ß-carbolines that enhance GABAA receptor response expressed in oligodendrocytes promote remyelination in an in vivo rat model of focal demyelination.

Demyelination is typically followed by a remyelination process through mature oligodendrocytes (OLs) differentiated from precursor cells (OPCs) recruited into the lesioned areas, however, this event usually results in uncompleted myelination. Potentiation of the remyelination process is an important target for designing effective therapeutic strategies against white matter loss. Here, it was evaluated the remyelinating effect of different ß-carbolines that present differential allosteric modulation on the GABAA receptor expressed in OLs. For this, we used a focalized demyelination model in the inferior cerebellar peduncle (i.c.p.) of rats (DRICP model), in which, demyelination by ethidium bromide (0.05%) stereotaxic injection was confirmed histologically by staining with Black-Gold II (BGII) and toluidine blue. In addition, a longitudinal analysis with diffusion-weighted magnetic resonance imaging (dMRI) was made by computing fractional anisotropy (FA), apparent diffusion coefficient (ADC) and diffusivity parameters to infer i.c.p. microstructural changes. First, dMRI analysis revealed FA decreases together with ADC and radial diffusivity (RD) increases after demyelination, which correlates with histological BGII observations. Then, we evaluated the effect produced by three allosteric GABAA receptor modulators, the N-butyl-ß-carboline-3-carboxylate (ß-CCB), ethyl 9H-pyrido [3,4-b]indole-3-carboxylate (ß-CCE), and 4-ethyl-6,7-dimethoxy-9H-pyrido [3,4-b]indole-3-carboxylic acid methyl ester (DMCM). The results indicated that daily systemic ß-CCB (1 mg/Kg) or ß-CCE (1 mg/Kg) administration for 2 weeks, but not DMCM (0.35 mg/Kg), in lesioned animals increased FA and decreased ADC or RD, suggesting myelination improvement. This was supported by BGII staining analysis that showed a recovery of myelin content. Also, it was quantified by immunohistochemistry both NG2+ and CC1+ cellular population in the different experimental sceneries. Data indicated that either ß-CCB or ß-CCE, but not DMCM, produced an increase in the population of CC1+ cells in the lesioned area. Finally, it was also calculated the g-ratio of myelinated axons and observed a similar value in those lesioned animals treated with ß-CCB or ß-CCE compared to controls. Thus, using the DRICP model, it was observed that either ß-CCB or ß-CCE, positive modulators of the GABAA receptor in OLs, had a potent promyelinating effect.

Transcriptional and bioinformatic analysis of GABAA receptors expressed in oligodendrocyte progenitor cells from the human brain.

Introduction: Oligodendrocyte progenitor cells (OPCs) are vital for neuronal myelination and remyelination in the central nervous system. While the molecular mechanisms involved in OPCs' differentiation and maturation are not completely understood, GABA is known to positively influence these processes through the activation of GABAA receptors (GABAARs). The molecular identity of GABAARs expressed in human OPCs remains unknown, which restricts their specific pharmacological modulation to directly assess their role in oligodendrocytes' maturation and remyelination. Methods: In this study, we conducted a transcriptomic analysis to investigate the molecular stoichiometry of GABAARs in OPCs from the human brain. Using eight available transcriptomic datasets from the human brain cortex of control individuals, we analyzed the mRNA expression of all 19 known GABAARs subunit genes in OPCs, with variations observed across different ages. Results: Our analysis indicated that the most expressed subunits in OPCs are α1-3, ß1-3, γ1-3, and ε. Moreover, we determined that the combination of any α with ß2 and γ2 is likely to form heteropentameric GABAARs in OPCs. Importantly, we also found a strong correlation between GABAAR subunits and transcripts for postsynaptic scaffold proteins, suggesting the potential postsynaptic clustering of GABAARs in OPCs. Discussion: This study presents the first transcriptional-level identification of GABAAR subunits expressed in human OPCs, providing potential receptor combinations. Understanding the molecular composition of GABAARs in OPCs not only enhances our knowledge of the underlying mechanisms in oligodendrocyte maturation but also opens avenues for targeted pharmacological interventions aimed at modulating these receptors to promote remyelination in neurological disorders.

Potential contributions of the intrinsic retinal oscillations recording using non-invasive electroretinogram to bioelectronics.

Targeted electric signal use for disease diagnostics and treatment is emerging as a healthcare game-changer. Besides arrhythmias, treatment-resistant epilepsy and chronic pain, blindness, and perhaps soon vision loss, could be among the pathologies that benefit from bioelectronic medicine. The electroretinogram (ERG) technique has long demonstrated its role in diagnosing eye diseases and early stages of neurodegenerative diseases. Conspicuously, ERG applications are all based on light-induced responses. However, spontaneous, intrinsic activity also originates in retinal cells. It is a hallmark of degenerated retinas and its alterations accompany obesity and diabetes. To the extent that variables extracted from the resting activity of the retina measured by ERG allow the predictive diagnosis of risk factors for type 2 diabetes. Here, we provided a comparison of the baseline characteristics of intrinsic oscillatory activity recorded by ERGs in mice, rats, and humans, as well as in several rat strains, and explore whether zebrafish exhibit comparable activity. Their pattern was altered in neurodegenerative models including the cuprizone-induced demyelination model in mice as well as in the Royal College of Surgeons (RCS-/-) rats. We also discuss how the study of their properties may pave the way for future research directions and treatment approaches for retinopathies, among others.

Sea anemone Bartholomea annulata venom inhibits voltage-gated Na+ channels and activates GABAA receptors from mammals.

Toxin production in nematocysts by Cnidaria phylum represents an important source of bioactive compounds. Using electrophysiology and, heterologous expression of mammalian ion channels in the Xenopus oocyte membrane, we identified two main effects produced by the sea anemone Bartholomea annulata venom. Nematocysts isolation and controlled discharge of their content, revealed that venom had potent effects on both voltage-dependent Na+ (Nav) channels and GABA type A channel receptors (GABAAR), two essential proteins in central nervous system signaling. Unlike many others sea anemone toxins, which slow the inactivation rate of Nav channels, B. annulata venom potently inhibited the neuronal action potential and the Na+ currents generated by distinct Nav channels opening, including human TTX-sensitive (hNav1.6) and TTX-insensitive Nav channels (hNav1.5). A second effect of B. annulata venom was an agonistic action on GABAAR that activated distinct receptors conformed by either α1ß2γ2, α3ß2γ1 or, ρ1 homomeric receptors. Since GABA was detected in venom samples by ELISA assay at low nanomolar range, it was excluded that GABA from nematocysts directly activated the GABAARs. This revealed that substances in B. annulata nematocysts generated at least two potent and novel effects on mammalian ion channels that are crucial for nervous system signaling.

Therapeutic Potential of GABAergic Signaling in Myelin Plasticity and Repair.

Oligodendrocytes (OLs) produce myelin to insulate axons. This accelerates action potential propagation, allowing nerve impulse information to synchronize within complex neuronal ensembles and promoting brain connectivity. Brain plasticity includes myelination, a process that starts early after birth and continues throughout life. Myelin repair, followed by injury or disease, requires new OLs differentiated from a population derived from oligodendrocyte precursor cells (OPCs) that continue to proliferate, migrate and differentiate to preserve and remodel myelin in the adult central nervous system. OPCs represent the largest proliferative neural cell population outside the adult neurogenic niches in the brain. OPCs receive synaptic inputs from glutamatergic and GABAergic neurons throughout neurodevelopment, a unique feature among glial cells. Neuron-glia communication through GABA signaling in OPCs has been shown to play a role in myelin plasticity and repair. In this review we will focus on the molecular and functional properties of GABA A receptors (GABA A Rs) expressed by OPCs and their potential role in remyelination.

GABAA Receptors Expressed in Oligodendrocytes Cultured from the Neonatal Rat Contain α3 and γ1 Subunits and Present Differential Functional and Pharmacological Properties.

Oligodendrocytes (OLs) express functional GABAA receptors (GABAARs) that are activated by GABA released at synaptic contacts with axons or by ambient GABA in extrasynaptic domains. In both instances, the receptors' molecular identity has not been fully defined. Furthermore, data on their structural diversity in different brain regions and information on age-dependent changes in their molecular composition are scant. This lack of knowledge has delayed access to a better understanding of the role of GABAergic signaling between neurons and OLs. Here, we used functional, and pharmacological analyses, as well as gene and protein expression of GABAAR subunits, to explore the subunit combination that could explain the receptor functional profile expressed in OLs from the neonate rat. We found that GABAAR composed of α3ß2γ1 subunits mimicked the characteristics of the endogenous receptor when expressed heterologously in Xenopus laevis oocytes. Either α3 or γ1 subunit silencing by small interfering RNA transfection changed the GABA-response characteristics in oligodendrocyte precursor cells, indicating their participation in the endogenous receptor conformation. Thus, α3 subunit silencing shifted the mean EC50 for GABA from 75.1 to 46.6 µM, whereas γ1 silencing reduced the current amplitude response by 55%. We also observed that ß-carbolines differentially enhance GABA responses in oligodendroglia as compared with those in neurons. These results contribute to defining the molecular and pharmacological properties of GABAARs in OLs. Additionally, the identification of ß-carbolines as selective enhancers of GABAARs in OLs may help to study the role of GABAergic signaling during myelination. SIGNIFICANCE STATEMENT: GABAergic signaling through GABAA receptors (GABAARs) expressed in the oligodendroglial lineage contributes to the myelination control. Determining the molecular identity and the pharmacology of these receptors is essential to define their specific roles in myelination. Using GABAAR subunit expression and silencing, we identified that the GABAAR subunit combination α3ß2γ1 conforms the bulk of GABAARs in oligodendrocytes from rat neonates. Furthermore, we found that these receptors have differential pharmacological properties that allow specific positive modulation by ß-carbolines.

Expression and Function of GABA Receptors in Myelinating Cells.

Myelin facilitates the fast transmission of nerve impulses and provides metabolic support to axons. Differentiation of oligodendrocyte progenitor cells (OPCs) and Schwann cell (SC) precursors is critical for myelination during development and myelin repair in demyelinating disorders. Myelination is tightly controlled by neuron-glia communication and requires the participation of a wide repertoire of signals, including neurotransmitters such as glutamate, ATP, adenosine, or γ-aminobutyric acid (GABA). GABA is the main inhibitory neurotransmitter in the central nervous system (CNS) and it is also present in the peripheral nervous system (PNS). The composition and function of GABA receptors (GABARs) are well studied in neurons, while their nature and role in glial cells are still incipient. Recent studies demonstrate that GABA-mediated signaling mechanisms play relevant roles in OPC and SC precursor development and function, and stand out the implication of GABARs in oligodendrocyte (OL) and SC maturation and myelination. In this review, we highlight the evidence supporting the novel role of GABA with an emphasis on the molecular identity of the receptors expressed in these glial cells and the possible signaling pathways involved in their actions. GABAergic signaling in myelinating cells may have potential implications for developing novel reparative therapies in demyelinating diseases.

P2X7 Receptors as a Therapeutic Target in Cerebrovascular Diseases.

Shortage of oxygen and nutrients in the brain induces the release of glutamate and ATP that can cause excitotoxicity and contribute to neuronal and glial damage. Our understanding of the mechanisms of ATP release and toxicity in cerebrovascular diseases is incomplete. This review aims at summarizing current knowledge about the participation of key elements in the ATP-mediated deleterious effects in these pathologies. This includes pannexin-1 hemichannels, calcium homeostasis modulator-1 (CALHM1), purinergic P2X7 receptors, and other intermediaries of CNS injury downstream of ATP release. Available data together with recent pharmacological developments in purinergic signaling may constitute a new opportunity to translate preclinical findings into more effective therapies in cerebrovascular diseases.

Demyelination-Remyelination of the Rat Caudal Cerebellar Peduncle Evaluated with Magnetic Resonance Imaging.

Remyelination is common under physiological conditions and usually occurs as a response to a pathological demyelinating event. Its potentiation is an important goal for the development of therapies against pathologies such as multiple sclerosis and white matter injury. Visualization and quantification in vivo of demyelination and remyelination processes are essential for longitudinal studies that will allow the testing and development of pro-myelinating strategies. In this study, ethidium bromide (EB) was stereotaxically injected into the caudal cerebellar peduncle (c.c.p.) in rats to produce demyelination; the resulting lesion was characterized (i) transversally through histology using Black-Gold II (BGII) staining, and (ii) longitudinally through diffusion-weighted magnetic resonance imaging (dMRI), by computing fractional anisotropy (FA) and diffusivity parameters to detect microstructural changes. Using this characterization, we evaluated, in the lesioned c.c.p., the effect of N-butyl-ß-carboline-3-carboxylate (ß-CCB), a potentiator of GABAergic signaling in oligodendrocytes. The dMRI analysis revealed significant changes in the anisotropic and diffusivity properties of the c.c.p. A decreased FA and increased radial diffusivity (λ⊥) were evident following c.c.p. lesioning. These changes correlated strongly with an apparent decrease in myelin content as evidenced by BGII. Daily systemic ß-CCB administration for 2 weeks in lesioned animals increased FA and decreased λ⊥, suggesting an improvement in myelination, which was supported by histological analysis. This study shows that structural changes in the demyelination-remyelination of the caudal cerebellar peduncle (DRCCP) model can be monitored longitudinally by MRI, and it suggests that remyelination is enhanced by ß-CCB treatment. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Inwardly Rectifying K+ Currents in Cultured Oligodendrocytes from Rat Optic Nerve are Insensitive to pH.

Inwardly rectifying K+ (Kir) channel expression signals at an advanced stage of maturation during oligodendroglial differentiation. Knocking down their expression halts the generation of myelin and produces severe abnormalities in the central nervous system. Kir4.1 is the main subunit involved in the tetrameric structure of Kir channels in glial cells; however, the precise composition of Kir channels expressed in oligodendrocytes (OLs) remains partially unknown, as participation of other subunits has been proposed. Kir channels are sensitive to H+; thus, intracellular acidification produces Kir current inhibition. Since Kir subunits have differential sensitivity to H+, we studied the effect of intracellular acidification on Kir currents expressed in cultured OLs derived from optic nerves of 12-day-old rats. Unexpectedly, Kir currents in OLs (2-4 DIV) did not change within the pH range of 8.0-5.0, as observed when using standard whole-cell voltage-clamp recording or when preserving cytoplasmic components with the perforated patch-clamp technique. In contrast, low pH inhibited astrocyte Kir currents, which was consistent with the involvement of the Kir4.1 subunit. The H+-insensitivity expressed in OL Kir channels was not intrinsic because Kir cloning showed no difference in the sequence reported for the Kir4.1, Kir2.1, or Kir5.1 subunits. Moreover, when Kir channels were heterologously expressed in Xenopus oocytes they behaved as expected in their general properties and sensitivity to H+. It is therefore concluded that Kir channel H+-sensitivity in OLs is modulated through an extrinsic mechanism, probably by association with a modulatory component or by posttranslational modifications.

GAT-1 mediated GABA uptake in rat oligodendrocytes.

Stimulated by the results of a recent paper on the effects of tiagabine, a selective inhibitor of the main GABA transporter GAT-1, on oligodendrogenesis, we verified the possibility that GAT-1 may be expressed in oligodendrocytes using immunocytochemical methods and functional assays. Light microscopic analysis of the subcortical white matter of all animals revealed the presence of numerous GAT-1+ cells of different size (from 3 to 29 µm) and morphology. An electron microscope analysis revealed that, besides fibrous astrocytes and interstitial neurons, GAT-1 immunoreactivity was present in immature and mature oligodendrocytes. Co-localization studies between GAT-1 and markers specific for oligodendrocytes (NG2 and RIP) showed that about 12% of GAT-1 positive cells in the white matter were immature oligodendrocytes, while about 15% were mature oligodendrocytes. In vitro functional assays showed that oligodendrocytes exhibit tiagabine-sensitive Na+ -dependent GABA uptake. Although relationships between GABA and oligodendrocytes have been known for many years, this is the first demonstration that GAT-1 is expressed in oligodendrocytes. The present results on the one hand definitely closes the era of "neuronal" and "glial" GABA transporters, on the other they suggest that oligodendrocytes may contribute to pathophysiology of the several diseases in which GAT-1 have been implicated to date. GLIA 2017;65:514-522.

Electrophysiological activity of a neurotoxic fraction from the venom of box jellyfish Carybdea marsupialis.

Carybdea marsupialis is a widely distributed box jellyfish found in the Mediterranean and in the tropical waters of the Caribbean Sea. Its venom is a complex mixture of biologically active compounds that are used to catch prey. In order to evaluate the activity of the neurotoxins in the venom, bioassays were carried out using the marine crab Ocypode quadrata. The proteins with neurotoxic effect were partially purified using low-pressure liquid chromatography techniques. Gel filtration (Sephadex G-50M) was used as the first step and the active fraction in crabs was passed through a QAE Sephadex A-25 column. Finally, the active fraction was run onto a Fractogel EMD SO3- column. No further purification step could be carried out due to the loss of neurotoxic activity. The Fractogel EMD SO3- fraction was analyzed electrophysiologically using the voltage-clamp technique in Xenopus laevis oocytes expressing membrane proteins from rat brain through mRNA injection. The crude venom and a fraction were observed to affect crustaceans and showed at least two types of bioactivity in oocytes expressing brain proteins. The effects were dose-dependent and completely reversible. These results evidence the presence of neurotoxins in Carybdea marsupialis venom that act on membrane proteins of the vertebrate nervous system.

Axon-to-Glia Interaction Regulates GABAA Receptor Expression in Oligodendrocytes.

Myelination requires oligodendrocyte-neuron communication, and both neurotransmitters and contact interactions are essential for this process. Oligodendrocytes are endowed with neurotransmitter receptors whose expression levels and properties may change during myelination. However, only scant information is available about the extent and timing of these changes or how they are regulated by oligodendrocyte-neuron interactions. Here, we used electrophysiology to study the expression of ionotropic GABA, glutamate, and ATP receptors in oligodendrocytes derived from the optic nerve and forebrain cultured either alone or in the presence of dorsal root ganglion neurons. We observed that oligodendrocytes from both regions responded to these transmitters at 1 day in culture. After the first day in culture, however, GABA sensitivity diminished drastically to less than 10%, while that of glutamate and ATP remained constant. In contrast, the GABA response amplitude was sustained and remained stable in oligodendrocytes cocultured with dorsal root ganglion neurons. Immunochemistry and pharmacological properties of the responses indicated that they were mediated by distinctive GABAA receptors and that in coculture with neurons, the oligodendrocytes bearing the receptors were those in direct contact with axons. These results reveal that GABAA receptor regulation in oligodendrocytes is driven by axonal cues and that GABA signaling may play a role in myelination and/or during axon-glia recognition.

Differential role of STIM1 and STIM2 during transient inward (T in) current generation and the maturation process in the Xenopus oocyte.

BACKGROUND: The Xenopus oocyte is a useful cell model to study Ca2+ homeostasis and cell cycle regulation, two highly interrelated processes. Here, we used antisense oligonucleotides to investigate the role in the oocyte of stromal interaction molecule (STIM) proteins that are fundamental elements of the store-operated calcium-entry (SOCE) phenomenon, as they are both sensors for Ca2+ concentration in the intracellular reservoirs as well as activators of the membrane channels that allow Ca2+ influx. RESULTS: Endogenous STIM1 and STIM2 expression was demonstrated, and their synthesis was knocked down 48-72 h after injecting oocytes with specific antisense sequences. Selective elimination of their mRNA and protein expression was confirmed by PCR and Western blot analysis, and we then evaluated the effect of their absence on two endogenous responses: the opening of SOC channels elicited by G protein-coupled receptor (GPCR)-activated Ca2+ release, and the process of maturation stimulated by progesterone. Activation of SOC channels was monitored electrically by measuring the T in response, a Ca2+-influx-dependent Cl- current, while maturation was assessed by germinal vesicle breakdown (GVBD) scoring and electrophysiology. CONCLUSIONS: It was found that STIM2, but not STIM1, was essential in both responses, and T in currents and GVBD were strongly reduced or eliminated in cells devoid of STIM2; STIM1 knockdown had no effect on the maturation process, but it reduced the T in response by 15 to 70%. Thus, the endogenous SOCE response in Xenopus oocytes depended mainly on STIM2, and its expression was necessary for entry into meiosis induced by progesterone.

Paracrine stimulation of P2X7 receptor by ATP activates a proliferative pathway in ovarian carcinoma cells.

P2X7 is a purinergic receptor-channel; its activation by ATP elicits a broad set of cellular actions, from apoptosis to signals for survival. Here, P2X7 expression and function was studied in human ovarian carcinoma (OCA) cells, and biopsies from non-cancerous and cancer patients were analyzed by immunohistochemistry. Ovarian surface epithelium in healthy tissue expressed P2X7 at a high level that was maintained throughout the cancer. The cell lines SKOV-3 and CAOV-3 were used to investigate P2X7 functions in OCA. In SKOV-3 cells, selective stimulation of P2X7 by 2'(3')-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate (BzATP) induced a dose-dependent increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) but not cell death. Instead, BzATP increased the levels of phosphorylated ERK and AKT (pERK and pAKT), with an EC(50) of 44 ± 2 and 1.27 ± 0.5 µM, respectively; 10 µM BzATP evoked a maximum effect within 15 min that lasted for 120 min. Interestingly, basal levels of pERK and pAKT were decreased in the presence of apyrase in the medium, strongly suggesting an endogenous, ATP-mediated phenomenon. Accordingly: (i) mechanically stimulated cells generated a [Ca(2+)](i) increase that was abolished by apyrase; (ii) apyrase induced a decrease in culture viability, as measured by the MTS assay for mitochondrial activity; and (iii) incubation with 10 µM AZ10606120, a specific P2X7 antagonist and transfection with the dominant negative P2X7 mutant E496A, both reduced cell viability to 70.1 ± 8.9% and to 76.5 ± 5%, respectively, of control cultures. These observations suggested that P2X7 activity was auto-induced through ATP efflux; this increased pERK and pAKT levels that generated a positive feedback on cell viability.

A3 Adenosine receptors mediate oligodendrocyte death and ischemic damage to optic nerve.

Adenosine receptor activation is involved in myelination and in apoptotic pathways linked to neurodegenerative diseases. In this study, we investigated the effects of adenosine receptor activation in the viability of oligodendrocytes of the rat optic nerve. Selective activation of A3 receptors in pure cultures of oligodendrocytes caused concentration-dependent apoptotic and necrotic death which was preceded by oxidative stress and mitochondrial membrane depolarization. Oligodendrocyte apoptosis induced by A3 receptor activation was caspase-dependent and caspase-independent. In addition to dissociated cultures, incubation of optic nerves ex vivo with adenosine and the A3 receptor agonist 2-CI-IB-MECA(1-[2-Chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-D-ribofuranuronamide)-induced caspase-3 activation, oligodendrocyte damage, and myelin loss, effects which were prevented by the presence of caffeine and the A3 receptor antagonist MRS 1220 (N-[9-Chloro-2-(2-furanyl)[1,2,4]-triazolo [1,5-c]quinazolin-5-yl]benzene acetamide). Finally, ischemia-induced injury and functional loss to the optic nerve was attenuated by blocking A3 receptors. Together, these results indicate that adenosine may trigger oligodendrocyte death via activation of A3 receptors and suggest that this mechanism contributes to optic nerve and white matter ischemic damage.

Cloning and functional analysis of P2X1b, a new variant in rat optic nerve that regulates the P2X1 receptor in a use-dependent manner.

P2X receptors are trimeric, ATP-gated cation channels. In mammals seven P2X subtypes have been reported (P2X1-P2X7), as well as several variants generated by alternative splicing. Variants confer to the homomeric or heteromeric channels distinct functional and/or pharmacological properties. Molecular biology, biochemical, and functional analysis by electrophysiological methods were used to identify and study a new variant of the P2X1 receptor named P2X1b. This new variant, identified in rat optic nerve, was also expressed in other tissues. P2X1b receptors lack amino acids 182 to 208 of native P2X1, a region that includes residues that are highly conserved among distinct P2X receptors. When expressed in Xenopus oocytes, P2X1b was not functional as a homomer; however, when co-expressed with P2X1, it downregulated the electrical response generated by ATP compared with that of oocytes expressing P2X1 alone, and it seemed to form heteromeric channels with a modestly enhanced ATP potency. A decrease in responses to ATP in oocytes co-expressing different ratios of P2X1b to P2X1 was completely eliminated by overnight pretreatment with apyrase. Thus, it is suggested that P2X1b regulates, through a use-dependent mechanism, the availability, in the plasma membrane, of receptor channels that can be operated by ATP.

Agonist-activated Ca2+ influx and Ca2+ -dependent Cl- channels in Xenopus ovarian follicular cells: functional heterogeneity within the cell monolayer.

Xenopus follicles are endowed with specific receptors for ATP, ACh, and AII, transmitters proposed as follicular modulators of gamete growth and maturation in several species. Here, we studied ion-current responses elicited by stimulation of these receptors and their activation mechanisms using the voltage-clamp technique. All agonists elicited Cl(-) currents that depended on coupling between oocyte and follicular cells and on an increase in intracellular Ca(2+) concentration ([Ca(2+) ](i)), but they differed in their activation mechanisms and in the localization of the molecules involved. Both ATP and ACh generated fast Cl(-) (F(Cl)) currents, while AII activated an oscillatory response; a robust Ca(2+) influx linked specifically to F(Cl) activation elicited an inward current (I(iw,Ca)) which was carried mainly by Cl(-) ions, through channels with a sequence of permeability of SCN(-) > I(-) > Br(-) > Cl(-). Like F(Cl), I(iw,Ca) was not dependent on oocyte [Ca(2+) ](i) ; instead both were eliminated by preventing [Ca(2+) ](i) increase in the follicular cells, and also by U73122 and 2-APB, drugs that inhibit the phospolipase C (PLC) pathway. The results indicated that F(Cl) and I(iw,Ca) were produced by the expected, PLC-stimulated Ca(2+) -release and Ca(2+) -influx, respectively, and by the opening of I(Cl(Ca)) channels located in the follicular cells. Given their pharmacological characteristics and behavior in conditions of divalent cation deprivation, Ca(2+) -influx appeared to be driven through store-operated, calcium-like channels. The AII response, which is also known to require PLC activation, did not activate I(iw,Ca) and was strictly dependent on oocyte [Ca(2+) ](i) increase; thus, ATP and ACh receptors seem to be expressed in a population of follicular cells different from that expressing AII receptors, which were coupled to the oocyte through distinct gap-junction channels.

Electrophysiological characterization of a novel small peptide from the venom of Conus californicus that targets voltage-gated neuronal Ca2+ channels.

Conus californicus belongs to a genus of marine gastropods with more than 700 extant species. C. californicus has been shown to be distantly related to all Conus species, but showing unusual biological features. We report a novel peptide isolated from C. californicus with a significant inhibitory action over neuronal voltage-gated calcium channels. The new toxin is formed by 13-amino acid residues with two disulfide bonds, whose sequence (NCPAGCRSQGCCM) is strikingly different from regular ω-conotoxins. In the HPLC purification procedure, the venom fraction eluted in the first 10-15 min produced a significant decrease (54% ± 3%) of the Ca(2+) current in Xenopus laevis oocytes transfected with purified rat-brain mRNA. A specific peptide obtained from the elution at 13 min decreased the Ca(2+) current in the adult rat dorsal-root ganglion neurons in a primary culture by 34% ± 2%. The cysteine pattern of this peptide corresponds to the framework XVI described for the M-superfamily of conopeptides and is unprecedented among Conus peptides acting on Ca(2+) channels.

Functional expression and intracellular signaling of UTP-sensitive P2Y receptors in theca-interstitial cells.

BACKGROUND: Purinergic receptors are expressed in the ovary of different species; their physiological roles remain to be elucidated. UTP-sensitive P2Y receptor activity may regulate cell proliferation. The aim of the present work was to study the functional expression of these receptors in theca/interstitial cells (TIC). METHODS: TIC were isolated by centrifugation in a Percoll gradient. P2Y receptors and cellular markers in TIC were detected by RT-PCR and Western blot. Intracellular calcium mobilization induced by purinergic drugs was evaluated by fluorescence microscopy, phosphorylation of MAPK p44/p42 and of cAMP response element binding protein (CREB) was determined by Western blot and proliferation was quantified by [3H]-thymidine incorporation into DNA. RESULTS: RT-PCR showed expression of p2y2r and p2y6r transcripts, expression of the corresponding proteins was confirmed. UTP and UDP, agonists for P2Y2 and P2Y6 receptors, induced an intracellular calcium increase with a maximum of more than 400% and 200% of basal level, respectively. The response elicited by UTP had an EC50 of 3.5 +/- 1.01 microM, while that for UDP was 3.24 +/- 0.82 microM. To explore components of the pathway activated by these receptors, we evaluated the phosphorylation induced by UTP or UDP of MAPK p44 and p42. It was found that UTP increased MAPK phosphorylation by up to 550% with an EC50 of 3.34 +/- 0.92 and 1.41 +/- 0.67 microM, for p44 and p42, respectively; these increases were blocked by suramin. UDP also induced p44/p42 phosphorylation, but at high concentrations. Phosphorylation of p44/p42 was dependent on PKC and intracellular calcium. To explore possible roles of this pathway in cell physiology, cell proliferation and hCG-induced CREB-phosphorylation assays were performed; results showed that agonists increased cell proliferation and prevented CREB-phosphorylation. CONCLUSION: Here, it is shown that UTP-sensitive P2Y receptors are expressed in cultured TIC and that these receptors had the ability to activate mitogenic signaling pathways and to promote cell proliferation, as well as to prevent CREB-phosphorylation by hCG. Regulation of TIC proliferation and steroidogenesis is relevant in ovarian pathophysiology since theca hyperplasia is involved in polycystic ovarian syndrome. Purinergic receptors described might represent an important new set of molecular therapeutic targets.