Intracellular Distribution of Adenylate Cyclase in HumanCardiocytes Determined by Electron MicroscopicCytochemistrySHOJI YAMAMOTO,1 KEISHIRO KAWAMURA,2 AND THOMAS N. JAMES1*1World Health Organization Cardiovascular Center and the Department of Medicine and Department of Pathologyat the University of Texas Medical Branch, Galveston, Texas, USA2Osaka Medical College, Osaka, Japan

KEY WORDS sarcoplasmic reticulum; nuclear envelope; intracellular compartmentalization

ABSTRACT Subcellular localization of adenylate cyclase (AC) in human cardiocytes wasstudied by electron microscopic cytochemistry using ventricular biopsies from various diseasedhearts. In addition to the weak enzyme activity on the sarcolemma, the intense reaction products ofAC were demonstrated within distinctive morphologic components of sarcoplasmic reticulum,nuclear envelope, and other internal membranes such as parallel lamellar structures and interlacedtubular structures in the perinuclear regions and stacked membranous structures beneathsarcolemma in cardiocytes. The distribution and intensity of cytochemical activity within differentorganelles was variable among biopsy cases. The reaction products of AC cytochemistry within thesarcoplasmic reticulum could be related to signal transduction targeting Ca21 handling by theorganella. Cytochemical activity within the nuclear envelope and perinuclear internal membranespossibly reflects AC participation in a signal function to regulate nuclear activity, such as geneexpression. Cytochemical distribution of the enzyme in membranous structures beneath thesarcolemma is most likely related to hormone receptors and the linked activity of AC. Thesubcellular distribution of AC on various internal membrane structures in cardiocytes may reflectcompartmentalization of the enzyme at individual intracellular sites to regulate a preferentialspecific signal function among multiple potential signal transductions by a cascade of AC, cyclicAMP, and cyclic AMP-dependent protein kinase. Alternatively, subcellular localization of thereaction products may reflect local enzyme synthesis or represent sites of enzyme transport, e.g., toterminal localization beneath the sarcolemma. Microsc. Res. Tech. 40:479–487, 1998. r 1998 Wiley-Liss, Inc.

INTRODUCTIONCytochemical studies have demonstrated intracellu-

lar adenylate cyclase (AC) activity within the sarcoplas-mic reticulum or beneath the sarcolemma in animal(Revis, 1979; Schulze et al., 1972; Slezak and Geller,1979, 1984; Wollenberger and Schulze, 1976) and hu-man (Yamamoto et al., 1991) cardiocytes. Despite vari-ous attempts to improve cytochemical methods (Cutler,1975; Howell and Whitfield, 1972; Schulze et al., 1977;Schulze, 1982; Slezak and Geller, 1979; Wagner et al.,1972), there are still some questions about the reliabil-ity of AC cytochemistry (Kempen et al., 1978; Lemayand Jarett, 1975; Richards, 1994; Schulze, 1982). Never-theless, electron microscopic cytochemistry has becomeboth the most sensitive and the most widely usedmethod to demonstrate a subcellular distribution of AC(Richards, 1994).

Recently, cloning of cDNAs has identified a family ofat least eight mammalian AC isoforms which differ intheir amino acid sequence, tissue distribution, andregulatory features of enzyme activity, including re-sponse to Ca21.They also differ in chromosomal locationof coding genes (Espinasse et al., 1995; Gaudin et al.,1994; Haber et al., 1994; Iyengar, 1993). Among thedifferent isoenzymes, type V and VI are the major ACisozymes expressed in mammalian heart (Espinasse et

al., 1995; Katsushika et al., 1992; Yu et al., 1995). Bymeans of an antiserum that recognizes a peptide se-quence shared by all known mammalian AC isoforms,studies with immunohistochemistry by electron micros-copy have demonstrated in nerve cells a selectivesubcellular distribution of AC protein in the cytosol andcertain internal membranes (Mons et al., 1995). Asmore sensitive methods are now available for thedemonstration of AC localization, the credibility of thesubcellular distribution of AC has increased.

AC, along with cyclic AMP (cAMP) and cAMP-dependent protein kinase, mediates a signal transduc-tion which regulates a variety of physiological re-sponses by cells. To control a specific signal functionamong multiple potential signal transductions, compart-mentalization of cAMP and cAMP-dependent proteinkinase has been suggested as a regulatory mechanism(Coghlan et al., 1993; Hausken et al., 1994; McCartneyet al., 1995; Oquist et al., 1992; Smith et al., 1993;Yabana et al., 1995). In the present study, we demon-

Contract grant sponsor: Pegasus Fund of the University of Texas MedicalBranch, Galveston, Texas.

*Correspondence to: Thomas N. James, M.D., Office of the President, Univer-sity of Texas Medical Branch, Galveston, Texas 77555-0129 USA.

Received 30 July 1996; accepted in revised form 13 November 1996.

MICROSCOPY RESEARCH AND TECHNIQUE 40:479–487 (1998)

r 1998 WILEY-LISS, INC.

strate cytochemical localization of AC at several differ-ent intracellular sites in addition to the sarcoplasmicreticulum and sarcolemma in human cardiocytes.

MATERIALS AND METHODSMaterials

Sixteen myocardial biopsies (six from the right ven-tricle and ten from the left ventricle) were obtainedfrom 16 patients by means of a Konno-Sakakibarabioptome (Sakakibara and Konno, 1962; Konno andSakakibara, 1963) at catheterization or by Vim Silver-man needle during open-heart surgery or permanentpacemaker implantation. The patients included 14 menand two women, age 34 to 63 years (mean, 49); sevenpatients had congenital or acquired heart diseases andnine had idiopathic cardiomyopathies (five cases di-lated and four hypertrophic). Informed consent forbiopsy was obtained from every patient and all proce-dures were conducted according to the principles of theDeclaration of Helsinki.

MethodsIn addition to conventional light and electron micros-

copy studies, parts of each biopsy sample served for

cytochemical study to demonstrate AC activity (Slezakand Geller, 1979, 1984).

Immediately after biopsy, tissues were fixed for 15minutes at 4°C with 2% paraformaldehyde and 0.25%glutaraldehyde in 0.1 mol/L sodium cacodylate buffer(pH 7.4) containing 8% sucrose and 5% DMSO. Tissuesof sufficient size were cut with a Vibratome (TechnicalProducts International, St. Louis, MO) into 40 µm thicksections to facilitate diffuse infiltration of the tissueswith incubation medium. Smaller tissues were treatedas a block. After fixation, tissues were rinsed in thesame buffer for 1 to 2 hours at 4°C. Incubation in freshlyprepared medium was for 45 minutes at 37°C todemonstrate AC activity. The assay medium consistedof 80 mmol/L Tris-maleate buffer (Sigma Chemical, St.Louis, MO), pH 7.4, with 8% sucrose, 5% DMSO, 2mmol/L theophylline (Nakarai, Kyoto, Japan), 4 mmol/LMgSO4 (Nakarai), 2 mmol/L lead nitrate as tracer, andpurified 0.5 mmol/L adenylate imidodiphosphate (AMP-PNP; Sigma) as substrate. Then 20 mmol/L NaF (Naka-rai) and 10 mmol/L DL-isoproterenol HCl (Nakarai)were added as activators of AC. In some controls,ouabain (G-strophanthin; Nakarai) 10 mmol/L wasadded as an inhibitor of (Na1, K1)ATPase, and 1

Fig. 1. Electron microscopic cytochemistry of AC in a left ventricu-lar myocardial biopsy. AC activity is demonstrated as electron-densedeposits of reaction products on well-preserved fine structures of acardiocyte. In addition to the weak activity within sarcolemma, theintense activity is seen in association with various intracellular

organelles (arrowheads). More details of AC activity in boxed areas (A,B, and C) are shown in Figures 2 and 3. All micrographs are electronmicroscopic cytochemistry of AC and stained with uranyl acetateand/or lead citrate.

480 S. YAMAMOTO ET AL.

mmol/L levamisole HCl (Aldrich Chemical., Milwau-kee, WI) was added as an inhibitor of alkaline phospha-tase in the assay medium. Tissues for cytochemicalcontrol were incubated in the medium without AMP-PNP. After incubation, tissues were rinsed severaltimes in 0.1 mol/L sodium cacodylate buffer (pH 7.4)containing 8% sucrose and postfixed for 10 minutes at4°C with 1% osmium tetroxide in 0.1 mol/L sodiumcacodylate buffer (pH 7.4). Serial dehydration in gradedalcohols and propylene oxide was followed by embed-ding in Epon resin (Luft, 1961).

Semithin (1 µm) sections were cut with an LKB 8800Ultratome III and stained with toluidine blue to use forselection purpose. Ultrathin sections were cut with adiamond knife, stained with aqueous uranyl acetateand/or lead citrate, and examined with either a Hitachi300 or H-7000 electron microscope.

RESULTSAC activity was apparent as deposits of coarse or fine

granules of electron-dense reaction product. Fine struc-tures of tissues were well preserved so that a localiza-tion of the enzyme could be studied in relation to

intracellular organelles (Figs. 1–7). In controls in whichtissues were incubated in the assay medium withouabain and levamisol HCL, lesser amounts of nonspe-cific dispersion of deposits were seen. However, therewas no difference in the enzyme localization in associa-tion with specific intracellular structures. No reactionproducts of the enzyme activity were seen in tissues ofthe cytochemical controls.

Weak activity of AC, seen as spotty or uneven distri-bution of small amounts of reaction deposits, was foundin association with sarcolemma in most biopsy speci-mens. However, intense enzyme activity with homog-enous or dense distribution of abundant deposits wasdemonstrated only on different intracellular organelles.The intensity and distribution of the cytochemicalenzyme activity on individual organelles differed amongbiopsy specimens but had no apparent correlation withthe clinical diagnosis.

The most frequently observed localizations of ACcytochemical activity were in the sarcoplasmic reticu-lum, nuclear envelope, perinuclear lamellar and tubu-lar structures, and subsarcolemmal membranous struc-tures.

Fig. 2. Higher magnification of boxed areas A (2a) and B (2b) fromFigure 1. (a) The reaction products of AC are seen within tubularelements of sarcoplasmic reticulum associated with myofibrils (ar-rows) and cobular element of sarcoplasmic reticulum (arrow heads).

(b) The reaction products are seen within internal (arrows) andperipheral (arrow heads) junctional sarcoplasmic reticulum. Refer-ence bars 5 1 µm. Mf, myofibril; T, T tubule: IS, interstitial space.

481AC DISTRIBUTION IN CARDIOCYTES

Sarcoplasmic ReticulumAC activity was present in several morphologically

distinct components of sarcoplasmic reticulum (Fig. 2).The reaction products were seen on both the internaland peripheral junctional sarcoplasmic reticulum inmany cardiocytes of most biopsy specimens. In somespecimens, the enzyme activity was localized on addi-tional tubular elements of free sarcoplasmic reticulumassociated with myofibrils and mitochondria. Reactionproducts were also seen on corbular elements of sarco-plasmic reticulum. The localization patterns of theenzyme activity among the different components ofsarcoplasmic reticulum were variable among indi-vidual cardiocytes.

Perinuclear Parallel Cisternae and InterlacedTubular Structures

In some biopsy specimens, the enzyme activity waspresent in parallel lamellar structures in the peri-

nuclear regions (Fig. 3). The membrane structuresconsisted of stacks of parallel cisternae. The reactionproduct was most intense in individual cisternae. In theperinuclear regions of some cardiocytes, the enzymeactivity was also demonstrated in association withinterlaced tubular structures (Fig. 4).

Nuclear EnvelopeAC activity was demonstrated on nuclear envelope in

some cardiocytes (Fig. 5). These reaction products werefound mainly in association with the perinuclear cis-terna, a space between the two parallel (outer andinner) membranes of the nuclear envelope.

Membranous Structures at the Cell PeripheryEnzyme activity was occasionally demonstrated in

association with membranous structures at the cellperiphery (Fig. 6). A few membranous structures be-

Fig. 3. Higher magnification of boxed area C from Figure 1. Thecytochemical activity is demonstrated in association with parallellamellar structures in the perinuclear region (arrows). The cisternaeare stacked in parallel with narrow cytoplasmic interspaces. Thereaction products are seen within individual cistern structures. Theenzyme activity is also seen within tubular structures near thenucleus (arrow heads). Reference bar 5 1 µm. N, nucleus; Lf,lipofuscin granule.

Fig. 4. Electron micrograph of a right ventricular myocardialbiopsy. The enzyme activity is localized within tubular structures(arrows) which present an interlaced network in the perinuclearregion of a cardiocyte. The tubular structures appeared to be endoplas-mic reticulum, even though not clear whether rough or smoothcomponents of the reticulum. Reference bar 5 1 µm.

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neath the sarcolemma were stacked in parallel withnarrow cytoplasmic interspaces. The reaction productswere localized on individual membranes. The membra-nous structures were flattened cisternae with twomembranes opposed tightly. Coated vesicles and endo-cytotic pits were occasionally seen in the same vicinity.

Other OrganellesAdditional intracellular organelles in which enzyme

activity was demonstrated (Fig. 7) included vesicular,tubular, and membranous structures of undeterminedorigin or derivation.

DISCUSSIONIntracellular Localization of AC in CardiocytesCytochemical demonstration of AC activity has often

been done in various mammalian tissues, includingheart. Questions about the validity of cytochemistryhave been based upon several criticisms of histochemi-cal procedures such as substrate specificity, non-enzymatic hydrolysis of AMP-PNP by lead, and inhibi-tion of AC activity by lead or by fixation procedures(Kempen et al., 1978; Lemay and Jarett, 1975; Rich-

ards, 1994; Schulze, 1982). Various methodologic im-provements have been developed in response to thiscriticism (Cutler, 1975; Howell and Whitfield, 1972;Schulze et al., 1977; Slezak and Geller, 1979; Wagner etal., 1972) and quantitative analysis of the effect ofindividual cytochemical procedures on AC has beenstudied in different tissues, including heart (Schulze,1982). Detailed efforts to standardize optimal proce-dures among many improved cytochemical methodswere also conducted in animal tissues, such as epider-mal epithelium (Richards, 1994).

In the present study, methodologic improvementsincluded the use of AMP-PNP as substrate, ouabainand levamisol HCl as other enzyme inhibitors, and lowlead concentration of tracer. Considering the possibleinhibition of basal activity of AC by lead (Schulze, 1982)and the stimulation of the enzyme activity by isoproter-enol and NaF in the incubation medium in the presentstudy, the localization of AC presented here could beassociated with hormone-stimulated enzyme activity,not solely with the basal enzyme activity. The localiza-tion of AC activity we found in human cardiocytes isconsistent with the cytochemical distribution of theenzyme in other mammalian cardiocytes (Schulze et al.,1972, 1977; Slezak and Geller, 1979, 1984; Wollen-berger and Schulze, 1976). As we previously reportedusing similar biopsy materials of human myocardium(Yamamoto et al., 1991), AC was again cytochemicallydemonstrated within sarcoplasmic reticulum in addi-tion to weak enzyme activity on sarcolemma. But wehave now demonstrated the intracellular distributionof enzyme activity in association with a wide spectrumof other membrane structures. The cytochemical distri-bution of AC among intracellular organelles was vari-able among biopsy cases, irrespective of their clinicaldiagnosis. The cytochemical variations could reflectintrinsic involvement of the AC system in diseasedcardiocytes, but also the various alterations of enzymeactivity in response to multiple extrinsic regulation oncardiocyte functions.

Even with the methodologic improvements used inthe present study, the results may not be totally free ofthe problems of cytochemical procedures. For example,the validity of the distribution of enzyme activity oninternal membranes is still controversial (Richards,1994). Sarcoplasmic reticulum, which exhibited themost intense enzyme activity in the present study, isknown as the crucial regulatory site of cytosolic Ca21,which is a principal target of hormonal signal transduc-tion in cardiocytes. The localization of other mediatorssuch as cAMP and cAMP-dependent protein kinase onsarcoplasmic reticulum has already been suggested(Coghlan et al., 1993; Hausken et al., 1994; McCartneyet al., 1995; Oquist et al., 1992; Smith et al., 1993;Yabana et al., 1995). Thus, we believe that the cyto-chemical localization within sarcoplasmic reticulumand other internal membranes in the present study isclosely related to subcellular distribution of in vivoactivity of AC.

Cyclic AMP has been long recognized as a secondmessenger (Gilman, 1987; Pastan, 1972; Schramm andSelinger, 1984; Sutherland, 1972). The cytochemicallocalization of AC on cell membranes is consistent withthe enzyme distribution linked with hormone receptorson cell membranes. Conversely, the cytochemical local-

Fig. 5. Electron micrograph of a right ventricular myocardialbiopsy. AC activity is demonstrated in the nuclear envelope (arrows).The reaction products are localized within the perinuclear cisternae ofthe envelope. The enzyme activity is also seen in association with theparallel lamellar structures (arrowheads). Reference bar 5 1 µm. Mt,mitochondria.

483AC DISTRIBUTION IN CARDIOCYTES

ization of AC within intracellular organelles might bequestioned on the basis of inconsistency with thesecond-messenger theory. In a recent immunohisto-chemical study of AC in nerve cells (Mons et al., 1995),some reaction products were detectable in associationwith endoplasmic reticulum or microtubules in cellbodies in addition to the most intense immunoreactiv-ity in dendritic spines. Even though these findings werereported in neurons and not in cardiocytes, the consis-tency of enzyme distribution on the internal mem-branes in this immunohistochemical study and in an-other cytochemical study of similar nerve tissue(Drescher et al., 1995) supports the likelihood of thelocalization of AC within the intracellular organelles ofcardiocytes in animal and human myocardium. A num-ber of biochemical studies have also suggested thatthere is AC activity within the sarcoplasmic reticulumin mammalian myocardium (Katz et al., 1974; Sulakheand Dhalla, 1973).

Significance of the Enzyme Distributionon Internal Membranes of Cardiocytes

The signal transduction mediated by a cascade of AC,cAMP, and cAMP-dependent protein kinase regulates avariety of biochemical events. The phosphorylation of

target substrates by cAMP-dependent protein kinaseinitiates several different physiological responses. Ithas been suggested that this protein kinase is compart-mentalized with its substrates so that individual stimu-lation can preferentially result in phosphorylation ofthe specific substrates (Coghlan et al., 1993). Forexample, the differential localization of type II cAMP-dependent protein kinase is achieved by an interactionof the regulatory subunit of the enzyme with A-kinaseanchor proteins, which are localized in the sarcoplas-mic reticulum of rat cardiocytes (Hausken et al., 1994;McCartney et al., 1995). Compartmentalization of cAMPis also suggested by the localization of cyclic GMP-inhibited cAMP phosphodiesterase in sarcoplasmic re-ticulum (Oquist et al., 1992). Beta 1-adrenoceptor ago-nists and beta 2-adrenoceptor agonists cause differentialcompartmentalization of cAMP and cAMP-dependentprotein kinase in cardiac muscle (Yabana et al., 1995).

In addition to the compartmentalization of cAMP-dependent protein kinase and cAMP, preferential acti-vation of a specific signal function following individualstimulation could also be regulated by a compartmental-ization of AC to subcellular locations. In a previousbiochemical study which indicated compartmentaliza-tion of cAMP and cAMP-dependent protein kinase in

Fig. 6. Electron micrographs of a left ventricular biopsy. ACactivity is present within membranous structures at the cell periphery(arrows). Those beneath the sarcolemma are stacked in parallel withnarrow cytoplasmic interspaces. Insert is electron micrograph from adifferent serial ultrathin section of the same cardiocytic area. Similar

structures presented at different levels in these serial sections indicatethat the enzyme activity is present in membranous structures situatedparallel to the sarcolemma. Coated vesicle (large arrowhead) andcoated pit (small arrowhead) are seen in the vicinity of enzymelocalization. Reference bars 5 1 µm.

484 S. YAMAMOTO ET AL.

rabbit cardiocytes (Buxton and Brunton, 1983), it wasproposed that the compartmentalization begins at thelevel of hormone-receptor AC complex, whereas thelocation of the compartment was speculated to be onsarcolemma. The distribution of cytochemical enzymeactivity within a variety of intracellular membranes inthe present study may reflect exactly such compartmen-talization of AC within cardiocytes. AC activity withinthe sarcoplasmic reticulum most likely represents aregulatory function by signal transduction for calciumhandling by the sarcoplasmic reticulum (calcium up-take, release or binding) (Yamamoto et al., 1991).Nuclear trafficking of the catalytic subunit of cAMP-dependent protein kinase is critical for a regulation ofgene expression and the localization of both catalyticunit and regulatory subunits of protein kinase has beendemonstrated in cell nuclei of various rat tissues (Trinc-zek et al., 1993). Active or passive mechanisms havebeen suggested for nuclear import and export of thisenzyme protein (Harootunian et al., 1993; Wen et al.,1995). AC distributed on the nuclear envelope in cardio-cytes of the present study could regulate nuclear func-tions, such as gene expression, through a cascade

mediated by cAMP and cAMP-dependent protein ki-nase. The localization of AC in association with peri-nuclear parallel cisternae and interlaced tubular struc-tures could also reflect a signal transduction, whichcontrols nuclear functions through cAMP-dependentprotein kinase. The perinuclear region is a site ofprotein synthesis and AC distributed in this subcellularlocation could also serve to regulate specific proteinsynthesis in cardiocytes.

AC found within membranous structures at the cellperiphery is most likely related to the enzyme activitylinked to hormone receptors on the sarcolemma. Thecoated vesicles or pits in the vicinity of this enzymelocalization may support the relation to the hormonereceptors. This enzyme distribution possibly reflects aputative phenomenon of recycling of hormone receptorsand AC localized on or near the sarcolemma.

Diversity of ACs, such as different amino acid se-quence, mode of enzyme activity regulation, and chromo-somal location of coding genes among eight isoforms ofthe enzyme (Espinasse et al., 1995) may well representother key elements for the regulation of preferentialactivation of individual signals among multiple poten-

Fig. 7. Electron micrographs of ventricular myocardial biopsies. The AC reaction products are seen inassociation with various subcellular structures (arrows). Some of the structures are vesicles of differentsize (a, b) and others are membranous (c) or tubular (d) structures. They are often unclear as to structuralorigin.

485AC DISTRIBUTION IN CARDIOCYTES

tial signal transductions. The enzyme activity of type Vand VI ACs in mammalian hearts is inhibited bysubmicromolar concentrations of Ca21 and, conse-quently, serve to regulate cardiocyte contractility. Thespecific physiologic significance of the differential car-diac expression of these two AC isoforms in relation totheir similar biochemical properties remains unknown(Espinasse et al., 1995; Katsushika et al., 1992; Yu etal., 1995).

Another explanation of the localization of the cyto-chemical enzyme activity within some organelles maylie in the synthesis of the enzyme or its transportdirected to the sarcolemma. The cytochemical demon-stration of AC may represent not only functional activ-ity of the enzyme on the sarcolemma, but collectively allits potential activity (such as transport) which could becaused by exposure to NaF and isoproterenol in theincubation medium.

It has been shown that AC localized on cell mem-branes is closely linked to cell surface receptors, whichbind to extracellular signals, including hormones (Gil-man, 1987; Pastan, 1972; Schramm and Selinger, 1984;Sutherland, 1972). In the present study of humancardiocytes, the cytochemical localization of AC wasdemonstrated for several different intracellular mem-branes and the potential significance as a regulatorymechanism for a specific signal function is postulated.An essential question now becomes what could be theupstream signals to regulate AC activity within theseintracellular organelles.

CONCLUSIONSIntracellular distribution of AC within human cardio-

cytes was demonstrated with electron microscopic cyto-chemistry. The enzyme localization in association withvarious internal membranes may reflect compartmen-talization of AC to sarcoplasmic reticulum, nuclearenvelope, sarcolemma, and other membrane structuresto regulate a preferential signal processing for Ca21

handling, gene expression, and hormone actions withinthese cardiocytes.

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487AC DISTRIBUTION IN CARDIOCYTES