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Phe-Met-Arg-Phe-NH2 (FMRFa)-related peptides inhibit Na(+)-Ca2+ exchange in cardiac sarcolemma vesicles
D Khananshvili1, D C Price, M J Greenberg
1Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Israel.
Abstract:
The molluscan cardioexcitatory tetrapeptide FMRF-amide (Phe-Met-Arg-Phe-NH2) and related peptides inhibit Na(+)-Ca2+ exchange in calf cardiac sarcolemma vesicles. FMRFa itself has a low inhibitory potency (IC50 = 750 microM) which completely resides in its COOH-terminal RFa portion. The physiologically active analog FLRFa is 10-fold more potent (IC50 = 60 microM). Two other substitutions of the Met2 in FMRFa, by either Ile or Lys increase inhibitory potency 7- and 50-fold, respectively. The inhibitory potency increases 300-500-fold if the NH2-terminal Phe1 in FMRFa is substituted by either Val or His (IC50 = 1-2 microM). The inhibitory activity of WnLRFa (IC50 = 40 microM) is lost when either the NH2-terminal amino group is acylated or the NH2-terminal Trp1 is deleted. These data suggest that the COOH-terminal portion is essential for the basic low potency inhibition of Na(+)-Ca2+ exchange, whereas the NH2-terminal portion is important for the potentiation of the inhibitory activity. Although the IC50 values of various peptides range widely (10(-6)-10(-3) M), all of them induce a complete inhibition. The dose-response pattern of the peptide-induced inhibition is identical for the Na(+)-Ca2+ exchange and its partial reaction, the Ca(2+)-Ca2+ exchange. The inhibitory effect is reversible and affects both Nai(or Cai)-dependent 45Ca uptake and Nao-dependent 45Ca efflux, suggesting that the bidirectional movements of ions are altered. A mild pretreatment of vesicles with trypsin augments the Na(+)-Ca2+ exchange 1.5-fold but diminishes the inhibitory potency 3-4-fold, suggesting that the inhibition is mediated by an extravesicular membrane protein. The characteristics of the peptide-induced inhibition resemble the effect of opiates on Na(+)-Ca2+ exchange. FLRFa and dextrorphan (a non-opioid stereoisomer of an opiate agonist) are mutually exclusive inhibitors, suggesting that they may bind to the same site. This putative site lacks the pharmacological properties of opiate receptors and may be located either on the Na(+)-Ca2+ exchanger or at its vicinity. Endogenous analogs of FMRFa may regulate intracellular calcium via Na(+)-Ca2+ exchange.
Insights
Molluscan cardioexcitatory tetrapeptides, like FMRF-amide, inhibit cardiac Na(+)-Ca2+ exchange. Structural modifications significantly enhance this inhibition, suggesting a regulatory role in intracellular calcium.
Area of Science:
- Biochemistry
- Cardiovascular Physiology
- Neuroendocrinology
Background:
- The molluscan cardioexcitatory tetrapeptide FMRF-amide (Phe-Met-Arg-Phe-NH2) and its analogs are known to influence cardiac function.
- Na(+)-Ca2+ exchange is a critical mechanism for regulating intracellular calcium levels in cardiac cells.
Purpose of the Study:
- To investigate the inhibitory effects of FMRF-amide and related peptides on Na(+)-Ca2+ exchange in calf cardiac sarcolemma vesicles.
- To determine the structure-activity relationships of these peptides concerning their inhibitory potency.
Main Methods:
- Inhibition assays using calf cardiac sarcolemma vesicles to measure Na(+)-Ca2+ exchange.
- Radiolabeled ion flux measurements (45Ca uptake and efflux) to assess inhibition.
- Structure-activity relationship studies involving systematic modifications of the FMRF-amide peptide sequence.
Main Results:
- FMRF-amide and its analogs inhibit Na(+)-Ca2+ exchange, with potency varying significantly based on peptide structure.
- The COOH-terminal RFa portion is essential for basic inhibition, while NH2-terminal modifications greatly potentiate activity.
- Inhibition affects both Na(+)-Ca2+ and Ca(2+)-Ca2+ exchange, is reversible, and appears mediated by an extravesicular membrane protein.
- The inhibitory mechanism shares similarities with opiate effects, suggesting a potential common binding site on or near the Na(+)-Ca2+ exchanger.
Conclusions:
- The NH2-terminal and COOH-terminal regions of FMRF-amide analogs play distinct roles in modulating Na(+)-Ca2+ exchange inhibition.
- Endogenous FMRF-amide analogs may regulate intracellular calcium homeostasis through interaction with the Na(+)-Ca2+ exchanger.
- The findings suggest a novel regulatory pathway for cardiac calcium handling involving FMRF-amide-like peptides.