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Regular Article |
a Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614
b Department of Physiology, Hong Kong Chinese University, Shatin, Hong Kong
c Phoenix Pharmaceuticals, Inc., Mountain View, California 94043
ABSTRACT
Cocaine- and amphetamine-regulated transcript (CART) is a novel family of peptides, of which CART peptide fragments 55–102 and 62–102 are reported to be the endogenous, physiologically active peptides. Immunohistochemical studies with an antiserum directed against the CART peptide fragment 55–102 revealed CART-like immunoreactive (CART-LI) nerve fibers in the rat epididymis. The number was highest in the cauda epididymis and became progressively fewer toward the caput epididymis; the vas deferens exhibited an abundance of CART-LI fibers. Injection of the retrograde tracer Fluorogold (Fluorochrome, Inc., Englewood, CO) to the junction between the vas deferens and cauda epididymis labeled a large number of neurons in the major pelvic ganglion, some of which were CART-positive. Double-labeling the ganglion sections with tyrosine hydroxylase (TH) and CART antisera revealed that CART-LI and TH-LI were expressed in two distinct populations of ganglion cells. Some of the TH-LI cells in the ganglia, however, were covered with web-like CART-LI endings. The effects of CART peptide 55–102, referred to herein as CART, on anion secretion in the form of short circuit currents (Isc) were assessed in cultured epithelia. The CART (1 to 5 µM) applied to the basolateral or apical side of the cultured epithelia caused no significant responses on Isc, whereas lys-bradykinin (1 µM) produced a large Isc response in the same preparations. Our results show that CART-LI is present in a population of rat pelvic ganglion cells, which may give rise to CART-LI nerve fibers as observed in the vas deferens and the epididymis. The biological function of CART in the rat epididymis is not known, but it apparently is not involved in ion secretion across the epithelium.
epididymis, male reproductive tract, male sexual function, vas deferens
INTRODUCTION
The epithelium lining the epididymis plays an active role in creating an environment conducive for sperm maturation and storage [1]. Results from several studies suggest that neurohumoral agents, either released from nerve endings close to or within the epithelium or via circulation, modulate the secretory process [2–10]. For example, bradykinin, serotonin, endothelin, or angiotensin II interact with specific receptors located on the basal membrane of the principal cells, and they effectively alter anion secretion across the epithelium [4, 8, 11, 12]. Further, there is evidence that bradykinin-stimulated anion secretion is mediated by prostaglandin E2, which is formed from arachidonic acid by COX-1 in the basal cells. Prostaglandin E2 diffuses into the interstitial space and acts on the prostaglandin EP2/4 receptors on the principal cells to increase intracellular cAMP, which then activates the apical cAMP-activated chloride channels, resulting in secretion of anions and, secondarily, of water [9].
Several neurohumoral agents (e.g., norepinephrine, serotonin, and acetylcholine) that regulate fluid and electrolyte secretion are present in autonomic nerves to the epididymis [8, 13, 14]. Here, we report the detection of a novel peptide in autonomic nerve fibers to the rat epididymis by immunohistochemical methods. The peptide in question is the cocaine- and amphetamine-regulated transcript (CART), which was first identified by reverse transcription-polymerase chain reaction in the rat striatum after administration of cocaine and amphetamine [15]. Subsequent in situ hybridization and immunohistochemical studies showed that mRNA and CART-like immunoreactivity (CART-LI) are constitutively expressed in the rat brain, pituitary, spinal cord, adrenal gland, and enteric ganglia [16–19]. More recently, CART-LI was shown to be distributed extensively in the rat sympathoadrenal axis [20]. The present study was undertaken to explore whether CART-LI could be extended to the epididymis, which is richly innervated by autonomic nerves. If present, what might be the biological function of CART with respect to fluid secretion? Because agents that increase or decrease chloride secretion in cultured epithelia also correspondingly affect blood to luminal flux of chloride in the intact epididymis of anesthetized rats in vivo [3, 10], the cultured epididymal epithelium appears to be an appropriate in vitro model to investigate the secretory process in intact epithelium.
MATERIALS AND METHODS
Male Sprague-Dawley rats, weighing from 150–250 g, were used in this study. Experimental protocols were approved by the Animal Care and Use Committees of the respective institutions.
Immunohistochemistry
Rats were anesthetized with urethane (1.2 g/kg i.p.) and intracardially perfused with 0.1 M PBS, followed by freshly prepared, 4% paraformaldehyde in PBS. The epididymis, vas deferens and major pelvic ganglia were removed, postfixed for 2 h, and immersed in 30% sucrose/PBS overnight. Tissues were sectioned to 40 µm with a Vibratome (Technical Products International, Inc., St. Louis, MO) and processed for CART-LI by the avidin-biotin complex (ABC) or fluorescent techniques, as described elsewhere [21, 22]. In addition, some sections were set aside for double-labeling experiments, in which only the fluorescent method was used [21, 22].
In the ABC method, tissues were first treated with 3% H2O2 to quench endogenous peroxidase, washed several times in Tris-buffered saline, and blocked with 10% normal goat sera (Vector Laboratories, Burlingame, CA). Tissues were incubated in the primary antibody to CART peptide fragment 55–102 (1:10 000 dilution with 0.4% Triton X-100 and 1% BSA in PBS) for 48 h at 4°C with gentle agitation. The CART antiserum, a rabbit polyclonal from Phoenix Pharmaceuticals, Inc. (Mountain View, CA), exhibits 100% cross-reactivity with the rat CART peptide 55–102 (Phoenix Pharmaceuticals). After thorough rinsing, sections were incubated with biotinylated antirabbit immunoglobulin (Ig) G (1:150 dilution; Vector Laboratories) for 2 h. Sections were rinsed with PBS and incubated in ABC solution for 1 h (1:100 dilution; Vector Laboratories). After several rinses in Tris-buffered saline, sections were developed in diaminobenzidine-H2O2 solution and washed for at least 2 h with Tris-buffered saline. Sections were mounted on slides with 0.25% gel alcohol, air-dried, dehydrated with absolute alcohol followed by xylene, and coverslipped with Permount (Fisher Scientific, Pittsburgh, PA).
For the fluorescent method, tissues were first blocked with 10% normal goat sera and then incubated with CART antisera (1:2 000 dilution with 0.4% Triton X-100 and 1% BSA in PBS) for 48 h in a cold room with gentle agitation. After several washes with PBS, sections were incubated with biotinylated antirabbit IgG (1:50 dilution; Vector Laboratories) for 2 h. After several washes in PBS, tissues were incubated with Fluorescein Avidin D (1:50 dilution; Vector Laboratories). Lastly, tissues were washed for 30 min with PBS, mounted in Citifluor (Ted Pella, Redding, CA), and coverslipped.
In the case of double-labeling studies, the technique of sequential labeling with primary antisera from two different hosts was used [21, 22]. Tissues were first processed for fluorescent CART-LI as described earlier. Thereafter, tissues were washed with PBS for at least 2 h, blocked with normal horse sera, and then incubated with tyrosine hydroxylase (TH) antisera (1:500 dilution with 0.4% Triton X-100 and 1% BSA in PBS) for 48 h in a cold room with gentle agitation. The TH antiserum was a mouse monoclonal from Chemicon International, Inc. (Temecula, CA), and the specificity of the antibody has been extensively evaluated [21, 22]. After washing with PBS for 30 min, tissues were incubated with Avidin Texas Red (Vector Laboratories) for 4 h, washed for 30 min with PBS, mounted in Citifluor, and coverslipped. Sections were examined with a Nikon EC600 fluorescent microscope and photographed.
Fluorogold Injection
Rats were anesthetized with urethane as described previously. A small incision was made in the lower abdomen under aseptic conditions, and the junction between the vas deferens and the cauda epididymis was gently lifted into view. Approximately 2 µl of Fluorogold solution (2.5% in aqueous solution; Fluorochrome, Inc., Englewood, CO) was slowly injected into the tissue junction between the vas deferens and cauda epididymis. After withdrawing the needle, the area was washed several times with saline to remove any Fluorogold solution. The incision was closed, and the animal was allowed to recover from anesthesia before returning to the cage. Two to three days later, the animal was anesthetized with urethane and intracardially perfused with PBS, followed by paraformaldehyde as described earlier. Left and right major pelvic ganglia were removed and processed for CART-LI using the fluorescent method with Texas Red as the chromogen. Bright Fluorogold particles within the cytoplasm of major pelvic ganglion cells were visualized with a UV-2A barrier filter and Texas Red-labeled cells with a Y-2E/C barrier filter.
Control Experiments
Several sections from each experiment were set aside for control preparation. Two sets of control experiments were performed. First, the primary antibody to CART or TH was omitted from the staining procedure. Second, sections were processed with CART antisera preabsorbed with the CART peptide 55–102 (10 µg/ml; Phoenix Pharmaceuticals) overnight.
Epididymal Cell Culture
Procedures for obtaining cultured cauda epididymal cells from rats weighing 150 g were similar to those described elsewhere [2, 8, 9]. Epididymides removed from rats were cut into small pieces and placed in sterile Hanks balanced salt solution (HBSS) containing 0.25% trypsin. After incubating for 1 h at 32°C with vigorous shaking, tissues were separated by low-speed centrifugation (800 x g, 5 min). The supernatant was discarded, and the pellet was resuspended in HBSS containing 0.1% collagenase for 2 h at 32°C with vigorous shaking. Cells were separated by centrifugation at 800 x g for 5 min. The pellet was resuspended in Eagle minimum essential medium containing nonessential amino acids (0.1 mM), sodium pyruvate (1 mM), glutamine (4 mM), 10% fetal bovine serum, streptomycin (100 µg/ml), and penicillin (100 IU/ml). The cell suspension was stored for 4 h in a flask incubated at 32°C in 5% CO2 to eliminate smooth muscle cells and fibroblasts, which settled on the bottom of the flask more readily than epithelial cells. Epithelial cells in suspension were seeded onto the wells of Matrigel-coated, Millipore filter (Millipore Co., Bedford, MA) assemblies with a window area of 0.2 cm2. Monolayers reached confluency after 3 days in culture and were ready for measurement of anion secretion using the short circuit current (Isc) technique.
Measurement of Isc
Confluent epididymal monolayers were clamped between two halves of Ussing chambers with a 0.6-cm2 window and incubated on both sides with oxygenated (95% O2/5% CO2) Krebs-Henseleit solution maintained at 32°C. Epithelia were short circuited by a voltage clamp amplifier (DVC-1000; World Precision Instruments, New Haven, CT). The Isc was displayed continuously on a pen recorder (Kipp and Zonen, Pelft, The Netherlands). Transepithelial resistance was measured by transiently commanding the clamp to set the voltage at 0.2 or 0.4 mV away from zero. The resulting changes in transepithelial current allowed calculation of the resistance using the Ohm law. Generally, confluent epithelia had a basal potential difference of 1–2 µAcm-2 and a transepithelial resistance of 500 
cm-2.
The CART peptide and bradykinin were added directly to the apical or basolateral side of the epithelium. The basal Isc was measured after monolayers were allowed to equilibrate for 20 min. Responses to CART and bradykinin were measured as changes at the peak of the response. The Isc was expressed in terms of microamperes per square-centimeter area of the epithelium (µA/cm2).
Materials
The CART and TH antisera were from Phoenix Pharmaceuticals and Chemicon International, respectively. The CART peptide 55–102 and lys-bradykinin employed in the electrophysiological studies were purchased from Peptides International (Louisville, KY) and Research Biochemicals International (Natick, MA), respectively.
RESULTS
Immunohistochemical studies were performed on epididymides from 12 rats. Longitudinal sections were cut from various regions of the epididymis and from the vas deferens.
CART-LI in Vas Deferens and Epididymis
In longitudinal sections of the vas deferens, numerous CART-LI fibers were seen surrounding the lumen (Fig. 1, A and B). In the cauda epididymis, CART-LI fibers were distributed throughout the intertubular space, many of which showed bead-like varicosities (Fig. 1, C and D). In addition, a plexus of CART-LI fibers was seen surrounding the epithelium of individual tubules (Fig. 1, C and D). With respect to the corpus section, CART-LI fibers were less numerous in the intertubular space, and fewer fibers were seen underneath the epithelium (Fig. 1E). The CART-LI fibers were nearly undetectable in the caput (Fig. 1F). Thus, the density of CART-LI nerve fibers was region dependent, such that they were abundant in the cauda epididymis, moderate in the corpus, and sparse in the caput.
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In control experiments, epididymal tissues incubated with CART antisera preabsorbed with the CART peptide 55–102 (10 µg/ml) overnight showed no positive staining in any of the sections (data not shown).
CART-LI in Major Pelvic Ganglia
The rat epididymis is innervated by autonomic nerves arising from neurons in the major pelvic ganglion [23]. To explore whether the major pelvic ganglion may provide CART-LI fibers to the epididymis, the presence of CART-LI was examined in the major pelvic ganglia from five rats.
The CART-LI was detected in a population of ganglion cells (Fig. 2A). In the case of positively labeled somata, immunoreactivity was distributed throughout the cytoplasm, excluding the nucleus (Fig. 2B). Most of these ganglion cells had a small diameter, measuring 15–25 µm (Fig. 2B). Ganglion cells of larger diameter (>25 µm) were very often CART negative (Fig. 2B). Interestingly, some of the CART-negative somata were covered with web-like, varicose CART-LI endings (Fig. 2B, arrows). In addition, several clusters of small-diameter (<10 µm) cells were intensely labeled (Fig. 2C, boxes). The number of cells in each cluster varied from several to more than 30 (Fig. 2C). On the basis of their size and morphology, these CART-LI cells may correspond to the small, intensely fluorescent "SIF" cells described elsewhere [20, 24].
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In control experiments, ganglion sections incubated with CART antisera preabsorbed with the peptide (10 µg/ml) showed no staining in any of the sections (Fig. 2D).
Double-Labeling Studies
Neurons in the rat major pelvic ganglion can be grouped into two types according to their transmitter phenotypes: adrenergic, or cholinergic [25]. The next series of studies was performed to determine whether TH, which is a marker for adrenergic neurons, and CART-LI are present in the same populations of ganglion cells. Pelvic ganglion sections from four rats were double-labeled with CART and TH antisera.
The TH-LI and CART-LI were present in two populations of ganglion cells (Fig. 3, A and B). Further, these two types of ganglion cells differed in their diameters. As noted earlier, CART-LI was detected in smaller-diameter ganglion cells (Fig. 3A). Consistent with our earlier finding [21], TH-LI was noted in larger-diameter (>30 µm) pelvic ganglion cells (Fig. 3B). However, clusters of small-diameter cells, which are referred to as SIF cells, were labeled by both antisera (Fig. 3, A and B, boxes).
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Fluorogold/CART-LI Neurons in Major Pelvic Ganglia
In three rats, unilateral injection of Fluorogold to the junction between the vas deferens and cauda epididymis resulted in labeling of a population of pelvic ganglion cells (Fig. 3D). Double-labeling the sections with CART antisera revealed that many Fluorogold-containing ganglion cells were CART positive (Fig. 3, C and D). Some of the Fluorogold-containing somata were CART negative, and vice versa (Fig. 3, C and D). Interestingly, some of the CART-negative somata, which presumably were TH positive, were surrounded by CART-LI endings (Fig. 3C).
Effects of CART on Isc
Application of CART peptide (1 to 5 µM) to either the basolateral or apical side of cultured epididymal cells resulted in no apparent changes of Isc. On the other hand, application of lys-bradykinin to either the basolateral or the apical side of the same preparations resulted in a large Isc response (Fig. 4).
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DISCUSSION
The CART is a family of recently identified peptides [26]. The predicted translation products are either 129 or 116 amino acids in length in the rat and 116 amino acids in the human [26]. Western blot analysis has identified six peptides from rat tissues, of which the fragments 55–102 and 62–102 are thought to be the endogenously active peptides [26, 27]. In this regard, the CART antisera used in our studies were directed against the fragment 55–102. The specificity of the antisera was ascertained by a preabsorption study in which tissues incubated with CART antisera preabsorbed with the peptide exhibited no staining. In a previous study, the CART antisera were directed against the fragment 106–129 [19]. Despite the differences, these two antisera appear to recognize similar neural elements in the rat brain and spinal cord. For example, fibers in superficial layers of the dorsal horn and lateral horn neurons in the rat spinal cord were labeled by both CART antisera [19, 20].
With respect to the epididymis, CART-LI is confined to fiber-like elements, which are most abundant in the cauda epididymis, fewer in the corpus, and virtually absent in the caput. The vas deferens contains a dense network of CART-LI fibers. A similar regional difference with respect to the distribution of nitric oxide synthase (NOS) immunoreactivity is also noted in the rat epididymis [21]. Within the cauda epididymis, the most prominent feature is a ring-like plexus of CART-LI fibers surrounding tubules. Contrary to the serotonin-immunoreactive fibers in the rat epididymis [8], CART-LI fibers do not seem to penetrate into the epithelium.
Insofar as the origin of CART-LI fibers is concerned, results of immunohistochemical and tract-tracing studies are consistent with the notion that these fibers may originate from pelvic ganglion cells. First, CART-LI is present in a population of pelvic ganglion cells, which are known to provide autonomic nerves to the epididymis [23, 28]. Second, injection of the retrograde tracer Fluorogold into the junction between the vas deferens and cauda epididymis consistently labeled a population of pelvic ganglion cells. More importantly, some of the Fluorogold-containing pelvic ganglion cells were CART positive, indicating that the latter send their axons to the vas deferens and epididymis. The possibility that dorsal root ganglion cells, some of which are CART positive (unpublished results), may contribute CART-positive fibers to the epididymis cannot be entirely ruled out.
Rat pelvic ganglion cells can be distinguished on the basis of their transmitter phenotypes: cholinergic, and adrenergic [25, 28]. Cholinergic neurons in the pelvic ganglion have also been shown to express vasoactive intestinal polypeptide (VIP) and NOS immunoreactivity [21, 25]. In addition, cholinergic neurons generally are of smaller diameter compared with adrenergic neurons [21, 28, 29]. Results of our double-labeling experiments show that CART-LI and TH immunoreactivity was expressed in two distinct populations of pelvic ganglion cells, and that CART-positive cells were smaller compared with TH-positive ganglion cells. It follows that CART-LI ganglion cells are cholinergic and, by inference, should also express VIP and NOS immunoreactivity. Interestingly, some of the CART-negative ganglion cells, which presumably are adrenergic, are covered with web-like CART-LI endings. The latter may derive from CART-LI ganglion cells within the pelvic ganglion or be extrinsic in origin. The close apposition of CART-LI endings and the presumed adrenergic ganglion cells suggests a functional role of CART on these neurons.
In addition to CART-LI ganglion cells, clusters of small-diameter cells, which were intensely labeled, are scattered throughout the ganglion. On the basis of their size and morphology, these small, intensely labeled cells may correspond to the SIF cells described by others in a variety of sympathetic ganglia [24]. The physiological role of these small cells remains elusive.
Several putative neurotransmitters/neurohumorals modulate anion secretion by acting on receptors located on the basolateral and/or apical side of the epithelium [2–10]. The presence of CART-LI fibers underneath the epithelium provided the impetus to examine a possible modulatory role of the peptide with respect to anion secretion. Application of CART in concentrations of 1–5 µM to either the basolateral or apical side of cultured epithelial cells caused no apparent Isc response, whereas bradykinin at the concentration of 1 µM produced a large Isc response in the same preparations. On the basis of these results, it may be concluded that CART, at a concentration of 5µM or less, does not significantly modify anion secretion. Thus, the biological role of CART in the epididymis remains to be explored. As CART-LI fibers are most abundant in the vas deferens and the cauda epididymis, the peptide may modulate tubular contractility.
FOOTNOTES
1 Supported by NIH grants NS18710 and NS39646 from the Department of Health and Human Services (NJD) and CUHK4293/99M from the Research Grant Council Hong Kong (P.Y.D.W.). 
2 Correspondence: Nae J. Dun, Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, P.O. Box 70577, Johnson City, TN 37614. FAX: 423 439 8773; dunnae{at}etsu.edu
Accepted: June 26, 2000.
Received: May 2, 2000.
REFERENCES
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