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

M. S. RAO 0 1 PAUL H. PATTERSON 1 S. C. LANDIS 0 0 Department of Neurosciences, Case Western University , Cleveland, OH 44106 , USA 1 Division of Biology, California Institute of Technology , Pasadena, CA 91125 , USA *Author for correspondence - 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 22-26 103 Mr 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. While the sympathetic innervation of adult rat sweat glands is cholinergic, the axons that initially innervate the sweat gland are noradrenergic (Landis and Keefe, 1983; Leblanc and Landis, 1986; Stevens and Landis, 1987). Thus, there is a switch in the neurotransmitter phenotype of these sympathetic neurons. Cross-innervation studies in vivo indicate that this switch is target-directed. Neurons that normally innervate sweat glands fail to acquire cholinergic properties when they are made to innervate the parotid gland, a target whose sympathetic innervation is ordinarily noradrenergic (Schotzinger and Landis, 1990). Furthermore, normally noradrenergic sympathetic neurons that innervate hairy skin become cholinergic when they are made to innervate sweat glands (Schotzinger and Landis, 1988). Acquisition of immunoreactivity for vasoactive intestinal peptide (VIP), a neuropeptide present in mature sweat gland innervation, is also target-dependent (Landis et al., 1988; Schotzinger and Landis, 1988; Stevens and Landis, 1990 and unpublished observations). Although the evidence for target control of transmitter phenotype is compelling, the identity of the molecules involved is not known. We and others previously found that extracts of rat footpads contain factor(s) that induce choline acetyltransferase (ChAT) and VIP, and reduce catecholamine and neuropeptide Y (NPY) content in cultured sympathetic neurons (Rao and Landis, 1990; Rohrer, 1992). This activity was not detected in extracts of parotid gland or hairy skin, tissues that receive noradrenergic sympathetic innervation. The cholinergic differentiation activity is evident in developing footpads as early as postnatal day 5 (P5) and increases over a period of two weeks to reach adult levels. Therefore, it is present at the appropriate time in development to be responsible for the target-mediated noradrenergic-to-cholinergic switch. Several purified proteins elicit the same noradrenergicto-cholinergic switch in cultured sympathetic neurons and are therefore candidates for the differentiation signal in sweat glands. The cholinergic differentiation factor (CDF), originally purified from heart-cell-conditioned medium (Patterson and Chun, 1977; Fukada, 1985), is identical to leukemia inhibitory factor (LIF; Yamamori et al., 1989). Recombinant CDF/LIF also influences neuropeptide expression in cultured sympathetic neurons in a manner very similar to that of the footpad factor (Rao and Landis, 1990; Nawa et al., 1990). Studies using the reverse transcriptase/polymerase chain reaction (RT-PCR) revealed that CDF/LIF mRNA is selectively localized in postnatal rat footpads (Yamamori, 1991). Another candidate for the sweat gland cholinergic factor is ciliary neurotrophic factor (CNTF; Lin et al., 1989; Stockli et al., 1989). This protein regulates cholinergic, noradrenergic and neuropeptide properties in cultured sympathetic neurons in the same fashion as CDF/LIF (Ernsberger et al., 1989; Saadat et al., 1989; Rao et al., 1992a). A third cholinergic factor, MANS (membrane associated neurotransmitter stimulating factor), was partially purified from rat spinal cord (Wong and Kessler, 1987; Adler et al., 1989). MANS appears to be related to CNTF since it supports the survival of chick ciliary neurons (Rao et al., 1990) and antibodies to CNTF immunoprecipitate the cholinergic inducing activity of MANS and detect a band of 24 103, the relative molecular mass of CNTF (Carnow et al., 1985; Manthorpe et al., 1986), on immunoblots of MANS preparations (Rao et al., 1992b). Finally, a cholinergic differentiation factor, which is biochemically distinct from CDF/LIF and CNTF, has been purified from skeletal muscle. It enhances the survival of motor neurons in vitro and in vivo and increases levels of ChAT in spinal cord cultures (McManaman et al., 1988, 1989, 1990). The ability of the skeletal muscle cholinergic differentiation factor to induce cholinergic properties in sympathetic neurons has not (...truncated)