[Intravenous thrombolysis for acute ischemic stroke. A four years’ experience in a Chilean public hospital]. / Evolución del protocolo de trombolisis endovenosa en ataque cerebrovascular isquémico agudo.

BACKGROUND: Intravenous thrombolysis (IVT) with alteplase (tissue plasminogen activator) is the standard pharmacological treatment in acute ischemic stroke (AIS), reducing disability in patients. AIM: To report the results a thrombolysis protocol during four years in a regional public hospital. MATERIAL AND METHODS: Data from 106 consecutive patients aged 68 ± 13 years (57% men) who were treated with IVT, from May 2012 until April 2016, was analyzed. RESULTS: The median door-to-needle time was 80 minutes (interquartile range = 57-113). The median National Institute of Health Stroke Scale (NIHSS) scores on admission and at discharge were was 11.5 and 5 points respectively. At discharge, 27% of hospitalized patients had a favorable outcome (n = 99), defined as having 0 to 1 points in the modified Rankin scale. Symptomatic intracerebral hemorrhage and mortality rates were 5.7 and 13.1%, respectively. The thrombolysis rate rose from 0.7% in 2012 to 6% in 2016. CONCLUSIONS: The implementation of 24/7 neurology shifts in the Emergency Department allowed us to increase the amount and quality of IVT in our hospital, as measured by the rate of thrombolysis and by process indicators such as door-to-needle time.

Evolución del protocolo de trombolisis endovenosa en ataque cerebrovascular isquémico agudo / Intravenous thrombolysis for acute ischemic stroke. A four years’ experience in a Chilean public hospital

Background: Intravenous thrombolysis (IVT) with alteplase (tissue plasminogen activator) is the standard pharmacological treatment in acute ischemic stroke (AIS), reducing disability in patients. Aim: To report the results a thrombolysis protocol during four years in a regional public hospital. Material and Methods: Data from 106 consecutive patients aged 68 ± 13 years (57% men) who were treated with IVT, from May 2012 until April 2016, was analyzed. Results: The median door-to-needle time was 80 minutes (interquartile range = 57-113). The median National Institute of Health Stroke Scale (NIHSS) scores on admission and at discharge were was 11.5 and 5 points respectively. At discharge, 27% of hospitalized patients had a favorable outcome (n = 99), defined as having 0 to 1 points in the modified Rankin scale. Symptomatic intracerebral hemorrhage and mortality rates were 5.7 and 13.1%, respectively. The thrombolysis rate rose from 0.7% in 2012 to 6% in 2016. Conclusions: The implementation of 24/7 neurology shifts in the Emergency Department allowed us to increase the amount and quality of IVT in our hospital, as measured by the rate of thrombolysis and by process indicators such as door-to-needle time.

Review: Placental syncytiotrophoblast membranes--domains, subdomains and microdomains.

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.

Programa piloto para pacientes beneficiarios de Fonasa, que padecen esclerosis multiple: tratamiento con inmunomoduladores en el sistema público de salud de Chile: Informe del primer año, 10 de julio 2008-30 de Junio 2009 / Pilot national treatment program for multiple sclerosis patients belonging to the public health care system of Chile (FONASA) with immunomodulating disease-modifying therapies: first year report from July 2008 to June 2009

Multiple Sclerosis (MS) is a chronic disease of the central nervous system. It is manifested in the young adult who presents at the beginning alternation between transient neurological dysfunction and normality, followed by a progressive level of disability. MS affects the quality of life in the young adults in their full productive and creative age limiting not only in their personal lives but also affects to the whole society in terms of "dreams and life projects". Besides, this illness also influences the family group who has to assume progressively the help and care for the patient. In healthcare aspect MS implies intensive and progressive resources. In Chile, although we don't have epidemiological studies that indicate which is the MS prevalence it exist a projection that states 14 per 100.000 inhabitants. Considering a population of 16.5 million of inhabitants our expectative of patients with MS is of 2310 cases in our country. The MS immunomodulating injectable disease-modifying therapies are of high cost and were not available in a regular way in the state health care system of Chile (FONASA) that attends the 70 percent of the population; the other 30 percent has different private health insurances. In 2008 the ministry of health decided to initiate and pilot (exploratory) program which had a great meaning and impact concerning to start offering immunomodulating therapies to relapsing remitting MS, for patients belonging to FONASA system. The pilot program was thought with a double mission, on the one hand to achieve that a very limited group of MS patients belonging to FONASA system (80 cases) from all over the country had access to immunomodulating injectable disease-modifying therapies of high cost in a regular way. The second objective was to obtain clinical and epidemiological information which let us to evaluate the clinical and administrative obstacles generated by the incorporation of this treatment in the public health...

Introducción El presente documento corresponde al informe del primer año de trabajo operativo del "Programa piloto de tratamiento con inmunomoduladores, para pacientes beneficiarios de Fonasa1, que padecen esclerosis múltiple (EM)", elaborado por el equipo del centro de referencia nacional, para este programa, con sede en el Servicio de Neurología del Complejo Asistencial Barros Luco (CABL) del SSMS2. Dado su origen no incluye antecedentes del proceso de gestión ni toma de decisiones del nivel Minsal3 o Fonasa. Este trabajo, no es ni aspira ser: un ensayo clínico, una guía de práctica clínica, una revisión bibliográfica, ni una puesta al día sobre el tratamiento de la Esclerosis Múltiple (EM), es simplemente el informe anual de un centro de referencia, para una tarea específica, a la autoridad ministerial competente. El informe incluye algunos antecedentes generales y referencias presentadas como "notas al pie", sólo para contextualizar la información presentada4. La EM es una enfermedad crónica del SNC, de origen incierto, inmunológicamente mediada, bien definida en sus características inmunopatogénicas, patológicas, imagenológicas y clínicas. Se expresa en el adulto joven, quien presenta inicialmente alternancia entre disfunción neurológica transitoria y normalidad y cuya progresión determina múltiples efectos discapacitantes. La EM afecta la calidad de vida de adultos jóvenes en plena edad productiva y creativa limitando tanto los "sueños y proyectos de vida" como el desarrollo laboral, social y afectivo. Además trasciende al grupo familiar, cuando deben asumir la asistencia del paciente. En lo sanitario, la EM, genera uso intensivo y progresivo de recursos. Las terapias inmunomoduladores para la EM, que tienen la posibilidad de detener o reducir la evolución de la modalidad recurrente remitente de la EM, no se encontraban disponibles en forma regular en el sector público de salud de nuestro país, por esto el presente programa piloto...

Placental chloride channels: a review.

The human placental syncytiotrophoblast (hSTB) is a polarized epithelial structure, that forms the main barrier to materno-fetal exchange. The chloride (Cl(-)) 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(-) channels to these functions are demonstrated by certain diseases and knock-out animal models. There are multiple lines of evidence for the presence of Cl(-) 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(-) 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.

Significado de la histerectomía para un grupo de mujeres chilenas / Meaning of hysterectomy for a group of Chilean women

Antecedentes: La histerectomía por patología benigna del útero, es una de las cirugías más frecuente después de la operación cesárea. Objetivo: Evaluar el significado que tiene la histerectomía para mujeres chilenas, beneficiarías de un hospital público de la Región Metropolitana de Santiago, Chile. Método: Estudio transversal descriptivo, de tipo cualitativo, sobre el significado que 120 mujeres histerectomizadas asigna a esa cirugía. Se describen 3 temas que reflejan el significado de la extracción del útero: pérdida, tristeza-dolor, y mejoría de una enfermedad. Resultados: El promedio de edad fue 56 +/- 11,9 años, y de escolaridad de 7,8 +/- 4,3 años. El 63 por ciento de las mujeres fue histerectomizada por patología ginecológica benigna. El significado que la mujer atribuye a la extracción del útero se encuentra relacionado a una experiencia de pérdida, que si bien conlleva un alivio de la enfermedad, también implica dolor, tristeza y soledad. Conclusión: Sensibilizar a los profesionales de la salud, que trabajan con mujeres histerectomizadas, a comprender y respetar el significado que para ellas tiene la histerectomía y propiciar el ambiente adecuado para contenerlas y educarlas pre y pos cirugía.

Background: Elective hysterectomy, after caesarian section, is one of the most frequent surgeries. Objective: To evaluate the significance that the hysterectomy has for Chilean women from a public hospital of the Metropolitan Region of Santiago, Chile. Method: Qualitative and cross-sectional design study of the significance of hysterectomy for 120 women. Three themes were derived from interviews: loss, sadness - sorrow, recovery from illness. Results: The mean of age was 56 +/- 11.9 years; the educational level was 7.8 +/- 4.3 years. A total of 63 percent of women had planned elective hysterectomy preformed. The significance that the women attribute to the hysterectomy is related to the experience of loss, and although it includes recovery from illness, it also signifies sorrow, sadness, and loneliness. Conclusion: The health care providers, who work with these women, need to be respectful and understanding of the significance that the women give to this surgery, and to give them the best setting for supporting and educating them before and after the surgery.

Barium, TEA and sodium sensitive potassium channels are present in the human placental syncytiotrophoblast apical membrane.

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(+)) channels in the placental apical membrane; which could contribute to membrane potential and volume regulation. We have therefore examined the K(+) 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(+) channels in the hSTB microvillous membrane sensitive to Tetraethylammonium (TEA) and Barium (Ba(+2)). Patch current activity was diminished 50% and 70% by 20 mmol/L TEA and 5 mmol/L Ba(+2) respectively. The more frequent conductance was approximately 73pS, however several levels of current were detected suggesting the presence of more than one type of K(+) channel. In addition, sodium (Na(+)) sensitivity was detected in the patch current thus, over 10 mmol/L Na(+) reduced the seal current to 38%. These results were corroborated by the total current experiments where the K(+) current elicited in injected oocytes with apical purified membrane was blocked by Ba(+2) and TEA. The total current was also affected by Na(+), becoming larger when a Na(+)-free solution was used. Our results show the existence of at least two types of Ba(+2)-sensitive K(+) channels including a TEA sensitive sub-population, and some of them Na(+) sensitive K(+) 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(+) channel recordings in systems suitable for electrophysiological studies of isolated apical membranes.

Functional and structural demonstration of the presence of Ca-ATPase (PMCA) in both microvillous and basal plasma membranes from syncytiotrophoblast of human term placenta.

It is known that human syncytiotrophoblast (hSCT) actively transports more than 80% of the Ca2+ that goes from maternal to fetal circulation. Transepithelial transport of Ca2+ is carried out through channels, transporters and exchangers located in both microvillous (MVM) and basal (BM) plasma membranes. The plasma membrane Ca-ATPase (PMCA) is the most important mechanism of Ca2+ homeostasis control in the human placenta. In this work, we reexamined the distribution of PMCA in isolated hSCT of term placenta. The PMCA activity was determined in isolated hSCT plasma membranes. A partial characterization of the PMCA activity was performed, including an evaluation of the sensitivity of this enzyme to an in vitro induced lipid peroxidation. Expression of the PMCA in hSCT plasma membranes and tissue sections was investigated using Western blots and immunohistochemistry, respectively. Our study demonstrates, for the first time, a correlation between the activity and structural distribution of PMCA in both MVM and BM of hSCT. It also demonstrates a higher PMCA activity and expression in MVM as compared to BM. Finally, PMCA4 seems to be preferentially distributed in both hSCT plasma membranes, while PMCA1 is shown to be present in the hSCT homogenate. However, the membrane fractions did not show any PMCA1 labeling. Our results must be taken into account in order to propose a new model for the transport of calcium across the hSCT.

Satisfacción con la educación recibida en un grupo de mujeres histerectomizadas / Satisfaction with the education received in group of hysterectomized women

La histerectomía es una de las intervenciones quirúrgicas mas frecuentes en nuestro país, por lo que resulta importante conocer las necesidades educativas de las mujeres para mejorar la calidad del cuidado otorgándoles una educación oportuna y atingente durante este proceso quirúrgico. El objetivo de este estudio es conocer la satisfacción de un grupo de mujeres histerectomizadas con la educación recibida. Es un estudio descriptivo, transversal, con 120 mujeres de un hospital público de Santiago. Se realizo una entrevista a cada mujer, previo a la cual se solicitó consentimiento informado. El 46,7 por ciento recibió educación del médico, el 75 por ciento de manera verbal-individual. El 70 por ciento recibe educación antes de ser histerectomizada, principalmente durante la hospitalización y el nivel secundario de atención. Los contenidos abordados están centrados en la cirugía y cuidados posteriores; ninguna mujer recibió educación respecto de sexualidad. El 100 por ciento de ellas declara que sus parejas no recibieron educación. El 23,3 por ciento quedó muy satisfecha con el profesional que las educó; el 10 por ciento con la forma utilizada; el 10,8 por ciento con el lugar donde recibió educación; el 18,3 por ciento con el momento en que recibe educación y el 7 por ciento con los contenidos recibidos. Se concluye que el grado de satisfacción de las mujeres con la educación recibida es bajo, por lo que se destaca la importancia de orientar nuestra intervención a las necesidades educativas de las mujeres histerectomizadas, de manera de implementar programas educativos orientados a lo que ellas realmente quieren y necesitan saber sobre los cuidados antes, durante y después de esta cirugía.

Hysterectomy is one of the most important surgeries in our country, therefore it is very important to know what the women need to know to improve the quality of care, by giving them a right and timely education during the surgical process. The aim of this research is to know the satisfaction with the education in a group of histerecto-mized women. The methodology is cross sectional study, with 120 women in a public hospital of Santiago. Data compilation was done by means of an interview, before a consent inform was applied. 46.7 percent of women received education from the physician, 75 percent with verbal and individual way; 70 percent of the women received education before the surgery, at hospital and in the second level. The topics were related with the surgery and the different cares before it. None of the women received education about sexuality. 100 percent of the women say that their part-ners did not received education. 23.3 percent of the women were very satisfied with the professional that gave them education, 10 percent with the way, 10.8 percent with the place, 18.3 percent with the time and 7 percent with the topics. The satisfaction with the education was very low, therefore it is very important that our intervention will be related with the real needs that women want about the surgery.

The maxi-chloride channel in human syncytiotrophoblast: a pathway for taurine efflux in placental volume regulation?

Taurine (Tau), the most abundant amino acid in fetal blood, is highly concentrated in human placenta. During pregnancy, Tau is involved in the neurological development of the fetus, and in volume regulation of the placenta. The placenta may release taurine in parallel with K(+) and Cl(-) in response to an increase in cell volume. However, the pathway for the volume-activated taurine efflux is unknown. One candidate is a voltage-dependent Maxi-chloride channel from apical syncytiotrophoblast membrane (MVM), with a conductance over 200pS and multiple subconductance states. Our aim was to study whether this channel could be a Tau conductive pathway in the MVM. Purified human placental MVM were reconstituted into giant liposomes suitable for patch clamp recordings. Typical Maxi-chloride channel activity was detected in symmetrical chloride (Cl(-)) solutions, and then taurine (Tau), Aspartate (Asp), and glutamate (Glu) solutions were used in the bath of excised patches to detect single channel currents carried by these anions. The relative permeabilities (P), estimated from the shift in reversal potential of current-voltage curves after anion replacement, were as follows: Chloride>Taurine=Glutamate=Aspartate. In Tau symmetric conditions using equivalent Cl(-) concentrations, the slope conductance was 62.4+/-7.3pS. The data shows that Tau and other amino acids diffuse through the Maxi-chloride channel, which could be of great importance as part of the mechanism involved in the volume regulation process in human placenta.

Diverse calcium channel types are present in the human placental syncytiotrophoblast basal membrane.

The functional expression of calcium channels has been scarcely studied in human placental syncytiotrophoblast. We have presently sought to characterize Ca(2+) 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(2+) (relative PCa/PK up to 99.5) using K(+) solutions in symmetric conditions. Recordings performed in Ba(2+) gradients showed Ba(2+)-conducting channels in 100% of experiments. Ba(2+) total patch currents were consistently blocked by addition of NiCl(2), Nifedipine (L-type voltage-gated calcium channel blocker) or Ruthenium Red (TRPV5-TRPV6 channel blocker); Nifedipine and Ruthenium Red exerted a synergic blocking effect on Ba(2+) total patch currents. Immunohistochemistry of placental villi sections evidenced presence of alpha(1) subunit of voltage-gated calcium channels and TRPV5-TRPV6 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(2+) transport, but could participate in other relevant trophoblast processes, such as exocytosis and Ca(2+) transport regulation.

Educación de mujeres histerectomizadas: ¿qué desean saber? / Educational needs of hysterectomized women

Antecedentes: La histerectomía (HT) es una de las intervenciones más frecuentes en ginecología, por lo tanto resulta de relevancia evaluar lo que las mujeres desean para enfrentar este proceso. Objetivo: Conocer las necesidades educativas de un grupo de mujeres sometidas a histerectomía. Método: Estudio descriptivo, de corte transversal, con una muestra de 120 usuarias. Se realizó una entrevista para conocer la educación recibida y la deseada, y así establecer la necesidad educativa de las mujeres. Resultados: El 100 por ciento de ellas señala que sus parejas no recibieron educación acerca de la HT. El 46,7 por ciento declara recibir la educación por parte del médico y el 80 por ciento desearía hubiese sido la matrona. El 45 por ciento recibe la educación al fijar la fecha de la cirugía y el 78 por ciento desearía haberla recibido antes de este momento. El 47 por ciento recibe la educación en el hospital y el 46 por ciento desea recibirla ahí. El 75 por ciento refiere recibir educación en forma verbal-individual y el 47,5 por ciento le hubiese gustado de manera audiovisual-individual. Se educa a más del 50 por ciento de las mujeres sólo en 3 tópicos de los 16 evaluados. Los principales temas en los cuales fueron educadas no coinciden con aquellos deseados por las mujeres. Conclusión: Se concluye que las necesidades de las mujeres no son satisfechas en su totalidad y se destaca la importancia del rol educativo por parte de los profesionales del área.

Influence of C-terminal protein domains and protein-lipid interactions on tetramerization and stability of the potassium channel KcsA.

KcsA is a prokaryotic potassium channel formed by the assembly of four identical subunits around a central aqueous pore. Although the high-resolution X-ray structure of the transmembrane portion of KcsA is known [Doyle, D. A., Morais, C. J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280, 69-77], the identification of the molecular determinant(s) involved in promoting subunit tetramerization remains to be determined. Here, C-terminal deletion channel mutants, KcsA Delta125-160 and Delta120-160, as well as 1-125 KcsA obtained from chymotrypsin cleavage of full-length 1-160 KcsA, have been used to evaluate the role of the C-terminal segment on the stability and tetrameric assembly of the channel protein. We found that the lack of the cytoplasmic C-terminal domain of KcsA, and most critically the 120-124 sequence stretch, impairs tetrameric assembly of channel subunits in a heterologous E. coli expression system. Molecular modeling of KcsA predicts that, indeed, such sequence stretch provides intersubunit interaction sites by hydrogen bonding to amino acid residues in N- and C-terminal segments of adjacent subunits. However, once the KcsA tetramer is assembled, its remarkable in vitro stability to detergent or to heat-induced dissociation into subunits is not greatly influenced by whether the entire C-terminal domain continues being part of the protein. Finally and most interestingly, it is observed that, even in the absence of the C-terminal domain involved in tetramerization, reconstitution into membrane lipids promotes in vitro KcsA tetramerization very efficiently, an event which is likely mediated by allowing proper hydrophobic interactions involving intramembrane protein domains.

Multisensory convergence in the mushroom bodies of ants and bees.

The mushroom bodies, central neuropils in the arthropod brain, are involved in learning and memory and in the control of complex behavior. In most insects, the mushroom bodies receive direct olfactory input in their calyx region. In Hymenoptera, olfactory input is layered in the calyx. In ants, several layers can be discriminated that correspond to different clusters of glomeruli in the antennal lobes, perhaps corresponding to different classes of odors. Only in Hymenoptera, the mushroom body calyx also receives direct visual input from the optic lobes. In bees, six calycal layers receive input from different classes of visual interneurons, probably representing different parts of the visual field and different visual properties. Taken together, the mushroom bodies receive distinct multisensory information in many segregated input layers.

Isolation and purification of human placental plasma membranes from normal and pre-eclamptic pregnancies. a comparative study.

Human placental syncytiotrophoblast is the main barrier for materno-fetal exchange. Analysis of transplacental transport involves the study of ion channels in both the maternal-facing microvillous membrane (MVM) and the fetal-facing basal membrane (BM). Difficulties in having access to intact placenta with conventional electrophysiological methods favour alternative methodologies, such as isolation and reconstitution of membranes in artificial lipid systems. Pre-eclampsia is a major health problem of human pregnancy. The search for altered physiological processes in pre-eclamptic placentae requires the investigation of events at both the microvillous and basal surfaces. The aim of this study was to obtain reliable syncytiotrophoblast plasma membranes from human normal (N) and pre-eclamptic (PE) pregnancies. We describe a protocol which allows for the simultaneous isolation of MVM and BM. The purity of the membranes isolated was evaluated using enzymatic assays, binding studies, Western blotting and immunohistochemistry. Enrichment of alkaline phosphatase activity for MVM was 17 to 21-fold, with 13-16 per cent protein recovery, for both N and PE. Enrichment of adenylate cyclase activity for BM was 9-fold for N, and enrichment of dihydroalprenolol binding to beta-adrenergic receptors was 12-fold for N and 6-fold for PE, with 14 per cent protein recovery for both N and PE. Cross contamination was low and mitochondrial membrane contamination was negligible. We conclude that MVM and BM isolated from placentae of pre-eclamptic women are similar in enrichment and purity to those of healthy women, thus allowing their use in comparative electrophysiological studies.

Large chloride channel from pre-eclamptic human placenta.

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.

17beta-estradiol and tamoxifen regulate a maxi-chloride channel from human placenta.

Steroid hormones have been implicated in the modulation of several transport processes, including conductive chloride transport in epithelial cells. Micromolar concentrations of these hormones have been determined in blood of pregnant women. The purpose of this work was to explore the effects of 17beta-Estradiol, a steroid hormone, on the biophysical properties of the Maxi chloride channel present in apical membranes from human placental syncytiotrophoblast. Apical membrane chloride channels from human term placentas were reconstituted in giant liposomes suitable for electrophysiologic studies by the patch-clamp method. Low micromolar concentrations of 17beta-Estradiol inhibit the Maxi chloride channels in excised patches in a potential-dependent manner. The addition of 1 mM 17beta-Estradiol to the bath solution decreased the total current in the patch from 100% control to 71% at -40 mV holding potential and the current was not affected by 17beta-Estradiol at + 40 mV. However, the presence of the hormone did not affect the single-channel conductance, therefore its effect must be due to modulation of its open probability (Po). Interestingly, 17alpha-Estradiol did not change the total current in the patch. Tamoxifen, an antiestrogen, also showed inhibition, but in a voltage-independent manner. Our results suggest that the Maxi Cl- channel from human term placenta may be regulated by direct interaction of both compounds with the channel. From a functional point of view, the control of these channels by steroid hormones may be of great importance in placental physiology and their regulation may help to unravel their possible role in transplacental transport.

Functional transplantation of chloride channels from the human syncytiotrophoblast microvillous membrane to Xenopus oocytes.

The materno-fetal transfer of metabolites and nutrients requires the operation of specific transport mechanisms through syncytiotrophoblast membranes. Electrophysiological studies on these cells are scarce and, because of their syncytial nature, whole-cell current recordings have not been carried out. We have now studied whether or not ion channels from the human syncytiotrophoblast microvillous (hSM) membrane can be transplanted to Xenopus oocytes. Sixty-two percent of hSM-injected oocytes displayed lower resting potential and higher membrane conductance than uninjected cells. The increased membrane conductance was due to the incorporation of Cl(-) channels, because neither replacing Na(+) in the bathing solution by N-methyl- D-glucamine or K(+), nor withdrawing Ca(2+) had any significant effect on the currents elicited by voltage pulses. In contrast, substitution of Cl(-) by different anions markedly affected the membrane conductance, giving an anion selectivity sequence of I(-)>Br(-)>Cl(-)>methanosulfonate congruent with gluconate. In addition, disulfonic stilbenes and gluconate, but not anthracene-9-carboxylic acid, blocked the transplanted channels. These properties are compatible with those of placental Cl(-) "maxi" channels. It is concluded that functional Cl(-) channels from the hSM become effectively incorporated into the Xenopus oocyte membrane, where their function can be studied in detail.

A low conductance, non-selective cation channel from human placenta.

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.

Effect of the inactivating "ball" peptide of Shaker B on intermediate conductance Ca(2+)-dependent inwardly rectifying K+ channels of HeLa cells.

The patch-clamp technique was used to study the effect of intracellularly added inactivating "ball" peptide (BP) of the Shaker B K+ channel upon Ca(2+)-dependent inwardly rectifying K+ channels of the intermediate conductance type expressed in HeLa cells. Intracellular BP caused only moderate inhibition of outward K+ currents when assayed at an intracellular Ca2+ concentration of 100 nmol/l. Increasing intracellular Ca2+ levels led in itself to some voltage-dependent blockade of K+ currents, which was absent when high extracellular K+ was used. An additional strong blockade by intracellular BP was nevertheless observed both in Na(+)- and K(+)-rich extracellular solutions. A non-inactivating BP analogue had no effect. At this higher intracellular Ca2+ concentration the inhibition of these intermediate conductance Ca(2+)-dependent channels by BP was voltage-dependent, being absent at hyperpolarizing potentials, and could be relieved by increasing extracellular K+. These data suggest that BP acts at an internal pore site in Ca(2+)-dependent intermediate conductance K+ channels of HeLa cells, and that these might possess a receptor site for the peptide similar to that of other K+ channels such as Ca(2+)-activated maxi-K+ channels.