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Melatonin is a key hormone involved in the photoperiodic signaling pathway. In both teleosts and mammals, melatonin produced in the pineal gland at night is released into the blood and cerebrospinal fluid, providing rhythmic information to the whole organism. Melatonin acts via specific receptors, allowing the synchronization of daily and annual physiological rhythms to environmental conditions. The pituitary gland, which produces several hormones involved in a variety of physiological processes such as growth, metabolism, stress and reproduction, is an important target of melatonin. Melatonin modulates pituitary cellular activities, adjusting the synthesis and release of the different pituitary hormones to the functional demands, which changes during the day, seasons and life stages. It is, however, not always clear whether melatonin acts directly or indirectly on the pituitary. Indeed, melatonin also acts both upstream, on brain centers that control the pituitary hormone production and release, as well as downstream, on the tissues targeted by the pituitary hormones, which provide positive and negative feedback to the pituitary gland. In this review, we describe the known pathways through which melatonin modulates anterior pituitary hormonal production, distinguishing indirect effects mediated by brain centers from direct effects on the anterior pituitary. We also highlight similarities and differences between teleosts and mammals, drawing attention to knowledge gaps, and suggesting aims for future research.

Accumulating evidence indicates that some pituitary cell types are organized in complex networks in both mammals and fish. In this study, we have further investigated the previously described cellular extensions formed by the medaka (Oryzias latipes) luteinizing hormone gonadotropes (Lh cells). Extensions, several cell diameters long, with varicosity-like swellings, were common both in vitro and in vivo. Some extensions approached other Lh cells, while others were in close contact with blood vessels in vivo. Gnrh further stimulated extension development in vitro. Two types of extensions with different characteristics could be distinguished, and were classified as major or minor according to size, origin and cytoskeleton protein dependance. The varicosity-like swellings appeared on the major extensions and were dependent on both microtubules and actin filaments. Immunofluorescence revealed that Lhβ protein was mainly located in these swellings and at the extremity of the extensions. We then investigated whether these extensions contribute to network formation and clustering, by following their development in primary cultures. During the first two days in culture, the Lh cells grew long extensions that with time physically attached to other cells. Successively, tight cell clusters formed as cell somas that were connected via extensions migrated towards each other, while shortening their extensions. Laser photolysis of caged Ca2+ showed that Ca2+ signals originating in the soma propagated from the soma along the major extensions, being particularly visible in each swelling. Moreover, the Ca2+ signal could be transferred between densely clustered cells (sharing soma-soma border), but was not transferred via extensions to the connected cell. In summary, Lh gonadotropes in medaka display a complex cellular structure of hormone-containing extensions that are sensitive to Gnrh, and may be used for clustering and possibly hormone release, but do not seem to contribute to communication between cells themselves.

Perfluoroalkyl acids (PFAAs) are persistent man-made chemicals, ubiquitous in nature and present in human samples. Although restrictions are being introduced, they are still used in industrial processes as well as in consumer products. PFAAs cross the blood-brain-barrier and have been observed to induce adverse neurobehavioural effects in humans and animals as well as adverse effects in neuronal in vitro studies. The sulfonated PFAA perfluorooctane sulfonic acid (PFOS), has been shown to induce excitotoxicity via the N-methyl-D-aspartate receptor (NMDA-R) in cultures of rat cerebellar granule neurons (CGNs). In the present study the aim was to further characterise PFOS-induced toxicity (1–60 μM) in rat CGNs, by examining interactions between PFOS and elements of glutamatergic signalling and excitotoxicity. Effects of the carboxylated PFAA, perfluorooctanoic acid (PFOA, 300–500 μM) on the same endpoints were also examined. During experiments in immature cultures at days in vitro (DIV) 8, PFOS increased both the potency and efficacy of glutamate, whereas in mature cultures at DIV 14 only increased potency was observed. PFOA also increased potency at DIV 14. PFOS-enhanced glutamate toxicity was further antagonised by the competitive NMDA-R antagonist 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) at DIV 8. At DIV 8, PFOS also induced glutamate release (9–13 fold increase vs DMSO control) after 1−3 and 24 h exposure, whereas for PFOA a large (80 fold) increase was observed, but only after 24 h. PFOS and PFOA both also increased alanine and decreased serine levels after 24 h exposure. In conclusion, our results indicate that PFOS at concentrations relevant in an occupational setting, may be inducing excitotoxicity, and potentiation of glutamate signalling, via an allosteric action on the NMDA-R or by actions on other elements regulating glutamate release or NMDA-R function. Our results further support our previous findings that PFOS and PFOA at equipotent concentrations induce toxicity via different mechanisms of action.

Perfluoroalkyl acids (PFAAs) are persistent compounds used in many industrial as well as consumer products. Despite restrictions, these compounds are found at measurable concentrations in samples of human and animal origin. In the present study we examined whether the effects on cell viability of two sulfonated and four carboxylated PFAAs in cultures of cerebellar granule neurons (CGNs), could be associated with deleterious activation of the N-methyl-d-aspartate receptor (NMDA-R). PFAA-induced effects on viability in rat CGNs and unstimulated PC12 cells were examined using the MTT assay. Cells from the PC12 rat pheochromocytoma cell line lack the expression of functional NMDA-Rs and were used to verify lower toxicity of perfluorooctanesulfonic acid (PFOS) in cells not expressing NMDA-Rs. Protective effects of NMDA-R antagonists, and extracellular as well as intracellular Ca2+ chelators were investigated. Cytosolic Ca2+ ([Ca2+]i) was measured using Fura-2. In rat CGNs the effects of the NMDA-R antagonists MK-801, memantine and CPP indicated involvement of the NMDA-R in the decreased viability induced by PFOS and perfluorohexanesulfonic acid (PFHxS). No effects were associated with the four carboxylated PFAAs studied. Further, EGTA and CPP protected against PFOS-induced decreases in cell viability, whereas no protection was afforded by BAPTA-AM. [Ca2+]i significantly increased after exposure to PFOS, and this increase was completely blocked by MK-801. In PC12 cells a higher concentration of PFOS was required to induce equivalent levels of toxicity as compared to in rat CGNs. PFOS-induced toxicity in PC12 cells was not affected by CPP. In conclusion, PFOS at the tested concentrations induces excitotoxicity in rat CGNs, which likely involves influx of extracellular Ca2+ via the NMDA-R. This effect can be blocked by specific NMDA-R antagonists.

Accumulating evidence in the scientific literature indicates that some pituitary cell types are organized in complex networks. Previous observations have indicated that this may also be the case in medaka (Oryzias latipes), where long cellular extensions with varicosity-like swellings are formed by luteinizing hormone (Lh)-producing gonadotropes expressing green fluorescent protein. In this study, immunofluorescence of intact pituitaries reveal that Lh beta polypeptides are mainly located in the varicosity-like swellings and at the extremity of the extensions. Some extensions approach nearby Lh-producing cells, and other extensions are in close contact with blood vessels. To investigate whether these extensions may contribute to network formation, we followed their development using confocal and fluorescent microscopy on primary cultures. During the first two days in culture, the extensions initiated the formation of homotypic cellular networks and clustering. The extensions were classified as either major or minor. Major extensions were several cell diameters long, dependent on microtubules, and displaying varicosity-like swellings at regular intervals. Minor extensions typically protruded from the major, were significantly shorter and thinner, and dependent on actin. The swellings were dependent on both microtubules and actin. Flash photolysis of caged Ca2+showed that the signal was propagated along the major extensions, intensifying in each swelling, indicating a continuous structure. However, the Ca2+signal did not transfer to the next cell in the network, but was transferred between cells merged at their somas. In summary, Lh-producing gonadotropes in medaka display a complex cellular structure of extensions, possibly linked to communication with blood vessels and/or other gonadotrope cells.

Through the action of cortisol, stress can affect reproductive biology with behavioural and physiological alterations. Using mixed sex primary pituitary cultures from Atlantic cod (Gadus morhua), the present study aimed to investigate potential direct effects of basal and stress level cortisol on the pituitary in terms of cell viability and reproduction-related gene expression at different stages of sexual maturity. Stress level of cortisol stimulated cell viability in cells derived from sexually maturing and mature fish. In cells from spent fish, high cortisol levels did not affect cell viability in terms of metabolic activity, but did stimulate viability in terms of membrane integrity. Basal cortisol levels did not affect cell viability. Ethanol, used as solvent for cortisol, decreased cell viability at all maturity stages, but did generally not affect gene expression. Genes investigated were fshb, lhb and two Gnrh receptors expressed in cod gonadotropes (gnrhr1b and gnrhr2a). Cortisol had dual effects on fshb expression; stimulating expression in cells from mature fish at stress dose, while inhibiting expression in cells from spent fish at both doses. In contrast, cortisol had no direct effect on lhb expression. While gnrhr2a transcript levels largely increased following cortisol treatment, gnrhr1b expression decreased in cells from spent fish and was unaffected at other maturity stages. These findings demonstrate that cortisol can act directly and differentially at the pituitary level in Atlantic cod and that factors facilitating these actions are dose-dependently activated and vary with level of sexual maturity.

Often referred to as "the master gland", the pituitary is a key organ controlling growth, maturation, and homeostasis in vertebrates. The anterior pituitary, which contains several hormone-producing cell types, is highly plastic and thereby able to adjust the production of the hormones governing these key physiological processes according to the changing needs over the life of the animal. Hypothalamic neuroendocrine control and feedback from peripheral tissues modulate pituitary cell activity, adjusting levels of hormone production and release according to different functional or environmental requirements. However, in some physiological processes (e.g. growth, puberty, or metamorphosis), changes in cell activity may be not sufficient to meet the needs and a general reorganization of cell composition and pituitary structure may occur. Focusing on gonadotropes, this review examines plasticity at the cellular level, which allows precise and rapid control of hormone production and secretion, as well as plasticity at the population and structural levels, which allows more substantial changes in hormone production. Further, we compare current knowledge of the anterior pituitary plasticity in fishes and mammals in order to assess what has been conserved or not throughout evolution, and highlight important remaining questions.

Pituitary endocrine cells fire action potentials (APs) to regulate their cytosolic Ca2+ concentration and hormone secretion rate. Depending on animal species, cell type, and biological conditions, pituitary APs are generated either by TTX-sensitive Na+ currents (INa), high-voltage activated Ca2+ currents (ICa), or by a combination of the two. Previous computational models of pituitary cells have mainly been based on data from rats, where INa is largely inactivated at the resting potential, and spontaneous APs are predominantly mediated by ICa. Unlike in rats, spontaneous INa-mediated APs are consistently seen in pituitary cells of several other animal species, including several species of fish. In the current work we develop a computational model of gonadotropin releasing cells in the teleost fish medaka (Oryzias latipes). The model stands out from previous modeling efforts by being (1) the first model of a pituitary cell in teleosts, (2) the first pituitary cell model that fires sponateous APs that are predominantly mediated by INa, and (3) the first pituitary cell model where the kinetics of the depolarizing currents, INa and ICa, are directly fitted to voltage-clamp data. We explore the firing properties of the model, and compare it to the properties of previous models that fire ICa-based APs. We put a particular focus on how the big conductance K+ current (IBK) modulates the AP shape. Interestingly, we find that IBK can prolong AP duration in models that fire ICa-based APs, while it consistently shortens the duration of the predominantly INa-mediated APs in the medaka gonadotroph model. Although the model is constrained to experimental data from gonadotroph cells in medaka, it may likely provide insights also into other pituitary cell types that fire INa-mediated APs., Author summary Excitable cells elicit electrical pulses called action potentials (APs), which are generated and shaped by a combination of ion channels in the cell membrane. Since one type of ion channels is permeable to Ca2+ ions, there is typically an influx of Ca2+ during an AP. Pituitary cells therefore use AP firing to regulate their cytosolic Ca2+ concentration, which in turn controls their hormone secretion rate. The amount of Ca2+ that enters during an AP depends strongly on how long it lasts, and it is therefore important to understand the mechanisms that control this. Pituitary APs are generally mediated by a combination of Ca2+ channels and Na+ channels, and the relative contributions of from the two vary between cell types, animal species and biological conditions. Previous computer models have predominantly been adapted to data from pituitary cells which tend to fire Ca2+-based APs. Here we develop a new model, adapted to data from pituitary cells in the fish medaka, which APs that are predominantly Na+-based, and compare its dynamical properties to the previous models that fire Ca2+-based APs.

Bisphenol A (BPA) and tetrabromobisphenol A (TBBPA) are widely used industrial chemicals, ubiquitously present in the environment. While BPA is a well-known endocrine disruptor and able to affect all levels of the teleost reproductive axis, information regarding TBBPA on this subject is very limited. Using primary cultures from Atlantic cod (Gadus morhua), the present study was aimed at investigating potential direct effects of acute (72 h) BPA and TBBPA exposure on cell viability and the expression of reproductive-relevant genes in the pituitary. The results revealed that both bisphenols stimulate cell viability in terms of metabolic activity and membrane integrity at environmentally relevant concentrations. BPA had no direct effects on gonadotropin gene expression, but enhanced the expression of gonadotropin-releasing hormone (GnRH) receptor 2a, the main gonadotropin modulator in Atlantic cod. In contrast, TBBPA increased gonadotropin transcript levels but had no effect on GnRH receptor mRNA. In conclusion, both anthropogenic compounds display endocrine disruptive properties and are able to directly interfere with gene expression related to reproductive function in cod pituitary cells at environmentally relevant concentrations in vitro.

We have previously characterized the response to gonadotropin-releasing hormone (Gnrh) 2 in luteinizing hormone ( lhb )-expressing cells from green fluorescent protein (Gfp)-transgenic medaka ( Oryzias latipes ), with regard to changes in the cytosolic Ca 2+ concentration. In the current study we present the corresponding responses to Gnrh1 and Gnrh3. Ca 2+ imaging revealed three response patterns to Gnrh1 and Gnrh3, one monophasic and two types of biphasic patterns. There were few significant differences in the shape of the response patterns between the three Gnrh forms, although the amplitude of the Ca 2+ signal was considerably lower for Gnrh1 and Gnrh3 than for Gnrh2, and the distribution between the two different biphasic patterns differed. The different putative Ca 2+ sources were examined by depleting intracellular Ca 2+ stores with thapsigargin, or preventing influx of extracellular Ca 2+ by either extracellular Ca 2+ depletion or the L-type Ca 2+ -channel blocker verapamil. Both Gnrh1 and 3 relied on Ca 2+ from both intracellular and extracellular sources, with some unexpected differences in the relative contribution. Furthermore, gene expression of Gnrh-receptors ( gnrhr ) in whole pituitaries was studied during development from juvenile to adult. Only two of the four identified medaka receptors were expressed in the pituitary, gnrhr1b and gnrhr2a , with the newly discovered gnrhr2a showing the highest expression level at all stages as analyzed by quantitative PCR. While both receptors differed in expression level according to developmental stage, only the expression of gnrhr2a showed a clear-cut increase with gonadal maturation. RNA sequencing analysis of FACS-sorted Gfp-positive lhb -cells revealed that both gnrhr1b and gnrhr2a were expressed in lhb -expressing cells, and confirmed the higher expression of gnrhr2a compared to gnrhr1b . These results show that although lhb -expressing gonadotropes in medaka show similar Ca 2+ response patterns to all three endogenous Gnrh forms through the activation of two different receptors, gnrhr1b and gnrhr2a , the differences observed between the Gnrh forms indicate activation of different Ca 2+ signaling pathways.

Pituitary endocrine cells fire action potentials (APs) to regulate their cytosolic Ca2+ concentration and hormone secretion rate. Depending on animal species, cell type, and biological conditions, pituitary APs are generated either by TTX-sensitive Na+ currents (INa), high-voltage activated Ca2+ currents (ICa), or by a combination of the two. Previous computational models of pituitary cells have mainly been based on data from rats, where INa is largely inactivated at the resting potential, and spontaneous APs are exclusively mediated by ICa. As a part of the previous modeling studies, a paradoxical role was identified for the big conductance K+ current (IBK), which was found to prolong the duration of ICa-mediated APs, and sometimes give rise to pseudo-plateau bursts, contrary to what one would expect from a hyperpolarizing current. Unlike in rats, spontaneous INa-mediated APs are consistently seen in pituitary cells of several other animal species, including several species of fish. In the current work we develop the, to our knowledge, first computational model of a pituitary cell that fires INa-mediated APs. Although we constrain the model to experimental data from gonadotrope cells in the teleost fish medaka (Oryzias latipes), it may likely provide insights also into other pituitary cell types that fire INa-mediated APs. In the current work, we use the model to explore how the effect of IBK depends on the AP generating mechanisms of pituitary cells. We do this by comparing simulations on the medaka gonadotrope model (two versions thereof) with simulations on a previously developed model of a rat pituitary cell. Interestingly, we find that IBK has the opposite effect on APs in the two models, i.e. it reduces the duration of already fast INa-mediated APs in the medaka model, and prolongs the duration of already slow ICa-mediated APs in the rat model.Author summaryExcitable cells elicit electrical pulses called action potentials (APs), which are generated and shaped by a combination of ion channels in the cell membrane. While neurons use APs for interneuronal communication and heart cells use them to generate heart-beats, pituitary cells use APs to regulate their cytosolic Ca2+ concentration, which in turn controls their hormone secretion rate. The amount of Ca2+ that enters the pituitary cell during an AP depends strongly on how long it lasts, and it is therefore important to understand the mechanisms that control this. Depending on animal species and biological conditions, pituitary APs may be initiated either by Ca2+ channels or Na+ channels. Here, we explore the differences between the two scenarios by comparing simulations on two different computer models: (i) a previously developed model which fires Na+-based APs, adapted to data from pituitary cells in rats, and (ii) a novel model that fires Ca2+-based APs, adapted to data from pituitary cells in the fish medaka. Interestingly, we find that the role of big conductance K+ (BK) channels, which are known to affect the duration of the AP, are opposite in the two models, i.e., they act to prolong Ca2+-based APs while they act to shorten Na+-based APs.

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled environment. Despite vast differences in the physiology between mammals and fish, primary cell culture protocols from fish are often based on mammalian culture conditions, often with only minor modifications. The environmental differences affect not only body temperature, but also blood serum parameters such as osmolality, pH, and pH buffer capacity. As cell culture media and similar working solutions are meant to mimic characteristics of the extracellular fluid and/or blood serum to which a cell is adapted, it is crucial that these parameters are adjusted specifically to the animal in question. The current protocol describes optimized primary culture conditions for medaka (Oryzias latipes). The protocol provides detailed steps on how to isolate and maintain healthy dissociated pituitary cells for more than one week and includes the following steps: 1. the adjustment of the osmolality to the values found in medaka blood plasma, 2. the adjustment of the incubation temperature to normal medaka temperature (here in the aquarium facility), and 3. the adjustment of the pH and bicarbonate buffer to values comparable to other fish species living at similar temperatures. The results presented using the described protocol promote physiologically meaningful results for medaka and can be used as a reference guide by scientists making primary cell cultures from other non-mammalian species.

Depending on the stage of gonad maturation, as well as other factors, gonadal steroids can exert either a positive or negative feedback at the brain and pituitary level. While this has been demonstrated in many teleost species, little is known about the nature of steroid feedback in Gadiform fish. Using an optimized

Lactobacillus plantarum C11 releases plantaricin A (PlnA), a cationic peptide pheromone that has a membrane-permeabilizing, antimicrobial effect. We have previously shown that PlnA may also permeabilize eukaryotic cells, with a potency that differs between cell types. It is generally assumed that cationic antimicrobial peptides exert their effects through electrostatic attraction to negatively charged phospholipids in the membrane. The aim of the present study was to investigate if removal of the negative charge linked to glycosylated proteins at the cell surface reduces the permeabilizing potency of PlnA. The effects of PlnA were tested on clonal rat anterior pituitary cells (GH 4 cells) using patch clamp and microfluorometric techniques. In physiological extracellular solution, GH 4 cells are highly sensitive to PlnA, but the sensitivity was dramatically reduced in solutions that partly neutralize the negative surface charge of the cells, in agreement with the notion that electrostatic interactions are probably important for the PlnA effects. Trypsination of cells prior to PlnA exposure also rendered the cells less sensitive to the peptide, suggesting that negative charges linked to membrane proteins are involved in the permeabilizing action. Finally, pre-exposure of cells to a mixture of enzymes that split carbohydrate residues from the backbone of glycosylated proteins also impeded the PlnA-induced membrane permeabilization. We conclude that electrostatic attraction between PlnA and glycosylated membrane proteins is probably an essential first step before PlnA can interact with membrane phospholipids. Deviating glycosylation patterns may contribute to the variation in PlnA sensitivity of different cell types, including cancerous cells and their normal counterparts.

The onset of puberty and reproduction are tightly controlled by extrinsic and intrinsic inputs combined with genetically determined biological blueprints. Environmental inputs are then mediated by the brain– pituitary–gonad endocrine axis resulting in a unified output. In fish, one of the primary factors controlling the timing of sexual maturation is light, although how these signals are mediated in the brain and pituitary is not well understood. We therefore aimed to elucidate the molecular basis of the control of reproduction during the first spawning season in two year old female Atlantic cod. To this end, we measured GnRH and GnRH-R variant gene expression in brains and pituitaries collected from cod kept under four different photoperiod regimes: natural light (NL), continuous light (LL) and combined treatment of NL–LL and LL–NL. LL inhibited sexual development and spawning and LL–NL delayed sexual development and spawning. LL inhibited the spawning-related increase in brain GnRH3 and pituitary GnRH-R2a gene expression found under NL conditions, and the expression of these genes were delayed in concert with spawning of LL–NL cod. This study indicates that regulation of brain GnRH3 and pituitary GnRH-R2a genes likely mediates photoperiod induced changes in cod gonadal maturation.

Gonadotropin releasing hormone (GnRH) is a key regulator of sexual development and reproduction in vertebrates. Fish have either two or three pre-pro-GnRH genes, encoding structurally distinct peptides. We identified three pre-pro-GnRH genes in Atlantic cod (Gadus morhua, gmGnRH) using RT-PCR, RACE-PCR and BAC DNA library clone sequencing based on synteny searching. Gene identity was confirmed by sequence alignment and subsequent phylogenetic analysis. The expression of these genes was measured by quantitative PCR in the brain and pituitary of female cod throughout their reproductive cycle and in peripheral tissues. All three gmGnRH genes have highly conserved deduced decapeptide sequences, but sequence and phylogenetic data for gmGnRH1 suggest that this is a pseudogene. gmGnRH1 shares low identity with all fish GnRH variants and grouped with the GnRH3 clade. Although gmGnRH1 is a putative pseudogene, it is transcribed in multiple tissues but at low levels in the brain, indicating the loss of conserved hypophysiotrophic function. Phylogenetic analysis reveals that gmGnRH2 and gmGnRH3 variants are located in variant-specific clades. Both gmGnRH2 and gmGnRH3 transcripts are most abundant in the brain, with lower expression in pituitaries and ovaries. Brain gmGnRH3 gene expression increases in spawning fish and is expressed in the pituitary during puberty. Brain gmGnRH2 transcripts are highly expressed relative to gmGnRH3 before and during spawning. Sequence and expression data suggest that gmGnRH1 is a pseudogene and that gmGnRH3 is likely the hypophysiotrophic form of GnRH in Atlantic cod.

The nonhemolytic enterotoxin (Nhe) produced by Bacillus cereus is a pore-forming toxin consisting of three components, NheA, -B and -C. We have studied effects of Nhe on primate epithelial cells (Vero) and rodent pituitary cells (GH(4)) by measuring release of lactate dehydrogenase (LDH), K(+) efflux and the cytosolic Ca(2+) concentration ([Ca(2+)](i)). Plasma membrane channel events were monitored by patch-clamp recordings. Using strains of B. cereus lacking either NheA or -C, we examined the functional role of the various components. In both cell types, NheA + B + C induced release of LDH and K(+) as well as Ca(2+) influx. A specific monoclonal antibody against NheB abolished LDH release and elevation of [Ca(2+)](i). Exposure to NheA + B caused a similar K(+) efflux and elevation of [Ca(2+)](i) as NheA + B + C in GH(4) cells, whereas in Vero cells the rate of K(+) efflux was reduced by 50% and [Ca(2+)](i) was unaffected. NheB + C had no effect on either cell type. Exposure to NheA + B + C induced large-conductance steps in both cell types, and similar channel insertions were observed in GH(4) cells exposed to NheA + B. In Vero cells, NheA + B induced channels of much smaller conductance. NheB + C failed to insert membrane channels. The conductance of the large channels in GH(4) cells was about 10 nS. This is the largest channel conductance reported in cell membranes under quasi-physiological conditions. In conclusion, NheA and NheB are necessary and sufficient for formation of large-conductance channels in GH(4) cells, whereas in Vero cells such large-conductance channels are in addition dependent on NheC.

Certain antimicrobial peptides from multicellular animals kill a variety of tumor cells at concentrations not affecting normal eukaryotic cells. Recently, it was reported that also plantaricin A (PlnA), which is a peptide pheromone with strain-specific antibacterial activity produced by Lactobacillus plantarum, permeabilizes cancerous rat pituitary cells (GH(4) cells), whereas normal rat anterior pituitary cells are resistant to the peptide. To examine whether the preferential permeabilization of cancerous cells is a general feature of PlnA, we studied its effect on primary cultures of cells from rat liver (hepatocytes, endothelial, and Kupffer cells) and rat kidney cortex, as well as two epithelial cell lines of primate kidney origin (Vero cells from green monkey and human Caki-2 cells). The Vero cell line is derived from normal cells, whereas the Caki-2 cell line is derived from a cancerous tumor. The membrane effects were studied by patch clamp recordings and microfluorometric (fura-2) monitoring of the cytosolic concentrations of Ca(2+) ([Ca(2+)](i)) and fluorophore. In all the tested cell types except Kupffer cells, exposure to 10-100 microM PlnA induced a nearly instant permeabilization of the membrane, indicated by the following criteria: increased membrane conductance, membrane depolarization, increased [Ca(2+)](i), and diffusional loss of fluorophore from the cytosol. At a concentration of 5 microM, PlnA had no effect on any of the cell types. The Kupffer cells were permeabilized by 500 microM PlnA. We conclude that the permeabilizing effect of PlnA is not restricted to cancerous cells.

Plantaricin A (PlnA) is a 26-mer peptide pheromone with membrane-permeabilizing, strain-specific antibacterial activity, produced by Lactobacillus plantarum C11. We investigated the membrane-permeabilizing effects of PlnA on cultured cancerous and normal rat anterior pituitary cells using patch-clamp techniques and microfluorometry (fura-2). Cancerous cells displayed massive permeabilization within 5 s after exposure to 10–100 μm PlnA. The membrane depolarized to nearly 0 mV, and the membrane resistance decreased to a mere fraction of the initial value after less than 1 min. In outside-out membrane patches, 10 μm PlnA induced membrane currents reversing at 0 mV, which is compatible with an unspecific conductance increase. The d and l forms of the peptide had similar potency, indicating a nonchiral mechanism for the membrane-permeabilizing effect. Surprisingly, inside-out patches were insensitive to 1 mm PlnA. Primary cultures of normal rat anterior pituitary cells were also insensitive to the peptide. Thus, PlnA differentiates between plasma membranes and membrane leaflets. Microfluorometric recordings of [Ca2+] i and cytosolic concentration of fluorochrome verified the rapid permeabilizing effect of PlnA on cancerous cells and the insensitivity of normal pituitary cells.

The aim of the present study was to explore the electrophysiological properties of pituitary cells from Atlantic cod (Gadus morhua), as a basis for future studies of the signaling pathways involved in the control of pituitary secretion in this species. Primary cultures of pituitary cells from maturing Atlantic cod were prepared by trypsin treatment and mechanical dispersion. Electrophysiological recordings were performed using the perforated patch clamp method. A subpopulation of large cells were selected for recordings. Spontaneous action potentials were observed in about 30% of the cells. The action potentials displayed a fast initial spike followed by a prolonged plateau. Correspondingly, the inward current elicited by depolarizing steps consisted of both a transient, tetrodotoxin-sensitive Na+ component and a nifedipine-sensitive Ca2+ component that was sustained when Ba2+ replaced Ca2+ as current carrier. The outward current was partially blocked both by 5 mM tetraethylammonium and 10 mM 4-aminopyridine. The voltage-activated ion channels present in these cells largely correspond to the ion channels of pituitary cells in other teleosts (goldfish, Carassius auratus, and tilapia, Oreochromis mossambicus) and mammals, although differences exist regarding the shape and duration of action potentials.

Aim: Membrane stretch due to cell swelling may cause a minute leakage of adenosine triphosphate (ATP) that stimulates endogenous purinergic receptors. The following elevation of the cytosolic-free Ca 2+ concentration ([Ca 2+ ]i) may then participate in cell volume regulation. The aim of the present study was to test if purinergic receptors and large conductance Ca 2+ activated K + (BK) channels are activated in response to hypotonic stress in clonal kidney cells (Vero cells). Methods: The methods used are fura-2 microfluorometry, cell-attached patch clamp and reverse-transcriptase polymerase chain reaction (RT-PCR). Results: Subjecting cells to hypotonic stress for 10 s by exposure to a solution with 45% reduced osmolality induced a transient rise in [Ca 2+ ]i. This response persisted in virtually Ca 2+ -free extracellular solution, demonstrating that Ca 2+ was mainly released from intracellular stores. The hypotonically induced elevation of [Ca 2+ ]i was completely inhibited by the P2 receptor antagonists suramine (100 lm) and pyridoxalphosphate-6-azophenyl-2¢4¢-disulphonate (PPADS; 20 lm), indicating that extracellular ATP is crucial for the [Ca 2+ ]i increase. RT-PCR revealed the expression of mRNA for P2Y1 receptors in Vero cells. The putatively selective P2Y1 antagonist PPADS did completely block Ca 2+ responses to both ATP and hypotonic stress, suggesting that P2Y1 receptors are mediating the response. Furthermore, patch clamp recordings in cell-attached configuration revealed that BK channels are activated in response to hypotonic stress. Conclusion: Vero cells express functional purinergic receptors, presumably of the P2Y1 subtype. These receptors are responsible for the elevation of [Ca 2+ ]i evoked by hypotonic stress. The concurrent activation of BK channels

Aims: Thyrotropin-releasing hormone (TRH) induces biphasic changes in electrical activity, cytosolic free Ca 2 + level ([Ca 2 + ] i ), and prolactin secretion from both clonal GH cells and native lactotrophs. The first phase of the TRH response is characterized by hyperpolarization because of activation of Ca 2 + -activated K + channels (K C a ). In the present study, the relative contribution of BK, SK, and IK channels to the first phase of the TRH response in GH 4 cells was assessed. Methods: The expression of IK channels was confirmed by PCR with specific primers for SK4 (IK). The response to TRH was studied using the perforated patch technique and Ca 2 + microfluoromety (fura-2). The involvement of different K C a channels was estimated by employing the specific channel blockers iberiotoxin (BK), apamin (SK) and clotrimazole (IK). Results: Application of 100 nM iberiotoxin, 1 μM apamin, and 10 μM clotrimazole reduced the peak value of the outward K + current during the first phase of the TRH response by 33, 26, and 33%, respectively. Clotrimazole also shortened the duration of the outward current response by 60%, causing a reduction of total charge movement by 73%. All these toxin-induced reductions were significant (P < 0.05). A combination of all three toxins abolished the current response almost completely. Conclusion: All the three main types of K C a channels are involved in the first phase of the TRH response, with IK as the major contributor. This is the first demonstration of a dominant role of IK compared with BK and SK channels in excitable cells.

Aim: Thyrotropin-releasing hormone (TRH) induces biphasic changes in the electrical activity, the cytosolic free Ca 2+ concentration ([Ca 2+ ] i ), and prolactin secretion from both GH cells and native lactotrophs. It is well established that inhibition of erg channels contributes to the second phase of the TRH response. We have investigated if BK channels are also involved. Results: The BK channels may be active at the resting membrane potential (open probability, P o = 0.01) in clonal rat anterior pituitary cells (GH 4 ), which makes it possible that inhibition of these channels may contribute to the reduced K + conductance during the TRH response. The specific BK channel blocker iberiotoxin (IbTx, 100 nM) had no effect on the resting conductance at holding potentials negative to -40 mV, but significantly reduced the conductance at shallower membrane potentials. This corresponds to the voltage dependency of the sustained [Ca 2+ ] i . Furthermore, IbTx increased the action potential frequency by 36% in spontaneously firing cells. During the second phase of the TRH response, the action potential frequency increased by 34%, concomitantly with 61% reduction of the P o of single BK channels. The protein kinase C (PKC)-activating phorbol ester TPA had no significant effect on BK channel P o within the normal range of the resting potential. Conclusion: The BK channels may contribute to the resting membrane conductance, and they are partially inhibited by TRH during the second phase. This modulation seems not to depend on PKC. We propose that inhibition of erg and BK channels acts in concert to enhance the cell excitability during the second phase of the response to TRH.

RNA-Seq has become a widely used method to study transcriptomes, and it is now possible to perform RNA-Seq on almost any sample. Nevertheless, samples obtained from small cell populations are particularly challenging, as biases associated with low amounts of input RNA can have strong and detrimental effects on downstream analyses. Here we compare different methods to normalize RNA-Seq data obtained from minimal input material. Using RNA from isolated medaka pituitary cells, we have amplified material from six samples before sequencing. Both synthetic and real data are used to evaluate different normalization methods to obtain a robust and reliable pipeline for analysis of RNA-Seq data from samples with very limited input material. The analysis outlined here shows that quantile normalization outperforms other more commonly used normalization procedures when using amplified RNA as input and will benefit researchers employing low amounts of RNA in similar experiments.

We have studied modulation of the slow Ca2+-activated K+current ( IsAHP) in CA1 hippocampal pyramidal neurons by three peptide transmitters: corticotropin releasing factor (CRF, also called corticotropin releasing hormone, CRH), vasoactive intestinal peptide (VIP), and calcitonin gene–related peptide (CGRP). These peptides are known to be expressed in interneurons. Using whole cell voltage clamp in hippocampal slices from young rats, in the presence of tetrodotoxin (TTX, 0.5 μM) and tetraethylammonium (TEA, 5 mM), IsAHPwas measured after a brief depolarizing voltage step eliciting inward Ca2+current. Each of the peptides CRF (100–250 nM), VIP (400 nM), and CGRP (1 μM) significantly reduced the amplitude of IsAHP. Thus the IsAHPamplitude was reduced to 22% by 100 nM CRF, to 17% by 250 nM CRF, to 22% by 400 nM VIP, and to 40% by 1 μM CGRP. We found no consistent concomitant changes in the Ca2+current or in the time course of IsAHPfor any of the three peptides, suggesting that the suppression of IsAHPwas not secondary to a general suppression of Ca2+channel activity. Because each of these peptides is known to activate the cyclic AMP (cAMP) cascade in various cell types, and IsAHPis known to be suppressed by cAMP via the cAMP-dependent protein kinase (PKA), we tested whether the effects on IsAHPby CRF, VIP, and CGRP are mediated by PKA. Intracellular application of the PKA-inhibitor Rp-cAMPS significantly reduced the suppression of IsAHPby CRF, VIP, and CGRP. Thus with 1 mM Rp-cAMPS in the recording pipette, the average suppression of IsAHPwas reduced from 78 to 26% for 100 nM CRF, from 83 to 32% for 250 nM CRF, from 78 to 30% for 400 nM VIP, and from 60 to 7% for 1 μM CGRP. We conclude that CRF, VIP, and CGRP suppress the slow Ca2+-activated K+current, IsAHP, in CA1 hippocampal pyramidal neurons by activating the cAMP-dependent protein kinase, PKA. Together with the monoamine transmitters norepinephrine, serotonin, histamine, and dopamine, these peptide transmitters all converge on the cAMP cascade modulating IsAHP.

Pedarzani, Paola, Michael Krause, Trude Haug, Johan F. Storm, and Walter Stühmer. Modulation of the Ca2+-activated K+current s IAHPby a phosphatase-kinase balance under basal conditions in rat CA1 pyramidal neurons. J. Neurophysiol. 79: 3252–3256, 1998. The slow Ca2+-activated K+current, s IAHP, underlying spike frequency adaptation, was recorded with the whole cell patch-clamp technique in CA1 pyramidal neurons in rat hippocampal slices. Inhibitors of serine/threonine protein phosphatases (microcystin, calyculin A, cantharidic acid) caused a gradual decrease of s IAHPamplitude, suggesting the presence of a basal phosphorylation-dephosphorylation turnover regulating s IAHP. Because selective calcineurin (PP-2B) inhibitors did not affect the amplitude of s IAHP, protein phosphatase 1 (PP-1) or 2A (PP-2A) are most likely involved in the basal regulation of this current. The ATP analogue, ATP-γ-S, caused a gradual decrease in the s IAHPamplitude, supporting a role of protein phosphorylation in the basal modulation of s IAHP. When the protein kinase A (PKA) inhibitor adenosine-3′,5′-monophosphorothioate, Rp-isomer (Rp-cAMPS) was coapplied with the phosphatase inhibitor microcystin, it prevented the decrease in the s IAHPamplitude that was observed when microcystin alone was applied. Furthermore, inhibition of PKA by Rp-cAMPS led to an increase in the s IAHPamplitude. Finally, an adenylyl cyclase inhibitor (SQ22,536) and adenosine 3′,5′-cyclic monophosphate-specific type IV phosphodiesterase inhibitors (Ro 20–1724 and rolipram) led to an increase or a decrease in the s IAHPamplitude, respectively. These findings suggest that a balance between basally active PKA and a phosphatase (PP-1 or PP-2A) is responsible for the tonic modulation of s IAHP, resulting in a continuous modulation of excitability and firing properties of hippocampal pyramidal neurons.

Activation of large conductance Ca 2+ -activated K + channels (BK channels) in intact clonal rat pituitary cells (GH 4 cells) was investigated using the cell-attached patch-clamp configuration. This method prevents loss of intracellular factors which might influence channel activity. BK channels are generally considered to be inactive at the resting membrane potential in excitable cells. However, at the resting potential (0 mV pipette potential), 40% of the cell-attached patches displayed spontaneously active BK channels, which remained active even at 20 mV hyperpolarization. The peptide thyroliberin (TRH) elevates the cytosolic Ca 2+ concentration (Ca 2+ 1,1 in GH cells by IP 3 -induced release of Ca 2+ from intracellular stores. This rise in (Ca 2+ 1, occurs concomitantly with membrane hyperpolarization. TRH stimulation caused activation of BK channels in nine out of 30 silent cell-attached patches, and caused enhanced channel activity in seven out of 29 cell-attached patches containing spontaneously active BK channels. The Ca 2+ ionophore ionomycin activated silent BK channels in three out of 10 cell-attached patches, and increased the activity of spontaneously active BK channels in seven out of 16 cell-attached patches. The pipette potential was clamped to 0 mV in all these experiments. We conclude that the BK channels in GH 4 cells may be active at the resting membrane potential and more negative membrane potentials. The channels may also be activated further by physiological elevations of [Ca 2+ ) ; in the same potential range. Our results point towards new possible physiological roles for the BK channels in GH 4 cells. This is in agreement with the emerging picture of BK channels highly sensitive to [Ca 2+ ], in a wide variety of cell types.

Pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are key regulators of vertebrate reproduction. The differential regulation of these hormones, however, is poorly understood and little is known about gonadotrope embryonic development. The different cell types in the vertebrate pituitary develop from common progenitor cells just after gastrulation. Proper development and merging of the anterior and posterior pituitary is dependent upon carefully regulated cell-to-cell interactions, and a suite of signaling pathways with precisely organized temporal and spatial expression patterns, which include transcription factors and their co-activators and repressors. Among the pituitary endocrine cell types, the gonadotropes are the last to develop and become functional. Although much progress has been made during the last decade regarding details of gonadotrope development, the coordinated program for their maturation is not well described. FSH and LH form an integral part of the hypothalamo-pituitary-gonad axis, the main regulator of gonad development and reproduction. Besides regulating gonad development, pre- and early post-natal activity in this axis is thought to be essential for proper development, especially of the central nervous system in mammals. As a means to investigate early functions of FSH and LH in more detail, we have developed a stable transgenic line of medaka with the LH beta subunit gene (lhb) promoter driving green fluorescent protein (Gfp) expression to characterize development of lhb-expressing gonadotropes. The lhb gene is maternally expressed early during embryogenesis. lhb-Expressing cells are initially localized outside the primordial pituitary in the developing gut tube as early as 32 hpf. At hatching, lhb-Gfp is clearly detected in the gut epithelium and in the anterior digestive tract. lhb-Gfp expression later consolidates in the developing pituitary by 2 weeks post-fertilization. This review discusses status of knowledge regarding pituitary morphology and development, with emphasis on gonadotrope cells and gonadotropins during early development, comparing main model species like mouse, zebrafish and medaka, including possible developmental functions of the observed extra pituitary expression of lhb in medaka.

Synthesis and release of FSH and LH are differentially regulated by GnRH, but the mechanisms by which this regulation is achieved are not well understood. Teleost fish are powerful models for studyingthisdifferentialregulationbecausetheyhavedistinctpituitarycellsproducingeitherFSH or LH. By using pituitary cultures from Atlantic cod (Gadus morhua), we were able to investigate and compare the electrophysiological properties of fshb- and lhb-expressing cells, identified by single-cell quantitative PCR after recording. Both cell types fired action potentials spontaneously. The relative number of excitable cells was dependent on reproductive season but varied in opposing directions according to season in the 2 cell types. Excitable and quiescent gonadotropes displayed different ion channel repertoires. The dynamics of outward currents and GnRH-induced membrane responses differed between fshb- and lhb-expressing cells, whereas GnRH-induced cytosolic Ca 2 responses were similar. Expression of Ca 2 -activated K channels also differed with cell type and showed seasonal variation when measured in whole pituitary. The differential presence of these channels corresponds to the differences observed in membrane response to GnRH. We speculate that differences in ion channel expression levels may be involved in seasonal regulation of hormone secretion as well as the differential response to GnRH in LHand FSH-producing gonadotropes, through differences in excitability and Ca 2 influx. (Endocrinology 154: 3319–3330, 2013)

Background: Luteinizing hormone (LH) and follicle stimulating hormone (FSH), produced in gonadotrope cells in the adenohypophysis are key regulators of vertebrate reproduction. The differential regulation of these hormones, however, is poorly understood and little is known about gonadotrope embryonic development. We developed a stable transgenic line of medaka with the LH beta subunit gene (lhb) promotor driving green fluorescent protein (gfp) expression to characterize development of LH-producing gonadotropes in whole larvae and histological sections. Additionally, developmental and tissue-specific gene expression was examined. Results: The lhb gene is maternally expressed during early embryogenesis. Transcript levels increase by stage 21 (36 hours post fertilization [hpf]) and then decrease during continued larval development. Examination of the expression of pituitary marker genes show that LH-producing cells are initially localized outside the primordial pituitary, and they were localized to the developing gut tube by 32 hpf. At hatching, lhb-GFP is clearly detected in the gut epithelium and in the anterior digestive tract. lhb-GFP expression later consolidate in the developing pituitary by 2 weeks postfertilization. Conclusions: During embryonic development, lhb is primarily expressed outside the central nervous system and pituitary. The novel expression of lhb in the embryonic gut suggests that LH has a hitherto unidentified developmental function. Developmental Dynamics 241:1665–1677, 2012. © 2012 Wiley Periodicals, Inc.

Gonadotropin releasing hormones (GnRH) are an important part of the brain–pituitary–gonad axis in vertebrates. GnRH binding to its receptors (GnRH-R) stimulates synthesis and release of gonadotropins in the pituitary. GnRH-Rs also mediate other processes in the central nervous system such as reproductive behavior and neuromodulation. As many as five GnRH-R genes have been identified in two teleost fish species, but the function and phylogenetic relationship of these receptors is not fully understood. To gain a better understanding of the functional relationship between multiple GnRH-Rs in an important aquaculture species, the Atlantic cod (Gadus morhua), we identified four GnRH-Rs (gmGnRH-R) by RT-PCR, followed by full-length cloning and sequencing. The deduced amino acid sequences were used for phylogenetic analysis to identify conserved functional motifs and to clarify the relationship of gmGnRH-Rs with other vertebrate GnRH-Rs. The function of GnRH-R variants was investigated by quantitative PCR gene expression analysis in the brain and pituitary of female cod during a full reproductive cycle and in various peripheral tissues in sexually mature fish. Phylogenetic analysis revealed two types of teleost GnRH-Rs: Type I including gmGnRH-R1b and Type II including gmGnRH-R2a, gmGnRH-R2b and gmGnRH-R2c. All four gmGnRH-Rs are expressed in the brain, and gmGnRH-R1b, gmGnRH-R2a and gmGnRH-R2c are expressed in the pituitary. The only GnRH-R differentially expressed in the pituitary during the reproductive cycle is gmGnRH-R2a such that its expression is significantly increased during spawning. These data suggest that gmGnRH-R2a is the most likely candidate to mediate the hypophysiotropic function of GnRH in Atlantic cod.

The aim of the present study was to explore the electrophysiological properties of pituitary cells from Atlantic cod (Gadus morhua), as a basis for future studies of the signaling pathways involved in the control of pituitary secretion in this species. Primary cultures of pituitary cells from maturing Atlantic cod were prepared by trypsin treatment and mechanical dispersion. Electrophysiological recordings were performed using the perforated patch clamp method. A subpopulation of large cells were selected for recordings. Spontaneous action potentials were observed in about 30% of the cells. The action potentials displayed a fast initial spike followed by a prolonged plateau. Correspondingly, the inward current elicited by depolarizing steps consisted of both a transient, tetrodotoxin-sensitive Na(+) component and a nifedipine-sensitive Ca(2+) component that was sustained when Ba(2+) replaced Ca(2+) as current carrier. The outward current was partially blocked both by 5 mM tetraethylammonium and 10 mM 4-aminopyridine. The voltage-activated ion channels present in these cells largely correspond to the ion channels of pituitary cells in other teleosts (goldfish, Carassius auratus, and tilapia, Oreochromis mossambicus) and mammals, although differences exist regarding the shape and duration of action potentials.

Neuronal excitability is regulated by neurotransmitters through modulation of ion channels. The channels can be modulated via two basic mechanisms: by ligand binding or by covalent modification by kinases, phosphatases and other enzymes (Hille 1992). The archetypal second messenger, cyclic AMP (cAMP) is thought primarily to act indirectly, by activating protein kinase A (PKA) which in turn can phosphorylate ion channels or their regulatory proteins. In the brain, in particular, cAMP effects have been considered to be almost exclusively PKA-mediated (Walaas and Greengard 1991). Here we review our results concerning the modulation of two types of ion channels in hippocampal pyramidal cells, indicating that monoamine transmitters acting via cAMP can exert both kinasedependent and kinase-independent regulation of neuronal excitability in the brain.

ATP-sensitive potassium (K ATP ) channels in the pancreatic β cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca 2+ influx and exocytosis. Metabolic regulation of K ATP channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The β cell K ATP channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotide-binding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate K ATP channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg 2+ caused an apparent reduction in the inhibitory action of ATP on wild-type K ATP channels, and MgATP actually activated K ATP channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates K ATP channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg 2+ .

Background Omega-3 PUFAs are known to have anti-inflammatory properties, and different mechanisms are involved. GPR120 is a G-protein coupled receptor that has recently received attention because of its anti-inflammatory signalling properties after binding omega-3 PUFAs. However, both omega-3 and omega-6 PUFAs are natural GPR120 ligands. The aim of this study was to study possible differences in GPR120-mediated signalling events after treatment with different long-chain PUFAs in intestinal epithelial cells. We also investigated possible GPR120-mediated anti-inflammatory effects of different long-chain PUFAs that may be relevant in the understanding of how dietary PUFAs influence inflammatory responses in inflammatory diseases such as IBD. Methods We used Caco-2 cells as a model system to study GPR120-mediated signalling events because we found this cell line to express GPR120, but not GPR40, another plasma membrane receptor for medium- and long chain fatty acids. Increase in cytosolic Ca2+concentration, activation of MAP kinase ERK1/2 and the inhibition of IL-1β induced NF-κB activity were studied to reveal potential differences in the activation of GPR120 by the omega-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the omega-6 PUFA arachidonic acid (AA). Results We found that EPA, DHA and AA enhanced the cytosolic concentration of the second messenger Ca2+ with the same efficiency, but with different kinetics. Both omega-3 and omega-6 PUFAs activated MAP kinase ERK1/2, but differences regarding kinetics and intensity were also observed in this pathway. ERK1/2 activation was shown to be dependent upon EGFR and Raf-1. We further investigated the ability of EPA, DHA and AA to inhibit NF-κB activity in Caco-2 cells. All PUFAs tested were able to inhibit IL-1β induced breakdown of IκBα after binding to GPR120, but with different potency. Conclusions Our results show that EPA, DHA and AA elicit the same signalling events, but with different kinetics and efficiency through GPR120 in Caco-2 cells. We show, for the first time, that both omega-3 and omega-6 PUFAs inhibit NF-κB activation in intestinal epithelial cells. Our results may be important for understanding how dietary PUFAs influence inflammatory processes relevant in delineating effects of PUFAs in the treatment of IBD.