[Comparative effects of menthol and icilin on the induced contraction of the smooth muscles of the vas deferens of normal and castrated rats].

Non-specific TRPM8 agonist menthol was shown to inhibit voltage- and agonist-evoked contractions of the smooth muscle (SM) of rat vas deferens. Here we compared the action of menthol with the action of more specific TRPM8 agonist icilin on depolarization- (60 mM KCl), carbachol-(CCh) and noradrenalin-(Nor)-evoked contractions of the SM strips from the prostatic and epididymal portions of the vas deferens of normal and castrated (60-137 days) rats. Inhibitory action of menthol (100 microM) and icilin (10 microM) on the amplitude of KCl-, CCh- and Nor-induced contractions of normal as well as castrated rats was similar consisting about 50%, despite castration per se strongly potentiated CCh- and Nor-evoked contractions compared to the control animals. In the epididymal portion of the control animals menthol suppressed KC 1- and CCh-evoked contractions by 46 +/- 5% and 32 +/- 3% and icilin by only 14 +/- 4% and 6 +/- 7%, respectively, whilst after castration both compounds became virtually ineffective. Considering that TRPM8 may localize in the sarcolemma and sarcoplasmic reticulum (SR) membrane and that menthol can also block voltage-gated calcium channels (VGCCs), our data indicate that in the prostatic portion TRPM8 modulates contractility by primarily decreasing the SR Ca2+ stores content, whilst in the epididymal one by both decreasing the SR filling and supporting Ca2+ entry. Drop in the circulation androgens as a result of castration changes the menthol- and icilin-mediated modulation of the rat vas deferens SM contractility via the decrease of the expression of L-type VGCCs and increase of the expression of TRPM8.

[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.

[Effects of N-stearoyl- and N-oleoylethanolamine on cardiac voltage-dependent sodium channels].

The group of N-acylethanolamines (NAE) includes lipids that are capable of modulating plasma membrane ion channels without involvement of cannabinoid receptors. However, the action of various members of NAE on voltage-gated Na+ channels (VGSC) in cardiac tissue is still not fully elucidated. Here using patch-clamp technique we have studied the modulation of biophysical properties of VGSC of neonatal cardiomyocytes by saturated N-stearoylethanolamine (NSE) and monounsaturated N-oleoylethanolamine (OEA). NSE in 1-200 tM concentration range did not significantly alter the amplitude of inward Na+ current (I(Na)), but 100 microM NSE shifted its steady-state activation and inactivation curves in hyperpolarization direction by 2.4 mV and 10.6 mV, respectively. Activation kinetics of the current was not changed by NSE, but its inactivation was accelerated by about 1.2-fold in the -60 - -30 mV range of membrane potentials. Unlike NSE, OEA dose-dependently inhibited I(Na) with kappa(D) = 11.4 +/- 1.6 microM and maximal block at saturating concentration of 30 +/- 3%. It also stronger than NSE shifted current's steady-state activation and inactivation curves (-6.4 mV and -14.0 mV, respectively, at 100 microM) in hyperpolarization direction. The effect of OEA on I(Na) activation kinetics was negligible, but it more pronouncedly than NSE accelerated inactivation of the current. Thus, both members of NAE influence the voltage-dependence of activation, inactivation and kinetics of I(Na). These effects were more prominent for monounsaturated OEA, which also partially blocked I(Na). The discovered effects of NSE and OEA on VGSCs may in part be responsible for the decrease of cardiomycytes' excitability by these lipids under normal as well as pathologic conditions.

[Gender differences in cold sensitivity: role of hormonal regulation of TRPM8 channel].

We have studied the gender differences in the perception of cutaneous cold stimuli in the innocuous temperature range (5-30 degrees C) in mice and rats. In the behavioral tests using two variable temperature plates technique female subjects displayed lower threshold for the sensation of cooling temperatures in the range of 15-25 degrees C compared to males. Patch-clamp experiments carried out on dorsal root ganglion (DRG) neurons from male and female rats maintained in the short-term cultures in the presence of testosterone or 17beta-estradiol, respectively, have revealed gender- and hormone-related differences in the electrophysiological properties of cold/menthol-sensitive TRPM8 channel: average density of menthol-activated I(TRPM8) current density in females' DRG neurons was higher compared to males', and the steady-state voltage-dependent activation curve of TRPM8 in females was shifted towards hyperpolarized potentials compared to males. These distinctive TRPM8 properties vanished upon withdrawal of testosterone and 17beta-estradiol from the culture mediums. We conclude that the observed differences in the behavioural sensitivity to innocuous cold and in functional properties of TRPM8 cold receptor are due to differential regulation of TRPM8 by sex steroid hormones, testosterone and/or 17beta-estradiol.

[Regulation of the excitability of neonatal cardiomyocytes by N-stearoyl- and N-oleoyl-ethanolamines].

N-acylethanolamines (NAE) are biologically active lipids able of modulating ion transport through the cellular plasma membrane, however specific targets of their action and signalling mechanisms involved in cardiac tissue are still poorly understood. Physiological activity of NAEs is known to depend on the level of unsaturation. Therefore, here we investigated the effects of saturated N-stearylethanolamine (NSE) and monounsaturated N-oleylethanolamine on electric excitability of neonatal rat cardiomyocytes. 1 microM of either NSE or OEA decreased the duration of cardiac action potential (AP) from all parts of heart muscle. Shortening of AP was partially reversible, though the reversibility of AP duration upon washout of substances was more complete for endocardial ventricular compared to epicardial and atrial cardiomyocytes. I microM NSE depolarized resting membrane potential (RMP) of epicardial and of 65% of endocardial cells, whilst other cells types showed weakly reversible hyperpolarization. 1 microM OEA caused reversible RMP hyperpolarization of all studied cell types. NSE and OEA decreased the amplitude and upstroke velocity of AP that suggests their effect on sodium channels. NSE and to a lesser extent OEA inhibited the amplitude of AP phase 2 (plateau) which may indicate an inhibition of high-voltage-activated calcium channels. Effects of NSE and OEA on RMP and repolarization phase of AP (phase 3) depended on cardiac cell type suggesting differential regulation of inward rectifier Kir and voltage-gated delayed rectifier potassium channels by these lipids. We cannot also exclude interaction of NSE and OEA with anion channels, backgound K+ channels and ion transporters of the cardiomyocytes' plasma membrane. Overall, NSE-induced changes of AP parameters were less reversible than those induced by OEA, suggesting a slower degradation/ convertion of NSE in plasma membrane compared to OEA.

[Expression of the cold receptor TRPM8 in the smooth muscles of the seminal ejaculatory ducts in rats].

By means of real-time RT-PCR and immunofluorscent staining combined with confocal microscopy we show for the first time the expression ofmRNA and protein ofthe cold/menthol-sensitive cationic channel, TRPM8, in the smooth muscle cells (SMC) from the epididimal and prostatic portions of the rat vas deferens. Expression of TRPM8 mRNA correlated with the expression ofmRNA for androgen receptor (AR): orchidectomy of the animals resulted in the enhancement of the expression of both mRNAs, which likely reflects specific for the vas deferens compensatory response to the decreasing levels of circulating androgens. TRPM8 protein in the SMC from both parts of the vas deferens primarily localized outside the plasma membrane (PM), however, in the SMC from prostatic portion we observed higher TRPM8 protein targeting specifically the endoplasmic reticulum and PM, where it can form functional cold/menthol-sensitive cationic channel capable of modulating contractile activity of the smooth muscle.

[Modulation of the smooth muscle contractions of the rat vas deferens by TRPM8 channel agonist menthol].

TRPM8 is nonselective, Ca2- permeable cationic channel, which is activated by innocuous cold and by chemical drugs imitators of cooling, menthol, icilin and cucalyptol. TRPM8 expression was detected in the smooth muscle cells of the rat vas deference with preferential localization of the TRPM8 protein to the membrane of sarcoplasmic reticulum (SR). In the present work we have studied the effects of TRPM8 channel agonist, menthol, on the contractions of the smooth muscle strips of the epididimal and prostatic portions of the rat vas deferens evoked by potassium rich (KCl) Krebs solution and by muscarinic or adrenergic agonists carbachol (CCh) or noradrenalin (Nor). Menthol (0.1-1 mmol/l) per se virtually unaffected the basal tone, but inhibited in a dose-dependent manner KCl-, CCh- and Nor-evoked contractions of both parts of the vas deference by 30-50%. Blockade of the Ca2+ -ATPase of the SR with cyclopiazonic acid (CPA, 10 micromol/l) enhanced inhibitory action of menthol on KCl-induced contractions, but slightly decreased inhibition by menthol of agonist-induced ones. Nonspecific TRPM8 blocker, capsazepine (10 micromol/l), did not eliminate, but augmented inhibitory action of menthol on all types of contractions. Our data propose that menthol inhibits contractions via two mechanisms: partial blockade of Ca2+ entry via the voltage-gated, L-type calcium channels and a decrease of the calcium storage capacity of the SR. The latter mechanism at least in part is mediated by the SR-resident TRPM8 channel, which by activation of menthol leads to the enhancement of passive leak of Ca2+ from the SR and reduction in the amount of the releasable calcium during activation of contractions.

[A hyperpolarization-activated current in rat menthol-sensitive sensory neurons].

In the present study we have investigated the correlation between hyperpolarization-activated current (1(h)) and menthol-activated current (I(TRPM8)) in rat dorsal root ganglion (DRG) neurons. We showed that I(h) is present in 89% of menthol-sensitive neurons which makes its presence reliable, though not absolute, criterion for pre-selection of such neurons. Endogenous I(h) recorded from different neurons exhibited variable density and activation kinetics. Based on the analysis of I(h) activation kinetics we hypothesize that the population of hyperpolarization-activated channels in menthol-sensitive DRG neurons is mainly represented by HCN1, HCN2 and HCN3 channels. The expression of HCN4 isoform in these cells is very low.

[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.

[Calcium signaling in carcinogenesis].

Malignant transformation of the cells in cancer is caused by excessive proliferation accompanied by diminished ability for apoptosis due to the loss of normal control on these processes. Despite their ubiquity calcium ions are central to both processes, serving as major signalling agents. The present review examines what changes in calcium distribution among various compartments: extracellular space, cytoplasm, endoplasmic reticulum and mitochondria take place during transition from normal to pathological cell growth and death, what Ca2+-handling proteins are involved, and how this affects spatial and temporal characteristics of Ca2+ signals aimed at regulating specific cellular responses. Uncovering the underlying molecular events may help elaborating new strategies for cancers treatment.

[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.

[Alterations in ATP-dependence of swelling-activated Cl- current associated with neuroendocrine differentiation of LNCaP human prostate cancer epithelial cells].

Increasing population of malignant, apoptosis resistant neuroendocrine (NE) cells due to differentiation of prostate epithelial/basal cells is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. Acquisition of apoptosis resistance involves alterations in the mechanisms of cell volume homeostasis, of which volume-regulate anion channels (VRAC) that carry swelling-activated Cl- current (I(Cl,swell)) represent one of the key determinants. Given that VRAC function is generally known to be ATP-dependent, here we investigated how such dependence may evolve during NE differentiation of LNCaP prostate cancer epithelial cells. In the whole-cell patch-clamp recording mode I(Cl,swell) could be activated in response to hypotonicity-induced cell swelling in control and NE-differentiated (by incubation in membrane-permeable cAMP analogs) LNCaP cells even following total depletion of intracellular ATP using a cocktail of metabolic inhibitors. However, this basal I(Cl,swell) had about 30% higher density and was less inactivating in NE-differentiated cells. Inclusion of 5 mM Mg-ATP in the patch pipette caused I(Cl,swell) augmentation in both cell types. The augmentation in the control cells was more prominent and occurred mostly at the expense of a non-inactivating current component. We conclude that I(Cl,swell) in LNCaP cells consists of a non-inactivating, ATP-dependent and inactivating, ATP-independent components. NE differentiation promotes the increase of non-inactivating component and partial loss of its ATP sensitivity making the whole I(Cl,swell) less ATP-sensitive as well. By largely avoiding the ATP metabolic control I(Cl,swell) may contribute to better control of cell volume under metabolic stress and thus enhance the survival rates of apoptosis-resistant NE cells.

[Adenosine triphosphate-dependence of volume sensitive chloride current in LNCaP cell line of human prostate cancer].

Although sensitivity to intracellular ATP is considered to be one of the hallmarks of swelling activated Cl- current (I(Cl,swell)) involved in regulatory volume decrease (RVD) following hypotonic stress, the type and manner of such sensitivity seems to vary in different cell types. Here by using whole-cell patch-clamp recording we investigated ATP sensitivity of I(Cl,swell) in LNCaP human prostate cancer cell line. Suppression of endogenous ATP production with metabolic inhibitors (oligomycin, iodoacetate and rotenone) during cell dialysis with ATP- and Mg2+-free pipette solution did not prevent I(Cl,swell) in response to hypotonic exposure. However, supplementing this solution with 5 mM Na-ATP led to the development of I(Cl,swell) with nearly 305 higher density and less pronounced voltage-dependent inactivation (manifested mainly by the increase of non-inactivated current component) at positive potentials. On the contrary, inclusion of 1 mM Mg2+ in the patch pipette resulted in even smaller I(Cl,swell) (30% lower density compared to Mg2+-free conditions), which inactivated completely on prolonged depolarization. The presence of 5 mM Mg-ATP in the pipette did not affect I(Cl,swell) density. Neither intervention significantly altered the rate of I(Cl,swell) development in response to hypotonicity. We conclude that intracellular ATP, a positive modulator of I(Cl,swell)-carrying volume-regulated anion channel (VRAC) in LNCaP cells most likely acts via binding rather than hydrolysis and/or phosphorylation reactions, whereas intracellular Mg2+ is VRAC inhibitor.

[Calcium-dependent programmed cell death in prostate cancer]. / Kal'tsiieva zalezhnist' zaprohramovanoï klitynnoï zahybeli pry raku prostaty.

In the present review we describe the major molecular determinants of calcium homeostasis in prostate cancer cells and establish their role in the transformation to apoptosis-resistant cell phenotypes typical of advanced androgen-independent prostate cancer. We show that the hallmark of such transformation is complete loss of apoptosis pathway associated with endoplasmic reticulum calcium store depletion.

[Effect of neuroendocrine cell differentiation of the human prostatic neoplasm cell line LNCaP on the characteristics of volume-sensitive chloride content]. / Vplyv neiroendokrynnoho dyferentsiiuvannia klityn raku prostaty liudyny liniï LNCaP na kharakterystyky ob'iemchutlyvoho khlornoho strumu.

By means of patch-clamp technique we examined changes in volume-regulated chloride current (ICl,swell) at neuroendocrine differentiation of androgen-dependent LNCaP prostate cancer cells. In those cells with neuroendocrine differentiation resulted from an increase in the intracellular cAMP, ICl,swell became much faster in response to applying external hypotonic solution and cell swelling. Changes in final rectification and voltage-dependent inactivation were not detected, as compared to the control cells. The differentiation also diminished ICl,swell blockade by Ca2+ transported via store-operated channels (SOC). On the base of our data we suggest that potentiation of the current at neuroendocrine differentiation, at least in part, resulted from a decrease in an inhibitory effect of Ca2+, transported into a cell through SOC, on volume-sensitive chloride current. Accelerated current in those cells might be induced by cytoskeleton rearrangement at the neuron-like growth.

[The influence of extracellular pH on volume-activated chloride current in the prostate cancer epithelial cells]. / Vpliv zovnishn'oklitynnoho pH na ob'iemaktivovanyi khlornyi strum v epitelial'nykh klitynakh kartsynomy prostaty.

The influence of extracellular pH on characteristics of volume-activated chloride current, ICl,swell, in the human prostate cancer epithelial cell line, LNCaP, was studied using the patch-clamp technique. Acidification of the extracellular hypotonic solution used to develop the current shortened the temporal parameters of ICl,swell development and reduced its maximal density. Sudden shifts of the pH towards acidification caused fast, transient potentiation of the current followed by its sustained inhibition. Voltage-dependent inactivation of ICl,swell was the most pronounced in the narrow range of pH = 6-7. Based on the analysis of our data we hypothesize that volume-regulated anion channels underlying ICl,swell possess in their structure at least two pH-sensitive molecular groups with similar pK = 6, titration of which modulates the current amplitude and two additional proton-sensitive groups that determine channel's inactivation.

[Testosterone modulation of HERG potassium channel blockade induced by neuroleptics]. / Moduliatsiia testosteronom blokady kaliievykh kanaliv HERG neiroleptykamy.

The repolarisation phase of cardiac action potential is characterized by sexual dimorphism suggesting the role of sex steroid hormones in the regulation of K+ channels. Here we report on the effect of testosterone on blockade of HERG-encoded K+ channels induced by neuroleptics. These compounds are used in clinics to treat psychiatric disorders, but reportedly have proarrhythmic side effects, on HERG-encoded K+ channels responsible for the rapid component of cardiac delayed rectifier K+ current, IKr. HERG was expressed in Xenopus oocytes, HERG-expressing oocytes were preincubated in 1 microM of testosterone from 3 to 8 hours before experiments. The extent of the blockade by neuroleptics in control oocytes increased with depolarization correlating with channels activation consistent with open-channel blocking mechanism. The IC50 and A (maximal block) values for the haloperidol-, pimozide- and fluspirilen-induced blockade of fully activated IKr were 1.36 microM and 73%, 1.74 microM and 76%, 2.34 microM and 65% respectively. Testosterone decreased extent of maximal block and significantly diminished block voltage-dependance of IKr inhibition, it also decreased the efficiency of block, with IC50 and A values of 2.73 microM and 65%, 2.08 microM and 59%, 5.04 microM and 64% for haloperidol, pimozide and fluspirilen respectively. Testosterone treatment increased IC50 and decreased A for all three agents. The largest decrease in A was with pimozide and the largest increase in IC50 was with fluspirilen. Our results suggest protective role of testosterone (androgens) against proarrhythmic side effects of some compounds.

[Effects of sex hormones on HERG potassium channels expressed in Xenopus oocytes]. / Vplyv statevykh hormoniv na kaliievi kanaly HERG, ekspresovani v ootsytakh Xenopus

The repolarisation phase of cardiac action potential is characterized by sexual dimorphism suggesting the role of sex steroid hormones in the regulation of K+ channels. Here we report on the effects of testosterone and 17 beta-estradiol on HERG-encoded K+ channels, expressed in Xenopus oocytes. At 1M concentration testosterone decreased the amplitude of HERG-directed IKr (rapid component of cardiac delayed rectifier K+ current) by 30% within 30 min of exposure, while 17-estradiol had no effect. Testosterone did not alter the HERG channels kinetics, voltage-dependence, steady-state activation and inactivation properties suggesting that its action most probably involves the decrease of channels open probability and/or the level of their expression. The signaling pathways mediating testosterone-induced down-regulation of HERG channels in Xenopus oocytes remains to be elucidated. The effect of testosterone on HERG channels in Xenopus oocytes is opposite to what one might expect from shorter cardiac action potential duration and electrocardiographic QT-interval in males compared to females.