Pedro Verdugo

Spontaneous assembly of marine dissolved organic matter into polymer gels

A large pool of organic carbon resides in the worlds oceans in the form of dissolved organic matter (DOM),. DOM is operationally defined as the fraction of organic matter that passes through a filter with a given pore size (which can range from less than 0.1 μm to 0.46 μm). This fraction has a longer oceanic residence time — and is generally less biodegradable — than particulate organic matter (POM). Processes transforming DOM into POM are therefore crucial for our understanding of the cycling of organic material in the oceans. The aggregation of marine colloids, which constitute 10–40% of DOM,,, is thought to be an important step in the transformation of DOM into POM. It has been suggested that colloids, as well as transparent exopolymer particles and large aggregates (‘marine snow’) can be viewed as polymer gels. Whether free DOM polymers can indeed spontaneously assemble to form polymer gels has, however, not yet been shown. Here we present experimental observations that demonstrate that marine polymer gels can assemble from free DOM polymers, and that their formation mechanism, physical characteristics and mineralization can be understood in terms of polymer gel theory. The principles and methods of polymer gel physics thus have the potential to provide profound new insights into the processes controlling the exchange between the DOM and POM pools and the cycling of marine organic matter.

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 μM). 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.

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 slugs 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 poly ionic mucins, and that the bursting discharge of mucins from secretory granules must result from the release of calcium from the intragranular compartment. Calcium release would unshield the polyionic charges of mucins, driving the mutual repulsion of polymer chains and triggering a quick expansion of the mucin network (resembling a Jack-in-the-box mechanism). The existence of a poly ion associated with a shielding cation seems to be a common feature in a large variety of secretory granules. Thus, the proposed spring-loaded release system based on the unshielding of a condensed polyion may serve as a general model for explaining the molecular mechanism of product release in secretion.

Unpacking a gel-forming mucin: a view of MUC5B organization after granular release.

Gel-forming mucins are the largest complex glycoprotein macromolecules in the body. They form the matrix of gels protecting all the surface epithelia and are secreted as disulfide-bonded polymeric structures. The mechanisms by which they are formed and organized within cells and thereafter released to form mucus gels are not understood. In particular, the initial rate of expansion of the mucins after release from their secretory granules is very rapid (seconds), but no clear mechanism for how it is achieved has emerged. Our major interest is in lung mucins, but most particularly in MUC5B, which is the major gel-forming mucin in mucus, and which provides its major protective matrix. In this study, using OptiPrep density gradient ultracentrifugation, we have isolated a small amount of a stable form of the recently secreted and expanding MUC5B mucin, which accounts for less than 2% of the total mucin present. It has an average mass of approximately 150 x 10(6) Da and size Rg of 150 nm in radius of gyration. In transmission electron microscopy, this compact mucin has maintained a circular structure that is characterized by flexible chains connected around protein-rich nodes as determined by their ability to bind colloidal gold. The appearance indicates that the assembled mucins in a single granular form are organized around a number of nodes, each attached to four to eight subunits. The organization of the mucins in this manner is consistent with efficient packing of a number of large heavily glycosylated monomers while still permitting their rapid unfolding and hydration. For the first time, this provides some insight into how the carbohydrate regions might be organized around the NH(2)- and COOH-terminal globular protein domains within the granule and also explains how the mucin can expand so rapidly upon its release.

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.

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.

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 μM and at 100 μM 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 μM and 100 μM 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‐γ‐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.

Cardiac Muscle Models An Overextension of Series Elasticity

Quick-stretch and quick-release experiments were performed on right ventricular cat papillary muscles to test the applicability of the Hill model to cardiac mechanics. Series elastic component (SEC) force-length curves were calculated from stretches and releases carried out at various times during the contractile cycle. At any SEC force, the SEC elastic modulus depended on the time during the contractile cycle at which it was measured. When measured at the same time and at the same SEC force, elastic moduli obtained by releases of less than 1% of muscle length differed from those obtained by corresponding stretches. Larger stretches, in fact, appeared to yield negative elastic moduli. Thus, a unique SEC modulus could not be identified at any level of SEC force. It is concluded that the concept of the SEC as a passive elasticity appears unsatisfactory and, as a consequence, that the quantitative validity of the Hill model for cardiac muscle is questionable. Moreover, since an anatomical counterpart of the SEC has not been identified, the Hill model also appears unsatisfactory from a structural point of view.

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.

Supramolecular Dynamics of Mucus

Our purpose here is not to address specific issues of mucus pathology, but to illustrate how polymer networks theory and its remarkable predictive power can be applied to study the supramolecular dynamics of mucus. Avoiding unnecessary mathematical formalization, in the light of available theory, we focus on the rather slow progress and the still large number of missing gaps in the complex topology and supramolecular dynamics of airway mucus. We start with the limited information on the polymer physics of respiratory mucins to then converge on the supramolecular organization and resulting physical properties of the mucus gel. In each section, we briefly discuss progress on the subject, the uncertainties associated with the established knowledge, and the many riddles that still remain.