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P Verdugo

Publications and source records attributed to P Verdugo.

At least 19 recordsLinked to original sources
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↗
PubMed1987

Molecular mechanism of mucin secretion: I. The role of intragranular charge shielding.

Mucus is an ubiquitous polymer hydrogel that functions as a protective coat on the surface of integument and mucosa of species ranging from simple animals (such as coelenterates) to mammals. The polymer matrix of mucus is made out of long-chain glycoproteins called mucins that are tangled together, forming a randomly woven, highly polyionic network (Lee et al., 1977; Verdugo et al., 1983). Mucin-containing granules, produced by mammalian goblet cells in vitro, undergo massive post-exocytotic swelling. Their swelling kinetics is similar to the swelling of condensed artificial polymer gels (Verdugo, 1984; Tanaka and Fillmore, 1979). We had proposed that mucins must be condensed in the secretory granule and expand by hydration during or after exocytosis (Verdugo, 1984; Tam and Verdugo, 1981). However, the polyionic charges of mucins prevents condensation unless they (the mucins) are appropriately shielded. The present experiments were designed to assert the presence of an intragranular shielding cation and its role in secretion. Giant mucin granules of the slug (Ariolimax columbianus) are released intact from mucus-secreting cells of the slug's skin. They burst spontaneously outside the cell, forming, upon hydration, the typical slug mucus (Deyrup-Olsen et al., 1983). We report here that these granules contain from 2.5 to 3.6 moles calcium/kg dry material, and that calcium is released from the granules immediately before the burst that discharges their secretory product. Therefore, we propose that calcium functions as a shielding cation of polyionic mucins, and that the bursting discharge of mucins from secretory granules must result from the release of calcium from the intragranular compartment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Molecular mechanism of product storage and release in mucin secretion. II. The role of extracellular Ca++.

Mucin-containing granules, produced by mammalian goblet cells in vitro, undergo massive post-exocytotic swelling (23). Their swelling kinetics are similar to the swelling of condensed artificial polymer gels (22). Earlier, we proposed that mucins are condensed in the secretory granule and expand by swelling during or after exocytosis (21). The swelling of mucus is affected by ionic influences, as it is governed by a Donnan equilibrium process (21). However, the effect of cations on the swelling of newly released mucins had not yet been investigated. Calcium has been found in high concentration inside secretory granules of mucin-secreting cells (18, 9, 25), and is also elevated in the mucus of cystic fibrosis patients (17). The present experiments were designed to study the effect of extracellular Ca++ concentration on the swelling kinetics of the newly released secretory product of respiratory goblet cells in vitro. The data show that extracellular Ca++, in concentrations similar to those found in the mucus of cystic fibrosis patients (2 to 4 mM) can produce a four-fold decrease in the diffusivity of the newly released mucin polymer network, resulting in a slow rate of swelling, and a mucus that remains thick for long periods of time. The present findings are in agreement with the Donnan equilibrium hypothesis for the regulation of mucus swelling and rheology (21), and bear important implications for the pathophysiology of cystic fibrosis.

Animals↗
PubMed1986

The oviductal cilia and Kartagener's syndrome.

Women who have Kartagener's syndrome (primary ciliary dyskinesia) may or may not be fertile. The bronchial mucociliary clearance is reduced markedly in most of these women; this has led investigators to the conclusion that the cilia in the respiratory tract are immotile, and that "beating cilia may have no indispensable role in the female reproductive tract." Yet motile cilia are considered by many workers to be essential for normal ovum transport. More recently, bizarre ciliary motion has been described in the respiratory cilia of Kartagener's women. Our hypothesis was that the dyskinetic ciliary activity (or immotility) would be the same in both the respiratory and reproductive tracts and thus explain the fertility (or lack of it) in Kartagener's women. This report shows an identical ultrastructure and absolute immotility of cilia in both the respiratory tract and reproductive tract of a woman with Kartagener's syndrome who has never conceived. From this concordance, we suggest that the fertility of Kartagener's women is explained by the dyskinetic motion of oviductal cilia, and that the ciliated endosalpinx is essential for human reproduction.

Adult↗
PubMed1984

Hydration kinetics of exocytosed mucins in cultured secretory cells of the rabbit trachea: a new model.

Experiments have been done to test the idea that mucins undergo postexocytotic swelling. Previous work led to the hypothesis that the glycoprotein network of the mucus gel is probably held together by entanglements and low energy bonds, rather than by interchain covalent bonding. Since glycoproteins and other proteins in the mucus are polyions, it was further proposed that mucus must be capable of swelling, with its swelling properties depending on the pH and ionic strength of the medium hydrating the mucus. Experiments using oestrous cervical mucus from cows as a model confirmed this prediction. Observations on tissue cultures of respiratory secretory cells reported here show that freshly secreted mucins also undergo swelling during and after exocytosis. This evidence supports the hypothesis that the rheological properties of mucus may be physiologically regulated by hydration via control of the transepithelial movement of water, ions and soluble proteins, rather than by variations in the degree of covalent cross-linking between glycoprotein chains as proposed earlier.

Animals↗
PubMed1983

Conformational structure of respiratory mucus studied by laser correlation spectroscopy.

Samples of respiratory mucus were obtained from the trachea of dogs and patients undergoing bronchoscopy. The samples were studied by laser correlation spectroscopy. The autocorrelation function of laser light scattered by both human and canine respiratory mucus was a single exponential in thick mucus or a double exponential in thin watery mucus. This finding suggests that, as in the case of uterine cervical mucus, the molecular structure of respiratory mucus is an ensemble of entangled, randomly-coiled glycoproteins forming a loose network of variable density rather than a covalently cross-linked molecular network as proposed in earlier studies.

Animals↗