Further examination of these vmat IR cells shows that
Further examination of these vmat-IR cells shows that they are also positive for vimentin (vim-IR) (Fig. 3A). However, within any one cell there may be a region of weaker vmat-IR, suggesting sub-cellular localization. As also shown in Fig. 3B and C not all vim-IR cells are vmat-IR suggesting that there is a sub-population of catecholamine related interstitial cells. These vim-IR and vmat-IR cells appear to form an interconnecting network of heterogeneous interstitial cells. No vmat-IR cells were seen to run within the muscle bundles. However, vim-IR cells and processes were found within muscle bundles (see also Fig. 4).
These observations suggest the possibility of the storage and release of catecholamines within the interstitial cell network. The question then arises: ‘What is the target for this putative signaling system?’ Since functional studies have led to the suggestion that β1 dependent mechanisms (Gillespie et al., 2015b), rather than β2 or β3, are operating in the rat bladder wall, experiments were done to determine the presence and location of β1-adrenoceptor immunoreactivity (β1AR-IR). Fig. 4 shows sections stained to locate β1AR-IR and vim-IR. In Fig. 4A the network of vim-IR cells is readily seen (panels (a) and (b)). β1AR-IR is found within this network located principally within the cell bodies of the vim-IR cells (panel (c) arrows). It would appear that the majority of vim-IR interstitial cells show β1AR-IR. The β1AR-IR does not appear to be distributed throughout the entire interstitial cell and regional intense β1AR staining can be seen (Fig. 4B).
The signal interactions of these cells were explored further by combining staining for vmat-IR and β1AR-IR. A key observation is shown in Fig. 5. It appears that the majority if not all of vmat-IR cells are also β1AR-IR suggesting an autocrine signaling system.
The idea that the interstitial cells are involved in such an autocrine signaling system is dependent on the specificity of the m-Chlorophenylbiguanide hydrochloride to detect the underlying signaling elements. In order to check the specificity, vmat and β1AR experiments were done using the blocking peptides for each antibody. Fig. 6 shows examples using serial sections to determine the extent of both vmat-IR and β1AR-IR with and without the respective blocking peptides. Fig. 6A shows that the staining of the interstitial cells with the β1AR antibody is removed by the blocking peptide. Fig. 6B shows that the staining with the vmat antibody, identifying adrenergic nerve fibers surrounding blood vessels, is removed by the blocking peptide. The blocking peptide also removed the vmat-IR of the interstitial cells (Fig. 6C). Thus, it would appear that the antibodies are detecting specifically vmat and β1AR in the interstitial cells.
Discussion The sympathetic innervation to the bladder has been proposed to play a significant role in bladder physiology and pathology (Andersson and Arner, 2004). However, reports that significant areas of the detrusor have no or only a sparse adrenergic innervation (Gosling et al., 1999; Watanabe and Yamamoto, 1979), raises questions about the presence and nature of any sympathetic control (Levin and Wein, 1979). The present study, using immunoreactivity to nerves containing tyrosine hydroxylase (TH-IR) and vesicular monoamine transporter (vmat-IR), has confirmed that, in the rat, there is almost certainly a sparse adrenergic innervation of the detrusor in the lateral wall and base. However, there is a significant innervation in the bladder neck and proximal urethra (Gosling et al., 1999). In the bladder neck and urethra, these adrenergic nerves may be responsible for activation of α-adrenoceptors, resulting in contraction of the smooth muscle in the wall of the bladder neck and urethra and the maintenance of continence during bladder filling (Levin and Wein, 1979). Therefore, a significant sympathetic nerve mediated regulation of the detrusor body seems unlikely. This has important implications for our understanding of the physiology of the bladder. Relaxation involving sympathetic nerves is unlikely. Also, the therapeutic mode of action of sympathomimetic drugs, such as the selective β3-adrenoceptor agonists, that are purported to mimic an adrenergic dependent relaxation, must now be open to question.