Structure and Nanomechanics of Dry and Hydrated Intermediate Filament Films and Fibers Produced from Hagfish Slime Fibers.

Intermediate filaments (IFs) are known for their extensibility, flexibility, toughness, and their ability to hydrate. Using keratin-like IFs obtained from slime fibers from the invertebrate Atlantic hagfish ( Myxine glutinosa), films were produced by drop-casting and coagulation on the surface of a MgCl2 buffer. Drop-casting produced self-supporting, smooth, and dense films rich in ß-sheets (61%), whereas coagulation formed thin, porous films with a nanorough surface and a lower ß-sheet content (51%). The films hydrated and swelled immediately when immersed in water and did not dissolve. X-ray diffraction showed that the ß-crystallites remained stable upon hydration, that swelling presumably happens in the amorphous C-terminal tail-domains of the IFs, and that high salt conditions caused a denser network mesh size, suggesting polyelectrolyte behavior. Hydration resulted in a roughly 1000-fold decrease in apparent Young's modulus from 109 to 106 Pa as revealed by atomic force microscopy nanoindentation. Nanoindentation-based power-law rheology and stress-relaxation measurements indicated viscoelasticity and a soft-solid hydrogel character for hydrated films, where roughly 80% of energy is elastically stored and 20% is dissipated. By pulling coagulation films from the buffer interface, macroscopic fibers with highly aligned IF ß-crystals similar to natural hagfish fibers were produced. We propose that viscoelasticity and strong hydrogen bonding interactions with the buffer interface are crucial for the production of such long biomimetic fibers with aligned ß-sheets. This study demonstrates that hagfish fiber IFs can be reconstituted into functional biomimetic materials that are stiff when dry and retain the ability to hydrate to become soft and viscoelastic when in water.

Effect of ionic strength and seawater cations on hagfish slime formation.

The defensive slime of hagfish consists of a polyanionic mucin hydrogel that synergistically interacts with a fiber network forming a coherent and elastic hydrogel in high ionic strength seawater. In seawater, the slime deploys in less than a second entrapping large quantities of water by a well-timed thread skein unravelling and mucous gel swelling. This rapid and vast hydrogel formation is intriguing, as high ionic strength conditions generally counteract the swelling speed and ratio of polyelectrolyte hydrogels. In this work we investigate the effect of ionic strength and seawater cations on slime formation dynamics and functionality. In the absence of ionic strength skeins swell radially and unravel uncontrolled, probably causing tangling and creating a confined thread network that entraps limited water. At high ionic strength skeins unravel, but create a collapsed and dense fiber network. High ionic strength conditions therefore seem crucial for controlled skein unraveling, however not sufficient for water retention. Only the presence of naturally occurring Ca2+ or Mg2+-ions allowed for an expanded network and full water retention probably due to Ca2+-mediated vesicle rupture and cross-linking of the mucin. Our study demonstrates that hagfish slime deployment is a well-timed, ionic-strength, and divalent-cation dependent dynamic hydrogel formation process.

Hagfish slime exudate stabilization and its effect on slime formation and functionality.

Hagfish produce vast amounts of slime when under attack. The slime is the most dilute hydrogel known to date, and is a highly interesting material for biomaterial research. It forms from a glandular secrete, called exudate, which deploys upon contact with seawater. To study slime formation ex vivo and to characterize its material properties, stabilization of the sensitive slime exudate is crucial. In this study, we compared the two main stabilization methods, dispersion in high osmolarity citrate/PIPES (CP) buffer and immersion in oil, and tested the influence of time, temperature and pH on the stability of the exudate and functionality of the slime. Using water retention measurements to assess slime functionality, we found that CP buffer and oil preserved the exudate within the first 5 hours without loss of functionality. For longer storage times, slime functionality decreased for both stabilization methods, for which the breakdown mechanisms differed. Stabilization in oil likely favored temperature-sensitive osmotic-driven swelling and rupture of the mucin vesicles, causing the exudate to gel and clump. Extended storage in CP buffer resulted in an inhibited unraveling of skeins. We suggest that a water soluble protein glue, which mediates skein unraveling in functional skeins, denatures and gradually becomes insoluble during storage in CP buffer. The breakdown was accentuated when the pH of the CP buffer was raised from pH 6.7 to pH 8.5, probably caused by increased denaturation of the protein glue or by inferior vesicle stabilization. However, when fresh exudate was mixed into seawater or phosphate buffer at pH 6-9, slime functionality was not affected, showing pH insensitivity of the slime formation around a neutral pH. These insights on hagfish exudate stabilization mechanisms will support hagfish slime research at a fundamental level, and contribute to resolve the complex mechanisms of skein unraveling and slime formation.

Improvement of the closed cranial window model in rats by intracarotid infusion of signalling molecules implicated in migraine.

Intravital microscopy on a closed cranial window allows one to measure change in the diameter of cranial blood vessels after intravenous (i.v.) administration of pharmacodynamic substances. Putative targets being pursued in migraine are large vasodilating peptide molecules such as calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase polypeptide (PACAP)-38. High i.v. doses are required to study their craniovascular pharmacology. Unfortunately, this leads to a drop in blood pressure (BP) that subsequently causes blood vessels to dilate by autoregulation. Hence it is difficult to decipher what effect is caused by direct receptor agonist interaction or contributed by autoregulation. In the present study we infused substances with an ingenious indwelling catheter in the common carotid artery in rats. Intracarotidly seven-, 12- and 17-fold lower doses of CGRP, PACAP-38 and capsaicin were required, respectively, compared with i.v. infusion to induce the same dilation in dural artery. Dilating intracarotid (i.c.) doses caused no or a minimal fall in BP, whereas equi-responsive i.v. doses caused a marked BP reduction. The CGRP blocking potential of olcegepant was amplified by > 20 times on i.c. infusion. Pial artery responses to CGRP did not change with i.c. infusion, demonstrating that dilations after i.v. CGRP are mediated by autoregulation rather than through specific receptors. We applied CGRP topically, which induced concentration-dependent dural vasodilation, but no effect on pial artery or on BP. In conclusion, this new approach offers an improvement of the existing model by allowing more accurate assessment of effects of pharmaca on the cranial vasculature without inducing significant systemic effects.

The in vivo effect of VIP, PACAP-38 and PACAP-27 and mRNA expression of their receptors in rat middle meningeal artery.

The parasympathetic nervous system is probably involved in migraine pathogenesis. Its activation releases a mixture of signalling molecules including vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), which subsequently stimulate VPAC(1), VPAC(2) and PAC(1) receptors. The objective of the present study was to investigate the in vivo effect of VIP, PACAP-27, PACAP-38, the selective VPAC(1) agonist ([Lys15, Arg16, Leu27]-VIP(1-7)-GRF(8-27)) and a PAC(1) agonist, maxadilan on rat middle meningeal artery (MMA) diameter using the closed cranial window model. Selective antagonists were used for further characterization of the responses. Reverse transcriptase-polymerase chain reaction experiments were also conducted to determine expression of mRNA of PACAP receptors in the MMA. The results showed that VIP, PACAP-38, PACAP-27 and the VPAC(1) specific agonist evoked significant dilations with the rank order of potency; VIP = PACAP-38 > PACAP-27 = [Lys15, Arg16, Leu27]-VIP(1-7)-GRF(8-27). Significant inhibition of dilation was only observed for the VPAC(1) antagonist PG97-269 on PACAP-38-induced dilation of MMA. The VPAC(2) antagonist PG99-465 and PAC(1) antagonist PACAP(6-38) did not significantly block VIP- or PACAP-induced dilation. Expression of mRNA of all three receptors was detected in the MMA. In conclusion, the VPAC(1) receptor seems to be predominant in mediating MMA dilation. A selective VPAC(1) antagonist may be a candidate molecule in the treatment of migraine headache.

K(ATP) channel expression and pharmacological in vivo and in vitro studies of the K(ATP) channel blocker PNU-37883A in rat middle meningeal arteries.

BACKGROUND AND PURPOSE: Dilatation of cerebral and dural arteries causes a throbbing, migraine-like pain, indicating that these structures are involved in migraine. Clinical trials suggest that adenosine 5'-triphosphate-sensitive K(+) (K(ATP)) channel opening may cause migraine by dilatating intracranial arteries, including the middle meningeal artery (MMA). We studied the K(ATP) channel expression profile in rat MMA and examined the potential inhibitory effects of the K(ATP) channel blocker PNU-37883A on K(ATP) channel opener-induced relaxation of the rat MMA, using the three K(ATP) channel openers levcromakalim, pinacidil and P-1075. EXPERIMENTAL APPROACH: mRNA and protein expression of K(ATP) channel subunits in the rat MMA were studied by quantitative real-time PCR and western blotting, respectively. The in vivo and in vitro effects of the K(ATP) channel drugs on rat MMA were studied in the genuine closed cranial window model and in myograph baths, respectively. KEY RESULTS: Expression studies indicate that inwardly rectifying K(+) (Kir)6.1/sulphonylurea receptor (SUR)2B is the major K(ATP) channel complex in rat MMA. PNU-37883A (0.5 mg kg(-1)) significantly inhibited the in vivo dilatory effect of levcromakalim (0.025 mg kg(-1)), pinacidil (0.38 mg kg(-1)) and P-1075 (0.016 mg kg(-1)) in rat MMA. In vitro PNU-37883A significantly inhibited the dilatory responses of the three K(ATP) channel openers in rat MMA at 10(-7) and 3 x 10(-7) M. CONCLUSIONS AND IMPLICATIONS: We suggest that Kir6.1/SUR2B is the major functional K(ATP) channel complex in the rat MMA. Furthermore, we demonstrate the potent in vivo and in vitro blocking potentials of PNU-37883A on K(ATP) channel opener-induced relaxation of the rat MMA.