PMC

Immunofluorescence staining for vWF in HUVECs (a) and HMVEC-L cells (b). Cells were fixed, permeabilized, and stained using anti-vWF antibodies as described in Figure . Both cell types contain similar numbers of typical WP bodies. Bar, 20 μm.

Expression of V2R.EGFP or A295.EGFP in HUVECs and immunofluorescence staining of the cells for vWF. HUVECs were transfected with pV2R.EGFP or pA295.EGFP. Twenty-four hours later they were fixed, permeabilized, and stained using anti-vWF antibodies followed by rhodamine-conjugated goat anti-rabbit antibodies. Images show EGFP fluorescence from HUVECs expressing V2R.EGFP (a) or A295.EGFP (b) and distribution of vWF in WP bodies in the same cells (c) and (d), respectively. Membrane labeling with EGFP was seen only in cells expressing V2R.EGFP, whereas perinuclear labeling was seen with both constructs. Bar, 20 μm.

DDAVP-mediated vWF secretion from cells expressing V2R.EGFP. HUVECs were transfected with pV2R.EGFP or pA295.EGFP. Forty-eight hours later, EGFP-positive and -negative cells were sorted by FACS and seeded into 48-well plates. Seventy-two hours later they were incubated for 1 hour in KRBH containing 100 μM IBMX alone (open bars) or in the presence of either 0.1 μM DDAVP (shaded bars), or 10 μM forskolin (filled bars). Then, vWF in the media was measured by ELISA. The results are shown as a percentage of the vWF secreted in basal conditions (IBMX alone). The data are means ± SEM for six independent experiments. (^AP < 0.01; ^BP < 0.001 by Student’s paired t test).

Vasopressin-induced, cAMP-dependent vWF secretion from HMVEC-L cells. (a) Confluent HMVEC-L grown in 12-well plates were incubated in KRBH for 30 minutes in the presence of DDAVP (1 μM), AVP (1 μM), forskolin (Forsk; 10 μM), or KRBH alone (Contr). The experiments were performed in the absence or the presence of 100 μM IBMX, and vWF released in the incubation medium was assayed by ELISA. Results are shown as relative values, release from control cells being defined as 100%. The data are the mean ± SEM of 10 (DDAVP and forskolin) or four (AVP) experiments. (^AP < 0.05 by Student’s paired t test). (b) Confluent HMVEC-L grown in 12-well plates were incubated in KRBH for 30 minutes at 37^oC in the presence or absence of 0.2 mM Rp-8CPT-cAMPS (Rp). Then, 100 μM IBMX (I), 1 μM DDAVP, 10 μM forskolin (Forsk), or 0.5 U/mL thrombin (Thr) were added and the incubation continued for 30 minutes. Results are expressed in relative values, release from control cells being defined as 100%. The vWF released during the preincubation was subtracted, assuming continuous basal release over 1 hour. Data are the mean ± SEM of five experiments. (^AP < 0.02 by Student’s paired t test).

DDAVP mediates vWF secretion by an increase in [cAMP]_i in cells expressing V2R.EGFP. (a) DDAVP-mediated increase in [cAMP]_i in cells expressing V2R.EGFP. HUVECs transfected with V2R were handled as in Figure . Sorted EGFP-negative cells were used as controls. At the end of the incubations (with 100 μM IBMX, alone [open bars], in the presence of 0.1 μM DDAVP [shaded bars], or of 10 μM forskolin [filled bars]) cAMP was extracted and quantified by radioimmunoassay. The results are shown as a percentage of the [cAMP]_i in basal conditions (IBMX alone). The data are means ± SEM for four independent experiments. (^AP < 0.05; ^BP < 0.01 by Student’s paired t test). (b) Inhibition of DDAVP-mediated vWF secretion from cells expressing V2R.EGFP by the PKA inhibitor Rp-8CPT-cAMPS. Seventy-two hours after FACS sorting, V2R.EGFP-positive cells were preincubated for 30 minutes in the absence (filled bars) or presence (shaded bars) of 0.5 mM Rp-8CPT-cAMPS in KRBH. They were then incubated for 1 hour in the presence of 100 μM IBMX alone or together with either 0.1 μM DDAVP or 10 μM forskolin (as in Figure ). Then, vWF in the media was measured by ELISA. The results are shown as a percentage of the vWF secreted in basal conditions (IBMX alone). The data are means ± SEM for five independent experiments. (^AP < 0.05 by Student’s paired t test).

Expression of V2R in HMVEC-L and human lung. (a) RT-PCR amplification from human kidney, HMVEC-L, HUVECs, and human lung polyA⁺. The expected 857-bp fragment (arrow) was amplified from V2R mRNA using primers V2Rc and V2Rd. The RT-PCR reaction was performed in absence or presence of RT. First lane: HindIII/EcoRI lambda marker. The gel was stained with ethidium bromide. (b) Southern blot analysis of the RT-PCR products amplified from kidney, lung, HUVECs, and HMVEC, using a probe derived from V2R cDNA. Negative control: RT-PCR reaction performed in absence of template. The arrow indicates V2R transcript (857 bp); the band visible just above represents genomic V2R DNA. Five times less of the RT-PCR reaction was loaded on the gel for both kidney and lung compared with HUVECs and HMVEC-L. (c) RT-PCR amplification of reversed transcribed polyA⁺ RNA from human lung from two different donors (lanes 3 and 4) using primers STV2 and V2RBamHI. The RT-PCR reaction was performed without (lanes 3 and 4) or with prior pretreatment of the samples with RNase-free DNaseI (lane 5). The size of the fragment expected to be amplified from V2R mRNA is 1,144 bp, as verified by amplification of plasmid DNA carrying the full-length coding region of the V2R (lane 2). Lane 1: RT-PCR amplification of plasmid DNA harboring the genomic DNA of the V2R (expected size: 1,611 bp). The gel was stained with ethidium bromide.