MICROSCOPY RESEARCH AND TECHNIQUE 40:488–491 (1998) Immunocytochemical Localization of Adenylyl Cyclase in Human Myometrium PETER D.G. RICHARDS,1* ANDREW J. TILTMAN,1 AND PENELOPE A. RICHARDS2 1Department 2Department of Anatomical Pathology, SAIMR, Johannesburg, Republic of South Africa of Anatomy, University of Pretoria, Pretoria, Republic of South Africa KEY WORDS adenylyl cyclase V/VI; smooth muscle; relaxation ABSTRACT The enzyme adenylyl cyclase (AC) plays a pivotal role in smooth muscle relaxation. Biochemical evidence suggests that AC is predominantly located in the outer layers of the myometrium; however, neither immunocytochemical nor histochemical studies have been undertaken to demonstrate the specific cellular distribution of the enzyme in this tissue. As part of an ongoing study of the human myometrium, a polyclonal antibody against types V and VI AC was used to detect the presence of these isoforms in sections of formalin-fixed, wax-embedded myometrial tissue. A positive reaction was seen in the cytoplasm of the smooth muscle cells with the midmyometrial area having the greatest number of positive cells, when compared to the subserosal and subendometrial areas. It is hypothesized that AC isoform type VI is the predominant isoform present in the myometrium and that the percentage distribution of positive cells reflects the area of highest myometrial activity during parturition. Microsc. Res. Tech. 40:488–491, 1998. r 1998 Wiley-Liss, Inc. INTRODUCTION The human myometrium consists of interlacing bundles of smooth muscle in a loose connective tissue matrix. The myometrial wall itself can be histologically divided into three layers of variable distinction: the inner subendometrium, the midmyometrium, and the outer subserosal layer. Recent immunocytochemical studies have elucidated that estrogen receptors are not uniformly distributed through the depth of the myometrial wall (Richards and Tiltman, 1995). Similarly, biochemical evidence suggests that adenylyl cyclase (AC), an enzyme, is also differentially distributed in the myometrium (Fortier and Krall, 1983); however, this distribution does not appear to have been studied by either histochemical or immunocytochemical means and, thus, the exact cellular localization of AC remains unknown. AC is a membrane-bound enzyme that cyclisizes adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). In the context of smooth muscle, agents that induce relaxation, such as βadrenergic agonists, act directly through the AC-cAMP system (Lincoln and Cornwell, 1991). Since the elucidation of the structure and amino acid sequence of AC by Krupinski et al. (1989), at least eight isoforms have been cloned (Watson and Arkinstall, 1994). In situ hybridization studies and Western blot analysis using the cloned isoforms have determined their distribution in a number of tissues. Types I, II, III, VII, and VIII are usually located in mammalian neural tissue (Bakalyar and Reed, 1990; Cali et al., 1994; Cooper et al., 1995; Feinstein et al., 1991; Krupinski et al., 1989;), while types IV, V, and VI have a wider distribution in tissues such as heart, liver, and lung (Gao and Gilman, 1991; Ishikawa et al., 1992; Premont et al., 1992). Unfortunately, there has been little indepth immunocytochemical analyses of any of these tissues (Mons et al., 1995), with few studies having r 1998 WILEY-LISS, INC. screened uterine tissue in order to ascertain the isoform present. As both in situ hybridization and Western blot analysis have shown isoform type I to be absent from uterine tissue (Xia et al., 1993), it would be unlikely for the other predominantly neurally expressed isoforms to be present in myometrial cells. Similarly, it is questionable whether type IV occurs in the myometrium, for although it is present in human tissue, its occurrence is rare (Cooper et al., 1995). Therefore, as part of a wider study of the human myometrium, a polyclonal antibody against the C-terminal amino acids of the more widely distributed human isoforms, types V and VI, was used to determine 1) whether these isoforms are present in myometrial tissue and 2) if present, what the distribution of AC is within the myometrial wall. MATERIALS AND METHODS Samples of uterine tissue were obtained from eight patients undergoing hysterectomy for either menorrhagia or dysmenorrhoea. The patients ranged in age from 34 to 56 years and had no history of exogenous hormone treatment. Uteri were opened in the sagittal plane within 10 minutes of surgical excision from the patients. A transmural block of tissue was dissected from the fundus of each uterus, with both the endometrium and serosa intact. Following fixation in 10% formalin, the tissue was processed for routine histology. Wax sections stained with Mayer’s haematoxylin and eosin were used to determine the normality of the tissue and the phase of the endometrial cycle. Contract grant sponsor: Medical Research Council of South Africa. *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 10 September 1996; accepted in revised form 6 January 1997. ADENYLYL CYCLASE IN MYOMETRIUM The presence of AC types V and/or VI was immunocytochemically determined using a streptavidin-biotin technique. Briefly, sections of 4 µm thick were dewaxed and placed in a 3% methanolic solution of hydrogen peroxide for 30 minutes at room temperature in order to block endogenous peroxidase activity. Secondary antibody binding sites were blocked by placing the sections in a solution of 5% normal horse serum for 30 minutes at room temperature. The sections were then incubated with the primary antibody, a polyclonal rabbit antibody against AC V and VI (2 µg/ml) (Santa Cruz Biotech, USA), for 60 minutes at room temperature. A biotinylated horse anti-rabbit (1:200) secondary antibody (Vecta, USA) was applied for 30 minutes, following which the sections were treated with a streptavidin-peroxidase conjugate kit (Vecta, USA) for a period of 45 minutes. The peroxidase was visualized using a diaminobenzidine (DAB)-hydrogen peroxide solution (0.1 g/100 ml) (5 minutes at room temperature) enhanced with cobalt chloride. Each application of antibody was followed by three changes of phosphate buffer with 0.5% bovine serum albumin (pH 7.2). This buffer was also used for the dilution of the antibodies. Liver sections known to contain AC V and VI (Watson and Arkinstall, 1994) were used as positive controls and processed along with the myometrial sections. Negative control sections were processed in exactly the same manner, except that they were placed in normal horse serum instead of the primary antibody against AC V and VI. All the sections were counterstained with Mayer’s haematoxylin, dehydrated, and mounted. Sections were viewed and photographed using bright-field microscopy. For counting purposes, the myometrial sections were subdivided into three regions of equal width; these being the subendometrium, midmyometrium, and subserosa. The total cell population of 10 oil immersion fields (0.28 mm2) was counted in each region per slide, where a cell was counted as positive when it showed evidence of DAB reaction product in the cytoplasm or nucleus. RESULTS Examination of the haematoxylin and eosin-stained sections confirmed the histological normality of each specimen. All phases of the endometrial cycle were represented by the selected myometrial samples (secretory n 5 4; proliferative n 5 3; menstrual n 5 1). The DAB reaction product, associated with all positive myometrial cells, usually obscured the entire cytoplasmic component, although sporadic nuclei also stained positive. Staining intensity was inconsistent, with some fibres showing distinctive reaction product while others were only lightly stained (Fig. 1). Mast cells and vascular smooth muscle cells were occasionally positive (Fig. 2). Control sections of liver that had not been incubated in the primary antibody were negative (Fig. 3a), while sections incubated in the presence of the primary antibody acted as positive controls (Fig. 3b). Table 1 shows the average number of cells counted per high-power field, as well as the percentage positivity for each of the three regions. The midmyometrial region demonstrates the highest percentage positivity when compared with the subserosal and subendometrial regions. Despite the differences in the number of cells between the three regions, the subserosal and 489 Fig. 1. Light micrograph of the midmyometrial region stained with an antibody against type V and VI AC. There is a large number of positive cells present with some staining darker (arrows) than others. Scale bar 5 100 µm. Fig. 2. Light micrograph of the midmyometrial area stained with an antibody against type V and VI AC. The prominent blood vessel is strongly positive (arrows). Scale bar 5 100 µm. subendometrium are both significantly less positive than the midmyometrium (analysis of variance P , 0.0001). There is no significant difference in positivity between the subserosal and subendometrial areas, although the latter is the least positive. Age appears to be an influencing factor in the positivity of the midmyometrium, where there is a significant decrease in positivity with advancing age (analysis of variance P , 0.001). No statistically significant relationship was demonstrated between the phase of the endometrial cycle and the positivity of the specific regions of the myometrium. DISCUSSION The positive reaction obtained with the AC V/VI antibody, indicates the presence of AC isoform type V and/or VI in human myometrial cells. As these two isoforms are 93% similar with respect to the amino 490 P.D.G. RICHARDS ET AL. Fig. 3. Light micrograph of a section of liver. (a) Negative control and (b) positive control. Scale bar 5 100 µm. TABLE 1. The average number of positive cells counted per high power field (HPF) and the percentage positivity for each region of the myometrium irrespective of the stage of the endometrial cycle Average 1ve cells per HPF % positivity SE (n 5 80) MM (n 5 80) SS (n 5 80) 108 27.10 84 69.74* 56 56.57 *Significantly higher percentage positivity in the midmyometrial (MM) region when compared to the subendometrial (SE) and subserosal (SS) regions. acids at the C-terminal end (Watson and Arkinstall, 1994) it is not possible, using this antibody, to determine which of the two isoforms predominates. In terms of their specific distribution in tissues of muscular origin, type V occurs only in cardiac muscle, while type VI has been isolated in both cardiac and smooth muscle (Watson and Arkinstall, 1994). Considering that the most abundant source of mRNA for AC type VI is cardiac muscle, while for all of the other isoforms it is from tissues of neural origin (Cooper et al., 1995), it is probable that the AC isoform present in myometrial tissue is type VI. Even though the specific AC isoform in myometrial tissue is not known, its pivotal role in the relaxation of smooth muscle by β-adrenergic agonists has been established (Carsten and Miller, 1987). Smooth muscle contraction and relaxation are determined by the intracellular calcium ion concentration ([Ca21]I) (Somolyo and Somolyo, 1994), where initiation of contraction results from an increase in [Ca21]I, due to the release of Ca21 from intracellular stores. Relaxation is the consequence of a lowering of [Ca21]I due to the activation of plasmaand sarcolemmal Ca21 adenosine triphosphatase (Ca21 ATPase) pumps. Relaxation is induced by agents such as β-adrenergic agonists which activate AC, thus increasing the levels of cAMP in the cell. The rise in cAMP levels in turn activates protein kinase A and cyclic guanosine monophosphate kinase, these being two enzymes that activate the Ca21 ATPase pumps (Lincoln and Cornwell, 1991). In the nongravid uterus, myometrial activity increases through the menstrual cycle to a peak in the late secretory period (Bell et al., 1968). In the gravid uterus, myometrial contractions start in earnest from the 20th week with an increasing frequency towards term, with total contraction of the uterus taking place at the end of the third stage of labour (Bell et al., 1968). The percentage distribution of the AC protein as revealed by this study implicates the midmyometrial region as being the most sensitive to relaxation stimuli and, thus, probably the area of the myometrium with the highest work potential during periods of contraction. The sustained contraction of the uterus at the end of the third stage of labour is probably achieved by the inhibition of AC (types V and VI) following the increase in [Ca21]I that results from exogenous Ca21 influx via L type channels (Yu et al., 1993). The myometrium changes as it ages, atrophying after the climacteric as a result of the loss of the bulk and tone, which are normally maintained by estrogen (Llewllyn-Jones, 1982). If AC positivity reflects areas of high muscular activity, then any loss in such activity as a result of aging or disease should be reflected by a decrease in AC positivity. The decrease in AC positivity seen with increasing age in this study is probably due to decreasing estrogen stimulation and myometrial bulk in the midmyometrial region with advancing age. In conclusion, it is probable that AC isoform type VI is present in myometrial tissue, its main distribution in the myometrium being in the midmyometrial area of the muscle, where it probably plays a major role in the relaxation of myometrial smooth muscle during parturition. ACKNOWLEDGMENTS The authors wish to acknowledge the technical assistance of Ms. Z. Haffejee and Ms. L. Taylor as well as Dr. A. Bener for his assistance with the statistical analysis. This work forms part of a MSc thesis being undertaken at the University of the Witwatersrand, Republic of South Africa. REFERENCES Bakalyar, H.A., and Reed, R.R. (1990) Identification of a specialized adenylyl cyclase that may mediate odorant detection. Science, 250:1403–1406. Bell, G.H., Davidson, J.N., and Scarborough, H. (1968) Textbook of Physiology and Biochemistry. E.&S. Livingstone, Edinburgh, pp. 1171–1174. Cali, J.J., Zwaagstra, J.C., Mons, N., Cooper, D.M.F., and Krupinski, J. (1994) Type VIII adenylyl cyclase. A Ca21/calmodulin-stimulated enzyme expressed in discrete regions of rat brain. J. Cell Biol., 269:12190–12195. Carsten, M.E., and Miller, J.D. (1987) A new look at uterine muscle contraction. Am. J. Obstet. Gynecol., 157:1303–1305. Cooper, D.M.F., Mons, N., and Karpen, J.W. (1995) Adenylyl cyclases and the interaction between calcium and cAMP signalling. Nature, 374:421–424. Feinstein, P.G., Schrader, K.A., Bakalyar, H.A., Tang, W.J., Krupinski, J., Gilman, A.G., and Reed, R.R. (1991) Molecular cloning and characterization of a Ca21/calmodulin-insensitive adenylyl cyclase from rat brain. Proc. Natl. Acad. Sci. USA, 88:10173–10177. Fortier, M., and Krall, J.F. (1983) Adenylate cyclase activity of circular and longitudinal muscle layers of rat myometrium. Biochem. Pharmacol., 32:2118–2120. Gao, B., and Gilman, A.G. (1991) Cloning and expression of a widely distributed (type IV) adenylyl cyclase. Proc. Natl. Acad. Sci. USA, 88:10178–19182. Ishikawa, Y., Katsushika, S., Chen, L., Halnon, N.J., Kawabe, J., and Homcy, C.J. (1992) Isolation and characterization of a novel cardiac adenylylcyclase cDNA. J. Biol. Chem., 267:13553–13557. Krupinski, J., Coussen, F., Bakalyar, H.A., Tang, W-J., Feinstein, P.G., ADENYLYL CYCLASE IN MYOMETRIUM Orth, K., Slaughter, C., Reed, R.A., and Gilman, A.G. (1989) Adenylyl cyclase amino acid sequence: Possible channel- or transporter-like structure. Science, 244:1558–1564. Lincoln, T.M., and Cornwell, T.L. (1991) Toward an understanding of the mechanism of action of cyclic AMP and cyclic GMP in smooth muscle relaxation. Blood Vessels, 28:1129–1137. Llewellyn-Jones, D. (1982) Fundamentals of Obstetrics and Gynaecology. Volume Two: Gynaecology. 3rd Edition. Faber and Faber, London, pp. 282–289. Mons, N., Harry, A., Dubourg, P., Premont, R.T., Iyengar, R., and Cooper, D.M.F. (1995) Immunohistochemical localization of adenylyl cyclase in rat brain indicates a highly selective concentration at synapses. Proc. Natl. Acad. Sci. USA, 92:8473–8477. Premont, R.T., Chen, J., Ma, H-W., Ponnapalli, M., and Iyengar, R. (1992) Two members of a widely expressed subfamily of hormone- 491 stimulated adenylyl cyclases. Proc. Natl. Acad. Sci. USA, 89:9809– 98113. Richards, P.A., and Tiltman, A.J. (1995) Anatomical variation of oestrogen receptor in normal myometrium. Virchows Arch., 427:303– 307. Somolyo, A.P, Somolyo A.V. (1994) Signal transduction and regulation in smooth muscle. Nature, 372:231–236. Watson, S., and Arkinstall, S. (1994) The G-Protein Linked Receptor Factsbook. Academic Press, London, pp. 391–407. Xia, Z., Choi, E-J., Wang, F., Blazynski, C., and Storm, D.R. (1993) Type I calmodulin-sensitive adenylyl cyclase is neural specific. J. Neurochem., 60:305–311. Yu, H.J., Ma, H., and Green, R.D. (1993) Calcium entry via L-type calcium channels acts as a negative regulator of adenylyl cyclase activity and cyclic AMP levels in cardiac myocytes. Mol. Pharmacol., 44:689–693.