34.0±5.1% (n=6) in 8 week-old SHR as compared to their age-matched WKY. The levels of Gia-3 like Gia-2 were also not different in younger SHR as compared to their age-matched WKY as shown in Figure 1(c). Similar increase in Gia-2 and Gia-3 protein expression was also observed in aorta from 2 weeks and older SHR (data not shown). We extended our studies further to investigate if mRNA levels change concomitantly with protein levels and therefore examined the mRNA levels by Northern blot analysis using specific cDNA probes encoding for Gsa, Gia-2 and Gia-3 (Fig. 2). The Gsa cDNA probe detected a message of 1.8 kb in

1014

J Marcil et al. Table 2 Summary of the quantification of immunoblot by densitometric scanning G-protein levels (Arbitrary unit) Strain

Age

Gia-2 (40 kDa)

Gia-3 (40/41 kDa)

Gsa (45 kDa)

Gsa (47 kDa)

WKY SHR WKY SHR WKY SHR WKY SHR

3–5 days 3–5 days 2 weeks 2 weeks 4 weeks 4 weeks 8 weeks 8 weeks

4.33 4.70 2.82 4.94 0.58 1.57 1.52 2.98

3.39 3.50 1.78 2.87 0.62 0.98 0.75 0.96

1.82 1.84 1.54 1.58 2.55 2.67 3.72 3.36

10.11 11.91 13.14 14.16 16.08 18.16 13.43 13.75

The membrane proteins (50 lg) from 3–5 day-old, 2 week-old, 4 week-old and 8 week-old SHR and WKY were separated on a SDS/PAGE and transferred to nitrocellulose which was then immunoblotted by using Gsa, Gia-2 and Gia-3 antibodies as described in Materials and Methods. Quantification of G-proteins was performed by densitometric scanning using an enhanced laser densitometer (LKB). The values presented in this table are taken from the immunoblots shown in Figure 1.

heart from both WKY and SHR and the amount of Gsa mRNA was not different at all the ages studied as compared to their age-matched WKY [Fig. 2(a)]. However, the Gia-2 probe detected a message of 2.3 kb in heart from SHR and the amount of Gia-2 mRNA was slightly increased by 15.3±5.2% (n= 5) in younger SHR and was significantly increased by 63.9±8.6% (n=5) in 2 week-old SHR, 134.6±7.3% (n=5) in 4 week-old SHR and 184.3±9.8% (n=5) in 8 week-old SHR as compared to their counterparts WKY as shown in Figure 2(b). In addition, the Gia-3 cDNA probe detected a message of 3.5 kb in heart from SHR and WKY; however, the amount of Gia-3 mRNA was significantly increased by 135.2±11.2% (n=5) in 1 week-old SHR, 268.4±17.5% (n=5) in 2 weekold SHR, 138.4±8.4% (n=5) in 4 week-old SHR and 553.1±23.6% (n=5) in 8 week-old SHR as compared to their age-matched WKY as shown in Figure 1(c). The alteration in Gia-2 and Gia-3 mRNA levels in heart from SHR may not be attributed to the variation in the amounts of total RNA in individual samples applied to the gels, due to the fact that the hybridization with an oligonucleotide that recognize a highly conserved region of the 28S rRNA showed a similar amount of 28S rRNA loaded from WKY and SHR on the gels.

Effect of guanine nucleotides on adenylyl cyclase activity Since the expression of G-protein was enhanced at the protein and mRNA levels in SHR as compared

to WKY at younger age when the blood pressure is still normal, it was of interest to investigate if these alterations are reflected in G-protein functions, we examined the ability of guanine nucleotides to stimulate adenylyl cyclase in heart and aorta from SHR and their age-matched WKY rats. As shown in Table 3, GTP and GTPcS stimulated adenylyl cyclase activity in both WKY and SHR to different extents, however, the percent stimulation in SHR was significantly decreased in 2 weeks and older SHR as compared to their age-matched WKY, whereas no significant decrease was observed in 3–5 days old SHR as compared to age-matched WKY rats. Similar results were observed in mesenteric arteries from 3–5 days up to 8 week-old SHR (Data not shown). Similarly, the extent of stimulation of adenylyl cyclase by GTP or GTPcS in aorta was significantly decreased in 2, 4 and 8 week-old SHR and not in 3–5 days old SHR (Table 3). These results suggest that the decreased stimulation of adenylyl cyclase by GTPcS in SHR as compared to WKY when Gsa was unaltered, may be due to the increased levels of Gia-2 and Gia-3 and not of Gsa.

Hormonal regulation of adenylyl cyclase Since the levels of Gsa were not altered and the levels of Gia-2 and Gia-3 were increased in heart from 2 week-old SHR up to 8 week-old SHR, it was of interest to investigate if the increased level of Gia could modulate the Gs-mediated functions, we studied the effect of isoproterenol, NECA (N-ethylcarboxamideadenosine) and glucagon on adenylyl

G Proteins and Hypertension (a) 3–5 days

2 weeks

4 weeks

8 weeks

WKY SHR

WKY SHR

WKY SHR

WKY SHR

28 s -

18 s -

(b) 3–5 days

2 weeks

4 weeks

8 weeks

WKY SHR

WKY SHR

WKY SHR

WKY SHR

28 s -

18 s -

(c) 3–5 days

2 weeks

4 weeks

8 weeks

WKY SHR

WKY SHR

WKY SHR

WKY SHR

28 s -

18 s -

Figure 2 mRNA expression of Gsa (a), Gia-2 (b) and Gia-3 (c) in hearts from 1 week, 2 week-, 4 week-, and 8 week-old WKY and age-matched SHR. Total RNA (5 lg in WKY and SHR 3–5 days, 2 weeks and 10 lg in WKY and SHR 4 weeks and 8 weeks) isolated from the hearts of WKY and SHR were subjected to 1% agarose gel electrophoresis and transferred to nylon membranes, the blots were then probed with full length radiolabelled cDNA probe encoding Gsa (a), Gia-2 (b) and Gia-3 (c) (Upper panels) and further re-hybridized with an oligonucleotide recognizing the 28S rRNA (lower panel) as described in Materials and Methods. The autoradiogram is representative of five separate experiments.

cyclase activity in heart and aorta from SHR and age-matched WKY and the results are shown in Figure 3. Isoproterenol, NECA and glucagon stimulated adenylyl cyclase activity to various degrees in SHR and WKY at all the ages studied, however, the stimulations exerted by these hormones were

1015

significantly decreased in hearts from 2, 4 and 8 week-old SHR as compared to their age-matched WKY rats. Similar decreases were also observed in aorta from 2, 4 and 8 week-old SHR as compared to their age-matched WKY. We also observed similar results in mesenteric arteries from 2, 4 and 8 weekold SHR (data not shown). On the other hand a decreased stimulation of adenylyl cyclase by isoproterenol (30–40%) was observed in heart as well as in aorta from 3–5 days old SHR as compared to WKY; whereas, NECA-stimulated enzyme activity was not altered, and glucagon was unable to stimulate adenylyl cyclase activity in younger rats. We also investigated the relationship between the enhanced expression of Gia proteins and their functions by examining the effect of inhibitory hormones on adenylyl cyclase in control and SHR. As shown in Figure 4, C-ANF4–23 (a ring deleted analog of atrial natriuretic factor), that has been reported to inhibit adenylyl cyclase through Gia (AnandSrivastava et al., 1990, 1991a), inhibited adenylyl cyclase activity in heart and aorta from SHR and age-matched WKY rats at all the ages studied, however, the extent of inhibition was not different in 3–5 days old SHR as compared to WKY, but was greater in hearts and aorta from 2, 4 and 8 weekold SHR as compared to their age-matched WKY rats. Similarly, oxotremorine inhibited adenylyl cyclase activity to a greater extent in heart and aorta from 2, 4 and 8 week-old SHR as compared to their age-matched WKY rats. However, at early ages (3–5 days), oxotremorine was unable to inhibit adenylyl cyclase activity in heart from WKY or SHR.

Forskolin and NaF-stimulated adenylyl cyclase activity Figure 5 shows the stimulatory effect of forskolin on adenylyl cyclase in heart and aorta from SHR and age-matched WKY. Forskolin stimulated adenylyl cyclase in heart and aorta from SHR and WKY at various ages, however, the percent stimulation was significantly decreased in heart and in aorta in 2, 4 and 8 week-old SHR as compared to their age-matched WKY [Figs 5(a), (b)]. Similarly, the stimulatory effect of NaF on adenylyl cyclase (% stimulation) was also significantly decreased in hearts from 2, 4 and 8 week-old SHR and not from 3–5 day-old SHR as compared to their age-matched WKY [Fig. 5(c)]. In order to investigate if the decreased stimulation is attributed to the decreased levels of catalytic subunit [type V and VI enzyme expressed in heart (Krupinski et al., 1992; Ishikawa et al., 1992)], the

1016

J Marcil et al.

Table 3 GTP and GTPcS-stimulated adenylyl cyclase activity in heart and aorta from 3–5 day-, 2 week-, 4 week- and 8 week-old WKY and SHR. Adenylyl cyclase activity [pmol cAMP (mg protein. 10 min)] Age

Basal

GTP (10 lM)

% Stimulation (GTP/basal)

GTPcS (10 l)

% Stimulation (GTPcS/basal)

3–5 days 3–5 days 2 weeks 2 weeks 4 weeks 4 weeks 8 weeks 8 weeks

25.7±1.3 29.5±1.5 25.0±2.6 22.7±0.8 29.1±4.6 34.3±0.2 21.6±3.3 18.4±2.2

121.9±5.3 130.9±6.5 131.6±3.5 113.5±2.4§ 183.7±3.0 171.9±4.3‡ 139.6±7.9 103.9±7.3§

374.3±20.0 343.7±22.0 426.4±14.0 400.0±10.6† 531.3±10.3 401.2±12.5§ 546.3±36.6 464.7±39.7†

163.1±15.1 185.3±9.5 397.5±10.6 261.1±12.4§ 250.7±8.3 168.1±6.1§ 185.7±9.0 111.3±20.0‡

534.6±164.4 528.1±126.8 1490.0±42.4 1050.2±54.6§ 761.5±58.5 390.1±47.8§ 759.7±41.7 504.9±53.8§

3–5 days 3–5 days 2 weeks 2 weeks 4 weeks 4 weeks 8 weeks 8 weeks

19.1±4.5 24.8±2.7 27.7±4.7 21.7±9.5 34.5±1.7 38.1±2.4 46.8±7.7 51.8±5.9

131.9±15.4 155.2±14.3 135.6±13.1 96.6±7.0‡ 128.1±3.0 107.6±5.9‡ 206.7±23.5 162.9±11.1‡

590.6±50.6 525.8±37.6 389.5±27.3 345.2±12.3† 271.3±8.7 182.4±15.4§ 341.6±32.2 214.5±11.4‡

235.8±12.3 255.4±11.6 429.3±18.7 227.9±3.3§ 511.1±10.5 294.4±15.5§ 407.3±18.2 304.9±17.4§

1134.5±64.4 929.8±46.8§ 1449.8±67.5 950.2±55.2§ 1381.4±30.4 672.7±40.7§ 770.3±38.9 488.6±33.5§

Strain Heart WKY SHR WKY SHR WKY SHR WKY SHR Aorta WKY SHR WKY SHR WKY SHR WKY SHR

The adenylyl cyclase activity was determined in the absence of presence of 10 l GTP or GTPcS as described in Materials and Methods. Values are mean ± ... of four separate experiments. Four animals were used for each experiment. Statistical analysis was performed with Student’s t test for comparison between SHR and WKY rats. †P<0.05, ‡P<0.01, §P<0.001.

mRNA levels of type V enzyme were determined in hearts by using a cDNA probe encoding type V enzyme. As shown in Figure 6(a), the cDNA probe detected a message of about 6.2 kb in heart from both WKY and SHR. However, the amount of mRNA type V enzyme mRNA was significantly decreased by about 25%, 30% and 60% in SHR at 2, 4 and 8-weeks of age and was unaltered in 3–5 day-old SHR as quantified by densitometric scanning [Fig. 6(b)].

Discussion We have previously reported an increased expression of inhibitory guanine nucleotide regulatory proteins (Gia-2 and Gia-3) and not of stimulatory guanine nucleotide regulatory proteins (Gsa) in hearts and aorta from 12 week-old SHR as compared to their age-matched WKY (Anand-Srivastava, 1991; Anand-Srivastava et al., 1992). The results presented in the present studies demonstrate a possible relationship between the levels of Giproteins and elevation of blood pressure in SHR at various ages of development of blood pressure. The blood pressure in 3-week-old SHR was not

different as compared to their age-matched WKY rats but was significantly elevated at 4 weeks of age and older rats. However, the levels of Gia-2 and Gia-3 proteins as quantified by immunoblotting analysis were significantly higher in 2 week-old SHR as compared to their age-matched WKY. The levels of Gia-2 and Gia-3 mRNA levels were also increased in 1 and 2 week-old SHR as compared to their age-matched WKY and suggests that the genes for Gia-2 and Gia-3 are overexpressed in prehypertensive state and may partly be responsible for the observed increase in protein levels. This increase in mRNA levels of Gia-2 and Gia-3 is not due to the amount of RNA loaded since there is no difference in the amount of the oligonucleotide 28S. The increase in mRNA levels did not parallel with an increase in protein levels and is in accordance with our previous results reported in hearts and aorta from 12 week-old SHR (Thibault and AnandSrivastava, 1992), where Gia-2 and Gia-3 mRNA levels were enhanced by 60 and 120% respectively and protein levels were increased by about 50 and 30% respectively (Anand-Srivastava, 1992; Thibault and Anand-Srivastava, 1992). Luetje et al. (1988) have also reported differences between the level of Gia-2 protein and Gia-2 mRNA levels in their studies on the developmental changes in

1017

G Proteins and Hypertension Heart

Aorta

Isoproterenol 350

300

300

250

†

250 200

†

*

200 †

†

150

†

150 100

100

50

50

Adenylyl cyclase activity (% stimulation)

0

†

†

3–5 days

2 weeks

4 weeks

8 weeks

0

3–5 days

2 weeks

4 weeks

8 weeks

NECA 70

200

60 *

50

150 *

40 *

30

†

†

20

*

100 50

10 0

3–5 days

2 weeks

4 weeks

8 weeks

0

3–5 days

2 weeks

4 weeks

8 weeks

Glucagon 250

40 35

200 * 150

30 25 †

20 100

15 *

50 0

† 3–5 days

† 2 weeks

†

10 5

4 weeks

8 weeks

0

† 3–5 days

2 weeks

4 weeks

8 weeks

Figure 3 Effect of stimulatory hormones on adenylyl cyclase activity in heart and aorta from 3–5 day-, 2 week-, 4 week- and 8 week-old WKY and SHR. Adenylyl cyclase activity was determined in WKY (Φ) or SHR (Γ) as described in Materials and Methods in the presence of 5 U/ml ADA and 10 l GTP alone (Basal), or in combination with 50 l isoproterenol, or 10 l (NECA), or 1 l glucagon. Basal enzyme activity in heart and aorta membranes in the presence of GTP (10 l) are presented in Table 3. Values are mean ± ... of four different experiments. Four animals were used for each experiment. In these studies, IBMX was replaced by ROCHE, Ro 20–1724. Statistical analysis was performed by Student’s t-test for comparison between WKY and SHR and it was considered significant when ∗P<0.05, †P<0.01, ‡P<0.001.

Gi proteins in heart ventricle. In the present studies, a developmental decrease in Gia-2 and Gia-3 levels in WKY as well as in SHR is in agreement with the results reported previously in rabbit (Kumar et al., 1994) and rat hearts (Tobise and Homcy, 1994). However, an enhanced expression of Gia-2 and Gia-

3 at protein and mRNA levels in younger SHR as compared to age-matched WKY before the onset of hypertension has not been reported earlier and thus is the first study suggesting that increased expression of Gia-2 and Gia-3 protein may participate in the pathogenesis of hypertension. The

1018

J Marcil et al. Heart

Aorta

C-ANF4-23 60

†

†

50

†

50 *

40

40

†

*

30

Adenylyl cyclase activity (% inhibition)

30 20

20

10

10 0

3–5 days

2 weeks

4 weeks

8 weeks

0

3–5 days

2 weeks

4 weeks

8 weeks

Oxotremorine 50

50 †

* 40

40

*

† *

30 20

20

10

10

0

3–5 days

2 weeks

4 weeks

†

30

8 weeks

0

3–5 days

2 weeks

4 weeks

8 weeks

Figure 4 Effect of inhibitory hormones on adenylyl cyclase activity in heart and aorta from 3–5 day-old, 2 week-old, 4 week-old and 8 week-old WKY and SHR. Adenylyl cyclase activity was determined in heart or aorta from WKY (Φ) or SHR (Γ) as described in Materials and Methods in presence of 10 l GTPcS alone (Basal) and in combination with 50 l oxotremorine and 10−7  C-ANF4–23. Basal enzyme activity in heart and aorta membranes in the presence of GTPcS in WKY and SHR are presented in Table 3. Values are mean ± ... of four different experiments in heart and three different experiments in aorta. Three or four animals were used for each experiment. Statistical analysis was performed by Student’s test for comparison between WKY and SHR and it was considered significant when ∗P<0.05, †P<0.01, ‡P<0.001.

increased levels of Gia-2 and Gia-3 in younger rats may not be attributed to the hypertrophy of the heart, because of the fact that heart weight/body weight ratio was not different in SHR as compared to WKY. Whether the increase in G-proteins in prehypertensive state is attributed to the intrinsic hormonal factors is not known and has to be explored. However, the plasma level of catecholamines and ANF have been shown to be elevated in adult SHR and may contribute to the increased levels of Gia. However, no studieas have been reported to indicate an increased level of these hormones in early ages of development of hypertension. The increased expression of Gia-2 and Gia-3 proteins as well as mRNA was reflected in Gimediated functions. Concurrent to the unaltered levels of Gia-2 and Gia-3 in 3–5 day-old SHR as compared to the age-matched WKY, the extent of

C-ANF4–23-mediated inhibition of adenylyl cyclase was also unchanged in 3–5 day-old rats. These results also suggest the presence of ANP-C receptor in neonatal rats and are in agreement with our previous results demonstrating ANP-mediated inhibition of adenylyl cyclase in ventricular cardiocytes from neonatal rats (Anand-Srivastava and Cantin, 1983). On the other hand, the inability of oxotremorine to inhibit cardiac adenylyl cyclase in younger rats may be due to the possibility that the muscarinic receptors although increased at an early age (McMahon, 1989) may not be functional or the coupling of the receptor to Gi-protein may not be efficient. In this context, the Gi-proteinmuscarinic receptor interaction in cardiac tissue has been shown to be more efficient in adult than in neonate tissue (McMahon, 1989). The enhanced inhibition of adenylyl cyclase by inhibitory hormones in 2-weeks of age and older SHR as compared

1019

G Proteins and Hypertension (b)

Adenylyl cyclase activity (% inhibition)

(a) Forskolin

Aorta

Heart 8000

8000

7000

7000

6000

6000

5000

5000

†

4000

*

4000

3000

3000

2000

2000

1000

1000

0

3–5 days

2 weeks

0

8 weeks

4 weeks

†

†

3–5 days

2 weeks

†

4 weeks

8 weeks

Adenylyl cyclase activity (% stimulation)

(c) NaF Heart 800 700 600 †

*

500 400

†

300 200 100 0

3–5 days

2 weeks

4 weeks

8 weeks

Figure 5 Effect of forskolin and NaF on adenylyl cyclase activity in heart and aorta from 3–5 day-, 2 week-, 4 weekand 8 week-old WKY and SHR. Adenylyl cyclase was determined as described in Materials and Methods in the absence or presence of 50 l forskolin in heart (a), aorta (b) or 10 m NaF in heart (c) from WKY (Φ) or SHR (Γ). GTP or GTPcS was omitted from the reaction mixture. Basal enzyme activity in heart and aorta membranes from WKY and SHR are presented in Table 3. Values are means ± ... of four different experiments. Four animals were used for each experiment. Statistical analysis was performed by Student’s t test for comparison between WKY and SHR and was considered statistically different when ∗ P<0.05, † P<0.01, ‡ P<0.001.

28 s -

18 s -

Heart Catalytic subunit type V

(b) Catalytic subunit type V

3–5 days

2 weeks

4 weeks

8 weeks

WKY SHR

WKY SHR

WKY SHR

WKY SHR

120 mRNA (% of control)

(a)

100 80 60 40 20 0

3–5 days

2 weeks

4 weeks

8 weeks

Figure 6. Expression of the catalytic subunit type V enzyme mRNA in heart from 3–5 day-old, 2 week-old, 4 weekold and 8 week-old WKY and SHR. (a) Total RNA (10 lg) isolated from the heart of the four different groups were separated on a 1% agarose and transferred to nylon membranes, which were then hybridized with full length cDNA probe (upper panel). These filters were re-hybridized with an oligonucleotide recognizing the 28S rRNA (lower panel). The autoradiogram is representative of four separate experiments. (b) Densitometric scanning of the autoradiogram shown in (a). The results are expressed as percent mRNA in SHR over their age-matched WKY (control) which has been taken as 100%.

1020

J Marcil et al.

to age-matched WKY is in agreement with our previous studies conducted in 12-week-old SHR (Anand-Srivastava et al., 1991b; Anand-Srivastava, 1992). As suggested previously (Anand-Srivastava, 1992), the enhanced inhibition of adenylyl cyclase by inhibitory hormones may be attributed to the upregulation of receptor or to the post receptor modifications. However, ANF and muscarinic receptors have been reported to be down regulated in various tissues from hypertensive rats (Delcayre and Swynghedauw, 1991; Anand-Srivastava and Trachte, 1993). Taken together, it may be suggested that the enhanced expression of Gia-2, Gia-3 and decreased expression of type V enzyme in SHR may be responsible for the enhanced responsiveness of adenylyl cyclase to inhibitory hormones in SHR. This is further supported by the fact that parallel to the increased levels of Gia-2 and Gia-3 and decreased levels of type V enzyme in 2-week-old SHR as compared to age-matched WKY rats, the Gia-mediated inhibition of adenylyl cyclase by C-ANF4–23 and oxotremorine was also augmented in heart as well as in aorta. Our results on unaltered expression of Gsa47 and Gsa45 in heart and aorta from SHR as compared to WKY at all the ages studied, are in agreement with our previous studies performed in 12 weeks old SHR (Anand-Srivastava, 1992; Thibault and Srivastava, 1992) and suggest that Gsa may not be playing a role in the development of high blood pressure. The decreased stimulation of adenylyl cyclase by GTPcS in hearts and aorta from 2-weeks of age and older SHR and not in 3–5 day-old SHR as compared to WKY may be attributed to the decreased levels of type V enzyme and not to the decreased levels or activity of Gsa (Anand-Srivastava, 1992). However, the decreased stimulation of adenylyl cyclase by isoproterenol, NECA and glucagon in 2-weeks of age and older SHR as compared to WKY may be attributed to the down regulation of hormone receptors (Limas et al., 1978) or to the decreased levels of type V enzyme. On the other hand, the decreased stimulation of adenylyl cyclase by isoproterenol in 3–5 day-old SHR as compared to age-matched WKY, when the levels of Gsa, Gia and type V enzyme were not altered, may be explained by the desensitization of the receptors at younger age. However, an unaltered stimulation of adenylyl cyclase by NECA may not be due to the down-regulation of adenosine receptors in 3–5 dayold SHR, whereas, the lack of responsiveness of adenylyl cyclase to glucagon in 3–5 day-old rats (SHR and WKY) may be due to the possibility that glucagon receptors are not expressed at that younger age.

The modulation of Gsa functions by Gia has been reported by several investigators (Cerione et al., 1985; Feldman et al., 1988). An increased expression of Gia-2 and Gia-3 has been shown to be associated with attenuated responsiveness of adenylyl cyclase to stimulatory hormones (AnandSrivastava, 1992) whereas a decreased expression of Gia-2 resulted in the augmentation of stimulatory responses of hormones on adenylyl cyclase (AnandSrivastava, 1993). Taken together, it is possible that the enhanced levels of Gia-2 and Gia-3 in aorta and hearts from 2-weeks of age and older SHR as compared to age-matched WKY may also be responsible for the diminished responsiveness of adenylyl cyclase to isoproterenol, NECA and glucagon stimulation. The decreased sensitivity of adenylyl cyclase to FSK (forskolin) stimulation in heart and aorta from 2-weeks and older SHR in comparison to age-matched WKY may be due to the defective catalytic subunit per se or the overexpression of Gia or both. This is further supported by our results observed in 3–5 day-old SHR, where unaltered responsiveness of adenylyl cyclase to FSK stimulation was associated with unaltered expression of Gi proteins as well as with unaltered expression of mRNA of catalytic subunit type V enzyme. However, in 2-weeks of age and older SHR, a decreased stimulation of adenylyl cyclase by forskolin was associated with an increased expression of Gia-2 and Gia-3 and decreased expression of type V enzyme. Whether the expression of adenylyl cyclase type VI enzyme is also decreased in hearts from SHR has to be investigated. The involvement of Gi in FSK-mediated stimulation has also been shown (Anand-Srivastava et al., 1987). In addition, the requirement of Gs and guanine nucleotide for FSK-activation of adenylyl cyclase has been reported (Hildebrandt et al., 1982). Since the present studies do not demonstrate any alterations in Gs, the diminished stimulation of adenylyl cyclase by FSK in SHR cannot be attributed to the impaired Gs levels or activity. In conclusion, we have demonstrated for the first time that the increased expression of Gia-2 and Gia-3 genes and translated proteins precedes the development of hypertension. The increased expression of Gia proteins results in the decreased levels of cAMP that may be responsible for the increased vascular resistance and thereby development of hypertension. It may thus be suggested that increased expression of Gia-2 and Gia-3 proteins may be one of the factors participating in the pathogenesis of hypertension.

G Proteins and Hypertension

Acknowledgements We are grateful to Dr Randall Reed and Dr Hiroshi Itoh for their kind gift of cDNAs of G-proteins, Dr. Yoshihiro Ishikawa for the kind gift of cDNA of adenylyl cyclase type V enzyme and 32-mer oligonucletoide. We would like to thank Christiane Laurier for her valuable secretarial help.

References A MS, 1975. Cyclic nucleotide in disease; on the biochemical etiology of hypertension. Life Science 17: 1021–1038. A-S MB, 1988. Altered responsiveness of adenylate cyclase to adenosine and other agents in the myocardial sarcolemma and aorta of spontaneously hypertensive rats. Biochem Pharmacol 37: 3017–3022. A-S MB, 1992. Enhanced expression of inhibitory guanine nucleotide regulatory protein in spontaneously hypertensive rats: relationship to adenylate cyclase inhibition. Biochem J 288: 79–85. A-S MB, 1993. Platelets from spontaneously hypertensive rats exhibit decreased expression of inhibitory guanine nucleotide regulatory protein: relation with adenylyl cyclase activity. Circ Res 73: 1032–1039. A-S MB, C M, 1983. Regulation of adenylate cyclase in cultured cardiocytes from neonatal rats by adenosine and other agonists. Arch Biochem Biophys 223: 468–476.43. A-S MB, T J, 1993. Atrial natriuretic factor receptors and signal transduction mechanisms. Pharmacol Rev 45: 455–497. A-S MB, S AK, C M, 1987. Pertussis toxin attenuates atrial natriuretic factor-mediated inhibition of adenylate cyclase. Involvement of inhibitory guanine nucleotide regulatory protein. J Biol Chem 262: 4931–4934. A-S MB, S MR, C M, 1990. Ring deleted analogs of atrial natriuretic factor inhibits adenylyl cyclase/cAMP system: possible coupling of clearance atrial natriuretic factor receptors to adenylate cyclase/cAMP signal transduction system. J Biol Chem 265: 8566–8572. A-S MB, G J, C M, 1991a. The presence of atrial natriuretic factor receptors of ANF-R2 subtype in rat platelets: coupling to adenylate cyclase/ cAMP signal transduction system. Biochem J 278: 211–217. A-S MB, P S, T C, 1991b. Altered expression of inhibitory guanine nucleotide regulatory proteins (Gia) in Spontaneously hypertensive rats. Am J Hypertens 4: 840–843. A-S MB,  C J, T C, 1993. DOCA-salt hypertensive rat hearts exhibit altered expression of G-proteins. Am J Hypertens 6: 72–75. B M, G P, K A, L K, W K, E E, 1992. Desensitization of adenylate cyclase and increase of Gia in cardiac hypertrophy due to acquired hypertension. Hypertension 20: 103–112.

1021

B P, C A, S C, G V, P C, H J, S A, N H, 1986. Human cDNA clones for fours species of Gsa signal transduction protein. Proc Natl Acad Sci U.S.A. 83: 8893–8897. C RA, S C, C MG, L RJ, C J, B L, 1985. A role for Ni in the hormonal stimulation of adenylate cyclase. Nature 318: 293–295. C F, S N, 1987. Single-step method of RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction. Anal Biochem 162: 156– 159. D EE, 1981. Vasodilators, Vanhoutte PM, Leusen I, eds, New York: Raven Press, 381–390. D V M, I R, 1994. G protein pathways: signal processing by effectors. Mol Cell Endocrinol 100: 65–70. D C, S B, 1991. Biological adaptation and dysadaptation of the heart to chronic arterial hypertension: a review. J Hypertens 9 (Suppl.): S23–S28. F AP, V B, 1983. A technique for radiolabeling DNA restriction endonuclease fragment to high specific activity. Anal Biochem 132: 6–13. F AM, C AE, V WB, H RE, B MR, B KL, B WA, D CV, 1988. Increase of the 40,000 mol wt pertussis toxin substrate (G-protein) in the failing human heart. J Clin Invest 82: 189–197. F CM, P IH, 1978. Current views concerning cardiac output in the genesis of experimental hypertension. Circ Rec 43: 821–831. F JW, W PL, W AM, 1992. Signal transduction by G proteins in cardiac tissues. Circulation 85: 420–433. F B, H M, L Y, S R, W L, 1973. Importance of adaptive changes in vascular design for establishment of primary hypertension, studies in man and in spontaneously hypertensive rats. Circ Res 32(Suppl. 1):2–16. G AG, 1984. G protein and dual control of adenylate cyclase. Cell 36: 577–579. H JD, H J, B J, 1982. Guanine nucleotide inhibition of cyc-S49 mouse lymphoma cell membrane adenylyl cyclase. J Biol Chem 257: 14723–14725. I TP, S J, 1992. G-protein-coupled receptors. Curr Opin Cell Biol 4: 195–202. I T, T R, I T, Changes in the content of atrial natriuretic factor with the progression of hypertension in spontaneously hypertensive rats. Biochem Biophys Res Commun 133: 759–765. I Y, K S, C L, H NJ, K J-I, H CJ, 1992. Isolation and characterization of a novel cardiac adenylyl cyclase cDNA. J Biol Chem 267: 13553–13557. I H, T R, K T, T T, M M, K Y, 1988. Presence of three distinct molecular species of Gi protein: a subunit structure of rat cDNA and human genomic DNAs. J Biol Chem 263: 6656– 6664. I H, K T, M S, N S, K T, U M, I S, O E, K H, S K, K Y, 1986. Molecular cloning and sequence determination of cDNAs for a subunits of the guanine nucleotide-binding proteins Gs, Gi and Go from brain. Proc Natl Acad Sci USA 83: 3776–3780.

1022

J Marcil et al.

J DT, R R, 1987. Molecular cloning of five GTPbinding protein cDNA species from rat olfactory neuroepithelium. J Biol Chem 262: 14241–14249. J WV, W AM, M WR, A BS, Y PI, 1979. Sympathetic nerve activity and blood pressure in normotensive back-cross rats genetically related to the spontaneously hypertensive rat. Hypertension 1: 598–604. K S, C L, K J-I, N R, H NJ, H CJ, I Y, 1992. Cloning and characterization of a sixth adenylyl cyclase isoform: types V and VI constitute a subgroup within the mammalian adenylyl cyclase family. Proc Natl Acad Sci USA 89: 8774–8778. K J, T CL, F CD, Z JC, W PA, 1992. Molecular diversity in the adenylyl cyclase family: evidence for eight forms of the enzyme and cloning of type VI. J Biol Chem 267: 24858–24862. K R, J RW, H HC, E D, R F, E DC, L C, A T, 1994. Postnatal changes in the G-proteins, cyclic nucleotides and adenylyl cyclase activity in rabbit heart cells. J Mol Cell Cardiol 26: 1537–1550. L C, L CJ, 1978. Reduced number of b-adrenergic receptors in the myocardium of spontaneously hypertensive rats. Biochem Biophys Res Commun 83: 710–714. L OM, R NJ, F A, R RJ, 1951. Protein measurement with the folin phenol reagent. J Biol Chem 193: 265–275. L CW, T KM, C JL, M NM, 1988. Differential tissue expression and developmental regulation of guanine nucleotide regulatory proteins and their messenger RNAs in rat heart. J Biol Chem 263:13357–13365. ML AR, M G, H MD, C MC, 1993. G-protein in experimental hypertension. J Hypertens 11: 365–372. MM KK, 1989. Developmental changes of the Gproteins-muscarinic cholinergic receptor interactions in rat heart. J Pharmacol Exp Therapeutics 251: 372– 377. M T, Y H, 1986. Rat heart cell membranes contain three substrate for cholera toxin-catalyzed ADP-ribosylation and a single substrate for Pertussis toxin-catalyzed ADP-ribosylation. Biochem Biophys Res Commun 138: 1355–1361. M MC, B OE, I PA, 1993. Are cardiac

G-proteins altered in rat models of hypertension. J Hypertens 11: 355–363. N EJ, 1995. Heterotrimeric G proteins: organizers of transmembrane signals. Cell 80: 249–257. P SK, A-S MB, 1996. Modulation of G-protein expression by the angiotensin converting enzyme inhibitor captopril in hearts from spontaneously hypertensive rats: relationship with adenylyl cyclase. Am J Hypertens 9: 833–837. P RT, C J, M H-W, P M, I R, 1992. Two members of a widely expressed subfamily of hormone-stimulated adenylyl cyclases. Proc Natl Acad Sci U.S.A. 89: 9809–9813. R JD, S MD, G AG, 1986. Molecular basis for two forms of the G-protein that stimulates adenylate cyclase. J Biol Chem 261: 9587–9590. S Y, L C, R M, 1974. A highly sensitive adenylyl cyclase assay. Anal Biochem 58: 541– 548. S J, F EF, M T, 1989. Labeling the 5′ terminus of DNA with bacteriophage T4 polynucleotide kinase. In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, NY; Cold Spring Harbor Laboratory, 10.59–10.61. S TL, H PM, 1979. Central hemodynamics in the developmental stage of spontaneous hypertension in the unanesthetized rat. Hypertension 1: 508– 517. T R, Q LM, G AG, 1993. Regulation of purified type I and type II adenylyl cyclases by G protein bc subunits. J Biol Chem 268: 9–12. T C, A-S MB, 1992. Altered expression of G-protein mRNA in spontaneously hypertensive rats. FEBS Letters 313: 160–164. T K, I Y, H SR, I M-J, N JB, Y H, F M, S EE, H CJ, 1994. Changes in type VI adenylyl cyclase isoform expression correlate with a decreased capacity for cAMP generation in the aging ventricle. Circ Res 74: 596–603. T MJ, M E, B L, 1987. Inhibitory regulation of adenylyl cyclases. Evidence inconsistent with bc-complexes of Gi proteins mediating hormonal effects by interfering with activation of Gs. Mol Endocrinol 1: 669–676. T L, V Y, V M, H DV, N VV, 1992. Life Science 11: 817–824. W YH, C BB, B HR, 1992. G2-mediated hormonal inhibition of cyclic AMP accumulation. Science 255: 339–342.