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Tyrosine hydroxylase gene expression in ventrolateral medulla oblongata of WKY and SHR: a quantitative real-time polymerase chain reaction study
Autonomic Neuroscience: Basic and Clinical 98 (2002) 79 – 84 www.elsevier.com/locate/autneu
Tyrosine hydroxylase gene expression in ventrolateral medulla oblongata of WKY and SHR: a quantitative real-time polymerase chain reaction study Valin Reja a, Ann K. Goodchild a, Jacqueline K. Phillips b, Paul M. Pilowsky a,* a
Hypertension and Stroke Research Laboratories, Department of Physiology, University of Sydney, and Department of Neurosurgery, Royal North Shore Hospital, 2065 Sydney, Australia b The Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, 6150 Western Australia, Australia
Abstract The aims of this study were, first, to determine quantitatively the levels of tyrosine hydroxylase (TH) gene expression in both peripheral and central sites related to blood pressure regulation, and to compare the level of expression in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Second, to see if any relationship exists between TH gene expression and systolic arterial blood pressure. Total RNA was isolated from adrenal glands and from tissue punches taken from the C1 and A1 cell groups in the rostral and caudal ventrolateral medulla oblongata of the brainstem, respectively. Total RNA was reverse-transcribed into cDNA followed by quantitative fluorescence detection polymerase chain reaction for TH cDNA. The levels of TH gene expression measured as a percentage of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in SHR, were significantly higher ( f 2.5-fold) compared to WKY in all sites examined ( P < 0.01). There was a positive and significant relationship between systolic blood pressure and TH gene expression in the C1 area of the brainstem in both WKY (n = 5, P < 0.05) and SHR (n = 6, P < 0.05). Taken together, these results suggest that elevated gene expression of the TH gene is associated with the phenotypic characteristic of SHR. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Catecholamines; C1 adrenaline cells; A1 noradrenaline cells; Adrenal gland; Baroreflex; Sympathetic
1. Introduction It has long been known that the catecholamines dopamine, noradrenaline and adrenaline, both peripherally and centrally, are involved in the regulation of arterial blood pressure (Guyenet and Cabot, 1981; Goodchild et al., 1984; Pilowsky et al., 1986). Catecholamines are synthesized from tyrosine through a pathway of enzymes. The initial and ratelimiting enzyme of the pathway is tyrosine hydroxylase (TH), which converts tyrosine into dihydroxyphenylalanine (DOPA) (Carlsson and Waldeck, 1972). Expression of the TH enzyme is restricted to catecholaminergic neurons and chromaffin cells (Chamba and Renaud, 1983). The chromaffin cells are primarily located in the adrenal gland where the noradrenaline, but not the adrenaline-secreting cells, are under the control of the baroreflex (Morrison et al., 1991). * Corresponding author. Hypertension and Stroke Research Laboratories, Ground Floor, Block 3, Royal North Shore Hospital, 2065 Sydney, Australia. Tel.: +61-2-9926-8080; fax: +61-2-9926-6483. E-mail address: [email protected] (P.M. Pilowsky).
Centrally, catecholaminergic neurons, which play key roles in blood pressure control, are localised to specific areas of the pons and medulla oblongata. The C1 cell group, which in addition to TH also expresses the adrenaline-synthesising enzyme phenylethanolamine-N-methyltransferase (PNMT), is located in the rostral ventrolateral medulla (Goodchild et al., 2000; Phillips et al., 2001). Rostral C1 neurons project directly to sympathetic preganglionic neurons (SPN) in the spinal cord (Lipski et al., 1995), whereas caudal C1 neurons innervate neurons within the hypothalamus and forebrain (Verberne et al., 1999). The A1 noradrenaline containing cell group lies caudal to the C1 neurons and provides a major excitatory drive to the vasopressin-releasing neurons of the hypothalamus (Day et al., 1980). An increased level of TH gene expression has been associated with hypertension. Indeed, Kumai et al. (1994, 1996), using Northern blot analysis, reported increased amounts of TH mRNA within the adrenal medulla and medulla oblongata of SHR compared to their WKY counterparts, and correlated the increases in mRNA to increased TH activity.
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Although increased amounts of TH mRNA in SHR have been reported, it is not known if these differences are ‘‘global’’ or if they are specifically regional. Furthermore, earlier methodologies may be less sensitive in determining precise levels of mRNA (Medhurst et al., 2000). The aims of this study were, first, to quantitate the amount of TH mRNA in both peripheral and central sites; these were the adrenal gland, C1 cell group and A1 cell group. Second, to compare TH gene expression levels between WKY and SHR strains to determine if any differences were sitespecific or ‘‘global’’. Third, to determine if a relationship exists between systolic arterial blood pressure and TH gene expression in both WKY and SHR animals. In order to obtain accurate measurements of TH expression, relative quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was used (Heid et al., 1996).
paraformaldehyde solution then cut into 50-Am-thick slices. Immunocytochemistry was conducted according to Goodchild et al. (2000). Slices were incubated for 48 h at room temperature with sheep anti-TH (1:1000, Pelfreez, Rogers, AR, USA) in Tris phosphate-buffered saline (TPBS, pH 7.4) containing 0.05% merthiolate with 5% normal horse serum (NHS). Sections were then washed 3 for 20 min and incubated for 12– 16 h with biotinylated donkey anti-sheep secondary antibody (1:500) containing 2% NHS in TPBS. Following washing, sections were incubated in ExtrAvidin (Sigma, St. Louis, MO, USA) conjugated to horseradish peroxidase (HRP) at 1:1000 for 4 h. Sections were washed and reacted with nickel-intensified diaminobenzidine (DAB), mounted onto gelatin slides, dehydrated, coverslipped and viewed under a bright-field microscope at 40 magnification. 2.3. Real-time RT-PCR
2. Methods 2.1. Animals and tissue preparation All experiments were conducted in accordance with the Australian code of practice for the care and use of animals for scientific purposes as endorsed by the National Health and Medical Research Council of Australia. The Animal Care and Ethics Committee of the Royal North Shore Hospital approved all protocols. Male, 20 –25 weeks old, WKYand SHR were used. Systolic arterial blood pressure was measured at 18 weeks of age using the tail-cuff method (n = 11). This approach was validated in our laboratory using simultaneous femoral artery recording and shown to be an accurate reflection of true systolic pressure with a variance of less than 1%. Individual animals were used to remove each targeted area. Tissue sections that contained the C1 (adrenaline synthesising) cells (n = 11) and the A1 (noradrenaline synthesising) cells (n = 6) were removed from euthanased (sodium pentobarbitone, 90 mg kg 1) animals. Tissue punches of the above-mentioned cell groups were taken from 1-mm-thick sections using an 18-gauge needle as described by Comer and Lipski (1997) (Fig. 1A). Although the sections were thicker compared to previous studies (Comer and Lipski, 1997), both the A1 and C1 cell groups were specifically isolated. This was confirmed by viewing thinner-sliced sections (50 Am), of the 1-mm sections, under a light microscope. Punches were also taken from a region 1 mm dorsal to the RVLM, known not to contain any TH-synthesising neurons and were used as negative controls (punch negatives, n = 6). The adrenal gland was removed and used for TH mRNA relative quantitation (n = 6). 2.2. Immunocytochemistry Immunocytochemistry for TH in punched tissue sections was conducted to ensure tissue punches contained TH positive cells. Punched sections were post-fixed in 4%
Total RNA was extracted using an RNA isolation reagent (ABgene, Epsom, Surry, UK) with chloroform and centrifuging the mixture for 10 min at 12,000 rpm before removing the aqueous phase containing the total RNA. Total RNA was precipitated with isopropanol and 0.3 M sodium acetate (pH 4), and then washed in 70% (v/v) ethanol before re-suspending the RNA pellet into RNasefree water. An RQ1 DNase (1 U Ag 1 RNA) was used according to the manufacturers protocol (Promega, Madison, WI, USA) to remove any genomic DNA contamination in the RNA samples. RNA was assayed using a UV spectrophotometer (Beckman Coulter, Fullerton, CA, USA) at a wavelength of 260 nm, where 1 absorbance unit at 260 nm is equal to 40 Ag ml 1 RNA. The RNA was reversetranscribed (RT) using a Reverse Transcription System (Promega) according to the manufacturers’ protocol. RT negative controls were run to determine if any genomic contamination was present. Relative quantitative PCR was achieved using a fluorogenic intercalating dye and intron spanning primers to TH cDNA published by Comer et al. (1999). Each 20-Al reaction contained 3.5 mM MgCl2, 1 PCR buffer, 0.5 AM each primer, SYBRGreen (1:106, Molecular Probes, Eugene, OR, USA), 0.625 U Taq polymerase (Promega) and template cDNA. No template controls (NTC) were run to determine the level of primer dimer formation. Amplification was performed in 0.2-ml tubes on a Rotor Gene 2000 real-time PCR machine (Corbett Research, Sydney, Australia, http://www.corbettresearch.com). PCR parameters were an initial denature at 94 jC for 180 s followed by 45 cycles of 94 jC for 15 s, 57 jC for 20 s and 72 jC for 25 s. Fluorescence data were acquired using a low gain at 85 jC, where only specific products were assumed to be present. A melt analysis on resulting PCR products supported this for all samples measured. In addition, PCR products were run on 2% Tris borate ethylenediaminetetraacetic acid (TBE) agarose gels to confirm that correct band sizes were present (Fig. 1B). A standard curve was gen-
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Fig. 1. Panel A: Nickel-intensified DAB labelling of TH on a punched tissue section showing TH positive cells on the right-hand side with only a few cells present around the punch on the left hand side. Panel B: Real-time RT-PCR amplification plot showing GAPDH products (dash and/or dotted lines) from serial dilutions of cDNA obtained from the adrenal gland of an SHR, TH product (solid line) taken from undiluted samples of the same cDNA, and a no template control (NTC, dashed line). Threshold cycle values (Ct) were taken at an equal point where all samples were within the exponential phase of amplification. Panel C: Agarose gel electrophoresis of RT-PCR products. Amplification of GAPDH internal cDNA standard was achieved using 10 , 1 , 0.1 and 0.01 concentrations of cDNA taken from an SHR adrenal gland and GAPDH primers to yield a 239-bp product (lanes 1 – 4). Lane 5 was a GAPDH primer no template control (NTC) and lane 6 was a negative RT control for genomic GAPDH. Amplification of TH cDNA yielded a 322-bp product from the same tissue sample (adrenal gland, lane 8). Lane 9 was the TH primer NTC and lane 10 was a negative RT control for genomic TH. Lane 7 = pGEMR molecular weight marker (Promega). Panel D: Graph comparing levels of TH mRNA, expressed as percentage TH cDNA/GAPDH cDNA, from the adrenal gland, C1 cell group, A1 cell group and punch negatives of WKY (black) and SHR (white) rat strains ( ** P < 0.01; *** P < 0.001). Panel E: Graph showing a positive and significant linear relationship ( P < 0.05, r2 = 0.87, n = 5) between systolic arterial pressure and TH gene expression (as a percentage of GAPDH gene expression) in the C1 cell group of WKYanimals (black boxes). Dashed lines represent 95% confidence intervals. Panel F: Graph showing a positive and significant linear relationship ( P < 0.05, r2 = 0.66, n = 6) between systolic arterial blood pressure and TH gene expression (as a percentage of GAPDH gene expression) in the C1 cell group of SHR animals (white boxes).
erated by amplifying, in serially diluted cDNA samples, the endogenous cDNA standard glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using primer sequences published
by Comer and Lipski (1997). Although it has been reported that in cell cultures systems (including cancer cell lines) the level of GAPDH gene expression can vary (Zhong and
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Simons, 1999), most studies find GAPDH to be expressed in equal concentrations between different tissues whatever the experimental conditions (e.g. partial nerve ligation) (Medhurst et al., 2000; Macdonald et al., 2001). Furthermore, changing conditions do not apply to this study as it only focuses on basal levels of gene expression in WKY and SHR. By plotting the Ct values (cycle threshold set where the exponential phase of the PCR reaction began) ( y) against the concentration of GAPDH cDNA (x), a standard curve was generated from which the unknown amount of TH gene expression within the sample was determined (Heid et al., 1996). 2.4. Data analysis Data were normalized and expressed as TH cDNA (as a relative percentage of GAPDH cDNA in sample) F S.E.M. for each group of animals. An unpaired Student’s t-test was used to determine any significance between groups (a = 0.05). A correlation coefficient between systolic blood pressure and TH cDNA (as a relative percentage of GAPDH cDNA in sample) was determined in WKY and SHR animals.
3. Results Tail-cuff measurements showed, as expected, that SHR have a significantly higher systolic blood pressure than WKY for those animals measured (SHR, 180 F 5 mm Hg, n = 6; WKY, 103 F 2 mm Hg, n = 5, P < 0.001). This also showed that the SHR hypertensive phenotype was being appropriately expressed within our colony. Tissue punches of the C1 and A1 cell groups within the medulla oblongata of separate animals were removed and total RNA extracted. Immunocytochemistry on the remaining tissue sections revealed that a good proportion of the TH positive cells within these cell groups was collected (Fig. 1A). RT-PCR was conducted on all RNA samples with positive amplification of the GAPDH cDNA internal standard fragment (239 bp) and TH fragment (322 bp) (Fig. 1B and C). Very little nonspecific product was observed (Fig. 1C), which was also confirmed by a melt analysis of each PCR (data not shown). There was no evidence seen of genomic DNA amplification within RT negative controls (Fig. 1C). Although some primer dimer formation was present within PCR products, this did not affect quantitation as the Ct values were obtained at the beginning of the exponential phase where only specific product amplification was above the detection threshold. Quantitation of TH cDNA within the three sites examined gave the results shown in Fig. 1D. The amount of TH gene expression within the adrenal gland of the SHR was significantly higher than in WKY with a ratio of 2.5:1 (SHR, 25.1 F 1 TH cDNA as a relative percentage of GAPDH cDNA in sample, n = 3; WKY, 10.4 F 2 TH cDNA as a relative percentage of GAPDH cDNA in sample, n = 3, P < 0.01). TH gene expression within the C1 cell group of
the SHR was also significantly higher than in WKY (SHR, 3.6 F 0.2 TH cDNA as a relative percentage of GAPDH cDNA in sample, n = 6; WKY, 1.6 F 0.1 TH cDNA as a relative percentage of GAPDH cDNA in sample, n = 5, P < 0.001). Indeed, the amounts of TH cDNA within the C1 cells of the SHR were 2.2 times that of the WKY, a similar ratio to that obtained between strains for the adrenal gland. Levels of TH gene expression within the A1 cell group of the SHR was significantly higher than in WKY (SHR, 1.5 F 0.1 TH cDNA as a relative percentage of GAPDH cDNA in sample, n = 3; WKY, 0.5 F 0.1 TH cDNA as a relative percentage of GAPDH cDNA in sample, n = 3, P < 0.01). The ratio of TH cDNA between strains was 3:1. Tissue punches taken from regions known not to contain any TH (punch negatives) showed background levels of TH gene expression that were not significantly different between strains (SHR, 0.1 F 0.1 TH cDNA as a relative percentage of GAPDH cDNA in sample, n = 3; WKY, zero TH cDNA as a relative percentage of GAPDH cDNA in sample, n = 3). There was no significant difference between the ratios obtained for SHR and WKY at each of the sites studied (SHR/ WKY, C1 = 2.2, A1 = 3, adrenal gland = 2.5). Finally, there was a positive and significant correlation between systolic arterial blood pressure and TH cDNA as a relative percentage of GAPDH cDNA in sample in C1 cells of both WKY (n = 5, r2 = 0.87, P < 0.05) and SHR (n = 6, r2 = 0.66, P < 0.05) (Fig. 1E and F).
4. Discussion Differences in the levels of TH gene expression have been reported previously in the adrenal gland and in the whole medulla oblongata (Kumai et al., 1994, 1996; Goc and Stachowiak, 1994; Chan and Sawchenko, 1995; Sterling and Tank, 2001). Most of these studies examined changes in TH gene expression in normotensive rats at rest, or cultured cells, in response to a stimulus. For example, Goc and Stachowiak (1994) showed that upon addition of angiotensin II to cultured cells obtained from the medulla oblongata, there was an increase in TH gene expression. In two earlier studies, Kumai et al. (1994, 1996) reported an up-regulation of TH gene expression in the adrenal medulla and in the whole medulla oblongata of WKY and SHR aged 25 weeks. However, in these studies, the TH gene expression was determined in whole medulla oblongata. This approach would include all of the catecholamine cell groups in this brain region: including the A1, A2, C1, C2, C3 and TH-expressing neurons in the area postrema. Furthermore, these authors used Northern blot analysis as a ‘‘quantitative’’ technique, although without demonstrating that the changes were linear over the range used for measurement. Nevertheless, this work demonstrated a clear difference in TH gene expression between WKY and SHR when comparing whole medulla oblongata and adrenal medulla.
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In the present study, we extend the findings of Kumai et al. (1994, 1996) by examining specific brain regions that are known to be involved in the regulation of arterial blood pressure and determined stationary levels of TH gene expression in WKY and SHR. Our results demonstrate, for the first time, that there is an up-regulation of TH gene expression in SHR within sitespecific regions of the medulla oblongata involved in blood pressure regulation viz., the C1 and A1 cell groups. Interestingly, the ratio of TH gene expression at a given site between the species was not significantly different to that found at other sites. In fact, at all three sites (adrenal gland, A1 and C1), SHR expressed about 2.5 times as much TH mRNA as WKY rats. This result demonstrates that the up-regulation of TH gene expression is ‘‘global’’ rather than being restricted to individual groups of catecholamine-synthesising cells. This result raises the question of whether or not the increase in TH gene expression is a cause or consequence of the hypertensive phenotype. In addition, analysis of the data revealed a positive and significant correlation between systolic arterial blood pressure and TH gene expression (as a percentage of GAPDH gene expression) in C1 cells of WKY (n = 5, P < 0.05) and SHR (n = 6, P < 0.05) animals. Indeed, there is considerable evidence that changes in TH gene expression result in changes in systolic blood pressure. For example, Kumai et al. (2001) have shown that administration (i.v.) of a TH antisense to SHR causes a decrease in systolic blood pressure along with decreases in TH protein, TH activity and catecholamine synthesis. However, administration of the TH antisense to WKY had no effect on systolic blood pressure even though there were decreases in TH protein, TH activity and catecholamine synthesis. Furthermore, Schreihofer et al. (2000) have demonstrated that almost complete destruction of C1 neurons with DBH-saporin in normotensive animals also causes no change in resting blood pressure. These latter findings conflict with the data of this study, which shows a significant correlation between TH gene expression and systolic blood pressure in the C1 neurons of both hypertensive and normotensive rats. Presumably, this reflects a smaller role for TH in normotensive rats compared with SHR. Clearly, this issue requires further investigation. Alternatively, it is conceivable that the increase in TH gene expression is driven by an abnormality in expression of other genes such as c-fos or the angiotensin II type 1 (AT1) receptor that can in turn regulate TH gene expression (Yu et al., 1996a). These possibilities remain to be explored. Furthermore, future studies could be directed at investigating the levels of TH gene expression in individual C1 cells, through the use of single cell RT-PCR, rather than whole sites (Phillips and Lipski, 2000). In this study, we use, for the first time, the technique of real-time RT-PCR to detect levels of gene expression at sites that are critical for blood pressure regulation. A key advantage of this method as shown by Medhurst et al. (2000) is that it is capable of detecting and accurately quantitating much smaller amounts of mRNA (50 –100 copies) compared to
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other methods such as Northern blot analysis. For example, Kumai et al. (1994, 1996) used whole tissue from the medulla oblongata from which they used 20,000 ng per analysis as opposed to our methods, which used less than 200 ng of cDNA per analysis. Thus, we were able to investigate smaller site-specific regions within the brainstem known to control arterial pressure. Real-time RT-PCR also exhibits far less intra- and inter-assay variability compared to Northern blot analysis. Furthermore, quantitation using Northern blot analysis relies on densitometry, which is notoriously inaccurate (Heid et al., 1996; Medhurst et al., 2000). Finally, real-time RT-PCR, which follows the reaction from start to finish, quantitates products in the exponential phase of the PCR where the efficiency of the reaction is constant between samples and there is a very low abundance of nonspecific product as opposed to the plateau phase occurring at the end of amplification. 5. Conclusion In the present study, we have demonstrated that the levels of TH gene expression within SHR are significantly and consistently higher than in their WKY counterparts in all three sites studied viz., the adrenal gland, the C1 cell group and the A1 cell group. Secondly, the differences in TH gene expression between strains in all three sites were not significantly different, suggesting that the level of TH up-regulation within SHR is ‘‘global’’. Finally, we demonstrated a positive and significant relationship between systolic arterial blood pressure and TH gene expression in C1 cells of both SHR and WKY. The combination of these results suggests that the hypertensive phenotype in SHR is associated with expression of the TH gene and/or other factors influencing TH gene expression. The possibility that an increase in TH gene expression is a causative factor in the hypertensive phenotype in SHR remains to be determined. Acknowledgements Work in the authors laboratories is supported by grants from the National Health and Medical Research Council (No. 980077), National Heart Foundation of Australia (Nos. G00S0716 and G99S0472), Ramaciotti Foundation (No. RN025/00), North Shore Heart Research Foundation (Nos. 04-97/98, 15-00/01 and 17-00/01), Northern Area Health Service, the University of Sydney and Murdoch University Special Research Grant (MU.AVE.D.411). Valin Reja is in receipt of an Australian Postgraduate Award.
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Tyrosine hydroxylase gene expression in ventrolateral medulla oblongata of WKY and SHR: a quantitative real-time polymerase chain reaction study Valin Reja a, Ann K. Goodchild a, Jacqueline K. Phillips b, Paul M. Pilowsky a,* a
Hypertension and Stroke Research Laboratories, Department of Physiology, University of Sydney, and Department of Neurosurgery, Royal North Shore Hospital, 2065 Sydney, Australia b The Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, 6150 Western Australia, Australia
Abstract The aims of this study were, first, to determine quantitatively the levels of tyrosine hydroxylase (TH) gene expression in both peripheral and central sites related to blood pressure regulation, and to compare the level of expression in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Second, to see if any relationship exists between TH gene expression and systolic arterial blood pressure. Total RNA was isolated from adrenal glands and from tissue punches taken from the C1 and A1 cell groups in the rostral and caudal ventrolateral medulla oblongata of the brainstem, respectively. Total RNA was reverse-transcribed into cDNA followed by quantitative fluorescence detection polymerase chain reaction for TH cDNA. The levels of TH gene expression measured as a percentage of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in SHR, were significantly higher ( f 2.5-fold) compared to WKY in all sites examined ( P < 0.01). There was a positive and significant relationship between systolic blood pressure and TH gene expression in the C1 area of the brainstem in both WKY (n = 5, P < 0.05) and SHR (n = 6, P < 0.05). Taken together, these results suggest that elevated gene expression of the TH gene is associated with the phenotypic characteristic of SHR. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Catecholamines; C1 adrenaline cells; A1 noradrenaline cells; Adrenal gland; Baroreflex; Sympathetic
1. Introduction It has long been known that the catecholamines dopamine, noradrenaline and adrenaline, both peripherally and centrally, are involved in the regulation of arterial blood pressure (Guyenet and Cabot, 1981; Goodchild et al., 1984; Pilowsky et al., 1986). Catecholamines are synthesized from tyrosine through a pathway of enzymes. The initial and ratelimiting enzyme of the pathway is tyrosine hydroxylase (TH), which converts tyrosine into dihydroxyphenylalanine (DOPA) (Carlsson and Waldeck, 1972). Expression of the TH enzyme is restricted to catecholaminergic neurons and chromaffin cells (Chamba and Renaud, 1983). The chromaffin cells are primarily located in the adrenal gland where the noradrenaline, but not the adrenaline-secreting cells, are under the control of the baroreflex (Morrison et al., 1991). * Corresponding author. Hypertension and Stroke Research Laboratories, Ground Floor, Block 3, Royal North Shore Hospital, 2065 Sydney, Australia. Tel.: +61-2-9926-8080; fax: +61-2-9926-6483. E-mail address: [email protected] (P.M. Pilowsky).
Centrally, catecholaminergic neurons, which play key roles in blood pressure control, are localised to specific areas of the pons and medulla oblongata. The C1 cell group, which in addition to TH also expresses the adrenaline-synthesising enzyme phenylethanolamine-N-methyltransferase (PNMT), is located in the rostral ventrolateral medulla (Goodchild et al., 2000; Phillips et al., 2001). Rostral C1 neurons project directly to sympathetic preganglionic neurons (SPN) in the spinal cord (Lipski et al., 1995), whereas caudal C1 neurons innervate neurons within the hypothalamus and forebrain (Verberne et al., 1999). The A1 noradrenaline containing cell group lies caudal to the C1 neurons and provides a major excitatory drive to the vasopressin-releasing neurons of the hypothalamus (Day et al., 1980). An increased level of TH gene expression has been associated with hypertension. Indeed, Kumai et al. (1994, 1996), using Northern blot analysis, reported increased amounts of TH mRNA within the adrenal medulla and medulla oblongata of SHR compared to their WKY counterparts, and correlated the increases in mRNA to increased TH activity.
1566-0702/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 6 - 0 7 0 2 ( 0 2 ) 0 0 0 3 7 - 1
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Although increased amounts of TH mRNA in SHR have been reported, it is not known if these differences are ‘‘global’’ or if they are specifically regional. Furthermore, earlier methodologies may be less sensitive in determining precise levels of mRNA (Medhurst et al., 2000). The aims of this study were, first, to quantitate the amount of TH mRNA in both peripheral and central sites; these were the adrenal gland, C1 cell group and A1 cell group. Second, to compare TH gene expression levels between WKY and SHR strains to determine if any differences were sitespecific or ‘‘global’’. Third, to determine if a relationship exists between systolic arterial blood pressure and TH gene expression in both WKY and SHR animals. In order to obtain accurate measurements of TH expression, relative quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was used (Heid et al., 1996).
paraformaldehyde solution then cut into 50-Am-thick slices. Immunocytochemistry was conducted according to Goodchild et al. (2000). Slices were incubated for 48 h at room temperature with sheep anti-TH (1:1000, Pelfreez, Rogers, AR, USA) in Tris phosphate-buffered saline (TPBS, pH 7.4) containing 0.05% merthiolate with 5% normal horse serum (NHS). Sections were then washed 3 for 20 min and incubated for 12– 16 h with biotinylated donkey anti-sheep secondary antibody (1:500) containing 2% NHS in TPBS. Following washing, sections were incubated in ExtrAvidin (Sigma, St. Louis, MO, USA) conjugated to horseradish peroxidase (HRP) at 1:1000 for 4 h. Sections were washed and reacted with nickel-intensified diaminobenzidine (DAB), mounted onto gelatin slides, dehydrated, coverslipped and viewed under a bright-field microscope at 40 magnification. 2.3. Real-time RT-PCR
2. Methods 2.1. Animals and tissue preparation All experiments were conducted in accordance with the Australian code of practice for the care and use of animals for scientific purposes as endorsed by the National Health and Medical Research Council of Australia. The Animal Care and Ethics Committee of the Royal North Shore Hospital approved all protocols. Male, 20 –25 weeks old, WKYand SHR were used. Systolic arterial blood pressure was measured at 18 weeks of age using the tail-cuff method (n = 11). This approach was validated in our laboratory using simultaneous femoral artery recording and shown to be an accurate reflection of true systolic pressure with a variance of less than 1%. Individual animals were used to remove each targeted area. Tissue sections that contained the C1 (adrenaline synthesising) cells (n = 11) and the A1 (noradrenaline synthesising) cells (n = 6) were removed from euthanased (sodium pentobarbitone, 90 mg kg 1) animals. Tissue punches of the above-mentioned cell groups were taken from 1-mm-thick sections using an 18-gauge needle as described by Comer and Lipski (1997) (Fig. 1A). Although the sections were thicker compared to previous studies (Comer and Lipski, 1997), both the A1 and C1 cell groups were specifically isolated. This was confirmed by viewing thinner-sliced sections (50 Am), of the 1-mm sections, under a light microscope. Punches were also taken from a region 1 mm dorsal to the RVLM, known not to contain any TH-synthesising neurons and were used as negative controls (punch negatives, n = 6). The adrenal gland was removed and used for TH mRNA relative quantitation (n = 6). 2.2. Immunocytochemistry Immunocytochemistry for TH in punched tissue sections was conducted to ensure tissue punches contained TH positive cells. Punched sections were post-fixed in 4%
Total RNA was extracted using an RNA isolation reagent (ABgene, Epsom, Surry, UK) with chloroform and centrifuging the mixture for 10 min at 12,000 rpm before removing the aqueous phase containing the total RNA. Total RNA was precipitated with isopropanol and 0.3 M sodium acetate (pH 4), and then washed in 70% (v/v) ethanol before re-suspending the RNA pellet into RNasefree water. An RQ1 DNase (1 U Ag 1 RNA) was used according to the manufacturers protocol (Promega, Madison, WI, USA) to remove any genomic DNA contamination in the RNA samples. RNA was assayed using a UV spectrophotometer (Beckman Coulter, Fullerton, CA, USA) at a wavelength of 260 nm, where 1 absorbance unit at 260 nm is equal to 40 Ag ml 1 RNA. The RNA was reversetranscribed (RT) using a Reverse Transcription System (Promega) according to the manufacturers’ protocol. RT negative controls were run to determine if any genomic contamination was present. Relative quantitative PCR was achieved using a fluorogenic intercalating dye and intron spanning primers to TH cDNA published by Comer et al. (1999). Each 20-Al reaction contained 3.5 mM MgCl2, 1 PCR buffer, 0.5 AM each primer, SYBRGreen (1:106, Molecular Probes, Eugene, OR, USA), 0.625 U Taq polymerase (Promega) and template cDNA. No template controls (NTC) were run to determine the level of primer dimer formation. Amplification was performed in 0.2-ml tubes on a Rotor Gene 2000 real-time PCR machine (Corbett Research, Sydney, Australia, http://www.corbettresearch.com). PCR parameters were an initial denature at 94 jC for 180 s followed by 45 cycles of 94 jC for 15 s, 57 jC for 20 s and 72 jC for 25 s. Fluorescence data were acquired using a low gain at 85 jC, where only specific products were assumed to be present. A melt analysis on resulting PCR products supported this for all samples measured. In addition, PCR products were run on 2% Tris borate ethylenediaminetetraacetic acid (TBE) agarose gels to confirm that correct band sizes were present (Fig. 1B). A standard curve was gen-
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Fig. 1. Panel A: Nickel-intensified DAB labelling of TH on a punched tissue section showing TH positive cells on the right-hand side with only a few cells present around the punch on the left hand side. Panel B: Real-time RT-PCR amplification plot showing GAPDH products (dash and/or dotted lines) from serial dilutions of cDNA obtained from the adrenal gland of an SHR, TH product (solid line) taken from undiluted samples of the same cDNA, and a no template control (NTC, dashed line). Threshold cycle values (Ct) were taken at an equal point where all samples were within the exponential phase of amplification. Panel C: Agarose gel electrophoresis of RT-PCR products. Amplification of GAPDH internal cDNA standard was achieved using 10 , 1 , 0.1 and 0.01 concentrations of cDNA taken from an SHR adrenal gland and GAPDH primers to yield a 239-bp product (lanes 1 – 4). Lane 5 was a GAPDH primer no template control (NTC) and lane 6 was a negative RT control for genomic GAPDH. Amplification of TH cDNA yielded a 322-bp product from the same tissue sample (adrenal gland, lane 8). Lane 9 was the TH primer NTC and lane 10 was a negative RT control for genomic TH. Lane 7 = pGEMR molecular weight marker (Promega). Panel D: Graph comparing levels of TH mRNA, expressed as percentage TH cDNA/GAPDH cDNA, from the adrenal gland, C1 cell group, A1 cell group and punch negatives of WKY (black) and SHR (white) rat strains ( ** P < 0.01; *** P < 0.001). Panel E: Graph showing a positive and significant linear relationship ( P < 0.05, r2 = 0.87, n = 5) between systolic arterial pressure and TH gene expression (as a percentage of GAPDH gene expression) in the C1 cell group of WKYanimals (black boxes). Dashed lines represent 95% confidence intervals. Panel F: Graph showing a positive and significant linear relationship ( P < 0.05, r2 = 0.66, n = 6) between systolic arterial blood pressure and TH gene expression (as a percentage of GAPDH gene expression) in the C1 cell group of SHR animals (white boxes).
erated by amplifying, in serially diluted cDNA samples, the endogenous cDNA standard glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using primer sequences published
by Comer and Lipski (1997). Although it has been reported that in cell cultures systems (including cancer cell lines) the level of GAPDH gene expression can vary (Zhong and
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Simons, 1999), most studies find GAPDH to be expressed in equal concentrations between different tissues whatever the experimental conditions (e.g. partial nerve ligation) (Medhurst et al., 2000; Macdonald et al., 2001). Furthermore, changing conditions do not apply to this study as it only focuses on basal levels of gene expression in WKY and SHR. By plotting the Ct values (cycle threshold set where the exponential phase of the PCR reaction began) ( y) against the concentration of GAPDH cDNA (x), a standard curve was generated from which the unknown amount of TH gene expression within the sample was determined (Heid et al., 1996). 2.4. Data analysis Data were normalized and expressed as TH cDNA (as a relative percentage of GAPDH cDNA in sample) F S.E.M. for each group of animals. An unpaired Student’s t-test was used to determine any significance between groups (a = 0.05). A correlation coefficient between systolic blood pressure and TH cDNA (as a relative percentage of GAPDH cDNA in sample) was determined in WKY and SHR animals.
3. Results Tail-cuff measurements showed, as expected, that SHR have a significantly higher systolic blood pressure than WKY for those animals measured (SHR, 180 F 5 mm Hg, n = 6; WKY, 103 F 2 mm Hg, n = 5, P < 0.001). This also showed that the SHR hypertensive phenotype was being appropriately expressed within our colony. Tissue punches of the C1 and A1 cell groups within the medulla oblongata of separate animals were removed and total RNA extracted. Immunocytochemistry on the remaining tissue sections revealed that a good proportion of the TH positive cells within these cell groups was collected (Fig. 1A). RT-PCR was conducted on all RNA samples with positive amplification of the GAPDH cDNA internal standard fragment (239 bp) and TH fragment (322 bp) (Fig. 1B and C). Very little nonspecific product was observed (Fig. 1C), which was also confirmed by a melt analysis of each PCR (data not shown). There was no evidence seen of genomic DNA amplification within RT negative controls (Fig. 1C). Although some primer dimer formation was present within PCR products, this did not affect quantitation as the Ct values were obtained at the beginning of the exponential phase where only specific product amplification was above the detection threshold. Quantitation of TH cDNA within the three sites examined gave the results shown in Fig. 1D. The amount of TH gene expression within the adrenal gland of the SHR was significantly higher than in WKY with a ratio of 2.5:1 (SHR, 25.1 F 1 TH cDNA as a relative percentage of GAPDH cDNA in sample, n = 3; WKY, 10.4 F 2 TH cDNA as a relative percentage of GAPDH cDNA in sample, n = 3, P < 0.01). TH gene expression within the C1 cell group of
the SHR was also significantly higher than in WKY (SHR, 3.6 F 0.2 TH cDNA as a relative percentage of GAPDH cDNA in sample, n = 6; WKY, 1.6 F 0.1 TH cDNA as a relative percentage of GAPDH cDNA in sample, n = 5, P < 0.001). Indeed, the amounts of TH cDNA within the C1 cells of the SHR were 2.2 times that of the WKY, a similar ratio to that obtained between strains for the adrenal gland. Levels of TH gene expression within the A1 cell group of the SHR was significantly higher than in WKY (SHR, 1.5 F 0.1 TH cDNA as a relative percentage of GAPDH cDNA in sample, n = 3; WKY, 0.5 F 0.1 TH cDNA as a relative percentage of GAPDH cDNA in sample, n = 3, P < 0.01). The ratio of TH cDNA between strains was 3:1. Tissue punches taken from regions known not to contain any TH (punch negatives) showed background levels of TH gene expression that were not significantly different between strains (SHR, 0.1 F 0.1 TH cDNA as a relative percentage of GAPDH cDNA in sample, n = 3; WKY, zero TH cDNA as a relative percentage of GAPDH cDNA in sample, n = 3). There was no significant difference between the ratios obtained for SHR and WKY at each of the sites studied (SHR/ WKY, C1 = 2.2, A1 = 3, adrenal gland = 2.5). Finally, there was a positive and significant correlation between systolic arterial blood pressure and TH cDNA as a relative percentage of GAPDH cDNA in sample in C1 cells of both WKY (n = 5, r2 = 0.87, P < 0.05) and SHR (n = 6, r2 = 0.66, P < 0.05) (Fig. 1E and F).
4. Discussion Differences in the levels of TH gene expression have been reported previously in the adrenal gland and in the whole medulla oblongata (Kumai et al., 1994, 1996; Goc and Stachowiak, 1994; Chan and Sawchenko, 1995; Sterling and Tank, 2001). Most of these studies examined changes in TH gene expression in normotensive rats at rest, or cultured cells, in response to a stimulus. For example, Goc and Stachowiak (1994) showed that upon addition of angiotensin II to cultured cells obtained from the medulla oblongata, there was an increase in TH gene expression. In two earlier studies, Kumai et al. (1994, 1996) reported an up-regulation of TH gene expression in the adrenal medulla and in the whole medulla oblongata of WKY and SHR aged 25 weeks. However, in these studies, the TH gene expression was determined in whole medulla oblongata. This approach would include all of the catecholamine cell groups in this brain region: including the A1, A2, C1, C2, C3 and TH-expressing neurons in the area postrema. Furthermore, these authors used Northern blot analysis as a ‘‘quantitative’’ technique, although without demonstrating that the changes were linear over the range used for measurement. Nevertheless, this work demonstrated a clear difference in TH gene expression between WKY and SHR when comparing whole medulla oblongata and adrenal medulla.
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In the present study, we extend the findings of Kumai et al. (1994, 1996) by examining specific brain regions that are known to be involved in the regulation of arterial blood pressure and determined stationary levels of TH gene expression in WKY and SHR. Our results demonstrate, for the first time, that there is an up-regulation of TH gene expression in SHR within sitespecific regions of the medulla oblongata involved in blood pressure regulation viz., the C1 and A1 cell groups. Interestingly, the ratio of TH gene expression at a given site between the species was not significantly different to that found at other sites. In fact, at all three sites (adrenal gland, A1 and C1), SHR expressed about 2.5 times as much TH mRNA as WKY rats. This result demonstrates that the up-regulation of TH gene expression is ‘‘global’’ rather than being restricted to individual groups of catecholamine-synthesising cells. This result raises the question of whether or not the increase in TH gene expression is a cause or consequence of the hypertensive phenotype. In addition, analysis of the data revealed a positive and significant correlation between systolic arterial blood pressure and TH gene expression (as a percentage of GAPDH gene expression) in C1 cells of WKY (n = 5, P < 0.05) and SHR (n = 6, P < 0.05) animals. Indeed, there is considerable evidence that changes in TH gene expression result in changes in systolic blood pressure. For example, Kumai et al. (2001) have shown that administration (i.v.) of a TH antisense to SHR causes a decrease in systolic blood pressure along with decreases in TH protein, TH activity and catecholamine synthesis. However, administration of the TH antisense to WKY had no effect on systolic blood pressure even though there were decreases in TH protein, TH activity and catecholamine synthesis. Furthermore, Schreihofer et al. (2000) have demonstrated that almost complete destruction of C1 neurons with DBH-saporin in normotensive animals also causes no change in resting blood pressure. These latter findings conflict with the data of this study, which shows a significant correlation between TH gene expression and systolic blood pressure in the C1 neurons of both hypertensive and normotensive rats. Presumably, this reflects a smaller role for TH in normotensive rats compared with SHR. Clearly, this issue requires further investigation. Alternatively, it is conceivable that the increase in TH gene expression is driven by an abnormality in expression of other genes such as c-fos or the angiotensin II type 1 (AT1) receptor that can in turn regulate TH gene expression (Yu et al., 1996a). These possibilities remain to be explored. Furthermore, future studies could be directed at investigating the levels of TH gene expression in individual C1 cells, through the use of single cell RT-PCR, rather than whole sites (Phillips and Lipski, 2000). In this study, we use, for the first time, the technique of real-time RT-PCR to detect levels of gene expression at sites that are critical for blood pressure regulation. A key advantage of this method as shown by Medhurst et al. (2000) is that it is capable of detecting and accurately quantitating much smaller amounts of mRNA (50 –100 copies) compared to
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other methods such as Northern blot analysis. For example, Kumai et al. (1994, 1996) used whole tissue from the medulla oblongata from which they used 20,000 ng per analysis as opposed to our methods, which used less than 200 ng of cDNA per analysis. Thus, we were able to investigate smaller site-specific regions within the brainstem known to control arterial pressure. Real-time RT-PCR also exhibits far less intra- and inter-assay variability compared to Northern blot analysis. Furthermore, quantitation using Northern blot analysis relies on densitometry, which is notoriously inaccurate (Heid et al., 1996; Medhurst et al., 2000). Finally, real-time RT-PCR, which follows the reaction from start to finish, quantitates products in the exponential phase of the PCR where the efficiency of the reaction is constant between samples and there is a very low abundance of nonspecific product as opposed to the plateau phase occurring at the end of amplification. 5. Conclusion In the present study, we have demonstrated that the levels of TH gene expression within SHR are significantly and consistently higher than in their WKY counterparts in all three sites studied viz., the adrenal gland, the C1 cell group and the A1 cell group. Secondly, the differences in TH gene expression between strains in all three sites were not significantly different, suggesting that the level of TH up-regulation within SHR is ‘‘global’’. Finally, we demonstrated a positive and significant relationship between systolic arterial blood pressure and TH gene expression in C1 cells of both SHR and WKY. The combination of these results suggests that the hypertensive phenotype in SHR is associated with expression of the TH gene and/or other factors influencing TH gene expression. The possibility that an increase in TH gene expression is a causative factor in the hypertensive phenotype in SHR remains to be determined. Acknowledgements Work in the authors laboratories is supported by grants from the National Health and Medical Research Council (No. 980077), National Heart Foundation of Australia (Nos. G00S0716 and G99S0472), Ramaciotti Foundation (No. RN025/00), North Shore Heart Research Foundation (Nos. 04-97/98, 15-00/01 and 17-00/01), Northern Area Health Service, the University of Sydney and Murdoch University Special Research Grant (MU.AVE.D.411). Valin Reja is in receipt of an Australian Postgraduate Award.
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