Dual posttranscriptional targets of retinoic acid-induced gene expression.

Retinoic acid-induced differentiation of the pre-osteoblastic cell line, UMR 201, is associated with a marked increase in the proficiency of posttranscriptional nuclear processing of alkaline phosphatase mRNA. In this study we attempted to correlate the posttranscriptional actions of retinoic acid with changes in phosphorylation, or abundance of spliceosome components, or both. Treatment with retinoic acid for periods of < or = 4 h resulted in dephosphorylation of nuclear U1 70K protein without affecting its abundance. Peptide mapping showed that U1 70K dephosphorylation was related to the disappearance of one specific phosphopeptide out of four major U1 70K phosphopeptides. A twofold decrease in mRNA expression of an isoform of alternative splicing factor that inhibits splicing was also observed over the same period. Tumor necrosis factor-alpha, which enhances the posttranscriptional action of retinoic acid, reduced U1 70K mRNA expression, while an inhibition of retinoic acid action by transforming growth factor-beta was associated with a marked increase in U1 70K mRNA levels. Our results draw attention to the complex interactions between short- and long-term alterations in the abundance and functional status of U1 70K, as well as SR proteins by growth and/or differentiation factors in the regulation of spliceosome formation and function.

Steroid regulation of parathyroid hormone-related protein expression and action in the rat uterus.

The gene encoding parathyroid hormone-related protein (PTHrP), an autocrine/paracrine inhibitor of vascular and nonvascular smooth muscle contractility, is regulated by hormonal steroids including estrogens (E2), 1,25-dihydroxy vitamin D (Vit D3) and glucocorticoids. While E2 increases PTHrP gene expression, Vit D3 and glucocorticoids inhibit transcriptional activity of this gene. In the uterus of ovariectomized rats, E2-treatment increases both PTHrP mRNA levels and smooth muscle sensitivity to the action of PTHrP(1-34). To examine the action(s) of Vit D3 and glucocorticoids on these parameters, OVX rats were treated with E2, Vit D3 or the synthetic glucocorticoid, dexamethasone (Dex), alone, or with E2 following a 1 h pretreatment with Vit D3 or Dex. PTHrP and PTH/PTHrP receptor mRNA were measured by blot hybridization analysis of RNA prepared from uteri collected 2, 4 and 24 h after treatment. Uterine horns were used to measure the effect of the steroids on the ability of PTHrP(1-34) to inhibit spontaneous myometrial contraction. When E2, Vit D3 and Dex were given alone, only E2 altered PTHrP mRNA levels in the uterus, however, a 1 h pretreatment with Dex but not Vit D3 markedly diminished this effect of E2. The temporal decline in uterine PTH/PTHrP receptor mRNA levels measured 2 and 4 h after E2 treatment inversely correlated to changes in sensitivity of the tissue to PTHrP(1-34) measured at 24 h after E2 administration. In comparison to E2 alone, treatment with Vit D3 and E2 augmented the uterine responsiveness to PTHrP(1-34) while pretreatment with Dex (1 mg/kg) and E2 decreased this response. These data indicate that in the uterus, Dex opposes the positive effect of E2 on PTHrP gene activity and differentially modulates the action of PTHrP on myometrial tone. Moreover, elevations in the circulating levels of cortisol at term may serve to decrease both the uterine expression of PTHrP and the local action of PTHrP on the myometrium prior to parturition, therefore promoting myometrial contraction associated with labor.

Foscarnet inhibits vascular smooth muscle contraction.

Foscarnet inhibited noradrenaline and calcium-mediated contractions of the isolated perfused tail artery of the rat. When the noradrenaline contractile response was split into two components, where the first was due to the release of calcium from intracellular stores and the second to the influx of calcium from the extracellular fluid, foscarnet (30 microM) inhibited only the first component of the response. Foscarnet did not inhibit the calcium influx component of the noradrenaline contraction, nor did it affect the inhibition of this component by the L-type calcium channel antagonists verapamil and nicardipine. These results indicate that foscarnet inhibits vascular smooth muscle contraction by inhibiting calcium release from intracellular stores.

Oestrogen enhancement of the myometrial response to exogenous parathyroid hormone-related protein (PTHrP), and tissue localization of endogenous PTHrP and its mRNA in the virgin rat uterus.

Classical pharmacological studies have shown that oestrogen dominance in humans and other animals can increase the responsiveness of the uterus to many locally acting peptides. Parathyroid hormone-related protein (PTHrP) has been shown to be expressed in the pregnant and non-pregnant rat uterus and exogenous PTHrP is known to relax uterine contraction in vitro. We investigated whether oestrogen dominance can influence the responsiveness of the uterine horn to PTHrP, and further studied the localization of PTHrP mRNA and protein in the rat uterine horn using in-situ hybridization and immunohistochemistry. Exogenous PTHrP(1-34) inhibited spontaneous and electrically induced contractions in uteri isolated from non-cycling rats. Pretreatment of non-cycling rats with oestradiol-17 beta increased uterine sensitivity to PTHrP: EC50 values for inhibition of spontaneous contractions by PTHrP were 0.33 nmol/l, 1.1 nmol/l, 2.6 nmol/l and 7800 nmol/l in uteri from animals treated for 2 days with oestradiol-17 beta alone, 2 days with oestradiol-17 beta + 1 day progesterone, 1 day with oestradiol-17 beta alone and in untreated rats respectively. Similar EC50 values were obtained for electrically stimulated uteri. In agreement with these findings, uterine horns from cycling rats in pro-oestrous and oestrous phases of the cycle showed a higher responsiveness to PTHrP(1-34) when compared with uterine horns taken from rats in metoestrus and dioestrus. PTHrP mRNA and protein were detected in the endometrial epithelium lining of the lumen and the endometrial glands, as well as in the myometrium of rats which were either pretreated for 2 days with oestradiol-17 beta or untreated. This study suggests that PTHrP may act in an autocrine and/or paracrine manner to modulate uterine motility and function.

Clodronate inhibits contraction and prevents the action of L-type calcium channel antagonists in vascular smooth muscle.

Clodronate (dichloromethylenebisphosphonate) decreased vasoconstriction of the isolated perfused rat tail artery mediated by norepinephrine and by Ca2+ in a K(+)-depolarizing solution. The norepinephrine contractile response was divided into two components by sequential manipulation of the composition of the perfusion fluid, where the first component is due to the release of Ca2+ from intracellular stores and the second to the influx of Ca2+ from extracellular fluid. Clodronate (20 microM) decreased only the first component of the response at a norepinephrine concentration of 50 nM, and both components of the response at a higher norepinephrine concentration (100 nM). The L-type Ca2+ channel blocking drugs, nicardipine (10 nM) and verapamil (1 microM), reduced the second component of the norepinephrine-mediated vasoconstriction, but in the presence of clodronate (20 microM) this blocking action was prevented. These results were confirmed by examining the interaction between clodronate and nicardipine on norepinephrine and K(+)-mediated lanthanum (La(3+)-resistant unidirectional 45Ca uptake. Nicardipine (1-10 nM) decreased the norepinephrine (100 nM) and K(+)-induced (60 mM) La(3+)-resistant unidirection 45Ca uptake in a concentration-dependent manner, but in the presence of clodronate (20 microM) this concentration-dependent response was abolished. Thus, clodronate not only reduced agonist-induced Ca2+ release from intracellular stores and Ca2+ influx through L-type Ca2+ channels but also prevented L-type Ca2+ channel antagonists from exerting their effect. These results indicate clodronate has two sites of action during vascular smooth muscle contraction: the first on intracellular mobilization of Ca2+ and the second on L-type Ca2+ channels.