, Volume 83, Issue 1, pp 41–46 | Cite as

Ultrastructural immunocytochemical co-localization of serotonin and PNMT in adrenal medullary vesicles

  • M. S. Brownfield
  • B. C. Poff
  • M. A. Holzwarth


Previous immunocytochemical studies at the light microscopic level have demonstrated serotonin immunoreactivity in rat adrenal epinephrine-containing cells. In this study we have used electron microscopic immunocytochemical methods to study the subcellular distribution of serotonin and the enzyme responsible for epinephrine biosynthesis, phenylethanolamine-N-methyltransferase (PNMT). The distribution of the immunostaining was compared in adjacent serial thin sections using a post-embedding method in conjunction with peroxidase-antiperoxidase (PAP) immunocytochemistry. Serotonin immunoreactivity was associated with the limiting membrane as well as with the core of the chromaffin vesicles. In adjacent sections PNMT immunoreactivity was also seen in the serotonin-containing vesicles. However, its intravesicular distribution was different from that of serotonin; PNMT occupied the eccentric zone of the vesicles between the serotonin immunoreactive sites.

These results are interpreted to be in support of biochemical studies claiming a serotonin uptake and storage capacity of adrenal chromaffin vesicle fractions as well as those which suggest serotonin is synthesized by chromaffin cells. The relative contribution of uptake and synthesis to the pool of serotonin that is stored in the vesicles is an open question. The co-localization of serotonin and PNMT in the same vesicle is suggestive of a capacity for co-release of serotonin and epinephrine by the adrenal medulla.


Serotonin Epinephrine Microscopic Level Chromaffin Cell Subcellular Distribution 
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  1. Brusco A, Peressini S, Saavedra JP (1983) Serotonin-like immunoreactivity and anti-5-hydroxytryptamine (5HT) antibodies. J Histochem Cytochem 31:524–530Google Scholar
  2. Carlsson A, Hillarp N-A, Waldeck P (1963) Analysis of the Mg-ATP dependent storage mechanism in the amine granules of the adrenal medulla. Acta Physiol Scand 59:(Suppl 215):1–38Google Scholar
  3. Coupland RE, Hopwood D (1966) The mechanism of the differential staining reaction for adrenaline and noradrenaline-storing granules in tissues fixed in glutaraldehyde. J Anat 100:227–243Google Scholar
  4. De May J (1983) Immunogold probes in immunocytochemistry. In: Polak JM, Van Noorden S (eds) Immunocytochemistry, Wright, Boston, pp 82–112Google Scholar
  5. Farnum CE, Wilsman N (1984) Lectin-binding histochemistry of non decalicified growth plate cartilage: a post-embedding method for light microscopy of epon-embedded tissue. J Histochem Cytochem 32:593–607Google Scholar
  6. Gershon MD, Ross LL (1966a) Radioisotope studies of the binding exchange, and distribution of 5-hydroxytryptamine synthesized form its radioactive precursor. J Physiol 186:451–476Google Scholar
  7. Gershon MD, Ross LL (1966b) Location of sites of 5-hydroxytryptamine storage and metabolism by radioautography. J Physiol 186:477–492Google Scholar
  8. Holzwarth MA, Brownfield MS (1985) Serotonin coexists with epinephrine in rat adrenal medulla. Neuroendocrinology (in press)Google Scholar
  9. Holzwarth MA, Sawetawan C, Brownfield MS (1984) Serotonin-immunoreactivity in the adrenal medulla: Distribution and response to pharmacological manipulation. Brain Res Bull 13:299–308Google Scholar
  10. Huang WM, Gibson SJ, Facer P, Gu J, Polak JM (1983) Improved section adhesion for immunocytochemistry using high molecular weight polymers of l-lysine as a slide coating. Histochemistry 77:275–279Google Scholar
  11. Kent C Coupland RE (1984) On the uptake and storage of 5-hydroxy-tryptamine, 5-hydroxytryptophan and catecholamines by adrenal chromaffin cells and nerve endings. Cell Tissue Res 236:189–195Google Scholar
  12. Nagatsu I, Karasawa N, Kondo Y, Inagaki S (1979) Immunocytochemical localization of tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase in the adrenal glands of frog and rat by a peroxidase-antiperoxidase method. Histochemistry 64:131–144Google Scholar
  13. Ritzen M, Hammarstrom L, Ullbery S (1965) Autoradiographic distribution of 5-hydroxytryptamine and 5-hydroxytryptophan in the mouse. Biochem Pharmacol 14:313–332Google Scholar
  14. Schipper J, Tilders FJH (1983) A new technique for studying specificity of immunocytochemical procedures. J Histochem Cytochem 31:12–18Google Scholar
  15. Slotkin TA, Kirschner N (1971) Uptake, storage and distribution distribution of amines in bovine adrenal medullary vesicles. Mol Pharmacol 7:581–592Google Scholar
  16. Snyder SH, Axelrod J, Zweig M (1965) A sensitive and specific fluorescence assay for tissue preparation. Biochem Pharmacol 14:831–835Google Scholar
  17. Steinbusch HWM, Verhofstad AAJ, Joosten HWJ (1978) Localization of serotonin in the central nervous system by immunohistochemistry: description of a specific and sensitive technique and some applications. Neuroscience 3:811–819Google Scholar
  18. Sternberger LA (1979) Immunocytochemistry. 2 Edn. John Wiley & Sons, New YorkGoogle Scholar
  19. Ungar A, Phillips JH (1983) Regulation of the adrenal medulla. Physiol Rev 63:787–843Google Scholar
  20. Van de Kar LD, Wilkinson CW, Ganong WF (1981) Pharmacological evidence for a role of brain serotonin in the maintenance of plasma renin activity unanaesthetized rats. J Pharmacol Exp Ther 219:85–90Google Scholar
  21. Van Orden LS, III, Burke JP, Redick JA, Rybarczyk KE, Van Orden DE, Baker HA, Hartman BK (1977) Immunocytochemical evidence for particulate localization of phenylethanolamine-N-methyltransferase in adrenal medulla. Neuropharmacology 16:129–133Google Scholar
  22. Verhofstad AAJ, Jonsson G (1983) Immunohistochemical and neurochemical evidence for the presence of serotonin in the adrenal medulla of the rat. Neuroscience 10:1443–1453Google Scholar
  23. Winkler H (1977) The biogenesis of adrenal chromaffin granules. Neuroscience 2:657–683Google Scholar
  24. Winkler H, Westhead E (1980) The molecular organization of adrenal chromaffin granules. Neuroscience 5:1803–1823Google Scholar

Copyright information

© Springer-Verlag 1985

Authors and Affiliations

  • M. S. Brownfield
    • 1
  • B. C. Poff
    • 1
  • M. A. Holzwarth
    • 2
  1. 1.Department of Structural and Functional Sciences, School of Veterinary MedicineUniversity of WisconsinMadisonUSA
  2. 2.Department of Anatomical SciencesUniversity of IllinoisUrbanaUSA

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