The normal physiological contraction of the urinary bladder, which is required for voiding, is predominantly mediated by muscarinic receptors, primarily the M 3 subtype, with the M 2 subtype providing a secondary backup role. Bladder relaxation, which is required for urine storage, is mediated by β-adrenoceptors, in most species involving a strong β 3 -component. An excessive stimulation of contraction or a reduced relaxation of the detrusor smooth muscle during the storage phase of the micturition cycle may contribute to bladder dysfunction known as the overactive bladder. Therefore, interference with the signal transduction of these receptors may be a viable approach to develop drugs for the treatment of overactive bladder. The prototypical signaling pathway of M 3 receptors is activation of phospholipase C (PLC), and this pathway is also activated in the bladder. Nevertheless, PLC apparently contributes only in a very minor way to bladder contraction. Rather, muscarinic-receptor-mediated bladder contraction involves voltage-operated Ca 2+ channels and Rho kinase. The prototypical signaling pathway of β-adrenoceptors is an activation of adenylyl cyclase with the subsequent formation of cAMP. Nevertheless, cAMP apparently contributes in a minor way only to β-adrenoceptor-mediated bladder relaxation. BK Ca channels may play a greater role in β-adrenoceptor-mediated bladder relaxation. We conclude that apart from muscarinic receptor antagonists and β-adrenoceptor agonists, inhibitors of Rho kinase and activators of BK Ca channels may have potential to treat an overactive bladder.

In this study we investigated the effect of NF‐kB signaling blockade on polymethylmethacrylate (PMMA) particle‐induced osteoclastogenesis in vitro. We first established effective blockade of NF‐kB activity as tested by electrophoretic mobility shift assays (EMSA). Particle‐induced NF‐kB activation in murine osteoclast precursor cells (CSF‐1‐dependent bone marrow macrophages) was markedly reduced by co‐treatment of the cells with the NF‐kB inhibitors N‐tosyl‐L‐phenylalanine chloromethyl ketone (TPCK) and Calpain Inhibitor I (CPI). This inhibition of NF‐kB activity was associated with blockade of p50 NF‐kB subunit nuclear trans‐location. We then established a direct NF‐kB inhibition approach by utilizing a TAT‐bound, mutant IkB (TAT:IkB46‐317), and demonstrated an inhibitory effect evidenced by decreased NF‐kB DNA binding activity. Having established that these strategies (TPCK, CPI, TAT: IkB46‐317) effectively block NF‐kB activation, we next investigated the effect of these agents on particle‐stimulated osteoclast formation. PMMA particle stimulation of mature osteoclast formation from RANKL‐primed osteoclast precursor cells was blocked by all three inhibitors. To further test the efficacy of NF‐kB blockade, experiments were performed with the TAT:IkB46‐317 mutant peptide in whole bone marrow cultures that contain supporting stromal cells. Again, this inhibitor efficiently blocked particle‐induced osteoclastogenesis. Thus, we have shown that pharmaceutical and molecular blockade of NF‐kB activation inhibits PMMA particle‐directed osteoclastogenesis in vitro. © 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Cyclic AMP is the prototypical second messenger of ␤-adrenergic receptors, but recent findings have questioned its role in mediating smooth muscle relaxation upon ␤-adrenergic receptor stimulation. We have investigated the signaling mechanisms underlying ␤-adrenergic receptor-mediated relaxation of rat urinary bladder. Concentration-response curves for isoproterenolinduced bladder relaxation were generated in the presence or absence of inhibitors, with concomitant experiments using passive tension and KCl-induced precontraction. The adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536; 1 M), the protein kinase A inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; 10 M), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89; 1 M), and Rp-adenosine 3Ј,5Ј-cyclic monophosphorothioate (Rp-cAMPS; 30 M), and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 3 M) produced only minor if any inhibition of relaxation against passive tension or KClinduced precontraction. Among various potassium channel inhibitors, BaCl 2 (10 M), tetraethylammonium (3 M), apamin (300 nM), and glibenclamide (10 M) did not inhibit isoproterenol-induced relaxation. Some inhibition of the isoproterenol effects against KCl-induced tone but not against passive tension was seen with inhibitors of calcium-dependent potassium channels such as charybdotoxin and iberiotoxin (30 nM each). A combination of SQ 22,536 and ODQ significantly inhibited relaxation against passive tension by about half, but not that against KCl-induced tone. Moreover, the combination failed to enhance inhibition by charybdotoxin against KCl-induced tone. We conclude that cAMP and cGMP each play a minor role in ␤-adrenergic receptor-mediated relaxation against passive tension, and calcium-dependent potassium channels play a minor role against active tension.During the storage phase of the micturition cycle, the urinary bladder must accommodate increasing amounts of urine without major elevation of intravesical pressure. This enhancement of bladder compliance requires relaxation of smooth muscle cells of the detrusor, which is controlled by reflex pathways involving an efferent activity of the sympathetic nervous system, particularly the hypogastric nerve originating from spinal cord segments Th12-L2 (Michel and Peters, 2004). Norepinephrine released from the hypogastric nerves primarily acts upon ␤-adrenergic receptors in the urinary bladder to promote relaxation during the storage phase. Therefore, ␤-adrenergic receptor activation is considered to be the most important physiological mechanism mediating urinary bladder relaxation during the filling/storage phase of the micturition cycle (Yamaguchi, 2002;Andersson, 2004).Several recent reports have investigated the ␤-adrenergic receptor subtypes mediating urinary bladder relaxation in several species. Atypical ␤-adrenergic receptors, i.e., ␤ 3 and/or other non-␤ 1 -non-␤ 2 subtypes, seem to be important for bladder relaxation in all species, b...

Inflammatory arthritis is associated with devastating joint tissue destruction and periarticular bone erosion. Although secreted products of infiltrating immune cells perpetuate the inflammatory response, the osteolytic component of this disease is a direct result of localized recruitment and activation of osteoclasts. Given that NF-κB plays a central role in both processes, the function of this transcription factor was examined. Using a mouse model of autoreactive Ig transfer that engenders inflammatory arthritis, we show numerous osteoclasts in the articular joint tissue associated with progressive periarticular osteolytic lesions. Moreover, cells retrieved from these joints exhibit heightened NF-κB activity. Importantly, direct administration of dominant negative∗I-κB or tyrosine 42-mutated I-κB (Y42F∗I-κB) proteins into mice before induction of the disease attenuates in vivo activation of the transcription factor. More importantly, these I-κB mutant forms significantly inhibit in vivo production of TNF and receptor activator of NF-κB ligand, and block joint swelling, osteoclast recruitment, and osteolysis. Thus, NF-κB appears to be the centerpiece of inflammatory-osteolytic arthritis and direct inhibition of this transcription factor by unique and novel I-κB mutant proteins blocks manifestation of the disease.