In 1963 moved to the United States on a one-year research fellowship of the Deutsche Forschungsgemeinschaft in the Department of Pharmacology (with L.J. Roth) at the University of Chicago to develop a method for the localization of drugs in the brain. Extended research with completion of a Ph.D. in Pharmacology (1967). Continued at the University of Chicago as Assistant Professor in Pharmacology (1967-1970).
From 1970 to 1995 at the University of North Carolina Chapel Hill as Professor of Cell Biology and Pharmacology, with appointments in the Department of Anatomy (Cell Biology and Anatomy) and in the Department of Pharmacology. Member of the Laboratories for Reproductive Biology (with H.S. Bennett) and Neurobiology. Teaching in Histology and Neuroendocrinology. Research on sites and mechanisms of action of estrogens, androgens, adrenal steroids, thyroid hormone, others, with focus on brain, spinal cord, and female and male reproductive organs. Providing brain maps of target neurons and circuits for estradiol, progestin, androgen, corticosterone, aldosterone, ecdysteroid, vitamin D, 2-deoxyglucose. From the applications of receptor microautoradiography and related follow-up many discoveries and new concepts resulted.
Between 1992 to 1995 part-time leave from the university, working as in-house research advisor at Chugai Pharmaceutical Company in Tokyo, Japan, for the study of vitamin D analogues.
Development of methods and applications
Localization of drugs and other diffusible compounds with dry-mount and thaw-mount autoradiography
For the in vivo cellular and subcellular localization of non-covalently bound drugs (diffusible compounds) no method existed. Development of a suitable histochemical method was considered very difficult (C.P. Leblond), even impossible (H. Levi). Adequate preservation of tissue structure and simulataneous retention of diffusible constituents and drugs posed seemingly insurmountable problems.
As a clinical neurologist and psychiatrist, being confronted with severe side effects of newly introduced psychotropic drugs, I recognized the need for better understanding sites and related mechanisms of action. I abandoned work at the bedside in order to focus on research of drug receptor binding and deposition.
In association with Lloyd Roth at the University of Chicago, during a three-year period, I developed two approaches: dry-mount and thaw-mount autoradiography that excluded any fluid phase during tissue preparation, preserving tissue structure for cellular resolution, and retaining tissue constituents at original in vivo sites. Several technical innovations and breakthroughs provided the foundation, such as, simultaneous freeze-mounting on tissue holders, avoiding disruptive ice crystal formation and translocation, cutting thin frozen sections at low temperatures previously considered impossible. Common multi-step dehydration-infiltration with liquid fixation and embedding, considered necessary by histologists, were eliminated. A brief single-step staining replaced the common multistep H&E staining. Tissue treatment was kept minimal with the goal to study ‘unmolested tissue' in order to gain authentic in vivo information.
Freezing of tissue
Development of procedure to prevent or minimize ice crystal disruption of tissue structure, considering tissue size, temperature and speed of freezing in experiments with liquified propane, isopentane, liquid nitrogen, or dry ice. For details see monograph (Stumpf, Drug Localization in Tissues and Cells).
Frozen sectioning at low and ultra-low temperatures
First demonstration of feasibility of frozen sectioning at temperatures below-45° C, as low as -105° C. Recognition of relationship between temperature and section thickness (Nature 1965), providing a basis for the development of ultra-cryotomy (acknowledged by U. Fernandez-Moran). Frozen sectioning below -45° C has been previously considered impossible (A.G.E. Pearse, 1963, 1968).
Freeze drying of frozen sections
First demonstration of freeze-dried frozen sections suitable for microscopy. Development of conditions for freezing and freeze-drying (Stumpf and Roth 1966). Design of a portable Cryosorption-Pump. Freeze-dried sections have been used for biochemical studies (O. Lowry) and whole body autoradiography (S.Ullberg), however, without preserving microscopic structure.
Dry-mounting on microscopic slides of freeze-dried frozen section – without exposure to fluids or moisture – was considered essential in order to exclude loss or translocation of any tissue constituents. Dry-mounted freeze-dried frozen sections served as a basis for authentic (representative in vivo) microscopic localization of drugs and as a control for less pristine.approaches. Diffusible compounds known localized served as test substances, including estradiol in the uterus and mesobilirubinogen in the liver (Stumpf and Roth, 1964-1967). In addition, the “extracellular space indicator” inulin was tested and found not exclusively restricted to extracellular space.
Thaw-mounting of frozen sections
Results obtained with freeze-dried sections were compared with those from thaw-mounted sections and found similar if sections 4 micrometer thin or less used, thus possible artifacts due to interaction of tissue components with nuclear emulsion avoided or minimized. Large scale use of the thaw-mount procedure during several decades established its utility as a standard approach designated as Receptor Microscopic Autoradiography. Cutting and thaw-mounting of thin frozen sections are delicate steps that require skill and experience. Attention to detail is required in the execution of all steps of any method of high sensitivity and resolution.
Receptor microautoradiography can be combined with other histochemical techniques. For instance: combination of high resolution autoradiography with fluorescence microscopy (e.g., 3H-estradiol and catecholamines) or immunocytochemistry (e.g., 3H-vitamin D and antibodies to receptor protein; thyrotropin, insulin) for characterization of receptor binding sites.
Receptor Microscopic Autoradiography presently is the method of choice for the localization (high-resolution imaging) of drugs at the cellular-subcellular and tissue-organ level. Discoveries and changes of paradigm resulted from information gained that is difficult or impossible to obtain otherwise. Important leads for new drug development have been provided.
Because of time and effort required, some consider such methods non-expedient. However, evidence indicates, results with high-resolution histochemical methods may turn out highly informative, essential, time and money saving, compared to quick “expedient” procedures alone that often turn out as costly failures.
Discoveries and new concepts
With receptor microscopic autoradiography – as the method of thaw-mount autoradiography with 4 micrometer sections has been named – many discoveries were made with radiolabeled steroid hormones, drugs, and metabolic indicators. New targets were identified, characterized by their topographic location and through combined autoradiography-immunocytochemistry with radiolabeled compounds and antibodies to cellular products. Detailed target maps of brain, spinal cord and other organs provided. Discoveries gave rise to new concepts, e.g.:
Applications that require high resolution and high sensitivity
Receptor Microautoradiography currently is the general method of choice, providing high sensitivity and high resolution with cellular-subcellular information simultaneously with tissue and organ overviews.
Regulatory agencies and pharmaceutical companies are challenged to seek information on target identification and target pharmacokinetics for the approval of new drugs. Such information is essential for the understanding of mechanisms of action (see article: Memo to the FDA and ICH, 2007).
Reviews by others
Roth LJ and Stumpf WE, eds. AUTORADIOGRAPHY OF DIFFUSIBLE SUBSTANCES. 371 pp, Academic Press, New York, 1969.
Stumpf WE and Grant LD, eds. ANATOMICAL NEUROENDOCRINOLOG,
472 pp, S. Karger, Basel, 1975.
Stumpf WE and Solomon H, eds. AUTORADIOGRAPHY AND CORRELATIVE IMAGING. Academic Press, San Diego, 1995.
Stumpf WE. DRUG
LOCALIZATION IN TISSUES AND CELLS. IDDC Press, Chapel Hill, NC,
300 + (Medline recorded), 550 + (total)
StumpfWE. “Vitamin D: Beyond Bone.” Address to Conference. New York Academy of Science, September 21, 2012.
StumpfWE. Vitamin D and the scientific calcium dogma: understanding the ‘Panacea’ of the sun. Editorial. Europ. J. Clinic. Nutr., 2012; 66: 1080-1081.
StumpfWE. Vitamin D in Relations to the Dao – Life, Death, and Return. Metaphysics of the one- and two- phasic actions toward maintenance of life (Yang Sheng) and Cyclicity (Huan[Yuan]-Tao[Dao] ‘Go-around’ des Lueshih chunqiu (Spring and Autumn Analects).
StumpfWE. Vitamin D Beziehungen zur Philosophie des Dao – Leben, Tod und Wiederkehr. Zur Metaphysik der ein- und zweiphasischen Aktionen zur Erhaltung des Lebens (Yang Sheng) und Zyklizitaet (Huan[Yuan] –Tao[Dao] ‘Go-around’ [circle] des Lueshi chunqiu (Fruehling und Herbst Analekte).
StumpfWE. Drugs in the brain – cellular imaging with receptor microscopic autoradiography. Progress in Histochemistry and Cytochemistry 47 (2012) 1–26.
Stumpf WE. In vivo target recognition with high-resolution imaging: significance for drug development. Eur J Drug Metab Pharmacokinet. 2010 Sep;35(1-2):15-22.
Stumpf WE., Hayakawa, N., Bidmon, H-J. Skin research and drug localization with receptor
Stumpf WE., Hayakawa, N. Salivary glands epithelial and myoepithelial cells are major vitamin D targets. European Journal of Drug Metabolism and Pharmacokinetics 2007, Vol. 32, No. 3, pp. 123-129.
Stumpf WE. Memo to the FDA and ICH: appeal for in vivo drug target identification and target pharmacokinetics Recommendations for improved procedures and requirements. Drug Discov Today. 2007 Aug;12(15-16):594-8.
Stumpf WE. Editorial. The main role of vitamin D: seasonal regulation of vital functions. High-resolution target recognition leads to a new paradigm and advanced drug development. Eur J Drug Metab Pharmacokinet. 2007 Jan-Mar;32(1):1-6.
Stumpf WE. Drug
localization and targeting with receptor microscopic autoradiography.
Hayakawa N, Kubota N, Imai N, Stumpf WE. Receptor microscopic autoradiography for the study of percutaneous absorption, in vivo skin penetration, and cellular-intercellular deposition. J Pharmacol Toxicol Methods. 2004 Sep-Oct;50(2):131-7.
Stumpf WE. Corpora
non agunt nisi in loco. Interactions between things do not "take place"
unless at "proper locus".
Stumpf WE. Vitamin
D sites and mechanisms of action: a histochemical perspective.
Reflections on the utility of autoradiography and cytopharmacology for
drug targeting. Histochem Cell Biol. 1995 Dec;104(6):417-27.
Stumpf WE, Bidmon HJ, Li L, Pilgrim C, Bartke A, Mayerhofer A, Heiss C. Nuclear receptor sites for vitamin D-soltriol in midbrain and hindbrain of Siberian hamster (Phodopus sungorus) assessed by autoradiography. Histochemistry 1992 98:155-164.
Stumpf WE, Bidmon HJ, Murakami R. Retinoic acid binding sites in adult brain, pituitary, and retinaNaturwissenschaften. 1991 Dec;78(12):561-2.
Stumpf WE, Privette TH. The steroid hormone of sunlight soltriol (vitamin D) as a seasonal regulator of biological activities and photoperiodic rhythms. J Steroid Biochem Molec Biol 1991 39(2):283-289.
Bidmon HJ, Gutkowska J, Murakami R, Stumpf WE. Vitamin D receptors in heart: effects on atrial natriuretic factor. Experientia. 1991 Sep 15;47(9):958-62.
Duncan GE, Kaldas RG, Mitra KE, Breese GR, Stumpf WE. High activity neurons in the reticular formation of the medulla oblongata: a high-resolution autoradiographic 2-deoxyglucose study. Neuroscience. 1990;35(3):593-600.
Stumpf WE, Privette TH. Light, vitamin D and psychiatry. Role of 1,25 dihydroxyvitamin D3 (soltriol) in etiology and therapy of seasonal affective disorder and other mental processes. Psychopharmacology (Berl). 1989;97(3):285-94.
Stumpf WE. Vitamin D-Soltriol. The heliogenic steroid hormone: somatotrophic activator and modulator. Discoveries from histochemical studies lead to new concepts. 89:209-219, 1988
Shrughrue PJ, Stumpf WE and Sar M. The distribution of progesterone receptor in the 20-day old fetal mouse: An autoradiographic study with [125I] progestin. Endocrinol. 123:2382-2389, 1988.
Stumpf WE and O'Brien LP. 1,25 (OH)2 Vitamin D3 sites of action in the brain: an autoradiographic study. Histochem.87:393-406, 1987.
Stumpf WE, Clark, SA, O'Brien, LP and Reid, FA. 1,25 (OH)2 vitamin D3
Stumpf WE, Jennes, L. The A-B-C (Allocortex-Brainstem-Core) Circuitry of Endocrine-Autonomic Integration and Regulation. Peptides. Vol. 5, Suppl 1, pp 221-226, 1984.
Stumpf WE, Jennes, L. The A-B-C (Allocortex-Brainstem-Core) Circuitry - schematic
Stumpf WE, Sar M, Narbaitz R, Huang S, DeLuca HF. Autoradiographic localization of 1,25-dihydroxyvitamin D3 in rat placenta and yolk sac. Horm Res. 1983;18(4):215-20.
Heritage AS, Stumpf WE, Sar M, Grant LD. Brainstem catecholamine neurons are target sites for sex steroid hormones. Science. 1980 Mar 21;207(4437):1377-9.
Stumpf WE. "Peer"
Stumpf WE, Sar M, Reid FA, Tanaka Y, DeLuca HF. Target cells for 1,25-dihydroxyvitamin D3 in intestinal tract, stomach, kidney, skin, pituitary, and parathyroid. Science. 1979 Dec 7;206(4423):1188-90.
Sar, M and Stumpf, WE. Neurons of the hypothalamus concentrate3H
progesterone or metabolites of it. Science. 182:1266 1268, 1973.
Examples of autoradiograms prepared by Receptor Microscopic Autoradiography after injection of tritium-labeled vitamin D. The same preparation yields low-resolution surveys and high-resolution cellular-subcellular detail with quantitative differences of uptake and retention of radiolabeled compound. Among the over fifty target tissues identified and characterized by autoradiography a hierarchy of uptake and retention is recognizable. (For details see Stumpf: Drug Localization in Tissues and Cells, 2003; Drug localization and targeting with receptor microscopic autoradiography, 2005.)
Brain amygdala central nucleus (upper picture) and spinal cord
lamina IX (lower picture) with labeled target neurons. Note the
differential nuclear concentration of radio-labeled compound. Most but
not all motor neurons are labeled.
Pituitary target cell population survey (upper picture). Colocalization with TSH-antibodies (brown cytoplasm in lower picture) characterizes many of the heavily labeled target cells as thyrotropes.
Duodenum with strong nuclear concentration of radiolabeled compound in absorptive epithelial cells – but not in Goblet cells (upper picture). In the low-magnification survey (lower picture) labeling exists in the nuclear regions of epithelium of intestinal villi and crypts but not in the muscularis. High level of radioactivity in the intestinal lumen - probably mostly liver-bile derived metabolites - with barrier between luminal content and villi.
Adrenal with radioactive labeling in medullary cells and blood
vessels, recognizable at low magnification (upper picture). At high
magnification (lower picture) nuclear concentration in medullary cells
can be clearly identified. Also relatively high radioactivity in
capillary sinusoids. In cortex zona reticularis (dark cells at left) no
nuclear concentration of labeled compound exists under the conditions of the
Target identification derived from the application of receptor microautoradiography allows composition of a “drug homunculus” for drug-specific finger-printing, overviews, links for functional detail, facilitating functional and clinical follow-up, prediction of actions, side-effects, and toxicity.
The results thus obtained and insights gained suggest a change of concept. Although systemic calcium homeostasis is an important function of vitamin D, calcium binding proteins cannot be viewed as general guide to vitamin D sites and mechanisms of actions – as indicated in our comparative studies. Our data suggest:
These include regulation of growth, reproduction, survival stress response; endocrine and exocrine secretion, cell proliferation, cognition and mood; neuro-motor, neuro-endocrine, and neuro-sensory functions, immune response, cardio-vascular and gastro-intestinal functions, regulation of calcium and other mineral levels, cell proliferation and protein synthesis-differentiation, others.
Comparative data with different steroid hormones and target-overlap further suggest interactive (cooperative and/or antagonistic) regulation of DNA by steroid messengers (Stumpf, 1995):
From the microautoradiographic target recognition and related actions it follows that vitamin D has healing potential for prevention and treatment of various deficiencies and ailments, including old age: a PANACEA? If there is any compound that deserves being designated a panacea, the multifunctional heliogenic vitamin D appears a suitable candidate.
Philosophical consideration: “Vitamin D”, the term does not reflect its significance. I have used instead SOLTRIOL in several publications as a more appropriate designation. – Is there not a link to HERACLITUS' emanation of “ ever-living fire ”? The cosmic solar fire as the sustaining life force, providing wave length energies for Temperature , Visible Light , and Ultraviolet B (Soltriol).
Fec. April 2005
Updated November 2012