Vascular NK-1 receptor occurrence in normal and chronic painful Achilles and patellar tendons:: studies on chemically unfixed as well as fixed specimens
Introduction
Substance P (SP), isolated from horse brain and gut as early as 1931 [1] and consisting of 11 amino acids, is one member of the tachykinin family of peptides. The other members in this family are neurokinin A and neurokinin B. Three different tachykinin receptors have been identified; neurokinin-1 receptor (NK-1R), neurokinin-2 receptor (NK-2R) and neurokinin-3 receptor (NK-3R). Substance P has a high affinity for the NK-1R and binds to NK-2R with low affinity [2], [3]. The NK-1R has been cloned by Yokota et al. [4] and Hershey and Krause [2], has 407 amino acids and belongs to the family of G-protein-coupled receptors [5]. The N-terminal end and extracellular loops are responsible for the binding of SP [6], [7] and the intracellular C-terminus mediates the phosphorylation and internalization of the receptor after agonist binding and activation [8], [9].
A large number of immunohistochemical studies on NK-1R distribution in various tissues have been performed. However, varying descriptions have often been presented, presumably related to methodological aspects [10], [11] and the occurrence of different subtypes of the NK-1R [12], [13]. Another puzzling fact is that there often is a mismatch between occurrence of labelling with antibodies directed against the C-terminus of the NK-1R and SP-innervation [14], which may not only be related to the fact that neuropeptides like SP can diffuse for distances in the extracellular fluid [15]. Thus, interestingly, antibodies against the N-terminal end of the NK-1R have been found to label structures that are not labeled by antibodies against the C-terminus but which nevertheless respond to SP [16]. A truncated form of the NK-1R, lacking the intracellular C-terminus, exists [cf. Refs. [6], [16]], and that might be responsible for effects not mediated via the C-terminus of the full-length receptor. Thus, it has been found that neurons lacking immunoreactivity to the C-terminus respond to SP via receptors that pharmacologically show similarities to NK-1 receptors [17].
There is a presence of sensory neuropeptides, including SP, in the Achilles tendon of the rat [18], [19], [20], and it has been suggested that SP can be involved in the healing processes of ruptured rat Achilles tendons [21]. We have in a previous study noted the presence of SP-containing nerve fibers in the proximal insertion of another tendon, the tendon of extensor carpi radialis brevis (ECRB) muscle [22]. Recently, by using an antibody against the C-terminus of NK-1R, we found that specific immunoreactions were detected at the proximal insertion of the ECRB muscle [23]. The reactions were, however, restricted to nerve fascicles and to some freely coursing nerve fibers. Immunoreactions were not detected in blood vessel walls, a finding that is somewhat puzzling as it is well-known that SP in other parts of the body has vasoregulatory properties [24], [25].
There is no information on the possible occurrence of NK-1R in patellar or Achilles tendons, neither in man nor in animals. Thus, morphologic correlates for SP effects in these tissues are completely lacking. This is a drawback for the understanding of SP effects in the normal situation and for the understanding of the recent observations, made by the use of ultrasound and color Doppler, that dilated blood vessels occur in the regions of the structural changes in painful tendon disorders (“tendinosis”) [26]. The blood vessels were interpreted to represent neovascularization [26]. Therefore, the aim of the present study was to examine the pattern of distribution of NK-1R in the normal and tendinosis Achilles and patellar tendons of man. Chemically fixed and unfixed tissue samples, PAP and TRITC stainings and five different NK-1R antibodies, one of which was directed against the N-terminal end of the NK-1R, were utilized.
Section snippets
Individuals
In this investigation, samples from altogether 36 tendons were examined. Some of the individuals had normal tendons, while others were patients with a long duration of pain symptoms, and who by ultrasonography or MRI displayed structural tendon changes corresponding to the painful part of the tendon, and were classified to suffer from “tendinosis”. Achilles tendinosis was localized to the mid-portion of the tendon, while patellar tendinosis was localized to the proximal part of the tendon. In
Results
Specific immunoreactions were mainly detected in two of the structural components in the specimens; blood vessel walls and nerve fascicles/nerve fibers. To some extent, immunoreactions were also observed for tenocytes. Extensive initial stainings and continuous control stainings were performed in order to clarify the importance of different procedures in showing the specific immunoreactions in these structures. The protocols that gave a clear outcome for the different antibodies are shown in
Discussion
The present study shows that NK-1R immunoreaction was detectable in human patellar and Achilles tendons. The reactions that were clearly the most frequent were reactions seen in blood vessel walls. Sometimes, immunoreaction was also detected in nerve fibers occurring singly or being parts of nerve fascicles and in tenocytes. The levels of NK-1R immunoreaction in the blood vessels were found to show interindividual variations in the tendinosis groups, the reactions being most pronounced in
Acknowledgements
The authors wish to thank Ulla Hedlund and Lena Jonsson for excellent technical services. Financial support has been obtained from the Faculty of Medicine and Odontology, Umeå University, and the Swedish Research Council for Sports.
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