Læknablaðið : fylgirit - 01.09.1993, Blaðsíða 42

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LÆKNABLAÐIÐ/FYLGIRIT 24
Transient Increase in ÍCa2+N in Rat Cardiac
V 18 Ventricular Myocytes Induced by Extracellular
Adenine Nucleotides & Electrical Stimulation:
A Study of ATP-induced Desensitization.
'O.G. Bjc5rnssonf I.R. Siemens, J.R. Williamson
Metab. Res. Lab., Radcliffe Inf. Oxford, U.K.
Dept. Biochem.S Biophys. Univ. of Penn. U.S.A
The [Ca2+]i response of cardiac ventricul-
ar myocytes stimulated by extracellular aden-
ine nucleotides (or nucleotide analogues) was
monitored in bulk suspensions of myocytes or
in single cells loaded with fura-2, and the
desensitization studied which developed to-
wards repeated applications of ATP. A great
variety of different [Ca2+]i response patterní
was observed upon stimulation by extracellulai
ATP (10“4 M) in both cell preparations. The
[Ca2+]i response was usually transient with
an initial fast phase (< 1 s), followed by a
slower phase of higher amplitude (time-to-
peak, 14.2 ± 1.2 s, SEM, n = 36, range 5.9 -
23.0; to.5~off = 32.2 ± 3.9 s, range 7.5 -
99.0, n = 24). The rank-order of potency of
adenine nucleotides and slowly hydrolyzable
ATP analogues to (desensitize) suppress el-
ectrically-induced [Ca2+]i transients was
similar to their order of potency to initiall)
depolarize the plasma membrane(increase bis-
oxonol fluorescence) and raise [Ca2+]i (in-
crease fura-2 fluorescence): ATP > 2-methyl-
thio-ATP » ATP^S » AMPPNP > ot,£-methylene-
ATP = /3 ,^-methylene-ATP; and ATP > ADP > AMP,
with adenosine having no effect. Forskolin
Is Hydrolysis of High-Energy Phosphate Bonds
V 19 Essential for Extracellular Adenine Nucleo-
tide Induced [Ca2+]j Transients in Isolated
Cardiac Ventricular Myocytes of the Rat?
O.G. Bjb'rnsson, J.R. Williamson. Metab. Res.
Lab., Oxford, U.K., Dept. of Biochem. & Bio-
phys., Univ. of Penn., Philadelphia, U.S.A.
Extracellular adenine nucleotides have for
long been known to have potent inotropic
effects on the mammalian heart, and these
effects are exerted directly on the myocard-
ium rather than as a consequence of the vas-
cular effects of these compounds [1]. Previ-
ous reports have also shown that extracellu-
lar ATP depolarizes (bisoxonol) the plasma
membrane of cardiac ventricular myocytes and
increases the [Ca2+]i (fura-2) [2], but chang-
es in [Ca2+]j[ are considered to be an import-
ant determinant of muscle contraction. In the
present work we have shown in bulk suspensions
of myocytes, or in single myocytes, that the
rank-order of potency of adenine nucleotides
and slowly hydrolyzable ATP analogues to de-
polarize (bisoxonol) the sarcolemma and to
raise [Ca2+]i (fura-2) was: ATP > ADP > AMP,
adenosine being without an effect; and ATP >
2-methylthio-ATP » ATP^S > AMPPNP >
methyleneATP = /2,^-methyleneATP, i.e. the more
hydrolyzable the compounds were, or the more
high energy phosphate bonds they had, the more
potent they were in depolarizing the sarco-
lemma and in raising [Ca2+]i. The effects of
ATP on [Ca2+]i were enhanced by magnesium, but
(5 x 10“6 M) (but not isoproterenol) partiall>
restored ATP suppressed [Ca2+]i transients,
while the 1,4-dihydropyridine calcium agonist
BAY K 8644 (5 x 10“6 M) enhanced the ATP-in-
duced [Ca2+]i signal, but without having an
effect on the ATP suppressed electrical trans-
ients, In cells placed in a Na+ free buffer
(Na+ replaced by Li+), extracellular ATP had
similar effects on [Ca2+]i as in normal (Na+)
buffer, while pretreatment of the cells with
tetrodotoxin suppressed both electrically
and ATP-induced [Ca2+]i transients.- The pre-
sent data show a correlation between the po-
tency of adenine nucleotides to depolarize the
sarcolemma of cardiac ventricular myocytes and
to increase [Ca2+]i and to cause desensitiz-
ation of the cells towards subsequent electri-
cal stimulation. Failure of isoproterenol, but
partial restoration by forskolin of suppressed
electrical [Ca2+]i transients, suggests un-
coupling of y5-adrenergic receptors and aden-
ylate cyclase. Like electrically stimulated
increase of [Ca2+]i, the effects of extra-
cellular ATP depend on early activation of
sodium channels of the sarcolemma.
suppressed by verapamil, nifedipine or EGTA.
Tetrodotoxin, or replacement of Na+ of the
incubation buffer with the impermeant cation
N-methyl-D-glucamine, blocked the effects of
ATP.- We hypothesize that free chemical en-
ergy and phosphate groups released upon hydro*
lysis of adenine nucleotides are responsible
for depolarization, phosphorylation and event-
ually activation of cation channels of the
sarcolemma of cardiac ventricular myocytes.
Such a hypothesis might involve activation of
ectonucleotides and ectoprotein kinases and
phosphorylation of ion channels from the out-
er surface of the plasma membrane.- [1] Green
H.N., and H.B. Stoner: Biological Actions of
the Adenine Nucleotides, H.K. Lewis, London,
1950; [2] Eur. J. Biochem. 186:395-404, 1989.