Christine Delporte

Abnormal Distribution of Aquaporin-5 Water Channel Protein in Salivary Glands from Sjögren’s Syndrome Patients

Patients with Sjögren’s syndrome (SS) suffer from deficient secretion of saliva due to an autoimmune destruction of salivary glands, however, glandular dysfunction also occurs without destruction. Based upon its abnormal distribution in SS salivary glands, a potential role for the water channel protein aquaporin-5 (AQP5) is proposed in the pathogenesis of SS. The immunohistochemical distribution of AQP5 was compared in minor salivary gland biopsies obtained from women after informed consent: primary SS (53.2 ± 14 years old, n = 10), healthy volunteers (46.2 ± 17 years old, n = 10), patients with sarcoidosis (37 and 48 years old), and patients with non-specific sialoadenitis (54 and 61 years old). Biopsies from normal subjects revealed AQP5 primarily at the apical membrane of the salivary gland acinus. In contrast, biopsies from SS patients revealed AQP5 primarily at the basal membranes of the acinus. The AQP5 distribution in biopsies from patients with other dry mouth disorders, such as non-specific sialoadenitis or sarcoidosis, was similar to biopsies from control subjects. Computer-assisted microscopy was performed to quantitatively evaluate AQP5 distribution in the immunoreactive acini of both SS and control subjects. Biopsies from SS patients had higher labeling indices (percentage of acinus area immunoreactive for AQP5) at the basal membrane when compared with biopsies from control subjects. In contrast, biopsies of SS patients exhibited lower labeling indices at the apical membrane when compared with biopsies from control subjects. To verify the specificity of the AQP5 antibody, Western blot analysis was performed on membranes from Xenopus oocytes injected with AQP5 cRNA or on membranes from minor salivary glands of control subjects and SS patients. In each case, the immunoblots had a 27 kd band, corresponding to the expected molecular weight of AQP5. Abnormal distribution of AQP5 in salivary gland acini is likely to contribute to the deficiency of fluid secretion, which is a defining feature of Sjögren’s syndrome.

Cloning of Trp1β isoform from rat brain: immunodetection and localization of the endogenous Trp1 protein

The Trp gene product has been proposed as a candidate protein for the store-operated Ca2+channel, but the Trp protein(s) has not been identified in any nonexcitable cell. We report here the cloning of a rat brain Trp1β cDNA and detection and immunolocalization of the endogenous and expressed Trp1 protein. A 400-bp product, with >95% homology to mouse Trp1, was amplified from rat submandibular gland RNA. Rat-specific primers were used for cloning of a full-length rat brain Trp1β cDNA (rTrp1), encoding a protein of 759 amino acids. Northern blot analysis demonstrated the transcript in several rat and mouse tissues. The peptide (amino acids 523-536) was used to generate a polyclonal antiserum. The affinity-purified antibody 1) immunoprecipitated human Trp1 (hTrp1) from transfected HEK-293 cells, 2) reacted with a protein of ∼92 kDa, but not with hTrp3, in membranes of hTrp3-expressing HEK-293 cells, and 3) reacted with proteins of 92 and 56 kDa in human and rat brain membranes. Confocal microscopy and cell fractionation demonstrated that endogenous and expressed hTrp1 and expressed hTrp3 proteins were localized in the plasma membrane of HEK-293 cells, consistent with their proposed role in Ca2+ influx. The data demonstrate for the first time the presence of Trp1 protein in a nonexcitable cell.

The pituitary adenylate cyclase activating polypeptide (PACAP I) and VIP (PACAP II VIP1) receptors stimulate inositol phosphate synthesis in transfected CHO cells through interaction with different G proteins.

The PACAP receptor (PACAP I receptor, selective for PACAP) and the PACAP II VIP1 receptor (recognizing PACAP and VIP with the same high affinity) were stably expressed in Chinese Hamster Ovary (CHO) cells. Cell lines expressing different receptor densities, as measured by binding saturation curves, were selected. Inositol phosphate production was stimulated dose dependently in all the cell lines by PACAP and VIP, and the order of potency of the agonists was identical to that of high affinity receptor occupancy. The stimulatory effect of a saturating peptide concentration was proportional to the total receptor density. At similar receptor densities, however, the PACAP receptor mediated stimulation was higher than the VIP receptor-mediated stimulation. Pretreatment of the cells with pertussis toxin for 8 h had no effect on receptor densities, did not alter the PACAP stimulated inositol phosphate synthesis by the cells expressing the PACAP I receptor but markedly inhibited the response of the cells expressing the PACAP II VIP1 receptor. Thus, the present results indicate that the two G(s)-coupled PACAP I and PACAP II VIP1 receptors may stimulate IP production. The maximal stimulation depended on the number of receptor expressed; the PACAP I and PACAP II VIP1 receptors probably activated the phospholipase C through G proteins of the G(q), and of the G(i)/G(o) families, respectively.

Murine 3T3-L1 adipocyte cell differentiation model: validated reference genes for qPCR gene expression analysis.

Background Analysis of gene expression at the mRNA level, using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), mandatorily requires reference genes (RGs) as internal controls. However, increasing evidences have shown that RG expression may vary considerably under experimental conditions. We sought for an appropriate panel of RGs to be used in the 3T3-L1 cell line model during their terminal differentiation into adipocytes. To this end, the expression levels of a panel of seven widely used RG mRNAs were measured by qRT-PCR. The 7 RGs evaluated were ß-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl-transferase I (HPRT), ATP synthase H+ transporting mitochondrial F1 complex beta subunit (ATP-5b), tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein, zeta polypeptide (Ywhaz), Non-POU-domain containing octamer binding protein (NoNo), and large ribosomal protein L13a (RPL). Methodology/Principal Findings Using three Excel applications, GeNorm, NormFinder and BestKeeper, we observed that the number and the stability of potential RGs vary significantly during differentiation of 3T3-L1 cells into adipocytes. mRNA expression analyses using qRT-PCR revealed that during the entire differentiation program, only NoNo expression is relatively stable. Moreover, the RG sets that were acceptably stable were different depending on the phase of the overall differentiation process (i.e. mitotic clonal expansion versus the terminal differentiation phase). RPL, ACTB, and Ywhaz, are suitable for terminal differentiation, whereas ATP-5b and HPRT, are suitable during mitotic clonal expansion. Conclusion Our results demonstrate that special attention must be given to the choice of suitable RGs during the various well defined phases of adipogenesis to ensure accurate data analysis and that the use of several RGs is absolutely required. Consequently, our data show for the first time, that during mitotic clonal expansion, the most suitable RGs are ATP-5b, NoNo and HPRT, while during terminal differentiation the most suitable RGs are, NoNo, RPL, ACTB and Ywhaz.

Discovery of a potent atrial natriuretic peptide antagonist for ANPA receptors in the human neuroblastoma NB-OK-1 cell line

The effects of seven competitive atrial natriuretic peptide (ANP) receptor antagonists were compared on cultured human neuroblastoma NB-OK-1 cells expressing exclusively ANPA receptors, by evaluating their capacity to inhibit [125I]ANP binding and to suppress ANP-stimulated cyclic GMP elevation. In ANP analogues with a shortened Cys7-Cys18 bridge, Asp13 and a hydrophobic Tic residue at position 16 expressed antagonistic activity, while Ala16 provoked lower antagonistic potency and Phe16 induced receptor activation. The binding affinity of A71915 ([Arg6, Cha8]ANP-(6-15)-D-Tic-Arg-Cys-NH2), the most potent antagonist (with a pKi of 9.18 and a pA2 of 9.48) was only 22 times less lower than that of the agonist ANP-(1-28).

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide stimulate two signaling pathways in CHO cells stably transfected with the selective type I PACAP receptor

The properties of the pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor were studied on a clone of Chinese hamster ovary cells (CHO) stably transfected with the recombinant receptor. PACAP(1-27), PACAP(1-38) and VIP inhibited [125I-acetyl-His1]PACAP (1-27) binding, stimulated cyclic AMP and inositol phosphates production and induced [Ca2+]i increase with the same order of potency: PACAP(1-27) = PACAP(1-38) > VIP. The concentrations required for half maximal receptor occupancy, IP3- and [Ca2+]i increase were not different for both PACAPs (1 nM) and 100-fold higher than those required for cyclic AMP increase (0.010 nM). These data suggest that the occupancy of a portion of the total receptors available was sufficient for maximal cyclic AMP production but not for maximal IP3 production. It is concluded that the possibility of the type I PACAP receptor being coupled to a transduction pathway is not located at the level of the ligand but rather at the level of the G-proteins.

Contrasting effects of glycerol and urea transport on rat pancreatic beta-cell function.

Background/aims: Pancreatic β-cell function is influenced by changes in cell volume. Such volume changes depend on water permeability of the plasma membrane, conferred in part by aquaporins. Islet cells express aquaporin 7 (AQP7), which is permeable to urea and glycerol in addition to water. We therefore investigated the effects of glycerol and urea on rat pancreatic β-cell function. Methods: Electrical activity and whole-cell current were studied using the perforated patch technique. Cell volume was measured by video-imaging and insulin release by radioimmunoassay. Aquaporin 7 expression was studied by RT-PCR, Western blot and double fluorescent immunolabelling. Results: The isosmotic addition of glycerol and urea resulted in depolarization of the plasma membrane and electrical activity, accompanied by β-cell swelling, activation of the volume-regulated anion channel (VRAC) and insulin release. However, the effects of glycerol, in contrast to urea, persisted throughout exposure to the osmolyte. Glycerol also caused β-cell activation when added hyperosmotically. A non-metabolizable glycerol analogue had comparable effects to urea on β-cells. The expression of AQP7 was demonstrated in rat β-cells. Conclusion: Glycerol and urea can activate β-cells via their rapid uptake across the β-cell plasma membrane, possibly via AQP7. This results in cell swelling, VRAC activation, electrical activity and insulin release. Glycerol appears to exert an additional effect, possibly related to its intracellular metabolism.

Contrasting effects of PACAP and carbachol on [Ca2+]i and inositol phosphates in human neuroblastoma NB-OK-1 cells

The effects of PACAPs on [Ca2+]i were compared to those of carbachol in human neuroblastoma NB-OK-1 cells. PACAP(1-27) and PACAP(1-38) increased [Ca2+]i in a biphasic manner: a transient rise and a secondary plateau. The transient phase reflected the mobilization of [Ca2+]i pool(s) via the inositol phosphate pathway. The modest sustained plateau required extracellular Ca2+. Carbachol also increased [Ca2+]i in a biphasic manner, but it mobilized intracellular Ca2+ pool(s) with a higher efficacy than PACAPs, then greatly increased Ca2+ entry, this being accompanied by a more marked and prolonged elevation of IP3 and IP4 than with PACAPs. It is likely that cAMP-mediated phosphorylations due to PACAPs facilitated desensitization at the PACAP receptor-phospholipase C level, so that there was less Ca2+ handling through PACAP receptors than with muscarinic M1 receptors.

Atrial natriuretic peptide binds to ANP-R1 receptors in neuroblastoma cells or is degraded extracellularly at the Ser-Phe bond

ANP-R1 receptors for atrial natriuretic peptide (ANP) showed the following rank order of affinity in intact human neuroblastoma cells NB-OK-1: human ANP-(99-126) approximately human ANP-(102-126) approximately rat ANP-(99-126) (K1 17-32 pM) > human ANP-(103-126) > porcine brain natriuretic peptide (BNP). Analogues truncated at the C-terminal extremity or devoid of a disulphide bridge, such as rat ANP-(103-123), rat C-ANP-(102-121), rat ANP-(111-126), rat ANP-(99-109) and rat [desCys105,Cys121]ANP-(104-126) and chicken C-type natriuretic peptide, were not recognized. The occupancy of these high affinity ANP-R1 receptors led to marked cyclic GMP accumulation in the presence of 3-isobutyl 1-methylxanthine. An ectoenzymic activity, partly shed in the incubation medium, provoked the stepwise release of Phe-Arg-[125I]Tyr, Arg-[125I]Tyr and [125I]Tyr from rat [125I]ANP-(99-126), at an optimal pH of 7.0. Its inhibition by 1,10-phenanthroline, EDTA and bacitracin but not by thiorphan suggests the contribution of at least one neutral metalloendopeptidase, distinct from EC 3.4.24.11, for which ANP showed high affinity.

A new role for aquaporin 7 in insulin secretion.

Bacgrouns/Aims: Several insulinotropic agents were recently reported to cause β-cell swelling. The possible participation of AQP7 to water transport was investigated in AQP7+/ + or AQP7-/- mice. Methods: Aquaporin expression, insulin secretion, cell swelling and electrical activity were investigated in pancreatic islets. Results: RT-PCR revealed the expression of AQP5 and AQP8 mRNA. Double immunofluorescent labeling indicated their presence in β-cells. Whilst basal insulin release from isolated pancreatic islets incubated at 2.8 mM D-glucose did not differ between AQP7+/ + or AQP7-/- mice, the secretion of insulin evoked by the omission of 50 mM NaCl, the substitution of 50 mM NaCl by 100 mM glycerol or a rise in D-glucose concentration to 8.3 mM and 16.7 mM was severely impaired in the islets from AQP7-/- mice. Yet, exposure of β-cells to either the hypotonic medium or a rise in D-glucose concentration caused a similar degree of swelling and comparable pattern of electrical activity in cells from AQP7+/ + and AQP7-/- mice. Both the cell swelling and change in membrane potential were only impaired in AQP7-/- cells when exposed to 50 mM glycerol. Conclusion: It is proposed, therefore, that AQP7 may, directly or indirectly, play a role at a distal site in the exocytotic pathway.