somatogenic and lactogenic binding sites in rat brain and liver: quantitative autoradiographic...

E L S E V I E R Neuroscience Research 20 (1994) 257-263

NEUROSCIENCE RESERRCH

Somatogenic and lactogenic binding sites in rat brain and liver: quantitative autoradiographic localization

Amged Mustafa a'b, Fred Nyberg a, Nenad Bogdanovic c, Atiqul Islam c, Paul RoosO, Abdu Adem *c

aDepartment of Pharmaceutical Biosciences, bDepartment of Biochemistry, University of Uppsala, Uppsala. Sweden CDepartment of Geriatric Medicine, Karolinska Institute, Huddinge Hospital, 141 86 Huddinge, Stockholm, Sweden

Received 2 March 1994; revision received 23 June 1994; accepted 23 June 1994

Abstract

The distribution of somatogenic and lactogenic binding sites in female and male rat brain as well as in liver was studied by quantitative receptor autoradiography using 125I-human growth hormone (t25I-hGH) as a iigand. Quantitative measurement of binding sites for 125I-hGH showed differences in the levels of these sites in the female and male brain and liver. Moreover, regional differences in the brain were also observed in each sex. In the female brain high levels of J25I-hGH binding sites were found in the choroid plexus. Intermediate levels were observed in the striatum, the hypothalamus and the hippocampus, whereas low levels of these sites were found in the central gray, the temporal, the piriform and the entorhinal cortices. In the male brain high levels of 1251- hGH binding sites were detected in the choroid plexus. Intermediate levels were observed in the parietal cortex, the hypothalamus and the hippocampus, whereas low levels were found in the tegmentum, the temporal cortex and the striatum. Quantification of 125I-hGH binding sites in the liver revealed higher levels in the female than in the male liver. In general, higher levels of binding sites (16%-77%) were observed in the female than in the male tissues. The quantification of rat growth hormone (rGH) by radioimmunoassay was also performed in this study. Varying amounts of rGH immunoreactivity were detected in the different brain regions, with the highest levels of rGH-like material being found in the midbrain and cortex of both sexes. Moreover, higher levels of rGH-like material were observed in the female than in the male brain. Thus, sex differences in the levels of both J251-hGH binding sites and rGH-like material were observed between the female and male rat brain.

Keywords: Somatogenic; Lactogenic; Binding sites; Quantitative autoradiography; Brain; Liver; Rats

1. Introduction

The ability of the pituitary growth hormone (GH) and prolactin (PRL) to elicit diverse effects including growth promotion, metabolic regulation, mammary gland de- velopment and milk production, has long been known (Cheek and Hill, 1974; NicoU, 1974; Nicoll et al., 1986; Davidson, 1987). It is widely accepted that both G H and PRL act through binding to specific receptors localized in cell membranes (Isaksson et al., 1985), and are widely distributed in different tissues (Posner et al., 1974; Djiane et al., 1977). The effects of G H are known to be medi-

* Corresponding author. Fax: +46 8 7111751.

ated by the somatogenic receptors, while those of PRL are mediated by the lactogenic receptors. The molecular events that take place following the hormone-receptor binding are yet to be known. However, recent studies have demonstrated that the molecular mechanism of G H signaling involves tyrosyl phosphorylation of the receptors and of an associated protein kinase (JAK2), which is present in a complex with G H receptors (Carter-Su et al., 1989; Stred et al., 1992; Campbell et al., 1993; Lawrence et al., 1993; Wang et al., 1993).

It is thought that G H and PRL induce only peripheral effects; however, central effects of the hormones have recently been observed. Several studies provide convincing evidence that both hormones are able to affect some central mechanisms. Examples are the ability of G H and

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258 ,4. MustaJa et al. / Neurosci. Res. 20 (1994) 257-263

PRL to autoregulate their own secretion from the pituitary gland (Ikrelowitz et al., 1981; Andersson et al., 1983; Murai et al., 1987; Chomczynski et al., 1988) and the ability of PRL to influence water and solute balance (Nicoll, 1980) and behaviour (Bridges et al., 1985; Butin and Tesch, 1985). Consistent with these observations, GH and PRL immunoreactivity was detected in the rat brain (Ho- jvat et al., 1982; Harlan et al., 1989). Moreover, GH receptor immunoreactivity (Lobie et al., 1989; Waters et al., 1990) as well as GH- and PRL-receptors mRNAs (Bur- ton et al., 1992; Sufen et al., 1992) were identified in different regions of the rat brain.

Different methods have been used to characterize receptors for GH and PRL in various brain regions obtained from several species (Posner et al., 1983; Walsh et al., 1990). 125I-human growth hormone Q25I-hGH), which is known to bind to somatogenic as well as to lactogenic receptors in rat tissues (Gluckman et al., 1990), was used in these studies. Recently, using rat brain homogenates we reported sex differences with respect to the levels and properties of hGH binding sites (Mustafa et al., 1994). In the present study quantitative receptor autoradiography was performed to localize binding sites for 125I- hGH in the female and male rat brain and liver. Our results demonstrate, in agreement with our previous findings, higher levels of 125I-hGH binding sites in the female than in the male tissues.

2. Materials and methods

2.1. Materials

125I-hGH (specific activity 111 t~Ci//~g) was purchased from Dupont (NEN-USA), unlabeled hGH and hGH- RIA Kit were from Pharmacia (Stockholm, Sweden). Rat pituitary growth hormone was prepared according to the procedure outlined by Roos et al. (1987). Male and female Sprague-Dawley rats were purchased from ALAB (Sollentuna, Sweden).

2.2. Receptor autoradiography

Five-week-old male and female Sprague-Dawley rats (210-240 g) were decapitated, the brains and livers were removed and mounted for sectioning and frozen onto chucks by slow immersion in liquid nitrogen. Frozen rat brains and livers which were mounted for sectioning were cut in a Leitz cryostat. Sections of 10 t~m were cut and thaw-mounted onto cold microscope slides coated with a chrome/gelatin solution. Slide-mounted sections were used for receptor autoradiography as described previously (Adem, 1987). Briefly, slide-mounted sections were prein- cubated in cold 0.025 M Tris-HCl buffer (pH 7.4) con- raining 0.01 M MgCI 2 and 1% BSA (w/v) for 30 min. For autoradiographic studies, the sections were incubated with the radiolabeled iigand (2 nM, 5 x 10Scounts/min 125I-

hGH; specific activity 111/zCi/#g) in the above buffer for 16 h at 4°C. Nonspecific binding was defined by inhibi- tion in the presence of unlabeled hGH (1 ~M). At the end of the incubation period the sections were rinsed in the buffer to terminate the assay and dipped in distilled water to remove buffer salts. Subsequently, sections were dried under a stream of cold air and tightly juxtaposed to tritium-sensitive films (Hyper film 3H, Amersham, UK) in X-ray cassettes for 72 h at -20°C. Following the ex- posure period the films were removed and developed. For calibration radioactive 'standards' were exposed and developed with the sections to permit conversion of op- tical densities to molar quantities of receptor-bound radioligand. The quantitative analysis of autoradiographs was performed using a Zeiss-Kontron 1BAS image analyzer for the densitometric analysis of the film sheets as described previously by Adem (1987). Quantification of 125I-hGH binding to each region of the brain was performed in six sections from three animals (two sections per animal). Specific binding was obtained by subtrac- ting nonspecific binding from total binding. The specific binding was expressed as femtomoles per milligram tissue wet weight.

Student's T-test was used for statistical analysis.

2.3. Radioimmunoassay

Extraction of GH was carried out as described previously (Hojvat et al., 1982). Briefly, following decapita- tion of the female and male rats, brains were removed and immediately dissected. The dissected brain regions (pool of 4 animals) were homogenized separately in 1 ml of ice-cold PBS buffer (pH 7,6) for 30 s using a motor driven teflon pestle homogenizer. Samples were centrifug- ed at 3000 rev/min for 30 min at 4°(2, supernatants removed and the pellets resuspended in the above buffer overnight at 4°C. The samples were recentrifuged and the supernatants used for radioimmunoassay using rat growth hormone (rGH) antibodies(kindly donated by Prof. Olle Isaksson, University of Gothenburg, Sweden) as described by Hojvat et al. (1982). Radioimmunoassay was also performed using hGH RIA-Kit as described by the manufac- turer. The amount of rGH-like material was expressed as ng/mg protein. Protein concentrations were measured according to Lowry et al. (1951).

3. Results

The distribution of 12SI-hGH binding sites in the female and male rat brain and liver was studied by quantitative receptor autoradiography. Figs. l and 2 show the topographical distribution of these sites in the brain and liver, respectively. Quantitative measurement of 1251- hGH binding in the female brain revealed high levels of binding sites (9.4 fmol/mg tissue wet weight) in the choroid plexus; whereas intermediate levels (3.2-4.7 fmol/mg

A. Mustafa et al./ Neurosci. Res. 20 (1994) 257-263 259

Fig. I. Representative photomicrographs of the autoradiographic distribution of 125I-hGH binding sites in cryosection from female and male rat brain. Total binding of 125I-hGH (2 nM, 5 x l0 5 counts/min) in the absence of excess unlabeled ligand in female A (bregma +1.5) and D (bregma -3.5) and male B (bregma +1.5) and E (bregma -3.5). Nonspecific binding in the presence of excess unlabeled hGH (l /zM), in male C (bregma +1.5) and female F (bregma -3.5).

tissue wet weight) were observed in the hippocampus, the tegmentum, the septum, the hypothalamus and the striatum. Low levels (1.7-2.9 fmol/mg tissue wet weight) were detected in the central gray, the temporal, the piriform and the entorhinal cortices (Fig. 1; Table l). In the male brain high levels of I25I-hGH binding sites (7.9 fmol/mg tissue wet weight) were found in the choroid plexus,

whereas intermediate levels (2.1-3.3 fmol/mg tissue wet weight) were observed in the hippocampus, the parietal cortex and the hypothalamus. Low levels (0.9-1.8 fmol/mg tissue wet weight) of 125I-hGH binding sites were detected in the temporal and the piriform cortices, the striatum and the tegmentum (Fig. 1; Table 1). In- terestingly, intermediate levels of 125I-hGH binding sites

26O

Table i Distribution of 1251-hGH and liver

A. MustaJa et al. /Neurosci.

binding sites in the female and male rat brain

R e , o n 125I-hGH binding sites (fmol/mg tissue wet weight)

Male Female*

Cortex: Frontal 1.1 ± 0.01 3.6 4- 0.03 Parietal 2.7 4- 0.02 3.7 4- 0.03 Temporal 1.7 4- 0.02 2.3 4- 0.04 Occipital 1.2 ± 0.01 3.2 4- 0.02 Retrosplenial 1.2 4- 0.02 3.3 4- 0.03 Piriform 1.6 4- 0.04 2.0 4- 0.03 Entorhinal 1.1 ± 0.01 1.7 4- 0.01 Cingular 0.9 4- 0,02 4,0 4- 0.03

Striatum 1.3 4- 0,01 4.7 4- 0.03 Septum 1.0 4- 0,01 4.1 4- 0.02 Hippocampus 2.1 4- 0.02 3.2 4- 0.03 Hypothalamus 3.3 4- 0,01 4.4 4- 0.02 MammiUary bodies 1.6 4- 0.01 3.2 4- 0.03 Tegraentum 1.8 4- 0.02 4.0 4- 0.04 Central gray 1.1 4- 0.01 2.9 4- 0.03 Capsula interna 3.2 4- 0.03 4.3 4- 0.02 Choroid plexus 7.9 4- 0.02 9.4 4- 0.01 Liver 4.4 4- 0.01 9.6 4- 0.01

Results represent the mean values -~- S.E.M. of 3 animals. *The female values were all significantly higher (P < 0.001) than the male values.

Res. 20 (1994) 257-263

were observed in the female and male white matter. Spe- cific binding to the white matter of the brain has also been reported for other labeled ligands (Ekblom et al., 1992; Ekblom, 1993). In their study, Ekblom et al. (1993) demonstrated that the binding of 3H-L-deprenyl to the white matter is due to its binding to glial cells. Thus, the binding of 125I-hGH to the white matter warrants further study to determine the cellular localization of these binding sites in the white matter in order to understand their physiological relevance.

Measurement of 125I-hGH binding sites in cryosec- tions from female and male liver revealed high levels of these sites in the female liver (9.6 fmol/mg tissue wet weight) compared to the male liver (4.4 fmol/mg tissue wet weight) (Fig. 2; Table 1).

The levels of the binding sites were found to be significantly higher (P < 0.001) in all the female tissues than in the male tissues (Table l). Moreover, from Fig. 3 it can be seen that 12Sl-hGH binding sites in the male brain regions were found to be from 23% to 84% of those in the female brain. Similar to the brain, the male liver showed lower levels of the binding sites (46%) than the female liver (Table l; Fig. 3). The binding of 125I-hGH to its binding sites in the rat brain and liver was almost com- pletely inhibited (75%) in the presence of excess unlabeled hGH.

!

I

i

Fig. 2. Representative photomicrographs of the autoradiographic distribution of t251-hGH binding sites in cryosection from rat liver. Total binding of 1251-hGH (2 nM, 5 x 105 counts/min) in the absence of excess unlabeled ligand in female (A) and male (B). Nonspecific binding in the presence of excess unlabeled hGH (I #M), in female (C) and male (D).

A. Mustafa et al. / Neurosci. Res. 20 (1994) 257-263 261

I00

80

60

40

20

H E-

Fig. 3. Rat brain regional distribution of 1251-hGH binding sites in male as percentage of female binding sites. CTX, cortex.

The distribution of rGH immunoreactivity in the female and male rat brain was measured using a radioimmunoassay for rGH (Table 2). As can be seen from Table 2, rGH-like material was present in different regions of the female and male brain. The amounts of rGH-like material were significantly higher in the female midbrain (P < 0.05) and cortex (P < 0.01) than in the corresponding male brain regions. Using hGH antibodies we also detected GH-like material in the different rat brain regions, and the highest amounts were found in the midbrain and cortex (data not shown).

4. Discussion

The presence of somatogenic and lactogenic receptors in brain and liver homogenates from several species was

Table 2 Distribution of rat GH immunoreactivity in the female and male rat brain

Region a Rat GH immunoreactivity (ng/mg protein)

Male Female

Midbrain 0.92 ± 0.04 1.22 4. 0.11' Cortex 0.64 4- 0.05 0.78 4. 0.08**

Hippocampus 0.58 4. 0.02 0.62 4. 0.04 Striatum 0.38 4. 0.04 0.41 4. 0.03 Olfactory bulb 0.21 4. 0.02 0.31 4. 0.04 Cerebellum 0.03 4. 0.01 0.05 4. 0.02

Results represent mean apool of 4 animals.

*P < 0.05. **P < 0.01.

values ± S.E.M. of 3 experiments.

reported previously (Walsh et al., 1978, 1990; Posner et al., 1983; Muccioli et al. 1989; Di Carlo et al., 1992; Lai et al., 1993). Recently, using 125I-hGH as a ligand we further investigated these binding sites in the rat brain and liver, and observed sex differences with respect to the levels (higher in females) and properties of these sites. Moreover, by using various unlabeled ligands that possess either somatogenic or lactogenic properties we showed that these binding sites display predominantly lactogenic properties in the female brain and almost exclusively somatogenic properties in the male brain (Mustafa et al., 1994). The methodology used in these studies, however, did not allow the precise anatomical localization of J25I- hGH binding sites in the rat brain. In the present study using in vitro receptor autoradiography we provide the regional distribution as well as a quantification of 125I- hGH binding sites in the female and male rat brain and liver. Analysis of 125I-hGH binding in the tissues exam- ined showed a wide distribution of specific binding sites for 125I-hGH in these tissues. Moreover, the presence of rGH immunoreactivity was demonstrated in both female and male rat brain. The quantitative measurement of 125I-hGH binding sites indicated higher levels in the female tissues than in the male. In the brain the highest levels of hGH binding sites were found in the choroid plexus, which showed higher levels of the binding sites (2 to 8 times) than the other brain regions of the same sex. Our results are in agreement with earlier studies demonstrating high levels of 125I-hGH binding sites in the choroid plexus from several species (Posner et al., 1983; Walsh et al., 1990; Lai et al., 1991). The choroid plexus contains a monolayer of cuboidal epithelial cells with three main

262 A. Mustq/~l et aL / Neurosci. Res. 20 { 1994) 257-263

functions: to serve as the blood-cerebrospinal fluid (CSF) barrier, to regulate the CSF production, and to modulate the CSF composition (Cserr, 1975). The choroid plexus is enriched in a variety of m R N A species encoding transport proteins (Dickson et al., 1985; Aldred et al., 1987a,b). Although the physiological relevance of 125I-hGH binding sites in this tissue is not fully understood, it could be suggested that these binding sites may represent binding proteins which transport G H and PRL from the system- ic circulation to the CSF.

Recently, the presence of G H and PRL receptor mRNAs in rat hypothalamus was reported (Burton et al., 1992; Sufen et al., 1992). Our results that 125I-hGH binding sites are present in the hypothalamus further support these findings. Taken together these studies show not only the presence of the message for somatogenic and lactogenic binding sites in the hypothalamus but also of the proteins that bind J25I-hGH. However, Walsh et al. (1990) failed to demonstrate 125I-hGH binding in rat hypothalamus. The discrepancy between our results and those of Walsh et al. (1990) could possibly be due to methodological differences and/or differences in age and sex of the animals used. The somatogenic binding sites in this brain area could possibly be involved in regulating the level of G H by a negative feedback mechanism (Shep- pard et al., 1978; Patel, 1979; Bereiowitz et al., 1981; Andersson et al., 1983). Hypothalamic G H receptors may also be involved in regulating low levels of G H by increas- ing the synthesis of growth hormone-releasing hormone (GHRH) (Chomczynski et al., 1988). Recent studies have provided convincing evidence that G H deficiency is fre- quently caused by impaired synthesis or release of hypothalamic G H R H (Frohman and Jansson, 1986), The presence of lactogenic binding sites in the hypothalamus is consistent with its known relations to PRL. PRL is capable of influencing the hypothalamic components con- cerned with the regulation of PRL secretion from the anterior pituitary gland (Walsh et al., 1990).

The wide distribution of somatogenic and lactogenic binding sites in the brain raises the question as to the or- igin(s) of the hormones that interact with these binding sites. Similar to Hojvat et al. (1982) we detected G H immunoreactivity in the rat brain. De Vito et al. (1987) and De Vito (1988) detected PRL immunoreactivity in the rat brain. Moreover, PRL immunoreactivity was found in the network of nerve terminals in the rat hypothalamus which was unaffected by hypophysectomy (Fuxe et al., 1977). Hojvat et al. (1982) and Gossard et al. (1987) suggested that G H in the brain is endogenously produced since hypophysectomy did not affect the brain content of GH. On the other hand, Login and Macleod (1977) and Walsh et al. (1987) demonstrated a prolactin blood-to-CSF transport mechanism at the choroid plexus. Taken together our present study as well as the above-mentioned studies, it is possible to speculate that 125I-hGH binding sites in the brain interact with endogenously produced and/or blood-borne hormones.

A major site of action for G H appears to be the liver.

It has been reported that the liver is one of the richest tissues containing binding sites for 1251-hGH (Posner et al., 1974). In this study we observed high levels of 125I- hGH binding sites in the liver. Moreover, the female showed higher levels of binding sites (54°/~) than the male liver. The prevalence of G H binding sites in the liver imply that G H induces specific effects in this tissue. It has been demonstrated that G H stimulates the liver cells to syn~ thesize and secrete the insulin-like growth factors (IGFs) which are known to be responsible for most if not all of the anabolic actions of G H (Daughaday et al., 1972).

In conclusion, the present study is the first to quan- titatively demonstrate the localization of ~25I-hGH binding sites in the female and male rat brain and liver. These sites were found to be higher in the female than in the male tissues. The broad distribution of 125I-hGH binding sites in the rat brain provides evidence of the influence of G H and PRL on the brain.

Acknowledgement

This study was supported by Konung Gustaf V:s och Drottning Victorias Stiftelse, Swedish Medical Research Council (Grant 9459 and K93-12P-09288-04B), KI fonder, Stiftelsen f6r Gamla tj/inarinnor, Elsa Goljes Minnes Foundation and Loo och Hans Ostermans Foundation and Pharmacia, Kabi Peptide Hormones, Stockholm.

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somatogenic and lactogenic binding sites in rat brain and liver: quantitative autoradiographic...

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