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Endocrinology Vol. 147, No. 4 2051-2062
Copyright © 2006 by The Endocrine Society

Regulation of Spontaneous Contractile Activity in the Bovine Epididymal Duct by Cyclic Guanosine 5'-Monophosphate-Dependent Pathways

Marco Mewe, Christiane K. Bauer, Dieter Müller and Ralf Middendorff

Institut für Anatomie (M.M., R.M.), Institut für Angewandte Physiologie (C.K.B.), Zentrum für Experimentelle Medizin, Universitätsklinikum Hamburg-Eppendorf, Universität Hamburg, D-20246 Hamburg, Germany; and Institut für Anatomie und Zellbiologie (D.M., R.M.), Justus-Liebig-Universität Giessen, Aulweg 123, D-35385 Giessen, Germany

Address all correspondence and requests for reprints to: Dr. Marco Mewe, Institut für Anatomie II: Experimentelle Morphologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany. E-mail: mewe{at}uke.uni-hamburg.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Passage of spermatozoa through the epididymis is obligatory for sperm maturation processes and is based on spontaneous phasic contractions (SC) of the epididymal duct. Here, the functional role of cyclic GMP (cGMP) signaling in modulating SC in the bovine epididymal caput and corpus region was examined by muscle tension recording and immunological and autoradiographic techniques. The cGMP-analog 8-bromo (Br)-cGMP, as well as the nitric oxide (NO) donor sodium nitroprusside and the natriuretic peptides (NPs) atrial NP and C-type NP, displayed distally increasing SC-relaxant effects. In agreement, a distally increasing epididymal expression of the cGMP-dependent protein kinase I (PKG I), endothelial NO synthase (eNOS), and the atrial NP receptor was found. Immunoreactivity for PKG, soluble guanylate cyclase, and eNOS could be localized to the epididymal muscle cells as well as to the epithelial basal cells only at the corpus level. The SC-relevant action of NO and the NPs was cGMP dependent, and the action of 8-Br-cGMP, in turn, was modified by epithelial and luminal factors. The NOS inhibitor L-NAME (N{omega}-nitro-L-arginine methyl ester) caused an increase in SC frequency, indicating basal activity of NO generating enzymes. The SC-inhibitory effect of 8-Br-cGMP was clearly reduced by the PKG inhibitor Rp-8-Br-cGMPS as well as by iberiotoxin, thapsigargin, and indomethacin, pointing to PKG as main SC-relevant target of cGMP, and to large-conductance calcium-activated K+ channels, the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase and cyclooxygenase-1 as possible targets of PKG. These data support an essential role of cGMP signaling in the control of epididymal peristalsis, thereby enabling fine tuning of sperm transport and maturation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAMMALIAN epididymal duct is a single, highly convoluted tubule, which serves to propel spermatozoa from the testes to the cauda epididymidis, where they are stored until ejaculation (1). During the 8–17 d of passage through the tubule, mammalian spermatozoa are exposed to a constantly changing environment, which ensures their maturation, including the acquisition of fertilizing potential and progressive motility (2, 3, 4, 5). The propulsion of the immotile spermatozoa from the caput via the corpus to the epididymal cauda region occurs by means of spontaneous phasic contractions (SC) of the surrounding muscle layer of the duct, whereas the contractions in the richly innervated cauda are predominantly neurogenic in origin (see Ref.6 for review). Transport of sperm is characterized by pendular movements providing stirring of the intraluminal content (7, 8). The bovine epididymal SC are triggered by an epithelial cyclooxygenase (COX)-2-dependent prostaglandin production and the activation of L-type Ca2+ channels in the peritubular contractile cells (Mewe, M., C. K. Bauer, J. R. Schwarz, and R. Middendorff, manuscript in preparation). However, knowledge about the precise mechanisms underlying epididymal autorhythmicity is still limited. In particular, little attention has been directed to the mechanisms of epididymal muscle relaxation, which prevents excessive propulsion of sperm through the duct, thereby ensuring time-dependent sperm maturation processes.

Smooth muscle relaxation, in general, is known to be predominantly mediated by cyclic GMP (cGMP)-dependent signaling pathways (see Ref.9 for review). Likewise, a significant role of cGMP in the control of epididymal sperm transport has been suggested (10). The messenger molecule cGMP can be generated by different pathways, involving either the soluble (cytosolic) or the particulate (plasma membrane-localized) guanylate cyclase (sGC and pGC, respectively) (11). Nitric oxide (NO) represents the most important stimulator of sGC. The generation of NO is catalyzed by three isoforms of NO synthases (NOS), designated as neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS). Whereas nNOS and eNOS are constitutively expressed in a Ca2+/calmodulin-dependent manner, iNOS activity is regulated by gene expression inducible by immunological stimuli (see Ref.12 for review). Atrial natriuretic peptide (ANP), which acts as a hormone in the regulation of blood pressure and fluid volume homeostasis, and C-type natriuretic peptide (CNP), which exerts local activities, bind to the enzyme-coupled pGCs, designated as GC-A and GC-B, respectively (see Ref.11 for review). A third NP receptor, designated as clearance receptor (NPCR), reveals an equal affinity to all natriuretic peptides, but lacks GC activity. It has been proposed to mediate metabolic clearance of the circulating NPs by internalization (13), but there is also evidence for implications in signal transduction (14).

The NO/cGMP system has been shown to induce relaxation in smooth muscle by attenuating the intracellular Ca2+ concentration ([Ca2+]i) as well as the sensitivity of the contractile apparatus to [Ca2+]i. The activity of cGMP is terminated by allosteric binding and the hydrolytic action of cGMP-dependent phosphodiesterases (PDEs), including the cGMP-specific PDE5 and the cGMP-inhibited cAMP-specific PDE3. The systems regulated by PDE5 include contraction-relaxation cycles in smooth muscle (15), and PDE3 is suggested to participate in NO-induced vasodilation (16). The key enzyme in mediating cGMP-induced relaxation in smooth muscle is the cGMP-dependent protein kinase I (PKG I), acting by diverse mechanisms. These involve activation of Ca2+-dependent K+ channels and the sarcoplasmic-endoplasmic Ca2+ ATPase (SERCA), as well as inhibition of the sarcolemmal inositol 1,4,5-tris-phosphate (IP3) receptors (IP3R) via the IP3R-associated PKG I substrate (17, 18).

With regard to male reproductive organs, a functionally active NO/cGMP system has been detected in the tunica albuginea of the testis (19), the lamina propria of the testicular seminiferous tubules (20) and in the regulation of penile erection (21). In the epididymis, localization of specific receptors for NPs in the epididymal duct of the turtle has been described (10). In addition, NOS expression was found in the epididymis of different species (22, 23, 24). However, data on the functional role of both NO/ and NP/cGMP signaling pathways in the epididymal duct are missing. In the present study, we investigated the potential role of cGMP signaling in regulating epididymal spontaneous motility as part of our efforts to understand the molecular mechanisms of sperm transport. We demonstrate the localization and functional activity of components of an epididymal NO/ and NP/cGMP system as well as its regional effectiveness in modulating epididymal muscle activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue preparation
Epididymal tissue was obtained from sexually mature (at least 50 wk old; see Ref.25) bulls in a local slaughterhouse, 10–15 min after stunning followed by exsanguination. The epididymides were immediately placed in ice-cold Ca2+-free Hanks’ balanced salt solution (HBSS; Life Technologies, Inc., Karlsruhe, Germany) for transport to the laboratory.

Tissue samples of different regions of the duct (caput-corpus-cauda; see Fig. 1Go) were either immediately frozen in liquid nitrogen and stored at –80 C (for subsequent protein preparation), fixed in Bouin’s fluid for 24 h at 20 C (for immunohistochemical analyses) or chilled on dry ice before storage at –80 C (for use in receptor autoradiography). Cryostat tissue sections (20 µm) were thaw-mounted onto chrome-gelatin-coated slides and stored at –20 C until use. For tension studies, segments of the epididymal duct were separated in ice-cold Ca2+-free physiological salt solution by carefully dissecting the surrounding tissue with fine forceps and scissors under a stereomicroscope. For those tension studies, which were performed in the absence of sperm and other intraluminal factors, the segments were perfused with Ca2+-free HBSS by the use of a Hamilton-syringe to wash out the original epididymal fluid. Removal of the epididymal epithelium was carried out with 1% Triton X-100 (Merck, Darmstadt, Germany) in PBS (3 min). Until use in tissue bath assays, segments were stored for at least 2 h in DMEM (Life Technologies, Inc.) at 4 C.


Figure 1
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  		FIG. 1. 8-Br-cGMP effects in different parts of the bovine epididymal duct. Schematic illustration of different regions of the epididymis and typical isometric force recordings of the effect of 8-Br-cGMP (100 µM) on SC in segments from the different epididymal duct regions. The inset shows the concentration dependence of the SC-inhibitory effect of 8-Br-cGMP (100 µM) in the corpus region. Data calculated for the relative decrease in SC frequency averaged over a period of 5 min starting directly after application of the agonist (means ± SEM).

 
Tension studies
For contractility studies, segments (around 1.5 cm in length) of different regions of the ductus epididymidis were mounted with silk thread on two stainless-steel hooks in a double-jacketed organ bath (volume, 15 ml). Preparations were equilibrated in modified Krebs bicarbonate Ringer (continuously gassed with carbogen to provide oxygenation and pH of 7.3–7.4) at 35 C. The incubation temperature was somewhat above the in vivo situation to ensure a regular spontaneous contraction pattern leading to more standardized testing conditions with regard to the pharmacological treatments. One of the two hooks was connected to a SG4-90 force-displacement transducer (Hugo Sachs, Freiburg, Germany) for isometric tension recording. The output of the transducer was displayed on a Linseis (Seeb, Germany) L 6510 writer and digitized at 1 Hz using a Metrabyte DAS 1202 (Keithley Instruments, Cleveland, OH) interface. Segments were stretched incrementally to a preload tension of around 6 mN. Preparations were then allowed to equilibrate for 60 min and to relax to a steady-state resting tension. The Ringer solution was changed once after 30 min to avoid accumulation of metabolites in the organ bath.

Immunohistochemistry
Immunohistochemical analyses were performed essentially as described previously (20). In brief, paraffin sections (6 µm) were mounted on chrome-gelatin-coated slides and incubated with the mouse monoclonal antibody anti-eNOS (Transduction, Lexington, KY; 1:100) or with the rabbit polyclonal antisera anti-sGC (Calbiochem, San Diego, CA; 1:800) and anti-PKG I (Stressgen, Victoria, Canada; 1:200) overnight. For visualization of immunoreactivity, a combination of the peroxidase-antiperoxidase technique and the avidin-biotin-peroxidase complex method (26) was used. Biotinylated antimouse or antirabbit IgG (Dako, Hamburg, Germany) were used as secondary antibodies. Peroxidase activity was visualized by the nickel glucose oxidase technique (27). For negative controls, sections were used in which the primary antibodies were replaced by PBS.

Protein preparation
Frozen tissue was pulverized under liquid nitrogen in a mortar. One hundred micrograms of tissue were each suspended in 1 ml homogenization buffer [25 mM NaH2PO4/Na2HPO4 (pH 7.2) containing 5 mM EGTA, 0.1 mM dithiotreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride] and homogenized by three strokes in a Potter-Elvehjem homogenizer (Neolab, Heidelberg, Germany). After centrifugation at 3000 x g for 8 min to remove cell debris and nuclei, the supernatant fractions were stored at –70 C. Protein concentration was determined using a bicinchoninic acid kit from Pierce (Rockford, IL) and an Ultrospec photometer (Pharmacia, Cambridge, UK).

For extraction of membrane proteins Tris-HCl buffer [50 mM Tris-HCl (pH 7.5) containing 10 mM EDTA, 10 mM DTT, and 0.1 mM phenylmethylsulfonyl fluoride] was used as homogenization buffer. The initial procedure was as described before. After the first centrifugation step the supernatant fractions were centrifuged at 100,000 x g for 30 min. The crude pellets, referred to as membrane protein fractions, were washed once each in homogenization buffer plus 0.6 M KCl, in salt-free homogenization buffer, and finally resuspended in 50 mM Tris-HCl buffer (pH 7.5). The resulting membrane suspensions were frozen in liquid nitrogen and stored at –80 C. Protein concentration was determined with a Bio-Rad (Munich, Germany) kit using BSA (fraction V; purchased from Sigma, Deisenhofen, Germany) as standard.

Immunoblotting
The immunoblotting procedure was performed essentially as described previously (28). Separation of proteins by SDS-PAGE was performed in 12% acrylamide gels; for visualization of immunoreactivity, ECL(-plus) kits (Amersham, Braunschweig, Germany) were used. Rabbit polyclonal antibody anti-PKG I (Stressgen; 1:1000) and mouse monoclonal antibody anti-eNOS (Transduction; 1:100) were used as primary antibodies, and peroxidase-linked antirabbit or antimouse IgG (Pierce, Rockford, IL) as secondary antibodies.

Affinity cross-linking of GC-A by 125I-ANP
Membranes (10 µg of protein) were incubated for 15 min at 22 C with 125I-ANP (0.5 nM) in a total volume of 40 µl of 20 mM HEPES buffer (pH 7.5) containing 5 mM MgCl2, 125 mM NaCl, and the protease inhibitors parahydroxymercury benzoate (60 µg/ml), bacitracin (1 mg/ml), bestatin (50 µg/ml), phosphoramidon (50 µg/ml), and 1,10-phenanthroline (1 mM). Samples were then irradiated in the dark with UV light (peak wavelength 302 nm) followed by chilling and immediate addition of 20 µl of 3x SDS-PAGE sample buffer consisting of 0.3 M Tris-HCl (pH 6.8), 200 mM dithiotreitol, 30% glycerin, 15% sodium dodecyl sulfate (SDS), and 0.06% Bromphenol blue. Before analysis by SDS-PAGE under reducing conditions in 7.0% polyacrylamide separation gels, samples were boiled for 3 min. For visualization of molecular weight marker proteins (SDS-6H; Sigma), gels were stained with Coomassie brilliant blue, then dried and exposed for 5 d to x-ray film (Kodak XAR-5; Eastman Kodak, Rochester, NY) between intensifying screens at –70 C.

Affinity cross-linking of cGMP-binding by fluorescein-labeled cGMP
Based on photoaffinity labeling assays with 125I-labeled and 32P-labeled (see Ref.19) ligands, a novel procedure for nonradioactive cross-linking was developed using 8-fluorescein-cGMP. Tissue homogenates (80 µg of protein each) were incubated in darkness at 22 C for 30 min with 25 mM HEPES, 100 mM KCl, 2.5 mM EGTA, 20 µM 3-isobutyl-1-methylxanthine, 1.25 mM DTT, and 1 µM 8-fluorescein-cGMP. For competition experiments, 8-bromo (Br)-cGMP was added to a final concentration of 1 mM. Samples were irradiated for 6 min on an UV table (peak 312 nm, maximum output; Vilber Lourmat TFX-20 M; Marine la Vallee, France) followed by administration of 20 µl 3x Western blot buffer containing 0.375 M Tris-HCl (pH 6.8), 0.2 M DTT, 15% SDS, 20% glycerol, and 0.6 mg/dl Bromphenol blue. After separation of proteins by SDS-PAGE without preceding heat denaturation, samples were used in Western blot assays using sheep antifluorescein antibody conjugated to horseradish peroxidase (NEN Life Science Products, Boston, MA; 1:1000).

Receptorautoradiography
Tissue sections were brought to room temperature and preincubated for 5 min in 50 mM HEPES buffer (pH 7.5), containing 150 mM NaCl, 5 mM MgCl2 and 0.1% BSA. The sections were then incubated in a humid chamber with 125I-(Tyr0)-CNP (2.0 nM) in the same buffer (70 µl total volume per section) for 60 min at 4 C. In case of control reactions with the unlabeled peptide (CNP) or the clearance-receptor-ligand c-ANF, these substances were present at 200 nM. The sections were washed twice for 5 min each in the same buffer, dipped in distilled water, and dried in air before direct exposition to RX films (Fuji, Tokyo, Japan) for 6–12 d at 4 C. After autoradiography, sections were fixed by incubations with methanol (–20 C, 10 min) and acetone (5 sec) and finally stained with hemalaun/eosin for histological analyses.

Chemicals and solutions
The Ca2+-free physiological salt solution contained (mM): NaCl 118, KCl 6, HEPES 10; pH was adjusted to 7.4 with NaOH. The modified Krebs bicarbonate Ringer contained (mM): NaCl 118, KCl 4.75, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, NaHCO3 25, D-glucose 11; bubbled with carbogen to establish a pH of 7.3–7.4. All these reagents were purchased from Sigma and of the highest analytical grade.

ANP and CNP were from Bachem (Heidelberg, Germany). 125I-ANP (2 kCi/mmol) and the ring-deleted ANP analog c-ANF were obtained from Amersham Pharmacia Biotech (Braunschweig, Germany). (Tyr0)-CNP, purchased from Peninsula (Belmont, CA), was radiolabeled (Amersham) with sodium 125iodide via the chloramine-T method. The product, purified by reverse-phase chromatography, was supplied at a specific activity of 2 kCi/mmol. 8-Br-cAMP, 8-Br-cGMP, Rp-8-Br-cGMPS, and Rp-cAMPS were from Biolog (Bremen, Germany). Sodium nitroprusside (SNP) was obtained from Fluka (Buchs, Switzerland). The general NO synthase inhibitor N{omega}-Nitro-L-arginine methyl ester (L-NAME), iberiotoxin, indomethacin, milrinone, niflumic acid and norepinephrine (NE) were purchased from Sigma. The GC-B blocker HS-142–1 was kindly provided by Dr. Y. Matsuda (Tokyo, Japan). The sGC blocker, 1H-[1, 2, 4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), was from Alexis (San Diego, CA). Thapsigargin was obtained from Calbiochem (Bad Soden, Germany).

Rp-8-Br-cGMPS, milrinone, niflumic acid, and thapsigargin were dissolved in dimethylsulfoxide. Stocks of ANP, CNP, and HS-142–1 were prepared in 0.1% trifluoro-acetic acid. Indomethacin, NE and ODQ were dissolved in ethanol. The final concentrations of these solvents never exceeded 0.3%. The solvents were tested in their highest final concentration and showed no effect on the spontaneous contractions of the epididymal duct. All other substances were dissolved in distilled water or buffer.

Data analysis
Data collection was carried out using a DOS program developed by P. Bassalay (UKE, Hamburg, Germany). Experimental data are given as means ± SEM, where n represents the number of samples of epididymidal tissue, derived from different animals each. Data calculated for changes in contraction frequency and amplitude induced by agonists and antagonists were averaged over periods of 5 min immediately before and after application of the drugs. Further data processing was performed with Excel (Microsoft, Redmond, WA) and Sigma Plot 4.01 (SPSS, Chicago, IL). For calculation of EC50 and IC50 values concentration response curves were analyzed using GraphPad Prism 4 (GraphPad Software, San Diego, CA). Student’s two-tailed paired t test was used to assess statistical significance. P values ≤ 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Following earlier studies (see Ref.4), the bull epididymis was subdivided into a caput, corpus, and cauda region as depicted in Fig. 1Go. Here, the cauda was further subdivided into a proximal [cauda (p)] and medial subregion [cauda (m)]. The bovine epididymal duct exhibits regular spontaneous phasic contractions (SC) from the caput to the cauda (p). The contraction profiles are characterized by a decrease in SC frequency from proximal to distal and an increase in SC amplitude at the level of the cauda (Fig. 1Go).

Regional effects of 8-Br-cGMP
The membrane-permeable cGMP analog 8-Br-cGMP at a concentration of 100 µM suppressed phasic activity in the epididymal duct with increasing efficiency from proximal to distal (n = 3/region; Fig. 1Go). In the caput, SC frequency was reduced to 25.3 ± 0.9% and SC amplitude to 57.7 ± 1.3% (n = 3; P ≤ 0.05 each) of the control values. Within the corpus region, an EC50 value for 8-Br-cGMP of 1.9 x 10–5 M (n = 3) was calculated (Fig. 1Go). In all experiments, the SC-inhibitory effect of the cGMP analog was accompanied by an abrupt decrease in basal tone, which was particularly pronounced at the level of the caput.

Endogenous NO production
To assess the physiological relevance of a basal endogenous NO production in modulating spontaneous phasic activity, L-NAME (1 mM) was used. In the caput, application of the NOS inhibitor resulted in an immediate increase in SC frequency by 15.3 ± 2.1% (n = 3; Fig. 2AGo). In the corpus, the L-NAME-induced acceleration in SC frequency amounted to 73.8 ± 2.3% (n = 5; P ≤ 0.01). In contrast to the caput, the L-NAME-effect in the corpus was preceded by an initial transient suppression of phasic activity by 2–3 min (n = 5; Fig. 2BGo).


Figure 2
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  		FIG. 2. Endogenous NO production in the epididymis. Representative recordings of the effect of the NOS inhibitor L-NAME (1 mM) on the spontaneous activity in the caput (panel A) and corpus region (panel B). Panel C, Immunohistochemical analysis of eNOS expression in cross-sections of the indicated regions. Immunoreactivity for eNOS is localized to endothelial cells of blood vessels (v) and contractile cells of the epididymal duct (thick arrows) as well as to the perinuclear region of the basal cells in the corpus (small arrow). Calibration bars: 50 µm; E, epithelium. Panel D, Comparative immunoblot analysis of eNOS expression in the caput and corpus by the use of two protein preparations each. Immunoreactivity is associated with a protein of about 140 kDa (arrow), consistent with the size of the antigen.

 
Immunohistochemical analysis revealed immunoreactivity against eNOS in the muscle wall of the epididymal duct and in endothelial cells of associated blood vessels in both the caput and corpus region (Fig. 2CGo). In addition, immunoreactivity was localized to the perinuclear region of the epithelial basal cells; however, only at the level of the corpus. Immunoblot experiments confirmed the expression of eNOS (140 kDa) in both the caput and corpus region (Fig. 2DGo). Hence, a functional role of constitutive endogenous NO production in modulating epididymal sperm transport seems likely.

Regional cGMP-dependent effects of the NO donor SNP
Like 8-Br-cGMP, SNP concentration-dependently attenuated SC generation in the epididymal duct segments with increasing efficiency from proximal to distal. Application of SNP (1 mM) had no distinct modulatory effect on SC frequency in the caput (n = 3; Fig. 3AGo), but resulted in a temporary suppression (3.4 ± 0.41 min) of spontaneous contractions in the corpus region (n = 4; Fig. 3BGo). When recovered, the spontaneous activity in the corpus was characterized by a decrease in SC frequency by 12.2 ± 1.2% and an increase in SC amplitude by 12.5 ± 0.6% (n = 4; P ≤ 0.05 each). SNP also induced an increase in SC amplitude by 10.0 ± 0.3% in the caput (n = 3; P ≤ 0.05). Using concentrations of 1 µM–1 mM, an EC50 value for SNP in the corpus of 9.3 x 10–5 M (n = 3) was calculated on the basis of ability to reduce SC frequency. In addition, the NO donor induced a reduction in muscle tone in both the caput and corpus region (Fig. 3Go, A and B).


Figure 3
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  		FIG. 3. cGMP-dependent action of NO. Typical SC-modulatory effects of the NO donor SNP (1 mM) in the caput (panel A) and corpus region (panel B). Panel C, SNP effect after prior application of the sGC blocker ODQ (10 µM) at the corpus level. Panel D, Immunohistochemical analysis of sGC expression in cross-sections of the indicated regions. Immunoreactivity for sGC is localized to the muscle wall of blood vessels (v) and the epididymal duct (thick arrows) as well as to the perinuclear region of the basal cells in the corpus region (thin arrow). Calibration bars: 50 µm; E, epithelium.

 
The SC-suppressive, but not the tone-reducing effect of SNP (1 mM) in the corpus was virtually abolished by prior application of the specific sGC blocker ODQ (10 µM, n = 3; Fig. 3CGo), indicating that the SC-relevant action of the NO donor is dependent on the synthesis of cGMP. Immunohistochemical analysis of sGC expression revealed immunoreactivity in the epididymal muscle wall in both the caput and the corpus region and, as an internal control, in vascular smooth muscle cells as well as in the perinuclear region of the basal cells at the level of the corpus (Fig. 3DGo).

Regional cGMP-dependent effects of natriuretic peptides
The regional effects of the natriuretic peptides ANP and CNP (each used at a final concentration of 1 µM) on SC generation (Fig. 4Go, A and B) resembled the corresponding relaxation profiles of 1 mM SNP. Both NPs displayed a distally increasing SC-inhibitory effect. Contrary to SNP, the NPs did not induce an increase in SC amplitude. In addition, ANP did not reduce muscle tone in the caput (Fig. 4AaGo). CNP clearly reduced SC frequency and disequilibrated tone in both the caput and the corpus region (Fig. 4Go, Ba and Bb). The SC-modulatory effects of both NPs in the corpus could be inhibited by prior application of the specific particulate GC (pGC) blocker HS-142–1 (100 µg/ml, n = 3; Fig. 4CGo).


Figure 4
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  		FIG. 4. cGMP-dependent action of NPs. A and B, Representative recordings of the effects of ANP (1 µM; A) and CNP (1 µM; B) on the spontaneous contractile activity in the caput (a) and corpus region (b). C, Lack of SC-modulatory effects of both NPs in the corpus after prior application of the pGC blocker, HS-142–1 (100 µg/ml). D, Identification of the ANP receptor GC-A in the caput and corpus (two protein preparations each) by affinity cross-linking to 125I-labeled ANP. GC-A, migrating at about 130 kDa, is marked by an arrow. E, Autoradiographic localization of 125I-Tyr0-CNP binding sites in sections of the corpus. Serial cryostat sections were incubated with 2 nM 125I-Tyr0-CNP in the absence (a) and presence of c-ANF (200 nM; b), a synthetic peptide binding only to NPCR, or unlabeled CNP (200 nM; c). The tubular muscle wall is indicated by arrowheads. Calibration bars: 500 µm.

 
By affinity cross-linking to radiolabeled ANP (125I-ANP, 0.5 nM), we could demonstrate expression of the ANP receptor GC-A on epididymal membranes from the corpus region (Fig. 4DGo). SDS-PAGE analysis revealed a single specifically labeled protein of about 130 kDa, consistent with the size of GC-A (29). In the caput region, GC-A was barely detectable by this method (Fig. 4DGo). This agrees with the absence of a SC-modulatory effect of ANP in this region.

For demonstration of the CNP receptor GC-B, whose visualization generally is more complicated than that of GC-A, receptor autoradiography was used, known to be more sensitive than cross-linking analyses (30). Expression of GC-B was demonstrated by this method on cryostat cross sections of the epididymis. Figure 4EaGo shows a high density of binding sites for radiolabeled CNP [125I-(Tyr0)-CNP, 2 nM] in peripheral structures of the duct at the level of the corpus. The reaction was proved to be specific as unlabeled CNP (200 nM) blocked receptor labeling (Fig. 4EcGo). Further controls carried out in the presence of the specific NP clearance receptor ligand c-ANF (200 nM) revealed clear receptor binding of 125I-(Tyr0)-CNP (Fig. 4EbGo) and thereby also confirmed the specificity of the reaction and ruled out that NPCR have been involved significantly in binding reactions.

Putative cGMP target proteins
As contraction-relevant target of cGMP in smooth muscle, the cGMP-inhibited cAMP-degrading PDE3 is often discussed (16, 18). We therefore tested the SC-modulatory effect of the specific inhibitor of this cGMP-dependent PDE, milrinone, in the corpus. Application of 20 µM milrinone caused a long-term suppression of SC (n = 3; Fig. 5AaGo), which is indicative of a basal PDE3 activity and a relevance of cAMP levels for spontaneous contractions in this region. The SC-blocking effect of milrinone resembled the effect of 8-Br-cGMP in the corpus, pointing to PDE3 inhibition by cGMP in vivo. The relaxation profile of the membrane-permeable cAMP-analog 8-Br-cAMP (2 mM), in turn, was very similar to that of SNP in the corpus region (n = 4; Fig. 5AbGo).


Figure 5
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  		FIG. 5. Epididymal receptor groups of cGMP. Panel A, Representative SC-modulatory effects of the PDE3 inhibitor milrinone (20 µM; a) as well as of 8-Br-cAMP (2 mM) and cumulatively applied 8-Br-cGMP (100 µM; b) in the corpus. Panel B, SC-modulatory effects of 8-Br-cAMP (2 mM) and 8-Br-cGMP (100 µM) after prior application of the PKA inhibitor Rp-A (50 µM; a) and the PKG I inhibitor Rp-G (25 µM; b) in the corpus region. Panel C, Comparative immunoblot analysis of the expression of PKG I in the caput, corpus and cauda region. Immunoreactivity is associated with a protein of about 74 kDa (arrow), consistent with the size of the antigen. Panel D, Detection of cGMP-binding proteins in the regions indicated by affinity cross-linking to fluorescein-labeled cGMP. Incubations were carried out in the absence (–) or presence (+) of unlabeled cGMP to confirm specificity of the reaction. A cGMP-binding protein of 74 kDa (indicated by an arrow) is detectable, consistent with the size of PKG I. Panel E, Immunohistochemical analysis of PKG I expression in the proximal duct regions. Immunoreactivity is localized to the epididymal duct (thick arrows) and the muscle wall of blood vessels (v) as well as to the perinuclear region of the basal cells in the corpus (thin arrows). Calibration bars: 50 µm; E, epithelium.

 
To assess the significance of the different cyclic nucleotide kinases for the cyclic nucleotide-induced SC-modulatory effects in the corpus region, the specific inhibitors of the cGMP-dependent protein kinases (PKG) I{alpha} and Iß, Rp-8-Br-cGMPS (Rp-G), and the cAMP-dependent protein kinases (PKA) I and II, Rp-cAMPS (Rp-A), were used. The PKA antagonist Rp-A hardly showed any inhibitory effect on the 8-Br-cAMP (2 mM)-induced relaxation at a concentration of about 50 µM, at which the 8-Br-cGMP (100 µM) effect was already clearly attenuated (Fig. 5BaGo), indicating beginning cross-inactivation of PKG I by Rp-A and cross-activation of the PKG I by 8-Br-cAMP. An IC50 value for Rp-A with respect to the 8-Br-cGMP (100 µM) effect of 4.5 x 10–5 M (n = 3) and to the 8-Br-cAMP (2 mM) effect of 6.9 x 10–5 M (n = 3) was calculated. The SC-inhibitory effects of both, 8-Br-cGMP (100 µM) and 8-Br-cAMP (2 mM), on SC generation were restricted to a slight reduction in tone and SC frequency after prior application of 25 µM Rp-G (Fig. 5BbGo). An IC50 value for Rp-G with respect to the 8-Br-cGMP (100 µM) effect of 4.3 x 10–6 M (n = 3) and to the 8-Br-cAMP (2 mM) effect of 1.4 x 10–5 M (n = 3) was calculated. These data point to the PKG I as predominant SC-relevant target of both, cGMP and cAMP.

Comparative immunoblotting assays confirmed expression of PKG I in all regions of the epididymal duct (Fig. 5CGo). Immunoreactivity for PKG I was associated with a protein of about 74 kDa, consistent with the size of the antigen (31). Also by affinity cross-linking to fluorescein-labeled cGMP, a cGMP-binding protein with an apparent molecular mass of 74 kDa was detected in the soluble fractions of epididymal homogenates from the caput and corpus region (Fig. 5DGo). Immunohistochemical analysis of PKG I expression in the proximal duct regions revealed reactivity for the kinase in the muscle wall of the epididymal duct and associated blood vessels (Fig. 5EGo). In addition, staining of the perinuclear region of the basal cells only at the level of the corpus could be detected.

Cellular targets of PKG I
In the following, we examined potential cellular target proteins of PKG I in the corpus. Application of the large-conductance calcium-activated potassium (BK) channel blocker iberiotoxin (100 nM) increased SC frequency and tone and decreased the 8-Br-cGMP (100 µM)-induced SC suppression (n = 4; Fig. 6AGo). By contrast, the main effects of the SERCA blocker thapsigargin (2 µM) were a reduction in tone accompanied by an increase in SC amplitude (n = 4; Fig. 6BGo). In the presence of thapsigargin, 8-Br-cGMP was also unable to completely block the phasic contractions in all experiments. Similarly, the COX-1 inhibitor indomethacin (20 µM), which reduced basal tone and SC amplitude, clearly attenuated SC suppression by the cGMP analog (n = 3; Fig. 6CGo).


Figure 6
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  		FIG. 6. Putative targets of cGMP signaling. A–C, Representative isometric force recordings showing the effects of 8-Br-cGMP (100 µM) after prior application of the BK channel blocker iberiotoxin (100 nM; A), the SERCA blocker thapsigargin (2 µM; B) and the COX-1 inhibitor indomethacin (20 µM; C) at the level of the corpus.

 
Involvement of luminal and epithelial factors in NO/cGMP signaling
We finally investigated whether intraluminal factors and the epithelium can influence the effectiveness of the cGMP system in the epididymal duct. Proximal duct segments, which had previously been perfused with HBSS to remove sperm and luminal factors, exhibited burst-like instead of regular spontaneous phasic contractions. In addition, the effects of 8-Br-cGMP (100 µM) in perfused segments of the caput and the corpus region were changed (n = 3/region; Fig. 7Go, Aa and Ab). The cGMP-analog completely and promptly abolished SC in the caput (a), but hardly affected SC in the corpus (b). In addition, only a slight gradual decrease in tone could be observed in both regions. In conjunction, these data suggest regional variations of the 8-Br-cGMP action triggered by luminal factors.


Figure 7
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  		FIG. 7. NO/cGMP signaling independent of luminal factors/epithelium. A, Contraction profile of Ringer-perfused proximal duct segments and representative effects of 8-Br-cGMP (100 µM) in the caput (a) and corpus region (b). B, Typical effects of SNP (1 mM; a) and 8-Br-cGMP (100 µM; b) on NE-induced contraction in epithelium-denuded corpus segments.

 
Experiments on Triton X-100 perfused, epithelium-denuded corpus segments, which were devoid of spontaneous contractions, revealed a clearly stronger relaxing effect of SNP (1 mM) in comparison to 8-Br-cGMP (100 µM) on NE-precontracted material. The SNP effect was characterized by an abrupt termination of the NE-induced contraction (n = 3; Fig. 7BaGo), whereas the cGMP analog caused only incomplete relaxation (n = 3; Fig. 7BbGo), indicative of an (additional) cGMP- and epithelium-independent action of NO in excited epididymal contractile cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The bovine epididymal duct exhibits autorhythmicity from the caput to the proximal cauda, which is characterized by regular phasic contractions with distally decreasing frequency. Contrary to the neurogenic origin of contractions in the mid-cauda and ductus deferens, local factors seem to play a crucial role in triggering epididymal peristalsis. The present results demonstrate for the first time the presence and possible mechanisms of NO-/NP-cGMP signaling in the proximal duct contractile cells and epithelial basal cells at the level of the corpus (see Fig. 8Go). The relaxing effects of ANP and CNP as well as SNP and 8-Br-cGMP in the bovine epididymis were characterized by a distally increasing suppression of phasic activity indicating a different significance of relaxation-inducing mechanisms in the course of the epididymal duct. Especially in the corpus region, the cGMP system may prevent excessive epididymal smooth muscle activity (e.g. during ejaculation) and guarantee that time-dependent sperm maturation processes can take place.


Figure 8
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  		FIG. 8. Scheme showing possible cGMP signaling pathways in the corpus epididymidis. cGMP is generated as a consequence of ANP/CNP- and NO-induced activation of pGC and sGC, respectively. The SC-inhibitory effect of cGMP at least partially bases on a PKG I-mediated activation of BK channels and the COX-1, as well as an increased release or sequestration of intracellular Ca2+ via activation of ryanodin receptors (RyR) and the SERCA, respectively. Cross talk between these putative targets of PKG I may constitute the mechanisms underlying the cGMP-induced relaxation. In addition, the PDE3 as target of cGMP and cross-activation of PKG I by cAMP, as well as direct contraction-relevant targets of NO, can be assumed.

 
Epididymal basal NO production
The findings of the organ-bath assays revealed a significant role of the constitutively expressed eNOS with regard to modulation of the spontaneous phasic and agonist-induced contraction in the bovine epididymis. The immunological results indicated a weak NO production in the epithelial principal cells and a clearly stronger one in the periductual muscle wall. In the corpus, an additional eNOS immunoreactivity within the epithelial basal cells was observed, confirming a functional significance of an epithelium-associated NO-cGMP system particularly in this region. In human, eNOS could also be localized to the epididymal epithelial cells, however, not to the surrounding contractile cells (24). It remains to be elucidated whether the partially different localization of the eNOS is species-dependent or due to methodological reasons. The distinct staining of the endothelial, but not the muscle cells of subepithelial vessels confirmed specific binding of the monoclonal antibody used in the current experiments. In addition, the expression of NOSs was also shown within the myofibroblasts of the testicular seminiferous tubules (20).

The physiological relevance of epididymal epithelial NOS is still unknown, but its activity seems to be regulated by androgens (32). The SC-accelerating action of the NOS inhibitor L-NAME points to a constitutive production and muscle-associated effect of NO in both the caput and corpus region. Due to the similar regional effects of L-NAME and iberiotoxin and because the PKG I inhibitor Rp-G lacked any SC-modulatory effect, NO possibly activates BK channels directly in the epididymal contractile cells. A cGMP-independent activation of Ca2+-dependent K+ channels (KCa) by NO in (vascular) smooth muscle is amply verified (33, 34, 35, 36). By contrast, the mechanism(s) underlying the L-NAME-induced transient SC suppression in the corpus remain(s) to be elucidated.

SNP effects
Increasing the NO level by the NO donor SNP hardly affected spontaneous phasic activity in the caput region, but clearly suppressed SC in the corpus. This SC-inhibitory effect of SNP was abolished after application of the sGC blocker ODQ, indicating a predominantly cGMP-dependent action of NO.

Remarkably, SNP exhibited a much stronger inhibitory effect on norepinephrine-induced contraction with epithelium-denuded corpus segments than 8-Br-cGMP. This indicates additional cGMP-independent relaxation-inducing effects of NO in the muscle cells. Apart from BK channels, NO may directly act on SERCA. In arterial smooth muscle, NO-derived peroxynitrite was found to directly increase SERCA activity, resulting in a decreased intracellular Ca2+ concentration and relaxation (37). In addition, peroxynitrite is known to stimulate prostaglandin H synthase and COX activity (38), and it has been shown that NO directly stimulates uterine (39) and ileal PGE2 [prostaglandin (PG) E2] production (40) to induce relaxation.

The SNP-induced relaxation of NE-precontracted epithelium-denuded corpus segments could also involve Gi- and/or Gs-proteins as additional cGMP-independent targets of NO (see Refs.41 and 42). In the vascular system, NO is suspected to directly cause ADP-ribosylation of G{alpha}i and G{alpha}s, resulting in an elevation of the intracellular cAMP level and vasodilation (41).

Guanylate cyclases and natriuretic peptide effects
The evidence of functionally active NP receptors (GC-A, GC-B) in the bovine ductus epididymidis suggests a substantial role of NP/cGMP in addition to NO/cGMP signaling in modulating epididymal sperm transport. Both natriuretic peptides, ANP and CNP, induced SC suppression and a reduction in tone in the corpus. These SC-inhibitory effects of the NPs were effectively inhibited by the pGC blocker HS-142-1, indicating cGMP-dependent actions as in case of SNP. In contrast, ANP was without effect in the caput, pointing to a lack of ANP receptors in this region. Accordingly, clear expression of GC-A could be identified in the corpus, but not the caput region. This suggests a physiological significance of the circulating ligands of GC-A (ANP/brain NP) only at the level of the corpus.

The effect of CNP was much stronger in the corpus than in the caput region, where it exerted only a tone-reducing effect. At the level of the corpus, receptor autoradiography revealed an abundant expression of the CNP receptor GC-B in the outer zone of the duct. The cellular localization of GC-B was difficult due to the detection limit of the method. However, binding sites for radiolabeled CNP seem to be present in the basal cells and the peritubular muscle wall. In contrast, the studies showed no significant expression of the so-called NPCR, which lacks GC activity and is involved in metabolic clearance of NPs (13). A role of NPs as humoral and autocrine or paracrine factors in regulating smooth muscle tone and thereby sperm transport has previously been suggested in the epididymal duct of the freshwater turtle (10). In addition, CNP was found to exert relaxation of the rabbit penis via augmentation of cGMP production (43).

Mechanisms of cGMP signaling
The SC-modulating effects of the NPs, SNP, and 8-Br-cGMP/-cAMP in the bovine epididymis turned out to be predominantly PKG I-dependent as they were blocked by HS-142-1, ODQ, and Rp-8-Br-cGMPS, respectively. The inhibitory PKG I analog Rp-8-Br-cGMPS itself hardly affected the SC profile of corpus segments, confirming that the SC-relevant L-NAME effects in this region can be put down to PKG I-independent basal NO effects.

As a possible target of PKG I in the bovine epididymis, BK channels were determined in our experiments. The PKG-induced activation of BK channels is a well-known mechanism (44), leading to a hyperpolarization of the membrane potential and consecutive closure of L-type Ca2+ channels in smooth muscle (45). Due to the clearly reduced relaxation potency of 8-Br-cGMP after prior application of the BK channel blocker iberiotoxin, direct PKG phosphorylation of these channels seems also likely in the epididymal contractile cells. However, the fact that 8-Br-cGMP failed to decrease SC amplitude after emptying the sarcoplasmic reticulum-Ca2+ stores with thapsigargin points to an additional indirect activation of BK channels. In smooth muscle, an indirect amplification of BK channel activity via increased genesis of Ca2+ sparks is described resulting from PKG-mediated phosphorylation of ryanodin-sensitive Ca2+ channels (46).

A fall in the intracellular free Ca2+ concentration ([Ca2+]i) by PKG I-dependent stimulation of SERCA activity in the peritubular contractile cells may also account for part of the SNP-/NP-/8-Br-cGMP-induced SC suppression in the corpus. A decrease in [Ca2+]i by NO-cGMP-induced SERCA activation is discussed in aorta smooth muscle (47) and endothelial cells (48). Remarkably, Ca2+ release from the sarcoplasmic reticulum was not essential for maintenance of epididymal phasic activity. In addition, SNP, as well as the NPs, caused SC suppression only in the corpus, but not the caput region, indicating that the SC-relevant effects of these agents originate from mechanisms occurring in the epithelial basal cells. In agreement, all components of the NO/cGMP system could be localized to these cells in the corpus, but not the caput region.

The SC-inhibitory effect of 8-Br-cGMP was clearly reduced by indomethacin, suggesting the COX-1 as another direct or indirect epididymal target of the PKG I. COX-1 activity was previously found in rat epididymal basal cells (49, 50), and a distally increasing COX-1 mRNA expression was determined in the epididymal epithelium of the mouse (51). The predominant PG synthesized by this COX-isozyme seems to be PGE2, which was found to reduce contraction in the epididymal caput of the rat (52). In contrast, in the present study, the selective blockade of the COX-1 led to a decrease in SC amplitude and tone in the corpus pointing to an excitatory net effect of the COX-1 products. An agonist/cGMP-induced increase in PGE2 synthesis, on the other hand, may act as a negative inotrope. In the seminiferous tubules of the testis, PGE1 acts stimulatory in low concentrations, but exerts inhibitory effects on contractility in high concentrations (53). In myofibroblasts, a serotonin-induced increase in PG synthesis has a positive inotropic effect, which changes into an inhibition of contraction at higher PG concentrations (54). Hence, it can be hypothesized that in the epithelial basal cells at the level of the corpus, a PKG I-induced amplification of the Ca2+-activated constitutive COX-1 activity leads to an increased PGE2 production and consecutive cAMP-mediated relaxation of the surrounding muscle cells. Interestingly, the SC-modulatory effects of indomethacin and thapsigargin as well as the consecutive relaxation profiles of 8-Br-cGMP were similar. This may indicate that in the corpus basal cells, PKG I increases [Ca2+]i by increasing the release of Ca2+ from thapsigargin-sensitive stores and thereby stimulates COX-1-activity. A cGMP-mediated increase in COX-1 activity and PGE2 production is already described in the duodenum (55).

As a direct target of cGMP in the proximal epididymal duct the PDE3 was identified in our experiments, where the specific PDE3 inhibitor milrinone caused long-term suppression of SC in the corpus. The cGMP/PDE3 pathway modulates the intracellular cAMP-level in various cell types and is suggested to be involved in the NO-induced vasodilation (16, 56). In conjunction with the very similar SC-modulatory effects of the NPs, SNP, and the cAMP-analog 8-Br-cAMP in the corpus region, it can be hypothesized that part of the NO-/NP-induced SC inhibition physiologically bases on a cGMP/PDE3-mediated cAMP synthesis. Because the SC-suppressive effect of 8-Br-cAMP was abolished by Rp-8-Br-cGMPS, and high cAMP concentrations can cross-activate the PKG (57), a consecutive activation of the PKG I/COX-1 pathway in the basal cells of this region seems likely.

In general, application of 8-Br-cGMP exerted much stronger SC-modulatory effects than SNP and the NPs. This could be explained by the metabolically stable nature of 8-Br-cGMP (detail of the manufacturer) as well as the absence of a feedback-inhibition of the sGC by PKG (see Ref.58). In addition, compartmentalization in cellular microdomains of the physiological cGMP effects are conceivable.

Significance of luminal factors for regional 8-Br-cGMP effects
The organ-bath assays using proximal duct segments, which were cleared of the physiological epididymal fluid, revealed that the generation of regular phasic contractions as well as the regional 8-Br-cGMP-effects are closely connected with the presence of intraluminal factors in the duct. As luminal factors modulating epididymal muscle activity, angiotensin II (59), arachidonic acid (60) and androgens (61, 62) are discussed. Like ILs (63) and reactive oxygen species, which are produced by sperm (64), these substances may modulate epididymal tone and SC generation by regulating epithelial COX-activity (see Refs.49 and 50) or directly act on the peritubular muscle cells. In addition, they may determine the effectiveness of the epididymal cGMP system.

Contrary to the control, 8-Br-cGMP completely and promptly abolished SC in sperm-deficient caput, but hardly affected SC in sperm-deficient corpus segments. Hence, in the sperm-deficient caput segments, a (muscle-associated) relaxation-inducing component may predominate over a (epithelium-associated) contraction-promoting component of the 8-Br-cGMP action. In contrast, the high efficiency of the cGMP system in intact corpus segments may depend on the presence of luminal factors and a COX-1-dependent synthesis of (an) SC-inhibitory prostaglandin(s) in the epithelial basal cells.

Physiological significance
In the course of the epididymal duct, we determined a distally increasing relaxing potency of 8-Br-cGMP, SNP and the NPs ANP and CNP, which was accompanied by an increased expression of GC-A and the presence of eNOS, sGC, and PKG I in the epithelial basal cells. Combined with the lack of SC-modulating effects of SNP and the NPs in the caput and the clearly reducable 8-Br-cGMP effect by indomethacin in the corpus, a distally increasing effectiveness of the cGMP-system via PKG I-induced and COX-1-mediated PGE2 synthesis in the epithelial basal cells seems likely (see Fig. 8Go). From the present findings, a distally increasing importance of cGMP signaling in the spontaneously contractile regions of the epididymal duct can be derived. It can be speculated, that the epithelial relaxation-inducing cGMP system provides a relatively slow and steady flow of sperm in the corpus, thereby ensuring physiologically important time-dependent sperm maturation processes in this region. This assumption is confirmed by the dependence of cGMP-induced relaxation on the presence of intraluminal factors, including sperm. The cGMP production in the corpus region may be stimulated by local or systemic factors, enabling appropriate sperm transport regardless of the filling of the epididymal lumen and of neuronal or hormonal stimulation, respectively. A stretch-induced increase in muscle contraction and thereby accelerated sperm transport in case of sperm accumulation might be prevented by stimulation of the stretch-activated eNOS (65) in the basal cells. Increased NO levels may also prevent excessive propulsion of sperm by systemic factors like noradrenaline during ejaculation. In this context, NO could act cGMP dependently or directly as indicated by the high efficiency of SNP in comparison to 8-Br-cGMP in the relaxation of noradrenaline-induced contraction. Hence, the cGMP system seems to enable fine-tuning of sperm transport and sperm maturation.


   Footnotes
 
The funding of this work by grants from the Deutsche Forschungsgemeinschaft (Mi 637/1-1) is gratefully acknowledged.

M.M., C.K.B., D.M., and R.M. have nothing to declare.

First Published Online January 26, 2006

Abbreviations: ANP, Atrial NP; BK, large-conductance calcium-activated K+ channels; Br, bromo; cGMP, cyclic GMP; CNP, C-type NP; COX, cyclooxygenase; DTT, dithiothreitol; eNOS, endothelial NOS; GC-A and GC-B, enzyme-coupled pGCs; HBSS, Hanks’ balanced salt solution; iNOS, inducible NOS; IP3, inositol 1,4,5-tris-phosphate; L-NAME, N{omega}-nitro-L-arginine methyl ester; NE, norepinephrine; NO, nitric oxide; NOS, NO synthase; nNOS, neuronal NOS; NP, natriuretic peptide; NPCR, NP clearance receptor; ODQ, 1H-[1, 2, 4]oxadiazole[4,3-a]quinoxalin-1-one; PDE, phosphodiesterase; PG, prostaglandin; pGC, plasma membrane-localized guanylate cyclase; PGE2, prostaglandin E2; PKA, protein kinase A; PKG I, cGMP-dependent protein kinase I; Rp-A, Rp-cAMPS; Rp-G, Rp-8-Br-cGMPS; SCs, spontaneous phasic contractions; SDS, sodium dodecyl sulfate; SERCA, sarcoplasmic-endoplasmic Ca2+ ATPase; sGC, soluble guanylate cyclase; SNP, sodium nitroprusside.

Received October 18, 2005.

Accepted for publication January 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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