Long‐lasting activation of cation current by low concentration of endothelin‐1 in mouse fibroblasts and smooth muscle cells of rabbit aorta

Abstract

1 Recombinant human ETA receptors were expressed in a mouse fibroblast cell line (Ltk-cell) and functional coupling of the receptors with Ca24 permeable channels at low concentrations of endothelin-l (ET-1) was investigated using whole-cell recordings and monitoring the changes in intracellular free Ca2" concentrations ([Ca24] evoked an initial transient peak and a subsequent sustained elevation in [Ca24]i whereas a lower concentration of ET-1 (10-10 M) evoked only a sustained elevation of [Ca2"i. After removal of extracellular Ca2", ET-l evoked only an initial peak without a sustained elevation of [Ca24]i. The sustained elevation induced by 101 M ET-1 was blocked by 300 pM mefenamic acid (a cation channel blocker) but not by 10 JIM nifedipine (a blocker of voltage-operated Ca24 channel). 3 In whole-cell recordings with Ltk-cells, a brief (3-5 min) application of ET-l (1010 M) induced a sustained inward current at a holding potential of -60 mV. The current-voltage relationship revealed that the reversal potential of the ET-l-induced current was close to 0 mV (1.9 mV) and was not altered by reducing the concentration of Cl-in the bath solution, indicating that the current is carried by cations. The current was reversibly blocked by 300 JiM mefenamic acid, and it persisted after all cations in the bath solution had been replaced by Ca2" (5 or 30 mM) and nonpermeant cation N-methyl-Dglucamine, indicating that the ET-1-activated channel is permeable to Ca24. Activation of the current was independent of membrane potential and the current was induced even after addition of a high concentration (10 mM) of a Ca2" chelator, EGTA, to the pipette solution.4 In whole-cell recordings from rabbit aortic VSMCs, ET-l (101-M) induced a sustained inward current at a holding potential of -60 mV. The reversal potential was -12 mV and was not altered when the concentration of Cl-in the pipette solution was decreased, indicating that the current is carried by cations

ETA receptors are the primary mediators of myofilament calcium sensitization induced by ET‐1 in rat pulmonary artery smooth muscle: a tyrosine kinase independent pathway

“…When one considers the possible role of ET-1 in mediating HPV, it is of some interest to note that endothelin receptor activation can induce constriction of arterial smooth muscle not only by raising the intracellular Ca 2+ concentration (Highsmith et al, 1992;Pang et al, 1989;Kasuya et al, 1989;Goto et al, 1989;Enoki et al, 1995), but by increasing the sensitivity of the contractile apparatus to Ca 2+ (Nishimura et al, 1992;Ohanian et al, 1997). The signi®cance of this observation becomes apparent when taken together with the fact that calcium sensitization has also been implicated in the development of HPV .…”

Mitogen-Activated Protein Kinases Partially Regulate Endothelin-1-Induced Contractions through a Myosin Light Chain Phosphorylation- Independent Patyway.

“…The mechanism by which contractions are evoked by ET-1 in isolated vascular preparations is complicated. The increase in the sensitivity of the contractile apparatus to intracellular Ca 2+ ([Ca 2+ ] i ), as well as the increase of [Ca 2+ ] i concentration, may be involved in ET-1-induced vasoconstriction [8,26,28,29]. On the other hand, ET-1 evoked an increase of myofilament Ca 2+ sensitivity by a mechanism(s) independent of tyrosine kinase in the rat pulmonary artery [10].…”

Pharmacological characterization of receptor-mediated Ca2+ entry in endothelin-1-induced catecholamine release from cultured bovine adrenal chromaffin cells

“…[Ca 2+ ] i was determined using the fluorescent probe fluo-3 instead of fura-2 as described previously (Enoki et al 1995;Iwamuro et al 1998), because LOE 908 was found to disturb measurement of [Ca 2+ ] i by absorbing the light at excitation wavelengths for fura-2. For loading of the probes, the cultured cells were incubated in Ca 2+ -free Krebs-HEPES buffer containing 5 µM fluo-3/acetoxymethyl ester for 45 min at 37°C.…”

“…Immediately before measurement of [Ca 2+ ] i , CaCl 2 was added to the suspension at the final concentration of 1 mM, and 1-ml aliquots of the suspension were used for measurement of fluorescence by a CAF 110 spectrophotometer (JASCO, Tokyo, Japan). Wavelengths of excitation and emission for fluo-3 were 490 nm and 540 nm, respectively, and [Ca 2+ ] i was calculated as described previously (Enoki et al 1995). The ET-1-induced increases in [Ca 2+ ] i were determined by subtracting the basal [Ca 2+ ] i from the peak value following addition of ET-1, and unless otherwise specified, the data were expressed as the ET-1-induced increases in [Ca 2+ ] i .…”

“…Several researchers have shown that Ca 2+ -permeable nonselective cation channels (NSCC) are activated by stimulation of native ET A receptor in vascular smooth muscle cells (van Renterghem et al 1988;Chen and Wagoner 1991;Enoki et al 1995;Minowa et al 1997) and of recombinant human ET A receptors expressed in Ltk -cells (Enoki et al 1995). We have recently found that in A7r5 cells derived from rat thoracic aortic smooth muscle cells, ET-1 can activate three types of voltage-independent Ca 2+ entry channels: type-1 Ca 2+ -permeable NSCC (NSCC-1), type-2 Ca 2+ -permeable NSCC (NSCC-2) and store-operated Ca 2+ channel (SOCC), in addition to VOCC (Iwamuro et al 1998(Iwamuro et al , 1999.…”