Histochemical and elemental localization of calcium in the granular cell subapical granules of the amphibian urinary bladder epithelium.код для вставкиСкачать
THE ANATOMICAL RECORD 218:229-236 (1987) Histochemical and Elemental Localization of Calcium in the Granular Cell Subapical Granules of the Amphibian Urinary Bladder Epithelium WALTER L. DAVIS, RUTH GWENDOLYN JONES, H.K. HAGLER, GENE R. FARMER, AND DAVID B.P. GOODMAN Department of Anatomy, Baylor College of Dentistry, Dallas, TX 75246 (WLD., R.G.J., G.RX), Department of Pathology, The University of Texas Health Science Center, Southwestern Medical School, Dallas, TX 75212 (H.K.H.),Department of Pathology and Laboratory Medicine, The University of Pennsylvania Medical School, Philadelphia, PA 19104 @. B. P. G.) ABSTRACT The ultrahistochemical analysis of apical granules in the epithelial cells, i.e., granular cells, of the amphibian urinary bladder using the N,N-naphthaloylhydroxylamine procedure identified the presence of calcium in these structures. Subsequent analytical microscopy employing fresh-frozen ultrathin cryosections for X-ray microanalysis of the granules further confirmed the above histochemical findings. In addition to calcium, elemental analysis indicated the presence of magnesium, phosphorus, sulfur, silicon, potassium, and chlorine either within or in close proximity to the granules. The possibility that these granules function as subcellular compartments for the uptake and storage of calcium ions, in a way similar to mitochondria, and thus function in intracellular calcium homeostasis, is discussed. Additionally, a role for this cation in the secretion of granular glycoproteins, i.e., stimulus-secretion coupling, is hypothesized. The amphibian urinary bladder has been used exclusively as a simple in vitro model system for the mammalian distal nephron (Leaf, 1965). The peptide hormone vasopressin (antidiuretic hormone or ADH) produces two well-established physiological effects on the toad urinary bladder: 1)the hormone induces a n increase in the permeability of the tissue to both water and low molecular weight solutes (urea, etc.) allowing for the bulk movement of fluid (water) down a mucosal to a serosal osmotic gradient, and 2) ADH stimulates active transepithelial sodium transport (Leaf, 1965,1967). The mucosal epithelium is a transitional epithelium and has been the subject of numerous fine structural studies (Peachey and Rasmussen, 1961; Choi, 1961; Davis et al., 1974). It is this epithelium that represents the major permeability barrier to water (Leaf, 1967; Parisi and Piccinni, 1973). The epithelium is comprised of four cell types: 1)granular cells, 2) mitochondria-rich (MR) cells, 3) goblet cells, and 4) basal cells (Peachey and Rasmussen, 1961). Only the basal cells lack a luminal face. Of these four cells, the granular cells are the most numerous, comprising approximately 90-95% of the epithelial cell population (Peachey and Rasmussen, 1961; DiBona et al., 1969a,b,; Davis et al., 1974). The next most abundant cell of the mucosal epithelium is the MRcell (10-15%) (Peachey and Rasmussen, 1961; DiBona et al., 1969a,b; Davis et al., 1974). The granular cells are believed to be the major site of vasopressin action in this tissue (DiBona et al., 1969a,b; Davis et al., 1974). Granular cells, as their name implies, are characterized by the presence of numerous membrane delimited, electron dense granules, which are located often in clusters or “stacks” immediately subadjacent to the luminal 0 1987 ALAN R. LISS, INC. (apical) microvillated border (Choi, 1963; Masur et al., 1972; Gronowicz et al., 1980). Both ultrastructural and cytochemical studies have indicated that these granules contain complex carbohydrates (glycoconjugates, glycoproteins), which represent precursor molecules that are secreted by exocytosis apically to become components of the PAS-positive, luminal membrane associated glycocalyx (Choi, 1963; Masur et al., 1972; Pisam and Ripoche, 1973; Strum and Ekblad, 1977; Gronowicz et al., 1980; Masur et al., 1986). All of these observations prompted our present investigation of the ionic composition of the apical granules, particularly the calcium content of these structures since this cation is known to be involved in a variety of cellular secretory processes (Rasmussen and Goodman, 1977). MATERIALS AND METHODS Large female toads (Bufo marinus) were obtained from National Reagents Inc., Bridgeport, CT, and maintained at room temperature on a bed of moistened peat. The urinary hemibladders were excised from doubly pithed animals and placed immediately in Ling-Ringer’s phosphate buffer of the following composition (mM): 1) NaC1, 92.7, 2) KC1, 2.5, 3) CaC12, 1.0, 4) MgS04, 1.2, 5) NaHC03, 7.8, 6) NaH2P04, 1.8, 7) Na2HP04, 1.2, pH 7.47.6; osmolarity, 200-220 mosmolkg H2O (Ling, 1962). The hemibladders were then gently stretched and mounted, mucosal surface up, while under buffer to tefReceived September 3, 1986; accepted March 2, 1987. Address reprint requests to Dr. Walter L. Davis, Baylor College of Dentistry, Dallas, TX 75246. 230 W.L. DAVIS ET AL. lon screens. After mounting, the buffer was replenished with fresh solution. The mounted hemibladders were maintained in plastic dishes containing 25 ml of fullstrength buffer. The solution was continuously aerated during this preincubation period. Buffer was changed each 30 min. For this study, hemibladders from 6 toads were used for each phase of the project. Thus, matched hemibladders from 6 toads were used for the histochemical study (see below) and another 6 were used for elemental analysis (see below). Continuing, one block of embedded tissue from each hemibladder (12 blocks), or one block of fresh-frozen tissue from each hemibladder (12 blocks) were sectioned. Two grids from each sample were evaluated, photographed, andor analyzed. For the selective ultrahistochemical localization of cellular calcium, the procedure of Zeichmeister (1979) was employed. This technique uses the sodium salt of N,Nnaphthaloylhydroxylamine (1,8-CloH6CON(ONa)CO), NHA, to localize subcellular calcium in the form of the electron dense precipitates of the NHA-calcium salt. Potassium pyroantimonate and potassium hydroxyantimonate procedures have been used extensively for the ultrastructural localization of calcium ions (Simson and Spicer, 1975). However, the specificity of this technique has been criticized (Klein et al., 1972). A much more specific ultrahistochemical technique for the localization of calcium, employing NHA, was utilized in the present study. The NHA method appears to have a greater affinity, a greater sensitivity, a greater selectivity, and the potential to detect sites with tightly bound calcium (Zeichmeister, 1979; Slocum and Wang, 1982) than the previously employed antimonate procedures, which are not specific, selective reagents since sodium and magnesium can also participate in the reaction. NHA has been used to detect calcium in microanalytical chemistry as well a s microscopic demonstration of this cation (Voight, 1957). NHA is characterized by its sensitivity for calcium, which is approximately 100 times higher than other detection reagents currently employed in ultrahistochemistry, i.e., potassium pyroantimonate, potassium oxalate, and murexide (Zeichmeister, 1979). For this procedure, the physiologic buffer covering the mounted hemibladders was decanted and replaced with freshly prepared, cacodylate-buffered (0.05) M, pH 7.2) 2.5% glutaraldehyde. Tissues were incubated in this prefixation medium for 30-60 min at 4°C. After this primary fixation step, the tissues were washed in cacodylate buffer prior to incubation in 0.01 mM NHA (Sigma Chemical Co., St. Louis, MO) for 5 min a t RT. A secondary fixation was then carried out for 60 min a t 4°C in the previously mentioned glutaraldehyde. After several washes in cacodylate buffer, the tissues were postfixed in 1.0% osmium tetroxide buffered with cacodylate (0.05 M, pH 7.2) for 60 min. After osmication, the tissues were washed several times in cacodylate buffer. While in the last buffer wash, the hemibladders were removed from their frames and diced into longitudinal strips approximately 2 mm in width. The tissues were then dehydrated and subsequently placed into a 1:l mixture of propylene 0xide:Durcupan (Fluka AG, Buchs SG, Switzerland) for 120 min. This was followed by pure Durcupan (14 hr) and finally by embedment in Durcupan (12-24 hr) at 37OC-45"C. Ultrathin sections were cut with freshly prepared glass knives, mounted to cop- per grids, and examined unstained in a Philips 300 transmission electron microscope (Philips Electronics Inc., Mount Vernon, NY). Subsequently, sections were doubly stained with uranyl acetate and lead citrate and photographed. For the microanalysis of the reaction products, dark gold sections were prepared, mounted to copper grids, and subsequently carbon coated prior to elemental analysis (see below). For the preparation of fresh-frozen cryosections for analytical X-ray microanalysis, the techniques of Hagler et al. (1983, 1984) were employed. Hemibladders were removed from the buffer wash still attached to their screens and placed directly into dissection dishes under liquid nitrogen (LN). While under LN, the hemibladders were sectioned into strips with LN-cooled razor blades. A single specimen was then mounted in a specimen holder and subsequently transferred and mounted in the specimen arm of a DuPont-Sorvall FS-1000 cryostem (DuPont Instruments, Sorvall Operations, Newtown, CT) attachment for the MT-5000 ultramicrotome. The specimen and knife temperatures were maintained a t - 120°C or colder in order to prevent the freeze drying of sections within the cryosystem. Dark gold sections (1,000-1,200 A in thickness) were cut on 45" glass knives using a clearance angle of 6". Sections were picked up on 50 mesh, formvar-carbon coated copper grids. The grids were then transferred to a holding block within the cryosystem. While in the holding block, the sections were "sandwiched" beneath a formvar film. The cold stage was then used to transfer the frozen-hydrated ultrathin sections to the electron microscope. Due to the lack of recognizeable morphology in the hydrated state, all sections were freeze-dried prior to X-ray microanalysis. This was accomplished in the electron microscope by "warming" the cold stage to -100°C. Usually, after 30 min the sections were dry as judged by a stable continuum and the recognition of fine structural detail. For analytical electron microscopy, a JEOL lOOC TEMSCAN (JEOL, U.S.A., Medford, MA) equipped with a 32 mm2, 158 eV resolution, lithium-drifted, silicon, energy dispersive X-ray detector was used. The X-ray spectra were collected and analyzed by a Tracor-Northern TN-2000 multichannel analyzer. The microscope was operated in the STEM mode a t 80 keV, 50 microamps emission current, 30" specimen tilt, with a beam diameter of 20 nm. X-ray spectra were collected over the energy range of 0-20 keV for 50 sec a t a resolution of 20 evlchannel. The selected-area raster mode was used to collect spectra from each sample. Ten granules were analyzed per sample for 20 sec each and their spectra summed. The latter were then subjected to a multipleleast squares fitting routine using reference spectra obtained by the analysis of single crystal specimens. The analysis of spectra were performed with the DEC PDP 11/05 computer-based Tracor-Northern NS 880 unit. RESULTS In routinely fixed and prepared ultrathin sections of the amphibian urinary bladder for transmission electron microscopy, the granular cells of the epithelium are characterized by the presence of numerous subapical dense granules (Fig. 1). These structures are round to ovoid (ellipsoid) in shape and are located immediately subadjacent to the lumina1 plasmalemma. A few morphologically similar granules were occasionally ob- TOAD BLADDER GRANULAR CELL 231 Fig. 1. Transmission electron micrograph (TEM) of the mucosal epi- dense, ovoid secretory granules (arrows). Several of these granules thelium from a hemibladder fixed in cacodylate-buffered paraformal- seem to possess eccentric densities (arrowheads). A few of the granules dehyde, osmicated, and subsequently embedded in low viscosity resin. appear to be in the process of exocytosis (e). L, bladder lumen; M, Section double-stained with uranyl acetate and lead citrate. Several microvilli; g, glycocalyx; N, nuclei. The morphology of the epithelial granular cells are seen (G). Note the numerous subapical, electron cells is identical when the tissues are fixed in glutaraldehyde. X11,500. served in mitochondria-rich cells. Fine structural observations showed the granular cell granules to be membrane delimited with varying degrees of electron density. A few showed a n eccentric electron dense structure or core. Some granular membranes were in close proximity to the luminal cell membrane remniscent of ongoing exocytosis. In tangential sections, the ovoidshaped granules were organized into clusters or aggregates. Cytochemically, a n electron dense reaction product was seen within the granules in tissues exposed to the sodium salt of NHA (Fig. 2). Apical and basolateral cell membranes, mitochondria, nuclei, and nucleoli also showed evidence of a positive reaction. Direct evidence for the presence of calcium, as calcium N,N-naphthaloylhydroxylamine, in the electron dense reaction product seen within the subapical granules of these cells was confirmed by X-ray microanalysis of semithin sections (Fig. 3). This X-ray spectrum represents a summed analysis of 10 separate granules, each analyzed for 20 sec. The latter procedure was performed a total of 10 times. The identifiable peaks shown in this spectrum are: 1) silicon (Si-K,), 2) phosphorus (P-K,), 3) cholorine (Cl-Ka), 4) calcium (Ca-K,, Kb), 5) copper (Cu-K,, Kb), and 6) osmium (OS-K,). The presence of osmium, chlorine, and copper is the direct result of the preparative techniques utilized for the tissues. Thus, osmium is from the postosmication step, chlorine is from the embedding medium, and copper is from the grid. From this the granules appear to contain silicon, calcium, and phosphorus. Using STEM, freeze-dried cryosections photographed at - 100°C showed good preservation and retention of the fine structure of the hemibladder epithelium (Fig. 4). Large areas showing minimal ice damage were seen. Under these conditions, the granules appeared a s ovoid to elongate structures of varying density (Figs. 4, 5). Xray microanalysis of the granules in freeze-dried sections showed the presence of the following ions: 1)magnesium (Mg-K,), 2) silicon (Si-Ka), 3) phosphorus (P-K,), 4) sulfur (S-K,), 5 ) chlorine (Cl-K,), potassium (K-K,), 6) calcium (Ca-K,, Kb), and 7) copper (not shown) (Fig. 6). Generally the more dense granules contained considerably more calcium and phosphorus than did the light, less dense granules. The granules were analyzed as described above, i.e., 10 granules were analyzed for 20 sec each and their spectra summed. This procedure was repeated 10 times. All granules analyzed showed the presence of calcium. Quantitative analyses, using the Hall peak-to-continuum approach (Hall et al., 1973), indicate that the granules contain calcium in a concentration of (mM/Kg dry weight f S.D.) 10.1 f 8.9 (N = 10). These quantitative results reflect a random sampling of both light and dark granules. DISCUSSION The granular cells of the amphibian urinary bladder mucosal epithelial cells represent the most numerous cell type of this tissue (Peachey and Rasmussen, 1961; DiBona et al., 1969a,b,; Davis et al., 1974). Ultrastructural studies have shown the granular cells to be characterized by a n “inverted pyramid” morphology with a narrow basal aspect and a n expansive, microvillated luminal surface (Peachey and Rasmussen, 1961; Choi, 1963; DiBona et al., 1969a,b; Davis et al., 1974). These, W.L. DAVIS ET AL. 232 2000- j I 4 I t I 15004 tI i cu t I C 0 P ; ,ooo-: t I 1 s t t + 500-4 4 ? Si , ' ... . . 'C. . . Cu.0~ . . ..: :\ 3 0- .ooo 2.000 4 .ooo 6.000 9.000 10.000 EWERCT ( K E V ) Fig. 2. TEM of a granular cell from a hemibladder reacted by the NHA-method for the histochemical localization of calcium. Section stained with uranyl acetate only. An electron dense reaction product is seen along the lumina1 microvillated border and within the subadjacent secretory granules. These precipitates contain calcium (see below). X42,500. Fig. 3.X-ray microanalysis of secretory granules from a hemibladder incubated by the NHA-method for the localization of cell calcium. This spectrum shows the presence of: 1) magnesium (Mg), 2) silicon (Si), 3) phosphorus (PI, 4) chlorine (Cl), 5) calcium (Ca), 6) copper (Cu), 7) osmium (OS). C1 is from the embedding medium; Cu is from the copper grid; OS is from the postosmication step following primary fixation. Summed spectrum from the analysis of 10 granules. TOAD BLADDER GRANULAR CELL 233 Fig. 4. Low magnification TEM of a freeze-dried section of the hemibladder epithelium. The morphology of the two granular cells (G)shown is well preserved with excellent retention of fine structure. Minimal ice damage is apparent. L, bladder lumen; MV, microvilli; N, nucleus; Arrows, secretory granules. x21,000. plus additional morphological studies, have all described the presence of regular aggregates of membrane delimited, ovoid to ellipsoid-shaped, electron dense granules immediately subadjacent to the apical plasmalemma of the granular cells (Masur et al., 1971, 1972, 1986). Continuities between the granule membrane and the surface cell membrane, perhaps indicative of granular secretion or exocytosis, have been described (Peachey and Rasmussen, 1961; Masur et al., 1972; Gronowicz et al., 1980). Despite the numerous ultrastructural studies on the granular cells, the precise nature and function of the subapical granules is only now beginning to be appreciated. Early electron microscopic studies on the bladder mucosal cells described a similarity in the morphology, texture, etc., of the granule contents to that of the lu- 234 W.L. DAVIS ET AL. 1 4mm v) c K iam z 3 0 0 I oa 6 a. t . 2 2 .. 3. 4. s. ENERGY ( K E V 1) Fig. 5. High magnification STEM from a freeze-dried cryosection prepared as in Figure 4. Sample photographed and analyzed at -100°C. A cluster of secretory granules is seen (1-4). Note the difference in density between individual secretory granules. Generally, the denser the granule, the greater the calcium concentration. X144,OOO. Fig. 6. X-ray spectrum from the subapical secretory granules shown in Figure, 5. The granules contain: 1)Mg, 2) Si, 3) P, 4) sulfur (S), 5) C1, 6) potassium, K, 7 ) Ca. All analyses were performed at the higher magnification where it was possible to thoroughly analyze a single granule. The presence of K, the intracellular ion, is indicative that the cells were adequately preserved and not disrupted by rapid freezing and the subsequent freeze-drying. TOAD BLADDER GRANULAR CELL mina1 fibrous glycocalyx (Choi, 1963). This observation was subsequently supported by both light microscopic and transmission electron microscopic histochemical procedures, which showed both the granules and the glycocalyx to be: 1) PAS-positive (Masur et al., 19721, and 2) silver methenamine positive using the procedure of Rambourg (1967), a technique performed on ultrathin sections (Pisam and Ripoche, 1976; Gronowicz, 1980). Such information indicates that both the subapical granules as well a s the glycocalyx are rich in carbohydrate moieties. The source of the glycocalyx associated with the surface membrane of the granular cells has been investigated. Histochemical and autoradiographic studies employing tritiated-fucose (a precursor of glycoproteins) have both indicated that the carbohydrate-rich glycocalyx is derived from the subapical granules via secretion or exocytosis (Masur et al., 1972; Pisam and Ripoche, 1976; Strum and Ekblad, 1977).Variations in the nature of these surface glycoconjugates can be further ascertained by even more specific light and electron microscopic histochemical procedures (Spicer et al., 1981). In the present study, X-ray microanalysis of freeze-dried sections confirms the presence of complex carbohydrates and glycoconjugates. Thus, the sulfur peak (Fig. 6) probably identifies sulfated glycosaminoglycans (mucopolysaccharides) and even more complex proteoglycans within the granular matrix. It is conceivable that at least a portion of these glycoconjugates may indicate the presence of a glycoprotein, perhaps a calcium-binding glycoprotein with a high affinity for this cation. Such proteins have been identified in mitochondria (Carafoli, 1980). This may help to explain the qualitative analytical data reported here, which shows the granules to contain considerable calcium. This is supported by quantitative data as well (see below). Granular calcium may be associated with a calcium-binding protein located within the granular matrix. Thus, these granules may play a role in intracellular calcium homeostasis similar to the well-established ion transport capabilities of mitochondria, smooth endoplasmic reticulum, etc. (Rasmussen, 1966, 1970; Rasmussen and Goodman, 1977; Carafoli, 1980). In the amphibian urinary bladder, ADH induces a change in the cytosolic calcium ion concentration (Thorn and Schwartz, 1965; Schwartz and Walter, 1969; Rasmussen, 1970; Cuthbert and Wong, 1974; Berridge, 1975). The granular cells of the bladder epithelium are believed to be the primary site of action for ADH (DiBona et al., 1969a,b). Our quantitative elemental analyses indicate that the granules contain approximately 10.1 f 8.9 m W g dry weight + S.D., N = 10. In comparison, the concentration of calcium in normal cardiac myocyte mitochondria, a s determined by X-ray microanalysis of similarly prepared freeze-dried tissue sections analyzed at -1OO"C, was determined to be 10.5 & 9.2 ( m W g dry weight calcium k S.D., N = 10) (Hagler et al., 1984). Quantitatively, the calcium content for these very different organelles appears to be similar. Additionally, the ionic profile for the secretory granules is comparable to that of normal cardiac myocyte mitochondria (Hagler et al., 1984). The presence of calcium in association with cytoplasmic secretory granules is not a new observation. This relationship has been repeatedly demonstrated in many diverse systems: 1)cortical (secretory) granules of 235 unfertilized sea urchin eggs (Cardasis et al., 1976)., 2) chromaffin cells of the rat adrenal medulla (Ravazzola, 1976), 3) in endocrine cells of the rat pars distalis (Schecter, 1976),4)in the zymogen granules of the guinea pig exocrine pancreas (Clemente and Meldolesi, 1975), and 5) in the secretory granules of rat odontoblasts (Reith, 1976). The majority of these studies have utilized cation localization with osmium pyroantimonate coupled with selective calcium chelation with EGTA and/or microprobe analysis. It has been firmly established that calcium plays a n essential role in the secretory process (Rasmussen, 1970; Rasmussen and Goodman, 1977). With regard to cell secretory mechanisms, this cation appears to be involved in one or more of the following ways: 1)calcium may be involved in the packaging of secretory materials (Clemente and Meldolesi, 1969), 2) calcium appears to be involved in regulating plasma membrane permeability, including those changes in membrane permeability that occur after the stimulation of secretion (Rasmussen, 1970; Dean and Matthews, 1970); the latter may be associated with the known lumina1 membrane permeability changes that take place in the toad urinary bladder following exposure to ADH, and 3) calcium seems to be involved in the mechanism of stimulus-secretion coupling (Douglas and Rubin, 1961; Rasmussen, 1970; Rasmussen and Goodman, 1977). Since the pioneering work of Douglas on the adrenal medulla (Douglas and Rubin, 1961; Douglas, 1966), it was generally assumed that the calcium involved in excitation-secretion coupling was of extracellular origin (Robberecht and Christophe, 1971). However, recent kinetic studies have determined that the calcium involved in this phenomenon is derived from a n intracellular calcium pool (Case and Clausen, 1973; Matthews et al., 1973). Thus, the mechanism involved in stimulus-secretion coupling is similar to that established for excitation-contraction coupling in skeletal muscle with regard to the intracellular source for calcium ions (Ebashi and Endo, 1968). In the amphibian urinary bladder, the calcium associated with the subapical granules may function in the exocytosis of glycocalyx components via a mechanism not unlike calcium-dependent excitation-secretion coupling. Several reports have adduced that ADH stimulates the exocytosis of these granules (Masur et al., 1972; Gronowicz et al., 1980). In addition to calcium, magnesium was also identified in the subapical granules by Xray microanalysis (Fig. 6). Like calcium, magnesium may also be involved in cellular secretion methods (Douglas and Rubin, 1961; Douglas, 1968). The phosphorus (phosphate) seen in the subapical granules is probably involved in the mechanism of calcium uptake by the secretory granules. A similar mechanism is present in mitochondria (Rasmussen, 1970; Rasmussen and Goodman, 1977). Also present in the granular matrix was silicon. Although the precise function of this ion cannot be explained at the present time, silicon has been identified in all toad tissues thus far analyzed. These include urinary bladder, intestine, skeletal muscle, kidney, and liver (Davis, unpublished observations). The presence of silicon may simply reflect the habitat in which these animals normally live, i.e., swamps. There is little doubt that the various precipitation techniques for the ultrahistochemical localization of cal- 236 W.L. DAVIS ET AL cium ions present potential problems and artifacts. The initial precipitation of electron dense calcium salts no doubt serve as potential nucleation sites for additional calcium epitaxis. The latter probably involves the artifactual translocation of this cation from other sources. In the study reported here, the NHA-technique is more than likely inducing the translocation of calcium from extragranular sources. This can effectively explain the increase in calcium seen in the NHA spectrum when the latter is compared to the calcium in the spectrum obtained from freeze-dried sections. Despite this, histochemical procedures have proven themselves to be valuable in assessing the distribution of cations within cells and tissues. ACKNOWLEDGMENTS We thank Rhonda B. Davis for excellent manuscript preparation. This research was supported in part by a grant from Baylor College of Dentistry, Dallas, TX, and by grants from Mr. and Mrs. Thomas Bedford and by the General Dynamics Corporation of Fort Worth, TX, to W.L. Davis. LITERATURE CITED Berridge, M.J. (1975) The interaction of cyclic nucleotides and calcium in the control of cellular activity. Adv. Cyc. Nuc. Res., 6:l-98. 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