De novo synthesis and axoplasmic transport of [35S]methionine-substance P in explants of nodose ganglion/vagus nerve

Abstract

The synthesis and transport of substance P, the widely distributed undecapeptide, was studied in the vagus nerve of the guinea pig. In preliminary in vivo studies, the cervical vagus nerve was ligated 2 cm distal to the nodose ganglion. Twenty-four hours later, the content of immunoreactive substance P (IR-SP) in the 3-mm nerve segment proximal to ligature was 2147 +/- 207 pg (mean +/- S.E.M.) vs 133 +/- 31 pg in an equal segment of unligated nerve or 243 +/- 55 pg in the nodose ganglion. When the vagus nerve was crushed above the ganglion and simultaneously ligated 2 cm distally, the IR-SP content proximal to the ligature was reduced 50% to 1131 +/- 99 pg (P less than 0.01), while nodose ganglion content increased to 420 +/- 140 pg (n.s.). To confirm that residual transport following supranodose crush was derived from nodose ganglion-synthesized SP, SP synthesis and transport were studied in explants of nodose ganglion and attached distal vagus nerve removed from perfused animals and maintained in vitro for up to 24 h. At the time of resection, nerves were ligated 1.5 cm distal to the ganglion. Twenty-four hours following explantation, IR-SP content in proximal segments was 1022 +/- 142 pg vs 155 +/- 22 pg in unligated segments and 560 +/- 72 pg in the nodose ganglion. Accumulation in the proximal segment was time dependent. In separate experiments, [35S]methionine was added to explant medium and the explants maintained for varying time intervals. Nerve tissue was extracted and subjected to either serial reverse phase high performance liquid chromatography (HPLC), or immunoprecipitation with SP antiserum followed by a single HPLC separation. By 4 h, radiolabeled SP was present in nodose ganglia and lesser amounts in the proximal segments. By 12 h, [35S]SP was present equally in ganglia and proximal segments whereas by 18 h, two-thirds or more of the newly synthesized peptide was present in proximal segments. At 18 h, the quantity of radiolabeled SP covaried with IR-SP content in the individual nerve segments. The addition of cycloheximide to explant medium reduced [35S]SP synthesis by 90%. These studies demonstrate that: (1) approximately 50% of immunoreactive SP transported efferently within the vagus nerve of the guinea pig is derived from the nodose ganglion, (2) de novo SP synthesis within and export from the nodose ganglion occurs within 4 h, (3) the changes in IR-SP content demonstrated in in vivo and in vitro ligation studies accurately reflect ongoing SP synthesis within the nodose ganglion.(ABSTRACT TRUNCATED AT 400 WORDS)