New insights into pharmacological tools to TR(i)P cancer up.

The aim of this review is to address the recent advances regarding the use of pharmacological agents to target transient receptor potential (TRP) channels in cancer and their potential application in therapeutics. Physiologically, TRP channels are responsible for cation entry (Ca(2+) , Na(+) , Mg(2+) ) in many mammalian cells and regulate a large number of cellular functions. However, dysfunction in channel expression and/or activity can be linked to human diseases like cancer. Indeed, there is growing evidence that TRP channel expression is altered in cancer tissues in comparison with normal ones. Moreover, these proteins are involved in many cancerous processes, including cell proliferation, apoptosis, migration and invasion, as well as resistance to chemotherapy. Among the TRP superfamily, TRPC, TRPV, TRPM and TRPA1 have been shown to play a role in many cancer types, including breast, digestive, gliomal, head and neck, lung and prostate cancers. Pharmacological modulators are used to characterize the functional implications of TRP channels in whole-cell membrane currents, resting membrane potential regulation and intracellular Ca(2+) signalling. Moreover, pharmacological modulation of TRP activity in cancer cells is systematically linked to the effect on cancerous processes (proliferation, survival, migration, invasion, sensitivity to chemotherapeutic drugs). Here we describe the effects of such TRP modulators on TRP activity and cancer cell phenotype. Furthermore, the potency and specificity of these agents will be discussed, as well as the development of new strategies for targeting TRP channels in cancer.

F-actin as a functional target for retro-retinoids: a potential role in anhydroretinol-triggered cell death.

The retro-retinoids, metabolites of vitamin A (retinol), belong to a family of lipophilic signalling molecules implicated in regulation of cell growth and survival. Growth-promoting properties have been ascribed to 14-hydroxy-retro-retinol (14HRR), while anhydroretinol (AR) was discovered to act as a natural antagonist triggering growth arrest and death by apoptosis. Based on morphological studies and inhibition of apoptosis by the kinase blocker, herbimycin A, it has been suggested that retro-retinoids exhibit their function in the cytosolic compartment. F-actin emerged as a functional target for retro-retinoid action. By FACS analysis and fluorescence microscopy of phalloidin-FITC labeled cells we demonstrated that F-actin reorganization was an early event in AR-triggered apoptosis. Fluorescence images of AR-treated fibroblasts displayed short, thick, stick-like and punctate structures, and membrane ruffles at the cell periphery along with an increased diffuse staining pattern. Reversal of the AR effect by 14HRR or retinol indicates that F-actin is a common site for regulation by retro-retinoids. Inhibition of both cell death and actin depolymerisation by bcl-2 implies that cytoskeleton reorganization is downstream of bcl-2-related processes. Furthermore, stabilization of microfilaments by jasplakinolide increased the survival potential of AR treated cells, while weakening the cytoskeleton by cytochalasin B abetted apoptosis. Thus the cytoskeleton is an important way station in a communication network that decides whether a cell should live or die.

Aldosterone modulates both the Na/H antiport and Cl/HCO3 exchanger in cultured neonatal rat cardiac cells.

Mineralocorticoid hormones regulate many physiological functions in the cardiovascular system. Although high affinity binding sites for aldosterone have been found in myocardium, aldosterone effects on pHi regulatory systems in cardiac cells have not been described. We have addressed this issue by using microspectrofluorimetric monitoring of intracellular pH in developing neonatal rat cardiomyocytes cultured for 2 weeks. Developmental changes in cell morphology were controlled by anti-myosin light chain antibody staining of the sarcomeric units using confocal laser scanning microscopy. The data obtained demonstrate that from early stages of the development, pHi in neonatal cardiac cells is regulated by three ion transporting mechanisms, namely, Na/H antiport, Na- and HCO3-dependent transporter and Cl/HCO3 exchanger. A 24-h treatment of the cells with aldosterone increases the activity of the Cl/HCO3 exchanger at day 6 of cell culture while the Na/H antiport activity is enhanced in the cells treated with the hormone at days 9 and 13 of culture. Thus, by affecting the activity of ionic transporters, aldosterone modulates acid-base balance in cardiac cells.

Cl- -HCO3- exchange in developing neonatal rat cardiac cells. Biochemical identification and immunolocalization of band 3-like proteins.

The Cl- -HCO3- exchanger is the main anionic exchanger (AE) that alleviates alkaline loads in cardiac cells. We recently identified in adult ventricular cells two membrane proteins (80 and 120 kD) immunologically related to the erythroid band 3 and likely to mediate the anion exchange. In the present study, we further investigated the Cl- -HCO3- exchanger activity concomitantly with the expression and intracellular localization of the band 3-like proteins during the development of neonatal rat cardiac cells maintained in culture for 17 days. Microspectrofluorometric measurements of pHi in single cells show that neonatal rat cardiomyocytes display a fully functional DIDS-sensitive Cl- -HCO3- exchanger at early stages of development. Neither basal pHi nor the anion exchange activity changes with different stages of the culture. In Western blotting with an anti-whole erythroid band 3 antibody, we found both the 80- and the 120-kD band 3-like proteins in whole heart and cultured neonatal cardiac cells. The 80-kD protein was also recognized by an anti-AE1 antiserum, whereas the 120-kD protein was specifically detected by an anti-cardiac AE3 antibody. Thus, we propose that the proteins are encoded by two different genes, AE1 and AE3, respectively. Subcellular fractionation of isolated and cultured cardiomyocytes revealed the presence of both proteins in the membrane, nuclear, and myofibril fractions. The results obtained in biochemical experiments corroborate the confocal images of immunostained neonatal cells, which demonstrate perinuclear location of band 3-like proteins at an early stage of development and their appearance within myofilaments after cell maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of band 3-like proteins and Cl-/HCO3- exchange in isolated cardiomyocytes.

The identification of the protein that exerts the function of Cl-/HCO3- exchange is still unresolved in cardiac tissue. We have addressed this issue by using a multiple technical approach. Western blotting analysis with an antibody raised against human erythroid whole band 3 protein, the so-called protein that mediates the Cl-/HCO3- exchange in erythrocytes, showed that adult cardiomyocytes expressed two proteins immunologically related to the erythroid band 3. These proteins migrated in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of 80 and 120 kDa. They were specifically found in the membrane but not in the cytosolic or the myofibril fractions of adult cardiomyocytes. Confocal microscopy further indicated that the immunostained proteins were mainly located at the sarcolemma and along T-tubules, typical membrane structures of adult cardiomyocytes. Using an antibody raised against a cardiac amino-terminal domain of rat AE3, we found that the 120-kDa protein is the translation product of the AE3 gene specifically expressed in heart and brain. Using an antiserum raised against a specific domain of mouse erythroid band 3 (AE1), which is not shared by AE3, we showed that the 80-kDa protein is likely to be a truncated translation product of the AE1 gene. Microinjection of the anti-human erythroid whole band 3 antibody into single isolated cardiac cells significantly inhibited the Cl-/HCO3- exchange activity. Furthermore, the anti-AE1 antibody strongly decreased the efficiency of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate to inhibit the ionic exchange. We thus suggest that the 80-kDa or both the 80- and the 120-kDa proteins immunologically related to the erythroid band 3 protein perform the anionic exchange in rat cardiomyocytes.

Different effects of endothelin-3 on the Ca2+ discharge induced by agonists and Ca(2+)-ATPase inhibitors in human platelets.

1. The present study demonstrates that endothelin-3 (ET-3), previously shown to attenuate thrombin-evoked aggregation of human platelets, delayed the dose-dependent aggregatory response to thapsigargin (Tg). As this Ca(2+)-ATPase inhibitor induces platelet activation in part through the depletion of internal Ca(2+)-stores, we examined the influence of ET-3 on Ca2+ discharge from internal pools. 2. Cytosolic Ca2+ concentration was evaluated with Fura-2 in the absence of Ca2+ influx. Platelet preincubation for 15 min with 5 x 10(-7) M ET-3 decreased the Ca2+ release evoked by thrombin and U46619, a thromboxane-mimetic. However, ET-3 did not affect Ca2+ movements induced by 1 microM ADP. Addition of Tg (0.5 to 5 microM) to resting platelets induced a cytosolic [Ca2+] rise with concentration-dependent increase of the initial rate and decrease of the time to reach the peak. ET-3 slowed down these dose-dependent effects with a more marked influence on the responses induced by low concentrations of Tg. 3. ET-3 did not modify the Ca2+ response to another Ca(2+)-ATPase inhibitor, 2,5-di-(tert-butyl)-1,4-benzohydroquinone(tBuBHQ). The thromboxane A2 receptor antagonist, SQ 29548, reduced by 53% the calcium signal evoked by 1 microM Tg, which became similar to that induced by 15 microM tBuBHQ. Under these conditions, the ET-3 effects were suppressed. A subsequent addition of thrombin induced a substantial further Ca2+ increase which was again sensitive to ET-3. 4. ET-3 attenuates Ca2+ mobilization from an internal pool dependent on the stimulation of thrombin and thromboxane A2 receptors and insensitive to the direct effect of Ca2+-ATPase inhibitors. The small but significant inhibitory effect of ET-3 leads us to propose that endothelin-3 acts as a modulator of platelet activation.

Alterations in beta-adrenoceptor density on T-lymphocytes upon activation with interleukin-2 and phytohaemagglutinin.

Cultivation of human peripheral blood T-lymphocytes in the presence of interleukin-2 (IL-2) and phytohaemagglutinin (PHA) caused biphasic alterations in the beta 2-adrenoceptor density (Bmax) and cAMP content of these cells. The increase in Bmax after 18 h incubation with IL-2 and PHA was due to the expression of the receptors in a low-affinity state. The stimulatory effect of isoproterenol on adenylate cyclase activity and its effect on cAMP content remained unchanged, indicating uncoupling between the expressed receptors and regulatory Gs-proteins. The addition of phorbolmyristateacetate (PMA), a protein kinase C activator, also caused a biphasic change in beta 2-adrenoceptor density on the surface of the cells. The data point to the involvement of protein kinase C in the mechanism responsible for the increase and subsequent decrease in beta 2-adrenoceptor density seen after activation of T-lymphocytes.

Interleukin-2- and phytohemagglutinin-activated proliferation of human T-lymphocytes is accompanied by stimulation of phosphoinositide turnover.

Interleukin-2 (IL-2) was more effective than phytohemagglutinin (PHA) in increasing the proliferative activity of human T-lymphocytes. Unlike PHA, IL-2 stimulated phosphoinositide turnover (PI turnover) only in those T-lymphocytes which had been preactivated by PHA with IL-2 and expressed the IL-2 receptors. The effect of PHA on PI turnover was, in general, stronger than that of IL-2. These results indicate that IL-2 and PHA-activated proliferation of human T-lymphocytes is accompanied by stimulation of PI turnover. However, IL-2-induced proliferative response may not be a direct consequence of PI turnover stimulation in these cells.

[Dependence of kinetic constants of intact human lymphocyte beta2-adrenoreceptors on the nature of the radioligands used]. / Zavisimost' kineticheskikh konstant beta2-adrenoretseptorov intaktnykh limfotsitov cheloveka ot prirody primeniaemykh radioligandov.

An analysis of human peripheral blood intact mononuclear lymphocyte beta 2-adrenoreceptors showed that the hydrophilic radioligand 3H-CGP-12177 binds at various temperatures only to surface receptors. The density of receptors as determined by the binding of the lipophilic antagonist 125I-CYP at 37 degrees C is 2 times as high as that at 4 degrees C. The affinity of lymphocyte beta 2-adrenoceptors for 1-isoproterenol measured by the labeled ligand displacement at low (4 degrees C) temperatures is by two or three orders of magnitude higher than that at 37 degrees C. The thiol-alkylating agent, N-ethylmaleimide (NEM) causes oppositely directed changes in the density of beta 2-adrenoceptors on intact lymphocytes depending on their original density. NEM increases the affinity of beta 2-adrenoreceptors for the hormone (1.5-12-fold). The state and regulation of human peripheral blood lymphocyte beta 2-adrenoreceptors depend on the hormonal status of patients at the moment of blood sample collection.

[Effect of physical load and brief monotherapy with obsidan on the characteristics of beta 2-adrenoreceptors of intact lymphocytes in patients with hypertension]. / Vliianie fizicheskoi nagruzki i korotkogo kursa monoterapii obzidanom na kharakteristiki beta 2-adrenoretseptorov intaktnykh limfotsitov bol'nykh gipertonicheskoi bolezn'iu.

Lymphocytes beta 2-receptor density (LBRD) was studied by binding with 125ICYP in 18 male patients with mild/moderate essential hypertension (EH) before and after propranolol monotherapy. In 5 of these patients LBRD was determined before and after dynamic exercise also. Propranolol therapy evoked different changes in LBRD in patients with EH. According to these results, all the patients were divided into 2 groups: the patients who respond to propranolol administration by an increase in the LBRD were attributed to the 1 group. If the LBRD decreased after propranolol, the patients were attributed to the 2 group. Propranolol had pronounced hypotensive effect in the 2 group and no hypotensive effect in the 1 group. Heart rate was significantly higher initially and decreased significantly more after treatment in the 2 group. In this group initial values of LBRD were significantly higher than in the 1 group. Exercise produced different changes in LBRD, which correlated with the changes produced by propranolol treatment. The positive correlations were found between LBRD and left ventricular myocardial mass and interventricular septum thickness. The data obtained indicate that LBRD is regulated in a qualitatively different manner in the two groups of patients with EH which appears to be related to baseline renin or perhaps catecholamine levels.

[The density of lymphocyte beta 2-adrenoreceptors and blood plasma catecholamines in patients with dilated cardiomyopathy]. / Plotnost' beta 2-adrenoretseptorov limfotsitov i katekholaminy plazmy krovi u bol'nykh dilatatsionnoi kardiomiopatiei.

27 patients with dilated cardiomyopathy (DCM) complicated by heart failure (HF) of stages I-IIB were studied. The density of lymphocyte beta 2-adrenoceptors decreased significantly as HF progressed. The level of circulating catecholamines (epinephrine, norepinephrine) was significantly higher in patients were severe HF, which probably caused more evident decrease in lymphocyte beta 2-adrenoceptors density in these patients. This point of view was confirmed by the data of correlation-regression analysis between the general beta 2-adrenoceptors density and hemodynamics data. The identified changes in the number of adrenoceptors reflect the process of cellular receptors desensitization. They should be considered as a possible mechanism of HF development on a molecular level that cause development of refractoriness to routine therapy.

Characteristics of lymphocyte beta-adrenoceptors in essential hypertension: effects of propranolol treatment and dynamic exercise.

The density of beta 2-receptors in intact lymphocytes was studied by binding with 125iodo-cyanopindolol in 18 male patients with mild or moderate hypertension before and after monotherapy with propranolol. The density of these receptors was also determined in 5 patients before and after dynamic exercise. We found that propranolol therapy evoked different changes in the density of beta-receptors in patients with essential hypertension. Based on these results, all patients were divided into two groups: (a) patients who responded to the administration of propranolol by an increase in receptor density and (b) patients who responded with a decrease in receptor density. Propranolol therapy had a pronounced hypotensive effect in the second group and no hypotensive effect in the first group. In the second group, heart rate was significantly higher initially and showed a significantly greater decrease after treatment. The initial values for beta 2-receptor density were also significantly higher in this group than in the first group. Mean values of baseline plasma renin activity (PRA) were higher in the second group, but this difference was nonsignificant. The 5 patients who participated in dynamic exercise exhibited different changes in beta-receptor density, which correlated with the changes observed with propranolol treatment. There was no correlation between PRA and the density of beta 2-receptors in lymphocytes. Positive correlations were found between the density of these receptors and left-ventricular myocardial mass and interventricular septal thickness. The data indicate that the density of these receptors in lymphocytes is regulated in a qualitatively different manner in the two groups of patients with essential hypertension; this difference appears to be related to baseline renin levels or, perhaps, catecholamine levels. Additional studies are needed to clarify the regulation of beta 2-receptors in essential hypertension.

[Effect of a physical load on the adrenergic receptor function of peripheral blood lymphocytes and thrombocytes in healthy donors]. / Vliianie fizicheskoi nagruzki na sostoianie adrenergicheskikh retseptorov limfotsitov i trombotsitov perifericheskoi krovi zdorovykh donorov.

Beta 2-adrenergic receptor density and platelet adenylate cyclase activity were determined in the peripheral blood of healthy donors during short-term physical exercise and compared to changes in biochemical parameters. The data obtained indicate the activation of sympathetic-adrenal system in general and suggest phasic regulation of catecholamine beta-adrenergic receptors.