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Neuropeptide Y-like immunoreactivity of plasma during hypoglycaemia in man
Journal of the Autonomic Nervous System, 26 (1989) 85-88 Elsevier
85
JAN 00899
Short Communication
Neuropeptide Y-like immunoreactivity of plasma during hypoglycaemia in man A.E. Russell, M.D. Cain, V. Kapoor, M.J. Morris and J.P. Chalmers The Department of Medicine and Centre for Neuroscience, Flinders Medical Centre, Adelaide, South Australia (Australia) (Received 26 July 1988) (Revised version received 27 October 1988) (Accepted 11 November 1988)
Key words: Plasma neuropeptide Y; Plasma noradrenaline; Plasma adrenaline; Adrenal medulla; Hypoglycaemia
Abstract Previous studies have demonstrated that plasma Neuropeptide Y-like immunoreactivity (NPY-LI) increases after activation of sympathetic nerves. To test the hypothesis that the adrenal medulla may also be a significant source of circulating plasma NPY-LI and to determine if NPY is co-released with adrenal catecholamines, we have measured the peripheral venous concentrations of NPY-LI, adrenaline and noradrenaline in six patients, before and after induction of hypoglycaemia as part of pituitary function tests that also tested gonadotrophin and thyroid stimulating hormone release. The plasma adrenaline concentration was increased approximately 15 times (p < 0.05) relative to baseline at 30 rains and remained elevated for the 90 minutes of the study. The plasma concentration of both noradrenaline and NPY-LI remained unchanged. These results failed to demonstrate an increase in the amount of NPY-LI released into the plasma during stimulation of the adrenal medulla with hypoglycaemic stress in man. They do not support significant co-release of NPY with adrenaline from the adrenal medulla in man, nor a physiological role for NPY as an adrenal hormone in human subjects in this situation.
Neuropeptide Y (NPY) is a 36 amino acid peptide that has potent vasoconstrictor actions, that also increases vascular responses to noradrenaline [4]. It is widely distributed throughout the central and peripheral nervous systems and is present in the adrenal medulla [7,12,16]. In the peripheral components of the sympathetic nervous system NPY has been found co-localised with noradrenaline in nerve terminals [5]. In the adrenal medulla NPY has been identi-
Correspondence: J.P. Chalmers, The Department of Medicine, Flinders Medical Centre, Bedford Park, South Australia, 5042, Australia.
fled in catecholamine-containing cells [1] but it is unclear if NPY is co-localised with adrenaline [10] or noradrenaline [19] or both. The relationship between plasma noradrenaline concentration and the plasma concentration of NPY-like immunoreactivity (NPY-LI) in the human during sympathoadrenal activation [16] and in the rat after haemorrhage [15] has suggested a predominantly neuronal source for circulating NPY-LI. However, studies in patients with pheochromocytoma [3] and in rats after insulin [18] have raised the possibility that the adrenal medulla may be a significant source of circulating NPY-LI and that NPY might act as a hormone of adrenal origin [18]. This study was undertaken to test this by-
0165-1838/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
86 pothesis by examining the circulating concentration of N P Y - L I before and after adrenal catecholamine release was stimulated during hypoglycaemia induced with i.v. insulin as part of triple axis pituitary function tests. Six patients (5 males and one female), aged from 24 to 66 years (mean _+ SD: 47 _+ 19 years) having triple axis pituitary function studies for the investigation of pituitary tumors (5) or hypothyroidism (1) agreed to additional blood samples being taken for this study. Subsequently, 3 were found to have significantly abnormal pituitary function. Five had a pituitary tumor confirmed at h y p o p h y s e c t o m y ( a d e n o m a 3, eosinophilic granuloma 1) a n d / o r computed tomography (1). The subjects rested supine throughout the study after a cannula had been placed into an arm vein for blood sampling and i.v. injection. Blood samples for measurement of blood glucose, plasma cateeholamines and plasma NPY-LI were taken before and 30, 60 and 90 min after the subject received 0.1 U / k g of Actrapid MC insulin (Novo), 100 /~g of thyrotropin-releasing hormone (TRH, Roche) and 200 /~g of gonadotrophin-releasing hormone (LHRH, Hoechst) i.v. Approximately 140 ml of blood was taken during the 90 rain of the study. Blood samples were placed into chilled tubes containing lithium heparin for measurement 5~
i'
1000~
{mmol.l-1)
/
'c- f ~
***~g
2!
400!
1]
200 ~
.! O'
O'
/'
600~
I
600q
NA (pg.ml-l,~
-V--+
_4
2003
NPY-LI
p0m,
~,~ ~ ~. - ~
T
~,-
200 J Pre
30
60
90
Pre
30
60
90
Fig. 1. Blood glucose (Glu) and the plasma concentration of adrenaline (Adr), noradrenaline (NA) and Neuropeptide Y like
immunoreactivity (NPY-LI) at baseline, and at 30. 60 and 90 rain after i.v. insulin, TRH and LHRH. * P < 0.05; * * * P < 0.001.
of adrenaline and noradrenaline (5 ml) and tubes containing potassium EDTA and 5000 KIU of aprotinin (Bayer) for measurement of NPY-LI (5 ml). Samples were placed on ice before being centrifuged at 4 ° C. Separated plasma was stored at - 8 0 °C until assay. The plasma concentration of NPY-LI was measured by specific direct radioimmunoassay and the free concentrations of adrenaline and noradrenaline by high performance liquid chromatography with electrochemical detection as has previously been described [16]. The chromatographic elution pattern of human plasma NPY-LI measured in this assay is similar to that obtained with NPY standard. Results are expressed as the mean _+ S.E.M. Differences between baseline and 30, 60, and 90 rain were examined using paired t-tests on a preplanned basis and were regarded as significant if P was less than 0.05 [19]. The plasma concentrations of adrenaline, noradrenaline and NPY-LI and the blood glucose at the various sampling times are displayed in Fig. 1. All subjects had a fall in blood glucose and an increase in plasma adrenaline concentration. Blood glucose fell from 4.7 _+ 0.27 mmol • 1-1 at baseline to 2.2_+0.32 m m o l - I 1 ( P < 0 . 0 0 1 ) at 30 min after i.v. injection of insulin, T R H and L H R H . The plasma adrenaline concentration was increased approximately 15 times from 48 _+ 8 pg- ml-1 at baseline to 706 _ 243 pg. ml- 1 ( p < 0.05) at 30 rain. Blood glucose remained reduced and plasma adrenaline concentration remained increased throughout the study period whereas the plasma concentrations of both noradrenaline and NPY-LI did not change. Hypoglycaemia such as that produced with i.v. insulin is a potent stimulus to secretion of catecholamines from the adrenal medulla and in man adrenaline is the major adrenal catecholamine [6]. We have been unable to demonstrate increases in the plasma NPY-LI concentration during significant hypoglycaemia despite a marked increase in the plasma concentration of adrenaline. This is in contrast to the increase in plasma NPY-LI concentration that has been found during dynamic exercise and cold pressor tests [16] when noradrenaline concentration was increased. This suggests that NPY is not released in significant
87 q u a n t i t i e s w h e n the h u m a n a d r e n a l m e d u l l a is stimulated. T h o u g h N P Y has b e e n localised to c a t e c h o l a m i n e - c o n t a i n i n g cells of the a d r e n a l m e d u l l a , it is u n c l e a r if N P Y is co-localised with a d r e n a l i n e o r n o r a d r e n a l i n e . L u n d b e r g et al. f o u n d N P Y - L I to b e p r e s e n t in s o m e c h r o m a f f i n cells c o n t a i n i n g the e n z y m e n e e d e d for a d r e n a l i n e synthesis, p h e n y l e t h a n o l a m i n e N - m e t h y l transferase ( P N M T ) [10] in the a d r e n a l g l a n d of the rat, mouse, cat, g u i n e a pig a n d m a n . O n the o t h e r h a n d V a r n d e l l et al. localised N P Y - L I to n o r a d r e n a l i n e - c o n t a i n i n g cells o f the a d r e n a l m e d u l l a in a n u m b e r of species i n c l u d i n g the c o w [13,19]. O t h e r workers have r e p o r t e d t h a t the h u m a n a d r e n a l m e d u l l a c o n t a i n s relatively little N P Y in r e l a t i o n to s y m p a t h e t i c g a n g l i a a n d tissues with s y m p a t h e t i c i n n e r v a t i o n [11] a n d to the a d r e n a l m e d u l l a in o t h e r species [2,8]. It is p o s s i b l e that this N P Y is p r e s e n t in n o r a d r e n a l i n e c h r o m a f f i n cells o r even a d r e n a l i n e c o n t a i n i n g cells that are n o t a c t i v a t e d b y the stress of h y p o g l y c a e m i a o f this degree [9]. It m a y n o t b e p o s s i b l e to d i f f e r e n t i a t e release of N P Y - L I with n o r a d r e n a l i n e f r o m the a d r e n a l m e d u l l a f r o m t h a t f r o m p e r i p h e r a l s y m p a t h e t i c nerves. I n a d d i t i o n to insulin o u r subjects received T R H a n d L H R H . D e s p i t e T R H b e i n g p r e s e n t in the a d r e n a l m e d u l l a of the rat [14] its effect on N P Y secretion has as yet n o t been e x a m i n e d . N o n e the less o u r results d o n o t reveal a n y signific a n t c o n t r i b u t i o n o f the a d r e n a l m e d u l l a to circ u l a t i n g N P Y - L I n o r a n y significant co-release of N P Y with a d r e n a l i n e in man. T h e y d o n o t s u p p o r t a p h y s i o l o g i c a l role for N P Y as a h o r m o n e of a d r e n a l origin in h u m a n subjects in r e s p o n s e to hypoglycaemia.
W e wish to t h a n k Sue T a y l o r for assistance with statistical analysis a n d illustrations, D r S. J u d d for access to his p a t i e n t s a n d Lily M i c h a i l o v a n d Sue F i l m e r for their c o o p e r a t i o n . N P Y a n t i b o d y was k i n d l y p r o v i d e d b y D r s C. M a c c a r r o n e a n d B. Jarrott. A . R . is the recipient o f a L i o n s P o s t g r a d u a t e M e d i c a l R e s e a r c h Scholarship. M . M . was an N H & M R C A u s t r a l i a n P o s t d o c t o r a l Fellow.
1 Allen, J.M., Adrian, T.E., Polak, J.M. and Bloom, S.R., Neuropeptide Y in the adrenal gland, J. Auton. Nero. Syst., 9 (1983) 559-563. 2 Allen, J.M., Bircham, P.M.M., Bloom, S.R. and Edwards, A.V., Release of neuropeptide Y in response to splanchnic nerve stimulation in the conscious calf, J. Physiol. (Lond.), 357 (1984) 401-408. 3 Corder, R., Lowry, P.J., Emson, P.C. and Galliard, R.C., Chromatographic characterisation of the circulating neuropeptide Y immunoreactivity from patients with pheochromocytoma, Regul. Pept., 10 (1985) 91-97. 4 Edvinsson, L., Ekblad, E., Hakanson, R. and Wahlestedt, C., Neuropeptide Y potentiates the effect of various vasoconstrictor agents on rabbit blood vessels, Br. J. Pharmacol., 83 (1984) 519-525. 5 Ekblad, E., Edvinsson, L., Wahlestedt, R., Uddman, R., Hakanson, R. and Sundler, F., Neuropeptide Y co-exists and cooperates with noradrenaline in perivascular nerve fibres, Regul. Pept., 8 (1984) 225-235. 6 Garber, A.J., Cryer, P.E., Santiago, J.V., Haymond, M.W., Pagliara, A.S. and Kipnis, D.M., The role of adrenergic mechanisms in the substrate and hormonal response to insulin-induced hypoglycemia in man. J. Clin. Invest., 58 (1976) 7-15. 7 Gray, T.S. and Morley, J.E., Neuropeptide Y: anatomical distribution and possible function in mammalian nervous system, Life Sci., 38 (1986) 389-401. 8 Hexum, T.D., Majane, E.A., Russett, L.R. and Yang, H.Y.T., Neuropeptide Y release from the adrenal medulla after cholinergic receptor stimulation, J. Pharmacol. Exp. Ther., 243 (1987) 927-930. 9 Hillarp, N. and HSkfelt, B., Cytological demonstration of noradrenaline in the suprarenal medulla under conditions of varied secretory activity, Endocrinology, 55 (1954) 255-260. 10 Lundberg, J.M., HSkfelt, T., Hemsen, A., TheodorssonNorheim, E., Pernow, J., Hamberger, B. and Goldstein, M., Neuropeptide Y-like immunoreactivity in adrenal cells of adrenal medulla and in tumors and plasma of pheochromocytoma patients, Regul. Pept., 13 (1986) 169-182. 11 Lundberg, J.M., Terenius, L., H~kfelt, T. and Goldstein, M., High levels of Nenropeptide Y in peripheral noradrenergic neurons in various mammals including man, Neurosci. Lett., 42 (1983) 167-172. 12 Maccarrone, C. and Jarrott, B., Neuropeptide Y: a putative neurotransmitter, Neurochem. Int., 8 (1986) 13-22. 13 Majane, E.A., Alho, H., Kataoka, Y., Lee, C.H. and Yang, H.-Y.T., Neuropeptide Y in bovine adrenal glands: distribution and characterization, Endocrinology, 117 (1985) 1162-1168. 14 Mitsuma, T., Sun, D.H., Nogimori, T., Ohtake, K. and Hirooka, Y., Dopamine inhibits thyrotropin releasing hormone release from rat adrenal gland in vitro, Hormone Res., 25 (1987) 223-227. 15 Morris, M., Kapoor, V. and Chalmers, J., Plasma Neuropeptide Y concentration is increased after haemorrhage in conscious rats: relative contribution of sympathetic nerves
88 and the adrenal medulla, J. Cardiovasc. Pharmacol., 9 (1987) 541-545. 16 Morris, M.J., Russell, A.E., Kapoor, V., Cain, M.D., Elliott, J.M., West, M.J., Wing, L.M.H. and Chalmers, J.P., Increases in plasma neuropeptide Y concentrations during sympathetic activation in man. J..4uton. Nerv. Syst., 17 (1986) 143-149. 17 O'Donohue, T.L., Chronwall, B.M., Pruss, R.M., Mezey, E., Kiss, J.Z., Eiden, L.E., Massari, V.J., Tessel, R.E., Pickel, V.M., Dimaggio, D.A., Hotchkiss, A.J., Crowley, W.R. and Zukowska-Grojec, Z., Neuropeptide Y and Peptide YY
neuronal and endocrine systems, Peptides. 6 (1985) 755-768. 18 de Quidt, M.E. and Emson, P.C., Neuropeptide Y in the adrenal gland: Characterisation distribution and drug effects, Neuroscience, 19 (1986) 1011-1022. 19 Steel, R.G.D. and Torrie, J.H., Principles and Procedures of Statis, McGraw-Hill, Sydney, 1981, 174 pp. 20 Varndell, I.M., Polak, J.M., Allen, J.M., Terenghi, G. and Bloom, S.R., Neuropeptide tyrosine (NPY) immunoreactivity in norepinephrine containing cells of the mammalian adrenal gland, Endocrinology, 114 (1984) 1460-1462.
85
JAN 00899
Short Communication
Neuropeptide Y-like immunoreactivity of plasma during hypoglycaemia in man A.E. Russell, M.D. Cain, V. Kapoor, M.J. Morris and J.P. Chalmers The Department of Medicine and Centre for Neuroscience, Flinders Medical Centre, Adelaide, South Australia (Australia) (Received 26 July 1988) (Revised version received 27 October 1988) (Accepted 11 November 1988)
Key words: Plasma neuropeptide Y; Plasma noradrenaline; Plasma adrenaline; Adrenal medulla; Hypoglycaemia
Abstract Previous studies have demonstrated that plasma Neuropeptide Y-like immunoreactivity (NPY-LI) increases after activation of sympathetic nerves. To test the hypothesis that the adrenal medulla may also be a significant source of circulating plasma NPY-LI and to determine if NPY is co-released with adrenal catecholamines, we have measured the peripheral venous concentrations of NPY-LI, adrenaline and noradrenaline in six patients, before and after induction of hypoglycaemia as part of pituitary function tests that also tested gonadotrophin and thyroid stimulating hormone release. The plasma adrenaline concentration was increased approximately 15 times (p < 0.05) relative to baseline at 30 rains and remained elevated for the 90 minutes of the study. The plasma concentration of both noradrenaline and NPY-LI remained unchanged. These results failed to demonstrate an increase in the amount of NPY-LI released into the plasma during stimulation of the adrenal medulla with hypoglycaemic stress in man. They do not support significant co-release of NPY with adrenaline from the adrenal medulla in man, nor a physiological role for NPY as an adrenal hormone in human subjects in this situation.
Neuropeptide Y (NPY) is a 36 amino acid peptide that has potent vasoconstrictor actions, that also increases vascular responses to noradrenaline [4]. It is widely distributed throughout the central and peripheral nervous systems and is present in the adrenal medulla [7,12,16]. In the peripheral components of the sympathetic nervous system NPY has been found co-localised with noradrenaline in nerve terminals [5]. In the adrenal medulla NPY has been identi-
Correspondence: J.P. Chalmers, The Department of Medicine, Flinders Medical Centre, Bedford Park, South Australia, 5042, Australia.
fled in catecholamine-containing cells [1] but it is unclear if NPY is co-localised with adrenaline [10] or noradrenaline [19] or both. The relationship between plasma noradrenaline concentration and the plasma concentration of NPY-like immunoreactivity (NPY-LI) in the human during sympathoadrenal activation [16] and in the rat after haemorrhage [15] has suggested a predominantly neuronal source for circulating NPY-LI. However, studies in patients with pheochromocytoma [3] and in rats after insulin [18] have raised the possibility that the adrenal medulla may be a significant source of circulating NPY-LI and that NPY might act as a hormone of adrenal origin [18]. This study was undertaken to test this by-
0165-1838/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
86 pothesis by examining the circulating concentration of N P Y - L I before and after adrenal catecholamine release was stimulated during hypoglycaemia induced with i.v. insulin as part of triple axis pituitary function tests. Six patients (5 males and one female), aged from 24 to 66 years (mean _+ SD: 47 _+ 19 years) having triple axis pituitary function studies for the investigation of pituitary tumors (5) or hypothyroidism (1) agreed to additional blood samples being taken for this study. Subsequently, 3 were found to have significantly abnormal pituitary function. Five had a pituitary tumor confirmed at h y p o p h y s e c t o m y ( a d e n o m a 3, eosinophilic granuloma 1) a n d / o r computed tomography (1). The subjects rested supine throughout the study after a cannula had been placed into an arm vein for blood sampling and i.v. injection. Blood samples for measurement of blood glucose, plasma cateeholamines and plasma NPY-LI were taken before and 30, 60 and 90 min after the subject received 0.1 U / k g of Actrapid MC insulin (Novo), 100 /~g of thyrotropin-releasing hormone (TRH, Roche) and 200 /~g of gonadotrophin-releasing hormone (LHRH, Hoechst) i.v. Approximately 140 ml of blood was taken during the 90 rain of the study. Blood samples were placed into chilled tubes containing lithium heparin for measurement 5~
i'
1000~
{mmol.l-1)
/
'c- f ~
***~g
2!
400!
1]
200 ~
.! O'
O'
/'
600~
I
600q
NA (pg.ml-l,~
-V--+
_4
2003
NPY-LI
p0m,
~,~ ~ ~. - ~
T
~,-
200 J Pre
30
60
90
Pre
30
60
90
Fig. 1. Blood glucose (Glu) and the plasma concentration of adrenaline (Adr), noradrenaline (NA) and Neuropeptide Y like
immunoreactivity (NPY-LI) at baseline, and at 30. 60 and 90 rain after i.v. insulin, TRH and LHRH. * P < 0.05; * * * P < 0.001.
of adrenaline and noradrenaline (5 ml) and tubes containing potassium EDTA and 5000 KIU of aprotinin (Bayer) for measurement of NPY-LI (5 ml). Samples were placed on ice before being centrifuged at 4 ° C. Separated plasma was stored at - 8 0 °C until assay. The plasma concentration of NPY-LI was measured by specific direct radioimmunoassay and the free concentrations of adrenaline and noradrenaline by high performance liquid chromatography with electrochemical detection as has previously been described [16]. The chromatographic elution pattern of human plasma NPY-LI measured in this assay is similar to that obtained with NPY standard. Results are expressed as the mean _+ S.E.M. Differences between baseline and 30, 60, and 90 rain were examined using paired t-tests on a preplanned basis and were regarded as significant if P was less than 0.05 [19]. The plasma concentrations of adrenaline, noradrenaline and NPY-LI and the blood glucose at the various sampling times are displayed in Fig. 1. All subjects had a fall in blood glucose and an increase in plasma adrenaline concentration. Blood glucose fell from 4.7 _+ 0.27 mmol • 1-1 at baseline to 2.2_+0.32 m m o l - I 1 ( P < 0 . 0 0 1 ) at 30 min after i.v. injection of insulin, T R H and L H R H . The plasma adrenaline concentration was increased approximately 15 times from 48 _+ 8 pg- ml-1 at baseline to 706 _ 243 pg. ml- 1 ( p < 0.05) at 30 rain. Blood glucose remained reduced and plasma adrenaline concentration remained increased throughout the study period whereas the plasma concentrations of both noradrenaline and NPY-LI did not change. Hypoglycaemia such as that produced with i.v. insulin is a potent stimulus to secretion of catecholamines from the adrenal medulla and in man adrenaline is the major adrenal catecholamine [6]. We have been unable to demonstrate increases in the plasma NPY-LI concentration during significant hypoglycaemia despite a marked increase in the plasma concentration of adrenaline. This is in contrast to the increase in plasma NPY-LI concentration that has been found during dynamic exercise and cold pressor tests [16] when noradrenaline concentration was increased. This suggests that NPY is not released in significant
87 q u a n t i t i e s w h e n the h u m a n a d r e n a l m e d u l l a is stimulated. T h o u g h N P Y has b e e n localised to c a t e c h o l a m i n e - c o n t a i n i n g cells of the a d r e n a l m e d u l l a , it is u n c l e a r if N P Y is co-localised with a d r e n a l i n e o r n o r a d r e n a l i n e . L u n d b e r g et al. f o u n d N P Y - L I to b e p r e s e n t in s o m e c h r o m a f f i n cells c o n t a i n i n g the e n z y m e n e e d e d for a d r e n a l i n e synthesis, p h e n y l e t h a n o l a m i n e N - m e t h y l transferase ( P N M T ) [10] in the a d r e n a l g l a n d of the rat, mouse, cat, g u i n e a pig a n d m a n . O n the o t h e r h a n d V a r n d e l l et al. localised N P Y - L I to n o r a d r e n a l i n e - c o n t a i n i n g cells o f the a d r e n a l m e d u l l a in a n u m b e r of species i n c l u d i n g the c o w [13,19]. O t h e r workers have r e p o r t e d t h a t the h u m a n a d r e n a l m e d u l l a c o n t a i n s relatively little N P Y in r e l a t i o n to s y m p a t h e t i c g a n g l i a a n d tissues with s y m p a t h e t i c i n n e r v a t i o n [11] a n d to the a d r e n a l m e d u l l a in o t h e r species [2,8]. It is p o s s i b l e that this N P Y is p r e s e n t in n o r a d r e n a l i n e c h r o m a f f i n cells o r even a d r e n a l i n e c o n t a i n i n g cells that are n o t a c t i v a t e d b y the stress of h y p o g l y c a e m i a o f this degree [9]. It m a y n o t b e p o s s i b l e to d i f f e r e n t i a t e release of N P Y - L I with n o r a d r e n a l i n e f r o m the a d r e n a l m e d u l l a f r o m t h a t f r o m p e r i p h e r a l s y m p a t h e t i c nerves. I n a d d i t i o n to insulin o u r subjects received T R H a n d L H R H . D e s p i t e T R H b e i n g p r e s e n t in the a d r e n a l m e d u l l a of the rat [14] its effect on N P Y secretion has as yet n o t been e x a m i n e d . N o n e the less o u r results d o n o t reveal a n y signific a n t c o n t r i b u t i o n o f the a d r e n a l m e d u l l a to circ u l a t i n g N P Y - L I n o r a n y significant co-release of N P Y with a d r e n a l i n e in man. T h e y d o n o t s u p p o r t a p h y s i o l o g i c a l role for N P Y as a h o r m o n e of a d r e n a l origin in h u m a n subjects in r e s p o n s e to hypoglycaemia.
W e wish to t h a n k Sue T a y l o r for assistance with statistical analysis a n d illustrations, D r S. J u d d for access to his p a t i e n t s a n d Lily M i c h a i l o v a n d Sue F i l m e r for their c o o p e r a t i o n . N P Y a n t i b o d y was k i n d l y p r o v i d e d b y D r s C. M a c c a r r o n e a n d B. Jarrott. A . R . is the recipient o f a L i o n s P o s t g r a d u a t e M e d i c a l R e s e a r c h Scholarship. M . M . was an N H & M R C A u s t r a l i a n P o s t d o c t o r a l Fellow.
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