Effects of Sialagogues on Ornithine Decarboxylase Induction and Proto-oncogene Expression in Murine Parotid Gland

Abstract

The mechanism of a sialagogue-induced increase in ornithine decarboxylase (ODC) activity and the expressions of proto-oncogenes in murine parotid gland were investigated by use of isoproterenol (IPR), carbachol (CC), and methoxamine (MTX). The results were as follows: (1) The three sialagogues had similar effects on the parotid in vivo (mouse parotid after a single injection of IPR) and/or in vitro (rat parotid explants cultured on siliconized lens paper floating on 199 medium containing IPR, CC, or MTX), the order of their effectiveness being IPR > CC > MTX. (2) Northern/dot and Western blot analyses revealed that the sialagogues elevated the steady-state levels of ODC mRNA and ODC protein to maxima at two h and six h, respectively, after stimulation. The increases were roughly proportional to those in ODC activity, suggesting that sialagogue-dependent enzyme induction is regulated at the transcriptional level. (3) The mRNAs of four of nine proto-oncogenes examined showed sialagogue-dependent increases to maxima at 30 min (c-fos) or 60 min (c-jun, c-myc, and c-src) after the beginning of stimulation. These increases were all transient, with the levels returning to the control values (without sialagogue) within 60 min. (4) The IPR-dependent elevations of ODC activity and the mRNAs of ODC, c-fos, and c-jun were inhibited by monensin, but not by polymyxin B. On the other hand, the CC-dependent increases in these parameters were inhibited by polymyxin B but not by monensin.(ABSTRACT TRUNCATED AT 250 WORDS)

Constitutive expression of thrombospondin 1 in MC3T3‐E1 osteoblastic cells inhibits mineralization

“…After stripping antibodies using a Western Blot recycling Kit (Alpha Diagnostic, San Antonio, TX) according to the manufacturer's instructions, the blot was reprobed with rabbit anti-b-actin peptide antibody (Santa Cruz) (1:200 diluted) as a control of protein loading. Quantification was then achieved by densitometric scanning as described previously (Kawano et al, 1992).…”

“…Total RNA was isolated with RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and Northern blot analysis was performed as described previously (Kawano et al, 1992). Denatured RNA samples (5 mg RNA per lane) were loaded on 1% agarose gels containing 6.5% formaldehyde and transferred to Hybond-N þ nylon filters (Amersham).…”

The Role of Phosphotyrosine Signaling Pathway in Parotid Gland Proliferation and Function

“…The signals initiated by p2\ ms GTP are further enhanced by several events leading to activation of the serine and threonine kinases Ra/-1 and MAP-kinase . Here again, both isoproterenol-and EGF-stimulated acinar cell growth involve activation of these kinases, which ultimately leads to cell division through activation of transcription factors such as Rb 110 , myc, fos, and jun (Kousvelari et al, 1988(Kousvelari et al, , 1990Mirels et al, 1989;Kawano et al, 1992;Lee et al, 1992;Pulverer et al, 1993). Inoue and co-workers (Kawano et al, 1992) have additionally shown isoproterenol treatment to cause increased mRNA and protein levels for ornithine decarboxylase (an enzyme required for DNA synthesis) and the sre proto-oncogene.…”

Osteotropic factor‐stimulated synthesis of thrombospondin in rat dental pulp cells

“…Total RNA was isolated from guanidine thiocyanate-disrupted RPC-C2A cells by ultracentrifugation in cesium chloride solution [14]. A sample of 5 gg of RNA was fractionated in 1% agarose gel containing 6.5% formaldehyde [15]. After transfer to a Hybond-N + membrane (Amersham), and hybridization with cDNA probe (1-5X 109 cpm/lag), the membrane was washed with 1 × SSPE/ 0.1% SDS for 1 h and then with 0.1× SSPE/0.1% SDS for 30 min [15].…”

“…A sample of 5 gg of RNA was fractionated in 1% agarose gel containing 6.5% formaldehyde [15]. After transfer to a Hybond-N + membrane (Amersham), and hybridization with cDNA probe (1-5X 109 cpm/lag), the membrane was washed with 1 × SSPE/ 0.1% SDS for 1 h and then with 0.1× SSPE/0.1% SDS for 30 min [15]. The hybridized RNA was located by exposure to Fuji RX film at -80°C for 2 h with intensifying screens (DuPont).…”