Rogelio O. Arellano

Electrophysiological and hemolytic activity elicited by the venom of the jellyfish Cassiopea xamachana.

In this study, we determined hemolysis activity in human and sheep erythrocytes, and characterized the electrical responses in Xenopus oocyte membrane elicited by the venom of the jellyfish Cassiopea xamachana (Cx). The Cx venom produced hemolysis in both species, being more potent on human red cells. The electrophysiological study showed that the Cx venom elicited three different responses in the oocytes. One current was generated in all the oocytes tested and corresponded with a slow inward current (I(Cx)) associated with an increase in membrane conductance. I(Cx) was concentration-dependent and had a reversal potential of -10.3+/-0.4 mV. Ionic substitution studies indicated that the conductive pathway was mainly permeable to cations and non-selective. The oocyte membrane resistance was completely recovered after washout of the venom, this suggested that the effect was due to generation of a specific membrane conductance as opposed to a possible non-specific membrane breakdown. A comparative study with three distinct native cationic channels present in the oocyte membrane [i.e. (1) hemi-gap-junction channels, (2) mechanosensitive channels, and (3) the ouabain-sensitive channel activated by palytoxin], showed that I(Cx) might correspond to opening of mechanosensitive channels or to activation of an unknown cationic channel located in the oocyte membrane. The bioactive fraction eliciting I(Cx) were peptides and was separated from two other peptidic hemolytic fractions by chromatography.

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 Ca2+ concentration ([Ca2+]i) but not cell death. Instead, BzATP increased the levels of phosphorylated ERK and AKT (pERK and pAKT), with an EC50 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 [Ca2+]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. J. Cell. Biochem. 115: 1955–1966, 2014.

Ionic Currents Activated via Purinergic Receptors in the Cumulus Cell-Enclosed Mouse Oocyte

Abstract Several chemical signals synthesized in the ovary, including neurotransmitters, have been proposed to serve as regulators of folliculogenesis, however, their mechanisms of action have not been completely elucidated. Here, electrophysiological and molecular biology techniques were used to study responses generated via purinergic stimulation in cultured mouse cumulus cell-enclosed oocytes (CEOs). Application of extracellular ATP elicited depolarizing responses in CEOs. Using the voltage clamp technique by impaling oocytes with two microelectrodes, we determined that these responses were mainly due to activation of two distinct ionic currents. The first corresponded to the opening of Ca2+-dependent Cl− channels (ICl(Ca)) and the second to the opening of Ca2+-independent channels that are permeable to Na+ (Ic+). The potency order for different nucleotides (50 μM) was UTP > ATP > 2meS-ATP > ADP, and α,βme-ATP and adenosine were found to be inactive. Suramin (100 μM) blocked the response elicited by ATP or UTP. In addition, voltage dependent K+ currents activated by depolarization of CEOs were characterized. All CEO ionic currents recorded from the oocyte were completely inhibited by octanol (1 mM), a gap junction blocker. Thus, purinergic responses and K+ currents originate mainly in the membrane of cumulus cells. Transcripts of the purinergic receptor P2Y2 subtype were amplified by polymerase chain reaction from the cDNA of granulosa cells or cumulus cells. This study shows that P2Y2 receptors are expressed in CEOs, and that their stimulation opens at least two different types of ion channels. Both the ion channels and the receptors seemed to be located in the cumulus cells, which transmit their corresponding electrical signals to the oocyte via gap junction channels.

Cl− currents activated via purinergic receptors inXenopus follicles

Ionic currents elicited via purinergic receptors located in the membrane of Xenopus follicles were studied using electrophysiological techniques. Follicles responded to ATP-activating inward currents with a fast time course ( F in). In Ringer solution, reversal potential ( E rev) of F in was -22 mV, which did not change with external substitutions of Na+ or K+, whereas solutions containing 50 or 5% of normal Cl-concentration shifted E rev to about +4 and +60 mV, respectively, and decreased F in amplitude, indicating that F in was carried by Cl-. F in had an onset delay of ∼400 ms, measured by application of a brief jet of ATP from a micropipette positioned near the follicle (50 μm). F in was inhibited by 50% in follicles pretreated with pertussis toxin. This suggests a G protein-mediated receptor channel pathway. F in was mimicked by 2-MeSATP and UTP, the potency order (half-maximal effective concentration) was 2-MeSATP (194 nM) > UTP (454 nM) > ATP (1,086 nM). All agonists generated Cl- currents and displayed cross-inhibition on the others. F in activation by acetylcholine also cross-inhibited F in-ATP responses, suggesting that all act on a common channel-activation pathway.

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. GLIA 2014;62:199–216

Activation of volume‐regulated Cl− channels by ACh and ATP in Xenopus follicles

1 Osmolarity‐dependent ionic currents from follicle‐enclosed Xenopus oocytes (follicles) were studied using electrophysiological techniques. Whole follicle currents were monitored using a two‐electrode voltage clamp and single‐channel activity was measured using the patch‐clamp technique. 2 In follicles held at ‐60 mV two chloride currents were activated in external hyposmotic solutions. One was the habitual volume‐regulated current elicited by external hyposmolarity (ICl,swell), and the second was a slow and smooth current (Sin) generated by ACh or ATP application. 3 In follicles, the permeability ratios for different anions with respect to Cl− were similar for both ICl,swell and Sin, with a sequence of: SCN− > I− > Br−≥ NO3−≥ Cl− > gluconate ≥ cyclamate > acetate > SO42−. 4 Extracellular ATP blocked the outward component of Sin. Also, extracellular pH modulated the inactivation kinetics of Sin elicited by ACh; e.g. inactivation at +80 mV was ∼100% slower at pH 8.0 compared with that at pH 6.0. 5 Lanthanides inhibited ICl,swell and Sin. La3+ completely inhibited ICl,swell with a half‐maximal inhibitory concentration (IC50) of 17 ± 1.9 μm, while Sin was blocked up to 55% with an apparent IC50 of 36 ± 2.6 μm. 6 Patch‐clamp recordings in follicular cells showed that hyposmotic challenge opened inward single‐channel currents. The single channel conductance (4.7 ± 0.4 pS) had a linear current‐voltage relationship with a reversal membrane potential close to −20 mV. This single‐channel activity was increased by application of ACh or ATP. 7 The ICl,swell generation was not affected by pirenzepine or metoctramine, and did not affect the purinergic activation of the chloride current named Fin. Thus, ICl,swell was not generated via neurotransmitters released during cellular swelling. 8 All together, equal discrimination for different anions, similar modulatory effects by extracellular pH, the blocking effects by ATP and La3+, and the same single‐channel activity, strongly suggest that ICl,swell and Sin currents depend on the opening of the same type or a closely related class of volume‐regulated chloride channels.

MUSCARINIC RECEPTOR HETEROGENEITY IN FOLLICLE-ENCLOSED XENOPUS OOCYTES

1 Ionic current responses elicited by acetylcholine (ACh) in follicle‐enclosed Xenopus oocytes (follicles) were studied using the two‐electrode voltage‐clamp technique. ACh generated a fast chloride current (Fin) and inhibited K+ currents gated by cAMP (IK,cAMP) following receptor activation by adenosine, follicle‐stimulating hormone or noradrenaline. These previously described cholinergic responses were confirmed to be of the muscarinic type, and were independently generated among follicles from different frogs. 2 Inhibition of IK,cAMP was about 100 times more sensitive to ACh than Fin activation; the half‐maximal effective concentrations (EC50) were 6.6 ± 0.4 and 784 ± 4 nm, respectively. 3 Both responses were blocked by several muscarinic receptor antagonists. Using the respective EC50 concentrations of ACh as standard, the antagonist 4‐diphenylacetoxy‐N‐methylpiperidine methiodide blocked the two effects with very different potencies. Fin was blocked with a half‐maximal inhibitory concentration (IC50) of 2.4 ± 0.07 nm, whilst the IC50 for IK,cAMP inhibition was 5.9 ± 0.2 μm. 4 Oxotremorine, a muscarinic agonist, preferentially stimulated IK,cAMP inhibition (EC50= 15.8 ± 1.4 μm), whilst Fin was only weakly activated. In contrast, oxotremorine inhibited Fin generated by ACh with an IC50 of 2.3 ± 0.7 μm. 5 F in elicited via purinergic receptor stimulation was not affected by oxotremorine, indicating that the inhibition produced was specific to the muscarinic receptor, and suggesting that muscarinic actions do not exert a strong effect on follicular cell‐oocyte coupling. 6 Using reverse transcription‐PCR, transcripts of a previously cloned muscarinic receptor from Xenopus (XlmR) were amplified from the RNA of both the isolated follicular cells and the oocyte. The pharmacological and molecular characteristics suggest that XlmR is involved in IK,cAMP inhibition. 7 In conclusion, follicular cells possess two different muscarinic receptors, one resembling the M2 (or M4) subtype and the other the M3 subtype. These receptors are coupled to distinct membrane mechanisms leading to independent regulation of two membrane conductances.

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

BackgroundPurinergic 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).MethodsTIC 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.ResultsRT-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 μM, while that for UDP was 3.24 +/- 0.82 μM. 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 μM, 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.ConclusionHere, 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.

Functional interaction between native G protein-coupled purinergic receptors in Xenopus follicles

Purinergic receptors are expressed in the membrane of the follicular cell layer that communicates with the Xenopus oocyte. Adenosine (Ado) generates a cAMP-dependent K+ current (IK,cAMP), whereas ATP activates a Cl− current (FCl) and has a dual effect on IK,cAMP, provoking both its activation and inhibition. Here, purinergic responses were studied electrophysiologically, first in the whole follicle (w.f.), and then in the same follicle after removal of its epithelium/theca layers (e.t.r. follicle). Responses were analyzed as the ratio of the current amplitudes (ietr/iwf) in the two preparations. For ATP activation of IK,cAMP and FCl, the ratios ietr/iwf were 0.053 and 22, respectively, whereas that for Ado was 0.75. Thus, epithelium/theca removal drastically altered the ATP response, suggesting a change in the signaling pathway that correlated with changes in the pharmacological characteristics: the half-maximal effective concentration for activation of the main current in w.f. (IK,cAMP) was 14 ± 3.8 μM [Hill coefficient (nH) = 2.7 ± 0.61], and that in e.t.r. follicles (FCl) was 1.8 ± 0.68 μM (nH = 0.76 ± 0.09), whereas Ado-response parameters did not change. Responses to UTP and β,γ-methylene-ATP, specific agonists for IK,cAMP inhibition and activation, respectively, indicated that in e.t.r. follicles inhibition increased and activation decreased drastically. Thus, purinergic responses were not independent; instead, they were functionally linked. We hypothesize that this property was due to direct interactions between receptors for Ado (A2 subtype) and ATP (P2Y subtype) in the Xenopus follicle.

Epithelium and/or theca are required for ATP-elicited K+ current in follicle-enclosed Xenopus oocytes

The Xenopus follicular cell membrane is endowed with ATP‐sensitive K+ channels, which are operated by various transmitters. These generate the ionic response named IK,cAMP via a mechanism that involves intracellular cAMP synthesis. It is known that opening these K+ channels favors oocyte maturation. Follicle stimulation by adenosine (Ado) or ATP consistently generates a strong IK,cAMP via activation of P1 and P3 purinergic receptors; however, ATP can also inhibit IK,cAMP, apparently acting on a third receptor type. Here, we show that IK,cAMP might be elicited by ATP released within the follicle, and that current activation by ATP was entirely dependent on the presence of epithelial and/or theca layers. Morphological studies confirmed that removal of epithelium/theca in these follicles (e.t.r.) was complete, and activation of fast Cl− (Fin) currents by ATP in e.t.r. follicles confirmed that communication between oocyte and follicular cells remained unchanged. Thus, dependence on epithelium/theca was specific for ATP‐elicited K+ current. Using UTP and βγ‐MeATP as specific purinergic agents for IK,cAMP inhibition and activation, respectively, it was found that inhibition of IK,cAMP elicited by ATP or UTP was robustly present in e.t.r. follicles but was absent or strongly decreased in whole follicles (w.f.). Accordingly, this indicated that in w.f., epithelium and/or theca downregulated the IK,cAMP inhibition evoked by ATP, and that this control mechanism was absent in e.t.r. follicles. We suggest that this notable action on follicular cells involves one or both of two mechanisms, a paracrine transmitter released from epithelial and/or theca layers and action of ecto‐ATPases.