[Changes in calcium-dependent potassium channels of isolated smooth muscle cells of the bladder in rats with experimental diabetes].

Activation of large conductance Ca2+-dependent potassium channels (BK channels) influences repolarization of the action potential and the level of the resting potential of detrusor smooth muscle cells (SMC). Overactive bladder syndrome (OBS) is one of the complications of diabetes. Here using whole-cell patch clamp technique we show sizable reduction of depolarization-evoked BK current (lBK) and decrease in the amplitude and frequency of spontaneous transient outward currents (STOCs) in isolated SMC from detrusor of rats with streptozocin-induced diabetes compared to control animals. Under the diabetes IBK density at step depolarization to +50 mV decreased from control value of 15.0+/-0.4 pA/pF to 10.0+/-0.5 pA/pF, whereas the mean values of the STOCs' frequency and amplitude at holding potential -20 mV were reduced from 12.0+/-1.5 Hz to 2.4+/-0.6 Hz and from 0.9+/-0.1 pA/pF to 0.510.1 pA/pF, respectively. Using real time RT-PCR it was found that the expression of mRNA for the BK-channel primary pore-forming KCa1.1-subunit increases under the diabetes, whilst that for the auxiliary BKCabeta1-subunit decreases. It is concluded that the observed changes in the BK-channel currents can enhance excitability of the detrusor SMCs thereby promoting myogenic OBS. However, further studies are needed to determine how the decrease in BKCabeta1 expression under the diabetes impairs functional properties of BK channels and to establish possible changes in calcium signals that modulate channel activation.

[Urothelium-dependent modulation of urinary bladder smooth muscle contractions by menthol].

TRPM8 cold receptor/channel is considered amongst the variety of receptors that support and modulate sensory function of urothelium, although the information regarding this is still quite contradictory. Here we have studied the effects of nonspecific TRPM8 activator menthol on the contractions of the smooth muscle strips of the rat bladder with intact and removed urothelium, and assessed the expression in them of TRPM8 mRNA using semi-quantitative RT-PCR. Menthol (100 microM) decreased the basal tone and the amplitude of spontaneous contractions only in the strips with intact urothelium. Irrespective of the presence of urothelium it similarly inhibited (by approximately 45 %) the contractions evoked by high-potassium depolarization. Contractions induced by muscarinic agonist carbachol (1 microM) were inhibited by menthol much stronger (by approximately 63%) if the urothelium was present than without it (by approximately 12%). Expression of TRPM8 mRNA in urothelium was not detected, whilst in detrusor smooth muscle it was found very low. We conclude that modulation of contractile responses by menthol is most likely explained by its blocking action on voltage-gated calcium channels ofdetrusor smooth muscle cells (SMC) and by menthol-stimulated release from urothelium of some factor(s) with relaxant effects on SMCs. Stimulation of the secretion of these factors from urothelial cells most likely involves menthol-induced, TRPM8-independent mobilization of calcium.

[Expression of genes encoding vanilloid receptors type 1 and type 2 in the cultured hippocampal neurons].

Expression of mRNA of 1 and 2 type (TRPVI and TRPV2) vanilloid receptors in the cultured hippocampal neurons was determined. With the use of reverse transcription polymerase chain reaction (RT-PCR) of single neurons (single-cell RT-PCR), was shown the TRPV1 and TRPV2 receptors expression in both neurons and glia cell. With use of real-time (real-time PCR) we determined that the level of these genes expression in different neurons is identical. It was also shown that the level of mTRPVI in GABAergic neurons was considerably higher, than the level of mTRPV2, while expression of genes in the glutamatergic neurons did not differ. Findings about expression of TRPV 1 and TRPV2 allow to suppose that vanilloid receptors can play an important functional role in the hippocampal neurons.

[Functional identification of the TRPM8 cold receptor in rat prostate epithelial cells].

For the first time by means of electrophysiology we demonstrate functional expression of cold/menthol sensitive TRPM8 channel in secretory epithelial cells plasma membrane from rat prostate. Under whole-cell patch-clamp technique rapid cooling from 33 to 21 degrees C of freshly isolated rat prostate epithelial cells elicited the significant outward rectifying membrane current with close to 0 mV reversal potential characteristic of TRPM8 activation. Extracellular application of chemical analog of cooling, menthol (100 microM) at 33 degrees C evoked similar current responses. Cold- and menthol-evoked currents could be detected only in elongated epithelial cells, which in prostate slices were labeled with anti-cytokeratin 8, suggesting that they belong to the apical secretory phenotype. No such current could be detected in round apparently basal epithelial cells. We conclude that in normal rat prostate functional expression of TRPM8 is restricted to plasma membrane of luminal apical epithelial cells.

[Identification of E-4031-sensitive potassium current component in murine P19 embryonic carcinoma cell line differentiated in cardiomyocytes].

Pluripotent mouse P19 embryonic carcinoma cells represent a convenient in vitro model for studying various aspects of cardiac differentiation. Here by using whole-cell patch-clamp recording we have identified the rapid delayed rectifier K+ current, I(Kr) in P19 cell induced to differentiate into cardiac phenotype by DMSO (1%). Cardiac differentiation was confirmed by the appearance of spontaneously beating cells, their morphological features, ultrastructural clusterization of mitochondria around contraction elements, expression of cardiac actin mRNAs and MLC2v, and by the presence of inward sodium and calcium currents. I(Kr) was isolated based on the sensitivity to the specific blocker, E-4031, which at concentration of 1 MM blocked more than 50% of the total outward K+ current. However, in contrast to I(Kr) in native cardiac myocytes and in heterologous systems expressing I(Kr)-carrying ERG1 potassium channel, E-4031-sensitive K+ current in cardiac-like P19 cells lacked characteristic inward rectification, suggesting specific regulation and/or subunit composition of endogenous ERG -based channel in these cells. Establishing the reason(s) for this phenomenon will advance the understanding of the mechanisms of I(Kr)-channel rectification. Cardiac-differentiated P19 cells might also be useful for studying pharmacological modulation of I(Kr), which is recognized target for cardiotoxic side effects of numerous drugs.