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Substance P and opiate-like peptides in human adrenal medulla
Neuroscience Letters, 20 (1980) 195-200
195
© Elsevier/North-Holland Scientific Publishers Ltd.
SUBSTANCE MEDULLA
P AND
OPIATE-LIKE P E P T I D E S
IN H U M A N
ADRENAL
A. SARIA, S.P. WILSON, A. MOLNAR, O.H. VIVEROSand F. LEMBECK* Institut far experimentelle und klinische Pharmakologie, Universitat Graz, Universittitsplatz 4, A-8010 Graz (Austria)
and (S.P. IV. and O.H.V.) Department of Medicinal Biochemistry, Wellcome Research Laboratories, Research Triangle Park, N C 27709 (U.S.A .)
(Received July 25th, 1980; Accepted July 30th, 1980)
Opiate-like peptides (OLP), substance P (SP) and catecholamines (CA) were measured in 15 human adrenal medullae. Two groups of subjects were investigated. Group a consisted of subjects who died after traffic accidents and Group b consisted of subjects with other causes of death. OLP levels in Group a were only about 13%, and SP and CA levels about 50% of Group b. It is suggested that these differences might be due to massive premortal adrenal medullary discharge rather than post mortem degradation.
Opiate-like peptides (OLP) were demonstrated in the adrenal medullae of 8 mammalian species by chemical assay [25, 26]. Using immunofluorescence techniques Schultzberg et al. [23, 24] showed enkephalins to be localized in nerve terminals and cell bodies of sympathetic ganglia and in nerve fibres and chromaffin cells of the adrenal medulla. Lundberg et al. [17] showed the presence of enkephalins as well as of somatostatin in human adrenal medulla and pheochromocytomas. Evidence for adrenomedullary biosynthesis, storage in chromaffin vesicles and calcium-dependent release of enkephalins and other O L P was reported [25, 26, 28]. O L P in sympathetic nerves are also co-stored with catecholamines in large dense-core noradrenergic vesicles [13, 27]. The undecapeptide substance P (SP) was found in sympathetic fibres and ganglia of various species by bioassay [20], immunofluorescence technique [12] and radioimmunoassay [11, 14]. Role et al. [21] reported the presence of immunoreactive SP in guinea pig adrenal chromaffin cells and Saria et al. [22] found immunoreactive SP in adrenal glands of rat, rabbit, guinea pig and cow. SP was shown to inhibit the acetylcholine-evoked norepinephrine release from cultured bovine adrenal medullary cells [16]. In this paper we report post mortem levels of * To whom reprint requests should be addressed.
196
OLP supplementing earlier findings [25], and show the presence of SP in human adrenal medulla. Adrenal glands were removed at autopsy between 4 and 26 h after death and immediately frozen on dry ice. Cortex and medulla were separated. For estimation of OLP the tissue (300 mg maximum) was homogenized in 2 ml 1 N acetic acid, centrifuged for 15 min at 1000 × g and the supernatant was lyophilized. OLP were determined by displacement of [125I]u-Ala2,D-LeuS-enkephalin from rat brain membrane receptors [3, 18]. For SP determination the tissue was frozen in liquid nitrogen and homogenized in a Braun Dismembrator and extracted with acetone-0.1 N HC1 (100:3, by volume) [4]. SP was determined by radioimmunoassay with [125I]TyrS-Sp as tracer [19]. Pooled and lyophilized acetone-HC1 extracts were chromatographed on a Sephadex G-25 column (1.2 × 95 cm) [9]. For catecholamine (CA) assay the tissue (100 mg maximum) was extracted with 10 vol. 0.4 N HC104 and separated by adsorption on alumina [1]. Epinephrine and norepinephrine were estimated by reverse phase H P L C . The ' H P L C system consisted of an Altex 110A pump, an Altex microprocessor M-420, a Kontron ASI-45 autosampler, a Rheodyne injection valve with a 20 /A loop, a reverse phase column (Waters #Bondapak C~8, 3.9 x 300 mm) and a Kontron SFM 23 spectrofluorimeter. CA were eluted with 0.1 M NaH2PO4 containing 0.1 mM 'EDTA at a flow rate of 1.5 ml/min (500-800 psi) and detected fluorimetrically at 254 nm excitation and 320 nm emission wavelength [2]. The values for O L P , SP and CA in human adrenal medullae are shown in Table I. The table is divided into two groups. The values in Group a were obtained from adrenal medullae of 8 persons who had died by cranial trauma or internal haemorrhage after traffic accidents. Adrenal medullae of Group b were obtained from 6 persons who had died of various internal diseases and, in one case, of death by hanging (1 b). Norepinephrine comprised 34°70 of total CA in Group a and 26°70 in Group b; the difference was statistically not significant. O L P , SP and CA levels, but also the time between death and autopsy, were significantly different in the two groups. While CA and SP levels were approximately 2-fold higher in Group b, OLP levels were approximately 10-fold higher in Group b than in Group a. The value for O L P in Group b was comparable to that reported by Viveros et al. [25] for one human adrenal medulla. In Group b, SP levels were only about 1/100 of O L P levels in contrast to 1/30 in Group a. Pooled extracts of adrenal medullae from Group a separated on Sephadex G-25 showed two immunoreactive peaks of which 9007o eluted at the position of synthetic SP and 10% appeared in the void volume (Fig. 1). The differences between CA, SP and O L P in Groups a and b could be due to two factors: (1) the longer time between death and autopsy in Group a which might favour degradation of biological substances, or (2) the cause of death. All persons of Group a died by trauma, followed by hypovolaemic shock and massive adrenomedullary discharge if there was enough time between accident and death. In the second group, death followed prolonged disease which may have led to
12.1"* 1.5
8.6** 1.9
aTime between death and autopsy.
S.E.M.
15.62 8.64 11.29 7.73 19.33 11.16 11.71
10 5 19 8 4 6 8
lb 2b 3b 4b 5b 6b 7b
Death by hanging Poisoning (unknown compound) Myocardial infarction Unknown Cerebrovascular insufficiency Cirrhosis of the liver Emphysema, cardiac insufficiency
6.4 1.6
19.9 2.6
S.E.M.
0~mol/g)
1985"** 324
1820 1520 3610 1150 1320 1890 2590
262 61
592 154 355 81 91 180 339 302
OLP (pmol/g)
E + NE 5.97 2.02 2.27 2.41 8.00 5.19 11.51 13.70
Time (h) a 23 23 23 24 25.5 24.5 5 11.5
Cranial Cranial Cranial Cranial Internal Internal Internal Internal
la 2a 3a 4a 5a 6a 7a 8a
trauma trauma trauma trauma haemorrhage haemorrhage haemorrhage haemorrhage
Cause of death
P<0.05;** P<0.01;*** P<0.0OI.
14.8" 2.9
25.4 13.4 7.5 19.7 4.2 12.0 21.2
7.7 2.2
20.4 6.1 6.9 2.1 1.7 3.0 12.8 8.7
(pmol/g)
SP
+ N E ) , S U B S T A N C E P ( S P ) A N D O P I A T E - L I K E P E P T I D E S ( O L P ) OF H U M A N
werecompar~ bvtheSmdent'stwosamplet-test.*
No.
Groupaandgroupb
C O N T E N T S OF E P I N E P H R I N E PLUS N O R E P I N E P H R I N E ( E ADRENAL MEDULLAE
TABLEI
198
$P pg ISP/fractron
500[ 400~ 300 i
J
200t !oo~ L
J
L_____
lO
2~0
....
ooo?o " 30
! I o o-o.ooo
o ooj
40 50 |roctroN number
Fig. 1. Gel filtration of pooled extracts of adrenal medullae (acetone-HCl-extracts) on Sephadex G-25. ISP = immunoreactive SP. Flow rate was 12 ml/h, fraction volume 2 ml. The void volume was estimated by blue dextran, synthetic SP was used as reference substance.
adaptation of medullary response, or death was so fast (case lb) that there was no time for adrenomedullary depletion of CA and peptides. Three facts favour the assumption of premortally depleted adrenal glands in Group a: the high post mortem stability of SP and enkephalins in other nervous tissue; the marked depletion of OLP from adrenal medulla obtained after intense splanchnic stimulation [26], and the results from gel filtration of SP. The elution profile of adrenal medullary SP-immunoreactivity from the Sephadex G-25 column is very similar to the profile obtained from cat vagus nerve [9] and from rat spinal cord superfusates [10]. It showed the main peak of immunoreactivity eluting at the position of synthetic SP, therefore it is assumed that measured SP levels represent material of a similar size as SP and not breakdown products or other crossreacting material. The presence of enkephalins in human adrenal glands supplemented the findings made in 8 mammalian species [25, 27] and confirmed that humans are a species with a high OLP content. If there is no decrease of enkephalins and SP within 25 h after death, it should be possible to investigate the levels of these peptides in the adrenal medulla in various pathological conditions. Recently, human pheochromocytoma tumours were also shown to contain enkephalins [17, 27]. Recent experimental work suggests a functional connection between CA and enkephalins [5, 15, 25-27]. The additional presence of SP poses questions concerning its function in the adrenal medulla. Dun and Karczmar [6] suggested that SP may be the endogenous transmitter mediating the slow non-cholinergic depolarization in sympathetic ganglia. Inhibition of norepinephrine release from cultured adrenal medullary cells by high concentrations of SP was shown [16]. The present findings do not permit conclusions concerning the functional role of SP in the adrenal medulla; further experimental work is needed to clarify this point.
199
The authors wish to thank Dr. HOfler from the Institute of Pathological Anatomy, University of Graz and Prof. Dr. Maresch and his staff from the Institute of Forensic Medicine, University of Graz for collecting the adrenal glands. We also thank Dr. S. Leeman, Harvard Medical School, Boston for donating the SP antibody and R. Schuligoi and I. Obrecht for technical assistance. This study was supported by the Pain Research Commission of the Austrian Academy of Sciences.
1
2
3 4 5 6 7 8
10 11 12
13 14 15 16 17
Anton, A.H. and Sayre, D.F., A study of the factors affecting the aluminium oxide-trihydroxyindole procedure for the analysis of catecholamines, J. Pharmacol. exp. Ther., 138 (1962) 360-375. Baker, H.F., Joseph, M.H. and Ridley, R.M., HPLC analysis of tryptophan, 5-HIAA and HVA using fluorescence and electrochemical detection; the effect of probenecid studied in primate ventricular CSF, Proc. Brit. Pharmacol. Soc., Apr. (1980) 97-98. Chang, K.J. and Cuatrecasas, P., Multiple opiate receptors: enkephalins and morphine bind to receptors of different specificity, J. biol. Chem., 254 (1979) 2610-2618. Chang, M.M. and Leeman, S.E., Isolation of a sialogogic peptide from bovine hypothalamus and its characterishtion as substance P, J. biol. Chem., 245 (1970) 4784-4790. Dashwood, M.R. and Feldberg, W., Release of opioid peptides in anaesthetized cats, Brit. J. Pharmacol., 68 (1980) 697-704. Dun, N.J. and Karczmar, A.G., Actions of substance P on sympathetic neurons, Neuropharmacology, 18 (1979) 215-218. Emson, P.C., Peptides as neurotransmitter candidates in the mammalian CNS, Progr. Neurobiol., 13 (1979) 61-116. Gale, I.S., Bird, E.D., Spokes, E.G., lversen, L.L. and Jessel, T., Human brain substance P: distribution in controls and Huntington's chorea, J. Neurochem., 30 (1978) 633-634. Gamse, R., Lembeck, F. and Cuello, A.C., Substance P in the vagus nerve. Immunochemical and immunohistochemical evidence for axoplasmic transport, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 306 (1979) 37 44. Gamse, R., Molnar, A. and Lembeck, F., Substance P release from spinal cord slices by capsaicin, Life Sci., 25 (1979) 659-636. Gamse, R., Wax, A., Zigmond, R.E. and Leeman, S.E., lmmunoreactive substance P in sympathetic ganglia: Distribution and sensitivity towards capsaicin, Neuroscience, submitted. HOkfelt, T., Elfvin, L.G., Schultzberg, M., Goldstein, M. and Nilsson, G., On the occurrence of substance P containing fibers in sympathetic ganglia, lmmunohistochemical evidence, Brain Res., 132 (1977) 29 41. Klein, R., Gasparis, M., Yang, N., Viveros, O. and Wilson, S., Noradrenergic vesicles 'hydrophilic' D~H and opiate contents, Trans. Amer. Soc. Neurochem., 2 (1980) 230. Konishi, S., Tsunoo, A. and Otsuka, M., Substance P and noncholinergic excitatory synaptic transmission in guinea pig sympathetic ganglia, Proc. Jap. Acad., 55B (1979) 525-530. Kumakura, K., Karoum, F., Guidotti, A. and Costa, E., Modulation of nicotinic receptors by opiate receptor agonists in cultured adrenal chromaffin cells, Nature (Lond.), 283 (1980) 489-491. Livett, B.G., Kozousek, V., Mizobe, F. and Dean, D.M., Substance P inhibits nicotinic activation of chromaffin cells, Nature (Lond.), 278 (1979) 256-257. Lundberg, J.M., Hamberger, B., Schultzberg, M., Hokfelt, T., Granherg, P.O., Efendic, S., Terenius, L., Goldstein, M. and Luft, R., Enkephalin- and somatostatin-like immunoreactivities in human adrenal medulla and pheochromocytoma, Proc. nat. Acad. Sci. (Wash.), 76 (1979) 4079-4083.
200 18 Miller, R.T., Chang, K.J., Cooper, B. and Cuatrecasas, P., Radioimmunoassay and characterization of enkephalins in rat tissues, J. biol. Chem., 253 (1978) 531-538. 19 Mroz, E.A., Brownstein, M.J. and Leeman, S.E., Distribution of immunoassayable substance P in the rat brain. Evidence for the existing of substance P containing tracts. In U.S. yon Euler and B. Pernow (Eds.), Raven Press, New York, 1977, pp. 121-137. 20 Pernow, B., Studies on substance P, Acta physiol, scand., 29, Suppl. 105 (1953) 1-90. 21 Role, L.W., Perlman, R.L. and Leeman, S.E., Somatostatin and substance P inhibit catecholamine secretion from guinea pig chromaffin cells, Neurosci. Abstr., 5 (1979) abstract 2031. 22 Saria, A., Molnar, A. and Lembeck, F., lmmunoreactive substance P in the adrenal medulla, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., in press. 23 Schultzberg, M., Lundberg, J.M., H6kfelt, T., Terenius, L., Brandt, J., Elde, R.P. and Goldstein, M., Enkephalin-like immunoreactivity in gland cells and nerve terminals of the adrenal medulla, Neuroscience, 3 (1978) 1169-1186. 24 Schultzberg, M., Hokfelt, T., Lundberg, J.M., Terenius, L., Elfvin, L.G. and Elde, R., Enkephalinlike immunoreactivity in nerve terminals in sympathetic ganglia and adrenal medulla and in adrenal medullary gland cells, Acta physiol, stand., 103 0978) 475-477. 25 Viveros, D.H., Diliberto, E.J., Hazum, E. and Chang, K.J., Opiate-like materials in the adrenal medulla: evidence for storage and secretion with catecholamines, Molec. Pharmacol., 16 (1979) 1101-1108.
26 Viveros, O.H., Diliberto, E.J., Hazum, E. and Chang, K.J., Enkephalins as possible adrenomedullary hormones: storage, secretion and regulation of synthesis. In E. Costa and M. Trabucchi (Eds.), Advances in Biochemical Psychopharmacology, Raven Press, New York, 1980, pp. 191-204. 27 Viveros, O.H., Wilson, S.P., Diliberto, E.S., Hazum, E. and Chang, K.-T., Enkephalins in adrenomedullary chromaffin cells and sympathetic nerves. In Effects of Endogenous Opioid Peptides on the Neuroendocriue System, Proc. XVIII Int. Congr. Physiol. Sci. Syrup., Hungarian Academy of Sciences, Budapest, in press. 28 Wilson, S.P., Chang, K.J. and Viveros, O.H., Synthesis of enkephalins by adrenal medullary chromaffin cells: reserpine increases incorporation of radiolabelled amino acids, Proc. nat. Acad. Sci. (Wash.), 77 (1980) 4364-4368.
195
© Elsevier/North-Holland Scientific Publishers Ltd.
SUBSTANCE MEDULLA
P AND
OPIATE-LIKE P E P T I D E S
IN H U M A N
ADRENAL
A. SARIA, S.P. WILSON, A. MOLNAR, O.H. VIVEROSand F. LEMBECK* Institut far experimentelle und klinische Pharmakologie, Universitat Graz, Universittitsplatz 4, A-8010 Graz (Austria)
and (S.P. IV. and O.H.V.) Department of Medicinal Biochemistry, Wellcome Research Laboratories, Research Triangle Park, N C 27709 (U.S.A .)
(Received July 25th, 1980; Accepted July 30th, 1980)
Opiate-like peptides (OLP), substance P (SP) and catecholamines (CA) were measured in 15 human adrenal medullae. Two groups of subjects were investigated. Group a consisted of subjects who died after traffic accidents and Group b consisted of subjects with other causes of death. OLP levels in Group a were only about 13%, and SP and CA levels about 50% of Group b. It is suggested that these differences might be due to massive premortal adrenal medullary discharge rather than post mortem degradation.
Opiate-like peptides (OLP) were demonstrated in the adrenal medullae of 8 mammalian species by chemical assay [25, 26]. Using immunofluorescence techniques Schultzberg et al. [23, 24] showed enkephalins to be localized in nerve terminals and cell bodies of sympathetic ganglia and in nerve fibres and chromaffin cells of the adrenal medulla. Lundberg et al. [17] showed the presence of enkephalins as well as of somatostatin in human adrenal medulla and pheochromocytomas. Evidence for adrenomedullary biosynthesis, storage in chromaffin vesicles and calcium-dependent release of enkephalins and other O L P was reported [25, 26, 28]. O L P in sympathetic nerves are also co-stored with catecholamines in large dense-core noradrenergic vesicles [13, 27]. The undecapeptide substance P (SP) was found in sympathetic fibres and ganglia of various species by bioassay [20], immunofluorescence technique [12] and radioimmunoassay [11, 14]. Role et al. [21] reported the presence of immunoreactive SP in guinea pig adrenal chromaffin cells and Saria et al. [22] found immunoreactive SP in adrenal glands of rat, rabbit, guinea pig and cow. SP was shown to inhibit the acetylcholine-evoked norepinephrine release from cultured bovine adrenal medullary cells [16]. In this paper we report post mortem levels of * To whom reprint requests should be addressed.
196
OLP supplementing earlier findings [25], and show the presence of SP in human adrenal medulla. Adrenal glands were removed at autopsy between 4 and 26 h after death and immediately frozen on dry ice. Cortex and medulla were separated. For estimation of OLP the tissue (300 mg maximum) was homogenized in 2 ml 1 N acetic acid, centrifuged for 15 min at 1000 × g and the supernatant was lyophilized. OLP were determined by displacement of [125I]u-Ala2,D-LeuS-enkephalin from rat brain membrane receptors [3, 18]. For SP determination the tissue was frozen in liquid nitrogen and homogenized in a Braun Dismembrator and extracted with acetone-0.1 N HC1 (100:3, by volume) [4]. SP was determined by radioimmunoassay with [125I]TyrS-Sp as tracer [19]. Pooled and lyophilized acetone-HC1 extracts were chromatographed on a Sephadex G-25 column (1.2 × 95 cm) [9]. For catecholamine (CA) assay the tissue (100 mg maximum) was extracted with 10 vol. 0.4 N HC104 and separated by adsorption on alumina [1]. Epinephrine and norepinephrine were estimated by reverse phase H P L C . The ' H P L C system consisted of an Altex 110A pump, an Altex microprocessor M-420, a Kontron ASI-45 autosampler, a Rheodyne injection valve with a 20 /A loop, a reverse phase column (Waters #Bondapak C~8, 3.9 x 300 mm) and a Kontron SFM 23 spectrofluorimeter. CA were eluted with 0.1 M NaH2PO4 containing 0.1 mM 'EDTA at a flow rate of 1.5 ml/min (500-800 psi) and detected fluorimetrically at 254 nm excitation and 320 nm emission wavelength [2]. The values for O L P , SP and CA in human adrenal medullae are shown in Table I. The table is divided into two groups. The values in Group a were obtained from adrenal medullae of 8 persons who had died by cranial trauma or internal haemorrhage after traffic accidents. Adrenal medullae of Group b were obtained from 6 persons who had died of various internal diseases and, in one case, of death by hanging (1 b). Norepinephrine comprised 34°70 of total CA in Group a and 26°70 in Group b; the difference was statistically not significant. O L P , SP and CA levels, but also the time between death and autopsy, were significantly different in the two groups. While CA and SP levels were approximately 2-fold higher in Group b, OLP levels were approximately 10-fold higher in Group b than in Group a. The value for O L P in Group b was comparable to that reported by Viveros et al. [25] for one human adrenal medulla. In Group b, SP levels were only about 1/100 of O L P levels in contrast to 1/30 in Group a. Pooled extracts of adrenal medullae from Group a separated on Sephadex G-25 showed two immunoreactive peaks of which 9007o eluted at the position of synthetic SP and 10% appeared in the void volume (Fig. 1). The differences between CA, SP and O L P in Groups a and b could be due to two factors: (1) the longer time between death and autopsy in Group a which might favour degradation of biological substances, or (2) the cause of death. All persons of Group a died by trauma, followed by hypovolaemic shock and massive adrenomedullary discharge if there was enough time between accident and death. In the second group, death followed prolonged disease which may have led to
12.1"* 1.5
8.6** 1.9
aTime between death and autopsy.
S.E.M.
15.62 8.64 11.29 7.73 19.33 11.16 11.71
10 5 19 8 4 6 8
lb 2b 3b 4b 5b 6b 7b
Death by hanging Poisoning (unknown compound) Myocardial infarction Unknown Cerebrovascular insufficiency Cirrhosis of the liver Emphysema, cardiac insufficiency
6.4 1.6
19.9 2.6
S.E.M.
0~mol/g)
1985"** 324
1820 1520 3610 1150 1320 1890 2590
262 61
592 154 355 81 91 180 339 302
OLP (pmol/g)
E + NE 5.97 2.02 2.27 2.41 8.00 5.19 11.51 13.70
Time (h) a 23 23 23 24 25.5 24.5 5 11.5
Cranial Cranial Cranial Cranial Internal Internal Internal Internal
la 2a 3a 4a 5a 6a 7a 8a
trauma trauma trauma trauma haemorrhage haemorrhage haemorrhage haemorrhage
Cause of death
P<0.05;** P<0.01;*** P<0.0OI.
14.8" 2.9
25.4 13.4 7.5 19.7 4.2 12.0 21.2
7.7 2.2
20.4 6.1 6.9 2.1 1.7 3.0 12.8 8.7
(pmol/g)
SP
+ N E ) , S U B S T A N C E P ( S P ) A N D O P I A T E - L I K E P E P T I D E S ( O L P ) OF H U M A N
werecompar~ bvtheSmdent'stwosamplet-test.*
No.
Groupaandgroupb
C O N T E N T S OF E P I N E P H R I N E PLUS N O R E P I N E P H R I N E ( E ADRENAL MEDULLAE
TABLEI
198
$P pg ISP/fractron
500[ 400~ 300 i
J
200t !oo~ L
J
L_____
lO
2~0
....
ooo?o " 30
! I o o-o.ooo
o ooj
40 50 |roctroN number
Fig. 1. Gel filtration of pooled extracts of adrenal medullae (acetone-HCl-extracts) on Sephadex G-25. ISP = immunoreactive SP. Flow rate was 12 ml/h, fraction volume 2 ml. The void volume was estimated by blue dextran, synthetic SP was used as reference substance.
adaptation of medullary response, or death was so fast (case lb) that there was no time for adrenomedullary depletion of CA and peptides. Three facts favour the assumption of premortally depleted adrenal glands in Group a: the high post mortem stability of SP and enkephalins in other nervous tissue; the marked depletion of OLP from adrenal medulla obtained after intense splanchnic stimulation [26], and the results from gel filtration of SP. The elution profile of adrenal medullary SP-immunoreactivity from the Sephadex G-25 column is very similar to the profile obtained from cat vagus nerve [9] and from rat spinal cord superfusates [10]. It showed the main peak of immunoreactivity eluting at the position of synthetic SP, therefore it is assumed that measured SP levels represent material of a similar size as SP and not breakdown products or other crossreacting material. The presence of enkephalins in human adrenal glands supplemented the findings made in 8 mammalian species [25, 27] and confirmed that humans are a species with a high OLP content. If there is no decrease of enkephalins and SP within 25 h after death, it should be possible to investigate the levels of these peptides in the adrenal medulla in various pathological conditions. Recently, human pheochromocytoma tumours were also shown to contain enkephalins [17, 27]. Recent experimental work suggests a functional connection between CA and enkephalins [5, 15, 25-27]. The additional presence of SP poses questions concerning its function in the adrenal medulla. Dun and Karczmar [6] suggested that SP may be the endogenous transmitter mediating the slow non-cholinergic depolarization in sympathetic ganglia. Inhibition of norepinephrine release from cultured adrenal medullary cells by high concentrations of SP was shown [16]. The present findings do not permit conclusions concerning the functional role of SP in the adrenal medulla; further experimental work is needed to clarify this point.
199
The authors wish to thank Dr. HOfler from the Institute of Pathological Anatomy, University of Graz and Prof. Dr. Maresch and his staff from the Institute of Forensic Medicine, University of Graz for collecting the adrenal glands. We also thank Dr. S. Leeman, Harvard Medical School, Boston for donating the SP antibody and R. Schuligoi and I. Obrecht for technical assistance. This study was supported by the Pain Research Commission of the Austrian Academy of Sciences.
1
2
3 4 5 6 7 8
10 11 12
13 14 15 16 17
Anton, A.H. and Sayre, D.F., A study of the factors affecting the aluminium oxide-trihydroxyindole procedure for the analysis of catecholamines, J. Pharmacol. exp. Ther., 138 (1962) 360-375. Baker, H.F., Joseph, M.H. and Ridley, R.M., HPLC analysis of tryptophan, 5-HIAA and HVA using fluorescence and electrochemical detection; the effect of probenecid studied in primate ventricular CSF, Proc. Brit. Pharmacol. Soc., Apr. (1980) 97-98. Chang, K.J. and Cuatrecasas, P., Multiple opiate receptors: enkephalins and morphine bind to receptors of different specificity, J. biol. Chem., 254 (1979) 2610-2618. Chang, M.M. and Leeman, S.E., Isolation of a sialogogic peptide from bovine hypothalamus and its characterishtion as substance P, J. biol. Chem., 245 (1970) 4784-4790. Dashwood, M.R. and Feldberg, W., Release of opioid peptides in anaesthetized cats, Brit. J. Pharmacol., 68 (1980) 697-704. Dun, N.J. and Karczmar, A.G., Actions of substance P on sympathetic neurons, Neuropharmacology, 18 (1979) 215-218. Emson, P.C., Peptides as neurotransmitter candidates in the mammalian CNS, Progr. Neurobiol., 13 (1979) 61-116. Gale, I.S., Bird, E.D., Spokes, E.G., lversen, L.L. and Jessel, T., Human brain substance P: distribution in controls and Huntington's chorea, J. Neurochem., 30 (1978) 633-634. Gamse, R., Lembeck, F. and Cuello, A.C., Substance P in the vagus nerve. Immunochemical and immunohistochemical evidence for axoplasmic transport, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 306 (1979) 37 44. Gamse, R., Molnar, A. and Lembeck, F., Substance P release from spinal cord slices by capsaicin, Life Sci., 25 (1979) 659-636. Gamse, R., Wax, A., Zigmond, R.E. and Leeman, S.E., lmmunoreactive substance P in sympathetic ganglia: Distribution and sensitivity towards capsaicin, Neuroscience, submitted. HOkfelt, T., Elfvin, L.G., Schultzberg, M., Goldstein, M. and Nilsson, G., On the occurrence of substance P containing fibers in sympathetic ganglia, lmmunohistochemical evidence, Brain Res., 132 (1977) 29 41. Klein, R., Gasparis, M., Yang, N., Viveros, O. and Wilson, S., Noradrenergic vesicles 'hydrophilic' D~H and opiate contents, Trans. Amer. Soc. Neurochem., 2 (1980) 230. Konishi, S., Tsunoo, A. and Otsuka, M., Substance P and noncholinergic excitatory synaptic transmission in guinea pig sympathetic ganglia, Proc. Jap. Acad., 55B (1979) 525-530. Kumakura, K., Karoum, F., Guidotti, A. and Costa, E., Modulation of nicotinic receptors by opiate receptor agonists in cultured adrenal chromaffin cells, Nature (Lond.), 283 (1980) 489-491. Livett, B.G., Kozousek, V., Mizobe, F. and Dean, D.M., Substance P inhibits nicotinic activation of chromaffin cells, Nature (Lond.), 278 (1979) 256-257. Lundberg, J.M., Hamberger, B., Schultzberg, M., Hokfelt, T., Granherg, P.O., Efendic, S., Terenius, L., Goldstein, M. and Luft, R., Enkephalin- and somatostatin-like immunoreactivities in human adrenal medulla and pheochromocytoma, Proc. nat. Acad. Sci. (Wash.), 76 (1979) 4079-4083.
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