Evidence for two endothelin Et(A) receptor subtypes in rabbit arteriolar smooth muscle.

1. Effects of endothelin-1 (Et-1) were studied on membrane currents in choroidal arteriolar smooth muscle by using perforated patch-clamp recordings. 2. Et-1 (10 nM) activated oscillatory Ca(2+)-activated Cl(-)-currents (I(Cl(Ca))) which could not be reversed by washing out. 3. Currents through L-type Ca(2+) channels were resolved in a divalent free medium (I(Ca(L)Na)). Et-1 reduced I(Ca(L)Na) by 75 +/- 7% within 30 s and this effect faded over 5 min, when the depression remained constant. On washing out Et-1, I(Ca(L)Na) almost completely recovered within 10 s. 4. BQ123 (1 microM), a peptide Et(A) receptor blocker, prevented the activation of I(Cl(Ca)), but failed to inhibit I(Cl(Ca)) transients once they had been initiated. In contrast, BQ123 not only prevented but also reversed the inhibition of I(Ca(L)Na) by Et-1. BQ788 (1 microM), an Et(B) receptor antagonist, did not prevent the activation of I(Cl(Ca)) or the inhibition of I(Ca(L)Na) by Et-1. 5. ABT-627 (10 nM), a non-peptide Et(A) receptor antagonist also blocked the activation of I(Cl(Ca)). However, on I(Ca(L)Na), ABT-627 (10 nM) mimicked the action of Et-1 an effect blocked by BQ123 suggesting that ABT-627 acted as an agonist. 6. The data are consistent with choroidal arteriolar smooth muscle cells having two types of Et(A) receptor, one where BQ123 is an antagonist and ABT-627 an agonist, where ligands dissociate freely and this receptor is coupled to inhibition of L-type Ca(2+) channels. In the other, BQ123 and ABT-627 are both antagonists and with Et-1 the receptor converts to a high affinity state producing the classical irreversible activation I(Cl(Ca)).

The smooth muscle pharmacology of maximakinin, a receptor-selective, bradykinin-related nonadecapeptide from the venom of the Chinese toad, Bombina maxima.

Structural homologues of vertebrate regulatory peptides found in defensive skin secretions of anuran amphibians often display enhanced bioactivity and receptor binding when compared with endogenous mammalian peptide ligands. Maximakinin, a novel N-terminally extended bradykinin (DLPKINRKGPRPPGFSPFR) from the skin venom of a Chinese toad (Bombina maxima), displays such activity enhancement when compared with bradykinin but is additionally highly selective for mammalian arterial smooth muscle bradykinin receptors displaying a 50-fold increase in molar potency in this smooth muscle type. In contrast, a 100-fold decrease in molar potency was observed at bradykinin receptors in intestinal and uterine smooth muscle preparations. Maximakinin has thus evolved as a “smart” defensive weapon in the toad with receptor/tissue selective targeting. Natural selection of amphibian skin venom peptides for antipredator defence, through inter-species delivery by an exogenous secretory mode, produces subtle structural stabilisation modifications that can potentially provide new insights for the design of selectively targeted peptide therapeutics.

Kinestatin: a novel bradykinin B2 receptor antagonist peptide from the skin secretion of the Chinese toad, Bombina maxima

We have isolated a novel bradykinin B2-receptor antagonist peptide, kinestatin, from toad (Bombina maxima) defensive skin secretion. Mass spectroscopy established a molecular mass of 931.56 Da and a provisional structure: pGlu-Leu/Ile-Pro-Gly-Leu/Ile-Gly-Pro-Leu/Ile-Arg.amide. The unmodified sequence, -QIPGLGPLRG-, was located at the C-terminus of a 116-amino-acid residue open-reading frame following interrogation of a sequenced B. maxima skin cDNA library database. This confirmed the presence of appropriate primary structural attributes for the observed post-translational modifications present on the mature peptide and established residue 2 as Ile and residues 5/8 as Leu. Kinestatin represents a prototype novel peptide from amphibian skin.

Distribution of the Receptor for Advanced Glycation End Products (RAGE) in the Human Male Reproductive Tract. Prevalence in Men with Diabetes Mellitus.

BACKGROUND: Diabetics have a significantly higher percentage of sperm with nuclear DNA (nDNA) fragmentation and increased levels of advanced glycation end products (AGEs), in their testis, epididymis and sperm. As the receptor for AGEs (RAGE) is important to oxidative stress and cell dysfunction, we hypothesise, that it may be involved in sperm nDNA damage. METHODS: Immunohistochemistry was performed to determine the presence of RAGE in the human testis and epididymis. A comparison of the receptor's incidence and localisation on sperm from 10 diabetic and 11 non-diabetic men was conducted by blind semi-quantitative assessment of the immunostaining. ELISA analysis ascertained RAGE levels in seminal plasma and sperm from 21 diabetic and 31 non-diabetic subjects. Dual labelling immunolocalisation was employed to evaluate RAGE's precise location on the sperm head. RESULTS: RAGE was found throughout the testis, caput epididymis, particularly the principle cells apical region, and on sperm acrosomes. The number of sperm displaying RAGE and the overall protein amount found in sperm and seminal plasma were significantly higher in samples from diabetic men (p