Hypoxia and detrusor muscle function

The previous sections have described the normal voiding function of the bladder, the biomechanics of the organ and the physiology of how the bladder contracts to expel urine. It has been shown that bladder outlet obstruction has an influence on

the metabolism of detrusor smooth muscle (Hsu, Levin, Wein, Haugaard. 1994) it is likely that these changes are the result of hypoxia and this hypothesis is borne out by in vitro studies (Thomas & Fry 1996). Therefore it is important in the context of this project to consider how the metabolic state of freshly isolated cells and cell cultures could influence their function.

Detrusor contraction, mediated by second messengers, is an expensive metabolic process (Bilgen, Wein, Zhao & Levin. 1992) and tonic tension relies on aerobic metabolism to supply ATP for contraction (Levin, Haugaard, Hypolite, Wein & Buttyan. 1999). Moreover, evidence has indicated that bladder contraction is accompanied by a reduction of blood flow (Batista, Wagner, Azadzoi, Krane & Siroky. 1996) generating hypoxia, acidosis and hyperkalaemia (Azadzoi, Tarcan, Kozlowski, Krane & Siroky. 1999). The balance between aerobic and anaerobic metabolism in detrusor muscle can be further varied; for example anaerobic metabolism becomes increasingly important under conditions such as bladder outflow obstruction (Azadzoi, Pontari, Vlachiotis & Siroky. 1996). This is significant as it may provide an aetiological route for the development of pathologies such as detrusor overactivity (Azadzoi et al. 1999).

Part of the aim of this project has been to examine the physiology of cultured detrusor myocytes, how their phenotype may have changed during the culture process and how this may affect future clinical application e.g. if one were to use these cells for the creation of a functional, tissue engineered graft to augment or

replace a bladder With this in mind the next consideration is, that, unless the cells used to generate an implant are sufficiently oxygenated, both in culture and once they have been implanted that this could lead to a degradation of cell function and hence a poor graft. Once the graft had been implanted it would be unlikely to survive without adequate nutrient supply to all cells. If this were not the case for the subsequent decay would be either simply by necrosis and graft disintegration or by a chronic ischaemia inducing cellular dysfunction.

In the production of cultured cells this presents a particular problem. It is established that most cells in culture rely on anaerobic glycolysis and any oxygen they receive is generally dissolved - this in itself can create toxicity due to the generation of free radicals. There have been attempts to incorporate oxygen carriers in the culture medium - but this is not readily available (Freshney 2000). The suggestion is that for adult organ culture the optimal conditions are in an elevated oxygen environment ie. 95% (Freshney 2000). However, if one is to produce viable grafts that allow normal cell function it will be essential to incorporate some means of oxygenating the tissue. Ideally, this would be by creation of a capillary network that could be anastamosed to the endogenous vascular system, creating a blood supply to the new tissue. Various strategies for angiogenesis exist (Soker, Machado & Atala. 2000) but have not been employed in our collagen matrix or in the tissue engineered bladder implants to date.

If cultured cells are to be used in implants their biochemical profile must be measured, to determine their susceptibility to hypoxia, and whether ATP is produced, by aerobic metabolism, in similar quantities to that in normal tissue. The ability of cultured cells to regulate [Ca^^]i in the face of hypoxia will be an important factor to evaluate.

1.7 The cellular physiology of cultured detrusor myocytes.

The process of culturing cells will change their phenotype - the extent of this depends on the type of cells and the conditions under which they are cultured. Membrane receptor types may alter considerably during the culture of different smooth muscle cells - in canine airway smooth muscle culture, for example, the responses seen in fresh tissue to important autocoids (eg leukotrienes and thromboxanes) that are implicated in human airway disease are lost when these cells are cultured. However, in human airway smooth muscle cultures these responses were maintained. This indicates the importance of species selection when aiming to apply such techniques to the creation of a tissue model or tissue engineered graft (Panettieri, Murray, DePalo, Yadvish & Kotlikoff. 1989). Similarly, in the examination of colonic smooth muscle; receptor binding studies in freshly isolated tissue for vasoactive intestinal peptide (VIP) receptors (VIP normally functions through the VIP receptor to cause smooth muscle relaxation in the colon) and M2 receptor binding studies using /V-methylscopolamine (NMS) (a muscarinic

receptor antagonist - acetylcholine is the mediator of contraction in colonic smooth muscle via M2 receptors) it was found that once the cells were cultured the VIP

receptor population was maintained, however the muscarinic receptor population dropped substantially to <10% of its preculture value at 48hrs after culture. This was despite the employment of such techniques as sonication and serum withdrawal; which have previously been shown to increase muscarinic receptor expression (Yang 1990). It should also be noted that the muscarinic receptors that were present were found to be of comparable affinity (fresh cell Ky = 0.94 ± 0.35 nM and cultured cell Kd = 0.71 ± 0.25 nM) (Ennes, McRoberts, Hyman & Snape. 1992). It is therefore important to establish which functional properties are retained by detrusor smooth muscle cell cultures so that they may be useful both as potential implant material and as a biological system model.

It is also important to establish the cell type that is being cultured since it has been suggested that there may be more than one morphological cell type, as found in rabbit detrusor muscle (Lau & Chacko 1996) and also in human tracheal smooth muscle cultures (Panettieri, Murray, DePalo, Yadvish & Kotlikoff. 1989). In both instances the authors describe two morphological subtypes one of which appears to be dormant and non-dividing. The second type appears characteristically spindle shaped and abundantly proliferative; it is this type that overgrows the culture dish and forms the characteristic ‘hill and valley' distribution (Chamley- Campbell, Campbell, Ross. 1979). Whilst in the human bladder there may be extensive fibrous tissue in the specimens that are harvested (Levin, Haugaard, Hypolite, Wein, Buttyan. 1999) there has been no suggestion that there is more than one type of muscle cell. However, it has been suggested that there is a

Spectrum of cells from fibroblast through to smooth muscle cell (Lau & Chacko 1996) as both cell types express smooth muscle myosin. In this paper they described two morphological types of cell a long spindle shaped cell and a shorter, fatter cell. The long cells express vimentin (normally expressed by mesenchyme) but the short cells do not. It was suggested that such variability could be explained by conformational change to cellular vimentin during the cell cycle. Studies examining myofibroblasts have also suggested that this may be the case (Serini & Gabbiani 1999). Although there does not appear to be any definitive evidence to substantiate it, the possibility of such a spectrum of cell types exists, and that there may be specific subsets of fibroblasts that under certain conditions will alter their phenotype to that of a smooth muscle cell or myofibroblast. The likely candidates for the control of any such changes include growth factors, cytokines, adhesion

molecules and extracellular matrix (Serini & Gabbiani 1999).