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Endocrinology Vol. 140, No. 9 4342-4350
Copyright © 1999 by The Endocrine Society

ARTICLES

Parathyroid Hormone-Related Protein in Rat Penis: Expression, Localization, and Effect on Cavernosal Pressure1

Hervé Lang, Nicole Endlich, Véronique Lindner, Karlhans Endlich, Thierry Massfelder, Andrew F. Stewart, Christian Saussine and Jean-Jacques Helwig

Renovascular Physiology and Pharmacology (CJF INSERM 9409-EA MENRT 2307), Louis Pasteur University Medical School (H.L., N.E., V.L., K.E., T.M., C.S., J.-J.H.), 67085 Strasbourg, France; the Department of Urology, University Hospital (H.L., C.S.), 67091 Strasbourg, France; Institut of Pathology, Medical School (V.L.), 67064 Strasbourg, France; and the Section of Endocrinology, University of Pittsburgh Medical Center (A.F.S.), Pittsburgh, Pennsylvania 15213

Address all correspondence and requests for reprints to: Jean-Jacques Helwig, Ph.D., Pharmacologie et Physiologie Rénovasculaires (CJF INSERM 9409-EA MENRT 2307), 11 rue Humann, Bâtiment 4, 1er étage, 67085 Strasbourg Cedex, France. E-mail: jean-jacques.helwig{at}pharmaco-ulp.u-strasbg.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although PTH-related protein-(1–36) [PTHrP-(1–36)] is known to be expressed in smooth muscle and to exert potent myorelaxant effects, its tonic effects on cavernosal smooth muscle has not yet been explored. Using the RT-PCR technique, the present study establishes that PTHrP messenger RNA is present in microdissected corpus cavernosa in the rat. In immunohistochemical studies using affinity-purified antibodies to middle regions of PTHrP, immunostaining was localized throughout the penile structures, including vessels, cavernosal smooth muscle, and trabecular fibroblasts. Strong immunostaining for PTHrP was also detected in the dorsal nerve bundles. In anesthetized rats, intracavernosally injected boluses of increasing doses of PTHrP-(1–36) (0.3–30 pmol in 100 µl saline) had little effect on intracavernosal pressure. However, they markedly potentiated the dilatory response to papaverine (8–800 nmol), increasing the papaverine-induced intracavernous pressure by 2.5-fold, close to the mean arterial pressure. In conclusion, the cavernosal expression of PTHrP messenger RNA, the distribution of immunoreactive PTHrP throughout the structuro-functional components of the erectile apparatus and its strong potentiating action on papaverine-induced cavernosal relaxation, collectively suggest that PTHrP participates in the control of cavernosal tone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MAMMALIAN species, the corpus cavernosum (CC) is composed of a meshwork of sinusoidal spaces surrounded by the noncompliant tunica albuginea. As in vascular tissue, the cavernosal sinusoidal spaces are lined by endothelial and smooth muscle cells lining trabeculae, which contain vessels, nerves, connective tissue, and fibroblasts. Relaxation of the CC smooth muscle (CCSM) and the resulting synergistic increases in sinusoidal blood pressure and venous outflow resistance are generally acknowledged as the major hemodynamic events in penile erection (1). As in the arterial system, the modulation of CCSM tone involves the local release of dilatory and constrictor neurotransmitters. Acetylcholine (1, 2), nitric oxide (NO) (1, 3), and vasoactive peptides [e.g. vasoactive intestinal polypeptide and related peptides (1, 4, 5, 6, 7)] have been proposed to be major endogenous dilatory neurotransmitters contributing to the tumescent, dilated state of the CC. Conversely, norepinephrine acting on postjunctional {alpha}1-adrenoceptors plays a well established role in keeping the CC tone in the flaccid, detumescent, contracted state (8). Neuropeptide Y (1, 6) has also been suggested to be a possible constrictor neurotransmitter. In addition to the control by the autonomic nervous system, a number of vasoactive compounds released by CCSM cells and endothelial cells, including NO (1, 9, 10), PGs (1, 11, 12), endothelins (1, 13, 14), and angiotensin II (15), are likely to exert similar functions as in classical vascular beds. In smooth muscle cells, the responses to all of the above-mentioned dilatory factors are jointly mediated by the activation of either adenylyl cyclase [e.g. PGE1 or vasointestinal peptide (VIP)] or guanylyl cyclase (e.g. NO), which results in an increase in cytosolic cAMP or cGMP. In support of this, PGE1 as well as inhibitors of cyclic nucleotide phosphodiesterases (PDEs) such as papaverine (16, 17) alone or in combination with {alpha}-adrenergic blockade (18) are now commonly used in the therapy and diagnosis of erectile dysfunction.

Over the past decade, it has become apparent that additional peptides may also serve as modulators of smooth muscle tone. One such substance is PTH-related peptide (PTHrP), a single chain peptide containing 141 amino acids. The reader is referred to recent detailed reviews that describe the role of PTHrP in the humoral hypercalcemia of malignancy syndrome, the structure of the PTHrP gene, the regulation of its expression, the receptors of PTHrP, and its normal physiological roles (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). Briefly, the cloning of PTHrP in 1987 was the result of the search for the hypercalcemic PTH-like factor produced by a number of neoplastims causing the so-called humoral hypercalcemia of malignancy syndrome. Soon after its discovery, it became clear that PTHrP was actually a paracrine factor normally produced by almost every cell and tissue that have been tested. The sequence homology between PTHrP and PTH in the early N-terminal region explains the ability of both peptides to bind to a common heptahelical PTH/PTHrP receptor with similar affinity as well as their capacity to produce similar actions in bone and the renal tubule. PTHrP is abundantly expressed throughout the cardiovascular system and is likely to be involved in the regulation of smooth muscle tone (28, 29). Unlike PTH, however, PTHrP is produced in the vascular smooth muscle cell and the endothelial cell and exerts profound systemic and peripheral vasodilatory effects in vivo and in vitro (28, 29). PTHrP has also been shown to be produced and to be relaxant in hollow organ smooth muscles, including uterus, gastrointestinal tract, and trachea (28, 29). In the urogenital system, PTHrP has been detected in the kidney, bladder, testis, and prostate. In most of these organs, as well as in heart and vessels, mechanical stretch, distention, and vasoconstrictors such as angiotensin II or endothelin are potent up-regulators of PTHrP (28, 29). Up-regulation of PTHrP has been proposed as an adaptive mechanism to increase vascular wall compliance to lumenal filling and increases in systemic or local pressure.

By extrapolation from the above information, it appeared conceivable that PTHrP could be one of the numerous locally produced factors involved in the control of CCSM tone. Accordingly, the goals of the present study were 2-fold: first, to determine whether PTHrP messenger RNA (mRNA) and protein are expressed in the structuro-functional components of the rat penis, and second, to assess whether intracavernosal injected PTHrP-(1–36) is able to modulate CCSM tone in vivo. As it has been documented that drugs such as papaverine (PPV), VIP, and acetylcholine may play cooperative roles in relaxing CCSM (31, 32), the effect of PTHrP-(1–36) on intracavernous pressure (ICP) was tested either alone or in combination with PPV, which is currently considered an archetypal CC myorelaxant.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
In vivo isolation of rat corpus cavernosum
All animal studies were approved by and in compliance with the Louis Pasteur University animal use committee. Seven adult Wistar rats, weighing between 300–350 g, with free access to water and food were anesthetized with ip thiobarbitane (100 mg/kg) and placed on a servo-controlled heated operating table to maintain body temperature between 37–38 C throughout the experiment. A tracheotomy was performed, and the trachea was cannulated to facilitate respiration. The skin overlying the penis was incised, and the prepuce was degloved to fully expose both CC. The crus of each CC was carefully exposed by incision of the ischio-cavernous muscle, avoiding damage to the deep penile artery. The fibro-elastic membranes (Colles and Buck’s fasciae) surrounding the CC were fully resected to further expose the CC without altering the tunica albuginea. As illustrated in Fig. 1Go, the CC were dissected free by removing the bundle of dorsal vessels and the ventral corpus spongiosum surrounding the urethra, including the glans penis and the penile bone. Vascularization was maintained throughout the dissection, which was conducted with a maximum of aseptic care to prevent contamination by external RNA and ribonuclease. The CC exposed as shown in Fig. 1Go was finally excised in one piece distally from their crura, which contain the deep penile artery. The cavernous tissue was immediately frozen in liquid nitrogen and stored at -80 C.



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  		Figure 1. Detection of PTHrP transcripts in rat corpus cavernosum. The CC was isolated in vivo by microdissection in anesthetized rats. After removal of the dorsal vessel bundle (DV) and the corpus spongiosum (CS) with the uretra including the glans penis (G), the corpus cavernosum was cut above the crura for total RNA extraction. The inner panel shows a representative ethidium bromide staining of RT-PCR products using PTHrP-specific primers. Bands at the expected size of 315 bp were obtained by RT-PCR amplification of 1 µg total RNA from microdissected CC, as shown for three representative independent CC preparations (lanes 3–5). Total RNA (0.5 µg) of PTHrP-overexpressing WCS 256 cells, which served as a positive control, underwent RT-PCR amplification under the same conditions (lane 2). Water and omission of reverse transcriptase were used as negative controls (lanes 6 and 7), indicating that RT-PCR products were not the result of contamination or genomic DNA amplification. A DNA size marker is shown in lane 1.

 
Corpus cavernosum RNA extraction and RT-PCR analysis
After thawing, the CC was homogenized in a guanidine isothiocyanate buffer [4 M guanidine isothiocyanate, 25 mM sodium citrate (pH 7), 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol]. Total RNA was extracted by the method of Chomczynski and Sacchi (33). Walker carcinosarcoma cells (WCS 256, provided by Dr. T. Schilling, Department of Internal Medicine I, University of Heidelberg, Heidelberg, Germany) were used as a positive control for PTHrP expression. These cells overexpress PTHrP and induce humoral hypercalcemia in rats (34). Suspended WCS 256 cells were grown in RPMI 1640 (Life Technology, Cergy Pontoise, France) supplemented with 10% FCS (Boehringer Ingelheim GmbH, Gagny, France) at 37 C in a humidified atmosphere containing 10% CO2 in air. Cells were separated from medium by centrifugation, vigorously mixed with guanidine isothiocyanate buffer, and processed for extraction of total RNA as described above.

The following primers, which were derived from those described by Pirola et al. (35), were used for RT-PCR: sense, 5'-AC ACC AAA AAC CAC CCT GTG CGG T-3'; and antisense, 5'-GAATCCTGTAACGTGTCTTGG-3'. The primers cover the coding region of rat PTHrP-(52–141) contained in exons 3 and 4. RT was performed at 42 C with 1 µg total RNA from cavernous tissue or with 0.5 µg total RNA from WCS 256 cells in the presence of 25 U avian myeloblastoma virus reverse transcriptase (Roche Molecular Biochemicals, Mannheim, Germany), 1 µM antisense primer, 0.4 mM deoxy-NTP, 1.5 mM MgCl2, 500 mM KCl, and 0.1% gelatin in a Tris-HCl buffer (pH 8.3) for 45 min. PCR was subsequently started by the addition of 1 µM sense primer, 5 U TaqI DNA polymerase (Perkin Elmer Corp., Roissy, France), and two drops of mineral oil to prevent evaporation. The final reaction volume was 100 µl. The samples were first denatured at 92 C for 4 min. The PCR cycle was programmed as follows: 92 C for 1 min (melt), 60 C for 1 min (anneal), and 72 C for 1 min (extend). PCR was run for 35 cycles. PCR products were separated by agarose (2%) gel electrophoresis in a Tris-borate EDTA buffer containing 0.5 µg/ml ethidium bromide to visualize bands by UV illumination. PCR products were identified by their expected size of 315 bp. PCR product identity was confirmed by restriction digestion with SmaI, yielding two fragments of the expected sizes (107 and 208 bp). PCR products originating from genomic DNA or contamination were excluded by negative controls, in which RT was omitted or ultrapure water was used instead of the total RNA sample.

Immunohistochemistry of PTHrP in rat penis
After anesthesia, the abdominal aorta was cannulated for infusion of 4% formalin at a rate of 2 ml/min. After 90 min, the system was decompressed by incising the liver and by cavatomy, and infusion of fixative was continued at a rate of 4.5 ml/min for 180 min. The skin overlying the penis was incised, and the whole penis body, including the CC crura and the bulbospongiosum covered by the ischiocavernosus and bulbospongiosus skeletal muscles, was excised in one piece and further immersed in fixative for 12 h. The piece was then rinsed of fixative, dehydrated in an ascending series of alcohols, passed through xylene, sectioned at 3 µm, and embedded in paraffin. For immunodetection of PTHrP, paraffin was removed, and sections were stained using two different immunoaffinity-purified rabbit polyclonal primary antibodies recognizing either residues 37–74 (36, 37, 38) or residues 34–53 (Calbiochem France, Meudon, France) diluted to 1–4 µg/ml. Avidin-biotin immunoperoxidase complex/labeled streptavidin biotin kit (BioGenex Laboratories, Inc., San Ramon, CA) was used for detection. As a competition control, sections stained with either antibody were preincubated overnight at room temperature with 10-5–10-6 M PTHrP-(1–74) or PTHrP-(34–53). As an additional control, some sections were processed in the presence of nonimmune serum in the place of primary antibody. For better identification of the structures, adjacent slices were immunostained with a primary antismooth muscle {alpha}-actin antibody diluted to 2 µg/ml at room temperature (DAKO Corp., Trappes, France). The immunohistochemical reaction was performed with avidin-biotin peroxidase complex (DAKO Corp.), using 3-amino-9-ethyl carbazole as the chromogen. Both PTHrP- and {alpha}-actin-immunostained sections were counterstained with hematoxylin.

Action of PTHrP-(1–36) on ICP
Animal preparation and microdissections. Twenty-seven adult Wistar rats, weighing between 300–350 g, were anesthetized with ip thiobarbitane (100 mg/kg), placed on a heated operating table and tracheotomized, and penile crura were exposed as described above (CC isolation). ICP determinations were based on the method of Pineiro et al. (39). A 24-gauge catheter filled with saline was inserted into each of the CC crura. As both CC communicate in the rat, the right catheter was used for ICP measurements and was connected to a pressure transducer (Statham P23Db, Statham Laboratories, Inc., Hato Rey, Puerto Rico), whereas the left catheter was used for intracavernous injection of the drugs. To ascertain the correct position of the catheters in the CC, it was confirmed that a bolus injection of a small volume of saline resulted in fleeting sec penile erection with a simultaneous transient increase in ICP. A purse suture (8/0 prolene) was placed around both catheters to prevent leakage. With the same thread the catheters were further secured to avoid ejection during bolus injection of the drugs. For mean arterial pressure (MAP) measurement, the right carotid artery was exposed and cannulated with a catheter filled with heparinized saline (200 U/ml) and connected to a pressure transducer. Systemic and ICP values were continuously recorded with a WindowGraf 980 Gould recorder (Gould Instrument Systems Ltd., Akron, OH) that registers both pulsatile and MAP.

Experimental protocols illustrated in Fig. 3Go. In preliminary experiments, continuous infusion of PPV at 100 µl/min, rather than bolus injections, at concentrations of 0.027, 0.08, 0.27, 0.8, 2.7, and 8 mM demonstrated no effect on ICP, but decreased MAP by 30–40 mm Hg in a concentration-dependent fashion, indicating a rapid spillover of hypotensive PPV into the systemic circulation. This spillover into the systemic circulation was confirmed using cavernography studies (not shown), employing the continuous infusion of contrast medium (Omnipaque 300, Nycomed, Paris, France) at the same flow rate (100 µl/min). Cavernography weakly and partly opacified the left injected CC, with immediate venous leakage and no tumescence.



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  		Figure 3. Time course of the relaxant effect induced by sequential increasing concentrations of PPV in the absence or presence of PTHrP-(1–36). After a 60-min equilibration period, successive doses of PPV (0–800 nmol in 100 µl saline, as indicated) were injected as boluses in the absence (filled symbols; n = 8) or presence of 30 pmol PTHrP-(1–36) (hollow symbols; n = 4). ICP values (circles) were recorded every 0.5 min. The initial strong and transient increase in ICP coincides with the bolus injection of the drugs. MAP values were recorded at the midpoint of each period (squares). Both ICP values and MAP values are shown as the mean ± SEM.

 
By contrast, bolus injection of 100 µl contrast medium produced an immediate transient tumescence with a complete and dense opacification of both CC without any venous leakage and reflux toward the deep penile artery. The penis returned to the flaccid state within seconds after the bolus injection, with simultaneous opacification of the venous return. Accordingly, in all results reported herein, the drugs were injected as a 100-µl bolus. Under these conditions, a bolus injection of 100 µl saline caused an abrupt rise in ICP that rose to as high as 180 mm Hg, but returned to baseline value within 3 sec. The fact that ICP rapidly returned to the baseline value after the initial bolus injection of saline documented that the injection of fluid in the CC was not a confounding factor in the present studies.

After a 60-min equilibration period during which saline was continuously infused at a flow rate of 3 µl/min to prevent the blood reflux into the catheters, successive doses of PPV or PTHrP-(1–36) in saline, alone or in combination, were then injected as 100-µl boluses in six independent series of experiments. The first series (n = 8) consisted of seven successive bolus injections of 100 µl saline containing 0 (basal ICP), 2.7, 8, 27, 80 270, and 800 nmol PPV (Sigma Chemical Co., St. Louis, MO). The second series (n = 3) consisted of five successive bolus injections of saline containing 0 (basal ICP), 0.03, 0.3, 3, and 30 pmol PTHrP-(1–36) (Bachem, Bubbendorf, Switzerland). The third to sixth series (n = 4) consisted of a bolus injection of saline (basal ICP) followed by seven successive boluses containing a fixed amount of PTHrP-(1–36), i.e. 0.03, 0.3, 3, and 30 pmol in series 3, 4, 5, and 6, respectively, combined with 0 (PTHrP alone), 2.7, 8, 27, 80 270, and 800 nmol PPV. ICP and MAP were monitored during the 12 min that followed bolus injections.

Calculations and statistics. As MAP was stable over the successive 12-min periods, MAP (in millimeters of mercury) was measured at the midpoint of each period, i.e. 6 min after the bolus injection. ICP values in millimeters of mercury were measured from 0.5 min up to 12 min after bolus injections, every 0.5 min. The mean ICP responses to PPV and PTHrP-(1–36) were calculated from the area under the curve over the 12 min that followed drug injection and expressed as millimeters of mercury per min. All values are shown as the mean ± SEM. Effects of increasing doses of PPV, either alone or in combination with PTHrP-(1–36), were tested statistically by two-way ANOVA followed by Student-Newman-Keuls test for multiple comparisons. Differences with P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PTHrP mRNA expression in isolated rat CC
Total RNA was extracted from isolated cavernous tissue after in vivo microdissection of the rat penis (Fig. 1Go). PTHrP mRNA was detected in CC RNA by RT-PCR (Fig. 1Go). In three independent RT-PCR reactions, PTHrP mRNA was present in all seven CC preparations tested.

Immunohistological localization of PTHrP in rat penis
As shown in Fig. 2Go, PTHrP was detected in multiple sites within the rat penis using PTHrP-(34–53) antiserum. Similar results were observed using PTHrP-(37–74) antiserum. The CCSM lining the cavernosal spaces stained strongly not only for {alpha}-actin (Fig. 2CGo), but also for PTHrP (Fig. 2Go, B and D). Staining for PTHrP also occurred in cells embedded in the connective trabecular structure as a perinuclear halo in the cytoplasm (Fig. 2DGo). These cells are most likely fibroblasts, as they did not stain for {alpha}-actin (Fig. 2CGo). The other tissue components within the penis, including the corpus spongiosum and the urethra, stained weakly (not shown). Within the neurovascular components (Fig. 2Go, B, E, and F), staining for PTHrP was obvious in the vascular smooth muscle of all vessels, including the dorsal vein, the penile arteries, and the plexal subalbugineal venous system. As expected, vascular smooth muscle also stained for {alpha}-actin (Fig. 2EGo). Although staining for {alpha}-actin was clearly absent from endothelium (Fig. 2CGo), endothelial staining for PTHrP was present with variable intensity according to the antiserum (Fig. 2DGo). Intense staining for PTHrP was particularly apparent in the dorsal and subalbugineal large nerve bundles, including the perineurium, which, as expected, did not stain for {alpha}-actin (Fig. 2EGo). At discrete locations, staining for PTHrP was apparent in small nerve bundles close to the CCSM. For the sake of clarity, semiquantification of the immunostaining in the various penile sites is shown in Table 1Go. PTHrP staining was specific in that, first, preincubation of the primary antibody with PTHrP-(1–74) or PTHrP-(34–53) virtually abolished staining in a dose-dependent fashion (not shown), and, second, replacement of the primary antisera with nonimmune antisera led to the absence of staining (Fig. 2AGo).



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  		Figure 2. Immunohistochemical staining pattern of PTHrP with affinity-purified PTHrP-(34–53) antibody in cross-sections of rat penile corpus cavernosum counterstained with hematoxylin (B, D, and F). As a control, the penile section in A was processed in the presence of nonimmune serum in the place of primary antibody. For better localization of the muscle network and for better identification of the penile structures, serial sections were immunostained with purified smooth muscle {alpha}-actin antiantibody and counterstained with hematoxylin (C and E). Magnification, x45 in A and B and x320 in C–F. a, Arteriole; al, albuginea; av, subalbugineal venous plexus; cc, corpus cavernosum; dv, dorsal vein; e, endothelium; n, nerve bundles; s, cavernosal sinusoids; sm, smooth muscle; v, venule.

 

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  		Table 1. Semiquantitative distribution of immunoreactive PTHrP within rat penile structures

 
Effect of PTHrP-(1–36) on ICP
Figure 3Go illustrates the overall time course of cumulative changes in ICP in response to sequential increasing doses of PPV, either alone or combined with 30 pmol PTHrP-(1–36). After the initial bolus-induced spike, PPV alone delayed in a dose-dependent fashion the return of ICP to the baseline value, which occurred within 12 min with every dose of PPV. However, no penile tumescence (penile swelling or change in penile angle) was observed. On the average, in the absence of PPV, intracavernous injections of PTHrP-(1–36) in doses up to 30 pmol had a slight, but insignificant, effect on ICP over the time course, compared with the ICP changes induced by bolus injection of saline. Given that the cavernosal volume in rat could be approximated as 100 µl, the PTHrP concentration was approximately 0.1–0.3 µmol/liter in the CC. Such concentrations have been shown to produce maximal dilatory effects in the vasculature (38).

As it has previously been shown that vasodilatory drugs such as VIP, which alone produce poor erectile responses, greatly potentiated the erectile responses to PPV (31), we decided to ask whether PTHrP-(1–36) could potentiate the relaxant response to PPV. When 30 pmol PTHrP were combined with the lowest (2.7 and 8 nmol) doses of PPV, after the initial bolus-induced rise, ICP reached an intermediate maximum of about 35 mm Hg at 2–3 min before returning to baseline value within 12 min. This ICP increase was not accompanied by a perceptible change in penile appearance. At doses between 8–27 nmol PPV, there was a qualitative change in the postinjection time course of ICP. This was accompanied by a visible initiation of penile tumescence and swelling. At 27 nmol PPV, ICP suddenly achieved higher values and did not return to the baseline value within 12 min, presumably reflecting the fact that the threshold of CC relaxation required to block venous outflow had been reached, in turn allowing ICP to increase in a sustained fashion. Indeed, at higher (80, 270, and 800 nmol) doses of PPV, striking sustained dose-related increases in ICP as well as clear sustained rigidity of the penis were induced. Thus, 80–800 nmol PPV combined with 30 pmol PTHrP-(1–36) produced profound sustained cavernosal dilation, increasing ICP to approximately 80% of the MAP value. Importantly, none of these responses, significantly lowered MAP (Fig. 3Go).

These studies demonstrate that although PTHrP alone did not led to changes in ICP, the combination of both agents produced dramatic cavernosal hemodynamic responses. To characterize these responses in quantitative terms, mean ICP changes over the 12 min that followed administration of the drugs were calculated by integration of the area under the trace between 0.5 min (immediately after the initial bolus-induced ICP rise) and 12 min. Mean values of ICP at increasing doses of PPV alone or in combination with 30 pmol PTHrP are shown in Fig. 4Go. The basal level of ICP was 14.0 ± 2 mm Hg. ICP increased significantly at a threshold dose of PPV (27 nmol) and reached a maximum of only 30 ± 5 mm Hg at the highest dose (800 nmol). The dose of PPV that produced the half-maximal effect (ED50) was about 80 nmol (or 30 µg). In marked contrast to the results observed with PPV alone, the combination of PPV with 30 pmol PTHrP resulted in dramatic ICP increases, which reached a maximum of 78 ± 8 mm Hg for a MAP value of 104 ± 9 mm Hg. As shown in Fig. 5Go, the potentiating effect of PTHrP was dose dependent. Again, PTHrP-(1–36) alone, at any dose, had virtually no effect (Fig. 5BGo). On the other hand, PTHrP-(1–36) dose dependently augmented the effects of 27, 80, 270, and 800 nmol. It is noteworthy that at 270 or 800 nmol PPV, a maximum potentiating effect of PTHrP was reached. These ICP changes were accompanied by slight, albeit insignificant, decreases in MAP (Fig. 5AGo), for instance, from 114 ± 5 mm Hg under basal conditions to 104 ± 9 mm Hg 6 min after the last bolus injection of 800 nmol PPV with 30 pmol PTHrP-(1–36).



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  		Figure 4. Dose-dependent effect of PPV on ICP in the absence or presence of 30 pmol PTHrP-(1–36). After a 60-min equilibration period, successive doses of PPV (0–800 nmol in 100 µl saline) were injected as boluses in the absence (-PTHrP; n = 8) or presence of 30 pmol (+PTHrP; n = 4) PTHrP-(1–36). Mean ICP values have been calculated from the area under the curve over the 12-min period that followed bolus injection of the drugs. Values are the mean ± sem. *, P < 0.05, significant effect of PPV compared with saline (Ctl). #, P < 0.05, significant potentiating effect of PTHrP compared with PPV alone.

 


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  		Figure 5. Dose dependency of the potentiating effect of PTHrP on the cavernosal relaxant effect induced by PPV. After a 60-min equilibration period, successive doses of PPV (0–800 nmol in 100 µl saline, as indicated) were injected as boluses in the absence (n = 8) or presence of 0.3 (n = 4), 3 (n = 4), and 30 pmol (n = 4) PTHrP-(1–36). A shows the MAP recorded at the midpoint of each 12-min period. B shows the ICP calculated from the area under the curve over the 12-min period that followed injection of the drugs. Values are the mean ± SEM. *, P < 0.05, significant potentiating effect of PTHrP compared with PPV alone. #, P < 0.05, significant difference between 3 and 30 pmol PTHrP.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although PTHrP is present in most other systems, until the current study it was not known whether PTHrP was produced in the penis. Using RT-PCR to study the expression of PTHrP mRNA, these studies establish that PTHrP is produced in the rat CC. The cavernosal expression of PTHrP mRNA prompted us to perform a survey of PTHrP distribution in the rat penis using immunohistochemical techniques. These studies reveal a previously undocumented but intriguing distribution pattern of PTHrP immunoreactivity in the main structuro-functional components of the rat penis, including the vascular network, the cavernous smooth muscle, and the penile nerves.

PTHrP was found throughout the entire intrapenile vascular network in the rat, including the deep penile artery branches, the dorsal veins, the cavernous arterioles and veins, and the subalbugineal venous plexus. Further, within the CC, the trabecular smooth muscle displayed a strong PTHrP immunohistochemical signal. With the exception of the protruding nuclei, the cavernosal and vascular endothelial cell bodies were flattened against the underlying smooth muscle. Therefore, immunostaining for PTHrP in these cells was difficult to assess, but given that PTHrP has been shown to be expressed in every other endothelial system in which its presence has been sought, its presence within the penile endothelial systems seems extremely likely. For instance, it has been convincingly demonstrated that endothelial cells obtained from bovine carotid artery constitutively produce PTHrP (40). This has also been proven to be true in the human intrarenal arterial tree (38). Nevertheless, further studies are required to verify the endothelial localization of PTHrP in rat penis. Together, these observations strongly suggest that PTHrP may act as an autocrine/paracrine relaxant factor on the caverno-vascular system contributing to the modulation of intracavernous pressure.

There is evidence that both PTH and PTHrP are produced in the central nervous system (41, 42), where most attention has focused upon the hypothalamus. On the other hand, PTHrP has not been detected to date in any nerve fibers innervating peripheral tissues. An unexpected finding of the present study was the detection of PTHrP immunoreactivity in dorsal, cavernosal, and subalbugineal nerve bundles. It is interesting to note that small nerve bundles displaying immunoreactivity for PTHrP were tentatively identified beneath and close to cavernosal vessels and smooth muscle. Thus, PTHrP might belong to the family of neuropeptides with a functional role as a neurotransmitter or a modulator of neurotransmission in rat penile erection. In support of such a hypothesis, it has been demonstrated that PTHrP behaves as a neuropeptide in cultured cerebellar granule cells whose PTHrP gene expression is activity dependent and is controlled by depolarization-induced calcium entry via L-type voltage-sensitive calcium channels (43). It has been shown that PTH-(1–34), which binds to the same PTH-1 receptor as PTHrP-(1–36), reduced calcium influx through L-type voltage-sensitive calcium channels in the mouse neuroblastoma cell line N1E-115 (44), indicating that PTHrP could modulate neuronal cell activity. That PTHrP may play a fundamental role in normal neuronal function has been elegantly documented recently by Nagao and co-workers (45). In this study, intracerebroventricular injection of PTHrP produced a systemic pressor action through the activation of the sympathetic nervous system in conscious rats. In the present study, however, the exact localization of PTHrP to neuronal or glial structures and to sympathetic, parasympathetic, or sensitive nervous fibers and the functional significance of PTHrP in penile nerves must be subjected to much further evaluation.

The findings of immunopositive structures within the rat penis prompted us to ask whether intracavernosal injection of PTHrP would be able to modulate cavernosum tone in vivo. We used PTHrP-(1–36), which has been demonstrated to exhibit myorelaxant properties through interaction with the PTH/PTHrP receptor in vascular as well as in extravascular smooth muscle (28). Although PTHrP-(38–94) has been proven to be a potent stimulator of the intracellular calcium signaling pathway in A10 cells (46), this middle region peptide did not exhibit any systemic or local hemodynamic effect, despite extensive attempts (Massfelder, T., et al., unpublished results). We used quantitative assessment of ICP as an index of CC tone in anesthetized rats. The reliability of this method has been described, and it has been clearly documented that ICP measurement may represent a suitable index for the evaluation of penile erection in rat (47). For this purpose, the CC were approached from the crura, which allows reproducible and accurate insertion of a needle in each crura for separate ICP measurement and drug injection. This method, first described by Pineiro et al. (39), is far simpler than inserting a single needle in the narrow penis shaft, which often leads to penetration of the CC or to protrusion during the course of the experiment (47). Cavernography studies also revealed that bolus injections, rather than continuous intracavernous infusion, of the drugs induced compression of subtunical emissary venules against the tunica albuginea required to transiently occlude the venous outflow. In this way, the drugs were allowed to reach a high local concentration throughout the trabecular network, and spillover into the systemic circulation was prevented. Most importantly, the absence of arterial reflux strongly indicates that the increase in ICP in response to bolus administration of PPV and PTHrP cannot be attributed to upstream dilation of penile artery and to a resulting increase in CC blood flow. The basal ICP reported herein was around 15 mm Hg, a value quite similar to that reported by others in the rat (39, 48).

The net ICP increase (~26 mm Hg at 800 nmol or 0.3 mg PPV) and ED50 values (~80 nmol) for the response to PPV were quite comparable to those reported by others in the rat (Emax = 25–45 mm Hg; ED50 = 280 nmol or ~0.4 mg PPV) (39, 48). It therefore appears that in the rat, the maximal effect of PPV on ICP averages only 30–50% of the MAP. This value is significantly lower than the ICP changes recorded by telemetric devices in freely moving rats, in which ICP measured during intromission was close to the MAP (48, 49). Thus, PPV alone is actually a poor CC relaxant in anesthetized rats. In contrast, although PTHrP has been repeatedly shown to be a potent myorelaxant, in the present study it was unable to appreciably increase ICP. It has been suggested that the cavernosal smooth muscle relaxes up to a certain limit without altering the ICP due to the mechanical buffering function of the tunica albuginea (50). It therefore is conceivable that in the present studies PTHrP was actually able to relax the CCSM, but that the decreased CCSM tone was without effect on ICP, presumably reflecting that the threshold of CC relaxation required to block venous outflow was not reached.

On the other hand, this study demonstrates that PTHrP can markedly potentiate the erectile response to intracavernosal PPV. Maximum potentiating effect was reached with a dose of PTHrP as low as 30 pmol with an ICP value close to MAP value. The fact that in these experiments a maximum potentiating effect was reached also eliminates the possibility that the dosage of PTHrP was insufficient to decrease ICP on its own. Thus, although PPV alone was poor at inducing CC relaxation, PPV in combination with picomolar doses of PTHrP was able to increase ICP by 2.6-fold close to the physiological erectile value and to normal MAP. Such synergistic effects between drugs have been seen in other studies. For example, in men, PPV has been tested alone or in combination with VIP (31). In this study, intracavernosal injection of VIP alone was poorly erectogenic. By contrast, VIP potentiated the erectogenic activity of PPV, as evidenced by the induction of full penile rigidity. These effects in humans have not been quantified in terms of ICP. ICP has been measured in the dog in response to intracavernous injection of a combination of VIP and acetylcholine (32). In this study, the net effect of simultaneous injection of VIP and acetylcholine was not additive but synergistic, increasing the ICP 2.4 times more than the sum of the net ICP increases induced by each drug alone. By comparison, in our study the net effect of simultaneous injection of PPV and PTHrP was 3.3 times higher than the sum of the net ICP increases induced by each drug alone. From a physiological point of view, although the direct effect of PTHrP on CC tone may vary according to species, our findings strongly suggest that PTHrP may be involved in CCSM relaxation.

The precise mechanism of the cooperative relationship between PPV and PTHrP on ICP is unclear at the moment. PPV-induced smooth muscle relaxation has been proven to be associated with increases in the level of cAMP and cGMP due to inhibition of the corresponding PDEs (51). The cyclic nucleotides cAMP and cGMP also appeared to be important second messengers in mediating the relaxation of cavernous smooth muscle (52, 53). In human CCSM, three different PDE isoenzymes have been identified: cGMP-inhibited, cAMP/cGMP-specific PDE III, cAMP-specific PDE IV, and cGMP-specific PDE V. Moreover, PPV has been proven to be a potent inhibitor of all forms of PDE (54). In other respects, N-terminal PTHrP peptides stimulate adenylyl cyclase in vascular smooth muscle (28, 46, 55, 56) and induce cAMP-dependent vasorelaxation in rabbit renal vessels (38). In this latter study, nitric oxide is likely, together with cAMP, to play a key role in the mediation of relaxation in response to PTHrP (38, 57). Moreover, in these vessels, PTH-(1–34) and PTHrP-(1–34) have been shown to bind to common receptors (58). It is therefore tempting to speculate that the PTH/PTHrP receptor-mediated accumulation of cAMP and/or cGMP in response to PTHrP and the cooperative effect of PPV-induced inhibition of cyclic nucleotide degradation could be responsible for the synergistic action of the drugs in increasing ICP. In any case, the presence of cavernosal receptors for PTHrP and the exact cellular mechanism by which PTHrP potentiates the relaxant action of PPV will require further studies.

In conclusion, the immunohistochemical localization of PTHrP within all of the anatomical components of the erectile apparatus, together with its marked potentiating action on PPV-induced cavernosal relaxation suggest that PTHrP contributes to the control of CCSM tone. The exact role of the peptide in normal penile physiology and its therapeutic potential remain to be established.


   Acknowledgments
 
We warmly thank Prof. Didier Jacqmin (Chief of the Department of Urology, University Hospital, Strasbourg, France) for constant support, Prof. J. M. Vetter (Chief of the Department of Pathological Anatomy, University Hospital), for invaluable help in performing and evaluating the immunohistochemical preparations, Mrs. Jeannine Krill and Suzanne Wendling for skilled technical assistance, and Mrs. Danièle Kuhlwein and Sylvie Rothhut for outstanding manuscript preparation.


   Footnotes
 
1 This work was supported by the French National Institute of Health (INSERM; Grant CJF 9409), the French Ministry of Higher Education (EA 2307), and the French Foundation for Medical Research (Endowment FRM 20000337). Back

Received January 8, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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