Penile neuronal nitric oxide synthase and its regulatory proteins are present in hypothalamic and spinal cord regions involved in the control of penile erection

Animals and tissue processing

Young (3-months) and old (24-months) male Brown Norway rats were obtained from the NIH/NIA colony (Harlan Sprague-Dawley, Inc., San Diego, CA) and maintained under controlled temperature and lighting, according to NIH regulations. Young animals were anesthetized with Pentothal (thiopental sodium; 25 mg/kg body weight, intraperitoneal), pretreated with heparin, and perfused through the left ventricle with saline followed by 10% formalin buffered solution [pH 7.4] (Ferrini et al., 2001a,b). The brain was removed and post-fixed overnight in 10% formalin, washed with phosphate buffered saline (PBS, 140 mM NaCl, 10 mM phosphate salts, [pH 7.4]), and stored in PBS at 4°C until further processing within the first 24 hours. Additional young and old animals were used for the dissection of the hypothalamus, mainly hypothalamus and preoptic area, and of the lumbosacral spinal cord (L4–S1), without prior heparinization and perfusion with fixative. The fresh tissues were immediately frozen in liquid nitrogen and stored at −80°C. The experimental procedure was approved by the IUCAC of the Harbor UCLA Research and Education Institute.

Detection of mRNA and protein expression

Total RNA was isolated from the nonfixed preoptic area-hypothalamus (POA-HT) and spinal cord tissue by the Trizol procedure (Gibco BRL, Gaithersburg, MD), and submitted (1 μg) to reverse transcription by using Superscript II RNase H^- reverse transcriptase (Gibco BRL) and random primers (0.25 μg), followed by polymerase chain reaction (PCR) with the respective gene-specific 20-mer primers spanning an intron to exclude DNA contamination (González-Cadavid et al., 2000a; Ferrini et al., 2001a,b). Primers used for PCR were (1) for nNOS brain variant and PnNOS, on nucleotides (nt) 2561-2580 (forward) and 2788-2807 (reverse) of the rat PnNOS cDNA (Genbank accession no. U67309), as the source of the expected 247-bp (PnNOS) and 145-bp (nNOS, brain variant) bands; (2) for PIN, on nt 74-93 (forward) and nt 401-420 (reverse) of the rat PIN cDNA (Genbank accession no. U66461), as the source of the expected 347-bp band; (3) for NMDAR-1, nt 2522-2541 (forward) and nt 2785-2804 (reverse) of the rat NMDAR-1 cDNA (Genbank accession no. X63255), as the source of the expected 283-bp (NMDAR1, brain variant) and 334-bp (NMDAR1-T, penile variant) bands. PCR products were separated by electrophoresis on 1.5% agarose gels and stained with ethidium bromide. For densitometry, normalization was performed against the β-actin (rat β-actin control amplifier set, Clontech, Palo Alto, CA) housekeeping gene fragment generated in the same PCR reaction, giving a band of 764 bp.

Total RNA was also analyzed for PIN mRNA expression by Northern blot on formaldehyde 1% agarose gels with hybridization with a cDNA probe corresponding to a 347-bp full length rat PIN cDNA labeled with ³²P[dCTP] by random priming (Ferrini et al., 2001b).

For the detection of protein expression, tissue extracts were either obtained as side-products from the Trizol procedure for RNA extraction or by homogenizing fresh tissue in a buffer containing 0.32 M sucrose/20 mM HEPES pH 7.2/0.5 mM ethylenediaminetetraacetic acid/1 mM dithiothreitol and protease inhibitors (3 μM leupeptin, 1 μM pepstatin A, 1 mM phenylmethyl sulfonyl fluoride), and obtaining the supernatant at 12,000 × g for 60 minutes (González-Cadavid et al., 2000a). Equal amounts of protein (30 μg) were run on 7.5% polyacrylamide gels and submitted to Western blot immunodetection with either (1) a monoclonal anti-mouse nNOS brain variant immunoglobulin G (IgG; amino acids 1095-1289; BD Transduction Laboratories, San Jose, CA), at 1:500 dilution, detecting both nNOS and PnNOS as 155- to 160-kDa (α-form) or 130- to 135-kDa (β-form) bands, followed by a secondary polyclonal horse anti-mouse IgG linked to horseradish peroxidase (BD Transduction) at 1:2,000 dilution; (2) polyclonal anti-rat PnNOS variant IgG (16 amino acids in the 34 amino acid insert; González-Cadavid et al., 2000a) at 1:1,000 dilution, detecting PnNOS as 155- to 160-kDa (α-form) or 130- to 135-kDa bands (β-form), (3) anti-rat PIN IgG (amino acids 24-39), at 1:1,000 dilution, detecting a 12- to 15-kDa band (both custom-made, Bethyl Laboratories, Montgomery, TX), followed by secondary polyclonal horse anti-mouse IgG as above; (4) monoclonal anti-rat PIN IgG (whole length 89 amino acids), at 1:1,000 dilution (BD Transduction), detecting the same PIN band as (3), followed by an anti-mouse goat IgG linked to horseradish peroxidase (BD Transduction). Visualization of the bands was performed by a luminol reaction (Pierce Endogen, Rockford, IL). Negative controls were performed without primary antibody.

Cloning and expression of rat PIN cDNA and purification of fusion PIN protein

A construct of the full-length rat PIN cDNA was prepared by reverse transcribing penile total RNA followed by PCR using primers flanking the PIN coding region (Jaffrey and Snyder, 1996). The PIN fragment was then subcloned in both forward and reverse orientations into the TA cloning, mammalian expression vector pCR3.1 (Invitrogen, Carlsbad, CA), thereby originating two constructs named pCMV-PIN and pCMV-PIN (AS), respectively. The inserted cDNAs were sequenced and protein expression verified by transfection of HEK-293 cells cultures on six-well plates (0 or 2 μg plasmid DNA and 8 μl lipofectamine) for 5 hours, followed by cell growth in the presence of DMEM/10% fetal calf serum for 3 days. After growth, the cells were washing in PBS, and directly lysed in the wells by using an sodium dodecyl sulfate (SDS)-based buffer as before. PIN expression was detected by Western blot as above.

For the preparation of the recombinant GST-PIN fusion protein (Jaffrey and Snyder, 1996), PIN cDNA was cloned into pGEX-4T-1 (Amersham Pharmacia Biotech, Piscataway, NJ) by restriction digest of pCMV-PIN with EcoRI and NotI, agarose gel purification, and ligation into the fusion vector between EcoRI and NotI. Correct cloning of PIN in both forward and reverse direction was verified by DNA sequencing. Plasmids were grown in the protease-deficient Escherichia coli strain BL21 and fusion protein expression induced with 1 mM IPTG for 1.5 hours. Lysates were prepared and GST-PIN fusion protein purified with GST-agarose columns as per manufacturer's instructions (GST purification module protocol, Amersham Pharmacia Biotech). Purified protein was quantitated by a Bradford protein assay and synthesis of pure protein determined by Coomassie blue staining after polyacrylamide gel electrophoresis and by Western blotting as above with antibodies against PIN and against GST (Jaffrey and Snyder, 1996). In addition to GST-PIN, GST protein was purified in the same manner for use as a negative control for nNOS binding assays.

Immunohistochemical detection

The tissue detection of PnNOS, nNOS, and PIN was carried out on 5-μm paraffin-embedded coronal sections collected onto gelatin-coated slides (Ferrini et al., 2001a). Adjacent coronal sections were obtained at the level of the medial septum (9.20–8.74 mm interaural), organum vasculosum of the lamina terminalis (9.00–8.80 mm interaural), medial preoptic area (9.20–8.60 mm interaural), supraoptic nucleus (7.70–7.60 mm interaural), paraventricular nucleus (7.20–6.88 mm interaural), septohypothalamic nucleus (8.20–8.08 mm interaural), and frontal cortex according to the atlas of Paxinos and Watson (1997).

After deparaffinization and rehydration, sections were quenched for endogenous peroxidase activity by using 3% H₂O₂, blocked with 10% normal goat serum or normal horse serum in PBS containing 0.15% Triton X-100, and incubated with the purified primary antibodies (listed under Western blot method section), except that the dilutions were 1:1,000 (nNOS, PnNOS) and 1:600 (PIN) diluted in PBS + 0.15% Triton X-100 + 1% normal horse serum or normal goat serum, respectively. Negative controls were done by preabsorbing the nNOS antibody, at the concentration used previously, with 1 μM nNOS blocking peptide in PBS (Calbiochem, San Diego, CA). For PnNOS and PIN antisera, the immunogenic peptides (Bethyl) were used at 2 μM in PBS. After 1-hour incubation at room temperature, the mixtures were centrifuged in a microfuge at full speed for 15 minutes and the supernatants were used as primary antisera at the same dilutions used previously. Each slide assayed had a negative control. The first antibodies were incubated overnight at 4°C. After rinsing in PBS, the slides were incubated for 40 minutes with a secondary biotinylated anti-rabbit goat IgGs (1:200; Calbiochem), followed by the ABC complex (1:100; Calbiochem) and 3,3′-diaminobenzidine (DAB; Sigma). In the case of the monoclonal nNOS antibody, the secondary was a biotinylated anti-mouse horse IgG (1:200; Calbiochem). Sections were counterstained with hematoxylin.

Sections were photographed with an Olympus BHS microscope equipped with a SPOT-RT digital camera and acquisition software (Diagnostic Instruments, Sterling Heights, MI). Photographs were taken at 350 dpi resolution. Images were imported into Adobe Photodeluxe 1.0 (Adobe Systems, San Jose, CA). The images were cropped and adjusted for brightness and contrast. Labels and scale bars were added. Immunoreactivity was analyzed by Image Pro 4.01 image analysis system (Media Cybernetics, Silver Spring, MD; Ferrini et al., 2001a, b; Liu et al., 1997). At least 10 anatomically matched sections per animal were analyzed for medial septum, preoptic area (including organum vasculosum of the lamina terminalis, medial preoptic area, and septohypothalamic nucleus), and six sections for paraventricular nucleus and supraoptic nucleus. For the analysis of the paraventricular nucleus subregions, the description of the paraventricular nucleus atlas from Kiss and Palkovits was followed (Kiss et al., 1991).

Double-labeling immunodetection

Colocalization of nNOS and PnNOS neurons with PIN in different sections of the brain was performed on frozen sections (Ferrini et al., 2001b). Brains were perfused and fixed as described above, except that after the overnight fixation the tissues were immersed in 20% sucrose until submerged and frozen. Coronal sections (16 μm) were cut with a Leica CM 3050 S cryostat (Leica Microsystem, Buffalo, NY) and placed onto gelatin-coated slides. Anatomically matched sections of the chosen hypothalamic regions were preincubated with 10% normal goat serum in 0.15% Triton-PBS and then in a 1/300 dilution of anti-PIN antiserum (Bethyl, custom made), followed by a 1/20 dilution of biotinylated anti-rabbit goat IgGs (Calbiochem). Sections were then incubated in 10 μg/ml streptavidin-Texas Red (Vector Laboratories, Burlingame, CA). After several washes in PBS, the sections were incubated in 10% normal goat serum and then in a 1/1,000 dilution of PnNOS or nNOS antibodies for 1 hour at room temperature. Fluorescence labeling was performed with FITC-conjugated anti-rabbit (for PnNOS) or anti-mouse (for nNOS) IgG (13 μg/ml, Vector Laboratories). After several washes in PBS, the sections mounted in prolong antifade (Molecular Probes, Eugene, OR) were examined by using a Leica TCS SP confocal laser-scanning microscope equipped with argon and HeNe lasers coupled to acquisition software. Images were imported to Adobe Photodeluxe 1.0, cropped and adjusted for brightness and contrast only, and saved as TIFF files.

Colocalization of PnNOS and oxytocin was performed on frozen sections as described above on anatomically matched sections of the supraoptic nucleus and paraventricular nucleus. The sections were preincubated with 10% normal goat serum and then with a 1:1,000 dilution of the guinea pig oxytocin antiserum (Peninsula Laboratories, Belmont, CA) followed by a 1:100 dilution of the biotinylated anti-guinea pig goat IgGs (Vector Laboratories). Sections were then incubated in streptavidin-Texas Red (10 μg/ml; Vector Laboratories). After several washes in PBS, sections were re-incubated in 10% normal goat serum, followed by a 1:500 dilution of anti-PnNOS antiserum for 1 hour at room temperature, and finally with FITC-conjugated anti-rabbit goat IgGs. Sections were mounted and processed as in PIN colocalization.

Colocalization of PIN with glial fibrillar acidic protein (GFAP) was done in the adjacent frozen sections used for PIN colocalization with nNOS or PnNOS. Briefly, after blocking with 10% goat serum, sections were incubated overnight at 4°C with a 1:300 dilution of PIN antibody in PBS containing 1% normal goat serum and 0.15% Triton X-100. After several washes with PBS, sections were incubated with biotinylated anti-rabbit goat IgGs followed by streptavidin-FITC diluted in PBS. After washing, sections were blocked with 10% normal horse serum and incubated with a 1:800 dilution of anti-GFAP monoclonal antibody (Sigma) in PBS containing 1% normal horse serum and 0.15% Triton-X100, 1 hour at room temperature. After rinsing with PBS, sections were incubated with Texas Red conjugated anti-mouse horse IgGs (13 μg/ml) diluted in PBS containing 1% normal horse serum and 0.15% Triton-X100 for 40 minutes. Slides were washed with PBS and mounted and examined as above.

Test of PIN on NOS activity in tissue homogenates

NOS activity was measured by a modification of methods previously described (Vernet et al., 1998). Briefly, tissues were homogenized in a 1:6 wt/vol ratio in the medium described below for Western blotting. The particulate and cytosolic fractions were obtained by centrifugation at 12,000 × g for 60 minutes, and the soluble fraction was passed through Dowex AG50WX-8 (Na+) resin to remove endogenous arginine. Samples were then incubated at 37°C for 30 minutes with (3-H) arginine (L-[2,3,4,5-3H]arginine monohydrochloride, 63 μCi/nmol, from Amersham Life Science; final 3 μCi/ml), NADPH (2 mM), L-arginine (0.1 mM) and calcium (0.45 mM). Controls included the addition of a NOS inhibitor, L-NAME (2 mM), a Ca⁺⁺ chelator, EGTA (5 mM), or the omission of Ca⁺⁺. The recombinant PIN-GST or cleaved PIN proteins were added at increasing concentrations and incubated for 30 minutes at 0°C before the determination of NOS activity. At completion, the residual (3-H)L-arginine was eliminated through the resin, and (3-H)citrulline was counted in the trichloroacetic acid-ether–extracted supernatant. Background activity was corrected by accounting for time zero incubations. NOS activity was expressed as picomoles of L-citrulline per minute per milligrams of protein.

PIN/PnNOS protein interaction

GST-PIN (20 μg in 100 μl PBS) was incubated with tissue extracts from rat hypothalamus and cerebellum (100 μl each) for 1 hour at 4°C. All subsequent procedures were done at 4°C. The reaction was passed over a GST-Sepharose 4B column (200-μl bed volume), washed extensively with HNTG buffer (20 mM Hepes [pH 7.4], 500 mM NaCl, 10% glycerol, and 0.1% Triton X-100), and eluted with three 400-μl aliquots of glutathione elution buffer (100 mM Tris [pH 8.0], 10 mM glutathione; Jaffrey and Snyder, 1996). In addition, purified GST protein was incubated and processed in the same manner as GST-PIN and served as a negative control for PIN binding. One tenth of each eluted aliquot was run on an SDS- polyacrylamide gel electrophoresis (SDS-PAGE) denaturating polyacrylamide gel and submitted for Western blotting as described above.

Statistical analysis

Values were expressed as mean ± standard error of the mean (SEM). The normality distribution of the data was established by using the Wilk-Shapiro test, and the outcome measures between two groups were compared by the t test. Differences between two groups were considered significant at P < 0.05. Multiple comparisons among the different groups were analyzed by a single factor analysis of variance, followed by post hoc comparisons with the Student-Newman-Keuls test, according to the Graph Pad Prism V3.0 program. A P < 0.05 was considered significant.

Expression of PnNOS, PIN, and NMDAR mRNA and protein

To determine whether PnNOS and the genes encoding two of the interacting proteins, PIN and the subunit 1 of the NMDA receptor (NMDAR1), were expressed in the hypothalamus and spinal cord areas involved in the control of penile erection, RNA was isolated from different brain regions and from the L4–S2 spinal cord of two young rats. Reverse transcriptase-PCR (RT-PCR) was then performed with a set of primers that differentiates PnNOS from the brain-type nNOS by the size of the amplified band (247 vs. 145 bp). Additionally, RT-PCR was performed to identify PIN (347 bp), and a third set that distinguishes between the wild-type mRNA and the variant named NMDAR1-T expressed in the penis (283 vs. 334 bp; Fig. 1A). The band expected for the brain-type nNOS was clearly visible in all regions, although it was fainter in the hypothalamus, and the larger band from PnNOS was also detectable in all regions. The expression of PIN appeared to be considerably higher than the nNOS variants, as denoted by the intense band generated from all RNAs. NMDAR1 was observed in all tissue RNAs, except that only in the penis was the NMDAR1-T variant detected (Fig. 1B).

Protein expression was also investigated by immunoblotting of the tissue lysates (Fig. 1C). A custom-made antibody against PnNOS that differentiates this variant from the cerebellar nNOS shows expression of the 155-kDa band corresponding to the full-length PnNOS (α form), although at a much lower level than in the penis. No truncated nNOS/PnNOSβ form (130 kDa) was detected in this set of specimens.

For PIN, another custom-made antibody against a synthetic peptide of rat PIN was applied, which was validated by cloning rat PIN from penile RNA and expressing the respective sense and antisense cDNA constructs in HEK293 cells. As can be expected from a ubiquitously expressed protein, a 10-kDa PIN band was detected in the control cells transfected with the empty vector (endogenous PIN), but the corresponding band for the cells transfected with the sense construct was much more intense. The antisense PIN gave only a faint band, indicating that it blocks the expression of the endogenous PIN gene in the cells to some extent. The fainter band above (23–25 kDa) may have been a dimer form of PIN. The antibody detected the same band in all the brain and spinal cord regions examined, and expression in the cerebellum appeared to be the lowest (Fig. 1D). For NMDAR expression, the available subunit 1 antibody does not differentiate the penile and brain variants detected by RT-PCR, and because NMDAR2b has been extensively described in these tissues (Curras-Collazo et al., 2000; Herman et al., 2000), no Western blots were performed for NMDAR.

To determine whether the expression of PnNOS, nNOS, and PIN mRNAs vary with aging, the same RT-PCR analysis was performed on RNA isolated from the hypothalamus from four 5-month-old (young) and four 24-month-old (old) rats. Figure 2 shows that there is no significant difference with age in the levels of any of these mRNAs and that the ratio between PnNOS and nNOS is high, because approximately 40% of the nNOS mRNA is PnNOS (Fig. 2 left, top and bottom).

Immunohistochemical detection of PnNOS and PIN in different hypothalamic regions

The expression of both PnNOS and PIN detected above in extracts of the whole hypothalamus may occur specifically in certain areas critical for the control of erectile function, in association with either nNOS and NMDAR that are widely distributed in this organ. Based on this assumption, we examined systematically the localization of PnNOS, nNOS, and PIN to compare the respective distributions and compare them with the detection of NMDAR studied by other authors (see Discussion section). Figure 3A,D indicated that nNOS, as detected by an antibody common to both the brain-type and penile variants, was expressed in multiple cells, presumably neurons, scattered along the medial preoptic area. At least half of them stained with the antibody specific for PnNOS only (Fig. 3B,E). PnNOS staining was more concentrated in the medial region of the medial preoptic nucleus than in the central region of the medial preoptic nucleus, where only very faint staining was seen. PIN staining (Fig. 3C,F) was more restricted and showed two patterns: one faint and diffuse, which in some cells showed cytoplasmic expression extending to cell processes, and another more intense in a few isolated cells, that were nuclear or perinuclear with a certain polarization (right). These dark cells are presumably glial cells (see below, Fig. 6E–H) or small interneurons, and in some cases there appeared to be colocalization of PnNOS and PIN.

The nNOS and PnNOS distribution in the paraventricular nucleus had a strikingly different pattern from the one observed in the medial preoptic area. nNOS (Fig. 3G) was clearly present in the magnocellular and parvocellular regions, but PnNOS was predominant in what appeared to be the posterior magnocellular region where oxytocin neurons have been mapped (Sawchenko and Swanson, 1982), and extended to the medial parvocellular region, identified as posterior by Kiss et al. (1991; Fig. 3H). Few scattered PnNOS-positive cells were seen in the dorsal parvocellular region, suggesting that the medial parvocellular region expresses mainly the brain-type nNOS variant. Therefore, both nNOS variants appeared to be located in two neighboring discrete cell populations. PIN expression, as in the medial preoptic area, was fainter (Fig. 3I), and more scattered around all the paraventricular nucleus regions. Some cells stained intensively. No PnNOS/PIN colocalization was immediately obvious. PIN appeared also to be expressed in the ependymal cells of the third ventricle.

The expression of both nNOS variants in the supraoptic nucleus (Fig. 4A,B) resembled the one in the paraventricular nucleus in the sense that there was a concentration of nNOS-positive neurons along a defined area in the magnocellular neurons (Fig. 4A), where most of the cells stained also with the PnNOS antibody (Fig. 4B), indicating a PnNOS-rich area. The scattered neurons outside this area appeared to be of mixed origins but with a predominance of the brain nNOS variant. In the septohypothalamic nucleus (bottom), there was only a very scattered distribution of nNOS neurons (Fig. 4C), and PnNOS neurons were even more restricted (Fig. 4D). PIN staining (not shown) was virtually undetectable in both the supraoptic nucleus and septohypothalamic nucleus.

The medial septum showed scattered nNOS staining with approximately one third of the positive cells staining for PnNOS at a lower intensity (Fig. 4E,F). In both regions, PIN staining resembled medial preoptic area distribution (not shown). An interesting situation occurred in the organum vasculosum of the lamina terminalis, where cerebellar nNOS appeared to be concentrated around the third ventricle and was the only nNOS variant expressed in many of the neurons (Fig. 4G,H). PnNOS was virtually negative throughout. In the cortex (Fig. 5), PnNOS was detected in almost the same number of nNOS-positive neurons (Fig. 5 A,C), but the intensity was much less pronounced (see higher magnification, Fig. 5B,D). PIN (Fig. 5E,F) expression, as in the medial preoptic area, stained many cells in the cytoplasm and cellular processes. Also, certain glial cells stained for PIN in the perinuclear area, and some appeared to be nNOS-positive.

Colocalization of PnNOS with PIN and expression in oxytocinergic neurons

To determine whether neurons that harbor PnNOS also express PIN, we conducted a dual localization study, where PnNOS was tagged with a fluorescent green marker, PIN was identified with a red fluorescent stain, and the coexistence of both proteins in the same cell was denoted by yellow fluorescence. Figure 6 shows that in the medial preoptic area (Fig. 6A), many of the cells coexpressed PnNOS and PIN, and the colocalization was also evident in some cells of the medial septum and in the cortex. However, no colocalization was apparent in either the supraoptic nucleus or the paraventricular nucleus (not shown). Instead, PIN was more localized around the neuronal bodies. The latter is consistent with the putative absence of PIN from PnNOS-positive cells.

To determine whether those cells that presented a strong perinuclear expression of PIN are astrocytes, a colocalization with GFAP was performed. Figure 6E–H shows the overlay of PIN and GFAP (as a marker of astrocytes) in the medial preoptic area (Fig. 6E), cortex (Fig. 6F), and supraoptic nucleus (Fig. 6G,H). We found a clear expression of PIN in GFAP-positive cells, which was very strong around the perinuclear area, resembling the situation seen with peroxidase staining in what appeared to be glial cells. PIN expression was also seen in small interneurons. The staining with PIN was more concentrated at the level of the cell processes.

Dual labeling shows that, in the paraventricular nucleus, the region more involved in the control of penile erection, PnNOS was expressed in some oxytocinergic neurons which are crucial for the dopaminergic/nitrergic pathways operating in this process but was also present in nonoxytocinergic cells (Fig. 7A,C,E). This finding was replicated in the supraoptic nucleus (Fig. 7B,D,F).

PIN effects on hypothalamic NOS

To determine whether PIN indeed has the capacity to inhibit hypothalamic NOS activity, pure recombinant PIN-GST and GST protein were purified and characterized by Coomassie blue staining and Western blotting (Fig. 8, top). Increasing amounts of pure recombinant PIN-GST protein or PIN antibody were added to hypothalamic tissue extracts, incubated for 60 minutes, and the L-arginine/citrulline conversion assay was performed to assess NOS activity. PIN had only a very moderate dose-dependent effect on NOS activity, with an inhibition of 10 to 32% obtained at peak inhibition (not shown). Higher concentrations (100 μg) did not increase the inhibition. PIN antibody (purified IgG) was not efficient in neutralizing endogenous PIN, because virtually no increase of NOS activity was observed, compared with the effect of control IgG. The GST protein had no effect, as previously shown (Jaffrey and Snyder, 1996).

The ability of PIN to interact with hypothalamic nNOS was demonstrated by binding experiments, where PIN-GST was incubated with hypothalamic and cerebellar tissue extracts and then passed through a GST-Sepharose 4B column to retain the PIN-GST protein. As a negative control, tissue extracts were also incubated with GST and run over GST-Sepharose 4B columns. After washing, proteins bound to the columns were eluted and subjected to electrophoresis under denaturing conditions on PAGE gels followed by Western blotting. Both nNOS and PIN antibodies were used for immunodetection. Figure 8 (bottom) shows that nNOS from both hypothalamus and cerebellum bound GST-PIN but not GST alone, indicating a specific interaction in these tissues. This finding was evidenced by the immunodetection of nNOS in the fractions retained by the PIN-binding column and eluted as fraction C1 in Figure 8 (bottom).