Murine embryonic stem cell in vitro differentiation: applications to the study of vascular development.

The present review summarizes knowledge accumulated during the last decade concerning in vitro endothelial differentiation from embryonic stem (ES) cells. There is now growing evidence that ES cells may provide a powerful model system to determine the cellular and molecular mechanisms of vascular development. ES cells differentiate into the endothelial lineage by successive maturation steps recapitulating in vivo events observed in the embryo. Further maturation of ES-derived embryoid bodies either in three dimensional gels or in confrontation cultures with tumor spheroids can also provide a model of physiological or tumoral angiogenesis. The data obtained from experimental in vitro differentiation of genetically modified mouse ES cells highlight the potential and the complementarity of this model system to in vivo gene knock out studies. We also consider and discuss some of the potential applications of ES cell technology in vascular biology for future directions in basic research and medicine, by manipulation of differentiation and the generation of cell populations for analysis and transplantation for therapeutic use.

Embryonic stem cell-derived embryoid bodies development in collagen gels recapitulates sprouting angiogenesis.

The formation of new blood vessels proceeds by both vasculogenesis and angiogenesis. The development of models, which fully recapitulate spatio-temporal events involved during these processes, are crucial to fully understand their mechanisms of regulation. In vitro differentiation of murine embryonic stem (ES) cells has been shown to be a useful tool to investigate factors and genes potentially involved in vasculogenesis (Hirashima et al, 1999; Risau et al, 1988; Vittet et al, 1996; Wang et al, 1992; Wartenberg et al, 1998). We asked here whether this model system can also recapitulate angiogenesis, which may offer new means to study mechanisms involved in this process. ES-derived embryoid bodies (EBs) obtained after 11 days of differentiation, in which a primitive vascular network had formed, were then subcultured into a type I collagen matrix. In the presence of angiogenic growth factors, EBs rapidly developed branching pseudopods. Whole mount immunostainings with a PECAM antibody revealed that more than 75% EBs displayed, within a few days, a large number of endothelial outgrowths that can give tube-like structures with concomitant differentiation of alpha-smooth muscle actin positive cells, thus evoking sprouting angiogenesis. High expression levels of flk1 (VEGFR2), flt1 (VEGFR1), tie-1, and tie-2 are also found, indicating that budding endothelial cells displayed an angiogenic phenotype. The endothelial sprouting response was specifically induced by angiogenic factors with a major contribution of vascular endothelial growth factor (VEGF). Known angiostatic agents, such as platelet factor 4 (PF4), angiostatin, and endostatin inhibited the formation of endothelial sprouts induced by angiogenic factors. Moreover, consistent with the in vivo phenotype, VE-cadherin deficient EBs failed to develop angiogenesis in this model. ES cell differentiation can then recapitulate, in addition to vasculogenesis, the early stages of sprouting angiogenesis. This model system, in which genetic modifications can be easily introduced, may be of particular interest to investigate unsolved questions and molecular mechanisms involved in blood vessel formation.

ACTH-regulated expression of vascular endothelial growth factor in the adult bovine adrenal cortex: a possible role in the maintenance of the microvasculature.

Endothelial cells lining vessels of endocrine tissues are fenestrated. Interactions with the local environment via either soluble factors or cell-cell interactions appear to govern this terminal endothelial differentiation. Adrenocorticotropin (ACTH) has previously been reported to modulate endothelial fenestration in the rat adrenal cortex. Since vascular endothelial growth factor (VEGF) has been characterized as a potent inducer of endothelial fenestration, we aimed to characterize the status of VEGF expression in the bovine adult adrenal cortex and asked whether ACTH may regulate VEGF expression. By immunohistochemical analysis, we observed VEGF expression in steroidogenic cells from both zona glomerulosa and zona fasciculata of the bovine adrenal cortex. Double-labeling experiments performed on isolated cells in primary culture revealed VEGF immunoreactivity, essentially colocalized with the Golgi apparatus. The expression of two predominant VEGF isoforms, VEGF(121) and VEGF(165), was observed by RT-PCR analysis. ACTH (10 nM) was found to rapidly (within 2-4 h) increase the abundance of these VEGF transcripts, as assessed by both RT-PCR and Northern blot analysis. In parallel, ACTH significantly induced VEGF secretion into the medium of fasciculata cells in primary culture. Thus, our data are consistent with the involvement of ACTH, through its regulation of VEGF expression, in the maintenance of the adult adrenal cortex endothelium.

Paracrine control of the adult adrenal cortex vasculature by vascular endothelial growth factor.

The extensive vascular network that irrigates the adult adrenal cortex is essential for both the delivery of oxygen and nutrients to glandular steroidogenic cells and for the rapid and efficient export of corticosteroid products from these cells into the blood flow. During experimental and pathological changes in adrenal cortex size caused by ACTH overproduction or deficiency, the vasculature evolves in a coordinated manner with the mass of glandular cells so that blood vessel formation/regression and cortical gowth/atrophy are respectively synchronized. In addition to its previously reported expression in human fetal adrenocortical cells, the angiogenic factor VEGF-A appears also to be strongly expressed by both glomerulosa and fasciculata cells of the adult bovine adrenal cortex, when the endothelium is quiescent. Moreover, the expression of the two major transcripts encoding the 121 and the 165 amino acid-long isoforms of VEGF-A was observed to be rapidly (within 2-4 h) up-regulated (2-3 fold) by ACTH in primary cultures of bovine fasciculata and glomerulosa cells. The expression of the signalling VEGF receptors R1 (flt-1) and R2 (flk-1) was restricted to the endothelial cells of the cortex whereas neuropilin-1 was expressed by both endothelial and steroidogenic cells. This suggests that, under the control of the pituitary hormone ACTH, VEGF exerts a paracrine control over the vasculature of the adult adrenal cortex. Given its known effects as an anti-apoptotic agent and an inducer of endothelial fenestration, VEGF is likely to play a role in the maintenance of the dense and fenestrated vascular bed of the adrenal cortex. The vasculature thus appears as an important secondary target of ACTH action in the physiological control of adrenal cortex homeostasis.

Antiproliferative effects of imidazo[1,2-a]pyrazine derivatives on the Dami cell line.

Since cyclic 3',5'-adenosine monophosphate (cAMP) is involved in cell proliferation and as previous data showed that imidazo[1,2-alpha]pyrazine derivatives (PAB12, PAB30, PAB40, SCA40, SCA41, and SCA44) inhibited cAMP breakdown by a phosphodiesterase (PDE)-inhibitory effect, the aim of the present study was to investigate the effects of these derivatives on proliferation of the Dami cell line in relation with their actions on cAMP content and on PDE isoenzymes isolated from Dami cells. SCA41 and SCA44 inhibited cell growth in a dose-dependent manner, while SCA40 and PAB40 induced a weak inhibition. Growth inhibitions were 40%, 91%, and 60% for SCA41, SCA44 (at 100 microM), and IBMX (at 100 microM), respectively, and could not be related to their effects on cAMP levels. In addition, although all compounds potentiated cAMP formation by prostaglandin E1 (PGE1), no potentiations were observed when the antiproliferative effects of SCA41 and SCA44 were considered. Investigation of derivatives on PDE isoenzymes III, IV, and V indicated non-selective PDE inhibitory effects for SCA41 and SCA44, while SCA40 elicited preferences for type III, and PAB30 and PAB40 preferences for type IV isoenzymes. These effects could not totally explain the antiproliferative activity of the derivatives. The activation of P2 purinoceptors by imidazo[1,2-a]pyrazine did not lead to their antiproliferative effects. Thus, the mechanism of the antiproliferative effects of the compounds remains to be determined. It does, however, depend on the chemical substitutions of the imidazo[1,2-a]pyrazine skeleton and in particular on the 2-carbonitrile presence and the length of the 8-aminoaliphatic group.

Characterization of MEC 14.7, a new monoclonal antibody recognizing mouse CD34: a useful reage for identifying and characterizing blood vessels and hematopoietic precursors.

Endothelial cell-specific molecules are potential targets for new therapeutic strategies in the control of inflammatory reactions, immune responses and neoangiogenesis. We describe the production and characterization of MEC 14.7, a monoclonal antibody directed to murine endothelial cells recognizing a glycosylated protein with an apparent molecular mass of about 100 kDa in cultured endothelioma cell lysate and about 80 kDa in lung lysate. MEC 14.7 antigen was selectively expressed by the endothelium in vivo, particularly in small vessels and neoformed capillaries and by developing vascular structures in embryonal bodies. Deglycosylation of the molecule with neuraminidase, O- and N-glycanase showed that the MEC 14.7 epitope is neuraminidase-sensitive. MEC 14.7 antigen was purified from lung lysates by chromatographic techniques, and sequenced internal peptides indicated it was identical with murine CD34. Thus the apparent molecular mass of CD34 is heterogeneous, depending on the glycosylation state in the different cell types. Immunomagnetic isolation and culture of MEC 14.7-positive bone marrow cells showed that this antibody recognizes hematopoietic progenitors (particularly myelomonocytic) and can be used in murine models of bone marrow reconstitution.

Modulation of the megakaryoblastic Dami cell line differentiation by phosphodiesterase inhibitors and imidazo[1,2-a]pyrazine derivatives.

Phosphodiesterase inhibitors have been shown to modulate cell differentiation. We have previously shown that a series of imidazo[1,2-a]pyrazine derivatives displayed inhibitory effects on phosphodiesterase isoenzymes types III. IV and V isolated from Dami cells and on Dami cell growth. In the present study we have investigated the effect of these derivatives on the expression of two differentiation markers, glycoproteins Ib and IIb/IIIa of the human megakaryoblastic leukaemic Dami cell line in comparison to those elicited by 3-isobutyl-1-methylxanthine and selective phosphodiesterase inhibitors of types 1 (8-methoxymetyl-1-methyl-3-(2-methylpropyl) xanthine), III (Milrinone), IV (RO-201724) and V (Zaprinast). Imidazo[1,2-a]pyrazine derivatives, 3-isobutyl-1-methylxanthine and selective phosphodiesterase inhibitors, except 8-methoxymethyl-1-methyl-3-(2-methylpropyl) xanthine, decreased glycoprotein Ib expression. SCA40, SCA41, SCA44 and 3-isobutyl-1-methylxanthine-but not the other compounds affected the expression of glycoprotein IIb/IIIa in a positive manner. The effects of imidazo[1,2-a]pyrazine derivatives on glycoprotein expression appeared to be related to their phosphodiesterase inhibitory potency.

Targeted null-mutation in the vascular endothelial-cadherin gene impairs the organization of vascular-like structures in embryoid bodies.

Vascular endothelial-cadherin (VE-cadherin) is exclusively expressed in endothelial cells and is strictly located at cell-to-cell junctions. As the other members of the cadherin family, VE-cadherin is able to mediate a homotypic type of cellular interaction in a Ca2+-dependent manner. In the mouse embryo, VE-cadherin transcripts are detected at the earliest stages of vascular development. To ascertain if VE-cadherin expression is required for the assembly of endothelial cells into vascular structures, we generated VE-cadherin-negative mouse embryonic stem cells (VE-cadherin-/- ES cells) by gene targeting and examined the consequences on vascular development of ES-derived embryoid bodies (EBs). In contrast to wild-type EBs, we observed that endothelial cells remained dispersed and failed to organize a vessel-like pattern in VE-cadherin-/- ES-derived EBs. However, dispersed VE-cadherin-/- ES-derived endothelial cells expressed a large range of other endothelial markers. Moreover, the targeted null-mutation in the VE-cadherin locus did not interfere with the hematopoietic differentiation potential of ES cells. These in vitro experiments are consistent with a pivotal role of VE-cadherin in vascular structure assembly.

Dissociation between phosphodiesterase inhibition and antiproliferative effects of phosphodiesterase inhibitors on the Dami cell line.

Phosphodiesterase (PDE) inhibitors were shown to inhibit proliferation of various cell types. The present investigation was designed to study the activity of selective PDE inhibitors (8-MeoMIX, milrinone, trequinsin, rolipram, RO-201724, zaprinast, and MY-5445) on the proliferation of the Dami cell line in relation to their effects on cAMP levels and PDE isoenzymes isolated from Dami cells. All compounds, except 8-MeoMIX, elicited antiproliferative effects. Trequinsin, RO-201724, and MY-5445 (100 microM) were found to inhibit cell growth up to 60%, 83%, and 85%, respectively; milrinone, rolipram and zaprinast elicited only weak effects (19-21% at 100 microM). Their growth-inhibitory effects could not be related to their effects on cAMP levels. In addition, although PDE type III and IV inhibitors potentiated cAMP formation due to adenylycyclase activation, no potentiation could be observed when considering their antiproliferative effect. Separation and characterization of PDE of Dami cells revealed the existence of types III, IV, and V isoenzymes. The PDE inhibition found for the PDE inhibitors could not explain their antiproliferative effects. The lack of correlation with cAMP concentrations or PDE inhibition and the high concentrations needed to elicit antiproliferative effects suggest the implication of other parameters, such as cytotoxicity or lipophilicity, or other targets in addition to PDE for the PDE inhibitors tested. Lipophilicity did not seem to be of importance in antiproliferative effects. In contrast, cytotoxic effects, in particular those of trequinsin and MY-5445, could partially explain their negative action on cell growth.

Endothelin expression in human megakaryoblastic leukemia cell lines and normal platelet precursors.

The aim of the present investigation was to determine whether endothelin (ET) could be expressed in and released from the human leukemia megakaryoblastic cell lines HEL, MEG-01, DAMI and the normal human platelet progenitors. Using the reverse transcriptase-polymerase chain reaction (RT-PCR) on total RNA isolated from the cells, we amplified a cDNA of the expected size (453 bp). Southern-blotting hybridization revealed that RT-PCR products from the cell lines were specific of ET-1 mRNA. Immunocytochemical analyses highlighted immunoreactive ET-1 in the cytoplasm of these cells which also released the mature peptide. ET-1 release from the three cell lines was increased by thrombin exposure. Although MEG-01 cells express ET receptors, ET-1, the selective ETB agonist sarafotoxin 6C and the non-selective ET-receptor antagonist PD 142893 showed no proliferative or antiproliferative action in basal or stimulating medium. This indicated a lack of autocrine ET-mediated effect on growth. These results demonstrate for the first time that human megakaryoblastic leukemia cell lines and normal bone marrow platelet precursors express ET-1 mRNA and release the mature peptide.

Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps.

The mechanisms involved in the regulation of vasculogenesis still remain unclear in mammals. Totipotent embryonic stem (ES) cells may represent a suitable in vitro model to study molecular events involved in vascular development. In this study, we followed the expression kinetics of a relatively large set of endothelial-specific markers in ES-derived embryoid bodies (EBs). Results of both reverse transcription-polymerase chain reaction and/or immunofluorescence analysis show that a spontaneous endothelial differentiation occurs during EBs development. ES-derived endothelial cells express a full range of cell lineage-specific markers: platelet endothelial cell adhesion molecule (PECAM), Flk-1, tie-1, tie-2, vascular endothelial (VE) cadherin, MECA-32, and MEC-14.7. Analysis of the kinetics of endothelial marker expression allows the distinction of successive maturation steps. Flk-1 was the first to be detected; its mRNA is apparent from day 3 of differentiation. PECAM and tie-2 mRNAs were found to be expressed only from day 4, whereas VE-cadherin and tie-1 mRNAs cannot be detected before day 5. Immunofluorescence stainings of EBs with antibodies directed against Flk-1, PECAM, VE-cadherin, MECA-32, and MEC-14.7 confirmed that the expression of these antigens occurs at different steps of endothelial cell differentiation. The addition of an angiogenic growth factor mixture including erythropoietin, interleukin-6, fibroblast growth factor 2, and vascular endothelial growth factor in the EB culture medium significantly increased the development of primitive vascular-like structures within EBs. These results indicate that this in vitro system contains a large part of the endothelial cell differentiation program and constitutes a suitable model to study the molecular mechanisms involved in vasculogenesis.

Cyclic-AMP inhibits cell growth and negatively interacts with platelet membrane glycoprotein expression on the Dami human megakaryoblastic cell line.

Intracellular signaling processes by which hematopoietic growth factors regulate megakaryocytopoiesis remain incompletely understood. Cyclic AMP (cAMP) has been shown to be implicated in the regulation of growth and differentiation in various normal and malignant cell types. Since a few studies have suggested the possible involvement of the cAMP pathway as one of the intracellular mechanisms whereby megakaryocytopoiesis may be regulated, we investigated the functional effects of cAMP on the human megakaryoblastic Dami cell line. We observed that exposure of Dami cells to cAMP analogs or to agents elevating intracellular cAMP levels yielded dose-dependent cell growth inhibition. Cell cycle progression analysis of cells predominantly synchronized at the G1/S boundary by prior treatment with hydroxyurea revealed that cAMP transiently accumulated cells in the G2/M phase, then slowing down cell cycle. On the other hand, immunofluorescence and Northern blot analysis of megakaryocytic differentiation marker expression showed that probes we have used significantly inhibited GPIb expression. Moreover, although these agents used alone did not affect GPIIb/IIIa expression, they markedly reversed phorbol ester-induced GPIIb/IIIa expression increase. These inhibitory cAMP actions on glycoprotein expression were not the result of cell cycle perturbation since we observed that GPIb and GPIIb/IIIa expression were not cell cycle dependent. All these data may then be consistent with a potential negative regulatory role of the cAMP intracellular signaling pathway during megakaryocytopoiesis.

Thrombin interactions with cells of the megakaryocytic lineage.

Thrombin is a potent physiological platelet agonist, critical for haemostasis and thrombosis. Several studies have recently demonstrated that thrombin also displays functional effects on bone marrow platelet progenitors, i.e. the megakaryocytes, and possibly on megakaryocyte precursors. Following a brief description of the different cell systems used for these investigations, this paper focuses on data related to thrombin receptors, their coupling mechanisms, and biological responses due to their activation on cells of the megakaryocytic lineage. In spite of some differences, whose significance remains to be established, most data reported give evidence of considerable homologies in thrombin receptors and signal transduction mechanisms between platelets and cells belonging to the megakaryocytic lineage. In addition, a potential functional role for thrombin in the regulation of megakaryocytopoiesis is emerging. The physiological relevance of these observations are discussed.

Effects of endothelins on the human megakaryoblastic cell line MEG-01.

Some effects of endothelin-1 (ET-1) were studied on the megakaryoblastic cell line MEG-01. ET-1 induced an elevation of the intracellular levels of Ca2+([Ca2+]i) as measured with the fluorescent indicator indo-1, which consists of an initial transient increase and an ensuing sustained plateau. The plateau phase was abolished by removal of extracellular Ca2+. In addition, ET-1 induced a rapid (within 5 s) increase in the accumulation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and more delayed increases in Ins(1,3,4)P3 and Ins(1,3,4,5)P4, but did not modify cAMP levels. ET-1 homologues (ET-2, ET-3, sarafotoxin 6b and vasointestinal constrictor) also induced biphasic effects on [Ca2+]i. The Ca2+ elevation was concentration dependent, the order of potency being sarafotoxin 6b > ET-1 > ET-2 = vasointestinal constrictor > ET-3. The actions of ET analogs in raising [Ca2+]i were mutually exclusive, suggesting that they act through the same mechanism. These results suggest that cells of the megakaryoblast/megakaryocyte lineage are targets for endothelins.

Platelet receptor expression on three human megakaryoblast-like cell lines.

Platelets, the progeny of bone marrow megakaryocytes, are nonnucleated cells; many platelet proteins, including platelet membrane receptors, are believed to be derived from megakaryocytes. Several hematopoietic cell lines that exhibit megakaryocytic characteristics have been established as models for the study of megakaryocyte biology. We report here the screening of platelet receptor expression, in terms of functional coupling with the formation of two second messengers, calcium and cAMP, in three cell lines exhibiting megakaryoblastic properties: HEL, MEG-01, and DAMI. We show that all these cell lines respond to thrombin, ADP, epinephrine, and prostaglandin E1 (PGE1). However, transmembrane signaling pathways appear partly different from those present in mature platelets, because the action of thrombin was found to be positively coupled with the cAMP pathway, in addition to that of calcium, and because PGE1, which interacts with the cAMP pathway, also raises intracellular calcium levels in the three cell lines studied. Furthermore, an endothelin-1-induced increase in intracellular calcium level was observed in MEG-01 cells, strongly suggesting the expression of endothelin receptors on platelet precursors cells, whereas the presence of such receptors is controversial on platelets. These cell lines should prove useful in further studies of the expression and molecular pharmacology of platelet receptors on platelet precursor cells, as well as for the investigation of functional roles for platelet receptors on megakaryoblastic cells.

Thrombin inhibits proliferation of the human megakaryoblastic MEG-01 cell line: a possible involvement of a cyclic-AMP dependent mechanism.

Thrombin, a potent platelet activating agent, has previously been found to increase intracellular calcium levels and/or thromboxane A2 synthesis in leukemic cell lines exhibiting specific markers of the megakaryocyte/platelet lineage. However, its functional role on these cells has not been defined. As thrombin is implicated in the regulation of cellular proliferation or differentiation in various other cell types, we investigated the functional effects of thrombin on the megakaryoblastic MEG-01 cell line, and further explored its receptor coupling mechanisms on these cells. We observed that thrombin caused in 1% serum containing culture medium, a reduction in the proliferation of MEG-01 cells, without affecting their differentiation stage as determined by the expression of platelet glycoproteins GPIIb/IIIa and GPIb, FVIII-related-antigen and cell-size measurement, which are specific markers for megakaryocyte maturation. In addition, incubation of MEG-01 cells with thrombin resulted in dose-dependent increases in cAMP levels, and in inositol-trisphosphate formation and intracellular Ca2+ levels. All these responses required thrombin proteolytic activity. The lipoxygenase inhibitor, nordihydroguaiaretic acid, blunted thrombin-induced calcium increase without affecting thrombin-induced increase in cAMP levels, suggesting different thrombin coupling mechanisms with these two second messenger pathways. In addition, the inhibitory effect of thrombin on MEG-01 cell growth was mimicked by cAMP level enhancing agents such as forskolin, prostaglandin E1 and Bt2cAMP. These results suggest the involvement of a cAMP-dependent mechanism in the thrombin-induced reduction in MEG-01 cell growth.

Homologous regulation of human platelet vasopressin receptors does not occur in vivo.

Large variations in the functional responsiveness of human platelets to arginine vasopressin (AVP), related to maximal platelet AVP-binding capacity, have been observed among individuals. The effects of dehydration and overhydration on maximal platelet AVP-binding capacity, plasma AVP, platelet-associated AVP, and AVP-induced platelet aggregation were examined in healthy human volunteers to determine whether homologous regulation of platelet AVP receptors occurs in vivo within the physiological range of circulating AVP fluctuations. Although significant variations of plasma AVP were observed under both conditions, no correlation could be found in the same individual with any change in 1) the maximal AVP-binding capacity, 2) platelet aggregatory responses to AVP, or 3) the platelet AVP fraction. Moreover, there was no relationship between the number of detectable platelet AVP receptors and the amount of AVP associated with platelets. These data show that homologous regulation of platelet AVP receptors by AVP does not occur in vivo over the time investigated, at least within the physiological range of AVP. Nonetheless, regulation at the platelet precursor stage appears to be highly probable in view of the relationship between plasma AVP and platelet AVP binding capacity observed among different individuals.

Properties of vasopressin-activated human platelet high affinity GTPase.

Effect of 8-arginine vasopressin (AVP) was examined on human platelet membrane GTPase activity as an index of a G-protein involvement. AVP stimulated a high-affinity GTPase activity in a dose-dependent manner (Kact = 1.1 +/- 0.2 nM). This stimulation was blocked by a V1a antagonist, thus confirming the V1a nature of the platelet AVP receptor. There were important variations among individuals in the AVP-induced stimulation of GTPase activity, that were in relation with the AVP-maximal binding capacity. These data suggest a causal relationship between the binding of AVP to its receptor and transduction elicited by a G-protein, without amplification. In addition, in view of the variable AVP responsiveness observed among individuals, platelet AVP-receptor appears to be subject to regulation.

Vasopressin inhibition of human platelet adenylate cyclase: variable responsiveness between donors and involvement of a G-protein different from Gi.

There is controversy concerning the inhibitory effect of arginine-vasopressin (AVP) on human platelet adenylate cyclase activity, which putatively involves Gi as the G-protein. To clarify this point, the effects of AVP on human platelet membranes were studied by measuring the activities of the high-affinity GTPase, as an index of G-protein involvement, and of adenylate cyclase. AVP stimulated GTPase activity in a dose-dependent fashion (KAct = 1.1 +/- 0.2 nM) and caused a parallel adenylate cyclase inhibition (KAct = 1.3 +/- 0.7 nM). The extent of these AVP-induced responses varied considerably from one subject to another but they were linearly related, suggesting a causal relationship between the two activities. Moreover, a difference in responsiveness to the inhibitory effects to epinephrine on adenylate cyclase was also observed between donors. Since the AVP- and epinephrine-stimulated GTPase activities were additive at their respective maximal effect, and in view of the lack of linear relationship between AVP- and epinephrine-induced adenylate cyclase inhibition, our results suggest, that in spite of the AVP inhibitory action on platelet adenylate cyclase, the G-protein involved in this effect is different from Gi.