BK channel regulation by phosphodiesterase type 1: a novel signaling pathway controlling human detrusor smooth muscle function.

Large-conductance Ca(2+)-activated K(+) (BK) channels are critical regulators of detrusor smooth muscle (DSM) function. We aimed to investigate phosphodiesterase type 1 (PDE1) interactions with BK channels in human DSM to determine the mechanism by which PDE1 regulates human urinary bladder physiology. A combined electrophysiological, functional, and pharmacological approach was applied using human DSM specimens obtained from open bladder surgeries. The perforated whole cell patch-clamp technique was used to record transient BK currents (TBKCs) and the cell membrane potential in freshly isolated human DSM cells in combination with the selective PDE1 inhibitor, 8-methoxymethyl-3-isobutyl-1-methylxanthine (8MM-IBMX). Isometric DSM tension recordings were used to measure spontaneous phasic and electrical field stimulation-induced contractions in human DSM isolated strips. Selective pharmacological inhibition of PDE1 with 8MM-IBMX (10 µM) increased TBKC activity in human DSM cells, which was abolished by subsequent inhibition of protein kinase A (PKA) with H-89 (10 µM). The stimulatory effect of 8MM-IBMX on TBKCs was reversed upon activation of muscarinic acetylcholine receptors with carbachol (1 µM). 8MM-IBMX (10 µM) hyperpolarized the DSM cell membrane potential, an effect blocked by PKA inhibition. 8MM-IBMX significantly decreased spontaneous phasic and nerve-evoked contractions of human DSM isolated strips. The results reveal a novel mechanism that pharmacological inhibition of PDE1 attenuates human DSM excitability and contractility by activating BK channels via a PKA-dependent mechanism. The data also suggest interactions between PDE1 and muscarinic signaling pathways in human DSM. Inhibition of PDE1 can be a novel therapeutic approach for the treatment of overactive bladder associated with detrusor overactivity.

Quantification of phosphorus in DNA using capillary electrophoresis hyphenated with inductively coupled plasma mass spectrometry.

We have analyzed phosphorus in an enzymatically digested DNA molecule using capillary electrophoresis (CE) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS). The DNA concentration was quantified by the phosphorus value obtained in the CE-ICP-MS analysis. The CE-ICP-MS measurement, for which the interface device AIF-01 equipped three layered nebulizer was adopted, was achieved with limited µL/min nebulizing without loss of sample in the vaporizing chamber. The samples of nucleotides and free phosphate were separated well in the CE-ICP-MS measurement, and the calibration curve (0.1-10µg/mL) of the phosphorus showed a linear (R(2)=0.999) increase in intensity. After digestion of the 100-bp double-strand DNA sample to deoxyribonucleotide-5'-monophosphates (dNMPs) by phosphodiesterase-I, phosphorus was detected by CE-ICP-MS without further purification steps. In this study, we applied two calculation schemes of DNA analysis using a dNMP concentration obtained from CE-ICP-MS. Comparative CE-ICP-MS analysis with DNA digested to dNMPs showed that the assay gave an equal value obtained from the total DNA quantification using fluorescence detection. The detection limits of the DNA sample obtained from these species and phosphorus in nucleotides using CE-ICP-MS were 3.1-26ng/mL. These LOD values were equal to the conventional fluorescence determination of DNA.

Boronic acid-based inhibitor of autotaxin reveals rapid turnover of LPA in the circulation.

Autotaxin (ATX) is a secreted nucleotide pyrophosphatase/phosphodiesterase that functions as a lysophospholipase D to produce the lipid mediator lysophosphatidic acid (LPA), a mitogen, chemoattractant, and survival factor for many cell types. The ATX-LPA signaling axis has been implicated in angiogenesis, chronic inflammation, fibrotic diseases and tumor progression, making this system an attractive target for therapy. However, potent and selective nonlipid inhibitors of ATX are currently not available. By screening a chemical library, we have identified thiazolidinediones that selectively inhibit ATX-mediated LPA production both in vitro and in vivo. Inhibitor potency was approximately 100-fold increased (IC(50) approximately 30 nM) after the incorporation of a boronic acid moiety, designed to target the active-site threonine (T210) in ATX. Intravenous injection of this inhibitor into mice resulted in a surprisingly rapid decrease in plasma LPA levels, indicating that turnover of LPA in the circulation is much more dynamic than previously appreciated. Thus, boronic acid-based small molecules hold promise as candidate drugs to target ATX.

Optimization of a pipemidic acid autotaxin inhibitor.

Autotaxin (ATX, NPP2) has recently been shown to be the lysophospholipase D responsible for synthesis of the bioactive lipid lysophosphatidic acid (LPA). LPA has a well-established role in cancer, and the production of LPA is consistent with the cancer-promoting actions of ATX. Increased ATX and LPA receptor expression have been found in numerous cancer cell types. The current study has combined ligand-based computational approaches (binary quantitative structure-activity relationship), medicinal chemistry, and experimental enzymatic assays to optimize a previously identified small molecule ATX inhibitor, H2L 7905958 (1). Seventy prospective analogs were analyzed via computational screening, from which 30 promising compounds were synthesized and screened to assess efficacy, potency, and mechanism of inhibition. This approach has identified four analogs as potent as or more potent than the lead. The most potent analog displayed an IC(50) of 900 nM with respect to ATX-mediated FS-3 hydrolysis with a K(i) of 700 nM, making this compound approximately 3-fold more potent than the previously described lead.