The Antisecretory Factor (AF, Mw 41kD) is an endogenous protein, found in all mammalian tissues. AF acts in vivo by counteracting intestinal hypersecretion and various forms of inflammations. The detailed in vitro AF action on the cellular level is unknown. Using an in vitro neuronal model (granular cells from rat small brain) we studied AF action on ionotropic GABAA receptors. A facilitatory

activity, due to increased GABAA receptors expression, was demonstrated on the neuronal plasma membrane in response to AF influence. This effect may result in inhibition of intestinal secretomotor cells

Gatta, E.

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DOI 10.1393/ncc/i2018-18115-7Communications: SIF Congress 2017IL NUOVO CIMENTO 41 C (2018) 115GABAA receptor modulation by the Antisecretory Factoron cerebellar granule cellsE. Gatta(∗)Dipartimento di Fisica, Università di Genova - Genova Italyreceived 29 January 2018Summary. — The Antisecretory Factor (AF, Mw 41kD) is an endogenous protein,found in all mammalian tissues. AF acts in vivo by counteracting intestinal hyper-secretion and various forms of inflammations. The detailed in vitro AF action on thecellular level is unknown. Using an in vitro neuronal model (granular cells from ratsmall brain) we studied AF action on ionotropic GABAA receptors. A facilitatoryactivity, due to increased GABAA receptors expression, was demonstrated on theneuronal plasma membrane in response to AF influence. This effect may result ininhibition of intestinal secretomotor cells.1. – IntroductionThe antisecretory factor (AF), a 41 kDa endogenous protein produced and stored inthe pituitary gland, was initially described by Stefan Lange and Ivar Lonnroth in themid 1980s. The AF protein has been cloned and sequenced by [1]. The discovery of AFwas the result of searching for a substance that improved the intestinal immune defenceto diarrhoea caused by cholera toxin [2-4]. In fact endogenous AF secretion increases af-ter exposure to bacterial toxins and an increase in AF secretion in combination with aninflammatory reaction may be a part of normal defense against the secretory and inflam-matory component in diarrhoea and other pathological processes involving membraneleakage and inflammation. AF is excreted in plasma and other tissue fluids in mammals,its immunohistological distribution suggests a role in the immune system [5]. The AFprotein has a powerful ability to counteract different types of secretion. The clinicalimportance has been studied in different diseases where a disturbed fluid homeostasisis of vital importance, e.g. different bowel diseases, diarrhoea and Meniere’s syndrome.The active site of AF (16 amino acids derived from amino acids no. 36-51 of the fulllength AF protein) is located in the amino terminal part of the AF protein [6]. Thedetailed, cellular mechanism of AF action in vivo remains to be described, but some(∗) E-mail: gatta@fisica.unige.itCreative Commons Attribution 4.0 License (http://creativecommons.org/licenses/by/4.0) 12 E. GATTAof previous experimental work tentatively suggests either a direct block of cell volumesensitive anionic channels [7] or an action via the enteric nervous system [8,9].On the other hand, influences of AF on GABAA receptor mediated inhibition havebeen demonstrated in the rat hippocampus [10, 11]. The possibility is that this proteinmay exert effects on the central nervous system as well, via interaction with neuronalGABAA receptors. Thus, it is of interest to investigate the effects of AF on native neu-ronal GABAA receptors of a defined subunit composition. The aim of the present studywas to describe the effects of AF-16 on the GABAA receptors present on rat cerebellargranule cells. In particular, the patch clamp technique is used and the effect of AF-16on the chloride current activated by the neurotransmitter is determined. GABA neu-rons and GABAA receptors are abundantly present in the enteric nervous system [12,13].However, in contrast to the central nervous system, the enteric nervous system GABAAreceptor activation often gives rise to excitation of the target neurons [12-14]. For thisreason and for the rather intricate circuit connections within the enteric plexuses [15,16],it is not straightforward to translate modifications of enteric GABAA receptor activityinto final, functional effects on the intestinal secretion. It is, however, important to ver-ify the effects of AF-16 on GABAA receptors of defined subtypes in the enteric nervoussystem, in order to formulate plausible hypotheses of its final, regulative action on thewater and ion transport in the small intestine.In any case, the positive or negative modulating activity on the GABAA receptorsmight result in different end effects concerning inhibition or excitation of neurons, sincethe final influence is determined by the electrochemical gradient for chloride ions in thesecells.2. – MethodsAF-16 peptide production. – The AF-16 peptide consisting of the amino acid sequence36-51 (VCHSKTRSNPENNVGL) of the AF protein, was synthesized with solid phasesynthesis by Ross-Petersen AS, Copenhagen, Denmark, as previously described [7].Granule cell cultures. – Granule cells were prepared from eight-day-old Sprague-Dawley rats as described previously [17]. The cells were studied from the 6th to the12th day in vitro. Experimental procedures and care of the animals were accordingto the EU Parliament and Council of September 22nd 2010 (2010/63/EU), they wereapproved by the Italian Ministry of Health (protocol number 2207) according to D.M.116/1992. All efforts were made in order to minimize animal suffering and the numberof animals necessary in order to obtain reliable results.Whole-cell patch-clamp studies. – The membrane currents were measured with thestandard whole-cell patch-clamp as previously described [17].The internal and external solutions for electrophysiological measurements were per-formed as previously reported [18]. In all experiments, the peak currents evoked by10 μM GABA, according to the various experiments, in the presence of AF-16 were re-ferred to those activated by plain GABA in the same cell. Thus, the averages reported inthe figures represent the percent changes of currents in the presence of AF-16 at variousconcentrations (1 pM–10 μM) in comparison with the control (plain GABA) currents. Inparticular, the enhancement E% were expressed as(1) E% = 100IAF,GABA − IGABAIGABA,GABAA RECEPTOR MODULATION ETC. 3where IAF,GABA represents the chloride current in the presence of AF-16 plus GABAand IGABA is the one with plain GABA.Dose response curves were fitted to the following equation:(2) E%(CAF ) =E%Max · CnAF(CnAF + kn),where CAF is AF-16 concentration and E%Max represents the saturation level of E%.In the experiments, AF-16 was preincubated 3 min before application of GABA.Evaluation of the control chloride current peak due to plain GABA was before AF-16application and after its washout. Experiments were taken into account when the twowere consistent.AF-16 was always co-applied with GABA after the preincubations. In some experi-ments, however, 3 min 0.01 μM AF-16 preincubation was followed by 2 min washout ofAF-16, and after this washout, application of plain GABA took place. In a few experi-ments, AF-16 was in the intracellular solution at a concentration of 1μM in order to testwhether it had any effect on GABA activated current from the cell’s inside.All chemicals, apart from AF-16, were purchased from Sigma Chemicals Co., St Louis,MO, USA.Immunocytochemistry . – After incubation with 1μM AF-16 at 37 ◦C for 1 h the cells,seven days in vitro, 7 DIV, were fixed in 4% paraformaldehyde in PBS buffer from SigmaAldrich (Milano, Italy) for 15 min at room temperature, washed three times with PBSsolution, permeabilized with 0.1 Triton X-100 from Sigma Aldrich in PBS + BSA (BovineSerum Albumine, from Sigma Aldrich, Milano, Italy) for 5 min and incubated overnightat 4 ◦C with primary antibody and successively with immunofluorescent labeling as pre-viously described [18].Fluorescence image acquisition was performed by a multi-channel Leica TCS SP5laser-scanning confocal microscope, equipped with 458, 476, 488, 514, 543 and 633 nmexcitation lines. Images were taken through a plan-apochromatic oil immersion objective63 × /1.4. Leica “LAS AF” software package was used for acquisition, storage andvisualization.The density of subunit γ2 of GABAA receptor in control and treated samples wasevaluated by immunocytochemistry using the same concentrations and time of incubationof primary and secondary antibodies for both samples and analyzed with Leica TCS SP5microscopy.Many images of different granule cells of control and treated with AF peptide weretaken for each sample.Acquired images were analysed with Fiji Is Just Imagej software.The receptor density was evaluated by fluorescence average, estimated in two differentways: choosing regions of interest (ROIs) that include the whole granule cells or selectingmarginal plasma membrane neuronal regions.Statistical analysis. – Final data are given as mean ± SEM. Statistical comparisonswere made by Students t-test.3. – ResultsElectrophysiological results: peak component of the GABA activated current . – Pre-treatment of the cells with AF-16 for 3 min caused a statistically significant increase4 E. GATTAFig. 1. – Dose-response curve for AF-16 enhancement (E%) of 10 μM GABA induced peakchloride current. The closed circles represent effects after 3 min perfusion of AF-16. Vertical barsrepresent SEM’s. Number of experiments was at least three in all cases. Statistical significanceof E% increase is reported as asterisks: ∗p < 0.05; ∗ ∗ p < 0.01; ∗ ∗ ∗p < 0.001. Adaptedfrom [18].of the peak chloride current at all the concentrations studied in the 1 pM–10μM range(always: p < 0.01 or p < 0.001; only for 1μM AF-16: p < 0.05, fig. 1). No effect wasfound on the basal (GABA independent) chloride current.Figure 2 presents an example of the recordings obtained with a 3 min long pre-treatment with 0.01 μM AF-16. After washout of the AF-16 peptide, the GABA currentwent back to its original level. This occurred in all experiments performed.A saturating dose-response curve was obtained for the stimulatory effect of AF-16 inthat range after a 3 min long incubation period and co-application with GABA (fig. 1).The fit of the data with eq. (2) yielded a low k of 41 pM, an E%Max of 37% and n = 0.64.In part of the experiments, AF-16 was pre-incubated 30 s at concentrations of 0.01–10μMbefore application of GABA. In this case there is a statistically significant increase of thepeak chloride current starting from 0.1μM. However, this effect is much lower than theone with 3 min incubation (p < 0.01) (Data not shown).Fig. 2. – Example of the effect of preincubation of cells with 0.01 μM AF-16 for 3 min on thepeak chloride current evoked by 10 μM GABA. The recording to the left is the current activatedby plain 10 μM GABA (a); the recording to the right is that of application of AF together withGABA after 3min perfusion with AF 0.01 μM AF-16 (b). Adapted from [18].GABAA RECEPTOR MODULATION ETC. 5Fig. 3. – Confocal image showing the γ2 subunit containing GABAA receptors distribution ingranule cells permeabilized with 0.1% Triton X-100 for 5 min before incubating with the primaryantibody. Bar = 10 μm.In another set of experiments, 1μM AF-16 was only added to the intracellularmedium. After such an addition, the chloride current evoked by 10μM GABA wasrecorded after breaking of the neuronal membrane in the whole cell configuration. Thishigh AF-16 concentration was used so as to favour its rapid diffusion into the cell body. Inthese experimental conditions, no increase of the GABA current was found (−3.0±2.0%,n = 3, ns). Furthermore, a 3 min long preincubation with external 0.01μM AF-16 fol-lowed by a 2 min washout did not result in a disappearance of the effect. Instead, theeffect persisted (+27.0 ± 8.0%, n = 5, p < 0.01).Immunocytochemical results. – Membrane expression of GABAA receptor γ2 subunitwas quantified by immunolabeling (fig. 3). The distribution of the γ2 subunit in con-trol granule cells and in cells pretreated with 1μM AF-16 was quantified in terms ofimmunoreactivity density on cells’ membranes. Indeed the membrane distribution of theγ2 subunit of GABAA receptors yielded a highly significant (p < 0.001) average increaseby +17.4 ± 0.4% (n = 32) after AF-16 treatment. When the calculations were extendedto comprehend also the cells’ interior, the level of γ2 subunit immunoreactivity resultedstill increased by AF (+16.2 ± 0.3%, n = 37; p < 0.001) (Data not shown).In fig. 4 immunofluorescence pictures demonstrate the expression of flotillin-1 in cere-bellar granule cells: the distribution of patches of flotillin-1 localized in the plasmamembrane.4. – DiscussionIn the present study we observed an increase, induced by AF-16, of the peak chloridecurrent evoked by GABA in cultured cerebellar granule cells. This effect of AF-16 is de-pendent on concentration and incubation time, and demonstrates a low k of 41 pM. Thefact that the AF-16 stimulated action persists for some minutes after washout tentatively6 E. GATTAFig. 4. – Confocal image showing flotillin-1 distribution in granule cells permeabilized with 0.1%Triton X-100 for 5 min before incubating with the primary antibody. Bar = 5 μm.suggests that a biochemical cascade reaction is started, which results in an increasedfunction or presence of GABAA receptors at the cellular membrane. The immunocyto-chemical data demonstrate an increased presence of the GABAA receptor γ2 subunit inthe cells, as receptors on their way to membrane expression but particularly as expressedreceptors on the cell plasma membrane. This subunit is characteristic of the synapticGABAA receptor population which generates the peak component of the GABA evokedcurrent [19, 20]. Therefore there is evidence that the AF effect is due to an increasedexpression/function of GABAA receptors at the cellular membrane surface. This actionmost probably involves a biochemical cascade of events. A possibility to be investigatedis the involvement of flotillin-1 which was shown to participate in neurotransmitter recep-tors’ membrane insertion at synapses [21]. In fact, AF has been shown to interact withit [22] and indeed flotillin-1 is expressed in patches on the membranes of the cerebellargranule cells (fig. 4).Flotillins have been implicated in myriad processes that include endocytosis, signaltransduction and regulation of the cortical cytoskeleton, yet the molecular mechanismsthat underlie flotillin function in these different cases are still poorly understood [23].Our data also show that AF-16 does not seem to act on the cellular inside, but evi-dently interacts with membrane structures or proteins located on the external part ofthe plasma membrane. The level of increase by AF of the peak GABA activated current(around 40%) and the lower increase of the γ2 subunit expression as evaluated by im-munocytochemistry (16–18%) suggest that the increase of the maximal current evokedby GABA is partly due to increased membrane expression of the receptors and partlydue to increased functionality.The AF-16 mediated modulation of GABAA receptor activity in central nervous sys-tem neurons has been studied in hippocampal slices [10,11].Moreover in [10] evidence has been provided that AF could be a negative modulatorof GABA ergic transmission in the hippocampus and that this protein may be involvedin a gut-brain loop controlling intestinal secretion and inflammation.It is becoming increasingly evident that bidirectional signalling exists between thegastrointestinal tract and the brain. This relationship, commonly dubbed the gut-brain axis involves various afferent and efferent pathways such as the vagus nerve andthe hypothalamic-pituitary-adrenal pathway to regulate aspects of homeostasis such asGABAA RECEPTOR MODULATION ETC. 7satiety and hunger, and inflammation. Disruption of the gut-brain axis has been shownto be involved in the pathogenesis of a diverse range of diseases, including Parkinsondisease and irritable bowel syndrome. Although the majority of data to-date is fromanimal models, this emerging area of research is evolving quickly and with promisingvalue.In that case, as in ours, the possibility that these effects are aspects of a gut-brainaxis cannot be neglected. There is a different end result by AF action resulting in lessinhibition in hippocampal pyramidal cells and more inhibition in cerebellar granule cells.This may be explained in terms of different cellular mechanisms and function withinthe relevant circuits in which the two types of cells are involved. In conclusion, theAF system has been shown to counteract intestinal hypersecretion [24,25], inflammatoryevents [26-28] and raises in intracranial pressure [29, 30]. It has also probably a rolein counteracting endolymph hyperproduction in the inner ear as implied by results inclinical trials in Meniere’s disease [31,32]. In all cases a prominent feature is the controlof fluid balance in different body organs. Further work is needed in order to establishwhether the AF system [24] is also involved in brain activity modulation.∗ ∗ ∗This work was supported by MIUR University of Genoa.REFERENCES[1] Johansson E., Lönnroth I., Lange S., Jonson J., Jennische E. and Lönnroth C.,J. Biol. Chem., 270 (1995) 20615.[2] Lönnroth I. and Lange S., FEBS Lett., 177 (1984) 104.[3] Lönnroth I. and Lange S., Biochim. Biophys. Acta., 883 (1986) 138.[4] Johansson E., Jennische E., Lange S. and Lönnroth I., Gut, 41 (1997) 642.[5] Lange S., Jennische E., Johansson E. and Lönnroth I., Cell Tissue Res., 296 (1999)607.[6] Johansson E., Lange S. and Lönnroth I., Biochim. Biophys. Acta., 1362 (1997) 177.[7] Rapallino M. V., Cupello A., Lange S. and Lönnroth I., Acta Physiol. Scand., 179(2003) 367.[8] Lönnroth I., Lange S. and Skadhauge E., Comp. Biochem. Physiol., 90A (1988) 611.[9] Grøndahl M. L., Sørensen H., Unmack M. A., Holm A. and Skadhauge E., J.Comp. Biochem. Physiol. A Neuroethol. Sens. Neural Behav. Physiol., 188 (2002) 589.[10] Kim M., Wasling P., Xiao M.-Y., Jennische E., Lange S. and Hanse E., Regul.Pept., 129 (2005) 109.[11] Strandberg J., Lindquist C., Lange S., Asztely F. and Hanse E., Front. CellNeurosci., 8 (2014) 1.[12] Krantis A., News Physiol. 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GABAA receptor modulation by the Antisecretory Factor on cerebellar granule cells

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GABAA receptor modulation by the Antisecretory Factor on cerebellar granule cells

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