SEARCH

SEARCH BY CITATION

Keywords:

  • bladder;
  • urothelium;
  • prostaglandin;
  • ATP;
  • nitric oxide

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

OBJECTIVE

To use an isolated preparation of the guinea-pig bladder lamina propria (LP) to investigate the effects of adenosine tri-phosphate (ATP) and nitric oxide (NO) on the release of prostaglandin E2 (PGE2).

MATERIALS AND METHODS

The bladders of female guinea-pigs (200–400 g) were isolated and opened to expose the urothelial surface. The LP was dissected free of the underlying detrusor muscle and cut into strips from the dome to base. Strips were then incubated in Krebs buffer at 37 °C. Each tissue piece was then exposed to the stable ATP analogue, BzATP, and a NO donor, diethylamine-NONOate (DEANO), and the effect on PGE2 output into the supernatant determined using the ParameterTM PGE2 enzyme immunoassay kit (R & D Systems, Abingdon, UK). Experiments were repeated in the presence of purinergic receptor and cyclooxygenase (COX) enzymes, COX I and COX II, antagonists. The cellular location of COX I, COX II and neuronal NO synthase (nNOS) within the bladder LP was also determined by immunohistochemistry.

RESULTS

PGE2 production was significantly increased by BzATP. Antagonist studies showed the purinergic stimulation involved both P2X and P2Y receptors. The BzATP response was inhibited by the COX inhibitor indomethacin (COX I >COX II) but not by DUP 697 (COX II >COX I). Thus, BzATP stimulation occurs because of COX I stimulation. NO had no effect on PGE2 production over the initial 10 min of an exposure. However, PGE2 output was increased 100 min after exposure to the NO donor. In the presence of NO, the BzATP stimulation was abolished. Immunohistochemistry was used to confirm the location of COX I to the basal and inner intermediate urothelial layers and to cells within the diffuse layer of LP interstitial cells. In addition, nNOS was also located in the basal urothelial layers whilst COX II was found in the interstitial cell layers.

CONCLUSIONS

There is complex interaction between ATP and NO to modulate PGE2 release from the bladder LP in the un-stretched preparation. Such interactions suggest a complex interrelationship of signals derived from this region of the bladder wall. The importance of these interactions in relation to the physiology of the LP remains to be determined.


Abbreviations
LP

lamina propria

PG(E2)

prostaglandin (E2)

ATP

adenosine tri-phosphate

(n)NO(S)

(neuronal) nitric oxide (synthase)

COX

cyclooxygenase

TBS(-T)

Tris-buffered saline (containing 0.3% (v/v) Triton X-100)

DEANO

diethylamine-NONOate.

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

When the urothelium is stretched it responds by releasing prostaglandins (PG) [1–4], adenosine triphosphate (ATP) [5], nitric oxide (NO) [6] and acetylcholine [7–9]. The detailed mechanisms that generate the release of these signals are not known. It is also not known which cells in the lamina propria (LP) are responsible for producing and releasing these signals. Furthermore, the physiological systems involving these signals target are poorly defined.

Current thinking suggests that the release of these substances is an integral part of a sensory system assessing bladder volume [10–12]. Upon stretch, ATP, acetylcholine and NO are released from the urothelium where they then act directly or indirectly upon suburothelial afferent nerves and so affect sensation. There is direct evidence showing a direct modulation of afferent nerve activity by ATP [13] and indirect action of NO [14]. However, a major complication with this concept is that there are relatively few sensory nerves adjacent to the bulk of the urothelium particularly in the lateral wall and dome.

In previous studies on the guinea-pig bladder it was noted that the basal urothelial cells expressed neuronal NO synthase (nNOS) and that the umbrella cells and the suburothelial interstitial cells responded to NO with a rise in cGMP [15,16]. These data led to the suggestion that there might be interactions between the urothelial-derived signals. Recently, it has also been shown that the intermediate and basal cells of the guinea-pig urothelium express cyclooxygenase (COX) I [17]. As these cells lie in close proximity to the NO-producing cells this further suggests possible interactions between urothelial signals. This possibility has not been examined experimentally. An understanding of such possible interactions may shed light on additional complex functions of the urothelium.

In the present study, we developed an isolated preparation of the LP to examine the mechanisms regulating the output of PGE2. We also used immunohistochemical techniques to confirm which cells are responsible for producing PGE2 in this system.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

In all, 24 female guinea-pigs (200–400 g) were used in this study. The guinea-pigs were killed by cervical dislocation and the bladders were removed and placed in modified Krebs-Henseleit solution (Sigma-Aldrich, UK) gassed with 5% CO2. The bladder was opened from base to dome to expose the urothelial surface and pinned to the bottom of a Sylgard-coated dish (RS Components, Corby, UK). The LP (defined as the tissue layers including the urothelium, suburothelial interstitial cells, diffuse cell layer space and the microvasculature) was then dissected free of the underlying detrusor muscle as a complete sheet, taking care in that no underlying muscle was attached. This was confirmed in two preparations by histological examination. By maintaining the LP as a complete sheet the micro-anatomical relationships of the different cell layers was maintained. The tissue sheet was then cut into strips, with each strip including dome, lateral wall and base. Each strip was ≈5 mm wide and 10 mm long. Up to six identical strips could be isolated from one bladder. In this way, tissue from the different regions was included in each of the experimental incubation conditions. Single tissue pieces were placed in wells of standard tissue culture plates. Each tissue piece was left unstretched.

INCUBATION CONDITIONS AND TREATMENT PROTOCOLS

At the start of each experiment (t = 0 min) strips of LP were incubated in 0.5 mL modified Krebs-Henseleit solution (Sigma-Aldrich) at 37 °C with 5% CO2 and washed four times over the next 110 min in buffer without agonists or antagonists. After a buffer change at 110 min the strips were incubated for a further 10 min (110–120 min) and then the bathing media removed and immediately stored at −20 °C. This was defined as unstimulated sample 1 (us1). The buffer was then replaced with fresh solution containing exogenous agonist (purinergic or nitrergic). The tissue was incubated for a further 10 min (120–130 min) and the buffer removed and stored at −20 °C. In experiments using receptor antagonists, the drug of interest was added 20 min before stimulation (100–120 min) and maintained in all buffer changes. After stimulation with the receptor agonists the tissue strips were again washed with 0.5 mL Krebs at 37 °C with 5% CO2 for a further 110 min (130–240 min: four buffer changes). After the change at 240 min the strips were incubated for a further 10 min (240–250 min) before the surrounding buffer was removed and immediately stored at −20 °C as a second unstimulated sample (us2). The buffer was then replaced for a second time with fresh solution containing agonists. The tissue was incubated for a further 10 min (250–260 min) and the surrounding buffer removed and immediately stored at −20 °C. Finally, the tissue was washed for a further 110 min (260–370 min) with four buffer changes. After 370 min the strips were incubated for 10 min (370–380 min), the buffer was then removed and stored as a final unstimulated sample (us3). After each experiment, the strips of LP were weighed wet to calculate PGE2 release per mg of tissue.

The drugs used were from Tocris (UK) or Sigma-Aldrich (UK). The required concentration of each drug was prepared fresh in Krebs buffer and added to the strips of LP when required.

PGE2 ANALYSIS

PGE2 levels in supernatants were determined using the ParameterTM PGE2 enzyme immunoassay kit (R & D Systems, Abingdon, UK). The assay was performed following the regular sensitivity option according to the manufacturer’s instructions. This assay is based on the competitive binding technique in which the PGE2 present in a sample competes with a fixed amount of horseradish peroxidase-labelled PGE2 for a PGE2-specific antibody. The assay was performed in a 96-well plate format and absorbance determined at 450 nm on a Bio-tek FL600 microplate fluorescence reader. The intensity of the colour in each well was inversely proportional to the concentration of PGE2 in each sample. A control assay using, either Krebs buffer alone, or Krebs buffer containing the agonists and antagonists used in this study was performed to confirm that none interfered with the assay principle.

IMMUNOHISTOCHEMISTRY

Bladder tissue was immersed in freshly prepared ice-cold 4% depolymerized paraformaldehyde for 120 min at 4 °C. Tissues were then fixed at 4 °C in 0.1 m phosphate buffer; with a 10–30% sucrose gradient over a period of 72 h. The fixed tissues were then snap-frozen in a block of Tissue-Tek O.C.T. compound using isopentane cooled in liquid nitrogen. Cryostat sections (10 µm) were then cut and thawed on to chrome-alumn-gelatin-coated slides. The sections were prepared for immunohistochemistry by drying for 15 min at room temperature followed by three washes with Tris-buffered saline (TBS; pH 7.6). The slides were then incubated overnight with primary antibodies at 4 °C. To visualize vimentin a mouse anti-vimentin antibody (Sigma-Aldrich) was used at a 1:5000 dilution. COX I and COX II were visualized using well-characterized goat polyclonal anti-COX I and anti-COX II antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) used at a 1:2000 and 1:200 dilution, respectively; the specific cross reactivity of these antibody with guinea-pig COX I and II has been previously determined using blocking peptides [18]) After overnight incubation with the primary antibodies diluted in TBS containing 0.3% (v/v) Triton X-100 (TBS-T), sections were washed in TBS, TBS-T and TBS; each wash step lasted 15 min. Rabbit primary antibodies were visualized using Alexa Fluor 488 donkey anti-rabbit IgG (H + L) conjugate (Molecular Probes, Invitrogen Ltd, Paisley, UK), diluted 1:100 in TBS-T. Mouse primary antibodies were visualized with Alexa Fluor 488 donkey anti-mouse IgG conjugate (Molecular Probes), diluted 1:100. Goat primary antibodies were visualized with Alexa Fluor 594 donkey anti-goat IgG conjugate (Molecular Probes). Sections were incubated with the secondary antibodies for 60 min at room temperature in the dark. After washing with TBS-T, and TBS, sections were mounted with TBS-glycerol. Sections were analysed and photographed using an Olympus AX70 microscope using a ×4, ×10, ×20 and ×40 objectives. For the detection of Alexa 488 fluorescence we used a narrow band-pass MNIBA-filter and for the detection of Alexa 594 we used a filter with a narrow excitation band, the U-M41007A filter (both filters from Chroma Technologies, Rockingham, VT, USA). The microscope was equipped with a cooled CCD Olympus Digital video camera F-view. Images were stored digitally as 16-bits images by using the computer program analySIS® Vers.3.0. (Soft Imaging System, Münster, Germany).

STATISTICS

Shapiro–Wilk testing for normal distribution and Levene testing for homogeneity of variance were performed before repeated measures anova. Standard natural log transformations were applied to achieve normal distribution and homogeneity of variance where required. A Student’s t-test was used for post hoc analysis. Benjamini–Hochberg corrections of P values were applied to adjust for multiple comparisons. A P < 0.05 was considered to indicate statistical significance.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

BzATP-INDUCED PGE2 RELEASE

In the absence of any stimulus, low levels of PGE2 (≈45 pg/mg tissue/10 min) could be detected in the medium bathing the LP tissue indicating a basal release (us1: Fig. 1A). Addition of the stable ATP analogue BzATP (100 µm) for 10 min increased significantly the amount of PGE2 released to a mean (sd) of 106 (14) pg/mg/10 min (P < 0.05). Upon washing, the PGE2 output returned to basal levels (us 2: Fig. 1B) and a second challenge with BzATP, some 120 min later, gave a similar significant stimulation. In the absence of stimulation with BzATP no change in basal PGE2 production was detected over the same time period (Fig. 1B). Thus, the isolated LP preparation remained viable and stable and so could be used to study PGE2 production over a period of several hours.

image

Figure 1. The production of PGE2 from the isolated LP layer of the guinea-pig bladder. The effect of 100 µm BzATP on PGE2 release from the isolated strips of LP: (A) the effect of 100 µm BzATP on PGE2 release during two 10 min stimulations 120 min apart (n = 12) and (B) the basal levels of PGE2 released from the LP in 10-min periods over a total of 380 min (n = 8). Where marked by the horizontal lines a Student’s t-test was carried out; *P < 0.05. Values indicated by the solid bars are mean values. Error bars are +1 sd.

Download figure to PowerPoint

BzATP-stimulated PGE2 production was inhibited by indomethacin (a predominantly COX I inhibitor) (Fig. 2B) but the more selective COX II inhibitor DUP697 had little or no effect (Fig. 2C). From these results, it can be tentatively concluded that BzATP-stimulated PGE2 production is via a COX I-regulated pathway.

image

Figure 2. The role of COX 1 and COX 2 in PGE2 release from the LP: (A) the control BzATP response as shown in Fig. 1A (n = 3), (B) the BzATP response in the presence of 10 µm of the predominantly COX I inhibitor indomethacin (n = 3), and (C) the BzATP response in the presence of 10 µm of the predominantly COX II inhibitor DUP 697 (n = 3). Where marked by the horizontal lines a Student’s t-test was carried out; *P < 0.05. Values indicated by the solid bars are mean values. Error bars are +1 sd.

Download figure to PowerPoint

The involvement of purinergic receptors in the stimulation of PGE2 production was assessed using the nonspecific P2 purinergic antagonists; PPADS and suramin. Incubation with PPADS (Fig. 3B) and suramin (Fig. 3C) reduced significantly the BzATP-induced PGE2 production to 70% and 54%, respectively (P < 0.05). However, if PPADS and suramin were added together the BzATP-induced response was dramatically reduced to basal levels (Fig. 3D; P < 0.01). Thus, it can be concluded that both P2X and P2Y receptors are involved in the BzATP-induced PGE2 release. These results give no specific information on which specific subtypes of purinergic receptors are involved, this must await further experiments.

image

Figure 3. The effects of the purinergic receptor antagonists on PGE2 release from the LP: (A) the control BzATP response as shown in Fig. 1A (n = 5), (B) the BzATP response in the presence of 100 µm PPADS (n = 5), (C) the BzATP response in the presence of 100 µm suramin (n = 5), and (D) shows the BzATP response in the presence of a combination of PPADS and suramin (n = 5). Where marked by the horizontal lines a Student’s t-test was carried out; *P < 0.05, **P < 0.01. Values indicated by the solid bars are mean values. Error bars are +1 sd.

Download figure to PowerPoint

THE EFFECTS OF NO ON BASAL AND BzATP-STIMULATED PGE2 RELEASE

A brief exposure of the tissues for 10 min, to the NO-donor, diethylamine-NONOate (DEANO, Sigma-Aldrich) resulted in no change in basal PGE2 production (Fig. 4A) However, after 110 min this brief stimulus significantly increased PGE2 output from the mean (sd) basal level of 30 (9) to 57 (7) pg/mg tissue/10 min (P < 0.05). This late rise in PGE2 production appears to be a direct consequence of the exposure to DEANO, as there was no effect in matched unstimulated preparations (us1–3; Fig. 4B). Subsequent stimulation with BzATP resulted in a further significant increase in PGE2 release to 97 (9) pg/mg/10 min (P < 0.05), indicating that the tissues remained responsive. In a further series of experiments, DEANO and BzATP were added together to the bathing medium. Under these circumstances, no increase in PGE2 was detected (Fig. 4C). In fact, the data show that there was a small but significant fall in basal PGE2 production compared with control levels, from 28 (3) to 11 (1) pg/mg tissue/10 min (P < 0.05). After washing for a further 110 min (us2) the output of PGE2 was again higher than control (us1: 54 (14) pg/mg tissue/10 min: P < 0.05) which was also further significantly augmented by the addition of BzATP (104 (16) pg/mg tissue/10 min: P < 0.05). Thus, NO appears to have complex effects on this preparation. In the short-term NO abolishes the effects of added BzATP. However, this brief exposure also appears to initiate processes that result in an increase in PGE2≈100 min later.

image

Figure 4. The effects of NO on the basal production and BzATP-stimulated release of PGE2 from the isolated guinea-pig LP. (A) The effect of 10 min stimulation with 5 µm of the NO donor DEANO (NoNoate) on PGE2 release from the isolated LP and subsequent repeat stimulation for 10 min with 100 µm BzATP at 250 min (n = 3). (B) Basal levels of PGE2 released from the LP in 10-min periods over a total of 380 min (n = 3). (C) The effect of 10 min stimulation with a combination of 5 µm of DEANO and 100 µm BzATP on PGE2 release from the isolated LP and a subsequent repeat stimulation for 10 min with 100 µm BzATP alone at 250 min (n = 3). Where marked by the horizontal lines a Student’s t-test was carried out; *P < 0.05, **P < 0.01. Values indicated by the solid bars are mean values. Error bars are +1 sd.

Download figure to PowerPoint

IDENTIFICATION OF THE CELLS RESPONSIBLE FOR PGE2 PRODUCTION

The functional data suggest that the cells responsible for the BzATP-stimulated PGE2 release express COX I rather than COX II. Sections of the lateral bladder wall were immunostained using antibodies against COX I, COX II, and vimentin (a marker for interstitial cells) (Fig. 5). COX I immunoreactivity is located primarily in the basal and lower intermediate layers of the urothelium (Fig. 5A). It is also found within a sparse population of interstitial cells in the LP but is not located within the dense layer of suburothelial interstitial cells. In contrast, COX II (Fig. 5B) is located to a few of the umbrella cells where it is closely associated with the nuclei. There was no COX II in the basal or intermediate layers of the urothelium, only in the suburothelial interstitial cells. These data strongly suggest that the bulk of the PGE2 production measured in the present study may originate from the basal and lower intermediate layers of the urothelium, although there may be a small contribution from the sparse network of deep LP interstitial cells. Figure 5C shows the basal cell layer of the urothelium that also expresses nNOS. The distribution of nNOS is restricted to the basal layer, while COX I is present in the basal layer but also in the intermediate urothelial cell layers.

image

Figure 5. Localization of COX I and COX II immunoreactivity in the urothelium and suburothelium. (A) Shows an area from a representative section of the urothelium and suburothelium stained with antibodies to COX I (red) and vimentin (green). The adjacent panels show the individual images showing the location of COX I and vimentin confirming that that there cells expressing COX I in the urothelium but not in the suburothelial interstitial cell layer. (B) Shows a region of the urothelium and suburothelium stained with antibodies to COX II (red) and vimentin (green) showing that the COX II immunoreactivity is now associated with a few umbrella cells (nuclear staining) but predominantly with the cells in the suburothelial interstitial cell layer (vimentin-positive cells). (C) Shows a region of the urothelium and suburothelium stained with antibodies to COX I (red) and nNOS (green) showing that nNOS immunoreactivity is in the same basal COX I-expressing cells and adjacent to the COX I expressing intermediate layers of the urothelium. iuc, intermediate urothelial cells, buc, basal urothelial cells, suics, suburothelial interstitial cells, lpics, LP interstitial cells. Calibration bars 80 µm.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

It has been recognised for many years that the urothelium is capable of releasing several substances in response to stretch: PG [1–4], ATP [5], NO [6] and acetylcholine [7–9]. The physiological systems that involve these substances are not known. It has been considered that the stretch-generated output of PG may have a role in modulating bladder smooth muscle activity. Indeed, the detrusor expresses receptors to PGs and activation of these receptors results in contraction [18–23]. Similarly, ATP released from the urothelium has the potential to activate the muscle directly via purinergic receptors on their surface [24–26].

Much work has been done to explore the concept that the urothelium has a sensory role [5–8,10–13]. Here the concept is that substances released from the urothelium in response to stretch as the bladder fills activate afferent nerve fibres, thus conveying information to the CNS related to bladder volume [10–13]. There is now good direct evidence supporting this idea. Application of ATP to the bladder has a direct modulatory effect on afferent nerve discharge [13]. Also, there is indirect evidence that NO may have a similar role, as interference with NO synthesis interferes with the micturition cycle [14]. An interesting development of this sensory concept has been the suggestion that the urothelium activates afferent nerves indirectly via a specialized cell type: the myofibroblast [22,27–29]. Here, the idea is that urothelial-derived substances, principally ATP, act upon the myofibroblasts to cause a contraction. This local contraction then distorts afferent nerve endings resulting in an increased discharge. Although an interesting concept, there is as yet no physiological data to substantiate such a system. However, despite for the most part these roles for urothelial-derived substances being demonstrable they cannot be the entire story. Firstly, for substances released from the urothelium, diffusion distances to the muscle layers, typically 1 mm in the guinea-pig lateral wall, are sufficiently large to make this a slow signalling process. Secondly, particularly in the lateral wall and dome of the bladder, the density of afferent nerves associated with the urothelium is considerably lower than in the base [30–33]. Thus, urothelial-derived signals in these regions are unlikely to encounter afferent fibres in close proximity. If this is so then there may be additional physiological systems involving the release of urothelial-derived substances yet to be discovered.

It is clear that the urothelium and the suburothelial structures may be involved in complex physiological systems. Understanding this complexity has been compounded by the fact that detailed microanatomy involving cell types and cell systems differ in different species. For example, the umbrella cells and suburothelial cells of the human and guinea-pig LP respond to NO with a rise in cGMP [15,16], while those in the mouse and rat do not [34]. Similarly, nNOS is located to the basal layer in the guinea-pig bladder but not the mouse [16,35]. Based on the observations in the guinea-pig, including the presence of NO-producing cells in the basal layers of the urothelium and the activation of cGMP production in the umbrella cells and suburothelial interstitial cells, it was therefore suggested that complex signalling processes occur within the urothelium [16,35].

Using the guinea-pig model, the present study shows further complexities in both the physiology of the urothelium and in the specialization of different cell types and layers in the generation and modulation of urothelial-derived signals. In the present study, we concentrated on the output of PG and its regulation by ATP and NO. In the published literature, it has been tacitly assumed that stretch activates the release of the different signals. The possibility that there may be interactions between the urothelial signals not associated with stretch has not been widely considered. The present data show that, in the absence of an overt stretch ATP and NO influence PGE2 output. This of course does not imply that PGE2 production is not activated by stretch. The data simply suggest a complex interaction of signals regulating PGE2 production. In other cell systems it is well documented that there can be interactions between signals, such that NO influences PGE2 production and that PGE2 affects NO production [36,37]. The present data suggest that such interactions occur in the guinea-pig urothelium.

It is clear that ATP, in this case the stable analogue BzATP, can activate PGE2 production by a complex mechanism that involves both P2X and P2Y receptors. It was beyond the scope of the present study to elaborate further on the subtypes of purinergic receptors involved; however, this is the subject of ongoing research in our laboratory. Based on the inhibitory actions of indomethacin (COX I >COX II inhibitor) and DUP697 (COX II > COX I inhibitor) it is apparent that the ATP-induced PGE2 production is via a COX I system. That most of the COX I enzyme is located to the basal and intermediate cell layers of the urothelium point to these as being the cells involved.

NO has a short-term inhibitory action on PGE2 production. It has previously been reported that the basal urothelial cell layer expresses nNOS and that this cell layer may be a potential physiological source of NO [35,38]. These cells are in close proximity to those expressing COX I suggesting that NO released by this cell layer could affect COX I activation, either directly or indirectly. A more detailed analysis of the interplay of signals and an understanding of the complexity of this system must await further experimentation.

The present data suggest a complex interaction of signals generating and modulating PGE2 output from the urothelium. The reasons for this complexity are not known but it must reflect a key function of the urothelium. If the output of PGE2 is regulated by ATP and NO, it is also possible that the output of these signals may be similarly and complexly regulated. The cellular targets for the different urothelial-derived signals may be the urothelium itself but may also be other structures and systems. As discussed above the derived output may be targeted to afferent fibres. However, an alternative may be that the urothelial output is targeted to the cells of the suburothelium, the suburothelial interstitial cells. In the guinea-pig these cells respond to NO with a rise in cGMP [15,16]. There are also emerging data suggesting they also express PG type 2 receptors [39] and P2X7 purinergic receptors [unpublished observations]. Thus, these cells are a potential target for the urothelial-derived output and they also have the capacity to further integrate such signals. The physiology of this system remains an enigma and a major challenge to functional urology.

Figure 6 summarizes these findings and ideas. The signals acting upon the cells producing PGE2 are shown in Fig. 6A, together with possible intracellular signalling pathways that might be involved. Figure 6B shows the cell types and cell layers found in the LP. It specifically shows the different cell types in the urothelium and the signals they produce. There can be no doubt that the urothelium subserves more complex functions than just a simple barrier between the urine and the body tissues. The present data point the way for future studies to unravel this complexity.

image

Figure 6. Cartoons showing the cells and cellular processes that might be involved to influence the production and regulation of PG synthesis in the guinea-pig urothelium. (A) shows the excitatory input (ATP) and inhibitory input (NO) on COX I-regulated PG production. The ATP response occurs via both P2X and P2Y purinergic receptors. The mechanism of the inhibitory action of NO is to date not clear. (B) Shows the cell types in the urothelium and suburothelium of the guinea-pig bladder. The cell layers identified in the urothelium are the umbrella cells, the intermediate layers (two cell layers) and the basal layer. The location of the possible ATP-secreting cells (synaptic vesicle protein 2-positive), COX I and nNOS cells are shown. The interstitial cells in the LP are also shown. The suburothelial interstitial cells (su-ics) are associated with COX II while there is a subpopulation in the LP (lp-ics) that are associated with COX I. GMP, guanosine monophosphate, EP2, PG type 2 receptor, M3, muscarinic type 3 receptor.

Download figure to PowerPoint

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

We gratefully acknowledge the support of BJUI who supported this work with an International Collaborative Award. We are also indebted to Simone Grol and Rick de Jongh for their contribution to the immunohistochemistry.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES