High calcium transport by Polycystin-2 (TRPP2) induces channel clustering and oscillatory currents.

The regulation by Ca2+ of Ca2+-permeable ion channels represents an important mechanism in the control of cell function. Polycystin-2 (PC2, TRPP2), a member of the TRP channel family (Transient Potential Receptor), is a Ca2+ permeable non-selective cation channel. Previous studies from our laboratory demonstrated that physiological concentrations of Ca2+ do not regulate in vitro translated PC2 (PC2iv) channel activity. However, the issue as to PC2's Ca2+ permeability and regulation remain ill-defined, in particular because Ca2+ transport is usually observed in the presence of other ionic gradients. In this study, we assessed Ca2+ transport by PC2iv in a lipid bilayer reconstitution system in a high Ca2+ gradient (CaCl2 100 mM cis, CaCl2 10 mM trans) in the presence of either 3:7 or 7:3 1-palmitoyl-2-oleoyl-choline and ethanolamine lipid mixtures. Reconstituted PC2iv showed spontaneous Ca2+ currents in both lipid mixtures, with a maximum conductance of 63 ± 13 pS (n = 19) and 105 pS ± 9.8 (n = 9), respectively. In both cases, we best fitted the experimental data with the Goldman-Hodgkin-Katz equation, observing a reversal potential (Vrev âˆ¼ -27 mV) consistent with strict Ca2+ selectivity. The R742X mutated PC2 (PC2R742X), lacking the carboxy terminal domain of the channel showed no differences with wild type PC2. Interestingly, we also observed the onset of spontaneous Ca2+ current oscillations whenever PC2-containing samples were reconstituted in the 3:7, but not 7:3 POPC:POPE lipid mixture. The amplitude and frequency of the ionic oscillations were highly dependent on the applied voltage, the imposed Ca2+ gradient, and the presence of high Ca2+, which induced PC2 channel clustering as observed by atomic force microscopy (AFM). We also used the QuB suite to kinetically model the PC2 channel Ca2+ oscillations based on the presence of subconductance states in the channel. The encompassed evidence supports a high Ca2+ permeability by PC2, and a novel oscillatory mechanism dependent on the presence of Ca2+ and phospholipids that provides the first evidence for the relation between stochasticity and deterministic processes mediated by ion channels.

Reduction of the P5A-ATPase Spf1p phosphoenzyme by a Ca2+-dependent phosphatase.

P5 ATPases are eukaryotic pumps important for cellular metal ion, lipid and protein homeostasis; however, their transported substrate, if any, remains to be identified. Ca2+ was proposed to act as a ligand of P5 ATPases because it decreases the level of phosphoenzyme of the Spf1p P5A ATPase from Saccharomyces cerevisiae. Repeating previous purification protocols, we obtained a purified preparation of Spf1p that was close to homogeneity and exhibited ATP hydrolytic activity that was stimulated by the addition of CaCl2. Strikingly, a preparation of a catalytically dead mutant Spf1p (D487N) also exhibited Ca2+-dependent ATP hydrolytic activity. These results indicated that the Spf1p preparation contained a co-purifying protein capable of hydrolyzing ATP at a high rate. The activity was likely due to a phosphatase, since the protein i) was highly active when pNPP was used as substrate, ii) required Ca2+ or Zn2+ for activity, and iii) was strongly inhibited by molybdate, beryllium and other phosphatase substrates. Mass spectrometry identified the phosphatase Pho8p as a contaminant of the Spf1p preparation. Modification of the purification procedure led to a contaminant-free Spf1p preparation that was neither stimulated by Ca2+ nor inhibited by EGTA or molybdate. The phosphoenzyme levels of a contaminant-free Spf1p preparation were not affected by Ca2+. These results indicate that the reported effects of Ca2+ on Spf1p do not reflect the intrinsic properties of Spf1p but are mediated by the activity of the accompanying phosphatase.

Menthol inhibits detrusor contractility independently of TRPM8 activation.

Agonists such as icilin and menthol can activate the cool temperature-sensitive ion channel TRPM8. However, biological responses to menthol may occur independently of TRPM8 activation. In the rodent urinary bladder, menthol facilitates the micturition reflex but inhibits muscarinic contractions of the detrusor smooth muscle. The site(s) of TRPM8 expression in the bladder are controversial. In this study we investigated the regulation of bladder contractility in vitro by menthol. Bladder strips from wild type and TRPM8 knockout male mice (25-30 g) were dissected free and mounted in organ baths. Isometric contractions to carbachol (1 nM-30 µM), CaCl2 (1 µM to 100 mM) and electrical field stimulation (EFS; 8, 16, 32 Hz) were measured. Strips from both groups contracted similarly in response to both carbachol and EFS. Menthol (300 µM) or nifedipine (1 µM) inhibited carbachol and EFS-induced contractions in both wild type and TRPM8 knockout bladder strips. Incubation with the sodium channel blocker tetrodotoxin (1 µM), replacement of extracellular sodium with the impermeant cation N-Methyl-D-Glucamine, incubation with a cocktail of potassium channel inhibitors (100 nM charybdotoxin, 1 µM apamin, 10 µM glibenclamide and 1 µM tetraethylammonium) or removal of the urothelium did not affect the inhibitory actions of menthol. Contraction to CaCl2 was markedly inhibited by either menthol or nifedipine. In cultured bladder smooth muscle cells, menthol or nifedipine abrogated the carbachol or KCl-induced increases in [Ca2+]i. Intravesical administration of menthol increased voiding frequency while decreasing peak voiding pressure. We conclude that menthol inhibits muscarinic bladder contractions through blockade of L-type calcium channels, independently of TRPM8 activation.

Relaxant effects of the essential oil of Mentha pulegium L. in rat isolated trachea and urinary bladder.

OBJECTIVES: We evaluated the relaxant activity of the essential oil of Mentha pulegium L. (EOMP) and pulegone in rat isolated tracheal and bladder smooth muscles. METHODS: ISOMETRIC contractions of isolated tracheal and bladder strips from male Wistar rats were induced by KCl (K60; 60 mm) or acetylcholine (ACh; 10 µm). EOMP and its majory compound pulegone were incubated, after contracting agent, with the tissues in cumulating concentrations. KEY FINDINGS: EOMP (3-300 µg/ml) inhibited the contractions induced by ACh and K60 in both tissues, but was more effective against the contractions induced by K60 in trachea (IC50 = 40.47 ± 3.27 µg/ml) compared with ACh. Its relaxant action rules out ganglia and NO participation. Pulegone (10(-7) to 10(-3 ) m) inhibited the contractions induced by ACh and K60 in both tissues. EOMP concentration-dependently inhibited the contractions evoked by addition of CaCl(2) in depolarised trachea, suggesting inhibition of extracellular calcium entry. CONCLUSIONS: These findings suggests that EOMP induced relaxant responses in pre-contracted smooth muscles of rat trachea and bladder, which are likely to be mediated via inhibition of calcium entry, mainly by its major compound, pulegone. These effects are coherent with the popular use of EOMP as an antispasmodic agent.

Luminal calcium concentration controls intestinal calcium absorption by modification of intestinal alkaline phosphatase activity.

Intestinal alkaline phosphatase (IAP) is a brush-border phosphomonoesterase. Its location suggests an involvement in the uptake of nutrients, but its role has not yet been defined. IAP expression parallels that of other proteins involved in Ca absorption under vitamin D stimulation. Experiments carried out in vitro with purified IAP have demonstrated an interaction between Ca and IAP. The gut is prepared to face different levels of Ca intake over time, but high Ca intake in a situation of a low-Ca diet over time would cause excessive entry of Ca into the enterocytes. The presence of a mechanism to block Ca entry and to avoid possible adverse effects is thus predictable. Thus, in the present study, Sprague-Dawley rats were fed with different amounts of Ca in the diet (0.2, 1 and 2 g%), and the percentage of Ca absorption (%Ca) in the presence and absence of L-phenylalanine (Phe) was calculated. The presence of Phe caused a significant increase in %Ca (52.3 (SEM 6.5) % in the presence of Phe v. 31.1 (sem 8.9) % in the absence of Phe, regardless of the amount of Ca intake; paired t test, P = 0.02). When data were analysed with respect to Ca intake, a significant difference was found only in the group with low Ca intake (paired t test, P = 0.03). Additionally, IAP activity increased significantly (ANOVA, P < 0.05) as Ca concentrations increased in the duodenal lumen. The present study provides in vivo evidence that luminal Ca concentration increases the activity of IAP and simultaneously decreases %Ca, acting as a minute-to-minute regulatory mechanism of Ca entry.

Polysaccharide-based hydrogels: preparation, characterization, and drug interaction behaviour.

Oxidized alginate (ADA) and oxidized alginate blended with chitosan (ADA-Chit) were prepared in the presence of borax and CaCl 2, and their interactions with an antifolate drug, pyrimethamine (PYR), have been investigated. Tablets with a mean diameter of 1.2 +/- 0.06 cm were produced and drug interactions were performed in dimethyl sulfoxide (DMSO) using isothermal titration calorimetry (ITC). From ITC responses, the enthalpy changes of interaction PYR/materials, Delta int H, have been determined and were found to be -11.73 +/- 0.517 kJ mol (-1) for ADA and -4.86 +/- 0.156 kJ mol (-1) for ADA-Chit. The PYR encapsulation of approximately 75% was achieved for both materials, as measured by UV spectrometer.

Characterisation of an ATP diphosphohydrolase (Apyrase, EC 3.6.1.5) activity in Trichomonas vaginalis.

In the present report the enzymatic properties of an ATP diphosphohydrolase (apyrase, EC 3.6.1.5) in Trichomonas vaginalis were determined. The enzyme hydrolyses purine and pyrimidine nucleoside 5'-di- and 5'-triphosphates in an optimum pH range of 6.0--8.0. It is Ca(2+)-dependent and is insensitive to classical ATPase inhibitors, such as ouabain (1 mM), N-ethylmaleimide (0.1 mM), orthovanadate (0.1 mM) and sodium azide (5 mM). A significant inhibition of ADP hydrolysis (37%) was observed in the presence of 20 mM sodium azide, an inhibitor of ATP diphosphohydrolase. Levamisole, a specific inhibitor of alkaline phosphatase, and P(1), P(5)-di (adenosine 5'-) pentaphosphate, a specific inhibitor of adenylate kinase, did not inhibit the enzyme activity. The enzyme has apparent K(m) (Michaelis Constant) values of 49.2+/-2.8 and 49.9+/-10.4 microM and V(max) (maximum velocity) values of 49.4+/-7.1 and 48.3+/-6.9 nmol of inorganic phosphate x min(-1) x mg of protein(-1) for ATP and ADP, respectively. The parallel behaviour of ATPase and ADPase activities and the competition plot suggest that ATP and ADP hydrolysis occur at the same active site. The presence of an ATP diphosphohydrolase activity in T. vaginalis may be important for the modulation of nucleotide concentration in the extracellular space, protecting the parasite from the cytolytic effects of the nucleotides, mainly ATP.