Advances in the pharmacological control of the bladder.
(57/20616)
To effectively control bladder activity, and to treat urinary incontinence caused by bladder overactivity, identification of suitable targets for pharmacological intervention is necessary. Such targets may be found in the central nervous system (CNS) or peripherally. The causes of bladder overactivity are not known, but theoretically increased afferent activity, decreased inhibitory control in the CNS and/or peripheral ganglia, and increased sensitivity of the detrusor to efferent stimulation may be involved. Several CNS transmitters may modulate voiding, but few drugs with a defined CNS site of action have been developed for treatment of voiding disorders. Potentially, drugs affecting GABA, opioid, 5-HT, noradrenaline, dopamine, or glutamatergic receptors and mechanisms can be developed, but a selective action on the lower urinary tract may be difficult to obtain. Traditionally, drugs used for treatment of bladder overactivity have had a peripheral site of action, mainly the efferent neurotransmission or the detrusor muscle itself. Antimuscarinic drugs, beta-adrenoceptor agonists, alpha-adrenoceptor antagonists, drugs affecting membrane channels, prostaglandin synthetase inhibitors and several other agents have been used. However, none of them has been developed specifically for treatment of bladder disorders, and their efficacy, as judged from controlled clinical trials (where performed), is often limited. Recent information on the alpha-adrenoceptor, beta-adrenoceptor (beta 3), and muscarinic receptor subtypes of the human detrusor and outflow region can be the basis for the development of compounds with effect on bladder overactivity and with improved tolerance. New ways of decreasing acetylcholine release may represent a promising way of controlling bladder contraction. Potassium channel (KATP) openers are theoretically attractive, but the drugs available so far have targeted vascular rather than bladder smooth muscle, which has limited their clinical use. However, new drugs belonging to these groups with an interesting profile of action have been developed. Drugs decreasing afferent activity represent an attractive therapeutic approach and transmitters of afferent nerves and their receptors are possible targets for pharmacological interventions. Tachykinins, such as substance P, neurokinins A and B, and other neuropeptides have been demonstrated in nerves of the lower urinary tract and have been shown to influence bladder function. Agents affecting these nerves by causing release of tachykinins, such as capsaicin and resiniferatoxin, given intravesically can be effective in some cases of bladder overactivity, and agents antagonizing tachykinin receptors may also be of therapeutic interest. New drugs specifically directed for control of bladder activity are under development and will hopefully lead to improved treatment of urinary incontinence. (+info)
Identification of a Frizzled-like cysteine rich domain in the extracellular region of developmental receptor tyrosine kinases.
(58/20616)
In Drosophila, members of the Frizzled family of tissue-polarity genes encode proteins that appear to function as cell-surface receptors for Wnts. The Frizzled genes belong to the seven transmembrane class of receptors (7TMR) and have on their extracellular region a cysteine-rich domain that has been implicated as the Wnt binding domain. This region has a characteristic spacing of ten cysteines, which has also been identified in FrzB (a secreted antagonist of Wnt signaling) and Smoothened (another 7TMR, which is involved in the hedgehog signalling pathway). We have identified, using BLAST, sequence similarity between the cysteine-rich domain of Frizzled and several receptor tyrosine kinases, which have roles in development. These include the muscle-specific receptor tyrosine kinase (MuSK), the neuronal specific kinase (NSK2), and ROR1 and ROR2. At present, the ligands for these developmental tyrosine kinases are unknown. Our results suggest that Wnt-like ligands may bind to these developmental tyrosine kinases (+info)
Activated notch inhibits myogenic activity of the MADS-Box transcription factor myocyte enhancer factor 2C.
(59/20616)
Skeletal muscle gene expression is dependent on combinatorial associations between members of the MyoD family of basic helix-loop-helix (bHLH) transcription factors and the myocyte enhancer factor 2 (MEF2) family of MADS-box transcription factors. The transmembrane receptor Notch interferes with the muscle-inducing activity of myogenic bHLH proteins, and it has been suggested that this inhibitory activity of Notch is directed at an essential cofactor that recognizes the DNA binding domains of the myogenic bHLH proteins. Given that MEF2 proteins interact with the DNA binding domains of myogenic bHLH factors to cooperatively regulate myogenesis, we investigated whether members of the MEF2 family might serve as targets for the inhibitory effects of Notch on myogenesis. We show that a constitutively activated form of Notch specifically blocks DNA binding by MEF2C, as well as its ability to cooperate with MyoD and myogenin to activate myogenesis. Responsiveness to Notch requires a 12-amino-acid region of MEF2C immediately adjacent to the DNA binding domain that is unique to this MEF2 isoform. Two-hybrid assays and coimmunoprecipitations show that this region of MEF2C interacts directly with the ankyrin repeat region of Notch. These findings reveal a novel mechanism for Notch-mediated inhibition of myogenesis and demonstrate that the Notch signaling pathway can discriminate between different members of the MEF2 family. (+info)
Oxidative stress can activate the epidermal platelet-activating factor receptor.
(60/20616)
Platelet-activating factor (1-alkyl-2-acetyl-glycero-phosphocholine) is a lipid mediator that has been implicated in keratinocyte function and cutaneous inflammation. Keratinocytes both synthesize platelet-activating factor and express functional platelet-activating factor receptors linked to calcium mobilization. Oxidative stress to various cells including keratinocytes can also result in the mobilization of intracellular Ca2+, a known stimulus for platelet-activating factor biosynthesis. The ability of the epidermal platelet-activating factor receptors to modulate oxidant-induced signaling was investigated using a unique model system created by retroviral-mediated transduction of the platelet-activating factor receptor-negative epithelial cell line KB with the platelet-activating factor receptor. Treatment of KB cells with the lipid pro-oxidant tert-butyl hydroperoxide induced transient increases in intracellular Ca2+ in a concentration-dependent fashion. Expression of the platelet-activating factor receptor in KB cells lowered the threshold for tert-butyl hydroperoxide-induced Ca2+ flux by an order of magnitude (10 microM in control KB versus 1 microM in KB cells expressing the platelet-activating factor receptors) and increased the peak change in intracellular Ca2+ concentration in response to this lipid hydroperoxide. This augmentation of tert-butyl hydroperoxide-induced Ca2+ mobilization was inhibited by pretreatment with the two competitive platelet-activating factor receptor antagonists CV-6209 and WEB 2086, as well as by the antioxidants vitamin E and 1,1,3,3-tetramethyl-2-thiourea. KB cells synthesized platelet-activating factor and the platelet-activating factor receptor agonist 1-palmitoyl-2-acetyl-glycero-phosphocholine in response to tert-butyl hydroperoxide treatment, suggesting the augmentation of oxidative stress-induced signaling seen in platelet-activating factor receptor-expressing cells was due in part to endogenous platelet-activating factor biosynthesis. These studies suggest involvement of the epidermal platelet-activating factor receptors in oxidant-mediated signaling. (+info)
A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida.
(61/20616)
In this study, we have mapped and characterized a B cell epitope of sulfated glycoprotein ZP2 (ZP2) as a step toward the development of a multi-epitope zona pellucida (ZP) vaccine. Recombinant polypeptides expressed by random deoxyribonuclease-digested fragments of ZP2 cDNA were screened for binding to IE-3, a monoclonal antibody to murine ZP2. Positive clones contained cDNA inserts encoding polypeptide corresponding to ZP2(103-134). When normal or ovariectomized female mice were immunized with three overlapping peptides that span this region of ZP2 (101-120, 111-130, 121-140), only ZP2(121-140) elicited IgG antibodies that reacted with mouse ovarian ZP, indicative of the presence of native B epitope and helper T cell epitope in ZP2(121-140). To more finely map the ZP2 B cell epitope, a random peptide display library was screened with the IE-3 antibody, and a consensus tetramer sequence VxYK that matched the ZP2(123-126) sequence VRYK was located. Competitive immunofluorescence analysis with single alanine-substituted VxYK peptides ranked the relative contribution of the three critical B cell epitope residues as Y > V > K. A chimeric peptide was constructed that contained the YRYK motif of ZP2 and a bovine RNase T cell epitope. Although (C57BL/6xA/J) F1 (B6AF1) female mice immunized with the chimeric peptide developed ZP antibody response, this peptide elicited antibody only in mice of the histocompatibility complex (MHC) H-2(k or b) haplotype. In contrast, ZP2(121-140) peptide elicited antibody in inbred mice with three additional mouse MHC haplotypes. Moreover, although ZP2(121-140) contained a T cell epitope, no oophoritis was observed after immunization of B6AF1 mice with ZP2(121-140) in complete Freund's adjuvant (CFA). In a preliminary trial, female B6AF1 mice immunized with ZP2(121-140) in CFA had reduced litter sizes as compared with mice injected with CFA alone. (+info)
Tissue plasminogen activator and its receptor in the human amnion, chorion, and decidua at preterm and term.
(62/20616)
The plasminogen activator system consists of two proteins: tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which act upon their specific receptors to generate plasmin from plasminogen located on the cell surface. Plasmin then acts directly and indirectly to degrade the components of the extracellular matrix (ECM). This process is likely to be important in the normal turnover of the ECM of fetal membranes and in its premature weakening in preterm premature rupture of the fetal membranes. Quantitative Northern analysis and in situ hybridization have shown that the decidua expresses mRNA for tPA. However, the immunolocalized tPA protein was most strongly associated with the amnion and chorion, as was its receptor annexin II, suggesting that the amnion and chorion are the targets for decidual tPA. At term, decidual tPA expression was unaffected by labor, and the tPA receptor was elevated both before and after labor. At preterm, the converse was found: decidual tPA expression was significantly (p < 0. 05) up-regulated by labor, but the tPA receptor was not. The results suggest that the generation of plasmin at term would be controlled by an increased concentration of the tPA receptor in the amnion and chorion, whereas at preterm a pathological increase in plasmin would be generated by an overexpression of tPA, initiated by labor. (+info)
Cripto-1 indirectly stimulates the tyrosine phosphorylation of erb B-4 through a novel receptor.
(63/20616)
Cripto-1 (CR-1) is a recently discovered protein of the epidermal growth factor family that fails to directly bind to any of the four known erb B type 1 receptor tyrosine kinases. The present study demonstrates that CR-1 indirectly induces tyrosine phosphorylation of erb B-4 but not of the epidermal growth factor-related receptors erb B-2 and erb B-3 in different mouse and human mammary epithelial cell lines. In addition, down-regulation of erb B-4 in NMuMG mouse mammary epithelial cells and in T47D human breast cancer cells, using an anti-erb B-4 blocking antibody or a hammerhead ribozyme vector targeted to erb B-4 mRNA, impairs the ability of CR-1 to fully activate mitogen-activated protein kinase. Finally, chemical cross-linking of 125I-CR-1 to mouse and human mammary epithelial cell membranes results in the labeling of two specific bands with a molecular weight of 130 and 60 kDa, suggesting that the CR-1 receptor represents a novel receptor structurally unrelated to any of the known type I receptor tyrosine kinases. In conclusion, these data demonstrate that CR-1, upon binding to an unknown receptor, can enhance the tyrosine kinase activity of erb B-4 and that a functional erb B-4 receptor is required for CR-1-induced MAPK activation. (+info)
Internalization of the TXA2 receptor alpha and beta isoforms. Role of the differentially spliced cooh terminus in agonist-promoted receptor internalization.
(64/20616)
Thromboxane A2 (TXA2) potently stimulates platelet aggregation and smooth muscle constriction and is thought to play a role in myocardial infarction, atherosclerosis, and bronchial asthma. The TXA2 receptor (TXA2R) is a member of the G protein-coupled receptor family and is found as two alternatively spliced isoforms, alpha (343 residues) and beta (407 residues), which share the first 328 residues. In the present report, we demonstrate by enzyme-linked immunosorbent assay and immunofluorescence microscopy that the TXA2Rbeta, but not the TXA2Ralpha, undergoes agonist-induced internalization when expressed in HEK293 cells as well as several other cell types. Various dominant negative mutants were used to demonstrate that the internalization of the TXA2Rbeta is dynamin-, GRK-, and arrestin-dependent in HEK293 cells, suggesting the involvement of receptor phosphorylation and clathrin-coated pits in this process. Interestingly, the agonist-stimulated internalization of both the alpha and beta isoforms, but not of a mutant truncated after residue 328, can be promoted by overexpression of arrestin-3, identifying the C-tails of both receptors as necessary in arrestin-3 interaction. Simultaneous mutation of two dileucine motifs in the C-tail of TXA2Rbeta did not affect agonist-promoted internalization. Analysis of various C-tail deletion mutants revealed that a region between residues 355 and 366 of the TXA2Rbeta is essential for agonist-promoted internalization. These data demonstrate that alternative splicing of the TXA2R plays a critical role in regulating arrestin binding and subsequent receptor internalization. (+info)