Over the past few years an enormous amount of information about the transport mechanisms has been accumulated in the so-called tight epithelia. Various morphological and electrophysiological techniques have been used to examine the transporting membrane structures, membrane shuttling process, trans-membrane signalling, membrane electrical parameters and the major transport properties in urinary bladder of freshwater turtle(Nagel et al., 1981 Gluck et al., 1982 ; Schwartz et al., 1982 ; Steinmetz et al., 1981 ; Reeves et al., 1983 ; Stetson and Steinmetz, 1983, 1985 ; Clausen and Dixon, 1986 ; Dixon et al., 1986, 1988). The turtle bladder epithelium has been known to reabsorb sodium chloride and secrete proton to dilute and acidify the urine, thus contributing to the maintenance of osmotic and acid-base balance of the body. Moreover, turtle bladder has been known to possess the secretory mechanism of bicarbonate to alkalinize the urine (Steinmetz and Stetson, 1986). Recent studies have been concerned with the relationship between the membrane transport and its relations to the intramembrane structural specialization, namely intramembrane particles which are believed to be components of especially proton pump. In particular, freeze-fracture microscopy is essentially the only method available for detailed examination of the membrane structures and the full three-dimentional organization of cells, as well as the surface area of intracellular organelles. In recent years, the á and β type of carbonic anhydrase-rich cells were described within the turtle bladder tissues by Stetson and Steinmetz(1985). Both cell types are believed to be responsible for proton transport, but in opposite directions. At present, the developmental relationship of the two cell types is an important topic : are they the same cell type in different functional states or are they distinctly different cells using the same specialized transport system? The present work is to attempt to further describe the major cell types that is believed to be responsible for a single transport system, with particular attention to the proton secretion. In addition, some aspects of the cellular characteristics of the turtle bladder cells, as examined by means of conventional and freeze-fracture electron microscope, will be described. In the present work, three major cell types can be distinguished on the basis of electron microscopic observations of cells in the bladder mucosa. They are the granular cells, the a and β type of carbonic anhydraxe (CA) -rich cells. When looked at under electron micrographs, basal cells are observed being interposed between the basal lamina and the overlying mucosal lining cells. The goblet cells are also found within this bladder tissue but it is known to secrete only a mucous substance over the luminal surface. Figure 1, modified from the direct experimental results, diagrams the features of bladder mucosa. The following descriptions of three distinct cell types apply to cells of turtle, Pseudemys scripta, urinary bladder and are listed in Table(Distinguishing features of urinary bladder cells in Pseudemys scripta).
1. The granular cells
The granular cells, which are found predominantly in the bladder, are the site of sodium transport and are characterized by relative short microvilli on their apical membrane. Grarlules are abundantly seen in the apical cytoplasm. When examined with freeze-fracture microscopy(Figs. 12, 13), the cytoplasm contains ER, few mitochondria, lysosome, and Golgi complex occurrs immediately above the nucleus.
The granular cells perform most of the sodium transport. Its apical plasma membrane has specific amiloride-binding sodium channels, while the basolateral plasma membrane has potassium channels and the ouabain-binding Na-K pump. It has also known that the potassium channels of the basolateral rnembrance are closed by addition. of Ba2+ to the serosal bath. The transepithelial sodium transport in the granular cell(G) is revealed in Figure 2. As shown in this figure, the entry of sodium across the mucosal membrane is believed to be rate-limiting for overall sodium reabsorption, implying that this pathway is a key regulatory point for electrolyte and water balance. The active step in transepithelial sodium transport is energy-dependent process, which is catalyzed by the Na-K ATPase. It has also known that the granular cells are the target cells for the two hormones, ADH and aldosterone, that regulate salt and water transport in many sodium-transporting epithelia.
2. The á type of CA cells
In turtle bladder á type of CA cells contain numerous mitochondria, which are heavily concentrated in the infranuelear cytoplasm. This type of cells are also characterized by irregular microvilli on their apical surface area(Fig. 3) and usually round in shape. In á CA cells, the apical plasma membrane shows a distinctive rod-shaped intramembrane particle, possibly the integral membrane components of the proton pump. The apical inner membrane of a-type cell, as viewed in freeze-fractur electron micrograph, is illustrated in Figure 5. These intramembrane particles on P-face of the apical membrane are the mostly prominent structure, but these particles do not appear on the basolateral membrane of the a cell.
The á type of CA cells are responsible for the proton secretion using the proton pump on the apical plasma membrane. In contrast, the basolateral membranes possess chloride channel, as well as exchangers for bicarbonate transport.
3. The β type of CA cells
The epithelial cell in Figure 4 is â type of cell(B) which has a regular fingerlike microvilli on their apical surface area. As shown in this figure, the apical surface area is small, and mitochondria are widely distributed in the infranuclear cytoplasm. The basal surface of the p type of cell usually faces the serosal side which have very different transport properties from the apical surface. When looked at under freeze-fracture electron micrograph, any rod-shaped particles do not appear on the apical surface membrane unlike in the a type of CA cell(Fig. 6).
The turtle bladder â type of epithelial cell, as illustrated in Figure 2, is believed to be responsible for bicarbonate secretion via an oppositely-directed proton pump on the basolateral plasma membranes.
The urinary bladder tissues have become a model system for examining the epithelial transport mechanisms. In the bladder of freshwater turtle, three main cell types have been identified on their mucosal surface. They are the granular cells, á CA cells, and b CA cells. These cells have the ability through active transport systems to absorb sodium, secrete proton, and exchange chloride and bicarbonate(Schwartz, 1982). However, the basal cells do not reach the mucosal surface and are not involved in active transport properties unlike the other types of cells that are exposed to the surface. It appears that these types of cells are similar to those in toad bladder. For example, several investigators have shown that mitochondria~rich cells in toad bladder are characterized by small' area of luminal surface and differ from the adjacent granular cells(Wade et al., 1975 ; Wade, 1976 ; Dibona, 1978).
In turtle bladder, the granular cells have the active sodium transport systems and play a fundamental role in regulation of the ionic environment between mucosal side and serosal side. In the early work of sodium transporting epithelia, the twomembrane theory, as proposed by KoefoedJohnsen and Ussing(1958), has been suggested that the active sodium transport across the frog skin allows for the passive entry of sodium at the apical membrane and its active extrusion at the basolateral surface. They also revealed that the basolateral membrane has potassium channels, but that it was recycled across the basolateral membrane of the cells, while the apical membrane was virtually tight to potassium. In fact, two-membrane theory has served well to explain many of the transport properties of especially tight epithelia. However, it is now known that the apical membrane has sodium channels, but that this membrane is thought to be always tight to chloride.
On the basis of the two-membrane theory, it is evident that sodium passively enters the granular cell across the apical membrane and is subsequently transported to the serosal solution by an active pro~ cess across the basolateral membrane. It is also known that the active sodium transport is carried out by a Na-K pump having a coupling ratio of 1.5(Nielsen, 1979). In addition, a key enzyme to provide energy for active sodium transport, Na-K ATPase, is usually located on baso~ lateral membrane. In turtle bladder granular cells, the apical surface possesses an amiloride -sensitive sodium channel, while the basolateral surface possesses an ouabainsensitive Na-K pump. Conclusively, it appears that active sodium transport occurs via a two-step process, implying that first, sodium diffuses into the cells, followed by an ener gy-de pendent efflux step, which is catalyzed by the ouabain-sensitive Na-K ATPase(Sariban-Sohraby and Benos, 1986 ; Garty and Benos, 1988).
The á and β type of cells have been described in case of turtle bladder tissues by Stetson and Steinmetz(1985). In either types of cell, they possess a small area of mucosal surface in contrast to the granular cells. In both cell types, they have a high level of carbonic anhydrase in their cytoplasm and densely packed mitochondria in especially infranulear cytoplasm. It is well known that cytoplasmic carbonic anhydrase generates proton and bicarbonate from water and carbon dioxide. Therefore, it has become apparent that each is believed to be responsible for a single major transport mechanism, implying that a type of cell is responsible for the proton secretion using the proton pumps on the apical plasma membrane, while the p type of cell secretes bicarbonate via an oppositely-directed proton pumps in their basolateral plasma membrane.
When examined by the freeze-fracture techniques, the apical surface membranes of a cells have a characteristic population of rod-shaped intramembranous particles which are believed to be components of the proton pumps. Moreover, distinctive rod-shaped intramembrane particles has been described in freeze-fracture electron micrographs of plasma membranes from many cells thought to be responsible for acid transport(Humbert et al., 1975 ; Stetson et al., 1980 ; Stetson and Steinmetz, 1985). In turtle bladder a CA cells, this particle is the only prominent structure in the apical membrane. Conversely, β CA cells show rod-shaped particles in their basolateral membranes, which is consistent with the proton absorptive, bicarbonate secretory mechanism proposed by Stetson et al.(1985) and Stetson and Steinmetz (1985).
In the turtle bladder it has been demonstrated that C02 stimulates proton secretion, at least in part, by causing fusion of vesicles enriched in proton pumps with the apical plasma membrane(Cohen et al., 1980 ; Husted et al., 1981 ; Cohen and Steinmetz, 1980 ; Stetson and Steinmetz, 1983). Stetson and Steinmetz(1986) reported that the addition of exogenous C02 to the media caused a large increase in the CA cells apical area compared with bladders maintained without C02 and that this increase was brought about by the fusion of the membrane of cytoplasmic vesicles to the apical membrane. It has been also described that C02 addition increased the transport rate, presumably by lowering intracellular pH, whereas intracellular alkalinizaion inhibited the rate(Cohen and Steinmetz, 1980).
In many transporting epithelia, the mechanisms by which cells regulate the composition of their plasma membrane are the focus of current research on the membrane transport. Since the work of Brown and Goldstein(l986) on the receptor-mediated endocytosis, it has become established that this process, referred to as membrane recycling or vesicle recycling, involves the exocytotic fusion of cytoplasmic vesicles with the plasma and the endocytosis of specialized region of the plasma membrane. More recently, Brown(l989) reported that in the intercalated cells of the kidney. collecting duct, hydrogen ion secretion was controlled by the recycling of vesicles carrying proton pumps to and from the plasma membrane. These similarities are of interest because the collecting duct intercalated cell is a functional and morphological analogue of the acid-secreting a cell in turtle bladder. This means that the a CA cells of turtle bladder possess intracellular vesicles carrying proton pumps which are recycling back to the apical plasma membrane, but whether the recycling of vesicles stimulates acid transport remains to be determined.
β CA cells