Molecular forms of rat angiotensinogen in plasma and brain: identification by isoelectric focusing and immunoblot analysis

ELSEVIER

Regulatory Peptides 59 (1995) 31-41

Molecular forms of rat angiotensinogen in plasma and brain: identification by isoelectric focusing and immunoblot analysis Walter G. Thomas *, David Kerr, Conrad Sernia Neuroendocrine Laboratory, Department of Physiology and Pharmacology, University of Queensland, Brisbane, 4072 Qld., Australia Received 25 January 1995; revised 11 May 1995; accepted 12 May 1995

Abstract

Angiotensinogen (Ao) is the glycoprotein precursor of the vasoactive peptide angiotensin II. While Ao is synthesized as multiple molecular forms, the biochemical characteristics of this protein in blood and other tissues have not been defined. In this study, the charge heterogeneity of Ao in rat plasma, cerebrospinal fluid and that secreted by astrocyte and neuronal cultures was examined using analytical isoelectric focusing in combination with immunoblotting and quantitative image analysis. Normal rat male plasma Ao separated into 9 isoforms in the pl range 4.34-4.92 (1, 4.34; 2, 4.41; 3, 4.48; 4, 4.58; 5, 4.61; 6, 4.66; 7, 4.68; 8, 4.81; 9, 4.92); the percentage contribution of each to total plasma Ao was 13, 20, 23, 18, 2, 7, 10, 5, and < 1, respectively. A similar isoelectric focusing pattern was observed in female rat plasma with the exception that the relative contribution of isoform 6 was reduced to 2% of total Ao. Cerebrospinal fluid Ao displayed a more diverse charge heterogeneity than plasma Ao, focusing over a broader p l range of 4.42-5.24. Astrocytes and neurons secreted Ao isoforms in the pl range 4.44-5.29 and 4.42-4.95, respectively, with the astrocyte cultures showing additional bands towards the cathode. It was concluded that rat Ao is secreted as multiple charged forms that are regulated in a sex- and cell-specific manner. These differences between plasma and brain Ao suggest a functional diversity, a view which is supported by recent evidence linking Ao variants to hypertension. Keywords: Angiotensinogen; Isoelectric focusing; Renin-angiotensin system; Tissue-specific expression

1. Introduction

Angiotensin II (AlI), the principal effector hormone of the renin-angiotensin system (R_AS), regulates blood pressure and water and electrolyte balance, and thus plays a major role in the maintenance of cardiovascular physiology [1]. The octapeptide

* Corresponding author. Present address: Weis Center for Research, Geisinger Clinic, Danville, PA 17822, USA. Fax: + 1 (717) 2716701.

AII is also a t r o p h i c hormone with hypertrophic actions on the blood vessels and heart [2,3]. These roles of AII have implicated an overactivity of the RAS in cardiovascular dysfunction and hypertension. Generation of All in blood and tissues is by enzymatic cleavage from its obligate precursor protein, angiotensinogen (Ao), a glycoprotein found in relatively abundant amounts in plasma and cerebrospinal fluid [4,5]. Plasma Ao is the product of synthesis and constitutive release into the circulation from hepatocytes [6], while its origin in CSF is from astrocytes [7,8] and, to a lesser extent, from neurons [9].

0167-0115/95/$09.50 © 1995 Elsevier Science B.V. All fights reserved SSDI 0 1 6 7 - 0 1 1 5 ( 9 5 ) 0 0 0 7 1 - 2

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W.G. Thomas et al. / Regulatory Peptides 59 (1995) 31-41

Ao is coded by a single gene [10] which shows some polymorphism [11-13]. Rat plasma Ao occurs as two molecular weight forms, 56,000 and 54,000, termed Aol and Ao2 [14], which result from variable glycosylation at two asparagine residues on the 453 amino acid sequence. Studies in a number of species [5,15-19] have shown that plasma and brain Ao are also heterogeneous with respect to molecular charge. Thus, Ao in blood and tissues exists as multiple forms, differing from each other in molecular mass and carbohydrate composition. Interest in the biochemistry of Ao has recently been renewed by studies on the molecular variants of the human Ao gene which show an association between the expression of a particular Ao genotype and the development of hypertension [20]. Further support has been provided by studies in transgenic hypertensive mice which over-express the Ao gene in the liver and brain [21], and from spontaneously hypertensive rats which show high hypothalamic, but not hepatic, Ao mRNA content [22]. As well as supporting a role for Ao in hypertension, these animal studies suggest that tissues other than the liver may have decisive roles. It is also possible that the secretion of a different spectrum of Ao forms by different tissues is important in determining the local activity of tissue renin-angiotensin systems. Thus, the over-expression of the Ao gene may be the first of several events which lead to the secretion of a particular Ao form(s) that eventually precipitate a pathological state. In order to test such a hypothesis, it is first necessary to define the multiple forms of Ao secreted by relevant tissues such as the liver and brain. It was, therefore, the aim of this study to investigate the charge heterogeneity of rat Ao in plasma, CSF, and astrocyte- and neuronal-conditioned culture media using analytical isoelectric focusing (IEF) and immunoblotting with specific Ao antisera.

2. Materials and methods

(Sigma, St. Louis, MO) was iodinated with Na125I (Amersham, Arlington Heights, IL) by the chloramine-T method. Acrylamide, ampholines, PAG film, pH 4 - 6 isoelectric focusing markers, a mono S H R 5 / 5 FPLC column and chromatography system, and a 2000/150 power pack were obtained from Pharmacia LKB Biotechnology (North Ryde, NSW). Centriprep-10 and Microcon-30 concentrators were from Amicon (Beverly, MA), while all cell culture media and additives were from Cytosystems Pty (Castle Hill, NSW). Nitrocellulose Trans-Blot Transfer Medium was purchased from Biorad (Richmond, CA), and biotinylated anti-rabbit IgG streptavidin linked enzymes were from Dako (Bioscientific, Gymea, NSW). The MD30 + Image Analysis System was from Wild-Leitz (QLD). All other chemicals were obtained from Sigma. 2.2. Preparation of animals and collection of blood

and CSF Untreated male and female Wistar rats (250-300 g) were killed with chloroform and blood samples obtained by cardiac puncture. Blood was immediately placed in heparinized tubes on ice and then centrifuged at 4°C to yield plasma which was stored at - 2 0 ° C until used for isoelectric focusing. Repeated freeze-thawing of samples was avoided as it was found to alter immunoblot patterns. Cerebrospinal fluid (CSF) was carefully removed via puncture of the cisterna magna. Only CSF without traces of contaminating blood was retained. To determine the level of plasma contamination in such CSF samples, 4 rats were injected (i.v. via a jugular catheter) with 125I-labeled rat albumin (2 x 10 6 cpm) in 50 mM phosphate-buffered saline containing 1% BSA and after 2 min they were killed. One hundred microliters of blood and 20 /xl CSF were sampled and radioactivity measured in a LKB gamma-counter. No radioactivity could be measured in the CSF samples, even though the blood contained 12,400 _+ 600 (mean _ S.D.) cpm/100 /.tl. CSF samples were stored at - 2 0 ° C until use.

2.1. Materials 2.3. Isoelectric focusing/immunoblot procedure Wistar rats were obtained from the Central Animal Breeding House, University of Queensland (Pinjara Hills, QLD). Bovine serum albumin (BSA)

Isoelectric focusing was performed in 0.5 mm thick acrylamide gels (5% T / 3 % C) containing 10%

W.G. Thomas et al. /Regulatory Peptides59 (1995) 31-41

glycerol and an equal mixture of pH 4-6 and pH 4.2-4.9 ampholines to a final concentration of 8% (v/v). Polymerization (22°C, 1 h) was initiated by addition of ammonium persulfate and N , N , N ' , N ' - t e tramethylethylenediamine, and the mixture was cast onto gel-bond PAG film (25.8 x 12.4 cm) between two glass plates with 0.5-mm spacers. Sample application wells (2 cm from anode) were formed at the time of casting with 4 mm square pieces of 0.25 mm thick tape attached to the upper glass plate. Gels were prefocused for 20 min under powerlimiting conditions (12 W). The anolyte was 0.04 M glutamine and the catholyte was 0.1 M NaOH. During prefocusing, the amperage typically dropped from 45 to 14 mA, the voltage increased from 250 to 750 V and 200 Vh elapsed. Samples were then loaded 2 cm from the anode and focused for 3000 Vh (12 W, 7 mA, 2600 V). Gel temperature was maintained at 10°C by a recirculating cooling system attached to the LKB Ultrophor isoelectric focusing system. In preliminary experiments, anodal sample application and the run parameters described were found to be optimal for equilibration of banding and separation of Ao forms. At the end of focusing, gels were print-blotted [23] to nitrocellulose membranes (24 × 10.5 cm) for 30 min at 22°C. The nitrocellulose was then separated from the gel, washed in distilled-deionized water and dried. After rehydration in TST (50 mM Tris-HCl, pH 8.8, 0.5 M NaCI, 0.1% Tween 20), excess protein binding sites on the nitrocellulose were blocked by a 2 h incubation at 37°C in TST containing 1% Tween 20, 5% milk powder and 2% heat-inactivated horse serum. The membrane was then sequentially incubated in a rabbit anti-angiotensinogen antibody (R817 [24], 1:1000, 1 h, 22°C), a biotinylated anti-rabbit IgG antibody (1:2000, 1 h, 22°C) and a streptavidin-linked alkaline phosphatase complex (1:2000, 30 min, 22°C). Between steps, the nitrocellulose was washed 4 times for 5 min each with TST. The chromogen solution was nitro blue tetrazolium (0.033%) and 5-bromo-4-chloro-3-indolyl phosphate (0.017%) in 100 mM Tris-HC1, pH 9.5 containing 0.1 M NaCI and 5 mM MgCI 2. Isoelectric points (pI) were determined by comparison with standards (glucose oxidase, pl 4.15; soybean trypsin inhibitor, pI 4.55; fl-lactoglobulin A, pI 5.20; bovine carbonic anhydrase B, 5.85; human

33

carbonic anhydrase B, pI 6.55). The intra-assay variability of pI determinations was < 0.01 of a pH unit, while the between-assay variability, determined by including a quality control sample in all IEF runs, was between 0.01 and 0.02 of a pI unit. 2.4. SDS-PAGE electrophoresis

SDS-PAGE was performed under denaturing conditions in 10% acrylamide gels according to the procedure of Laemmli [25]. Gels were then electroblotted to nitrocellulose as previously described [26,27]. Visualization of Ao was as above for isoelectric focusing, with the exception that a streptavidin-linked horseradish peroxidase was used as the enzyme detection system and the chromogen solution was 0.1 M citrate-phosphate buffer, pH 5.0 containing 0.05% diaminobenzidine and 0.02% hydrogen peroxide. 2.5. Mono S chromatography procedure

Fifty microliters of male rat plasma was separated on a Mono S column as previously described [9] to separate the two molecular weight species, Aol and Ao2. Fractions containing Aol and Ao2 were desalted and concentrated by spin-dialysis in Microcon-30 microconcentrators. Samples were diluted to 500 ~1 with distilled-deionized H20, loaded in concentrators, and centrifuged at 14,000 g for 60 min at 4°C. The dilution and centrifugation procedure was repeated. The samples were then applied to isoelectric focusing. 2.6. Astrocyte and neuronal cell culture

Separate purified cultures of rat astrocytes and neuronal cells were prepared in 75-cm 2 flasks as previously described [9]. Astrocyte cultures, at confluence, and neuronal cultures, after 6 days, were changed to serum-free media (1:1 (v/v) Dulbecco's modification of Eagle's medium and Ham's F12, containing 0.8 g / l NaHCO 3 and 25 mM HEPES buffer) supplemented with 5 /~g/ml insulin and 100 ~ g / m l transferrin, and the Ao secreted into the media collected over a 3-day period. Samples were concentrated and desalted through Centriprep-10 concentrators, following the manufacturer's instruc-

W.G. Thomas et al. / Regulatory Peptides 59 (1995) 31-41

34

tions, and Microcon-30 concentrators as detailed above.

2. 7. Renin generation Male Wistar rats were anesthetized (Nembutal, Abbot, QLD), bilaterally nephrectomized and injected (s.c.) with 5 m g / k g body weight dexamethasone. They were sacrificed 24 h later and blood (1 ml) taken by cardiac puncture. Plasma, rich in angiotensinogen and low in renin activity, was obtained by centrifugation and stored at -20°C. Hog renin was prepared by the procedure of Corvol et al. [28]. Samples of plasma were diluted in 0.1 M phosphate buffer, pH 6.5, containing peptidase inhibitors (20 mM EDTA, 5 mM /3-mercaptopropanol and 0.3 mM phenylmethylsulphonyl fluoride) and sufficient renin to exhaustively cleave all angiotensin I in a 3-h incubation at 37°C.

l

2.8. Image analysis To semi-quantitate the IEF data a MD30 + Image Analysis system was used to digitally capture images of the immunoblots and to quantify individual bands. The integrated intensity of each band was corrected for background on a per area basis and expressed as a percentage of the sum total of intensity of all the bands. Linearity of detection was observed between 0.25 and 2 /zl plasma, a range encompassing the typical sample loading of 1 /xl plasma.

3. Results

The specificity of Ao antisera and the molecular size of Ao in plasma, CSF and brain cultures were determined by SDS-PAGE immunoblotting (Fig. 1). Purified plasma Ao (prepared as described in [24]), and Ao in crude plasma, were visualized as two

// -

92-5

-66

AO1 AO2" -45

- 31 Fig. 1. Immunoblot analysis for Ao of SDS-PAGE-separated proteins from rat pure Ao, plasma and CSF, and from astrocyte- and neuronal-conditioned media (indicated above each lane). The positions of A o l and Ao2 are labeled to the left and the position of marker proteins are shown (in kDa) on the fight.

W.G. Thomas et al. / Regulatory Peptides 59 (1995) 31-41

major bands of molecular weights 56,000 and 54,000, corresponding to Aol and Ao2 respectively. No additional bands were detected. In normal rat plasma, Aol is the predominant form, being present at concentrations two to three times that of Ao2 (see immunoblot in Fig. 2 and radioimmunoassay data in Fig. 3). CSF Ao was of a similar molecular size to plasma Ao, but appeared as a broad band from 57,000 to 54,000, suggesting a more heterogeneous phenotype. Ao secreted into the serum-free media supplying astrocyte and neuronal cultures was present as bands of molecular weight 57,000-54,000 and 56,000-55,000, respectively. When male rat plasma proteins were separated by analytical IEF and the forms of Ao identified by immunoblotting, multiple bands were observed. Fig. 2 shows a comparison of rat plasma Ao separated by SDS-PAGE and IEF. The two molecular weight forms of Ao (Aol and Ao2) observed with SDSPAGE were resolved into 9 separate forms, labeled 1-9 from anode to cathode. The isoelectric points of these 9 Ao forms encompassed the pH range 4.344.92 and the variation in stain intensity between the

5

35

Aol '

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il~ll .......

~

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~0

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37

42

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Fig. 2. Immunoblot analysis for Ao in normal rat plasma separated by SDS-PAGE or by analytical isoelectric focusing in a pH 4 - 6 gradient. The 9 isoforms of plasma Ao, separated by isoelectric focusing, are labeled anode ( + ) to cathode ( - ) and their isoelectric points indicated to the right.

6b

- ~-I 6

Fig. 3. Identification of the An isoforms of Aol and An2. Normal rat plasma proteins were separated by MonoS chromatography and the elution of Aol and An2 monitored by a radioimmunoassay (upper panel). Fractions containing Aol (3"/) and An2 (42), as well as unfractionated plasma, were then subjected to isoelectric focusing and An immunoblotting (lower panel). The position of focused p I markers is shown to the left of the plasma lane and the 9 plasma An isoforms are identified to the right of Aol and An2, respectively. Similar results were obtained in 3 additional experiments.

isoforms indicated that they were present in different amounts. To determine which of these 9 IEF forms of plasma Ao corresponds to the major molecular weight forms Aol (56,000) and Ao2 (54,000), normal rat plasma Ao was separated into Aol and Ao2 by Mono S FPLC chromatography [9]. Fractions containing Aol and Ao2 were identified by radioimmunoassay [9] and then subjected to IEF immunoblotting. As shown in Fig. 3, plasma Ao is separated into 2 major peaks corresponding to Aol and Ao2 by MonoS (cation exchange) chromatography; the more highly glycosylated, and thus more

36

W.G. Thomas et al. / Regulatory Peptides 59 (1995) 31-41

3o]

Male

C Q

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%

2 3 4 f5 =6 =7 'a '9 ' anglotenalnogenform number

4.58) represent the principal Ao isoforms, together comprising ~ 60% of total plasma Ao. The major isoform of A o l was band 3 ( p I 4.41), whereas band 7 (pI 4.68) was the principal component of Ao2. Also shown in Fig. 4 are the relative contributions of the various Ao IEF forms to total Ao in female rat plasma. The profiles were similar with the exception that band 6 ( p I 4.66) in female rat plasma is reduced 73% (7.1 to 1.9% of total plasma Ao) compared to male plasma. To demonstrate that the 9 forms identified by IEF immunoblotting all represented authentic Ao, plasma

RENIN 30"

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Fig. 4. Quantitationby image analysis of the isoelectric focusing immunoblot patterns for normal male (upper panel) and female (lower panel) rat plasma. Captured images were scanned and the integrated intensitiesfor each of the 9 forms were expressed as a percentage of the total Ao. This quantification is based on the assumption that all isoforms are detected equally by the polyclonal antisera(R817) which was raised against highly purifiedrat plasma Ao containingall 9 isoforms. Resultsare the mean+ S.E.M. from 8 animals. ~', significance difference (< 0.001, Student's t-test) from correspondingisoform in male plasma.

negatively charged, A o l eluting first. By IEF immunoblotting, A o l comprised 5 (1, 2, 3, 4 and 6) of the 9 bands of plasma Ao, while Ao2 corresponded to the other 4 bands (5, 7, 8 and 9) (Fig. 3). The relative contribution of each form to the total plasma Ao, as well as the forms corresponding to A o l and Ao2, was determined by image analysis; the results are shown in Fig. 4. In normal male rat plasma, bands 2 ( p I 4.41), 3 ( p I 4.48) and 4 ( p I

4.55

-

5.20

-

5'85 I

Fig. 5. Isoelectric shift in plasma Ao immunoblotpattern after cleavage by renin. Plasma from a nephrectomizedand dexamethasone-treated rat (low renin, high Ao), with or without exhaustive cleavage by hog renin, was isoelectricfocused and immunoblotted for Ao. The 9 Ao isoforms (bracketed) shifted about 0.15 of a pH unit towards the anode. The anode (+) and cathode ( - ) , and the position of the isoelectricpoint markers are shown to the left. The arrow indicatesthe position of sample applicationwells.

W.G. Thomas et al. / Regulatory Peptides 59 (1995) 31-41

+

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37

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Fig. 6. Comparison of the IEF immunoblot pattern for plasma and brain Ao. Samples of normal male rat plasma, astrocyte- and neuronal-conditionedmedia, and rat CSF were subjected to isoelectric focusing and immunoblottedfor Ao. Plasma Ao shows the typical 9-band pattern, while the heterogeneityof brain Ao differed from that of plasma. The position of pl markers is shown to the left.

from nephrectomized-dexamethasone treated (low renin, high Ao) male rats was incubated with, and without, an excess of hog renin to fully cleave all angiotensin I peptides from plasma Ao. The samples were then subjected to IEF immunoblotting, as shown in Fig. 5. In the absence of renin, the IEF Ao profile for nephrectomized plasma was in the same p I range

(4.3--4.9) as normal male plasma, although slightly different in relative composition of forms (presumably due to the effects of dexamethasone and nephrectomy on Ao isoform expression). Removal of the decapeptide (AI) resulted in an anodal shift in the Ao banding pattern by approximately 0.15 of a pH unit. This shift is due to the removal of the decapeptide

Table 1 Contributionof Ao isoforms to total Ao secreted by astrocytes and neurons Form number Astrocyte Ao pl % of total Ao NeuronalAo pI % of total Ao

1

2

3

4

5

6

7

8

9

4.44 2.5

4.51 4.8

4.55-4.61 5.0

4.63-4.68 13.7

4.70-4.78 14.6

4.82-4.88 37.8

4.99 18.2

5.17 2.6

5.29 0.8

4.42-4.44 16.6

4.50 18.6

4.54 5.1

4.60-4.66 40.7

4.68 10.7

4.82 6.4

4.95 1.7

Astrocyte- and neuronal-conditionedmedia were concentrated and desalted as described in the text. Samples were subjected to IEF, immunoblottedfor Ao and the bandingpatterns quantifiedby image analysis.Ao isoforms are numberedanode to cathode. Some forms are expressed as pl ranges to indicate multiple, closely spaced Ao bands or diffuse bands.

38

W.G. Thomas et al. / Regulatory Peptides 59 (1995) 31-41

Al sequence from Ao with the resultant protein residue, termed des-Al-Ao, carrying a more negative charge and therefore focusing closer to the anode. The movement of all 9 immunoreactive bands, in response to renin cleavage, identifies these as authentic Ao. In addition, another 3 Ao antisera, described in a recent immunocytochemical study [24], produced identical IEF immunoblot patterns and banding intensities (data not shown), confirming the identity of the 9 IEF forms. Brain Ao showed a distinctly different profile compared to plasma Ao. Fig. 6 shows a comparison of IEF immunoblotting for Ao from plasma, CSF and astrocyte- and neuronal-conditioned media. While plasma Ao displayed the typical 9-band pattern between pI 4.3 and 4.9, brain Ao focused differently. CSF Ao was consistently identified as multiple diffuse and discrete bands in the pI range 4.4-5.2; the majority of CSF Ao resolved as a number of forms in the pI range 4.42-4.65. Ao secreted by astrocytes and neurons also resolved into multiple forms. Astrocyte Ao displayed 9 forms in the p l range 4.44 to 5.29, while neuronal Ao consisted of 7 forms in the pI range 4.42 to 4.95. The pI values and percentage contribution to total Ao are listed in Table 1. In contrast to the 3 major forms (forms 2, 3 and 4) of plasma Ao (pl 4.41, 4.48 and 4.58), the predominant forms of astrocyte Ao, together comprising more than 50% of total, were forms 6 (pl 4.82-4.88, 37.8%) and 7 (pI 4.99, 18.2%). The major component of neuronal Ao was form 4 (pI 4.60-4.66, 40.7%). The IEF immunoblotting profile for CSF appeared to be a composite of the astrocyte and neuronal Ao patterns (Fig. 6).

4. Discussion

The combination of analytical IEF, immunoblotting and image analysis is a powerful technique for the identification and quantitation, in complex biological mixtures, of specific protein components in defined pH (pI) ranges. The present study was designed to examine the heterogeneity of Ao in rat plasma and CSF and that secreted by astrocytes and neurons. Our results confirm that plasma and brain Ao are present as multiple forms. We extended these observations to include: (1) the identification of 9

separate isoforms of plasma Ao, including a sexspecific regulation of one form; (2) the authentication of all 9 isoforms by renin generation; (3) the observation of a charge heterogeneity for brain Ao that differs from plasma Ao, reflecting tissue-specific post-translational processes; and (4) the description of differences in Ao isoforms secreted by separate highly purified cultures of astrocytes and neurons, that suggest multiple pathways for Ao synthesis and, by inference All generation, within a single tissue. IEF has been used previously to provide evidence for multiple Ao isoforms in plasma. Genain et al. [5], using column IEF, identified 8 human plasma Ao isoforms in the pI range, 4.3-4.9; des-AI-Ao was more negatively charged than intact Ao; and CSF Ao was expressed as multiple forms of more basic pI than plasma Ao. Despite the difference in species and type of IEF used, very similar results were obtained in the present study. Hilgenfeldt and Schott [17] examined the charge heterogeneity of highly purified rat plasma Ao and although there are similarities between their study and ours, clear differences are also apparent. Using IEF, Hilgenfeldt and Schott resolved pure rat plasma Ao, as well as purified Aol and Ao2, into multiple isoforms in the p l range 4.72-5.13. They showed three major and two minor forms of Aol, and two major and two minor forms of Ao2, but some of the bands overlapped and they were not able to identify these as distinct forms. We also identified 5 isoforms for Aol and 4 isoforms for Ao2, yet these focused in a lower pI range 4.34-4.92 and were resolved into clearly distinct bands. Both studies also used the specific cleavage by renin to confirm the authenticity of Ao isoforms. Deglycosylated pure Ao shifted 0.1 pH units towards the anode in the study of Hilgenfeldt and Schott [17], and we demonstrated that all 9 plasma Ao forms shifted 0.15 pH units upon renin cleavage. The discrepancy over the pI ranges for plasma Ao and our enhanced separation of the 9 Ao isoforms can be explained by procedural differences between the two studies. In preliminary experiments, we observed that application of samples near the cathode, as used by Hilgenfeldt and Schott [17], caused heavy precipitation of samples and that their focusing conditions (1 h focusing time at 2.5 mA, 550 V) were insufficient for equilibrium of Ao banding. In our hands, optimal Ao focusing required

W.G. Thomas et al. / Regulatory Peptides 59 (1995) 31-41

prefocusing of the gel, anodal sample application, and longer focusing times (3000 Vh, ~ 2 h) under higher field strengths (12 W, 7 mA, and 750 V increasing to 2600 V during the run). Our use of an enhanced pH 4-6 gradient, extended in the pH range 4.2-4.9, also afforded better separation of Ao isoforms. Moreover, our studies differed with respect to the source of Ao. While we examined Ao isoforms in unfractionated rat plasma, they used Ao from the plasma of nephrectomized rats, purified by multiple biochemical separation processes. The influence of nephrectomy, multiple chromatography steps, including freeze-thawing, on the integrity of Ao focusing is uncertain, but it is our experience that discrete Ao banding is compromised by excessive handling. Hence, we feel that the deliberate use of fresh plasma, combined with the optimized IEF and immunoblotting procedures outlined in this report, has given the most reliable determination of rat plasma Ao isoforms yet. Moreover, the application of image analysis, to quantify these isoforms, has provided the platform to investigate the regulation of Ao phenotype expression. Brain Ao has a number of biochemical similarities to plasma Ao. In studies by ourselves and others (see [24] and [26]), antibodies raised against purified plasma Ao readily detect the protein in brain. Both plasma and brain Ao appear to be translated from the same gene [29], and based on the SDS/PAGE/immunoblotting, in this study, brain (CSF, astrocyte and neuronal) and plasma Ao are of similar (54,00057,000), yet not identical, molecular weight. In contrast, these sources of Ao are distinctly different with respect to charge heterogeneity, presumably due to tissue- and cell-specific post-translational modifications, such as glycosylation and phosphorylation. While plasma Ao focused in the pl range 4.3-4.9 with ~ 70% attributable to isoforms in the pI range 4.41 to 4.58, CSF Ao focused over a broader, higher pI, as did the major isoforms of astrocyte Ao. The expression of an overall more basically charged brain Ao is supported by previous studies in the rat [9], human [5] and dog [19]. Also, CSF Ao appears to be the mixture of Ao isoforms from both astrocytes and neurons. This is particularly interesting since it supports the idea [9,29] that Ao synthesis in the brain is a complex process involving both cell types. That the pattern of neuronal Ao was distinct from that of the

39

other sources, especially astrocyte Ao, indicates that neuronal Ao is not merely the result of astrocyte contamination in culture, although the possibility of selective degradation by the separate cultures cannot be entirely dismissed. The exact mechanisms and modifications responsible for these differences, as well as their function, remain to be elucidated. The regulation of the individual forms of Ao in a sex-dependent manner and the tissue-specificity of the Ao isoform pattern strongly suggests that the heterogeneity of the Ao phenotype has a functional significance. Several observations support this view. Firstly, the rate of clearance for Aol and Ao2 from the circulation differs, and the ratio of Aol and Ao2 varies with nephrectomy [30]. Secondly, enhanced Ao synthesis, or overactivity of the RAS, appear to be major contributors to cardiovascular diseases and hypertension [21,22,31]. Thirdly, Jeunemaitre et al. [20] have shown that molecular variants of Ao are associated with a predisposition to hypertension in humans, and that people with the Ao variant display elevated plasma Ao levels. These observations raise the possibility that all 9 plasma Ao forms are not equally effective substrates for renin and/or susceptible to degradation and clearance. The use of isoelectric focusing to separate and isolate each of Ao isoforms should permit the investigation of these possibilities. The heterogeneity of Ao isoforms and the differences in expression between tissues are also apparent with other components of the RAS. Renin, the aspartyl protease that cleaves the decapeptide angiotensin I from Ao, is synthesized as a preprorenin which requires cleavage to yield active renin, is variably glycosylated into multiple isoforms, and its mRNA is present in a number of tissues [32]. Recently, renin isoforms have been demonstrated by IEF in human [33] and rat [34]. In the rat, isolated isoforms differed in their enzymatic activity and capacity to cause natrnresis. Angiotensin converting enzyme (ACE), the enzyme that converts AI to All, is found in multiple tissues, is variably glycosylated [35] and has distinctly different isoforms in different tissues [36]. All receptors show tissue-specific expression and developmental regulation, and have been subtyped on the basis of selectivity for various peptide, and non-peptide, agonists and antagonists, as well as molecular cloning [37]. This multi-level het-

40

W.G. Thomas et al. / Regulatory Peptides 59 (1995) 31-41

erogeneity and regulation of all components of the RAS endows it with the flexibility to tailor its activity for specific actions at multiple target tissues.

Acknowledgements We thank Kevin Bell, Helen Authur and Scott Robinson from the Equine Research Centre, University of Queensland, for assistance in establishing the isoelectric focusing procedure. We also thank Trixie Shinkel, and Karen Greenland for technical assistance. This work was supported by Grants from the National Health and Medical Research Council (NHMRC) of Australia to C.S.W.G.T. is the recipient of a NHMRC C.J. Martin Fellowship.

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