0022-534 7 /93/1496-1568$03.00/0 THE JOURNAL OF UROLOGY Copyright© 1993 by AMERICAN UROLOGICAL ASSOCIATION, INC. Vol. 149, 1568-1575, June 1993 Printed in U.S.A. GAP JUNCTIONS FORMED OF CONNEXIN43 ARE FOUND BETWEEN SMOOTH MUSCLE CELLS OF HUMAN CORPUS CA VERNOSUM A. C. CAMPOS DE CARVALHO, C. ROY, A. P. MORENO, A. MELMAN, E. L. HERTZBERG, G. J. CHRIST AND D. C. SPRAY* From the Departments of Neuroscience, Urology and Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York ABSTRACT Despite sparse autonomic innervation, the smooth muscle cells of the corpus cavernosum relax and contract synchronously to achieve penile erection and flaccidity. As with other smooth muscle cell types, the excitation process in the corpora is presumably propagated through gap junctions to allow the diffusion of current-carrying ions and second messenger molecules from cell to cell. Using both molecular and immunocytochemical techniques, we have identified gap junctions between human corporal smooth muscle cells in situ and in culture. Northern analyses demonstrated that corporal smooth muscle cells express the gap junction protein connexin43, but not connexin26 mRNA. Immunoblots showed the presence of connexin43 isoforms, whereas connexin32 was not detected. Immunocytochemical studies in cultured cells identified prominent connexin43 immuno- reactive puncta between cells, as well as within the cytoplasm. In addition, gap junction membranes both in situ and in culture were labelled in thin section by anti-connexin43 antibodies using the immunogold technique. We conclude that the presence and distribution of gap junctions in this sparsely innervated tissue may provide an important mechanism of intercellular communication among the smooth muscle cells, and thus play a major role in coordinating tissue contraction and relaxation. KEY WORDS: intercellular junctions; immunohistochemistry; penis; microscopy, electron Smooth muscle cells in the corpus cavernosum play a fun- damental role in the erectile process in that relaxation of these cells is required for penile tumescence and rigidity. 1 -s Moreover, recent observations suggest that altered corporal reactivity to adrenergic, 1 - 3 cholinergic 7 • 8 and/or nonadrenergic, noncholi- nergic neurotransmittersa- 11 may contribute to the heightened tissue tone and impaired relaxation that are characteristic of erectile dysfunction in some patients. As in other smooth muscle cell types, the excitation process in the corpora is presumably propagated through gap junctions to allow the spread of electronic currents and second messenger molecules from cell to cell. As such, it is conceivable that pathological alterations in corporal reactivity may be due, at least in part, to altered intercellular communication. Although gap junctions have not been reported previously between these cells, electro- myographic studies support a role for synchronization of elec- trical activity in the corpus cavernosum in vivo; 12 moreover, pharmacological uncoupling of corporal smooth muscle cells with heptanol alters both the kinetic and steady-state charac- teristics of adrenergic contraction in isolated corporal strips (see Discussion). In addition, sympathetic innervation of this tissue is sparse, and the rapid and robust relaxation response to microgram quantities of vasoactive intestinal polypeptide (VIP) and prostaglandin E 1 (PGE1) further implies the presence of an intrinsic mechanism of intercellular coordination. Characterization of the distribution and types of gap junction proteins (connexins) involved in junctional communication in this tissue is therefore of interest in studying the possible involvement of gap junctions in the physiological regulation of penile erection as well as in certain pathologic states leading to erectile dysfunction. Furthermore, corpus cavernosum smooth Accepted for publication November 17, 1992. * Requests for reprints: Albert Einstein College of Medicine, De- partment of Neuroscience, Kennedy Center, Bronx, New York 10461. Supported in part by NIH grants DK-42027, NS-07512, HL-38449 and GM-30667. Dr. Spray is the recipient of Grant-in-Aid and Partic- ipating Laboratory Awards from the New York Affiliate of the Ameri- can Heart Association and Dr. Hertzberg, of an Irma T. Hirschi Charitable Trust Career Scientist Award. muscle may be a valid model for the study of junctional com- munication in human vascular smooth muscle cells; this is particularly important in view of the ready availability of corporal tissue and the reported differences in primary se- quence between connexin43 from human and rat heart.13 In this study we have characterized the connexin type present in corporal smooth muscle cells in tissue culture by Northern and Western blot analysis and have demonstrated the presence of the human cardiac gap junction protein, connexin43. 13 We have identified gap junctions between corporal smooth muscle cells in situ and in culture and have localized connexin43 to these junctional regions at the electron microscopic level. Given the size of these intercellular junctions and the high input resistance of the corporal cells, we hypothesize that the direct pathway provided for intercellular communication through gap junctions plays a major role jn coordinating tissue response. MATERIALS AND METHODS Cell cultures. Human corpus cavernosum material was ob- tained from 15 impotent patients undergoing surgery for im- plantation of penile prostheses; radial sections approximately 3 x 3 x 10 mm. were excised from the mid-penile shaft of each patient. 2 - 4 Patients with Peyronie's disease, a generalized fi- brotic disease of the corpora, were excluded from this study. Tissue was washed and cut into 1 to 2 mm. pieces and placed in tissue culture dishes with minimal volume of Dulbecco's medium (DME, Gibco, Grand Island, New York). After the explants attached to the substrate (usually in 1 to 2 days), additional medium was added. Cells migrated from the explant and underwent division. 14 Cells were detached using 0.05% trypsin, 0.02% EDTA at 37C for 5 minutes to establish secondary cultures from the explants. Cultures were maintained for no more than 4 pas- sages; importantly, during this time all measured pharmacolog- ical properties observed in the intact tissue are retained. 15 The population is thus functionally homogeneous, although some progressive loss is detected in smooth muscle a-actin immu- noreactivity (see below). 1568