Chloride current activated by cyclic AMP and parathyroid hormone in rat osteoblasts

In primary cultures of rat osteoblasts, studied with the whole-cell configuration of the patch-clamp technique, 8-bromo-cyclic AMP (8BrcAMP) forskolin (FS) and 1-34 parathyroid hormone (PTH) were shown to activate a Cl conductance. This conductance shows a pronounced outward rectification, even with symmetrical Cl concentrations. It is blocked partially and reversibly by 4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid (DIDS) or diphenylcarboxylate (DPC). The blockade induced by DIDS is time- and voltage-dependent. The Cl responses to FS and PTH develop slowly, after a delay of several seconds and are very slowly reversible. These responses were observed only in a fraction of the cells tested and their detection was favoured by cell dialysis. This Cl current should be taken into account for studying possible modulations of the voltage-gated Ca currents of osteoblasts. It is suggested that its physiological role may be related to the well-known morphological changes induced by PTH in osteoblasts. The cyclic AMP-sensitivity, the outward rectification and the sensitivity to dialysis of this Cl current are reminiscent of the properties of the cystic fibrosis-sensitive Cl channels of epithelial cells.     

Blockade of Cl channels by organic and inorganic blockers in vascular smooth muscle cells

The effects of Cl channel blockers on large-conductance (LC-type) Cl channels of cultured vascular smooth muscle cells (VSMC) were studied in order to characterize the channel pharmacologically. Intracellular disulphonic stilbene derivatives, such as 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene-2,2-disulphonic acid (SITS) inhibited Cl channel activity in a dose-dependent manner. An obvious inhibitory effect of DIDS in this condition was obtained at concentrations higher than 5 M, and the complete inhibition was obtained at around 100 M, which was almost 10 times less than the effective dose of SITS. The inhibitory effect of DIDS was reversible at a drug concentration of lower than 50 M. Single-channel conductance decreased as the concentration of DIDS increased. This decrease in the conductance was a consequence of unresolved openings of the channel due to fast blocking and unblocking rates of the drug. The Cl channel was also obviously inhibited by extracellular DIDS at a concentration of 1 mM. In addition, in cell-attached patches, 500 M DIDS applied extracellularly inhibited Cl channel activated by the application of polymyxin B. We also investigated the effect of Zn on Cl channels in VSMC. Intracellular Zn dose dependently and reversibly blocked the channel at the positive intracellular potential range, whereas at the negative intracellular potential range it did not block the channel activity. Results in this study suggest the diversity of Cl channels among various tissues.     

Activation of the basolateral Cl− conductance by cAMP in rabbit renal proximal tubule S3 segments

The regulatory mechanism of basolateral Cl^– conductance in rabbit renal proximal tubule S3 segments was investigated with conventional and Cl^– sensitive microelectrodes. After the basolateral Cl^–/HCO ₃ ^– exchanger was blocked by 4,4-diisothiocyanatostilbene-2, 2-disulphonic acid (DIDS) we increased the bath K⁺ concentration from 5 mmol/l to 20 mmol/l, which depolarized the cells and thereby increased intracellular Cl^– activity ([Cl^–]_i). This [Cl^–]_i response was enhanced by +63% in the presence of forskolin (20 mol/l), by +40% in the presence of dibutyryl adenosine 3,5-cyclic monophosphate (db-cAMP) (1 mmol/l) and by +44% in the presence of parathyroid hormone (PTH, 10 nmol/l), whereas it was inhibited by a Cl^– channel blocker, indanyl-oxyacetic acid (IAA-94, 0.3 mmol/l). In addition, forskolin, PTH and chlorophenylthio-cAMP enhanced the electrogenic response to removal of bath Cl^– after the blockade of K⁺ conductance, and this activation was also sensitive to IAA-94. On the other hand, 2 mol/l ionomycin and 0.5 mol/l phorbol myristate failed to activate the [Cl^–]_i response to elevation of bath K⁺ concentration and the electrogenic response to Cl^– removal, and ionomycin had no effect even in the absence of DIDS. These results indicate that this basolateral Cl^– conductance can be activated by cAMP, while neither the increase in cytosolic Ca²⁺ nor the activation of protein kinase C has direct effects on this conductance.     

Intracellular pH in renal tubules in situ: single-cell measurements by confocal laserscan microscopy

Confocal laserscan microscopy with a dual-excitation device was used to record intracellular pH (pH_i) regulation in rat proximal convoluted tubules microperfused in vivo. Cells were loaded with the pH-sensitive, fluorescent dye 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Single cells could be distinguished within the tubules and separate measurements were possible. Application of an NH₄Cl pulse by peritubular perfusion caused an immediate increase in intracellular pH. Intraluminal injection of NH₄Cl led to a slower increase in intracellular pH. In both cases, cessation of perfusion led to an immediate acidification. Peritubular perfusion with 300 M 4,4-diisothiocanatodihydrostilbene-2, 2-disulphonic acid (H₂DIDS) caused an intracellular alkalinisation. Microperfusion, pH-sensitive dyes and confocal laserscan microscopy provide a new non-invasive method to measure intracellular pH effectively in individual cells of near-surface structures of the intact kidney.     

Characterization of the Cl− conductance in the granular duct cells of mouse mandibular glands

We have previously shown that mouse mandibular granular ducts contain a hyperpolarization-activated Cl^– conductance. We now show that the instantaneous current/voltage (I/V) relation of this Cl^– conductance is inwardly rectifying with a slope conductance of 15.4±1.8 nS (n=4) at negative potentials and of 6.7±0.9 nS (n=4) at positive potentials. Thus, the inward rectification seen in the steady-state I/V relation is due, not only to voltage activation of the Cl^– conductance, but also to the intrinsic conductance properties of the channel. We show further that the ductal Cl^– conductance is not activated by including ATP (10 mmol/l) in the pipette solution. Finally, we show that the conductance is not blocked by the addition of any of the following compounds to the extracellular solution: anthracene-9-carboxylate (A9C, 1 mmol/l), diphenylamine-2-carboxylate (DPC, 1 mmol/l), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 mol/l), 4,4-diisothiocyanato-stilbene-2,2-disulphonate (DIDS, 100 mol/l), indanyloxyacetic acid (IAA-94, 100 mol/l), verapamil (100 mol/l), glibenclamide (100 mol/l) and Ba²⁺ (5 mmol/l). The properties of the ductal Cl^– conductance most nearly resemble those of the ClC-2 channel. Both channel types have instantaneous I/V relations that are slightly inwardly rectifying, are activated by hyperpolarization with a time-course in the order of hundreds of milliseconds, have a selectivity sequence of Br^–>Cl^–>I^–, and are insensitive to DIDS. The only identified difference between the two is that the ClC-2 channel is 50% blocked both by DPC and A9C (1 mmol/l), whereas the ductal Cl^– conductance is insensitive to these compounds.     

Phosphorylation-regulated low-conductance Cl− channels in a human pancreatic duct cell line

A low-conductance Cl^– channel has been identified in the apical membrane of the human pancreatic duct cell Capan-1 using patch-clamp techniques. Cell-attached channels were activated by the vasoactive intestinal polypeptide (VIP, 0.1 mol/l), dibutyryl-adenosine 3,5-cyclic monophosphate (db-cAMP, 1 mmol/l), 8-bromo adenosine 3,5-cyclic monophosphate (8-BrcAMP, 1 mmol/l), 3-isobutyl-1-methyl-xanthine (IBMX, 100 mol/l) and forskolin (10 mol/l). No channel activity was observed in non-stimulated control cells. In both cell-attached and excised inside-out patches, the channel had a linear current/voltage relationship and a unitary conductance of 9 pS at 23°C and 12 pS at 37°C. Its opening probability was not voltage dependent although pronounced flickering was induced at negative potentials. Anionic substitution led to the selectivity sequence Cl^–>I^–>HCO₃ ^–>gluconate. In insideout excised patches, the channel activity declined spontaneously within a few minutes. Reactivation of silent excised channels was achieved by adding protein kinase A (PKA, in the presence of ATP, cAMP and Mg²⁺). Conversely, active channels were silenced in the presence of alkaline phosphatase. The PKA-activated Cl^– channel was 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS, 100 mol/l) and 4-acetamido-4-isothiocyanatostilbene-2, 2-disulphonic acid (SITS, 100 mol/l) insensitive, but was blocked by diphenylamine-2-carboxylic acid (DPC, 100 mol/l). These results demonstrate that the apical low-conductance Cl^– channel in Capan-1 is regulated on-cell by VIP receptors via cAMP and off-cell by PKA and phosphatases. They provide evidence that this channel is closely related to the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel.     

A volume-activated anion conductance in insulin-secreting cells

The whole-cell patch-clamp recording technique was used to measure volume-activated currents in K⁺-free solutions in RINm5F and HIT-T15 insulinoma cells and in dispersed rat islet cells. Cell swelling, induced by intracellular hypertonicity or extracellular hypotonicity, caused activation of an outwardly rectifying conductance which could be subsequently inactivated by hypertonic extracellular solutions. The conductance required adenosine 5-triphosphate (ATP) in the pipette solution but was Ca²⁺ independent. Na⁺ and Cl^– substitution studies suggested that the swelling-activated current is Cl^– selective with a halide permeability sequence of Br > Cl > 1. The conductance was reversibly inhibited by the anion channel inhibitors 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) and by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Further evidence for a volume-activated anion conductance was provided by studies of volume regulation in insulin-secreting cells. When RINm5F cells were exposed to a hypotonic medium, the initial cell swelling was followed by a regulatory volume decrease (RVD). This RVD response was also inhibited by DIDS and by NPPB. These data therefore provide evidence for a volume-activated anion conductance in insulin-secreting cells which could be involved in the RVD following osmotic stress. A possible role for the conductance in hypotonically induced insulin release is also discussed.     

Co-expression of an anion conductance pathway with Na+-glucose cotransport in rat renal brush-border membrane vesicles

Brush-border membrane vesicles were prepared from superficial rat renal cortex by a Mg²⁺-precipitation technique. The initial (20 s) [¹⁴C]glucose uptake rate from solutions containing 100 mmol/l Na (salt) was found to be dependent upon the anion composition of the medium; in comparison to gluconate-containing medium (0.46±0.05 nmol/mg protein), Cl^– accelerated the initial rate to 1.47±0.21 nmol/mg protein (n=4 preparations, ± SEM). This enhancement was reduced by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.5 mmol/l), but was unaffected by 4,4-diisothiocyanatostilbene 2,2-disulphonate (DIDS, 0.5 mmol/l). When membrane vesicles were pre-equilibrated with 100 mmol/l K (salt) and 100 mmol/l mannitol and glucose uptake was measured from a solution containing 100 mmol/l Na gluconate and 100 mmol/l mannitol in the presence of 80 mol/l valinomycin (to generate an outward K⁺ diffusion electrical p. d.), it was found that intravesicular KCl depressed the initial glucose uptake compared to K gluconate. NPPB (0.5 mmol/l) increased the initial glucose uptake with intravesicular KCl towards values seen in K gluconate vesicles. In conditions where the only driving force for glucose uptake was established by an inward anion gradient (Na_o=Na_i) it was found that inward Cl^– gradients could drive uphill glucose transport and that this was sensitive to NPPB (0.5 mmol/l), but insensitive to DIDS. We conclude that a Cl^– conductance co-exists with Na-cotransport in rat renal brush-border membrane vesicles prepared from superficial renal cortex and this may function to regulate the activity of electrogenic transport systems at this membrane.     

Interferon action in human fibroblasts: Induction of 2′, 5′-oligoadenylate synthetase in the absence of detectable protein kinase activity

Summary In several strains of human fibroblasts, treatment with human - or -interferon (IFN) resulted in an induction of 2, 5-oligoadenylate synthetase but no protein kinase activity. The antiviral and anticellular effects of IFN are elicited in these cells in the absence of detectable IFN-induced protein kinase activity.With 2 Figures     

The 5′ untranslated region of PVY RNA,even located in an internal position,enables initiation of translation

Potato virus Y (PVY) is the type member of the potyvirus group. Potyviruses, like picorna-, como-, and nepoviruses, belong to the picornavirus-like supergroup. All these viral RNAs have a VPg at their 5 end, and for four picornaviruses and one comovirus internal initiation of translation has been reported. To know if such a translational mechanism holds true for potyviral RNAs, the 5 nontranslated region (NTR) of PVY RNA was placed in an internal position, either by adding 91 bases upstream of the PVY 5NTR or by inserting the PVY 5NTR into an intercistronic region. The addition of extra bases stimulates translation in a rabbit reticulocyte lysate, and the presence of the PVY 5NTR in the intercistronic region allows the synthesis of the second cistron. These findings strongly suggest that PVY RNA initiates translation by an internal ribosome-binding mechanism. Furthermore, the use of antisense oligodeoxynucleotides indicates that the entire 5NTR seems to be involved in such a mechanism.     

In wheat ctDNA,segments of ribosomal protein genes are dispersed repeats,probably conserved by nonreciprocal recombination

Summary Some dispersed repeated sequences and their flanking regions from wheat and maize ctDNAs have been characterized. Two sets of wheat ctDNA repeats were found to be the chloroplast ribosomal protein genesrpl2 andrpl23, plus nonfunctional segments of them, designatedrpl2 andrpl23. Pairwise comparisons were made between the wheatrp123 andrpl23, and the maizerp123 sequences. The precise patterns of homology suggest that the divergence of the wheat and maize nonfunctional (rpl23) sequences is being retarded by nonreciprocal recombination, biased by selection for individuals with functional (rpl23) sequences. The implied involvement of these sequences in mechanisms of homologous recombination, and therefore in the creation and spread of new ctDNA variants, is discussed.     

Catechol oxidase activity associated with sporogenesis inHaplosporidium malacobdellae,a balanosporidan endoparasite of the hoplonemerteanAmphiporus lactifloreus

Catechol oxidase (E.C. 1.10.3.1. 1,2-benzenediol: oxygen oxidoreductase) and an enzyme exhibiting true peroxidatic activity have been demonstrated in tissues undergoing sporogenesis ofHaplosporidium malacobdellae, an Ascetosporan parasite of the marine nemerteanAmphiporus lactifloreus. Both enzymes have been investigated using standard histochemical techniques, employing a wide range of different substrates and metabolic effectors.The catechol oxidase appears to be copper-containing and relatively heatstable, acting upon a wide range of 1,2- and 1,3-benzenediol-containing substrates. The enzyme is completely inhibited by 10 mM sodium diethyldithiocarbamate and incompletely by 1,10-phenanthroline (2 mg/ml) and 50 mM sodium azide. Low concentrations (10 mM) of copper (II) ions activate the catechol oxidase activity, higher concentrations (100 mM) reduce the enzyme activity considerably. The peroxidase is a heat-labile enzyme, sensitive to sulphydryl binding agents and unaffected by high (100 mM) diethyldithiocarbamate concentrations. A possible function for the enzymes in the quinone tanning process of spore wall formation is proposed.     

Messenger RNA 3′-end formation of a DNA fragment from the human c-myc 3′-end region in Saccharomyces cerevisiae

We have tested the functioning of the human c-myc polyadenylation signal in Saccharomyces cerevisiae. A DNA fragment containing the two AATAAA polyadenylation signals of the c-myc gene was inserted into a plasmid designed for the in-vivo testing of polyadenylation signals in yeast. The c-myc fragment had a partial capacity for directing mRNA 3-end formation in yeast. The 3-endpoints were 50–100 bp distant from the mRNA 3-ends mapped in humans. This human DNA fragment is therefore unspecifically functional in yeast, indicating that other sequence elements than the human polyadenylation signal, AATAAA, are necessary for 3-end formation.     

3′-Terminal sequences of influenza C virion RNA

Summary The 3-terminal nucleotides of the genome segments of influenza C/Taylor/47, C/Bavaria/79 and C/Johannesburg/1/66 were identified by two RNA sequencing techniques. These comprised 11 nucleotides (3 UCGUCUUCGUC) which were found to be conserved among the genome segments of each virus.With 3 Figures     

On the secretagogue effect of dibutyryl cyclic AMP in the rat exocrine pancreas

Summary DbcAMP0.1 mM induces the discharge of exportable enzymes from rat pancreas fragments incubated in vitro. This effect is qualitatively similar to the action of physiological secretagogues acting via hormone receptors: 1) it is accompanied by the appearance of exocytotic images at the acinar cell apex; 2) it is energy dependent but energy supply is low while that required for the carbamylcholine or caerulein response is high and can only be afforded by oxidative phosphorylation; 3) it is calcium dependent, but no alteration of inward or outward calcium movement can be observed; 4) it is altered by agents known to disrupt the microfilamentous microtubular system [41]. However, the secretory response to DbcAMP is quantitatively less than that obtained with hormonal stimuli. A damaging effect of DbcAMP on pancreatic acinar cells is ruled out on histological and biochemical grounds: there is no significant leakage of LDH; protein synthesis, 2-deoxy-d-glucose andl-leucine uptake are unaltered. The secretagogue effect of DbcAMP is reversible, dose-related and specific. It is not mediated by neuro-transmitter release or by interaction with their receptors. The evidence presented points to a direct interaction of DbcAMP on the pancreatic acinar cell and suggests the last step of the secretory cycle as the most probable site of action of the nucleotide derivative.Abbreviations cAMP Adenosine-3,5-monophosphate - cyclic DbcAMP: N⁶-2-O-Dibutyryl-adenosine-3,5-monophosphate, cyclic - DbcGMP N²-2-O-Dibutyryl-guanosine-3,5-monophosphate, cyclic - 5-AMP Adenosine-5-monophosphate - TCA Trichloracetic acid - ATP Adenosine triphosphate - NADH Nicotinamide-adenine dinucleotide - Tris Tris-(hydroxy-methyl) amino-methane - EGTA Ethylene glycol-bis ( aminoethylether)-NN-tetraacetic acid - LDH Lactic dehydrogenase Part of this work has been presented in abstract from at the VIIIth Symposium of the European Pancreatic Club, Toulouse, France, October, 1975, 23rd–25th and at the Biochemical Society of Belgium [40]This work was partially carried out under contracts from the Ministère de la Politique Scientifique within the framework of the Association Euratom—University of Brussels—University of Pisa, and the Institut National de la Santé et de la Recherche Médicale (France)     

Antiidiotypic antibodies against anti-DNA antibodies in sera of families of lupus patients

We searched for antiidiotypes directed against anti-DNA in sera of healthy family members of lupus patients. Controls were healthy individuals without a personal or family history of lupus. No significant differences were noted between the family members' and the control group's sera with respect to binding to DNA or to non-anti-DNA F(ab)₂ fragments. Family members' sera had higher binding to anti-DNA F(ab)₂ and to normal IgG F(ab)₂ fragments (P<0.01). Sera of the family members had significantly higher binding to anti-DNA F(ab)₂ than to normal IgG F(ab)₂ fragments (P<0.0036). Inhibition experiments have shown that the antiidiotype is directed against the framework determinants and not against the antigen binding sites of the idiotype. The anti-idiotypic antibodies were directed against cross-reactive anti-DNA idiotypes and were not restricted to the idiotypes of the lupus proband. Age, sex, and blood relationship to the lupus patient did not influence the presence of antiidiotypes in the family members. The possible role of environmental factors in the induction of antiidiotypes and the role of the latter in regulating anti-DNA antibodies are discussed.     

Ein Flip-Flop-Modell des Chlorid-Kanal-Komplexes erklärt die Fehlregulation des Chloridflusses am Plasmalemm von Zellen bei der cystischen Fibrose

Summary The basic defect in cystic fibrosis is the chloride impermeability of the plasmalemm in different cells. A candidate for the chloride channel, thought to be affected in the syndrome, is Porin 31HL recently described by us. The molecule is i) expressed in the plasmalemm of different cells, it has ii) a molecular mass of 31000 Daltons, it shows iii) high conductance in artificial membranes and it can be iv) modified by 4,4-Diisothiocyana-tostilben-2,2-disulfonat. A porin in the outer membrane of cells should furthermore v) be regulated by modulators. All these characters of Porin 31HL correspond to those given in literature for chloride channels. The regulation of the channels can be explained by a two component flip flop model.

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Abkürzungsverzeichnis CF Cystische Fibrose - CFTR Cystic Fibrosis Transmembrane Conductance Regulator - DIDS 4,4-Diisothiocyanatostilben-2,2-disulfonat - kDa kilo Daltons - NPPB 5-Nitro-2-(3-Phenylpropylamino)benzoat - SDS-PAGE Natriumdodecylsulfat Polyacrylamid Gelelektrophorese - 2D-PAGE Zweidimensionale Polyacrylamid Gelelektrophorese - VDAC voltage dependent anion channel The model was presented for the first time in the 12th Magdeburg Colloquium on Mitochondrial Functions, 9–10 November 1990, in Magdeburg and will be discussed here more detailed.     

Two polymorphicAvaI andHhaI sites in a differentially methylated region of the human H19 gene

Summary The H19 gene is paternally imprinted both in the human and mouse (Bartolomeiet al., 1991; Zhang and Tycko, 1992), although its expression pattern seems somewhat different between the two species (Jinno,et al., 1995). DNA-methylation is a promising candidate for a parent-of-origin mark of the gene, and a paternal allele-specific methylation imprint was recently identified at the mouse H19 locus (Tremblayet al., 1995). We found a 50% methylated region in the human H19 gene (Jinno, unpublished data). A search for polymorphisms in this region revealed two novelAvaI andHhaI RFLPs, which contribute to the detection of allele-specific methylation at the human H19 locus.PCR primers for the AvaI-site PANL2 5-GAGCCTGCCAAGCAGAGCG-3 - PANR2 5-CACATAAGTAGGCGTGACTTGA-3 PCR primers for the HhaI-site ASMA 5-CAATGAGGTGTCCCAGTTCCA-3 - PANR2 5-CACATAAGTAGGCGTGACTTGA-3     

Adenosine 3′,5′-cyclic monophosphate in rabbit cerebrospinal fluid during fever induced byE. coli-endotoxin

In rabbit cerebrospinal fluid during fever induced byE. coli-endotoxin adenosine 3,5-cyclic monophosphate concentrations are about 2-fold higher in comparison to normal values. In addition to prostaglandins of the E series adenosine 3,5-cyclic monophosphate might act as another mediator in the genesis of fever during infectious diseases.This investigation was supported by the Deutsche Forschungsgemeinschaft.     

Characterization of human larynx carcinoma cell lines HLaC′79 and HLaC′82: a common origin but diverged malignancies

As part of a study on the relationship of tumour phenotype and behaviour, we have characterized two head and neck squamous cell carcinoma cell lines, derived from human laryngeal carcinomas and designated HLaC79 and HLaC82. Cytogenetic analysis revealed that HLaC79 and HLaC82 shared 10 major chromosome rearrangements indicating that the cell lines had a common origin. In the extremely complex chromosomal patterns, abnormalities were found in chromosomes 1, 3 (surplus 3q) and 5 (i(5p) × 2). Both cell lines displayed constitutive expression of vimentin and were capable of anchorage-independent growth in agarose gels. However, in spite of their common origin specific differences were found. Cells of HLaC79 were spindle shaped and formed tumours in athymic mice. In contrast, cells of HLaC82 had a compact morphology, contained less vimentin, were more contact inhibited and were not tumorigenic. These results indicate that malignant transformation in HLaC82 was partially reversed.