1 Three fully-de®ned a 1 -adrenoceptors (a 1A , a 1B and a 1D ) have been established in pharmacological and molecular studies. A fourth a 1 -adrenoceptor, the putative a 1L -adrenoceptor, has been de®ned in functional but not molecular studies, and has been proposed to mediate contraction of human lower urinary tract tissues; its relationship to the three fully characterized a 1 -adrenoceptors is not known. 2 In the present study, binding a nities were estimated by displacement of [ 3 H]-prazosin in membrane homogenates of Chinese hamster ovary (CHO-K1) cells stably expressing the human a 1A -, a 1B -and a 1D -adrenoceptors and were compared with a nity estimates obtained functionally in identical cells by measuring inhibition of noradrenaline (NA)-stimulated accumulation of [ 3 H]-inositol phosphates. 3 For the a 1A -adrenoceptor, binding studies revealed a pharmacological pro®le typical for the classically de®ned a 1A -adrenoceptor, such that prazosin, RS-17053, WB 4101, 5-methylurapidil, Rec 15/ 2739 and S-niguldipine all displayed subnanomolar a nity. A di erent pro®le of a nity estimates was obtained in inositol phosphates accumulation studies: prazosin, WB 4101, 5-methylurapidil, RS-17053 and S-niguldipine showed 10 to 40 fold lower a nity than in membrane binding. However, a nity estimates were not`frameshifted', as tamsulosin, indoramin and Rec 15/2739 yielded similar, high a nity estimates in binding and functional assays. 4 In contrast, results from human a 1B -and a 1D -adrenoceptors expressed in CHO-K1 cells gave antagonist a nity pro®les in binding and functional assays that were essentially identical. 5 A concordance of a nity estimates from the functional (inositol phosphates accumulation) studies of the a 1A -adrenoceptor in CHO-K1 cells was found with estimates published recently from contractile studies in human lower urinary tract tissues (putative a 1L -adrenoceptor). These data show that upon functional pharmacological analysis, the cloned a 1A -adrenoceptor displays pharmacological recognition properties consistent with those of the putative a 1L -adrenoceptor. Why this pro®le di ers from that obtained in membrane binding, and whether it explains the a 1L -adrenoceptor pharmacology observed in many native tissues, requires further investigation.
The present study was designed to characterize an "atypical" 5-hydroxytryptamine (5-HT) receptor mediating relaxation of the rat oesophageal tunica muscularis mucosae. All experiments were performed under equilibrium conditions, using pargyline to inhibit the oxidative deamination of indoleamines, and cocaine and corticosterone to inhibit neuronal and extraneuronal uptake. Under these conditions 5-HT (0.3-1000 nmol/l) produced a concentration-dependent relaxation of carbachol-induced tension. The concentration-effect curve to 5-HT was unaffected by potent antagonists for 5-HT1, 5-HT2, 5-HT3 and so called 5-HT1P receptors (metergoline, methysergide, ketanserin, ondansetron, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide), but was antagonized competitively by ICS 205-930 (pA2 = 6.7). Responses to 5-HT were mimicked by other indoleamines and substituted benzamides with the following order of potency: 5-HT greater than or equal to 5-methoxytryptamine greater than cisapride = alpha-methyl-5-HT = (S)-zacopride = renzapride greater than (RS)-zacopride greater than 5-carboxamido-tryptamine = metoclopramide = (R)-zacopride greater than tryptamine greater than 2-methyl-5-HT. ICS 205-930 afforded similar pA2 values (6.0-6.7) against each agonist, indicating a common site of action. Concentration-effect curves to 5-HT were not affected by tetrodotoxin or indomethacin, suggesting that 5-HT-induced relaxation of the tunica muscularis mucosae was mediated via a post-junctional receptor, independent of endogenous prostanoids. The pharmacological profile of the 5-HT receptor in the rat oesophageal tunica muscularis mucosae correlates well with the 5-HT4 receptor characterized recently in both the CNS and gastro-intestinal tract.
Self-assembled biomaterials are an important class of materials that can be injected and formed in situ. However, they often are not able to meet the mechanical properties necessary for many biological applications, losing mechanical properties at low strains. We synthesized hybrid hydrogels consisting of a poly(γ-glutamic acid) polymer network physically cross-linked via grafted self-assembling β-sheet peptides to provide non-covalent cross-linking through β-sheet assembly, reinforced with a polymer backbone to improve strain stability. By altering the β-sheet peptide graft density and concentration, we can tailor the mechanical properties of the hydrogels over an order of magnitude range of 10–200 kPa, which is in the region of many soft tissues. Also, due to the ability of the non-covalent β-sheet cross-links to reassemble, the hydrogels can self-heal after being strained to failure, in most cases recovering all of their original storage moduli. Using a combination of spectroscopic techniques, we were able to probe the secondary structure of the materials and verify the presence of β-sheets within the hybrid hydrogels. Since the polymer backbone requires less than a 15% functionalization of its repeating units with β-sheet peptides to form a hydrogel, it can easily be modified further to incorporate specific biological epitopes. This self-healing polymer−β-sheet peptide hybrid hydrogel with tailorable mechanical properties is a promising platform for future tissue-engineering scaffolds and biomedical applications.
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