A sensitive bioassay for enzymes that synthesize retinoic acid

doi:10.1016/S1385-299X(96)00034-7

Brain Research Protocols

Volume 1, Issue 3, August 1997, Pages 232-236

https://doi.org/10.1016/S1385-299X(96)00034-7 Get rights and content

Abstract

Retinoic acid (RA) is a potent regulator of gene transcription and it plays a pivotal role in neural development. As the compound is active at nanomolar concentrations, standard RA detection methods based on high-pressure liquid chromatography (HPLC) are poorly suited for neurodevelopmental questions and single RA measurements require pooling of tissues from very large numbers of embryos. An alternative approach is to determine the potential for RA synthesis by assaying for the enzymes that catalyze the last step of RA synthesis, the irreversible oxidation of retinaldehyde to RA. In a quantitative comparison of retinaldehyde dehydrogenase levels with endogenous RA levels, we found a good concordance between the two parameters. For the detection of retinaldehyde dehydrogenases we have developed an assay which involves analyses of protein fractions, separated by isoelectric focusing (IEF), with a RA responsive cell line; operationally, this technique is a zymography bioassay. This method is exquisitely sensitive: tissue volumes as small as a sugar grain can be assayed, and it can be used on all species. Separation by IEF allows in a single run the identification and relative quantitation of several enzyme isoforms.

Section snippets

Type of research

Relevant published studies:

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McCaffery et al. [7]. Zymography bioassay is used to describe the retinaldehyde dehydrogenases that set up a gradient of RA in the dorsal/ventral axis of the developing retina.
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McCaffery and Dräger [10]. Zymography bioassay is used to show RA synthesis in the corpus striatum and olfactory bulb.
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Marsh-Armstrong et al. [6]. The specificities of the retinaldehyde dehydrogenase inhibitors citral and disulfiram are demonstrated using zymography bioassay.
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Yamamoto et al. [14].

Time required

Two days (approximately 4 h of experimental manipulation, the remaining time required for incubations).

Special equipment

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Flat bed thermostatting unit for agarose IEF gels (e.g., Isobox from Hoefer, Scientific Instruments).
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Power supply with automatic constant power and a voltage maximum of over 1000 V (e.g., OSP-4000 from Owl Scientific Plastic Inc.).
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Agarose slab gels pH 3–10 (Resolve, Isolab). These are thin agarose gels attached to a flexible plastic backing.
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Gel cutting device made from 40 Exacto knife blades (Fig. 1). The single 5.3 mm wide Exacto blades are separated by 1.8 mm thick, wide-bore nuts as spacers,

Detailed procedures

(A) Depending on the enzyme activity in the sample, between 1 and 10 mg wet weight of tissue is homogenized. Embryonic tissue can be easily homogenized by trituration with a 10–200 μl pipette tip in a volume of buffer equal to the tissue. Less friable tissue is homogenized in a microfuge tube with a Teflon pestle or a sonicator. More complete lysis is achieved by freeze/thawing the tissue twice in liquid nitrogen and a 37°C water bath. The sample is spun at 16 000×g for 10 s in order to remove

Result sample

Fig. 3 shows zymography traces of retinas from mice of different ages: embryonic day 15.5 (E15.5), postnatal day 0.5 (P0.5), P2.5, P4.5, P11 and adult. The tissue samples were normalized for protein content and processed in parallel, in order to illustrate the decline in retinaldehyde dehydrogenase activity with age. The retinas contain two enzymes: AHD2, located in the dorsal retina, and V1, located in the ventral retina. AHD2 focuses at pH 7.6 and V1 at pH 5.7. In addition, the traces show a

Troubleshooting

The zymography bioassay is generally a reliable and robust technique. With this technique we have identified several novel retinaldehyde dehydrogenases which had not been detected in the most comprehensive HPLC-based search for such enzymes in the mouse 4, 5, 11. If problems occur, they are most likely a result of either: (1) poor protein separation by the IEF gel, or (2) a loss of responsiveness of the RA reporter cells.

(1) Poor protein separation by the IEF gel. This may be caused by (i) high

Quick procedure

•
(A) Preparation of tissue: 1–10 mg wet weight of embryonic tissue is homogenized by trituration in 10 μl of homogenization buffer and two rounds of freeze/thawing in liquid nitrogen and a warm water bath; the sample is spun at 16 000×g in a microfuge for 10 s.
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(B) Between 4 and 7 μl of sample are loaded onto an agarose gel lane on a flat bed cooling apparatus. The gel is run by gradually stepping up the power from 1 to 15 W over a total period of 45 min.
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(C) Single lane strips are cut out with

Essential literature references

References [7], [13].

Acknowledgements

We thank M. Wagner for the RA reporter cells. This work was supported by NIH Grants HD05515 and EY01938, and a gift from Johnson and Johnson.

References (15)

Chen, Y., Huang, L., Russo, A.F. and Solursch, M., Retinoic acid is enriched in Hensen's node and is developmentally...
De Thé, H., Del Mar Vivanco-Ruiz, M., Tiollais, P., Stunnenberg, H. and Dejean, A., Identification of a retinoic acid...
Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970)...
Lee, M.-O., Manthey, C.L. and Sladek, N.E., Identification of mouse liver aldehyde dehydrogenases that catalyze the...
Manthey, C.L., Landkamer, G.J. and Sladek, N.E., Identification of the mouse aldehyde dehydrogenases important in...
Marsh-Armstrong, N., McCaffery, P., Dowling, J.E., Gilbert, W. and Dräger, U.C., Retinoic acid is necessary for...
McCaffery, P., Lee, M.-O., Wagner, M.A., Sladek, N.E. and Dräger, U.C., Asymmetrical retinoic acid synthesis in the...

There are more references available in the full text version of this article.

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The present study provides a novel and rapid method to detect RAR ligand distribution in the body and brain after drug administration. Sil-15 cells have been used in multiple studies to detect endogenous RA levels, for instance by co-culturing reporter cells with tissue samples (Kelley, Xu, Wagner, Warchol, & Corwin, 1993; McCaffery, Lee, Wagner, Sladek, & Drager, 1992; Stoney et al., 2016; Wagner et al., 1992), adding conditioned medium to Sil-15 reporter cells from cultured embryonic spinal cord to measure RA released into the medium (McCaffery & Drager, 1994), adding zymography gel slices to reporter cells to measure RA synthetic enzyme activity (McCaffery & Dräger, 1997) or co-culturing neurospheres obtained from adult spinal cord stem cells with Sil-15 reporter cells (Ababon et al., 2016). Here we developed for the first time a bioassay using the Sil-15 reporter cell line that can map the tissue distribution of synthetic ligands to RAR injected into rodents.
The retinoic acid (RA) signaling pathway is crucial for the control of embryonic development and also regulates function of several organ systems in the adult, including the central nervous system. The retinoic acid receptors (RARs) that mediate the majority of the functions of RA can promote proliferation, differentiation, morphogenesis and cell survival. Dysregulation of this signaling pathway has been considered in the pathophysiology of various diseases including neurodegenerative disorders such Alzheimer's disease and amyotrophic lateral sclerosis. Thus, drugs targeted to the RARs have been proposed as treatments for such diseases. Understanding how these drugs distribute in the body is essential to determine their potential effectiveness. However measuring tissue levels of what are often lipophilic drugs can be difficult. Here we describe an indirect measurement of RAR ligand tissue distribution after intraperitoneal injection into rodents that uses a sensitive RA reporter cell line.
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Taken together, these experiments show definite interaction between ethanol and vitamin A, contributing to explain the mechanisms of prenatal ethanol consumption embryopathy (Zachman and Grummer, 1998). Three members of the RALDH family (RALDH1, 2, and 3) can account for all of the all‐trans RA generated in the early embryo (McCaffery and Dräger, 1997). RALDH2 plays a crucial role in the synthesis of RA and RALDH2 null mutant mice die in utero before 10.5 dpc (Niederreither et al., 1999).
Retinol (vitamin A) is a fat‐soluble nutrient indispensable for a harmonious mammalian gestation. The absence or excess of retinol and its active derivatives [i.e., the retinoic acids (RAs)] can lead to abnormal development of embryonic and extraembryonic (placental) structures. The embryo is unable to synthesize the retinol and is strongly dependent on the maternal delivery of retinol itself or precursors: retinyl esters or carotenoids. Before reaching the embryonic tissue, the retinol or the precursors have to pass through the placental structures. During this placental step, a simple diffusion of retinol can occur between maternal and fetal compartments; but retinol can also be used in situ after its activation into RA¹ or stored as retinyl esters. Using retinol‐binding protein knockout model, an alternative way of embryonic retinol supply was described using retinyl esters incorporated into maternal chylomicrons. In the embryo, the principal metabolic event occurring for retinol is its conversion into RAs, the active molecules implicated on the molecular control of embryonic morphogenesis and organogenesis. All these placental and embryonic events of retinol transport and metabolism are highly regulated. Nevertheless, some genetic and/or environmental abnormalities in the transport and/or metabolism of retinol can be related to developmental pathologies during mammalian development.
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2006, Advances in Developmental Biology
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Raldh2 is the first RALDH to be expressed during embryogenesis. Its induction during gastrulation (∼E7.0 in the mouse) in the posterior embryonic mesoderm (Fig. 2A) correlates with the stage at which RA can first be detected by HPLC analysis or enzymatic bioassays (McCaffery and Dräger, 1997; Ulven et al., 2000). From E8.5, Raldh2 is expressed in a dynamic and spatially restricted manner in many mesodermal derivatives including the somites, the cervical and posterior branchial arch mesenchyme, the posteriormost region of the heart tube, and the lateral plate mesenchyme of the limb‐forming region (Fig. 2B) (Niederreither et al., 1997).
This chapter explores the activation and restriction of retinoic acid (RA) signaling during embryonic and fetal development. It discusses the work performed on a variety of RA deficiencies in vivo or ex vivo animal models. It focuses on mouse models generated by gene targeting techniques, as these allow studying the function of specific molecular players in the context of the whole animal. Knowledge on the developmental functions of proteins implicated in the maternal–embryonic transfer of vitamin A, the intracellular metabolism of RA, and its signaling by nuclear receptors is reviewed. A number of developmental events in which the involvement of RA has been established are considered in the chapter. All of these involve region-specific patterns of embryonic RA synthesis by a single enzyme, the retinaldehyde dehydrogenase 2 (RALDH2). Data obtained through the analysis of Raldh2-null or “conditional” mouse mutants are reviewed in the context of additional knowledge obtained by experimentally interfering with RA levels and/or signaling in other vertebrate species.
Ethanol increases retinoic acid production in cerebellar astrocytes and in cerebellum
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Several characteristics of fetal alcohol syndrome (FAS) are similar to the teratogenic effects of retinoic acid (RA) exposure. It has been suggested that FAS may result from ethanol-induced alteration in endogenous RA synthesis, leading to abnormal embryonic concentrations of this morphogen. We examined whether ethanol may interfere with RA synthesis in the postnatal cerebellum, as a region of the developing CNS particularly vulnerable to both ethanol and RA teratogenesis. It was found that astrocytes are the predominant source of postnatal RA synthesis in the cerebellum. They express both retinaldehyde dehydrogenase 1 and 2. In vitro cytosolic preparations of astrocytes, as well as live cell preparations, have an increased capacity to synthesize RA in the presence of ethanol. A mechanism by which ethanol could stimulate RA synthesis is via the ethanol-activated short-chain retinol dehydrogenases, which we show to be present in the postnatal cerebellum. To determine whether ethanol stimulated RA synthesis in vivo, a sensitive and highly specific HPLC/MSⁿ technique was used to measure cerebellar RA after administration of ethanol to postnatal day 4 rat pups. Cerebellar RA levels climbed significantly after such treatment. These results suggest that the cerebellar pathology exerted by ethanol may occur, at least in part, through increased production of RA.
Retinal dehydrogenase-2 is inhibited by compounds that induce congenital diaphragmatic hernias in rodents
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After IEF, parallel lanes of the gel were cut into 16 or 24 consecutive slices 2.25 mm apart. These IEF fractions were distributed into the wells of microtiter plates, where proteins were eluted and assayed for retinoic acid (RA) synthesis from 50 nmol/L all-trans retinaldehyde in the presence of 0.6 mg/ml dithiothreitol and 0.8 mg/ml NAD+.21 After 4 hours of incubation at 37°C in darkness, 50 μl/well of reaction products were removed and tested with RA-sensitive reporter cells.
Currently, the etiology of the serious developmental anomaly congenital diaphragmatic hernia (CDH) is unknown. We have used an animal model of CDH to address this issue. We characterized four separate teratogens that produced diaphragmatic defects in embryonic rats that are similar to those in infants with CDH. We then tested the hypothesis that all these agents share the common mechanism of perturbing the retinoid-signaling pathway. Specifically, inhibition of retinal dehydrogenase-2 (RALDH2), a key enzyme necessary for the production of retinoic acid and that is expressed in the developing diaphragm, was assayed by measuring retinoic acid production in cytosolic extracts from an oligodendrocyte cell line. The following compounds all induce posterolateral defects in the rat diaphragm; nitrofen, 4-biphenyl carboxylic acid, bisdiamine, and SB-210661. Importantly, we demonstrate that they all share the common mechanism of inhibiting RALDH2. These data provide an important component of mounting evidence suggesting that the retinoid system warrants consideration in future studies of the etiology of CDH.
Sources and sink of retinoic acid in the embryonic chick retina: Distribution of aldehyde dehydrogenase activities, CRABP-I, and sites of retinoic acid inactivation
2001, Developmental Brain Research
Previous experiments in mice and zebrafish led to the hypothesis that an asymmetric distribution of the transcriptional activator retinoic acid (RA) causes ventral–dorsal polarity in the vertebrate eye anlage. A high concentration of RA in the ventral retinal neuroepithelium has been suggested to induce developmental events that finally establish topographic order in the retinotectal projection along the vertical eye axis. In the present study we have investigated potential sources and sinks of RA during embryonic development of the chick retina. At embryonic day (E)1 to E2, when the spatial determination of the eye primordia takes place, no RA synthesis by aldehyde dehydrogenases was detectable, and neither immunoreactivity for retinaldehyde dehydrogenase RALDH-2 nor for cellular retinoic acid binding protein CRABP-I was observed. These components of RA signal transduction appeared in the eye between E3 and E5. At later stages, RA-measurements with a reporter cell line showed highest synthesis in the retinal pigment epithelium (RPE) and at the ventral and dorsal poles of the retina. RA degradation occurred mostly in a horizontal region in the middle of the retina with only small differences along the nasal–temporal axis. CRABP-I immunoreactivity appeared first in differentiating retinal ganglion cells with no indication of a spatial gradient across the ventral–dorsal eye axis. RA-production depended on three NAD⁺-dependent enzyme activities, which could be competitively inhibited by citral. One enzyme, located in the dorsal retina (corresponding to mouse RALDH-1), and one enzyme in the RPE (RALDH-2) were aldehyde dehydrogenases of the same molecular weight (monomers about 55 kDa) but with different isoelectric points (6.5–6.9; 4.9–5.4). The third RA-synthesizing activity (pI 6.0–6.3) was limited to the ventral retina, and likely corresponded to mouse RALDH-3. The restricted localization of retinoid-metabolizing activities along the dorsal–ventral axis of the embryonic chick retina does support the idea that RA is involved in dorsal–ventral eye patterning. However, the late time of appearance of aldehyde dehydrogenase activities and CRABP-I points to functions in cellular differentiation that are distinct from the initiation of the dorsal–ventral polarity.

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Brain Research Protocols

ProtocolA sensitive bioassay for enzymes that synthesize retinoic acid

Abstract

Section snippets

Type of research

Time required

Special equipment

Detailed procedures

Result sample

Troubleshooting

Quick procedure

Essential literature references

Acknowledgements

Tissue localization of retinoic acid receptor (RAR) active drugs

Metabolism of Retinol During Mammalian Placental and Embryonic Development

Molecular mediators of retinoic acid signaling during development

Ethanol increases retinoic acid production in cerebellar astrocytes and in cerebellum

Retinal dehydrogenase-2 is inhibited by compounds that induce congenital diaphragmatic hernias in rodents

Sources and sink of retinoic acid in the embryonic chick retina: Distribution of aldehyde dehydrogenase activities, CRABP-I, and sites of retinoic acid inactivation

Protocol
A sensitive bioassay for enzymes that synthesize retinoic acid