Immunoreactive forms of natriuretic peptides in ovine brain: response to heart failure

Peptides 23 (2002) 2235–2244

Immunoreactive forms of natriuretic peptides in ovine brain: response to heart failure Chris J. Pemberton∗ , Tim G. Yandle, Eric A. Espiner Christchurch Cardioendocrine Research Group, Christchurch School of Medicine, University of Otago, Christchurch 8001, New Zealand Received 6 May 2002; accepted 15 July 2002

Abstract In order to elucidate how brain natriuretic peptides (NPs) are affected by experimentally induced heart failure, we have measured the immunoreactive (IR) levels of the NP in extracts from 10 regions of ovine brain, including pituitary, and clarified their molecular forms using high performance liquid chromatography (HPLC). Using species-specific radioimmunoassay (RIA), atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were all detected in extracts taken from control animals and sheep that had undergone rapid ventricular pacing for 7 days to induce heart failure. CNP was the most abundant NP as assessed by specific RIA, and the pituitary contained the highest IR levels for all three NP. Compared with control animals, the pituitary content of BNP in animals with heart failure was reduced by 40% (control, 0.26±0.02 pmol/g wet weight versus heart failure 0.16±0.01; P < 0.01, n = 7). No other significant changes were observed. The molecular forms of ANP and CNP in whole brain extracts as assessed by HPLC were proANP and CNP22, CNP53 and proCNP, respectively. BNP in pituitary extracts was assessed to be primarily proBNP with a minor component of mature BNP26. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Natriuretic peptide; Heart failure; Brain concentrations; Molecular form; HPLC

1. Introduction The natriuretic peptides (NPs) are cardiovascular-based hormones affecting body fluid and blood pressure regulation [9]. The NP is composed of at least three known members, atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). ANP and BNP both circulate in the blood and serve to antagonize the actions of the renin–angiotensin–aldosterone system (RAAS) and enhance natriuresis, diuresis and vasorelaxation. CNP, however, is primarily found in the vasculature where it serves to regulate vessel tone and the growth of endothelial and vascular smooth muscle cells [27]. In mammalian central nervous tissue, ANP is universally present in amino terminal truncated forms, namely ANP(102–126) and ANP(103–126), which differ from the circulating form—ANP(99–126) predominating in plasma [21]. Greater heterogeneity occurs across species in respect of BNP forms within nervous tissues. In porcine brain BNP26 and BNP32 forms are observed, both of which also circulate in porcine plasma [1]. In rat [1] and human [32] spinal cord extracts, BNP is present as BNP45 and BNP32, ∗

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respectively, which are also the predominant circulating forms in both species. CNP is present in brain as CNP22 [31] or CNP53 [20]; these same forms are also found in vascular tissue extracts [40]. Of the NP, CNP is by far the most abundant in brain tissue extracts. For example, CNP immunoreactivities (-IR) in porcine [35], canine [15] and human [32] brain extracts are approximately 10-fold higher than BNP and ANP. Thus, like the circulatory system, central nervous tissue appears to contain all three NP, albeit at levels much lower than those observed in plasma and cardiac tissue extracts [40]. Coupled with the presence of immunoreactive NP, natriuretic peptide receptors (NPR-A, NPR-B and NPR-C) in brain tissue extracts have also been well documented [4,37]—findings which confirm the presence of a bona fide NP system within central nervous tissue. Immunocytochemical, gene transcript localization and immunoreactive peptide studies indicate that at least ANP and CNP are prominent in the anterior-ventral region of the third ventricle (AV3V) of the brain, as well as the basal medial hypothalamus, brainstem and circumventricular organs of the human [20] and pig [35]. The presence of the NP in these key brain nuclei sub-serving cardiovascular regulation [14], and their inhibitory actions on the central effects of angiotensin II [6] point to important physiological roles. In contrast, although

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BNP was first identified from porcine brain tissue [30] subsequent work has shown that it is not universally found in mammalian brain tissue. Thus, BNP-IR and mRNA expression have not been demonstrated in rat [17] or mouse [29] brain and it is still unclear as to whether BNP-IR or transcript is present in human brain tissue [10,14]. Thus, in contrast to the findings relating to ANP and CNP, the presence of BNP appears to be a more species-specific phenomenon and its role in central cardiovascular regulation is less clear. Accordingly, this report sought to establish whether BNP-IR was present in the ovine brain, how its regional distribution compared with that of ANP and CNP and what the molecular forms responsible for IR were for each of the NP. Because few previous studies have examined brain NP changes accompanying systemic alterations in NP hormones, we also sought to determine how central tissue concentrations of NP were affected by stimuli that greatly increase circulating hormones levels, using an ovine model of pacing-induced heart failure.

induced in group 2 animals by rapid ventricular pacing at 225 beats/min for 7 days as previously reported [23,25]. At 7 days, whole brains plus the pituitary were collected on ice from normal sheep and those with pacing-induced heart failure, washed immediately in ice-cold isotonic (0.9%) saline and stored at −80 ◦ C until extraction and analysis. In all cases, the time from death until storage of brain tissue was less than 30 min. 2.2. Neural tissue extraction Stored whole brains were removed from −80 ◦ C, placed on ice and dissected into eight discrete regions as described in Table 1. The spinal cord and whole pituitary were also collected. Individual regions were diced and boiled in 10 ml distilled water containing 0.1% Triton X-100 (Sigma, Penrose, Auckland, New Zealand) for 10 min to remove intrinsic protease activity. After cooling on ice, glacial acetic acid and 1 M HCl were added to bring final concentrations to 1 M and 20 mM, respectively. Samples were homogenized at 4 ◦ C using an Ultra-Turrax T-25 dispersing homogenizor (IKA-Labortechnik, Staufen, Germany) for 3–5 min at the highest speed possible. After centrifugation at 18,000 × g/4 ◦ C for 30 min, the resulting supernatant was diluted two-fold with 1 M acetic acid and extracted further using SepPak C18 cartridges (Waters, Milford, USA) as described previously [23]. The recoveries of synthetic mature forms of the NP using this procedure were as follows: ANP 68%, BNP 56% and CNP 66%. Tissue concentrations presented here are not adjusted for recovery.

2. Materials and methods 2.1. Ovine tissue preparation and collection Two groups of age and weight matched Coopworth ewes were studied: (1) normal controls (n = 7) and (2) sheep with pacing-induced heart failure (n = 7). The study protocol was approved by the Christchurch School of Medicine Animal Ethics Committee. Animals with pacing-induced heart failure were prepared as previously described [23]. Briefly, sheep were anaesthetized with 17 mg/kg i.v. thiopentone sodium (Intraval, May and Baker, Dagenham, UK), an incision made in the left jugular vein and a 7-French His bundle electrode pacing wire advanced to the right ventricle. The position and stability of the wire was confirmed under X-ray. Animals were given i.m. pethidine and streptomycin, transferred to metabolic crates and allowed to recover for 48 h with free access to food and water. Heart failure was then

2.3. Radioimmunoassay (RIA) of central natriuretic peptide concentrations Natriuretic peptide concentrations in ovine brain extracts were determined by previously described, species-specific RIA, for ANP [38], BNP [23], amino terminal BNP [24] and CNP [39]. The detection limit (in pmol/g wet weight of tissue) for each assay was as follows: ANP 0.06, BNP 0.04,

Table 1 Regional distribution of natriuretic peptide-like immunoreactivity (-LI) in ovine brain extracts from control animals and those with pacing-induced heart failure Brain region

Olfactory bulb Posterior cortex Anterior cortex Septum/striatum Hypothalamus Pituitary Pons Medulla Cerebellum Spinal cord

Control (group 1)

Heart failure (group 2)

ANP-LI

BNP-LI

<0.06 <0.06 <0.06 0.08 0.10 0.25 0.09 0.08 <0.06 0.08

0.13 0.11 0.12 0.19 0.13 0.26 0.13 0.12 0.09 0.15

± ± ± ± ±

0.01 0.02 0.03 0.03 0.04

± 0.05

± ± ± ± ± ± ± ± ± ±

CNP-LI 0.01 0.02 0.02 0.02 0.01 0.02 0.02 0.01 0.01 0.02

0.26 0.18 0.20 1.05 0.52 15.84 0.64 0.70 0.57 0.81

± ± ± ± ± ± ± ± ± ±

ANP-LI 0.03 0.05 0.04 0.22 0.04 3.58 0.07 0.07 0.08 0.07

Values are mean ± S.E.M. (pmol/g wet weight) for n = 7 sheep in each group. a Significantly lower than BNP-IR in control pituitary; P < 0.01.

0.11 <0.06 <0.06 0.08 0.06 0.21 0.15 0.15 <0.06 0.09

BNP-LI

± 0.03 ± ± ± ± ±

0.02 0.02 0.03 0.04 0.06

± 0.04

0.14 0.07 0.08 0.13 0.09 0.16 0.11 0.11 0.08 0.09

± ± ± ± ± ± ± ± ± ±

CNP-LI 0.01 0.02 0.01 0.02 0.02 0.01a 0.02 0.01 0.01 0.01

0.30 0.11 0.15 0.57 0.26 14.94 0.38 0.46 0.29 0.43

± ± ± ± ± ± ± ± ± ±

0.07 0.01 0.02 0.07 0.05 3.82 0.05 0.06 0.05 0.06

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Fig. 1. Representative standard and extract dilution curves for ANP, BNP and CNP radioimmunoassays. Dark circles are the standard curve and open circles are the respective dilution of extract in each assay. Numbers indicate the dilution factor for each curve. Dilution curves were generated from brain extracts as outlined in Section 2.

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N-BNP 0.05 and CNP 0.02. Intra-assay coefficients of variation were as follows: ANP, 5–8%; BNP, 5–11%; N-BNP, 5–9% and CNP, 6%. Inter-assay coefficients of variation for each RIA were: ANP, 9%; BNP, 12%; N-BNP, 11% and CNP, 12%. 2.4. High performance liquid chromatography (HPLC) analysis of tissue extracts Initial molecular characterization of the immunoreactive (IR) species of natriuretic peptides present in ovine brain was carried out using a 22-cm C18 reversed phase HPLC column (Brownlee, Applied Biosystems, CA). Pooled extracts from whole brain and pituitary were dissolved in 20% acetonitrile/0.1% trifluoroacetic acid (TFA), loaded onto the column and eluted at 40 ◦ C with a gradient of acetonitrile/0.1% TFA from 20 to 60% over 40 min at 1 ml/min flow rate. Fractions were collected at 0.5 min intervals containing 10 ␮l 0.1% Triton X-100 and aliquots of each fraction diluted in RIA buffer for specific RIA measurement of IR-ANP, BNP, N-BNP and CNP. The remaining portion of each fraction was

stored at −80 ◦ C for further analysis. After initial RIA, appropriate fractions containing IR-ANP, BNP and CNP were pooled, dried under a stream of air, reconstituted in 20% acetonitrile/0.1% TFA/0.15 M NaCl and further subjected to size exclusion HPLC. Briefly, samples were loaded onto a G2000 SW size exclusion HPLC column (Toyosoda, Japan) and eluted at room temperature under isocratic conditions of 20% acetonitrile/0.1% TFA/0.15 M NaCl over 60 min at a flow rate of 0.5 ml/min. The eluate was collected in 0.5 ml fraction volumes containing 10 ␮l of 0.1% Triton X-100, dried under a stream of air at 37 ◦ C and stored at −20 ◦ C until analysis by specific RIA for ANP, BNP, N-BNP and CNP. 2.5. Data presentation and statistical analysis Data is presented as the mean (pmol/g wet weight tissue) ± S.E.M. Statistically significant differences in natriuretic peptide immunoreactivity between discrete brain regions were determined by ANOVA with post-hoc Tukey– Kramer analysis for multiple comparisons. Significance was defined as P < 0.05.

Fig. 2. Immunoreactive ANP in ovine brain extracts identified by HPLC. Panels A and B are from control animals, whereas C and D are from animals with heart failure. Panels B and D are reversed phase HPLC profiles which were obtained first. Panels A and C are subsequent size exclusion profiles of IR-ANP in peak 2 (fractions 47–50) of panels B and D. Arrows indicate the following elution times: Vo = void volume, 1 = cytochrome c (Mr = 12,384), 2 = synthetic ANP(99–126), 3 = synthetic porcine CNP22, Vt = total column volume.

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3. Results 3.1. Immunoreactive levels of natriuretic peptides in response to heart failure The concentration of IR-ANP, BNP and CNP in eight discrete brain regions, spinal cord and pituitary of control animals (group 1) and those with heart failure (group 2) are shown in Table 1. Serial dilution curves of tissue

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extracts for each natriuretic peptide are shown in Fig. 1, and indicate that the material detected in the extracts was immunologically similar to that generating each standard curve. In control animals, IR-ANP was detected in the septum/striatum, hypothalamus, pons, pituitary, medulla and spinal cord. Highest levels were found in the pituitary. Levels of IR-ANP in the olfactory bulb, posterior cortex, anterior cortex and cerebellum were below assay detection limit (Table 1). Compared with control animals, no significant

Fig. 3. Immunoreactive BNP in normal ovine pituitary. Panel A is reversed phase HPLC in pituitary extract whereas panel B shows two different size exclusion HPLC runs of fractions 25–27 (low molecular weight) and 49–51 (high molecular weight) from Panel A IR-BNP. No qualitative or quantitative differences were observed between normal and heart failed extracts. Filled circles indicate N-BNP-IR (N-BNP) whereas open circles indicate mature BNP26-IR (C-BNP). Arrows indicate the elution positions of: Vo = void volume, 1 = bovine albumin (Mr = 65,000), 2 = carbonic anhydrase (Mr = 29,000), 3 = cytochrome c (Mr = 12,384), 4 = aprotinin (Mr = 6500), 5 = synthetic porcine BNP26, Vt = total column volume.

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differences in IR-ANP concentration were found in animals with pacing-induced heart failure. However, the average level of IR-ANP in the olfactory bulb of group 2 animals rose above the assay detection limit, suggesting an increase

in ANP concentration in this region associated with heart failure. IR-BNP was detectable in all regions of the ovine brain, with concentrations generally exceeding those of ANP

Fig. 4. Representative HPLC profiles of CNP from extracts of control animals and those with heart failure. Panel A is a size exclusion profile of a whole brain extract from a control animal, panel B is a size exclusion profile of a pituitary extract from a normal animal and panel C is a reversed phase HPLC profile from a whole brain extract from an animal with heart failure. Arrows indicate: Vo = void volume, 1 = aprotinin, 2 = synthetic CNP22, Vt = total column volume. Note the absence of CNP22-like immunoreactivity in pituitary extract (panel B).

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(Table 1). Compared with group 1 animals, there was a significant reduction in the IR-BNP level of the pituitary body in group 2 animals (P < 0.01). No other significant changes were observed. CNP levels in all areas were higher (1.5–60-fold) than corresponding values of ANP and BNP. The CNP concentration in the normal ovine brain was highest in the pituitary (10-fold higher than that of the next highest region, septum/striatum, Table 1). No significant changes in regional CNP concentration were found in sheep with heart failure, even though the levels in the septum/striatum, hypothalamus and cerebellum decreased by 50%, compared with control (Table 1). 3.2. Molecular forms of brain natriuretic peptides Initial reversed phase HPLC revealed two immunoreactive peaks of ANP in both normal sheep and those with heart failure (Fig. 2, panels B and D). Neither of these two peaks co-eluted with synthetic ANP(99–126) standard. Peak one eluted in the void volume of the column; peak two eluted in a position consistent with high molecular weight proANP(1–126) as observed previously when ovine cardiac tissue extracts were analyzed using reversed phase HPLC [23]. When analyzed further on size exclusion HPLC (Fig. 2, panels A and C) these peaks did not resolve clearly, although immunoreactivity consistent with a proANP-like molecular weight form (fractions 26–28) was seen but not ANP(99–126) or its truncated forms (101–126 and 102–126). There were no significant differences observed in the molecular forms of ANP between normal animals and those with heart failure. BNP immunoreactivity in whole brain extracts was not clearly defined by reversed phase or size exclusion HPLC (data not shown). However, the majority of IR-BNP (filled circles) in pituitary extracts eluted on reversed phase (Fig. 3, panel A) and size exclusion (Fig. 3, panel B) HPLC in a position consistent with high molecular weight proBNP previously observed in cardiac extracts [23]. A minor component of BNP26 immunoreactivity (open circles) was observed, consistent with the mature form observed in ovine plasma [23]. Interestingly, the high molecular weight BNP form was more clearly detected using the N-BNP RIA compared with the BNP26 directed RIA on both forms of HPLC. Size exclusion HPLC coupled with N-BNP RIA also revealed a small peak (fractions 41–43, Fig. 3, panel B) immediately after the elution of proBNP like-IR (fraction 37) consistent with the elution position of N-BNP observed in ovine plasma extracts [24]. However, this was a minor component of the total IR observed using this RIA. When analyzed on size exclusion HPLC, CNP-IR from whole brain extracts gave an elution profile containing three peaks (fractions 26, 31 and 37, Fig. 4A), consistent in size with proCNP-, CNP53- and CNP22-like species, respectively. In contrast with whole brain—and in agreement with previous reports [39]—pituitary extracts contained only one

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molecular form, that of CNP53-like species (fraction 31, Fig. 4B) eluting at Mr 6500. When whole brain extracts were further analyzed using reversed phase HPLC, three immunoreactive peaks (fractions 44, 47/48 and 52) were observed, one of which co-eluted with synthetic CNP22 standard (fraction 44, Fig. 4C).

4. Discussion Radioimmunoassay measurement of tissue extracts revealed CNP to be the most abundant natriuretic peptide in the ovine brain (particularly in the pituitary), with BNP the next most abundant. ANP was not detectable by RIA in the cerebellum or posterior/anterior cortex. On average, BNP concentrations in whole brain extracts exceeded ANP by two-fold whereas the ratio of CNP:ANP/BNP was approximately 10:1. In the pituitary gland, the ratio of CNP:ANP/BNP was 60:1. Similar ratios were observed in extracts from animals with heart failure. Only the BNP concentration in the pituitary was altered (decreased) by pacing-induced heart failure. While the molecular form of BNP in whole brain extracts was not clearly resolved by HPLC, both high and low molecular weight forms of BNP were found in pituitary extracts, the high molecular weight form being similar to that observed in ovine cardiac tissue [23]. The involvement of the natriuretic peptides in the central regulation of cardiovascular control is most likely restricted to the cellular level, as the neural content of these peptides is not high enough to make a significant contribution to circulating plasma levels [14]. In keeping with these findings the current results show that central ANP and BNP concentrations were 10,000- and 1000-fold lower than cardiac levels, respectively [23]. These ratios compare well with those reported from porcine [34], canine [15] and human studies [20]. With regards to the regional distribution of the NP within the brain, and in agreement with previous reports [15,34] the septum/striatum and hypothalamus contained higher levels of BNP and CNP when compared with ANP. The ovine pituitary was observed to contain the highest amounts of each peptide in prepared extracts (especially CNP) and this is also in agreement with previously reported distributions [11,16]. ANP-containing perikarya are densely located in the region known as the anterior-ventral wall of the third ventricle (AV3V) and their axons have been shown to terminate in the median eminence and neural lobe of the pituitary [19,28]. Electrolytic lesion of all these above sites suppresses volume-induced cardiac release of ANP resulting in changed plasma levels of the peptide [2,26]. Similarly, i.c.v. administration of ANP antiserum in sheep also blocks the plasma volume-induced increase in plasma ANP levels [7]. In the present study neither the hypothalamic or pituitary concentration of ANP was altered in response to pacing-induced heart failure. However, significant depletion

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of ANP peptide has been demonstrated in the medial preoptic nucleus, the supraoptic nucleus (SON), the subfornical organ and the locus cerelus of rats with myocardial infarction [13]. This latter study employed micro-dissection techniques to identify these changes in individual regions. Compared with the extraction technique presently employed (which identified trends of decreasing ANP concentration in the hypothalamic region), the use of more sensitive micro-dissection techniques by Hu et al. may explain the difference in results. The “appearance” of detectable ANP in the olfactory bulb of sheep with pacing-induced heart failure is surprising, but may arise from changes in axonal secretion patterns of ANP into this region. In this regard, CNP gene expression is greatly increased in the olfactory region of rats that have undergone 48 h of salt loading [5] and a similar expression pattern for ANP may possibly underpin the peptide changes observed here. BNP levels in the septum/striatum and hypothalamus tended to decrease with heart failure, but pontine and medullary concentrations were unaltered. The tendency of hypothalamic and septum/striatum BNP concentrations to decrease in the heart failure group of animals may have important implications as in vitro studies have suggested BNP can modulate noradrenergic transmission at the presynaptic level in this region [36]. This latter study observed BNP stimulated enhancement of noradrenaline uptake in rat hypothalamic slices, which in turn reduced nor-adrenergic transmission and diminished the synthesis, turnover and presynaptic output of noradrenaline. However, the levels of BNP (high picomolar) required to achieve this and the fact that BNP mRNA or peptide has not been detected in rat brain extracts [17,22] suggest caution in interpreting the physiological relevance of these results. In the present study, we observed a significant (and unique) decrease of BNP concentration in the pituitary of the heart failure group. The molecular forms responsible for this change were identified as both high and low molecular weight BNP, with the greater component being the high molecular weight form. Like previous reports [16], the present study observed the highest CNP concentration in the pituitary. However, although no distinction was made in our study between anterior and posterior regions it has been shown that CNP peptide and mRNA is primarily located in the anterior region [3]. The trend for CNP immunoreactivity to decrease in the hypothalamus, pons, medulla, septum/striatum and cerebellum of the pacing-induced heart failure group, suggests that these regions of the ovine brain may be sensitive to changes in systemic blood pressure and/or neurohumoral modulation associated with heart failure. Interestingly, in contrast to the other nine tissues examined—all of which were reduced—CNP-LI in the olfactory bulb tended to increase in the heart failure group. This finding is consistent with the observation made by Cameron et al. [5] whereby increased CNP gene expression was observed in olfactory tissues of rats that had undergone 48 h of systemic salt loading or i.c.v. angiotensin II. Taken together, these studies

highlight the confusing nature of CNP (and BNP and ANP) action in the brain and the lack of contemporary understanding. Further, it is likely that complex interactions between each peptide exist and these need to be considered [8]. Reversed phase and size exclusion HPLC suggest ANP is present in the ovine brain primarily as a high molecular weight species, consistent with proANP-like material as observed in ovine cardiac tissue extracts [23]. ProANP-like forms are present in rat, human and monkey brain extracts [21] but the physiological relevance of this is not known. Furthermore, like the present study, previous work has not been able to distinguish if ANP forms are differentially represented within specific brain regions [12]. No mature ANP(99–126) forms, or truncated variants such as ANP(102–126) or ANP(103–126) which constitute the bulk of mature ANP forms in the brain [21] were observed in the present study. The RIA antiserum employed recognizes the ring structure of ANP—hence would detect the amino truncated variants—but we cannot definitively rule out their presence. Although we did not clearly determine the identity of BNP forms in whole brain extracts, pituitary preparations contained both mature BNP and proBNP forms, as well as N-BNP-like material, with the major species identified being proBNP. BNP exists as mature BNP26 and BNP32 in porcine brain [34] and as BNP32 in canine brain [15], forms which are similar to those observed in the circulation of each species [40]. The fact that mature BNP26 and N-BNP-like forms were observed in pituitary extracts here suggests that the processing of proBNP in this tissue may be carried out by enzymes similar to those proposed to process proBNP in cardiac extracts, i.e. furin [40]. The precise nature of BNP in ovine whole brain will need to be determined by studies using larger quantities of brain tissue as employed in porcine [34] and canine [15] studies. Three molecular forms of IR-CNP were defined in ovine brain, the majority of which comprised a CNP22-like form and (most likely) and CNP53-like form. The majority of CNP immunoreactivity in human [20,33] and porcine brain extracts [34] is CNP53 and/or CNP22 material, and like the present study a small amount of high molecular weight CNP (probably proCNP) is observed. Thus, CNP forms observed in the brain compare well with those observed in the vasculature [40]. A significant point raised by the HPLC data is the variable distribution of CNP forms between the pituitary and the rest of the brain. In accordance with the report of Yandle et al. [39], this study confirmed the presence of only CNP53-like material in pituitary extracts, whereas the whole brain (including the hypothalamus) contained CNP22, CNP53 and a high molecular weight form consistent with proCNP. The functional significance of this is unclear but it may relate to differential processing of CNP within these regions. A large amount of CNP immunoreactivity is present in the rat anterior pituitary, but the relative content of the posterior region appears to be very low [18]. Whether similar patterns are present in the ovine pituitary remain to be

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determined but it is clear that there exist marked differences in the regional distribution of natriuretic peptide molecular forms in brain tissue. In conclusion, utilizing species-specific RIA, we determined that immunoreactive ANP, BNP and CNP were all detectable in ovine brain extracts, including the pituitary. The most abundant NP was CNP, particularly in pituitary. In response to pacing-induced heart failure, the pituitary content of BNP was significantly decreased. Specific RIA coupled with HPLC revealed the molecular forms of ANP and CNP in whole brain to be proANP and proCNP, CNP22 and CNP53, respectively. The predominant molecular form of BNP in pituitary extracts was proBNP with a minor component of BNP26. The physiological relevance of a significant decrease in BNP-IR in the ovine pituitary in response to pacing-induced heart failure remains to be clarified. Furthermore, due to the low levels of peptides observed in this study, particularly those of ANP and BNP, further gene expression studies are clearly required to assess the role of central NP’s in the regulation of systemic volume overload.

Acknowledgments This work was supported by the National Heart Foundation and Health Research Council of New Zealand. We thank Drs. Miriam Rademaker and Chris Charles for assistance with surgical preparation and tissue collection.

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