Neuronal and endothelial nitric oxide synthase isoforms: Quanti cation of protein and mrna in the normal rat penis

Transcription

1 International Journal of Impotence Research (1999) 11, 301±308 ß 1999 Stockton Press All rights reserved /99 $ Neuronal and endothelial nitric oxide synthase isoforms: Quanti cation of protein and mrna in the normal rat penis RM Seyam 1 *, HT Huynh 2 and GB Brock 2 1 Department of Urology, Suez Canal University, Ismailia, Egypt; 2 Division of Urology, Department of Surgery, McGill University, Montreal, Quebec, Canada Nitric oxide synthase (NOS) is an important enzyme for erection. We evaluated the content of neuronal (nnos) and endothelial (enos) isoforms and their mrna in the penis and major pelvic ganglion (MPG) of adult male rats by Western and Northern blot analysis. The cerebellum was evaluated as a control. nnos protein and its mrna were detected in abundance in the MPG, cerebellum, pelvic urethra and within the crura of the penis. In contrast, the penile urethra, neurovascular bundle and the shaft of penis contained smaller amounts of this protein. enos protein was most abundant in the penile and pelvic parts of the urethra, whereas a moderate level was found in the penile shaft, crura, neurovascular bundle, MPG and cerebellum. Similarly enos mrna was abundant in the penile and pelvic parts of the urethra, MPG and cerebellum. Penile shaft, crura and neurovascular bundle showed moderate amounts of enos mrna. In conclusion, nnos and its mrna are most abundant in the MPG and crura of penis whereas enos is most abundant in the urethra and to a lesser extent present in the penis. Importantly enos protein and mrna were demonstrated in the MPG, where enos function has to be studied. Keywords: nitric oxide synthase; mrna; penis; major pelvic ganglion; urethra Introduction Nitric oxide synthase is thought to have a diverse group of physiologic effects with an important role as a neuromodulator of lower urinary tract function, with particularly profound roles in erection 1, 2 and micturition 3±5 The penis and proximal urethra normally demonstrate high levels of NOS; 1 however in the presence of conditions that contribute to erectile dysfunction as diabetes, 6 aging, 7 chronic smoking 8 and hypogonadism, 9, 10 enzyme activity was reduced. Three isoforms of NOS have been identi ed. nnos and enos are constitutive forms that require calmodulin=nadph for their activity and are involved in smooth muscle regulation and neural transmission. Inducible NOS (inos) is produced by cytokine stimulation of gene expression and has cytotoxic functions. Enzyme activity assays, 1 NADPH diaphorase histochemistry 1 11, 12 and immunohistochemistry have been used to localize NOS within the lower urinary tract in health and disease states. Recently, NOS isoforms have been characterized at the protein *Correspondence: Dr R Seyam, P.O. Box 55, Ismailia, 41511, Egypt Received 28 April 1998; revised 30 October 1998; accepted 24 May 1999 and gene levels. 13±15 Until now, few studies utilized Western blot analysis of nnos in the penis and showed controversial results pertaining to quantitative changes in certain pathological states. 6, 10, 16 In addition, no data is available on enos or nnos mrnas levels in relation to the different components of the penis. Detailed information on NOS proteins and the genes controlling their synthesis in the lower urinary tract is of great value. This will serve as a basis for understanding pathologies affecting NOS and the compensatory mechanisms that come into play at the translational and transcriptional levels. These mechanisms may play a major role in maintaining normal function after signi cant damage to the nnos pathway in the lower urinary tract as that seen in nnos knockout mice. 17, 18 In an attempt to evaluate more completely the normal role of NOS isoforms in erection, herein we describe in male adult rats the content of protein and mrna of nnos and enos in the penile shaft, crura, dorsal neurovascular bundle and penile urethra. Methods Six adult male Sprague-Dawley rats 24±26 weeks old were used. All surgical procedures and tissue harvesting were carried out under general anesthesia

2 302 using pentobarbital (45 mg=kg intraperitoneally). Animals were sacri ced by an overdose of pentobarbital (200 mg=kg i.p.). Tissue harvesting A lower abdominal incision and symphysiotomy were made exposing the bladder, prostate, MPG, pelvic urethra from the prostate apex to the perineal membrane, the crura of the penis at their origin from the pubic rami, the urethral bulb and the penis. Through a lower abdominal incision with optical magni cation (8) both major pelvic ganglia were identi ed and excised. The MPG was identi ed posterolateral to the dorsolateral prostate, dissected from accompanying blood vessels and excised. The penis was dissected free from integument and the glans removed. The dorsal neurovascular bundles, distal penile urethra and the urethral bulb were dissected free from the penile corpora and excised. The penile shaft and crura were excised at their pelvic bony attachment, divided and each cut into 4±5 pieces. To avoid loss of delicate cavernous tissue that was adherent to tunica albuginea, no attempt was made to dissect the tunica albuginea away from the cavernous tissue. The urinary bladder, ventral prostate and dorsolateral prostate were excised. The pelvic urethra was excised from its attachment to the perineal membrane caudally to its attachment to the prostatic urethra cranially. With the rat prone, a craniotomy was carried out, the cerebellum identi ed and both lobes excised. All tissues were immediately frozen in liquid nitrogen. Generation of rat nnos probe cdna was synthesized from the rat cerebellum RNA by reverse transcription. To obtain a 700 bp fragment of rat nnos cdna (from position 1±700), a set of primers (5 0 -ACGTCT- GACAAGCTGGTGACC-3 0 and 5 0 -GATGGTCTTGG- GGGTTCCATC-3 0 ) was used. The PCR primers were designed based on the published sequences of the rat brain NOS. 13 Each PCR was performed in a 100 ml reaction mixture containing 10 ml RT reaction mixture, 1PCR buffer [50 mm KCl, 10 mm Tris- HCl (ph 9.0), 1% triton-x 100, and 1.2 mm MgCl2], 0.1 mm concentration of each dntp, 2.5 units of Taq polymerase and 3.6 mm NOS primers. Thermal cycling was performed in a PTC-100TM Programmable Thermal Controller DNA ampli cation system by using the following ampli cation pro le: an initial denaturation at 95 C for 5 min; 40 cycles of denaturation at 95 C for 30 s, and annealing at 59 C for 30 s, and extension at 72 C for 45 s; and nal elongation step at 72 C for 10 min. The identity of the rat nnos was con rmed by sequencing using the dideoxy chain termination method with an automated Applied Biosystem Model 373A sequencer. Probe was randomly labeled with 32 P dctp (Mandel) using a T-7 Quick-Prime Kit (Pharmacia) according to the supplier's instructions. This probe was used to screen the rat uterine cdna libraries. Positive clones were isolated and recon rmed by hybridizing with rat NOS cdna fragment. These clones were used to detect nnos mrna in the penis and MPG. Northern blotting RNA extraction and hybridization. Total RNA was isolated from the six pooled tissues specimens of either MPG, dorsal prostate, ventral prostate, pelvic urethra, urinary bladder, penile urethra, cerebellum, crura and shaft of penis, or dorsal neurovascular bundle using RNA Zol premix solution and RNA Zol B method (Tel-Test, Friendswood, Texas). For Northern blots, 20 mg of total RNA was used per lane. RNA was subjected to electrophoresis through 1.2% agarose gels containing 2.2% formaldehyde. The RNA was transferred onto Zeta-probe membrane (Bio-Rad) in 50 mm NaOH. The blots were hybridized overnight with nick translated 32 P- labelled rat nnos cdna, enos cdna, S rrna cdna (ATCC), or GAPDH cdna, 10% dextran sulphate, 1% sodium dodecyl sulphate (SDS), 500 mg=ml herring sperm DNA, 0.9 M NaCl, 50 mm Na 2 HPO 4.7H 2 O, 5 mm EDTA for rat nnos and enos were hybridized at 42 C and 50 C respectively. The membranes were subjected to three washes at 42 C 15 min each in solution A (2saline sodium citrate (SSC); 0.1% SDS), solution B (0.5SSC; 0.1% SDS), solution C (0.1SSC; 0.1% SDS), respectively. Final wash was done at 42 C for nnos and 60 C for enos in solution C. The blots were air-dried and subjected to autoradiography from 5 h to 3 d with intensifying screen at 7 80 C. The mrna extraction and blots were repeated once to con rm our ndings. Western blotting For immunoblot analysis, the pooled six tissue specimens of either MPG, dorsal prostate, ventral prostate, pelvic urethra, urinary bladder, penile urethra, cerebellum, crura and shaft of penis, or dorsal neurovascular bundle were homogenized in homogenization buffer (20 mm Tris-HCl, ph 7.5, 1% Triton X-100, 100 mm NaCl, 50 mm NaF, 2 mm PMSF and 0.5% Nonidet P-40). The lysate was clari ed by centrifugation at g for 15 min.

3 303 Figure 1 Distribution of nnos in various parts of the lower urinary tract. Separation and excision of various components [major pelvic ganglion (MPG), dorsal prostate (DP), ventral prostate (VP), pelvic urethra (PvU), urinary bladder (UB), penile urethra (PnU), cerebellum (Cb), penile crura (Cr), shaft of the penis (Sft) and dorsal penile neurovascular bundle (NVB)] of the lower urinary tract; RNA extraction and Northern blots were performed as described in Materials and Methods. Blots were hybridized with rat nnos cdna (A) or 18 S rrna cdna (ATCC) (B) probes. A representative densitometric scanning of the band corresponding to nnos mrna (C). Representative western immunoblotting of nnos (D) and control Tubulin (E) are shown from various components of the urinary tract. Tissue lysates and western blots were performed as described in Material and Methods. (F) A representative densitometric scanning of nnos.

4 304 Protein concentrations were determined and equal amount of protein (50 mg for MPG or 150 mg for other tissues) was electrophoresed on 8% sodium dodecyl sulfate-polyacrylamide gels using the Protein II system (Bio-Rad) and electrophoretically transferred to a nitrocellulose membrane (Bio-Rad) in 25 mm Tris-base, 190 mm glycine and 20% methanol. The blots were blocked with 5% skim milk in TBS (25 mm Tris-HCl, ph 7.6, 500 mm NaCl) for 2 h at room temperature and incubated with either a polyclonal rabbit antiserum against rat nnos (1 : 500 dilution, Transduction Laboratories), mouse monoclonal anti-ecnos (endothelial) (1 : 2500 dilution, Transduction Laboratories) or mouse anti-beta tubulin antibody (1 : 5000 dilution, CALBIOCHEM, Hornby, Ontario, Canada) in TBST (TBS containing 0.1% Tween-20), for 3 h. After extensive washing in TBST, the lters were incubated for 1 h in a horseradish peroxidase-conjugated anti-rabbit or anti-mouse antiserum (1 : 2500 dilution, Amersham, Oakville, Ontario, Canada). Filters were washed three times with TBST and once with TBS before Western blots were visualized by a chemiluminescence-based photoblot system (ECL; Amersham). The protein extraction and blots were repeated once to con rm our ndings. Results anti-tubulin antibody indicates equal loading of protein (Figure 1E). The relative content of enos mrna: The distribution of enos mrna is shown in Figure 2A,C. The penile urethra expressed the highest levels of enos mrna. High levels of enos mrna were also detected in the MPG, pelvic urethra and cerebellum while the penile crura, shaft of penis, neurovascular bundle and urinary bladder expressed moderate levels of enos mrna. No enos mrna was detected in the prostate. Incubation with GAPDH cdna indicates equal loading of wells with mrna (Figure 2B). The relative content of enos protein: Similarly, enos protein detection in all tissues was in agreement with mrna levels (Figure 2D,F). The level of enos protein was abundant in the urethra where the penile part had more enos protein than the pelvic part. The urinary bladder showed a moderate level of enos protein. Low levels of enos protein were detected in the shaft of penis, crura, MPG, neurovascular bundle and cerebellum. No enos protein was detected in the prostate. Incubation with anti-tubulin antibody indicates equal loading of well with protein (Figure 2E). Extraction of nnos and enos mrna and protein as well as Northern and Western blotting were repeated once and showed similar ndings that con rmed our results. At the time of sacri ce the body weight of rats was g (mean SE). Dissection under the microscope facilitated separation and excision of the various components of the lower urinary tract. The relative content of nnos mrna: Among all components of the lower urinary tract, the MPG had the highest nnos mrna levels (Figure 1A). Densitometric scanning revealed that nnos mrna in the MPG was about 10 fold more than that found in the cerebellum (Figure 1C). The level of nnos mrna in the penile crura was approximately half of that detected in the cerebellum (Figure 1A). The pelvic urethra showed a moderate level of nnos mrna. No nnos mrna was detected in the shaft of the penis, the neurovascular bundle and the penile urethra (Figure 1A). Hybridization with 18S rrna cdna indicates equal loading of mrna (Figure 1B). The relative content of nnos protein: The content of nnos protein is shown in Figure 1D,F. Similar to the pattern of nnos mrna distribution, nnos protein was found abundantly in the MPG that had more protein content than the cerebellum. The level of nnos protein in penile crura was slightly less than in the cerebellum. The pelvic urethra showed high levels of nnos protein. Very little nnos protein was found in the penile urethra, neurovascular bundle and the shaft of penis. Incubation with Discussion Nitric oxide synthase containing nerve bers form an anatomically and functionally distinct population of nerves supplying the lower urinary tract. 19 The urethral sphincter and MPG demonstrated the highest NOS activity, with lower levels found in the proximal penis and lowest levels in the distal penis. 1 Arginine citrulline assays collectively estimate the enzyme activity of NOS constitutive isoforms (enos and nnos) therefore failing to provide speci c physiologic information. Furthermore, the inclusion of the penile urethra and dorsal penile neurovascular bundle, in previously reported penile NOS activity studies may have masked the actual enzyme activity within the corpus cavernous tissue. NADPH-diaphorase histochemistry is useful in mapping nnos protein in the lower urinary tract. Although most authorities agree that NADPH diaphorase and NOS immunohistochemistry correlate in penile tissue samples, there is still a degree of nonspeci city with these techniques. 11,20 Immunostaining can differentiate between isotypes of NOS protein in the penis with variable intensity in different anatomical locations. The neuronal isoform is detected in nerve cells, in the nerves arising

5 305 Figure 2 Distribution of enos in various parts of the lower urinary tract. Separation and excision of various components [major pelvic ganglion (MPG), dorsal prostate (DP), ventral prostate (VP), pelvic urethra (PvU), urinary bladder (UB), penile urethra (PnU), cerebellum (Cb), penile crura (Cr), shaft of the penis (Sft) and dorsal penile neurovascular bundle (NVB)] of the lower urinary tract; RNA extraction and Northern blots were performed as described in Materials and Methods. Blots were hybridized with rat enos (A) and GAPDH (B) cdna probes. A representative densitometric scanning of the band corresponding to enos mrna (C). Representative western immunoblotting of enos (D) and Tubulin (E) from various components of the urinary tract. Tissue lysates and western blots were performed as described in Material and Methods. (F) A representative densitometric scanning of enos.

6 306 from the MPG supplying the penile vasculature and in the urethra. 1,11 The endothelial isoform is mainly found within the penile endothelium of blood vessels. 12 The quality of immunohistochemistry however is affected by the af nity of antibodies to target proteins, methods of tissue xation and interpretation of staining. In our study Western blot analysis of nnos and enos proteins is a speci c and sensitive technique that allows separation of the proteins and their quanti cation. Northern blots further evaluated NOS isoforms at the transcription level by quanti- cation of their mrna. These data accurately describe NOS in different anatomically and functionally distinct locations within the penis. The penis: nnos protein Nerve bers containing nnos are important in initiating the cascade of events that lead to vascular and cavernous smooth muscle relaxation and erection. 1 Penile nnos protein location in our work is similar to that shown by immunohistochemistry in rat 1,11 and man, 12 following the course of the cavernous nerves. Western blot analysis indicates that the highest content of nnos protein within the penis is present proximally in the crura, where cavernous nerves penetrate into the cavernous bodies at the hilum of penis and arborize to supply the various structures of the penis. 1 Crural nnos protein levels approach what is seen in the cerebellum, known to have high nnos protein content. 21 Reduced amounts of nnos protein level are found along the shaft of the penis extending distally from the crura. This is in agreement with immunohistochemistry studies that demonstrate fewer nerve bers within the shaft of the penis probably because there is less cavernous muscle within the shaft, therefore less innervation. Considering the reported histologic structure of the tunica albuginea it seems likely that the tunica albuginea is a NOS poor structure. The inclusion of whole penile shaft (tunica albuginea corpus cavernous smooth muscle) likely produces lower NOS protein levels per mg tissue compared to a pure corpus cavernous sample. A low level of nnos protein was found in the dorsal neurovascular bundle and penile urethra, re ecting the paucity of nnos positive nerve bers supplying these structures. The penis: nnos mrna mrna for nnos was only detectable in the crura of the penis. This is probably because mrna synthesis can take place only at the nerve cell bodies that may be present around the crura. In support of our observation, recently Vanhatalo et al 22 reported the presence of NADPH diaphorase positive nerve cell bodies within the rat penis. Nerve bers within the penile shaft, urethra and neurovascular bundle are not capable of mrna synthesis. These structures contain nnos protein that probably reached them via axonal transport from their parent cell bodies mainly in the MPG and to a lesser degree within the crura of penis. The penis: enos In comparison to nnos protein, enos protein is equally detected in the crura, shaft and dorsal neurovascular bundle. Previous immunohistochemistry studies indicate that our ndings may re ect 11, 12 vascular rather than cavernous endothelial enos. Similarly, nearly equal levels of enos mrna are seen in these structures indicating that the machinery to synthesize enos protein is present locally. Therefore, enos protein synthesis may play a local role in modifying the erectile response in health and disease. The penile urethra The penile urethra shows enos protein and mrna levels three times more than the penile crura, shaft or dorsal neurovascular bundle with minimal nnos protein and no nnos mrna. The signi cance of the relatively high level of enos in penile urethra is not known. Our ndings expand observations by other investigators 12,23 on the presence of subepithelial urethral NADPH-diaphorase positive cells that may be involved in sensory innervation or periurethral gland secretion. The presence of a high content of enos protein in the penile urethra likely alters the results of studies that do not exclude the urethra from tissues studied for penile NOS activity, and enos protein and mrna determination. The major pelvic ganglion: nnos The most abundant quantity of nnos protein is found in the MPG. The level of nnos there exceeds that of the cerebellum by 30%. Similarly the level of nnos mrna in the MPG considerably exceeds that of the cerebellum reaching a ten fold increase. These ndings are consistent with those of Schirar et al 24 and imply the important role of nnos in the regulation of pelvic organs supplied by nerve bers originating from the cell bodies in the MPG. Compared to the crura of penis, the MPG has twice as much nnos protein and 25 times more nnos

7 mrna. The shaft of penis has the smallest amount of nnos and no nnos mrna, emphasizing the downstream distribution of nnos and its role as a mediator of central nervous system control over the erectile machinery. Acknowledgements We would like to thank Dr Kenneth D Bloch, Charlestown, MA who kindly provided us with enos cdna. 307 The major pelvic ganglion: enos Surprisingly, enos was found in the MPG. Careful dissection under the microscope prevented inadvertent inclusion of blood vessels into the sample. enos was recently described in the cerebellum. 21 We reproduced this nding and showed that the level of enos protein in the cerebellum is nearly equal to that in the MPG. Similarly we report high levels of enos mrna in the MPG and cerebellum. The presence of high levels of mrna of enos in neural tissue likely to have high population of cell bodies, re ects the ability of these structures to generate enos protein. While the role of enos in neural tissues is not known, it may function as a neurotransmitter. 21 One viable explanation for the ability of nnos knockout mice to maintain erection is that enos synthesis is recruited. 17 This same mechanism may be an important compensatory one in pathologic states that target nnos. Other structures The pelvic urethra showed high levels of nnos protein that may re ect an important contribution to urethral function. The relative low level mrna suggests that the source of nnos is nerve endings rather than cell bodies. enos protein and mrna are abundant in the pelvic and penile parts of the urethra. The role of enos in urethral function is not known. Functional, histochemical, immunohistochemical and enzyme activity studies strongly indicate that NOS is important in erection, bladder function and urethral sphincter activity. Evaluation of the normal distribution of NOS isoforms and their transcription machinery using direct and quantitative techniques is an important step towards understanding the physiological and the pathological states that involve NOS dependent pathways and affect lower urinary tract function. Although the neuronal isoform is the predominant NOS in the penis and MPG, yet the presence of enos in these structures warrants evaluation of its role, particularly in pathologic states that impair nnos. Further evaluation of the balancing forces of nnos and enos in health and disease is needed to develop new medications that target molecular sites affected in erectile dysfunction. References 1 Burnett AL et al. Nitric oxide: a physiologic mediator of penile erection. Science 1992; 257: 401± Rajfer J et al. Nitric oxide as a mediator of relaxation of the corpus cavernosum in response to nonadrenergic, noncholinergic neurotransmission. N Engl J Med 1992; 326: 90±94. 3 Smet PJ, Jonavicius J, Marshall VR, De Vente J. Distribution of nitric oxide synthase-immunoreactive nerves and identi cation of the cellular targets of nitric oxide in guinea-pig and human urinary bladder by cgmp immunohistochemistry. Neuroscience 1996; 71: 337± Pinna C, Ventura S, Puglisi L, Burnstock G. A pharmacological and histochemical study of hamster urethra and the role of urothelium. Br J Pharmacol 1996; 119: 655± Garcia-Pascual A et al. Characterization of nitric oxide synthase activity in sheep urinary tract: functional implications. Br J Pharmacol 1996; 118: 905± Vernet D et al. Reduction of penile nitric oxide synthase in diabetic BB=WORdp (type I) and BBZ=WORdp (type II) rats with erectile dysfunction. Endocrinology 1995; 136: 5709± Garban H et al. Restoration of normal adult penile erectile response in aged rats by long-term treatment with androgens. Biol Reprod 1995; 53: 1365± Xie Y et al. Effect of long-term passive smoking on erectile function and penile nitric oxide synthase in the rat. J Urol 1997; 157: 1121± Lugg JA, Rajfer J, Gonzalez-Cadavid NF. Dihydrotestosterone is the active androgen in the maintenance of nitric oxide-mediated penile erection in the rat. Endocrinology 1995; 136: 1495± Chamness SL et al. The effect of androgen on nitric oxide synthase in the male reproductive tract of the rat. Fertil Steril 1995; 63: 1101± Dail WG, Barba V, Leyba L, Galindo R. Neural and endothelial nitric oxide synthase activity in rat penile erectile tissue. Cell Tissue Res 1995; 282: 109± Burnett AL et al. Immunohistochemical localization of nitric oxide synthase in the autonomic innervation of the human penis. J Urol 1993; 150: 73± Bredt DS, Snyder SH. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 1990; 87: 682± Janssens SP et al. Cloning and expression of a cdna encoding human endothelium-derived relaxing factor=nitric oxide synthase. J Biol Chem 1992; 267: 14519± Marsden PA et al. Molecular cloning and characterization of human endothelial nitric oxide synthase. FEBS Lett 1992; 307: 287± Lugg J, Ng C, Rajfer J, Gonzalez-Cadavid N. Cavernosal nerve stimulation in the rat reverses castration-induced decrease in penile NOS activity. Am J Physiol 1996; 271: E354±E Burnett AL et al. Nitric oxide-dependent penile erection in mice lacking neuronal nitric oxide synthase. Mol Med 1996; 2: 288± Sutherland RS, Kogan BA, Piechota HJ, Bredt DS. Vesicourethral function in mice with genetic disruption of neuronal nitric oxide synthase. J Urol 1997; 157: 1109± Persson K et al. Morphological and functional evidence against a sensory and sympathetic origin of nitric oxide synthase-containing nerves in the rat lower urinary tract. Neuroscience 1997; 77: 271±281.

8 Zhou Y, Tan CK, Ling EA. Distribution of NADPH-diaphorase and nitric oxide synthase-containing neurons in the intramural ganglia of guinea pig urinary bladder. J Anat 1997; 190: 135± Dinerman JL et al. Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity. Proc Natl Acad Sci USA 1994; 91: 4214± Vanhatalo S, Klinge E, Sjostrand NO, Soinila S. Nitric oxidesynthesizing neurons originating at several different levels innervate rat penis. Neuroscience 1996; 75: 891± McNeill DL, Papka RE, Harris CH. CGRP immunoreactivity and NADPH-diaphorase in afferent nerves of the rat penis. Peptides 1992; 13: 1239± Schirar A, Bonnefond C, Meusnier C, Devinoy E. Androgens modulate nitric oxide synthase messenger ribonucleic acid expression in neurons of the major pelvic ganglion in the rat. Endocrinology 1997; 138: 3093±3102.