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1 Three fully-de®ned a 1 -adrenoceptors (a 1A , a 1B and a 1D ) have been established in pharmacological and molecular studies. A fourth a 1 -adrenoceptor, the putative a 1L -adrenoceptor, has been de®ned in functional but not molecular studies, and has been proposed to mediate contraction of human lower urinary tract tissues; its relationship to the three fully characterized a 1 -adrenoceptors is not known. 2 In the present study, binding a nities were estimated by displacement of [ 3 H]-prazosin in membrane homogenates of Chinese hamster ovary (CHO-K1) cells stably expressing the human a 1A -, a 1B -and a 1D -adrenoceptors and were compared with a nity estimates obtained functionally in identical cells by measuring inhibition of noradrenaline (NA)-stimulated accumulation of [ 3 H]-inositol phosphates. 3 For the a 1A -adrenoceptor, binding studies revealed a pharmacological pro®le typical for the classically de®ned a 1A -adrenoceptor, such that prazosin, RS-17053, WB 4101, 5-methylurapidil, Rec 15/ 2739 and S-niguldipine all displayed subnanomolar a nity. A di erent pro®le of a nity estimates was obtained in inositol phosphates accumulation studies: prazosin, WB 4101, 5-methylurapidil, RS-17053 and S-niguldipine showed 10 to 40 fold lower a nity than in membrane binding. However, a nity estimates were not`frameshifted', as tamsulosin, indoramin and Rec 15/2739 yielded similar, high a nity estimates in binding and functional assays. 4 In contrast, results from human a 1B -and a 1D -adrenoceptors expressed in CHO-K1 cells gave antagonist a nity pro®les in binding and functional assays that were essentially identical. 5 A concordance of a nity estimates from the functional (inositol phosphates accumulation) studies of the a 1A -adrenoceptor in CHO-K1 cells was found with estimates published recently from contractile studies in human lower urinary tract tissues (putative a 1L -adrenoceptor). These data show that upon functional pharmacological analysis, the cloned a 1A -adrenoceptor displays pharmacological recognition properties consistent with those of the putative a 1L -adrenoceptor. Why this pro®le di ers from that obtained in membrane binding, and whether it explains the a 1L -adrenoceptor pharmacology observed in many native tissues, requires further investigation.

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Glucocorticoid in excess suppresses bone formation in vivo and disrupts bone matrix protein synthesis by osteoblasts in vitro. In contrast, transforming growth factor ␤ (TGF-␤) potently enhances bone matrix apposition. The rat TGF-␤ type I receptor gene promoter contains cis-acting elements for transcription factor CBFa1, which increases in parallel with osteoblast differentiation. Here we present molecular data linking these events. We show that previously unexplained effects of glucocorticoid on bone loss may be mediated in part by suppression of CBFa1, with a resultant decrease in the expression and activity of the TGF-␤ type I receptor on matrix-producing bone cells.Glucocorticoid-dependent bone loss by disregulated hormone expression or pharmacologic excess causes clinically significant osteoporosis in approximately 50% of affected individuals. Although changes in calcium absorption and effects on nonskeletal tissues contribute to the disease, striking effects occur directly on osteoblasts and at sites of active skeletal matrix deposition and remodeling (1, 2). A chronic reduction in osteoblast activity without corresponding changes in resorption would uncouple normal bone remodeling and decrease skeletal durability. Important genes targeted by glucocorticoid and molecular mediators for these events remain uncertain.Transforming growth factor ␤ (TGF-␤) 1 enhances bone matrix synthesis and repair, and bone contains perhaps the largest store of TGF-␤ in the body (reviewed in Ref. 3). Bone cells exhibit conventional type II and type III TGF-␤ receptors (TRII and TRIII) that influence TGF-␤ binding to type I receptor (TRI) or its activation, both essential for TGF-␤-dependent events (4 -6). There are few systems where regulation of TRI expression has been examined in detail and where functional changes correlate with these variations. We found TRI levels specifically maintained on differentiated bone cells in vitro, despite decreases in TRII and TRIII in response to bone morphogenetic protein 2 (5). In contrast, high levels of glucocorticoid rapidly reduce the proportion of TGF-␤ binding to TRI on bone cells and correspondingly decrease TGF-␤ activity (6).To understand these events further, we cloned the rat TRI promoter and observed higher promoter activity in osteoblastlike cells compared with undifferentiated bone cells or dermal fibroblasts (7). The TRI promoter includes a CpG island, several transcription factor Sp1 binding sites consistent with constitutive expression by many cells, and six cis-acting elements for transcription factors, termed CBFa (7-8).2 Whereas CBFa2 and CBFa3 are important gene regulators in lymphoid cells (9), CBFa1 expression increases in parallel with osteoblast differentiation in vitro (10).2 Moreover, targeted disruption of the CBFa1 gene eliminates osteogenesis in mice, and insertion, deletion, or missense mutations in CBFa1 occur in humans with the skeletal disorder cleidocranial dysplasia (11). Genes directly affected by CBFa1, especially those important for skeletal developme...

The interaction of tumor necrosis factor a (TNF) with its two membrane-bound receptors initiates intracellular events in which arachidonic acid and its derivatives are involved. In HeLa cells, TNF treatment induces an arachidonic acid-selective, Ca2+-dependent cellular phospholipase A2 (cPLA2). By itself, TNF causes a modest increase in cPLA2 activity, but with the Ca2+ ionophore A23187 it provides a strong synergistic action. Within minutes in response to TNF, cPLA2 becomes phosphorylated and in the presence of Ca2+ produces a 3-to 4-fold increase in activity. TNF also increases cPLA2 mRNA and protein expression, an estimated 5-fold increase in an 8-hr period. This increase in cPLA2 activity occurs, therefore, in a biphasic time-dependent manner. Dexamethasone, known to antagonize the action of TNF, is here shown to inhibit TNF-induced gene expression and to prevent the second phase of increase in cPLA2 activation. Our results suggest that the cPLA2 activation may provide a regulatory function and may explain the proinflammatory action of TNF.Tumor necrosis factor alpha (TNF) is a multifunctional cytokine produced mainly by monocytes and macrophages (1). TNF is now considered to be one of the major "inflammatory" cytokines, playing an essential role as an immunostimulant to increase host defense mechanisms against infections (2). In this role, TNF induces the production of prostaglandins, leukotrienes, and platelet-activating factor, which serve as inflammatory mediators. Therefore, a variety of events are initiated through the interaction of TNF with its membrane-bound receptors. The presence of two divergent receptors (3-6) and the multiple effects of TNF suggest that various functions of TNF may be mediated by coupled or independent receptor-activated signal transduction pathways (7).Early events of TNF-mediated signal transduction appear to involve lipids as mediators (8-16). For example, TNF stimulates the release of arachidonic acid (AA) from a variety of cell types (11-13), and AA-depleted cells are less susceptible to the cytotoxic effects ofTNF (14). The induction ofthe protooncogene c-fos by TNF is mediated through lipoxygenase products of AA (15), which are also required for TNF-cycloheximide-induced cytotoxicity (16). Since AA metabolites such as prostaglandins and leukotrienes play an important role in inflammation (17, 18), the proinflammatory properties of TNF could be ascribed to its ability to release AA. Of the several enzymatic pathways responsible for the release of AA, which is esterified to the sn-2 position of phospholipids, phospholipase A2 is an important enzyme for this hydrolytic cleavage reaction (18,19). Thus, one possibility is that AA release by TNF is mediated through its effect on PLA2.Phospholipase A2 enzymes can be grouped into two classes based on their molecular weight and cellular distribution. The "'14-kDa small molecular weight granule-associated secretory PLA2 (sPLA2) enzymes are rich in disulfide bonds, require millimolar levels of Ca2+ for activity, but display...