Regulation of transforming growth factor beta by sex steroids.
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Animals↗
Biomedical subjects
S Hering
Publications and source records attributed to S Hering.
Interleukin 1 beta, but not tumor necrosis factor, enhances neurogenic vasodilatation in the rat skin: involvement of nitric oxide.
In phenobarbitone-anesthetized rats the effects of interleukin 1 beta (IL-1 beta) and tumor necrosis factors (TNFs) were examined on the capsaicin-induced increase of plantar cutaneous blood flow in the rat hind paw as measured by laser Doppler flowmetry. IL-1 beta (0.5-500 pg) or TNF alpha or TNF beta (50-500 pg) was injected subcutaneously into the left paws, while the right paws received vehicle (10 microL) only. IL-1 beta was without effect on blood flow by its own but dose dependently enhanced the hyperemia due to capsaicin (0.3 microgram). TNFs failed to enhance the capsaicin-induced vasodilatation although 5000 pg TNF alpha produced a transient increase of local blood flow. Indomethacin (10 mg/kg, i.p.) did not alter the capsaicin-induced vasodilatation but prevented IL-1 beta (50 pg) from augmenting the hyperemic response to capsaicin. Likewise, blockade of nitric oxide formation by NG-nitro-L-arginine methyl ester (L-NAME) failed to affect the capsaicin-evoked vasodilatation but abolished its amplification by IL-1 beta. Systemic pretreatment with a neurotoxic dose of capsaicin reduced the capsaicin-induced hyperemia and prevented the facilitatory effect of IL-1 beta. The hyperemia evoked by intraplantar calcitonin gene related peptide (0.038-3.8 ng) was not altered by IL-1 beta (50 pg). These data indicate that IL-1 beta but not TNF enhances the cutaneous hyperemic response to capsaicin. This proinflammatory action arises from sensitization of afferent nerve endings and depends on nitric oxide and cyclooxygenase products as essential intermediates.
Animals↗
Cloning and functional expression of a neuronal calcium channel beta subunit from house fly (Musca domestica).
The primary structure of a calcium channel beta subunit (beta M) from housefly (Musca domestica) has been deduced by cDNA cloning and sequence analysis. The open reading frame encodes a 441-amino acid polypeptide with a calculated molecular mass of 48,755 Da. Whole-mount in situ hybridization indicates that beta M mRNA is predominantly expressed in neuronal tissues. Transcription of beta M mRNA is evident from stage 13/14 of embryogenesis up to adulthood. Different expression patterns of splice variants were found in larvae and in adult fly heads. Amino acid identity between beta M and mammalian beta subunits is lower (66-68%) than within mammalian beta subunits (74-80%). Calculation of a phylogenetic tree indicates that beta M is an ancestral form of the four distinct beta subunit gene products identified in mammalian tissues so far. Despite these sequence differences, beta M is able to enhance endogenous calcium channel activity in Xenopus laevis oocytes as well as dihydropyridine binding to membranes from COS 7 cells transfected with rabbit heart alpha 1 cDNA in the same manner as was previously shown for mammalian beta subunits.
Alternative Splicing↗
A light stabilizer (Tinuvin 770) that elutes from polypropylene plastic tubes is a potent L-type Ca(2+)-channel blocker.
A pharmacologically active agent was easily extracted by aqueous or organic solvents from laboratory plastic tubes (Falcon Blue Max) and has been chemically identified as bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate. This compound (approximately 12 micrograms per tube approximately 25 nmol) blocked 1,4-dihydropyridine-sensitive 45Ca2+ uptake into GH3 cells with an IC50 value of 3.6 microM, inhibited Sr2+ currents through L-type Ca2+ channels in A7r5 smooth-muscle cells in whole-cell patch-clamp experiments after extracellular application, and affected the high-affinity binding of Ca2+ entry-blocker ligands to a variety of preparations. Bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate is a highly potent (IC50 values < 10 nM) inhibitor at the phenylalkylamine- and benzothiazepine-selective drug-binding domains of the alpha 1 subunit of L-type Ca2+ channels. This compound behaves as a heterotropic allosteric regulator for the 1,4-dihydropyridine-selective domain in purified Ca(2+)-channel preparations from rabbit skeletal muscle. (+)-Tetrandrine stimulation of 1,4-dihydropyridine binding to the membrane-bound L-type Ca2+ channel is inhibited by the compound in a competitive manner (Ki value = 6.8 nM). Bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate is therefore classified as the prototype of another class of L-type Ca(2+)-channel blockers that binds to the alpha 1 subunit at the drug-binding domains selective for (+)-tetrandrine or (+)-cis-diltiazem. This compound is identical to Tinuvin 770, which is used worldwide as a light stabilizer for polyolefins.
Alkaloids↗
Modulation of calcium channels in arterial smooth muscle cells by dihydropyridine enantiomers.
The actions of the optical enantiomers of BAY K 8644 and Sandoz 202,791 were studied on barium inward currents recorded using the whole-cell configuration of the patch clamp technique from enzymatically isolated smooth muscle cells from the rabbit ear artery. The enantiomers were applied by bath perfusion or rapidly by a concentration jump technique, which enabled the study of drug action under equilibrium and nonequilibrium conditions. A larger effect of agonists was seen on peak inward current in 110 mM Ba when small rather than large depolarizations were applied. The midpoint voltage of the steady-state inactivation curve of IBa was -12.8 +/- 1.9 mV (n = 4) in the absence of drug, -16.4 +/- 2.5 mV (n = 4) in 1 microM (+)202,791, and -31.4 +/- 0.4 mV (n = 4) in 1 microM (-)202,791. The rate of onset of action of the agonist and antagonist enantiomers of BAY K 8644 and Sandoz 202,791 was studied by rapid application during 20-ms depolarizing steps from different holding potentials to +30 mV at 1 or 0.2 Hz. The drugs were applied as concentration jumps between two single pulses of a pulse train. The rates of onset of drug action on peak IBa during a 1-Hz pulse train were concentration dependent over the range of 100 nM-3 microM for both (+) and (-)202,791. The rate of onset of inhibition of peak current by antagonist enantiomers was not significantly influenced by the test pulse frequency. At a holding potential of -60 mV, the onset rate of the increase in peak IBa on application of 1 microM of agonist enantiomers (+)202,791 or (-)BAY K 8644 during a train of pulses occurred with mean time constants of 2.1 +/- 0.7 s (n = 7) and 2.3 +/- 0.2 s (n = 4), respectively. The onset of current increase on application of 1 microM (+)202,791 during a single voltage clamp step to 20 mV was faster, with a mean time constant of 380 +/- 80 ms (n = 3).
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Evidence for an external location of the dihydropyridine agonist receptor site on smooth muscle and skeletal muscle calcium channels.
1. The location of the binding domain for agonist dihydropyridines (DHP) has been studied by comparing the action of (+)-202,791 and (-)-Bay K 8644 on Ba2+ currents (IBa) in whole cell patch clamp experiments. Drug effects were examined upon internal and external (extracellular) application in A7r5 smooth muscle cells and BC3H1 cells, a cell line expressing Ca channels of the skeletal muscle type. 2. Efficiency of internal drug application in the whole cell studies was demonstrated by inhibition of potassium currents and barium currents (IBa) upon internal perfusion with tetraethylammonium (TEA+) (10 mM) and the permanently charged phenylalkylamine, D 890 (100 microM) respectively. The uncharged DHP, (-)-STBODIPY-DHP (2 microM) was used to estimate the time course of internal perfusion by monitoring its fluorescence. 3. Intracellular application of (+)-202,791 and (-)-Bay K 8644 (5 microM) in patch clamp experiments was ineffective in stimulating Ca2+ channel currents in both cell lines. In contrast a 50 fold lower agonist concentration (0.1 microM (-)-Bay K 8644) applied to the external face of the membrane induced typical changes in tail currents and a current increase under conditions when up to 10 microM of the agonist was present in the intracellular perfusion solution. 4. In cell-attached patches in A7r5 cells, (-)-Bay K 8644 increased and (+)-PN 200,110 inhibited single channel activity when applied via the bath solution. This suggests partitioning and lateral diffusion of the DHPs in the lipid of the plasma membrane. 5. We conclude that the binding site for agonist DHPs on Ca2+ channels in A7r5 and BC3H1 cells is located close to the external surface of the membrane. The DHP binding domain can be reached by agonists and antagonists from the extracellular but not from the intracellular face of the membrane.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Extracellular localization of the benzothiazepine binding domain of L-type Ca2+ channels.
To determine which side of L-type Ca2+ channels forms the benzothiazepine binding domain, we tested the effects of a membrane-impermeable, diltiazem-like, Ca2+ antagonist, SQ32,428 [(cis)-1,3,4,5-tetrahydro-4-(4-methoxyphenyl)-3-methyl-6- (trifluoromethyl)-1-[2-trimethylammonio)ethyl]-2H-1-benzazepin-2-o ne], on Ca2+ channels in smooth muscle-like cells (A7r5 cells) and skeletal muscle-like cells (differentiated BC3H1 cells). This permanently charged, quaternary benzazepine bound to the benzothiazepine-selective domain of skeletal muscle Ca2+ channels with a Ki of 1.2 +/- 0.1 microM. Extracellular application of SQ32,428 reversibly blocked whole-cell barium currents through L-type Ca2+ channels in A7r5 and BC3H1 cells with similar potencies (A7r5, IC50 = 86 microM; BC3H1, IC50 = microM). Block was fully reversible, was independent of stimulation frequency, and did not affect steady state inactivation of the channel in A7r5 cells. Intracellular dialysis of the cells with 100 microM SQ32,428 was without effect, but the same concentration of the quaternary phenylalkylamine D890 blocked channel activity from the cytoplasmic side. Our data demonstrate that the benzothiazepine binding domain of L-type Ca2+ channels binds diltiazem-like benzazepine Ca2+ antagonists and is formed by amino acid residues exposed to the extracellular channel surface.
Animals↗
A method for estimation of drug affinity constants to the open conformational state of calcium channels.
The affinity of D600 to calcium channels in the open state has been examined in isolated smooth muscle cells of the rabbit ear artery. Calcium channel currents were measured in high external barium solution by means of the patch-clamp technique. The current inhibition in various D600 concentrations (3-100 microM) on application of trains of short test pulses (20-80 ms) has been studied in nonmodified calcium channels and in cells where the calcium channels were modified by the agonist dihydropyridine (+) 202,791 (100 nM). The kinetics of the peak current decay has been analyzed with a mathematical model which is based on the experimental finding that D600 interacts primarily with calcium channels in the open conformational state. The model approach allows the estimation of drug affinity constants of D600 to the calcium channel in the open conformation. An association rate constant to the open conformational state of D600 of 6.16 x 10(4) M-1 s-1 was estimated. The association rate of the drug was not significantly changed after the calcium channels have been modified with 100 nM (+) 202,791. A method for correction of rate constants for possible drug trapping is discussed.
Animals↗
[35S]sadopine, a novel high affinity, high specific activity, L-type Ca2+ channel probe: characterization of two equipotent diastereomers with opposite allosteric properties.
A novel 35S-labeled dihydropyridine (DHP), 1,4-dihydro-2,6-dimethyl-4-(2-trifluoromethylphenyl)-pyridine-3,5-dic arboxyl-3- [2-(N-tert-butoxycarbonyl-L-[35S]methionyl)-aminoethyl]-ester-5-ethyl ester, ([35S]sadopine) (800-1400 Ci/mmol), the respective (+)- and (-)-diastereomers, and unlabeled (+/- )-, (-)-, and (+)-sadopine were synthesized. [35S]Sadopine is an excellent high affinity, high specific activity radioligand to label selectively the DHP receptor of L-type Ca2+ channels in tissue sections as well as in membrane fragments. Both diastereomers bind to the DHP receptors in a saturable and reversible manner, with equal, subnanomolar, dissociation constants. Despite their similar affinities, (+)- and (-)-sadopine differ with respect to their kinetic properties [the association and dissociation rate constants are 10-fold higher for (+)-[35S]sadopine at 22 degrees] and their allosteric modulation of the phenylaklylamine or benzothiazepine binding domain. (+)-Sadopine is a negative but (-)-sadopine a positive allosteric modulator of (-)-[N-methyl-3H]LU49888 or (+)-cis-[3H] diltiazem binding at 30 degrees. Both diastereomers act as L-type Ca2+ channel blockers in cardiac and smooth muscle cells. Computer-based analysis of the electrostatic potentials of the two diastereomers and calculation of the interaction energies with a hypothetical DHP receptor model predicted not only the similar affinities of (+)- and (-)-sadopine but also their Ca2+ channel-blocking effects. The temperature-dependent allosteric differences between the diastereomers suggest that two distinct conformational states of the DHP receptor are stabilized in vitro, both corresponding to a nonconducting state of the channel. Our data indicate that access to the DHP receptor site, but not binding affinity, is a function of the opposite stereochemistry of the sadopine diastereomers. Therefore, labeled and unlabeled (+)- and (-)-sadopine will be useful probes to further characterize the molecular basis of DHP-Ca2+ channel interaction and the pharmacological and physiological significance of the different allosteric conformations of the channel induced by Ca2+ channel-active drugs.
Animals↗
Stimulation of cell division and fibroblast focus formation by antisense repression of retinoblastoma protein synthesis.
Circumstantial evidence supports a role for the retinoblastoma susceptibility gene, Rb-1, in the maintenance of normal cell growth, in that loss of its function results in abnormal growth and malignancy. Here we report that a high rate of mitosis and efficient dense focus formation in human embryonic lung fibroblasts (HEL cells) is induced by antisense oligonucleotide-directed inhibition of synthesis of p105-Rb, the product of the Rb-1 gene. mRNA specific for p105-Rb is truncated at the site of base pairing with the antisense oligonucleotide, and no synthesis of p105-Rb is observed. The rate of mitosis is considerably increased and the frequency of dense focus formation is extremely high in treated cells. However, although phosphothioate oligodeoxyribonucleotides taken up by the cells remain stable for at least 4 weeks, the recipient cells do not become immortal; nor are they able to induce tumor formation in nude mice. Thus, loss of Rb-1 function is not sufficient per se to allow malignant transformation.
Base Sequence↗
Immortalization of human fetal sinusoidal liver cells by polyoma virus large T antigen.
Fetal sinusoidal liver cells were isolated from human liver explant cultures and transfected with pCMVLT, a plasmid containing the immediate early promotor of cytomegalovirus (CMV) and the large tumor antigen (LT) coding part of the polyoma virus (py) genome. Whereas nontransfected cells stopped proliferating after 4 weeks, the transfected sinusoidal cells were stimulated to divide more quickly without changes in their morphology. Up to now, cells have been permanently cultured for more than 18 months and passaged over 130 times, corresponding to around 400 generations. This allows them to be regarded as "immortalized" cells. The presence of LT protein in the cells has been documented by means of immunoprecipitation and immunofluorescence. Expression of the v. Willebrandt factor VIII was the main criterion for classifying the cell population as endothelial cells. The presence of cytokeratins 7, 8, and 18 in these cells underlines their close ontogenic and functional relationship to mesothelial cells. Sinusoidal endothelial cells (SECs) synthesize vimentin and the typical extracellular matrix components collagen IV and fibronectin, but are negative for laminin and entactin. We used immortalized SEC's in co-culture experiments with fresh fetal human hepatocytes and adult mouse hepatocytes. They promoted survival of both types of hepatocytes over a period of 8-10 weeks. Control human fetal liver explant culture cells survived for only 3-4 weeks, whereas control adult mouse liver cells retained vitality for 8-10 days only.
Antigens, Polyomavirus Transforming↗
[Phosphoglucomutase subtyping by means of agarose thin-layer electrophoresis. A study on applying different buffer systems and agaroses].
For the routine subtyping of PGM1 isozymes by means of agarose gel thin-layer electrophoresis, a tris-maleic acid-buffer and an acetic acid-buffer, several agaroses with electroendosmosis up to -0, 16, and several papers are tested. With all these materials good results can be obtained, so that agarose without electroendosmosis is not necessary and FN 1 papers can be used.
Buffers↗
The action of caffeine on inward barium current through voltage-dependent calcium channels in single rabbit ear artery cells.
The effect of caffeine on inward current carried by barium ions through voltage-dependent calcium channels has been investigated in single rabbit ear artery cells using whole-cell voltage-clamp techniques. Caffeine (1-30 mM) caused a rapid and reversible concentration-dependent blockade of barium current and a related compound, 3-isobutyl-1-methylxanthine (IBMX), was a more potent inhibitor of barium current. Caffeine-induced inhibition of barium current showed no voltage- or use-dependence and caffeine did not alter the steady-state inactivation of barium current. The effect of caffeine was not blocked by extracellular or by intracellular ryanodine or inclusion of both 5 mM 1,2-bis(2-aminophenoxy)-ethane N,N,N',N',-tetraacetic acid (BAPTA) and 2 mM ethylene glycol-bis(beta-amino ethyl ether) N,N,N',N',-tetraacetic acid (EGTA) in the intracellular solution. Rolipram and M&B 22984, non-xanthine inhibitors of phosphodiesterase, did not diminish inward barium current. The data indicate that caffeine and IBMX block voltage-operated calcium channels and it is suggested that this is due to a direct interaction of methylxanthines with the calcium channel.
1-Methyl-3-isobutylxanthine↗
Thyroid hormone binding inhibition in critically ill patients--who is the inhibitor?
In 31 severely ill patients of our intensive care unit an increased thyroid hormone binding inhibition detected by quantitative measurement of the effect of ether extracts of patients' sera on thyroxine tracer binding to TBG was found. As free fatty acids in serum were diminished in these patients, their postulated role as binding inhibitors in the "low T4 syndrome" of critically ill patients must be questioned. It cannot be excluded that THBI assays demonstrating this inhibition measure artefacts caused by heparine-induced in vitro lipolysis.
Acute Disease↗
Evidence that agonist and antagonist enantiomers of the dihydropyridine PN 202-791 act at different sites on the voltage-dependent calcium channel of vascular muscle.
S(+)-PN 202-791, a calcium channel agonist, and its optical isomer R(-)-PN 202-791, a calcium channel antagonist, respectively increased and decreased inward current carried by barium through voltage-dependent calcium channels in isolated ear artery cells of the rabbit in a concentration-dependent manner. The EC50 or IC50 derived from the concentration-response relationship was unaltered in the presence of the other enantiomer indicating that no competitive antagonism exists between these enantiomers and suggesting that the actions of these two enantiomers involve two separate binding sites.
Animals↗
Immortalization and transformation of human fibroblasts by regulated expression of polyoma virus T antigens.
We have established conditions for the immortalization of human fibroblasts by the large T antigen of the rodent virus polyoma. This allows the mechanism of immortalization to be studied, without interference by transformation events, in cells with relatively stable chromosomes. Large T antigen could immortalize human fibroblasts if expression was driven by a heterologous promoter like the immediate early promoter/enhancer of cytomegalovirus or the inducible mouse mammary tumour virus (MMTV) promoter. Using the latter promoter and dexamethasone, three clones were obtained, the immortalized phenotype of which was strictly dependent on the induction of T-antigen expression. At least one of these clones became mortal after removal of the inducing agent. The expression of large T antigen was paralleled by PCNA gene expression, as shown by nuclear run-off transcription, whereas none of a number of other known proto-oncogenes was influenced in its activity. Immortalized fibroblasts were readily transformed by polyoma virus middle T antigen expressed from the MMTV promoter or by the activated c-Ha-ras oncogene. The reversibility of immortalization and transformation is considered.
Antigens, Polyomavirus Transforming↗
Characterization of the calcium channel state transitions induced by the enantiomers of the 1,4-dihydropyridine Sandoz 202 791 in neonatal rat heart cells. A nonmodulated receptor model.
The actions of the optical enantiomers of Sandoz 202 791 were studied in barium inward currents recorded from single cultured neonatal rat ventricular heart cells, using the whole-cell configuration of the patch clamp technique. The enantiomers were applied by bath perfusion or rapidly by the technique of concentration jumps during single voltage clamp steps. (1) (-)-202 791 reduced the barium current in response to depolarizations positive to 0 mV. The peak current amplitude in the threshold range (-40 to 0 mV) was either not affected or slightly increased by the substance. (2) The agonist enantiomer (+)-202 791 increased the inward current over the whole voltage range, where the increase in peak inward current amplitude was most prominent in the voltage range from -40 mV to 0 mV. (3) The antagonist enantiomer (10(-6) M) induced a 18.2 +/- 2.1 mV (n = 6) shift of the midpoint of the steady state inactivation curve in the hyperpolarizing direction; in contrast (+)-202 791 at the same concentration did cause only a small but not significant shift of the Ca-channel availability curve (n = 5). (4) Rapid extracellular application of (-)-202 791 (10(-6) M), during the sustained current component at a test potential of 0 mV was followed by a sudden acceleration in barium current decay. The drug-induced barium current block developed with a mean time constant of 214.7 +/- 20.6 ms (n = 5). (5) (+)-202 791 (10(-6) M) rapidly applied during test pulses to 0 and -20 mV caused an increase in barium current with a mono- or biexponential time course. The estimated mean time constant of the drug activated Ba2+ current at 0 mV membrane potential was 617.3 +/- 49.3 ms (n = 4). (6) The interaction of Sandoz 202 791 with the Ca-channels is discussed in terms of a "nonmodulated receptor" model.
Animals↗
Mechanism of calcium channel block by D600 in single smooth muscle cells from rabbit ear artery.
This study investigated the action of the calcium antagonist D600 on calcium channel currents recorded in high barium solution from single, enzymatically isolated smooth muscle cells from the rabbit ear artery using the whole cell configuration of the patch-clamp technique. D600 (1-100 microM) was applied by path perfusion or by a new technique that allowed a concentration jump during the current. Application of D600 at rest (holding potential, -60 mV) did not alter the peak inward current elicited on depolarization, but the activation of the channels led to a marked block and an acceleration of the current decay. The mechanism of block of calcium channels by D600 was studied by using pulse protocols with different pulse length and different interpulse intervals. The results were consistent with the hypothesis that D600 has a low affinity for the calcium channels in the resting state and that they have to pass to the open state before the drug affects the calcium channel current. A fast onset of the calcium channel block by D600 (time constant, 502 msec) could be shown by rapid application of D600 during the sustained current component of the barium inward current. However, experiments did not definitely distinguish whether binding occurred to the open or to the inactivated state (although there was some evidence of a long-lasting binding to an inactivated state).
Animals↗