pTH (3-34) (bovine)

Studies were performed to determine the effects of PTH and related compounds on phosphatidylcholine (PC) hydrolysis in UMR-106 cells and the pathway by which the PTH effects occurred. The responses were compared with those of phorbol 12,13-dibutyrate (PDBu). Both bovine PTH-(1-34) [bPTH-(1-34)] and PDBu stimulated PC hydrolysis within 10 min. Significant effects were elicited by concentrations of 0.3-1 nM bPTH-(1-34) and 5 nM PDBu. Dose-dependent increases were seen at higher concentrations of both compounds, however, the response to bPTH-(1-34) was reduced at 30 nM. Bovine or human PTH-(1-34) and human PTH-related peptide-(1-34) [hPTHrP-(1-34)] were equipotent in their effects, whereas bovine [Nle(8,18)Tyr34]PTH-(3-34) amide [bPTH-(3-34)] and hPTH-(1-31) amide [hPTH-(1-31)] were less potent than bPTH-(1-34). bPTH-(3-34) did not antagonize the effects of bPTH-(1-34). Down-regulation of protein kinase C isozymes by 24-h treatment with PDBu completely prevented the stimulatory effect of PDBu on PC hydrolysis, but did not significantly affect the stimulatory effect of bPTH-(1-34). Both bPTH-(1-34) and PDBu stimulated transphosphatidylation of PC, indicating a phospholipase D-stimulated mechanism. The results suggest that in the UMR-106 cell line PTH can stimulate activation of PLD by a mechanism other than through protein kinase C.

Cytoskeletal elements are critical for cell morphology and signal transduction, and are involved in many cellular processes including motility, intracellular transport, and differentiation. Small GTP-binding proteins (G proteins) of the Ras family, such as RhoA, influence various elements of the cytoskeleton. RhoA stabilizes the actin cytoskeleton and promotes formation of focal adhesions. We found previously that RhoA is expressed in osteoblastic cells and is translocated to the plasma membrane and activated by PTH 1-34 as well as by Nleu(8,18) Tyr(34) PTH 3-34 amide, a PTH analog that does not increase cAMP. We therefore investigated effects of manipulating RhoA on the actin cytoskeleton of osteoblastic MC3T3-E1 cells. Three inhibitors were used: 1) GGTI-2166, a geranylgeranyl transferase I inhibitor that prevents the isoprenylation and membrane translocation of RhoA, 2) Y-27632, a Rho kinase inhibitor, and 3) alendronate, a nitrogen (N)-containing bisphosphonate that reduces intracellular geranylgeranylpyrophosphate through inhibiting farnesyl pyrophosphate synthase. To increase RhoA activity, we used the geranylgeranyl group donor geranylgeraniol (GGOH), and a constitutively active RhoA. The F-actin cytoskeleton and focal adhesions (FA) were visualized with rhodamine-phalloidin and fluorescent anti-vinculin antibodies, respectively. Cells were imaged with confocal microscopy. Actin stress fiber density, edge actin bundle density, focal adhesion density, cellular area and circularity (a morphological descriptor relating area and perimeter) were quantified by a program developed with Matlab software. GGTI-2166, Y-27632, and alendronate reduced actin stress fibers, FA density, and FA size, but had no effect on edge actin bundle density, cellular area, or circularity. GGOH completely antagonized the effects of alendronate, but did not significantly affect responses to GGTI-2166 or Y-27632. Constitutively active RhoA antagonized the effects of alendronate and GGTI-2166, but not those of Y-27632. The effects of alendronate were also antagonized by Nleu(8,18) Tyr(34) PTH 3-34 amide, but not by PTH 1-34. The results indicate that RhoA is involved in the maintenance of stress fibers and focal adhesions in osteoblastic cells, that PTH can affect this pathway independently of cAMP, and that a N-containing bisphosphonate can affect the actin cytoskeleton and focal adhesions through actions on geranylgeranyl groups and potentially through RhoA. In view of the importance of the actin cytoskeleton, the findings constitute evidence that N-containing bisphosphonates, when they attain certain concentrations, have effects on osteoblasts that could influence bone remodeling.

Synthetic bovine parathyroid hormone containing the NH2 terminal 34 amino acids [bPTH-(1-34)] was recently demonstrated to inhibit oxytocin stimulated uterine contraction in vitro. The parathyroid hormone analogues [Nle8, Nle18, Tyr34]bPTH-(3-34)amide [NTA-(3-34)] and [Tyr34]bPTH-(7-34)amide [NTA-(7-34)] have been reported to act as inhibitors of antagonists of parathyroid hormone (PTH) in numerous assays. In the present study the effects of these PTH analogues on uterine contraction and the ability of these analogues to act as antagonists to the uterine inhibitory action of bPTH-(1-34) in vitro were investigated. The NTA-(3-34) fragment had no effect on oxytocin stimulated uterine contractions. However, the NTA-(3-34) fragment was able to alter the ability of bPTH (1-34) to reduce oxytocin stimulated uterine contraction in a dose-related manner. Bovine PTH(1-34) (0.3 microgram/ml) reduced the contractile response obtained with oxytocin (0.5 mU/ml) by 20%. A dose of 15 micrograms/ml) of NTA-(3-34) abolished this inhibitory action of bPTH-(1-34) on oxytocin stimulated uterine contraction. In contrast the NTA-(7-34) caused a change in itself, stimulated contraction of resting uterine horns in a dose-related manner; 3.0 micrograms/ml of NTA-(7-34) caused a change in gram tension of + 1.5 grams. Bovine PTH-(1-34) was able to reduce the uterine contraction stimulated by NTA-(7-34) and 0.3 microgram/ml of bPTH-(1-34) reduced the contractile response obtained with 3.0 micrograms/ml of NTA-(7-34) by as much as 70%.(ABSTRACT TRUNCATED AT 250 WORDS)