Distribution and origin of neuropeptide Y-immunoreactive fibers in the penis of the rat

Summary The present study investigated the distribution of neuropeptide Y-immunoreactive fibers to the penis of the rat. In the corpora cavernosa penis, a dense plexus of fibers was asociated with arteries, intrinsic cavernosal muscle, and veins including the deep dorsal vein. In the corpus spongiosum, immunoreactive fibers were present around vascular smooth muscle and at the periphery of the acini of the paraurethral glands. Immunohistochemistry of penile neurons identified by retrograde tracer injection into the penis indicates that about 5% of the penile neurons in the pelvic plexus contained the neuropeptide while larger percentages of penile neurons in the sympathetic chains were immunoreactive for neuropeptide Y. Chemical and surgical sympathectomy greatly reduced the neuropeptide Y- and catecholamine-containing fibers in the erectile tissue but had no clear effect on the neuropeptide Y fibers around the paraurethral glands; a tissue that is not innervated by adrenergic fibers. It is concluded that (1) the widespread distribution of neuropeptide Y indicates that it may function in the control of penile blood flow, (2) with the possible exception of the paraurethral glands, the sympathetic chain is the most likely source of neuropeptide Y fibers in both erectile bodies of the penis, and (3) this peptide may play a role in the secretory functions of the paraurethral glands.     

A large proportion of pelvic neurons innervating the corpora cavernosa of the rat penis exhibit NADPH-diaphorase activity

Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, which indicates the presence of neural nitric oxide synthase, the enzyme responsible for the generation of nitric oxide, was used in combination with retrograde labelling methods to determine, in whole-mounts and sections of rat major pelvic ganglia, whether neurons destined for the penile corpora cavernosa were able to produce nitric oxide. In whole-mount preparations of pelvic ganglia, among the 607±106 retrogradely labelled neurons innervating the penile corpora cavernosa, 84±7% were NADPH-diaphorase-positive, 30±7% of which were intensely histochemically stained. In serial sections of pelvic ganglia, out of a mean count of 451 retrogradely labelled neurons, 65% stained positively for NADPH-diaphorase. An average of 1879±363 NADPH-diaphorase positive cell bodies was counted in the pelvic ganglion. In the major pelvic ganglion, neurons both fluorescent for Fluorogold or Fast Blue and intensely stained for NADPH-diaphorase were consistently observed in the dorso-caudal part of the ganglia in the area close to the exit of the cavernous nerve and within this nerve. This co-existence was much less constant in other parts of the ganglion. In the rat penis, many NADPH-diaphorase-positive fibres and varicose terminals were observed surrounding the penile arteries and running within the wall of the cavernous spaces. This distribution of NADPH-diaphorase-positive nerve cells and terminals is consistent with the idea that the relaxation of the smooth muscles of the corpora cavernosa and the dilation of the penile arterial bed mediated by postganglionic parasympathetic neurons is attributable to the release of nitric oxide and that nitric oxide plays a crucial role in penile erection. Moreover, the existence in the pelvic ganglion of a large number of NADPH-diaphorase-positive neurons that are not destined for the corpora cavernosa suggests that nitric oxide is probably also involved in the function of other pelvic tissues.     

Protein inhibitor of nitric oxide synthase (NOS) and the N-methyl-D-aspartate receptor are expressed in the rat and mouse penile nerves and colocalize with penile neuronal NOS

Nitrergic neurotransmission triggering penile erection is mediated by nitric oxide (NO) synthesized in the cavernosal nerves of the penis by penile neuronal NO synthase (PnNOS). In the central nervous system, nNOS is activated by the N-methyl-D-aspartate receptor (NMDAR) and, presumably, is inhibited by the protein inhibitor of NOS (PIN). The PnNOS and NMDAR are expressed in the penis, and PnNOS has been localized in penile nerves. Both proteins colocalize with PIN in the hypothalamus and the spinal cord involved in the control of erection. The present study aimed to elucidate the relationship between PnNOS, PIN, and NMDAR in the penis. It was found that in the rat, PIN was expressed in the pelvic ganglion and the cavernosal nerve, and penile PIN cDNA was cloned, sequenced, and expressed. Immunohistochemistry localized PIN to the cavernosal and dorsal nerve of the penis, whereas NMDAR was not detected in the latter. Dual-fluorescence labeling showed that PnNOS colocalized with PIN in both nerves but with NMDAR only in the cavernosal nerve. Aging did not affect the mRNA levels of PnNOS, nNOS, NMDAR, and PIN. Both PIN and NMDAR were detected in penile nerves of the wild-type and nNOS(-/-) mouse. The PIN protein did not inhibit or bind NOS in penile extracts, and in vivo, PIN cDNA reduced the erectile response to electrical field stimulation. In conclusion, PIN and NMDAR colocalize with PnNOS in penile nerves, but the functional significance of these protein interactions for penile erection remains to be elucidated.     

The vasculature of the rat penis: a scanning electron microscopic and histologic study.

As a basis for understanding the mechanism of erection in an animal model frequently used in research in reproductive biology, the angioarchitecture of the penis of the rat has been described using scanning electron microscopy. Study of the penile vasculature of the rat indicates that the corpora cavernosa penis and the corpus spongiosum are independent erectile tissues, each with its own arterial and venous vessels. The large vascular spaces and abundant smooth muscle of the penile crura are compatible with its role in regulating blood flow to more distal penile tissues. Helicine arteries of the crura, but not the parent deep penile artery or arteries elsewhere, have muscular cushions in their walls. The venous drainage of the penile crura is via subtunical veins which are thought to be compressed during erection to elevate pressure within the penis. Large, paired cavernous veins drain the shaft of the penis. A unique method for inhibiting blood flow from the penis is indicated by the division of the cavernous veins into smaller channels prior to joining the subtunical venous plexus. Erectile tissue in the bifid origins of the corpus spongiosum has abundant cavernous muscle, while in the remainder of the corpus spongiosum little smooth muscle lines the cavernous spaces. The cavernous spaces on either side of the urethra coalesce to form vessels, each of which communicates with cavernous spaces in the glans. In addition, a bypass of the glans is effected by communication of these vessels directly with the deep dorsal vein. The apparent absence of muscular pads in vessels of the spongiosum, the relative paucity of cavernous smooth muscle, and the ample venous drainage provided by the deep dorsal vein may account for the lack of a venous occlusive mechanism similar to that of the corpora cavernosa penis.     

Antagonism of Rho-kinase stimulates rat penile erection via a nitric oxide-independent pathway

Relaxation of the smooth muscle cells in the cavernosal arterioles and sinuses results in increased blood flow into the penis, raising corpus cavernosum pressure to culminate in penile erection. Nitric oxide, released from non-adrenergic/non-cholinergic nerves, is considered the principle stimulator of cavernosal smooth muscle relaxation, however, the inhibition of vasoconstrictors (that is, norepinephrine and endothelin-1, refs. 5-9) cannot be ignored as a potential regulator of penile erection. The calcium-sensitizing rho-A/Rho-kinase pathway may play a synergistic role in cavernosal vasoconstriction to maintain penile flaccidity. Rho-kinase is known to inhibit myosin light chain phosphatase, and to directly phosphorylate myosin light-chain (in solution), altogether resulting in a net increase in activated myosin and the promotion of cellular contraction. Although Rho-kinase protein and mRNA have been detected in cavernosal tissue, the role of Rho-kinase in the regulation of cavernosal tone is unknown. Using pharmacologic antagonism (Y-27632, ref. 13, 18), we examined the role of Rho-kinase in cavernosal tone, based on the hypothesis that antagonism of Rho-kinase results in increased corpus cavernosum pressure, initiating the erectile response independently of nitric oxide. Our finding, that Rho-kinase antagonism stimulates rat penile erection independently of nitric oxide, introduces a potential alternate avenue for the treatment of erectile dysfunction.     

Neurturin is a neurotrophic factor for penile parasympathetic neurons in adult rat

Neurturin (NRTN), a member of the GDNF family of neurotrophic factors, promotes the survival and function of several neuronal populations in the peripheral and central nervous system. Recent gene ablation studies have shown that NRTN is a neurotrophic factor for many cranial parasympathetic and enteric neurons, whereas its significance for the sacral parasympathetic neurons has not been studied. NRTN signals via a receptor complex composed of the high-affinity binding receptor component GFRalpha2 and the transmembrane tyrosine kinase Ret. The aim of this study was to determine whether NRTN could be an endogenous trophic factor for penis-projecting parasympathetic neurons. NRTN mRNA was expressed in smooth muscle of penile blood vessels and corpus cavernosum in adult rat as well as in several intrapelvic organs, whereas GFRalpha2 and Ret mRNAs were expressed in virtually all cell bodies of the penile neurons, originating in the major pelvic ganglia. (125)I-NRTN injected into the shaft of the penis was retrogradely transported into the major pelvic and dorsal root ganglia. Mice lacking the GFRalpha2 receptor component had significantly less nitric oxide synthase-containing nerve fibers in the dorsal penile and cavernous nerves. In conclusion, these data suggest that NRTN acts as a target-derived survival and/or neuritogenic factor for penile erection-inducing postganglionic neurons.     

Androgen-dependent nitric oxide release in rat penis correlates with levels of constitutive nitric oxide synthase isoenzymes.

Androgens are known to influence penile erection and nitric oxide synthase (NOS) activity in cavernosal tissue homogenates. The present study was an assessment of the effects of castration and androgen replacement on the in vivo release of nitric oxide (NO), and of the simultaneously recorded intracavernosal pressure (ICP) changes elicited by electrostimulation of the cavernosal nerves (SCN) in the anesthetized rat. The extracellular levels of NO in the corpora were monitored electrochemically using porphyrin microsensors. The content of NOS isoenzymes in corporal homogenates was determined by immunoblotting. The responses of castrated rats with or without testosterone (T) implants were compared to those of intact animals. Castration virtually abolished both the NO and the ICP responses to SCN. There was a concomitant significant decrease in the content of both the neuronal (nNOS) and the endothelial (eNOS) isoenzymes in the cavernosal tissue. All these effects of castration were prevented by T replacement. The NO response to SCN was positively correlated with the levels of nNOS and eNOS, especially when the values of the two isoforms were added (r = 0.71, P < 0.001). These data suggest that the facilitatory action of androgens on penile erection involves the up-regulation of both constitutive NOS isoenzymes in the corpora cavernosa.     

Increased expression of arginase II in human diabetic corpus cavernosum: in diabetic-associated erectile dysfunction

Nitric oxide (NO) is the principal mediator of penile erection. NO is synthesized by nitric oxide synthase (NOS). It has been well documented that the major causative factor contributing to erectile dysfunction in diabetic patients is the reduction in the amount of NO synthesis in the corpora cavernosa of the penis resulting in alterations of normal penile homeostasis. Arginase is an enzyme that shares a common substrate with NOS, thus arginase may downregulate NO production by competing with NOS for this substrate, l-arginine. The purpose of the present study was to compare arginase gene expression, protein levels, and enzyme activity in diabetic human cavernosal tissue. When compared to normal human cavernosal tissue, diabetic corpus cavernosum from humans with erectile dysfunction had higher levels of arginase II protein, gene expression, and enzyme activity. In contrast, gene expression and protein levels of arginase I were not significantly different in diabetic cavernosal tissue when compared to control tissue. The reduced ability of diabetic tissue to convert l-arginine to l-citrulline via nitric oxide synthase was reversed by the selective inhibition of arginase by 2(S)-amino-6-boronohexanoic acid (ABH). These data suggest that the increased expression of arginase II in diabetic cavernosal tissue may contribute to the erectile dysfunction associated with this common disease process and may play a role in other manifestations of diabetic disease in which nitric oxide production is decreased.     

eNOS function and dysfunction in the penis

Endothelial nitric oxide (NO) synthase (eNOS) has an indispensable role in the erectile response. In the penis, eNOS activity and endothelial NO bioavailability are regulated by multiple post-translational molecular mechanisms, such as eNOS phosphorylation, eNOS interaction with regulatory proteins and contractile pathways, and actions of reactive oxygen species (ROS). These mechanisms regulate eNOS-mediated responses under physiologic circumstances and provide various mechanisms whereby endothelial NO availability may be altered in states of vasculogenic erectile dysfunction (ED). In view of the recent advances in the field of eNOS function in the penis and its role in penile erection, the emphasis in this review is placed on the mechanisms regulating eNOS activity and its interaction with the RhoA/Rho-kinase pathway in the physiology of penile erection and the pathophysiology of ED.     

Nitric oxide-dependent penile erection in mice lacking neuronal nitric oxide synthase.

BACKGROUND: Nitric oxide (NO) has been implicated as a mediator of penile erection, because the neuronal isoform of NO synthase (NOS) is localized to the penile innervation and NOS inhibitors selectively block erections. NO can also be formed by two other NOS isoforms derived from distinct genes, inducible NOS (iNOS) and endothelial NOS (eNOS). To clarify the source of NO in penile function, we have examined mice with targeted deletion of the nNOS gene (nNOS- mice). MATERIALS AND METHODS: Mating behavior, electrophysiologically induced penile erection, isolated erectile tissue isometric tension, and eNOS localization by immunohistochemistry and Western blot were performed on nNOS- mice and wild-type controls. RESULTS: Both intact animal penile erections and isolated erectile tissue function are maintained in nNOS mice, in agreement with demonstrated normal sexual behaviors, but is stereospecifically blocked by the NOS inhibitor, L-nitroarginine methyl ester (L-NAME). eNOS is abundantly present in endothelium of penile vasculature and sinusoidal endothelium within the corpora cavemosa, with levels that are significantly higher in nNOS- mice than in wild-type controls. CONCLUSIONS: eNOS mediates NO-dependent penile erection in nNOS- animals and normal penile erection. These data clarify the role of nitric oxide in penile erection and may have implications for therapeutic agents with selective effects on NOS isoforms.     

Immunoreactivities of eNOS,ET-1 and ETB-R in blood vessels of the uterine mesometrium during the estrous cycle in the pig

Cryostat sections of arterial and venous vessels from various size branches of the uterine artery and utero-ovarian vein of the pig mesometrium in different phases of the estrous cycle were stained immunohistochemically for endothelial nitric oxide synthase (eNOS), endothelin-1 (ET-1) and endothelin B receptor (ETB-R) using ABC method. Immunoreactivity was evaluated according to 6-point scale under light microscope. The differences in immunostaining intensity in the endothelium of the vessels studied at various levels of mesometrium suggest a correlation of eNOS, ET-1 and ETB-R expression with the estrous cycle. In the follicular phase, the highest eNOS immunoreactivity was noticed in arcuate arteries and veins, while immunoreaction of ET-1 was much lower, just as ETB-R. On the other hand, the highest ET-1 immunoreactivity was observed during first 2 days afterovulation, while ETB-R showed low immunoreactivity level during the whole luteal phase. Vessels from the middle part of mesometrium (I degrees and II degrees branches) and large vascular trunks revealed similar staining for eNOS during the cycle as compared to arcuate arteries. Those vessels showed very high immunoreactivity levels for ET-I and ETB-R during first 2 days after ovulation. Our results suggest that during the estrus eNOS, ET-I and ETB-R play a significant role in the regulatory process of blood flow through the mesometrial vessels, that are connected to the uterine horn.     

Cutaneous blood supply of the penis

Twelve male cadaver specimens were injected with a latex solution to define the cutaneous blood supply of the penis. The cutaneous blood supply of the penile shaft is derived solely from a pair of axial arteries running in the dartos layer. Additional deep perforating arteries from the dorsal penile artery and corporal vessels supply the glans and subcoronal region. An understanding of this anatomy allows one to develop safely a variety of penile skin flaps for difficult reconstructive problems.     

Study of neuropeptide Y-containing nerve fibers in the human penis

Summary Neuropeptide Y 1–36 (IR-NPY) immunoreactive nerve-fiber processes have been observed in tunicae of veins and arteries and in smooth muscles of the human penis taken at autopsy or during surgery by use of light-and electron-microscopic immunohistochemical techniques. Numerous IR-NPY nerve fibers were mostly concentrated in the inner part of the adventitia close to the media of the arterial and venous vessels and among the intracavernous smooth muscle cells. IR-NPY nerve fibers were less abundant in veins than in arteries. Positive somata were not observed in the penises. At the ultrastructural level, IR-NPY were localized exclusively in large, dense granules of nerve terminals by means of the postembedding immunogold technique. In the deep dorsal vein, IR-NPY nerve fibers were also located in the media formed by an outer circular and an inner longitudinal layer. In the intracavernous and dorsal arteries, they showed the highest density in the inner part of the adventitia. In the corpora cavernosa and in the corpus spongiosum, IR-NPY nerve processes were intermingled between the smooth-muscle fibers around the sinusoid spaces. IR-NPY nerve fibers were present in the cavernous nerves close to the central arteries. The urethra did not show any IR-NPY-positive nerve fibers. This peculiar distribution of IR-NPY nerve fibers suggested that they could participate in regulating arterial and venous blood flow and intracavernous smooth-muscle tone. NPY may therefore be of importance in some of the mechanisms of penile erection especially during detumescence.     

Expression of endothelial nitric oxide synthase in the vascular wall during arteriogenesis

Nitric oxide (NO) has been demonstrated to play an important role in angiogenesis, and also to be involved in collateral vessel growth. The expression of endothelial NO synthase (eNOS) is moderated partly by blood flow-induced mechanical factors, i.e., shear stress. The purpose of this study was to evaluate how the expression of eNOS correlates with the development of collateral vessels in dog heart, induced by chronic occlusion of the left circumflex artery. Immunoconfocal microscopy using an antibody against eNOS was used to detect expression of eNOS in different stages of arteriogenesis. Collateral vessels were classified into normal, growing and mature vessels by using the cytoskeleton marker desmin. Expression of the growth factors bFGF and metallproteinase-2 (MMP-2) was also examined. The data show that in normal arteriolar vessels, expression of eNOS is very low, but in growing collateral vessel there is a 6.2-fold increase, which, however, returned to normal levels in mature collateral vessels. The expression of eNOS was localized only in endothelium, either in normal or growing vessels. bFGF was very weakly stained in normal vessels, but highly expressed in growing collateral vessels. MMP-2 was strongly stained in neointima, but very weak in endothelium. In addition, we also examined expression of iNOS because iNOS may be induced in vessel injury or in disease states, but it was not detected in either normal or growing collateral vessels. Our findings indicate that the expression pattern of eNOS is closely associated with the development of collateral vessels, suggesting that eNOS plays an important role in arteriogenesis.     

Telmisartan Activates Endothelial Nitric Oxide Synthase via Ser1177 Phosphorylation in Vascular Endothelial Cells

Because endothelial nitric oxide synthase (eNOS) has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177) in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172) and eNOS and the concentration of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP). Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.     

CGRP immunoreactivity and NADPH-diaphorase in afferent nerves of the rat penis

Calcitonin gene-related peptide (CGRP)-immunoreactive afferent nerve fibers are abundant in the rat penis. In addition, NADPH-diaphorase, which stains for nitric oxide synthase, has been localized within both autonomic and sensory dorsal root ganglia (DRG) and may be part of an important biochemical pathway involved in penile tumescence. The purpose of this study was: 1) to examine the circuitry of afferent nerves that are CGRP immunoreactive from the L6 DRG, 2) to examine the possibility that there are NADPH-diaphorase-positive afferent fibers from the L6 DRG to the rat penis, and 3) to examine the localization and colocalization of CGRP and NADPH-diaphorase within L6 DRG afferent perikarya. Calcitonin gene-related peptide immunostaining in the penis was eliminated following a bilateral transection of the pudendal nerves, but was unchanged following a bilateral transection of the pelvic splanchnic or hypogastric nerves. The NADPH-diaphorase staining was not altered by any of the nerve transections. Injection of the retrograde axonal tracer fluorogold (FG) into the dorsum penis labeled perikarya in the L6 DRG. Although the majority of FG-labeled perikarya contained neither CGRP nor NADPH-diaphorase, small subpopulations of perikarya contained either CGRP immunoreactivity, NADPH-diaphorase, or both. A unilateral pudendal nerve transection virtually eliminated (>99%) FG labeling in the ipsilateral L6 DRG. These data suggest that NADPH-diaphorase and CGRP are present, either together or separately, within a subpopulation of penile afferent perikarya. In addition, CGRP-immunoreactive afferent nerve fibers reach the penis primarily via the pudendal nerves. Finally, NADPH-diaphorase-positive penile afferents may be another important source of nitric oxide (NO) for penile tumescence.     

Expression of endothelial nitric oxide synthase in the vascular wall during arteriogenesis

Nitric oxide (NO) has been demonstrated to play an important role in angiogenesis, and also to be involved in collateral vessel growth. The expression of endothelial NO synthase (eNOS) is moderated partly by blood flow-induced mechanical factors, i.e., shear stress. The purpose of this study was to evaluate how the expression of eNOS correlates with the development of collateral vessels in dog heart, induced by chronic occlusion of the left circumflex artery. Immunoconfocal microscopy using an antibody against eNOS was used to detect expression of eNOS in different stages of arteriogenesis. Collateral vessels were classified into normal, growing and mature vessels by using the cytoskeleton marker desmin. Expression of the growth factors bFGF and metallproteinase-2 (MMP-2) was also examined. The data show that in normal arteriolar vessels, expression of eNOS is very low, but in growing collateral vessel there is a 6.2-fold increase, which, however, returned to normal levels in mature collateral vessels. The expression of eNOS was localized only in endothelium, either in normal or growing vessels. bFGF was very weakly stained in normal vessels, but highly expressed in growing collateral vessels. MMP-2 was strongly stained in neointima, but very weak in endothelium. In addition, we also examined expression of iNOS because iNOS may be induced in vessel injury or in disease states, but it was not detected in either normal or growing collateral vessels. Our findings indicate that the expression pattern of eNOS is closely associated with the development of collateral vessels, suggesting that eNOS plays an important role in arteriogenesis. (Mol Cell Biochem 264: 193–200, 2004)     

Immunohistochemical analysis of smooth muscle cells and volumetric density of the elastic system fibers of wild boar (Sus scrofa) penis

The purpose of the present study was to verify the smooth muscle cell distribution and elastic system fibers volumetric density (Vv) in the corpus spongiosum and corpus cavernosum of the wild boar penis. Adult wild boars (n=13) were used. The penile mid shaft fragments were fixed with 4% phosphate buffered formalin solution and/or Bouin's liquid during 24-48 h, and processed using standard histological techniques. The sections were stained with Weigert's Resorcin-Fucsin with previous oxidation. The elastic system fibers Vv was determined in 25 random fields of each fragment using M42 test system. For immunohistochemical analysis, monoclonal anti-alpha actin smooth muscle was used. The histochemical methods detected elastic system fibers in both corpus spongiosum and corpus cavernosum of all animals. The elastic fibers Vv average was 36.6%+/-0.9 for corpus spongiosum and 11.7%+/-0.5 for corpus cavernosum. Through immunocitochemistry, a small quantity of smooth muscle cells was visualized in intimate relationship with blood vessels wall. The great amount of elastic fibers and the smooth muscle cell distribution beneath the endothelium suggest that these fibers may have an important role in penile erection process in the penis of wild boars.