Interactions between benzylamiloride and fura-2: Studies in vitro and in cardiac myocytes

Cathy A. Hudson, José D. Rojas, Narine Sarvazyan, Donald E. Wesson, Raul Martínez-Zaguilán

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

Amiloride derivatives are commonly used inhibitors of Na+/H+- and Na+/Ca2+-exchange. Because they are fluorescent molecules the use of benzylamiloride (BZA), an inhibitor of Na+/Ca2+ exchange, in conjunction with Fura-2, a commonly used fluorescent Ca2+ indicator, might complicate interpretation of fluorescence data obtained. In vitro data show that BZA decreases the Fura-2 fluorescence at all useful wavelengths in a concentration-dependent manner. The Fura-2 ratio 340/380 (used to estimate intracellular Ca2+ ([Ca2+](in))) also decreased with increasing BZA concentrations. The Stern-Volmer relation suggests that this phenomenon is due to either static or dynamic quenching. Varying temperatures from 4 to 37°C did not alter Stern-Volmer constants, consistent instead with fluorescence resonance energy transfer (FRET). The in situ relevance of these interactions was evaluated in adult rat cardiac myocytes which exhibit Na+/Ca2+ exchange reflected by rapid [Ca2+](in) increase following Na+ removal. Pretreatment with BZA ≤ 25 μM decreased the magnitude of Fura-2 changes induced by Na+ removal. Analysis of the individual Fura-2 useful wavelengths indicated that ≤ 25 μM BZA altered the Fura-2 signal in a manner consistent with the quenching effects noted in vitro. Together, these data show that BZA interacts with Fura-2 in vitro and in situ and suggest caution when interpreting Fura-2 fluorescence data derived in conjunction with BZA.

Original languageEnglish (US)
Pages (from-to)25-34
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume356
Issue number1
DOIs
StatePublished - Aug 1 1998
Externally publishedYes

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Keywords

  • Amiloride derivatives
  • Fluorescence
  • Na+/ Ca exchange
  • Quenching

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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