Postsynaptic densities (PSDs) were isolated from rat brain cortex and hippocampus, purified and i... more Postsynaptic densities (PSDs) were isolated from rat brain cortex and hippocampus, purified and incorporated into giant (5-80 mm in diameter) liposomes. Gigaohm seals were obtained with a patch-clamp pipette, and a giant liposome PSD-containing membrane patch, was excised and recorded. The PSD was always oriented in an inside-out configuration. This allowed receptor agonists or antagonists to be added from the interior of the recording pipette, and also the addition of different substances, such as ATP, calcium, calmodulin and others to the 'intracellular' side of the PSD, i.e. to the bath. a-Amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) receptor agonists such as quisqualate or AMPA induced in the PSD a complex pattern of electrical activity, that was blocked by 10 mM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but not by 2-aminophosphonovalerate (APV). The currents generated by 0.5-1 mM quisqualate were increased by about 100% when the PSDs were phosphorylated. Similar findings were obtained when the agonist was 0.2-2 mM kainate. These currents were also blocked by a non-N-methyl-d-aspartate (NMDA) receptor antagonist but not by APV, and were increased by about 70% by phosphorylation of the PSDs. Addition of 5-10 mM NMDA plus 1 mM glycine to the 'extracellular' side of the PSD, led to a characteristic pattern of activity, with the opening of multiple receptor ion channels. This was entirely blocked by 10 mM APV. Addition of extracellular Mg 2 + (1-2 mM) induced a voltage-dependent block of the currents. Phosphorylation of the PSD led to an increase of Mg 2 +blocked current of about 80%. The effect of phosphorylation on ion channel activity showed a markedly different requirement for calcium and for calmodulin among the AMPA, kainate and NMDA types of glutamate receptors, thus suggesting that each receptor type is coupled at the synapse with a unique complement of protein phosphokinases.
Reconstitution of the purified nicotinic acetylcholine receptor (AChR) from Torpedo into artifici... more Reconstitution of the purified nicotinic acetylcholine receptor (AChR) from Torpedo into artificial membranes has shown that the presence of lipids such as cholesterol and phosphatidic acid (PA), are important in preserving an optimal cation channel activity of the protein. However, no satisfactory explanation has been given for the molecular events by which specific lipids exert such effects on AChR activity. Several hypotheses have been entertained, including (i) indirect effects of lipids through alteration of bilayer properties, such as the fluidity or the membrane curvature and lateral pressure or (ii) direct effects through binding of lipids to transmembrane sites on the protein. The latter led to postulation of a role for lipids as peculiar 'allosteric' ligands of the AChR. Previous Fouriertransform infrared spectroscopy studies using AChR reconstituted into lipid mixtures containing dimyristoyl phospholipids as probes to explore the effects of the protein on phospholipid organization 3 suggested that the AChR specifically directs lateral phase separation of the monoanionic phosphoryl form of the dimyristoyl phosphatidic acid (DMPA) probe, causing the formation of phosphatidic acidrich domains that become segregated from the bulk lipids. We are now reporting on further details of the protein dependence of such phenomena. Differential scanning calorimetry (DSC) studies of AChR reconstituted at ~1:3500 protein to phospholipid molar ratio in egg phosphatidylcholine, cholesterol, and different dimyristoyl phosopholipids (2:1:1, by mole) show that in the absence of AChR protein, none of the vesicles exhibited thermal transitions within the temperature range studied, as expected from ideal (random) mixing of the lipid components. In the presence of AChR, only vesicles containing DMPA out of all the different dimyristoyl phospholipids attempted (DMPC, DMPS, DMPG, and DMPA) show a small and narrow endotherm, at ~41-43°C. A wider endotherm at lower temperatures (below 35-40°C), is also seen in these samples preceding the cooperative transition at 41-43°C (Fig. .1). At higher temperatures a very small and wide endotherm appears centered at 55-57°C resulting from thermal denaturation of the AChR protein which, because of its irreversible nature, disappears from the thermogram in a second thermal scan.
Regulation of human placental chloride channel by arachidonic acid and other cis unsaturated fatty acids
American Journal of Obstetrics and Gynecology, 1999
Arachidonic acid has been implicated in the modulation of various transport processes, including ... more Arachidonic acid has been implicated in the modulation of various transport processes, including conductive chloride transport in brush border membranes in the human placenta. The purpose of this work was to explore the effects of some cis unsaturated fatty acids on the electrophysiologic properties of the maxi chloride channels present in apical membranes from human placenta. Apical membrane chloride channels from human term placentas were reconstituted in giant liposomes. These cell-sized liposomes, generated by a cycle of dehydration and rehydration, are suitable for electrophysiologic studies by the patch-clamp method. Low micromolar concentrations of arachidonic acid reversibly inhibit maxi chloride channels in excised patches. Other cis unsaturated fatty acids, such as oleic and linoleic acids, show similar blockade. The inhibition was dose dependent. The maxi chloride channel can also be inhibited by 4,4 -diisothiocyanatostilbene-2,2 -disulfonic acid, a known chloride channel inhibitor. Our results identify the apical membrane maxi chloride channel as a possible electrophysi ologic counterpart of 4,4 -diisothiocyanatostilbene-2, 2 -disulfonic acid and cis unsaturated fatty acid-inhibited conductance previously described in brush border membranes of the human placenta. From a functional point of view the control of these channels by arachidonic acid may be of great importance in placental physiologic characteristics. Regulation of chloride channels could be important in the control of electrolyte and fluid transfer across the placenta. In addition, if these channels contribute to setting the membrane potential their regulation could have consequences for nutrient transport and delivery to the fetus. The electrophysiologic identification of these channels and their regulation might help to unravel their possible role in transplacental transport in normal and pathologic placental tissue.
The effects of short-term infusion of fenoldopam on systemic haemodynamics and indices of renal function in the normotensive neonatal foal
The effects of fenoldopam mesylate, a dopamine-1 receptor agonist, are unknown in the foal, as is... more The effects of fenoldopam mesylate, a dopamine-1 receptor agonist, are unknown in the foal, as is the distribution of dopamine1 receptors. The aim of this study was to document the effects of fenoldopam on the systemic circulation and renal function of the normotensive neonatal foal. Fenoldopam is licensed as an anti-hypertensive drug for humans in the USA. It decreases systolic and diastolic arterial pressure in hypertensive humans at doses of 0.04mcg/kg/min to 0.8mcg/kg/min (Taylor et al. 1999). However, no change in haemodynamics at a dose of 0.03mcg/kg/min were seen in normotensive humans, and a dose of 0.3mcg/kg/min caused a moderate decrease in diastolic pressure but no change in systolic pressure (Mathur et al. 1999). Both doses increased renal blood flow, but not creatinine clearance, in normotensive humans (Mathur et al. 1999). There are species differences in the renal response to fenoldopam. Dogs showed an increase in renal blood flow, but this effect could not be documen...
American Journal of Physiology-Cell Physiology, 1997
We have studied the effect of intracellular pH (pHi) shifts on the activity of Ca(2+)-dependent, ... more We have studied the effect of intracellular pH (pHi) shifts on the activity of Ca(2+)-dependent, inwardly rectifying K+ channels of HeLa cells. Recordings of macroscopic currents in symmetrical 145 mK K+ and internal pH of 7.4 gave moderate inward rectification of the current. At pH 6.4, inward rectification was more marked, whereas it switched to outward rectification at pH 8.2. In excised inside-out membrane patches, similar changes in pHi did not affect the single-channel conductance of the channels underlying the Ca(2+)-dependent K+ currents. At neutral pH, the open state probability (Po) was independent of voltage in the range from -70 to 70 mV. At alkaline pH, Po became voltage dependent, decreasing at negative potentials and increasing with depolarization compared with pH 7.4. These changes accounted for the pH-dependent changes in rectification of the macroscopic current. The possibility that voltage dependence might arise from the ionization of a thiol group was tested by u...
Comparative total currents between apical syncytiotrophoblast membrane in normal and pre-eclamptic human placenta transplanted to Xenopus laevis oocytes
Placental transfer involves specific transport mechanisms through both apical and basal plasma me... more Placental transfer involves specific transport mechanisms through both apical and basal plasma membranes. Pre-eclampsia is a pregnancy disease that affects multiple functions, among them transport function, with consistent fetal growth restriction. Our previous results showed that the open probability (Po) of the Maxi-chloride channel present in microvillous membrane from pre-eclamptic placentas becomes voltage independent, at difference from normal placentas. The aim of this study was to compare total currents of Xenopus laevis oocytes transplanted with microvillous membrane from normal (MVM) and pre-eclamptic (MVMpe) placentas. Methods: Two electrode voltage clamp technique was performed in Xenopus oocytes injected with MVM or MVMpe purified vesicles. Functional incorporation of foreign channels into the oocyte membrane was assessed by recording membrane currents (I) in response to voltage pulses from holding potential - 100mV up to +60mV, in 20mV steps in uninjected (control), MV...
The human placental syncytiotrophoblast (hSTB) is a polarized epithelial structure, without parac... more The human placental syncytiotrophoblast (hSTB) is a polarized epithelial structure, without paracellular routes, forming the main barrier for materno-fetal exchange. There is ample evidence suggesting the presence of potassium (K þ) channels in the placental apical membrane; which could contribute to membrane potential and volume regulation. We have therefore examined the K þ currents of isolated apical membranes from human term placenta using electrophysiological methods: reconstitution of ion channels from apical membranes into giant liposomes (single channel recordings, patch clamp method) or their functional transplantation into Xenopus laevis oocytes (total currents recording, voltage clamp method). Single channel recording experiments show the presence of K þ channels in the hSTB microvillous membrane sensitive to Tetraethylammonium (TEA) and Barium (Ba þ2). Patch current activity was diminished 50% and 70% by 20 mmol/L TEA and 5 mmol/L Ba þ2 respectively. The more frequent conductance was approximately 73 pS, however several levels of current were detected suggesting the presence of more than one type of K þ channel. In addition, sodium (Na þ) sensitivity was detected in the patch current thus, over 10 mmol/L Na þ reduced the seal current to 38%. These results were corroborated by the total current experiments where the K þ current elicited in injected oocytes with apical purified membrane was blocked by Ba þ2 and TEA. The total current was also affected by Na þ , becoming larger when a Na þ-free solution was used. Our results show the existence of at least two types of Ba þ2-sensitive K þ channels including a TEA sensitive sub-population, and some of them Na þ sensitive K þ channels. These channels could be the conductive pathways proposed previously for this cation in placental hSTB. Our novel contribution has been to successfully obtain K þ channel recordings in systems suitable for electrophysiological studies of isolated apical membranes.
A Low Conductance, Non-selective Cation Channel from Human Placenta
Placenta, 2002
Non-selective cation channels have been identified in the plasma membranes of many different cell... more Non-selective cation channels have been identified in the plasma membranes of many different cells. Previous research using fluorescent techniques has demonstrated the presence of cation conductances in membranes from human trophoblast. The purpose of this work was to explore, by electrophysiological methods, a non-selective cation channel in apical membranes from human placenta. Human placental apical membranes were purified by differential centrifugation and reconstituted in giant liposomes. These giant liposomes were then used for electrophysiological studies and were probed for the presence of cation channels by the patch-clamp method. The channel identified had a linear current-potential relationship with a conductance of around 16 pS in symmetrical Na(+) solution. Under asymmetrical conditions the reversal potential was close to the reversal potential for Na(+). The channel was equally permeable to sodium and potassium and the permeability sequence was NH+4>Cs(+) approximately Rb(+)>Na(+) approximately K(+)>Li(+). The channel also showed permeability to calcium and barium. The channel was insensitive to calcium but was blocked by millimolar concentration of Mg(2+). We have demonstrated the presence of a low conductance, non-selective cation channel in placental apical membranes. These channels share some properties with non-selective cation channels previously described in other different cells. The precise role of these channels in placental physiology has yet to be determined.
Large Chloride Channel from Pre-eclamptic Human Placenta
Placenta, 2003
Chloride transport involving conductive pathways participates in numerous epithelial functions, s... more Chloride transport involving conductive pathways participates in numerous epithelial functions, such as membrane voltage maintenance, solute transport and cell volume regulation. Evidence points to involvement of transepithelial chloride transport in such functions in placental syncytiotrophoblast. A molecular candidate for physiologic conductive chloride transport in apical syncytiotrophoblast membrane is a Maxi-chloride channel with distinct biophysical properties: conductance over 200 pS, multiple substates, voltage dependent open probability, and permeation to anionic amino acids. Pre-eclampsia, a high incidence pathology of pregnancy, exerts great impact on fetal morbi-mortality. This relies, among others, on intrauterine growth restriction (IUGR), thought to be mediated by diminished blood flow to the placenta, with growing knowledge regarding contribution of other factors. The Maxi-chloride channel's properties suggest it could be altered in this pathology. We have characterized the apical chloride channels from pre-eclamptic placentae, reconstituted in giant liposomes suitable for patch-clamp electrophysiological studies. In n=33 experiments from n=6 pre-eclamptic placentae we observed a chloride-permeable channel with similar biophysical properties to the channel from normal tissue (n=29 experiments from n=15 placentae). However, the main conductance state showed diminished magnitude (<150 pS), and the open probability versus voltage relationship exhibited a flattened curve instead of the bell-shaped curve of normal placentae. These results are the first evidence of a functionally altered ionic channel from placental syncytiotrophoblast in pre-eclampsia. Considering the abundance of chloride-conducting channel activity in human apical membrane and their relevance in epithelial function in general, these alterations could greatly disturb numerous placental functions that rely on syncytiotrophoblast integrity.
The human placental syncytiotrophoblast (hSTB) is a polarized epithelial structure, that forms th... more The human placental syncytiotrophoblast (hSTB) is a polarized epithelial structure, that forms the main barrier to materno-fetal exchange. The chloride (Cl À) channels in other epithelial tissues contribute to several functions, such as maintenance of the membrane potential, volume regulation, absorption and secretion. Additionally, the contributions of Cl À channels to these functions are demonstrated by certain diseases and knockout animal models. There are multiple lines of evidence for the presence of Cl À channels in the hSTB, which could contribute to different placental functions. However, both the mechanism by which these channels are involved in the physiology of the placenta, and their molecular identities are still unclear. Furthermore, a correlation between altered Cl À channels functions and pathological pregnancies is beginning to emerge. This review summarizes recent developments on conductive placental chloride transport, and discusses its potential implications for placental physiology.
Human placental syncytiotrophoblast (STB) is an epithelium responsible for materno-fetal exchange... more Human placental syncytiotrophoblast (STB) is an epithelium responsible for materno-fetal exchange. Ions play multiple roles in STB, as in other transport epithelia. We have been interested in the character and functional expression of ion channels in STB membrane fractions. Characterization of ion channels and their relationship with different domains, subdomains and microdomains of STB membranes is important to explain the intracellular mechanisms operating in the placental barrier. The aim of this paper is to summarize our work on this subject. We isolated and purified basal membrane (BM) and two fractions from the apical membrane, a classical fraction (MVM) and a light fraction (LMVM). They were used either for reconstitution into giant liposomes or for transplantation into Xenopus oocyte membranes followed by electrophysiological recordings to characterize chloride and cationic channels in STB from term human placenta. In addition, Western blot analysis, using ion channel antibodies, was performed on purified apical and basal membrane fractions. We also reported the presence of two functional microdomains (lipid rafts) in LMVM and MVM, using detergent resistant membranes (DRMs) and cholesterol-sensitive depletion. Moreover we found evidence of cytoskeletal participation in lipid rafts of different composition. Our results contribute to knowledge of the ion channels present in STB membranes and their participation in the physiology of this epithelium in normal and pathological pregnancies.
The functional expression of calcium channels has been scarcely studied in human placental syncyt... more The functional expression of calcium channels has been scarcely studied in human placental syncytiotrophoblast. We have presently sought to characterize Ca 2þ currents of the healthy syncytiotrophoblast basal membrane using purified basal membranes reconstituted in giant liposomes subjected to patch-clamp recordings. We detected presence of channels with high permeability to Ca 2þ (relative PCa/PK up to 99.5) using K þ solutions in symmetric conditions. Recordings performed in Ba 2þ gradients showed Ba 2þ-conducting channels in 100% of experiments. Ba 2þ total patch currents were consistently blocked by addition of NiCl 2 , Nifedipine (L-type voltage-gated calcium channel blocker) or Ruthenium Red (TRPV5eTRPV6 channel blocker); Nifedipine and Ruthenium Red exerted a synergic blocking effect on Ba 2þ total patch currents. Immunohistochemistry of placental villi sections evidenced presence of a 1 subunit of voltage-gated calcium channels and TRPV5eTRPV6 channels in basal and apical syncytiotrophoblast plasma membranes; these three calcium channels were also detected in purified basal and apical fractions using Western blot. These results show the presence of three types of calcium channels in the syncytiotrophoblast basal membrane by both functional and molecular means. These basal membrane calcium channels would not be directly involved in mother-to-fetus Ca 2þ transport, but could participate in other relevant trophoblast processes, such as exocytosis and Ca 2þ transport regulation.
Pfl�gers Archiv European Journal of Physiology, 1999
Tyrosine phosphorylation of botulinum neurotoxins augments their proteolytic activity and thermal... more Tyrosine phosphorylation of botulinum neurotoxins augments their proteolytic activity and thermal stability, suggesting a substantial modification of the global protein conformation. We used Fourier-transform infrared (FTIR) spectroscopy to study changes of secondary structure and thermostability of tyrosine phosphorylated botulinum neurotoxins A (BoNT A) and E (BoNT E). Changes in the conformationally-sensitive amide I band upon phosphorylation indicated an increase of the K K-helical content with a concomitant decrease of less ordered structures such as turns and random coils, and without changes in L L-sheet content. These changes in secondary structure were accompanied by an increase in the residual amide II absorbance band remaining upon H-D exchange, consistent with a tighter packing of the phosphorylated proteins. FTIR and differential scanning calorimetry (DSC) analyses of the denaturation process show that phosphorylated neurotoxins denature at temperatures higher than those required by non-phosphorylated species. These findings indicate that tyrosine phosphorylation induced a transition to higher order and that the more compact structure presumably imparts to the phosphorylated neurotoxins the higher catalytic activity and thermostability.
The plasma membrane distribution and related biological activity of nucleoside transporter protei... more The plasma membrane distribution and related biological activity of nucleoside transporter proteins (NTs) were investigated in human syncytiotrophoblast from term placenta using a variety of approaches, including nucleoside uptake measurements into vesicles from selected plasma membrane domains, NT immunohistochemistry, and subcellular localization (basal, heavy, and light apical membranes as well as raft-enriched membranes from the apical domain). In contrast with other epithelia, in this epithelium, we have identified the high-affinity pyrimidine-preferring human concentrative nucleoside transporter (hCNT) 1 as the only hCNT-type protein expressed at both the basal and apical membranes. hCNT1 localization in lipid rafts is also dependent on its subcellular localization in the apical plasma membrane, suggesting a complex cellular and regional expression. Overall, this result favors the view that the placenta is a pyrimidine-preferring nucleoside sink from both maternal and fetal sides, and hCNT1 plays a major role in promoting pyrimidine salvage and placental growth. This finding may be of pharmacological relevance, because hCNT1 is known to interact with anticancer nucleoside-derived drugs and other molecules, such as nicotine and caffeine, for which a great variety of harmful effects on placental and fetal development, including intrauterine growth retardation, have been reported.
Recent studies have shown that the epithelial sodium channel (ENaC) is expressed in vascular tiss... more Recent studies have shown that the epithelial sodium channel (ENaC) is expressed in vascular tissue. However, the role that ENaC may play in the responses to vasoconstrictors and NO production has yet to be addressed. In this study, the contractile responses of perfused pressurized small-diameter rat mesenteric arteries to phenylephrine and serotonin were reduced by ENaC blockade with amiloride (75.1±3.2% and 16.9±2.3% of control values, respectively; P <0.01) that was dose dependent (EC 50 =88.9±1.6 nmol/L). Incubation with benzamil, another ENaC blocker, had similar effects. α, β, and γ ENaC were identified in small-diameter rat mesenteric arteries using RT-PCR and Western blot with specific antibodies. In situ hybridization and immunohistochemistry localized ENaC expression to the tunica media and endothelium of small-diameter rat mesenteric arteries. Patch-clamp experiments demonstrated that primary cultures of mesenteric artery endothelial cells expressed amiloride-sensitive...
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Papers by G. Riquelme