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Background & Aims:Deletion of the codon for phenylalanine at position 508 (ΔF508) is the most frequent disease-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In heterologous cells, defective processing of the ΔF508 protein results in endoplasmic reticulum retention, proteolytic degradation, and absence of adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent plasma membrane Cl−conductance. However, data with respect to the processing block of ΔF508 protein in native epithelia are limited and conflicting. Methods:To characterize both the fate and function of ΔF508 protein in a native epithelium, we measured CFTR-mediated Cl−secretion, localization of the CFTR protein, and CFTR maturation in rectal biopsy specimens from normal individuals and ΔF508 homozygous patients with cystic fibrosis (CF). Results:Ussing chamber studies showed that cAMP-dependent and cholinergic Cl−secretion was absent from rectal tissues freshly excised from ΔF508 homozygous patients with CF. By immunohistochemistry, we detected wild-type but not ΔF508 CFTR at the luminal membrane of crypt colonocytes. By sequential immunoprecipitation and immunoblotting analyses, mature CFTR protein was detected in normal but not in ΔF508 homozygous tissues. Conclusions:Collectively, these data show that there is insufficient maturation and transport of ΔF508 CFTR from the endoplasmic reticulum to the apical membrane to support CFTR-mediated Cl−secretion in the CF colon.

Human airway epithelia express Ca2-activated Cl−channels (CaCC) that are activated by extracellular nucleotides (ATP and UTP). CaCC is preserved and seems to be up-regulated in the airways of cystic fibrosis (CF) patients. In the present study, we examined the role of basolateral Kchannels in CaCC-mediated Cl−secretion in native nasal tissues from normal individuals and CF patients by measuring ion transport in perfused micro Ussing chambers. In the presence of amiloride, UTP-mediated peak secretory responses were increased in CF compared with normal nasal tissues. Activation of the cAMP pathway further increased CaCC-mediated secretion in CF but not in normal nasal mucosa. CaCC-dependent ion transport was inhibited by the chromanol 293B, an inhibitor of cAMP-activated hKvLQT1 Kchannels, and by clotrimazole, an inhibitor of Ca2-activated hSK4 Kchannels. The Kchannel opener 1-ethyl-2-benzimidazolinone further increased CaCC-mediated Cl−secretion in normal and CF tissues. Expression of hSK4 as well as hCACC-2 and hCACC-3 but not hCACC-1 was demonstrated by reverse transcriptase PCR on native nasal tissues. We conclude that Ca2-activated Cl−secretion in native human airway epithelia requires activation of Ca2-dependent basolateral Kchannels (hSK4). Co-activation of hKvLQT1 improves CaCC-mediated Cl−secretion in native CF airway epithelia, and may have a therapeutic effect in the treatment of CF lung disease.

Human airway epithelia express Ca2+-activated Cl-channels (CaCC) that are activated by extracellular nucleotides (ATP and UTP). CaCC is preserved and seems to be up-regulated in the airways of cystic fibrosis (CF) patients. In the present study, we examined the role of basolateral K+channels in CaCC-mediated Cl-secretion in native nasal tissues from normal individuals and CF patients by measuring ion transport in perfused micro Ussing chambers. In the presence of amiloride, UTP-mediated peak secretory responses were increased in CF compared with normal nasal tissues. Activation of the cAMP pathway further increased CaCC-mediated secretion in CF but not in normal nasal mucosa. CaCC-dependent ion transport was inhibited by the chromanol 293B, an inhibitor of cAMP-activated hKvLQT1 K+channels, and by clotrimazole, an inhibitor of Ca2+-activated hSK4 K+channels. The K+channel opener 1-ethyl-2-benzimidazolinone further increased CaCC-mediated Cl-secretion in normal and CF tissues. Expression of hSK4 as well as hCACC-2 and hCACC-3 but not hCACC-1 was demonstrated by reverse transcriptase PCR on native nasal tissues. We conclude that Ca2+-activated Cl-secretion in native human airway epithelia requires activation of Ca2+-dependent basolateral K+channels (hSK4). Co-activation of hKvLQT1 improves CaCC-mediated Cl-secretion in native CF airway epithelia, and may have a therapeutic effect in the treatment of CF lung disease.

Glycogen storage disease type 1a (GSD 1a) is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase). A variant (GSD 1b) is caused by a defect in the transport of glucose-6-phosphate (G6P) into the microsome and is associated with chronic neutropenia and neutrophil dysfunction. Mutually exclusive mutations in the G6Pase gene and the G6P transport gene establish GSD 1a and GSD 1b as independent molecular processes and are consistent with a multicomponent translocase catalytic model. A modified translocase/catalytic unit model based on biochemical data in a G6Pase knockout mouse has also been proposed for G6Pase catalysis. This model suggests coupling of G6Pase activity and G6P transport. A 5-mo-old girl with hypoglycemia, hepatomegaly, and lactic acidemia was diagnosed with GSD 1a. She also developed neutropenia, neutrophil dysfunction, and recurrent infections characteristic of GSD 1b. Homozygous G188R mutations of the G6Pase gene were identified, but no mutations in the G6P translocase gene were found. We have subsequently identified a sibling and two unrelated patients with similar genotypic/phenotypic characteristics. The unusual association of neutrophil abnormalities in patients with homozygous G188R mutations in the G6Pase gene supports a modified translocase/catalytic unit model.

Glycogen storage disease type 1a (GSD 1a) is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase). A variant (GSD 1b) is caused by a defect in the transport of glucose-6-phosphate (G6P) into the microsome and is associated with chronic neutropenia and neutrophil dysfunction. Mutually exclusive mutations in the G6Pase gene and the G6P transport gene establish GSD 1a and GSD 1b as independent molecular processes and are consistent with a multicomponent translocase catalytic model. A modified translocase/catalytic unit model based on biochemical data in a G6Pase knockout mouse has also been proposed for G6Pase catalysis. This model suggests coupling of G6Pase activity and G6P transport. A 5-mo-old girl with hypoglycemia, hepatomegaly, and lactic acidemia was diagnosed with GSD 1a. She also developed neutropenia, neutrophil dysfunction, and recurrent infections characteristic of GSD 1b. Homozygous G188R mutations of the G6Pase gene were identified, but no mutations in the G6P translocase gene were found. We have subsequently identified a sibling and two unrelated patients with similar genotypic/phenotypic characteristics. The unusual association of neutrophil abnormalities in patients with homozygous G188R mutations in the G6Pase gene supports a modified translocase/catalytic unit model.

The flavonoid genistein has been shown to activate a Cl−conductance in various cell types expressing CFTR. We examined if similar effects can be observed when genistein is applied to native ex vivotissues from human respiratory tract and rectum. We further compared the effects when genistein was applied to oocytes of Xenopus laevisexpressing CFTR.In oocytes, both wtCFTR and ΔF508‐CFTR were activated by genistein while both cyclic AMP (KvLQT1) and Ca2+(SK4) activated K+channels were inhibited at high concentrations of genistein.Biopsies from nasal polyps and rectal mucosa were obtained from normal individuals (non‐CF) and CF patients and in the presence of amiloride (10 μmol l−1; mucosal side) the effects of genistein were assessed using a perfused Ussing chamber. In non‐CF airway epithelia, genistein (50 μmol l−1; mucosal side) increased lumen negative Iscbut had no additional effects on tissues pre‐stimulated with IBMX and forskolin (100 μmol l−1and 1 μmol l−1; both sides).In non‐CF rectal biopsies, in the presence of amiloride (10 μmol l−1; mucosal side) and indomethacin (10 μmol l−1; basolateral side), genistein increased lumen negative Iscand enabled cholinergic (carbachol; CCH, 100 μmol l−1; basolateral side) stimulation of Cl−secretion indicating activation of luminal CFTR Cl−channels. However, after stimulation with IBMX/forskolin, genistein induced opposite effects and significantly inhibited CCH activated Isc. In CF airway and intestinal tissues genistein failed to induce Cl−secretion.Thus, genistein is able to activate luminal CFTR Cl−conductance in non‐CF tissues and mutant CFTR in oocytes. However, additional inhibitory effects on basolateral K+conductance and missing effects in native CF tissues do not support the use for pharmacological intervention in CF.

Abstract: Congenital absence of the vas deferens (CAVD) is a frequent cause for obstructive azoospermia and accounts for 1%–2% of male infertility. A high incidence of mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has recently been reported in males with CAVD. We have investigated a cohort of 106 German patients with congenital bilateral or unilateral absence of the vas deferens for mutations in the coding region, flanking intron regions and promotor sequences of the CFTR gene. Of the CAVD patients, 75% carried CFTR mutations or disease-associated CFTR variants, such as the “5T” allele, on both chromosomes. The distribution of mutation genotypes clearly differed from that observed in cystic fibrosis. None of the CAVD patients was homozygous for ΔF508 and none was compound heterozygous for ΔF508 and a nonsense or frameshift mutation. Instead, homozygosity was found for a few mild missense or splicing mutations, and the majority of CAVD mutations were missense substitutions. Twenty-one German CAVD patients were compound heterozygous for ΔF508 and R117H, which was the most frequent CAVD genotype in our study group. Haplotype analysis indicated a common origin for R117H in our population, whereas another frequent CAVD mutation, viz. the “5T allele” was a recurrent mutation on different intragenic haplotypes and multiple ethnic backgrounds. We identified a total of 46 different mutations and variants, of which 15 mutations have not previously been reported. Thirteen novel missense mutations and one unique amino-acid insertion may be confined to the CAVD phenotype. A few splice or missense variants, such as F508C or 1716 G→A, are proposed here as possible candidate CAVD mutations with an apparently reduced penetrance. Clinical examination of patients with CFTR mutations on both chromosomes revealed elevated sweat chloride concentrations and discrete symptoms of respiratory disease in a subset of patients. Thus, our collaborative study shows that CAVD without renal malformation is a primary genital form of cystic fibrosis in the vast majority of German patients and links the particular expression of clinical symptoms in CAVD with a distinct subset of CFTR mutation genotypes.

AbstractPediatric reference ranges for osteocalcin measured by a new, fully automated, chemiluminescent immunometric assay on the Immulite®immunoanalyzer are presented. Samples from 627 children, ranging from newborns to 18 years of age, were measured. Osteocalcin values are generally higher in children than in adults, highest levels being reached during the puberty growth spurt at about 12 years in girls and 14 years in boys, thereafter rapidly declining towards adult levels.