Peptides of the corticotropin-releasing hormone/Urocortin (CRH/Ucn) family are known to suppress appetite primarily via CRH(2) receptors. In the rat hypothalamic supraoptic nucleus (SON), synthesis of both Ucn1 and CRH(2) receptors has been reported, yet little is known about the effects of Ucn1 in the SON on feeding behaviour. We first established the dose-related effects of Ucn1 injected into the SON on the feeding response in both freely fed and 24-h food-deprived rats. A conditioned taste avoidance paradigm was performed to investigate possible generalised effects of local Ucn1 treatment. Administration of Ucn1 into the SON at doses equal to or higher than 0.5 μg significantly decreased food intake in both freely fed and food-deprived rats. The Ucn1-mediated suppression of food intake was delayed in freely fed as compared to food-deprived animals. Conditioning for taste aversion to saccharine appeared at 0.5 and 1 μg of Ucn1. Both the early and the delayed onset of anorexia observed after intra-SON injection of Ucn1 under fasting and fed conditions, respectively, suggest the possible involvement of different CRH receptor subtypes in the two conditions, while the conditioned taste aversion seems to be responsible for the initial latency to eat the first meal in these animals.

Minocycline, a tetracycline antibiotic, has been reported to exert beneficial effects in models of Alzheimer's disease (AD). To characterize the mechanisms underlying the putative minocycline-related neuroprotection, we studied its effect in an in vitro model of AD. Primary hippocampal cultures were treated with β-amyloid peptide (Aβ) and cell viability was assessed by standard MTT-assay. Incubation with 10 μM Aβ for 24 h significantly inhibits cellular MTT-reduction without inducing morphological signs of enhanced cell death or increase in release of lactate dehydrogenase. This indicates that cell viability was not affected. The inhibition of MTT-reduction by Aβ was due to an acceleration of MTT-formazan exocytosis. Intriguingly, the Aβ-triggered increase in MTT-formazan exocytosis was abolished by co-treatment with minocycline. In vehicle-treated cells minocycline had no effect on formazan exocytosis. This hitherto unrecognized property of minocycline has to be noticed in the elucidation of the underlying mechanism of this promising neuroprotectant.

The semi-synthetic tetracycline derivative minocycline exerts neuroprotective properties in various animal models of neurodegenerative disorders. Although anti-inflammatory and anti-apoptotic effects are reported to contribute to the neuroprotective action, the exact molecular mechanisms underlying the beneficial properties of minocycline remain to be clarified. We analyzed the effects of minocycline in a cell culture model of neuronal damage and in single-channel measurements on isolated mitoplasts. Treatment of neuron-enriched cortical cultures with rotenone, a high affinity inhibitor of the mitochondrial complex I, resulted in a deregulation of the intracellular Ca2+-dynamics, as recorded by live cell imaging. Minocycline (100 microM) and cyclosporin A (2 microM), a known inhibitor of the mitochondrial permeability transition pore, decreased the rotenone-induced Ca2+-deregulation by 60.9% and 37.6%, respectively. Investigations of the mitochondrial permeability transition pore by patch-clamp techniques revealed for the first time a dose-dependent reduction of the open probability by minocycline (IC(50)=190 nM). Additionally, we provide evidence for the high antioxidant potential of MC in our model. In conclusion, the present data substantiate the beneficial properties of minocycline as promising neuroprotectant by its inhibitory activity on the mitochondrial permeability transition pore.

There is an ongoing debate on the presence of beneficial effects of minocycline (MC), a tetracycline-like antibiotic, on the preservation of mitochondrial functions under conditions promoting mitochondria-mediated apoptosis. Here, we present a multiparameter study on the effects of MC on isolated rat liver mitochondria (RLM) suspended either in a KCl-based or in a sucrose-based medium. We found that the incubation medium used strongly affects the response of RLM to MC. In KCl-based medium, but not in sucrose-based medium, MC triggered mitochondrial swelling and cytochrome c release. MC-dependent swelling was associated with mitochondrial depolarization and a decrease in state 3 as well as uncoupled respiration. Swelling of RLM in KCl-based medium indicates that MC permeabilizes the inner mitochondrial membrane (IMM) to K(+) and Cl(-). This view is supported by our findings that MC-induced swelling in the KCl-based medium was partly suppressed by N, N'-dicyclohexylcarbodiimide (an inhibitor of IMM-linked K(+)-transport) and tributyltin (an inhibitor of the inner membrane anion channel) and that swelling was less pronounced when RLM were suspended in choline chloride-based medium. In addition, we observed a rapid MC-induced depletion of endogenous Mg(2+) from RLM, an event that is known to activate ion-conducting pathways within the IMM. Moreover, MC abolished the Ca(2+) retention capacity of RLM irrespective of the incubation medium used, most likely by triggering permeability transition. In summary, we found that MC at low micromolar concentrations impairs several energy-dependent functions of mitochondria in vitro.

The impact of a lifelong absence of the neuronal nitric oxide synthase (nNOS) in the neuroendocrine stress response was investigated in nNOS knockout (KO) and wild type (WT) mice under basal conditions and in response to forced swimming. In the hypothalamic paraventricular nucleus oxytocin and corticotropin-releasing-hormone mRNA levels did not differ between these genotypes under resting conditions, whereas vasopressin mRNA levels were significantly lower in nNOS KO than in WT animals. Also, in the adrenal glands basal levels of tyrosine hydroxylase protein, the rate-limiting enzyme for catecholamine biosynthesis, and of phenylethanolamine N-methyltransferase, which converts norepinephrine to epinephrine, were significantly reduced in nNOS KO mice. Plasma adrenocorticotropin, corticosterone, norepinephrine and epinephrine levels were similar in the KO and WT genotypes under resting conditions. In response to forced swimming, a similar increase in plasma adrenocorticotropin and corticosterone was observed in KO and WT animals. Stressor exposure triggered also an increased epinephrine release in WT animals, but did not significantly alter plasma epinephrine levels in KO mice. These data suggest that the chronic absence of nNOS reduces the capacity of epinephrine synthesising enzymes in the adrenal gland to respond to acute stressor exposure with an adequate epinephrine release.

Nitric oxide (NO) is a free radical gas synthesised from arginine and oxygen by enzymes of the family of the nitric oxide synthase. In particular, the neuronal nitric oxide synthase (nNOS) is highly expressed by cells of the hypothalamic paraventricular nucleus, where the sympatho-adrenal system, the hypothalamic-pituitary-adrenal axis and the hypothalamic-neurohypophyseal system originate. These structures are deputed to regulate the neuroendocrine stress response. In the past years, evidence has been accumulated to suggest that NO of nNOS origin plays a significant role in modulating the activity of the above mentioned systems under acute stressor exposure. The availability of nNOS knock-out mice allowed to investigate not only the physiological consequences of a constitutive lack of NO of nNOS origin at the hormonal and molecular level, but also to examine possible behavioural alterations. In this review, we shall discuss and confront the current trends of research in this area, especially focusing on the latest findings gained from genetically modified mice.

Knock out mice deficient for the splice‐isoform αα of neuronal nitric oxide synthase (nNOSαα) display residual nitric oxide synthase activity and immunosignal. To attribute this signal to the two minor neuronal nitric oxide synthase splice variants, ββ and γγ, we generated isoform‐specific anti‐peptide antibodies against the nNOSαα specific ββ‐finger motif involved in PDZ domain scaffolding and the nNOSββ specific N‐terminus. The nNOSαα ββ‐finger‐specific antibody clearly recognized the 160‐kDa band of recombinant nNOSαα on Western blots. Using immunocytochemistry, this antibody displayed, in rats and wild‐type mice, a labeling pattern similar to but not identical with that obtained using a commercial pan‐nNOS antibody. This similarity indicates that the majority of immunocytochemically detectable nNOS is not likely to be complexed with PDZ‐domain proteins via the ββ‐finger motif. This conclusion was confirmed by the inhibition of PSD‐95/nNOS interaction by the nNOSαα ββ‐finger antibody in pull‐down assays. By contrast, nNOSαα ββ‐finger labeling was clearly reduced in hippocampal and cortical neuropil areas enriched in NMDA receptor complex containing spine synapses. In nNOSαα knock out mice, nNOSαα was not detectable, whereas the pan‐nNOS antibody showed a distinct labeling of cell bodies throughout the brain, most likely reflecting ββ/γγ‐isoforms in these cells. The nNOSββ antibody clearly detected bacterial expressed nNOSββ fusion protein and nNOSββ in overexpressing HEK cells by Western blotting. Immunocytochemically, individual cell bodies in striatum, cerebral cortex, and in some brain stem nuclei were labeled in knock out but not in wild‐type mice, indicating an upregulation of nNOSββ in nNOSαα deficient animals. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007

The effect of minocycline on nerve regeneration was studied in a rat model of acute sciatic nerve injury, in which the injury was caused by resection and reimplantation of the right sciatic nerve. Immunohistochemical and molecular biological methods, as well as morphometric and electron microscopic techniques, were used. Compared with uninjured and PBS-treated injured nerves, the minocycline-treated injured nerve showed: (i) a decrease in macrophage recruitment and activation, probably resulting from inhibition of blood-brain-barrier break-down via reduced MMP2 and MMP9 induction, inhibition of revascularization via additional reduction of VEGF induction, and inhibition of inducible NO synthase (iNOS) induction; (ii) reduced activation of phagocytic Schwann cells, probably by inhibition of iNOS, MMP2 and MMP9 expression; (iii) a slowed Wallerian degeneration; and subsequently, (iv) a diminished nerve regeneration. Macrophages, especially their function in the removal of cellular debris and formation of a microenvironment beneficial for nerve regeneration, are strongly implicated in constructive events after nerve injuries. Therefore, we suggest that additional research into optimizing minocycline intervention for treatment of neurodegenerative diseases is needed before further clinical trials are performed.

This study was undertaken to examine the importance of nitric oxide (NO) generated by the neural isoform of the nitric oxide synthase (nNOS) on the activity of the hypothalamic neurohypophyseal system in neural nitric oxide synthase knock-out (KO) and wild-type (WT) mice under basal conditions and in response to forced swimming. The intensity of the hybridisation signal for vasopressin (AVP) in the hypothalamic supraoptic nucleus (SON) was significantly higher in KO mice when compared with WT, whereas oxytocin (OXT) basal mRNA levels were similar in both groups. Although the basal peripheral release of AVP and OXT was equivalent in both genotypes, we observed in KO mice a significant drop of AVP and OXT plasma values 15min after stressor onset and a robust increase in the OXT plasma concentration at 60min. These findings suggest that in the male mouse, NO inhibits AVP gene transcription in magnocellular neurones of the SON and collaborates in maintaining constant AVP and OXT plasma levels following acute stressor exposure, exerting a bimodal regulatory action on OXT secretion. We conclude that NO is involved in the regulation of magnocellular neurones of the SON, and it is preferentially implicated in the attenuation of the peripheral release of OXT induced by acute stressor exposure.

Leber’s hereditary optic neuropathy (LHON) is a retinal neurodegenerative disorder caused by mitochondrial DNA point mutations. Complex I of the respiratory chain affected by the mutation results in a decrease in ATP and an increase of reactive oxygen species production. Evaluating the efficacy of minocycline in LHON, the drug increased the survival of cybrid cells in contrast to the parental cells after thapsigargin-induced calcium overload. Similar protection was observed by treatment with cyclosporine A, a blocker of the mitochondrial permeability transition pore (mPTP). Ratiometric Ca2+ imaging reveals that acetylcholine/thapsigargin triggered elevation of the cytosolic calcium concentration is alleviated by minocycline and cyclosporine A. The mitochondrial membrane potential of LHON cybrids was significantly conserved and the active-caspase-3/procaspase-3 ratio was decreased in both treatments. Our observations show that minocycline inhibits permeability transition induced by thapsigargin in addition to its antioxidant effects. In relation with its high safety profile, these results would suggest minocycline as a promising neuroprotective agent in LHON.

Leber's hereditary optic neuropathy (LHON) is a retinal neurodegenerative disorder caused by mitochondrial DNA point mutations. Complex I of the respiratory chain affected by the mutation results in a decrease in ATP and an increase of reactive oxygen species production. Evaluating the efficacy of minocycline in LHON, the drug increased the survival of cybrid cells in contrast to the parental cells after thapsigargin-induced calcium overload. Similar protection was observed by treatment with cyclosporine A, a blocker of the mitochondrial permeability transition pore (mPTP). Ratiometric Ca(2+) imaging reveals that acetylcholine/thapsigargin triggered elevation of the cytosolic calcium concentration is alleviated by minocycline and cyclosporine A. The mitochondrial membrane potential of LHON cybrids was significantly conserved and the active-caspase-3/procaspase-3 ratio was decreased in both treatments. Our observations show that minocycline inhibits permeability transition induced by thapsigargin in addition to its antioxidant effects. In relation with its high safety profile, these results would suggest minocycline as a promising neuroprotective agent in LHON.

You have accessJournal of UrologyModerated Poster 20, Monday, May 21, 2007, 8:00 - 10:00 am1 Apr 2007678: Expression of Cyclic AMP-Dependent Protein Kinase-1 Isoforms in Human Penile Erectile Tissue: Relation to Vasoactive Intestinal Polypeptide-Containing Nerves and PDE4 Eginhard S. Waldkirch, Stefan Ueckert, Udo Jonas, Kristina Langnaese, Karin Richter, Gerald Wolf, Michael Sohn, Christian G. Stief, Karl-Erik Andersson, and Petter Hedlund Eginhard S. WaldkirchEginhard S. Waldkirch More articles by this author , Stefan UeckertStefan Ueckert More articles by this author , Udo JonasUdo Jonas More articles by this author , Kristina LangnaeseKristina Langnaese More articles by this author , Karin RichterKarin Richter More articles by this author , Gerald WolfGerald Wolf More articles by this author , Michael SohnMichael Sohn More articles by this author , Christian G. StiefChristian G. Stief More articles by this author , Karl-Erik AnderssonKarl-Erik Andersson More articles by this author , and Petter HedlundPetter Hedlund More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)30918-2AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "678: Expression of Cyclic AMP-Dependent Protein Kinase-1 Isoforms in Human Penile Erectile Tissue: Relation to Vasoactive Intestinal Polypeptide-Containing Nerves and PDE4." The Journal of Urology, 177(4S), pp. 227–228 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 177Issue 4SApril 2007Page: 227-228 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Eginhard S. Waldkirch More articles by this author Stefan Ueckert More articles by this author Udo Jonas More articles by this author Kristina Langnaese More articles by this author Karin Richter More articles by this author Gerald Wolf More articles by this author Michael Sohn More articles by this author Christian G. Stief More articles by this author Karl-Erik Andersson More articles by this author Petter Hedlund More articles by this author Expand All Advertisement PDF downloadLoading ...

Repeated administration of beta-phenylalkylamines is known to produce neuronal changes in the central and peripheral nervous systems of mammals. It is suggested that various components of the cytoskeleton undergo profound alterations after amphetamine use and misuse, contributing to behavioral changes and neurotoxicity. Here we studied the expression of microtubule-associated protein 2 (MAP2) and beta-actin after repeated intraperitoneal applications with equimolar doses of p-chloroamphetamine (PCA), methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA) in the brain of male Wistar rats. Effective (molecular) pharmacological doses (ED) were derived and used for the calculation of (molecular) pharmacological indices (PI). Besides clear but different dose-response curves on the toxicity of the drugs, in situ hybridization and Western blot analysis revealed that repeated administration of these compounds resulted in different substance- and dose-dependent changes in MAP2 gene expression, e.g. in the frontoparietal somatosensoric cortex. In contrast, the expression of beta-actin was not influenced by any of the compounds at the dose levels tested. Lethal doses were determined with 2.1 (PCA), >5.1 (METH) and 8.4 mg/kg/day (MDMA). Linear and non-linear repeat-dose lethality was observed for MDMA and PCA, respectively, whereas METH was non-lethal in the dose range used. Values for ED(MAP2) were 0.3, 0.52 and >16.8 mg/kg/day, and therefore those for PI(MAP2) were 20, 4, and 0.5 for METH, PCA and MDMA, respectively. Although the results on mortality did not reflect changes in MAP2 gene expression, they suggest a remarkable difference for those amphetamines without substituents or with a halogen atom at the paraposition of the benzene ring, such as METH or PCA, when compared with MDMA-like substances.

Inhibition of the mitochondrial KATP (mitoKATP) channel abrogates the beneficial effects of preconditioning induced by a brief episode of sublethal ischemia. We studied the effect of 5-hydroxydecanoate, a well-known inhibitor of the mitoKATP channel, on swelling of isolated liver and brain mitochondria. Volume changes were determined by measurement of light absorbance at 540 nm. Mitochondrial swelling induced by adding Ca2+ ions correlated with opening of the permeability transition pore as shown by modulation by 1 μM cyclosporin A. In brain mitochondria, 5-hydroxydecanoate did not significantly affect Ca2+-induced swelling. In contrast, 50 or 500 μM 5-hydroxydecanoate increased swelling of liver mitochondria by 9.7 ± 5.1% (n = 6, P = 0.057) and 29.4 ± 1.4% (n = 5, P < 0.0001), respectively. The effect of 5-hydroxydecanoate was blocked by cyclosporin A and was dependent on the presence of potassium in the medium. In medium containing 200 μM ATP to inhibit the mitoKATP channel, 5–hydroxydecanoate did not further increase Ca2+-induced swelling. We conclude that inhibition of the mitoKATP channel exerts its detrimental effect by facilitation of permeability transition pore opening.

Pregnancy is accompanied by an array of adaptive changes that play an important role in pre- and postnatal events. In rats, urocortin 1, a corticotropin-releasing factor-like peptide, is expressed mainly in the non-preganglionic Edinger–Westphal nucleus. We investigated the number of neurons immunoreactive for urocortin 1 at three different levels of the Edinger–Westphal nucleus in female rats by immunohistochemistry. The number of urocortin 1 immunoreactive cells was found to be decreased in pregnant rats compared to virgin rats. These results indicate that the hormonal status of the female rat affects urocortin 1 immunoreactive neurons in the non-preganglionic Edinger–Westphal nucleus and its signaling to target brain areas.

The significance of the nitric oxide (NO) - cyclic GMP pathway in the control of human penile erectile tissue has been well established. Findings from experimental studies implicated that cyclic GMP might also be of importance in the maintenance of the normal function of the human prostate. The aim of the present study was to examine further the significance of cyclic GMP in the human prostate by means of a cell culture model using isolated human prostatic smooth muscle cells (PSMC), as well as by immunohistochemical methods.