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. 2009 Apr 1;513(4):385-98.
doi: 10.1002/cne.21982.

Coexpression of alpha 2A-adrenergic and delta-opioid receptors in substance P-containing terminals in rat dorsal horn

Maureen S Riedl  1 , Stephen A Schnell, Aaron C Overland, Anne-Julie Chabot-Doré, Anna M Taylor, Alfredo Ribeiro-da-Silva, Robert P Elde, George L Wilcox, Laura S Stone
Affiliations

Coexpression of alpha 2A-adrenergic and delta-opioid receptors in substance P-containing terminals in rat dorsal horn

Maureen S Riedl et al. J Comp Neurol. .

Erratum in

  • J Comp Neurol. 2009 Jun 20;514(6):674

Abstract

Agonists acting at alpha(2)-adrenergic and opioid receptors (alpha(2)ARs and ORs, respectively) inhibit pain transmission in the spinal cord. When coadministered, agonists activating these receptors interact in a synergistic manner. Although the existence of alpha(2)AR/OR synergy has been well characterized, its mechanism remains poorly understood. The formation of heterooligomers has been proposed as a molecular basis for interactions between neuronal G-protein-coupled receptors. The relevance of heterooligomer formation to spinal analgesic synergy requires demonstration of the expression of both receptors within the same neuron as well as the localization of both receptors in the same neuronal compartment. We used immunohistochemistry to investigate the spatial relationship between alpha(2)ARs and ORs in the rat spinal cord to determine whether coexpression could be demonstrated between these receptors. We observed extensive colocalization between alpha(2A)-adrenergic and delta-opioid receptors (DOP) on substance P (SP)-immunoreactive (-ir) varicosities in the superficial dorsal horn of the spinal cord and in peripheral nerve terminals in the skin. alpha(2A)AR- and DOP-ir elements were colocalized in subcellular structures of 0.5 mum or less in diameter in isolated nerve terminals. Furthermore, coincubation of isolated synaptosomes with alpha(2)AR and DOP agonists resulted in a greater-than-additive increase in the inhibition of K(+)-stimulated neuropeptide release. These findings suggest that coexpression of the synergistic receptor pair alpha(2A)AR-DOP on primary afferent nociceptive fibers may represent an anatomical substrate for analgesic synergy, perhaps as a result of protein-protein interactions such as heterooligomerization.

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Figures

Figure 1
Figure 1. Relationship between α2AAR-ir, α2CAR-ir, DOP-ir and MOP-ir in Rat Spinal Cord Dorsal Horn
Single confocal optical sections of lumbar rat spinal cord were double-labeled with combinations of guinea pig, rat or rabbit-derived antisera. The 1st column depicts immunoreactivity of antisera directed against α2AAR (A,E) and α2CAR (I,M). The 2nd column represents the same sections double-labeled with either DOP (B,J) or MOP (G,O) antisera. In the 3rd column the results of digitally merging images from the first two columns are shown (C,G,K,O). The 4th column contains higher magnification images of these combinations (D,H,L,P). In merged images, the appearance of white indicates probable co-localization. Antisera combinations were as follows: A-D: Rabbit-derived anti-α2AAR (A) with rat-derived anti-DOP (B), highly co-localized (C,D). E-H: Rabbit-derived anti-α2AAR (E) with guinea pig-derived anti-MOP (F), rarely co-localized (G,H). I-L: Guinea pig-derived anti-α2CAR (I) with rabbit-derived anti-DOP (J), rarely co-localized (K,L). M-P: Rabbit-derived anti-α2CAR (M) with guinea pig-derived anti-MOP (N), rarely co-localized (O,P). The lack of overlap between α2CAR and MOP was further supported by identical results obtained using rabbit-derived MOP paired with guinea pig-derived α2CAR (data not shown).
Figure 2
Figure 2. Close Association of DOP-ir and α2AAR-ir in Rat Spinal Cord Dorsal Horn
Single confocal optical sections of rat spinal cord dorsal horn double-labeled with rabbit-derived α2AAR and rat-derived DOP antisera. The 1st column depicts α2AAR-ir (A,D,G,J,M) and the 2nd column represents DOP-ir (B,E,H,K,N) in the same sections. Pairs of unmerged images illustrate the similarity in expression patterns of α2A AR and DOP. When the images in the 1st and 2nd columns are digitally merged (C,F,I,L,O), the large proportion of white suggests extensive co-localization. A-F: Coronal sections of rat dorsal horn at low (A-C) and higher (D-F) magnification. The insets in D-F are enlargements of the boxed area marked in D. G-I: A horizontal section at moderate magnification. J-L: Higher magnification images of the boxed area shown in G. M-O: Enlargements of the boxed region in J. Images in D-F (insets) and M-O demonstrate close associations of α2AAR-ir and DOP-ir in the same sub-cellular regions 0.25 μm or less in diameter (arrows: possible vesicles or clusters of vesicles).
Figure 3
Figure 3. Cross-reactivity Controls for DOP-ir and α2AAR-ir Co-localization
Single confocal optical sections show the results of double labeling in the presence or absence of cognate peptide absorption controls in three adjacent sections. Under normal conditions, α2AAR-ir (A) and DOP-ir (D) exhibit similar patterns of expression. In the presence of its cognate peptide, labeling for α2AAR is absent (B) whereas DOP-ir (E) is unaffected. Similarly, pre-incubation with the cognate peptide for the DOP antisera blocks DOP-ir (F), but not α2AAR-ir (C).
Figure 4
Figure 4. Triple Labeling of α2AAR-ir, DOP-ir and SP-ir in Rat Spinal Cord Dorsal Horn
A-D: Representative section of rat spinal cord double-labeled with rabbit-derived α2AAR (A, Red) and rat-derived SP (B, Blue) antibodies. When images A & B are digitally merged (C, D), instances of co-localization appears as fushia. E-H: Representative section of rat spinal cord double-labeled with rabbit-derived DOP (E, Green) and rat-derived SP (F, Blue) antisera. When images E & F are digitally merged (G, H), instances of co-localization appears as turquoise. I-P: A single confocal optical section of rat spinal cord triple-labeled with rabbit-derived α2AAR (I, Red), rat-derived DOP (J, Green), and guinea pig-derived SP (K, Blue) antisera. Each of the possible digital pairings of these images is shown where M = α2AAR-ir + SP-ir (co-localization = fuchsia); N = DOP-ir + SP-ir (co-localization = turquoise) and O = α2AAR-ir + DOP-ir (co-localization = yellow). Image L is the result of digital combination of Images I, J & K in which triple-labeled elements appear white. Enlargement of an area from L is shown in P. Q-U: Example of a triple-labeled single fiber for α2AAR-ir (Q, Red), DOP-ir (R, Green) and SP-ir (S, Blue). Image T is the result of digital combination of Images Q, R & S in which triple-labeled elements appear white. Image U is an enlargement the area indicated in T. The close association of all three markers along a single fiber suggests that α2AAR and DOP may be associated with SP-containing pre-synaptic vesicles.
Figure 5
Figure 5. Co-localization of α2AAR and DOP with SP in rat skin
A-C: Representative images of rat lower lip skin double-labeled with rabbit-derived α2AAR (A, Magenta) and rat-derived SP (B, Green) antisera. When images A & B are digitally merged (C), instances of co-localization appears as white. D-F: Representative section of rat spinal cord double-labeled with rabbit-derived DOP (D, Magenta) and guinea pig-derived SP (E, Green) antisera. When images D & E are digitally merged (F), instances of co-localization appears as white. The extensive co-localization observed between both α2AAR and DOP with SP suggests that α2AAR and DOP co-localize on SP-containing fibers in the periphery.
Figure 6
Figure 6. Labeling of α2AAR-ir, DOP-ir and SP-ir in Spinal Cord Synaptosome(s)
A-G: Nerve terminals were isolated from rat spinal cord and triple-labeled with rabbit-derived α2AAR (A, Red), rat-derived DOP (C, Green), and guinea pig-derived SP (E, Blue) antisera. Each of the possible digital pairings of these images is shown where B = α2AAR-ir + DOP-ir (co-localization = yellow); D = DOP-ir + SP-ir (co-localization = turquoise); F = α2AAR-ir + SP-ir (co-localization = fuchsia). Image G is the result of digital combination of Images A, C & E in which triple-labeled elements appear white. Arrows in G indicate several structural elements within the synaptosome that are triple-labeled. H-K: Enrichment of α2AAR-ir and DOP-ir in isolated nerve terminals. Subcellular fractions S1 (total protein), P2 (membrane fraction) and Syn (synaptosomes) were analyzed by Western blot. Immunoreactive bands for anti-α2AAR (∼50 kDa, L), anti-DOP (∼45 kDa, M) and the membrane marker pan-cadherin (∼135 kDa, N) were all enriched in the purified membrane and synaptosome fractions. The cytosolic marker GAPDH was most abundant in the S1 fraction (∼35 kDa, O).
Figure 7
Figure 7. Inhibition of neuropeptide release by α2AR and DOR agonists in spinal cord synaptosomes
Synaptosomes were exposed to vehicle, the α2AR agonist clonidine (circles), the DOP agonist deltorphin II (squares) or the combination of clonidine + deltorphin II (triangles) and stimulated with 60 mM K+. Clonidine and deltorphin II inhibited calcitonin gene-related peptide (CGRP) release in a concentration-dependent manner. Co-incubation with both agonists together resulted in enhanced effectiveness over either agonist alone. Error bars represent ±SEM for each concentration (n = 3 replicates/concentration).
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