Abstract
1. A giant patch method was used to study the stimulatory effect of
cytoplasmic MgATP on outward Na$^+$-Ca$^2+$ exchange current
in inside-out cardiac membrane patches (1-10 G omega seals with 14-24
microns pipette tip diameters) excised from guinea-pig, rabbit and
mouse myocytes. 2. To establish the validity of the method with respect
to structure, bleb formation was examined with electron microscopy
and with confocal fluorescence light microscopy. The blebs, which
form as the sarcolemma detaches, excluded intracellular organelles
and transverse tubules. The blebbed cells contained normal sarcomeres,
sarcoplasmic reticulum, triads and diads. 3. To further establish
the validity of the method for ion transport studies, measurements
of Na$^+$-K$^+$ pump currents and charge movements are described
briefly which demonstrate (i) free access to the cytoplasmic membrane
side, (ii) MgATP dependence comparable to reconstituted pump (Kd,
94 microns), (iii) fast, rigorous concentration control and (iv)
Na$^+$-K$^+$ pump densities in the range of whole-cell densities.
4. Stimulation of outward Na$^+$-Ca$^2+$ exchange current
by MgATP attenuated exchange current decay during step increments
of cytoplasmic sodium, shifted the secondary activation of outward
exchange current by cytoplasmic calcium to lower free calcium concentrations
and, particularly in mouse cardiac sarcolemma, induced cytoplasmic
calcium-independent current. 5. Upon removal of MgATP the stimulatory
effect usually decayed with a t50 (half-time) of about 3 min. However,
the reversal took place much more rapidly (t50, 5-20 s) in patches
from individual guinea-pig and rabbit myocyte batches. When decay
was rapid, secondary activation by cytoplasmic calcium was shifted
to higher free cytoplasmic calcium concentrations (Kd, 10-65 microns-free
calcium). 6. With repeated applications of MgATP the rate and magnitude
of the stimulatory effect progressively decreased. 7. The Kd for
MgATP of the initial rate of stimulation of outward exchange current
was 3 mM or greater. When decay was rapid, the steady-state dependence
of exchange current on MgATP also had a Kd of 3 mM or greater. 8.
Stimulation of Na$^+$-Ca$^2+$ exchange current by MgATP occurred
in the absence of cytoplasmic calcium with 9 mM-EGTA. 9. The stimulatory
effect of 2 mM-MgATP was not inhibited by up to 200 microM of the
protein kinase inhibitor 1-(5-isoquinoline sulphonyl)-2-methylpiperazine
(H7), or by peptide inhibitors of cyclic AMP-dependent protein kinase,
protein kinase C and calcium-calmodulin-dependent protein kinase
II.(ABSTRACT TRUNCATED AT 400 WORDS)
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