Preclinical effects of melanocortins in male sexual dysfunction.

The neurobiology of sexual behavior involves the interrelationships between sex steroids and neurotransmitters that result in both central nervous system (CNS) effects and effects in the genitalia. Tools such as positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) scanning can help determine what areas of the brain are activated under sexual stimulation. Our understanding of the role of various neurotransmitters, neurosteroids and other CNS-acting compounds is improving. The role of CNS-acting compounds such as dopamine agonists in the treatment of male sexual dysfunction is under active investigation. Melanocortins have CNS and peripheral roles in a wide variety of bodily functions. The melanocortin agonist bremelanotide appears to act in the CNS to promote erections in preclinical models, and may also stimulate behaviors that facilitate sexual activity beyond their erectogenic effects.

Evaluation of the safety, pharmacokinetics and pharmacodynamic effects of subcutaneously administered PT-141, a melanocortin receptor agonist, in healthy male subjects and in patients with an inadequate response to Viagra.

PT-141, a cyclic heptapeptide melanocortin analog, was evaluated following subcutaneous administration to healthy male subjects and to patients with erectile dysfunction (ED) who report an inadequate response to Viagra. An inadequate response was defined for this study by patient report indicating that achievement of an erection suitable for vaginal penetration occurred < or =50% of the time while taking 100 mg Viagra. Erectile responses were assessed by RigiScan in healthy subjects in the absence of visual sexual stimulation (VSS) and in ED patients in the presence of VSS. Doses ranging from 0.3 to 10 mg were administered to healthy male subjects, resulting in a statistically significant erectile response at doses greater than 1.0 mg. ED patients were treated with placebo, 4 or 6 mg PT-141 in a crossover design in the presence of VSS. The erectile response induced by PT-141 was statistically significant at both doses. PT-141 was safe and well tolerated in both studies. The erectogenic potential of PT-141, its tolerability profile and its ability to cause significant erections in patients who do not have an adequate response to a PDE5 inhibitor suggest that PT-141 may provide an alternative treatment for ED with a potentially broad patient base.

PT-141: a melanocortin agonist for the treatment of sexual dysfunction.

PT-141, a synthetic peptide analogue of alpha-MSH, is an agonist at melanocortin receptors including the MC3R and MC4R, which are expressed primarily in the central nervous system. Administration of PT-141 to rats and nonhuman primates results in penile erections. Systemic administration of PT-141 to rats activates neurons in the hypothalamus as shown by an increase in c-Fos immunoreactivity. Neurons in the same region of the central nervous system take up pseudorabies virus injected into the corpus cavernosum of the rat penis. Administration of PT-141 to normal men and to patients with erectile dysfunction resulted in a rapid dose-dependent increase in erectile activity. The results suggest that PT-141 holds promise as a new treatment for sexual dysfunction.

Nerve growth factor antiserum induces axotomy-like changes in neuropeptide expression in intact sympathetic and sensory neurons.

Axonal transection of adult sympathetic and sensory neurons leads to a decrease in their content of target-derived nerve growth factor (NGF) and to dramatic changes in the expression of several neuropeptides and enzymes involved in transmitter biosynthesis. For example, axotomy of sympathetic neurons in the superior cervical ganglion (SCG) dramatically increases levels of galanin, vasoactive intestinal peptide (VIP), and substance P and their respective mRNAs and decreases mRNA levels for neuropeptide Y (NPY) and tyrosine hydroxylase (TH). Axotomy of sensory neurons in lumbar dorsal root ganglia (DRG) increases protein and mRNA levels for galanin and VIP and decreases levels for substance P and calcitonin gene-related peptide (CGRP). To assess whether reduction in the availability of endogenous NGF might play an important role in triggering these changes, we injected nonoperated animals with an antiserum against NGF (alphaNGF). alphaNGF increased levels of peptide and mRNA for galanin and VIP in neurons in both the SCG and DRG. NPY protein and mRNA were decreased in the SCG, but levels of TH protein and mRNA remained unchanged. In sensory neurons the levels of SP and CGRP protein decreased after alphaNGF treatment. These data suggest that the reduction in levels of NGF in sympathetic and sensory neurons after axotomy is partly responsible for the subsequent changes in neuropeptide expression. Thus, the peptide phenotype of these axotomized neurons is regulated both by the induction of an "injury factor," leukemia inhibitory factor, as shown previously, and by the reduction in a target-derived growth factor.

Nerve growth factor inhibits sympathetic neurons' response to an injury cytokine.

Axonal damage to adult peripheral neurons causes changes in neuronal gene expression. For example, axotomized sympathetic, sensory, and motor neurons begin to express galanin mRNA and protein, and recent evidence suggests that galanin plays a role in peripheral nerve regeneration. Previous studies in sympathetic and sensory neurons have established that galanin expression is triggered by two consequences of nerve transection: the induction of leukemia inhibitory factor (LIF) and the reduction in the availability of the target-derived factor, nerve growth factor. It is shown in the present study that no stimulation of galanin expression occurs following direct application of LIF to intact neurons in the superior cervical sympathetic ganglion. Injection of animals with an antiserum to nerve growth factor concomitant with the application of LIF, on the other hand, does stimulate galanin expression. The data suggest that the response of neurons to an injury factor, LIF, is affected by whether the neurons still receive trophic signals from their targets.

Galanin induced in sympathetic neurons after axotomy is anterogradely transported toward regenerating nerve endings.

Peripheral neurons begin to express galanin after axotomy. When neurons in the superior cervical ganglion were axotomized near (about 2 mm) from the ganglion, galanin-like immunoreactivity (IR) was maximal within 72 h. Axotomy of neurons in the middle and inferior cervical ganglion complex (MICG), which could be performed 2 cm from the ganglia, led to an additional galanin increase 7 and 14 days later. This second increase was not accompanied by changes in galanin mRNA or the number of galanin-immunostained neurons. Galanin-IR was detectable in a postganglionic trunk of the MICG 2 days after axotomy. At this time, immunoreactive fibers were only seen near the lesion site, while later they were found throughout the trunk. The data suggest that galanin is actively transported toward the site of nerve crush/transection and that the second increase in galanin-IR found in the MICG may be due to a saturation of the axonal transport system.

Changes in neuropeptide phenotype after axotomy of adult peripheral neurons and the role of leukemia inhibitory factor.

Adult peripheral neurons undergo dramatic shifts in gene expression following axotomy that are collectively referred to as the cell body reaction. Changes in neuropeptide expression are a prominent feature of these axotomized neurons. For example, while sympathetic, sensory, and motor neurons do not normally express the neuropeptides galanin and vasoactive intestinal peptide, they begin to do so within days after axotomy. In contrast, the expression of other peptides, which these neurons normally express, such as neuropeptide Y in sympathetic neurons and substance P in sensory neurons, is decreased. Recent studies in sympathetic neurons have demonstrated that leukemia inhibitory factor plays an important role in triggering these changes in neuropeptide phenotype in adult neurons. Future studies will be directed at determining to what extent LIF triggers the many other changes in gene expression after sympathetic axotomy and whether this cytokine plays a similar role in sensory and motor neurons.

Changes in the macrophage population of the rat superior cervical ganglion after postganglionic nerve injury.

Following peripheral nerve transection, a series of biochemical changes occurs in axons and Schwann cells both at the site of the lesion and distal to it. Macrophages differentiated from monocytes that invade the area in response to transection (elicited macrophages) and, perhaps, also macrophages normally present in the tissue (resident macrophages) play important roles in these changes. In addition, nerve transection produces changes in the cell bodies of axotomized neurons and their surrounding glial cells, located at some distance from the lesion. To determine whether macrophages might play a role in the changes occurring in the superior cervical ganglion (SCG) after axotomy, we examined the presence of macrophages before and after axonal damage. The monoclonal antibodies ED1, ED2, and OX6 were used, each of which recognizes a somewhat different population of macrophages. Ganglia from normal rats contained a population of resident cells that were ED2+ but very few that were ED1+. Within 2 days after the post-ganglionic nerves were transected, the number of ED1+ cells increased substantially, with little change in immunostaining for ED2. These data, in combination with published studies on other tissues, suggest that ED1 in the SCG is selective for elicited macrophages and ED2 for resident macrophages. OX6 immunostaining was prominent in normal ganglia but also increased significantly after axotomy, suggesting that it reflects both macrophage populations. Systemic administration of 6-hydroxydopamine, a neurotoxin that causes the destruction of sympathetic nerve endings, also produced an increase in ED1 immunostaining. Thus, the change in ED1 immunostaining in the SCG does not require surgery, with the attendant severing of local blood vessels and connective tissue, but rather only the disconnection of sympathetic neurons from their end organs. The time course of the invasion of monocytes after axotomy indicates that this process is not required to trigger the biochemical changes occurring in the ganglion within the first 24 h. On the other hand, the existence of a resident population of macrophages raises the possibility that changes in those cells might be involved.

Galanin expression in sympathetic ganglia after partial axotomy is highly localized to those neurons that are axotomized.

The neuropeptide phenotype of adult sympathetic neurons changes dramatically after postganglionic nerve transection. Studies, thus far, have been done on the superior cervical ganglion; however, one limitation of this preparation is that it is necessary to transect the postganglionic axons quite close to the ganglion. In the present study, we examined the effects of axonal damage on galanin-like immunoreactivity in the middle and inferior cervical ganglion complex. With these ganglia, it is possible to transect postganglionic axons at a considerable distance from their cell bodies and, therefore, to examine the extent to which local tissue damage, rather than specific axonal transection, is required for these changes in neuropeptide phenotype to occur. The anatomy of this system also allowed us to determine the extent to which the changes in galanin expression are restricted to those neurons that have been axotomized. The axons of a small population of the neurons in the middle and inferior cervical ganglia complex project into the cervical sympathetic trunk. Within two days after this trunk was transected, there was an increase in the level of galanin-like immunoreactivity in the complex and in the number of immunostained principal neurons. These neurons were concentrated primarily in the most rostral part of the complex. An increase in galanin-like immunoreactivity also occurred in response to the systemic administration of the sympathetic neurotoxin 6-hydroxydopamine. In that case, many more neurons were affected than after transection of the cervical sympathetic trunk, and the neurons were distributed evenly throughout the ganglion complex.(ABSTRACT TRUNCATED AT 250 WORDS)

The interleukin-1-induced increase of substance P in sympathetic ganglia is not mediated by ciliary neurotrophic factor.

Interleukin-1 (IL-1) induction of substance P (SP) in cultured sympathetic ganglia requires a soluble intermediate molecule that is present in IL-1 conditioned medium (IL-1CM). One of the required intermediates is leukemia inhibitory factor (LIF; Shadiack et al., J Neurosci 13:2601-2609, 1993). In the present study we have examined the possibility that ciliary neurotrophic factor (CNTF) is another intermediate involved in the IL-1 induction of sympathetic SP. CNTF mimics the action of IL-1CM by raising both SP and choline acetyltransferase activity--actions that are blocked by a specific neutralizing antiserum for CNTF. However, IL-1CM and CNTF differ in their response to depolarizing agents: while KCl (40 mM) blocks the action of IL-1CM (and LIF), it enhances the action of CNTF. Furthermore, neither CNTF bioactivity nor CNTF protein is detected in IL-1CM. Neutralizing antiserum to CNTF fails to block the action of either IL-1 or IL-1CM, suggesting that neither a soluble nor a membrane-bound form of the molecule is active in direct response to IL-1 action. While Northern blots confirm the presence of both CNTF and CNTF receptor mRNA in neonatal ganglia, neither culturing nor IL-1 treatment alters these mRNA levels. These data taken together suggest that while CNTF is present and possibly active in sympathetic ganglia, it is not a mediator of the IL-1 induction of SP.

Lipopolysaccharide induces substance P in sympathetic ganglia via ganglionic interleukin-1 production.

The immune cytokine interleukin-1 (IL-1) causes a pronounced elevation in substance P (SP) immunoreactivity and the mRNA coding for its preprotachykinin precursor in cultured superior cervical (sympathetic) ganglia (SCG; Jonakait and Schotland, 1990; Freidin and Kessler, 1991; Hart et al., 1991). In this study we have investigated the possibility that the SCG can respond to other immune stimulators, notably lipopolysaccharide (LPS), a product of bacterial cell walls. LPS treatment of cultured SCG resulted in a dose-dependent increase in SP. However, LPS did not induce SP in the absence of non-neuronal cells, suggesting the necessity of a non-neuronal cell-derived intermediate. Since the LPS induction of SP was partially blocked by a specific IL-1 receptor antagonist (IL-1ra) and since LPS induced approximately an 8-fold increase in mRNA coding for IL-1 itself, we concluded that IL-1 is at least one of these LPS-induced intermediates. TNF-alpha, which also raises SP levels, may be another. IL-6, which may also be increased by LPS, does not increase levels of SP. The synthetic glucocorticoid hormone dexamethasone (DEX) blocks the LPS induction of SP with a Ki approximating 8 x 10(-11) M. The inhibition is due in part to the blockade of the LPS induction of ganglionic IL-1 mRNA. Moreover, inhibition of the LPS induction of SP by indomethacin implies mediation of the effect through prostaglandins. The inhibition by indomethacin suggests a non-monocytic cell source since prostaglandins are thought to restrict the LPS induction of monocytic IL-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 induces substance P in sympathetic ganglia through the induction of leukemia inhibitory factor (LIF).

It has become increasingly clear that immune cytokines perform growth and differentiation functions in the nervous system similar to those performed in the immune system. In previous studies we have shown that interleukin-1 beta (IL-1 beta) raises substance P (SP) and the mRNA coding for its preprotachykinin precursor in cultured sympathetic superior cervical ganglia (SCG) (Jonakait and Schotland, 1990; Hart et al., 1991a). The action of IL-1 is blocked both by depolarization of the ganglia and by glucocorticoid hormones (Hart et al., 1991a). In the present report, we have found that IL-1 does not act directly upon neurons to raise SP, but rather induces the production of a soluble intermediate molecule that raises both SP and the cholinergic-specific enzyme ChAT. Its induction by IL-1 is blocked by the synthetic glucocorticoid hormone dexamethasone; its action is compromised under depolarizing conditions. Because medium conditioned by IL-1 (IL-1CM) is functionally similar to leukemia inhibitory factor (LIF), we sought to determine whether this molecule might be an active constituent of IL-1CM. Immunoprecipitation with an antiserum directed against LIF eliminated large proportions of SP-inducing activity from IL-1CM. In addition, steady-state levels of mRNA coding for LIF are increased by IL-1 treatment of SCG. These data suggest that LIF, induced by IL-1, may ultimately be responsible for the IL-1 induction of SP.

An mRNA homologous to interleukin-1 receptor type I is expressed in cultured rat sympathetic ganglia.

Interleukin-1 (IL-1) induces substance P (SP) gene expression in cultured rat superior cervical ganglion (SCG) explants. In order to study the molecular mechanism of this action of IL-1, the presence of an interleukin-1 receptor (IL-1R) activity and the identity of an mRNA homologous to known IL-1R sequence was determined in SCG. The SP increase is blocked by recombinant IL-1 receptor antagonist protein, so IL-1 must be interacting with a specific receptor. We have cloned cDNA homologous to IL-1R type I from rat SCG using a reverse transcription-polymerase chain reaction (RT-PCR). The resulting cDNA sequence is strongly homologous with mouse and human IL-1R cDNA of the T cell and fibroblast type (type I; encoding an 80-kDa protein). mRNA specific for IL-1R can be readily detected in intact SCG by quantitative RT-PCR and S1 hybridization. However, the level of IL-1R mRNA increases 3-6-fold by 2 days in culture. This increase is independent of the presence of dexamethasone, IL-1 beta or IL-1 receptor antagonist protein ligands. The increase of IL-1R following explantation, a model of nerve injury, may provide a mechanism linking inflammatory signalling to neuronal phenotypic changes.

Agrin induces alpha-actinin, filamin, and vinculin to co-localize with AChR clusters on cultured chick myotubes.

Agrin induces discrete high-density patches of acetylcholine receptors (AChRs) and other synaptic components on cultured myotubes in a manner that resembles synaptic differentiation. Furthermore, agrin-like molecules are present at developing neuromuscular junctions in vivo. This provides us with a unique opportunity to manipulate AChR patching in order to examine the role of cytoskeletal components. Cultured chick myotubes were fixed and labeled to visualize the distributions of actin, alpha-actinin, filamin, tropomyosin, and vinculin. Overnight exposure to agrin caused a small amount of alpha-actinin, filamin, and vinculin to reorganize into discrete clusters. Double-labeling studies revealed that 78% of the AChR clusters were associated with detectable concentrations of filamin, 70% with alpha-actinin, and 58% with vinculin. Filamin even showed congruence to AChRs within clustered regions. By contrast, actin (visualized with fluorescein-phalloidin) and tropomyosin did not show specific associations with agrin-induced AChR clusters. The accumulation of cytoskeletal components at AChRs clusters raised the possibility that cytoskeletal rearrangements direct AChR clustering. However, a time course of agrin-induced clustering that focused on filamin revealed that most of the early AChR clusters (3-6 h) were not associated with detectable amounts of cytoskeletal material. The accumulation of cytoskeletal material at later times (12-18 h) may imply a role in maintenance and stabilization, but it appears unlikely that these cytoskeletal elements initiate AChR clustering on myotubes.

Substance P gene expression is regulated by interleukin-1 in cultured sympathetic ganglia.

We have investigated the effects of interleukin-1 beta (IL-1 beta) on the induction of substance P (SP) in cultured sympathetic ganglia. Northern blot analysis reveals that SP increases are secondary to an increase in mRNA coding for the preprotachykinin (PPT) precursor of SP. Nuclear transcription assays detect an early increase in PPT-specific nascent transcripts, suggesting that the ultimate effect of IL-1 is on transcription itself. Depolarizing agents, interferon-gamma, glucocorticoid hormones, and prostaglandin synthesis inhibitors all diminish the induction of SP and PPT mRNA by IL-1. Since SP has stimulatory effects on the immune system, the IL-1-induced increase in ganglionic SP may be one means by which the nervous and immune systems interact during an acute response to ganglionic injury.