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Biomedical subjects

D A Jans

Publications and source records attributed to D A Jans.

At least 109 records · Page 6Linked to original sources
PubMed1991

Overexpression of the alpha-type protein kinase (PK) C in LLC-PK1 cells does not lead to a proportional increase in the induction of two 12-O-tetradecanoylphorbol-13-acetate-inducible genes.

Phorbol esters, by activating protein kinase C (PKC), induce the expression of the urokinase-type plasminogen activator (uPA) gene and the proto-oncogene c-fos in LLC-PK1 (PK1) porcine kidney epithelial cells. To investigate the role of PKC in the regulation of these two 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible genes, the alpha-type PKC, the predominant subtype present in the PK1 cells, was overexpressed in this cell line. Two clonal PK1 derivatives overexpressing the alpha PKC 15- and 20-fold, respectively, were established. Compared with the parental and control cells, only a modest but substantially sustained (2- to 3-fold) increase in the accumulation of uPA as well as c-fos mRNAs were observed by TPA in these cells. These results indicate that the extent of induction of these genes mediated by TPA was not proportional to the amounts of alpha-type PKC stably overexpressed in these cells, suggesting that factor(s) downstream of the activation of the alpha PKC appear to be rate limiting for the induction of both TPA-inducible genes in PK1 cells.

Animals↗
PubMed1990

An inverse relationship between receptor internalization and the fraction of laterally mobile receptors for the vasopressin renal-type V2-receptor. An active role for receptor immobilization in down-regulation?

Lateral mobility of the vasopressin renal-type V2-receptor was investigated in LLC-PK1 porcine epithelial cells using the technique of fluorescence microphotolysis (photobleaching) and a rhodamine-labelled vasopressin analogue. At various times after ligand addition, cells were analyzed for both receptor lateral mobility and ligand internalization. The V2-receptor mobile fraction diminished from 0.9 to 0.43 over 60 min at 37 degrees C, whereas the apparent lateral diffusion coefficient remained essentially unchanged (2-3 X 10(-10) cm2/s). Interestingly, the fraction of immobile V2-receptors corresponded exactly with the fraction of internalized receptors, implying a functional relationship. These observations together with comparable results reported for other polypeptide hormone receptors indicate a possible mechanistic role for receptor immobilization in the desensitization of hormonal response.

Animals↗
PubMed1990

Derivatives of somatic cell hybrids which carry the human gene locus for nephrogenic diabetes insipidus (NDI) express functional vasopressin renal V2-type receptors.

The gene responsible for familial vasopressin-resistant nephrogenic diabetes insipidus (NDI) has been localized to a small region of the human X-chromosome (Xq28). A series of hamster lung fibroblast and mouse lymphocyte cell lines carrying fragments of the wild type human X-chromosome was analyzed for vasopressin renal-type V2 receptor expression, to test the hypothesis that the NDI locus may have identity with the V2 receptor gene. V2 receptor binding activity and induction of cAMP production in response to [Arg8] vasopressin (AVP) were exhibited by all cell lines carrying the wild type NDI locus, in contrast to control cell lines. AVP stimulation of cAMP production was concentration-dependent and could be almost completely inhibited by co-incubation with a V2-V1 receptor-specific antagonist. The V2-specific agonist [Mpa1,Val4,Sar7]AVP was as potent as AVP in inducing cAMP production by NDI-DNA-carrying cells, whereas no response was shown to other hormones such as calcitonin, oxytocin (less than 10(-8) M), isoproterenol, or an oxytocin-specific agonist. All results were consistent with the hypothesis that the V2 receptor gene co-localized with the NDI locus, supporting the view that the loci are one and the same.

Animals↗
PubMed1990

Biotinyl analogues of vasopressin as biologically active probes for vasopressin receptor expression in cultured cells.

Biotinyl analogues of [Arg8]vasopressin were synthesized with the biotinyl moiety at position 4. This involved the substitution of 2, 4-diaminobutyric acid (Dab) for Gln4 in [1-deamino-Arg8]vasopressin to give the parent peptide des-[Dab4,Arg8]vasopressin. Two biotinyl analogues with different spacers between the side chain of Dab4 and the biotinyl residue were then prepared and characterized in detail. The analogues retained high binding affinities for the V2-receptor in both bovine kidney membranes and LLC-PK1 renal epithelial cells and for the V1-receptor in rat liver membranes. Both analogues were as potent as [Arg8] vasopressin in stimulating the cAMP-dependent protein kinase and the production of urokinase-type plasminogen activator in LLC-PK1 cells, with concentration dependence consistent with receptor binding affinities. Avidin or streptavidin did not appear to reduce receptor binding or biological activity of the biotinyl analogues. The use of the biotinylated vasopressin analogue des-[Dab-(biotinylamido)hexanoyl4, Arg8]vasopressin together with fluorescein-labeled streptavidin as a fluorescent probe for the V2-receptor in LLC-PK1 cells demonstrated the following: 1) Specific binding of the biotinyl analogue shown by quantitative single-cell fluorescence measurements using the technique of fluorescence microphotolysis; 2) the V2-receptor visualized by fluorescence microscopy; and 3) the expression of the V2-receptor detected by flow cytometry.

Amino Acid Sequence↗
PubMed1990

Lateral mobility of the phospholipase C-activating vasopressin V1-type receptor in A7r5 smooth muscle cells: a comparison with the adenylate cyclase-coupled V2-receptor.

The present work examines lateral mobility of the vasopressin V1-type receptor, representing the first determination of lateral mobility of a hormone receptor coupled to phospholipase C activation. The V1-receptor of A7r5 smooth muscle cells was characterized for [Arg8] vasopressin (AVP) binding properties and affinity for the fluorescent vasopressin analogue 1-deamino[8-lysine (N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP). TR-LVP was biologically active in A7r5 cells, inducing inositol 1,4,5-trisphosphate turnover in similar fashion to AVP. TR-LVP was used to specifically label the V1-receptor of living A7r5 cells, and lateral mobility of the V1-receptor was measured using the technique of fluorescence microphotolysis. The apparent lateral diffusion coefficient (D) at 37 degrees C was 5.1 x 10(-10) cm2/s, falling to 2.9 x 10(-10) cm2/s at 13 degrees C. These D values are higher than comparable values for the adenylate cyclase-activating vasopressin V2-receptor of LLC-PK1 renal epithelial cells analysed with the same fluorescent ligand. In contrast to the V2-receptor, no marked temperature dependence was observed for the V1-receptor mobile fraction (f). From 37 degrees C to 13 degrees C, f was relatively low (between 0.4 and 0.5) consistent with V1-receptor immobilization through internalization, which is rapid even at room temperature in A7r5 cells. These differences between V1- and V2-receptor lateral mobility are discussed in terms of the implications for their respective signal transduction systems.

Adenylyl Cyclases↗
PubMed1990

Ammonium chloride affects receptor number and lateral mobility of the vasopressin V2-type receptor in the plasma membrane of LLC-PK1 renal epithelial cells: role of the cytoskeleton.

The acidotropic agent ammonium chloride (NH4Cl) not only affects receptor metabolism by inhibiting lysosomal acidification, but can also affect the targeting of proteins to specific membranes in polarized cells, possibly through effects mediated by the cytoskeleton. The present study examines the effects of NH4Cl and perturbers of cytoskeleton structure on vasopressin V2 receptor expression in LLC-PK1 renal epithelial cells. Surprisingly, long-term pretreatment of cells with NH4Cl or short-term treatment with the actin perturber cytochalasin B resulted in an up to 70% increase in specific Arg-8-vasopressin binding compared to control cells, which was independent of the presence of NH4Cl in the binding test, and apparently the result of increased V2 receptor expression. Perturbers of microtubules such as colchicine and vinblastine had no such effect. A rhodamine-labeled analog of vasopressin was used to fluorescently label the V2 receptor of LLC-PK1 cells, and microscopic measurements of membrane-localized fluorescence confirmed the increased V2 receptor expression in the basal plasma membrane subsequent to NH4Cl pretreatment. Lateral mobility of the V2 receptor was measured in living cells using the technique of microphotolysis (photobleaching). The fraction of mobile receptors was 0.2 in cells pretreated with NH4Cl, markedly reduced compared to that of 0.9 in untreated cells. The apparent lateral diffusion coefficient D was about 3 x 10(-10) cm2/s in both pretreated and untreated cells. Results for fluorescence labeling of the actin cytoskeleton indicate that NH4Cl pretreatment of LLC-PK1 cells results in perturbation of microfilament structure. All results imply that the cytoskeleton plays a central role in V2 receptor expression and lateral mobility.

Actins↗
PubMed1990

A system to select for mutant LLC-PK1 cells affected in cAMP mediated hormonal response using a photoactivatable analogue of vasopressin.

The photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenyl-amidino)lysine] vasopressin (apa-LVP) could be used to elicit stimulation of cAMP production in LLC-PK renal epithelial cells, detectable up to 24 h after photoactivation by flash photolysis. This is in contrast to cells treated with vasopressin, or apa-LVP without photoactivation, where cAMP synthesis is down regulated within 4 h. The prolonged stimulation of cAMP production induced by photoactivation of apa-LVP was demonstrated to be cytotoxic to LLC-PK1 cells, whereas the vasopressin receptor negative LLC-PK1 mutant M18 was resistant to the cytotoxic effect. A selection strategy was developed for mutants resistant to this long-term stimulation of cAMP production, whereby multiple cycles of treatment with apa-LVP and photoactivation were used. Mutants so selected were then characterized using a novel screening system for detection of the production of urokinase-type plasminogen activator in response to cAMP agonists. One mutant was examined and found to be impaired in hormonal responsiveness, whereby hormone and forskolin stimulated cAMP-mediated responses were markedly reduced. It exhibited resistance to the long-term stimulation of cAMP production elicited by apa-LVP and photoactivation. This implies that apa-LVP can be used to select for novel mutants specifically impaired in cAMP metabolism and in particular down-regulation of cAMP response.

Adenylyl Cyclases↗
PubMed1989

The adenylate cyclase-coupled vasopressin V2-receptor is highly laterally mobile in membranes of LLC-PK1 renal epithelial cells at physiological temperature.

The lateral mobility of membrane-associated hormone receptors has been proposed to play an important role in signal transduction. Direct measurements, however, have shown that the receptors for insulin, epidermal growth factor and beta-adrenergic antagonists exhibit low mobility at physiological temperature. The present study, which represents the first report of lateral mobility of a polypeptide hormone receptor coupled to adenylate cyclase, yielded quite different results. The lateral mobility of the vasopressin renal-type (V2)-receptor was measured in the basal plasma membrane of cells of the LLC-PK1 porcine epithelial line, using the technique of fluorescence microphotolysis (photobleaching) and a rhodamine-labelled analogue of vasopressin. The analogue, 1-deamino[8-lysine(N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP) was synthesized and shown to have binding properties and biological activities very similar to those of Arg8-vasopressin (AVP). TR-LVP could be used to label specifically the V2-receptor of living LLC-PK1 cells, whereby LLC-PK1 cells incubated with TR-LVP in the presence of a 100-fold excess of AVP, or cells from the LLC-PK1 V2-receptor-deficient line M18 incubated with TR-LVP could be used as controls for non-specific binding. Using optical sectioning, specific receptor mobility could be measured both in the absence and presence of free TR-LVP. The V2-receptor was found to be largely mobile at 37 degrees C: the mobile fraction (f) was approximately 0.9, and the apparent lateral diffusion coefficient (D) approximately 3.0 X 10(-10) cm2/s. V2-receptor mobility greatly decreased with decreasing temperature: at 10 degrees C f was reduced to approximately 0.1.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1988

Codominant expression of a mutation affecting the cAMP-dependent protein kinase catalytic subunit in somatic cell hybrids of LLC-PK1 cells.

The LLC-PK1 mutant cell lines FIB4 and FIB6 are affected in the catalytic (C) subunit of cAMP-dependent protein kinase (cAMP-PK) such that they possess less than 10% parental activity. However, by Western blot analysis they were shown to possess normal levels of C subunit protein. Somatic cell hybrids were derived between mutant and LLC-PK1 cells, and examined for complementation of the cAMP-PK lesion. Codominant expression of mutant and normal alleles was observed, in that somatic cell hybrids between FIB4 and LLC-PK1, and between FIB6 and LLC-PK1 cells, exhibited cAMP-PK activity 60-75% that of LLC-PK1 cells, intermediate between mutant and normal parental cell lines. The cAMP-PK of the FIB6 x LLC-PK1 and FIB4 x LLC-PK1 hybrids was examined by ion exchange chromatography. In contrast to the FIB6 and FIB4 mutants which lack an active Type I cAMP-PK, the hybrids retained levels of active Type I cAMP-PK greater than 30% that of LLC-PK1, concomitant with the retention of catalytic activity. It was concluded that the loss of Type I kinase in the FIB6 and FIB4 mutants is most likely a consequence of the lesion in the cAMP-PK C subunit. All somatic cell hybrids examined showed levels of cAMP-PK C subunit (as determined by Western blot analysis), and in vivo regulation of cAMP-PK activation (in response to hormonal or nonreceptor-mediated stimulation of adenylate cyclase), completely comparable to those of the parental LLC-PK1 cells. Hence, no aberrant regulation of either cAMP-PK subunit levels or cAMP-PK activities was evident in the somatic cells hybrids. All data were consistent with the hypothesis that FIB4 and FIB6 contain a structural mutation affecting the cAMP-PK catalytic subunit that is expressed phenotypically in the presence of the normal allele.

Animals↗
PubMed1988

Electroporation of cultured adult rat hepatocytes with the c-myc gene potentiates DNA synthesis in response to epidermal growth factor.

The human c-myc gene was introduced and transiently expressed in adult rat hepatocyte cultures by the technique of electroporation and its effect on DNA synthesis was examined. Epidermal growth factor (EGF) has been found to stimulate a wave of DNA synthesis in electroporated rat hepatocytes. Hepatocyte cultures electroporated with the c-myc gene showed a potentiation of this EGF effect exhibiting rates of DNA synthesis up to 50% greater than those of control electroporated cultures, as determined by [3H]thymidine labeling of cell nuclei. This potentiation was dependent on the amount of c-myc DNA transfected. The potentiation was due neither to an alteration in the dose-response of the stimulatory effect of EGF nor to a change in the time course of the DNA synthesis wave.

Animals↗
PubMed1987

Long-term stimulation of cAMP production in LLC-PK1 pig kidney epithelial cells by salmon calcitonin or a photoactivatable analogue of vasopressin.

A photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenylamidino)lysine]-vasopressin, was compared to salmon calcitonin and [8-arginine]-vasopressin with respect to stimulation of cAMP synthesis in the LLC-PK1 pig kidney epithelial cell line. Without photoactivation, the vasopressin analogue-elicited responses were identical to those induced by vasopressin, in that cAMP synthesis returned to the basal, unstimulated level about 4 h after hormonal treatment. In contrast, the levels of activation of cAMP-dependent protein kinase induced by salmon calcitonin returned to basal approx. 12 h after hormone addition. When activated by ultraviolet irradiation, the vasopressin analogue induced 'permanent' stimulation of adenylate cyclase, whereby cAMP production could be detected even 12.5 h after treatment. Both salmon calcitonin and the photoactivated vasopressin analogue inhibited growth of LLC-PK1 cells, in contrast to vasopressin or the nonactivated analogue. Growth inhibition appeared to be a consequence of the prolonged stimulation of adenylate cyclase. This conclusion was supported by the fact that a LLC-PK1 cell mutant in cAMP-dependent protein kinase was resistant to growth inhibition by salmon calcitonin and activated vasopressin analogue. The results imply that the cAMP-dependent protein kinase is the mediator of the hormone-stimulated growth inhibition.

Animals↗
PubMed1987

Isolation of a cDNA and characterization of the 5' flanking region of the gene encoding the type I regulatory subunit of the cAMP-dependent protein kinase.

A bovine cDNA probe for the type I regulatory subunit of the cAMP-dependent protein kinase [Lee et al. (1983) Proc. Natl Acad. Sci. USA 80, 3608-3612] was used to screen two lambda gt11 libraries constructed from poly(A)-rich RNA from the porcine kidney cell line, LLC-PK1. A series of overlapping clones were isolated and characterized. The largest clone, lambda RI15, of 1426 bp was found to code for the entire RI protein but was apparently missing the 3' end of the mRNA. The porcine cDNA codes for a protein of 389 amino acids that shows 99% homology to bovine RI and hybridizes to two major mRNA transcripts of approximately 2.0 kb and 4.5 kb from LLC-PK1 cells. The porcine cDNA for RI was used to screen a genomic library of LLC-PK1 DNA constructed in the EMBL-3 vector and several clones were isolated and characterized. By using a probe from the 5' end of the RI cDNA we isolated the 5' end of the gene and 700 bp of the promoter region of the gene were sequenced. The promoter region lacks a characteristic TATA box but contains two inverted CAAT boxes and is rich in G + C residues. Several sequence motifs were identified in the 5' promoter region which could be responsible for the regulation of synthesis of this gene. Multiple transcription initiation sites were identified by S1 nuclease mapping.

Animals↗
PubMed1987

Recombinant type I regulatory subunit of the cAMP-dependent protein kinase is biologically active.

The cDNA for the porcine type I regulatory subunit (RI) of the cAMP-dependent protein kinase (cAMP-PK) was cloned into two different bacterial expression vectors: pKK223 and pUC18. Recombinant RI was produced by bacteria transformed with either construct, and purified by affinity chromatography. Both the native RI from the pKK223 construct and the RI with an amino terminal extension of eight amino acids from the pUC18 construct were found to be completely native with regard to inhibition of the catalytic subunit activity and cAMP binding.

Animals↗
PubMed1987

Dependence of urokinase-type-plasminogen-activator induction on cyclic AMP-dependent protein kinase activation in LLC-PK1 cells.

The activation of cyclic AMP-dependent protein kinase (cAMP-PK) in vivo was studied in LLC-PK1 pig kidney cells and the mutant cell lines M18 and FIB5, which have total levels of cAMP-PK catalytic-subunit and regulatory-subunit activities comparable with those of parental cells. The extent of cAMP-PK activation (release of active catalytic subunit from the holoenzyme) was directly correlated with the cellular cyclic AMP concentration in LLC-PK1 cells. In LLC-PK1 cells, as well as in the mutants M18 and FIB5, the extent of the induction of urokinase-type plasminogen activator (uPA) by the cyclic AMP-mediated effectors calcitonin, vasopressin and forskolin was directly correlated with the levels of activated catalytic subunit. The 'receptorless' mutant M18, which is impaired in calcitonin- and vasopressin-receptor function, did not show any activation of cAMP-PK or uPA production in response to either hormone, whereas cAMP-PK and uPA responses to forskolin were about 35% higher than in parental cells. Analysis of the FIB5-cell line revealed a lesion affecting the regulation of adenylate cyclase activity, whereby basal and stimulated (both receptor- and non-receptor-mediated) adenylate cyclase levels were less than 36% of those in parental cells. The activation of cAMP-PK in response to cyclic AMP effectors was similarly reduced, and uPA induction was concomitantly lower than that in parental cells. The results demonstrate the dependence of uPA induction by cyclic AMP effectors on dissociation of the cAMP-PK holoenzyme, implying the importance of activated free cAMP-PK catalytic subunit in this process. Thus it is concluded that the mutations in the cellular cyclic AMP-generating apparatus of the M18 and FIB5 cell lines impair uPA induction by preventing cAMP-PK activation.

Adenylyl Cyclases↗
PubMed1987

Characterization of two mutants of the LLC-PK1 porcine kidney cell line affected in the catalytic subunit of the cAMP-dependent protein kinase.

The catalytic (C) subunit activity of the cAMP-dependent protein kinase (cAMP-PK) from the mutant cell lines, FIB4 and FIB6, is only 10% compared with the parent cell line, LLC-PK1 [Jans and Hemmings (1986) FEBS Lett. 205, 127-131]. In order to understand the nature of the mutant phenotypes the cAMP-PK from parent and mutant cell lines was studied in more detail. Analysis of mutant cAMP-PK activity by ion-exchange chromatography revealed that kinase activity associated with type I holoenzyme of both FIB4 and FIB6 was only 5% parental, and the activity of the type II holoenzyme was about 20% parental. The type I regulatory (RI) subunits associated with the type I were also found to be reduced by 70-80% in both mutants, whereas the type II R subunit levels were similar to that of the parent. The residual kinase activity associated with the type I holoenzyme from FIB4 and FIB6 could not be activated by cAMP whereas the type II holoenzyme was activated by cAMP (Ka of 5.5 X 10(-8) M), and showed normal affinities for Kemptamide and ATP. A polyclonal antibody to the catalytic subunit was used to quantify the level of this protein in wild-type and mutant cells. This analysis showed that FIB4 and FIB6 had nearly normal levels of C subunit, suggesting that the C subunit synthesized by the mutants was mostly inactive. As both type I and type II cAMP-PK holoenzymes were abnormal, the most likely explanation of the mutant phenotype is a defect either in the structural gene for the C subunit or in an enzyme involved in its posttranslational processing. However, a second lesion affecting the RI subunit cannot be ruled out at this moment.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Complementation between LLC-PK1 mutants affected in polypeptide hormone-receptor function.

The mutant LLC-PK1 cell lines FIB6 and FIB5/N4 were examined for responsiveness to the polypeptide hormones calcitonin and vasopressin. Both mutants exhibited little or no activation of adenylate cyclase or cAMP-dependent protein kinase (cAMP-PK) in response to calcitonin, but responded to vasopressin. Analysis of calcitonin receptor function demonstrated that both mutants bound less than 9 fmol 125I-labeled salmon calcitonin/mg cellular protein, which was about 1% of parental activity (642 fmol calcitonin bound/mg). Concomitant with reduced calcitonin binding, both mutants exhibited increased vasopressin binding (greater than 272 fmol [[3H]Arg]vasopressin bound/mg) compared to parental (166 fmol bound/mg). The concentration of vasopressin for half-maximal stimulation of adenylate cyclase in both mutants was comparable to that for LLC-PK1 cells (40 pM) and hence the increased binding activity was concluded to be due to increased numbers of functional vasopressin receptors in the mutants. Somatic cell hybrids formed between each mutant and LLC-PK1 cells exhibited normal hormone binding and activation of cAMP-PK in response to both vasopressin and calcitonin. The mutations affecting receptor function in FIB6 and FIB5/N4 were accordingly concluded to be recessive. Somatic cell hybrids between FIB6 and FIB5/N4 showed no complementation of the mutant phenotype, indicating that both cell lines were affected in the same gene. In contrast, somatic cell hybrids between FIB5/N4 and the 'receptorless' mutant M18 (which lacks functional calcitonin and vasopressin receptors) exhibited approximately the same responsiveness to vasopressin and to calcitonin as LLC-PK1. Complementation between two different mutations affecting polypeptide receptor function was thus observed. The results are discussed in terms of a proposed common pathway for processing of calcitonin and vasopressin receptors.

Adenylyl Cyclases↗
PubMed1987

Pathway of urokinase-type plasminogen activator induction in the T47D and LLC-PK1 cell lines.

Induction of urokinase-type plasminogen activator (uPA) in response to either reagents activating cAMP-dependent protein kinase (cAMP-PK) or the calcium ion phospholipid-dependent kinase (C-kinase) was compared in the LLC-PK1 and T47D cell lines. The two cell lines exhibited quantitatively different responses to calcitonin, to the phosphodiesterase inhibitor isobutylmethylxanthine, and to the adenylate cyclase activator forskolin. Both showed activation of cAMP-PK in response to all these reagents, with T47D cells displaying a greater extent of activation. T47D cells, however, failed to produce uPA in response to calcitonin, forskolin, or the cAMP analog 8-bromo-cAMP, whereas LLC-PK1 cells produced high levels of uPA in response to all these agents. Both cell lines responded to phorbol esters in terms of uPA induction, though to differing extents. Phorbol myristate acetate (PMA) was shown conclusively not to activate cAMP-PK in either cell line, even at concentrations 10-fold higher than those promoting maximal uPA induction. It was concluded that phorbol ester-mediated induction of uPA does not involve cAMP or cAMP-PK activation. These results are discussed in relation to proposed models concerning the role of cAMP-PK in uPA induction.

Animals↗