The snake venom peptide Bj‐PRO‐7a is a M1 muscarinic acetylcholine receptor agonist

Abstract

Proline-rich peptides from Bothrops jararaca venom (Bj-PRO) were characterized based on the capability to inhibit the somatic angiotensin-converting enzyme. The pharmacological action of these peptides resulted in the development of Captopril, one of the best examples of a target-driven drug discovery for treatment of hypertension. However, biochemical and biological properties of Bj-PROs were not completely elucidated yet, and many recent studies have suggested that their activity relies on angiotensinconverting enzyme-independent mechanisms. Here, we show that Bj-PRO-7a (<EDGPIPP) specifically activates [Ca 21 ] i transients in CHO-M1 cells expressing heterologous rat M1 muscarinic subtype. The activation curve established by microfluorimetry in CHO-M1 cells using increasing concentrations of Bj-PRO-7a reached the maximum response in the presence of 3 lM Bj-PRO-7a (EC 50 5 0.25 6 0.07 lM). The variation observed by calcium imaging in these cells ranged from 52 to 1218 nM (EC 50 5 0.31 6 0.12 lM). [Ca 21 ] i responses in CHO-M1 cells were largely inhibited by pirenzepine, a specific M1 antagonist. Neural-differentiated P19 cells expressing endogenous M1 receptors were also responsive to Bj-PRO-7a application, whereas no such response was observed in undifferentiated P19 cells not expressing muscarinic receptors. As further support for its specific action on M1 receptors, the peptide did not activate M3 subtypes in transfected CHO cells. Our findings provide a novel M1 muscarinic receptor agonist that could be used for basic research and even for pharmacological applications. '

Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotes vasodilatation mediated by both bradykinin B2 and M1 muscarinic acetylcholine receptors

“…Thus, the hypothesis was raised that each Bj-PRO might act through distinct mechanism of action, probably involving different target proteins [9,23]. Recently, our group demonstrated that Bj-PRO-7a is a mAchR-M1 agonist [39], and Bj-PRO-10c action involves argininosuccinate synthase (ASS) activation, a key enzyme to provide substrate for NOS [13].…”

Modulation of Mouse Embryonic Stem Cell Proliferation and Neural Differentiation by the P2X7 Receptor

“…After loading with Fluo-3AM, the cells were incubated with extracellular buffer (140 mM NaCl, 3 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 10 mM HEPES, 10 mM glucose at pH 7.4) [25]. Ca 2+ imaging was performed by using the Inverted Research Microscope ECLIPSE-TiS (Nikon, Melville, NY) equipped with a 14 bit high-resolution CCD camera CoolSNAP HQ2 (Photometrics, Tucson, AZ) and analyzed with NIS-Element software (Nikon) using image acquisition rates of two frames per second.…”

The monoterpene (–)‐carvone: A novel agonist of TRPV1 channels

“…where K d is the dissociation constants of Fluo-3 (450 nM) or Fura-2 (224 nM), F is the variable fluorescence intensity, F max and F min are, respectively, the maximum and minimum fluorescence obtained in the presence of ionomycin (5 lM) or EGTA (20 mM) (16). F was substituted for R in the formula above when Fura-2 was used as fluorophore.…”