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Fluoride-resistant acid phosphatase in the rat adrenal gland
Brain Research, 253 (1982) 325-329
325
Elsevier Biomedical Press
Fluoride-resistant acid phosphatase in the rat adrenal gland S. R. VINCENT*, M. SCHULTZBERG and C.-J. DALSGAARD Departments of Histology and ( C.-J.D.) Anatomy, Karolinska lnstitutet, S-104 01 Stockholm (Sweden)
(Accepted August 10th, 1982) Key words: fluoride-resistant acid phosphatase (FRAP) - - adrenal gland - - chromaffin cell - -
adrenalin - - enkephalin - - immunohistochemistry
The adrenal medulla of the rat, but not of the mouse, guinea pig or cat, was shown to contain a fluoride-resistant acid phosphatase activity similar to that previously noted in rat spinal cord. By combined immunohistochemistry and FRAP histochemistry it was determined that the FRAP in the rat adrenal was confined to the adrenaline chromaffin cells. A unique fluoride-resistant acid phosphatase (FRAP) enzyme activity has been demonstrated histochemically in a population of primary afferent neurons in the ratT,15,16,19,~2. FRAP-positive fibres are found concentrated in lamina II of the spinal cord and trigeminal complex, and the small type-C cells in the trigeminal and spinal ganglia are also FRAP-reactive. These cells appear to be distinct from the sensory neurons containing somatostatin or substance pls,19 and thus represent a biochemically distinct nerve cell type, using an as yet unidentified transmitter substance. Peptides such as substance p1z,14, somatostatin~0, cholecystokinin 17 and vasoactive intestinal polypeptide (VIP) is, which have been reported in primary afferents have also been found in other nerve cell types and/or endocrine cells (see refs. 1, 12). In the present report we present histochemical evidence that F R A P activity is also contained in cell types other than primary afferents. We have found this enzyme activity in adrenaline chromaffin cells in the rat adrenal gland. Young adult male albino rats, mice and guinea pigs and an adult cat were examined. The animals were anesthetized with pentobarbital and perfused through the aorta with ice-cold calcium-free Tyrodes' solution followed by 1 0 ~ formalin in
phosphate buffer. The adrenal gland and spinal cord were removed, and immersed in the same fixative for a further 90 min. After rinsing at least 24 h in 5 sucrose in phosphate buffer the tissues were cut on a cryostat (Dittes, Heidelberg) at 14 #m. Sections of the adrenal gland and spinal cord were stained for the demonstration of F R A P activity according to Sz6nyi et al. 21 in 0 . 2 5 ~ sodium flglycerophosphate and 0.08 ~ lead nitrate in 20 m M Tris-maleate buffer, p H 5, containing 4 m M N a F 7 for 30 min at 37 °C. The sections were then rinsed in distilled water and treated with 1 ~ a m m o n i u m sulfide for 15 s, rinsed in water and mounted in glycerol-phosphate-buffered saline (PBS) (3:1). Some sections were stained immunohistochemically prior to histochemical staining for F R A P activity. In these experiments, the indirect immunofluorescence technique of Coons et al. was employed2,11. Adrenalin ceils were identified using antibodies raised against the enzyme phenylethanolamine-Nmethyl transferase (PNMT)S, 11, noradrenalin and adrenalin ceils were stained with antisera to dopamine-fl-hydroxylase (DBH)S, 11 and enkephalin immunoreactivity was detected with antisera raised against Met-enkephalin (rabbit 336) 21. The sections were incubated in the primary antisera at 4 °C overnight, then rinsed with PBS and incubated in fluo-
* To whom correspondence should be addressed at: Dept. of Physiology, University of British Columbia, Vancouver, B.C., V6T lW5 Canada. 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
326 rescene-isothiocyanate (FITC)-labelled swine antirabbit igG (diluted 1:10) (Dako, Copenhagen), for 30 min at 37 "C, rinsed again in PBS and mounted in glyceroI-PBS. All primary antisera were diluted (1:100) in PBS containing 0.3 ')i~ Triton X-100. The specimens were examined in a fluorescence microscope, and after p h o t o g r a p h y the coverslips were removed and the sections stained histochemically for F R A P activity. By this protocol the same section could be sequentially stained by immunohistochemistry and F R A P histochemistry. Control sections were incubated with non-immune sera or with antisera pretreated with an excess (10 nmol/ml diluted
antiserum) of Met-enkephalin. Within the rat spinal cord, intense F R A P activity was found in lamina I1 of the dorsal horn (Fig. IA). In contrast, the dorsal horn of the guinea pig (Fig. I B) and mouse contained no F R A P activity. When sections of the rat adrenal gland were incubated for F R A P activity, an intense reaction was observed in the medulla (Fig. IC). This reaction was present even in the presence of 100 mM NaF, indicating that this acid phosphatase was resistant to fluoride inhibition. The medulla o f the guinea pig (Fig. 1D), mouse and cat showed practically no F R A P activity.
Fig. 1. Micrographs of the spinal cord and adrenal gland of the rat (A, C) and guinea pig (B, D) stained for FRAP activity. Intense FRAP activity is found in the substantia gelatinosa of the rat dorsal horn (A), but the enzyme activity is not detected in the guinea pig (B). Similarly, many cells of the rat adrenal medulla (arrow) show intense FRAP activity (C), while the guinea pig adrenal medulla (arrow) is unstained, Bar indicates 50/*m for all figures.
327 In an attempt to determine which cell types in the rat adrenal gland contain FRAP activity, sections were first stained immunohistochemically, and then processed for FRAP. By this procedure it was found that only some of the DBH-positive cells contained F R A P (Fig. 2A and B). These were apparently the adrenalin cells since all FRAP-positive cells were stained with PNMT antisera (Fig. 2C and D). How-
ever, some additional PNMT-positive cells did not show FRAP activity (Fig. 2C and D). Some PNMT cells have been reported to contain enkephalin 2°,21, and we have observed FRAP in some enkephalin cells in the medulla (Fig. 2E and F); however, most enkephalin cells were not FRAP-positive (Fig. 2E and F). Erfink6 originally noted that certain cells in the
/
Fig. 2. lmmunofluorescence micrographs of the rat adrenal medulla stained with antisera raised against dopamine-fl-hydroxylase (DBH) (A), phenylethanolamine-N-methyl transferase (PNMT) (C), and methionine-enkephalin (ENK) (E). The same sections are shown in (B), (D) and (F), respectively, following histochemical staining for F R A P activity. All FRAP-positive cells are also positive for both DBH (A, B) and PNMT (C, D) (arrows); however, some cells that are positive for DBH or PNMT do not appear to contain F R A P activity (arrowheads). Some ENK-positive cells (E) are also FRAP-positive (F) (arrows), although some do not appear to contain F R A P activity (arrowheads). In addition, some FRAP-positive cells (double arrows) do not contain E N K immunoreactivity. Bar indicates 50/tm for all figures.
328 rat adrenal gland contain high acid phosphatase activityL By histochemical methods he was able to demonstrate that this enzyme activity was confined to the adrenaline cells of the medulla 6. In the present study we present evidence, from combined immunohistochemical and enzyme histochemical experiments, that the acid phosphatase in the adrenaline (PN MT-positive) chromaffin cells of the rat adrenal gland is a fluoride-resistant isoenzyme. FRAP activity appears to have a unique species specificity. We have been able to demonstrate intense F R A P activity in the adrenal gland of the rat, but not in the mouse, guinea pig or cat. In earlier studies, Er/ink6 could demonstrate intense acid phosphatase activity in the rat adrenal glandS,6; however, the enzyme could not be detected histochemically in mouse, cat, dog, guinea pig, rabbit 6, bovine 9, pig or human 3 adrenal gland. Similarly, we have found F R A P activity in the dorsal horn of the spinal cord in the rat, as have many othersV,lS, 16, 19,z2, but we could not detect this enzyme in the mouse or guinea pig. It has been previously reported that F R A P is not detectable in the dorsal horn of hamster, rabbit, cat, dog, monkey or human spinal cord x6. Thus the observations that the acid phosphatase activity in the adrenal gland and spinal cord are found only in rat, and that in both areas the enzyme is fluoride-resistant, suggest that these two tissues contain the same enzyme. The enzyme appears to have a similar subcellular localization in these two tissues as well. In the dorsal horn, F R A P is found in the cytoplasm around synaptic vesicles and mitochondria la. The acid phosphatase in the rat adrenal medulla has a similar appearance, being found in the cytoplasm around the chromaffin gra-
nules and organelles 4. The function of F R A P is not known. In sensory neurons FRAP appears to be in cells distinct from those containing somatostatin or substance p15,19 and is thus unrelated to the synthesis of these peptides. It is probably also not directly involved in adrenalin synthesis, since not all PNMT-positive cells contain FRAP. FRAP activity might be involved in the metabolism of high-energy phosphate compounds; however, we have found that adenosine triphosphate (ATP) is not a substrate in either the spinal cord or the adrenal medulla (unpublished observation). It has been suggested that the apparent species specificity found for F R A P is due to the histochemical procedure, and that a similar enzyme is in fact present in other species t6. Thus, F R A P may be involved in the synthesis or degradation of some unidentified substance important for the function of certain neurons and endocrine cells in many species. At present F R A P can serve as a useful histochemical marker for a unique population of cells. Whether other neurons and endocrine cells contain F R A P is currently under study.
1 Bloom, S. R. (Ed.), Gut Hormones, Churchill Livingstone, Edinburgh, 1978. 2 Coons, A. H., Fluorescent antibody methods In J. F. Danielli (Ed.), General Cytochemical Methods, Academic Press, New York, 1958, pp. 399422. 3 Coupland, R. E., The Natural History of the Chromaffin Cell, Longmans, London, 1965. 4 Coupland, R. E., Mastrolia, L. and Weakley, B. S., Localization of acid phosphatase in the adrenal medulla of the albino rat. In O. Er~nk6 (Ed.), Histochemistry of
adrenal medulla of the rat, Nature (Lond.), 168 (1951) 250-251. Er~nkb, O., Fluorescing islets, adrenaline and noradrenaline in tbe adrenal medulla of some common laboratory animals, Ann. Med. Exp. BioL Fennaei., 33 (1955) 278290. Gerebtzoff, M. A. and Maeda, T., Caracteres et localisation histochimique d'un isoenzyme fluororesistant de la phosphatase acide dans la moelle epiniere du rat, C.R. Soc. Biol. (Paris), 162 (1968) 2032-2035. Goldstein, M., Fuxe, K. and H6kfelt, T., Characterization and tissue localization of catecholamine synthesizing enzymes, Pharmacol. Rev., 24 (1972) 293-309. Hillarp. N.-A. and Falck, B.. Localization of acid phos-
The authors thank Ms. A. Peters and Ms. A. Edin for excellent technical assistance. S.R.V. supported by a fellowship from the M R C of Canada. This study was performed under a grant from the Swedish Medical Research Council (04X-2887:12X- 5189). We are grateful to Prof. M. Goldstein, New York University Medical Center, NY, U.S.A. and Prof. L. Terenius, Uppsala University, Uppsala, Sweden, for the generous supply of antibodies. We thank Prof. T. H6kfelt for support and helpful criticism.
6
7
Nervous Transmission, Progress in Brain Research, Vol. 34,
8
Elsevier, Amsterdam, 1971, pp. 455-464. 5 Er/inkb, O., Histochemical evidence of the presence of acid-phosphatase-positive and -negative cell islets in the
9
329 phatase in the adrenal medullary cell, Acta endocr., 22 (1956) 95-106. 10 H6kfelt, T., Elde, R., Johansson, O., Luft, R., Nilsson, G. and Arimura, A., Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat, Neuroscience, 1 (1976) 131-136. 11 H~Skfelt, T., Fuxe, K., Goldstein, M. and Joh, T. H., Immunohistochemical localization of three catecholamine synthesizing enzymes. Aspects on methodology, Histochemie, 33 (1973) 231-254. 12 H6kfelt, T., Johansson, O., Ljungdahl, A., Lundberg, J. M. and Schultzberg, M., Peptidergic neurones, Nature (Lond.), 284 (1980) 515-521. 13 H6kfelt, T., Kellerth, J.-O., Nilsson, G. and Pernow, B., Substance P: localization in the central nervous system and in some primary sensory neurons, Science, 190 (1975) 889-890. 14 H6kfelt, T., Kellerth, J.-O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Research, 100 (1975) 235-252. 15 H6kfelt, T., Vincent, S., Dalsgaard, C.-J., Skirboll, L., Johansson, O., Schultzberg, M., Lundberg, J. M., Rosell, S., Pernow, B. and Jancso, G., Substance P - - immunohistochemical aspects on its distribution in brain and periphery and its possible role as co transmitter. In Substance P in the Nervous System, Ciba Foundation Symposium.
16 Knyihar-Csillik, E. and Csillik, B., FRAP: histochemistry of the primary nociceptive neuron, Progr. Histochem. Cytochem., 14 (1981) 1-137. 17 Larson, L.-I. and Rehfeld, J. F., Localization and molecular heterogeneity of cholecystokinin in the central and peripheral nervous system, Brain Research, 165 (1979) 201-218. 18 Lundberg, J. M., H6kfelt, T., Nilsson, G., Terenius, L., Rehfeld, J., Elde, R. and Said, S., Peptide neurons in the vagus, splanchnic and sciatic nerves, Acta physioL scand., 104 (1978) 499-501. 19 Nagy, J. I. and Hunt, S. P., FRAP-containing primary sensory neurons are not identical to substance P and somatostatin neurons, Neuroscience, 7 (1982) 89-97. 20 Schultzberg, M., H6kfelt, T., Lundberg, J. M., Terenius, L., Elfvin, L.-G. and Elde, R. P., Enkephalin-like immunoreactivity in nerve terminals in sympathetic ganglia and adrenal medulla and in adrenal medullary gland cells, Acta physiol, scand., 103 (1978) 475~,77. 21 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) 11691186. 22 Sz6nyi, G., Knyihar, E. and Csillik, B., Extra-lysosomal, fluoride-resistant acid phosphatase-active neuronal system subserving nociception in the rat cornea, Z. mikrosk.-anat. Forsch., 93 (1979) 974-981.
325
Elsevier Biomedical Press
Fluoride-resistant acid phosphatase in the rat adrenal gland S. R. VINCENT*, M. SCHULTZBERG and C.-J. DALSGAARD Departments of Histology and ( C.-J.D.) Anatomy, Karolinska lnstitutet, S-104 01 Stockholm (Sweden)
(Accepted August 10th, 1982) Key words: fluoride-resistant acid phosphatase (FRAP) - - adrenal gland - - chromaffin cell - -
adrenalin - - enkephalin - - immunohistochemistry
The adrenal medulla of the rat, but not of the mouse, guinea pig or cat, was shown to contain a fluoride-resistant acid phosphatase activity similar to that previously noted in rat spinal cord. By combined immunohistochemistry and FRAP histochemistry it was determined that the FRAP in the rat adrenal was confined to the adrenaline chromaffin cells. A unique fluoride-resistant acid phosphatase (FRAP) enzyme activity has been demonstrated histochemically in a population of primary afferent neurons in the ratT,15,16,19,~2. FRAP-positive fibres are found concentrated in lamina II of the spinal cord and trigeminal complex, and the small type-C cells in the trigeminal and spinal ganglia are also FRAP-reactive. These cells appear to be distinct from the sensory neurons containing somatostatin or substance pls,19 and thus represent a biochemically distinct nerve cell type, using an as yet unidentified transmitter substance. Peptides such as substance p1z,14, somatostatin~0, cholecystokinin 17 and vasoactive intestinal polypeptide (VIP) is, which have been reported in primary afferents have also been found in other nerve cell types and/or endocrine cells (see refs. 1, 12). In the present report we present histochemical evidence that F R A P activity is also contained in cell types other than primary afferents. We have found this enzyme activity in adrenaline chromaffin cells in the rat adrenal gland. Young adult male albino rats, mice and guinea pigs and an adult cat were examined. The animals were anesthetized with pentobarbital and perfused through the aorta with ice-cold calcium-free Tyrodes' solution followed by 1 0 ~ formalin in
phosphate buffer. The adrenal gland and spinal cord were removed, and immersed in the same fixative for a further 90 min. After rinsing at least 24 h in 5 sucrose in phosphate buffer the tissues were cut on a cryostat (Dittes, Heidelberg) at 14 #m. Sections of the adrenal gland and spinal cord were stained for the demonstration of F R A P activity according to Sz6nyi et al. 21 in 0 . 2 5 ~ sodium flglycerophosphate and 0.08 ~ lead nitrate in 20 m M Tris-maleate buffer, p H 5, containing 4 m M N a F 7 for 30 min at 37 °C. The sections were then rinsed in distilled water and treated with 1 ~ a m m o n i u m sulfide for 15 s, rinsed in water and mounted in glycerol-phosphate-buffered saline (PBS) (3:1). Some sections were stained immunohistochemically prior to histochemical staining for F R A P activity. In these experiments, the indirect immunofluorescence technique of Coons et al. was employed2,11. Adrenalin ceils were identified using antibodies raised against the enzyme phenylethanolamine-Nmethyl transferase (PNMT)S, 11, noradrenalin and adrenalin ceils were stained with antisera to dopamine-fl-hydroxylase (DBH)S, 11 and enkephalin immunoreactivity was detected with antisera raised against Met-enkephalin (rabbit 336) 21. The sections were incubated in the primary antisera at 4 °C overnight, then rinsed with PBS and incubated in fluo-
* To whom correspondence should be addressed at: Dept. of Physiology, University of British Columbia, Vancouver, B.C., V6T lW5 Canada. 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
326 rescene-isothiocyanate (FITC)-labelled swine antirabbit igG (diluted 1:10) (Dako, Copenhagen), for 30 min at 37 "C, rinsed again in PBS and mounted in glyceroI-PBS. All primary antisera were diluted (1:100) in PBS containing 0.3 ')i~ Triton X-100. The specimens were examined in a fluorescence microscope, and after p h o t o g r a p h y the coverslips were removed and the sections stained histochemically for F R A P activity. By this protocol the same section could be sequentially stained by immunohistochemistry and F R A P histochemistry. Control sections were incubated with non-immune sera or with antisera pretreated with an excess (10 nmol/ml diluted
antiserum) of Met-enkephalin. Within the rat spinal cord, intense F R A P activity was found in lamina I1 of the dorsal horn (Fig. IA). In contrast, the dorsal horn of the guinea pig (Fig. I B) and mouse contained no F R A P activity. When sections of the rat adrenal gland were incubated for F R A P activity, an intense reaction was observed in the medulla (Fig. IC). This reaction was present even in the presence of 100 mM NaF, indicating that this acid phosphatase was resistant to fluoride inhibition. The medulla o f the guinea pig (Fig. 1D), mouse and cat showed practically no F R A P activity.
Fig. 1. Micrographs of the spinal cord and adrenal gland of the rat (A, C) and guinea pig (B, D) stained for FRAP activity. Intense FRAP activity is found in the substantia gelatinosa of the rat dorsal horn (A), but the enzyme activity is not detected in the guinea pig (B). Similarly, many cells of the rat adrenal medulla (arrow) show intense FRAP activity (C), while the guinea pig adrenal medulla (arrow) is unstained, Bar indicates 50/*m for all figures.
327 In an attempt to determine which cell types in the rat adrenal gland contain FRAP activity, sections were first stained immunohistochemically, and then processed for FRAP. By this procedure it was found that only some of the DBH-positive cells contained F R A P (Fig. 2A and B). These were apparently the adrenalin cells since all FRAP-positive cells were stained with PNMT antisera (Fig. 2C and D). How-
ever, some additional PNMT-positive cells did not show FRAP activity (Fig. 2C and D). Some PNMT cells have been reported to contain enkephalin 2°,21, and we have observed FRAP in some enkephalin cells in the medulla (Fig. 2E and F); however, most enkephalin cells were not FRAP-positive (Fig. 2E and F). Erfink6 originally noted that certain cells in the
/
Fig. 2. lmmunofluorescence micrographs of the rat adrenal medulla stained with antisera raised against dopamine-fl-hydroxylase (DBH) (A), phenylethanolamine-N-methyl transferase (PNMT) (C), and methionine-enkephalin (ENK) (E). The same sections are shown in (B), (D) and (F), respectively, following histochemical staining for F R A P activity. All FRAP-positive cells are also positive for both DBH (A, B) and PNMT (C, D) (arrows); however, some cells that are positive for DBH or PNMT do not appear to contain F R A P activity (arrowheads). Some ENK-positive cells (E) are also FRAP-positive (F) (arrows), although some do not appear to contain F R A P activity (arrowheads). In addition, some FRAP-positive cells (double arrows) do not contain E N K immunoreactivity. Bar indicates 50/tm for all figures.
328 rat adrenal gland contain high acid phosphatase activityL By histochemical methods he was able to demonstrate that this enzyme activity was confined to the adrenaline cells of the medulla 6. In the present study we present evidence, from combined immunohistochemical and enzyme histochemical experiments, that the acid phosphatase in the adrenaline (PN MT-positive) chromaffin cells of the rat adrenal gland is a fluoride-resistant isoenzyme. FRAP activity appears to have a unique species specificity. We have been able to demonstrate intense F R A P activity in the adrenal gland of the rat, but not in the mouse, guinea pig or cat. In earlier studies, Er/ink6 could demonstrate intense acid phosphatase activity in the rat adrenal glandS,6; however, the enzyme could not be detected histochemically in mouse, cat, dog, guinea pig, rabbit 6, bovine 9, pig or human 3 adrenal gland. Similarly, we have found F R A P activity in the dorsal horn of the spinal cord in the rat, as have many othersV,lS, 16, 19,z2, but we could not detect this enzyme in the mouse or guinea pig. It has been previously reported that F R A P is not detectable in the dorsal horn of hamster, rabbit, cat, dog, monkey or human spinal cord x6. Thus the observations that the acid phosphatase activity in the adrenal gland and spinal cord are found only in rat, and that in both areas the enzyme is fluoride-resistant, suggest that these two tissues contain the same enzyme. The enzyme appears to have a similar subcellular localization in these two tissues as well. In the dorsal horn, F R A P is found in the cytoplasm around synaptic vesicles and mitochondria la. The acid phosphatase in the rat adrenal medulla has a similar appearance, being found in the cytoplasm around the chromaffin gra-
nules and organelles 4. The function of F R A P is not known. In sensory neurons FRAP appears to be in cells distinct from those containing somatostatin or substance p15,19 and is thus unrelated to the synthesis of these peptides. It is probably also not directly involved in adrenalin synthesis, since not all PNMT-positive cells contain FRAP. FRAP activity might be involved in the metabolism of high-energy phosphate compounds; however, we have found that adenosine triphosphate (ATP) is not a substrate in either the spinal cord or the adrenal medulla (unpublished observation). It has been suggested that the apparent species specificity found for F R A P is due to the histochemical procedure, and that a similar enzyme is in fact present in other species t6. Thus, F R A P may be involved in the synthesis or degradation of some unidentified substance important for the function of certain neurons and endocrine cells in many species. At present F R A P can serve as a useful histochemical marker for a unique population of cells. Whether other neurons and endocrine cells contain F R A P is currently under study.
1 Bloom, S. R. (Ed.), Gut Hormones, Churchill Livingstone, Edinburgh, 1978. 2 Coons, A. H., Fluorescent antibody methods In J. F. Danielli (Ed.), General Cytochemical Methods, Academic Press, New York, 1958, pp. 399422. 3 Coupland, R. E., The Natural History of the Chromaffin Cell, Longmans, London, 1965. 4 Coupland, R. E., Mastrolia, L. and Weakley, B. S., Localization of acid phosphatase in the adrenal medulla of the albino rat. In O. Er~nk6 (Ed.), Histochemistry of
adrenal medulla of the rat, Nature (Lond.), 168 (1951) 250-251. Er~nkb, O., Fluorescing islets, adrenaline and noradrenaline in tbe adrenal medulla of some common laboratory animals, Ann. Med. Exp. BioL Fennaei., 33 (1955) 278290. Gerebtzoff, M. A. and Maeda, T., Caracteres et localisation histochimique d'un isoenzyme fluororesistant de la phosphatase acide dans la moelle epiniere du rat, C.R. Soc. Biol. (Paris), 162 (1968) 2032-2035. Goldstein, M., Fuxe, K. and H6kfelt, T., Characterization and tissue localization of catecholamine synthesizing enzymes, Pharmacol. Rev., 24 (1972) 293-309. Hillarp. N.-A. and Falck, B.. Localization of acid phos-
The authors thank Ms. A. Peters and Ms. A. Edin for excellent technical assistance. S.R.V. supported by a fellowship from the M R C of Canada. This study was performed under a grant from the Swedish Medical Research Council (04X-2887:12X- 5189). We are grateful to Prof. M. Goldstein, New York University Medical Center, NY, U.S.A. and Prof. L. Terenius, Uppsala University, Uppsala, Sweden, for the generous supply of antibodies. We thank Prof. T. H6kfelt for support and helpful criticism.
6
7
Nervous Transmission, Progress in Brain Research, Vol. 34,
8
Elsevier, Amsterdam, 1971, pp. 455-464. 5 Er/inkb, O., Histochemical evidence of the presence of acid-phosphatase-positive and -negative cell islets in the
9
329 phatase in the adrenal medullary cell, Acta endocr., 22 (1956) 95-106. 10 H6kfelt, T., Elde, R., Johansson, O., Luft, R., Nilsson, G. and Arimura, A., Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat, Neuroscience, 1 (1976) 131-136. 11 H~Skfelt, T., Fuxe, K., Goldstein, M. and Joh, T. H., Immunohistochemical localization of three catecholamine synthesizing enzymes. Aspects on methodology, Histochemie, 33 (1973) 231-254. 12 H6kfelt, T., Johansson, O., Ljungdahl, A., Lundberg, J. M. and Schultzberg, M., Peptidergic neurones, Nature (Lond.), 284 (1980) 515-521. 13 H6kfelt, T., Kellerth, J.-O., Nilsson, G. and Pernow, B., Substance P: localization in the central nervous system and in some primary sensory neurons, Science, 190 (1975) 889-890. 14 H6kfelt, T., Kellerth, J.-O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Research, 100 (1975) 235-252. 15 H6kfelt, T., Vincent, S., Dalsgaard, C.-J., Skirboll, L., Johansson, O., Schultzberg, M., Lundberg, J. M., Rosell, S., Pernow, B. and Jancso, G., Substance P - - immunohistochemical aspects on its distribution in brain and periphery and its possible role as co transmitter. In Substance P in the Nervous System, Ciba Foundation Symposium.
16 Knyihar-Csillik, E. and Csillik, B., FRAP: histochemistry of the primary nociceptive neuron, Progr. Histochem. Cytochem., 14 (1981) 1-137. 17 Larson, L.-I. and Rehfeld, J. F., Localization and molecular heterogeneity of cholecystokinin in the central and peripheral nervous system, Brain Research, 165 (1979) 201-218. 18 Lundberg, J. M., H6kfelt, T., Nilsson, G., Terenius, L., Rehfeld, J., Elde, R. and Said, S., Peptide neurons in the vagus, splanchnic and sciatic nerves, Acta physioL scand., 104 (1978) 499-501. 19 Nagy, J. I. and Hunt, S. P., FRAP-containing primary sensory neurons are not identical to substance P and somatostatin neurons, Neuroscience, 7 (1982) 89-97. 20 Schultzberg, M., H6kfelt, T., Lundberg, J. M., Terenius, L., Elfvin, L.-G. and Elde, R. P., Enkephalin-like immunoreactivity in nerve terminals in sympathetic ganglia and adrenal medulla and in adrenal medullary gland cells, Acta physiol, scand., 103 (1978) 475~,77. 21 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) 11691186. 22 Sz6nyi, G., Knyihar, E. and Csillik, B., Extra-lysosomal, fluoride-resistant acid phosphatase-active neuronal system subserving nociception in the rat cornea, Z. mikrosk.-anat. Forsch., 93 (1979) 974-981.