Effects of MCH/NEI in the striatum and interactions between them and with alpha-MSH on IP3 levels

In this study, we investigated: (a) the effect of melanocyte concentrating hormone (MCH) and neuropeptide glutamine (E)-isoleusine-(I) (NEI) on IP(3) production on an "in vitro" model using slices containing caudate putamen (CP) and accumbens nuclei (ACC); and (b) the interaction between these peptides and with alpha-melanocyte-stimulating-hormone (alpha-MSH) on the production of this second messenger. Only MCH at the highest dose studied (3.6 microM) increased the production of IP(3), whereas at the low concentration (0.6 microM) it did not affect IP(3) levels. NEI and alpha-MSH at both concentrations tested (0.6 and 3.6 microM), did not affect IP(3) production either. However, when NEI or alpha-MSH (at 3.6 microM) were added together with 3.6 microM MCH, the increase in the IP(3) content induced by this last peptide was blocked.     

Interaction between alpha-MSH and acetylcholinergic system upon striatal cAMP and IP(3) levels

The interaction between the neuropeptide alpha-MSH and the acetylcholinergic system as reflected by changes in cAMP and inositol 1-3-5 triphosphate(IP(3))production was investigated in an in vitro model of striatal slices. The possible involvement of D(1) receptors in cholinergic and alpha-MSH- stimulated cAMP and IP(3) production in slices of rat striatum was also examined, because it has been demonstrated that acetylcholinergic drugs induce endogenous dopamine release in the striatum. alpha-MSH, pilocarpine(PL) and the selective muscarinic M1 agonist McN-A-343 increased cAMP and IP(3) striatal levels, effects blocked by the D(1) antagonist SCH-23390, except for the effects of alpha-MSH on IP(3).The muscarinic M(2) antagonist gallamine (GL) brought about an increase in cAMP levels, an effect blocked by SCH-23390. The M(1) antagonist pirenzepine (Pz) induced a decrease both in cAMP and IP(3) content, and the nicotinic antagonist di-hydro-beta-eritroidine(DBE) only diminished cAMP production. When alpha-MSH and cholinergic agents were simultaneously added, cAMP and IP(3) levels were modified with respect to the values reached when these agents were added alone. An interaction between the acetylcholinergic system and alpha-MSH through M(1) and nicotinic receptors was also observed. These results suggest that the intracellular signaling pathways related to cAMP and IP(3) production gated by alpha-MSH and these cholinergic receptors are probably related. alpha-MSH striatum cAMP IP(3) muscarinic and nicotinic receptors an in vitro model.     

Interaction of melanin-concentrating hormone (MCH), neuropeptide E-I (NEI), neuropeptide G-E (NGE), and alpha-MSH with melanocortin and MCH receptors on mouse B16 melanoma cells.

Melanin-concentrating hormone (MCH) and alpha-melanocyte-stimulating hormone (alpha-MSH) are known to exhibit mostly functionally antagonistic, but in some cases agonistic activities, e.g., in pigment cells and in the brain. Neuropeptide E-I (NEI) displays functional MCH-antagonist and MSH-agonist activity in different behavioral paradigms; the role of neuropeptide G-E (NGE) is not known. This study addressed the question of possible molecular interactions between alpha-MSH, MCH and the MCH-precursor-derived peptides NEI and NGE at the level of the pigment cell MCH receptor subtype (MCH-Rpc) and the different melanocortin (MC) receptors. Radioreceptor assays using [125I]MCH, [125l]alpha-MSH and [125I]NEI as radioligands and bioassays were performed with MCI-R-positive and MC1-R-negative mouse B16 melanoma cells and with COS cells expressing the different MC receptors. The IC50s of alpha-MSH and NEI or NGE for [125I]MCH displacement from mouse MCH-Rpc were 80-fold and, respectively, >300-fold higher than that of MCH, and the IC50s for MCH and NEI or NGE for [125I]alpha-MSH displacement from mouse MC1-R were 50,000-fold and >200,000-fold higher than that of alpha-MSH. No high-affinity binding sites for NEI were detected on B16 melanoma cells and there was no significant displacement of [1251]alpha-MSH by MCH, NEI or NGE with MC3-R, MC4-R and MC5-R expressed in COS cells. At concentrations of 100 nM to 10 microM, however, MCH, NEI and NGE induced cAMP formation and melanin synthesis which could be blocked by agouti protein or inhibitors of adenylate cyclase or protein kinase A. This shows that mammalian MCH-precursor-derived peptides may mimic MSH signalling via MC1-R activation at relatively high, but physiologically still relevant concentrations, as e.g. found in autocrine/paracrine signalling mechanisms.     

Effects of morphine and beta-endorphin on basal and elevated plasma levels of alpha-MSH and vasopressin.

Morphine induced an increase of plasma α-MSH levels and a decrease of AVP levels after peripheral or intracerebroventricular administration. This increase of α-MSH levels and decrease of AVP levels after morphine treatment was observed in non-stimulated animals as well as in rats in which the hormone levels were elevated by water deprivation or by administration of hypertonic saline. These latter effects of morphine on plasma levels of α-MSH and AVP could be blocked by simultaneous administration of naltrexone.β-Endorphin also increased plasma α-MSH levels and lowered plasma AVP levels. From these effects only the increase of the plasma α-MSH level and not the decrease of plasma AVP could be blocked by naltrexone. Moreover PLG treatment was ineffective with respect to the endorphin-induced decrease in plasma AVP, but it partly blocked the increase of plasma α-MSH when this tripeptide was given in combination with β-endorphin.     

Correlation between intracellular cAMP levels and phospholipids of Microsporum gypseum.

Atropine, a modulator of cAMP has been used to examine the relationship between phospholipids and intracellular levels of cAMP in Microsporum gypseum. A decreased phospholipid content was observed in atropine grown cells as a result of reduced levels of intracellular cAMP. This decline was caused by the inhibitory effect of atropine on adenylate cyclase. Lowered phospholipid content was supported by decreased [14C]acetate incorporation as well as reduced activities of key enzymes of phospholipid biosynthesis. In vitro supplementation of atropine in control cells also caused inhibition in lipid synthesis indicating similar effects of atropine and its metabolites. These results in conjunction with our previous report, in which enhanced levels of cAMP resulted in increased phospholipid synthesis, suggest a direct correlation between phospholipid biosynthesis and intracellular levels of cAMP in M. gypseum.     

Effects of LSD and 2-bromo LSD on striatal DOPAC levels.

Administration of d-lysergic acid diethylamide (LSD) and its analogue 2-bromo lysergic acid diethylamide (BOL) resulted in a shortlasting increase of 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the rat striatum. BOL was more potent than LSD in the dose range 0.5–4.0 mg/kg. Since there was a concomitant increase in the striatal $\text{in}$$\text{vivo}$ tyrosine hydroxylation as measured by DOPA accumulation after decarboxylase inhibition, our findings suggest that LSD and BOL increase the impulse flow in the nigro-neostriatal pathway probably by central dopamine (DA) receptor antagonism. However, 4 hrs after LSD the DOPAC level was decreased, while the DOPA accumulation was not. Thus the effect of LSD on the dopaminergic system appears not to be limited to a pure receptor antagonism. The possibility also exists that the effect of LSD on the nigro-neostriatal DA pathway is secondary to its effect on the central 5-hydroxytryptaminergic system.     

cAMP levels within Mycobacterium tuberculosis and Mycobacterium bovis BCG increase upon infection of macrophages

Adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signal transduction is common in both prokaryotes and eukaryotes, and several bacterial pathogens modulate cAMP signaling pathways of their mammalian hosts during infection. In this study, cAMP levels associated with Mycobacterium tuberculosis and Mycobacterium bovis BCG were measured during macrophage infection. cAMP levels within both bacteria increased c . 50-fold during infection of J774.16 macrophages, relative to the cAMP levels within bacteria incubated in tissue culture media alone. cAMP levels also increased within the macrophage cytoplasm upon uptake of live, but not dead, mycobacteria. The presence of albumin in the absence of oleic acid significantly decreased cAMP secretion and production by both M. tuberculosis and M. bovis BCG. These results suggest that cAMP signaling plays a role in the interaction of tuberculosis-complex mycobacteria with macrophages during infection, and that albumin may be a physiological indicator differentiating host environments during infection.     

Effects of dopaminergic drugs and acute medial forebrain bundle lesions on striatal acetylcholine levels.

Placement of radio frequency lesions in the medial forebrain bundle resulted in a 50% depletion of striatal acetylcholine levels but did not change hippocampal levels. A similar result was obtained with the administration of chlorpromazine, haloperidol and pimozide. When these drugs were administered simultaneously with placement of lesions, there was the same 50% depletion of striatal acetylcholine. Apomorphine reversed the depletion due to lesions. These results suggest that the action of antipsychotic drugs on the cholinergic system in the striatum is primarily due to their action at dopamine receptors rather than a direct action on cholinergic receptors which would be due to their anticholinergic activity.     

Neuroendocrine-immune (NEI) circuitry from neuron-glial interactions to function: Focus on gender and HPA-HPG interactions on early programming of the NEI system

Bidirectional communication between the neuroendocrine and immune systems during ontogeny plays a pivotal role in programming the development of neuroendocrine and immune responses in adult life. Signals generated by the hypothalamic-pituitary-gonadal axis (i.e. luteinizing hormone-releasing hormone, LHRH, and sex steroids), and by the hypothalamic-pituitary-adrenocortical axis (glucocorticoids (GC)), are major players coordinating the development of immune system function. Conversely, products generated by immune system activation exert a powerful and long-lasting regulation on neuroendocrine axes activity. The neuroendocrine-immune system is very sensitive to preperinatal experiences, including hormonal manipulations and immune challenges, which may influence the future predisposition to several disease entities. We review our work on the ongoing mutual regulation of neuroendocrine and immune cell activities, both at a cellular and molecular level. In the central nervous system, one chief compartment is represented by the astroglial cell and its mediators. Hence, neuron-glial signalling cascades dictate major changes in response to hormonal manipulations and pro-inflammatory triggers. The interplay between LHRH, sex steroids, GC and pro-inflammatory mediators in some physiological and pathological states, together with the potential clinical implications of these findings, are summarized. The overall study highlights the plasticity of this intersystem cross-talk for pharmacological targeting with drugs acting at the neuroendocrine-immune interface.     

HIV-1 Tat and morphine interactions dynamically shift striatal monoamine levels and exploratory behaviors over time

Despite the advent of combination anti-retroviral therapy (cART), nearly half of people infected with HIV treated with cART still exhibit HIV-associated neurocognitive disorders (HAND). HAND can be worsened by co-morbid opioid use disorder. The basal ganglia are particularly vulnerable to HIV-1 and exhibit higher viral loads and more severe pathology, which can be exacerbated by co-exposure to opioids. Evidence suggests that dopaminergic neurotransmission is disrupted by HIV exposure, however, little is known about whether co-exposure to opioids may alter neurotransmitter levels in the striatum and if this in turn influences behavior. Therefore, we assayed motor, anxiety-like, novelty-seeking, exploratory, and social behaviors, and levels of monoamines and their metabolites following 2 weeks and 2 months of Tat and/or morphine exposure in transgenic mice. Morphine decreased dopamine levels, but significantly elevated norepinephrine, the dopamine metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the serotonin metabolite 5-hydroxyindoleacetic acid, which typically correlated with increased locomotor behavior. The combination of Tat and morphine altered dopamine, DOPAC, and HVA concentrations differently depending on the neurotransmitter/metabolite and duration of exposure but did not affect the numbers of tyrosine hydroxylase-positive neurons in the mesencephalon. Tat exposure increased the latency to interact with novel conspecifics, but not other novel objects, suggesting the viral protein inhibits exploratory behavior initiation in a context-dependent manner. By contrast, and consistent with prior findings that opioid misuse can increase novelty-seeking behavior, morphine exposure increased the time spent exploring a novel environment. Finally, Tat and morphine interacted to affect locomotor activity in a time-dependent manner, while grip strength and rotarod performance were unaffected. Together, our results provide novel insight into the unique effects of HIV-1 Tat and morphine on monoamine neurochemistry that may underlie their divergent effects on motor and exploratory behavior.     

alpha-MSH (melanocyte stimulating hormone) and MCH (melanin concentrating hormone) actions in Bufo ictericus ictericus melanophores

1. The darkening actions of MCH (melanin concentrating hormone), alpha-MSH and the synthetic analog [Nle4, D-Phe7]-alpha-MSH on the toad, Bufo ictericus ictericus, melanophores were studied regarding the role of calcium in the hormone receptor coupling, signal transduction and intracellular pigment translocation. 2. In the absence of external calcium, MCH and both melanotropins still elicit maximal skin darkening. 3. Verapamil, a calcium-channel blocker, completely abolishes the alpha-MSH-induced response and partially inhibits MCH-induced darkening, although the calcium carrier, ionophore A23187, was unable to promote any pigment translocation. 4. Since darkening responses promoted by cyclic nucleotides proceeded normally in the presence of verapamil and extracellular calcium was not necessary for melanotropin dispersing action, it is suggested that the blocking activity obtained with verapamil is probably due to an impairment of the Ca2+-dependent adenylate cyclase activity. 5. Reversal of melanotropin-induced darkening could be obtained with melatonin, in both normal and Ca2+-free Ringer, whereas MCH darkening is reversed by melatonin only in the absence of calcium. 6. The results seem to indicate that calcium is not required for hormone receptor binding and pigment migration, whereas it is specifically needed for signal transduction.     

Cerebellar cAMP levels following acute and chronic morphine administration.

Acute administration of morphine sulfate at 20 mg/kg decreased mouse cerebellar adenosine 3',5'-cyclic phosphate (cAMP) levels while not affecting cAMP phosphodiesterase (EC 3.1.4.17). The cAMP levels and cAMP phosphodiesterase activies were not affected by chronic treatment. However, cAMP levels increased during abrupt withdrawal both with and without naloxone precipitation, with cAMP phosphodiesterase activities being correspondingly decreased. Propanolol prevented the cAMP increase during abrupt withdrawal.     

Interbilayer interactions between sphingomyelin and sphingomyelin/cholesterol bilayers.

Pressure versus fluid spacing relations have been obtained for sphingomyelin bilayers in the gel phase and equimolar sphingomyelin/cholesterol in the liquid-crystalline phase by the use of X-ray diffraction analysis of osmotically stressed aqueous dispersions and oriented multilayers. For interbilayer separations in the range of 5-20 A, the repulsive hydration pressure decays exponentially with increasing fluid spacing. The decay length (lambda) of this repulsive pressure is about 2 A for both bovine brain and N-tetracosanoylsphingomyelin, similar to that previously found for phosphatidylcholine bilayers. However, both the magnitude of the hydration pressure and the magnitude of the dipole potential (V) measured for monolayers in equilibrium with liposomes are considerably smaller for sphingomyelin than for either gel or liquid-crystalline phosphatidylcholine bilayers. Addition of equimolar cholesterol increases both the magnitude of the hydration pressure and the dipole potential. These data suggest that the magnitude of the hydration pressure depends on the electric field at the interface as given by (V/lambda)2. For sphingomyelin bilayers, there is a sharp upward break in the pressure-fluid spacing relation at an interbilayer spacing of about 5 A, indicating the onset of steric hindrance between the head groups of apposing bilayers.     

Lateral hypothalamic lesions and striatal dopamine levels

Rats with bilateral lateral hypothalamic lesions were killed on the third day after surgery and their brains were assayed for tel-diencephalic norepinephrine and striatal dopamine. Lesion-induced weight loss was highly correlated with depletion of striatal dopamine but not with tel-diencephalic norepinephrine. In rats with severe dopamine depletions, the degree of weight loss was related more to the striatum with the highest remaining level of dopamine suggesting that a critical level of dopamine in one striatum may be essential for lateral hypothalamic recovery.     

Effects of UVB on interactions between Schistosoma mansoni and Biomphalaria glabrata

Ultraviolet B (UVB, 280-315 nm) radiation is detrimental to both of larvae of the digenetic trematode Schistosoma mansoni and its snail intermediate host, Biomphalaria glabrata. We explored effects of UVB on three aspects of the interaction between host and parasite: survival of infected snails, innate susceptibility and resistance of snails to infection, and acquired resistance induced by irradiated miracidia. Snails infected for 1 week showed significantly lower survival than uninfected snails following irradiation with a range of UVB intensities. In contrast to known immunomodulatory effects in vertebrates, an effect of UVB on susceptibility or resistance of snails to infection could not be conclusively demonstrated. Finally, exposure of susceptible snails to UVB-irradiated miracidia failed to induce resistance to a subsequent challenge with nonirradiated miracidia, a result similar to that reported previously with ionizing radiation.     

Competition between cell-substratum interactions and cell-cell interactions.

Clusterin, a glycoprotein which elicits the aggregation of a wide variety of cells (Fritz, I. B., and Burdy, K.:J. Cell Physiol., 140:18-28, 1989), has been utilized to investigate some of the factors modulating the competition between cell-substratum interactions and cell-cell interactions. We compared the responses to clusterin by anchorage-independent cells (erythrocytes) with those by anchorage-dependent TM4 cells (a cell line derived from neonatal mouse testis cells). Cells were maintained in culture in the presence of various substrata chosen to enhance cell-substratum interactions (laminin-coated wells), or to diminish cell-substratum interactions (agarose-coated wells). Results obtained showed that the aggregation of erythrocytes elicited by clusterin was independent of the nature of the substratum. In contrast, clusterin addition resulted in aggregation of anchorage-dependent TM4 cells only when TM4 cell-substratum interactions were weak. Thus, clusterin did not aggregate TM4 cells plated upon a laminin substratum, but readily aggregated TM4 cells plated upon an agarose-coated substratum, independent of the sequence of addition of cells and clusterin to the culture dish. We utilized YIGSR, a peptide which competes with laminin for laminin receptors, to determine the possible role of laminin receptors on TM4 cells in the competition between cell-substratum interactions and cell-cell interactions. The presence of YIGSR did not alter responses of erythrocytes to clusterin under all conditions examined. In contrast, the responses of TM4 cells to clusterin were greatly changed. YIGSR addition resulted in the inhibition of aggregation of TM4 cells otherwise elicited by clusterin. YIGSR also prevented attachment of TM4 cells to a laminin-coated surface, but this was reversed by the presence of clusterin. We discuss the possible roles of clusterin and laminin in altering the balance in the competition between cell to cell interactions and cell to substratum interactions.     

ACTH-(1-24) and alpha-MSH antagonize dopamine receptor-mediated inhibition of striatal dopamine and acetylcholine release

The effects of ACTH-(1-24), alpha-MSH and ACTH-(4-10) were studied on the electrically evoked release of 3H-dopamine and 14C-acetylcholine from striatal slices in the absence and presence of the dopamine receptor agonist TL-99. None of the peptides affected transmitter release when TL-99 was not present. ACTH-(1-24) and alpha-MSH concentration-dependently antagonized the inhibition of striatal transmitter release induced by dopamine receptor stimulation due to the presence of TL-99. ACTH-(1-24), 10(-7)M, reduced the TL-99-induced inhibition of the release of both dopamine and acetylcholine by approximately 50%, and 5 X 10(-6) M ACTH-(1-24) restored the release fully to control values. alpha-MSH was less effective by a factor 20-30 in counteracting the release-inhibiting effect of TL-99. ACTH-(4-10) had no effect at any of the concentrations tested. These results show that ACTH/MSH-like neuropeptides may act by modulating dopamine receptor functions in rat striatum.     

Neuropeptide-dopamine interactions. IV. Effect of thyrotropin-releasing hormone on striatal dopaminergic neurons

Many biologic effects of TRH seem to be mediated via a dopaminergic mechanism. The present study examined the effects of chronic TRH administration on the properties of nigrostriatal dopaminergic neurons. Ten days, continuous subcutaneous TRH administration via an osmotic minipump led to a significant rise in striatal level of 3,4-dihydroxyphenylacetic acid, but not of homovanillic acid or dopamine. These treatments also did not elicit any significant changes in the maximal binding capacity (Bmax) or affinity (KD) of either D1- or D2-dopamine receptor. By contrast, TRH administration led to a significant increase in both Bmax and KD of striatal mazindol binding. This effect of TRH, however, was not observed in in vitro studies. In conclusion, these data suggest that in vivo administration of TRH may modulate dopaminergic activities by altering, directly or indirectly, dopamine release and reuptake.     

Inhibitory actions of ceramide upon PKC-epsilon/ERK interactions

We have previously shown that interleukin-1receptor-generated ceramide induces growth arrest in smooth musclepericytes by inhibiting an upstream kinase in the extracellularsignal-regulated kinase (ERK) cascade. Here, we now report themechanism by which ceramide inhibits ERK activity. Ceramide renders thehuman embryonic kidney 293 cells (HEK 293) resistant to the mitogenicactions of growth factors and activators of protein kinase C (PKC). A role for PKC to mediate ceramide inhibition of growth factor-induced ERK activity and mitogenesis is suggested, as exogenous ceramide directly inhibits both immunoprecipitated and recombinant PKC- activities. To confirm that PKC- is necessary for ceramide-inhibited ERK activity, HEK 293 cells were transfected with a dominant-negative mutant of PKC- (PKC-). These transfected cells respond toinsulin-like growth factor I (IGF-I) with a significantly decreased ERKactivity that is not further reduced by ceramide treatment.Coimmunoprecipitation studies reveal that the treatment with IGF-Iinduces the association of ERK with PKC- but not with PKC-.Ceramide treatment significantly inhibits the IGF-I-induced PKC-interaction with bioactive phosphorylated ERK. Ceramide also inhibitsIGF-I-induced PKC- association with Raf-1, an upstream kinase ofERK. Together, these studies demonstrate that ceramide exertsanti-mitogenic actions by limiting the ability of PKC- to form asignaling complex with Raf-1 and ERK.