scholarly journals Stimulation of creatine kinase activity in rat skeletal tissue in vivo and in vitro by protease-resistant variants of parathyroid hormone fragments

1995 ◽  
Vol 309 (1) ◽  
pp. 85-90 ◽  
Author(s):  
D Sömjen ◽  
V Vargas ◽  
A Waisman ◽  
E Wingender ◽  
W Tegge ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Parathyroid Hormone ◽  
Double Mutant ◽  
Bone Cells ◽  
Specific Activity ◽  
Control Level ◽  
Maximal Stimulation ◽  
Stimulation Of

We have reported that mid-region fragments of human parathyroid hormone (hPTH), exemplified by hPTH-(28-48), stimulated [3H]thymidine incorporation into DNA and increased the specific activity of the brain-type isoenzyme of creatine kinase (CK) in both skeletal-derived cell cultures (ROS 17/2.8 cells) and immature rat epiphyseal cartilage and diaphyseal bone, without stimulating cyclic AMP synthesis which is a prerequisite for bone resorption. In the present study, substitution of amino acids in hPTH-(28-48), which resulted in increased resistance to proteolysis, produced variants that stimulated skeletal systems at two orders of magnitude lower concentration than the wild-type fragment. We modified hPTH-(28-48) at Leu-37 by replacement with Met, Thr or Val. Under conditions in which 20% of the native hPTH-(28-48) resisted proteolysis by cathepsin D for 6 h, approx. 40% of the L37V mutant and 70% of the L37T mutant remained intact. Substitution of Met for Phe-34 in addition to Thr for Leu-37, or the substitution of Met for Phe-34 alone, produced 100%-resistant fragments. These variants at residue 34 caused maximal stimulation of CK in ROS 17/2.8 cells at 0.24 nM compared with 24 nM for hPTH-(28-48). The double mutant stimulated CK activity significantly in immature rats, at a minimum dose of 12.5 ng/rat, and caused maximal stimulation at 125 ng/rat, a 10-fold lower dose than for hPTH-(28-48). The effect of the double mutant lasted up to 24 h which differs from the stimulation by hPTH-(28-48) in which CK specific activity returns to the control level at 24 h. This same dose also significantly stimulated CK activity in gonadectomized rats. These results show the advantage of using protease-resistant mid-region variants of hPTH-(28-48) to stimulate bone cells, in terms of lower doses and longer duration of effectiveness, both in vitro and in vivo.

Stimulation by insulin-like growth factor-I of creatine kinase activity in skeletal-derived cells and tissues of male and female rats

1994 ◽  
Vol 143 (2) ◽  
pp. 251-259 ◽  
Author(s):  
D Sömjen ◽  
A M Kaye
Keyword(s):  
Cell Cultures ◽  
Bone Cells ◽  
Specific Activity ◽  
Calvarial Bone ◽  
Factor I ◽  
Maximal Stimulation ◽  
Igf I ◽  
And Cartilage ◽  
Stimulation Of

Abstract Insulin-like growth factor-I (IGF-I) has been reported to mediate the effects of oestradiol-17β in the osteoblast-like osteosarcoma cell line ROS 17/2·8 and to stimulate directly cell proliferation in cell cultures derived from rat calvaria. Few data are available on the role of IGF-I in androgen stimulation of cultured skeletal cells and in oestrogen and androgen stimulation of bone and cartilage in vivo. The purpose of the present study was to compare the effect of IGF-I in rats in vivo with its effect in vitro on calvarial bone cells from females (responding only to oestrogens) and from males (responding only to androgens, such as testosterone and dihydrotestosterone). We found that IGF-I stimulated, in a dose- and time-dependent manner, the specific activity of creatine kinase (CK, a marker of skeletal cell division), in both female and male calvarial bone cells, in ROS 17/2·8 cells and in epiphyseal cartilage cell cultures. Maximal stimulation occurred at 30 or 100 nm within 1–2 h after stimulation. In ROS 17/2·8 cells, IGF-I stimulated [3H]thymidine incorporation, after 22 h of treatment, in parallel with CK activity. IGF-II, at higher doses than IGF-I (maximal stimulation at 300 nm), stimulated CK specific activity in female- and male-derived calvarial cell cultures. When IGF-I (50 nm) was applied together with oestradiol-17β (30 nm) or with dihydrotestosterone (300 nm) there was no additional response in the cultures. IGF-I injection (1·5 μg) into immature female or male rats resulted in a time-dependent stimulation of CK specific activity in epiphyses and diaphyses starting at 2 h. Injection of IGF-I (1·5 μg) together with either 5 μg oestradiol-17β to females or 50 μg dihydrotestosterone to males did not change the pattern or the extent of response to IGF-I. The response of both male- and female-derived bone cells to IGF-I suggests that it could play a role in the effects of androgens as well as oestrogens in bone and cartilage. Journal of Endocrinology (1994) 143, 251–259

Stimulation of creatine kinase specific activity in human osteoblast and endometrial cells by estrogens and anti-estrogens and its modulation by calciotropic hormones

1996 ◽  
Vol 150 (2) ◽  
pp. 275-285 ◽  
Author(s):  
B Fournier ◽  
S Häring ◽  
A M Kaye ◽  
D Sömjen
Keyword(s):  
Creatine Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
Bone Cells ◽  
Specific Activity ◽  
Human Bone ◽  
Endometrial Cells ◽  
Calcitropic Hormones ◽  
Stimulation Of

Abstract We have previously demonstrated sex-specific stimulation of creatine kinase specific activity (CK) in bone cells both in vivo and in vitro, in primary culture cells derived from rat and human bone and in established human bone-derived cell lines. We found that the female-derived cell line, SaOS-2, responded to 17β-estradiol (E2) by increased CK specific activity. The effects of E2 on the CK activity in SaOS-2 cells was inhibited by 100-fold excess of 4-hydroxytamoxifen (Tam) as well as by the other anti-estrogen, ICI 164,384. Tam by itself had some stimulatory effect whereas ICI 164,384 showed no estrogenic activity. We also demonstrated the estrogenic-like effect of another anti-estrogen, raloxifene (Ral), which is agonist only in the SaOS-2 osteoblast-like cells but not in the human endometrial, Ishikawa cell line. Ishikawa cells respond to E2 and to Tam by increased CK activity. In both osteoblasts and endometrial cell lines, Ral and Tam were inhibitory in the presence of E2. The effects of E2 on SaOS-2 cells are at least partially mediated by the estrogen receptor (ER) at the level of transcription as demonstrated by transient transfection experiments using the human creatine kinase promoter chloramphenicol acetyltransferase in these cells. Pretreatment of SaOS-2 with calcitropic hormones, either 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) or human parathyroid hormone (1–34) (hPTH(1–34)) increased the stimulation of CK by E2 by 40–60% relative to E2 alone and significantly increased the sensitivity of the cells to E2 by lowering the effective hormonal dose needed for stimulation of CK by E2 by 100-fold. This stimulatory effect of pretreatment of the cells with 1,25(OH)2D3 was due to a 2·5-fold increase in the level of ER expression as measured directly by enzyme immunoassay in the SaOS-2/1 subline. The increase in the responsiveness to E2 by hPTH(1–34) was not due to an increase in ER level in the cells. We can conclude that in cell cultures as in vivo, Ral shows different effects depending on the cell type, namely estrogenic-like activity in skeletal cells but not in uterine cells. We can also conclude that as with rat-derived cells, in bone cells derived from human bone 1,25(OH)2D3 increased the sensitivity to E2 due to an increase in the number of ER in the cells, whereas PTH(1–34) augmented the response to E2 without increasing ER, by another, as yet unknown, mechanism. These studies suggest that the treatment of pathological bone disorders may be improved by combined hormone therapy. Journal of Endocrinology (1996) 150, 275–285

Insulin sensitivity of rates of glycolysis and glycogen synthesis in soleus, stripped soleus, epitrochlearis, and hemi-diaphragm muscles isolated from sedentary rats

1983 ◽  
Vol 3 (7) ◽  
pp. 675-679 ◽  
Author(s):  
R. A. J. Challiss ◽  
J. Espinal ◽  
E. A. Newsholme
Keyword(s):  
Fatty Acid ◽  
Glucose Utilization ◽  
Glycogen Synthesis ◽  
Maximal Inhibition ◽  
Maximal Stimulation ◽  
Glucose Fatty Acid Cycle ◽  
Isolated Adipocytes ◽  
Stimulation Of

The effect of insulin concentrations on the rates of glycolysis and glycogen synthesis in four different in vitro rat muscle preparations (intact soleus, stripped soleus, epitrochlearis, and hemi-diaphragm) were investigated: the concentrations of insulin that produced half-maximal stimulation of the rates of these two processes in the four muscle preparations were similar – about 100 μunits/ml. This is at least 10-fold greater than the concentration that produced half-maximal inhibition of lipolysis in isolated adipocytes. Since 100 μunits/ml insulin is outside the normal physiological range in the rat, it is suggested that, in vivo, insulin influences glucose utilization in muscle mainly indirectly, via changes in the plasma fatty acid levels and the ‘glucose/fatty acid cycle’. Consequently the view that insulin stimulates glucose utilization in muscle mainly by a direct effect on membrane transport must be treated with caution.

scholarly journals Stimulation of creatine kinase BB activity by parathyroid hormone and by prostaglandin E2 in cultured bone cells

1985 ◽  
Vol 225 (3) ◽  
pp. 591-596 ◽  
Author(s):  
D Sömjen ◽  
A M Kaye ◽  
I Binderman
Keyword(s):  
Creatine Kinase ◽  
Parathyroid Hormone ◽  
Prostaglandin E2 ◽  
Bone Cells ◽  
Combined Treatment ◽  
Fold Increase ◽  
Bone Cell ◽  
Growth Promoters ◽  
Bone Cell Cultures ◽  
Stimulation Of

Bone cells in culture responded to parathyroid hormone (PTH) and prostaglandin E2 (PGE2) by a 2-fold increase in creatine kinase (CK) activity. Combined treatment resulted in a higher response than with PTH alone. Calcitonin (CT) failed to stimulate CK activity, did not affect the response of CK to PTH, but inhibited slightly the increase in CK activity by PGE2. Bone-cell cultures grown in low [Ca2+] (0.125 mM), enriched in PTH-responsive osteoblast-like cells, responded to PTH, but not to PGE2 or CT, by increased CK activity. In both normal and low-[Ca2+] cultures, 8-bromo cyclic AMP did not affect CK activity, nor did it change the response of the cells to PTH, PGE2 or CT. The increase in CK activity was time- and dose-dependent and inhibited both by cycloheximide and by actinomycin D. The isoenzyme of CK stimulated was the CKBB form, the isoenzyme induced by other hormones. This appears to be the first report of the stimulation of CK activity by a polypeptide hormone or a prostaglandin. We suggest that stimulation of CKBB can serve as a marker for the action of a variety of hormones and growth promoters.

scholarly journals Stimulation of cell proliferation in skeletal tissues of the rat by defined parathyroid hormone fragments

1991 ◽  
Vol 277 (3) ◽  
pp. 863-868 ◽  
Author(s):  
D Sömjen ◽  
K D Schlüter ◽  
E Wingender ◽  
H Mayer ◽  
A M Kaye
Keyword(s):  
Parathyroid Hormone ◽  
Bone Resorption ◽  
Thymidine Incorporation ◽  
Specific Activity ◽  
Fold Increase ◽  
Dependent Manner ◽  
Equimolar Concentration ◽  
Dose Dependent

We have found, in previous studies in vitro using skeletal derived cell cultures, that mid-region fragments of human parathyroid hormone (hPTH) stimulate [3H]thymidine incorporation into DNA and increase the specific activity of the brain-type isoenzyme of creatine kinase (CK). These changes occurred without an increase in cyclic AMP formation which is linked to bone resorption. In this study, we found that the mid-region fragment hPTH-(28-48) stimulated CK activity in diaphysis, epiphysis and kidney in a time- and dose-dependent manner, parallel to the effects of the whole molecule bovine (b)PTH-(1-84) and the fully active fragment hPTH-(1-34). The increase caused by hPTH-(28-48) at a dose of 1.25 micrograms/rat was not less than the 2-fold increase caused by a roughly equimolar concentration bPTH-(1-84). A significant increase was reached at 1 h after intraperitoneal injection in all cases. All three sequences of PTH caused an increase in [3H]thymidine incorporation into DNA in diaphysis and epiphysis, but not in kidney, 24 h after injection. A fragment further towards the C-terminal, hPTH-(34-47), was inactive compared with an equimolar concentration of the fragment hPTH-(25-39), which stimulated both CK activity and DNA synthesis. These results in vivo are in line with previous findings in vitro; they provide further support for the suggestion that mid-region fragments of the PTH molecule could be used to induce bone formation without incurring the deleterious effect of bone resorption.

DIFFERENTIAL STIMULATION BY PARATHYROID HORMONE OF BONE AND KIDNEY RIBONUCLEIC ACID SYNTHESIS

1971 ◽  
Vol 49 (3) ◽  
pp. 493-506 ◽  
Author(s):  
J. STEINBERG ◽  
G. NICHOLS
Keyword(s):  
Parathyroid Hormone ◽  
Specific Activity ◽  
Acid Synthesis ◽  
Hormonal Stimulation ◽  
Precursor Pool ◽  
Hormonal Effect ◽  
Parathyroid Extract ◽  
Apparent Shift ◽  
Stimulation Of

SUMMARY The effects of parathyroid extract (PTE) on the synthesis in vivo of free nucleotide and RNA were compared in rat metaphysial bone and kidney. The incorporation of 32P into chromatographically pure acid-soluble 5′-AMP and purified bulk RNA was examined at various times after PTE administration. Pulse-labelled RNA was further characterized by sedimentation in sucrose density gradients and by ribomononucleotide analysis. In both organs the labelling of 5′-AMP and its turnover were accelerated after administration of the hormone. The pool size of free AMP of kidney was approximately 3 times that of bone; neither was affected by PTE. The specific activity of pulse-labelled kidney AMP was always greater, and hormonal stimulation of its labelling was more rapid than in bone. Despite more extensive precursor labelling, the stimulation of renal RNA synthesis was negligible, and was delayed for several hours, the overall hormonal effect being inseparable from its effect on phosphate entry into the nucleotide precursor pool. In bone, the hormonal stimulation of RNA labelling was immediate, and continued to increase at a linear rate for up to 12 h. Initially, stimulation of RNA polymerization accounted for the total hormonal effect, while after 4 h an increasing proportion of the total increase in RNA labelling was attributable to enhanced precursor labelling. Newly synthesized bone RNA differed qualitatively from kidney RNA in its sedimentation properties and composition. Although the labelling of all RNA species and RNA-nucleotides in bone was stimulated by PTE, there was a proportionately greater effect on the labelling of ribosomal RNA, and an apparent shift towards GMP-rich molecules, neither change being manifest in kidney. It is concluded that while bone and kidney share certain mechanisms, they show changes in RNA biosynthesis in response to parathyroid hormone which are both quantitatively and qualitatively different and which are in accord with the RNA requirements for the respective physiological response of each.

Effect of parathyroid hormone-like peptides on 25-hydroxyvitamin D-1 alpha-hydroxylase activity in rodents

1990 ◽  
Vol 258 (2) ◽  
pp. E297-E303 ◽  
Author(s):  
A. T. Walker ◽  
A. F. Stewart ◽  
E. A. Korn ◽  
T. Shiratori ◽  
M. A. Mitnick ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Maximal Activity ◽  
Hydroxylase Activity ◽  
Humoral Hypercalcemia Of Malignancy ◽  
Vitamin D Metabolism ◽  
Control Activity ◽  
Humoral Hypercalcemia ◽  
Stimulation Of

The role of vitamin D metabolism in the humoral hypercalcemia of malignancy syndrome (HHM) is unclear. We studied in vivo and in vitro effects of synthetic parathyroid hormone-like peptides (PTH-LPs) on rodent renal 25-OHD-1 alpha-hydroxylase activity. Infusion of mice with PTH-LP-(1-36) at 10 pmol/h for 12 and 24 h showed significant (429 +/- 139% and 937 +/- 413%, respectively) stimulation of control enzyme activity. Infusion for 36 h demonstrated diminution of activity to levels nearer to the unstimulated state (228 +/- 36% of control). In that maximal activity was observed after 24 h of infusion, we examined 1 alpha-hydroxylase activity after variable dosages of PTH-LP-(1-36) at this time point. Animals infused with PTH-LP-(1-36) at dosages of 2.5, 10, and 30 pmol/h for 24 h demonstrated 1 alpha-hydroxylase activities of 0.71 +/- 0.12, 4.74 +/- 2.09, and 9.91 +/- 1.01 ng.mg protein-1.20 min-1 (means +/- SD), respectively, all significantly greater than control activity (0.51 +/- 0.20 ng.mg protein-1.20 min-1). PTH-LP-(1-36) and PTH-LP-(1-74) were comparable in potency to bovine (b)PTH-(1-34) in stimulating 1 alpha-hydroxylase. Direct in vitro incubation of PTH-LP-(1-36) with renal slices resulted in stimulation of 1 alpha-hydroxylase activity up to 200% of control levels, comparable to that seen with equimolar concentrations of bPTH-(1-34).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Tubulin tyrosinolation in human polymorphonuclear leukocytes: studies in normal subjects and in patients with the Chediak-Higashi syndrome.

1982 ◽  
Vol 95 (2) ◽  
pp. 519-526 ◽  
Author(s):  
J Nath ◽  
M Flavin ◽  
J I Gallin
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Specific Activity ◽  
Normal Subjects ◽  
In Vivo Experiments ◽  
Peritoneal Leukocytes ◽  
Chediak Higashi Syndrome ◽  
Human Polymorphonuclear Leukocytes ◽  
Stimulation Of

We have recently reported a specific dose-dependent stimulation of posttranslational incorporation of tyrosine into tubulin alpha-chains of rabbit peritoneal leukocytes as induced by the synthetic peptide chemoattractant formyl-methionyl-leucyl-phenylalanine (FMLP). The present study reports a similar, specific stimulation of tubulin tyrosinolation in human polymorphonuclear leukocytes (PMN). When compared to normal PMN, both the resting and FMLP-stimulated levels of posttranslational tyrosine incorporation were two- to threefold higher in PMN of three patients with the Chediak-Higashi syndrome (CHS). The concentration of cellular tubulin and the specific activity of tubulin tyrosine ligase were similar in PMN of CHS patients and normal donors and resembled that of other non-neuronal cells. The high levels of tyrosine incorporation in PMN of CHS patients were normalized by the administration of ascorbate, both in vitro and in in vivo experiments. In vitro addition of ascorbate also inhibited the FMLP-induced stimulation of tyrosine incorporation in both normal and CHS cells. Normalization of higher levels of tyrosine incorporation in PMN of CHS patients and the inhibition of FMLP-induced stimulation of tubulin tyrosinolation in normal and CHS cells as observed with ascorbate could also be affected by other reducing agents such as reduced glutathione, cysteine, or dithiothreitol. These results suggest a possible relationship between cellular redox and tubulin tyrosinolation in PMN.

Stimulation of adenylate cyclase in isolated rat glomeruli by prostaglandins

1978 ◽  
Vol 235 (5) ◽  
pp. F458-F464 ◽  
Author(s):  
D. Schlondorff ◽  
P. Yoo ◽  
B. E. Alpert
Keyword(s):  
Parathyroid Hormone ◽  
Adenylate Cyclase ◽  
Specific Activity ◽  
Fold Increase ◽  
Guanosine Triphosphate ◽  
Crude Homogenate ◽  
Maximal Stimulation ◽  
Camp System ◽  
Isolated Rat ◽  
Stimulation Of

The effect of prostaglandins and various agents on cyclic adenosine 3',5'-monophosphate generation was studied in isolated rat glomeruli. Specific activity of adenylate cyclase in the glomeruli showed a 12-fold increase over the crude homogenate and a fivefold increase over a tubular preparation. Prostacyclin (PGI2) preferentially stimulated adenylate cyclase of isolated glomeruli at a concentration as low as 10(-9) M. Prostaglandins (PGE1, PGE2, and PGA2) and parathyroid hormone (1–34 synthetic PTH fragment) increased adenylate cyclase in glomeruli with maximal stimulation at 2 X 10(-5) M and 2–4 microgram/ml, respectively. No inhibition of PGE2 on PTH stimulation was observed. Isoproterenol (2 X 10(-4) M) caused a small stimulation, while PGF2alpha, arginine vasopressin, and angiotensin II were ineffective. The presence of guanosine triphosphate in the adenylate cyclase assay enhanced basal and PGE2- and PTH-stimulated activity, but had no effect on NaF stimulation. These findings show an effect of prostaglandins and PTH on the glomerular cAMP system and raise the possibility of a physiological action of these agents on the glomerulus.

Sign in / Sign up

Export Citation Format

Share Document