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In addition to the somatodendritic region, myenteric motoneuron terminals are endowed with nicotinic autoreceptors. We aimed at investigating the effect of nicotinic receptor (nAChR) activation on [3H]-acetylcholine ([3H]-ACh) release from longitudinal muscle-myenteric plexus of the rat ileum and to evaluate whether this could be modulated by adenosine, an endogenous neuromodulator typically operating changes in intracellular cyclic AMP. The nAChR agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP, 1–30 μM, 3 min) increased [3H]-ACh release in a concentration-dependent manner. DMPP (30 μM)-induced [3H]-ACh outflow was attenuated by hexamethonium (0.1–1 mM), tubocurarine (1–5 μM), or by removing external Ca2+ (plus EGTA, 1 mM). In contrast to veratridine (0.2–10 μM)-induced [3H]-ACh release, the DMPP (30 μM)-induced outflow was resistant to tetrodotoxin (1 μM) and cadmium (0.5 mM). Pretreatment with adenosine deaminase (0.5 U/mL) or with the adenosine A2A-receptor antagonist, ZM 241385 (50 nM), enhanced nAChR-induced transmitter release. Activation of A2A receptors with CGS 21680C (3 nM) reduced the DMPP-induced release of [3H]-ACh. CGS 21680C (3 nM) inhibition was prevented by MDL 12,330A (10 μM, an adenylate cyclase inhibitor) and by H-89 (10 μM, an inhibitor of protein kinase A), but was potentiated by rolipram (300 μM, a phosphodiesterase inhibitor). DMPP-induced transmitter release was decreased by 8-bromo-cyclic AMP (1 mM, a protein kinase A activator), rolipram (300 μM), and forskolin (3 μM, an activator of adenylate cyclase). Both MDL 12,330A (10 μM) and H-89 (10 μM) facilitated DMPP-induced release of [3H]-ACh.The results indicate that nAChR-induced [3H]-ACh release is triggered by the influx of Ca2+, independent of voltage-sensitive calcium channels, presumably directly through nAChRs located on myenteric axon terminals. It was also shown that endogenous adenosine, activating A2A receptors coupled to the adenylate cyclase/cyclic AMP transducing system, is tonically downregulating this nAChR-mediated control of [3H]-ACh release. [Copyright &y& Elsevier]

Methylphenidate (MPH) is a first-line stimulant drug to treat attention deficit hyperactivity disorder (ADHD). Overdiagnosis of ADHD and MPH abuse lead to serious concerns about the possible long-term adverse consequences of MPH in healthy children and adolescents. We aimed to evaluate MPH effects in adolescent male Wistar rats (postnatal day 40) using an oral dose scheme (2 daily MPH doses 5 mg/kg in a 5% sucrose solution, 5 h apart, for 7 days) that mimics the therapeutic doses given to human adolescents. Twenty-four hours after the last MPH administration, rats were sacrificed and brain areas [cerebellum, prefrontal cortex (PFC), hippocampus, and striatum], peripheral organs (liver, heart, and kidneys), and blood were collected for biochemical and histological analysis. MPH treatment did not alter rats’ body temperature or weight, neither food or water intake throughout the experiment. The ratio of reduced glutathione/oxidized glutathione (GSH/GSSG) significantly increased in the PFC and hippocampus of MPH-treated rats, meanwhile protein carbonylation remained unchanged in the brain. In the heart, the GSH/GSSG ratio and GSH levels were significantly increased, with decreased GSSG, while histology revealed significant damage, namely interstitial edema, vascular congestion, and presence of a fibrin-like material in the interstitial space. In the kidneys, MPH treatment resulted in extensive necrotic areas with cellular disorganization and cell infiltration, and immunohistochemistry analysis revealed a marked activation of nuclear factor-ĸB. This study showed that clinically relevant oral MPH doses improve the GSH redox status in the brain and heart, but evoke heart and kidney tissue damage to adolescent rats. [ABSTRACT FROM AUTHOR]

Abstract: Adenosine plays a dual role on acetylcholine (ACh) release from myenteric motoneurons via the activation of high-affinity inhibitory A1 and facilitatory A2A receptors. The therapeutic potential of adenosine-related compounds for controlling intestinal motility and inflammation, prompted us to investigate further the role of low-affinity adenosine receptors, A2B and A3, on electrically-evoked (5Hz, 200pulses) [3H]ACh release from myenteric neurons. Immunolocalization studies showed that A2B receptors exhibit a pattern of distribution similar to the glial cell marker, GFAP. Regarding A1 and A3 receptors, they are mainly distributed to cell bodies of ganglionic myenteric neurons, whereas A2A receptors are localized predominantly on cholinergic nerve terminals. Using selective antagonists (DPCPX, ZM241385 and MRS1191), data indicate that modulation of evoked [3H]ACh release is balanced through tonic activation of inhibitory (A1) and facilitatory (A2A and A3) receptors by endogenous adenosine. The selective A2B receptor antagonist, PSB603, alone was devoid of effect and failed to modify the inhibitory effect of NECA. The A3 receptor agonist, 2-Cl-IB MECA (1–10nM), concentration-dependently increased the release of [3H]ACh. The effect of 2-Cl-IB MECA was attenuated by MRS1191 and by ZM241385, which selectively block respectively A3 and A2A receptors. In contrast to 2-Cl-IB MECA, activation of A2A receptors with CGS21680C attenuated nicotinic facilitation of ACh release induced by focal depolarization of myenteric nerve terminals in the presence of tetrodotoxin. Tandem localization of excitatory A3 and A2A receptors along myenteric neurons explains why stimulation of A3 receptors (with 2-Cl-IB MECA) on nerve cell bodies acts cooperatively with prejunctional facilitatory A2A receptors to up-regulate acetylcholine release. The results presented herein consolidate and expand the current understanding of adenosine receptor distribution and function in the myenteric plexus of the rat ileum, and should be taken into consideration for data interpretation regarding the pathophysiological implications of adenosine on intestinal motility disorders. [Copyright &y& Elsevier]

• Dissolvable pullulan microneedles were successfully prepared by solvent casting. • Pullulan-insulin microneedles successfully penetrate through the skin. • Storage at 4, 20 and 40 °C maintained the secondary structure of insulin. • Pullulan microneedles deliver 87% of insulin, 120 min after insertion. • Pullulan-insulin microneedles are non-cytotoxic towards human keratinocyte cells. The present study reports the fabrication of dissolvable microneedle (MN) patches using pullulan (PL), a water-soluble polysaccharide with excellent film-forming ability, for the transdermal administration of insulin, envisioning the non-invasive treatment of diabetes. PL MNs patches were successfully prepared by micromoulding and revealed good thermal stability (T dmax = 294 °C) and mechanical properties (>0.15 N needle−1), penetrating skin up to 381 μm depth, as revealed by in vitro skin tests. After application into human abdominal skin in vitro , the MNs dissolved within 2 h releasing up to 87% of insulin. When stored at 4, 20 and 40 °C for 4 weeks, insulin was able to retain its secondary structure, as shown by circular dichroism spectropolarimetry. The prepared PL MNs were non-cytotoxic towards human keratinocytes, being suitable for skin application. These findings suggest that PL MNs have potential to deliver insulin transdermally, thus avoiding its subcutaneous administration. [ABSTRACT FROM AUTHOR]