Cinzia Boselli

Depression and antidepressants: molecular and cellular aspects

Clinical depression is viewed as a physical and psychic disease process having a neuropathological basis, although a clear understanding of its ethiopathology is still missing. The observation that depressive symptoms are influenced by pharmacological manipulation of monoamines led to the hypothesis that depression results from reduced availability or functional deficiency of monoaminergic transmitters in some cerebral regions. However, there are limitations to current monoamine theories related to mood disorders. Recently, a growing body of experimental data has showed that other classes of endogenous compounds, such as neuropeptides and amino acids, may play a significant role in the pathophysiology of affective disorders. With the development of neuroscience, neuronal networks and intracellular pathways have been identified and characterized, describing the existence of the interaction between monoamines and receptors in turn able to modulate the expression of intracellular proteins and neurotrophic factors, suggesting that depression/antidepressants may be intermingled with neurogenesis/neurodegenerative processes.

Promiscuous or heterogeneous muscarinic receptors in rat atria? I. Schild analysis with simple competitive antagonists

Carbachol has been shown to produce a biphasic response in rat left atria. At low concentrations, carbachol depresses basal inotropy, while at high doses a positive inotropic effect is observed. The negative inotropic response can be selectively eliminated by pretreatment of rats with pertussis toxin. The aim of these studies was to determine whether or not evidence could be obtained to show that different muscarinic receptors produced these different biochemical responses to the agonist carbachol. Schild analysis was used to measure the equilibrium dissociation constant of the antagonist-receptor complex for antagonism of the negative inotropy to carbachol by atropine, scopolamine 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and AF-DX 116. The antagonism of the positive inotropic response to carbachol by these antagonists was studied in atria from rats pretreated with pertussis toxin where the negative inotropy was nearly completely abolished. In general, it was found that the antagonists did not produce simple competitive blockade of the positive inotropy but rather a nominal shift to the right of the dose-response curves followed by a depression of maximal responses. However, it was found that when pA2 or pKb values could be calculated, they coincided with those determined for the antagonism of the negative inotropy to carbachol. The conclusion drawn from these experiments was that no evidence was obtained to disprove the null hypothesis that a common receptor, interacting with two G-proteins, mediates these two effects of carbachol in rat left atria. The implications of these data for the classification of drug receptors with agonists is discussed.

Halloysite and chitosan oligosaccharide nanocomposite for wound healing

Halloysite is a natural nanotubular clay mineral (HNTs, Halloysite Nano Tubes) chemically identical to kaolinite and, due to its good biocompatibility, is an attractive nanomaterial for a vast range of biological applications. Chitosan oligosaccharides are homo- or heterooligomers of N-acetylglucosamine and D-glucosamine, that accelerate wound healing by enhancing the functions of inflammatory and repairing cells. The aim of the work was the development of a nanocomposite based on HNTs and chitosan oligosaccharides, to be used as pour powder to enhance healing in the treatment of chronic wounds. A 1:0.05 wt ratio HTNs/chitosan oligosaccharide nanocomposite was obtained by simply stirring the HTNs powder in a 1% w/w aqueous chitosan oligosaccharide solution and was formed by spontaneous ionic interaction resulting in 98.6% w/w HTNs and 1.4% w/w chitosan oligosaccharide composition. Advanced electron microscopy techniques were considered to confirm the structure of the hybrid nanotubes. Both HTNs and HTNs/chitosan oligosaccharide nanocomposite showed good in vitro biocompatibility with normal human dermal fibroblasts up to 300μg/ml concentration and enhanced in vitro fibroblast motility, promoting both proliferation and migration. The HTNs/chitosan oligosaccharide nanocomposite and the two components separately were tested for healing capacity in a murine (rat) model. HTNs/chitosan oligosaccharide allowed better skin reepithelization and reorganization than HNTs or chitosan oligosaccharide separately. The results suggest to develop the nanocomposite as a medical device for wound healing. STATEMENT OF SIGNIFICANCE The present work is focused on the development of halloysite and chitosan oligosaccharide nanocomposite for wound healing. It considers a therapeutic option for difficult to heal skin lesions and burns. The significance of the research considers two fundamental aspects: the first one is related to the development of a self-assembled nanocomposite, formed by spontaneous ionic interaction, while the second one is related to the possibility to find an effective treatment for cutaneous non healing lesions. The characterization of this hybrid system involves a multidisciplinary approach considering integrated techniques of solid state investigation and advanced electron microscopies, and in vitro/in vivo models to understand biocompatibility and proliferation properties (enhancement of in vitro fibroblast motility, proliferation and migration, and of in vivo burn healing), to understand safety and effectiveness of the developed nanocomposite.

Biphasic dose-response curves to arecoline in rat atria-mediation by a single promiscuous receptor or two receptor subtypes ?

SummaryArecoline produces a biphasic response in rat left atria, i. e., a depresion of basal inotropy at low doses and a positive inotropic effect at higher doses. These present studies were designed to determine whether it can be shown that the two separate responses to arecoline are mediated by two distinct cell surface muscarinic receptors. The antagonists scopolamine, 4-DAMP and AFDX 116 produced apparent simple competitive antagonism of the negative responses to arecoline. Schild analysis was used to measure the equilibrium dissociation constant of the antagonist-receptor complex for antagonism of this response to arecoline by these antagonists.In atria from rats treated with pertussis toxin, the negative inotropy to arecoline was abolished and only the positive inotropic effects were observed. The antagonism of the positive inotropic response to arecoline by these antagonists was studied separately in atria from rats treated with pertussis toxin by the Schild technique.The pKB estimates made from the Schild regressions indicated no evidence to suggest that the two responses to arecoline (negative and positive inotropy) were mediated by two separate receptors in rat left atria. These data are discussed in terms of a single muscarinic receptor in this tissue mediating these two responses by interaction with two G-proteins in the same cell membrane. These data also are discussed in terms of the use of agonist potency ratios for the classification of receptors.

Presence and passage dependent loss of biochemical M3 muscarinic receptor function in human detrusor cultured smooth muscle cells.

PURPOSE We explored morphological and biochemical aspects of human detrusor cells in culture as a tool for investigating the physiological mechanisms underlying bladder function and disturbances. MATERIALS AND METHODS Primary cultures of smooth muscle cells were derived from human bladder specimens of patients with an average age of 70 years undergoing cystectomy. Cultured cells were investigated by morphological, immunocytochemical and Western blot analysis. The alpha-actin content as well as the presence of muscarinic M2 and M3 receptors was determined in cell lysates and in fresh tissue homogenate for comparison. The functional response to muscarinic stimulation was assessed by measuring IP3 production induced by 1 mM. carbachol. RESULTS Cultured smooth muscle cells showed a characteristic spindle-shaped morphology at early passages. Similarly immunocytochemical and Western blot analysis revealed an alpha-actin cell content that was unmodified up to passage 3. Conversely this marker protein sharply decreased during further passages. M3 muscarinic receptor was present in cultured cells and fresh tissue homogenates, whereas the M2 subtype was evident only in homogenates. Carbachol produced a time dependent increase in IP3 cell content, reaching maximal production after 20 minutes of exposure. This response was passage sensitive. CONCLUSIONS Cultured human detrusor smooth muscle cells maintain their morphological and biochemical characteristics up to passage 3. With this caveat such cells can be an appropriate tool for investigating the molecular pathways underlying cholinergic activation in normal physiological and pathological bladders, and accordingly for detecting putative targets for pharmacological intervention.

Alcohol differentially affects noradrenergic and purinergic responses in the bisected rat vas deferens.

This study investigates the effect of acute in vivo or in vitro ethanol administration on the adrenergic and purinergic responses in the epididymal and prostatic portion of rat vas deferens. Acute in vivo ethanol treatment (3.0 g/kg, i.p.) selectively impaired the response to noradrenaline in the prostatic portion of rat vas deferens, leaving unaffected the epididymal portion. In addition, the response evoked by the maximal concentration of alpha, beta-methylene-ATP was significantly depressed by acute in vivo ethanol treatment in both the epididymal and prostatic segments. Ethanol 50 mM in vitro was devoid of any action on the response to exogenous noradrenaline in both tissues. Ethanol in vitro left unaltered the response to alpha,beta-methylene-ATP in the epididymal portion, while potentiated the contractile response in the prostatic one. These results provide, for the first time, evidence that ethanol in vitro and in vivo differentially affects the noradrenergic and purinergic responses in the bisected vas deferens, suggesting that this substance may alter the male reproductive tract function.

Determination of dissociation constants and relative efficacies of some potent muscarinic agonists at postjunctional muscarinic receptors

SummaryThis study was undertaken to determine dissociation constants (KA) and relative efficacies (er) of seven muscarinic agonists (methylfurtrethonium; dioxolane, oxathiolane, carbachol, muscarine, muscarone and oxotremorine) in three isolated tissues (guinea-pig ileum and atria and rat urinary bladder).The rank order of affinities (-log KA) of the various compounds varied depending on the tissue used. er values for the different agonists did not differ significantly from each other in any of the three tissues, except that the er of muscarine in the guinea-pig ileum was higher than those of the other compounds and that of oxotremorine in the rat urinary bladder was lower than those of the other agonists.Comparisons among tissues show that KA and er values were the same in different tissues for some compounds (muscarone, muscarine and methylfurtrethonium), while significant differences were found for the other compounds. This suggests the existence of a discrete receptor population recognized by some but not all agonists.For oxotremorine er as well as -log KA, is greater in atria than in smooth muscle: these factors combine to determine the cardioselectivity of this compound which can now ascribed to receptor selectivity.

Cromakalim blocks the purinergic response evoked in rat vas deferens by single-pulse electrical stimulation.

The present study was carried out to look at the influence of the K+ channel opener cromakalim, compared with suramin and prazosin, on the contractile response evoked by single-pulse field stimulation and exogenous agonists in epididymal and prostatic portions of rat vas deferens. In the epididymal portion suramin abolished the first phase of the response to single shock, prazosin deeply affected the second phase and a combination of both antagonists almost completely abolished both phases. Cromakalim was able to inhibit in a concentration-dependent manner the first purinergic phase (pD2 = 5.90 +/- 0.11), leaving practically unaffected the second, adrenergic phase. This inhibitory effect of cromakalim on the electrically evoked response was counteracted by glibenclamide. Cromakalim and prazosin, but not suramin, affected the response to exogenous noradrenaline. Suramin but not cromakalim was able to antagonize responses to alpha, beta-methylene-ATP. In the prostatic portion because of a less clear discrimination between adrenergic and purinergic phases of the electrically evoked response, the picture was less clear although the trend was identical. Cromakalim was not able to antagonize the response to ATP. It is concluded that in rat vas deferens cromakalim inhibits purinergic transmission by acting prejunctionally.

Cholinergic agents structurally related to furtrethonium. 1

Abstract A series of 5-substituted-2-(dimethylaminomethyl)-furyl derivatives 4 was prepared, with the aim of discovering novel antimuscarinic agents which are selective for smooth muscle as opposed to cardiac tissue. Both non-quaternary and quaternary ammonium compounds were synthesised. The agonist starting point, furtrethonium 3 , was gradually transformed into antagonist by introduction of lipophilic and bulky groups in position 5 of this molecule. In particular, the introduction of α-hydroxy-α-cyclohexylbenzyl moiety (compound 9b ), a lipophilic group characteristic of antimuscarinic agents, caused an appreciable increase of the antagonists potency, and the lengthening of the distance between this lipophilic group and the furan ring, obtained by introduction of an ester, ether or amide group, led to some selectivity towards smooth muscle (compounds 19, 21, 25 ). Interestingly, compound 19 , with an ester moiety as a spacer group, proved to be at least 20 times more potent in rat ileum (p K B = 7.3) and rat bladder (p K B = 7.2) than guinea-pig atria (p K B = 5.9).

An appraisal of recently patented compounds for bladder overactivity and urinary incontinence

Bladder overactivity and urinary incontinence are lower urinary tract syndromes for which there is no adequate pharmacological treatment. The latter condition is associated with an involuntary loss of urine causing a hygienic and social concern to the patient. Recently, central and peripheral mechanisms that control bladder storage and voiding processes have been partly addressed and clarified. Novel antimuscarinic agents, as well as newer formulations of available drugs and selective antagonists at α1D-receptors, have been patented, in addition to selective agonists at the β3-adrenoceptor and selective antagonists at 5-hydroxytryptamine, subtype 1A and 7 (5-HT1A and 5-HT7) receptors. Other potential therapeutic options include neurokinin type 1 and 2 (NK1/NK2) tachykinin receptor antagonists and potassium channel openers. The aforementioned agents may contribute to the pharmacological armamentarium in urology treatment, although their therapeutic effectiveness and safety profile remain to be validated in the clinical setting.