Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling.

Functional expression of voltage-gated Na(+) channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

Inactivation of a Gα(s)-PKA tumour suppressor pathway in skin stem cells initiates basal-cell carcinogenesis.

Genomic alterations in GNAS, the gene coding for the Gαs heterotrimeric G protein, are associated with a large number of human diseases. Here, we explored the role of Gαs on stem cell fate decisions by using the mouse epidermis as a model system. Conditional epidermal deletion of Gnas or repression of PKA signalling caused a remarkable expansion of the stem cell compartment, resulting in rapid basal-cell carcinoma formation. In contrast, inducible expression of active Gαs in the epidermis caused hair follicle stem cell exhaustion and hair loss. Mechanistically, we found that Gαs-PKA disruption promotes the cell autonomous Sonic Hedgehog pathway stimulation and Hippo signalling inhibition, resulting in the non-canonical activation of GLI and YAP1. Our study highlights an important tumour suppressive function of Gαs-PKA, limiting the proliferation of epithelial stem cells and maintaining proper hair follicle homeostasis. These findings could have broad implications in multiple pathophysiological conditions, including cancer.

RhoA and ROCK mediate histamine-induced vascular leakage and anaphylactic shock.

Histamine-induced vascular leakage is an integral component of many highly prevalent human diseases, including allergies, asthma and anaphylaxis. Yet, how histamine induces the disruption of the endothelial barrier is not well defined. By using genetically modified animal models, pharmacologic inhibitors and a synthetic biology approach, here we show that the small GTPase RhoA mediates histamine-induced vascular leakage. Histamine causes the rapid formation of focal adherens junctions, disrupting the endothelial barrier by acting on H1R Gαq-coupled receptors, which is blunted in endothelial Gαq/11 KO mice. Interfering with RhoA and ROCK function abolishes endothelial permeability, while phospholipase Cß plays a limited role. Moreover, endothelial-specific RhoA gene deletion prevents vascular leakage and passive cutaneous anaphylaxis in vivo, and ROCK inhibitors protect from lethal systemic anaphylaxis. This study supports a key role for the RhoA signalling circuitry in vascular permeability, thereby identifying novel pharmacological targets for many human diseases characterized by aberrant vascular leakage.

Differential regulation of endosomal GPCR/ß-arrestin complexes and trafficking by MAPK.

ß-Arrestins are signaling adaptors that bind to agonist-occupied G protein-coupled receptors (GPCRs) and target them for endocytosis; however, the mechanisms regulating receptor/ß-arrestin complexes and trafficking in endosomes, remain ill defined. Here we show, in live cells, differential dynamic regulation of endosomal bradykinin B2 receptor (B2R) complexes with either ß-arrestin-1 or -2. We find a novel role for MAPK in the B2R/ß-arrestin-2 complex formation, receptor trafficking and signaling mediated by an ERK1/2 regulatory motif in the hinge domain of the rat ß-arrestin-2 (PET(178)P), but not rat ß-arrestin-1 (PER(177)P). While the ERK1/2 regulatory motif is conserved between rat and mouse ß-arrestin-2, it is surprisingly not conserved in human ß-arrestin-2 (PEK(178)P). However, mutation of lysine 178 to threonine is sufficient to confer MAPK sensitivity to the human ß-arrestin-2. Furthermore, substitution for a phosphomimetic residue in both the rat and the human ß-arrestin-2 (T/K178D) significantly stabilizes B2R/ß-arrestin complexes in endosomes, delays receptor recycling to the plasma membrane and maintains intracellular MAPK signaling. Similarly, the endosomal trafficking of ß2-adrenergic, angiotensin II type 1 and vasopressin V2 receptors was altered by the ß-arrestin-2 T178D mutant. Our findings unveil a novel subtype specific mode of MAPK-dependent regulation of ß-arrestins in intracellular trafficking and signaling of GPCRs, and suggest differential endosomal receptor/ß-arrestin-2 signaling roles among species.

Somatic mutation of GRIN2A in malignant melanoma results in loss of tumor suppressor activity via aberrant NMDAR complex formation.

The ionotropic glutamate receptors (N-methyl-D-aspartate receptors (NMDARs)) are composed of large complexes of multi-protein subunits creating ion channels in the cell plasma membranes that allow for influx or efflux of mono- or divalent cations (e.g., Ca(2+)) important for synaptic transmissions, cellular migration, and survival. Recently, we discovered the high prevalence of somatic mutations within one of the ionotropic glutamate receptors, GRIN2A, in malignant melanoma. Functional characterization of a subset of GRIN2A mutants demonstrated a loss of NMDAR complex formation between GRIN1 and GRIN2A, increased anchorage-independent growth in soft agar, and increased migration. Somatic mutation of GRIN2A results in a dominant negative effect inhibiting the tumor-suppressive phenotype of wild-type (WT) GRIN2A in melanoma. Depletion of endogenous GRIN2A in melanoma cells expressing WT GRIN2A resulted in increased proliferation compared with control. In contrast, short-hairpin RNA depletion of GRIN2A in mutant cell lines slightly reduced proliferation. Our data show that somatic mutation of GRIN2A results in increased survival, and we demonstrate the functional importance of GRIN2A mutations in melanoma and the significance that ionotropic glutamate receptor signaling has in malignant melanoma.

Cross-desensitization and cointernalization of H1 and H2 histamine receptors reveal new insights into histamine signal integration.

G protein-coupled receptor signaling does not result from sequential activation of a linear pathway of proteins/enzymes, but rather from complex interactions of multiple, branched signaling routes, i.e., signaling networks. In this work we present an exhaustive study of the cross-talk between H1 and H2 histamine receptors (H1R and H2R) in U937 cells and Chinese hamster ovary-transfected cells. By desensitization assays we demonstrated the existence of a crossdesensitization between both receptors independent of protein kinase A or C. H1R-agonist stimulation inhibited cell proliferation and induced apoptosis in U937 cells following treatment of 48 hours. H1R-induced antiproliferative and apoptotic response was inhibited by an H2R agonist suggesting that the cross-talk between both receptors modifies their function. Binding and confocal microscopy studies revealed cointernalization of both receptors upon treatment with the agonists. To evaluate potential heterodimerization of the receptors, sensitized emission fluorescence resonance energy transfer experiments were performed in human embryonic kidney 293T cells using H1R-cyan fluorescent protein and H2R-yellow fluorescent protein. To our knowledge these findings may represent the first demonstration of agonist-induced heterodimerization of the H1R and H2R. In addition, we also show that the inhibition of the internalization process did not prevent receptor crossdesensitization, which was mediated by G protein-coupled receptor kinase 2. Our study provides new insights into the complex signaling network mediated by histamine and further knowledge for the rational use of its ligands.

PDZ-RhoGEF and LARG are essential for embryonic development and provide a link between thrombin and LPA receptors and Rho activation.

G protein-coupled receptors (GPCRs) linked to both members of the Gα12 family of heterotrimeric G proteins α subunits, Gα12 and Gα13, regulate the activation of Rho GTPases, thereby contributing to many key biological processes. Multiple Rho GEFs have been proposed to link Gα12/13 GPCRs to Rho activation, including PDZ-RhoGEF (PRG), leukemia-associated Rho GEF (LARG), p115-RhoGEF (p115), lymphoid blast crisis (Lbc), and Dbl. PRG, LARG, and p115 share the presence of a regulator of G protein signaling homology (RGS) domain. There is limited information on the biological roles of this RGS-containing family of RhoGEFs in vivo. p115-deficient mice are viable with some defects in the immune system and gastrointestinal motor dysfunctions, whereas in an initial study we showed that mice deficient for Larg are viable and resistant to salt-induced hypertension. Here, we generated knock-out mice for Prg and observed that these mice do not display any overt phenotype. However, deficiency in Prg and Larg leads to complex developmental defects and early embryonic lethality. Signaling from Gα11/q-linked GPCRs to Rho was not impaired in mouse embryonic fibroblasts defective in all three RGS-containing RhoGEFs. However, a combined lack of Prg, Larg, and p115 expression abolished signaling through Gα12/13 to Rho and thrombin-induced cell proliferation, directional migration, and nuclear signaling through JNK and p38. These findings provide evidence of an essential role for the RGS-containing RhoGEF family in signaling to Rho by Gα12/13-coupled GPCRs, which may likely play a critical role during embryonic development.

A synthetic biology approach reveals a CXCR4-G13-Rho signaling axis driving transendothelial migration of metastatic breast cancer cells.

Tumor cells can co-opt the promigratory activity of chemokines and their cognate G protein-coupled receptors (GPCRs) to metastasize to regional lymph nodes or distant organs. Indeed, the migration toward SDF-1 (stromal cell-derived factor 1) of tumor cells bearing CXCR4 [chemokine (C-X-C motif) receptor 4] has been implicated in the lymphatic and organ-specific metastasis of various human malignancies. Here, we used chimeric G proteins and GPCRs activated solely by artificial ligands to selectively activate the signaling pathways downstream of specific G proteins and showed that CXCR4-mediated chemotaxis and transendothelial migration of metastatic basal-like breast cancer cells required activation of Gα(13), a member of the Gα(12/13) G protein family, and of the small guanosine triphosphatase Rho. Multiple complementary experimental strategies, including synthetic biology approaches, indicated that signaling-selective inhibition of the CXCR4-Gα(13)-Rho axis prevents the metastatic spread of basal-like breast cancer cells.

Study of G protein-coupled receptor/ß-arrestin interactions within endosomes using FRAP.

ß-arrestins, through their scaffolding functions, are key regulators of G protein-coupled receptor (GPCR) signaling and intracellular trafficking. However, little is known about the dynamics of ß-arrestin/receptor interactions and how these complexes, and complexes with other regulatory proteins, are controlled in cells. Here, we use yellow fluorescent protein (YFP)-tagged ß-arrestin 2 and a fluorescence recovery after photobleaching (FRAP) imaging approach to probe the real-time interaction of ß-arrestin with a GPCR, the bradykinin type 2 receptor (B2R). We provide a detailed protocol to assess the avidity of ß-arrestin2-YFP for B2R within endosomes in HEK293 cells. ß-arrestin2-YFP associated with internalized receptors is photobleached with intense light, and fluorescence recovery due to the entry of nonbleached ß-arrestin2-YFP is monitored over time as a measure of the rate exchange of ß-arrestin2-YFP within the endosome. This approach can be extended to other GPCR/ß-arrestin complexes and their putative regulators to provide information about the kinetics of similar protein-protein interactions in cells. Moreover, these techniques should provide insight into the role of ß-arrestins in the intracellular trafficking and signaling of GPCRs.

Role of ßarrestins in bradykinin B2 receptor-mediated signalling.

G protein-coupled receptors (GPCRs) can engage multiple pathways to activate ERK1/2 via both G proteins and/or ßarrestin. Receptor recruitment of ßarrestin is also important for GPCR desensitization, internalization and resensitization. Modulation of the receptor/ßarrestin interaction through modification of either component would presumably alter the output generated by receptor activation. Here we examined how ßarrestins regulate bradykinin (BK) B2 receptor (B2R) signalling and desensitization by either truncating ßarrestin1 or ßarrestin2 or by alanine substitution of a serine/threonine cluster in the C-terminal tail of B2R (B2R-4A), conditions which all affect the avidity of the B2R/ßarrestin complex. We first demonstrate that BK-mediated ERK1/2 activation is biphasic containing an early peak (between 2-5min) followed by sustained activation for at least 60min. The early but not the sustained phase was predictably affected by inhibition of either Gαq/11 or Gαi/o, whereas loss of ßarrestin2 but not ßarrestin1 resulted in diminished prolonged ERK1/2 activation. ßarrestin2's role was further examined using a truncation mutant with augmented avidity for the agonist-occupied receptor, revealing an increase in both immediate and extended ERK1/2 signalling. We also show that ERK1/2 is recruited to the B2R/ßarrestin complex on endosomes as well as the plasma membrane. Moreover, we investigated ßarrestin's role using the B2R-4A, which is deficient in ßarrestin binding and does not internalize. We show that ERK1/2 signalling downstream of the receptor is entirely G protein-dependent and receptor-mediated intracellular calcium mobilization studies revealed a lack of desensitization. Functionally, the lack of desensitization resulted in increased cell growth and migration compared to the wild-type receptor, which was sensitive to MEK inhibition. These results highlight ßarrestin's crucial role in the maintenance of proper B2R signalling.

C5a- and ASP-mediated C5L2 activation, endocytosis and recycling are lost in S323I-C5L2 mutation.

UNLABELLED: C5L2, a G-protein-coupled receptor (GPCR), has been identified as an ASP (C3adesArg) and C5a receptor. Controversy exists regarding both ligand binding and functionality. ASP activation of C5L2 is proposed to regulate fat storage. C5L2 is also proposed as a decoy receptor for C5a, an inflammatory mediator, based on absence of Ca(2+) or chemotaxis changes. AIMS: (i) to evaluate C5L2 receptor activation and recycling using recombinant ASP (rASP) and rC5a and (ii) assess receptor trafficking of S323I-C5L2 mutation previously identified in a family and demonstrated to have altered functionality. RESULTS: stably transfected C5L2-HEK cells were sorted using fluorescent-ASP (Fluos-ASP) binding. Following 2-h serum-free pretreatment, C5L2 was typically localized to the cell-surface. beta-Arrestin-2-GFP transiently transfected C5L2-HEK cells demonstrated rASP and rC5a-dependent beta-arrestin-2-GFP translocation, which showed time-dependent intracellular colocalization with C5L2. Without ligand or C5L2 transfection, no translocation was identified at any time point. Ligand-dependent (rASP and rC5a) C5L2 endocytosis was time-dependent with a 1-h nadir, and was clathrin- and cholesterol-dependent. Transiently transfected Rab-GFP proteins (Rabs 5, 7 and 11) demonstrated time-dependent colocalization of Rab5, Rab7, and Rab11 with C5L2. In contrast to C5L2, a large proportion of stably transfected S323I-C5L2 did not localize to the cell-surface. While S323I-C5L2 was competent for Fluos-ASP and (125)I-ASP binding, although at a reduced level, there was no ligand-mediated receptor phosphorylation. Further, there was no ligand-mediated activation of beta-arrestin-2-GFP translocation, and no downstream functional activation of glucose transport or triglyceride synthesis. CONCLUSION: C5L2 is a functional metabolic receptor, and serine 323 is important for ASP induced functionality.

c-Src-mediated phosphorylation of AP-2 reveals a general mechanism for receptors internalizing through the clathrin pathway.

Clathrin-mediated endocytosis is a complex process regulated at many different levels. We showed previously that activation of the angiotensin type 1 receptor (AT1R), which belongs to the G protein-coupled receptor (GPCR) family, leads to c-Src-dependent tyrosine phosphorylation of beta2-adaptin, a subunit of the clathrin adaptor AP-2. The phosphorylation of beta2-adaptin on tyrosine residue 737 (Y737) negatively regulates its interaction with betaarrestin, another important clathrin adaptor for GPCR internalization. Here we sought to determine whether AP-2 phosphorylation represents a general mechanism for different receptors internalizing through the clathrin pathway. Using a specifically designed antibody against the phosphorylated form of Y737 on beta2-adaptin, we demonstrate that this residue is phosphorylated by AT1R in different cell types like HEK293, COS-7 and vascular smooth muscle cells. Using RNA interference approaches, we reveal that this agonist-mediated event is both betaarrestin- and c-Src-dependent, and that it occurs at the plasma membrane in clathrin-coated vesicles (CCVs). We further show that this is not only a common event employed by other GPCRs like the beta2-adrenergic, vasopressin V2, bradykinin type 2, platelet-activating factor and endothelin A receptors but that the epidermal growth factor receptor is capable of eliciting the phosphorylation of AP-2 in CCVs. Our results imply that tyrosine phosphorylation of Y737 on beta2-adaptin is a common regulatory mechanism employed by different receptors undergoing clathrin-dependent endocytosis, and suggest a wider function for this event than originally anticipated.

Inferring the lifetime of endosomal protein complexes by fluorescence recovery after photobleaching.

Cellular signal transduction is dynamic, with signaling proteins continually associating and dissociating into and from protein complexes. Here we present a fluorescence recovery after photobleaching technique to determine the lifetime of protein complexes on intracellular vesicles. We use Bayesian inference based on a model that includes the diffusion of cytosolic proteins and their interaction with membrane-bound receptors. Our analysis is general: we incorporate prior information on protein diffusion, measurement error in determining fluorescence intensities, corrections for photobleaching, and variation in the concentration of receptors between vesicles. We apply our method to the complexes formed on endosomes by G-protein-coupled receptors and the protein beta-arrestin. The lifetime of these complexes determines the recycling rate of the receptors. We find in mammalian cells that the bradykinin type 2 receptor and beta-arrestin2 complex has a lifetime of approximately 2 min, while the angiotensin II type 1A receptor and beta-arrestin2 complex has a lifetime of approximately 6 min. As well as allowing quantitative comparisons between experiments, our method provides in vivo parameters for systems biology simulations of signaling networks.

Src-dependent phosphorylation of beta2-adaptin dissociates the beta-arrestin-AP-2 complex.

Beta-arrestins are known to act as endocytic adaptors by recruiting the clathrin adaptor protein 2 (AP-2) complex to G-protein-coupled receptors (GPCRs), linking them to clathrin-coated pits (CCPs) for internalization. They also act as signaling molecules connecting GPCRs to different downstream effectors. We have previously shown that stimulation of the angiotensin II (Ang II) type 1 receptor (AGTR1, hereafter referred to as AT1R), a member of the GPCR family, promotes the formation of a complex between beta-arrestin, the kinase Src and AP-2. Here, we report that formation of such a complex is involved in the AT1R-mediated tyrosine phosphorylation of beta2-adaptin, the subunit of AP-2 involved in binding beta-arrestin. We identify a crucial tyrosine residue in the ear domain of beta2-adaptin and show in vitro that the phosphorylation of this site regulates the interaction between beta-arrestin and beta2-adaptin. Using fluorescently tagged proteins combined with resonance energy transfer and image cross-correlation spectroscopy approaches, we show in live cells that beta2-adaptin phosphorylation is an important regulatory process for the dissociation of beta-arrestin-AP-2 complexes in CCPs. Finally, we show that beta2-adaptin phosphorylation is involved in the early steps of receptor internalization. Our findings not only unveil beta2-adaptin as a new Src target during AT1R internalization, but also support the role of receptor-mediated signaling in the control of clathrin-dependent endocytosis of receptors.

Dissociation of beta-arrestin from internalized bradykinin B2 receptor is necessary for receptor recycling and resensitization.

Beta-arrestins are multifunctional adaptors that bind agonist-activated G protein-coupled receptors (GPCRs), mediate their desensitization and internalization, and control the rate at which receptors recycle back at the plasma membrane ready for subsequent stimulation. The activation of the bradykinin (BK) type 2 receptor (B2R) results in the rapid desensitization and internalization of the receptor. Little is known, however, about the role of beta-arrestin in regulating the intracellular trafficking and the resensitization of the B2R. Using confocal microscopy, we show that BK stimulation of COS-7 cells expressing B2R induces the colocalization of the agonist-activated receptor with beta-arrestin into endosomes. Fluorescent imaging and ligand binding experiments also reveal that upon agonist removal, beta-arrestin rapidly dissociates from B2R into endosomes, and that receptors return back to the plasma membrane, fully competent for reactivating B2R signaling as measured by NO production upon a second BK challenge. However, when the receptor is mutated in its C-terminal domain to increase its avidity for beta-arrestin, B2R remains associated with beta-arrestin into endosomes, and receptors fail to recycle to the plasma membrane postagonist wash. Similarly, the recycling of receptors is prevented when a beta-arrestin mutant exhibiting increased avidity for agonist-bound GPCRs is expressed with B2R. Stabilizing receptor/beta-arrestin complexes into endosomes results in the dampening of the BK-mediated NO production. These results provide evidence for the involvement of beta-arrestin in the intracellular trafficking of B2R, and highlight the importance of receptor recycling in reestablishing B2R signaling.

c-Src regulates clathrin adapter protein 2 interaction with beta-arrestin and the angiotensin II type 1 receptor during clathrin- mediated internalization.

Beta-arrestins are multifunctional adapters involved in the internalization and signaling of G protein-coupled receptors (GPCRs). They target receptors to clathrin-coated pits (CCPs) through binding with clathrin and clathrin adapter 2 (AP-2) complex. They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs. Here we sought to determine whether c-Src regulates the recruitment of AP-2 to beta-arrestin and the angiotensin II (Ang II) type 1 receptor (AT1R) during internalization. We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, beta-arrestins and AP-2. In vitro studies using coimmunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between beta-arrestin2 and the beta-subunit of AP-2 independently of the kinase activity of c-Src. However, although c-Src expression promoted the rapid dissociation of AP-2 from both beta-arrestin and AT1R after receptor stimulation, a kinase-inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor. Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in human embryonic kidney (HEK) 293 cells using a small interfering RNA strategy. Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization. Moreover, coimmunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and beta-arrestin, respectively. Together, our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and beta-arrestin during the clathrin-mediated internalization of receptors and suggest a novel function for c-Src kinase in the internalization of AT1R.

Involvement of a cytoplasmic-tail serine cluster in urotensin II receptor internalization.

Most G-protein-coupled receptors that undergo agonist-dependent internalization require the presence of specific cytoplasmic-tail residues to initiate interactions with proteins of the endocytic machinery. Here we show that the UT receptor (urotensin II receptor) undergoes internalization, and that specific serine residues of the receptor's cytoplasmic tail participate in this process. We first observed a time-dependent increase in internalization of the UT receptor expressed in COS-7 cells following binding of the agonist urotensin II. This sequestration was significantly reduced in the presence of sucrose, demonstrating that the agonist-activated UT receptor is internalized in part by clathrin-coated pits. Moreover, the sequestered receptor was co-localized in endocytic vesicles with beta-arrestin1 and beta-arrestin2. To assess whether specific regions of the receptor's cytoplasmic tail were involved in internalization, five UT receptor mutants were constructed. In four constructs the receptor's cytoplasmic tail was truncated at various positions (UTDelta367, UTDelta363, UTDelta350 and UTDelta336), and in the other four adjacent serine residues at positions 364-367 were replaced by Ala (Mut4S). Each mutant, except UTDelta367, demonstrated a significantly reduced internalization rate, thereby revealing the importance of specific serine residues within the cytoplasmic tail of the UT receptor for its ability to be internalized efficiently.

Calcium channels contribute to the decrease in blood pressure of pregnant rats.

Pregnancy is associated with hemodynamic changes such as reduced vascular resistance and blood pressure. We reported that, during late pregnancy, the activity of voltage-dependent calcium channels (VDCC) is altered in the adrenal cortex and vascular smooth muscle. These observations suggested that the late pregnancy-induced decrease in blood pressure is linked to diminished VDCC function. We attempted to prevent pregnancy-induced reduced blood pressure with a calcium channel activator (CGP 28392) in pregnant rats and to mimic it by administration of a calcium channel blocker (nifedipine) to nonpregnant rats. Treatment was given from the 15th day of gestation for 7 days. The systolic blood pressure of CGP 28392-treated pregnant rats rose transiently for 2 days and then declined toward values of nontreated pregnant controls, although remaining higher. However, nonpregnant rats maintained their high arterial pressure throughout CGP 28392 treatment. Nifedipine lowered the blood pressure in nonpregnant rats to values of nontreated term-pregnant controls. Both agents did not affect body weight, water or food intake, plasma renin activity, and plasma aldosterone or corticosterone levels. Nifedipine and CGP 28392 treatment of nonpregnant and pregnant animals, respectively, did not modify the response of aortic rings to KCl. These results show that VDCC activation caused hypertension, which modified the extent of the decrease in blood pressure at the end of pregnancy.