DDAVP is thought to raise plasma vWF levels by increased exocytosis from WP bodies. This is suggested by the rapid effect of the drug (less than 1 hour) and by the appearance of high-molecular-weight vWF multimers typically released from WP bodies (31
). The vWF is stored in WP bodies together with its propeptide (vWF:AgII) in a 1:1 molar ratio. DDAVP causes a simultaneous equimolar increase in vWF and propeptide, again suggesting exocytosis of the two moieties from WP bodies (32
). The most obvious explanation is a direct effect of vasopressin or DDAVP on ECs. However, several groups have failed to demonstrate vasopressin-induced vWF secretion from cultured HUVECs (13
), a finding confirmed in the present study. Further, several reports have shown that in HUVECs exocytosis from WP bodies is mediated by a rise in [Ca2+
). These findings were hard to reconcile with the features of the vasopressin V2R, which is known to activate cAMP-mediated signaling in principal cells of renal collecting ducts.
The present data demonstrate that vasopressin and DDAVP can directly induce vWF secretion from ECs by activation of V2 receptors. This effect appears to be mediated by a rise in [cAMP]i
. Previous work has shown that a rise in [cAMP]i
is sufficient to induce vWF secretion from WP bodies in HUVECs (13
). We therefore predicted that expression of V2R in HUVECs would be sufficient to reconstitute DDAVP-induced vWF release from these cells. Indeed, in HUVECs transfected with V2R (but not with the truncated, nonfunctional A295 receptor), DDAVP induced a striking increase in vWF secretion. DDAVP was as effective as forskolin, whereas activation of endogenous AC-coupled receptors (e.g., epinephrine and adenosine receptors) had a relatively smaller effect, as observed in our previous studies (13
). This difference is likely explained by the relative overexpression of the transfected V2R receptor. DDAVP-induced vWF secretion was accompanied by an increase in [cAMP]i
and was inhibited by the PKA inhibitor Rp-8CPT-cAMPS. These data indicate that DDAVP-induced vWF secretion from V2R-transfected HUVECs is mediated by cAMP-dependent signaling. These results strongly encouraged us to test the hypothesis that ECs from other vascular beds express V2R in addition to the same postreceptor molecular machinery for cAMP-dependent exocytosis.
In HMVEC-L cells, both vasopressin and DDAVP induced vWF secretion. This secretory response was observed within 30 minutes, suggesting release from preformed stores (i.e., WP bodies) rather than increased synthesis and constitutive release. The DDAVP response was potentiated by IBMX (added to inhibit endogenous phosphodiesterases) and was associated with a small but significant rise in [cAMP]i
. More importantly, the PKA inhibitor Rp-8CPT-cAMPS inhibited vWF secretion in response to DDAVP and forskolin, but not to thrombin. Taken together these results indicate that DDAVP-induced vWF secretion in HMVEC-L cells is mediated by cAMP-dependent signaling. Vasopressin-induced vWF secretion in HMVEC-L cells is due to activation of V2 receptors. This is indicated by the response to DDAVP and by the inhibitory effect of SR121463B, a selective inhibitor of the renal V2 receptors (25
). Finally, V2R expression was identified by RT-PCR in HMVEC-L cells but not in HUVECs, as predicted from the secretion studies. Our results firmly establish that vasopressin can directly induce endothelial vWF secretion and demonstrate — to our knowledge for the first time — that V2R are functionally expressed in vascular cells.
Our study identified the V2R in whole human lung tissue, thereby extending the results of Fay et al. (28
). Indeed, the full-length receptor was amplified from human lung RNA by RT-PCR with a nucleotide sequence identical to the published sequence of the renal V2R. This result supports our conclusion that vasopressin-induced, V2R-mediated vWF secretion in HMVEC-L cells reflects a physiological phenomenon rather than ectopic V2R expression in cultured ECs. We identified V2R expression in HMVEC-L cells but not in HUVECs, an observation that raises the possibility that V2R expression is restricted to specific vascular beds. It is possible that V2R is functionally expressed in additional tissues. A complete study of cell type- and tissue-specific expression of the V2R remains to be performed.
A cAMP-mediated secretion is most likely to be relevant for the physiological regulation of plasma vWF levels. Indeed, epinephrine infusion raises plasma vWF levels, and physical activity increases plasma vWF levels by catecholamines acting on β2-adrenergic, AC-coupled receptors (34
). Most [Ca2+
-raising agonists are mediators of inflammation and/or thrombosis that also induce cell retraction. In contrast, cAMP-raising agonists preserve cell-cell junctions, a property more compatible with the physiological, systemic regulation of vWF secretion (36
). The effects of vasopressin and DDAVP on vWF secretion in HMVEC-L cells were small and were best demonstrated in the presence of IBMX. In contrast, [Ca2+
-raising agents such as thrombin induce a much stronger vWF secretory response both in HMVEC-L (Figure b) and in HUVECs (20
). However, this weak secretory response (as well as the dependence on IBMX) is a general feature of cAMP-raising agents such as adenosine and epinephrine (13
). It is possible that V2R expression is low in HMVEC-L compared with ECs in vivo as a result of dedifferentiation in culture. Another possibility is that constitutive vWF secretion is increased in culture, which would lead to an underestimation of the relative increase in regulated secretion after DDAVP. Increased constitutive release is reported for several types of secretory cells, compared with the parent tissue (e.g., ref. 37
). Further, it is possible that increased levels of phosphodiesterases in cultured cells blunt the response to cAMP-raising agents, accounting for the potentiating effect of IBMX.
Several authors have proposed an indirect mechanism for DDAVP-induced vWF secretion: DDAVP activates an intermediate cell, which in turn secretes a vWF-releasing hormone that acts on ECs (6
). Our data make this hypothesis less attractive, although the existence of two parallel mechanisms cannot be excluded. Hashemi et al. have suggested that platelet-activating factor (PAF) released from macrophages in response to DDAVP induces vWF secretion from ECs (16
). However, these authors have not demonstrated V2R expression in macrophages. The observation that pretreatment with a PAF inhibitor does not suppress DDAVP-induced vWF secretion in dogs also argues against this hypothesis (38
DDAVP raises not only vWF levels, but also tissue-type plasminogen activator (t-PA) and FVIII levels (39
). The t-PA is synthesized, stored, and released from ECs. We and others have reported that t-PA can be stored in WP bodies in cultured HUVECs (41
). A distinct storage compartment (consisting of small, round granules) has also been described (43
). The endothelial storage compartment for t-PA remains to be confirmed in vivo and may vary between tissues. Colocalization of vWF and t-PA in WP bodies would imply that a single regulatory mechanism accounts for the increase in the plasma levels of the two proteins. Even if t-PA is stored in a distinct granule pool, it appears very likely that this pool also responds to V2R activation with cAMP-mediated secretion (22
). Similarly, Rosenberg et al. have shown that heterologous expression of FVIII, transfected into EC, colocalizes with vWF in WP bodies (44
). If this colocalization is confirmed in vivo, DDAVP-induced FVIII increase could also be explained by the same regulatory mechanism.
In addition to its hemostatic effects, DDAVP has vasodilator properties that are unlikely to result from renal V2R activation (9
). Our finding that V2R is functionally expressed in ECs raises the possibility that DDAVP induces the synthesis of endothelium-dependent vasodilators. One candidate is prostacyclin. However, we have shown that prostacyclin production is inhibited by cAMP (21
). DDAVP could induce endothelial nitric oxide (NO) production, but a link between activation of cAMP-dependent signaling pathways and NO production remains to be established.
In summary, we have shown that vasopressin and its analogue DDAVP induce vWF secretion from cultured ECs. This effect is due to V2R activation and is mediated by cAMP-dependent signaling. V2R expression was shown in cultured lung ECs and in whole lungs, providing evidence for the functional extrarenal expression of this receptor. These observations provide a cellular mechanism for the hemostatic effect of DDAVP.