Study of pro-opiomelanocortin mRNA expression in human postmortem pituitaries

Molecular Brain Research, 10 (1991) 129-137 © 1991 Elsevier Science Publishers B.V. 0169-328X/91/$03.50 ADONIS 0169328X9170288T

129

BRESM 70288

Study of pro-opiomelanocortin mRNA expression in human postmortem pituitaries G. Mengod 1, M.M. Vivanco L*, A. Christnacher 1, A. Probst 2 and J.M. Palacios 1 t Preclinical Research, Sandoz Pharma Ltd., Basle (Switzerland) and 2Department of Pathology, Institut of Pathology, University of Basle, Basle (Switzerland)

(Accepted 11 December 1990) Key words: Pro-opiomelanocortin mRNA; In situ hybridization; Human pituitary; Neurological disease

Complementary oligonucleotide probes specific for the human pro-opiomelanocortin (POMC) mRNA were used to analyze the expression of POMC gene in 56 human postmortem pituitaries by in situ hybridization histochemistry. POMC transcripts were visualized by autoradiography in anterior lobe of the pituitary where their distribution was in a 'patchy-like' pattern. No hybridization could be observed in the posterior lobe of the pituitary. We examined pituitaries from several controls and from patients dying with schizophrenia, Parkinson's disease, AIzheimer's disease, Wernicke's encephalopathy and depressive illness. Computer-assisted microdensitometric semiquantification of POMC mRNA using a complementary oligonucleotide as hybridization standard, revealed no statistically significant effect of postmortem delay (between 2.5 and 66 h), of gender, age (between 22 and 103) or cause of death in 56 human pituitary glands. A large variation in POMC levels was already observed among all 30 control cases. The levels of POMC mRNA observed in pituitaries from different pathologies did not show a significant variation when compared with control cases. INTRODUCTION Pro-opiomelanocortin (POMC)-derived peptides are found predominantly in pituitary gland, although they have also been reported in a variety of tissues, including pancreas, gastrointestinal tract, placenta, thyroid, male reproductive tract and central nervous system 18A9'28'29'35. Several groups 13'16 have used P O M C c D N A as a hybridization probe to study the differential expression of P O M C gene in the rat pituitary. In the anterior lobe of rat pituitary, approximately 3 - 5 % of the cells express P O M C gene. These cells (corticotrophs) posttranslationally cleave the precursor predominantly to adenocorticotropic h o r m o n e ( A C T H ) and fl-lipotropin (fl-LPH). In contrast, more than 95% of cells of intermediate lobe of the rat pituitary express P O M C gene. Although the same p r o h o r m o n e precursor is synthesized in both cell types, additional proteolytic cleavages occur in intermediate lobe. A C T H is further processed to a-melanocytestimulating h o r m o n e (a-MSH) and corticotropin-like intermediate lobe peptide; fl-lipotropin, to fl-endorphin and 6-1ipotropin 8. Due to the synthesis of melanocytestimulating hormones, these later cells are called melanotrophs. Several substances or physiological conditions can affect the levels of POMC-derived peptides. Gluco-

corticoids, which are synthesized and secreted from the adrenal cortex in an A C T H - d e p e n d e n t manner, inhibit P O M C peptide secretion and gene transcription in corticotrophs, but have no effect in melanotrophs 2'3°. In return, adrenalectomy elevates P O M C gene transcription in the anterior lobe, which can be reversed by administration of the synthetic glucocorticoid, dexamethasone 2. Transcription of the P O M C gene in the intermediate lobe is not altered by either of these treatments, probably due to a lack of functional glucocorticoid receptors 1. In contrast, the neurotransmitter dopamine is an effective inhibitor of the P O M C synthesis in the intermediate lobe 15. Both dopamine and its receptor agonists reduce P O M C m R N A levels in this tissue 5't°,15. No effect of dopamine occurs in anterior pituitary P O M C gene expression 5, indicating that different mechanisms negatively regulate P O M C gene expression in the two pituitary lobes. The role of hypothalamic factors, such as corticotropin-releasing h o r m o n e ( C R H ) and vasopressin, in maintaining high levels of P O M C m R N A in pituitary was shown by Bruhn and coUeagues 3. Stress, which stimulates the release of POMC-derived peptides in vivo, also increases P O M C m R N A levels 16. Using oligonucleotides complementary to P O M C m R N A we have studied the expression of this m R N A in

* Present address: EMBL, Heilderberg, ER.G. Correspondence: G. Mengod, Preclinical Research, Sandoz Pharma Ltd., CH-4002, Basle, Switzerland.

130 h u m a n p i t u i t a r y g l a n d s . B e c a u s e of t h e r e p o r t e d effects of dopaminergic drugs on POMC mRNA expression, we h a v e c a r r i e d o u t a s e m i q u a n t i t a t i v e s t u d y o f t h e levels of

min with undiluted D 19 Kodak developer. After the stop bath rinse, films were immersed in Kodak rapid film fixer at 20 °C for 5 min, rinsed in running water for 20 min at 20 °C and hung to dry. The sections were stained with Cresyl violet.

m R N A c o d i n g f o r P O M C in p i t u i t a r y g l a n d s f r o m s e v e r a l patients with schizophrenia (treated with neuroleptics), Parkinson's

disease

(treated

with L-DOPA).

Patients

with Alzheimer's disease or Wernicke's encephalopathy w e r e i n c l u d e d in t h e study. A p r e l i m i n a r y a c c o u n t o f s o m e o f t h e s e r e s u l t s h a s b e e n p r e s e n t e d e l s e w h e r e 26. MATERIALS AND METHODS

Specimens Pituitary glands were obtained at autopsy at different postmortem delays, from 56 patients (Table I), immediately frozen and kept at -70 °C until used.

A CTH immunohistochemistry The immunocytochemical reactions were performed on cryostat sections using the avidin-biotin complex method (ABL, VectastainKit, Vector Labs, Burlingame, U.S.A.). The primary antiserum was a rabbit antibody against porcine pituitary ACTH (1-35) (Immunonuclear Corporation, Stillwater, MN, U.S.A.) at an optimal dilution of 1/1000. Negative controls were obtained by using nonimmune serum as first layer, rabbit IgG as first or third layer and by omission of diaminobenzidine or hydrogen peroxide from the incubation medium for the peroxidase reaction.

Oligonucleotide probes The oligonucleotides were made on an Applied Biosystems DNA synthesizer 380A, and purified on a 20% polyacrylamide/8 M urea preparative sequencing gel. Oligomers were complementary to bases 7511-7542 (POMC/1) and 7037-7172 (POMC/2) of the human POMC gene 34. A sense oligonucleotide, used as a hybridization standard, was complementary to the POMC/1 oligonucleotide. GH oligonucleotide was complementary to bases 946-974 of the human growth hormone mRNA 33. PRL oligonucleotide was complementary to bases 257-305 of human prolactin mRNA 9. The oligomers were labelled at their 3" end with [a-32p]dCTP (>3000 Ci/mmol, New England Nuclear) and terminal deoxynucleotidyltransferase (Boehringer Manheim) to specific activities of 3-7 x 104 Ci/mmol H.

Preparation of tissue sections Frozen pituitaries were brought to -20 °C and mounted onto microtome chucks. Ten-/am-thick sections were cut using a cryostat (Leitz, 1720, from Leitz, Wetzlar, ER.G.) and mounted onto gelatin-coated glass slides. The protocol followed was essentially that described by Hafen and colleagues TM.

Hybridization of labelled DNA to tissue sections Labelled DNA probe was diluted in the following buffer: 50% formamide, 600 mM NaCI, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.02% Ficoll, 0.02% polyvinyipyrrolidone, 0.02% bovine serum albumine and 500/ag/ml yeast tRNA, to a final concentration of 1-2 X 107 cpm/ml. Each section was covered with 20-60 /al of hybridization solution and a parafilm coverslip to prevent evaporation. The slides were put in humid boxes and incubated at 37 °C for 17 h. Following hybridization, coverslips were removed by floatation in a solution containing 50% formamide, 600 mM NaCI, 20 mM Tris-HCl pH 7.5 and 1 mM EDTA. Slides were subsequently washed in the same formamide buffer at 37 °C for 20 h with 4 changes of buffer and dehydrated by incubating twice in 70% ethanol containing 0.3 M ammonium acetate pH 7.0, 5 min each. The slides were air dried, covered with fl-max film (Amersham, U.K.) for autoradiography and kept at -70 °C. To study film response to standards, exposure times were varied from a few hours to 2 days. After the exposure, films were developed at 20 °C for 5

Preparation of hybridization standards Hybridization standards were prepared from pig brain cortex which was ground to a paste. Brain paste provides reasonable standards because it has the same cutting, mounting and density characteristics as the tissue sections being studied. Brain paste was boiled for 5-10 rain in order to inactivate endogenous nucleases. Known amounts of increasing number of molecules of the 'sense' human POMC oligonucleotide were thoroughly mixed with the brain paste, stirred enough to uniformly distribute it throughout the tissue and degassed in a vacuum dessicator. Silicon tubing (0.5 cm diameter) was filled with tissue containing varying concentrations of DNA, and frozen immediately over dry-ice. Sections (10/am in thickness) were obtained as described above. Protein concentration in the tissue sections from standards were determined by the method of Lowry 23. Standard sections were processed for in situ hybridization histochemistry in the same way as human tissue sections.

Computer assisted image analysis Films were analyzed using a computerized image-analysis system (MCID, Imaging Research Inc., Ste. Caterine, Ont.). The autoradiographic image was digitized into a matrix of 512 × 512 picture units with 256 gray level resolution using a solid state TV camera, displayed on the screen of the TV monitor and the areas of interest were outlined on the screen with a PC mouse. Optical densities of these areas were computed and transformed into an arbitrary number of POMC copy number molecules by comparison with optical densities of hybridized standards, which provide a minimum copy number of sense oligomer per mg of protein.

RNA extraction and Northern blot analysis Total RNA was isolated from human pituitary and several brain regions according to Chomczynski and Sacchi 6. Poly(A) ÷ mRNA was selected on oligo(dT)-ceUulose (Bethesda Research Labs), denatured with 1 M Glyoxal zT, separated by electrophoresis through a 1% agarose gel. Transfer to a nylon membrane was carried out according to Thomas 35. Labelled probe was hybridized for 18 h at 37 °C in a buffer containing 600 mM NaCI, 80 mM Tris-HCl pH 7.5, 4 mM EDTA, 0.1% sodium pyrophosphate, 0.2% SDS, l x Denhardt's, 500/ag/ml yeast tRNA and 50% formamide. The filter was then washed twice for 5 min in 2x SSC ( l x SSC: 150 mM NaCI, 15 mM sodium citrate pH 7.0)/0.1% SDS, at room temperature, and twice for 15 min in 0.1x SSC/0.1% SDS, at 60 °C and exposed for 3h. RESULTS In o r d e r t o c o n f i r m t h e specificity o f t h e h y b r i d i z a t i o n signal o b s e r v e d in t i s s u e s e c t i o n s , w e p e r f o r m e d a s e r i e s o f c o n t r o l e x p e r i m e n t s : (1) T h e specificity o f t h e p r o b e was c o n f i r m e d b y N o r t h e r n b l o t analysis. P O M C m R N A was

detected

approximate

only

in

the

pituitary

size o f 1200 n u c l e o t i d e s ,

gland,

with

an

similar to that

r e p o r t e d b y o t h e r s 7 (Fig. 1). (2) H u m a n p i t u i t a r y s e c t i o n s were hybridized with saturating concentrations of unlab e l l e d P O M C o l i g o n u c l e o t i d e , in p r e s e n c e o f t h e l a b e l l e d p r o b e . O n l y b a c k g r o u n d signal w a s o b t a i n e d u n d e r t h e s e c o n d i t i o n s ( d a t a n o t s h o w n ) . (3) T w o d i f f e r e n t o l i g o m e r p r o b e s w e r e u s e d ( P O M C / 1 a n d P O M C / 2 ) in c o n s e c u t i v e sections obtaining the same pattern of hybridization (not s h o w n ) . (4) W h e n t i s s u e s e c t i o n s w e r e t r e a t e d f o r 1 h at

131 TABLE I

Sources of brain tissue Cases

Age

Sex

Postmortem

lmmediate cause of death Clinical diagnosis

Neuropathology

delay 86 42 84 85

F M F F

3.45 26.00 12.50 53.00

87 86

F M

2.30 3.50

Cardiac failure Myocardial infarction Pneumonia Pneumonia Pulmonary embolism Myocardial infarction Cardiac failure

7 8

69 84

F F

32.00 12.00

Pulmonary embolism Myocardial infarction

9 10 11 12 13 14 15 16 17 18 19

64 101 81 81 83 81 72 80 76 67 78

F M M F F M M F F F F

7.00 15.00 11.00 29.00 39.00 15.00 13.00 4.35 22.15 4.00 4.50

Pulmonary embolism Pneumonia Cardiac failure Cardiac failure Myocardial infarction Hemorragic shock Pulmonary embolism Myocardial infarction Cardiac failure Pneumonia Pulmonary embolism

20

86

M

22.00

21 22 23 24 25

70 75 77 80 22

M M F M M

15.30 16.00 13.30 11.00 13.00

Pneumonia Pulmonary embolism Acute Pyelonephritis Pulmonary embolism Pulmonary embolism Cardiac failure Cardiac failure

26 27 28 29 30 31 32

73 82 72 88 52 93 78

F F F F F M M

13.00 48.00 30.00 22.00 24.00 18.30 3.30

33 34

78 72

F M

20.00 13.30

Pulmonary embolism Pneumonia Pneumonia Cardiac failure Cardiac failure Pulmonary embolism Pneumonia Myocardial infarction Pneumonia Pneumonia

35 36 37 38

85 82 85 85

M M F F

8.00 27.00 29.00 16.00

Pneumonia Pneumonia Cardiac failure Pulmonary embolism

39

95

F

4.45

Cardiac failure

40 41

65 31

F F

29.00 14.00

Pneumonia Cardiac failure

42

68

M

2.50

Pneumonia

43

67

M

66.00

Pneumonia

44

86

M

16.00

Pneumonia

45

76

F

36.30

Cardiac failure

46

103

M

12.00

Peritonitis

POMC copy number

Ischemic heart disease Ischemic heart disease Ischemic heart disease

0.68 1.70 0.28 0.54

Ischemic heart disease Arterial hypertension Diabetes mellitus Carcinoma of the colon Ischemic heart disease Diabetes meilitus Ovarial carcinoma Mild dementia Ischemic heart disease Diabetes mellitus Ischemic heart disease Gastric ulcer Carcinoma of the stomach Ischemic heart disease Diabetes meUitus Carcinoma of the colon Carcinoma of the breast Diabetes mellitus Pulmonary emphysema

0.50 1.32

Carcinoma of the colon Chronic polyarthritis Spinal compression Carcinoma of the prostate Malignant fibrous histocytoma of the left leg Ovarial carcinoma Schizophrenia Schizophrenia Schizophrenia Schizophrenia Senile dementia Dementia, Arterial hypertension Parkinsonism Severe dementia Senile dementia, Diabetes mellitus Multiple cerebral insults Multiinfarct dementia Senile dementia Senile dementia Adipositas per magna, Senile dementia Diverticula of the colon, Senile dementia Dementia, Diabetes mellitus Down's syndrome with cardiac malformation Alcoholism Korsakoff's psychosis Carcinoma of the lung Alcoholism Korsakoff's psychosis Alcoholism Korsakoff's psychosis Alcoholism Korsakoff's psychosis Parkinsonism

1.50 1.10 2.30 0.90

1.82 0.77 1.50 6.60 1.36 0.60 1.08 2.50 0.60 2.00 0.50 0.30 2.20 2.00

SDAT Multiinfarct dementia

1.00 0.90 1.60 4.70 1.00 0.90 1.23 1.80

SDAT Multiinfarct dementia

0.17 1.60

SDAT SDAT SDAT SDAT

0.40 0.10 2.20 2.60

SDAT

1.20

SDAT Typical features of Down's syndrome Wernicke's encephalopathy

0.60 1.30 0.90

Wernicke's encephalopathy

0.33

Wernicke's encephalopathy

0.27

Wernicke's encephalopathy

1.60

Parkinson's disease

0.17

(continued)

132 TABLE I (continued)

Cases

Age

Sex

Postmortem delay

Immediate cause of death Clinicaldiagnosis

Neuropathology

POMC copy number

Parkinson's disease Parkinson's disease

1.80 1.40

Parkinson's disease

0.30 2.60

47 48

81 80

F F

12.30 5.00

Pulmonary embolism Pulmonary embolism

49 50

63 59

F F

21.45 29.00

Pneumonia Pulmonary embolism

51 52 53 54 55 56

75 86 75 76 83 63

M F F M M F

7.00 36.00 14.00 15.30 28.30 15.50

Pulmonary embolism Cardiac failure Pneumonia Cardiac failure Cardiac failure Pneumonia

Parkinsonism Symptomatic epilepsia Small cortical lesion Parkinsonism Progressive supranuclear palsy Oligophrenia and catatonia Drug induced Parkinsonism Diabetes mellitus, Depression Depression Bipolar psychosis Epilepsy Ataxia Amyotrophiclateralsclerosis(ALS)

r o o m t e m p e r a t u r e with 0.1 mg/ml of RNase, hybridization signal d i s a p p e a r e d (not shown). (5) W h e n a heterologous p r o b e such as a P O M C sense oligonucleotide was used as a labelled probe, no hybridization signal could be observed (not shown). (6) Immunohistochemical detection of the A C T H peptide revealed the same distribution observed by in situ hybridization (Fig. 2).

Standardization o f the quantification Fig. 3, u p p e r panel shows a typical hybridization a u t o r a d i o g r a m o b t a i n e d with the P O M C oligomer standards generated as described in Materials and Methods. 1

28S 23S

-

18S 16S

-

2

3

4

5

F~I

Fig. 1. Northern blot analysis of POMC mRNA in human brain regions. Each lane contained 10/~g of poly(A)÷ RNA, except lane 1 that contained 5/~g. The blot was hybridized with the 32p-labeled oligonucleotide POMC/1 and exposed to an X-ray film for 3 h. Molecular weight ribosomal RNA markers migration is indicated on the left. Lane 1: pituitary. Lane 2: putamen. Lane 3: hippocampus. Lane 4: frontal cortex. Lane 5: cerebellum.

ALS

2.76 2.10 0.30 0.92 1.30 1.73

Lower panel presents the relationship between optical densities and D N A concentration in standards as analyzed by computer-assisted microdensitometric analysis. The values o b t a i n e d are given as the n u m b e r of P O M C copies present in the tissue. We choose the term ' P O M C copy n u m b e r ' as being the most a p p r o p r i a t e , even if an absolute quantification can not be p e r f o r m e d . These results d e m o n s t r a t e that a linear relationship can be established between the a m o u n t of sense oligonucleotide present in the tissue standard and the autoradiographic signal. The use of these standards allows: (1) control of each individual hybridization reaction, (2) control response variability of the a u t o r a d i o g r a m and film processing, and (3) standardization for microdensitometry. The results shown below were o b t a i n e d with the POMC/1 probe.

Hybridization with human postmortem pituitaries Hybridization of the P O M C oligonucleotide with human pituitaries is shown in Fig. 2. In contrast with the results o b t a i n e d with rat pituitaries 13, in h u m a n pituitary, the m R N A coding for P O M C was found to be located in a scattered patchy-like m a n n e r in anterior lobe (Fig. 2A,C). No hybridization signal could be seen in posterior lobe. We did not find any increased hybridization signal in a discrete region which could c o r r e s p o n d to the intermediate lobe zS. The a u t o r a d i o g r a p h i c image obtained c o r r e s p o n d e d to the distribution of P O M C m R N A , since immunohistochemistry experiments performed in consecutive tissue sections with an antibody against A C T H , (Fig. 2 B , D ) showed a similar distribution for the peptide in the pituitary gland. Variation o f P O M C m R N A levels with age and postmortem delay Variations of P O M C m R N A expression were observed in the different human pituitaries. We a t t e m p t e d to

133

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/

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Fig. 2. Autoradiograms from human pituitaries showing the distribution of the POMC mRNA in a control case (A) and a dementia case (C). ACTH immunostaining bright-field illumination of consecutive sections of the same control (B) and dementia (D) cases. A perfect correlation between the localization of the mRNA and the ACTH peptide could be observed in both cases. PL, posterior lobe; AL, anterior lobe. Bar = 2mm.

correlate POMC m R N A concentration with parameters such as age, gender and postmortem delay using linear and multiple regression analysis. The results obtained are shown in Fig. 4. No effect of postmortem delay (range 2 h 30 min to 66 h). (Fig. 4A) could be observed in the levels of POMC m R N A . The same is true when the range is taken up to 36 h; a slight decrease could be seen from 39 to 66 h, but there are not enough data points to draw any conclusion. No changes in the POMC levels are seen with age variation from 60 to 90 years old, (Fig. 4B), or with gender (Fig. 4C). Because of the mentioned effects of dopaminergic drugs on POMC m R N A expression, we examined pituitary glands from patients with schizophrenia (treated with neuroleptics), and Parkinson's disease (treated with L-DOPA). In addition, we examined patients with Alzheimer's disease, depressive illness and Wernicke's encephalopathy. The results obtained are shown in Fig. 5. When compared with healthy controls, no significant variation could be observed in the levels of POMC m R N A expression in pituitary glands from patients with schizophrenia, depression, Parkinson's disease, Wernicke's encephalopathy or Alzheimer's disease. Very low levels of hybridization signal in some cases was unlikely to be due to m R N A degradation, since hybridization of consecutive tissue slides with oligomers com-

plementary to other pituitary mRNAs, such as prolactin and growth hormone, revealed a completely different pattern and intensity of hybridization (Fig. 6). DISCUSSION We have shown that the m R N A for POMC can be visualized autoradiographically using in situ hybridization histochemistry with oligonucleotides as hybridization probes. Control experiments, including Northern blot analysis, use of two oligonucleotides complementary to different regions of the same m R N A , cohybridization of labelled antisense probe with excess of the same unlabelled, lack of hybridization obtained with a sense probe and after pre-treatment of the tissue with RNase A, indicate the specificity of the hybridization signals observed. This specificity is also supported by the fact that unrelated probes produced different distribution and intensity of the signal as we have observed with oligonucleotides for prolactin and growth hormone. The main finding of the present study is that POMC m R N A was detected in the anterior lobe of human postmortem pituitaries where it showed a patchy-like distribution. The levels of POMC m R N A present in pituitary slides could be determined in a semiquantitative way taking as

134

9

8

7

standard curve the hybridization signal obtained with different amounts of a sense oligonucleotide. This variation was measured in 56 human pituitary glands (Table I). The effects of age, gender and postmortem delay on the levels of the POMC m R N A were studied. No differences could be observed between male and female. In our sample, no statistically significant variations in m R N A concentration could be measured as a function of postmortem delay (up to 36 h), or age of patients. When consecutive pituitary sections were hybridized with other oligonucleotide probes (prolactin and growth hormone), a completely different pattern of distribution and intensity in hybridization signal could be observed, indicating that the changes in POMC m R N A content are specific for this particular messenger, and are not due to a general degradation of the m R N A content in the pituitaries used. When the semiquantification of in situ hybridization was extended to pathological tissues no significant differences in POMC m R N A concentration could be obtained. This is probably due to the large variation found in the control population as well as the limited number of pituitary glands from pathological cases analyzed in this study. There are only scarce data on the relationship between POMC peptide concentration in pituitary gland and mental disorders, such as depression and schizophrenia. It has been recently shown 4'22 that basal A C T H and N-POMC (N-terminal proopiomelanocortin) concentrations were normal in depressed patients but elevated, compared with controls, after dexamethasone. In contrast, in Alzheimer's disease patients, A C T H was elevated after dexamethasone but not N-POMC. These levels were increased in the depressed group in response

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Fig. 3. Upper panel: autoradiographic image of brain paste-sense probe standards each containing known amounts of the sense human POMC oligonucleotide after hybridization with the 32p-labelled human POMC anti-sense oligonucleotide. (1) 2 x 10H, (2) 1011, (3) 5 × 10 ~°,(4) 2.5 × 101°, (5) 1.2 x 10 '°, (6) 6 x 10~, (7) 3 x 109, (8) 1.5 × 109, (9) 7.5 × 108 molecules of the sense oligomer/mg of protein. (0) contains no oligomer. Lower panel: relationship between the number of molecules of the sense oligomer and the optical densities measured on films similar to the one shown in the upper panel by computer-assisted microdensitometry. (O) 24 h exposure, (O) 48 h exposure.

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Fig. 4. Effects of (A) postmortem delay and (B) age on POMC mRNA concentration in human pituitaries. Observed values and regression line are illustrated. A: slope -0.03, P = 0.8. B: slope -0.04, P = 0.5. C: scattergram of the POMC mRNA concentration in the male (m), female (0 population. (O) control, (&) senile dementia of the Alzheimer type, (©) schizophrenia, ( A ) Wernicke encephalopathy, ( i ) Parkinson's disease, (*) depression.

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Fig. 5. Scattergram of the POMC mRNA concentration in the human pituitaries from different cases. C: control; S, schizophrenia; SDAT, senile dementia of the Alzheimer type; WE, Wernicke's encephalopathy; PD, Parkinson's disease; D, depression.

to a human corticotropin releasing hormone stimulation test 21. POMC m R N A levels measured in pituitary glands

"

B

......

from patients with Alzheimer's disease, Parkinson's disease and Wemicke's encephalopathy were very close to those measured for controls. The changes on peptide levels can be due not only to changes of the corresponding m R N A levels but also to variations in the posttranslational processing of the precursor polypeptide and/or an altered secretion of the bioactive peptide. A large variety of parameters have been found to regulate the levels of POMC m R N A in the rodent pituitary. Neurotransmitters and drug agonists and antagonists acting on their receptors, peptide hormones and corticosteroids are among the best characterized factors affecting the levels of POMC gene transcription 24 in experimental systems. In some cases the physiological significance of the effects of these factors is unknown, for example for glucocorticoids. Our results showing a large variability in levels of POMC m R N A in samples of postmortem human pituitaries which are not clearly related to age, gender or postmortem delay, suggest that a variety of factors are involved in the regulation of POMC gene expression in human pituitaries, such as

C

i

O

E

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G

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Fig. 6. Autoradiograms showing the different patterns and levels of hybridization of three control cases of human pituitaries hybridized with the POMC oligomer (A-C), prolactin oligomer (D-F) and growth hormone oligomer (G-I). Bar = 2 mm.

136 a g o n a l status, stress, p r e m o r t e m anoxia, etc. This variability contrasts with a smaller dispersion of the results

itary P O M C m R N A levels m e a s u r e d p o s t m o r t e m can be i n f l u e n c e d by m a n y different factors. T h e e s t a b l i s h m e n t

o b t a i n e d in o u r studies of o t h e r peptides in h u m a n p o s t m o r t e m brain 25"32.

of P O M C m R N A level a l t e r a t i o n s in n e u r o l o g i c a l dis-

In c o n c l u s i o n , we have shown that using in situ

eases will require the s e p a r a t e analysis of these factors a n d the use of large samples of p i t u i t a r y glands.

h y b r i d i z a t i o n histochemistry with labelled oligonucleotides as p r o b e s , it is possible to m e a s u r e in a s e m i q u a n titative way the c o n t e n t of P O M C m R N A in h u m a n p o s t m o r t e m pituitary glands. T h e large variability observed inside the control p o p u l a t i o n suggests that pitu-

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Acknowledgements. The authors wish to thank I.N. Ferrier for critical reading of the manuscript, K.-H. Wiederhold for the photographic assistance and A. Wanner for technical help.

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