Expression and distribution of key proteins of the endocannabinoid system in the human seminal vesicles.

The endocannabinoid system (ECS), comprising the cannabinoid receptors (CBR), their ligands, and enzymes controlling the turnover of endocannabinoids, has been suggested to be involved in male reproductive function. As information is scarce on the expression of the ECS in human male reproductive tissues, this study aimed to investigate by means of molecular biology (RT-PCR) and immunohistochemistry/immunofluorescence the expression and distribution of CB1 and CB2, GPR55 (an orphan G protein-coupled receptor that recognises cannabinoid ligands) and FAAH (isoforms 1 and 2) in the human seminal vesicles (SV). The specimens expressed PCR products corresponding to CB1 (66 bp), CB2 (141 bp), GPR55 (112 bp), FAAH1 (260 bp) and FAAH2 (387 bp). Immumohistochemistry revealed dense expression of CB1, CB2 and GPR55 located to the pseudo-stratified columnar epithelium and varicose nerves (also characterised by the expression of vasoactive intestinal polypeptide and calcitonin gene-related peptide). Cytosolic staining for FAAH1 and FAAH2 was seen in cuboidal cells of all layers of the epithelium. No immunoreactivity was detected in the smooth musculature or nerve fibres. CB1, CB2, GPR55, FAAH1 and FAAH2 are highly expressed in the human SV. Considering their localisation, the ECS may be involved in epithelial homeostasis, secretory function or autonomic mechano-afferent signalling.

Pelvic nerve injury negatively impacts female genital blood flow and induces vaginal fibrosis-implications for human nerve-sparing radical hysterectomy.

OBJECTIVE: This study sought to develop a novel animal model to study the impact of nerve-sparing radical hysterectomy (NSRH) on female genital blood flow. DESIGN: In vivo animal study. POPULATION: Thirty Sprague-Dawley female rats. MATERIALS AND METHODS: Female rats underwent either unilateral pelvic nerve (PN) crush (PNC; n = 9), or crush of both the PNs and all efferent nerves in the pelvic plexus ('clock-nerve crush', CNC; n = 9). Under anaesthesia, we electrically stimulated the crushed PN at 3 and 10 days after crush while monitoring blood pressure and recording clitoral and vaginal blood flows by laser Doppler. Uninjured PNs were stimulated as an internal control. Twelve additional rats were assigned either to bilateral PNC or sham surgery, and genital tissues were processed 10 days after injury for in vitro analysis. MAIN OUTCOME MEASURES: Genital blood flow, nNOS, eNOS, collagen I-III. RESULTS: Stimulation of the crushed PN in both groups subjected to PNC and CNC induced significantly lower peak genital blood flow at 3 and 10 days (P < 0.05) compared to stimulation of the non-crushed control PN. The immunofluorescence and Western blot analyses revealed that all injured rats exhibited more vaginal collagen III and collagen I than rats did that ad undergone sham surgeries (P < 0.05). PCN reduced nNOS expression in both clitoral and vaginal tissue. CONCLUSIONS: Based on our study it may be hypothesised that NSRH might cause reductions of genital blood flow and vaginal fibrosis due to neurapraxia of the pelvic nerve and reductions of nNOS nerve fibres in clitoral and distal vaginal tissue. TWEETABLE ABSTRACT: Pelvic nerve neurapraxia during nerve-sparing radical hysterectomy could lead to sexual arousal dysfunction.

C-kit-positive multipolar cells in human penile erectile tissue: expression of connexin 43 and relation to trabecular smooth muscle cells.

The aim of this study was to evaluate the hypothesis that interstitial cells might play a role in controlling and synchronizing via gap junctions the electrical activity of smooth muscle cells. The expression and distribution of interstitial cells in human penile erectile tissue was evaluated to determine whether or not cavernous interstitial cells express the gap junction protein connexin 43. Specimens of human corpus cavernosum were excised from full preparations of human penises. Cryostat sections (10 microm to 15 microm) of formaldehyde-fixated tissue segments were incubated using a double-labelling technique with antibodies directed against smooth muscle alpha-actin, c-kit, and connexin 43. Then, sections were exposed to secondary antibodies. Visualization was commenced by means of laser fluorescence microscopy. Double-staining techniques revealed immunosignals specific for c-kit (transmembrane receptor protein) and connexin 43 (gap junction protein) in multipolar cells located adjacent to smooth muscle cells. The number of c-kit-positive cells was significantly lower within the smooth musculature than within bundles of connective tissue surrounding smooth muscle cells of corpus cavernosum or cavernous arteries. Our findings demonstrate the distribution of c-kit- and connexin 43-positive interstitial cells in the connective tissue and smooth musculature of the corpus cavernosum. Additional studies are needed in order to evaluate further the ultrastructure of human penile erectile tissue and enable the identification of gap junctions mediating direct cell-to-cell communication.

Expression and distribution of the transient receptor potential cationic channel A1 (TRPA1) in the human clitoris-comparison to male penile erectile tissue.

The transient receptor potential cationic channel ankyrin 1 (TRPA1) is a channel protein assumed to act in various human tissues as mechano- and pain sensor and play a role in neurotransmission. The expression of TRPA has already been investigated in the human prostate and urethra, however, only very few studies have addressed the expression and distribution in the male and female genital tract. The present study aimed to investigate by means of immunohistochemistry (double-labeling technique, laser fluorescence microscopy) in the human clitoris and penile erectile tissue the localization of TRPA1 in relation to nNOS, the vasoactive intestinal polypeptide (VIP) and vesicular acetylcholine transporter (VAChT). In the clitoral tissue, TRPA1 was observed in basal epithelial cells and slender nNOS-positive nerve fibers transversing the subepithelial space. To a certain degree, in the clitoral epithelial cells, TRPA1 was found co-localized with vimentin. In human corpus cavernosum, immunoreactivity for TRPA1 was seen in nerves transversing the cavernous sinusoidal space and running alongside small arteries, these nerves also displayed expression of the vesicular acetylcholine transporter protein (VAChT). Varicose nerves containing nNOS or VIP were not immunoreactive for TRPA1. It seems likely that TRPA1 is involved in nitric oxide-mediated afferent sensory transmission in the clitoris while, in penile erectile tissue, a role for TRPA1 in cholinergic signaling might be assumed.

Protein kinase enzymes in the human vagina-relation to key mediators of the cyclic AMP and cyclic GMP pathways.

Aside from phosphodiesterase (PDE) isoenzymes, protein kinases (cAK=cyclic AMP-binding protein kinase, cGK=cyclic GMP-binding protein kinase) have also been identified as important receptors for cyclic nucleotides. A significance of protein kinases in the control of the function of the male and female reproductive tract has been suggested; however, up until today, only a few approaches have addressed these enzymes in female genital tissues. The present study aimed to investigate by means of biochemical and immunohistochemical methods the expression of cAK and cGK. The distribution of cAK(I) and cGK(I) in relation to the vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and PDE type 4 (PDE4) was also evaluated. Cytosolic supernatants prepared from specimens of vaginal wall smooth muscle or epithelium were subjected to anion exchange chromatography and the activities of cAK and cGK(I) measured. To evaluate the distribution of cAK(I) and cGK(I) in relation to VIP, CGRP and PDE4, immunohistochemistry was conducted in sections of the human vaginal wall (full-wall specimens). Activities representing cGK(I) and cAK(I) were resolved from the chromatography column. Staining specific for cAK(Iα) was identified in both vascular and non-vascular vaginal smooth musculature, immunoreactivity for cGK(Iß) was observed in the smooth muscle and endothelium of small arteries interspersing the sections. cAK(Iα)-positive vessels were found innervated by slender varicose nerve fibers presenting the expression of VIP and CGRP. These arteries also expressed PDE4. Localization of cAK and cGK in close relation to key mediators of the cyclic AMP (PDE4, VIP) and cyclic GMP (CGRP) pathways indicate that both signaling systems may synergistically work together in human vaginal tissue.

Muscarinic stimulation of the rat isolated whole bladder: pathophysiological models of detrusor overactivity.

1 Hypotheses as to the pathophysiological basis of bladder detrusor muscle overactivity (DO) have identified both central nervous and peripheral mechanisms as likely contributory factors. In this paper, we describe peripheral autonomous bladder activity in two animal models of DO and discuss how the differences observed between the two models support the likelihood that clinical DO has a multifactorial basis. 2 A total of 12 adult female Sprague-Dawley rats underwent obstruction or sham operation for 1 or 4 weeks. Six adult female spontaneously hypertensive rats (SHR) were compared with normal Wistar controls. Bladders were microsurgically removed and mounted in whole organ tissue baths. Recordings of intravesical pressure in response to the muscarinic receptor agonist arecaidine were performed under standardized conditions. 3 In the partially obstructed rat bladder, the amplitude of pressure fluctuations elicited by the muscarinic agonist arecaidine was significantly increased compared with sham-operated animals. The tonic component of the response was no different for the two groups. No difference from controls was apparent in the SHR. 4 We conclude that alterations in autonomous bladder activity in the obstructed rat model suggest that peripheral functional changes contribute to the pathophysiological abnormality. In contrast, the fundamental abnormality in the SHR appears to be at a more central level. The observations support the supposition that lesions at widely separate sites can give rise to apparently similar abnormalities of lower urinary tract function.

Expression and distribution of the transient receptor potential cationic channel ankyrin 1 (TRPA1) in the human vagina.

The transient receptor potential cationic channel type A1 (TRPA1), belonging to a superfamily of cationic membrane channels, has been suggested to act as mechano- and pain sensor and, thus, to play a role in neurotransmission in the human body, including the urogenital tract. While the expression of TRPA1 has been investigated in a variety of tissues, up until today, no study has addressed the expression and distribution in the female genital tract. The present study aimed to investigate the expression and distribution of TRPA1 protein in human vaginal tissue. Reverse transcriptase PCR (RT-PCR) was applied in order to identify messenger ribonuleic acid specifically encoding for TRPA/A1. The distribution of TRPA1 in relation to the neuronal nitric oxide synthase (nNOS) and the signaling peptide calcitonin gene-related peptide (CGRP) was examined by means of immunohistochemical methods (double-antibody technique, laser fluorescence microscopy). RT-PCR analysis revealed the expression of mRNA encoding sequences specific for TRPA in the vaginal wall and epithelium. Immunostaining related to TRPA1 was observed in the basal epithelium and in slender varicose nerve fibers transversing the subepithelial and stromal space of the vaginal sections. In addition, these fibers presented immunoreactivity specific for nNOS or CGRP. The smooth musculature of the vaginal wall and small vessels interspersing the tissue did not present signals related to TRPA1. The findings indicate that TRPA1 might be involved in afferent neurotransmission in the vagina and work synergistically together with the nitric oxide/cyclic guanosine monophosphate pathway.

Receptor-receptor interactions as an integrative mechanism in nerve cells.

Several lines of evidence indicate that interactions among transmission lines can take place at the level of the cell membrane via interactions among macromolecules, integral or associated to the cell membrane, involved in signal recognition and transduction. The present view will focus on this last subject, i.e., on the interactions between receptors for chemical signals at the level of the neuronal membrane (receptor-receptor interaction). By receptor-receptor interaction we mean that a neurotransmitter or modulator, by binding to its receptor, modifies the characteristics of the receptor for another transmitter or modulator. Four types of interactions among transmission lines may be considered, but mainly intramembrane receptor-receptor interactions have been dealt with in this article, exemplified by the heteroregulation of D2 receptors via neuropeptide receptors and A2 receptors. The role of receptor-receptor interactions in the integration of signals is discussed, especially in terms of filtration of incoming signals, of integration of coincident signals, and of neuronal plasticity.

Radioligand binding analysis of knockout mice reveals 5-hydroxytryptamine(7) receptor distribution and uncovers 8-hydroxy-2-(di-n-propylamino)tetralin interaction with alpha(2) adrenergic receptors.

In the present autoradiographic study, we took advantage of 5-hydroxytryptamine(7) (5-HT(7)) receptor knockout mice to analyze the brain distribution of 5-HT(7) receptor binding sites using [(3)H]5-carboxamidotryptamine (5-CT; a 5-HT(1A/1B/1D/5/7) receptor ligand) and [(3)H]8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; a 5-HT(1A/7) receptor ligand). Low to moderate densities of [(3)H]5-CT (2 nM) binding sites insensitive to pindolol (10 microM, for 5-HT(1A/1B) receptor blockade) and GR-127935 (1 microM; for 5-HT(1D) receptor blockade) were observed in wild-type mice (mainly in thalamus and hypothalamus) but not in 5-HT(7) receptor knockout mice. Surprisingly, moderate to high densities of [(3)H]8-OH-DPAT (10 nM) binding sites insensitive to pindolol (10 microM) remained in 5-HT(7) receptor knockout mouse brain. These non-5-HT(1A), non-5-HT(7) binding sites were found to be adrenergic alpha(2A) receptor binding sites. In alpha(2A) receptor knockout mice low to moderate densities of [(3)H]8-OH-DPAT binding sites insensitive to pindolol but sensitive to the selective 5-HT(7) receptor antagonist SB-269970 (300 nM) were observed mainly in thalamus and hypothalamus. Therefore, in addition to 5-HT(1A) and 5-HT(7) binding sites, [(3)H]8-OH-DPAT also binds to alpha(2A) receptor binding sites in wild-type mouse brain. [(3)H]8-OH-DPAT (in the presence of pindolol and 1 microM RX-821002 for alpha(2) receptor blockade) and [(3)H]5-CT (in the presence of pindolol and GR-127935) bind to a similar receptor binding population corresponding to 5-HT(7) binding sites. Detailed anatomical mapping of 5-HT(7) receptor binding sites in wild-type mouse brain was then performed using both radioligands in the presence of suitable pharmacological agents for non-5-HT(7) receptor binding sites blockade. The mapping revealed binding sites consistent with the mRNA distribution with the highest densities found in anterior thalamic nuclei.

The effect of prophylaxis with low dose heparin on blood coagulation parameters. A double blind study in connection with transvesical prostatectomy.

Heparin was administered subcutaneously 5.000 IU twice daily using a double blind method to ten of twenty-one patients undergoing transvesical prostatectomy. Platelet count, APTT, throbin time, Reptilase time, Normotest, fibrinogen, Factor-VIII, ethanol gelation test, antithrombin III, fibrinolytic degradation products, alpha1-antitrypsin and alpha2-macroglobulin were studied pre- and postoperatively up to the 10th postoperative day. Statistical analysis of parameters of blood coagulation and fibrinolysis showed no significant difference between the two groups. The mechanism by which low dose heparin exerts its thromboprophylactic effect could not be elucidated from the study of the investigated parameters. The laboratory data gave no indication to a possible increased risk of postoperative hemorrhage.

NO synthase in cholinergic nerves and NO-induced relaxation in the rat isolated corpus cavernosum.

1. In the rat corpus cavernosum (CC), the distribution of immunoreactivity for neuronal and endothelial NO synthase (nNOS and eNOS), and the pattern of NOS-immunoreactive (-IR) nerves in relation to some other nerve populations, were investigated. Cholinergic nerves were specifically immunolabelled with antibodies to the vesicular acetylcholine transporter protein (VAChT). 2. In the smooth muscle septa surrounding the cavernous spaces, and around the central and helicine arteries, the numbers of PGP- and tyrosine hydroxylase (TH)-IR terminals were large, whereas neuropeptide Y (NPY)-, VAChT-, nNOS-, and vasoactive intestinal polypeptide (VIP)-IR terminals were found in few to moderate numbers. 3. Double immunolabelling revealed that VAChT- and nNOS-IR terminals, VAChT- and VIP-IR terminals, nNOS-IR and VIP-IR terminals, and TH- and NPY-IR terminals showed coinciding profiles, and co-existence was verified by confocal laser scanning microscopy. TH immunoreactivity was not found in VAChT-, nNOS-, or VIP-IR nerve fibres or terminals. 4. An isolated strip preparation of the rat CC was developed, and characterized. In this preparation, cumulative addition of NO to noradrenaline (NA)-contracted strips, produced concentration-dependent, rapid, and almost complete relaxations. Electrical field stimulation of endothelin-1-contracted preparations produced frequency-dependent responses: a contractile twitch followed by a fast relaxant response. After cessation of stimulation, there was a slow relaxant phase. Inhibition of NO synthesis, or blockade of guanylate cyclase, abolished the first relaxant phase, whereas the second relaxation was unaffected. 5. The results suggest that in the rat CC, nNOS, VAChT- and VIP-immunoreactivities can be found in the same parasympathetic cholinergic neurons. Inhibitory neurotransmission involves activation of the NO-system, and the release of other, as yet unknown, transmitters.

Morphological and functional in vitro and in vivo characterization of the mouse corpus cavernosum.

1. In normal mice, the distribution of adrenergic, cholinergic, some peptidergic, and neuronal nitric oxide synthase (nNOS)-containing nerves were investigated. Functional in vitro correlates were obtained. An in vivo model was developed in which erectile haemodynamics in response to drugs or nerve-stimulation were studied. 2. Immunoreactivities for vesicular acetylcholine transporter protein (VAChT), nNOS-, and vasoactive intestinal polypeptide (VIP), co-existed in nerve fibres and terminal varicosities. Immunoreactivities for neuropeptide Y (NPY) and tyrosine hydroxylase (TH) were found in the same nerve structures. 3. Chemical sympathectomy abolished TH- and NPY-IR nerve structures in cavernous smooth muscle bundles. The distribution of calcitonin gene-related peptide (CGRP)-, nNOS-, VAChT- and VIP-IR nerve structures was unchanged. 4. In endothelial cells of the central and helicine arteries, veins and venules, intense immunoreactivity for endothelial NOS (eNOS) was observed. No distinct eNOS-IR cells were found lining the cavernous sinusoids. 5. In vitro, nerve-induced relaxations were verified, and endothelial NO/cyclic GMP-mediated relaxant responses were established. VIP and CGRP had small relaxant effects. A functioning adenylate cyclase/cyclic AMP pathway was confirmed. 6. Neuronal excitatory responses were abolished by prazosin, or forskolin. VIP and CGRP counteracted contractions, whereas NPY and scopolamine enhanced excitatory responses. 7. In vivo, erectile responses were significantly attenuated by L-NAME (50 mg kg(-1)) and facilitated by sildenafil (200 microg kg(-1)). 8. It is concluded that the mouse is a suitable model for studies of erectile mechanisms in vitro and in vivo.

Pituitary adenylate cyclase-activating polypeptide, helospectin, and vasoactive intestinal polypeptide in human corpus cavernosum.

1. The distribution and effects of pituitary adenylate cyclase-activating polypeptide (PACAP-27 and -38), helospectin (Hel-1 and Hel-2), and vasoactive intestinal polypeptide (VIP), were investigated in isolated preparations of human corpus cavernosum (CC). 2. Immunohistochemistry revealed coinciding profiles of nerve structures that showed immunoreactivities for VIP and PACAP, and VIP and Hel. Confocal microscopy showed the co-existence of VIP- and PACAP-immunoreactivities, and VIP- and Hel-immunoreactivities in most (90%) varicose nerve structures. 3. As determined by radioimmunoassay, the amounts of VIP, PACAP-27, and PACAP-38 in the preparations were 61.7 +/- 11.6, 0.1 +/- 0.05, and 3.7 +/- 0.5 pmol g-1 wet weight of tissue (pmol g-1 wet wt.), respectively. In tissue from patients with diabetes, the content of VIP was lower (13.7 +/- 0.5 pmol g-1 wet wt.), whereas that of PACAP (-27 and -38) was unchanged. 4. Cyclic nucleotide levels were determined in preparations exposed to PACAP-27, PACAP-38, Hel-1, Hel-2, and VIP. All the peptides, but Hel-2, significantly increased the concentrations of cyclic AMP, whereas the levels of cyclic GMP were unchanged. 5. The peptides concentration-dependently relaxed noradrenaline-contracted preparations. The order of potency was VIP > PACAP 27 > Hel-1 > Hel-2 > PACAP-38. 6. Hel-1, VIP and PACAP-27 effectively counteracted electrically induced contractions. At 10(-6) M, the highest peptide concentration used, the inhibitory effects obtained reached 96 +/- 3%, 87 +/- 6%, and 80 +/- 3%, respectively. 7. The results suggest that PACAP and Hel-1 are co-localized with VIP in nerve structures within the human cavernous tissue, and that the peptides are effective relaxants of CC preparations in vitro. The role of the investigated peptides for penile erection remains to be established.

Impaired relaxation of stomach smooth muscle in mice lacking cyclic GMP-dependent protein kinase I.

1. Guanosine 3', 5'-cyclic monophosphate (cyclic GMP)-dependent kinase I (cGKI) is a major receptor for cyclic GMP in a variety of cells. Mice lacking cGKI exhibit multiple phenotypes, including severe defects in smooth muscle function. We have investigated the NO/cGMP- and vasoactive intestinal polypeptide (VIP)/adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-signalling pathways in the gastric fundus of wild type and cGKI-deficient mice. 2. Using immunohistochemistry, similar staining patterns for NO-synthase, cyclic GMP- and VIP-immunoreactivities were found in wild type and cGKI-deficient mice. 3. In isolated, endothelin-1 (3 nM - 3 microM)-contracted, muscle strips from wild type mice, electrical field stimulation (1 - 16 Hz) caused a biphasic relaxation, one initial rapid, followed by a more slowly developing phase. In preparations from cGKI-deficient mice only the slowly developing relaxation was observed. 4. The responses to the NO donor, SIN-1 (10 nM - 100 microM), and to 8-Br-cyclic GMP (10 nM - 100 microM) were markedly impaired in strips from cGKI-deficient mice, whereas the responses to VIP (0.1 nM - 1 microM) and forskolin (0.1 nM - 1 microM) were similar to those in wild type mice. 5. These results suggest that cGKI plays a central role in the NO/cGMP signalling cascade producing relaxation of mouse gastric fundus smooth muscle. Relaxant agents acting via the cyclic AMP-pathway can exert their effects independently of cGKI.

Effects of adenosine A(2A) receptor stimulation in vivo on dopamine D3 receptor agonist binding in the rat brain.

To investigate if adenosine A2A receptor stimulation in vivo modulates dopamine D3 receptor binding, we analyzed the effects of 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxyamidoade nosine (CGS 21680) on the binding properties of the selective D3 receptor agonist [N-propyl-2,3,-3H]4aR,10bR-(+)-trans-3,4,4a,10b-tetrahydro-4 -n-propyl2H,5H-[1]benzopyrano[4,3-b]1,4-oxazin-9-ol ([3H]PD 128907) in the rat forebrain using quantitative autoradiography. Intraperitoneally administered CGS 21680 (0.1-3 mg/kg) increased the Kd and Bmax values of [3H]PD 128907 binding in the islands of Calleja and in subregions of the caudate-putamen. These results suggest that stimulation of adenosine A2A receptors in vivo causes alterations in the binding characteristics of dopamine D3 receptors in the basal ganglia, and that this effect may relate to the neuroleptic-like effect of adenosine A2A receptor agonists.

Allosteric regulation by oleamide of the binding properties of 5-hydroxytryptamine7 receptors.

Oleamide belongs to a family of amidated lipids with diverse biological activities, including sleep induction and signaling modulation of several 5-hydroxytryptamine (5-HT) receptor subtypes, including 5-HT1A, 5-HT2A/2C, and 5-HT7. The 5-HT7 receptor, predominantly localized in the hypothalamus, hippocampus, and frontal cortex, stimulates cyclic AMP formation and is thought to be involved in the regulation of sleep-wake cycles. Recently, it was proposed that oleamide acts at an allosteric site on the 5-HT7 receptor to regulate cyclic AMP formation. We have further investigated the interaction between oleamide and 5-HT7 receptors by performing radioligand binding assays with HeLa cells transfected with the 5-HT7 receptor. Methiothepin, clozapine, and 5-HT all displaced specific [3H]5-HT (100 nM) binding, with pK(D) values of 7.55, 7.85, and 8.39, respectively. Oleamide also displaced [3H]5-HT binding, but the maximum inhibition was only 40% of the binding. Taking allosteric (see below) cooperativity into account, a K(D) of 2.69 nM was calculated for oleamide. In saturation binding experiments, oleamide caused a 3-fold decrease in the affinity of [3H]5-HT for the 5-HT7 receptor, without affecting the number of binding sites. A Schild analysis showed that the induced shift in affinity of [3H]5-HT reached a plateau, unlike that of a competitive inhibitor, illustrating the allosteric nature of the interaction between oleamide and the 5-HT7 receptor. Oleic acid, the product of oleamide hydrolysis, had a similar effect on [3H]5-HT binding, whereas structural analogs of oleamide, trans-9,10-octadecenamide, cis-8,9-octadecenamide, and erucamide, did not alter [3H]5-HT binding significantly. The findings support the hypothesis that oleamide acts via an allosteric site on the 5-HT7 receptor regulating receptor affinity.

Regional and age-dependent differences in the affinity of dopamine D2 agonist binding in the rat brain.

In sagittal brain sections of newborn male rats (1-day-old) there were no regional differences in the IC50 values of dopamine at [125I]iodosulpride binding sites. In contrast, in 20- and 60-day-old rats, there was a selective increase in the IC50 values of dopamine in the nucleus accumbens, caudate-putamen, and olfactory tubercule. The IC50 values of these regions decreased in 262-day-old rats. Some of the other brain areas appeared to behave in a similar, but much less pronounced, fashion. Thus, there were significant regional differences in the IC50 values of young, adult, and old rats. In addition, there was a rapid increase in [125I]iodosulpride binding between the newborn and the 20-day-old rats, which leveled off thereafter, and selectively decreased in the substantia nigra of the 262-day-old rats. In conclusion, these results indicate that a biphasic decrease-increase in the affinity of D2 agonist binding sites occurs selectively in the basal ganglia. These findings may be of relevance for developmental diseases in which dopaminergic mechanisms have been implicated, such as schizophrenia and Parkinson's disease.

Possible mechanisms for the powerful actions of neuropeptides.

In order to understand the mechanisms underlying the powerful actions of neuropeptides, the present article has emphasized the unique ability of neuropeptides to act as VT signals, which via high-affinity G-protein coupled receptors can exert long-lasting actions and control synaptic transmission via receptor-receptor interactions. Also of substantial importance is the ability of neuropeptides to act as a set of signals via the formation of different types of active fragments, which can act as negative-feedback or positive-feedback signals to modulate the response elicited by the parent peptide and to give origin to syndromic responses. Also in the actions of the fragments on the neuronal network, receptor-receptor interactions may play an important role both by modulating the parent peptide receptors and by modulating other types of VT and/or WT receptors. Future work will have to evaluate the role of neuropeptides as transcellular signals and as regulators of neuronal excitabilities after the formation of carbamates, but certainly new important developments are within the horizon of today's research.

Receptor-receptor interactions and their relevance for receptor diversity. Focus on neuropeptide/dopamine interactions.

Receptor diversity in combination with receptor-receptor subtype specific interactions, which can be antagonistic or synergistic in character, markedly increase plasticity in WT and VT in the nervous system. In this way switching among transmission lines for the various DA receptor subtypes becomes possible. Some of these aspects are supported by our work on selective modulation of D2 receptors by CCK and NT. Selective regulation of D2 receptors via CCK-8 receptor subtypes and NT receptors may underlie CCK/DA interactions and NT/DA interactions in the basal ganglia. These studies underline the importance of receptor-receptor interactions exerted at the membrane level between neuropeptide receptors and D2 receptors, which are determined at least in part by the ongoing activity at D1 receptors. In the case of both CCK/D2 and NT/D2 receptor interactions, it has been possible, by means of intrastriatal and intraaccumbens microdialysis, to obtain a functional correlate to the receptor interactions found in the membrane preparations from the striatum. Schizophrenia may be in part related to reduced release of CCK and/or NT peptides or to alterations in their receptor interactions with the D2 receptor. This view may lead to new therapeutic approaches.