(Received for publication, September 6, 1994; and in revised form, November 14, 1994)
From the
The biological action of adrenomedullin, a novel hypotensive
peptide, on bovine aortic endothelial cells, was examined. The specific
binding of adrenomedullin to these cells was observed, and
adrenomedullin was found to induce intracellular cAMP accumulation in a
dose-dependent manner. EC for the cAMP accumulation was
about 100 times lower than the apparent IC
for the binding
assay. Adrenomedullin also induced increase of intracellular free
Ca
in endothelial cells in a dose-dependent manner.
The Ca
response to adrenomedullin was biphasic with
an initial transient increase due to the release from
thapsigargin-sensitive intracellular Ca
storage and a
prolonged increase by influx through the ion channel on the plasma
membrane. This intracellular free Ca
increase
resulted from phospholipase C activation and inositol
1,4,5-trisphosphate formation, and seemed to cause nitric oxide
synthase activation by monitoring intracellular cGMP accumulation. Both
cAMP accumulation and Ca
increased responses to
adrenomedullin were mediated by cholera toxin-sensitive G protein, but
the two signal transduction pathways were independent. Thus, the
results suggest that adrenomedullin elicits the hypotensive effect
through at least two mechanisms, a direct action on vascular smooth
muscle cells to increase intracellular cAMP and an action on
endothelial cells to stimulate nitric oxide release, with both leading
to vascular relaxation.
Adrenomedullin is a novel hypotensive peptide isolated from
human pheochromocytoma(1) . It consists of 52 amino acid
residues with one intracellular disulfide bond and is classified into
the calcitonin gene-related peptide (CGRP) ()family. It has
a highly conserved structure among mammals such as porcine (2) and rat (3) . Only a few pharmacological activities
of adrenomedullin have been revealed, such as a potent hypotensive
effect in anesthetized rat and cAMP accumulation in rat platelets and
aortic smooth muscle cells (RASMC)(1, 4) . The
mechanism of the strong hypotensive effect and other physiological
roles are still unclear. Adrenomedullin may act primarily on vascular
endothelial cells because it exists in plasma at comparable levels (5) with atrial natriuretic peptide, another circulating
hypotensive hormone secreted by the heart. To investigate the
physiological role(s) and mechanism of pharmacological effects, we
examined the response of bovine aortic endothelial cells (BAEC) to
adrenomedullin. Here we report the action of adrenomedullin on BAEC
through two independent signal transduction pathways, cAMP accumulation
and Ca
mobilization.
Figure 1:
Competitive binding assay of I-adrenomedullin. Procedures are described in detail
under ``Experimental Procedures.'' Briefly, BAEC were
incubated with 20 pM
I-adrenomedullin in the
presence of 0-5 µM cold adrenomedullin (opencircles) or CGRP (closedcircles) for 2
h at 20 °C. After washing twice with ice-cold Hanks'
solution, cells were lysed and counted for radioactivity of cell
lysates. The values are expressed as means ± S.E. (n = 4).
Figure 2: Response of intracellular cAMP level to adrenomedullin. A, dose response of intracellular cAMP accumulation to adrenomedullin. BAEC (opencircles) or RASMC (closedcircles) were incubated with 0-100 nM adrenomedullin for 15 min and then intracellular cAMP levels were measured by radioimmunoassay.**, significantly different (p < 0.01) from basal level by Dunnett multiple comparison. B, effect of toxins on cAMP response of BAEC to adrenomedullin. After treatment with each toxin (100 ng/ml) for 20 h, BAEC were incubated with (hatchedbars) or without (openbars) 0.1 µM adrenomedullin for 15 min, and then cAMP levels were measured. ##, significantly different (p < 0.01) from each control by Student's multiple range test. C, effect of cAMP-dependent protein kinase inhibition on the cAMP response deteriorated by CTX. After treatment with 10 µM H-89 for 30 min, BAEC were further treated with CTX (2 µg/ml) for 4 h. Then, cells were incubated with (hatchedbars) or without (openbars) 0.1 µM adrenomedullin for 15 min, and cAMP levels were measured. ##, significantly different (p < 0.01) from each control by Student's multiple range test.
Figure 3:
Dose response of
[Ca]
increase
to adrenomedullin. A, a representative series of traces is
shown. AM, adrenomedullin; BK, bradykinin. B, dose-response curve is expressed as means ± S.E. (n = 7) with values relative to the response to 1
µM adrenomedullin in each experiment as 100%.
[Ca
]
increase was
measured with Fura-2-loaded cells suspended in Hanks' solution by
monitoring fluorescence ratio of 340-380 nm (F
/F
).
For further investigation of
the Ca mobilization mechanism of adrenomedullin in
BAEC, we examined the effects of channel blockers and other inhibitory
reagents. First, EGTA, SK& (a receptor operating ion channel
blocker), and nifedipine (an L-type Ca
channel
blocker) did not affect the initial peak for
[Ca
]
increase by
adrenomedullin, but EGTA and SK& caused loss of the prolonged
plateau phase (Fig. 4A), suggesting that this prolonged
plateau phase was due to Ca
influx through the plasma
membrane ion channel (with the exception of the L-type channel). Next,
thapsigargin (3 µM), which is a microsomal
Ca
-ATPase inhibitor and known to inhibit the
bradykinin-induced Ca
mobilization(18) ,
significantly diminished the initial peak of
[Ca
]
increase by
adrenomedullin, while the second prolonged phase was retained (Fig. 4B). This retained prolonged phase was lost when
cells were successively treated with SK&, suggesting again
that this prolonged response was mediated by an ion channel.
Ziegelstein et al.(19) have recently reported the
existence of a ryanodine-sensitive intracellular Ca
storage site in vascular endothelial cells. Ryanodine at 5
µM did not affect the
[Ca
]
increase response to
adrenomedullin as shown in Fig. 4B. When the cells were
pretreated with 10 µM U-73122, an inhibitor of
agonist-induced phospholipase C activation(20) , adrenomedullin
did not induce either the initial or the prolonged
[Ca
]
increase, while
pretreatment with 10 µM U-73343, an inactive analog to
U-73122(20) , did not affect the
[Ca
]
increase response at all (Fig. 4C). Pretreatment of cells with 10 µM H-89 did not affect the [Ca
]
increase response. These results suggest that activation of
phospholipase C is required for an adrenomedullin-induced biphasic
[Ca
]
increase and that the
activation of cAMP-dependent protein kinase is not involved in the
[Ca
]
increase as is often the
case in hormone-secreting cells(21, 22) . In addition,
CGRP at 1 µM did not affect the successive
[Ca
]
response to adrenomedullin (Fig. 4C). This also suggests that the receptor
responsible for the [Ca
]
increase response to adrenomedullin is not the CGRP receptor, as
the adrenomedullin response would have been desensitized by CGRP
pretreatment. For further characterization of the G protein involved in
the [Ca
]
increase response to
adrenomedullin, the effects of PTX and CTX were again examined (Fig. 4D). The adrenomedullin-induced
[Ca
]
increase was not affected
at all by the PTX treatment (100 ng/ml, 20 h) but was greatly
diminished by the CTX treatment (2 µg/ml, 4 h). The addition of
H-89 (10 µM) at 30 min before CTX treatment did not alter
the deterioration of [Ca
]
response, suggesting again that the desensitization of the
adrenomedullin receptor through cAMP-dependent protein kinase
constitutively activated by CTX is unlikely. Thus, the G protein in
BAEC responsible for the [Ca
]
response to adrenomedullin showed similar toxin sensitivity to
that observed in the cAMP response (Fig. 2B).
Figure 4:
Effects of reagents possibly influencing
on [Ca]
increase response to adrenomedullin. A, effects of
EGTA and ion channel blockers. AM, adrenomedullin; Ni, nifedipine. B, effects of thapsigargin (TG) and ryanodine. C, effects of inhibitors and
CGRP. D, effects of pretreatment with toxins (PTX, 100 ng/ml,
20 h; CTX, 2 µg/ml, 4 h). In the experiments with H-89, cells were
preincubated with 10 µM H-89 for 30 min before further
treatment. The traces were obtained from one experiment but are
representative of three experiments.
Figure 5:
Time course of Ins-1,4,5-P
response of BAEC to adrenomedullin. After preincubation with
Hanks' solution, cells were incubated with the same solution in
the presence of 100 nM adrenomedullin, and then intracellular
Ins-1,4,5-P
levels were determined as described under
``Experimental Procedures.'' Values are expressed as means
± S.E. (n = 4).**, significantly different (p < 0.01) from the level at 0 min by Dunnett multiple
comparison.
Figure 6:
Intracellular cGMP increase response of
BAEC to adrenomedullin. After preincubation with or without 100 nMN-nitro-L-arginine methyl ester,
cells were treated with Hanks' solution in the absence (openbar) or presence (hatchedbar) of 100
nM adrenomedullin for 15 min, and then cGMP levels were
measured as described under ``Experimental Procedures.''
Results are expressed as means ± S.E. (n = 4). ##, significantly different (p < 0.01) from each
control by Student's multiple range
test.
Adrenomedullin acts as a hypotensive substance in
anesthetized rat, being as effective as CGRP(1) , although its
hypotensive mechanism has not been clarified. Recently, adrenomedullin
was found to be 10-fold more potent than CGRP for increasing the cAMP
level in RASMC, suggesting that adrenomedullin and CGRP may share the
same receptor (4) . In this report, we investigated the actions
of adrenomedullin on BAEC, because vascular endothelial cells may be
the primary target of the circulating hormones (adrenomedullin is
present in normal plasma at about 17 pg/ml(3) ), and BAEC
showed the most specific binding of I-adrenomedullin
among the several cultured cells examined (data not shown). Results
from the competitive binding assay and
[Ca
]
response indicated the
existence of an adrenomedullin-specific receptor in BAEC. We also found
that adrenomedullin could activate at least two signal transduction
pathways, cAMP accumulation and [Ca
]
mobilization, in BAEC. The cAMP accumulation by adrenomedullin
seemed to be mediated by CTX-sensitive PTX-insensitive G protein,
probably G
, which is involved in many systems of
receptor-operated cAMP increase. As the cAMP response to adrenomedullin
was not completely diminished by CTX treatment (Fig. 2, B and C), it could be possible that the cAMP increase
response to adrenomedullin is mediated in part by other mechanism(s),
such as facilitating the interaction between activated G
and adenyl cyclase as shown in the case of angiotensin II action
in RASMC (28) . The second signal by adrenomedullin,
[Ca
]
mobilization, was also
mediated by CTX-sensitive and PTX-insensitive subtype of G protein. In
relation to this, Gil-Longo et al.(29) have reported
that the [Ca
]
and cGMP increase
in BAEC by bradykinin was mediated by CTX-sensitive G protein and
proposed G
and G
as candidates for
the G protein. Using some inhibitors and blockers, we showed that the
[Ca
]
increase response of BAEC
to adrenomedullin was biphasic. Adrenomedullin activated phospholipase
C through its specific receptor and coupled CTX-sensitive G protein and
accelerated Ins-1,4,5-P
formation to stimulate
Ca
release from the intracellular Ca
storage, endoplasmic reticulum, through thapsigargin-sensitive
Ins-1,4,5-P
receptor on the one hand (the initial rapid
increase phase), and through the ion channel on the plasma membrane to
promote Ca
influx on the other hand (the prolonged
increase phase).
We also showed the possibility that the
[Ca]
increase response of BAEC
to adrenomedullin caused activation of NO synthase and NO release
monitored by cGMP formation. It has been reported that bradykinin
evokes a PTX-insensitive phosphoinositol response to increase
[Ca
]
and cGMP in endothelial
cells(29) . Endothelin 3 also elicited a
[Ca
]
increase response through
the receptor coupled with PTX-sensitive G protein in endothelial cells
causing NO release(30) . All of these peptide hormones were
thought to stimulate Ins-1,4,5-P
formation through
phospholipase C activation. The toxin sensitivity was similar in
[Ca
]
response to adrenomedullin
and to bradykinin but not to endothelin 3, suggesting that at least two
subtypes of G protein could be involved in the
[Ca
]
increase response.
Adrenomedullin was likely to open ion channel(s), because EGTA and
SK& diminished the prolonged phase in the
[Ca
]
response, while the
response was completely diminished by the addition of SK&
after treatment with thapsigargin. The activation of phospholipase C
was also involved in the ion channel opening, because U-73122
completely extinguished the adrenomedullin-induced biphasic
[Ca
]
response. In this respect,
Ins-1,4,5-P
and inositol 1,3,4,5-tetraphosphate are
possible candidates for direct action on the ion
channel(31, 32) . The two signal transduction pathways
activated by adrenomedullin seemed to be independent, because a strong
inhibitor of cAMP-dependent protein kinase, H-89, had no effect on
[Ca
]
response to adrenomedullin
at a concentration that caused great loss of cAMP-dependent hormone
release from GH3 cells(16) . In addition, cAMP-elevating
agents, such as forskolin, dibutylyl-cAMP, or
3-isobutyl-1-methylxanthine, did not increase
[Ca
]
in endothelial cells (data
not shown). It is not clear whether these two adrenomedullin-induced
independent signal transduction pathways are mediated by one receptor
or not. The big difference between the apparent IC
value
in binding assay (Fig. 1) and EC
value in cAMP
increase response (Fig. 2A) suggests the existence of
different receptor subtypes. However, there are several reports of one
receptor being coupled to two independent signal mechanisms, for
example, the prostacyclin receptor (33) and the endothelin
receptor(34) .
Our results suggest that the hypotensive
effect of adrenomedullin could be explained by at least two mechanisms.
First, adrenomedullin can directly act on vascular smooth muscle cells
to cause intracellular cAMP increase leading to relaxation, probably
through inactivation of myosin light chain kinase. Second,
adrenomedullin can act on vascular endothelial cells to cause a
[Ca]
increase leading to the
activation of NO synthase and NO release. The released NO could then
induce the relaxation of smooth muscle cells. In smooth muscle cells,
[Ca
]
increase should lead to
contraction, but we could not detect any
[Ca
]
increase in RASMC by
adrenomedullin even at 1 µM (data not shown), while
intracellular cAMP increased greatly (Fig. 2A).
Therefore, the signal transduction mechanism by adrenomedullin in
smooth muscle cells must differ from that in endothelial cells.
Recently, immunoreative adrenomedullin was detected in the conditioned
medium of cultured endothelial cells(35) , suggesting the
secretion of adrenomedullin from endothelial cells. Therefore,
adrenomedullin may act as a local modulator as well as a circulating
hormone in the cardiovascular system. The physiological role of
intracellular cAMP accumulation in endothelial cells is not clear. This
activity is seen to be important because the activity was 15-fold more
potent than [Ca
]
increasing
activity. Further investigation, for example, by altering the
expression of some genes, should be done to solve this problem.