MICROSCOPY RESEARCH AND TECHNIQUE 40:434–439 (1998) Microscopical Localization of Adenylate Cyclase: A Historical Review of Methodologies P.A. RICHARDS1 AND P.D.G. RICHARDS2* 1Department 2Electron of Anatomy, University of Pretoria, University of the North, Sovenga, Republic of South Africa Microscope Unit, University of the North, Sovenga, Republic of South Africa KEY WORDS adenyl cyclase; AMP-PNP; AMP-PCP; histocytochemistry ABSTRACT The histochemistry technique for localizing adenylate cyclase has been developed over the past two decades. Early efforts were directed at overcoming the criticism of the lead capture technique, the inhibition of the enzyme by fixation, and problems associated with the substrate. The introduction of alternative metal ions, strontium and cerium, offered solutions to the criticism of the lead capture technique. The inhibition of the enzyme by the various fixation methods used has been rarely overcome satisfactorily and the use of non-fixed material during incubation is one of the alternatives that has been suggested. The introduction of adenylate (β-g-methylene) diphosphate as an alternative substrate offers a solution to the problems associated with commercially available adenylyl imidodiphosphate. Although no standard medium or method has been accepted by all researchers, the histochemical technique still has a place in the arsenal of the modern cell biologist. The technique localizes the active enzyme, as opposed to the protein, active and nonactive, by immunocytochemistry and the precursors of the protein by in situ hybridization methods. Microsc. Res. Tech. 40:434–439, 1998. r 1998 Wiley-Liss, Inc. INTRODUCTION Over the last two and a half decades the enzyme adenylate cyclase (E.C. 188.8.131.52) (AC) has been localized to a variety of tissues by histochemical methods. AC was solely detected by biochemical assays (Sutherland and Rall, 1958; Weiss and Costa, 1968) until 1970, when the first attempt to localize AC histochemically at the ultrastructural level was made (Reik et al., 1970). The technique Reik and colleagues used was based on the Wachstein and Meisel (1957) lead precipitation method for adenosine triphosphatase. Reik et al. (1970) formulated an incubation medium using lead nitrate as the capture agent and AC’s natural substrate, adenosine triphosphate (ATP) (Table 1). They found that the enzyme’s activity was 10–50% greater when using fresh tissue as opposed to glutaraldehyde-fixed tissue. Over the next two decades, three components of the localization system (pre-localization conditions, substrate, and capture agent) were debated in the literature and the development of the technique, as a result of these debates, is outlined below. THE 1970s, DIFFICULT BEGINNINGS Adenosine triphosphate is, unfortunately, not a specific substrate for AC and thus the use of ATP in histochemical localizations of AC is nonspecific. In 1971, a more specific substrate, adenylyl imidodiphosphate (AMP-PNP), a synthetic ATP substitute, was described (Yount et al., 1971). Although AMP-PNP was also cleaved by phosphodiesterase (Yount et al., 1971), this enzyme’s specific inhibitor, theophylline, was already in use as part of the localization medium (Table 1). AMP-PNP was used in a histochemical medium by Howell and Whitfield (1972) to demonstrate AC in pancreatic tissue. The tissue was fixed in 1% glutaraldehyde, which led to only 40% of the enzyme’s activity r 1998 WILEY-LISS, INC. being retained. During incubation, AC was stimulated by using sodium fluoride and other hormonal stimulators (Table 1). No visible precipitate was formed in the incubation medium in the absence of tissue. Most researchers (60% of the literature surveyed) over the following two and a half decades based their localization attempts on the Howell and Whitfield medium. Wagner et al. (1972) attempted to limit the amount of inhibition in the histochemical approach by using dextran to chelate the lead ions. The lead precipitation method was severely criticized by Lemay and Jarret (1975) who, using biochemical tests, noted that a lead concentration of 0.5 mM totally inhibited the enzyme and that Wagner et al.’s method for the protection of the enzyme did not have the desired effect. They further suggested that AMP-PNP was non-enzymatically broken down by the lead and that this, along with the high concentrations of lead used in histochemical localizations, meant that the method was invalid (Lemay and Jarret, 1975). The histochemical method was dealt a further blow when it was shown that AMP-PNP was not only hydrolyzed by AC but by another major membrane enzyme, nucleotide pyrophosphatase, as well (Johnson and Welden, 1977). Nucleotide pyrophosphatase is optimally active at pH 9–10.5, with some inhibition occurring at lower pH’s. As nucleotide pyrophosphatase was inhibited by the use of dithiothreitol (DTT), Johnson and Welden (1977) proposed that, when attempting to localize AC, histochemical media should use DTT as one of the constituents. They also suggested that AC was *Correspondence to: P.D.G. Richards, Department of Anatomy, Faculty of Medicine University of Pretoria, PO Box 2034, Pretoria 0001, Republic of South Africa. Received 1 July 1996; accepted in revised form 25 September 1996. AC METHODOLOGIES TABLE 1. A comparison of the incubation mediums formulated for the demonstration of AC by Reik et al. (1970) and Howell and Whitfied (1972) Compound Buffer Sugar Substrate Activators Tris maleate pH 7.4 Glucose Dextrose Adenosine triphosphate Adenylyl imidodiphosphate a) Isoproterenol or b) Glucagon or c) Sodium fluoride Other: PDE* inhibitor Theophylline Ions Magnesium sulphate Capture agent Lead nitrate Concentration Reik et al. (1970) Concentration Howell & Whitfield (1972) 0.05 M 0.8 M — 8% 0.5 mM 8% — — — 0.5 mM 4 µM 0.15 µM 12.5 mM — — 10 mM 2 mM 4 mM 2 mM 5 mM 4.8 mM 2 mM *PDE 5 phosphodiesterase. inhibited by adenosine but that this was reduced by including adenosine deaminase. Lemay and Jarret’s findings (1975) were to direct the research effort, as far as the histochemistry of AC was concerned, for the next few years. In 1977, Schulze et al., while in general agreement with Lemay and Jarret, stated that the tris-maleate buffer, which was used as the vehicle for the localization medium, chelated the free lead. As a result, higher concentrations of lead could be used as part of the histochemical procedure. The inhibition of the enzyme by lead was also overridden by the use of sodium fluoride (Schulze et al., 1977). Despite this, and as a suggestion to overcome the problems associated with lead as the precipitating agent, they recommended the use of strontium at a pH of 8.8. Although Lemay and Jarret (1975) suggested that prefixation in paraformaldehyde and glutaraldehyde would totally destroy AC’s activity, Schulze et al. (1977) were able to retain 30–40% of the basal activity. They further advised that the characteristics of each tissue needed to be taken into account before any medium could be formulated. The use of the original Reik solution as a reliable medium for the localization of AC was further debated by Kempen et al. (1978), who were unable to obtain any localization with the medium. They attributed the deposits obtained by others to the release of a precipitate, resulting from the presence of calcium ions in the tissue and not due to an enzyme-specific reaction. The calcium ions were thought to act as nucleation sites for the precipitation of the lead deposit (Kempen et al., 1978). Using biochemical methods, they noted that the use of paraformaldehyde alone as a fixative retained 70% of AC basal activity, whereas 0.5% glutaraldehyde left only ,10% of this activity (Kempen et al., 1978). In agreement with Lemay and Jarret (1975), it was further suggested that lead was responsible for total inhibition of AC. Kempen and colleagues (1978) found that the medium became cloudy on addition of lead, which led them to the conclusion that lead non- 435 enzymatically converted AMP-PNP. As a result, it should not be used in cytochemical experiments and as an alternative they suggested the use of barium ions. However, barium ions are, unfortunately, electron translucent and unless they are converted to an electrondense precipitate, barium phosphate is not visible when examining the tissue with an electron microscope. These criticisms of the technique were investigated in some depth by Kvinnsland (1979). He observed that when ATP was used as a substrate, as in the original medium (Reik et al., 1970), the resulting deposit was not affected by alloxan, the specific inhibitor of AC (Cohen and Bitensky, 1969) and inactivation of the enzyme by heat. He therefore concluded that when ATP was used as a substrate, nonspecific staining occurred; when Kvinnsland purified the AMP-PNP, using a Dowex column, before use in the localization medium, he obtained a good repeatable precipitate, even in fixed specimens. Also, the medium did not take on the cloudy appearance that had been observed by Kempen et al. (1978). The hydrolysis of AC by nucleotide pyrophosphatase was discounted as a source of nonspecific precipitate as it had a high pH requirement and was possibly inhibited by alkaline phosphatase inhibitors, such as levamisole, which he had included in the medium (Kvinnsland, 1979). In general, the control incubations that were used for AC histochemistry included heat inactivation (where the sample was heated to 70°C or greater, thus destroying the enzyme) the absence of the substrate from the incubation medium, and a specific inhibitor. The specific inhibitor used was alloxan (Cohen and Bitensky, 1969) which had a reversible and in part competitive action. Londos and Wolff (1977) demonstrated that AC has two adenosine reactive sites, termed the R (stimulatory) and P (inhibitory) sites. This introduced the P site inhibitors of AC activity, such as 2858 dideoxyadenosine (Londos and Wolff, 1977), which became a more common specific inhibitor of AC than alloxan in the 1980s. THE 1980s, SOLVING THE PROBLEMS The decade started with an investigation of commercially supplied AMP-PNP by Cutler and Christiansen (1980). They column purified commercially available AMP-PNP using both a Dowex Ag-50 W-X4 column and a DEAE-52 cellulose column. These purification techniques revealed two contamination peaks, thought to be adenylylimido phosphate and AMP. The purified AMP-PNP and the compounds from the two contamination peaks were used in cytochemical experiments before being re-chromatographed over a Dowex column. The contaminating compounds shifted in their chromatographic pattern, indicating that they were probably interacting with the lead in the incubation medium. Cutler and Christiansen (1980) recommended, in line with Kvinnsland (1979), that the purification step be introduced before using commercially obtained AMP-PNP. Døskeland (1980) suggested that protection of the enzyme against inhibition by lead ions could be obtained if excess EGTA was used in the medium as the EGTA reversed most of the lead inhibition, leaving a small irreversible component. He further stated that guanine nucleotides, particularly guanyl-58-yl imidodi- 436 P.A. RICHARDS AND P.D.G. RICHARDS phosphate, protected the enzyme against lead inhibition, not as a result of the sequestration of lead ions, but through the enzyme’s lower affinity for guanyl proteins in the presence of lead. As an alternative method of dealing with the lead inhibition of AC, Fujimoto et al. (1981) proposed the use of dimethyl sulfoxide (DMSO) in the medium. They found that, when assayed biochemically, ,20% of the basal activity was left in the presence of 0.001 M lead nitrate and sections only retained 20% of the basal activity with 0.01 M lead ions. In the presence of 5% v/v DMSO the basal activity was enhanced by 30–50% (Fujimoto et al., 1981). Poeggel and colleagues (Poeggel and Bernstein, 1981; Poeggel et al., 1981) used biochemical and polyacrylamide gel analysis in an attempt to formulate an ideal medium for the localization of AC. As a result of these experiments, they suggested that strontium ions be used as the capture agent in AC histochemistry following an earlier suggestion by Schulze et al. (1977). Strontium ions, like the barium ions used by Kempen et al. (1978), do not form an electron-dense product and have to first be converted to an electron-opaque product, using lead. In their work with microgels, Poeggel et al. (1981) clearly demonstrated the need for the adenosine pyrophosphatase inhibitor, DTT, as suggested earlier by Johnson and Welden (1977) and the inclusion of an alkaline phosphatase inhibitor from the methyl xanthines (Sugimura and Mizutami, 1979), such as isobutyl methylxanthine (IBMX) or levamisole. Poeggel and his colleagues (1982) tested the medium thus formulated histochemically and found that they could retain 75% of the basal activity. A threefold increase in AC was obtained, up to a maximal 267% activity, provided DTT was included in the medium and the tissue was pre-incubated in a Tris/HCl buffer containing stimulants. Further evidence for the nonspecificity of AMP-PNP was obtained by Taylor (1981) who showed that sarcoplasmic reticulum adenosine triphosphatase was also capable of hydrolyzing the substrate. As this enzyme is Ca21-dependent, very few precautions are taken by researchers to prevent the action of this enzyme in localization experiments. Many localization experiments use the AC stimulant, sodium fluoride, as part of the localization medium. This stimulant acts through the ‘G’ protein stimulatory pathway, which was shown to be inhibited by strontium ions (Schulze, 1982). Forskolin, a diterpene, stimulates AC directly via the catalytic unit (Seamon and Daly, 1981). However, after further investigation it was found that forskolin required the ‘G’ protein subunits for maximal activation (Darfler et al., 1982). The literature reveals that the majority of published work on AC localization use stimulants that utilize the ‘G’ protein pathway as opposed to the direct stimulation of AC. This is perhaps a reflection of the requirements of the studies being undertaken, in that they are looking at the effects of specific hormonal stimulants on AC. Fine et al. (1982) obtained good localization using unfixed specimens to overcome the loss of AC basal activity as a result of glutaraldehyde fixation (Schulze, 1982). They used lead as a capture agent in their experiments which gave a specific result when the substrate had been freshly prepared (Fine et al., 1982). TABLE 2. The optimized incubation medium as recommended by Poeggel et al. (1984) Buffer Substrate Sugar Activator Other: PDE* inhibitor Pyrophosphatase inhibitor Ions Capture agent Compound Concentration Tris HCl pH 8.9 Adenylyl imidodiphosphate Sucrose Sodium fluoride Aminophylline Dithiothreitol 100 mM 1 mM Magnesium chloride Strontium chloride 250 mM 10 mM 5 mM 1 mM 10 mM 20 mM *PDE 5 phosphodiesterase. TABLE 3. The incubation medium proposed as a standard for the localization of AC by Richards (1994) Buffer Substrate Sugar Activator PDE* inhibitor Pyrophosphatase inhibitor ATPase** inhibitor Ions Capture agent Compound Concentration Tris maleate pH 8.2 Adenylyl imidodiphospate Glucose Forskolin Theophylline 80 mM 0.5 mM 8% 25 µM 2 mM Dithiothreitol Oubain Magnesium chloride Heavy metal ion 1 mM 0.5 mM 5 mM 2–5 mM *PDE 5 phosphodiesterase. **ATPase 5 adenosine triphosphatase. Although the contaminants in commercially supplied AMP-PNP do not interact with strontium, nonspecific deposits were found when using this heavy metal ion in the incubation medium (Zajic and Schact, 1983), possibly as a result of nucleotide pyrophosphatase activity. Zajic and Schact (1983) further showed that sodium fluoride stimulation was slow to produce precipitation and this could lead to artifacts. They also pointed out that in substrate omission, the control experiment most histochemists use, the capture ion reacts not only with endogenous substrate but also with the cell surfaces, which could lead to artifacts. They suggested that microwaves, to disrupt the enzyme and short incubation times of one minute or less, would be better control experiments. Neither of these suggestions have been investigated further or used by others. Several authors at this time suggested that preincubation manipulation of the specimen determines the outcome of the histochemical results and that these manipulations are in turn determined by the specimen (Cutler, 1983; Schulze, 1982). Both sets of authors argued that a general method could not be used as fixation conditions had to be determined for each tissue. Cutler (1983) recommended that biochemical checks for inhibition and fixation be performed prior to the histochemical procedures. Poeggel and colleagues (1984) queried whether the results obtained using fixed tissue were valid in light of the free floatation theory of De Haën (1976), as the receptors could not be free floating in fixed membranes and would therefore be unable to stimulate AC activity. They agreed with Cutler (1983) and Schulze (1982) regarding the influence of the tissue on fixative selection. Poeggel and 437 AC METHODOLOGIES TABLE 4. Inhibition of AC by various fixative protocols as reported in the literature Fixation 4% PF 3% PF 2% PF 1% PF 0.5% PF 2% PF 1 1.5% GA 2% PF 1 0.6% GA 2% PF 1 0.5% GA 2% PF 1 0.25% GA 1% PF 1 1% GA 0.5% PF 1 0.5% GA 2.5% GA 2% GA 1.25% GA 1% GA 0.5% GA Suberimacid dimethyl diimidoester 100 mM % Inhibition Method Time (mins) Temp (°C) 50 0–70 56 10 90 0 45 92 70–87 51 70–90 60–70 99 present 10–87 20 20 0 70–87 70–87 70 100 60–80 60–75 60–80 50–90 90–100 100 60 228 Immersion Immersion Perfusion Immersion Immersion Immersion Immersion Immersion Immersion Immersion Immersion Immersion Immersion Immersion Immersion Perfusion Perfusion Perfusion Immersion Immersion Immersion Immersion Immersion Immersion Immersion Perfusion Immersion Immersion Immersion Immersion 5 20 5 30 15–30 5 10 30 60 5 30 30 15–30 15–30 180 5 5 5 60 15 5 5 60 15–30 60 5 10 10 5 60 20 20–22 20–22 20–22 20–22 20 4 20–22 4 4 20–22 20–22 20–22 20–22 4 20–22 4 20–22 4 4 20–22 22 20–22 20–22 20–22 20–22 20–22 4 20 20–22 Author Palkama et al. (1986) Cutler (1983) Poeggel & Bernstein (1981) Fujimoto et al. (1981) Davis et al. (1987) Palkama et al. (1986) Kempen et al. (1978) Fujimoto et al. (1981) Cutler (1983) Fujimoto et al. (1981) Davis et al. (1987) Muller et al. (1985) Cutler (1983) Cutler (1983) Schulze (1982) Poeggel & Bernstein (1981) Cutler (1983) Palkama et al. (1986) Cutler (1983) Davis et al. (1987) Howell & Whitfield (1972) Reik et al. (1970) Richards & Els (1990) Kempen et al. (1978) Palkama et al. (1986) Poeggel & Bernstein (1981) PF 5 paraformaldehyde, GA 5 glutaraldehyde, 20–22°C taken as room temperature. colleagues (1984) produced an optimized procedure and medium (Table 2) which they felt would overcome the problems in the technique thus far encountered. They recommended a pre-incubation step in the incubation procedure without the substrate and alkaline phosphatase inhibitor (Poeggel et al., 1984). Slezak and Gellar (1984) dispelled the earlier suggestion that calcium was responsible for the deposits seen in histochemical localizations (Kempen et al., 1978) by examining the deposit with electron dispersive energy spectrum analysis to ensure that it was a phosphate product. Light microscopical demonstration of AC had also been attempted during this period (Nomura, 1978; Fujimoto et al., 1981; Nomura and Asanuma, 1982; Mizukami et al., 1982; Vorbrodt et al., 1984) but Poeggel and Luppa (1984) raised questions as to the validity of such localizations, as the AC’s activity was overlapped by nucleotide pyrophosphatase and no provision had been made for this enzyme’s activity in the procedures. Mayer and colleagues circumvented this in 1985 by using a novel substrate, adenylate (β-gmethylene) diphosphate (AMP-PCP), which gave no nonspecific deposit after inhibition with alloxan. AMP-PCP has a stronger bond arrangement than AMPPNP (Yount et al, 1971) and as such was unlikely to break down during storage. Poeggel and Luppa (1988) criticized the use of AMP-PCP in AC histochemistry, suggesting that it was used by other enzymes to the same extent as AMP-PNP and the relevant controls should be included. Their criticism was based on a reference by Yount et al. (1971) that AMP-PCP was not hydrolyzed by AC. Despite this, the new substrate was used by Yamamoto and Gay (1989) for electron histo- chemical demonstration of AC in chicken osteoclasts with no apparent problem. 1990 TO PRESENT—TOWARDS A STANDARD MEDIUM The problems of lead in the medium when AMP-PNP was used as the substrate had been addressed by the inclusion of cerium ions (Rechardt et al., 1985) following their use by Robinson and Karnovsky (1983). Rechardt and colleagues (1990) found that cerium gave a good deposit but there were problems associated with its penetration of the tissue and this was overcome by the use of solubilization techniques. Richards and Els (1990) followed the example of Fine and colleagues (1982) by recommending the use of unfixed material for the localization of AC in frog epithelium. Using electron dispersive energy spectrum analysis, Asanuma (1990) confirmed that the deposit seen in AC histochemistry, using AMP-PNP as the substrate, was a result of AMP-PNP hydrolysis by AC. Further investigations on the choice of substrate by Mayer and colleagues (1991) showed that, although used by other enzymes, the rate of hydrolysis was less for AMP-PCP than AMP-PNP. As there is no breakdown of AMP-PCP during storage, lead could be used for enzyme histochemistry at both light and electron microscope levels, without the time-consuming purification step. Others have since used AMP-PCP as a substrate very successfully (Fukushima et al., 1991) but AMP-PNP is still more frequently used. Metaye et al. (1992) reported that the alkaline phosphatase inhibitors levamisole and bromotetramisole, which Kempen (1978) and Fujimoto et al. (1981) had 438 P.A. RICHARDS AND P.D.G. RICHARDS recommended to be included in the medium for AC localization, had an inhibitory effect on AC. Richards (1994), building on earlier work (Richards and Els, 1990; Richards, 1992), formulated a possible standard medium which could be used with all tissues, taking into account the criticisms over the last two decades (Table 3). Richards (1994) reduced the amount of stimulant, forskolin, in the medium as a result of his own and Poeggel et al.’s (1984) earlier observation regarding the positive effect of DTT on AC. Richards (1994), however, ascribed this effect to DTT’s protection of AC in a similar fashion to its protective role of adenosine triphosphatase (Caspers and Siegel, 1980) rather than to a stimulatory effect, as this could not be shown electrophysiologically. As can be seen from Table 4, the efforts of researchers to determine the optimal fixation conditions have been confusing. No two experiments on fixation have been carried out under the same conditions and even under similar fixation conditions (Poeggel and Bernstein, 1981; Cutler, 1983) the degree of inhibition has varied by 20%. Richards (1994) reiterated the proposals of others (Schulze, 1982; Cutler, 1983) that the preincubation conditions are determined by the specimen but that exclusion of pre-fixation should be the method of choice. If fixation is advisable, then a Karnovskystyle fixative (Karnovsky, 1965) with a low glutaraldehyde content would be the alternative to no fixation, as suggested by Fujimoto et al. (1981). Pre-incubation stimulation of the specimen prior to fixation (Poeggel et al., 1984) was seen to have limited value, as the tissue could be incubated in the full medium at this time (Richards, 1994). CONCLUSION—THE FUTURE OF AC HISTOCHEMISTRY During this latest phase of AC histochemistry’s development the enzyme’s structure has been elucidated (Krupinski et al., 1989), as have several isoforms (Cooper et al., 1995). As a result, in situ hybridization and immunolocalization techniques appear to have come to the fore (Bookbinder et al., 1990; Villacres et al., 1993; Cali et al., 1994; Hellevuo et al., 1995). The consequence of this has been a reduction in the number of histochemical investigations carried out during the 1990s. Histochemistry, however, still has a valued place in the cell biologist’s repertoire, as it uses the enzymes own activity to localize itself. 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