Biochemical investigation of possible lesions in human aorta that predispose to dissecting aneurysms: pyridinoline crosslinks.

Pyridinoline, a collagen specific covalent crosslink, was quantified in acid hydrolysates of human aorta using a non-equilibrium inhibition ELISA. The study was based on specimens from seven cases of aortic dissection and from seven control subjects whose death was unrelated to thoracic aortic dissection. There were no significant differences in the amounts or concentrations of pyridinoline in aortas with dissecting aneurysms compared with normal tissue, thus excluding the possibility of a causative relation between the degree of pyridinoline crosslinking of collagen molecules and dissection of the thoracic aorta. In all cases, however, the number of pyridinoline crosslinks per molecule of collagen in the ascending aorta and arch approached the theoretical maximum for lysyl derivatives and was as high as that present in cartilage. Thus in this region of the vessel pyridinoline represents the major stabilising crosslink of collagen. In contrast, the number of pyridinoline crosslinks per collagen molecule decreased maximally by a factor of 10 between the arch and the proximal regions of the descending thoracic aorta. This suggests a possible correlation between the rigidity of collagen fibres and the forces exerted on the aortic wall during diastole and systole.

GH regulation of the Type 2 IGF receptor in regenerating skeletal muscle of rats.

GH enhances skeletal muscle growth, and IGF-II peptide is highly expressed during regeneration. We have therefore investigated the effect of GH administration on IGF-II binding and expression in regenerating rat skeletal muscle using the techniques of receptor autoradiography and in situ hybridisation. Notexin, a myotoxin, was injected into the right M. biceps femoris (day 0), causing affected fibres to undergo necrosis followed by rapid regeneration. Animals were administered either GH (200 micrograms/100 g body weight) or saline vehicle daily. Contralateral muscles were used as regeneration controls. GH administration during regeneration resulted in significant increases in body weight, and damaged and undamaged muscle weights (P < 0.001). IGF-II expression, which was examined in regenerating fibres, survivor fibres and undamaged fibres, varied according to tissue type (P < 0.001). Specifically, IGF-II expression in regenerating fibres was elevated relative to control and survivor fibres after day 3 (P < 0.05), with a peak on day 9 (P < 0.001). GH did not affect IGF-II message levels. 125I-IGF-II binding in regenerating muscle was examined in the same fibre types as well as in connective tissue. 125I-IGF-II binding in regenerating fibres was higher (P < 0.001) than in other tissue types on day 5. GH administration increased 125I-IGF-II binding in all damaged muscle tissues on day 5 (P < 0.001, regenerating fibres; P < 0.01, others). We believe that this shows for the first time an effect of GH on the Type 2 IGF receptor in regenerating skeletal muscle.

Sarcolemmal K+ channel activity in developing rat skeletal muscle membranes.

A method has been adapted to produce membrane vesicles suitable for routine membrane patch clamping from neonate rat skeletal muscle. Single K+ channel activity was recorded from cell-free inside-out patches. Most Ca2(+)-activated voltage sensitive channels had large conductances of up to 300 pS, as determined from their current/voltage relationship, and an open probability (Po) approaching unity at positive membrane potentials. A lower conductance K+ channel, probably responsible for inward rectification, had a lower conductance of about 100 pS. Outward rectifying K+ channels were also observed with the lowest conductance, about 40 pS. 0.1 mM ATP when applied to the inner membrane surface reduced or blocked activity, drastically reducing Po without altering single channel conductance. Such an effect has been reported in other preparations but was different in the neonate preparation in that it blocked channels with conductances as high as 300 pS. The simple preparation described, which we have also used successfully on mature rat and mouse skeletal muscle, has potential in the analysis of channel activities in various conditions and pathologies without the need for tissue culture to produce suitable membrane preparations.

Potassium channel activity in sarcolemmal vesicles formed from skeletal muscle fibres of normal and dystrophic mice.

Sarcolemmal vesicles were produced from adult mouse extensor digitorum longus muscle (EDL) by treating swollen muscle fibres with collagenase. Vesicles formed from dystrophic (C57BL/6J dy/dy) and phenotypically normal animals were patch clamped and the single channel activity was recorded. Three types of K+ channel were observed in excised patches taken from normal and dystrophic muscle. A large conductance (300 pS) Ca2(+)-dependent K+ channel (KCa) was the most frequently observed of the K+ channels in both types of muscle preparation. In a number of patches taken from dystrophic muscle the open probability-voltage relationship for the KCa channel was markedly different from that in normal muscle, suggesting a possible reduction in Ca2+ sensitivity. An ATP-sensitive K+ channel (90 pS) was common to both normal and dystrophic muscle vesicles and was present in a large number of patches. An inwardly rectifying K+ channel (40 pS) was also observed in both types of sarcolemmal vesicles. The properties of all three K+ channels types were broadly consistent with other observations of skeletal muscle K+ channels, though all had higher conductances than had previously been noted in other species.

Collagen in dissecting aneurysms of the human thoracic aorta. Increased collagen content and decreased collagen concentration may be predisposing factors in dissecting aneurysms.

Biochemical investigations were made into dissecting aneurysms of the thoracic aorta by taking samples at 12 specific sites from aortas of 10 patients who had dissected with fatal results in comparison with samples from 10 matched controls. When sites actually involved in dissection were compared with corresponding sites in controls, there were highly significant increases in dry weight, amount of collagen, and total protein (all p less than 0.001) and a significant decrease in collagen concentration (p = 0.016). In contrast, when samples from sites within dissected aortas, but not involved in dissection, were compared with corresponding sites in controls, there were no significant differences. We therefore conclude that in dissecting aneurysms of the thoracic aorta, there is localized expansion of the aortic matrix, due to deposition of collagen and other proteins, which decreases the concentration of matrix constituents, including collagen. Clearly, the deposition of collagen could be a cause but not a consequence of dissection. The altered composition means that the aortic wall becomes weaker and less able to withstand the mechanical stresses constantly imposed upon it.