The rat osteosarcoma cell line UMR-106 has an osteoblast-like phenotype and possesses parathyroid hormone (PTH)-responsive dual signal transduction systems [adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) and calcium-protein kinase C (Ca-PKC)]. These cells transport inorganic phosphate (Pi) by a Na(+)-dependent carrier under stimulation by PTH. The present study aimed to clarify PTH-responsive signal transduction mechanisms in the regulation of Na(+)-dependent Pi transport by PTH in UMR-106 cells. Exposure of these cells to 10(-7) mol/l PTH induced a significant increase in Pi uptake within 30 min of incubation and it became maximal after 2 h. Parathyroid hormone (10(-9)-10(-7) mol/l) stimulated Pi uptake dose dependently. Activation of PKC by 12-O-tetradecanoyl phorbol-13-acetate (TPA) also increased Pi uptake in time- and dose-dependent manners similar to PTH. In contrast, neither PKA activation by 10(-4) mol/l forskolin or by 10(-4) mol/l dibutyryladenosine 3',5'-cyclic monophosphate nor calcium ionophore treatment with 10(-7) mol/l A23187 or with 10(-7) mol/l ionomycin during 3-h incubations affect Pi uptake, except its increase by 10(-4) mol/l forskolin at a 3-h incubation. These agents had no influence on Pi uptake even in combined treatments with TPA. The PTH-induced increase in Pi uptake was abolished almost completely by pretreating cells with PKC inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H-7) (50 mumol/l) or staurosporin (10 and 50 nmol/l), and by down-regulating PKC with a prolonged TPA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

HydrolyJi,= ol'¢ndoth=lil~ l by rat kidney membran~ ~ats invcJtiilated min~ a r¢'~r,~-phz~ HPLC and an autonutted I~s.pham protein re.qa~r. F_.ndothelin I w~,= hydrolyz~;d into four major fragments which w=m d=t~:ted by HPL¢, Pho.phoramidon, an inhibitor of neutral endo~ptid~t,c 24,1 l, almost =ompl=t~y supp~_~d_ the production of thr¢~ fralrnrntr,, but on= fralr, mcnt was not affcrt~ by tl'~ inhibitor, Anallais ofN-terminal ~quence.s of the d¢l~radation products revcal~ that the pho~phoramidon-~cmlitiv¢ fralmenta were s~=rated by cl¢~vai~ at th~ Seal-Lea" bond of endoth¢lin I that wa~ ideation| with it~ clcavall~ ~it¢ by purified rat ¢ndoi~ptida~ 24,1 I, reported previously. Th= phozphoramidon.in~miti,,~ frallrnent was produc~.d by cl~val~ at ~u~=Asp '', which was distinct from the sites by endopeptida~ 24,11, but ¢orRtponded to that by a phosphoramidon.in~ndtivc m©tallo-endop~ptidar,~ recently it, elated from rat kidney mcmbran¢~ by us [(1992) Eur. J. Bioch~m, 204, 547-552]. Kinetic determination o1" endothclin I hydroly~lis by the i~lat~ eaaym¢ yielded walu~ of ,~,,=71.5 aM and ka,. = 1.49 s "~' Siv[n~ a ratio of A'.,/E,~=2,O8 x tO' tC',M", The/f~ value wutl much hisher and th¢ L'.d/~. value wa,~ much lower than tho~t £or rat ~ndo~ptida~ 24, I 1 reportad previou,,ly, Thus, endop.ptidarm 24,11 app*ar,, to h>,drolyz~ ¢ndoth~lin I more ¢fficicntly than the iml~t=d enzyme dot~. Both enzymes may play phyr, iologir.al rol~ in the rn¢taboli~m ofendothelin I by rat kidney m~ntbrun~ in vivo,

To characterize a chymotrypsin-like hydrolytic activity in the cell surface membranes of intact opossum kidney (OK) cells, we partially purified a protease from the membrane fractions of OK cells using Suc-Leu-Leu-Val-Tyr-MCA (Suc, succinyl; MCA, 4-methylcoumaryl-7-amide), a synthetic substrate for chymotrypsin, as the substrate. The semipure enzyme showed seryl chymotrypsin-like characteristics such as preferential hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA and inhibition by phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, and chymostatin. However, it clearly differed from alpha-chymotrypsin in its weak ability to hydrolyze Suc-Ala-Ala-Pro-Phe-MCA and in its high molecular mass (250-300 kDa). The enzyme also had an endopeptidase-like activity in that it cleaved human parathyroid hormone(1-84) at the Leu(37)-Gly(38) and Arg(52)-Lys(53) bonds. These results suggest that a high molecular mass chymotrypsin-like endopeptidase with unique characters is present in the membrane fractions of OK cells.

The mechanisms involved in parathyroid hormone (PTH) degradation by proximal renal tubule cells were studied using an opossum kidney cell line possessing PTH receptors as an in vitro model system. One hour incubation of 5 nmol/l human (h) PTH-(1-84) with intact opossum kidney cells (4.0\m=x\ 106 cells) resulted in about 70% degradation and disappearance of hPTH-(1-84) from the medium, as determined by a two-site immunoradiometric assay. Preincubation with 100 nmol/l h[Nle8, Nle18, Tyr34]PTH-(1-34)amide for 6, 24, 48 and 72 h caused a 26, 47, 62 and 73% decrease, respectively, in PTH degradation by opossum kidney cells. Binding studies with 125I-labeled h[Nle8, Nle18, Tyr34]PTH-(1\x=req-\ 34)amide as a radioligand showed that PTH receptor binding decreased with the time of pretreatment with the agonist. Pretreatments of the cells with monensin, an inhibitor of endocytosis, and the lysosomotropic agents such as chloroquine, ammonium chloride and leupeptin, inhibited degradation of hPTH-(1-84) by 87, 71, 76 and 72%, respectively. Concentrations of 5 nmol/l hPTH-(39-84) and hPTH-(39-68), which are known not to bind to PTH receptors appreciably, were not degraded by opossum kidney cells during 1 h incubations. Thus intact, biologically active PTH, but not its inactive fragments, is degraded by opossum kidney cells, by receptor-mediated endocytosis and lysosomal hydrolysis. A mechanism resembling the peritubular uptake of intact PTH by perfused kidneys reported previously appears to play a main role in PTH metabolism by cultured renal cells.

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