MICROSCOPY RESEARCH AND TECHNIQUE 40:473–478 (1998)

Adenylyl Cyclase in the Heart: An Enzymocytochemical

and Immunocytochemical Approach
WOLFGANG SCHULZE* AND IGOR B. BUCHWALOW
Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany

KEY WORDS cardiomyocytes; enzymocytochemistry; immunocytochemistry

ABSTRACT This review provides a discussion of the localization of adenylyl cyclase (AC) in
normal mammalian heart tissue employing enzymocytochemistry (detection of the catalytic activity
of AC by a metal precipitation technique) and immunocytochemistry (immunolabeling of the enzyme
protein with antibodies against AC subtypes). By the metal precipitation technique, AC activity was
localized in adult guinea pig cardiomyocytes along the sarcolemma and the T-tubule membranes.
This reaction can be enhanced by hormones and guanylyl imidodiphosphate, fluoride, and forskolin.
With this technique, no precipitates were detected at the sarcoplasmic reticulum. However, under
ischemic conditions, AC activity was also found in the junctional sarcoplasmic reticulum of rat
cardiomyocytes. Immunocytochemistry revealed AC in the plasma membrane of rat cardiomyocytes.
Detection of AC in the perinuclear space of cardiomyocytes might reflect initiation of synthesis and
processing of the enzyme protein. Colocalization of AC with cytoskeleton fibers of non-cardiomyocytes
emerging in the cell culture of neonatal rat cardiocytes imply a direct cytoskeletal-AC interaction.
Finally, it can be stated that the immunolabeling pattern of AC in cryosections of adult and new-born
rat hearts reveals a good correspondence with the localization of AC activity in cardiomyocytes
demonstrated by enzymocytochemistry. Microsc. Res. Tech. 40:473–478, 1998. r 1998 Wiley-Liss, Inc.

INTRODUCTION ization of AC using biochemical procedures based on

Cellular response to environmental changes is initi-
ated by interactions of various hormones, neurotrans-
mitters, and other regulatory molecules with specific
membrane-spanning cell surface receptors. Subsequent
coupling of activated receptors, with corresponding
signal transducing proteins, triggers a cascade of enzy-
matic reactions specific for the given regulatory mol-
ecule and cell type. The largest class of eukaryotic
receptors is represented by receptors coupled with
heterotrimeric G-proteins (G-proteins). In this receptor
system, the effector enzyme is adenylyl cyclase (AC),
which synthesizes the second messenger cAMP, a ubiq-
uitous signaling molecule that modifies cell function by
activating cAMP-depending protein kinase A (for re-
view, see Tang and Gilman, 1992; Iyengar, 1993; Taus-
sig and Gilman, 1995).
Mammalian ACs have molecular weights in the
range of 110–120 kDa and are integral transmembrane
proteins. It has been well established that the signal
transduction chain in eukaryotic cells is associated
with the cell membranes (for review, see Taussig and
Gilman, 1995; Iismaa et al., 1995). But a cell, such as
the cardiomyocyte, is a complex polymorphous struc-
ture containing various types of inner and outer mem-
branes dividing the cytoplasm into several functionally
different components. In most cases, it has not been
possible to isolate a given membrane structure from
heart muscle uncontaminated by other cell compo-
nents. Moreover, AC is a quite elusive enzyme, ex-
pressed at very low levels, generally 0.01–0.001% of
membrane protein, and very unstable during purifica-
tion (Taussig and Gilman, 1995). All of this has ham-
pered thorough documentation of the subcellular local-
tissue homogenization, extraction, and differential cen-
trifugation. To overcome these difficulties, cytochemical
techniques, which do not disrupt the normal in situ
organization of the cell, are required. This review deals
with the localization of AC in normal mammalian heart
tissue employing enzymocytochemistry (detection of
the catalytic activity of AC by a metal precipitation
technique) and immunocytochemistry (immunolabel-
ing of the enzyme protein with AC-subtype-specific
antibodies (AB)).
ENZYMOCYTOCHEMICAL LOCALIZATION
OF AC IN THE HEART BY METAL
PRECIPITATION TECHNIQUE
AC catalyzes the conversion of ATP into cAMP and
pyrophosphate. Enzymocytochemical localization of AC
is based on precipitation of the pyrophosphate with
metal ions according to the classic Gomori-Takamatsu
principle (Gomori, 1939; Takamatsu, 1939). Since ATP
as a substrate can also be cleaved by other phosphohy-
drolases and pyrophosphatases that may be present in
cells, an ATP analogue, adenylyl imidodiphosphate
(AMP-PNP), synthesized by Yount et al. (1971), was
used. AMP-PNP is not hydrolyzed by ATPases and
other phosphates with one exception (Poeggel et al.,
1984). Howell and Whitfield (1972) introduced
AMP-PNP for the localization of AC in rat islets of
Contract grant sponsor: Deutsche Forschungsgemeinschaft.
*Correspondence to: Dr. Wolfgang Schulze, Max-Delbrueck-Center for Molecu-
lar Medicine, Robert-Roessle-Strasse 10, 13125 Berlin, Germany. E-mail:
wschulze@mdc-berlin.de
Received 16 September 1996; accepted in revised form 5 November 1996.

r 1998 WILEY-LISS, INC.

474 W. SCHULZE AND I.B. BUCHWALOW
Langerhans and Wagner et al. (1972) in capillaries of
epididymal fat cells. The phosphoimidate (imidodiphos-
phate) formed by the enzymatic hydrolysis of AMP-PNP
can be precipitated by lead, cobalt (Rechardt and
Härkönen 1977), strontium (Schulze et al., 1977), or
cerium (Rechardt and Hervonen, 1985; Schulze et al.,
1986). On account of its electron density, the metal
precipitate can be easily detected in the electron micro-
scope.
Various incubation conditions have been described
for the demonstration of AC in situ (Wagner and
Bitensky, 1974; Poeggel et al., 1984; Schulze, 1984).
Our basic incubation solution consists of 80 mM tris-
maleate buffer (pH 7.4) containing 6% sucrose, 10 mM
magnesium sulfate, 4 mM aminophylline, 0.5 mM
58-adenylyl imidodiphosphate, and 2 mM lead nitrate.
For AC stimulation, 10 mM sodium fluoride, 0.01 mM
adrenaline, 0.05 mM histamine, in the presence of 0.01
mM guanylyl imidodiphosphate [Gpp(NH)p], or 0.05
mM forskolin were added to the basic medium. To
detect the stimulatory effect, the incubation time (nor-
mally 30 minutes at 37°C) was reduced to 10 minutes.
This reduction in time failed to demonstrate basal
activity and showed an increment of AC activity in-
duced by the addition of activators (Schulze, 1984).
Tissues incubated in a substrate-free medium and
tissues heated to 80°C prior to incubation were used as
controls. Further, an adenosine derivate, 2858-dideoxy-
adenosine, was used as a most potent inhibitor (Londos
and Wolff, 1977). At a concentration of 1 mM, 2858-
dideoxyadenosine completely inhibited the enzyme ac-
tivity and prevented its stimulation by fluoride, forsko-
lin, and isoproterenol (Schulze, 1982).
For localization of AC activity, it is necessary to
precipitate the PNP (one of the reaction products) at or
near the site of their production. This is possible under
optimal conditions, including an exact tissue prepara-
tion and a well-balanced incubation medium. The pres-
ervation of both enzymatic activity and ultrastructure
requires a compromise which has been established for
the heart tissue by quantitative analysis (Schulze,
1982) and electron microscopical examination (Schulze,
1984). The best way to preserve the enzyme activity is
to use fresh tissue. However, Cutler (1975, 1980) sug-
gested that the prefixation of the tissue provided a
definite degree of protection for the enzymes against
the inhibitory effect of lead. Besides preserving the
ultrastructure, the prefixation should enhance the mem-
brane permeability for substrates and effectors of the
incubation medium. Therefore, except Revis (1979),
who localized AC in an unfixed section of heart tissue,
all investigators concurred that aldehyde fixation offers
the best way for localization of AC in well-preserved
tissue. Some authors (Slezak and Geller, 1979; Fuji-
moto and Ogawa, 1982) recommended a mixture of
glutaraldehyde and formaldehyde supplemented with
dimethyl sulphoxide.
In our experiments, we routinely use perfusion with
cacodylate-buffered 2% glutaraldehyde (Serva). The
protocol for prefixation used in our experiments was:
perfusion with cold (4°C) Tyrode’s solution containing
25 iu/ml heparin for 1 to 3 minutes, with 2% glutaralde-
hyde in 0.1 M cacodylate (pH 7.4) for 5 minutes, and
thereafter with 0.25 M sucrose for 20 minutes. The
coronary flow was standardized to 3 to 6 ml per minute.
The quantitative analysis of AC in tissue pretreated in
the described manner showed that about 80% of the
enzyme activity was still retained (Schulze, 1982).
Further treatment with the full incubation medium
reduced AC activity to 20% in comparison to untreated
heart tissue. However, this basal activity could be
stimulated two- or threefold by NaF, forskolin, catechol-
amine, and histamine in the presence of Gpp(NH)p.
The activation induced by hormones can be prevented
by specific antagonists. These quantitative data indi-
cate that the function of the adenylyl cyclase system is
still reactive under the cytochemical conditions used in
our experiments (Schulze, 1982).
Fundamental to the second messenger theory is the
fact that AC, the target for the first messenger, is
localized in the plasma membrane. This was confirmed
for heart tissue among others using the cytochemical
technique (Wollenberger and Schulze, 1976; Slezak and
Geller, 1979, 1984; Revis, 1979; Fujimoto and Ogawa,
1982). But in experiments with cardiac microsomal
fractions, enriched in vesicles from the sarcoplasmic
reticulum (SR), AC activity was also detected (Sulakhe
and Dhalla, 1973; Katz et al., 1974). However, the
possibility could not be excluded that such microsomal
fractions were contaminated with fragments of plasma
membranes. Therefore, substantiation of this data with
cytochemical methods was obvious. The cytochemical
approach enabled us to localize AC in left ventricles of
guinea pig heart along the sarcolemma (Fig. 1a) and the
tubular invagination of the sarcolemma, the T-tubules
(Figs. 1a,b). Some parts of the intercalated discs, espe-
cially those parallel to the myofibrils, were likewise
covered with lead precipitates. In the guinea pig heart,
we did not see any reaction marker on the SR, neither
at the junctional SR (Figs. 1c,d arrows) nor at the
subsarcolemmal cisterns. Even activation of the AC by
hormones did not change this precipitation pattern
(Fig. 1b). As opposed to the guinea pig heart, we found
that in rat heart, especially after long-lasting ischemia,
a positive reaction in the junctional SR. In contrast to
the AC activity in the junctional SR, the enzyme
activity in the sarcolemma was completely abolished
after total ischemia (Schulze and Will-Shahab, 1984).
AC activity in all parts of the SR in the rat heart has
been described by Revis (1979) and Fujimoto and
Ogawa (1982), whereas Slezak and Geller (1984) de-
tected AC localization in addition to the sarcolemma
also in the junctional SR but not in the longitudinal
parts. The reason for the variability in the localization
of AC in the heart of these two species (rat and guinea
pig) is not clear. It might be supposed that the different
pretreatment of tissue specimens by various laborato-
ries results in a different penetration of the incubation
medium components. But it is also possible that various
isozymes located in the heart respond differently to the
prefixation and incubation conditions. Quantitative
experiments with specific antibodies against the vari-
ous isozymes should help to answer this question.
IMMUNOCYTOCHEMICAL LOCALIZATION
OF AC IN THE HEART AND CULTURED
CARDIOMYOCYTES
AC is currently shown to comprise a family of at least
eight isotypes with different tissue distribution and
 Fig. 1. Enzymocytochemical localization of AC in the left ventricle
of guinea pig heart. Epon-embedding. Ultrathin sections. (a) Longitu-
dinal section of left ventricle of guinea pig heart after incubation in the
basic AC detection medium. Fine lead precipitates cover the sarco-
lemma and the deep invagination of the sarcolemma. No other cell
structures reveal any reaction. Bar 5 0.5 µm. (b) Intracellular part of
a longitudinal section from guinea pig left ventricle. The tissue was
incubated in the basic incubation medium supplemented with hista-
mine and Gpp(NH)p. The incubation time was reduced to 10 minutes.
Strong precipitation located on the T-Tubuli membranes. Neither
junctional (arrow) nor longitudinal parts of the SR reveal any lead
precipitate. Bar 5 0.5 µm. (c) Guinea pig left ventricle section. The
basal AC activity marked by the lead precipitation is restricted to the
plasma membrane and its vesicles. At the SR (asterisk) no precipitate
is visible. Bar 5 0.5 µm. (d) A higher magnification reveals the
reaction product along the sarcolemma and not at the SR. Junctional
SR is marked with arrows, longitudinal with asterisks. Bar 5 0.25 µm.
476 W. SCHULZE AND I.B. BUCHWALOW
activation characteristics. Whereas they are all acti-
vated by the GTP-bound form of as (or aolf), there are
differences in their susceptibility to stimulation, inhibi-
tion, or are unaffected by other regulatory factors, such
as Ca21 (either directly, or in the form of the Ca21-
binding protein, calmodulin), various isozymes of pro-
tein kinase C (PKC), βg subunits, and a-subunits of
other isoforms of G-proteins, Gz and Go. Gq proteins are
involved
21
in AC regulation indirectly via the release of
Ca ions, which subsequently bind to and activate
calmodulin (CaM) or PKC (Iismaa et al., 1995). Isotypes
V and VI and a widespread isotype IV have been
identified in heart tissue biochemically (Quarmby and
Hartzell, 1995; Iismaa et al., 1995). Yu et al. (1995),
applying reverse transcription polymerase chain reac-
tion and in situ hybridization with high specific activity
cDNA probes to isolated embryonic chick ventricular
cardiocytes, showed that type V AC mRNA appeared to
be localized primarily, if not exclusively, in myocytes.
However, the signal for AC VI mRNA was stronger in
non-myocytes than in myocytes. In situ hybridization is
useful to judge the expression of definite mRNA in
given cell types but this sophisticated approach does
not provide any information on the subcellular location
of the enzyme protein. Immunocytochemistry permit-
ted labeling with specific antibodies (AB) adenylyl
cyclase (Menco et al., 1992) and the product of its
hydrolytic activity, cAMP (Steiner et al., 1976; De Vente
et al., 1993). However, limited availability of high-
affinity, isoform-specific AB, until recently, accounted
for the scarcity of reports on immunocytochemical
localization of AC.
Currently, Santa Cruz Biotechnology, Inc., USA, has
started the production of specific rabbit polyclonal AB
directed against different epitopes of the enzyme mol-
ecule for differential immunocytochemical localization
of AC subtypes, including AC IV and AC V/VI. These AB
react with respective isotypes of AC of mouse and rat
origin by Western blotting and immunocytochemistry
and are not cross-reactive with other AC isotypes. We
applied AB from this source to immunocytochemical
localization of AC IV and V/VI in the heart of adult and
new-born Wistar rats and in cultured adult and neona-
tal cardiocytes.
Heart cryostat sections were fixed in a cold mixture of
methanol/acetone (1/1). Cell monolayers of adult and
neonatal cardiomyocytes (Wallukat and Wollenberger,
1993) were fixed in methanol at room temperature after
a short rinse in PBS. Thereafter, cryosections and cell
monolayers were treated for 10 minutes with methanol
containing 0.6% H2O2 to quench endogenous peroxi-
dase, rinsed briefly in distilled water (1–2 minutes),
washed in three changes of PBS with 0.5% Tween 20
(PBST) for 10 minutes, preincubated in 10% normal
goat serum in PBST for 30 minutes, reacted with a
rabbit primary polyclonal AB that recognizes AC IV or
AC V/VI (2 µg/ml PBST) for 2 hours at room tempera-
ture or overnight at 4°C, and washed in three changes
of PBST for 10 minutes. Detection of bound primary AB
was performed employing an avidin-biotin-peroxidase
staining kit (Vectastain ‘‘Elite’’ ABC-kit, Camon Labor-
Service GmbH). For conventional bright-field micros-
copy, heart sections and cell monolayers were incubated
with a diaminobenzidine-H2O2 mixture to visualize the
peroxidase label, slightly counterstained with Ehrlich
haemalaun, and mounted with aqueous mounting me-
dium (Dianova). For fluorescence microscopy, bound
primary AB were visualized with goat-anti-rabbit AB
labeled with DTAF (Dianova). Nuclei were counter-
stained with propidium iodide. In control preparations,
substitution of primary antibodies for rabbit IgG (whole
molecule) diluted in PBST to a final concentration of 2
µg/ml as well as incubation in media devoid of primary
antibodies revealed no specific immunostaining.
DTAF and peroxidase labeling both yielded the same
pattern of immunostaining (Fig. 2). AC IV was revealed
in the perinuclear space and sarcolemma of cardiomyo-
cytes of adult and new-born rats (Figs. 2a–c), as well as
in intercalated discs in the heart tissue of adult animals
(Fig. 2a). Positive immunolabeling of AC IV was also
detected in the plasma membrane of cardiomyocytes
from neonatal and adult animals (Figs. 2e,f) and in the
perinuclear space of neonatal cardiomyocytes (Fig. 2e).
In non-cardiomyocytes emerging in the cell culture of
neonatal cardiomyocytes, positive immunolabeling of
AC IV was also visible in the perinuclear zone and
apparently bound to cytoskeleton fibres (Fig. 2d).
In cultured adult rat cardiomyocytes (Fig. 2f), the
pattern of immunolabeling and immunostaining inten-
sity was nearly equal for AC IV and AC V/VI. In the
epicardia of adult rats, AC V/VI was immunolabeled
stronger than AC IV, whereas endotheliocytes posi-
tively immunoreacted only with AB to AC IV. Cardio-
myocytes in cryostat sections of the hearts from adult
and new-born rats, as well as cardiomyocytes, demon-
strated a similar pattern of immunolabeling of AC IV
and AC V/VI. Immunostaining for AC V/VI in cardiomyo-
cytes in cryosections of the heart of adult and new-born
rats was less intense than for AC IV, which cannot,
however, reflect the real ratio of expression of respec-
tive antigens in cardiomyocytes. This may just reflect
only differences in the affinity of the AB applied.
AC immunolabeling not only on the plasma mem-
brane of cardiocytes but also in the perinuclear space is
consistent with earlier published enzymocytochemical
data on the localization of AC activity in the perinuclear
space, endoplasmic reticulum, and Golgi complex of
macrophages (Dini and Del Rosso, 1983) and thymo-
cytes (Buchwalow et al., 1981). Colocalization of AC IV
with cytoskeleton fibers of non-cardiomyocytes (Fig. 2d)
testifies to a direct cytoskeletal-AC interaction, which
was implied earlier from cell fractionation data
(Rasenick et al., 1981; Wolff and Cook, 1985). Finally, it
must be noted that the immunolabeling pattern of AC
in cryosections of adult and new-born rat hearts reveals
a good correspondence with enzymocytochemical dem-
onstration of AC activity in cardiomyocytes. More de-
tailed confirmation is awaited from electron micro-
scopic immunocytochemical studies which are now in
progress.
ACKNOWLEDGMENTS
The authors are grateful to Marianne Vannauer and
Ingeborg Ley for exellent technical assistance. We are
indebted to Dr. Rosemarie Morwinski and Dr. Gerd
Wallukat for providing us with cultivated neonatal and
adult cardiocytes.
 Fig. 2. Immunocytochemical localization of AC with rabbit poly-
clonal antibodies in Wistar rat cardiocytes. Positive immunolabeling
of AC IV in sarcolemma (arrowheads), nuclear envelope (arrows),
intercalated disc (black asterisk), and cytoskeleton (white asterisk) in
acetone-methanol fixed cryostat sections of adult (a, b) and neonatal
(c) hearts, in a non-cardiomyocyte (d) and cardiomyocytes (e) in the
cell culture of neonatal rat cardiocytes and in cultured adult rat
cardiomyocytes (f). Secondary antibodies were goat-anti-rabbit IgG
labeled with horseradish peroxidase (visualization with diaminobenzi-
dine-H202 procedure—a, e, f) or with DTAF (b, c, d). Nuclei counter-
stained with hematoxylin (a, e, f) or propidium iodide (c, d).
478 W. SCHULZE AND I.B. BUCHWALOW
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