Endothelial cells synthesize prostacyclin in response to a wide variety of vasoactive stimuli. The transduction pathway is dependent on elevation of intracellular.
ent. It has been suggested (15) that calponin phosphorylated by either protein kinase C or CaM-kinase II does not bind to actin and therefore is noninhibitory. However, this has not been demonstrated in intact tissue and must be regarded as speculative. In summary, many mechanisms have been suggested to play a regulatory role in SM. The only mechanism with extensive experimental support is the phosphorylation theory. Of the other mechanisms, possibly more than one is operative. It is reasonable that the Ca2+ dependence of the kinase-phosphatase couple is altered under some conditions, and this may be subject to regulation via a G protein-linked mechanism. Also, the thin-filament proteins caldesmon and calponin may be involved in some aspect of regulation, although this is not identified. Obviously, considerably more research is required before an integrated pattern of SM function is obtained. We thank Dr. R. A. Murphy (University of Virginia) and Dr. J. T. Stull [University of Texas) for their advice (not always heeded) on preparation of this article. D. Hartshorne is supported by National Heart, Lung, and Blood Institute Grants HL23615 and HL-20984.
References 1. Adelstein, R. S., and M. A. Conti. Phosphorylation of platelet myosin increases actin-activated myosin ATPase activity. Nature Lond. 256: 597-598, 1975. 2. Dillon, P. F., M. 0. Aksoy, S. P. Driska, and R. A. Murphy. Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science Wash. DC 211: 495-497, 1981. 3. Driska, S. P. High myosin light chain phosphatase activity in arterial smooth muscle: can it explain the latch phenomenon? Prog. Clin. Biol. Bes. 245: 387-398, 1987. 4. Edelman, A. M., W.-H. Lin, D. J. Osterhout, M. K. Bennett, M. B. Kennedy, and E. G. Krebs. Phosphorylation of smooth muscle myosin by type II Ca’+/calmodulin-dependent protein kinase. Mol. Cell. Biochem. 97: 87-98, 1990. 5. Hai, C. M., and R. A. Murphy. Crossbridge phosphorylation and regulation of the latch state in smooth muscle. Am. J. Physiol. 255 (Cell Physiol. 24): C86-C94, 1988. 6. Hartshorne, D. J. Biochemistry of the contractile process in smooth muscle. In: Physiology of the Gastrointestinal Tract
64
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7.
8.
9.
10.
(2nd ed.), edited by L. R. Johnson. New York: Raven, 1987, p. 423-482. Ikebe, M., and D. J. Hartshorne. Proteolysis and actin-binding properties of 10s and 6s smooth muscle myosin: identification of a site protected from proteolysis in the 10s conformation and by the binding of actin. Biochemistry 25: 6177-6185, 1986. Morgan, K. G. The role of calcium in the control of vascular tone as assessed by the Ca2+ indicator aequorin. Cardiovasc. DrugsTher. 4:1355-1362,199O. Olson, N. J., R. B. Pearson, D. S. Needleman, M. Y. Hurwitz, B. E. Kemp, and A. R. Means. Regulatory and structural motifs of chicken gizzard myosin light chain kinase. Proc. Nat]. Acad. Sci. USA 87: 2284-2288,199O. Sobue, K., K. Kanda, T. Tanaka, and N. Ueki. Caldesmon: a common actin-linked regulatory protein in the smooth muscle
11.
12.
13.
14.
15.
and nonmuscle contractile system. J. Cell. Biochem. 37:317-325,1988. Somlyo, A. P., and A. V. Somlyo. Smooth muscle in health and disease. Sci. Am. In press. Stull, J. T., P. J. Gallagher, B. P. Herring, and K. E. Kamm. Vascular smooth muscle contractile elements: cellular regulation. Hypertension Dallas. 17: 723-732, 1991. Stull, J. T., L.-C. Hsu, M. G. Tansey, and K. E. Kamm. Myosin light chain kinase phosphorylation in tracheal smooth muscle. J. Biol. Chem. 265: 16683-16690,199O. Takahashi, K., K. Hiwada, and T. Kokubu. Vascular smooth muscle calponin. A novel troponin T-like protein. Hypertension Dallas 11: 620-626, 1988. Winder, S. J., and M. P. Walsh. Smooth muscle calponin. Inhibition of actomyosin Mg ATPase and regulation by phosphorylation. J. Biol. Chem. 265: 10148-10155, 1990.
Regulation of Prostacyclin Synthesis in Endothelial Cells Thomas D. Carter and Jeremy D. Pearson Endothelial cells synthesize prostacyclin in response to a wide variety of vasoactive stimuli. The transduction pathway is dependent on elevation of intracellular Ca2+ and specifically desensitizes cells to the stimulus used, thus providing tight temporal regulation of the release of this potent vasodilator and inhibitor of platelet aggregation. Introduction Prostacyclin (PG12)is a labile prostanoid released from endothelium in response to a wide variety of hormonal, chemical, immunological, and physical stimuli. It is the most potent inhibitor of platelet aggregation yet discovered and is a powerful dilator of most, but not all, blood vessels. Both of these actions are mediated via specific receptors for PG12, linked to the stimulation of adenylate cyclase. In addition to its role in the local regulation of vascular tone and haemostasis, PGI, also has efT. D. Carter is in the Div. of Neurophysiology and Neuropharmacology at the National Institute for Medical Research, Mill Hill, London NW7 ZAA, UK. J. D. Pearson is Professor of Vascular Biology, Biomedical Sciences Division, King’s College, Kensington, London WB 7AHJK. 0886-17
fects on cholesterol metabolism and mitogenesis in vascular smooth muscle cells. PGIz is therefore an important local regulator of vascular homeostasis (10). Basal PGIz release in vivo is sufficiently low to be virtually undetectable. However, in response to infusion of an agonist or after experimentally induced vessel injury, PGIz is briefly present in biologically active concentrations. Particularly from experiments with endothelial cells cultured in vitro, several classes of agonist have been demonstrated to interact with surface receptors, leading to PGIz synthesis. These include proteins and peptides (thrombin and bradykinin at B2-receptors), amines (notably histamine at H,-receptors), eicosanoids (leukotriene C,), and purines (ATP and ADP at P,,-purinoceptors). 14/92 $2.00 0 1992 Int. Union
Physiol.
Sci./Am. Physiol.
Sot.
PGIz production in response to such agonists is characteristically rapid in onset (C60 s) and transient, being complete within ~5 min, even in the continued presence of the agonist. In addition to these chemical mediators, alterations of flow and shear forces produce transient PGI, production. PGI, synthesis is thus normally tightly temporally regulated. Intracellular Ca2+ and PGI, production
PGI, is synthesized from arachidonic acid via cyclic endoperoxide intermediates by the sequential action of the enzymes cyclooxygenase and PGI, synthase. Control of PGIz synthesis must be exerted at or before the level of the release of arachidonic acid from membrane phospholipids, since PG12 synthesis in response to exogenous arachidonate is not transient but can continue for many minutes or even hours. Earlier studies with cells in which phospholipids were radiolabeled had shown that the majority of the free fatty acid was liberated by the action of phospholipase AZ, so more recent research has focused on the manner in which receptor occupation is coupled to activation of phospholipase A,. A prime candidate as an intracellular messenger is cytoplasmic free Ca 2+ since Ca2+ ionophores cause PGI, release and phospholipase A2 is Ca2+ sensitive. For all of the receptor-mediated stimuli so far examined, studies of endothelial cells loaded with Ca2+-sensitive fluorescent dyes have demonstrated that activation leads to elevation of the concentration of Ca2+ [Ca2+]i. A typical time course illustrating the changes in [Ca2+]i and PGI, production found in a population of endothelial cells is shown in Fig. 1A. [Ca2+]i rises from a resting value of ~0.1 PM to a peak within lo-15 s and then declines to an elevated steady-state level that is maintained for many minutes. The peak and steady-state levels depend on the dose of agonist used; maximally, they are 2-4 and 0.4-0.8 PM, respectively. PGI, release is detected -30 s after addition of agonist and has stopped within 4 min. In the absence of
A
1mM
extracellular
Thrombin 5.01
BlmM
Ca2+
(0.5
EGTA
Thrombin
U/ml)
(0.5
No
extracellular
Ca2+
U/ml)
I
h =E
,'
1.0
11
&
0.5
a u -
0.1
u1 2
4
5
6
10
Time(min) FIGURE 1. Time course of changes in cytoplasmic free calcium ((Ca”‘)J and prostacyclin (PG12) release in endothelial cells. In these experiments, monolayers of human umbilical vein endothelial cells on glass cover slips were loaded with Ca2+ -sensitive dye fura- and placed diagonally in fluorimeter cuvette. Release of PG12 was monitored by radioimmunoassay in bathing medium as cumulative level of its stable hydrolysis product &oxo-prostaglandin F1, (PGF& (Ca2+)i traces are individual examples; 6-oxo-PGI la values are means from 4 experiments EGTA, ethylene glycol-bis(P-aminoethyl ether)-NJV,N’,N’-tetraacetic acid. [Redrawn from Hallam et al. (6))
extracellular Ca2+ (Fig. 1B) the initial peak [Ca2+]i response is little changed, although [Ca2+]i then returns rapidly to the prestimulated level. PG12 release is similar, perhaps ceasing slightly earlier, to that found when extracellular Ca2+ is present. This kind of experiment shows that the initial peak [Ca’+]i is due, at least predominantly, to the mobilization of Ca2+ from membranebound internal stores. This result is consistent with biochemical studies, which have shown that vasoactive hormones inducing PGI, synthesis activate phospholipase C to generate inositol 1,4,5-trisphosphate (IP,), widely accepted as the mediator causing release of Ca2+ from internal stores (1). The coupling of hormone ret eptors to phospholipase C is thought to involve a G protein (G,) because, in bovine endothelial cells, IP, generation is sensitive to pertussis toxin, and in human endothelial cells the stable GTP analogue guanosine V-O-(S-thiotriphosphate) induces IP, production (2). The requirement for extracellular Ca2+ to sustain the steady-state level of [Ca2+]i suggests that receptor OCcupation also leads to Ca2+ influx. This is strengthened by showing that adding divalent cations, such as Ni2+, which block Ca2+ entry, eliminates the steady-state elevation, whereas others, such as Mn’+, are
translocated in response to agonist, as shown directly by the quenching of fluorescence of the Ca2+-sensitive dye (7). The mechanism and consequences of Ca2+ influx in endothelium are currently under investigation, although it is clear from results like those in Fig. 1 that this phase of the Ca2+ response is not related to PGI, production. Elevation of [Ca2+]i is necessary and sufficient to cause PGI, release
Although the experiments described above demonstrate an appropriate temporal relationship between the initial rise in [Ca2+]i and PG12 release, they do not prove that they are causally related, nor do they indicate whether elevation of [Ca2+]i above a threshold concentration is required. Two types of experiments have been designed to address these points. In the first, endothelial cells were depleted of internal stores of Ca2+, by treatment with ionophore or an agonist in the absence of extracellular Ca2+, and then challenged with a second agonist (3, 6). Under these conditions, [Ca2+]i fails to rise, and no PG12 is formed, although IP, production can be detected. Thus an elevation of [Ca2+]i is indeed necessary for PG12 production. In the second type of experiment, Volume
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15
B
creased Ca2+ sensitivity of a phospholipase. Although these results demonstrate a novel potential regulatory role of PKC in PGI, synthesis, it is not clear from these experiments whether the effects of phorbol ester are due to a more prolonged activa-
l 0 /I
l
/
10
tion of PKC than occurs when en-
dogenous DAG is liberated sponse to a natural agonist. 5
in re-
Role of cyclic nucleotide-
dependent Many
protein
kinases
vasoactive stimuli
that in-
duce PGI, release alter the levels of adenosine 3’,5’-cyclic monophosphate (CAMP) and/or guanosine
0 I 0.1 [Ca++]i FIGURE 2. Dose-response ((Ca”‘),)
and
prostacyclin
relationship
between
(PG12) release
I I 0.2 0.3
I I 0.8 1
I 0.5
I 2
1 3
phosphorylation
p” peak
in endothelial
cytoplasmic
cells.
free calcium
Experiments
were
carried
peak
cellular neither
out in
be involved
absence of extracellular Ca2’. (Ca2+)i values are initial peak values; PG4 release was measured as total accumulated 5 min after addition of stimulus. A: identical curves are found when
(Ca”‘),
is raised
by
varying
doses
of ionomycin
(O),
ATP
(I),
or the
more
potent
analogue 2-chloro-ATP (A), B: increased sensitivity of PG12 synthesis to (Ca2+)i in cells preincubated 5 min with 10 nM phorbol 12-myristate 13-acetate, 0, Control untreated cells; 0, treated cells. [Redrawn from Carter et al. (3, 4) with permission.]
again
performed
of extracellular
in
the
absence
Ca2+, the ability
of
ionomycin and agonists to elevate [Ca2+]i and cause PGI, release were compared (3, 6). The addition of ion-
3’,5’-cyclic monophosphate (cGMP) in endothelial cells and cause the
periments
described
that activation
above suggest
of PKC by DAG does
not play a significant ing PGI, release. Nonetheless,
role in trigger-
brief
pretreatment
of a variety of intra-
proteins (2). Despite this, CAMP nor cGMP appears to in mediating the effects
of these stimuli on PG12 release, nor do they seem to contribute to the short-term regulation of PGI, release by endothelium. However, the possibility that cyclic nucleotide-independent
protein
kinases are in-
volved in the regulation of PGI, release should not be ignored. How does Ca2+ trigger PGI, production?
omycin in doses from 1 nM to 1 PM causes dose-related elevations in
with phorbol esters (exogenous activators of PKC, which under these
The liberation of arachidonate from membrane phospholipids is the
peak [Ca2+]i, but significant PGI, pro-
conditions
rate-limiting
duction is found only after [Ca2+]i reaches -0.8 PM (Fig. ZA).
lease) substantially nist-induced PGI,
potentiates production
Closer examination
reveals that this
lipids were radiolabeled had shown
is accompanied by an inof agonist-induced phos-
that the majority of free fatty acid was liberated by the action of phos-
Addition
of increasing concentra-
do not induce PGI, re-
tions of agonist produces an indistinguishable relationship between peak
treatment hibition
[Cazf]i
phoinositide
and PGIz release (Fig. ZA).
turnover,
ago(4).
IP3 produc-
step in the production
of PGI,. Although earlier studies with cells in which the phospho-
pholipase A2 on phosphatidylcholine
These results demonstrate that the elevations in [Ca2+]i caused by ad-
tion, and Ca2+ release, which appears paradoxical in view of the
and phosphatidylethanolamine, now clear that several other
dition of agonist are sufficient to ac-
results described above, indicating
ways for arachidonate release exist.
count for the amount duced.
that PGI, synthesis creases in [Ca2+]i.
First, it can be formed by the hydrolysis of DAG produced after
of PGI, pro-
The inhibition Role of protein
in regulating
kinase C PGI, release
is driven
by in-
of mobilization
of
receptor activation
it is path-
of phospholipase
Ca2+ is, however, more than compensated for by a leftward shift in
C. As noted above, Ca2+ ionophore does not activate phosphoinositide
the Ca2+ activation
turnover
dose-response
in endothelium
but yields
Calcium-mobilizing agonists are also potentially capable of activating
curve for PGI, release (Fig. 2B). Thus the threshold elevation of [Ca’+]i re-
a Ca2+-activation curve for PGI, release identical to that given by an
protein
quired to drive PG12 is substantially
agonist. Thus the generation of ar-
reduced. This second independent effect of PKC must occur at or before
achidonate from DAG is unlikely be an important pathway.
the release of arachidonate, since the conversion of exogenous arachidonate to PGI, is unaffected, which suggests that it is due to the in-
Second, it has been shown that, in endothelium, arachidonate can be released after agonist activation of a phosphatidylcholine-specific phos-
kinase C (PKC), because
phosphoinositide diacylglycerol
hydrolysis yields (DAG), an endoge-
nous activator of PKC, as well as IP, (1). However, since Ca2+ ionophore does not stimulate phosphoinositide turnover in endothelial cells, the ex66
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1992
to
pholipase C (PC-PLC) and/or the combined action of phosphatidylcholine-specific phospholipase D (PC-PLD) and phosphatidic acid phosphohydrolase (9, 11). However, PC-PLC appears to be relatively insensitive to Ca”+, whereas inhibition of PC-PLD does not reduce agonistevoked PGI, release. Third, bradykinin has been shown to release arachidonate from phosphatidylinositol and phosphatidylethanolamine in endothelium without the involvement of phospholipase C, perhaps by direct coupling of the receptor to phospholipase A, via a G, (8). Because this pathway is likely to be Ca2+ independent, it is unclear what role it plays in providing arachidonate for hormone-stimulated PG12 synthesis. In contrast to the pathways described above, phospholipase A, is well characterized as a Ca2+-activated enzyme. The current model for PG12 synthesis in response to a single challenge with an agent that binds to receptors at the endothelial surface thus involves coupling via phospholipase C, release of Ca2+ from intracellular stores leading to a transient increase in [Ca2+]i above the threshold necessary to activate phospholipase A2, and hence to transient PGI, synthesis and release. The mechanism by which transient PGI, release occurs in response to changes in physical forces is not understood but may be one consequence of the opening of shear- or stretch-activated ion channels in endothelium. This model, however, does not explain several other features of PGI, release. One of these is the ability of phorbol esters that activate protein kinase C to induce sustained PGI, release, after a lag period of several minutes, in the absence of elevated [Ca2+]i (4). The long-term enhancement of “basal” and agonist-stimulated PGI, release, which occurs over several hours in response to phorbol esters or cytokines such as interleukin-1, involves upregulation of the synthesis of cyclooxygenase and/or PGI, synthase (13). Desensitization
of PG12 production
An important feature of PGI, release is the induction of tachyphylaxis: cells respond transiently in the
4
50/t M r’
A
R
ATP
a
Bradykinin 100 nM rJ
4 100 nM 5OpM
10 nM I
\ I 0
a
BK 10 nM
BK 1OOnM 5
I
I
I
I
I
0
10
20
30
0
I 10
I 20
I 30
Time (mid 3. Desensitization of agonist-stimulated prostacyclin (PG12) release in endothelial cells. PGIz release was measured in superfused columns of human umbilical vein endothelial cells grown on microcarrier beads. A and B: PG12 release to 2-min superfusion of ATP or bradykinin (BK) is dramatically reduced if cells were previously exposed for 20 min to lower dose of agonist. C and D: control response to ATP or bradykinin (0) is unaffected by 20-min pretreatment with other agonist (0). PGF1,,, prostaglandin F1,. [From Toothill et al. (12)) FIGURE
continued presence of an agonist, and the response to a second challenge shortly after removal of the first is dramatically reduced (Fig. 3). This is not due, as originally supposed, to inactivation of PGI, synthase or cyclooxygenase, because it is agonist specific (Fig. 3) and takes place even if PG synthesis is blocked during the first challenge with the agonist (12). The competence of a second agonist to induce the expected PG12 release in the continued presence of the first also shows that the internal store of bound Ca2+ is rapidly refilled upon depletion, regardless of the
presence of the first agonist; several experiments, including those described above which prove the need for elevated [Ca2+]i for PG12 synthesis, indicate that there is a single store accessible to all agonists. In endothelium, as in many other cells, tachyphylaxis is associated with reduced ability of the second exposure to agonist to cause phosphoinositide turnover and release of Ca2+ from internal stores. Thus reduced PG12 synthesis in desensitized cells is likely to be a consequence of an inability to raise Ca2+ sufficiently to activate phospholipase A2. To test this hypothesis, cells were Volume
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desensitized by incubating with an agonist for a short time before its removal. Cells were then reexposed to the same agonist at an interval of 2-20 min after the end of the initial exposure, and their ability to raise [Ca2+]i and release PGI, (measured concomitantly) was assessed (5). Two minutes after the initial exposure, the cells produce a dramatically reduced Ca2+ response to the agonist. This response recovers to control levels after a ZO-min interval between challenges. PGI, release is also markedly reduced and only begins to recover 10-15 min after the initial exposure of agonist. Again, a control response is obtained 20 min after the initial exposure. The delay in the recovery of PGI, release corresponds closely to the time taken for the Ca2+ response to recover above the activation threshold of 0.8 PM. These results show that the inability of the cells to raise Ca”+ above the activation threshold accounts for the desensitization of PG12 release. Because there was no divergence between the ability of these cells to raise [Ca2+]i and produce PG12, unlike what happens when cells are pretreated with PKC activators, these results also suggest that PKC-mediated mechanisms are not operating to desensitize PGI, release, Further experiments showed directly that inhibition of PKC, under conditions where the effects of phorbol esters are reversed, does not prevent agonist-induced desensitization (5). Desensitization of PGI, synthesis thus results either from a rapid downregulation of receptor number or affinity or from uncoupling of the receptor and phospholipase C, resulting in reduced production of IP,. As noted above, this coupling is likely to involve a GTP-binding protein Whether this represents the site of modification during desensitization in these cells is not yet known, but, whatever the target, agonist-induced desensitization does not involve PKC-mediated feedback inhibition. Conclusions
The research reviewed here has focused on the cellular signaling pathways controlling PGI, synthesis (summarized in Fig. 4), where con68
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1992
FIGURE 4. Summary of transduction events linking vasoactive hormone receptors to prostacyclin (PG12) release in endothelial cells. Elevation of intracellular Ca2+, predominantly due to release of Ca2+ from internal stores, is major transduction signal leading to activation of phospholipase A2 (PLA2) and PG12 release. Although Ca2+ influx, by a mechanism not understood, is a consequence of receptor activation, this process does not contribute significantly to PG12synthesis. Similarly, although independent activation of protein kinase C (PKC) can enhance agonist-induced PG12 release and inhibit intracellular Ca2+ mobilization, there is evidence that PKC activation is not involved in either initial release of PG12 in response to an agonist or subsequent agonistspecific desensitization that takes place. A, agonist; R, receptor; G,, GTP-binding protein; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; 20:4, arachidonic acid; PLC, phospholipase C.
nin or ATP (which induce profound tachyphylaxis of PGI, release) is more long lasting and not easily desensitized. Release of NO in response to a single challenge with an agonist is, unlike PGI, release, highly dependent on extracellular Ca2+. Treatment with phorbol esters, in contrast to the stimulation it produces with PGI, release, blocks NO release. Because phorbol esters efficiently block the sustained elevation of [Ca2+]i that requires Ca2+ influx, these results taken together suggest, first, that this phase of the agonistinduced response may be important for the more prolonged release of NO and, second, that stimulated Ca2+ influx may not be subject to tachyphylaxis (2, 4, 5). Both of these contentions require further testing. A major challenge for vascular physiologists in the next few years will be to provide an integrated picture of the transduction pathways used by the endothelial cell to achieve its versatile control of vessel functions in response to external stimuli. References
siderable progress has recently been made. Endothelial cells are, however, important regulators of all aspects of vascular homeostasis, due to the production of a wide variety of mediators, ranging from labile compounds of low molecular mass such as PGI, and nitric oxide (NO, endothelium-derived relaxing factor) to high-molecular-weight secreted proteins such as von Willebrand factor and tissue plasminogen activator or cell surface proteins such as thrombomodulin and specific leukocyte adhesion molecules. The synthesis or secretion of these agents is induced or repressed by a variety of external stimuli, acting on surface receptors that give overlapping but distinct patterns of cellular response. For example, the fact that several agonists concomitantly induce the synthesis and secretion of both PGI, and NO, another potent dilator and inhibitor of platelet function, has led to suggestions that the release of these mediators is coupled. There are, however, several salient differences. Release of NO in response to agonists such as bradyki-
Berridge, M. J., and R. F. Irvine.
.
Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature Land. 312: 315-321,1984. Boeynaems, J.-M., and J. D. Pearson. P2 purinoceptors on vascular endothelial cells: physiological significance and transduction mechanisms. Trends Pharmacol. Sci. 11: 34-37, 1990. Carter, T. D., T. J. Hallam, N. J. Cusack, and J. D. Pearson. Regulation of P2,-purinoceptor-mediated prostacyclin release from human endothelial cells by cytoplasmic calcium concentration. Br. J. Pharmacol. 95: 429-436,1988. Carter, T. D., T. J. Hallam, and J. D. Pearson. Protein kinase C activation alters the sensitivity of agonist-stimulated endothelial cell prostacyclin production to intracellular ionised calcium. Biochem. J. 262: 431-437,1989. Carter, T. D., J. Newton, R. Jacob, and J. D. Pearson. Homologous desensitization of ATP-mediated elevations in cytoplasmic calcium and prostacyclin release in human endothelial cells does not involve protein kinase C. Biochem. J. 272: 217-221,199O. Hallam, T. J., J. D. Pearson, and L. Needham. Thrombin-stimulated elevation of endothelial cell cytoplasmic free calcium concentration causes prostacyclin production. Biochem. J. 257: 243-249, 1988.
7. Jacob, R. Agonist-stimulated divalent cation entry into single cultured human umbilical vein endothelial cells. J. Physiol. Lond. 421: 55-57, 1990. 8. Kaya, H., G. M. Patton, and S. L. Hong. Bradykinin-induced activation of phospholipase A, is independent of the activation of polyphosphoinositide-hydrolysing phospholipase C. J. Biol. Chem. 264:
10.
4972-4977,1989.
9. Martin, T. W., D. R. Feldman, K. E. Goldstein, and J. R. Wagner. Long-term phorbol ester treatment dissociates phospholipase D activation from phosphoinositide hydrolysis and prostacyclin synthesis in
11.
endothelial cells stimulated with bradykinin and ATP. Biochem. Biophys. Res. Commun. 165: 319-326, 1989. Moncada, S., R. M. J. Palmer, and E. A. Higgs. Prostacyclin and endothelium-derived relaxing factor: biological interactions and significance. In: Thrombosis and Haemostasis, edited by M. Verstraete, H. R. Lijnen, and J. Arnout. Leuven, Belgium: University Press, 1987, p. 597-618. Ragab-Thomas, J. M.-F., F. Hullin, H. Chap, and L. Douste-Blazy. Pathways of arachidonic acid liberation in thrombin and calcium ionophore A23187-stimulated human endothelial cells: respective
Interactions in the InsulinLike Growth Factor Signaling System Charles T. Roberts, Jr., and Derek LeRoith The insulin-like growth factors, IGF-I and IGF-II, are mitogenic peptides structurally related to insulin, which have widespread effects on growth and differentiation during development. These effects are mediated via activation of specific cell-surface receptors, and this activation is modulated by several species of IGF-binding proteins. The insulin-like growth factors (IGFI and IGF-II) are widely distributed mediators of growth, development, and differentiation (reviewed in Refs. 4 and 8). As their name implies, these peptides are related to insulin, consisting of B and A domains analogous to the B and A chains of insulin, but in the case of the IGFs these are joined in the mature molecule by a short C domain that is not cleaved during posttranslational processing as is the C-peptide of insulin (Fig. 1). The B and A domains of the IGFs exhibit >60% amino acid similarity to each other and -40% amino acid similarity to the B and A chains of insulin. Both IGFs contain short COOH-terminal D domains for
C. T. Roberts, Jr., and D. LeRoith are in the Section on Molecular and Cellular Physiology, Diabetes Branch, National Institutes of Health, Bldg. 1O/Room 8 s243, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892, USA.
which no counterpart exists in the insulin molecule. The IGFs are synthesized as preprohormones, with signal peptides that are cleaved after directing the nascent peptides into the lumen of the endoplasmic reticulum. The IGF precursors contain, in addition to the B, C, A, and D domains of the mature peptide, COOH-terminal E domains (Fig. 1). This domain is cleaved at some point during transit of pro-IGFs through the constitutive secretory pathway. These E-peptides are presumably secreted with mature IGF-I and IGF-II, but their role, if any, remains obscure. Although insulin production is primarily restricted to pancreatic ,8cells, the IGFs are synthesized by multiple tissues, albeit at somewhat different stages of development. Studies in both humans and animals have shown that IGF-II synthesis is particularly widespread early in development. In humans circulating levels of IGF-II are maintained
roles of phospholipid and triacylglycerol and the evidence for diacylglycerol generation from phosphatidylcholine. Biochim. Biophys. Acta 917: 388-397, 1987. 12. Toothill, V. J., L. Needham, J. L. Gordon, and J. D. Pearson. Desensitization of agonist-stimulated prostacyclin release in human umbilical vein endothelial cells. Eur. J. Pharmacol. 157: 189-196,1988. 13. Wu, K. K., H. Hatzakis, S. S. Lo, D. C. Seong, S. K. Sanduja, and H. H. Tai. Stimulation of de novo synthesis of prostaglandin G/H synthase in human endothelial cells by phorbol ester. J. Biol. Chem. 263: 19043-19047,1988.
through adulthood, whereas in rodents postnatal IGF-II production is limited to a small number of sources, including the choroid plexus and the leptomeninges. IGF-I production, while also widespread, tends to increase postnatally in several tissues, especially the liver, although prenatal expression is undoubtedly important, particularly in brain development. The almost ubiquitous presence of the IGFs throughout the body suggests that many of the actions of the IGFs may be mediated through both autocrine and paracrine modes of action in addition to their more classicial endocrine mechanisms. That is to say, either IGF-I or IGF-II may exert effects on the same cells in which they were produced or on adjacent cells, without entering the circulatory or lymphatic systems. In vitro, both IGF-I and IGF-II exhibit growth factor-like mitogenic effects, resulting in proliferation, differentiation, and maintenance of differentiated function in different cells in culture. In a number of cases the IGFs have been shown to have metabolic (insulin-like) effects. Because the majority of these data has come from studies in cultured cells, the relevance of these actions to whole body physiology remains to be established. In vivo, a primary role of IGF-I is to mediate the growth-promoting effects of growth hormone (GH), particularly during the pubertal growth spurt. This involves the GH-induced synthesis and release of IGF-I into the circulation by the liver. That the synthesis of IGF-I by numerous exVolume
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1992
NIPS
69
