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Multiple cholinergic differentiation factors are present in footpad extracts: comparison with known cholinergic factors

Rao, M. S. and Patterson, Paul H. and Landis, S. C. (1992) Multiple cholinergic differentiation factors are present in footpad extracts: comparison with known cholinergic factors. Development, 116 (3). pp. 731-744. ISSN 0950-1991.

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Sweat glands in rat footpads contain a neuronal differentiation activity that switches the phenotype of sympathetic neurons from noradrenergic to cholinergic during normal development in vivo. Extracts of developing and adult sweat glands induce changes in neurotransmitter properties in cultured sympathetic neurons that mimic those observed in vivo. We have characterized further the factors present in the extract and compared their properties to those of known cholinergic factors. When assayed on cultured rat sympathetic neurons, the major activities in footpad extracts from postnatal day 21 rat pups that induce choline acetyltransferase (ChAT) and vasoactive intestinal peptide (VIP) and reduce catecholamines and neuropeptide Y (NPY) are associated with a soluble protein of 22-26 x 10^(3) M(r) and a pI of 5.0. These properties are similar to those of ciliary neurotrophic factor (CNTF). Moreover, the purified fraction from footpads has ciliary neurotrophic activity. Antibodies to CNTF that immunoprecipitate all differentiation activity from sciatic nerve extracts, a rich source of CNTF, immunoprecipitate 80% of the cholinergic activity in the footpad extracts, 50% of the VIP and 20% of the NPY activities. Neither CNTF protein nor CNTF mRNA, however, can be detected in immunoblot and northern analysis of footpads even though both CNTF protein and mRNA are evident in sciatic nerve. CNTF-immunoreactivity is associated with a sparse plexus of sensory fibers in the footpad but not with sweat glands or the Schwann cells associated with them. In addition, in situ hybridization studies with oligonucleotide probes failed to reveal CNTF mRNA in sweat glands. Comparison of the sweat gland differentiation activity with the cholinergic differentiation factor from heart cells (CDF; also known as leukemia inhibitory factor or LIF) suggests that most of the cholinergic activity in foot pads is biochemically distinct from CDF/LIF. Further, antibodies that block the activity of CDF/LIF purified from heart-cell-conditioned medium do not block the ChAT-inducing activity present in footpad extracts of postnatal day 8 animals. A differentiation factor isolated from skeletal muscle did not induce cholinergic properties in sympathetic neuron cultures and therefore is unlikely to be the cholinergic differentiation factor produced by sweat glands. Taken together, our data suggest that there are at least two differentiation molecules present in the extracts and that the major cholinergic activity obtained from footpads is related to, but distinct from, CNTF. The second factor remains to be characterized. In addition, CNTF associated with sensory fibers may make a minor contribution to the cholinergic inducing activity present in the extract.

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Additional Information:Copyright © 1992 by Company of Biologists. (Accepted 4 August 1992) The authors wish to thank Dr Ken Neet for providing NGF, Regeneron Pharmaceuticals for recombinant CNTF and anti-CNTF antiserum, Dr Keiko Fukada for purified CDF/LIF and antiserum, Dr B. Samal of Amgen Pharmaceuticals for recombinant human LIF and Dr Chris Beattie for advice. We thank Drs Donna Morissey and Mark Furth of Regeneron Pharmaceuticals for the polyclonal antiserum to CNTF. This work was supported by NINDS Javits Neuroscience Investigator Awards to P.H.P. and S.C.L., a McKnight Foundation Neuroscience Research Award to P.H.P. and an AHA Grant-In-Aid to S.C.L. Dr Rao is the recipient of a postdoctoral fellowship from the Northeast Ohio Affiliate of the American Heart Association.
Funding AgencyGrant Number
National Institute of Neurological Disorders and Stroke (NINDS)UNSPECIFIED
McKnight FoundationUNSPECIFIED
American Heart AssociationUNSPECIFIED
Subject Keywords:CDF/LIF, CNTF, sympathetic neurons, neurotransmitter plasticity, neural crest
Issue or Number:3
Record Number:CaltechAUTHORS:RAOdev92
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:12968
Deposited By: Archive Administrator
Deposited On:12 Jan 2009 20:55
Last Modified:03 Oct 2019 00:33

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