Abstract
The release of arachidonic acid by phospholipases in response to cell surface receptor activation may be an important step in the initiation of inotropic events in cardiac muscle. Endothelin has been shown to activate phospholipase A2 and release arachidonic acid in isolated rat hearts. Endothelin also has a positive inotropic effect in cardiac muscle, suggesting that endothelin increases Ca2+ influx or the amount of Ca2+ released from the sarcoplasmic reticulum. We used suspensions of adult rat ventricular myocytes loaded with fura-2/AM to compare the effects of arachidonic acid and endothelin on Ca2+ transients evoked by extracellular ATP. We showed recently (Damron, D.S., and Bond, M. (1993) Circ. Res. 72, 376-386) that pretreatment of cardiac myocytes with arachidonic acid significantly potentiated the amplitude of the ATP-triggered Ca2+ transient. We now report that endothelin also enhances the ATP-triggered Ca2+ transient and that the effect of the combination of maximal doses of endothelin and arachidonic acid is additive. Neither endothelin nor arachidonic acid was found to affect the size of the sarcoplasmic reticulum Ca2+ store. The potentiating effects of both arachidonic acid and endothelin were sensitive to inhibitors of protein kinase C. Endothelin was also found to stimulate phospholipase C but not phospholipase A2. Application of arachidonic acid to individual cardiac muscle cells resulted in inhibition of the transient outward K+ current, whereas application of endothelin inhibited the delayed rectifier current. These effects of arachidonic acid and endothelin were additive, and both effects could be blocked by the protein kinase C inhibitor, staurosporine. Similarly, staurosporine inhibited endothelin-induced increases in isometric contractions in ventricular papillary muscle. We conclude that arachidonic acid and endothelin may be involved in the modulation of inotropic activity in cardiac muscle by means of protein kinase C-dependent inhibition of two distinct K+ channels. This would result in a prolongation of action potential duration and thus an increase in Ca2+ influx across the sarcolemma.
Highlights
Response to cell surface receptor activationmay be an important step in the initiation of inotropic events in cardiac muscle
We used the mechanisms bywhich endothelin causes an suspensions of adult rat ventricular myocytes loaded inotropic response in cardiac muscle remain unknown, it is with fura-2/AM to compare the effectsof arachidonic clear that endothelin stimulates various signaling pathways acid and endothelin on Ca2*transients evoked by ex- that may be associated with alterations in Ca2+cycling or in tracellular ATP
We reported previously that arachidonic acid andother polyunsaturated fatty acids potentiate the amplitude of the ATP-evoked Ca2+transient via a protein kinase C (PKC)-dependent pathway [19].In addition,we demonstrated that these same fatty acids stimulate a transient rise in [Ca2+Iifrom a caffeine-sensitive of this article were defrayed in partby the payment of page charges
Summary
The procedures for the preparation of adult rat ventricular myocytes have been described in detail inan earlier report [19] and area modification of the method described by Altschuld et al [20]. Theheart wasremoved, cannulated via the aorta, mounted ona modified Langendorffperfusion apparatus, and perfused with oxygenated (95% 0 2 , 5% COZ) Krebs Henseleit buffer (KHB), which contained (in mM) 118NaCl, 4.8 KC1,1.2 MgC12,1.2KHzPO,, 1.25 CaC12, 0.68 glutamine, 37.5 NaHC03, 16.5 dextrose, and 7.5 pyruvate, pH 7.4. The perfusion buffer was changed to a Ca2+-freeKHB containing collagenase (type 11, 237 units/ml, Worthington). Reactions were terminated by the addition of ice-cold perchloric acid. Cell lysis, following perchloric acid treatment, was achieved by freezing the cells on dry ice and thenallowing them to thaw. The resulting supernatant were applied to l-ml packed AG 1-X8 columns (100-200 mesh, formate form), and inositol phosphates were eluted with 1 M ammonium formate, 0.1 M formic acid, after first washing the column with 16 ml of 0.1 M formic acid. Perforated Whole Cell Voltage and Current Clump Recordings retaining a rod-shaped form was routinely between 75 and 90%
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