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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. 4.6.1.1) (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. Other techniques, such as
immunocytochemistry and in situ hybridization, give
information regarding the presence of the protein and
its precursors but do not give any information regarding its functionality. The combination of histochemical
techniques and in situ or immunocytochemical methods would not only indicate the presence of the protein
and its precursors but would also indicate the enzyme’s
ability to operate.
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