GDNF is abundant in the adult rat gut

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Abstract

Glial derived neurotrophic factor (GDNF) is essential for the development of the enteric nervous system (ENS). Although previous work has measured GDNF mRNA levels, little is known about the concentration of GDNF protein produced in developing or adult tissues. The aim of this study was to quantitate the concentration of GDNF protein in various tissues of the developing and adult rat and in adult human gut. A two site antibody immunoassay was used to quantitate GDNF using recombinant rat GDNF as a standard. In the adult rat gastrointestinal tract the intestine contained the highest concentration of GDNF while the stomach and esophagus have the lowest concentrations. The isolated muscular wall of the intestine has approximately four times the GDNF concentration of the intact intestine. Other tissues with smooth muscle such as the aorta and urinary bladder contain moderate GDNF concentrations. In contrast, GDNF is barely detectable in the adult kidney and liver. High concentrations of GDNF were also detected in human colon and jejunum. As development proceeds in the rat, there is a tendency for the concentration of GDNF to increase in the intestine but decrease in other tissues. Treatment of the jejunum with the cationic surfactant benzyldimethyltetradecylammonium chloride (BAC) results in an increase in the number of smooth muscle cells, a decrease in myenteric neurons, and an increase in the concentration of GDNF in homogenates of intestine. The observations that GDNF concentrations are high in the adult intestine suggest that this growth factor may be important for the maintenance of the adult ENS.

Introduction

Glial derived neurotrophic factor (GDNF), a member of the TGF-β superfamily, was isolated and cloned based on its ability to support the survival of midbrain dopaminergic neurons in culture (Lin et al., 1993). Subsequent investigations showed that injection of GDNF protected dopamine neurons in the substantia nigra from damage in models of Parkinson's disease (Beck et al., 1995; Tomac et al., 1995). Recently, a novel method for exogenous expression, injection of adenovirus producing GDNF, was found to protect rat nigral neurons after injection of the neurotoxin 6-hydroxydopamine (Choi-Lundberg et al., 1997).

The expression of GDNF mRNA in the adult rat brain was localized to a number of target sites for nigral neurons including the striatum and nucleus accumbens (Trupp et al., 1997). These observations showed a moderate labeling over some neurons and suggest that neurons are producing this growth factor. Although GDNF mRNA is found in the brain, the role of GDNF in the brain is not clear.

In contrast to the adult, GDNF has a critical role in embryogenesis and is essential for the development of the enteric nervous system and kidneys as shown by targeted mutations of the murine GDNF gene (Moore et al., 1996; Pichel et al., 1996; Sanchez et al., 1996). Mutant mice died shortly after birth and lacked the enteric nervous system (ENS) and kidneys. They also showed a 20–50% reduction in the number of spinal motor neurons, and sensory neurons in the nodose-petrosal and dorsal root ganglia, but surprisingly not a reduction in the number of nigral neurons (Granholm et al., 1997).

Studies of expression of GDNF have measured or localized mRNA, mostly in developing rodents (Poulsen et al., 1994; Hellmich et al., 1996). RNAase protection assays show substantial levels in the developing gut (Trupp et al., 1995) and in situ hybridization experiments show substantial label in the gut wall (Nostrat et al., 1996). However, little is known about the levels of GDNF protein in either developing or adult tissues, particularly the gut. We measured GDNF protein with an ELISA and report that the adult rat intestine contains substantial concentrations relative to other tissues. To gain insight into the cellular source of GDNF we have treated jejunum with a surfactant that destroys most myenteric neurons and increases the number of smooth muscle cells. This treatment resulted in an increase in GDNF and suggests that GDNF might be produced by smooth muscle cells.

Section snippets

Animals

All procedures involving animal use were approved by the Animal Care Committee of the University of Wisconsin, Madison. Adult Sprague–Dawley (Madison, WI) rats (all male except one) were euthanized with carbon dioxide gas. Tissues were removed and placed in cold phosphate-buffered saline (PBS), pH 7.4. After the small intestine was isolated, several pieces from throughout its length were removed to serve as the intact intestine sample. From the remainder of the intestine, the mucosa was

Results

We wished to determine whether acidification was necessary for measurement of GDNF and if so, to optimize acidification conditions. Intestine from 12 postnatal-day 6 (P6) rats was homogenized and extracted as described above. Standard conditions were acidification to pH 3, incubation at room temperature for 15 min, and neutralization to pH 7. From these standard conditions, we varied the following parameters independently: the pH of acidification, the duration of incubation, and the pH after

Discussion

This study was undertaken to determine the levels of GDNF in tissues of the adult and developing rat and in human gut tissue. We have used a two antibody ELISA to measure levels of GDNF immunoreactivity. To date investigators have measured GDNF mRNA in RNAase protection assays or localized it by in situ hybridization (Nostrat et al., 1996; Trupp et al., 1995). Most of the tissues examined have been from developing animals and only a few from mature animals. In order to determine the possible

Acknowledgements

We are grateful to Mr Jason Benjamin for his measurements on the BAC and control rats and to Mr David Potter for his assistance with the statistical analysis. Supported by NIH Grant NS 31385 and a Grant from the University of Wisconsin Graduate School.

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