Targeted locus amplification reveals heterogeneity between and within CFTR genotypes and association with CFTR function in patient-derived intestinal organoids.

BACKGROUND: Cystic fibrosis (CF) disease severity can be highly variable, even between people with CF (pwCF) with similar genotypes. Here we use patient-derived intestinal organoids to study the influence of genetic variation within the cystic fibrosis transmembrane conductance regulator (CFTR) gene on CFTR function. METHODS: Organoids of F508del/class I, F508del/S1251N and pwCF with only one detected CF-causing mutation were cultured. Allele-specific CFTR variation was investigated using targeted locus amplification (TLA), CFTR function was measured using the forskolin-induced swelling assay and mRNA levels were quantified using RT-qPCR. RESULTS: We were able to distinguish CFTR genotypes based on TLA data. Additionally, we observed heterogeneity within genotypes, which we were able to link to CFTR function for S1251N alleles. CONCLUSIONS: Our results indicate that the paired analysis of CFTR intragenic variation and CFTR function can gain insights in the underlying CFTR defect for individuals where the disease phenotype does not match the CFTR mutations detected during diagnosis.

The role of rectal chloride secretion in childhood constipation.

BACKGROUND: Disturbance in fluid secretion, driven by chloride secretion, might play a role in constipation. However, disturbed chloride secretion in those patients has yet to be evaluated. Therefore, the aim of this study was to compare chloride secretion in rectal biopsies of children with functional constipation (FC) to those without constipation. METHODS: To measure changes in short circuit current (I(sc) in µA cm(-2)) reflecting chloride secretion, intestinal biopsies from children with constipation, to either exclude or diagnose Hirschsprung's disease, and from children without constipation (controls) undergoing colonoscopy for screening of familial adenomatous polyposis, juvenile polyps or inflammatory bowel disease (IBD), were compared and studied in Ussing chambers. Following electrogenic sodium absorption blockade by amiloride, chloride secretory responses to calcium-linked (histamine, carbachol) and cAMP-linked (IBMX/forskolin) secretagogues were assessed. KEY RESULTS: Ninety-six patients (46 FC) participated; nine FC patients (n = 1 congenital syndrome and n = 8 technical problems) and 13 controls (n = 6 IBD; n = 7 technical problems) were excluded. No significant difference was found in mean (±SE) basal chloride currents between children with FC and controls (9.6 ± 1.1 vs 9.2 ± 0.8; P = 0.75, respectively). Responses to calcium-linked chloride secretagogues (histamine and carbachol) were significantly higher in controls (33.0 ± 3.0 vs 24.5 ± 2.3; P = 0.03 and 33.6 ± 3.4 vs 26.4 ± 2.7; P = 0.05 following histamine and carbachol, respectively). CONCLUSIONS & INFERENCES: Calcium-linked chloride secretion is disturbed in children with FC. Whether this defect occurs at the level of histamine receptors, components of receptor-linked signal transduction pathways or basolateral Ca(2+) -sensitive K(+) channels enhancing the electrical driving force for apical chloride secretion, remains to be explored.

New clinical diagnostic procedures for cystic fibrosis in Europe.

In the majority of cases, there is no difficulty in diagnosing Cystic Fibrosis (CF). However, there may be wide variation in signs and symptoms between individuals which encourage the scientific community to constantly improve the diagnostic tests available and develop better methods to come to a final diagnosis in patients with milder phenotypes. This paper is the result of discussions held at meetings of the European Cystic Fibrosis Society Diagnostic Network supported by EuroCareCF. CFTR bioassays in the nasal epithelium (nasal potential difference measurements) and the rectal mucosa (intestinal current measurements) are discussed in detail including efforts to standardize the techniques across Europe. New approaches to evaluate the sweat gland, future of genetic testing and methods on the horizon like CFTR expression in human leucocytes and erythrocytes are discussed briefly.

Assessment of CFTR function in homozygous R117H-7T subjects.

BACKGROUND: R117H is a frequent missense mutation included in most CFTR mutation panels. However knowledge about the residual function of R117H-CFTR channels in cystic fibrosis-affected organs, e.g. airways, intestines and sweat glands is presently lacking. METHODS: We evaluated clinical CF symptoms and assessed CFTR function by sweat tests, nasal potential difference and intestinal current measurements in 2 homozygous R117H individuals (7T variant). RESULTS: The CFTR activity in airways and intestine was within the normal range. However both individuals presented with a borderline sweat test and the male patient was infertile. CONCLUSIONS: The lack of impact of the R117H mutation on chloride secretion in intestine and nose contrasts with the ~80% loss of CFTR activity reported in patch clamp studies. Apparently CFTR activity is not rate-limiting for chloride secretion in both tissues at levels >20% of normal, or compensatory factors may operate that are absent in heterologous host cells in vitro.

Alterations in the proteome of the NHERF2 knockout mouse jejunal brush border membrane vesicles.

To identify additional potential functions for the multi-PDZ domain containing protein Na+/H+ exchanger regulatory factor 2 (NHERF2), which is present in the apical domain of intestinal epithelial cells, proteomic studies of mouse jejunal villus epithelial cell brush border membrane vesicles compared wild-type to homozygous NHERF2 knockout FVB mice by a two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-iTRAQ approach. Jejunal architecture appeared normal in NHERF2 null in terms of villus length and crypt depth, Paneth cell number, and microvillus structure by electron microscopy. There was also no change in proliferative activity based on BrdU labeling. Four brush border membrane vesicles (BBMV) preparations from wild-type mouse jejunum were compared with four preparations from NHERF2 knockout mice. LC-MS/MS identified 450 proteins in both matched wild-type and NHERF2 null BBMV; 13 proteins were changed in two or more separate BBMV preparations (9 increased and 4 decreased in NHERF2 null mice), while an additional 92 proteins were changed in a single BBMV preparation (68 increased and 24 decreased in NHERF2 null mice). These proteins were categorized as 1) transport proteins (one increased and two decreased in NHERF2 null); 2) signaling molecules (2 increased in NHERF2 null); 3) cytoskeleton/junctional proteins (4 upregulated and 1 downregulated in NHERF2 null); and 4) metabolic proteins/intrinsic BB proteins) (2 upregulated and 1 downregulated in NHERF2 null). Immunoblotting of BBMV was used to validate or extend the findings, demonstrating increase in BBMV of NHERF2 null of MCT1, coronin 3, and ezrin. The proteome of the NHERF2 null mouse small intestinal BB demonstrates up- and downregulation of multiple transport proteins, signaling molecules, cytoskeletal proteins, tight junctional and adherens junction proteins, and proteins involved in metabolism, suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.

Alterations in the proteome of the NHERF1 knockout mouse jejunal brush border membrane vesicles.

Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCß3, E-cadherin, p120, ß-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.

Defective jejunal and colonic salt absorption and alteredNa(+)/H (+) exchanger 3 (NHE3) activity in NHE regulatory factor 1 (NHERF1) adaptor protein-deficient mice.

We investigated the role of the Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na(+) absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.

Regulation of the cell swelling-activated chloride conductance by cholesterol-rich membrane domains.

AIM: The role of high cholesterol-containing microdomains in the signal transduction cascade leading to the activation of volume-regulated anion channels (VRACs) was studied. METHODS: Osmotic cell swelling-induced efflux of 125I- was determined in human epithelial Intestine 407 cells and in skin fibroblasts obtained from healthy controls or Niemann-Pick type C (NPC) patients. Cellular cholesterol content was modulated by pre-incubation with 2-hydroxypropyl-beta-cyclodextrin in the presence of acceptor lipid vesicles. RESULTS: Osmotic cell swelling of human Intestine 407 cells leads to the rapid activation of a compensatory anion conductance. Treatment of the cells with cyclodextrin enhanced the response to submaximal hypotonic stimulation by approx. twofold, but did not further increase the efflux elicited by a saturating stimulus. In contrast, the volume-sensitive anion efflux was markedly inhibited when cholesterol-loaded cyclodextrin was used. Potentiation of the response by cholesterol depletion was maintained in caveolin-1 deficient Caco-2 colonocytes as well as in sphingomyelinase-treated Intestine 407 cells, indicating that cholesterol-rich microdomains are not crucially involved. However, treatment of the cells with progesterone, an inhibitor of NPC1-dependent endosomal cholesterol trafficking, not only markedly reduced the hypotonicity-provoked anion efflux, but also prevented its potentiation by cyclodextrin. In addition, the volume-sensitive anion efflux from human NPC skin fibroblasts was significantly smaller when compared with control fibroblasts. CONCLUSIONS: The results support a model of regulatory volume decrease involving recruitment of volume-sensitive anion channels from intracellular compartments to the plasma membrane.

[Expression of the cystic fibrosis gene in the lungs]. / Expressie van het cystische-fibrose-gen in de longen.

Cystic fibrosis (CF) is one of the most common hereditary diseases with a potentially lethal outcome. CF is caused by mutations in a gene on chromosome 7 that encodes for a polypeptide called 'cystic fibrosis transmembrane conductance regulator' (CFTR). Pulmonary complications in CF are a direct consequence of a fault in the production or function of CFTR. The consequences of these faults are a change in the viscosity and volume of the air-surface liquid in the airways and possible malfunctioning of the immunological defence system. The result is stasis of mucus and obstruction of the smaller airways. Secondary to this process, a (chronic) infection with an uncontrolled inflammatory response, leads to destruction of lung tissue and a reduction in lung function. Mutations in CFTR can be classified in five types, which reflect the level of production or the function of CFTR. This classification permits discrimination between mild and severe mutations. The clinical image and the course of CF are often related to the combination of mutations present. This may explain, in part, why some CF patients are diagnosed at a later age with a normal sweat test and a clinical image that is predominantly limited to respiratory symptoms.

Chloride conductance and genetic background modulate the cystic fibrosis phenotype of Delta F508 homozygous twins and siblings.

To investigate the impact of chloride (Cl(-)) permeability, mediated by residual activity of the cystic fibrosis transmembrane conductance regulator (CFTR) or by other Cl(-) channels, on the manifestations of cystic fibrosis (CF), we determined Cl(-) transport properties of the respiratory and intestinal tracts in Delta F508 homozygous twins and siblings. In the majority of patients, cAMP and/or Ca(2+)-regulated Cl(-) conductance was detected in the airways and intestine. Our finding of cAMP-mediated Cl(-) conductance suggests that, in vivo, at least some Delta F508 CFTR can reach the plasma membrane and affect Cl(-) permeability. In respiratory tissue, the expression of basal CFTR-mediated Cl(-) conductance, demonstrated by 30% of Delta F508 homozygotes, was identified as a positive predictor of milder CF disease. In intestinal tissue, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid-insensitive (DIDS-insensitive) Cl(-) secretion, which is indicative of functional CFTR channels, correlated with a milder phenotype, whereas DIDS-sensitive Cl(-) secretion was observed mainly in more severely affected patients. The more concordant Cl(-) secretory patterns within monozygous twins compared with dizygous pairs imply that genes other than CFTR significantly influence the manifestation of the basic defect.

Regulated trafficking of the CFTR chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR), the ABC transporter encoded by the cystic fibrosis gene, is localized in the apical membrane of epithelial cells where it functions as a cyclic AMP-regulated chloride channel and as a regulator of other ion channels and transporters. Whereas a key role of cAMP-dependent phosphorylation in CFTR-channel gating has been firmly established, more recent studies have provided clear evidence for the existence of a second level of cAMP regulation, i.e. the exocytotic recruitment of CFFR to the plasma membrane and its endocytotic retrieval. Regulated trafficking of the CFTR Cl- channel has sofar been demonstrated only in a subset of CFTR-expressing cell types. However, with the introduction of more sensitive methods to measure CFTR cycling and submembrane localization, it might turn out to be a more general phenomenon that could contribute importantly to both the regulation of CFTR-mediated chloride transport itself and to the regulation of other transporters and CFTR-modulated cellular functions. This review aims to summarize the present state of knowledge regarding polarized and regulated CFTR trafficking and endosomal recycling in epithelial cells, to discuss present gaps in our understanding of these processes at the cellular and molecular level, and to consider its possible implications for cystic fibrosis.

Residual chloride secretion in intestinal tissue of deltaF508 homozygous twins and siblings with cystic fibrosis. The European CF Twin and Sibling Study Consortium.

BACKGROUND & AIMS: Cholinergic stimulation of chloride secretion is impaired in the intestines of patients with cystic fibrosis (CF). However, intestinal chloride secretion has been observed in patients with mild CF mutations. The aim of this study was to investigate residual Cl(-) secretion in the intestine of DeltaF508 homozygous CF patients, and examine the contribution of cystic fibrosis transmembrane conductance regulator (CFTR) and alternative Cl(-) conductances. Twins and siblings with identical CFTR genotypes were investigated to determine the impact of factors other than CFTR on chloride secretion. METHODS: Chloride secretion in rectal tissue was investigated by applying Ca(2+) and adenosine 3',5'-cyclic monophosphate (cAMP)-linked agonists before and after the inhibition of alternative Cl(-) conductances with 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS). RESULTS: cAMP-mediated Cl(-) secretion was observed in 73% of patients, and 20% showed DIDS-sensitive Ca(2+)-activated Cl(-) secretion. This DIDS-sensitive alternative chloride conductance was seen only in CF patients who also responded to cAMP agonists. Chloride secretion was more concordant within monozygous twins than within dizygous pairs. CONCLUSIONS: These results suggest the presence of CFTR-mediated Cl(-) secretion in a subgroup of patients, implying that a portion of deltaF508 CFTR can be processed in vivo and function as a chloride channel in the apical membrane of intestinal cells. Moreover, a considerable number of deltaF508 homozygous patients express chloride conductances other than CFTR in their intestinal epithelia.

Differential role of cyclic GMP-dependent protein kinase II in ion transport in murine small intestine and colon.

BACKGROUND & AIMS: The aim of this study was to determine the role of guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (cGK) type II in intestinal fluid homeostasis under basal conditions and following exposure to cGMP-linked secretagogues, e.g., Escherichia coli heat-stable enterotoxin (STa) and guanylin. METHODS: Fluid and ion transport was determined in different segments of the intestine of wild-type and cGK II-deficient mice by ligated loop assays in vivo, and by short-circuit current and isotope flux measurements in vitro. RESULTS: Small intestinal fluid absorption in vivo was enhanced in cGK II-deficient mice under basal conditions and in the presence of STa. Furthermore, STa, guanylin, and 8-pCPT-cGMP stimulation of electrogenic anion secretion and inhibition of Na(+) absorption in vitro were markedly reduced in the small intestine from cGK II -/- mice but not in proximal colon. The type III phosphodiesterase inhibitor amrinone mimicked STa action in cGK II -/- mice, and also stimulated ion secretion in humans. CONCLUSIONS: This study shows that the cGMP/cGK II pathway regulates fluid homeostasis in the small intestine under basal conditions and mediates STa effects by both increasing anion secretion and inhibiting Na(+) absorption. It also demonstrates the presence of a cGK II-independent pathway for STa/cGMP-provoked secretion predominantly in the colon, which possibly involves a cGMP-inhibitable phosphodiesterase and/or activation of the cAMP-dependent protein kinase pathway.

Pitfalls in the immunohistochemical localization of the cystic fibrosis transmembrane conductance regulator in paraffin embedded sweat glands.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the hereditary disease cystic fibrosis. The most frequent mutant deltaF508 has been shown in vitro to be retained in the endoplasmic reticulum. Ex vivo studies using immunohistochemical labelling in cryofixed skin biopsies have confirmed the mislocalization of deltaF508 CFTR in sweat glands. The purpose of this study was to test CFTR antibodies in paraffin-embedded skin biopsies to take advantage of the superior tissue preservation as compared to cryofixation. A panel of 7 CFTR antibodies was applied to skin sections of healthy controls and of cystic fibrosis patients homozygous for the deltaF508 mutation. Sweat gland labelling consistent with CFTR localization and different between control and cystic fibrosis tissue was obtained with 2 antibodies. Conventional staining controls confirmed the labelling specificity. The antibodies were subsequently tested in a series of 237 sections of 16 biopsy specimens. However, the sweat gland labelling pattern proved not to be dependent on CFTR genotype. This finding was the sole indicator of non-specificity of the staining which was revealed only by the size of our random sample. Our results emphasize that CFTR immunolabelling following formalin fixation has to be interpreted with the utmost caution.

Protein N-myristoylation: critical role in apoptosis and salt tolerance.

N-myristoylation is a covalent protein modification that can promote the association of proteins with membranes. De Jonge, Hogema, and Tilly discuss how N-myristoylation may be involved in triggering Fas ligand-induced apoptosis in mammals, and in adapting to conditions of high salt in plants. The pro-apoptotic protein BID is unique in that its proteolytic cleavage product, tBID, is posttranslationally myristoylated. In contrast, the plant accessory protein SOS3 undergoes "classical" cotranslational N-myristoylation. N-myristoylation is essential for the proper functioning of these proteins in regulating the signaling pathways (apoptosis and adaptation to salt stress, respectively) in which they are involved.

Osmotic cell swelling-induced ATP release mediates the activation of extracellular signal-regulated protein kinase (Erk)-1/2 but not the activation of osmo-sensitive anion channels.

Human intestine 407 cells respond to hypo-osmotic stress by the rapid release of ATP into the extracellular medium. A difference in the time course of activation as well as in the sensitivity to cytochalasin B treatment and BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester] loading suggests that ATP leaves the cell through a pathway distinct from volume-regulated anion channels. To evaluate a putative role for nucleotides as autocrinic/paracrinic factors in osmotic signalling, the effects of extracellular ATP on the regulation of volume-sensitive anion channels as well as on the hypotonicity-induced activation of extracellular signal-regulated protein kinases (Erk-1/2) were investigated. Micromolar concentrations of ATP were unable to elicit an isotope efflux from (125)I(-)-loaded cells by itself, but strongly potentiated the hypotonicity-provoked anion efflux through a Ca(2+)-dependent mechanism. The order of potency of nucleotides (ATP = UTP = ATP[S] > ADP = AMP >> adenosine = cAMP) indicated the involvement of P2Y(2) receptors. In contrast, millimolar concentrations of ATP markedly inhibited both the osmotically induced isotope efflux and whole-cell Cl(-) currents. Inhibition of whole-cell Cl(-) currents, not only by millimolar ATP but also by the purinoceptor antagonists suramin and reactive blue, was observed most prominently at depolarizing holding potentials, suggesting a direct interaction with volume-sensitive Cl(-) channels rather than interaction with purinoceptors. Both ATP and UTP, at submicromolar levels, were found to act as potent activators of Erk-1/2 in intestine 407 cells. Addition of the ATP hydrolase apyrase to the bath greatly reduced the hypotonicity-induced Erk-1/2 activation, but did not affect the swelling-induced isotope efflux or whole-cell Cl(-) currents. Furthermore, pre-treatment with suramin or reactive blue almost completely prevented the hypo-osmotic activation of Erk-1/2. The results indicate that extracellularly released ATP functions as an autocrinic/paracrinic factor that mediates hypotonicity-induced Erk-1/2 activation but does not serve as an activator of volume-sensitive compensatory Cl(-) currents.

LLC-PK1 cells as a model system to study proximal tubule transport of water and other compounds relevant for renal stone disease.

LLC-PK1 cells were cultured on a permeable support in a two-compartment culture system. Confluent monolayers received an ultrafiltrate-like solution at the apical side and a plasma-like solution at the basolateral side. The distribution of various solutes, including phosphate, calcium, and oxalate over both compartments was measured in time. The transport of water was monitored by alterations in fluid concentrations of radiolabeled inulin. Bicarbonate, glucose, and phosphate were transported rapidly from the apical to basolateral side of the monolayer. Sodium and chloride were reabsorbed without major consequences for the osmolality in the apical and basal fluid. Calcium and potassium were also reabsorbed, but to a smaller extent than sodium. The luminal concentration of oxalate gradually increased to values that were at least three times higher (12.0+/-0.4 micromol/l) than those in the contraluminal fluid (3.8+/-0.1 micromol/l). However, since the luminal rise of oxalate completely matched the rise of inulin in the apical fluid this appeared to be the passive consequence of active water reabsorption rather than of net directed oxalate transport. The LLC-PK1 model could prove useful to study the regulation of proximal tubule water transport and its effect on luminal stone salt concentrations under different physiological conditions.

Atrial natriuretic peptide-stimulated Ca2+ reabsorption in rabbit kidney requires membrane-targeted, cGMP-dependent protein kinase type II.

Atrial natriuretic peptide (ANP) and nitric oxide (NO) are key regulators of ion and water transport in the kidney. Here, we report that these cGMP-elevating hormones stimulate Ca2+ reabsorption via a novel mechanism specifically involving type II cGMP-dependent protein kinase (cGK II). ANP and the NO donor, sodium nitroprusside (SNP), markedly increased Ca2+ uptake in freshly immunodissected rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). Although readily increasing cGMP, ANP and SNP did not affect Ca2+ and Na+ reabsorption in primary cultures of these segments. Immunoblot analysis demonstrated that cGK II, and not cGK I, was present in freshly isolated CNT and CCD but underwent a complete down-regulation during the primary cell culture. However, upon adenoviral reexpression of cGK II in primary cultures, ANP, SNP, and 8-Br-cGMP readily increased Ca2+ reabsorption. In contrast, no cGMP-dependent effect on electrogenic Na+ transport was observed. The membrane localization of cGK II proved to be crucial for its action, because a nonmyristoylated cGK II mutant that was shown to be localized in the cytosol failed to mediate ANP-stimulated Ca2+ transport. The Ca2+-regulatory function of cGK II appeared isotype-specific because no cGMP-mediated increase in Ca2+ transport was observed after expression of the cytosolic cGK Ibeta or a membrane-bound cGK II/Ibeta chimer. These results demonstrate that ANP- and NO-stimulated Ca2+ reabsorption requires membrane-targeted cGK II.

Evidence for involvement of 17beta-estradiol in intestinal calcium absorption independent of 1,25-dihydroxyvitamin D3 level in the Rat.

The sex steroid 17beta-estradiol (17beta-E2) has a broad range of actions, including effects on calcium and bone metabolism. This study with 3-month-old Brown Norway rats was designed to investigate the role of 17beta-E2 in the regulation of calcium homeostasis. Rats were divided in four groups, sham-operated, ovariectomized (OVX), and OVX supplemented with either a 0.025-mg or 0.05-mg 17beta-E2 pellet implanted subcutaneously. After 4 weeks, in none of the groups was serum calcium, phosphate, or parathyroid hormone altered compared with the sham group, while only in the OVX rats was a significant reduction in urinary calcium found. Bone mineral density and osteocalcin were modified, as can be expected after OVX and 17beta-E2 supplementation. OVX resulted in a nonsignificant increase in serum 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Supplementation with either one of the 17beta-E2 dosages resulted in an 80% reduction of 1,25(OH)2D3 and only a 20% reduction in 25-hydroxyvitamin D3 levels. OVX, as well as supplementation with 17beta-E2, did not affect serum levels of vitamin D binding protein. As a consequence, the estimated free 1,25(OH)2D3 levels were also significantly decreased in the 17beta-E2-supplemented group compared with the sham and OVX groups. Next, the consequences for intestinal calcium absorption were analyzed by the in situ intestinal loop technique. Although the 1,25(OH)2D3 serum level was increased, OVX resulted in a significant decrease in intestinal calcium absorption in the duodenum. Despite the strongly reduced 1,25(OH)2D3 levels (18. 1 +/- 2.1 and 16.4 +/- 2.2 pmol/l compared with 143.5 +/- 29 pmol/l for the OVX group), the OVX-induced decrease in calcium absorption could partially be restored by supplementation with either 0.025 mg or 0.05 mg of 17beta-E2. None of the treatments resulted in a significant change in calcium handling in the jejunum, although the trends were similar as those observed in the duodenum. 17beta-E2 did not change the VDR levels in both the intestine and the kidney. In conclusion, the present study demonstrates that 17beta-E2 is positively involved in intestinal calcium absorption, and the data strengthen the assertion that 17beta-E2 exerts this effect independent of 1,25(OH)2D3. In general, 17beta-E2 not only affects bone turnover but also calcium homeostasis via an effect on intestinal calcium absorption. (J Bone Miner Res 1999;14:57-64)

Osmotic swelling-induced activation of the extracellular-signal-regulated protein kinases Erk-1 and Erk-2 in intestine 407 cells involves the Ras/Raf-signalling pathway.

Human Intestine 407 cells respond to hypo-osmotic stress with a rapid stimulation of compensatory ionic conductances accompanied by a transient increase in the activity of the extracellular-signal-regulated protein kinases Erk-1 and Erk-2. In this study, we examined the upstream regulators of hypotonicity-induced Erk-1/Erk-2 activation and their possible role in cell-volume regulation. The hypotonicity-provoked Erk-1/Erk-2 activation was greatly reduced in cells pretreated with the specific mitogen-activated/Erk-activating kinase inhibitor PD098059 and was preceded by a transient stimulation of Raf-1. Pretreatment of the cells with PMA, GF109203X, wortmannin or Clostridium botulinum C3 exoenzyme did not appreciably affect the hypotonicity-provoked Erk-1/Erk-2 stimulation, suggesting the osmosensitive signalling pathway to be largely independent of protein kinase C and p21(rho). In contrast, expression of dominant negative RasN17 completely abolished the hypotonicity-induced Erk-1/Erk-2 activation. Stimulation of the swelling-induced ion efflux was independent of activation of these mitogen-activated protein kinases, as revealed by hypotonicity-provoked isotope efflux from 125I-- and 86Rb+-loaded cells after pretreatment with PD098059 and after expression of RasN17. In addition, the epidermal-growth-factor-induced potentiation of the hypotonicity-provoked anionic response did not depend on the increase in Erk-1/Erk-2 activity but, instead, was found to depend on Ca2+ influx. Taken together, these results indicate that hypotonic stress induces Erk-1/Erk-2 activation through the Ras/Raf-signalling pathway, and argue against a direct role for this pathway in cell-volume control.