Activation of Maxi-K Channels by Parathyroid Hormone and Prostaglandin E2 in Human Osteoblast Bone Cells

Patch clamp experiments were performed on two human osteosarcoma cell lines (MG-63 and SaOS-2 cells) that show an osteoblasticlike phenotype to identify and characterize the specific K channels present in these cells. In case of MG-63 cells, in the cell-attached patch configuration (CAP) no channel activity was observed in 2 mm Ca Ringer (control condition) at resting potential. In contrast, a maxi-K channel was observed in previously silent CAP upon addition of 50 nm parathyroid hormone (PTH), 5 nm prostaglandin E₂ (PGE₂) or 0.1 mm dibutyryl cAMP + 1 μm forskolin to the bath solution. However, maxi-K channels were present in excised patches from both stimulated and nonstimulated cells in 50% of total patches tested. A similar K channel was also observed in SaOS-2 cells. Characterization of this maxi-K channel showed that in symmetrical solutions (140 mm K) the channel has a conductance of 246 ± 4.5 pS (n = 7 patches) and, when Na was added to the bath solution, the permeability ratio (P_K/P_Na) was 10 and 11 for MG-63 and SaOS-2 cells respectively. In excised patches from MG-63 cells, the channel open probability (P _o ) is both voltage- (channel opening with depolarization) and Ca-dependent; the presence of Ca shifts the P _o vs. voltage curve toward negative membrane potential. Direct modulation of this maxi-K channel via protein kinase A (PKA) is very unlikely since in excised patches the activity of this channel is not sensitive to the addition of 1 mm ATP + 20 U/ml catalytic subunit of PKA. We next evaluated the possibility that PGE₂ or PTH stimulated the channel through a rise in intracellular calcium. First, calcium uptake (⁴⁵Ca⁺⁺) by MG-63 cells was stimulated in the presence of PTH and PGE₂, an effect inhibited by Nitrendipine (10 μm). Second, whereas PGE₂ stimulated the calcium-activated maxi-K channel in 2 mm Ca Ringer in 60% of patches studied, in Ca-free Ringer bath solution, PGE₂ did not open any channels (n = 10 patches) nor did cAMP + forskolin (n = 3 patches), although K channels were present under the patch upon excision. In addition, in the presence of 2 mm Ca Ringer and 10 μm Nitrendipine in CAP configuration, PGE₂ (n = 5 patches) and cAMP + forskolin (n = 2 patches) failed to open K channels present under the patch. As channel activation by phosphorylation with the catalytic subunit of PKA was not observed, and Nitrendipine addition to the bath or the absence of calcium prevented the opening of this channel, it is concluded that activation of this channel by PTH, PGE₂ or dibutyryl cAMP + forskolin is due to an increase in intracellular calcium concentration via Ca influx. Received: 17 September 1995/Revised: 7 December 1995     

Differential expression of the epidermal K1 and K10 keratin genes during mouse embryo development.

Induction of genes coding for the K1 and K10 keratins during mouse development was studied by measuring the accumulation of their respective mRNAs in day 10 to 17 embryos using an RNase protection assay. Although these two keratins are coexpressed in the suprabasal layers of the epidermis, it was found that while K1 mRNA was detectable as soon as day 10, K10 mRNA was not detectable before day 12. The expression of these genes at this stage of development was not expected since they are specifically associated with keratinization, a process that does not begin before day 17 of gestation. Histological examination of the epidermis of day 10 to 17 embryos suggests that both genes are induced in cells committed to epidermal differentiation, after stratification has started but before the onset of keratinization. It was also found that the two mRNAs increased in abundance steadily and significantly until day 16 and that, in spite of the expectation that filaments should contain equivalent amounts of each subunit, K1 mRNA remained more abundant than K10 mRNA at all times including in adult epidermis. These observations indicate that the two genes are regulated independently during development.     

Cloning of the gene for Escherichia coli glutamyl-tRNA synthetase

The structural gene for the glutamyl-tRNA synthetase of Escherichia coli has been cloned in E. coli strain JP1449, a thermosensitive mutant altered in this enzyme. Ampicillin-resistant and tetracycline-sensitive thermoresistant colonies were selected following the transformation of JP1449 by a bank of hybrid plasmids containing fragments from a partial Sau3A digest of chromosomal DNA inserted into the BamHI site of pBR322. One of the selected clones, HS7611, has a level of glutamyl-tRNA synthetase activity more than 20 times higher than that of a wild-type strain. The overproduced enzyme has the same molecular weight and is as thermostable as that of a wild-type strain, indicating that the complete structural gene is present in the insert. These characteristics were lost by curing this clone of its plasmid with acridine orange, and were transferred with high efficiency to the mutant strain JP1449 by transformation with the purified plasmid. A physical map of the plasmid, which contains an insert of about 2.7 kb in length, is presented.     

Thermosensitive mutants of Escherichia coli K-12 altered in the catalytic Subunit and in a Regulatory factor of the glutamy-transfer ribonucleic acid synthetase.

The glutamyl-transfer ribonucleic acid synthetase (GluRS) of a partial revertants (ts plus or minus) of the thermosensitive (ts) mutant strain JP1449 (LOcus gltx) and of a ts mutant strain EM111-ts1 with a lesion in or near the locus gltx have been studied to find the relation between these two genetic loci known to influence the GluRS activity in vitro and the presence of a catalytic subunit and of a regulatory subunit in the GluRS purified from Escherichia coli K-12. The ts character of strain JP1449-18ts plus or minus is co-transduced with the marker dsdA at the same frequency as is the ts character of strain JP1449. Its purified GluRS is very thermolabile and its Km for glutamate is higher than that of a wild-type GluRS. These results indicate that the locus gltX is in the structural gene for the catalytic subunit of this enzyme. The location of the mutation causing the partial ts reversion in strain JP1449-18ts plus or minus is discussed. The GluRS purified from the ts mutant strain EM111-ts1 has the same stability as the wild-type enzyme, but its Km forglutamate increases with the temperature, suggesting that the locus gltE codes for a regulatory factor, possibly for the polypeptide chain that is co-purified with the catalytic subunit.     

Coronary hypertonia and angina

Coronary hypertonia was observed in a patient with unstable angina. It was possible on one occasion to reproduce the clinical picture, electrocardiographic changes, lactate production and coronary hypertonia by means of atrial pacing. He had a normal left coronary arteriogram when the diffuse spasm was relieved by nitroglycerin. Therefore hypertonia (or spasm) of the left coronary artery was believed to be the cause of his variant angina with subendocardial ischemia.     

Analysis of the androgenic activity of synthetic "progestins" currently used for the treatment of prostate cancer

In order to assess the androgenic activity of synthetic "progestins" currently used as "antiandrogens" for the treatment of prostate cancer in men, the effect of a series of these compounds has been measured following 14 days of treatment of adult castrated rats on specific and sensitive parameters of androgenic activity, namely ventral prostate weight and prostatic ornithine decarboxylase (ODC) activity. Medroxyprogesterone acetate (MPA) is almost equipotent with 5 alpha-dihydrotestosterone (DHT), a 49% increase in prostatic weight being observed at the low dose of 0.15 mg, twice daily (P less than 0.01). Megestrol acetate (Megace), chlormadinone acetate (CMA) and spironolactone were less potent but caused a 36-59% increase in prostatic weight at the highest dose used, namely 10 mg. At the 5 mg dose, cyproterone acetate (CPA) caused a 75% increase in prostatic weight. The androgenic activity of the compounds is even more clearly illustrated by their marked stimulatory effect on prostatic ornithine decarboxylase (ODC) activity. MPA, at the low dose of 0.15 mg, caused a 20-fold increase (relative to the effect of placebo) in the activity of the enzyme while the same dose of DHT caused a 15-fold stimulation of enzymatic activity. At the 10 mg dose, megestrol acetate, CMA and spironolactone caused 13.1, 11.8 and 8.6-fold stimulations of ODC activity, respectively. Flutamide, on the other hand, had no stimulatory effect on either ventral prostate weight or prostatic ODC activity. In agreement with glucocorticoid activity, MPA, megestrol acetate and CMA caused a marked inhibition (45-64%) of adrenal weight. The present data show that MPA is a highly potent androgen while megestrol acetate, CMA, CPA and spironolactone have lower but significant androgenic activity on all the parameters measured. It should be added that MPA, megestrol acetate and CMA are completely devoid of antiandrogenic activity while spironolactone shows weak antiandrogen action and CPA is a mixed agonist-antagonist. Flutamide, the compound used as reference, is the only compound devoid of any androgenic action and is thus acting as a pure antiandrogen on both ventral prostate weight and prostatic ODC activity. The present data have major implications for the choice of drug to be used for the treatment of androgen-sensitive diseases, especially prostate cancer. As shown by the present data, the synthetic "progestins" so-far available all possess variable levels of androgenic activity and are thus not recommended for the treatment of prostate cancer.     

Cloning of Azorhizobium caulinodans nicotinate catabolism genes and characterization of their importance in N2 fixation.

Twenty Azorhizobium caulinodans vector insertion (Vi) mutants unable to catabolize nicotinate (Nic- phenotype) were identified and directly cloned as pVi plasmids. These pVi plasmids were used as DNA hybridization probes to isolate homologous wild-type sequences. From subsequent physical mapping experiments, the nic::Vi mutants defined four distinct loci. Two, possibly three, of these loci are physically linked. A. caulinodans nic loci II and III encode the structural genes for nicotinate catabolism; nic loci I and IV encode nicotinate-driven respiratory chain components. Recombinant lambda bacteriophages corresponding to three of these loci were subcloned in pRK293; resulting plasmids were used for complementation tests with resolved nic::IS50 derivatives of the nic::Vi mutants. When wild-type A. caulinodans was cultured in defined liquid medium under 3% O2, nicotinate catabolism stimulated N2 fixation 10-fold. In these exponentially growing cultures, the entire (300 microM) nicotinate supplement was exhausted within 10 h. While nic::Vi mutants retained the ability to fix some N2, they did so at rates only 10% of that of the wild type: nitrogenase activity by nic::Vi mutants was not stimulated by 300 microM added nicotinate. Higher-level (5 mM) nicotinate supplementation inhibited N2 fixation. Because 5 mM nicotinate repressed nitrogenase induction in all nic::Vi mutants as well, this repression was independent of nicotinate catabolism. During catabolism, nicotinate is first oxidized to 6-OH-nicotinate by a membrane-bound nicotinate hydroxylase which drives a respiratory chain to O2. In A. caulinodans wild-type cultures, added 300 microM 6-OH-nicotinate stimulated N2 fixation twofold better than did added 300 microM nicotinate. Likewise, nic::Vi mutant 61302, defective in nicotinate hydroxylase, fixed N2 at wild-type levels when supplemented with 300 microM 6-OH-nicotinate. Therefore, nicotinate catabolism stimulates N2 fixation not by nicotinate hydroxylase-driven respiration but rather by some subsequent aspect(s) of nicotinate catabolism.     

Effects of tetrodotoxin-induced disuse on properties of developing rat gastrocnemius muscle

Our goal was to determine the influence of a complete lack of neuromuscular activity, during a period of rapid muscle growth, on muscle morphology and contractile function. Rats, 21 days old, had one hindlimb paralyzed for a period of 7-9 consecutive days by repetitive implantation of a silastic cuff containing tetrodotoxin (TTX), a specific nerve impulse conduction blocker, around the sciatic nerve. In situ isometric contractile properties of gastrocnemius were measured at 31 days of age, and muscles were subsequently examined histologically. Normal growth during this period resulted in a two- to three-fold increase in muscle weights, mean muscle fiber cross-sectional areas and increases in absolute twitch and tetanic tensions. After inactivity from 21 to 30 days of age, gastrocnemius muscles were smaller, and tetanically weaker, than age-matched controls. The normal cross-sectional area increase of fast-twitch fibers was preferentially affected. Inactive muscles also demonstrated significantly slower twitch responses, had higher twitch:tetanus ratios and relative tensions at 25 Hz than age-matched controls, suggesting a "slower" contractile response. On the other hand, maximum rate of tetanic tension development was elevated. These effects of inactivity appeared to be reversed by resumption of normal activity for 4 days. Neuromuscular inactivity during a relatively short period of rapid muscle growth causes significant muscle morphological and contractile changes, which are most likely reversible.     

Characterization of the Azorhizobium sesbaniae ORS571 genomic locus encoding NADPH-glutamate synthase.

Sixteen independent Azorhizobium sesbaniae ORS571 vector insertion (Vi) mutants defective in ammonium assimilation (Asm-) were selected; genomic DNA sequences flanking the insertion endpoints were cloned directly. Resulting recombinant plasmids were used to identify, by hybridization, corresponding wild-type DNA sequences from an A. sesbaniae lambda EMBL3 genomic library (lambda Asm phages). All 16 Asm- Vi mutants physically mapped to a single genomic locus. Plasmid subclones of recombinant phage lambda Asm152 were able to complement both Escherichia coli gltB and A. sesbaniae Asm- Vi mutants; NADPH-glutamate synthase activity was detected in all such strains complemented to Asm+. Heterologous and homologous complementations required both A. sesbaniae gltA+ and (inferred) gltB+ genes. Eleven A. sesbaniae Asm- Vi mutants mapped to a 4-kilobase-pair (kbp) DNA region that exhibited homology with Bacillus subtilis gltA+. In E. coli maxicell labeling experiments, this 4-kbp DNA region encoded a 165-kilodalton polypeptide that was inferred to be the product of the A. sesbaniae gltA+ gene (glutaminase NADPH-dependent L-glutamate synthase subunit). Site-directed Tn5-lacZ mutagenesis of a glt plasmid subclone identified a region that bisected this locus into (at least) two cistrons. Because the remaining five A. sesbaniae Asm- mutants mapped to a 1.5-kbp region adjacent to gltA+, these mutants probably define a single gltB+ gene (glutamate dehydrogenase NADPH-dependent L-glutamate synthase subunit); this region did not exhibit homology with the B. subtilis gltB+ gene.     

Spectroscopic Analyses of Manganese Ions Effects on the Conformational Changes of Inorganic Pyrophosphatase from Psychrophilic Shewanella sp. AS-11

Mn²⁺ ions influence the activity, temperature dependence, and thermostability of the psychrophilic Shewanella-PPase (Sh-PPase), and are required to function in cold environments. The functional characteristics of Sh-PPase on activation with Mn²⁺ ions are possibly related to conformational changes in the molecule. In this study, conformational changes of Sh-PPase on activation with Mn²⁺ ions were analyzed in solution by fluorescence spectroscopy analysis of intrinsic tryptophan residues, 1-anilino-8-naphthalene sulfonate fluorescence, and circular dichroism spectroscopy. For Sh-PPase, Mn²⁺ ions did not affect the flexibility of the tryptophan residues and secondary structure of the enzyme. However, the microenvironment of the tryptophan residues and surface area of Sh-PPase were more hydrophilic on activation with Mn²⁺ ions. These results indicate that activation with Mn²⁺ ions causes conformational changes around the aromatic amino acid residues and affects the hydrophobicity of the enzyme surface, which results in conformational changes. Substrate-induced conformational changes reflect that metal-free Sh-PPase in solution indicated an open structure and will be a close structure when binding substrate. In combination of our spectroscopic analyses on Sh-PPase, it can be concluded that activation with Mn²⁺ ions changes some conformation of Sh-PPase molecule in solution.