microM Clotrimazole Research Articles

Contribution of different Ca2+‐activated K+ channels to the first phase of the response to TRH in clonal rat anterior pituitary cells

Thyrotropin-releasing hormone (TRH) induces biphasic changes in electrical activity, cytosolic free Ca(2+) level ([Ca(2+)](i)), and prolactin secretion from both clonal GH cells and native lactotrophs. The first phase of the TRH response is characterized by hyperpolarization because of activation of Ca(2+)-activated K(+) channels (K(Ca)). In the present study, the relative contribution of BK, SK, and IK channels to the first phase of the TRH response in GH(4) cells was assessed. The expression of IK channels was confirmed by PCR with specific primers for SK4 (IK). The response to TRH was studied using the perforated patch technique and Ca(2+) microfluoromety (fura-2). The involvement of different K(Ca) channels was estimated by employing the specific channel blockers iberiotoxin (BK), apamin (SK) and clotrimazole (IK). Application of 100 nM iberiotoxin, 1 microM apamin, and 10 microM clotrimazole reduced the peak value of the outward K(+) current during the first phase of the TRH response by 33, 26, and 33%, respectively. Clotrimazole also shortened the duration of the outward current response by 60%, causing a reduction of total charge movement by 73%. All these toxin-induced reductions were significant (P < 0.05). A combination of all three toxins abolished the current response almost completely. All the three main types of K(Ca) channels are involved in the first phase of the TRH response, with IK as the major contributor. This is the first demonstration of a dominant role of IK compared with BK and SK channels in excitable cells.

Endothelial mediators of 17 beta-estradiol-induced coronary vasodilation in the isolated rat heart.

The present study was designed to determine relaxation in response to 17 beta-estradiol by isolated perfused hearts from intact normotensive male and female rats as well as the contribution of endothelium and its relaxing factors to this action. Baseline coronary perfusion pressure was determined and the vasoactive effects of 17 beta-estradiol (10 microM) were assessed by in bolus administration before and after endothelium denudation by infusion of 0.25 microM sodium deoxycholate or perfusion with 100 microM L-NAME, 2.8 microM indomethacin, 0.75 microM clotrimazole, 100 microM L-NAME plus 2.8 microM indomethacin, and 100 microM L-NAME plus 0.75 microM clotrimazole. Baseline coronary perfusion pressure differed significantly between males (84 +/- 2 mmHg, N = 61) and females (102 +/- 2 mmHg, N = 61). Bolus injection of 10 microM 17 beta-estradiol elicited a transient relaxing response in all groups, which was greater in coronary beds from females. For both sexes, the relaxing response to 17 beta-estradiol was at least in part endothelium-dependent. In the presence of the nitric oxide synthase inhibitor L-NAME, the relaxing response to 17 beta-estradiol was reduced only in females. Nevertheless, in the presence of indomethacin, a cyclooxygenase inhibitor, or clotrimazole, a cytochrome P450 inhibitor, the 17 beta-estradiol response was significantly reduced in both groups. In addition, combined treatment with L-NAME plus indomethacin or L-NAME plus clotrimazole also reduced the 17 beta-estradiol response in both groups. These results indicate the importance of prostacyclin and endothelium-derived hyperpolarizing factor in the relaxing response to 17 beta-estradiol. 17 beta-estradiol-induced relaxation may play an important role in the regulation of coronary tone and this may be one of the reasons why estrogen replacement therapy reduces the risk of coronary heart disease in postmenopausal women.

Blockage of intermediate-conductance Ca2+-activated K+ channels inhibit human pancreatic cancer cell growth in vitro.

Ion channels are important in controlling cell cycle progression and proliferation in a variety of cell types. Using the whole-cell recording mode of the patch-clamp technique, functional ion channels were electrophysiologically characterized in PANC-1 (K-ras G12D (+/-), p53 R273C, Deltap16), BxPC-3 (smad4-, p53 Y220C, Deltap16), and MiaPaCa-2 [transforming growth factor-beta receptor type II defect, K-ras G12C(-/-), p53 R248W, Deltap16] human pancreatic cancer cell lines. In BxPC-3 and the MiaPaCa-2 cells, we could identify approximately 600 or approximately 1200 functional Ca2+-activated K+ channels (IK) per cell, respectively, whereas PANC-1 cells expressed approximately 200 functional IK channels per cell. These channels were observed by using pipette solutions buffering [Ca2+]i to 1 microM. The channels were voltage-independent, blocked by charybdotoxin, clotrimazole, 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34), and blocked by Ba2+ in a voltage-dependent manner. In the presence of 10 microM clotrimazole or TRAM-34, proliferation of the BxPC-3 as well as the MiaPaCa-2 cells was completely stopped. In contrast, proliferation of PANC-1 cells was hardly affected by clotrimazole or TRAM-34. Proliferation in all three cell lines could be inhibited in the presence of the Ca2+ channel antagonists verapamil, diltiazem, and nifedipine. By quantitative RT-PCR, we could show that MiaPaCa-2 cells exhibit a 2.8-fold and BxPC3 cells a more than 8-fold elevated level of IK mRNA level compared with PANC-1 cells. Interestingly, in primary pancreatic tumors we found a tremendous up-regulation of IK mRNA. In eight of nine (or 89%) primary pancreatic tumor tissues, we found a 6- to 66-fold increase in IK mRNA. Our findings suggest that a certain amount of functional IK channels is crucial for the proliferation of some pancreatic cancer types. The blockade of IK channels may ultimately prove useful as a therapeutic option for some patients with ductal adenocarcinoma of the pancreas with an up-regulated IK channel expression.

Pharmacological investigation of the role of ion channels in salivary secretion.

The role of K+ and Cl- channels in salivary secretion was investigated, with emphasis on the potential role of Ca2+ -activated K+ channels. Ligand saturation kinetic assays and autoradiography showed large-conductance (BK) K+ channels to be highly expressed in rat submandibular and parotid glands, whereas low-conductance (SK) K+ channels could not be detected. To investigate the role of K+ and Cl- channels in secretion, intact rabbit submandibular glands were vascularly perfused and secretion induced by 10 microM ACh. Secretion was inhibited by 34+/-3% following perfusion with the general K+ channel inhibitor Ba2+ (5 mM), whereas organic inhibitors of BK (200 nM paxilline) or intermediate-conductance (IK) K+ channels (5 microM clotrimazole) had no effect. Secretion was strongly influenced by Cl- channel inhibitors, as 100 microM 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) completely abolished, while 10 microM NPPB, 20 microM NS1652 and 20 microM NS3623 reduced secretion by 34+/-3%, 23+/-3% and 59+/-4%, respectively. In conclusion, although high expression levels of BK channels were demonstrated, pharmacological tools failed to demonstrate any role for BK, IK or SK channels in salivary secretion in the rabbit submandibular gland. Other types of K+ channel, however, and particularly Cl- channels, are essential for ACh-induced salivary secretion.

The antifungal antibiotic clotrimazole alters calcium homeostasis of leukemic lymphoblasts and induces apoptosis

Clotrimazole is an antimycotic imidazole derivative that interferes with cellular Ca(2+) homeostasis. We investigated the effects of clotrimazole on acute lymphoblastic leukemia (ALL) cells. Treatment with 10 microM clotrimazole (a concentration achievable in vivo) reduced cell recovery from cultures of all nine ALL cell lines studied (B-lineage: OP-1, SUP-B15, RS4;11, NALM6, REH, and 380; T-lineage: MOLT4, CCRF-CEM, and CEM-C7). After 4 days of culture, median cell recovery was 10% (range, <1% to 37%) of cell recovery in parallel untreated cultures. Clotrimazole also inhibited recovery of primary ALL cells cultured on stromal feeder layers. After leukemic cells from 16 cases of ALL were cultured for 7 days with 10 microM clotrimazole, median cell recovery was <1% (range, <1% to 16%) of that in parallel untreated cultures. Clotrimazole was active against leukemic cells with genetic abnormalities associated with poor response to therapy and against multidrug-resistant cell lines. In contrast, mature T lymphocytes and bone marrow stromal cells were not affected. Clotrimazole induced depletion of intracellular Ca(2+) stores in ALL cells, which was followed by apoptosis, as shown by annexin V binding and DNA fragmentation. Thus, clotrimazole is cytotoxic to ALL cells at concentrations achievable in vivo.

The antifungal antibiotic clotrimazole potently inhibits L-type calcium current in guinea-pig ventricular myocytes.

The antimycotic agent clotrimazole (CLT) is a promising potential therapeutic agent for a variety of diseases including cancer. Although it is known that CLT alters calcium homeostasis in many cell types, its cardiac effects are virtually unknown. We investigated the effects of CLT on L-type calcium current (ICa,L) and action potentials in guinea-pig ventricular myocytes. CLT (5, 25 and 50 microM) inhibited basal ICa,L by 16, 59 and 93%, respectively. The inhibitory effect of CLT was rapid and the peak effect was attained within 3 min. At a concentration of 25 microM, the inhibitory effect of CLT was partially reversible whereas the response to 50 microM CLT persisted following drug withdrawal. CLT abbreviated action potential duration at 50 and 90% of repolarization and suppressed the plateau significantly. These results indicate that CLT may have important cardiac effects at concentrations used to induce the antiproliferative action of the drug.

Clotrimazole, an Antimycotic Drug, Inhibits the Sarcoplasmic Reticulum Calcium Pump and Contractile Function in Heart Muscle

Clotrimazole (CLT), an antimycotic drug, has been shown to inhibit proliferation of normal and cancer cell lines and its systemic use as a new tool in the treatment of proliferative disorders is presently under scrutiny (Benzaquen, L. R., Brugnara, C., Byers, H. R., Gattoni-Celli, S., and Halperin, J. A. (1995) Nature Med. 1, 534-540). The action of CLT is thought to involve depletion of intracellular Ca2+ stores but the underlying mechanism has not been defined. The present study utilized membrane vesicles of rabbit cardiac sarcoplasmic reticulum (SR) to determine the mechanism by which CLT depletes intracellular Ca2+ stores. The results revealed a strong, concentration-dependent inhibitory action of CLT on the ATP-energized Ca2+ uptake activity of SR (50% inhibition with approximately 35 microM CLT). The inhibition was of rapid onset (manifested in <15 s), and was accompanied by a 7-fold decrease in the apparent affinity of the SR Ca2+-ATPase for Ca2+ and a minor decrement in the enzyme's apparent affinity toward ATP. Exposure of SR to CLT in the absence or presence of Ca2+ resulted in irreversible inhibition of Ca2+ uptake demonstrating that the Ca2+-bound and Ca2+-free conformations of the Ca2+-ATPase are CLT-sensitive. Introduction of CLT to the reaction medium subsequent to induction of enzyme turnover with Ca2+ and ATP resulted in instantaneous cessation of Ca2+ transport indicating that an intermediate enzyme species generated during turnover undergoes rapid inactivation by CLT. The inhibition of Ca2+ uptake by CLT was accompanied by inhibition of Ca2+-stimulated ATP hydrolysis and Ca2+-induced phosphoenzyme intermediate formation from ATP in the ATPase catalytic cycle. Phosphorylation of the Ca2+-deprived enzyme with Pi in the reverse direction of catalytic cycle and Ca2+ release from Ca2+-preloaded SR vesicles were unaffected by CLT. It is concluded that CLT depletes intracellular Ca2+ stores by inhibiting Ca2+ sequestration by the Ca2+-ATPase. The mechanism of ATPase inhibition involves a drug-induced alteration in the Ca2+-binding site(s) resulting in paralysis of the enzyme's catalytic and ion transport cycle. CLT (50 microM) caused marked depression of contractile function in isolated perfused, electrically paced rabbit heart preparations. The contractile function recovered gradually following withdrawal of CLT from the perfusate indicating the existence of mechanisms in the intact cell to inactivate, metabolize, or clear CLT from its target site.

Characterization and modulation of EDHF-mediated relaxations in the rat isolated superior mesenteric arterial bed.

1. We have used the isolated, buffer-perfused, mesenteric arterial bed of the rat (preconstricted with methoxamine or 60 mM K+) to characterize nitric oxide (NO)-independent vasorelaxation which is thought to be mediated by the endothelium-derived hyperpolarizing factor (EDHF). 2. The muscarinic agonists carbachol, acetylcholine (ACh) and methacholine caused dose-related relaxations in preconstricted preparations with ED50 values of 0.18 +/- 0.04 nmol (n = 8), 0.05 +/- 0.02 nmol (n = 6) and 0.26 +/- 0.16 nmol (n = 5), respectively. In the same preparations NG-nitro-L-arginine methyl ester (1-NAME, 100 microM) significantly (P < 0.05) decreased the potency of all the agents (ED50 values in the presence of L-NAME: carbachol, 0.66 +/- 0.11 nmol; ACh, 0.28 +/- 0.10 nmol; methacholine, 1.97 +/- 1.01 nmol). The maximal relaxation to ACh was also significantly (P < 0.05) reduced (from 85.3 +/- 0.9 to 73.2 +/- 3.7%) in the presence of L-NAME. The vasorelaxant effects of carbachol were not significantly altered by the cyclo-oxygenase inhibitor indomethacin (10 microM; n = 4). 3. The K+ channel blocker, tetraethylammonium (TEA, 10 mM) also significantly (P < 0.001) reduced both the potency of carbachol (ED50 = 1.97 +/- 0.14 nmol in presence of TEA) and the maximum relaxation (Rmax = 74.6 +/- 3.2% in presence of TEA, P < 0.05, n = 3). When TEA was added in the presence of L-NAME (n = 4), there was a further significant (P < 0.001) decrease in the potency of carbachol (ED50 = 22.4 +/- 13.5 nmol) relative to that in the presence of L-NAME alone, and Rmax was also significantly (P < 0.05) reduced (74.6 +/- 4.2%). The ATP-sensitive K+ channel inhibitor, glibenclamide (10 microM), had no effect on carbachol-induced relaxation (n = 9). 4. High extracellular K+ (60 mM) significantly (P < 0.01) reduced the potency of carbachol (n = 5) by 5 fold (ED50: control, 0.16 +/- 0.04 nmol; high K+, 0.88 +/- 0.25 nmol) and the Rmax was also significantly (P < 0.01) reduced (control, 83.4 +/- 2.7%; high K+, 40.3 +/- 9.2%). The residual vasorelaxation to carbachol in the presence of high K+ was abolished by L-NAME (100 microM; n = 5). In preparations preconstricted with high K+, the potency of sodium nitroprusside was not significantly different from that in preparations precontracted with methoxamine, though the maximal response was reduced (62.4 +/- 3.4% high K+, n = 7; 83.1 +/- 3.1% control, n = 7). 5. In the presence of the cytochrome P450 inhibitor, clotrimazole (1 microM, n = 5 and 10 microM, n = 4), the dose-response curve to carbachol was significantly shifted to the right 2 fold (P < 0.05) and 4 fold (P < 0.001) respectively, an effect which was further enhanced in the presence of L-NAME. Rmax was significantly (P < 0.01) reduced by the presence of 10 microM clotrimazole alone, being 86.9 +/- 2.5% in its absence and 61.8 +/- 7.8% in its presence (n = 6). 6. In the presence of the cell permeable analogue of cyclic GMP, 8-bromo cyclic GMP (6 microM), the inhibitory effects of L-NAME on carbachol-induced relaxation were substantially enhanced (ED50: L-NAME alone, 0.52 +/- 0.11 nmol, n = 5; L-NAME + 8-bromo cyclic GMP, 1.42 +/- 0.28 nmol, n = 7, Rmax: L-NAME alone, 82.2 +/- 2.4%; L-NAME + 8-bromo cyclic GMP, 59.1 +/- 1.8%. P < 0.001). These results suggest that the magnitude of the NO-independent component of vasorelaxation is reduced when functional cyclic GMP levels are maintained, suggesting that basal NO (via cyclic GMP) may modulate EDHF activity and, therefore, on loss of basal NO production the EDHF component of endothelium-dependent relaxations becomes functionally greater. 7. The present investigation demonstrates that muscaranic receptor-induced vasorelaxation in the rat mesenteric arterial bed is mediated by both NO-dependent and independent mechanisms. The L-NAME-insensitive mechanism, most probably occurs via activation of a K+ conductance and shows the characteristics of EDHF-mediated responses. Finally, the results demonstrate that EDHF activity may become upregulated on inhibition of NO production and this may compensate for the loss of NO.