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P Verdugo

Publications and source records attributed to P Verdugo.

At least 19 recordsLinked to original sources
PubMed2001

Intracellular pathways regulating ciliary beating of rat brain ependymal cells.

1. The mammalian brain ventricles are lined with ciliated ependymal cells. As yet little is known about the mechanisms by which neurotransmitters regulate cilia beat frequency (CBF). 2. Application of 5-HT to ependymal cells in cultured rat brainstem slices caused CBF to increase. 5-HT had an EC50 of 30 microM and at 100 microM attained a near-maximal CBF increase of 52.7 +/- 4.1 % (mean +/- s.d.) (n = 8). 3. Bathing slices in Ca2+-free solution markedly reduced the 5-HT-mediated increase in CBF. Fluorescence measurements revealed that 5-HT caused a marked transient elevation in cytosolic Ca2+ ([Ca2+]c) that then slowly decreased to a plateau level. Analysis showed that the [Ca2+]c transient was due to release of Ca2+ from inositol 1,4,5-trisphosphate (IP3)-sensitive stores; the plateau was probably due to extracellular Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels. 4. Application of ATP caused a sustained decrease in CBF. ATP had an EC50 of about 50 microM and 100 microM ATP resulted in a maximal 57.5 +/- 6.5 % (n = 12) decrease in CBF. The ATP-induced decrease in CBF was unaffected by lowering extracellular [Ca2+], and no changes in [Ca2+]c were observed. Exposure of ependymal cells to forskolin caused a decrease in CBF. Ciliated ependymal cells loaded with caged cAMP exhibited a 54.3 +/- 7.5 % (n = 9) decrease in CBF following uncaging. These results suggest that ATP reduces CBF by a Ca2+-independent cAMP-mediated pathway. 5. Application of 5-HT and adenosine-5'-O-3-thiotriphosphate (ATP-gamma-S) to acutely isolated ciliated ependymal cells resulted in CBF responses similar to those of ependymal cells in cultured slices suggesting that these neurotransmitters act directly on these cells. 6. The opposite response of ciliated ependymal cells to 5-HT and ATP provides a novel mechanism for their active involvement in central nervous system signalling.

Adenosine Triphosphate↗
PubMed2001

Mouse mast cell secretory granules can function as intracellular ionic oscillators.

Fluorescent Ca2+ probes and digital photo-sectioning techniques were used to directly study the dynamics of Ca2+ in isolated mast cell granules of normal (CB/J) and beige (Bg(j)/Bg(j)) mice. The resting intraluminal free Ca2+ concentration ([Ca2+]L) is 25 +/- 4.2 microM (mean +/- SD, n = 68). Exposure to 3 microM inositol 1,4,5-trisphosphate (InsP3) induced periodic oscillations of luminal Ca2+ ([Ca2+]L) of approximately 10 microM amplitude and a period around 8-10 s. The [Ca2+]L oscillations were accompanied by a corresponding oscillatory release of [Ca2+]L to the extraluminal space. Control experiments using ruthenium red (2 microM) and thapsigargin (100 nM) ruled out artifacts derived from the eventual presence of mitochondria or endoplasmic reticulum in the isolated granule preparation. Oscillations of [Ca2+]L and Ca2+ release result from a Ca2+/K+ exchange process whereby bound Ca is displaced from the heparin polyanionic matrix by inflow of K+ into the granular lumen via an apamin-sensitive Ca2+-sensitive K+ channel (ASK(Ca)), whereas Ca2+ release takes place via an InsP3-receptor-Ca2+ (InsP3-R) channel. These results are consistent with previous observations of [Ca2+]L oscillations and release in/from the endoplasmic reticulum and mucin granules, and suggest that a highly conserved common mechanism might be responsible for [Ca2+]L oscillations and quantal periodic Ca2+ release in/from intracellular Ca2+ storage compartments.

Animals↗
PubMed2001

[Populational research of gastric cancer in digestive symptomatic patients, from 1996 to 2000].

BACKGROUND: Gastric cancer is the first cause of death due to malignant tumors in Chile. Its mortality rates have stabilized in the last two decades and its prognosis is closely associated to the degree of tumor invasion at the moment of surgery. AIM: To study the frequency of gastric cancer among symptomatic patients subjected to an upper gastrointestinal endoscopy at a secondary care health center. PATIENTS AND METHODS: All upper gastrointestinal endoscopies performed to patients derived from public primary care clinics were recorded. RESULTS: In the study period, 4,145 endoscopies were done to 818 men and 2,128 women. Seventy one percent of patients were aged over 40 years of age. Fifty one carcinomas and one lymphoma were detected. Of these, 10 tumors were incipient. Thirty one patients were operated on and in 22 a total gastrectomy was performed. One patient, that required an esophageal resection, died. CONCLUSIONS: Gastric cancer was detected in 1.2% of symptomatic adult patients subjected to an upper gastrointestinal endoscopy.

Adenocarcinoma↗
PubMed1998

Role of Ca2+/K+ ion exchange in intracellular storage and release of Ca2+.

Although fluctuations in cytosolic Ca2+ concentration have a crucial role in relaying intracellular messages in the cell, the dynamics of Ca2+ storage in and release from intracellular sequestering compartments remains poorly understood. The rapid release of stored Ca2+ requires large concentration gradients that had been thought to result from low-affinity buffering of Ca2+ by the polyanionic matrices within Ca2+-sequestering organelles. However, our results here show that resting luminal free Ca2+ concentration inside the endoplasmic reticulum and in the mucin granules remains at low levels (20-35 microM). But after stimulation, the free luminal [Ca2+] increases, undergoing large oscillations, leading to corresponding oscillations of Ca2+ release to the cytosol. These remarkable dynamics of luminal [Ca2+] result from a fast and highly cooperative Ca2+/K+ ion-exchange process rather than from Ca2+ transport into the lumen. This common paradigm for Ca2+ storage and release, found in two different Ca2+-sequestering organelles, requires the functional interaction of three molecular components: a polyanionic matrix that functions as a Ca2+/K+ ion exchanger, and two Ca2+-sensitive channels, one to import K+ into the Ca2+-sequestering compartments, the other to release Ca2+ to the cytosol.

Adenosine Triphosphate↗
PubMed1997

Kinetics of release of serotonin from isolated secretory granules. I. Amperometric detection of serotonin from electroporated granules.

We developed a method for measuring the efflux of 5-hydroxytryptamine (5-HT, serotonin) from isolated intact granules of the mast cell of the beige mouse. This method combines electroporation of the vesicle membrane with amperometric detection of 5-HT. A single secretory granule is placed between two platinum electrodes (distance approximately 100 microm) and positioned adjacent (<1 microm) to a carbon fiber microelectrode. A short (approximately 30 micros) high-intensity voltage pulse (electric field of approximately 5 kV/cm) is delivered to the electrodes to trigger the mechanical breakdown of the granule membrane, which activates the release of 5-HT. We observed concurrent swelling of the granule matrix with the oxidation of 5-HT at the carbon fiber electrode (overpotential + 650 mV). Similar to the release of secretory products during exocytosis, the oxidation current exhibits a spike-like time course with a noninstantaneous rising phase (time between onset of current and maximum flux, t(max)) with approximately 25% of the molecules released during this period. When the current reaches its maximum, the granule matrix attains its maximum swollen state. We found that the rising phase depends on the initial cross-sectional area of the granule (t(max) approximately 21r2) and reflects the time required for membrane rupture. The average t(1/2)spike of the amperometric spikes was found to be approximately 150 ms, which is 3-7 times faster than the t(1/2) measured during cellular exocytosis.

Animals↗
PubMed1997

Kinetics of release of serotonin from isolated secretory granules. II. Ion exchange determines the diffusivity of serotonin.

We measured the efflux of 5-hydroxytryptamine (5-HT, serotonin) from an intact secretory granule extracted from the mast cell of the beige mouse. The efflux was measured with amperometry after rupture of the granule membrane was triggered by electroporation. We determined the diffusivity of 5-HT within the secretory granule to be 2.0 x 10(-8) cm2 s(-1) when the granule is in contact with a physiological saline and found that this diffusivity depends on the valence of the cation in the external electrolyte. There is a fivefold increase in the diffusion coefficient of 5-HT determined in CsCl (150 mM, pH 7.2) at 3.7 x 10(-8) cm2 s(-1) compared to that determined in histamine dihydrochloride (Hi, 100 mM at pH 4.5) at 0.7 x 10(-8) cm2 s(-1). We found that the rate of expansion of the granule matrix observed in physiological medium correlates with the efflux of 5-HT, and that the rate of swelling of the matrix and the efflux depend on the microviscosity within the granule matrix and not the bulk viscosity of the external solution. The low diffusivity of 5-HT (approximately 500-fold less than in the bulk), the observation that the valence of the counterion affects this diffusivity, and the relationship between the volume changes of the matrix and the efflux suggest that 5-HT is released from the granule by ion exchange. We discuss the implications of this result for exocytotic release in mast cells and propose that an ion exchange mechanism could control the rate of release in other secretory systems.

Animals↗
PubMed1997

[Skin and mucous membrane hemorrhages: clinical assessment, study sequence and relative frequency of hereditary diseases of the hemostasis in a Chilean population].

BACKGROUND: Skin and mucous membrane hemorrhages are distinctive manifestations of hereditary diseases of primary hemostasis and, among them, the different types of von Willebrand disease and of platelet function disorders are the most prevalent. AIM: To know the relative frequency of these disorders and to know the clinical features of patients with mucocutaneous hemorrhages. PATIENTS AND METHODS: Five hundred eighty nine patients whose main symptom was the presence of mucocutaneous hemorrhages were studied. Bleeding time, platelet count, coagulant activity of factor VIII (FVIII:C), FvW: Ag and FvW: CoRis and ABO blood group were measured in all patients in a first stage. According to the results of these tests, further studies were decided. RESULTS: In patients younger than 13 years old, male predominated and, in older patients, females consulted with higher frequency. There was a higher proportion of individuals with O blood type than in the normal population. Bleeding time was abnormal in 330 patients (56%). One hundred ten patients (19%) had won Willebrand disease and, among them, one third had a normal bleeding time. Isolated reduction of factor WII activity was found in 66 patients (11%, 51 males) and 32 of these had normal bleeding time. Eighty one patients (14%) were considered to have an hereditary platelet function defect. A precise diagnosis was not achieved in 332 patients (56%). CONCLUSIONS: Among patients consulting for mucocutaneous hemorrhages, 19% had von Willebrand disease, 11 had an isolated reduction of factor VIII activity, 14% had platelet function defects and in 56%, a precise diagnosis was not reached.

ABO Blood-Group System↗
PubMed1993

Characterization of a marker of differentiation for tracheal ciliated cells independent of ciliation.

Although morphologic features have been used to follow cell lineage and differentiation, an objective assessment of differentiation can be best established by characterizing the expression of specific proteins that form the phenotypic profile of differentiated cells. Thus, specific markers or probes are required to unequivocally identify the various types of cells resulting from differentiation in a cell lineage. We report characterization of an IgM monoclonal antibody (5B4/H3), which recognized a surface antigen of approximately 130 kD unique to ciliated cells. The antibody reacted with the lumenal surface of the ciliated cells in transmission electron micrographs, in immunohistochemical staining of tracheal sections, and in cultured monolayers of tracheal epithelial cells. Flow cytometry, performed on enzymatically dispersed tracheal epithelial cells tagged with 5B4/H3 and fluorescent-labeled goat anti-mouse IgA/IgG/IgM, produced a population of fluorescent ciliated cells and a mixed nonfluorescent, nonciliated cell population. Ciliated cells were followed in vitro by time-lapse video microscopy for 48 to 72 h. Some of the ciliated cells lost their cilia under these culture conditions, but these cells were still found to react with the 5B4/H3 antibody. The antigen detected by this antibody remained on the surface of the cells after they lost their cilia. These results indicate that 5B4/H3 recognized a cell surface antigen that is specific to the ciliated cells and is independent of cell morphology. This marker will be useful in tissue culture studies of airway epithelial lineage, or differentiation, in which cell morphology is variable and cannot be used as a reliable marker of differentiation.

Animals↗
PubMed1991

Reversible condensation of mast cell secretory products in vitro.

We have investigated the mechanisms responsible for the condensation and decondensation of secretory products that occur in mast cell secretion. We show here that the hydrated matrix of an exocytosed secretory granule can be recondensed to its original volume by exposure to acidic solutions containing histamine at concentrations that mimic those found in vivo. Recondensation by acidic histamine began in the range of 1-10 mM with a dose response curve that was accurately predicted by a Hill type equation with four highly cooperative binding sites and a half maximum concentration of [Hi++] = 3.9 mM. Recondensation by histamine showed a sigmoidal dependency on pH (critical range pH 5.5-6.5) and was fully reversible. These experiments suggest that histamine, possibly by binding to anionic sites in the protein-heparin complex of the granule matrix, triggers a change in the polymeric structures of the granule matrix from an extended coil to a collapsed globular state. This may be a useful model for understanding the condensation of secretory products into dense core granules and their subsequent decondensation upon exocytosis.

Animals↗
PubMed1991

Mucin exocytosis.

Mucins produced by goblet cells of the respiratory mucosa are condensed while stored in secretory granules. Mucin condensation and its decondensation upon exocytosis can be explained by the theory of polymer gel phase transition. After the opening of a secretory pore, Ca2+ inside the granule is exchanged for extracellular Na+. Na/Ca exchange triggers a polymer gel phase transition whereby the mucin polymer matrix undergoes massive swelling and thereby changes from a condensed to a hydrated phase. Swelling of the granular content is driven by a Donnan potential and results in the release of secretory product and the formation of small mucin gels, which later anneal to each other to form the respiratory mucus. Because of the tangled rather than cross-linked topology of the mucin network, the rheologic properties of the respiratory mucus depend primarily on hydration. As mucins are polyionic, the hydration of mucus is controlled by a Donnan equilibrium. Hence, mucus hydration and rheology are determined by two factors: the quantity, chain length, and charge density of the secreted mucins, and the amount and the ionic and polyionic composition of the water transported across the respiratory mucosa.

Exocytosis↗
PubMed1991

Autoregulation of beat frequency in respiratory ciliated cells. Demonstration by viscous loading.

As opposed to the virtually constant load exerted by water on the cilia of ciliated protozoa, the hydrodynamic load on respiratory cilia can undergo broad variations because of changes in the rheologic properties of mucus. When water-rowing ciliated cells are exposed to increased viscosity (1 to 50 cP), their beat frequency decreases exponentially. According to Newton's fluid dynamic theory, this outcome is expected for an engine that generates constant force. However, the findings reported here indicate that when mucus-propelling respiratory ciliated cells are exposed to high viscous loads, ranging from 12 to 150 cP, the frequency of ciliary beat decreases only slightly, whereas the beat amplitude remains virtually constant. These observations suggest that ciliated cells of the respiratory tract have a functional reserve that allows them to autoregulate their mechanical output in response to the changes in viscosity to which they are normally exposed in the airway.

Animals↗
PubMed1991

Enrichment of subpopulations of respiratory epithelial cells using flow cytometry.

The application of flow cytometry to enrich airway epithelial cell subpopulations is described. A complementary epithelial cell preparative technique is also outlined. The ability of the airway epithelium to protect the lung from environmental insults results from a complex interaction among the different cells that form its matrix. The separation of the different epithelial cell types is an essential step in the studies of mechanisms of the controlling factors of cell repair, cell differentiation, and neoplastic transformation. Epithelial cells of the New Zealand white rabbit trachea are prepared using enzymatic digestion and microdissection. Small sections of tracheal wall are dissected into pieces approximately 10 mm2. The mucosa is dissected and placed in 0.15% hyaluronidase for 40 min at 22 degrees C. Mucus is removed, and the mucosa is then placed in 0.1% pronase at 37 degrees C for 30 min. With careful dissection, the epithelium can be dissected from the mucosa in 10-mm2 sheets. Sheets of epithelial cells are placed in 6 ml of an enzymatic solution containing collagenase, 0.2% bovine serum albumin, 0.04% soya bean trypsin inhibitor, 0.06 ml of 1 M Hepes buffer for 3 h at 37 degrees C. The cells are gently pipetted during the 3-h period, yielding a suspension of viable cells. Subpopulations of these different cell types are enriched using an Orthocytofluorograph 50111. A krypton ion laser was used for excitation of cells at 488 nm. Forward-angle and 90 degrees scatter were gated on the histogram. The purification of the ciliated, basal, and secretory cells was 90%, 97%, and 94%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Development of an in vitro model of primate cervical goblet cells.

Mucins, are densely packed in secretory granules of goblet cells. Upon exocytosis they undergo massive hydration that results in the formation of the mucus gel. Because the mucin polymer network is held together by tangles and low energy bonds, the rheological properties of this gel are mainly determined by the degree of postexocytotic hydration. Hydration in mucus is governed by a Donnan equilibrium as it is driven by electrostatic interaction among the polyionic charges of the mucins and other fixed polyions. Although, variations of charge density of the mucin polymer could be an efficient physiologic mechanism to control the rate of mucus hydration and rheology, this subject has not been investigated. In here we describe a primary tissue culture system of cervical goblet cells of the monkey uterus. This preparation allows to measure directly the kinetic of hydration of exocytosed mucins. Because the physicochemical parameters of the bathing medium can be effectively controlled, variations in the kinetic of mucins swelling upon exocytosis, can be used as a convenient indicator of fluctuations of charge density in secretory products. Since the cervical mucosa readily respond to endocrine influences, this preparation can provide a useful model to study the effect of hormones or other transmitters on polyionic charge density of secretory product.

Animals↗
PubMed1989

Stimulus-response coupling in mammalian ciliated cells. Demonstration of two mechanisms of control for cytosolic [Ca2+].

Changes of cytosolic [Ca2+] have been proposed to couple stimulation of ciliary movement, however, quantitative measurements of fluctuations of intracellular free [Ca2+] associated with stimulation of ciliated cells have not been investigated. In primary cultures of rabbit oviductal ciliated cells, the stimulation of ciliary activity produced by micromolar concentrations of adenosine triphosphate (ATP) and prostaglandin F2 alpha (PGF2 alpha) was associated with a transient increase of intracellular [Ca2+]. Whereas the increase of cytosolic [Ca2+] and beat frequency produced by ATP were inhibited by the Ca-channel blocker LaCl3, the rise of cytosolic [Ca2+] and frequency of ciliary beat produced by PGF2 alpha was not affected by LaCl3. These results are the first direct demonstration that fluctuations of cytosolic [Ca2+] are associated with increased ciliary beat frequency in mammalian epithelial cells. The present findings suggest two different calcium-dependent mechanisms for stimulus-coupling in ciliary epithelium: ATP acting via purinergic receptor coupled to transmembrane influx of Ca2+, and PGF2 alpha acting via receptor-mediated release of intracellular sequestered Ca.

Adenosine Triphosphate↗
PubMed1989

Donnan mechanism of mucin release and conditioning in goblet cells: the role of polyions.

Mucin, the principal polymeric species in the mucus matrix, is condensed inside secretory granules and undergoes massive swelling upon exocytosis. Mucin swelling is governed by a Donnan equilibrium. However, the effect of polyions such as soluble proteins on the swelling of mucin granules has not been investigated. The experiments reported here were designed to evaluate the effect of albumin on swelling kinetics of mucin granules exocytosed from respiratory goblet cells in culture. The kinetics of swelling was monitored by video-microscopy. The diffusivity of newly released mucins, in the presence of different concentrations of serum albumin, was evaluated using the expression: D = r2f/tau where tau is the characteristic time of the swelling, and rf is the final equilibrium radius of the swollen granule. Preliminary results indicate that serum albumin at concentrations of 10(-7) M, which are equivalent to those found in the bronchial mucus of asthmatic or cystic fibrosis patients, can produce up to a 90% decrease in the diffusivity of newly released mucins. Albumin is commonly present on the surface of the respiratory mucosa, and its concentration is increased in asthma and other bronchial inflammatory diseases. The evidence presented here shows that soluble proteins can strongly modulate the rate of swelling of newly released mucins, as predicted by Donnan equilibrium. Since swelling is a critical determinant of mucus rheology, the concentration of plasma proteins on the mucosa could play an important role in the regulation of the rheological properties of mucus. Also, the characteristically thick mucus found in chronic bronchial inflammatory diseases could be at least partially explained by the high levels of albumin found in the airways of these patients.

Animals↗