Estradiol triggers key biological responses in the endometrium, which rely on the presence and levels of its cognate receptors on target cells. Employing the receptor micro-autoradiography (RMAR) technique, we aimed to provide a temporal and spatial map of the functional binding sites for estradiol in the mouse endometrial stroma during early pregnancy. Uterine samples from days 1.5 to 7.5 of pregnancy were collected 1 h after tritiated- (3H-) estradiol administration and prepared for RMAR analysis. Autoradiographic incorporation of 3H-thymidine (after 1-h pulse) was evaluated over the same gestational interval. Combined RMAR with either histochemistry with Dolichus biflorus (DBA) lectin or immunohistochemistry for detection of the desmin further characterized 3H-estradiol binding pattern in uterine Natural Killer (uNK) and decidual cells, respectively. 3H-estradiol binding levels oscillated in the pregnant endometrial stroma between the mesometrial and antimesometrial regions as well as the superficial and deep domains. Although most of the endometrial stromal cells retained the hormone, a sub-population of them, as well as endothelial and uNK cells, were unable to do so. Rises in the levels of 3H-estradiol binding preceded endometrial stromal cell proliferation. 3H-estradiol binding and 3H-thymidine incorporation progressively decreased along the development of the antimesometrial decidua. Endothelial proliferation occurred regardless of 3H-estradiol binding, whereas pericytes proliferation was associated with high levels of hormone binding. Endometrial cell populations autonomously control their levels of 3H-estradiol binding and retention, a process associated with their proliferative competence. Collectively, our results illustrate the intricate regulatory dynamic of nuclear estrogen receptors in the pregnant mouse endometrium.

For cell and tissue localization of drugs, receptor microscopic autoradiography is reviewed, including its development history, multiple testing, extensive applications and significant discoveries. This sensitive high-resolution imaging method is based on the use of radiolabeled compounds (esp. tagged with 3H or 125I), preservation through freezing of in vivo localization of tissue constituents, cutting thin frozen sections, and close contact with the recording nuclear emulsion. After extensive testing of the utility of this method, the distribution of radiolabeled compounds has been identified and characterized for estradiol, progestagens, adrenal steroids, thyroid hormone, ecdysteroids, vitamin D, retinoic acid, metabolic indicators glucose and 2-deoxyglucose, as well as extracellular space indicators. Target cells and associated tissues have been characterized with special stains, fluorescing compounds, or combined autoradiography-immunocytochemistry with antibodies to dopamine-beta-hydroxylase, GABA, enkephalin, specific receptor proteins, or other cellular products. Blood-brain barrier and brain entries via capillary endothelium, ependyma, or circumventricular recess organs have been visualized for 3H-dexamethasone, 210Pb lead, and 3H-1,25(OH)2 vitamin D3. With this histopharmacologic approach, cellular details and tissue integrative overviews can be assessed in the same preparation. As a result, information has been gained that would have been difficult or impossible otherwise. Maps of brain drug distribution have been developed and relevant target circuits have been recognized. Examples include the stria terminalis that links septal-amygdaloid-thalamic-hypothalamic structures and telencephalic limbic system components which extend as the periventricular autonomic-neuroendocrine ABC (Allocortex-Brainstem-Circuitry) system into the mid- and hindbrain. Discoveries with radiolabeled substances challenged existing paradigms, engendering new concepts and providing seminal incentives for further research toward understanding drug actions. Most notable are discoveries made during the 1980s with vitamin D in the brain together with over 50 target tissues that challenged the century-old doctrine of vitamin D's main role as ‘the calcitropic hormone’, when the new data made it apparent that the main biological function of this multifunctional sunshine hormone rather is maintenance of life and adapting vital functions to the solar environment. In the brain, vitamin D, in close relation to sex and adrenal steroids, participates in the regulation of the secretion of neuro-endocrines, such as, serotonin, dopamine, nerve growth factor, acetyl choline, with importance in prophylaxis and therapy of neuro-psychiatric disorders. Histochemical imaging with high cellular-subcellular resolution is necessary for obtaining detailed information, as this review indicates. New spectrometric methods, like MALDI-MSI, are unlikely to furnish the same information as receptor microautoradiography does, but can provide important correlative molecular information.

An observation of sleep improvement with vitamin D supplementation led to a 2 year uncontrolled trial of vitamin D supplementation in 1500 patients with neurologic complaints who also had evidence of abnormal sleep. Most patients had improvement in neurologic symptoms and sleep but only through maintaining a narrow range of 25(OH) vitamin D3 blood levels of 60-80 ng/ml. Comparisons of brain regions associated with sleep-wake regulation and vitamin D target neurons in the diencephalon and several brainstem nuclei suggest direct central effects of vitamin D on sleep. We propose the hypothesis that sleep disorders have become epidemic because of widespread vitamin D deficiency. The therapeutic effects together with the anatomic-functional correspondence warrant further investigation and consideration of vitamin D in the etiology and therapy of sleep disorders.

The concept of systemic calcium regulation as the main action of vitamin D was developed nearly 1 century ago. It is still widely accepted, despite challenging evidence against it.

In vivo target identification is basic for understanding mechanisms of drug action. Target identification requires cellular resolution. Extrapolation from blood bioavailability, low-resolution scans, radioassays, or in vitro tests regularly produce false-negatives and false-positives. Common ADME procedures disrealize organ complexities. While low-specificity high-capacity sites of deposition are easily recognized, high-specificity low-capacity receptor sites remain hidden. Serious limitations of target recognition are revealed in comparative studies with three methods: high-resolution microscopic autoradiography, radioassay, and whole-body autoradiography. With radioassays and whole-body autoradiography, many targets are simply undetectable. For example, high-resolution microscopic target information for vitamin D, gained 20-30 years ago, was widely ignored. The narrow calcium focus for this multi-target and multi-function hormone was perpetuated until recently through deficient results from conventional assays together with related expert bias. Thus, follow-up has been delayed on discoveries from the use of unconventional histopharmacology methods, pointing at important actions and therapies beyond systemic calcium regulation. High-resolution 'in vivo' target identification with associated functional characterization is useful not only for understanding mechanisms of action, but also for providing leads for innovative and successful drug development and prediction.

For the localization of drugs and related functional characterization, cellular-subcellular resolution can be achieved with radiolabelled compounds of high specific activity and receptor microscopic autoradiography, which is the method of choice for high-resolution qualitative and quantitative imaging. Detailed information together with integrative surveys can be obtained which is impossible with other methods. The history of discoveries of drug targets testifies to the utility and potential of receptor microscopic autoradiography that was designed to preserve in vivo conditions by excluding any liquid treatment during tissue preparation and to avoid translocation and loss of diffusible compounds. Examples of autoradiograms include in vivo applications of H-3-vitamin D, H-3-OCT (oxacalcitriol), H-3-oestradiol and H-3-retinoic acid. For topical applications, routes of delivery and sites of deposition and retention are demonstrated.

Target tissues of in vivo receptor binding and deposition of 1,25(OH)2 vitamin D3 and its oxygen analog OCT are reviewed in rats, mice, hamsters and zebra finch, identified with high-resolution microscopic autoradiography. Throughout the digestive system numerous sites with nuclear receptor binding of3H-1,25(OH)2 vitamin D3 and3H-OCT exist: in the oral region, epithelial cells of the oral cavity, tongue and gingiva, teeth odontoblast and ameloblast precursor pulp and stratum intermedium cells; in the parotid, submandibular and sublingual salivary glands, epithelial cells of striated ducts and granular convoluted tubules, intercalated ducts and acinar cells, as well as myoepithelial cells; in the stomach, neck mucous cells of gastric glands, endocrine cells of the antrum, and muscle cells of the pyloric sphincter; in the small and large intestine, absorptive and crypt epithelial cells; in the pancreas, predominantly islet B-cells. Perisinusoidal stellate (Ito) cells in the liver concentrate and retain variable amounts of radiolabeled compound in regions of their cytoplasm after administration of3H-1,25(OH)2 vitamin D3 and3H-25(OH) vitamin D3, probably sites of specific storage, similar to vitamin A. Submucosa in stomach and intestine also retain variable amounts of radiolabel, however unspecific with all compounds studied. In pilot studies with3H-25(OH)2 vitamin D3 and 3H-24,25(OH)2 vitamin D3, no nuclear concentration was detectable. The reviewed data for vitamin D and its oxygen analogue OCT indicate genomic effects on multiple target tissues of the digestive system that involve cell proliferation and differentiation, endo- and exocrine secretion, digestion and absorption for maintaining optimal functions, with potentials for health prophylaxis and therapies.

Nuclear receptor binding of 1,25(OH)(2)-vitamin D-3 (vitamin D) in skin keratinocytes of epidermis, hair sheaths and sebaceous glands was discovered through receptor microscopic autoradiography. Extended experiments with H-3-1,25(OH)(2)-vitamin D-3 and its analog H-3-oxacalcitriol (OCT) now demonstrate nuclear receptor binding in sweat gland epithelium of secretory coils and ducts as well as in myoepithelial cells, as studied in paws of nude mice after i.v. injection. The results suggest genomic regulation of cell proliferation and differentiation, as well as of secretory and excretory functions, indicating potential therapies for impaired secretion as in hypohidrosis of aged and diseased skin.

This memorandum is addressed to members of regulatory agencies, as well as managers of pharmaceutical companies. Pharmacokineticists and toxicologists may consider this proposal, weigh its merits, and provide input for implementation. Experience from academic research and ADME experiments during drug development has prompted this appeal for improved drug target recognition. Similar demands have been made repeatedly in the literature. Such efforts are not new, but a renewed urgency has come from comparing results obtained with methods of different resolution and sensitivity, namely high-resolution receptor microscopic autoradiography compared and viewed in parallel to conventional low-resolution 'cut-and-count' radioassays and whole body autoradiography. Conflicting results reveal astounding deficiencies of current ADME approaches. False negatives and false positives of favored 'expedient' procedures allow drugs to reach the market with misleading and inaccurate information about the total drug effect.

Receptor binding with3H-1,25(OH)2 vitamin D3 (vitamin D) and its oxygen analog3H-OCT is demonstrated in rat, hamster, and mice submandibular, sublingual and parotid glands, using receptor microautoradiography high-resolution imaging. Nuclear uptake and retention of radiolabeled compound exist strongest in epithelial cells of striated ducts, granular convoluted tubules and in myoepithelial cells throughout, scattered in epithelial cells of intercalated ducts and relatively low in cells of serous and mucous acini. Deposition and retention of radiolabeled compound is also observed in interstitial spaces. The specific nuclear localization with vita-min D and its analogue OCT, which is absent with3H-(OH) vitamin D3 and in competition with excess non-radioactive vitamin D, indicates involvement of vitamin D in the multi-hormonal regulation of salivary gland secretion, excretion, and cell proliferation. These data — together with previously recognized similar receptor binding in esophagus, gastric glands, entero-endocrine cells, pyloric muscle, and generative and absorptive epithelium of the small intestine and colon, point to the importance of vitamin D for the digestive system regulation of functions and maintenance with related therapeutic potentials.

Dose and time considerations in the development and use of a drug are important for assessing actions and side effects, as well as predictions of safety and toxicity. This article deals with epistemological aspects of dose selection by probing into the linguistic and cultural roots for the measure of medicine mediated by the medical doctor. Because toxicity is related to dose, historic and recent views suggest that less can be more. At low, medium and high dose levels, effects can differ not only quantitatively but also qualitatively. Dose-related target activation and recognition of enantiodromic thresholds between beneficial and toxic effects require elucidation of underlying events. Such studies, including hormesis and microdosing, call for extended ADME procedures with high-resolution methods in addition to the current low-resolution approaches. Improved information of drug logistics and target pharmacokinetics enables effective drug selection, dose determination and prediction. It also allows considerations of systems biology [i.e. integral (gestalt) pharmacology] exemplified by the drug homunculus, as in the case of vitamin D, that might lead to new paradigms and drug design.

This review is an argument in favor of better drug target identification. It presents the many merits and feasibilities of drug localization and target identification through the use of a suitable technique: receptor microautoradiography. Studies of drug targets and target bioavailability require methods with high resolution and sensitivity to gain information for understanding mechanisms of action, sound modeling, prediction of effects, and toxicity. For in vivo localization of drugs in tissues and cells, receptor microautoradiography was specifically designed to preserve both tissue structure and deposition of noncovalently bound diffusible compounds and to enable microscopic viewing, quantitative analysis, and characterization of target sites. This method and its applications are explained here. Pictorial and quantitative data are provided together with a discussion of identified targets that document the utility of receptor microautoradiography. For example, when applied to quantitative studies of vitamin D compounds, pharmacokinetic data of blood differed from those of target tissues and even among target tissues. Many of the target tissues discovered and characterized with receptor microautoradiography remained unrecognized with common ADME procedures, radioassay–HPLC, and whole-body autoradiography. For a visual overview of the multiple vitamin D targets, a drug homunculus has been composed. Such a drug or target homunculus may be created for any drug, dose, and time to aid in documenting and fingerprinting. Receptor microautoradiography also is a sensitive method. It can be used for the study of low-dose stimulatory actions of toxic substances to show relationships of receptor binding to dose-dependent reversal of effects, known as hormesis. In addition, a combination of autoradiography and immunocytochemistry with radiolabeled drug and antibodies to receptor or other cellular product permits further target characterization. In its own league, receptor microautoradiography provides unique information. Through greater detail and certainty, it can validate and complement less-sensitive approaches, decrease the failure rates of current ADMET predictions, and serve as a diagnostic tool and guide for biochemical, functional, and clinical follow-up in drug research and development.

Target cells for 1,25-dihydroxyvitamin D3 were demonstrated in the adrenal medulla by frozen-section autoradiography. The appearance of these target cells was age-dependent in neonatal mice. Immunocytochemical staining for phenylethanolamine-N-methyltransferase revealed that both epinephrine and non-epinephrine cells concentrate 1,25-dihydroxyvitamin D3 in their nuclei. In contrast, immunocytochemical staining for "vitamin D-dependent calcium-binding protein" (D-CaBP) demonstrated that D-CaBP immunoreactivity is localized in only a small percentage of adrenomedullary cells, in mice and rats. Comparison of PNMT and D-CaBP immunoreactivities in sequential sections showed that epinephrine-producing cells do not contain D-CaBP. These results indicate that adrenal medullary cells have receptors for 1,25-dihydroxyvitamin D3 and that 1,25-dihydroxyvitamin D3 may directly affect certain functions of these endocrine cells.

Brains and subesophageal ganglia from day 3.5 fifth stadium larvae of Manduca sexta were incubated in vitro with 4 nM tritiated ponasterone A, a 20-hydroxyecdysone analog, to determine whether uptake and specific binding of ecdysteroids occur at a cellular level. These tissues, which were taken just prior to the commitment peak in the hemolymph-ecdysteroid titer, showed saturable uptake of 3H-ponasterone A after 40–60 min of incubation. Uptake was blocked by the addition of 400 nM unlabelled ponasterone A, or of 500 nM or 1000 nM 20-hydroxyecdysone. RH 5849, a synthetic 20-hydroxyecdysone agonist with a long half-life, for which ecdysteroid receptors have low affinity, also reduced ponasterone A uptake at a concentration of 10 μM. Autoradiographs of 4 μm sections of brains revealed distinct nuclear concentrations of silver grains over cell populations in the pars intercerebralis, pars lateralis, and ventral tritocerebrum. Nuclear labelling was also found in many small cells around the mushroom bodies and the neuropil, and between the inner and outer larval optic lobes. Nuclear labelling of cells in the subesophageal ganglion was observed in the fronto-medial and lateral regions, in small cells around the neuropil, and caudally in a few large neurons. In addition to cells with nuclear labelling, both brains and ganglia at this development stage contained cells with exclusively cytoplasmic or both nuclear and cytoplasmic labelling. None of these apparent binding sites were observed in the competition experiments, suggesting that the binding is specific.

Introduction: Microscopic autoradiography with cellular resolution and preservation of in vivo conditions is potentially the method of choice to gain detailed information about sites of deposition and retention in the epidermis and of penetration to the dermis after topical application of drugs. We tested this using 3H-Maxacalcitol. Methods: Dorsal skin of adult rats was treated in vivo with ointment containing 1 or 40 μg/kg body weight of the vitamin D analogue 3H-Maxacalcitol for periods of 0.5, 2, 8, 24, 48, or 168 h. Samples of skin exposed to the ointment and control samples remote from the treatment site were excised and freeze-mounted, and 4-μm frozen sections were exposed to nuclear emulsion. Results: Two penetration routes to the dermis could be distinguished: one via epidermal cell layers and the other via hair follicles. Highest uptake and retention of radiolabeled steroid was observed in stratum corneum and in intercellular spaces of stratum granulosum. By contrast, cell boundaries and intercellular spaces in the stratum spinosum and basale contained low levels of radioactivity. Keratinocytes in these layers showed high concentration in the cytoplasm at early time intervals, when surrounding radioactivity levels were high, but high nuclear and low or no cytoplasmic concentration at late time intervals, when surrounding radioactivity levels were low. Discussion: The autoradiographic method provides detailed information on time- and dose-related distribution of radiolabeled compound at the cellular level that is not obtainable with common radioassays and biochemical procedures. A sustained concentration and retention of radiolabeled steroid in the stratum corneum and intercellular space of the stratum granulosum indicate a selective deposition in components of secreted-membrane-coating granules and suggest a temporary barrier and depot for slow release. The differential cytoplasmic–nuclear distribution in the stratum Malpighi suggests functional correlation to a toxic–hormetic reversal of action on cell proliferation, from high-dose inhibitory effects associated with high extranuclear concentration as utilized in the treatment of psoriasis, to low-dose stimulatory effects associated with high nuclear and low cytoplasmic concentration as applicable in wound healing.

The presence and changes of estradiol nuclear binding and related functions in uterine luminal and glandular epithelium were studied before and after blastocyst implantation using receptor autoradiography with 3H-estradiol-17β in association with 3H-thymidine incorporation and immunocytochemical binding of antibody to estrogen receptor ER-α. 3H-estradiol nuclear binding is present but variable during days 1.5–7.5 of pregnancy. Sites of strong nuclear binding of 3H-estradiol exhibit strong immunocytochemical staining with ER-α antibody. Qualitative and quantitative evaluation of autoradiograms reveal that there is a general increase of nuclear 3H-estradiol binding during the first 3 days after fertilization in both luminal and glandular epithelium. The binding of estradiol is stronger in glandular epithelium from day 2.5 to day 7.5, paralleled by a rise in 3H-thymidine incorporation on day 2.5. By comparison, in the epithelium of the uterine lumen 3H-estradiol nuclear binding is low, but relatively high in epithelial cells at lateral branching of the lumen where the increase in 3H-estradiol binding corresponds to an increased labeling index with 3H-thymidine. A highly differentiated binding of 3H-estradiol to luminal and glandular epithelium was demonstrated with region- and time-specific changes of related effects on cell proliferation, differentiation, and secretion, probably involving involution and remodeling. The strong 3H-estradiol binding to glandular epithelium suggests that estradiol exerts pronounced effects on glandular activities in the periimplantation period.