Research Article 3,5-Di-t-Butylcatechol as a Ryanodine Receptor Agonist in Rat Intact Skeletal Muscle Fibers Caterina Lacava, Giampietro Sgaragli, and Fabio Fusi* Dipartimento di Neuroscienze, Università degli Studi di Siena, Siena, Italy Strategy, Management and Health Policy Enabling Technology, Genomics, Proteomics Preclinical Research Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics Clinical Development Phases I-III Regulatory, Quality, Manufacturing Postmarketing Phase IV ABSTRACT 3,5-Di-t-butylcatechol (DTCAT) stimulates the rat skeletal muscle sarcoplasmic reticulum ryanodine receptor (RyR). In the present study, its effects on the contractile response of diaphragm preparation were characterized using electrically stimulated phrenic nerve–diaphragm preparations and diaphragm strips. DTCAT reduced, concentration-dependently, twitch contraction of the phrenic nerve– diaphragm preparation evoked by both direct and indirect stimulation and increased spontaneous tone. Twitch amplitude reduction was irreversible, while the increase of spontaneous tone was only partially reversible upon DTCAT washout. In diaphragm strips, caffeine > 4-chloro-m-cresol >> 3,5- diisopropylcatechol @ ryanodine > DTCAT enhanced spontaneous tone, whereas quercetin reduced it with all the compounds reducing twitch amplitude. DTCAT-induced contracture was partly dependent on extracellular Ca 2+ influx and antagonized by a Cd 2+ /La 3+ mixture. In intact skeletal muscle preparations, DTCAT behaved as a RyR agonist. Drug Dev Res 73 : 138–145, 2012. © 2012 Wiley Periodicals, Inc. Key words: 3,5-Di-t-butylcatechol; ryanodine receptor agonist; ryanodine; caffeine; phrenic nerve-diaphragm INTRODUCTION Some pharmacological probes have gained wide- spread acceptance as reagents to reveal the functional consequences of changes in intracellular Ca 2+ concen- tration. Many xenobiotics modulate intracellular Ca 2+ homeostasis by acting on ryanodine-sensitive Ca 2+ release channels. However, their effects on the ryano- dine receptor (RyR) are often complex, sometimes incompletely elucidated, or both [Zucchi and Ronca- Testoni, 1997]. A wide variety of structurally unrelated substances have been demonstrated to release Ca 2+ from sarcoplasmic reticulum [Palade, 1987]. These include ryanodine, caffeine, as well as sulphydryl oxi- dizing agents and drugs that are functionally and struc- turally unrelated such as rapamycin, halothane, and doxorubicin. The effects of such substances on the RyR have been characterized to a variable extent and only a few are considered useful pharmacological or diagnos- tic tools in RyR studies. Ryanodine, a plant alkaloid, binds the RyR at specific, high- or low-affinity sites and sustains either stimulatory or inhibitory effects. In cell-free systems, ryanodine usually activates but also inhibits the channel when used at nanomolar and micromolar concentrations, respectively [Lai et al., 1989]. Its use is often problematic owing to its slow and essentially irreversible binding to the receptor. This is particularly evident with intact cells where the distinc- tion between its stimulatory and inhibitory actions may Grant sponsor: Università degli Studi di Siena (grant PAR servizi 2007) and Ministero degli Affari Esteri (Rome, Italy) as stipulated by Law 212 (26-2-1992). *Correspondence to: Fabio Fusi, Dipartimento di Neuroscienze, Università degli Studi di Siena, via A. Moro 2, 53100 Siena, Italy. E-mail: fabio.fusi@unisi.it Received 8 November 2011; Accepted 10 December 2011 Published online in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1002/ddr.21003 DRUG DEVELOPMENT RESEARCH 73 : 138–145 (2012) DDR © 2012 Wiley Periodicals, Inc.