Barbara M. Sanborn

Activation of Murine Lung Mast Cells by the Adenosine A3 Receptor

Adenosine has been implicated to play a role in asthma in part through its ability to influence mediator release from mast cells. Most physiological roles of adenosine are mediated through adenosine receptors; however, the mechanisms by which adenosine influences mediator release from lung mast cells are not understood. We established primary murine lung mast cell cultures and used real-time RT-PCR and immunofluorescence to demonstrate that the A2A, A2B, and A3 adenosine receptors are expressed on murine lung mast cells. Studies using selective adenosine receptor agonists and antagonists suggested that activation of A3 receptors could induce mast cell histamine release in association with increases in intracellular Ca2+ that were mediated through Gi and phosphoinositide 3-kinase signaling pathways. The function of A3 receptors in vivo was tested by exposing mice to the A3 receptor agonist, IB-MECA. Nebulized IB-MECA directly induced lung mast cell degranulation in wild-type mice while having no effect in A3 receptor knockout mice. Furthermore, studies using adenosine deaminase knockout mice suggested that elevated endogenous adenosine induced lung mast cell degranulation by engaging A3 receptors. These results demonstrate that the A3 adenosine receptor plays an important role in adenosine-mediated murine lung mast cell degranulation.

Antibodies From Preeclamptic Patients Stimulate Increased Intracellular Ca2+ Mobilization Through Angiotensin Receptor Activation

Background—Preeclampsia is a serious disorder of pregnancy characterized by hypertension, proteinuria, edema, and coagulation and vascular abnormalities. At the cellular level, abnormalities include increased calcium concentration in platelets, lymphocytes, and erythrocytes. Recent studies have shown that antibodies directed against angiotensin II type I (AT1) receptors are also highly associated with preeclampsia. Methods and Results—We tested the hypothesis that AT1 receptor–agonistic antibodies (AT1-AAs) could activate AT1 receptors, leading to an increased intracellular concentration of free calcium and to downstream activation of Ca2+ signaling pathways. Sera of 30 pregnant patients, 16 diagnosed with severe preeclampsia and 14 normotensive, were examined for the presence of IgG capable of stimulating intracellular Ca2+ mobilization. IgG from all preeclamptic patients activated AT1 receptors and increased intracellular free calcium. In contrast, none of the normotensive individuals had IgG capable of activating AT1 receptors. The specific mobilization of intracellular Ca2+ by AT1-AAs was blocked by losartan, an AT1 receptor antagonist, and by a 7-amino-acid peptide that corresponds to a portion of the second extracellular loop of the AT1 receptor. In addition, we have shown that AT1-AA–stimulated mobilization of intracellular Ca2+ results in the activation of the transcription factor, nuclear factor of activated T cells. Conclusions—These results suggest that maternal antibodies capable of activating AT1 receptors are likely to account for increased intracellular free Ca2+ concentrations and changes in gene expression associated with preeclampsia.

Phosphorylation of Serine 1105 by Protein Kinase A Inhibits Phospholipase Cβ3 Stimulation by Gαq

The mechanism by which protein kinase A (PKA) inhibits Gαq-stimulated phospholipase C activity of the β subclass (PLCβ) is unknown. We present evidence that phosphorylation of PLCβ3 by PKA results in inhibition of Gαq-stimulated PLCβ3 activity, and we identify the site of phosphorylation. Two-dimensional phosphoamino acid analysis of in vitro phosphorylated PLCβ3revealed a single phosphoserine as the putative PKA site, and peptide mapping yielded one phosphopeptide. The residue was identified as Ser1105 by direct sequencing of reverse-phase high pressure liquid chromatography-isolated phosphopeptide and by site-directed mutagenesis. Overexpression of Gαq with PLCβ3 or PLCβ3 (Ser1105 → Ala) mutant in COSM6 cells resulted in a 5-fold increase in [3H]phosphatidylinositol 1,4,5-trisphosphate formation compared with expression of Gαq, PLCβ3, or PLCβ3 (Ser1105 → Ala) mutant alone. Whereas Gαq-stimulated PLCβ3 activity was inhibited by 58–71% by overexpression of PKA catalytic subunit, Gαq-stimulated PLCβ3 (Ser1105→ Ala) mutant activity was not affected. Furthermore, phosphatidylinositide turnover stimulated by presumably Gαq-coupled M1 muscarinic and oxytocin receptors was completely inhibited by pretreating cells with 8-[4-chlorophenythio]-cAMP in RBL-2H3 cells expressing only PLCβ3. These data establish that direct phosphorylation by PKA of Ser1105 in the putative G-box of PLCβ3 inhibits Gαq-stimulated PLCβ3 activity. This can at least partially explain the inhibitory effect of PKA on Gαq-stimulated phosphatidylinositide turnover observed in a variety of cells and tissues.

HORMONES AND CALCIUM: MECHANISMS CONTROLLING UTERINE SMOOTH MUSCLE CONTRACTILE ACTIVITY

The regulation of myometrial contraction is of paramount importance for the maintenance of pregnancy and for parturition. Understanding this regulation involves delineating the pathways that control myometrial contraction and relaxation and defining the regulation of these pathways. The pathways can be broken down further into those signalling cascades controlling the concentration of intracellular free calcium (Ca2+i) and those controlling the contractile apparatus itself. This discussion focuses primarily on the former and their regulation during pregnancy. In particular, cross‐talk between the contractant and relaxant signalling pathways mediated through cyclic AMP is markedly changed at the end of pregnancy.

Ion channels and the control of myometrial electrical activity

Understanding the role of ion channels in the generation of slow waves and action potentials in the myometrium is critical in designing strategies to regulate uterine contractile activity. The development of the patch clamp technique has allowed the identification of specific types of channels in the myometrium and provided insights into their regulation by hormones and drugs. Specifically, new studies suggest that KATP and KCa channel openers could be important tools in the management of inappropriate uterine contractions, but peripheral effects will have to be controlled. Conversely, blockers of these same channels may have some effects on dystocia. The study of contractant-operated channels in the myometrium is still in its infancy, but promises new insights into possible modes of regulation as well. Myometrial activity is controlled at a number of levels. The regulation of ion channels is an important aspect, but receptor-mediated actions that do not appear to be voltage- or ion-dependent presumably are also important contributors and hence are sites of potential modulation as well. Clearly, future multifaceted approaches to tocolysis, and perhaps also dystocia, may well include agents targeting the activity of ion channels.

Molecular mechanisms regulating the effects of oxytocin on myometrial intracellular calcium.

Oxytocin stimulates an increase in intracellular calcium in uterine myometrium by several mechanisms. Several lines of evidence indicate that the oxytocin receptor is functionally coupled to GTP-binding proteins of the G alpha q/11 class which stimulate phospholipase C activity. The IP3 generated as a result of phospholipase C activation can trigger release of calcium from intracellular stores. The finding that the oxytocin-stimulated increase in intracellular calcium in myometrial cells is greater in the presence of extracellular calcium than that in its absence indicates that oxytocin also has effects on calcium entry. This action is nifedipine-insensitive but may involve indirect stimulation of calcium entry through release-operated channels. An anti-G alpha q/11 antibody inhibits both oxytocin-stimulated GTPase activity and phospholipase C activity in myometrial membranes. The stimulation by oxytocin of phosphoinositide turnover in COS cells transfected with a plasmid expressing the oxytocin receptor is enhanced by cotransfection of G alpha q. Co-transfection of intracellular domains of the oxytocin receptor causes varying degrees of interference with oxytocin-stimulated phosphoinositide turnover. The data suggest that more than one intracellular domain is involved in oxytocin receptor/G-protein coupling. Oxytocin receptor stimulation of phospholipase C is inhibited by cAMP. This occurs in myometrial cells and in COS cells transfected with a plasmid expressing the receptor. The inhibitory mechanism involves the action of protein kinase A and is probably targeted indirectly at the G alpha q/11 /phospholipase C coupling step.

Evidence for the Involvement of Several Intracellular Domains in the Coupling of Oxytocin Receptor to Gαq/11

In order to probe the nature of oxytocin receptor (OTR)/G alpha(q/11) protein coupling, we examined the effect of co-expression of OTR intracellular domains on oxytocin-stimulated phosphoinositide turnover in COSM6 cells overexpressing OTR and G alpha(q). Co-expression of G alpha(q) enhanced the oxytocin response maximally at a pOTR/pG alpha(q) plasmid transfection ratio of 1:0.16. In cells co-expressing OTR and G alpha(q/11), oxytocin stimulated phosphoinositide turnover with an EC50 of 48 nM. Co-transfection with plasmids expressing OTR intracellular domains inhibited oxytocin-stimulated phosphoinositide turnover by 23 +/- 6% (1i), 37 +/- 4% (2i), 55 +/- 6% (3i), and 40 +/- 6% (4i), respectively (P < 0.01). Expression of the 3i loop of the alpha(1B)-adrenergic receptor, which also couples to G alpha(q/11), inhibited phosphoinositide turnover by 35 +/- 2% (P < 0.01), while expression of the 3i loop of the dopamine 1A receptor, which couples to G alpha(s), had no effect. While these data indicate a functional role for the OTR 3i loop, they also suggest that interactions with more than one intracellular domain probably mediate the coupling of OTR to the G alpha(q/11) class of GTP-binding proteins.

KN-93 inhibition of G protein signaling is independent of the ability of Ca2+/calmodulin-dependent protein kinase II to phosphorylate phospholipase Cβ3 on 537-Ser

Stimulation of the phospholipase Cbeta (PLC) signaling pathway results in intracellular Ca2+ release and subsequent activation of calmodulin (CaM) and CaM kinase II (CaMK II). KN-93, an inhibitor of CaMK II, reduced the stimulation of phosphatidylinositide (PI) turnover by Galphai-coupled (formyl-Met-Leu-Phe, fMLP) or Galphaq-coupled [M1 muscarinic and oxytocin (OT)] receptors. The inhibitory effect of KN-93 was also observed when PLCbeta3 was stimulated directly by Galphaq or Gbetagamma in overexpression assays. CaMK II phosphorylated PLCbeta3 but not PLCbeta1 in vitro. Phosphorylation occurred exclusively on 537Ser in the X-Y linker region of PLCbeta3. 537Ser was also phosphorylated in the basal state in cells and phosphorylation was enhanced by ionomycin treatment. However, mutation of 537Ser to Glu had no effect on inhibition of Galphaq or Gbetagamma-stimulated PLCbeta3 activity by KN-93. KN-93 also inhibited Galphaq -stimulated PLCbeta1 activity, even though this enzyme is not a substrate for CaMK II. These data indicate that phosphorylation of PLCbeta3 by CaMK II is not directly involved in the inhibitory effect of KN-93 on phosphatidylinositide turnover.

FSH and the Sertoli Cell

Over a century ago, Sertoli (1865) discovered and described a non-germinal cell in the seminiferous epithelium of the testes. He visualized this cell to be a “supporting” element for the germ cells, a phagocytic element engaged in the removal of germ cell debris and other matter from the seminiferous tubule, and a “nurse” cell for the differentiating germinal elements. This cell was subsequently named the Sertoli cell, and both the morphological description and the suggestions for its function made originally by Sertoli have been supported by either direct or indirect observations.

Editorial: Increasing the Options—New 3′,5′ Cyclic Adenosine Monophosphate (cAMP)-Responsive Promoters and New Exons in the cAMP Response Element Modulator Gene

Both cAMP response element binding (CREB) and cAMP response element modulator (CREM) proteins bind to cAMP response elements (CRE) in genes regulated by cAMP (1). These proteins possess similar elements including regulatory regions responsive to phosphorylation, Gln-rich transactivation domains (t), and basic region Leu zipper (bZIP) domains responsible for dimerization and interaction with DNA (Fig. 1). The complexity of transcriptional responses to cAMP signals in a given cell is compounded by the expression of multiple isoforms, particularly of CREM. These result from the use of alternate promoters and transcription initiation sites, intron/exon junction splicing choices, poly-A sites affecting message stability, and translation initiation sites. CREM gene transcripts undergo extensive alternative splicing involving one or multiple exons, generating a large number of CREM messenger RNA (mRNA) isoforms. Although more than one isoform can be expressed in a given cell, the choices are not entirely random and can be both cell specific and regulated (1–10). Not surprisingly, CREM proteins transcribed from mRNAs lacking both exons encoding transactivation domains but retaining the DNA binding site determinants act as transcriptional repressors, whereas isoforms containing one or more transactivation domains can act as transcriptional activators of genes regulated by CREs (1, 2, 10). The P1 promoter responsible for generation of the large majority of CREM mRNA isoforms has been reported to be constitutive (2). Alterations in the concentration of full-length CREMt forms are determined, at least in part, by changes in mRNA stability (1, 4). An important alternative promoter (P2) contains multiple CREs (3). The products of transcription from this promoter, inducible cAMP early repressors (ICERs), are truncated molecules that retain the ability to bind to the CRE but not to activate transcription. Because the transcription of ICERs is enhanced by cAMP, the resulting proteins provide a potent negative feedback loop for CREM action that has been demonstrated to be important in a number of cell types (1). In an article in this issue by Daniel et al. (11), evidence is presented for use in the testis of two new promoters and two new exons containing translation initiation codons within the CREM gene. Both of these promoter regions (P3 and P4) and the respective exons u1 and u2 were mapped on genomic DNA between the exon containing a previously described translation start codon (Exon B in Fig. 1) and the exon containing most of the first transactivation domain (Exon C). In reporter assays, both rat P3 and P4 promoters were found to be cAMP responsive. Thus CREM mRNA forms potentially up-regulated by cAMP now include CREM u1 and CREM u2, as well as the ICERs. The data are consistent with significant expression of CREM u1 and CREM u2 mRNA in testis and in few or no other places. In testis, CREM gene transcripts undergo cellspecific, stage-dependent alternative splicing in the germ cells (1, 2, 12), and crem null mice exhibit an arrest in spermiogenesis (13, 14). Available data are consistent with the expression of limited amounts of truncated repressor forms of CREM mRNA generated from the P1 promoter in early germ cells and large amounts of the activator forms of CREM (CREMts) in haploid germ cells (2). The coactivator protein ACT, which facilitates CREMt action without the requirement for phosphorylation, is expressed concomitantly with CREMt (15). ICERs, generated from the cAMP-regulated P2 promoter, are not present in significant amounts in germ cells. The article by Daniel et al. provides evidence for differential expression of CREM u1 and CREM u2 mRNAs as a function of the stage of the seminiferous tubule cycle and during the first wave of spermatogenesis in testicular maturation. These data, while consistent with expression of CREM u2 and CREM u1 mRNA primarily in premeiotic and postmeiotic germ cells, respectively, await more direct confirmation. These studies expand the possibilities for regulation of CREM expression and raise some interesting questions. If both P3 and P4 promoters are cAMP-responsive, what accounts for the apparent differential expression of the two respective products? Daniel et al. suggest that the P3 promoter (CREM u1) is more sensitive to cAMP regulation than the P4 promoter (CREM u2). Expression of CREM u1 mRNA in postmeiotic germ cells would be consistent with the potential for regulation of P3 by CREMt, which is highest in these cells. Other elements may be more important for P4. Clearly, there is much more to be learned about the regulation of the P3 and P4 CREM promoters. Assuming that CREM u1 and CREM u2 proteins are expressed in significant amounts, what could be their functions? Exons u1 and u2 both contain translation initiation codons. Full-length complementary DNAs that use these ATGs and contain transactivation t and bZIP domains were prepared from testis mRNA. The resulting proteins presumably would activate CRE-directed transcription. Furthermore, inclusion of exon u1 introduces additional potential Received August 24, 2000. Address all correspondence and requests for reprints to: Barbara M. Sanborn, Department of Biochemistry and Molecular Biology, University of Texas Houston Medical School, Houston, Texas 77030. 0013-7227/00/