Camp. Eiochem. Physiol. Vol. IOZA,No. 1, pp. 4M8, Printedin Great Britain

0300-9629/92 $5.00 + 0.00 0 1992 PergamonPress plc

1992

MESOTOCIN AND OXYTOCIN IN THE BRAIN AND PLASMA OF AN AUSTRALIAN MARSUPIAL, THE NORTHERN BROWN BANDICOOT, ISOODON

MACROURUS

R. A. D. BATHGATE,*C. SEaNIA*t and R. T. GEMMELL~ *Nemoendocrine Laboratory, Department of Physiology and Pharmacology and SDepartment of Anatomy, University of Queensland, St. Lucia, Qld. 4072, Australia. Telephone: (07) 365-3132; Fax: (07) 365-1766 (Received

27 August 1991)

Abstraet-1. Mesotocin (MT) and oxytocin (OT) were measured in the brain and plasma of bandicoots using reverse phase high performance liquid chromatography and specific radioimmunoassays. 2. MT and OT were found in the pituitary (1.25 & 0.10 &MT; 0.725 f 0.077 pg/OT) and hypothalamus (38.37 f 6.46 ng/MT, 19.1 f 4.61 ng/OT). Smaller amounts were present in the cerebral cortex. 3. Basal plasma concentrations ranged from 1.5 to 8.1 pg/ml for both peptides (N = 14) and were elevated by stress. 4. It was concluded that both MT and OT are secreted by the bandicoot brain and that stress stimulates secretion.

INTRODUCTION

The proposal that OT receptors may have evolved an equal or greater affinity for MT in marsupials is not supported by experimental evidence (Sernia et al., 1990, 1991). Moreover, speculation on the adaptational significance of MT in marsupials is rendered difficult by the very limited data on the functional roles of MT and OT in marsupials (see Tyndale-Biscoe and Renfree, 1987). Thus, the aims of this study were to confirm the preliminary observations in the bandicoot (Rouille et al., 1988; Gemmell and Sernia, 1989) using methods previously established for the brushtail possum (Bathgate et al., 1990), and to obtain values for MT and OT in the brain and plasma which could form the basis of future functional studies.

The neurohypophysis of eutherian mammals secretes the nonapeptides oxytocin (OT) and, with the exception of the pig, arginine-vasopressin (AVP) (Heller, 1974). Marsupials secrete related peptide hormones in addition to, or instead of, OT and AVP (Heller, 1974; Acher, 1980, 1985). Of the four Australian marsupial families studied, three (Phalangeridae, Dasyuridae, Phascolarctidae) secrete AVP (Chauvet et al., 1987; Hurpet et al., 1982), whereas one (Macropodidae) secretes lys-8-vasopressin (LVP) and phe-2-arg-vasopressin [phenypressin (PP)] instead of AVP (Chauvet et al., 1983a, b, c). American opossums (Didelphidae) secrete LVP and AVP (Chauvet et al., 1984). For oxytocic peptides, the North American opossum secretes OT and the typically reptilian mesotocin (MT), while the South American opossums secrete only OT (Acher, 1980, 1985; Chauvet et al., 1984). Australian marsupials were thought to express exclusively MT, since the four families studied all expressed this peptide and not OT (Chauvet et al., 198lb, 1983a, b, c, 1987; Hurpet et al., 1982). However, Rouille et al. (1988), investigating a single posterior pituitary gland of the northern brown bandicoot (Isoodon macrourus), showed the presence of OT as well as MT, and Gemmell and Sernia (1989), using immunocytochemistry in the paraventricular and supraoptic nuclei of the bandicoot hypothalamus detected OT but not MT. These observations indicate that at least one family of Australian marsupials (Permalidae) secretes OT and the generalization about exclusive MT secretion was incorrect. A satisfactory explanation has yet to be found for the persistence of MT and the coexistence of MT and OT in marsupials.

MATERIALSAND METHODS Collection

of blood and tissues A total of 15 male (I. l-2.8 kg) and four female (0.9-l .3 kg) bandicoots were used in this study. Blood samples were taken from all 19 animals and tissue samples from five of the males. Bandicoots were kept in outdoor enclosures with access to food and water ad lib. In the five selected males, anaesthesia was induced by intramuscular injection of a mixture of 50 mg ketamine/kg (Jurox, NSW, Australia) and 10 mg xylazine/kg (‘Rompun’, Bayer, Australia). The chest cavity was opened and blood was collected from the left ventricle, decanted into lithium heparin tubes with EDTA to a final concentration of 5 mmol/l to decrease enzymatic degradation of peptides, and centrifuged at 15OOg for 20min. Plasma was collected and stored at -20°C. After death, the skull was removed and the pituitary, hypothalamus and one hemisphere of cortex were collected, homogenized in 10 ml of 1 mol/l HCl and centrifuged at lO,OOOgfor 20 min. The supematant was collected and stored at -20°C. In the remaining 10 male and four female bandicoots, blood was collected by heart puncture during light anaesthesia with a halothane (Fluothane, ICI Pharmaceuticals, Macclesfield, U.K.) and oxygen mixture.

tTo whom all correspondence should be addressed. 43

44

R. A. D. BATHGATEet al.

HPLC SEPARATIONOF N-SlAL

PEpTlDEs

‘9 AVP

I

I~...I~~.~I’.~‘I....I~...I......~..I

inject

10

20 FRACTION NUMBER

30

40

Fig. 1. HPLC profile of authentic arginine-vasopressin (AVP), lysine-vasopressin (LVP), mesotocin (MT), isotocin (IT) and oxytocin (OT) separated on a Cl8 reverse phase column by a 1 ml/min flow of 16.5% acetonitrile (ACN) in 50 mM triethylamine (pH 3 with orthophosphoric acid). Extraction of peptides

The procedures for extraction of peptides from plasma and tissues have been described previously (Bathgate et al., 1990). Dried extracts of tissues or plasma were resuspended in 80 ~1 of 16.5% ACN in 50 mmol/l triethylamine (pH 3.0 with orthophosphoric acid) and loaded onto a Phenomenex maxsil 10 C- 18 reverse phase HPLC column (250 x 4.6 mm, Torrance. CA. U.S.A.) and .neotides eluted bv HPLC usina . a flow rate of 1 ml/min of the above buffer. Fractions were collected at 30 set intervals, dried under a stream of air and resuspended in 1 ml of radioimmunoassay (RIA) buffer (100 mmol/l phosphate buffered saline, 0.3 mmol/l phenylmethylsulphonylfluoride, 0.2% bovine serum albumin) for RIA in duplicate for both OT and MT. Recovery of peptides from this procedure was 35% for both MT and OT; all results are adjusted for recovery. HPLC fractions from pituitary and hypothalamus resuspended in 1 ml of RIA buffer were divided into two 200 ~1 samples and to each was added 20,OOOcpm of iZ51-MTor ‘r’I-OT in 100~1 and 100~1 of anti-MT or anti-OT at a dilution of 1:4Oband 1: 3OO$OO,respectively. Plasma extracts and HPLC extracts of cortex and plasma were pre-incubated with antibody for 24 hr before addition of 10,000 cpm of trace. After an incubation period of 40 hr, 0.5 ml of bovine gamma globulin (4 mg/ml) and 1.5 ml of 22% polyethyleneglycol were added and the antibody-bound peptide precipitated by centrifugation at 1500g for 30 min. The radioactivity in the pellet was counted in a gamma counter (LKB model 1277) and the peptide concentrations calculated from fitted standard curves (Riacalc LM RIA programme, Wallac, Sweden). The peptides were radioiodinated by the chloramine-T method, as described previously (Bathgate et al., 1990) and separated by HPLC to an approximate activity of 2000 mCi/mol. The anti-MT (SM-75) and anti-OT (SO-44) antibodies were raised in sheep using limpet haemocyanin conjugates of each wutide (Gemmell and Semia. 1989). The crossreactivities bf SML75 with various peptides are: MT lOO%, deamino-OT 17.5%, OT 13.5%, isotocin (IT) 10.6%, AVP ~0.1%. angiotensin II (AID 0%. The cross-reactivity protile of So-44 is: OT IOO%, deamino-OT 42.9%, MT 12.5%. IT 1.1%. AVP 0%. AI1 0%. The sensitivitv of each RIA was 1 pg/tube for OT and 2 pg/tube for MT. Intraassay and inter-assay variabilities were 3.27 and 15% for OT and 3.88 and 18% for MT RIA. MT was purchased from Bachem (Torrance, CA) and OT from Sigma (St. Louis, MO). All data are presented as mean f SEM of N animals.

RESULTS

Brain content Figure 1 shows the separation by HPLC of a mixture of authentic LVP, AVP, MT, IT and OT with a 1 ml/min flow of 16.5% ACN in 50 mmol/l triethyl-

amine (pH 3). This system clearly separated AVP, MT and OT; the neurohypophysial peptides reported to be present in the bandicoot. It was used in conjunction with specific RIA to quantify MT and OT in RAT PITUITARY

4o1(W

Aulhenllc

15

20

Fraction

Ye~olocln

25 Number

30

35

40

(OSml/fraclion)

Fig. 2. Rat pituitary extract separated on HPLC and fractions assayed for (a) MT and (b) OT by specific radioimmunoassay (RIA). Retention times of authentic MT and OT (arrows) indicate that only OT is present.

45

Mesotocin and oxytocin in the bandicoot the brain tissues as well as the plasma. The effectiveness of this system and the specificity of the antisera are demonstrated in Figs 2 and 3. Figure 2a, b shows the separation and RIA of peptides in the rat pituitary, with the expected large peak in the OT assay where authentic OT elutes, and no peak in the MT assay were authentic MT elutes (Fig. 2b). The peak in the MT assay corresponding to OT is cross-reactivity (13.2%) to approximately the level expected from antibody characterization (I 3.5%). Figure 3a, b represents the separation and RIA of peptides in the brushtail possum pituitary which we have previously shown to contain MT only (Bathgate et al., 1990). A large peak corresponding to authentic MT is present in the MT assay (Fig. 2a), whereas the OT assay shows only cross-reactivity to MT and no immunoreactivity where authentic OT elutes. The cross-reactivity of the OT antibody to MT (13.0%) again correlates with antibody characterization data (12.5%). Figure 4 shows the MT and OT content of five bandicoot pituitaries after extraction of peptides and separation by HPLC. Large peaks corresponding to authentic MT and OT are present. The peaks added to a total of 1.25 k 0.10 pg (mean + SEM) MT and 0.725 f 0.077 pg OT; the OT being 58% of the MT peak. The results for five hypothalami (Fig. 5) reflect those of the pituitary with peaks of MT (Fig. 5a) and OT (Fig. 5b) present. The total peptides per hypothalamus are 38.37 + 6.46 ng MT and 19.10 f 4.61 ng OT; the proportion of OT to MT (49.8%) being comparable to the pituitary (58%). The smaller peaks

POSSUM 600

Aulhenllc (a)

present in both assays for both tissues represent cross-reactivity with antiserum. The levels of peptides in the cortex were lower and difficult to measure reliably, consequently only two samples of bandicoot cortex were measured successfully. Both peptides were found at 3 11 and 154 pg for MT and 360 and 92 pg for OT, respectively, per hemisphere of cortex. These levels are far lower than those in the pituitary and hypothalamus but are much higher than would be expected from blood contamination (see Table 1). HPLC-plasma Table 1 lists MT and OT levels in the plasma of three bandicoots and one pooled bandicoot plasma. To obtain accurate levels for both peptides without antibody cross-reactivity, peptides were extracted from plasma and separated by HPLC. Because of the losses involved in this procedure and the low amounts of peptide in the plasma, samples with elevated levels or large volumes of plasma were used. The three individual bandicoots listed Bl, B6 and B7 were exsanguinized under heavy anaesthesia as outlined previously (see Materials and Methods). The presence of both peptides was clearly shown in these samples. MT was found at 48.5 f 6.5 pg/ml and OT at 38.6 f 28.6 pg/ml, the proportion of each peptide being very similar to their pituitary content (Fig. 3). A pool of plasma from bandicoots sampled by cardiac puncture under light halothane anaesthesia was also extracted and separated by HPLC. The

PITUITARY

BANOICOOT

Mesolocln

Authentic

Mesolocin i

:a)

$

PITUITARY

I

F

/k

.; 400 3 ‘E Sl d .z 200

n=5

0 B f 0

7oh

15

1

250 (b)

20

Fraction

25 Number

30

35

40

(OSmllfraction)

Fig. 3. Brushtail possum pituitary extract separated on HPLC and the fractions assayed for (a) MT and (b) OT by specific RIA. Retention times of authentic MT and OT (arrows) indicate only MT is present.

15

20 Fraction

25 Number

30

35

40

(O.lml/fraction)

Fig. 4. Extracts of five bandicoot pituitaries separated by HPLC and assayed for (a) MT and (b) OT. Peaks corresponding to both authentic MT and OT (arrows) are present.

R. A. D. BATHGATE et al.

46 BANDICOOT

3

I5

2

1

Aulhenllc

(a)

HYPOTHALAMUS

Table 2. PlasmaMT and OT concentrationsin (a) 10 male and (b) four female bandicoots measured directly on plasma extracts

+

Ma8otocln

Weight (kg) (a) Male

Female

4.6 4.7 4.6 5.1 3.9 2.3 1.6 1.7 4.1 8.1 4.1 + 0.6

1.2

2.0 2.3 1.8 3.3 2.4f 0.4

2.5 2.0 1.5 3.4 2.4f 0.5

0.9 1.3 1.1 Mean f SEM

n=5

1-5 20 Fraction

25 Number

30 35 (0.5mMraction)

40

Fig. 5. Extracts of five bandicoot hypothalami separated by HPLC and assayed for (a) MT and (b) OT. Peaks corresponding to both authentic MT and OT (arrows) are present. concentrations obtained were 4.3 and 3.4 pg/ml of MT and OT, respectively, much lower than those levels obtained from exsanguinated bandicoots. Plasma concentrations

The normal range of MT and OT concentrations in the plasma taken from halothane anaesthetized bandicoots is shown in Table 2. MT and OT were measured directly on plasma extracts without HPLC separation. Concentrations of MT in 10 male bandicoots ranged from 1.9 to 8.1 pg/ml with an average concentration of 4.7 + 0.8 pg/ml. OT ranged in concentration from 1.6 to 8.1 pg/ml with an average of 4.1 f 0.6 pg/ml. In four female bandicoots MT ranged from 1.8 to 3.3 pg/ml with an average of 2.4 + 0.4 pg/ml and OT from 1.5 to 3.4 pg/ml with an average of 2.4 + 0.5 pg/ml. DISCUSSION

The nonapeptides MT and OT were shown to be present in the brain and plasma of the bandicoot using HPLC and specific RIA. The content of Table I. Plasma MT and OT concentrations in (a) pooled bandicoot and (b) exsanguinized bandicoot plasma measured from fractions seuarated by HPLC Animal (a) (b)

Weight (kg)

Bandicoot pool B1 86 87 [Mean (Bl, B6, 87) i

(low stress) 1.5 I.1 SD]

MT (pglml) 4.3 41.0 52.4 52. I 48.5 * 6.5

OT (pg/ml) 3.4 69.3 19.4 20.3 38.6 f 28.6

OT (pg/ml)

3.7 2.5 3.7 1.9 2.3 7.4 7.4 6.7 3.6 8.1 4.7 + 0.8

Mean + SEM (b)

MT (pglml)

1.9 1.7 2.8 2.0 1.5 1.3 2.1 1.5 1.5 1.5

MT and OT in the pituitary was 1.25 + 0.10 and 0.725 &-0.077 pg, respectively (means + SEM of five pituitaries). This is in close agreement with published data for a single pituitary of 1.2 and 0.7 pg (Rouille et al., 1988). To compare our results with those obtained by bioassays, a uterogenic activity of 290 U/ /rmol for MT and 430 U/ pmol for OT (Rouille et al., 1988) may be used. The North American opossum (Didelphis virginiana), the only other marsupial reported to secrete MT and OT, was calculated to contain 2.3 pg MT and 3.2 pg OT/pituitary (Chauvet et al., 1985). These levels are much higher than those from the bandicoot and the proportion of OT : MT is different (140 compared to 58%), whereas levels from the South American opossums, Didelphis marsupialis (1.4 pgg/OT/pituitary) and Philander opossum (1.04 pg/OT/pituitary) (Chauvet et al., 1984), are similar to the bandicoot as are those from some smaller Australian marsupials, the quokka (Setonix brachyrus, 1.6 pg/MT/pituitary) (Chauvet et aI., 1983b), kowari (Dasyurus byrnei, 3.0 pg/MT/pituitary) and koala (Phascolarctos cinereus, 2.3 pg/MT/pituitary) (Chauvet et al., 1987). Oxytocin but not MT has been localized in the paraventricular (PVN) and supraoptic (SON) nuclei of the bandicoot hypothalamus by immunocytochemistry (Gemmell and Sernia, 1989). In this study we found 38.37 f 6.46 and 19.10 & 4.61 ng/hypothalamus (mean &-SEM of five animals) of MT and OT, respectively; representing 3.1 and 2.6% of pituitary values. In the possum, the only other marsupial for which data are available, the hypothalamic MT content is only 0.45% of that in the pituitary (Bathgate et al., 1990). The failure to find MT staining in our immunocytochemical study indicates that, while immunocytochemistry has the advantage of cellular localization, HPLC and RIA should be used to confirm immunocytochemical observations. The cortex was found to contain only small amounts of MT (3 11 and 154 pg/hemisphere cortex) and OT (360 and 92pg/hemisphere cortex). Even though they are much lower than those found in the possum (10.5 ng/hemisphere cortex), they are considerably higher than could be explained by plasma contamination. The presence of these peptides in the cortex of two marsupial species may indicate the presence of MT and OT fibres in the cortex similar

41

Mesotocin and oxytocin in the bandicoot to OT fibres shown in eutherian mammals (Buijs et al., 1985; Sofroniew, 1985). Both MT and OT were identified in the plasma of the bandicoot by HPLC and RIA. The concentrations were influenced by the method of blood sampling. Consequently, blood collected under heavy anaesthesia during exsanguination (Table 1) showed high concentrations of MT (48.5 + 6.5 pg/ml) and of OT (36.3 f 28.6 pg/ml) (mean f SD), while pooled plasma collected from animals under light anaesthesia yielded concentrations of only 4.3 pg MT/ml and 3.4pg OT/ml. The sensitivity of our RIAs for MT and OT allowed us to measure accurately plasma concentrations down to 2 and 1 pg/ml for MT and OT, respectively. Thus, lightly anaesthetized animals could be measured in individual samples of plasma. In 10 male bandicoots (Table 2) the mean MT was 4.7 + 0.8 pg/ml and mean OT was 4.1 + 0.6 pg/ml. In a smaller group of four female bandicoots, the mean concentrations of MT (2.4 + 0.4pg/ml) and OT (2.4 f 0.4 pg/ml) were lower (P < 0.05, r-test) than the mean concentrations in males. However, the female group was too small to reach a firm conclusion in regards to possible sex differences. The higher plasma concentrations in exsanguinized animals compared to those in lightly anaesthetized bandicoots is in accordance with observations in the brushtail possum (Bathgate et al., 1990), and is similar to responses shown in eutherian mammals. Severe hypovolemia (as would occur during exsanguination) elicits an increase in OT secretion in rats (Onaka and Yagi, 1990) and dogs (Weitzman et al., 1978). Surgical stress has also been shown to stimulate OT secretion in rats (Nussey et al., 1988). Thus, the combination of heavy anaesthesia, surgical procedures, and major blood loss would explain the elevated concentrations of MT and OT seen during exsanguination in the bandicoot and the possum. These responses are associated with increases in plasma AVP whereas the increased OT concentrations achieved from immobilization, forced swimming and cold exposure in rats (Lang et al., 1983; Gibbs, 1984) are not. It is clear, therefore, that wherever possible MT and OT should be measured in plasma from conscious (or lightly anaesthetized), unrestrained animals that are accustomed to handling. The evolutionary significance of the simultaneous secretion of two oxytocin-like and two vasopressinlike peptides in marsupials has yet to be realised. The presence of MT in all non-mammalian tetrapods and the appearance of OT in the echidna, an egg-laying mammal, lead to the view that OT evolved with mammalian lactation (Acher, 1980, 1985; Berde and Boissonnas, 1968; Acher et al., 1973). The persistence of MT in marsupials does not appear to support this view. Furthermore, it is difficult to explain on the basis of lactation the presence of OT in such divergent species as the bandicoot and the American opossums and its absence in all other marsupials. We have recently examined the possibility that receptors for oxytocin-like peptides in marsupials were particularly selective for MT, thereby maintaining an evolutionary advantage for MT over OT (Sernia et al., 1990, 1991). However, both mammary and uterine OT receptors in the brushtail possum show a greater affinity for OT and are very similar to rat and sheep oxytocin

receptors in their ligand-binding properties. Thus, the persistence of MT in marsupials does not lie in the evolution of an MT receptor. It remains possible that selectivity for MT in marsupials resides in postreceptor events. This possibility should be tested in future studies by examining the biological potency of oxytocin-like peptides on marsupial tissues, such as the uterus and mammary gland. Finally, the presence of OT in numerous areas of the brain (Buijs et al., 1985; Sofroniew, 1985) and in the adrenals (Hawthorn et al., 1987) and gonads (Pickering et al., 1990) of eutherian mammals, indicates that selective pressure for either MT or OT would operate on a wide and diverse range of actions; lactation and parturition being only two of the many (Argiolas and Gessa, 1991; Pickering et al., 1990). A broader understanding of these actions may be necessary to provide an insight into the evolution of neurohypophysial hormones in marsupials. Acknowledgements-This research was supported by a grant from the Australian Research Council to R.T.G. and C.S.

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Sofroniew M. V. (1985) Vasopressin, oxytocin and their related neurophysins. In Handbook of Chemical Neuroanatomy, Vol. 4, pp. 93-165. Elsevier, Amsterdam. Tyndale-Biscoe H. and Renfree M. (1987) Reproductice Physiology of Marsupials, 1st edition. Cambridge University Press, Melbourne. Wietzman R. E., Glatz T. H. and Fisher D. A. (1978) The effect of haemorrhage and hypertonic saline upon plasma oxytocin and arginine vasopressin in conscious dogs. Endocrinology 103, 2 154-2 160.