- Email: [email protected]
Presence of pituitary adenylate cyclase-activating polypeptide (PACAP) in the plasma and milk of ruminant animals
General and Comparative Endocrinology 172 (2011) 115–119
Contents lists available at ScienceDirect
General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen
Presence of pituitary adenylate cyclase-activating polypeptide (PACAP) in the plasma and milk of ruminant animals Levente Czegledi a, Andrea Tamas b, Rita Borzsei c, Terez Bagoly c, Peter Kiss b, Gabriella Horvath b, Reka Brubel b, Jozsef Nemeth d, Balint Szalontai e, Krisztina Szabadfi f, Andras Javor a, Dora Reglodi b,⇑,1, Zsuzsanna Helyes c,1 a
Institute of Animal Science, Centre for Agricultural and Applied Economic Sciences, University of Debrecen, H-4015 Debrecen, P.O. Box 36, Hungary Department of Anatomy, University of Pecs, H-7624 Pecs, Szigeti Street 12, Hungary Department of Pharmacology and Pharmacotherapy, University of Pecs, H-7624 Pecs, Szigeti Street 12, Hungary d Department of Pharmacology and Pharmacotherapy, University of Debrecen, H-4012, P.O. Box 12, Hungary e Department of Plant Physiology, University of Pecs, H-7624 Pecs, Ifjusag Street 6, Hungary f Department of Experimental Zoology and Neurobiology, University of Pecs, H-7624 Pecs, Ifjusag Street 6, Hungary b c
a r t i c l e
i n f o
Article history: Available online 23 December 2010 Keywords: Immunohistochemistry Udder biopsy Radioimmunoassay Milk Serum
a b s t r a c t Milk contains a variety of proteins and peptides that possess biological activity. Growth factors, such as growth hormone, insulin-like, epidermal and nerve growth factors are important milk components which may regulate growth and differentiation in various neonatal tissues and also those of the mammary gland itself. We have recently shown that pituitary adenylate cyclase-activating polypeptide (PACAP), an important neuropeptide with neurotrophic actions, is present in the human milk in much higher concentration than in the plasma of lactating women. Investigation of growth factors in the milk of domestic animals is of utmost importance for their nutritional values and agricultural significance. Therefore, the aim of the present study was to determine the presence and concentration of PACAP in the plasma and milk of three ruminant animal species. Furthermore, the presence of PACAP and its specific PAC1 receptor were investigated in the mammary glands. Radioimmunoassay measurements revealed that PACAP was present in the plasma and the milk of the sheep, goat and the cow in a similar concentration to that measured previously in humans. PACAP38-like immunoreactivity (PACAP38-LI) was 5–20-fold higher in the milk than in the plasma samples of the respective animals, a similar serum/milk ratio was found in all the three species. The levels did not show significant changes within the examined 3-month-period of lactation after delivery. Similar PACAP38-LI was measured in the homogenates of the sheep mammary gland samples taken 7 and 30 days after delivery. PAC1 receptor expression was detected in these udder biopsies by fluorescent immunohistochemistry suggesting that this peptide might have an effect on the mammary glands themselves. These data show that PACAP is present in the milk of various ruminant domestic animal species at high concentrations, the physiological implications of which awaits further investigation. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal peptide (VIP)/secretin/glucagon peptide family, which was isolated from ovine hypothalamus as an activator of adenylate cyclase in the pituitary gland [24]. Since its discovery, the presence of PACAP has been shown to be widely distributed in endocrine glands and nervous system, and also in cardiovascular, gastrointestinal and respiratory tracts [24]. ⇑ Corresponding author. Fax: +36 72536393. 1
E-mail address: [email protected] (D. Reglodi). These authors made equal contribution to this work.
0016-6480/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2010.12.012
PACAP is found in two bioactive forms, PACAP27 and PACAP38, with PACAP38 being the major form in mammalian tissues [24]. The peptide has a very conserved structure: the amino acid sequence of PACAP is the same in all mammalian species [24]. PACAP is involved in the regulation of various physiological processes, such as feeding, reproduction, thermoregulation, catecholamine synthesis and motor activity [24]. Special attention has been paid to its developmental role not only in the nervous system but also in peripheral organs [6,18,20,22]. The specific PACAP receptor is PAC1, which can be coupled to both Gs and Gq proteins activating adenylate cyclase and phospholipase C signal transduction pathways, respectively. The other PACAP receptors VPAC1 and VPAC2, have similar binding affinities to both PACAP and VIP [22,24].
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Recently, we have provided mass spectrometric evidence that PACAP is present in human milk [3]. Radioimmunoassay (RIA) measurements revealed that PACAP concentrations exceeded those in plasma 5–20-fold [3]. High concentrations of VIP, structurally the closest to PACAP, have been measured in milk [25]. Milk contains a variety of proteins and peptides that possess biological activity. Several hormones have been described in the milk, including pituitary, hypothalamic, pancreatic, thyroid, adrenal, gonadal and gut hormones [11]. Growth factors, such as growth hormone, insulin-like growth factor (IGF), epidermal growth factor (EGF), nerve growth factor and transforming growth factor, are important milk components [4,11]. Bioactive substances in milk may regulate growth and differentiation in various neonatal tissues and also that of the mammary gland itself [4,11]. Milk-borne growth factors play an especially important role in the maturation of the alimentary tract [17]. Investigation of growth factors in the milk of domestic animals is of utmost importance for their nutritional values and agricultural significance [17]. Several studies have shown the presence of different growth factors in milk of cow, sheep and goats [1,5,7,9,15,17,26] and other animals with dietary importance [29]. The aim of the present study is to investigate whether PACAP, a trophic peptide, is present in the milk of the most commonly used ruminant domestic animals, the cow, the goat and the sheep, and to compare milk concentrations to plasma PACAP levels. PACAP and PAC1 receptor-like immunoreactivity in the mammary gland were also investigated.
2. Materials and methods 2.1. Sample collection Samples were collected from adult lactating 4–5-year-old Holstein–Friesian cows, Merino sheeps and Hungarian Milk Brown goats (n = 10 of each species) from April till July according to a protocol approved by the Institutional Ethic Committee. The animals were kept in stable during winter. Sheep and goat were grazing from April, but cows were still housed with free access to outdoor during the whole experiment. Blood, milk and biopsy samples were collected in the morning, between 8–10 h at each time. Blood (10 ml per animal) was taken from the jugular vein into ice-cold glass tubes containing EDTA (18 mg) and Trasylol (1200 U). Following centrifugation (1000 rpm for 10 min and 4000 rpm for 10 min at 4 °C) the plasma samples were removed and stored at 80 °C for further investigations. Milk (5 ml per animal) was obtained at the morning milking from the same animals. Blood and milk samples were collected on postpartum Days 7, 30 and 90. Udder biopsy samples were taken from lactating Merino ewes (n = 4) using a minimal invasive method on postpartum Days 7 and 30. Udder biopsy was taken by a Bard Magnum core biopsy system with a 130 mm legth and 15 mm penetration depth coaxial needle. Before sampling animals were fixed, udder cleaned and anesthetized locally by hypodermic lidocaine injection. One part of tissue samples from each animal were immediately placed into liquid nitrogen, transferred to laboratory, and stored at 80 °C. Other part of samples was fixed in 4% (v/v) formaline in PBS.
2.2. Radioimmunoassay (RIA) method PACAP38-like immunoreactivity (PACAP38-LI) in the plasma and milk whey was determined with a specific and sensitive RIA technique developed in our laboratory [3,12,16] and concentrations of the peptide were calculated with the help of a calibration curve.
The peptide was extracted from 3 ml plasma samples by addition of a double volume of absolute alcohol and 20 ll 96% acetic acid. After precipitation and a second centrifugation (3000 rpm for 20 min at 4 °C) the samples were dried under nitrogen flow and resuspended in 300 ll assay buffer before RIA determination to achieve a 10 times higher concentration for the RIA procedure [3,12,16]. Milk samples were collected into ice-cold tubes, then 10 ll 96% acetic acid was added to 1 ml milk and incubated in 40 °C water bath for 5 min to precipitate the protein content. Centrifugation was performed at 4000 rpm for 10 min at 4 °C to obtain a solid fat component on the top of the samples. The whey localized between the precipitated protein and fat fractions was then removed for RIA analysis. The ‘‘88111-3’’ PACAP38 antiserum was raised in rabbits with synthetic peptides conjugated to bovine serum albumin or thyroglobulin by glutaraldehyde or carbodiimide. The high specificity and C-terminal sensitivity of this antibody has been confirmed by cross-reactivity studies, and no cross-reactivity was found with PACAP27 or other neuropeptides [12]. The RIA tracers were mono-125I-labelled peptides prepared in our laboratory. Synthetic peptides were used as RIA standards ranging from 0 to 1000 fmol/ml. The assay was prepared in 1 ml 0.05 mol/l (pH 7.4) phosphate buffer containing 0.1 mol/l sodium chloride, 0.25% (w/ v) BSA and 0.05% (w/v) sodium azide. The antiserum (100 ll, 1:10,000 dilution), the RIA tracer (100 ll 5000 cpm/tube) and the standard (100 ll) or the samples (200 ll) were measured into polypropylene tubes with the assay buffer. After 48–72 h incubation at 4 °C, the antibody-bound peptide was separated from the free one by the addition of 100 ll separating solution (10 g charcoal, 1 g dextran and 0.5 g commercial fat-free milk powder in 100 ml distilled water). Following centrifugation (3000 rpm, 4 °C, 20 min) the tubes were gently decanted and the radioactivity of the precipitates was measured in a gamma counter (Gamma, type: NZ310). PACAP38-LI concentrations of the samples were read from a calibration curve created using the standard preparations. 2.3. PAC1 receptor-immunostaining The tissues were immediately dissected in ice-cold phosphatebuffered saline and fixed in 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (PB, pH 7.4) for 1 h at room temperature. Tissue was then washed in 0.1 M PB, and cryoprotected in 10% sucrose for 1 h, 20% sucrose in phosphate-buffered saline (PBS) overnight at 4 °C. For cryostat sectioning, tissues were embedded in tissue freezing medium (Tissue-Tek, OCT Compound, Sakura Finetech, NL), cut in a cryostat (Leica, Nussloch, Germany) at 10 lm radial sections. Sections were mounted on chrome–alum– gelatin coated subbed slides and stored at 20 °C until use. At least 10 sections were examined. Tissue sections were rinsed in PBS, permeabilized by incubation for 5 min in 0.1% Triton X-100 in PBS and incubated with 0.1% bovine serum albumin, 1% normal goat serum and 0.1% Na-azide in PBS for 1 h to minimize nonspecific labeling. Sections were incubated with anti-PAC1 receptor antibody raised in rabbit (Sigma, Hungary) for overnight at room temperature. After several washes in PBS, sections were incubated for 2 h at 37 °C in the dark with the corresponding Alexa Fluor ‘‘568’’ secondary antibody also raised in rabbit (Southern Biotech). Sections were then washed in PBS and were coverslipped using Fluoromount-G (Southern Biotech). For control experiments, primary antisera were omitted and after the protocol, specific cellular staining was not found. Digital photographs were taken with a Nikon Eclipse 80i microscope equipped with a cooled CCD camera. Images were taken with the Spot software package. Photographs were further processed with the Adobe Photoshop 7.0 program. Images were adjusted for contrast only, aligned, arranged and labeled using the functions of the above program.
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2.4. Statistical analysis Data are expressed as means ± SEM of n = 8 to 10 animals per group for the plasma and milk whey measurements, and of n = 4 sheep for the udder biopsies. Statistical analysis to compare PACAP38-Li in the plasma and milk of the same animals and also between the three postpartum time-points was performed with Student’s t-test for paired comparisons. For the evaluation of differences between the species Student’s t-test for unpaired comparison was used. In case of the udder biopsies the non-parametric Mann–Whitney U-test was used. In all cases p < 0.05 was considered statistically significant.
3. Results 3.1. PACAP38-LI in the plasma and milk whey of the sheep, goat and cow
Fig. 2. PACAP38-like immunoreactivity determined by RIA from the cow plasma and milk whey. Data are means ± SEM of n = 8 to 10 animals per group, ⁄⁄⁄p < 0.001 vs. respective plasma samples (Student’s t-test for paired comparison).
PACAP38 could be very accurately and reliably measured in the plasma with relatively small interindividual differences. Similarly to the plasma, this RIA technique also proved to be very sensitive and specific for the determination of PACAP38-LI in the milk of sheep and goat. The concentration of this peptide in the milk whey was almost 10 times higher than in the plasma of the respective animals. The levels did not show significant changes within the examined 3-month-period of lactation after birth (Fig. 1). The presence of PACAP38-LI was also demonstrated in the cow milk, with a serum/milk ratio similar to the other two ruminant animal species, but significantly lower that in the milk of goat and sheep. Since no significant changes were observed within the 3-month-period of lactation in the other species, in the cow, no time-dependence was investigated (Fig. 2).
3.2. PACAP38-LI in the homogenates of sheep udder biopsies Seven days after delivery 21.07 ± 3.39 fmol/mg PACAP38-LI was measured in the homogenates of the mammary gland biopsies. This value decreased to 12.92 ± 4.07 at the 30-day postpartum time-point, but this change did not prove to be statistically significant (Fig. 3).
Fig. 3. PACAP38-like immunoreactivity determined by RIA from the homogenates of sheep udder biopsy samples taken 7 and 30 days after delivery. Data are means ± SEM of n = 4 animals per group.
3.3. Immunolocalization of the PAC1 receptor in the mammary gland Immunolocalization of the PACAP-specific PAC1 receptor was clearly shown on the glandular epithelial cells of the lactating mammary gland of the sheep similarly 7 and 30 days after delivery. Negative control sections stained without the primary antibody (10 sections) did not show any specific signals (Fig. 4A). The distribution of PAC1 receptor immunolabeling was structure-dependent. The glandular epithelial cells expressed remarkable and intensive immunopositivity of the PAC1 receptor, particularly in the membrane of these cells, but granular immunostaining could also be detected in the cytoplasmic region (Figs. 4B–D). In contrast, the interlobular connective tissue was negative (Fig. 4C).
4. Discussion
Fig. 1. PACAP38-like immunoreactivity determined by RIA from the goat and sheep plasma and milk whey 7, 30 and 90 days after delivery. Data are means ± SEM of n = 8 to 10 animals per group, ⁄⁄⁄p < 0.001 vs. respective plasma samples (Student’s t-test for paired comparison).
In the present study we provided evidence that PACAP38 is present in the milk of domestic animals at concentrations comparable to human milk [3]. Highest levels were found in goat and sheep milk, while cow milk contained significantly lower levels. Furthermore, similarly to human milk, concentrations of PACAP
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Fig. 4. Representative sections of the lactating mammary gland samples of the sheep taken 7 days after delivery and stained with anti-PAC1 receptor-specific primary, and a fluorescent secondary antibody. Panel A shows a negative control of PAC1 receptor reactivity. The glandular epithelial cells express clear and intensive immunopositivity of the PAC1 receptor, particularly in the membrane of these cells (arrowheads), but granular immunostaining could also be detected in the cytoplasmic region (B)–(D). Panel C represents the typical segmental structure of the mammary gland where the main part of the tissue shows remarkable PAC1 receptor labeling, but the connective tissue sections between the segments are negative (arrows). Scale bars: 50 lm in Fig. 4A, C, D and 20 lm in Fig. 4B.
in the milk of these animals exceeded those of plasma by 5–20fold. The finding that cow milk contained the lowest levels of PACAP is in accordance with the observations of others showing that cow milk contains lower levels of growth factors than human or goat milk [28]. Various hormones and peptides have been described in the milk [11], like gastric inhibitory peptide, bombesin, peptide YY, neurotensin, gastrin, cholecystokinin, and peptide histidine methionine. A supposed function of the gastrointestinal peptides is the regulation of growth and maturation of the gastrointestinal system in neonates [2]. The list of other growth factors and hormones present in human milk is abundant, and several of these are present in higher concentrations in the milk than in the plasma, such as gonadotropin releasing hormone (GnRH), thyreotrop hormone (TRH), VIP, somatostatin, growth hormone releasing hormone (GHRH), relaxin, IGF-1, epidermal growth factor (EGF), transforming growth factor (TGF) and prostaglandins [11]. Multifunctional roles are attributed to several growth factors present in milk, like influencing lactation, mammary epithelial cell cycle and growth and the development of the newborn [4]. Although the exact function of PACAP in the milk is not known at the moment, several studies support one or more of the above-mentioned actions. PACAP influences lactation [14], and its presence has been shown in the mammary gland [21]. VIP, structurally the closest to PACAP, is also present at high concentrations in human and bovine milk [23,25] and it increases mammary blood flow during lactation [8]. The growth and apoptosis of mammary epithelial cells are tightly controlled during lactation and involution of the mammary gland, when massive apoptosis is present. High mammary concentrations of another growth factor, IGF, coincide with the period of very active mammary growth and development during late pregnancy [11]. PACAP has well-known antiapoptotic effects not only in the nervous system, but also in
peripheral organs. Although the antiapoptotic effect of PACAP has not been evidenced in the mammary gland yet, such a role is possible in this organ similarly to that described in the salivary gland [19]. The special chemical composition of milk together with the immature neonatal proteolytic activity and higher intestinal permeability for macromolecules allow milkborne factors acting on a diversity of neonatal tissues [28]. Although the function of PACAP in milk can only be hypothesized at the moment, its high-concentrations suggest that it might be required for the growth and development of the newborn. This is supported by the well-known roles of PACAP during not only pre-, but also postnatal nervous system development [27]. PACAP functions as a neurotrophic factor from very early stages of neuronal development influencing neurogenesis, differentiation and patterning, and it is also involved in processes that continue after birth, such as astrocytogenesis, myelination, cerebellar development and neuronal migration. Multifunctional milk components protect against several pathogens and also stimulate the development of the natural defensive mechanisms [11,13]. PACAP has a variety of immunomodulatory actions, both during the development of the immune system and in mature lymphocytes and macrophages [10]. Taken together, the present results provide evidence that PACAP is present in bovine, goat, and ovine milk. These data provide the basis for further experiments to elucidate the functions of PACAP in milk. Acknowledgments This work was supported by OTKA grants K72592, K73044, F67830, K75965, 78480, Richter Gedeon Centenary Foundation, Bolyai Scholarship ETT 278-04/2009, SROP-4.2.2/08/1/2008-0011 and PTE AOK Research Grant 2010.
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Contents lists available at ScienceDirect
General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen
Presence of pituitary adenylate cyclase-activating polypeptide (PACAP) in the plasma and milk of ruminant animals Levente Czegledi a, Andrea Tamas b, Rita Borzsei c, Terez Bagoly c, Peter Kiss b, Gabriella Horvath b, Reka Brubel b, Jozsef Nemeth d, Balint Szalontai e, Krisztina Szabadfi f, Andras Javor a, Dora Reglodi b,⇑,1, Zsuzsanna Helyes c,1 a
Institute of Animal Science, Centre for Agricultural and Applied Economic Sciences, University of Debrecen, H-4015 Debrecen, P.O. Box 36, Hungary Department of Anatomy, University of Pecs, H-7624 Pecs, Szigeti Street 12, Hungary Department of Pharmacology and Pharmacotherapy, University of Pecs, H-7624 Pecs, Szigeti Street 12, Hungary d Department of Pharmacology and Pharmacotherapy, University of Debrecen, H-4012, P.O. Box 12, Hungary e Department of Plant Physiology, University of Pecs, H-7624 Pecs, Ifjusag Street 6, Hungary f Department of Experimental Zoology and Neurobiology, University of Pecs, H-7624 Pecs, Ifjusag Street 6, Hungary b c
a r t i c l e
i n f o
Article history: Available online 23 December 2010 Keywords: Immunohistochemistry Udder biopsy Radioimmunoassay Milk Serum
a b s t r a c t Milk contains a variety of proteins and peptides that possess biological activity. Growth factors, such as growth hormone, insulin-like, epidermal and nerve growth factors are important milk components which may regulate growth and differentiation in various neonatal tissues and also those of the mammary gland itself. We have recently shown that pituitary adenylate cyclase-activating polypeptide (PACAP), an important neuropeptide with neurotrophic actions, is present in the human milk in much higher concentration than in the plasma of lactating women. Investigation of growth factors in the milk of domestic animals is of utmost importance for their nutritional values and agricultural significance. Therefore, the aim of the present study was to determine the presence and concentration of PACAP in the plasma and milk of three ruminant animal species. Furthermore, the presence of PACAP and its specific PAC1 receptor were investigated in the mammary glands. Radioimmunoassay measurements revealed that PACAP was present in the plasma and the milk of the sheep, goat and the cow in a similar concentration to that measured previously in humans. PACAP38-like immunoreactivity (PACAP38-LI) was 5–20-fold higher in the milk than in the plasma samples of the respective animals, a similar serum/milk ratio was found in all the three species. The levels did not show significant changes within the examined 3-month-period of lactation after delivery. Similar PACAP38-LI was measured in the homogenates of the sheep mammary gland samples taken 7 and 30 days after delivery. PAC1 receptor expression was detected in these udder biopsies by fluorescent immunohistochemistry suggesting that this peptide might have an effect on the mammary glands themselves. These data show that PACAP is present in the milk of various ruminant domestic animal species at high concentrations, the physiological implications of which awaits further investigation. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal peptide (VIP)/secretin/glucagon peptide family, which was isolated from ovine hypothalamus as an activator of adenylate cyclase in the pituitary gland [24]. Since its discovery, the presence of PACAP has been shown to be widely distributed in endocrine glands and nervous system, and also in cardiovascular, gastrointestinal and respiratory tracts [24]. ⇑ Corresponding author. Fax: +36 72536393. 1
E-mail address: [email protected] (D. Reglodi). These authors made equal contribution to this work.
0016-6480/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2010.12.012
PACAP is found in two bioactive forms, PACAP27 and PACAP38, with PACAP38 being the major form in mammalian tissues [24]. The peptide has a very conserved structure: the amino acid sequence of PACAP is the same in all mammalian species [24]. PACAP is involved in the regulation of various physiological processes, such as feeding, reproduction, thermoregulation, catecholamine synthesis and motor activity [24]. Special attention has been paid to its developmental role not only in the nervous system but also in peripheral organs [6,18,20,22]. The specific PACAP receptor is PAC1, which can be coupled to both Gs and Gq proteins activating adenylate cyclase and phospholipase C signal transduction pathways, respectively. The other PACAP receptors VPAC1 and VPAC2, have similar binding affinities to both PACAP and VIP [22,24].
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Recently, we have provided mass spectrometric evidence that PACAP is present in human milk [3]. Radioimmunoassay (RIA) measurements revealed that PACAP concentrations exceeded those in plasma 5–20-fold [3]. High concentrations of VIP, structurally the closest to PACAP, have been measured in milk [25]. Milk contains a variety of proteins and peptides that possess biological activity. Several hormones have been described in the milk, including pituitary, hypothalamic, pancreatic, thyroid, adrenal, gonadal and gut hormones [11]. Growth factors, such as growth hormone, insulin-like growth factor (IGF), epidermal growth factor (EGF), nerve growth factor and transforming growth factor, are important milk components [4,11]. Bioactive substances in milk may regulate growth and differentiation in various neonatal tissues and also that of the mammary gland itself [4,11]. Milk-borne growth factors play an especially important role in the maturation of the alimentary tract [17]. Investigation of growth factors in the milk of domestic animals is of utmost importance for their nutritional values and agricultural significance [17]. Several studies have shown the presence of different growth factors in milk of cow, sheep and goats [1,5,7,9,15,17,26] and other animals with dietary importance [29]. The aim of the present study is to investigate whether PACAP, a trophic peptide, is present in the milk of the most commonly used ruminant domestic animals, the cow, the goat and the sheep, and to compare milk concentrations to plasma PACAP levels. PACAP and PAC1 receptor-like immunoreactivity in the mammary gland were also investigated.
2. Materials and methods 2.1. Sample collection Samples were collected from adult lactating 4–5-year-old Holstein–Friesian cows, Merino sheeps and Hungarian Milk Brown goats (n = 10 of each species) from April till July according to a protocol approved by the Institutional Ethic Committee. The animals were kept in stable during winter. Sheep and goat were grazing from April, but cows were still housed with free access to outdoor during the whole experiment. Blood, milk and biopsy samples were collected in the morning, between 8–10 h at each time. Blood (10 ml per animal) was taken from the jugular vein into ice-cold glass tubes containing EDTA (18 mg) and Trasylol (1200 U). Following centrifugation (1000 rpm for 10 min and 4000 rpm for 10 min at 4 °C) the plasma samples were removed and stored at 80 °C for further investigations. Milk (5 ml per animal) was obtained at the morning milking from the same animals. Blood and milk samples were collected on postpartum Days 7, 30 and 90. Udder biopsy samples were taken from lactating Merino ewes (n = 4) using a minimal invasive method on postpartum Days 7 and 30. Udder biopsy was taken by a Bard Magnum core biopsy system with a 130 mm legth and 15 mm penetration depth coaxial needle. Before sampling animals were fixed, udder cleaned and anesthetized locally by hypodermic lidocaine injection. One part of tissue samples from each animal were immediately placed into liquid nitrogen, transferred to laboratory, and stored at 80 °C. Other part of samples was fixed in 4% (v/v) formaline in PBS.
2.2. Radioimmunoassay (RIA) method PACAP38-like immunoreactivity (PACAP38-LI) in the plasma and milk whey was determined with a specific and sensitive RIA technique developed in our laboratory [3,12,16] and concentrations of the peptide were calculated with the help of a calibration curve.
The peptide was extracted from 3 ml plasma samples by addition of a double volume of absolute alcohol and 20 ll 96% acetic acid. After precipitation and a second centrifugation (3000 rpm for 20 min at 4 °C) the samples were dried under nitrogen flow and resuspended in 300 ll assay buffer before RIA determination to achieve a 10 times higher concentration for the RIA procedure [3,12,16]. Milk samples were collected into ice-cold tubes, then 10 ll 96% acetic acid was added to 1 ml milk and incubated in 40 °C water bath for 5 min to precipitate the protein content. Centrifugation was performed at 4000 rpm for 10 min at 4 °C to obtain a solid fat component on the top of the samples. The whey localized between the precipitated protein and fat fractions was then removed for RIA analysis. The ‘‘88111-3’’ PACAP38 antiserum was raised in rabbits with synthetic peptides conjugated to bovine serum albumin or thyroglobulin by glutaraldehyde or carbodiimide. The high specificity and C-terminal sensitivity of this antibody has been confirmed by cross-reactivity studies, and no cross-reactivity was found with PACAP27 or other neuropeptides [12]. The RIA tracers were mono-125I-labelled peptides prepared in our laboratory. Synthetic peptides were used as RIA standards ranging from 0 to 1000 fmol/ml. The assay was prepared in 1 ml 0.05 mol/l (pH 7.4) phosphate buffer containing 0.1 mol/l sodium chloride, 0.25% (w/ v) BSA and 0.05% (w/v) sodium azide. The antiserum (100 ll, 1:10,000 dilution), the RIA tracer (100 ll 5000 cpm/tube) and the standard (100 ll) or the samples (200 ll) were measured into polypropylene tubes with the assay buffer. After 48–72 h incubation at 4 °C, the antibody-bound peptide was separated from the free one by the addition of 100 ll separating solution (10 g charcoal, 1 g dextran and 0.5 g commercial fat-free milk powder in 100 ml distilled water). Following centrifugation (3000 rpm, 4 °C, 20 min) the tubes were gently decanted and the radioactivity of the precipitates was measured in a gamma counter (Gamma, type: NZ310). PACAP38-LI concentrations of the samples were read from a calibration curve created using the standard preparations. 2.3. PAC1 receptor-immunostaining The tissues were immediately dissected in ice-cold phosphatebuffered saline and fixed in 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (PB, pH 7.4) for 1 h at room temperature. Tissue was then washed in 0.1 M PB, and cryoprotected in 10% sucrose for 1 h, 20% sucrose in phosphate-buffered saline (PBS) overnight at 4 °C. For cryostat sectioning, tissues were embedded in tissue freezing medium (Tissue-Tek, OCT Compound, Sakura Finetech, NL), cut in a cryostat (Leica, Nussloch, Germany) at 10 lm radial sections. Sections were mounted on chrome–alum– gelatin coated subbed slides and stored at 20 °C until use. At least 10 sections were examined. Tissue sections were rinsed in PBS, permeabilized by incubation for 5 min in 0.1% Triton X-100 in PBS and incubated with 0.1% bovine serum albumin, 1% normal goat serum and 0.1% Na-azide in PBS for 1 h to minimize nonspecific labeling. Sections were incubated with anti-PAC1 receptor antibody raised in rabbit (Sigma, Hungary) for overnight at room temperature. After several washes in PBS, sections were incubated for 2 h at 37 °C in the dark with the corresponding Alexa Fluor ‘‘568’’ secondary antibody also raised in rabbit (Southern Biotech). Sections were then washed in PBS and were coverslipped using Fluoromount-G (Southern Biotech). For control experiments, primary antisera were omitted and after the protocol, specific cellular staining was not found. Digital photographs were taken with a Nikon Eclipse 80i microscope equipped with a cooled CCD camera. Images were taken with the Spot software package. Photographs were further processed with the Adobe Photoshop 7.0 program. Images were adjusted for contrast only, aligned, arranged and labeled using the functions of the above program.
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2.4. Statistical analysis Data are expressed as means ± SEM of n = 8 to 10 animals per group for the plasma and milk whey measurements, and of n = 4 sheep for the udder biopsies. Statistical analysis to compare PACAP38-Li in the plasma and milk of the same animals and also between the three postpartum time-points was performed with Student’s t-test for paired comparisons. For the evaluation of differences between the species Student’s t-test for unpaired comparison was used. In case of the udder biopsies the non-parametric Mann–Whitney U-test was used. In all cases p < 0.05 was considered statistically significant.
3. Results 3.1. PACAP38-LI in the plasma and milk whey of the sheep, goat and cow
Fig. 2. PACAP38-like immunoreactivity determined by RIA from the cow plasma and milk whey. Data are means ± SEM of n = 8 to 10 animals per group, ⁄⁄⁄p < 0.001 vs. respective plasma samples (Student’s t-test for paired comparison).
PACAP38 could be very accurately and reliably measured in the plasma with relatively small interindividual differences. Similarly to the plasma, this RIA technique also proved to be very sensitive and specific for the determination of PACAP38-LI in the milk of sheep and goat. The concentration of this peptide in the milk whey was almost 10 times higher than in the plasma of the respective animals. The levels did not show significant changes within the examined 3-month-period of lactation after birth (Fig. 1). The presence of PACAP38-LI was also demonstrated in the cow milk, with a serum/milk ratio similar to the other two ruminant animal species, but significantly lower that in the milk of goat and sheep. Since no significant changes were observed within the 3-month-period of lactation in the other species, in the cow, no time-dependence was investigated (Fig. 2).
3.2. PACAP38-LI in the homogenates of sheep udder biopsies Seven days after delivery 21.07 ± 3.39 fmol/mg PACAP38-LI was measured in the homogenates of the mammary gland biopsies. This value decreased to 12.92 ± 4.07 at the 30-day postpartum time-point, but this change did not prove to be statistically significant (Fig. 3).
Fig. 3. PACAP38-like immunoreactivity determined by RIA from the homogenates of sheep udder biopsy samples taken 7 and 30 days after delivery. Data are means ± SEM of n = 4 animals per group.
3.3. Immunolocalization of the PAC1 receptor in the mammary gland Immunolocalization of the PACAP-specific PAC1 receptor was clearly shown on the glandular epithelial cells of the lactating mammary gland of the sheep similarly 7 and 30 days after delivery. Negative control sections stained without the primary antibody (10 sections) did not show any specific signals (Fig. 4A). The distribution of PAC1 receptor immunolabeling was structure-dependent. The glandular epithelial cells expressed remarkable and intensive immunopositivity of the PAC1 receptor, particularly in the membrane of these cells, but granular immunostaining could also be detected in the cytoplasmic region (Figs. 4B–D). In contrast, the interlobular connective tissue was negative (Fig. 4C).
4. Discussion
Fig. 1. PACAP38-like immunoreactivity determined by RIA from the goat and sheep plasma and milk whey 7, 30 and 90 days after delivery. Data are means ± SEM of n = 8 to 10 animals per group, ⁄⁄⁄p < 0.001 vs. respective plasma samples (Student’s t-test for paired comparison).
In the present study we provided evidence that PACAP38 is present in the milk of domestic animals at concentrations comparable to human milk [3]. Highest levels were found in goat and sheep milk, while cow milk contained significantly lower levels. Furthermore, similarly to human milk, concentrations of PACAP
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Fig. 4. Representative sections of the lactating mammary gland samples of the sheep taken 7 days after delivery and stained with anti-PAC1 receptor-specific primary, and a fluorescent secondary antibody. Panel A shows a negative control of PAC1 receptor reactivity. The glandular epithelial cells express clear and intensive immunopositivity of the PAC1 receptor, particularly in the membrane of these cells (arrowheads), but granular immunostaining could also be detected in the cytoplasmic region (B)–(D). Panel C represents the typical segmental structure of the mammary gland where the main part of the tissue shows remarkable PAC1 receptor labeling, but the connective tissue sections between the segments are negative (arrows). Scale bars: 50 lm in Fig. 4A, C, D and 20 lm in Fig. 4B.
in the milk of these animals exceeded those of plasma by 5–20fold. The finding that cow milk contained the lowest levels of PACAP is in accordance with the observations of others showing that cow milk contains lower levels of growth factors than human or goat milk [28]. Various hormones and peptides have been described in the milk [11], like gastric inhibitory peptide, bombesin, peptide YY, neurotensin, gastrin, cholecystokinin, and peptide histidine methionine. A supposed function of the gastrointestinal peptides is the regulation of growth and maturation of the gastrointestinal system in neonates [2]. The list of other growth factors and hormones present in human milk is abundant, and several of these are present in higher concentrations in the milk than in the plasma, such as gonadotropin releasing hormone (GnRH), thyreotrop hormone (TRH), VIP, somatostatin, growth hormone releasing hormone (GHRH), relaxin, IGF-1, epidermal growth factor (EGF), transforming growth factor (TGF) and prostaglandins [11]. Multifunctional roles are attributed to several growth factors present in milk, like influencing lactation, mammary epithelial cell cycle and growth and the development of the newborn [4]. Although the exact function of PACAP in the milk is not known at the moment, several studies support one or more of the above-mentioned actions. PACAP influences lactation [14], and its presence has been shown in the mammary gland [21]. VIP, structurally the closest to PACAP, is also present at high concentrations in human and bovine milk [23,25] and it increases mammary blood flow during lactation [8]. The growth and apoptosis of mammary epithelial cells are tightly controlled during lactation and involution of the mammary gland, when massive apoptosis is present. High mammary concentrations of another growth factor, IGF, coincide with the period of very active mammary growth and development during late pregnancy [11]. PACAP has well-known antiapoptotic effects not only in the nervous system, but also in
peripheral organs. Although the antiapoptotic effect of PACAP has not been evidenced in the mammary gland yet, such a role is possible in this organ similarly to that described in the salivary gland [19]. The special chemical composition of milk together with the immature neonatal proteolytic activity and higher intestinal permeability for macromolecules allow milkborne factors acting on a diversity of neonatal tissues [28]. Although the function of PACAP in milk can only be hypothesized at the moment, its high-concentrations suggest that it might be required for the growth and development of the newborn. This is supported by the well-known roles of PACAP during not only pre-, but also postnatal nervous system development [27]. PACAP functions as a neurotrophic factor from very early stages of neuronal development influencing neurogenesis, differentiation and patterning, and it is also involved in processes that continue after birth, such as astrocytogenesis, myelination, cerebellar development and neuronal migration. Multifunctional milk components protect against several pathogens and also stimulate the development of the natural defensive mechanisms [11,13]. PACAP has a variety of immunomodulatory actions, both during the development of the immune system and in mature lymphocytes and macrophages [10]. Taken together, the present results provide evidence that PACAP is present in bovine, goat, and ovine milk. These data provide the basis for further experiments to elucidate the functions of PACAP in milk. Acknowledgments This work was supported by OTKA grants K72592, K73044, F67830, K75965, 78480, Richter Gedeon Centenary Foundation, Bolyai Scholarship ETT 278-04/2009, SROP-4.2.2/08/1/2008-0011 and PTE AOK Research Grant 2010.
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