MICROSCOPY RESEARCH AND TECHNIQUE 40:479–487 (1998) Intracellular Distribution of Adenylate Cyclase in Human Cardiocytes Determined by Electron Microscopic Cytochemistry SHOJI YAMAMOTO,1 KEISHIRO KAWAMURA,2 AND THOMAS N. JAMES1* 1World Health Organization Cardiovascular Center and the Department of Medicine and Department of Pathology at the University of Texas Medical Branch, Galveston, Texas, USA 2Osaka Medical College, Osaka, Japan KEY WORDS sarcoplasmic reticulum; nuclear envelope; intracellular compartmentalization ABSTRACT Subcellular localization of adenylate cyclase (AC) in human cardiocytes was studied by electron microscopic cytochemistry using ventricular biopsies from various diseased hearts. In addition to the weak enzyme activity on the sarcolemma, the intense reaction products of AC were demonstrated within distinctive morphologic components of sarcoplasmic reticulum, nuclear envelope, and other internal membranes such as parallel lamellar structures and interlaced tubular structures in the perinuclear regions and stacked membranous structures beneath sarcolemma in cardiocytes. The distribution and intensity of cytochemical activity within different organelles was variable among biopsy cases. The reaction products of AC cytochemistry within the sarcoplasmic reticulum could be related to signal transduction targeting Ca21 handling by the organella. Cytochemical activity within the nuclear envelope and perinuclear internal membranes possibly reflects AC participation in a signal function to regulate nuclear activity, such as gene expression. Cytochemical distribution of the enzyme in membranous structures beneath the sarcolemma is most likely related to hormone receptors and the linked activity of AC. The subcellular distribution of AC on various internal membrane structures in cardiocytes may reflect compartmentalization of the enzyme at individual intracellular sites to regulate a preferential specific signal function among multiple potential signal transductions by a cascade of AC, cyclic AMP, and cyclic AMP-dependent protein kinase. Alternatively, subcellular localization of the reaction products may reflect local enzyme synthesis or represent sites of enzyme transport, e.g., to terminal localization beneath the sarcolemma. Microsc. Res. Tech. 40:479–487, 1998. r 1998 Wiley-Liss, Inc. INTRODUCTION Cytochemical studies have demonstrated intracellular adenylate cyclase (AC) activity within the sarcoplasmic reticulum or beneath the sarcolemma in animal (Revis, 1979; Schulze et al., 1972; Slezak and Geller, 1979, 1984; Wollenberger and Schulze, 1976) and human (Yamamoto et al., 1991) cardiocytes. Despite various 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 reliability of AC cytochemistry (Kempen et al., 1978; Lemay and Jarett, 1975; Richards, 1994; Schulze, 1982). Nevertheless, electron microscopic cytochemistry has become both the most sensitive and the most widely used method to demonstrate a subcellular distribution of AC (Richards, 1994). Recently, cloning of cDNAs has identified a family of at least eight mammalian AC isoforms which differ in their amino acid sequence, tissue distribution, and regulatory features of enzyme activity, including response to Ca21.They also differ in chromosomal location of coding genes (Espinasse et al., 1995; Gaudin et al., 1994; Haber et al., 1994; Iyengar, 1993). Among the different isoenzymes, type V and VI are the major AC isozymes expressed in mammalian heart (Espinasse et r 1998 WILEY-LISS, INC. al., 1995; Katsushika et al., 1992; Yu et al., 1995). By means of an antiserum that recognizes a peptide sequence shared by all known mammalian AC isoforms, studies with immunohistochemistry by electron microscopy have demonstrated in nerve cells a selective subcellular distribution of AC protein in the cytosol and certain internal membranes (Mons et al., 1995). As more sensitive methods are now available for the demonstration of AC localization, the credibility of the subcellular distribution of AC has increased. AC, along with cyclic AMP (cAMP) and cAMPdependent protein kinase, mediates a signal transduction which regulates a variety of physiological responses by cells. To control a specific signal function among multiple potential signal transductions, compartmentalization of cAMP and cAMP-dependent protein kinase has been suggested as a regulatory mechanism (Coghlan et al., 1993; Hausken et al., 1994; McCartney et 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 Medical Branch, Galveston, Texas. *Correspondence to: Thomas N. James, M.D., Office of the President, University of Texas Medical Branch, Galveston, Texas 77555-0129 USA. Received 30 July 1996; accepted in revised form 13 November 1996. 480 S. YAMAMOTO ET AL. Fig. 1. Electron microscopic cytochemistry of AC in a left ventricular myocardial biopsy. AC activity is demonstrated as electron-dense deposits of reaction products on well-preserved fine structures of a cardiocyte. In addition to the weak activity within sarcolemma, the intense 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 electron microscopic cytochemistry of AC and stained with uranyl acetate and/or lead citrate. strate cytochemical localization of AC at several different intracellular sites in addition to the sarcoplasmic reticulum and sarcolemma in human cardiocytes. cytochemical study to demonstrate AC activity (Slezak and Geller, 1979, 1984). Immediately after biopsy, tissues were fixed for 15 minutes 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. Tissues of sufficient size were cut with a Vibratome (Technical Products International, St. Louis, MO) into 40 µm thick sections to facilitate diffuse infiltration of the tissues with incubation medium. Smaller tissues were treated as a block. After fixation, tissues were rinsed in the same buffer for 1 to 2 hours at 4°C. Incubation in freshly prepared medium was for 45 minutes at 37°C to demonstrate AC activity. The assay medium consisted of 80 mmol/L Tris-maleate buffer (Sigma Chemical, St. Louis, MO), pH 7.4, with 8% sucrose, 5% DMSO, 2 mmol/L theophylline (Nakarai, Kyoto, Japan), 4 mmol/L MgSO4 (Nakarai), 2 mmol/L lead nitrate as tracer, and purified 0.5 mmol/L adenylate imidodiphosphate (AMPPNP; Sigma) as substrate. Then 20 mmol/L NaF (Nakarai) and 10 mmol/L DL-isoproterenol HCl (Nakarai) were added as activators of AC. In some controls, ouabain (G-strophanthin; Nakarai) 10 mmol/L was added as an inhibitor of (Na1, K1)ATPase, and 1 MATERIALS AND METHODS Materials Sixteen myocardial biopsies (six from the right ventricle and ten from the left ventricle) were obtained from 16 patients by means of a Konno-Sakakibara bioptome (Sakakibara and Konno, 1962; Konno and Sakakibara, 1963) at catheterization or by Vim Silverman needle during open-heart surgery or permanent pacemaker implantation. The patients included 14 men and two women, age 34 to 63 years (mean, 49); seven patients had congenital or acquired heart diseases and nine had idiopathic cardiomyopathies (five cases dilated and four hypertrophic). Informed consent for biopsy was obtained from every patient and all procedures were conducted according to the principles of the Declaration of Helsinki. Methods In addition to conventional light and electron microscopy studies, parts of each biopsy sample served for AC DISTRIBUTION IN CARDIOCYTES 481 Fig. 2. Higher magnification of boxed areas A (2a) and B (2b) from Figure 1. (a) The reaction products of AC are seen within tubular elements of sarcoplasmic reticulum associated with myofibrils (arrows) and cobular element of sarcoplasmic reticulum (arrow heads). (b) The reaction products are seen within internal (arrows) and peripheral (arrow heads) junctional sarcoplasmic reticulum. Reference bars 5 1 µm. Mf, myofibril; T, T tubule: IS, interstitial space. mmol/L levamisole HCl (Aldrich Chemical., Milwaukee, WI) was added as an inhibitor of alkaline phosphatase in the assay medium. Tissues for cytochemical control were incubated in the medium without AMPPNP. After incubation, tissues were rinsed several times in 0.1 mol/L sodium cacodylate buffer (pH 7.4) containing 8% sucrose and postfixed for 10 minutes at 4°C with 1% osmium tetroxide in 0.1 mol/L sodium cacodylate buffer (pH 7.4). Serial dehydration in graded alcohols and propylene oxide was followed by embedding in Epon resin (Luft, 1961). Semithin (1 µm) sections were cut with an LKB 8800 Ultratome III and stained with toluidine blue to use for selection purpose. Ultrathin sections were cut with a diamond knife, stained with aqueous uranyl acetate and/or lead citrate, and examined with either a Hitachi 300 or H-7000 electron microscope. intracellular organelles (Figs. 1–7). In controls in which tissues were incubated in the assay medium with ouabain and levamisol HCL, lesser amounts of nonspecific dispersion of deposits were seen. However, there was no difference in the enzyme localization in association with specific intracellular structures. No reaction products of the enzyme activity were seen in tissues of the cytochemical controls. Weak activity of AC, seen as spotty or uneven distribution of small amounts of reaction deposits, was found in association with sarcolemma in most biopsy specimens. However, intense enzyme activity with homogenous or dense distribution of abundant deposits was demonstrated only on different intracellular organelles. The intensity and distribution of the cytochemical enzyme activity on individual organelles differed among biopsy specimens but had no apparent correlation with the clinical diagnosis. The most frequently observed localizations of AC cytochemical activity were in the sarcoplasmic reticulum, nuclear envelope, perinuclear lamellar and tubular structures, and subsarcolemmal membranous structures. RESULTS AC activity was apparent as deposits of coarse or fine granules of electron-dense reaction product. Fine structures of tissues were well preserved so that a localization of the enzyme could be studied in relation to 482 S. YAMAMOTO ET AL. Fig. 3. Higher magnification of boxed area C from Figure 1. The cytochemical activity is demonstrated in association with parallel lamellar structures in the perinuclear region (arrows). The cisternae are stacked in parallel with narrow cytoplasmic interspaces. The reaction products are seen within individual cistern structures. The enzyme activity is also seen within tubular structures near the nucleus (arrow heads). Reference bar 5 1 µm. N, nucleus; Lf, lipofuscin granule. Sarcoplasmic Reticulum AC activity was present in several morphologically distinct components of sarcoplasmic reticulum (Fig. 2). The reaction products were seen on both the internal and peripheral junctional sarcoplasmic reticulum in many cardiocytes of most biopsy specimens. In some specimens, the enzyme activity was localized on additional tubular elements of free sarcoplasmic reticulum associated with myofibrils and mitochondria. Reaction products were also seen on corbular elements of sarcoplasmic reticulum. The localization patterns of the enzyme activity among the different components of sarcoplasmic reticulum were variable among individual cardiocytes. Perinuclear Parallel Cisternae and Interlaced Tubular Structures In some biopsy specimens, the enzyme activity was present in parallel lamellar structures in the peri- Fig. 4. Electron micrograph of a right ventricular myocardial biopsy. The enzyme activity is localized within tubular structures (arrows) which present an interlaced network in the perinuclear region of a cardiocyte. The tubular structures appeared to be endoplasmic reticulum, even though not clear whether rough or smooth components of the reticulum. Reference bar 5 1 µm. nuclear regions (Fig. 3). The membrane structures consisted of stacks of parallel cisternae. The reaction product was most intense in individual cisternae. In the perinuclear regions of some cardiocytes, the enzyme activity was also demonstrated in association with interlaced tubular structures (Fig. 4). Nuclear Envelope AC activity was demonstrated on nuclear envelope in some cardiocytes (Fig. 5). These reaction products were found mainly in association with the perinuclear cisterna, a space between the two parallel (outer and inner) membranes of the nuclear envelope. Membranous Structures at the Cell Periphery Enzyme activity was occasionally demonstrated in association with membranous structures at the cell periphery (Fig. 6). A few membranous structures be- AC DISTRIBUTION IN CARDIOCYTES Fig. 5. Electron micrograph of a right ventricular myocardial biopsy. AC activity is demonstrated in the nuclear envelope (arrows). The reaction products are localized within the perinuclear cisternae of the envelope. The enzyme activity is also seen in association with the parallel lamellar structures (arrowheads). Reference bar 5 1 µm. Mt, mitochondria. neath the sarcolemma were stacked in parallel with narrow cytoplasmic interspaces. The reaction products were localized on individual membranes. The membranous structures were flattened cisternae with two membranes opposed tightly. Coated vesicles and endocytotic pits were occasionally seen in the same vicinity. Other Organelles Additional intracellular organelles in which enzyme activity was demonstrated (Fig. 7) included vesicular, tubular, and membranous structures of undetermined origin or derivation. DISCUSSION Intracellular Localization of AC in Cardiocytes Cytochemical demonstration of AC activity has often been done in various mammalian tissues, including heart. Questions about the validity of cytochemistry have been based upon several criticisms of histochemical procedures such as substrate specificity, nonenzymatic hydrolysis of AMP-PNP by lead, and inhibition of AC activity by lead or by fixation procedures (Kempen et al., 1978; Lemay and Jarett, 1975; Rich- 483 ards, 1994; Schulze, 1982). Various methodologic improvements have been developed in response to this criticism (Cutler, 1975; Howell and Whitfield, 1972; Schulze et al., 1977; Slezak and Geller, 1979; Wagner et al., 1972) and quantitative analysis of the effect of individual cytochemical procedures on AC has been studied in different tissues, including heart (Schulze, 1982). Detailed efforts to standardize optimal procedures among many improved cytochemical methods were also conducted in animal tissues, such as epidermal epithelium (Richards, 1994). In the present study, methodologic improvements included the use of AMP-PNP as substrate, ouabain and levamisol HCl as other enzyme inhibitors, and low lead concentration of tracer. Considering the possible inhibition of basal activity of AC by lead (Schulze, 1982) and the stimulation of the enzyme activity by isoproterenol and NaF in the incubation medium in the present study, the localization of AC presented here could be associated with hormone-stimulated enzyme activity, not solely with the basal enzyme activity. The localization of AC activity we found in human cardiocytes is consistent with the cytochemical distribution of the enzyme in other mammalian cardiocytes (Schulze et al., 1972, 1977; Slezak and Geller, 1979, 1984; Wollenberger and Schulze, 1976). As we previously reported using similar biopsy materials of human myocardium (Yamamoto et al., 1991), AC was again cytochemically demonstrated within sarcoplasmic reticulum in addition to weak enzyme activity on sarcolemma. But we have now demonstrated the intracellular distribution of enzyme activity in association with a wide spectrum of other membrane structures. The cytochemical distribution of AC among intracellular organelles was variable among biopsy cases, irrespective of their clinical diagnosis. The cytochemical variations could reflect intrinsic involvement of the AC system in diseased cardiocytes, but also the various alterations of enzyme activity in response to multiple extrinsic regulation on cardiocyte functions. Even with the methodologic improvements used in the present study, the results may not be totally free of the problems of cytochemical procedures. For example, the validity of the distribution of enzyme activity on internal membranes is still controversial (Richards, 1994). Sarcoplasmic reticulum, which exhibited the most intense enzyme activity in the present study, is known as the crucial regulatory site of cytosolic Ca21, which is a principal target of hormonal signal transduction in cardiocytes. The localization of other mediators such as cAMP and cAMP-dependent protein kinase on sarcoplasmic reticulum has already been suggested (Coghlan et al., 1993; Hausken et al., 1994; McCartney et al., 1995; Oquist et al., 1992; Smith et al., 1993; Yabana et al., 1995). Thus, we believe that the cytochemical localization within sarcoplasmic reticulum and other internal membranes in the present study is closely related to subcellular distribution of in vivo activity of AC. Cyclic AMP has been long recognized as a second messenger (Gilman, 1987; Pastan, 1972; Schramm and Selinger, 1984; Sutherland, 1972). The cytochemical localization of AC on cell membranes is consistent with the enzyme distribution linked with hormone receptors on cell membranes. Conversely, the cytochemical local- 484 S. YAMAMOTO ET AL. Fig. 6. Electron micrographs of a left ventricular biopsy. AC activity is present within membranous structures at the cell periphery (arrows). Those beneath the sarcolemma are stacked in parallel with narrow cytoplasmic interspaces. Insert is electron micrograph from a different serial ultrathin section of the same cardiocytic area. Similar structures presented at different levels in these serial sections indicate that the enzyme activity is present in membranous structures situated parallel to the sarcolemma. Coated vesicle (large arrowhead) and coated pit (small arrowhead) are seen in the vicinity of enzyme localization. Reference bars 5 1 µm. ization of AC within intracellular organelles might be questioned on the basis of inconsistency with the second-messenger theory. In a recent immunohistochemical study of AC in nerve cells (Mons et al., 1995), some reaction products were detectable in association with endoplasmic reticulum or microtubules in cell bodies in addition to the most intense immunoreactivity in dendritic spines. Even though these findings were reported in neurons and not in cardiocytes, the consistency of enzyme distribution on the internal membranes in this immunohistochemical study and in another cytochemical study of similar nerve tissue (Drescher et al., 1995) supports the likelihood of the localization of AC within the intracellular organelles of cardiocytes in animal and human myocardium. A number of biochemical studies have also suggested that there is AC activity within the sarcoplasmic reticulum in mammalian myocardium (Katz et al., 1974; Sulakhe and Dhalla, 1973). target substrates by cAMP-dependent protein kinase initiates several different physiological responses. It has been suggested that this protein kinase is compartmentalized with its substrates so that individual stimulation can preferentially result in phosphorylation of the specific substrates (Coghlan et al., 1993). For example, the differential localization of type II cAMPdependent protein kinase is achieved by an interaction of the regulatory subunit of the enzyme with A-kinase anchor proteins, which are localized in the sarcoplasmic reticulum of rat cardiocytes (Hausken et al., 1994; McCartney et al., 1995). Compartmentalization of cAMP is also suggested by the localization of cyclic GMPinhibited cAMP phosphodiesterase in sarcoplasmic reticulum (Oquist et al., 1992). Beta 1-adrenoceptor agonists and beta 2-adrenoceptor agonists cause differential compartmentalization of cAMP and cAMP-dependent protein kinase in cardiac muscle (Yabana et al., 1995). In addition to the compartmentalization of cAMPdependent protein kinase and cAMP, preferential activation of a specific signal function following individual stimulation could also be regulated by a compartmentalization of AC to subcellular locations. In a previous biochemical study which indicated compartmentalization of cAMP and cAMP-dependent protein kinase in Significance of the Enzyme Distribution on Internal Membranes of Cardiocytes The signal transduction mediated by a cascade of AC, cAMP, and cAMP-dependent protein kinase regulates a variety of biochemical events. The phosphorylation of AC DISTRIBUTION IN CARDIOCYTES 485 Fig. 7. Electron micrographs of ventricular myocardial biopsies. The AC reaction products are seen in association with various subcellular structures (arrows). Some of the structures are vesicles of different size (a, b) and others are membranous (c) or tubular (d) structures. They are often unclear as to structural origin. rabbit cardiocytes (Buxton and Brunton, 1983), it was proposed that the compartmentalization begins at the level of hormone-receptor AC complex, whereas the location of the compartment was speculated to be on sarcolemma. The distribution of cytochemical enzyme activity within a variety of intracellular membranes in the present study may reflect exactly such compartmentalization of AC within cardiocytes. AC activity within the sarcoplasmic reticulum most likely represents a regulatory function by signal transduction for calcium handling by the sarcoplasmic reticulum (calcium uptake, release or binding) (Yamamoto et al., 1991). Nuclear trafficking of the catalytic subunit of cAMPdependent protein kinase is critical for a regulation of gene expression and the localization of both catalytic unit and regulatory subunits of protein kinase has been demonstrated in cell nuclei of various rat tissues (Trinczek et al., 1993). Active or passive mechanisms have been suggested for nuclear import and export of this enzyme protein (Harootunian et al., 1993; Wen et al., 1995). AC distributed on the nuclear envelope in cardiocytes of the present study could regulate nuclear functions, such as gene expression, through a cascade mediated by cAMP and cAMP-dependent protein kinase. The localization of AC in association with perinuclear parallel cisternae and interlaced tubular structures could also reflect a signal transduction, which controls nuclear functions through cAMP-dependent protein kinase. The perinuclear region is a site of protein synthesis and AC distributed in this subcellular location could also serve to regulate specific protein synthesis in cardiocytes. AC found within membranous structures at the cell periphery is most likely related to the enzyme activity linked to hormone receptors on the sarcolemma. The coated vesicles or pits in the vicinity of this enzyme localization may support the relation to the hormone receptors. This enzyme distribution possibly reflects a putative phenomenon of recycling of hormone receptors and AC localized on or near the sarcolemma. Diversity of ACs, such as different amino acid sequence, mode of enzyme activity regulation, and chromosomal location of coding genes among eight isoforms of the enzyme (Espinasse et al., 1995) may well represent other key elements for the regulation of preferential activation of individual signals among multiple poten- 486 S. YAMAMOTO ET AL. tial signal transductions. The enzyme activity of type V and VI ACs in mammalian hearts is inhibited by submicromolar concentrations of Ca21 and, consequently, serve to regulate cardiocyte contractility. The specific physiologic significance of the differential cardiac expression of these two AC isoforms in relation to their similar biochemical properties remains unknown (Espinasse et al., 1995; Katsushika et al., 1992; Yu et al., 1995). Another explanation of the localization of the cytochemical enzyme activity within some organelles may lie in the synthesis of the enzyme or its transport directed to the sarcolemma. The cytochemical demonstration of AC may represent not only functional activity of the enzyme on the sarcolemma, but collectively all its potential activity (such as transport) which could be caused by exposure to NaF and isoproterenol in the incubation medium. It has been shown that AC localized on cell membranes is closely linked to cell surface receptors, which bind to extracellular signals, including hormones (Gilman, 1987; Pastan, 1972; Schramm and Selinger, 1984; Sutherland, 1972). In the present study of human cardiocytes, the cytochemical localization of AC was demonstrated for several different intracellular membranes and the potential significance as a regulatory mechanism for a specific signal function is postulated. An essential question now becomes what could be the upstream signals to regulate AC activity within these intracellular organelles. CONCLUSIONS Intracellular distribution of AC within human cardiocytes was demonstrated with electron microscopic cytochemistry. The enzyme localization in association with various internal membranes may reflect compartmentalization of AC to sarcoplasmic reticulum, nuclear envelope, sarcolemma, and other membrane structures to regulate a preferential signal processing for Ca21 handling, gene expression, and hormone actions within these cardiocytes. REFERENCES Buxton, I.L.O., and Brunton, L.L. (1983) Compartments of cyclic AMP and protein kinase in mammalian cardiocytes. J. Biol. Chem., 258:10233–10239. 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