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Brain corticotropin-releasing factor mediates ‘anxiety-like’ behavior induced by cocaine withdrawal in rats

Anxiety is a key symptom of the cocaine withdrawal syndrome in human addicts, and it is considered to be one of the major factors in precipitating relapse to chronic cocaine abuse. Corticotropin-releasing factor (CRF) plays an important role in the pathophysiology of anxiety and depression, and it may also be involved in the acute behavioral and neuroendocrine actions of cocaine. The role of endogenous CRF in cocaine withdrawal-induced anxiety was investigated in the present study. Animals were subjected to chronic cocaine (20 mg/kg, intraperitoneally, once a day for 14 days) administration. Rats tested 30 min after the last cocaine injection did not show withdrawal anxiety on the elevated plus maze or any alterations in brain CRF levels. Withdrawal (48 h) from chronic cocaine administration produced an intense anxiety-like behavior characterized by decreased open arm exploration. Immunoreactive CRF (CRF-LI) levels were selectively altered in the hypothalamus, in the amygdala and in the basal forebrain structures at the time of the behavioral anxiety, reflecting an increased activity of brain CRF systems. Daily intracerebroventricular (i.c.v.) pretreatment with an immunoserum raised against CRF completely prevented the development of anxiety induced by cocaine withdrawal. These data suggest that extrahypothalamic-limbic CRF hypersecretion may be involved in the development of anxiety related to cocaine withdrawal and that the CRF system may be a useful target for new pharmacotherapies for cocaine withdrawal and relapse.

Evaluation of the cytotoxicity of β-cyclodextrin derivatives: evidence for the role of cholesterol extraction.

Several beta-cyclodextrin (beta-CD) derivatives have been synthesized recently to improve the physicochemical properties and inclusion capacities of the parent molecule, however, there is limited information available about their cytotoxic effects. In this study we investigated the cytotoxic and hemolytic properties of various beta-CDs in correlation with their cholesterol-solubilizing capacities to expose the mechanism of toxicity. MTT cell viability test, performed on Caco-2 cells showed significant differences between the cytotoxicity of beta-CD derivatives. Cell toxicity of methylated-beta-CDs was the highest, while ionic derivatives proved to be less toxic than methylated ones. Most of the second generation beta-CD derivatives, having both ionic and methyl substituents showed less cytotoxicity than the parent compounds both on Caco-2 cells and human erythrocytes. Inclusion of cholesterol into the ring of randomly methylated-beta-CD and heptakis(2,6-di-O-methyl)-beta-CD abolished the cell toxicity indicating the role of cholesterol extraction in cytotoxicity. These data demonstrate the correlation between the cytotoxic effect, hemolytic activity and the cholesterol complexation attributes of beta-CD derivatives and we propose that cholesterol-solubilizing properties can be a predictive factor for beta-CD cell toxicity.

Salsolinol is a Putative Endogenous Neuro-intermediate Lobe Prolactin-Releasing Factor

The isolation and identification of a prolactin‐releasing factor (PRF) from the neuro‐intermediate lobe of the pituitary gland has been pursued for over a decade. Using high‐pressure liquid chromatography with electrochemical detection (HPLC‐ECD) and gas chromatography/mass spectrometry (GC/MS) (R)‐salsolinol (SAL) (a dopamine‐related stereo‐specific tetrahydroisoquinoline) was found to be present in neuro‐intermediate lobe as well as median eminence extracts of male, intact‐, and ovariectomized female rats. Moreover, analysis of SAL concentrations in neuro‐intermediate lobe revealed parallel increases with plasma prolactin in lactating rats exposed to a brief (10 min) suckling stimulus following 4‐h separation. SAL appears to be a selective and potent stimulator of prolactin secretion in vivo and it was without effect on the secretion of other pituitary hormones. We have also found that SAL can elevate prolactin release, although to a lesser extent, in pituitary cell cultures as well as in hypophysectomized rats bearing anterior lobe transplants under the kidney capsule. Lack of interference of SAL with [3H]‐spiperone binding to AP homogenates indicates that SAL does not act at the dopamine D2 receptor. Moreover, [3H]‐SAL binds specifically to homogenate of AL as well as neuro‐intermediate lobe obtained from lactating rats. Taken together, our data clearly suggest that SAL is synthesized in situ and this compound can play a role in the regulation of pituitary prolactin secretion.

Evaluation of cytotoxicity of surfactants used in self-micro emulsifying drug delivery systems and their effects on paracellular transport in Caco-2 cell monolayer

The objective of this study was to examine the cellular effects of the members of two non-ionic amphiphilic tenside groups and their mixtures on human Caco-2 cell monolayers as dependent upon their chemical structures and physicochemical properties. The first group of polyethylene glycol esters is represented by Polysorbates and Labrasol alone and in blends, while the members of the second group. Capryol 90, Capryol PGMC, Lauroglycol 90 and Lauroglycol FCC were used as propylene glycol esters. They are increasingly used in SMEDDS as recent tensides or co-tensides to increase hydrophobic bioavailability of a drug. Critical micelle concentration was measured by determination of surface tension. CMC refers to the ability of solubilization of surfactants. Cytotoxicity tests were performed on Caco-2 cell monolayers by MTT and LDH methods. Paracellular permeability as a marker of the integrity of cell monolayers, was examined with Lucifer yellow assays combined with TransEpithelial Electrical Resistance (TEER) measurements. The effect of these surfactants on tight junctions as evidence for paracellular pathway was also characterized. The results of cytotoxicity assays were in agreement, and showed significant differences among the cytotoxic properties of surfactants in a concentration-dependent manner. Polysorbates 20, 60, 80 are the most toxic compounds. In the case of Labrasol, the degree of esterification and lack of sorbit component decreased cytotoxicity. If the hydrophyl head was changed from polyethylene glycol to propylene glycol the main determined factor of cytotoxicity was the monoester content and the length of carbon chain. In our CMC experiments, we found that only Labrasol showed expressed cytotoxicity above the CMC. It refers to good ability of micelle solubilization of Labrasol. In our paracellular transport experiments each of polyethylene glycol surfactants (Polysorbates and Labrasol) altered TEER values, but propylene glycol esters did not modify the monolayer integrity. Polyethylene glycol esters alone and in blends (0.05% Labrasol--0.001% Polysorbates 20, 60, 80) were able to increase Lucifer yellow permeability significantly below the IC₅₀ concentration. On the other hand Labrasol and Polysorbates 20 have expressed effect on tight junctions of Caco-2 monolayer. It could be concluded that polyethylene glycol ester-type tensides were able to enhance the paracellular permeability by the redistribution of junctional proteins. Our results might ensure useful data for selection of suitable tensides, co-tensides and tenside mixtures for SMEDDS formulations.

Effects of cocaine on the contents of neurohypophyseal hormones in the plasma and in different brain structures in rats

The effects of acute and chronic cocaine treatments on the levels of the neurohypophyseal hormones oxytocin (OXT) and vasopressin (AVP) in the plasma and in different brain structures in rats were measured by radioimmunoassay (RIA). Acute cocaine treatment had no effect on the level of OXT in the plasma or in the amygdala, but increased OXT contents were measured in the hypothalamus and in the hippocampus. The OXT levels in the basal forebrain structures (including the septum and the nucleus accumbens) were decreased by a single dose of cocaine. The acute injection of cocaine increased the level of AVP in the plasma, and decreased contents of OXT were measured in the amygdala and in the basal forebrain. Repeated treatment with cocaine decreased the level of OXT in the plasma, hypothalamus and hippocampus. The AVP contents were decreased in all of the brain structures investigated, but no change was caused in the plasma level of AVP by repeated injections of cocaine. These results demonstrate complex, region-specific interactions between cocaine and the neurohypophyseal hormones in the brain and in the periphery underlying the alteration in behavioral and autonomic functions caused by acute and chronic cocaine exposure.

P-glycoprotein inhibition by membrane cholesterol modulation

P-glycoprotein (Pgp) is a transmembrane protein that actively exports lipophilic chemotherapeutics from the cells causing multidrug resistance. Pgp molecules are partially localized in TX-100-resistant rafts, and the activity of the transporter is highly sensitive to the presence of cholesterol. To better understand these relationships, the influence of membrane cholesterol content on Pgp function, as measured via calcein accumulation, was studied in correlation with changes elicited in membrane structure. Membrane cholesterol was modulated by heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DIMEB) and the cholesterol inclusion complex of DIMEB (Chol-DIMEB). Changes in membrane cholesterol level were reflected by alterations in the overall lipid packing as measured by Merocyanine 540 (MC540) staining and were also accompanied by changes in the raft association of the pump. DIMEB and Chol-DIMEB treatments have also lead to increased permeability of the cell membrane in both directions, raising the possibility that the effects on pumping efficiency reflect leakage of ATP also from the non-permeabilized cells. However, the treatments did not influence the intracellular ATP levels of the non-permeabilized cells. Our data suggest that Pgp inhibition by cyclodextrin treatments arises through modulation of its membrane microenvironment, rather than as a result of concomitant cytotoxicity.

Alterations of corticotropin-releasing factor-like immunoreactivity in different brain regions after acute cocaine administration in rats

Corticotropin-releasing factor (CRF) may mediate some of the neuroendocrine and behavioral responses to cocaine. In this study, the distribution of CRF-like immunoreactivity (CRF-LI) was determined in the hypothalamus and in several extrahypothalamic brain regions after acute cocaine administration in handled rats. CRF-LI decreased dose-dependently with cocaine administration in the hypothalamus and in the basal-forebrain structures. A small dose of cocaine (7.5 mg/kg) decreased CRF-LI in the hippocampus and in the frontal cortex. A significant, selective, dose-dependent increase in CRF-LI was found in the amygdala after cocaine injection. None of the investigated doses of cocaine altered CRF-LI in the striatum. These results suggest that acute cocaine administration alters brain CRF systems to contribute behavioral and neuroendocrine responses to cocaine.

The effect of neurointermediate lobe denervation on hypothalamic neuroendocrine dopaminergic neurons

The contribution of tuberohypophyseal and periventricular-hypophyseal dopaminergic neurons to the regulation of the secretion of prolactin (PRL) has yet to be clarified. In this study, we used pituitary stalk compression to disrupt hypothalamic neural input to the neurointermediate lobe (NIL). Neurointermediate lobe denervation (NIL-D) selectively disrupts the axons of tuberohypophyseal and periventricular-hypophyseal dopaminergic neurons, while leaving tuberoinfundibular dopaminergic neurons and the vascular supply of the pituitary gland intact. NIL-D was performed in ovariectomized (OVX) rats. The concentration of DA and 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence (ME) and various regions of the pituitary gland of OVX and OVX+NIL-D rats were measured by HPLC-EC. The concentration of PRL, alpha-melanocyte stimulating hormone (alpha-MSH), and luteinizing hormone (LH) in serum were determined by radioimmunoassay. Successful NIL-D was confirmed by increased water intake. One week after NIL-D, serum PRL and alpha-MSH were elevated, but there was no change in the concentration of LH in serum. The concentration of DA was increased in the median eminence (ME), decreased in the outer zone of the anterior lobe (AL-OZ), as well as the intermediate (IL) and neural lobes (NL), and remained unchanged in the inner zone of the anterior lobe (AL-IZ). The concentration of DOPAC was increased in the ME and NL, decreased in the IL, and remained unchanged in both the AL-IZ and AL-OZ. These data confirm that pituitary stalk compression denervates the NIL. Moreover, decreases in the concentration of DA in the IL and AL-OZ, coupled with elevation of serum PRL and alpha-MSH indicate that DA from the NIL contributes to the increased inhibition of the secretion of PRL and alpha-MSH in OVX rats.

Increased Concentration of Atrial Natriuretic Factor in the Cerebrospinal Fluid of Patients with Aneurysmal Subarachnoid Hemorrhage and Raised Intracranial Pressure

Plasma and cerebrospinal fluid (CSF) atrial natriuretic factors/peptides (ANFs/ANPs) were measured in 26 patients with normal or raised intracranial pressure (ICP) by means of an instant radioreceptor assay. All 26 patients were suffering from aneurysmal subarachnoid hemorrhage (SAH), and 11 had also developed raised ICP (ICP greater than 20 mm Hg). In SAH patients with normal ICP, the plasma levels of ANF were 20 to 200 pg/ml (mean +/- SE, 89 +/- 68 pg/ml); in the 11 SAH patients with raised ICP, however, ANF levels were 14 to 262 pg/ml (mean 114 +/- 79 pg/ml). The difference was not statistically significant. The ANF/ANP plasma levels in 6 healthy volunteers were 15 to 167 pg/ml (mean 77 +/- 32 pg/ml). Although the ANF/ANP concentration in the CSF of patients with normal ICP did not reach the lower limit of detectability (i.e., 4 pg/ml) in any case, in those with elevated ICP it was 14 to 120 pg/ml (mean 49 +/- 37 pg/ml). This difference was statistically highly significant. The results of this preliminary study suggest that the ANF/ANP concentration in human CSF is 1 to 2 orders lower than that in the plasma and that there is no significant correlation between ANF/ANP levels in the CSF and the plasma. After SAH in patients with raised ICP, there was an accompanying increase in the ANF/ANP concentration in the CSF, but the ANF/ANP concentration in the plasma was not changed significantly. Accordingly, a central ANF/ANP release might be hypothesized to play a causative or adaptive role in the neuroendocrine regulation of ICP dynamics, although this may simply be an epiphenomenon.

Complete inhibition of p-glycoprotein by simultaneous treatment with a distinct class of modulators and the UIC2 monoclonal antibody

P-glycoprotein (Pgp) is one of the active efflux pumps that are able to extrude a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance. The conformation-sensitive UIC2 monoclonal antibody potentially inhibits Pgp-mediated substrate transport. However, this inhibition is usually partial, and its extent is variable because UIC2 binds only to 10 to 40% Pgp present in the cell membrane. The rest of the Pgp molecules become recognized by this antibody only in the presence of certain substrates or modulators, including vinblastine, cyclosporine A (CsA), and SDZ PSC 833 (valspodar). Simultaneous application of any of these modulators and UIC2, followed by the removal of the modulator, results in a completely restored steady-state accumulation of various Pgp substrates (calcein-AM, daunorubicin, and 99mTc-hexakis-2-methoxybutylisonitrile), indicating near 100% inhibition of pump activity. Remarkably, the inhibitory binding of the antibody is brought about by coincubation with concentrations of CsA or SDZ PSC 833 ∼20 times lower than what is necessary for Pgp inhibition when the modulators are applied alone. The feasibility of such a combinative treatment for in vivo multidrug resistance reversal was substantiated by the dramatic increase of daunorubicin accumulation in xenotransplanted Pgp+ tumors in response to a combined treatment with UIC2 and CsA, both administered at doses ineffective when applied alone. These observations establish the combined application of a class of modulators used at low concentrations and of the UIC2 antibody as a novel, specific, and effective way of blocking Pgp function in vivo.