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Ciliary neurotrophic factor induces preprotachykinin A gene expression in the rat carotid body
Neuroscience Letters 298 (2001) 95±98
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Ciliary neurotrophic factor induces preprotachykinin A gene expression in the rat carotid body Patrice R. Akins, Guimei Wu, Estelle B. Gauda* Department of Pediatrics, Johns Hopkins Medical School, Baltimore, MD 21287-3200, USA Received 21 July 2000; received in revised form 24 November 2000; accepted 27 November 2000
Abstract Substance P (SP), a translational product of preprotachykinin-A (PPT-A) mRNA plays an important role in hypoxic chemotransmission in the rat carotid body. Although hypoxic exposure has been associated with an increase in SP content in the carotid body, factors that cause induction, regulation and release of PPT-A and SP in the carotid body remain to be elucidated. The purpose of this study was to investigate whether ciliary neurotrophic factor (CNTF), a factor that has been shown to regulate neurotransmitter phenotype in tissue from neural crest origin, could induce PPT-A gene expression in the rat carotid body. We used in situ hybridization histochemistry with radioactive ribonucleotide probes to investigate the effect of CNTF on PPT-A gene induction in the carotid body. Exposure of the rat superior cervical ganglia and carotid body to increasing concentrations of CNTF in culture resulted in up-regulation and induction of PPT-A mRNA, respectively. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ciliary Neurotrophic Factor; In situ hybridization; Carotid body; Preprotachykinin mRNA; Substance P; Superior cervical Ganglia; mRNA traf®cking
Catecholaminergic cells including those in the sympathetic superior cervical ganglion (SCG), adrenal chromaf®n cells, and type 1 cells in the carotid body are of neural crest origin. Type 1 cells in the carotid body sense changes in arterial blood, carbon dioxide and oxygen tension. Although the mechanism of hypoxic chemoreception is not completely understood, release of neurotransmitters from type 1 cells is involved in chemotransmission of the hypoxic response [6]. Physiological and pharmacological experiments have demonstrated a role for substance P, an excitatory neuropeptide in the carotid body, in hypoxic chemotransmission in several animal species [1,3]. The mechanism by which substance P, a translational product of preprotachykinin-A (PPT-A) mRNA, is involved in hypoxic chemotransmission in the rat carotid body, however, remains to be elucidated. Factors that regulate neurotransmitter phenotype in tissues from neural crest origin include leukotrienes and growth factors, speci®cally ciliary neurotrophic factor (CNTF). CNTF changes the neurotransmitter phenotype of SCG neurons in culture * Corresponding author. Johns Hopkins Hospital 600 N. Wolfe St., CMSC 210 Baltimore, MD 21287-3200, USA. Tel.: 11-410-9555259; fax: 11-410-955-0298. E-mail address: [email protected] (E.B. Gauda).
from that of primarily catecholaminergic to a mixed cholinergic and tachykinin phenotype [10,12]. Although substance P has been shown to be extensively involved in hypoxic chemotransmission in the carotid body, we have shown that 4 h of hypoxic exposure failed to induce PPTA gene expression in the rat carotid [4]. In order to further investigate factors that might regulate PPT-A gene expression in the carotid body, we asked whether CNTF would upregulate and induce PPT-A gene expression in the SCG and rat carotid body, respectively. We studied the effect of CNTF on PPT-A gene expression in the carotid body since CNTF regulates PPT-A gene expression in the SCG and both, the SCG and the carotid body, are of neural crest origin. Sprague±Dawley female rats at 7 days postnatal age (n 12) were deeply anesthetized with 3% methoxy¯urane and immediately decapitated. The right and left bifurcation of the carotid artery, including the carotid body, superior cervical, nodose and petrosal ganglia were removed rapidly en bloc. The tissue bloc was pre-incubated in 95% O2/5% CO2 bubbled Opti-MEM I (Life Technologies, Rockville, MD) for 1 h with 100 mg/ml penicillin±streptomycin (Life Technologies, Rockville, MD). The tissue blocs were then placed in fresh 95% O2/5% CO2 bubbled Opti-MEM I at 378C containing 100 ng/ml 2.5S murine nerve growth factor
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 71 7- 1
96
P.R. Akins et al. / Neuroscience Letters 298 (2001) 95±98
(Promega, Madison, WI), antibiotics, and increasing concentrations (0, 50, 100 and 200 ng/ml) of CNTF (Promega, Madison, WI) (n 6 per dose). After 24 h, they were removed from the incubation medium, placed in tissue freezing medium, then quickly frozen on dry ice. The tissue was stored at 2708 C until further processing for in situ hybridization histochemistry. Using a cryostat, tissue blocs were cut into 12 mm sections and thaw-mounted onto gelatin-coated slides. In situ hybridization histochemistry was done according to [4]. Antisense ribonucleotide probes were generated by in vitro transcription using complementary DNA containing 465 base pairs of exon 7 of the rat PPT-A gene [9] as the template. The sections were hybridized at 558C, washed at 608C in 2£ sodium citrate as previously described [4]. Slides were then dipped in Kodak autoradiography emulsion (Eastman Kodak, Rochester, NY), dried and exposed in the dark at 2208 C for 6±7 weeks. The slides were developed, counterstained with thionin, dehydrated through a series of alcohols, defatted in xylenes and then coverslipped. In order to visualize the tissue section and the silver grains in the same ®eld, we used a combination of dark®eld and phase-contrast microscopy. Silver grains generated by 35S in the emulsion were analyzed using a Nikon microscope and Macintosh image analysis program (NIH Image, W. Rashnad, NIMH). Qualitative representation of the effect of CNTF on PPT-A gene induction is presented as photomicrographs showing the presence or absence of clusters of silver grains in tissue sections of the SCG, carotid body and nerve processes. Expression of PPT-A mRNA in the SCG was homogeneous in contrast to that of the carotid body. Expression of PPT-A mRNA in the carotid body was low and the distribution of clusters of silver grains was sparse. Therefore, semi-quantitation of PPT-A gene expression for the SCG and carotid body was performed differently. Semi-quantitation of silver grains in cells in the SCG was measured using a counting algorithm from the NIH
Image program, as previously described [4]. For the SCG, grains were counted from 20 areas selected at random of 60 mm diameter. The areas selected for analysis were always over ganglion cells. Three to four sections of the SCG were analyzed per animal. The mean grains/SCG per animal was then compared to determine the statistical difference between treatment groups. For the carotid body, semi-quantitative analysis of the level of PPT-A gene induction was determined by obtaining the percent of carotid body sections with PPT-A gene expression. For the entire carotid body, for each animal, we determined the number of 12 mm carotid body sections that showed clusters of silver grains and expressed that value as a percent of total number of carotid body sections per animal. Differences between treatment groups were determined by ANOVA. Statistical signi®cance was set at P , 0:05. Exposure of the SCG and the carotid body in tissue culture to CNTF resulted in up-regulation of PPT-A mRNA in the SCG (Fig. 1) and induction of PPT-A mRNA expression in the rat carotid body (Fig. 2). Clusters of silver grains were distributed in a linear pattern in the carotid body (Fig. 3A,B) and were also seen in sections of nerve processes (Fig. 3C,D) versus the diffuse pattern of gene expression seen in the ganglion cells in the SCG and nodose±petrosal±jugular ganglia complex. The number of grains/cell of PPT-A mRNA gene expression in the SCG was signi®cantly increased by CNTF exposure (24.3 ^ 1.9, 28.7 ^ 3.3, 36.0 ^ 2.0, and 51.3 ^ 6.1% at 0, 50, 100, and 200 ng/ml, respectively). Similarly, the percent of PPT-A positive carotid body sections was signi®cantly increased by CNTF exposure (0 ^ 0, 5.5 ^ 5.5, 41.9 ^ 5.5, and 61.1 ^ 2.2% at 0, 50, 100, and 200 ng/ml, respectively). The major ®ndings from this study are: (1) CNTF up-regulates PPT-A mRNA levels in the SCG in a dose dependent manner and (2) CNTF induces PPT-A mRNA expression in a small subpopulation of cells in the rat carotid body also in a dose dependent manner.
Fig. 1. Low-power, dark-®eld photomicrographs showing up-regulation of PPT-A gene expression in the SCG at time 0 (A), after 24 h in culture without (B), and with 200 ng/ml CNTF (C). Silver grains are depicted as clusters of white dots. Scale bar is 25 mm.
P.R. Akins et al. / Neuroscience Letters 298 (2001) 95±98
97
Fig. 2. High-power, phase-dark-®eld photomicrographs showing induction of PPT-A gene expression in the carotid body after 24 h in culture without (A) and with 50 ng/ml (B), 100 ng/ml (C) and 200 ng/ml (D) of CNTF. Silver grains are depicted as clusters of white dots. White arrows (B±D) depict PPT-A gene expression in the carotid body. Black arrows (B,C) depict gene expression in the SCG. Scale bar is 25 mm.
CNTF is a neurotrophin that supports the development, differentiation, maintenance and repair of sympathetic and parasympathetic ganglia of the peripheral nervous system (PNS) and neurons of the central nervous system (CNS) [7]. In addition, CNTF changes neurotransmitter phenotypes in sympathetic ganglia [12]. It is expressed in glial cells within
the CNS and PNS and promotes survival and differentiation through binding to the CNTF-binding protein, CNTFRa. The CNTF receptor is a complex structure that is composed of an extracellular binding subunit (CNTFRa) and two transmembrane proteins, leukemia inhibitory factor receptor ((LIFRb) and gp 130 [2]. Binding of CNTF to CNTFR (and
Fig. 3. Phase-dark-®eld photomicrographs demonstrating the pattern of PPT-A gene expression induced by CNTF in the carotid body (A,B) and nerve process (C,D). There is a linear pattern of PPT-A gene expression in the carotid body (A,B) and the nerve process (C,D). B and D are high-power photomicrographs of the carotid body depicted by the arrows in (A) and the nerve process depicted by the arrows in (C), respectively. Silver grains are depicted as clusters of white dots represent by the white arrows. Black arrows depict gene expression in the SCG (A,C). Scale bars are 25 mm.
98
P.R. Akins et al. / Neuroscience Letters 298 (2001) 95±98
the subsequent formation of an active receptor induces gene expression through a cascade of intracellular events thereby affecting neuronal differentiation [13]. Similar to other studies [10], we show that exposure of SCG neurons to CNTF up-regulates PPT-A gene expression presumably through binding to its receptor. CNTFa mRNA [8], and immunoreactivity [11] have been localized to ganglion cells in the SCG. Our study, however, is the ®rst to demonstrate induction of PPT-A gene expression in the carotid body by CNTF. Although our study design does not address the mechanism by which CNTF exposure induces PPT-A mRNA expression in the carotid body, the pattern of PPT-A expression suggest two possibilities. CNTF exposure (1) induces PPT-A transcription in a small subset of type 1 cells, or (2) induces PPT-A gene expression in nerve ®bers adjacent to type 1 cells. If CNTF induces PPT-A gene expression in type 1 cells, it is mostly likely through binding to the CNTFRa receptor. Although possible, it would be unusual for only a small subset of type 1 cells to express CNTFRa receptors, since other genes that have been identi®ed in the carotid body are usually expressed uniformly [5]. Furthermore, CNTFRa receptor mRNA is expressed in virtually all neurons in the sympathetic, sensory and parasympathetic ganglia [8]. Alternatively, the sparse distribution of clusters of PPT-A mRNA expression in the carotid body in response to CNTF exposure could suggest that PPT-A mRNA maybe in nerve ®bers adjacent to type 1 cells. The carotid body is innervated by both the ganglioglomerular nerve with cell bodies in the SCG and the carotid sinus nerve, a branch of the IX cranial nerve, with cell bodies in the petrosal ganglia (PG). PPT-A mRNA is abundantly expressed in the SCG in response to CNTF exposure and is also abundantly expressed in PG cell bodies with and without CNTF. Thus, PPT-A mRNA expression in the carotid body might be secondary to mRNA traf®cking down the carotid sinus or ganglioglomerular nerve. The observation of PPT-A gene expression in the IX cranial nerve and the linear pattern of gene expression in the carotid body that was frequently seen support this speculation. In summary, we show that although PPT-A gene expression is not normally expressed in the rat carotid body its expression is induced by exposure to CNTF in culture. Our data suggest that CNTF regulates PPT-A gene expression in the carotid body either by inducing PPT-A gene expression in type 1 cells or inducing PPT-A mRNA traf®cking in either carotid sinus or ganglioglomerular nerve ®bers. These results raise important questions about mechanisms of gene regulation and mRNA transport in peripheral arterial chemoreceptors by neurotrophic factors. The authors would like to thank Frances J. Northington
for reviewing the manuscript. This work was funded by HL03365 (E.B. Gauda).
[1] Czyzyk-Krzeska, M.F., Bayliss, D.A., Seroogy, K.B. and Millhorn, D.E., Gene expression for peptides in neurons of the petrosal and nodose ganglia in rat, Exp. Brain Res., 83 (1991) 411±418. [2] Davis, S., Aldrich, T.H., Stahl, N., Pan, L., Taga, T., Kishimoto, T., Ip, N.Y. and Yancopoulos, G.D., LIFR beta and gp130 as heterodimerizing signal transducers of the tripartite CNTF receptor, Science, 260 (1993) 1805±1808. [3] Dinger, B., Gonzalez, C., Yoshizaki, K. and Fidone, S.J., [3H] Spiroperidol binding in normal and denervated carotid bodies, Neurosci. Lett., 21 (1981) 51±55. [4] Gauda, E.B., Bamford, O.S. and Northington, F.J., Lack of induction of substance P gene expression by hypoxia and absence of neurokinin 1-receptor mRNAs in the rat carotid body, J. Auton. Nerv. Syst., 74 (1998) 100±108. [5] Gauda, E.B., Northington, F.J., Linden, J. and Rosin, D.L., Differential expression of A(2a), A(1)-adenosine and D(2)dopamine receptor genes in rat peripheral arterial chemoreceptors during postnatal development, Brain Res., 872 (2000) 1±10. [6] Gonzalez, C., Dinger, B.G. and Fidone, S.J., Mechanisms of carotid chemoreception, In J.A. Dempsey and A.I. Pack (Eds.), Regulation of Breathing, Marcel Dekker, New York, 1994, pp. 391±470. [7] Ip, N.Y., Maisonpierre, P., Alderson, R., Friedman, B., Furth, M.E., Panayotatos, N., Squinto, S., Yancopoulos, G.D. and Lindsay, R.M., The neurotrophins and CNTF: speci®city of action towards PNS and CNS neurons, J. Physiol. (Paris), 85 (1991) 123±130. [8] Ip, N.Y., McClain, J., Barrezueta, N.X., Aldrich, T.H., Pan, L., Li, Y., Wiegand, S.J., Friedman, B., Davis, S. and Yancopoulos, G.D., The alpha component of the CNTF receptor is required for signaling and de®nes potential CNTF targets in the adult and during development, Neuron, 10 (1993) 89± 102. [9] Krause, J.E., Chirgwin, J.M., Carter, M.S., Xu, Z.S. and Hershey, A.D., Three rat prepotachykinin mRNAs encode the neuropeptides substance P and neurokinin A, Proc. Natl. Acad. Sci. USA, 84 (1987) 881±885. [10] Lewis, S.E., Rao, M.S., Symes, A.J., Dauer, W.T., Fink, J.S., Landis, S.C. and Hyman, S.E., Coordinate regulation of choline acetyltransferase, tyrosine hydroxylase, and neuropeptide mRNAs by ciliary neurotrophic factor and leukemia inhibitory factor in cultured sympathetic neurons, J. Neurochem., 63 (1994) 429±438. [11] MacLennan, A.J., Vinson, E.N., Marks, L., McLaurin, D.L., Pfeifer, M. and Lee, N., Immunohistochemical localization of ciliary neurotrophic factor receptor alpha expression in the rat nervous system, J. Neurosci., 16 (1996) 621±630. [12] Saadat, S., Sendtner, M. and Rohrer, H., Ciliary neurotrophic factor induces cholinergic differentiation of rat sympathetic neurons in culture, J. Cell Biol., 108 (1989) 1807±1816. [13] Sleeman, M.W., Anderson, K.D., Lambert, P.D., Yancopoulos, G.D. and Wiegand, S.J., The ciliary neurotrophic factor and its receptor, CNTFR alpha, Pharm. Acta Helv., 74 (2000) 265±272.
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Ciliary neurotrophic factor induces preprotachykinin A gene expression in the rat carotid body Patrice R. Akins, Guimei Wu, Estelle B. Gauda* Department of Pediatrics, Johns Hopkins Medical School, Baltimore, MD 21287-3200, USA Received 21 July 2000; received in revised form 24 November 2000; accepted 27 November 2000
Abstract Substance P (SP), a translational product of preprotachykinin-A (PPT-A) mRNA plays an important role in hypoxic chemotransmission in the rat carotid body. Although hypoxic exposure has been associated with an increase in SP content in the carotid body, factors that cause induction, regulation and release of PPT-A and SP in the carotid body remain to be elucidated. The purpose of this study was to investigate whether ciliary neurotrophic factor (CNTF), a factor that has been shown to regulate neurotransmitter phenotype in tissue from neural crest origin, could induce PPT-A gene expression in the rat carotid body. We used in situ hybridization histochemistry with radioactive ribonucleotide probes to investigate the effect of CNTF on PPT-A gene induction in the carotid body. Exposure of the rat superior cervical ganglia and carotid body to increasing concentrations of CNTF in culture resulted in up-regulation and induction of PPT-A mRNA, respectively. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ciliary Neurotrophic Factor; In situ hybridization; Carotid body; Preprotachykinin mRNA; Substance P; Superior cervical Ganglia; mRNA traf®cking
Catecholaminergic cells including those in the sympathetic superior cervical ganglion (SCG), adrenal chromaf®n cells, and type 1 cells in the carotid body are of neural crest origin. Type 1 cells in the carotid body sense changes in arterial blood, carbon dioxide and oxygen tension. Although the mechanism of hypoxic chemoreception is not completely understood, release of neurotransmitters from type 1 cells is involved in chemotransmission of the hypoxic response [6]. Physiological and pharmacological experiments have demonstrated a role for substance P, an excitatory neuropeptide in the carotid body, in hypoxic chemotransmission in several animal species [1,3]. The mechanism by which substance P, a translational product of preprotachykinin-A (PPT-A) mRNA, is involved in hypoxic chemotransmission in the rat carotid body, however, remains to be elucidated. Factors that regulate neurotransmitter phenotype in tissues from neural crest origin include leukotrienes and growth factors, speci®cally ciliary neurotrophic factor (CNTF). CNTF changes the neurotransmitter phenotype of SCG neurons in culture * Corresponding author. Johns Hopkins Hospital 600 N. Wolfe St., CMSC 210 Baltimore, MD 21287-3200, USA. Tel.: 11-410-9555259; fax: 11-410-955-0298. E-mail address: [email protected] (E.B. Gauda).
from that of primarily catecholaminergic to a mixed cholinergic and tachykinin phenotype [10,12]. Although substance P has been shown to be extensively involved in hypoxic chemotransmission in the carotid body, we have shown that 4 h of hypoxic exposure failed to induce PPTA gene expression in the rat carotid [4]. In order to further investigate factors that might regulate PPT-A gene expression in the carotid body, we asked whether CNTF would upregulate and induce PPT-A gene expression in the SCG and rat carotid body, respectively. We studied the effect of CNTF on PPT-A gene expression in the carotid body since CNTF regulates PPT-A gene expression in the SCG and both, the SCG and the carotid body, are of neural crest origin. Sprague±Dawley female rats at 7 days postnatal age (n 12) were deeply anesthetized with 3% methoxy¯urane and immediately decapitated. The right and left bifurcation of the carotid artery, including the carotid body, superior cervical, nodose and petrosal ganglia were removed rapidly en bloc. The tissue bloc was pre-incubated in 95% O2/5% CO2 bubbled Opti-MEM I (Life Technologies, Rockville, MD) for 1 h with 100 mg/ml penicillin±streptomycin (Life Technologies, Rockville, MD). The tissue blocs were then placed in fresh 95% O2/5% CO2 bubbled Opti-MEM I at 378C containing 100 ng/ml 2.5S murine nerve growth factor
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 71 7- 1
96
P.R. Akins et al. / Neuroscience Letters 298 (2001) 95±98
(Promega, Madison, WI), antibiotics, and increasing concentrations (0, 50, 100 and 200 ng/ml) of CNTF (Promega, Madison, WI) (n 6 per dose). After 24 h, they were removed from the incubation medium, placed in tissue freezing medium, then quickly frozen on dry ice. The tissue was stored at 2708 C until further processing for in situ hybridization histochemistry. Using a cryostat, tissue blocs were cut into 12 mm sections and thaw-mounted onto gelatin-coated slides. In situ hybridization histochemistry was done according to [4]. Antisense ribonucleotide probes were generated by in vitro transcription using complementary DNA containing 465 base pairs of exon 7 of the rat PPT-A gene [9] as the template. The sections were hybridized at 558C, washed at 608C in 2£ sodium citrate as previously described [4]. Slides were then dipped in Kodak autoradiography emulsion (Eastman Kodak, Rochester, NY), dried and exposed in the dark at 2208 C for 6±7 weeks. The slides were developed, counterstained with thionin, dehydrated through a series of alcohols, defatted in xylenes and then coverslipped. In order to visualize the tissue section and the silver grains in the same ®eld, we used a combination of dark®eld and phase-contrast microscopy. Silver grains generated by 35S in the emulsion were analyzed using a Nikon microscope and Macintosh image analysis program (NIH Image, W. Rashnad, NIMH). Qualitative representation of the effect of CNTF on PPT-A gene induction is presented as photomicrographs showing the presence or absence of clusters of silver grains in tissue sections of the SCG, carotid body and nerve processes. Expression of PPT-A mRNA in the SCG was homogeneous in contrast to that of the carotid body. Expression of PPT-A mRNA in the carotid body was low and the distribution of clusters of silver grains was sparse. Therefore, semi-quantitation of PPT-A gene expression for the SCG and carotid body was performed differently. Semi-quantitation of silver grains in cells in the SCG was measured using a counting algorithm from the NIH
Image program, as previously described [4]. For the SCG, grains were counted from 20 areas selected at random of 60 mm diameter. The areas selected for analysis were always over ganglion cells. Three to four sections of the SCG were analyzed per animal. The mean grains/SCG per animal was then compared to determine the statistical difference between treatment groups. For the carotid body, semi-quantitative analysis of the level of PPT-A gene induction was determined by obtaining the percent of carotid body sections with PPT-A gene expression. For the entire carotid body, for each animal, we determined the number of 12 mm carotid body sections that showed clusters of silver grains and expressed that value as a percent of total number of carotid body sections per animal. Differences between treatment groups were determined by ANOVA. Statistical signi®cance was set at P , 0:05. Exposure of the SCG and the carotid body in tissue culture to CNTF resulted in up-regulation of PPT-A mRNA in the SCG (Fig. 1) and induction of PPT-A mRNA expression in the rat carotid body (Fig. 2). Clusters of silver grains were distributed in a linear pattern in the carotid body (Fig. 3A,B) and were also seen in sections of nerve processes (Fig. 3C,D) versus the diffuse pattern of gene expression seen in the ganglion cells in the SCG and nodose±petrosal±jugular ganglia complex. The number of grains/cell of PPT-A mRNA gene expression in the SCG was signi®cantly increased by CNTF exposure (24.3 ^ 1.9, 28.7 ^ 3.3, 36.0 ^ 2.0, and 51.3 ^ 6.1% at 0, 50, 100, and 200 ng/ml, respectively). Similarly, the percent of PPT-A positive carotid body sections was signi®cantly increased by CNTF exposure (0 ^ 0, 5.5 ^ 5.5, 41.9 ^ 5.5, and 61.1 ^ 2.2% at 0, 50, 100, and 200 ng/ml, respectively). The major ®ndings from this study are: (1) CNTF up-regulates PPT-A mRNA levels in the SCG in a dose dependent manner and (2) CNTF induces PPT-A mRNA expression in a small subpopulation of cells in the rat carotid body also in a dose dependent manner.
Fig. 1. Low-power, dark-®eld photomicrographs showing up-regulation of PPT-A gene expression in the SCG at time 0 (A), after 24 h in culture without (B), and with 200 ng/ml CNTF (C). Silver grains are depicted as clusters of white dots. Scale bar is 25 mm.
P.R. Akins et al. / Neuroscience Letters 298 (2001) 95±98
97
Fig. 2. High-power, phase-dark-®eld photomicrographs showing induction of PPT-A gene expression in the carotid body after 24 h in culture without (A) and with 50 ng/ml (B), 100 ng/ml (C) and 200 ng/ml (D) of CNTF. Silver grains are depicted as clusters of white dots. White arrows (B±D) depict PPT-A gene expression in the carotid body. Black arrows (B,C) depict gene expression in the SCG. Scale bar is 25 mm.
CNTF is a neurotrophin that supports the development, differentiation, maintenance and repair of sympathetic and parasympathetic ganglia of the peripheral nervous system (PNS) and neurons of the central nervous system (CNS) [7]. In addition, CNTF changes neurotransmitter phenotypes in sympathetic ganglia [12]. It is expressed in glial cells within
the CNS and PNS and promotes survival and differentiation through binding to the CNTF-binding protein, CNTFRa. The CNTF receptor is a complex structure that is composed of an extracellular binding subunit (CNTFRa) and two transmembrane proteins, leukemia inhibitory factor receptor ((LIFRb) and gp 130 [2]. Binding of CNTF to CNTFR (and
Fig. 3. Phase-dark-®eld photomicrographs demonstrating the pattern of PPT-A gene expression induced by CNTF in the carotid body (A,B) and nerve process (C,D). There is a linear pattern of PPT-A gene expression in the carotid body (A,B) and the nerve process (C,D). B and D are high-power photomicrographs of the carotid body depicted by the arrows in (A) and the nerve process depicted by the arrows in (C), respectively. Silver grains are depicted as clusters of white dots represent by the white arrows. Black arrows depict gene expression in the SCG (A,C). Scale bars are 25 mm.
98
P.R. Akins et al. / Neuroscience Letters 298 (2001) 95±98
the subsequent formation of an active receptor induces gene expression through a cascade of intracellular events thereby affecting neuronal differentiation [13]. Similar to other studies [10], we show that exposure of SCG neurons to CNTF up-regulates PPT-A gene expression presumably through binding to its receptor. CNTFa mRNA [8], and immunoreactivity [11] have been localized to ganglion cells in the SCG. Our study, however, is the ®rst to demonstrate induction of PPT-A gene expression in the carotid body by CNTF. Although our study design does not address the mechanism by which CNTF exposure induces PPT-A mRNA expression in the carotid body, the pattern of PPT-A expression suggest two possibilities. CNTF exposure (1) induces PPT-A transcription in a small subset of type 1 cells, or (2) induces PPT-A gene expression in nerve ®bers adjacent to type 1 cells. If CNTF induces PPT-A gene expression in type 1 cells, it is mostly likely through binding to the CNTFRa receptor. Although possible, it would be unusual for only a small subset of type 1 cells to express CNTFRa receptors, since other genes that have been identi®ed in the carotid body are usually expressed uniformly [5]. Furthermore, CNTFRa receptor mRNA is expressed in virtually all neurons in the sympathetic, sensory and parasympathetic ganglia [8]. Alternatively, the sparse distribution of clusters of PPT-A mRNA expression in the carotid body in response to CNTF exposure could suggest that PPT-A mRNA maybe in nerve ®bers adjacent to type 1 cells. The carotid body is innervated by both the ganglioglomerular nerve with cell bodies in the SCG and the carotid sinus nerve, a branch of the IX cranial nerve, with cell bodies in the petrosal ganglia (PG). PPT-A mRNA is abundantly expressed in the SCG in response to CNTF exposure and is also abundantly expressed in PG cell bodies with and without CNTF. Thus, PPT-A mRNA expression in the carotid body might be secondary to mRNA traf®cking down the carotid sinus or ganglioglomerular nerve. The observation of PPT-A gene expression in the IX cranial nerve and the linear pattern of gene expression in the carotid body that was frequently seen support this speculation. In summary, we show that although PPT-A gene expression is not normally expressed in the rat carotid body its expression is induced by exposure to CNTF in culture. Our data suggest that CNTF regulates PPT-A gene expression in the carotid body either by inducing PPT-A gene expression in type 1 cells or inducing PPT-A mRNA traf®cking in either carotid sinus or ganglioglomerular nerve ®bers. These results raise important questions about mechanisms of gene regulation and mRNA transport in peripheral arterial chemoreceptors by neurotrophic factors. The authors would like to thank Frances J. Northington
for reviewing the manuscript. This work was funded by HL03365 (E.B. Gauda).
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