Phoenixin: a novel peptide in rodent sensory ganglia

Abstract

Phoenixin-14 amide, herein referred to as phoenixin, is a newly identified peptide from the rat brain. Using a previously characterized rabbit polyclonal antiserum against phoenixin, enzyme-immunoassay detected a high level (>4.5 ng/g tissue) of phoenixin-immunoreactivity (irPNX) in the rat spinal cords. Immunohistochemical studies revealed irPNX in networks of cell processes in the superficial dorsal horn, spinal trigeminal tract and nucleus of the solitary tract; and in a population of dorsal root, trigeminal and nodose ganglion cells. The pattern of distribution of irPNX in the superficial layers of the dorsal horn was similar to that of substance P immunoreactivity (irSP). Double-labeling the dorsal root ganglion sections showed that irPNX and irSP express in different populations of ganglion cells. In awake mice, intrathecal injection of phoenixin (1 or 5 μg) did not significantly affect the tail-flick latency as compared to that in animals injected with artificial cerebrospinal fluid (aCSF). Intrathecal administration of phoenixin (0.5, 1.25 or 2.5 μg) significantly reduced the number of writhes elicited by intraperitoneal injection of acetic acid (0.6%, 0.3 ml/30 g) as compared to that in mice injected with aCSF. While not affecting the tail-flick latency, phoenixin antiserum (1:100) injected intrathecally 10 min prior to the intraperitoneal injection of acetic acid significantly increased the number of writhes as compared to mice pre-treated with normal rabbit serum. Intrathecal injection of non-amidated phoenixin (2.5 μg) did not significantly alter the number of writhes evoked by acetic acid. Our result shows that phoenixin is expressed in sensory neurons of the dorsal root, nodose and trigeminal ganglia, the amidated peptide is bioactive, and exogenously administered phoenixin may preferentially suppress visceral as opposed to thermal pain.

Keywords: ABC; ANOVA; BCA; DRG; EIA; FITC; G-protein-coupled receptor; GPCR; MALDI-TOF; PBS; PEPS; PNX; RP-HPLC; SDB-L; aCSF; analysis of variance; artificial cerebrospinal fluid; avidin–biotin complex; bicinchoninic acid protein assay; dorsal root ganglia; enzyme-immunoassay; fluorescein isothiocyanate; irPNX; irSP; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; nAmb; nucleus ambiguus; pericentral spikes; phoenixin immunoreactivity; phoenixin-14 amide; phosphate-buffered saline.; reverse-phase High-performance liquid chromatography; sensory neurons; spinal cord; styrene-divinylbenzene polymer; substance P immunoreactivity; thermal pain; visceral pain.

Fig.1

Isolation and identification of endogenous phoenixin in rat tissues. A, Mass spectrometry analysis of rat spinal cord extracts shows the major peak corresponds to the peptide phoenixin-14 amide; a small peak corresponding to oxidized phoenixin-20 amide (+16Da for Hydroxy-Trp) is also noted. B, measurement of irPNX and irSP in rat spinal cords and other tissue homogenates by EIA shows that rat spinal cords expressing the highest level of irPNX (4.5 ng/g tissues); rat hypothalamus and hearts yield about 2.5 ng/g and 1.7 ng/g tissues; irSP is about 9.22 ng/g tissues.

Fig. 2

Fluorescence images of rat spinal segments labeled with phoenixin antiserum or phoenixin antiserum pre-absorbed with the peptide. A, C and E, cervical, thoracic and thoracolumbar spinal segment where irPNX is detected in superficial layers of the dorsal horn. B and D, a high magnification of A and C, where several strands of irPNX cell processes extend into the deeper laminae. F, a thoracolumbar segment labeled with phoenixin-antiserum pre-absorbed with the peptide (1 μg/ml); irPNX is not detected in this spinal section. Scale bar: A, C E and F, 250 μm; B and D, 100 μm.

Fig. 3

Phoenixin immunoreactivity in the rat medulla. A, a segment of medulla where irPNX is present in cell processes occupying the superficial layer of the spinal trigeminal tract. B, a higher magnification of boxed area in panel A, where some of the irPNX cell processes extend toward the nucleus of the solitary tract. C, irPNX cell processes are detected in the medial nucleus of the solitary tract (SolM) and central nucleus of the solitary tract (SolC), and above the central canal (cc). D, irPNX cell processes from the spinal trigeminal tract (sp5) project towards the nucleus of ambiguus (nAmb). Scale bar: A, 250 μm, B, C, and D, 100 μm.

Fig. 4

Distribution of phoenixin- and substance P- immunoreactivity in the rat spinal cord. A, irPNX cell processes are distributed almost exclusively to the superficial layers of the dorsal horn of a thoracic segment; the lateral and ventral horn are practically devoid of irPNX cell processes. B and C are higher magnifications showing varicose cell processes in the superficial layer. D, a thoracic spinal section where dense irSP cell processes are detected in the superficial layers, deeper laminae, lateral horn and ventral horn. E and F, numerous irSP cell processes are seen in the deeper laminae, lateral horn, central cancel (cc) and the ventral horn. Calibration bar: A and D, 250 μm; B, E and F, 100 μm; C, 50 μm.

Fig. 5

Phoenixin immunoreactivity in neurons of the rat sensory ganglia. A, B and C, a section of dorsal root, trigeminal and nodose ganglion where some of the ganglion cells are irPNX. Scale bar: A and C, 40 μm and B, 30 μm.

Fig.6

Histogram of percent of phoenixin-immunoreactive ganglion cells against cell diameters. The diameter of the majority of ganglion cells is between 25 and 40 μm.

Fig.7

Tail flick latency not significantly affected by intrathecal administration of phoenixin in mice. Intrathecal injection of phoenixin 1 or 5 μg did not significantly change the tail flick latency as compared to that of mice injected with aCSF; p>0.05. Data were collected from 5 to 7 mice per group.

Fig.8

Phoenixin suppresses writhing induced by intraperitoneal injection of acetic acid in mice. Phoenixin at the doses of 1.25 μg and 2.5 μg significantly reduced the number of writhes as compared to that of aCSF injected mice; data were collected from 5 to 7 mice per group 5 to 15 min post-injection of acetic acid. * statistically significant, p<0.05; ** statistically significant, p<0.01.