In the epithelium of rat distal colon the acetylcholine analogue carbachol induces a transient increase of short-circuit current (Isc) via stimulation of cellular K+ conductances. Inhibition of the turnover of inositol-1,4,5-trisphosphate (IP3) by LiCl significantly reduced both the amplitude and the duration of this response. When the apical membrane was permeabilized with nystatin, LiCl nearly abolished the carbachol-induced activation of basolateral K+ conductances. In contrast, in epithelia, in which the basolateral membrane was bypassed by a basolateral depolarization, carbachol induced a biphasic increase in the K+ current across the apical membrane consisting of an early component carried by charybdotoxin- and tetraethylammonium-sensitive K+ channels followed by a sustained plateau carried by channels insensitive against these blockers. Only the latter was sensitive against LiCl or inhibition of protein kinases. In contrast, the stimulation of the early apical K+ conductance by carbachol proved to be resistant against inhibition of phospholipase C or protein kinases. However, apical dichlorobenzamil, an inhibitor of Na+/Ca2+ exchangers, or a Ca2+-free mucosal buffer solution significantly reduced the early component of the carbachol-induced apical K+ current. The presence of an apically localized Na+/Ca2+-exchanger was proven immunohistochemically. Taken together these experiments reveal divergent regulatory mechanisms for the stimulation of apical Ca2+-dependent K+ channels in this secretory epithelium, part of them being activated by an inflow of Ca2+ across the apical membrane.

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Ca2+-dependent secretagogues evoke only a transient Cl- secretion in intestinal epithelia, although they induce a prolonged increase in the intracellular Ca2+ concentration, suggesting that they may exert an additional antisecretory action. In order to study the mechanism of this antisecretory effect, Cl- secretion, measured as the increase in short-circuit current (Isc), was evoked by carbachol in the absence and presence of different inhibitors. Neither a calmodulin antagonist, calmidazolium, nor different inhibitors of the nitric oxide (NO) pathway, i.e. Nomega-nitro-L-arginine (L-NNA) and Nomega-nitro-L-arginine methylester (L-NAME), affected the carbachol-induced Isc. However, inhibition of phospholipases A2 (PLA2) by quinacrine or arachidonyltrifluoromethyl ketone (AACOCF3) enhanced the Isc response evoked by carbachol, suggesting a role of fatty acids in the downregulation of anion secretion. Neither econazole, a cytochrome P450 inhibitor, nor nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenases, mimicked the action of the PLA2 blockers. Conversely, short- or medium-chain fatty acids inhibited the carbachol- and forskolin-induced Isc with caprate (C10:0) being the most efficient water-soluble fatty acid. This fatty acid inhibited a Cl- current, which was driven across the apical membrane by a serosally to mucosally directed Cl- gradient after depolarization of the basolateral membrane. A second action site of fatty acids seems to be the basolateral membrane. After permeabilization of the apical membrane with the ionophore nystatin, a mucosally to serosally directed K+ gradient induced a K+ current, which was also inhibited by caprate. These results indicate that carbachol not only acts as a secretagogue but at the same time initializes downregulation by increasing the intracellular concentration of fatty acids, a mechanism limiting the resulting Cl- secretion.

The effect of cell swelling and cell shrinkage on K+ transport across the rat colonic epithelium was studied by measuring unidirectional fluxes, uptake and efflux of 86Rb+, a marker for K+. Exposure to a hypotonic medium stimulated the secretory, serosa-to-mucosa flux of K+, whereas exposure to a hypertonic medium inhibited the absorptive, mucosa-to-serosa flux of K+ in the distal, but not in the proximal colon. Neither manoeuvre had any effect on the uptake of K+ across the apical or the basolateral membrane. Cell swelling induced a sustained increase in the apical and basolateral K+ efflux from both colonic segments, whereas cell shrinkage reduced the efflux. Ba2+ (10(-2) mol l(-1)) inhibited the swelling-induced stimulation of the apical, quinine (10(-3) mol l(-1)) that of the basolateral K+ efflux in the distal colon. Incubation of the tissue in Ca2+-free buffer or La3+, which blocks Ca2+-influx into the epithelium, strongly reduced the basal K+ efflux across the basolateral membrane. The same was observed with brefeldin A, a blocker of the transport of newly synthesized proteins out of the endoplasmatic reticulum. Swelling-induced K+ efflux, however, was not reduced. In the presence of colchicine, an inhibitor of the polymerization of microtubules, swelling evoked only a transient increase in mucosal efflux, which, especially in the proximal colon, fell after 6 min to the level of the isotonic control period. These results demonstrate that the cell volume is involved in the regulation of transepithelial K+ transport across the rat colonic epithelium and suggest a role of the cytoskeleton in the control of a part of the volume-sensitive K+ channels.

Docente do Curso de Medicina Veterinária da UNOESTEPresidente Prudente -SP. RESUMOO microambiente do gameta feminino é o líquido folicular e, portanto, as proteínas ali presentes devem interferir na qualidade do oócito. Este experimento foi delineado com o objetivo de verificar o perfil proteico do líquido folicular de folículos em diferentes fases do ciclo estral de bovinos. Para tanto, os líquidos foliculares provenientes de ovários de vacas de abatedouro foram alocados em 5 diferentes fases do ciclo estral, levando em conta a presença ou ausência do corpo lúteo (CL) e sua caracterização morfológica. "Pools" de líquido folicular foram coletados de folículos de 2 a 7 mm de ovários em 5 diferentes fases do ciclo estral (1=CL inicial, hemorrágico; 2=CL em desenvolvimento; 3=CL maduro; 4=CL em regressão e 5=ausência de CL). O perfil proteico foi avaliado por eletroforese em gel de poliacrilamida e determinado em porcentagens nas amostras de cada fase. Os polipeptídeos de 35, 32, 30 e 18 KDa variaram consideravelmente nas amostras. O polipeptídeo de mesmo peso molecular que o IGFBP-2 está mais presente nas amostras de líquido folicular ao final do ciclo estral. Maior número de polipeptídeos foi observado em folículos antrais pequenos de ovários em que o CL está em regressão. Palavras-chave: eletroforese; IGFBP; polipeptídeos; proteínas; SDS-PAGE. PROTEIC PROFILE OF FOLLICULAR FLUID FROM OVARIES IN DIFFERENT PHASES OF BOVINE ESTROUS CYCLE. ABSTRACTThe female gamete's microenvironment is the follicular fluid. The proteins in the follicular fluid must interfere in the oocyte quality. The objective of this study was to verify the protein profile in the follicular fluid of different phases of bovine estrous cycle. The follicular fluid was taken from follicles of bovine ovaries obtained at slaughterhouse. The ovaries were classified into five different phases of estrous cycle, according to presence or absence of corpus luteum (CL) and the morphologic characteristics. Pools of follicular fluid were collected from follicles ranging from 2 to 7mm in diameter from ovaries in five different phases of estrous cycle (1=initial CL, hemorrhagic; 2= developing CL; 3= mature CL; 4=regressing CL and 5=absence CL). The protein profile was evaluated by poliacrilamide eletrophoresis and determined by percentages of samples from each phase. The polypeptides with molecular weigh of 35, 32, 30 and 18 KDa changed considerably in samples from different phases of estrous cycle. The polypeptide with the same molecular weight of IGFBP-2 is more observed in samples from follicular fluid in the end of estrous cycle. The most polypeptides were observed in small follicles from ovaries with regressing CL.