Department of Medicine, Brown University, Rhode Island Hospital, Providence, Rhode Island 02902-0001
THE CURRENT ARTICLE IN FOCUS by Murthy and Makhlouf
(Ref. 19; see p. C1199 in this issue) reports and
discusses in some detail cGMP-mediated
Ca2+ release in smooth muscle
cells isolated from the circular layer of the rabbit stomach. The
authors note that 8-bromo-cGMP, nitric oxide (NO), and vasoactive
intestinal peptide (VIP), agents that normally cause relaxation of
smooth muscle, can be converted to contractile agents when cAMP- and
cGMP-dependent protein kinases (PKA and PKG, respectively) are
inhibited. It has been reported elsewhere that sodium nitroprusside
(SNP) causes contraction of longitudinal esophageal muscle (10, 21)
through cGMP-dependent mechanisms. These agents (SNP, NO, VIP) have in
common the ability to increase the intracellular levels of cGMP. The
authors show that, when PKA and PKG are inhibited, cGMP causes
mobilization of Ca2+ from
intracellular stores. It is therefore reasonable to examine how cGMP,
and possibly other cyclic and noncyclic nucleotide phosphates, may
function as Ca2+-mobilizing
agents.
Mobilization of intracellular Ca2+
is a common signal transduction mechanism that can be triggered by
diverse signals such as hormones binding to specific surface receptors,
cell-cell fusion during fertilization, or membrane depolarization
during muscle contraction. This diversity of signals culminating in
Ca2+ mobilization is reflected by
diversity of Ca2+-mobilizing
messengers. To date several
Ca2+-release activators have been
discovered, including inositol trisphosphate (IP3) (3), sphingosine
1-phosphate (7), and several cyclic and noncyclic nucleotide phosphates
(14).
cGMP is a member of an expanding family of nucleotide phosphates that
act as Ca2+-mobilizing agents.
These include GTP, cGMP, cyclic ADP-ribose (cADPR), a metabolite of
GTP. GTP stimulates
Ca2+ release in neuronal, smooth
muscle, and fibroblast cell lines (see Refs. 5, 10, 14, and 40 of Ref.
19). GTP-induced Ca2+
release is additive to that of IP3
and appears to be mediated by a product of the hydrolysis of GTP, since
Ca2+ release could not be induced
by nonhydrolyzable analogs of GTP. The GTP-dependent mechanism, so far
observed in transformed cell lines, is different from the cGMP-mediated
Ca2+ release observed in the
present study on freshly dispersed smooth muscle cells. In smooth
muscle cells GTP did not cause
Ca2+ release.
cADPR. cADPR is a product of the
activity of ADP-ribosyl cyclase, which is homologous to the mammalian
CD38 ectoenzyme, first recognized as a lymphocyte antigen but
subsequently found to be present in numerous cell types (14). These
enzymes are bifunctional in that they are capable of catalyzing the
conversion of It is worth noting that production of cADPR, leading to
Ca2+ release, occurs through
different mechanisms in sea urchin eggs and in smooth muscle. In sea
urchin eggs ADP-ribosyl cyclase is found in a soluble form that is cGMP
and ATP dependent and in a membrane-bound form that is a homologue of
CD38 (8), fertilization is accompanied by production of cGMP and
activation of PKG, and the latter is thought to activate ADP-ribosyl
cyclase to generate cADPR and thus stimulate
Ca2+ release. In smooth muscle,
however, activation of ADP-ribosyl cyclase appears to depend on an
initial influx of Ca2+ into the
cell, which causes Ca2+-induced
Ca2+ release and activates
ADP-ribosyl cyclase. The resultant formation of cADPR enhances
Ca2+-induced
Ca2+ release (6, 11, 12, 17).
The enzyme responsible for synthesizing cADPR, ADP-ribosyl cyclase, is
found in numerous cell types (14, 16, 20). The wide distribution of the
enzyme and the complexity of the associated metabolic pathways suggest
that there may be multiple regulatory mechanisms controlling the
concentration of cADPR (8).
NAADP.
NAADP+ is a recently identified
metabolite of NADP+ that is as
potent as IP3 and cADPR in
mobilizing intracellular Ca2+ in
sea urchin eggs and microsomes (the usual model for studying Ca2+ release by these agents).
Like cADPR, NAADP is also a catalytic product of ADP-ribosyl cyclase
(or CD38), this time catalyzing the exchange of the nicotinamide group
of NADP with nicotinic acid to generate NAADP (2). The switch of the
catalysis to the exchange reaction requires acidic pH and nicotinic
acid (2). The mechanism of Ca2+
release activated by NAADP+ and
the Ca2+ stores it acts on are
different from those of IP3 and
cADPR. The NAADP+-sensitive
Ca2+ release is likely to be a new
pathway for mobilizing internal Ca2+, since it is unaffected by
pharmacological agents of known
Ca2+-release mechanisms (1). It is
insensitive to 8-amino-cADPR, a specific antagonist of the cADPR
receptor (22). Heparin, an antagonist of the
IP3 receptor, antagonists of the
ryanodine receptor, high concentrations of
Mg2+, procaine, and ruthenium red
(4, 5, 9, 15) also have no effect on the release. Fractionation studies
show that the Ca2+ stores
discharged by NAADP are different from those discharged by cADPR or
IP3 as well as from mitochondria
(5, 15). Furthermore, these stores are not thapsigargin sensitive and
may thus possess a distinct
Ca2+-ATPase uptake mechanism.
Binding studies show that the
NAADP+ receptor is distinct from
that of cADPR and that, at subthreshold concentrations,
NAADP+ can fully inactivate
subsequent binding to the receptor in a time-dependent manner; this
desensitization does not affect cADPR (1). Thus the
NAADP+-sensitive
Ca2+-release process has novel
regulatory characteristics, which are distinguishable from
Ca2+ release mediated by either
IP3 or cADPR.
cGMP. The ability of cGMP to stimulate
Ca2+ release was detected in
smooth muscle cells from the circular layer of the stomach after
blockade of PKA and especially PKG activity. In smooth muscle these
protein kinases act at different locations to attenuate the
intracellular levels of Ca2+. They
block Ca2+ release from
sarcoplasmic stores (phosphorylation of
IP3 and ryanodine receptors),
stimulate Ca2+ uptake by the
stores (phosphorylation of
Ca2+-ATPase), inhibit plasma
membrane Ca2+ channel activity
(block Ca2+ influx), and stimulate
plasma membrane K+ channel
activity (hyperpolarization and thus inhibition of
Ca2+ influx). When the effect of
these kinases is blocked, cGMP could be shown to induce
Ca2+ release. The release occurred
from thapsigargin-sensitive Ca2+
stores. These stores, however, were distinct from
IP3- or ryanodine-sensitive Ca2+ stores, since cGMP-induced
Ca2+ release could not be blocked
by either heparin or ruthenium red (the latter blocks ryanodine
receptor/Ca2+ channels, which are
found in intestinal longitudinal muscle rather than circular muscle).
Furthermore, Ca2+ release induced
by cGMP was additive to that induced by
IP3, consistent with an effect of
cGMP on a distinct store. The fact that kinase inhibitors were required
to demonstrate the effect of cGMP implies that kinases are able to
inhibit (presumably by phosphorylation) the cGMP-dependent
Ca2+-release pathway, just as they
inhibit the IP3-dependent pathway in circular muscle and the ryanodine-dependent pathways in longitudinal muscle.
It is important to note that, whereas PKA and PKG inhibit
Ca2+ release in vascular and
visceral smooth muscle and cerebellar neurons, they seem to stimulate
Ca2+ release in other cell types
(e.g., hepatocytes). Whether cGMP independently of its kinase is able
to stimulate Ca2+ release in
hepatocytes, for example, is not known.
The functional significance of some of these
Ca2+-mobilizing agents, in cells
other than sea urchin eggs, remains to be established. cADPR-induced
Ca2+ release appears to play a
physiological role in agonist-induced contraction of intestinal
longitudinal muscle (12) by causing Ca2+ release from
ryanodine-sensitive stores and amplifying
Ca2+-induced
Ca2+ release. cGMP-induced
Ca2+ release, while clearly
demonstrated in this article (19), occurs in smooth muscle
only after blockade of PKA and PKG, which are otherwise present and
functioning in cells. As the authors speculate, it is possible that
these kinases may not be present at some stage of development, when
cGMP may mediate release of cGMP-dependent Ca2+ stores. It is also possible
that SNP-induced contraction of esophageal longitudinal
muscle, which is cGMP dependent and mediated through an
indomethacin-dependent pathway (10), may in part depend on cGMP-induced
Ca2+ mobilization. This
possibility, however, has not yet been examined and remains
speculative.
It is becoming increasingly clear, however, that multiple and distinct
Ca2+ stores may be present in a
variety of cells and that these stores may be regulated by multiple and
distinct mechanisms. This battery of release mechanisms may provide
versatility of response to diverse signals requiring
Ca2+ mobilization to obtain a
specific result.
ARTICLE
Top
Article
References
NAD+, and nicotinic acid
adenine dinucleotide phosphate (NAADP). These agents differ in their
properties and in the Ca2+ stores
they are capable of discharging.
NAD+ to cADPR via
an ADP-ribosyl cyclase reaction and to catalyze the exchange of the
nicotinamide group of NADP with nicotinic acid to generate NAADP (2).
The ability of cADPR to stimulate Ca2+ release independently of
IP3 was first determined in sea
urchin eggs (5), a well-established and convenient model for studying Ca2+ release by these agents (14).
Subsequently, it was shown that cADPR-induced
Ca2+ release was pharmacologically
similar to Ca2+-induced
Ca2+ release (6), which is thought
to be mediated by ryanodine receptors (18). In smooth muscle cells of
the longitudinal muscle layer, clear evidence for the ability of
endogenous cADPR to induce Ca2+
mobilization was obtained by Kuemmerle and Makhlouf (12) as follows: a contractile agonist (CCK-8) was shown to
activate ADP-ribosyl cyclase and to stimulate the formation of cADPR in
longitudinal smooth muscle in a concentration-dependent manner; cADPR
bound to and selectively activated ryanodine
receptor/Ca2+ channels (13) and
had no effect on IP3
receptor/Ca2+ channels; and cADPR
mobilized Ca2+ and acted further
to enhance Ca2+-induced
Ca2+ release via ryanodine
receptor/Ca2+ channels (12).
![]() |
REFERENCES |
---|
![]() ![]() ![]() |
---|
1.
Aarhus, R.,
D. M. Dickey,
R. M. Graef,
K. R. Gee,
T. F. Walseth,
and
H. C. Lee.
Activation and inactivation of Ca2+ release by NAADP+.
J. Biol. Chem.
271:
8513-8516,
1996
2.
Aarhus, R.,
R. M. Graef,
D. M. Dickey,
T. F. Walseth,
and
H. C. Lee.
ADP-ribosyl cyclase and CD38 catalyze the synthesis of a calcium-mobilizing metabolite from NADP+.
J. Biol. Chem.
270:
30327-30333,
1995
3.
Berridge, M. J.,
and
R. F. Irvine.
Inositol trisphosphate, a novel second messenger in cellular signal transduction.
Nature
312:
317-321,
1984.
4.
Chini, E. N.,
K. W. Beers,
and
T. P. Dousa.
Nicotinate adenine dinucleotide phosphate (NAADP) triggers a specific calcium release system in sea urchin eggs.
J. Biol. Chem.
270:
3216-3223,
1995
5.
Clapper, D. L.,
T. F. Walseth,
P. J. Dargie,
and
H. C. Lee.
Pyridine nucleotide metabolites stimulate calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate.
J. Biol. Chem.
262:
9561-9568,
1987
6.
Galione, A.,
H. C. Lee,
and
W. B. Busa.
Ca2+-induced Ca2+ release in sea urchin homogenates: modulation by cyclic ADP-ribose.
Science
253:
1143-1146,
1991[Medline].
7.
Gosh, T. K.,
J. Bian,
and
D. L. Gill.
Intracellular calcium release mediated by sphingosine derivatives generated in cells.
Science
248:
1653-1656,
1990[Medline].
8.
Graeff, R. M.,
L. Franco,
A. De Flora,
and
H. C. Lee.
Cyclic GMP-dependent and -independent effects on the synthesis of the calcium messengers cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate.
J. Biol. Chem.
273:
118-125,
1998
9.
Graeff, R. M.,
R. J. Podein,
R. Aarhus,
and
H. C. Lee.
Magnesium ions but not ATP inhibit cyclic ADP-ribose-induced calcium release.
Biochem. Biophys. Res. Commun.
206:
786-791,
1995[Medline].
10.
Hirano, I.,
R. Kakkar,
J. K. Saha,
P. T. Szymanski,
and
R. K. Goyal.
Tyrosine phosphorylation in contraction of opossum esophageal longitudinal muscle in response to SNP.
Am. J. Physiol.
273 (Gastrointest. Liver Physiol. 36):
G247-G252,
1997
11.
Hua, S. Y.,
T. Tokimasa,
S. Takasawa,
Y. Furuya,
M. Nohmi,
H. Okamoto,
and
K. Kuba.
Cyclic ADP-ribose modulates Ca2+ release channels for activation by physiological Ca2+ entry in bullfrog sympathetic neurons.
Neuron
12:
1073-1079,
1994[Medline].
12.
Kuemmerle, J. F.,
and
G. M. Makhlouf.
Agonist-stimulated cyclic ADP ribose. Endogenous modulator of Ca2+-induced Ca2+ release in intestinal longitudinal muscle.
J. Biol. Chem.
270:
25488-25494,
1995
13.
Kuemmerle, J. F.,
K. S. Murthy,
and
G. M. Makhlouf.
Agonist-activated, ryanodine-sensitive, IP3-insensitive Ca2+ release channels in longitudinal muscle of intestine.
Am. J. Physiol.
266 (Cell Physiol. 35):
C1421-C1431,
1994
14.
Lee, H. C.
Calcium signaling by cyclic ADP-ribose and NAADP.
Cell Biochem. Biophys.
28:
1-17,
1998.[Medline]
15.
Lee, H. C.,
and
R. Aarhus.
A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cyclic ADP-ribose.
J. Biol. Chem.
270:
2152-2157,
1995
16.
Lee, H. C.,
and
R. Aarhus.
Wide distribution of an enzyme that catalyzes the hydrolysis of cyclic ADP-ribose.
Biochim. Biophys. Acta
1164:
68-74,
1991.
17.
Lee, H. C.,
R. Aarhus,
and
R. Graeff.
Sensitization of calcium-induced calcium release by cyclic ADP-ribose and calmodulin.
J. Biol. Chem.
270:
9060-9066,
1995
18.
Meissner, G.
Rayanodine receptor/Ca2+ release channels and their regulation by endogenous effectors.
Annu. Rev. Physiol.
56:
485-508,
1994[Medline].
19.
Murthy, K. S.,
and
G. M. Makhlouf.
cGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle.
Am. J. Physiol.
274 (Cell Physiol 43):
C1199-C1205,
1998
20.
Rusinko, N.,
and
H. C. Lee.
Widespread occurrence in animal tissues of an enzyme catalyzing the conversion of NAD+ into a cyclic metabolite with intracellular Ca2+ mobilizing activity.
J. Biol. Chem.
264:
11725-11731,
1989
21.
Saha, J. K.,
and
R. K. Goyal.
Biphasic effects of SNP on opossum esophageal longitudinal muscle: involvement of cGMP and eicosanoids.
Am. J. Physiol.
265 (Gastrointest. Liver Physiol. 28):
G403-G407,
1993
22.
Walseth, T. F.,
and
H. C. Lee.
Synthesis and characterization of antagonists of cyclic-ADP-ribose-induced Ca2+ release.
Biochim. Biophys. Acta
1178:
235-242,
1993[Medline].