Lung tissue mechanics and extracellular matrix composition in a murine model of silicosis.

The dynamic mechanical properties of lung tissue and its contents of collagen and elastic fibers were studied in strips prepared from mice instilled intratracheally with saline (C) or silica [15 (S15) and 30 days (S30) after instillation]. Resistance, elastance, and hysteresivity were studied during oscillations at different frequencies on S15 and S30. Elastance increased from C to silica groups but was similar between S15 and S30. Resistance was augmented from C to S15 and S30 and was greater in S30 than in S15 at higher frequencies. Hysteresivity was higher in S30 than in C and S15. Silica groups presented a greater amount of collagen than did C. Elastic fiber content increased progressively along time. This increment was related to the higher amount of oxytalan fibers at 15 and 30 days, whereas elaunin and fully developed elastic fibers were augmented only at 30 days. Silicosis led not only to pulmonary fibrosis but also to fibroelastosis, thus assigning a major role to the elastic system in the silicotic lung.     

Models of fibronectin.

The radius of gyration of human plasma fibronectin was determined by light scattering both under conditions in which the molecule is in an extended conformation (ionic strength 1.01 M, pH 8) and close to its native, more compact conformation (ionic strength 0.16 M, pH 8). These values were found to be 17.5 +/- 0.8 nm and 10.7 +/- 0.9 nm respectively, for a constant mol. wt of 533,000 +/- 8000, in excellent agreement with the value of 520,000 deduced from its known composition. A set of models, each made of two identical, end-to-end joined chains of 28 beads, was then constructed, and their calculated physico-chemical parameters were compared with those available for the whole fibronectin molecule and for some of its proteolytic fragments in both conformations. Two possible models for the circulating form are presented here: in both, the fibronectin molecule is in a compact, tangled conformation, with the amino-terminal end of one chain folded over to the carboxy end of itself or of the other chain either in a hairpin or in a circular fashion. With the exception of the carboxy-terminal fibrin(ogen)-binding domains, all the domains appear to be well exposed to the solvent, and thus free to interact with potential ligands.     

Coupling of voltage-dependent gating and Ba++ block in the high- conductance, Ca++-activated K+ channel

Voltage-dependent Ca++-activated K+ channels from rat skeletal muscle were reconstituted into planar lipid bilayers, and the kinetics of block of single channels by Ba++ were studied. The Ba++ association rate varies linearly with the probability of the channel being open, while the dissociation rate follows a rectangular hyperbolic relationship with open-state probability. Ba ions can be occluded within the channel by closing the channel with a strongly hyperpolarizing voltage applied during a Ba++-blocked interval. Occluded Ba ions cannot dissociate from the blocking site until after the channel opens. The ability of the closed channel to occlude Ba++ is used as an assay to study the channel's gating equilibrium in the blocked state. The blocked channel opens and closes in a voltage-dependent process similar to that of the unblocked channel. The presence of a Ba ion destabilizes the closed state of the blocked channel, however, by 1.5 kcal/mol. The results confirm that Ba ions block this channel by binding in the K+-conduction pathway. They further show that the blocking site is inaccessible to Ba++ from both the cytoplasmic and external solutions when the channel is closed.     

Role of surface electrostatics in the operation of a high-conductance Ca2+-activated K+ channel

This paper demonstrates that local electric fields originating from negatively charged groups on a K+-specific ion channel modify its behavior. Single high-conductance, Ca2+-activated K+ channels were studied in neutral phospholipid bilayers. The channel protein surface charges were manipulated experimentally by carboxyl group esterification using trimethyloxonium (TMO) or by electrolyte screening. Three channel properties--ion conduction, ion blockade, and voltage-dependent gating--are affected by surface electrostatics. Negative charges increase the affinity of cationic pore blockers by establishing a local negative potential at the pore entrance; these charges modify channel gating by establishing a potential gradient across the ion channel; finally, both effects influence ion permeation through the pore.     

Internal blockade of a Ca(2+)-activated K+ channel by Shaker B inactivating "ball" peptide.

Shaker B inactivating peptide ("ball peptide", BP) interacts with Ca(2+)-activated K+ (KCa) channels from the cytoplasmic side only, producing inhibition of channel activity. This effect was reversible and dose and voltage dependent (stronger at depolarized potentials). The inhibition of KCa channels by BP cannot be mimicked by an inactive point mutation of the BP, L7E. BP binds to KCa channels in a bimolecular reaction (dissociation constant of 95 microM at +40 mV). The binding site is probably located in the internal "mouth" or conduction pathway, since both external K+ and internal tetraethylammonium relieve BP-induced inhibition. These results suggest that KCa channels possess a binding site for the BP with some properties similar to the ball receptor found in Shaker B K+ channels.     

Four cases of direct ion channel gating by cyclic nucleotides

Four different nucleotide-gated ion channels are discussed in terms of their biophysical properties and their importance in cell physiology. Channels activated directly by cGMP are present in vertebrate and invertebrate photoreceptors. In both cases cGMP increases the fraction of time the channel remains in the open state. At least three cGMP molecules are involved in channel opening in vertebrate photoreceptors and the concentration of the cyclic nucleotide to obtain the half maximal effect is about 15 µM. The light-dependent channel of both vertebrates and invertebrates is poorly cation selective. The vertebrate channel allows divalent cations to pass through 10–15-fold more easily than monovalent ions. In agreement with their preference for divalent cations, this channel is blocked byl-cis Dialtazem, a molecule that blocks certain types of calcium channels. In olfactory neurons a channel activated by both cAMP and cGMP is found and, as in the light-dependent channel, several molecules of the nucleotide are needed to open the channel with a half maximal effect obtained in the range of 1–40 µM. The channel is poorly cationic selective. A K⁺ channel directly and specifically activated by cAMP is found inDrosophila larval muscle. At least three cAMP molecules are involved in the opening reaction. Half-maximal effect is obtained at about 50 µM. This channel is blocked by micromolar amount of tetraethylammonium applied internally. Interestingly, this channel has a probability of opening 10–20-fold larger in the mutantdunce, a mutant that possesses abnormally elevated intracellular cAMP level, than in the wild type.     

Effect of verapamil on the aldosterone-stimulating properties of metoclopramide: in vitro and in vivo studies

The role of calcium in the regulation of aldosterone secretion has been recently clarified. Angiotensin II and potassium stimulate aldosterone secretion through a calcium-entry dependent mechanism, while ACTH action is both calcium and cyclic AMP dependent. To establish whether also the so-called aldosterone dopaminergic regulatory system is calcium-dependent we have studied, in vitro and in vivo, the effect of verapamil, a calcium entry blocker agent, on the aldosterone-stimulating properties of the antidopaminergic drug, metoclopramide. In the rat adrenal cells perfusion system, verapamil blocked both angiotensin II and metoclopramide-stimulated aldosterone. This effect on metoclopramide action seems to be present also in vivo in normal subjects: in fact aldosterone response was slightly but significantly reduced after pretreatment with verapamil. In conclusion the results suggest that also the dopaminergic system could regulate aldosterone secretion through calcium-mediated mechanisms.     

Low temperature optical spectroscopy of cobalt-substituted hemocyanin from Carcinus maenas

In this work we report the optical absorption spectra of three cobalt-substituted derivatives of hemocyanin (He) from Carcinus maenas, in the temperature range 300–20 K. The derivatives studied are the mononuclear (Co²⁺)-He with a single cobalt ion in the Cu_A site, the binuclear (Co²⁺)₂-He and the binuclear mixed metal (Co²⁺-Cu¹⁺)-He. At low temperature three main bands are clearly resolved; the temperature dependence of their zeroth, first and second moments sheds light on the stereodynamic properties in the surroundings of the chromophore. Within the limits of the reported analysis, in the binuclear derivatives the motions coupled to the chromophore appear to be essentially harmonic in the whole temperature range investigated; moreover the data are consistent with the presence of an exogenous ligand strongly bound to the two metal ions. For the mononuclear derivative an essentially harmonic behavior is evident only up to 200 K where the data are consistent with the presence of an exogenous ligand much less strongly bound, while at higher temperatures the behavior of the spectra indicates the onset of very large anharmonic contributions to motions, that plausibly involve the above exogenous ligand and, quite likely, the entire active site.Abbreviations He Hemocyanin - M₀ zeroth moment - M₁ first moment - M₂ second moment - (Co^2–)₂-He binuclear bicobalt hemocyanin derivative - (Co²⁺)-He mononuclear monocobalt hemocyanin derivative - (Co²⁺-Cu¹⁺)-He binuclear mixed metals hemocyanin derivative - LFT ligand field theory - CT charge transfer - EPR electronic paramagnetic resonance - XANES X-ray absorption near edge structure Correspondence to: L. Cordone