Development of Mortars That Use Recycled Aggregates from a Sodium Silicate Process and the Influence of Graphene Oxide as a Nano-Addition.

This research analyses how different cement mortars behave in terms of their physical and mechanical properties. Several components were necessary to make seven mixes of mortars, such as Portland cement, standard sand, and solid waste from a factory of sodium silicate, in addition to graphene oxide. Furthermore, graphene oxide (GO) was selected to reduce the micropores and increase the nanopores in the cement mortar. Hence, some tests were carried out to determine their density, humidity content, water absorption capacity, open void porosity, the alkali-silica reaction, as well as flexural and mechanical strength and acid resistance. Thus, standard-sand-manufactured mortars' mechanical properties were proved to be slightly better than those manufactured with recycled waste; the mortars with this recycled aggregate presented problems of alkali-silica reaction. In addition, GO (in a ratio GO/cement = 0.0003) performed as a filler, improving the mechanical properties (30%), alkali-silica (80%), and acid resistance.

Cost-effectiveness analysis of spontaneous vaginal delivery vs elective cesarean delivery for maternal outcomes in Colombia.

BACKGROUND: In worldwide literature, it has been found that cesarean deliveries represent higher costs and are associated with maternal morbidity and other complications. OBJECTIVE: This study aimed to estimate the cost-effectiveness of elective cesarean delivery compared with spontaneous vaginal delivery in short-term maternal outcomes for low-risk obstetrical population in Colombia. STUDY DESIGN: A cost-effectiveness study using a healthcare-system perspective was performed in 2019 in Colombia. The reference population were women with full-term and low-risk pregnancy, either by spontaneous vaginal delivery or elective cesarean delivery under medical or nonmedical indications. An analytical decision model (decision tree) was designed for maternal outcomes. The time horizon was 42 days postpartum, and the health effects were measured by Quality Adjusted Life Years. A review of the literature and a validation process by a national expert committee were conducted to determine the maternal outcomes and estimate their probabilities. Costs were estimated with a top-down analysis, an incremental cost-effectiveness ratio was calculated, and finally, a sensitivity analysis was performed. RESULTS: Within a 42-day time horizon, it was found that spontaneous vaginal delivery is the less-expensive and more-effective mode of delivery, it showed a reduction in costs (324 USD) and a gain in Quality Adjusted Life Years (0.03) compared with elective cesarean delivery. Our analysis suggests that spontaneous vaginal delivery is the dominant alternative compared with elective cesarean delivery. CONCLUSION: Spontaneous vaginal delivery showed to be the cost-effective mode of delivery for low-risk obstetrical population in Columbia. These results are useful not only for obstetricians but for decision makers, who should encourage nationwide health policies in favor of spontaneous vaginal delivery.

Code Blue: Life-threatening Methemoglobinemia.

Most cases of acquired methemoglobinemia result from exposure to certain drugs or toxins. One of the more common and well-described causes in the literature is exposure to topical benzocaine during medical procedures. We present a case series of acute acquired methemoglobinemia from a food source that has not been previously described in the literature: a dessert. Three patients, ages 5, 33, and 86 years, were brought to our emergency department by ambulance after becoming extremely ill from ingesting a dessert containing nitre powder at a family gathering. They all presented with hypotension, cyanosis, and hypoxia that was not responsive to oxygen administration. The adult patients had major improvement of symptoms after a single dose of methylene blue. In contrast, the 5-year-old child who had the worst symptoms minimally improved with administration of two doses of methylene blue requiring intensive care admission and transfer to a tertiary care center.

Probabilistic Flexural Fatigue in Plain and Fiber-Reinforced Concrete.

The objective of this work is two-fold. First, we attempt to fit the experimental data on the flexural fatigue of plain and fiber-reinforced concrete with a probabilistic model (Saucedo, Yu, Medeiros, Zhang and Ruiz, Int. J. Fatigue, 2013, 48, 308-318). This model was validated for compressive fatigue at various loading frequencies, but not for flexural fatigue. Since the model is probabilistic, it is not necessarily related to the specific mechanism of fatigue damage, but rather generically explains the fatigue distribution in concrete (plain or reinforced with fibers) for damage under compression, tension or flexion. In this work, more than 100 series of flexural fatigue tests in the literature are fit with excellent results. Since the distribution of monotonic tests was not available in the majority of cases, a two-step procedure is established to estimate the model parameters based solely on fatigue tests. The coefficient of regression was more than 0.90 except for particular cases where not all tests were strictly performed under the same loading conditions, which confirms the applicability of the model to flexural fatigue data analysis. Moreover, the model parameters are closely related to fatigue performance, which demonstrates the predictive capacity of the model. For instance, the scale parameter is related to flexural strength, which improves with the addition of fibers. Similarly, fiber increases the scattering of fatigue life, which is reflected by the decreasing shape parameter.

Síndrome metabólico en conductores de servicio público en Armenia, Colombia / Metabolic syndrome in public service drivers in Armenia, Colombia

RESUMEN Objetivo Caracterizar y comparar la población de conductores de servicio público de una empresa de transporte en Armenia, Colombia. Materiales y Métodos Estudio analítico de corte transversal en el cual participaron conductores de taxi y colectivo en una empresa de servicio público de Armenia, Colombia. El análisis de las variables se realizó por medio de Statgraphics Centurion XVI. Se de criben las variables, se lleva a cabo una regresión múltiple y una regresión logística. Resultados Participaron 125 conductores, de los cuales, ocho se retiraron y finalmente quedaron 117 de sexo masculino como objeto de investigación. El 28,21 % ha conducido colectivo y 71,79 % taxi. Se observó un mayor consumo de alcohol respecto al de tabaco. El 60,69 % no realizaba ningún tipo de ejercicio físico. El promedio de IMC y perímetro abdominal fue de 28,03 kg/m2 y 100,09 cm, respectivamente. La media de triglicéridos y HDL-c en suero fue de 207,53 mg/dL y 33,12 mg/dL. Del total de conductores, el 49,57 % cumplía con los criterios diagnósticos de METS. Se encontraron diferencias estadísticamente significativas en los valores de IMC, perímetro abdominal, presión arterial sistólica, presión arterial diastólica, ejercicio semanal, triglicéridos y glicemia en ayunas entre individuos con Síndrome Metabólico y sin éste. Conclusiones Los conductores de servicio público presentan un perfil de riesgo aumentado para desarrollar Síndrome Metabólico. La obesidad, la hipertrigliceridemia y los bajos niveles de HDL-c fueron los principales parámetros de alarma para la presencia de Síndrome Metabólico.(AU)

ABSTRACT Objective To characterize and compare the population of public service drivers of a transport company in Armenia, Colombia. Materials and Methods Taxi and bus drivers from a transport company of Armenia, Colombia participated in an Analytical cross-sectional study. The analysis of the variables was made through Statgraphics Centurion XVI. The comparison of variables as well as multiple and logistic regression were carried out. Results 125 individuals participated, 8 were removed, and finally there were 117 male drivers as aim of research. The 28.21 % of individuals have driven small buses and the 71.79 % have driven cars. The increase in alcohol consumption was remarkable compared to tobacco. The 60.69 % of subjects did not get exercise. Besides, the BMI average and abdominal circumference was 28.03 kg/m2 and 100.09 cm respectively. The triglyceride average and serum HDL-C were 207.53 mg/dL to 33.12 mg/dL. 49.57 % of all drivers, fulfilled the Metabolic Syndrome diagnostic criteria. Statistically significant differences were found in the values of BMI, waist circumference, systolic blood pressure, diastolic blood pressure, weekly exercise, triglycerides and fasting glucose between individuals with and without Metabolic Syndrome. Conclusion Public service drivers have a profile risk for developing Metabolic Syndrome. Obesity, hypertriglyceridemia and low HDL-C were the main alarm parameters of the presence of Metabolic.(AU)Syndrome.

[Metabolic syndrome in public service drivers in Armenia, Colombia]. / Síndrome metabólico en conductores de servicio público en Armenia, Colombia.

OBJECTIVE: To characterize and compare the population of public service drivers of a transport company in Armenia, Colombia. MATERIALS AND METHODS: Taxi and bus drivers from a transport company of Armenia, Colombia participated in an Analytical cross-sectional study. The analysis of the variables was made through Statgraphics Centurion XVI. The comparison of variables as well as multiple and logistic regression were carried out. RESULTS: 125 individuals participated, 8 were removed, and finally there were 117 male drivers as aim of research. The 28.21 % of individuals have driven small buses and the 71.79 % have driven cars. The increase in alcohol consumption was remarkable compared to tobacco. The 60.69 % of subjects did not get exercise. Besides, the BMI average and abdominal circumference was 28.03 kg/m2 and 100.09 cm respectively. The triglyceride average and serum HDL-C were 207.53 mg/dL to 33.12 mg/dL. 49.57 % of all drivers, fulfilled the Metabolic Syndrome diagnostic criteria. Statistically significant differences were found in the values of BMI, waist circumference, systolic blood pressure, diastolic blood pressure, weekly exercise, triglycerides and fasting glucose between individuals with and without Metabolic Syndrome. CONCLUSION: Public service drivers have a profile risk for developing Metabolic Syndrome. Obesity, hypertriglyceridemia and low HDL-C were the main alarm parameters of the presence of Metabolic Syndrome.

OBJETIVO: Caracterizar y comparar la población de conductores de servicio público de una empresa de transporte en Armenia, Colombia. MATERIALES Y MÉTODOS: Estudio analítico de corte transversal en el cual participaron conductores de taxi y colectivo en una empresa de servicio público de Armenia, Colombia. El análisis de las variables se realizó por medio de Statgraphics Centurion XVI. Se de criben las variables, se lleva a cabo una regresión múltiple y una regresión logística. RESULTADOS: Participaron 125 conductores, de los cuales, ocho se retiraron y finalmente quedaron 117 de sexo masculino como objeto de investigación. El 28,21 % ha conducido colectivo y 71,79 % taxi. Se observó un mayor consumo de alcohol respecto al de tabaco. El 60,69 % no realizaba ningún tipo de ejercicio físico. El promedio de IMC y perímetro abdominal fue de 28,03 kg/m2 y 100,09 cm, respectivamente. La media de triglicéridos y HDL-c en suero fue de 207,53 mg/dL y 33,12 mg/dL. Del total de conductores, el 49,57 % cumplía con los criterios diagnósticos de METS. Se encontraron diferencias estadísticamente significativas en los valores de IMC, perímetro abdominal, presión arterial sistólica, presión arterial diastólica, ejercicio semanal, triglicéridos y glicemia en ayunas entre individuos con Síndrome Metabólico y sin éste. CONCLUSIONES: Los conductores de servicio público presentan un perfil de riesgo aumentado para desarrollar Síndrome Metabólico. La obesidad, la hipertrigliceridemia y los bajos niveles de HDL-c fueron los principales parámetros de alarma para la presencia de Síndrome Metabólico.

New clathrin-based nanoplatforms for magnetic resonance imaging.

BACKGROUND: Magnetic Resonance Imaging (MRI) has high spatial resolution, but low sensitivity for visualization of molecular targets in the central nervous system (CNS). Our goal was to develop a new MRI method with the potential for non-invasive molecular brain imaging. We herein introduce new bio-nanotechnology approaches for designing CNS contrast media based on the ubiquitous clathrin cell protein. METHODOLOGY/PRINCIPAL FINDINGS: The first approach utilizes three-legged clathrin triskelia modified to carry 81 gadolinium chelates. The second approach uses clathrin cages self-assembled from triskelia and designed to carry 432 gadolinium chelates. Clathrin triskelia and cages were characterized by size, structure, protein concentration, and chelate and gadolinium contents. Relaxivity was evaluated at 0.47 T. A series of studies were conducted to ascertain whether fluorescent-tagged clathrin nanoplatforms could cross the blood brain barriers (BBB) unaided following intranasal, intravenous, and intraperitoneal routes of administration. Clathrin nanoparticles can be constituted as triskelia (18.5 nm in size), and as cages assembled from them (55 nm). The mean chelate: clathrin heavy chain molar ratio was 27.04±4.8: 1 for triskelia, and 4.2±1.04: 1 for cages. Triskelia had ionic relaxivity of 16 mM(-1) s(-1), and molecular relaxivity of 1,166 mM(-1) s(-1), while cages had ionic relaxivity of 81 mM(-1) s(-1) and molecular relaxivity of 31,512 mM(-1) s(-1). Thus, cages exhibited 20 times higher ionic relaxivity and 8,000-fold greater molecular relaxivity than gadopentetate dimeglumine. Clathrin nanoplatforms modified with fluorescent tags were able to cross or bypass the BBB without enhancements following intravenous, intraperitoneal and intranasal administration in rats. CONCLUSIONS/SIGNIFICANCE: Use of clathrin triskelia and cages as carriers of CNS contrast media represents a new approach. This new biocompatible protein-based nanotechnology demonstrated suitable physicochemical properties to warrant further in vivo imaging and drug delivery studies. Significantly, both nanotransporters crossed and/or bypassed the BBB without enhancers. Thus, clathrin nanoplatforms could be an appealing alternative to existing CNS bio-nanotechnologies.

Effect of protein kinase C and Ca(2+) on p42/p44 MAPK, Pyk2, and Src activation in rat conjunctival goblet cells.

Conjunctival goblet cells synthesize and secrete mucins onto the ocular surface to lubricate it and protect it from bacterial infections. Mucin secretion is under neural control, and cholinergic agonists released from parasympathetic nerves are major stimuli of this secretion. The signal transduction pathways these agonists use to stimulate secretion involve activating protein kinase C (PKC) and increasing intracellular [Ca(2+)] to activate the non-receptor kinases Pyk2 and p60Src (Src) to transactivate the EGF receptor. Transactivation of the EGF receptor activates a kinase cascade culminating in the activation of p42/p44 MAPK (MAPK) and ultimately that leads to secretion of high molecular weight glycocongujates (HMWGC), including mucins. To further examine the roles of PKC and Ca(2+) in the activation of MAPK, Pyk2, and Src in mucin secretion, rat conjunctival pieces and cultured goblet cells were incubated with the PKC activator phorbol myristate acid (PMA), the cholinergic agonist carbachol, or the calcium ionophore, ionomycin for varying times. Conjunctival pieces were preincubated with PKC inhibitors 10min prior to addition of carbachol (10(-4)M) for 10min. The amount of phosphorylated (activated) MAPK, Pyk2 and Src was determined by Western blotting techniques using antibodies specific to the phosphorylated forms of each kinase. PMA significantly increased the activation of MAPK, Pyk2, and Src in a time and concentration-dependent manner. PMA-stimulated MAPK activity was completely inhibited by the EGF receptor inhibitor AG1478 (10(-7)M). Carbachol-stimulated MAPK activity was inhibited by three PKC inhibitors, calphostin C, chelethyrine, and staurosporine. Ionomycin (10(-6)M)-stimulated MAPK activity was inhibited 66% by AG1478 (10(-7)M). Ionomycin also significantly increased Pyk2 and Src in time dependent manner. PKC and ionomycin also activated p42/p44 MAPK, Pyk2, and Src in cultured conjunctival goblet cells. We conclude that PKC and intracellular Ca(2+) activate Pyk2 and Src and phosphorylate the EGF receptor leading to stimulation of MAPK in conjunctival goblet cells.

Roles of protein kinase C, Ca2+, Pyk2, and c-Src in agonist activation of rat lacrimal gland p42/p44 MAPK.

PURPOSE: Although p42/p44 mitogen-activated protein kinase (MAPK) negatively modulates protein secretion stimulated by cholinergic and alpha(1D)-adrenergic agonists, it does not play a role in epidermal growth factor (EGF)-stimulated protein secretion. Therefore, this study was conducted to determine the roles that protein kinase C (PKC), intracellular Ca(2+) ([Ca(2+)](i)), and nonreceptor tyrosine kinases Pyk2 and Src play in the activation of agonist- and EGF-stimulated MAPK activation. METHODS: Lacrimal gland acini were isolated by collagenase digestion and incubated with phorbol 12-myristate 13-acetate (PMA) to activate PKC or ionomycin, a Ca(2+) ionophore. Acini were preincubated with the PKC inhibitors calphostin C or Ro-31-8220, EGTA to chelate Ca(2+), or the c-Src inhibitor PP1 before stimulation with the cholinergic agonist carbachol, the alpha(1D)-adrenergic agonist phenylephrine, or EGF. Activated MAPK, Pyk2, and c-Src amounts were measured by Western blot analysis. RESULTS: PMA and ionomycin significantly increased the activation of MAPK in a time- and concentration-dependent manner. Inhibition of PKC partially inhibited carbachol-stimulated MAPK activation while completely inhibiting phenylephrine- and EGF-stimulated MAPK activation. Chelation of Ca(2+) also partially inhibited carbachol-stimulated MAPK with no effect on phenylephrine- and EGF-stimulated MAPK activation. Carbachol increased the phosphorylation of Pyk2 on tyrosine 402 and c-src on tyrosine 416 in a time-dependent manner. The c-src inhibitor PP1 inhibited carbachol-stimulated phosphorylation of Pyk2. CONCLUSIONS: It was concluded that cholinergic agonists use Ca(2+) and PKC to phosphorylate Pyk2 and c-Src, which subsequently stimulate MAPK activity. In contrast, alpha(1D)-adrenergic agonists and EGF do not use Pyk2 and Src but do use PKC to activate MAPK.

OPC-12759 increases proliferation of cultured rat conjunctival goblet cells.

PURPOSE: To determine if the gastroprotective drug OPC-12759 increased proliferation of rat conjunctival goblet cells in culture. METHODS: Cultured goblet cells were incubated with 10(-12) to 10(-8) M OPC-12759 for 1 to 7 days. Fetal bovine serum (FBS) was used as a positive control. Cell proliferation was determined by a MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay and by immunohistochemical staining with anti-Ki-67, a marker of cell division. Goblet cells were identified by double-labeling with anti-Ki-67, a marker of cell division, and Ulex europaeus agglutinin I lectin, anti-MUC5AC and anticytokeratin 7. Stratified squamous cells were identified by using Griffonia (Bandeiraea) simplicifolia lectin and anticytokeratin 4 antibody. RESULTS: As determined by MTT conversion to formazan, OPC-12579 at 10(-11) M induced an almost 2-fold increase in goblet cell proliferation on Days 1 and 3 of incubation but not on Days 5 and 7. The FBS at 10% increased cell proliferation by 2- to 3-fold at each time point. Daily replenishment of OPC-12579 for 3 consecutive days induced cell proliferation at all concentrations. Proliferation as determined by the number of Ki-67 positive cells increased by 4- and 3-fold at Days 1 and 3, respectively with addition of 10(-11) M OPC-12579. The FBS at 10% induced a 10-fold increase in goblet cell proliferation on Days 1, 3, and 5. Colocalization of Ulex europaeus agglutinin I, MUC5AC and anticytokeratin 7 with Ki-67 indicated that proliferating cells were goblet cells. Proliferating cells were negative for the nongoblet cell markers Bandeiraea lectin and anticytokeratin 4. CONCLUSIONS: The OPC-12759 stimulates proliferation of conjunctival goblet cells in primary culture.

Presence and localization of neurotrophins and neurotrophin receptors in rat lacrimal gland.

PURPOSE: To determine which of the neurotrophins (NTs)-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4/5 (NT4)-and their receptors (NTrs), TrkA, TrkB, TrkC, and p75, are present in the adult rat lacrimal gland. METHODS: RT-PCR was performed on RNA isolated from male rat lacrimal gland, using oligonucleotides specific to each NT and NTr. The presence of NT and NTr protein, was determined by Western blot analysis of lacrimal gland homogenate or membranes. The location of NTs and NTrs was determined by immunofluorescence histochemistry. Western blot analyses and immunofluorescence microscopy were performed using primary rabbit polyclonal antibodies raised against NTs and NTrs. RESULTS: RT-PCR showed positive bands at the appropriate sizes for NGF, BDNF, NT3, and NT4, and for the receptors TrkA, TrkB, TrkC, and p75. Western blot analysis confirmed these results, showing that the lacrimal gland expresses NGF, BDNF, NT3, and NT4 as well as the NTrs TrkA, TrkB, and TrkC and the p75 protein. NGF, BDNF, NT3, and NT4 were localized in the lacrimal gland acini with differing cellular distributions, whereas TrkA, TrkB, and TrkC, were localized in myoepithelial cell and ductal cell membranes. The protein p75 was expressed only on myoepithelial cell membranes. CONCLUSIONS: Members of the neurotrophin family of growth factors and their receptors are present in rat lacrimal gland, which suggests a role for NTs and their receptors in the lacrimal gland.

Isolation and characterization of cultured human conjunctival goblet cells.

PURPOSE: To isolate and characterize goblet cells from normal human conjunctival tissue to determine whether epidermal growth factor (EGF) receptors are present and whether EGF can influence goblet cell proliferation. METHODS: Goblet cells were isolated from explant cultures established from normal conjunctival tissue harvested from patients during periocular surgery. The cells were grown in RPMI culture medium supplemented with 10% fetal bovine serum and characterized using morphology, histochemistry, indirect immunofluorescence microscopy, molecular biology, and biochemistry. Proliferation was determined with a MTT proliferation assay after exposing goblet cells, which had been serum deprived for 48 hours, to increasing concentrations of epidermal growth factor (EGF; 0-80 ng/mL) for 24 hours. RESULTS: Goblet cells were isolated from conjunctival explants by scraping nongoblet cells from the culture dish. Human goblet cells exhibited positive reactivity with alcian blue-periodic acid Schiff (PAS) reagent, goblet cell-specific cytokeratin-7, HPA lectin, and MUC5AC, but negative reactivity to the stratified squamous epithelial cell marker, cytokeratin-4. The mRNA for MUC5AC was detected using RT-PCR. The presence of the EGF receptors EGFR, ErbB2, and ErbB3 was confirmed through Western blot analysis of cell lysates. EGF elicited a concentration-dependent increase in goblet cell proliferation of 160% +/- 0.5%, 188% +/- 0.45%, 293% +/- 1.3%, and 220% +/- 0.5% of control values with 10, 20, 40, and 80 ng/mL EGF, respectively. CONCLUSIONS: Human goblet cells that retain characteristics of goblet cells in vivo can be cultured. EGF receptors are present in human goblet cells, and EGF stimulates their proliferation.

Activation of mitogen-activated protein kinase by cholinergic agonists and EGF in human compared with rat cultured conjunctival goblet cells.

PURPOSE: To compare activation of the p42/p44 mitogen-activated protein kinase (MAPK) by cholinergic agonists and epidermal growth factor (EGF) in cultured human and rat goblet cells. METHOD: . Conjunctiva was removed from either humans during ocular surgery or male Sprague-Dawley rats and cultured in RPMI medium. These cells were incubated with the cholinergic agonist carbachol (10(-4) M) or EGF (10(-8) M) for various times. Before stimulation, cells were incubated with the EGF receptor (EGFR) inhibitor, AG1478 (10(-7) M) or the muscarinic M(3) receptor inhibitor, 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP; 10(-5) M) for 10 minutes. Proteins were analyzed by Western blot analysis, using antibodies specific to phosphorylated (activated) p42/44-MAPK or total p42-MAPK. Immunoreactive bands were quantified, and data were expressed as percentage of increase over basal. RESULTS: Carbachol (10(-4) M) increased MAPK activity in human and rat cultured goblet cells in a time-dependent manner, increasing pMAPK with a maximum at 10 minutes. EGF (10(-8) M) activated MAPK in human and rat goblet cells in a time-dependent manner with a maximum at 5 minutes. Carbachol- and EGF-induced activation of pMAPK was completely inhibited by AG1478 in cultured conjunctival goblet cells from both species. Carbachol-induced MAPK activity was also completely inhibited by 4-DAMP in both species. CONCLUSIONS: In human and rat cultured conjunctival goblet cells, cholinergic agonists and EGF activate MAPK with a similar time dependency, this activation is receptor mediated, and cholinergic agonists transactivate the EGF receptor. Thus, rat cultured conjunctival goblet cells can be used as a model to study human conjunctival goblet cells.

Cholinergic agonists transactivate EGFR and stimulate MAPK to induce goblet cell secretion.

Conjunctival goblet cells are the primary source of mucins in the mucous layer, the innermost layer of the tear film. Conjunctival goblet cell mucin secretion is under neural control because exogenous addition of parasympathetic agonists stimulates goblet cell secretion. To elucidate the intracellular signal pathways used by cholinergic agonists to stimulate goblet cell mucin secretion, we determined whether p42/p44 mitogen-activated protein kinase (MAPK) is activated during cholinergic agonist-stimulated mucin secretion. Rat conjunctiva was removed, preincubated with or without antagonists, and stimulated with the cholinergic agonist carbachol (10(-4) M). Carbachol statistically significantly stimulated the phosphorylation of MAPK in a time- and concentration-dependent manner. U-0126, an inhibitor of MAPK activation, completely inhibited both the activation of MAPK and goblet cell secretion stimulated by carbachol. The M(1) muscarinic antagonist pirenzepine, the M(2) muscarinic antagonist gallamine, and the M(1)/M(3) muscarinic receptor antagonist N-(3-chloropropyl)-4-piperidinyl diphenylacetate (4-DAMP) also inhibited carbachol-stimulated MAPK activation. Increasing the intracellular Ca(2+) concentration with a Ca(2+) ionophore increased MAPK activation, and chelation of extracellular Ca(2+) inhibited carbachol-stimulated activation. Carbachol also increased tyrosine phosphorylation of Pyk2, p60Src, and the epidermal growth factor receptor (EGFR). The Src inhibitor PP1 and the EGFR inhibitor AG-1478 completely inhibited carbachol-stimulated MAPK activation. AG-1478 also inhibited goblet cell secretion. We conclude that carbachol transactivates the EGFR to activate MAPK, leading to conjunctival goblet cell secretion. In addition, carbachol also activates Pyk2 and p60Src that could play a role in the transactivation of the EGFR.

Alpha 1-adrenergic and cholinergic agonists activate MAPK by separate mechanisms to inhibit secretion in lacrimal gland.

The purpose of this study was to determine the role of p42/p44 mitogen-activated protein kinase (MAPK) in alpha(1)-adrenergically and cholinergically stimulated protein secretion in rat lacrimal gland acinar cells and the pathways used by these agonists to activate MAPK. Acini were isolated by collagenase digestion and incubated with the alpha(1)-adrenergic agonist phenylephrine or the cholinergic agonist carbachol, and activation of MAPK and protein secretion were then measured. Phenylephrine and carbachol activated MAPK in a time- and concentration-dependent manner. Inhibition of MAPK significantly increased phenylephrine- and carbachol-induced protein secretion. Inhibition of EGF receptor (EGFR) with AG1478, an inhibitor of the EGFR tyrosine kinase activity, significantly increased phenylephrine- but not carbachol-induced protein secretion. Whereas phenylephrine-induced activation of MAPK was completely inhibited by AG1478, activation of MAPK by carbachol was not. Phenylephrine stimulated tyrosine phosphorylation of the EGFR, whereas carbachol stimulated p60(Src), and possibly Pyk2, to activate MAPK. We conclude that, in the lacrimal gland, activation of MAPK plays an inhibitory role in alpha(1)-adrenergically and cholinergically stimulated protein secretion and that these agonists use different signaling mechanisms to activate MAPK.