Tetraethylammonium, glibenclamide, and 4-aminopyridine modulate post-occlusive reactive hyperemia in non-glabrous human skin with no roles of NOS and COX.

OBJECTIVES: Post-occlusive reactive hyperemia (PORH) following arterial occlusion is widely used to assess cutaneous microvascular function, though the underlying mechanisms remain to be fully elucidated. We evaluated the hypothesis that Ca2+ -activated, ATP-sensitive, and voltage-gated K+ channels (KCa , KATP , and KV channels, respectively) contribute to PORH while nitric oxide synthase (NOS) and cyclooxygenase (COX) do not. METHODS: On separate occasions, cutaneous blood flow (laser Doppler flowmetry) was monitored before and following 5-min arterial occlusion at forearm skin sites treated via microdialysis with the following: Experiment 1 (n = 11): (a) lactated Ringer solution (Control), (b) 10 mM Nω -nitro-L -arginine (NOS inhibitor), (c) 10 mM ketorolac (COX inhibitor), and (d) combined NOS+COX inhibition; Experiment 2 (n = 14): (a) lactated Ringer solution (Control), (b) 50 mM tetraethylammonium (non-selective KCa channel blocker), (c) 5 mM glibenclamide (non-specific KATP channel blocker), and (d) 10 mM 4-aminopyridine (non-selective KV channel blocker). RESULTS: Separate and combined NOS and COX inhibition did not influence PORH. Conversely, tetraethylammonium and glibenclamide attenuated, whereas 4-aminopyridine augmented PORH. CONCLUSIONS: We showed that tetraethylammonium, glibenclamide, and 4-aminopyridine modulate PORH with no roles of NOS and COX in human non-glabrous forearm skin in vivo. Thus, cutaneous PORH changes could reflect altered K+ channel function.

Separate and combined effects of KCa and KATP channel blockade with NOS inhibition on cutaneous vasodilation and sweating in older men during heat stress.

Our objective in this study was to examine the separate and combined effects of potassium (K+) channels and nitric oxide synthase (NOS) on cutaneous vasodilation and sweating in older men during rest and exercise in the heat. In 13 habitually active men (61 ± 4 yr), cutaneous vascular conductance and local sweat rate were assessed at six dorsal forearm skin sites continuously perfused with either 1) lactated Ringer (control), 2) 10 mM NG-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor), 3) 50 mM tetraethylammonium (TEA; Ca2+-activated K+ channel blocker), 4) 5 mM glybenclamide (GLY; ATP-sensitive K+ channel blocker), 5) 50 mM TEA + 10 mM l-NAME, and 6) 5 mM GLY + 10 mM l-NAME via microdialysis. Participants rested in non-heat stress (25°C) and heat stress (35°C) conditions for ∼60 min each, followed by 50 min of moderate-intensity cycling (∼55% V̇o2peak) and 30 min of recovery in the heat. During rest and exercise in the heat, l-NAME, TEA + l-NAME, and GLY + l-NAME attenuated CVC relative to control (all P ≤ 0.05), although l-NAME was not different from TEA + l-NAME or GLY + l-NAME (all P > 0.05). TEA attenuated CVC during rest, whereas GLY attenuated CVC during exercise (both P ≤ 0.05). Additionally, whereas neither l-NAME nor TEA altered sweating throughout the protocol (all P > 0.05), combined TEA + l-NAME attenuated sweating during exercise in the heat (P ≤ 0.05). We conclude that in habitually active older men blockade of KCa and KATP channels attenuates cutaneous vasodilation during rest and exercise in the heat, respectively, and these effects are NOS dependent. Furthermore, combined NOS inhibition and KCa channel blockade attenuates sweating during exercise in the heat.

Evidence for the Involvement of Potassium Channel Inhibition in the Antidepressant-Like Effects of Hesperidin in the Tail Suspension Test in Mice.

The administration of hesperidin elicits an antidepressant-like effect in mice by a mechanism dependent on an interaction with the L-arginine-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway, whose stimulation is associated with the activation of potassium (K(+)) channels. Thus, this study investigated the involvement of different types of K(+) channels in the antidepressant-like effect of hesperidin in the mice tail suspension test (TST). The intracerebroventricular administration of tetraethylammonium (TEA, a nonspecific blocker of K(+) channels), glibenclamide (an ATP-sensitive K(+) channel blocker), charybdotoxin (a large- and intermediate-conductance calcium-activated K(+) channel blocker) or apamin (a small-conductance calcium-activated K(+) channel blocker) combined with a subeffective dose of hesperidin (0.01 mg/kg, intraperitoneally [i.p.]) was able to produce a synergistic antidepressant-like effect in the mice TST. Moreover, the antidepressant-like effect elicited by an effective dose of hesperidin (0.3 mg/kg, i.p.) in TST was abolished by the treatment of mice with pharmacological compounds K(+) channel openers (cromakalim and minoxidil). Results showed that the antidepressant-like effect of hesperidin in TST may involve, at least in part, the modulation of neuronal excitability through inhibition of K(+) channels and may act through a mechanism dependent on the inhibition of L-arginine-NO pathway.

The effects of methyl palmitate, a putative regulator from perivascular fat, on the contractility of pregnant human myometrium.

AIMS: Methyl palmitate is thought to cause relaxation in vascular smooth muscle by opening voltage-activated potassium channels. We have tested the hypothesis that methyl palmitate, a putative regulator from perivascular fat, is an inhibitor of the contractility of human pregnant myometrium and that its effects might partially explain the higher incidence of dysfunctional labor in obese women compared to those with normal body mass indices. MAIN METHODS: Strips of myometrium obtained with informed consent from women undergoing elective cesarean section at term were mounted in organ baths. Strips stimulated with oxytocin (1nM) or KCl (30mM) were exposed to cumulatively increasing concentrations of methyl palmitate up to 10µM. Similar strips were exposed to cumulative addition of the potassium channel blockers 4-aminopyridine and tetraethylammonium. The contractility of the strips was monitored and analyzed using conventional methods. KEY FINDINGS: Methyl palmitate failed to inhibit oxytocin- or KCl-induced contractions over the concentration range tested. In fact, it exerted a slight excitatory effect in the presence of KCl, though not in the presence of oxytocin. The contractility of naïve strips was unaltered by exposure to 1µM methyl palmitate. Both 4-aminopyridine and tetraethylammonium produced concentration-dependent contractions of human pregnant myometrium providing pharmacological evidence for the presence of voltage-activated potassium channels in this preparation. SIGNIFICANCE: Our findings do not support the hypothesis that methyl palmitate is an inhibitor of human pregnant myometrial contractility. Alternate hypotheses must be pursued to explain the higher incidence of dysfunctional labor in obese women.

Role of organic cation transporters in the ocular disposition of its intravenously injected substrate in rabbits: implications for ocular drug therapy.

The present study was conducted to test the hypothesis; OCT may be active from blood-to-vitreous for the uptake of its substrates. Ocular uptake of Tetraethylammonium (TEA) across blood ocular barriers and the tissue distribution was evaluated in vivo in New Zealand albino rabbits after intravenous administration. Quinidine (blocker) pretreatment resulted in a significant (p < 0.05) reduction in the Area Under the Curve (AUC) of TEA in vitreous (4.2 fold) and aqueous humor (1.8 fold) as compared to the control group which supports the role of OCT in uptake transport of its substrate across Blood ocular barrier. The blockade of OCT also affected the elimination of its substrate resulting in increased plasma levels. In most of the tissues, OCT are functionally present from apical to basolateral. The gene expression studies also showed the presence of OCT1, OCTN1 and OCTN2 in various ocular tissues studied. The present findings suggest that OCT are functionally active in blood ocular barriers and involved in the transport of its substrate from blood-to-vitreous humor.

Tuning the tetraethylammonium sensitivity of potassium channel Kcv by subunit combination.

Tetraethylammonium (TEA) is a potassium (K(+)) channel inhibitor that has been extensively used as a molecular probe to explore the structure of channels' ion pathway. In this study, we identified that Leu70 of the virus-encoded potassium channel Kcv is a key amino acid that plays an important role in regulating the channel's TEA sensitivity. Site-directed mutagenesis of Leu70 can change the TEA sensitivity by 1,000-fold from ∼100 µM to ∼100 mM. Because no compelling trends exist to explain this amino acid's specific interaction with TEA, the role of Leu70 at the binding site is likely to ensure an optimal conformation of the extracellular mouth that confers high TEA affinity. We further assembled the subunits of mutant and wt-Kcv into a series of heterotetramers. The differences in these heterochannels suggest that all of the four subunits in a Kcv channel additively participate in the TEA binding, and each of the four residues at the binding site independently contributes an equal binding energy. We therefore can present a series of mutant/wild-type tetramer combinations that can probe TEA over three orders of magnitude in concentration. This study may give insight into the mechanism for the interaction between the potassium channel and its inhibitor.

Competitive inhibition of the luminal efflux by multidrug and toxin extrusions, but not basolateral uptake by organic cation transporter 2, is the likely mechanism underlying the pharmacokinetic drug-drug interactions caused by cimetidine in the kidney.

Cimetidine, an H2 receptor antagonist, has been used to investigate the tubular secretion of organic cations in human kidney. We report a systematic comprehensive analysis of the inhibition potency of cimetidine for the influx and efflux transporters of organic cations [human organic cation transporter 1 (hOCT1) and hOCT2 and human multidrug and toxin extrusion 1 (hMATE1) and hMATE2-K, respectively]. Inhibition constants (K(i)) of cimetidine were determined by using five substrates [tetraethylammonium (TEA), metformin, 1-methyl-4-phenylpyridinium, 4-(4-(dimethylamino)styryl)-N-methylpyridinium, and m-iodobenzylguanidine]. They were 95 to 146 µM for hOCT2, providing at most 10% inhibition based on its clinically reported plasma unbound concentrations (3.6-7.8 µM). In contrast, cimetidine is a potent inhibitor of MATE1 and MATE2-K with K(i) values (µM) of 1.1 to 3.8 and 2.1 to 6.9, respectively. The same tendency was observed for mouse Oct1 (mOct1), mOct2, and mouse Mate1. Cimetidine showed a negligible effect on the uptake of metformin by mouse kidney slices at 20 µM. Cimetidine was administered to mice by a constant infusion to achieve a plasma unbound concentration of 21.6 µM to examine its effect on the renal disposition of Mate1 probes (metformin, TEA, and cephalexin) in vivo. The kidney- and liver-to-plasma ratios of metformin both were increased 2.4-fold by cimetidine, whereas the renal clearance was not changed. Cimetidine also increased the kidney-to-plasma ratio of TEA and cephalexin 8.0- and 3.3-fold compared with a control and decreased the renal clearance from 49 to 23 and 11 to 6.6 ml/min/kg, respectively. These results suggest that the inhibition of MATEs, but not OCT2, is a likely mechanism underlying the drug-drug interactions with cimetidine in renal elimination.

Endothelium-derived hyperpolarizing factor determines resting and stimulated forearm vasodilator tone in health and in disease.

BACKGROUND: We assessed the contribution of endothelium-derived hyperpolarizing factors to resting and agonist-stimulated vasodilator tone in health and disease. Tetraethylammonium chloride (TEA) was used to inhibit K(+)(Ca) channel activation and fluconazole was used to inhibit cytochrome P450 2C9-mediated epoxyeicosatrienoic acid synthesis. We hypothesized that endothelium-derived hyperpolarizing factors contribute to resting vascular tone by K(+)(Ca) channel activation and epoxyeicosatrienoic acid release and that endothelium-derived hyperpolarizing factors compensate for reduced nitric oxide bioavailability at rest and with endothelium-dependent vasodilators. METHODS AND RESULTS: In 103 healthy subjects and 71 nonhypertensive subjects with multiple risk factors, we measured resting forearm blood flow (FBF) using venous occlusion plethysmography before and after intra-arterial infusions of N(G)-monomethyl-l-arginine (L-NMMA), TEA, fluconazole, and their combination. The effects of these antagonists on resting FBF and on bradykinin- and acetylcholine-mediated vasodilation were studied. Resting FBF decreased with TEA and L-NMMA in all subjects (P<0.001); however, the vasoconstrictor response to L-NMMA was greater (P=0.04) and to TEA was lower (P=0.04) in healthy subjects compared with those with risk factors. Fluconazole decreased resting FBF in all subjects, and the addition of TEA further reduced FBF after fluconazole, suggesting that cytochrome P450 metabolites and other hyperpolarizing factor(s) activate K(+)(Ca) channels. Both L-NMMA and TEA attenuated bradykinin-mediated vasodilation in healthy and hypercholesterolemic subjects (P<0.001). In contrast, acetylcholine-mediated vasodilation remained unchanged with TEA in healthy subjects but was significantly attenuated in hypercholesterolemia (P<0.04). CONCLUSIONS: First, by activating TEA-inhibitable K(+)(Ca) channels, endothelium-derived hyperpolarizing factors, together with nitric oxide, contribute to resting microvascular dilator tone. The contribution of K(+)(Ca) channel activation compared with nitric oxide is greater in those with multiple risk factors compared with healthy subjects. Second, activation of K(+)(Ca) channels is only partly through epoxyeicosatrienoic acid release, indicating the presence of other hyperpolarizing mechanisms. Third, bradykinin, but not acetylcholine, stimulates K(+)(Ca) channel-mediated vasodilation in healthy subjects, whereas in hypercholesterolemia, K(+)(Ca) channel-mediated vasodilation compensates for the reduced nitric oxide activity. Thus, enhanced endothelium-derived hyperpolarizing factor activity in conditions of nitric oxide deficiency contributes to maintenance of resting and agonist-stimulated vasodilation. Clinical Trial Registration- URL: http://www.clinicaltrials.gov. Unique identifier: NCT00166166.

Iontophoretic transport of charged macromolecules across human sclera.

The mechanisms of transscleral iontophoresis have been investigated previously with small molecules in rabbit sclera. The objective of the present study was to examine transscleral iontophoretic transport of charged macromolecules across excised human sclera. Passive and 2mA iontophoretic transport experiments were conducted in side-by-side diffusion cells with human sclera. The effects of iontophoresis upon transscleral transport of model permeants bovine serum albumin (BSA) and polystyrene sulfonic acid (PSS) as well as a model drug bevacizumab (BEV) were determined. Passive and iontophoretic transport experiments of tetraethylammonium (TEA) and salicylic acid (SA) and passive transport experiments of the macromolecules served as the controls. The results of iontophoresis enhanced transport of TEA and SA across human sclera were consistent with those in a previous rabbit sclera study. For the iontophoretic transport of macromolecules BSA and BEV, higher iontophoretic fluxes were observed in anodal iontophoresis as compared to passive and cathodal iontophoresis. This suggests the importance of electroosmosis. For the polyelectrolyte PSS, higher iontophoretic flux was observed in cathodal iontophoresis compared to anodal iontophoresis. Both electroosmosis and electrophoresis affected iontophoretic fluxes of the macromolecules; the relative contributions of electroosmosis and electrophoresis were a function of molecular size and charge of the macromolecules.

Arterial stiffness is regulated by nitric oxide and endothelium-derived hyperpolarizing factor during changes in blood flow in humans.

Cytochrome-derived epoxyeicosatrienoic acids may be important endothelium-derived hyperpolarizing factors, opening calcium-activated potassium channels, but their involvement in the regulation of arterial stiffness during changes in blood flow in humans is unknown. In healthy volunteers, we measured arterial pressure, radial artery diameter, wall thickness, and flow (NIUS02) during hand skin heating in the presence of saline or inhibitors of NO synthase (N(G)-monomethyl-L-arginine), calcium-activated potassium channels (tetraethylammonium), and cytochrome epoxygenases (fluconazole). Arterial compliance and elastic modulus were calculated and fitted as functions of midwall stress to suppress the confounding influence of geometric changes. Under saline infusion, heating induced an upward shift of the compliance-midwall stress curve and a downward shift of the modulus-midwall stress curve demonstrating a decrease in arterial tone and stiffness when blood flow increases. These shifts were reduced by N(G)-monomethyl-L-arginine and abolished by the combinations of N(G)-monomethyl-L-arginine+tetraethylammonium and N(G)-monomethyl arginine+fluconazole. In parallel, in isolated mice coronary arteries, fluconazole and tetraethylammonium reduced the relaxations to acetylcholine. However, fluconazole did not affect the relaxations to the openers of calcium-activated potassium channels of small- and intermediate-conductance NS309 and of large-conductance NS1619 excluding a direct effect on these channels. Moreover, tetraethylammonium reduced the relaxations to NS1619 but not to NS309, suggesting that the endothelium-derived hyperpolarizing factor involved mainly acts on large-conductance calcium-activated potassium channels. These results show in humans that, during flow variations, arterial stiffness is regulated by the endothelium through the release of both NO and cytochrome-related endothelium-derived hyperpolarizing factor.

The influence of porosity changes in human epidermal membrane during iontophoresis on the permeability enhancement of a model peptide.

PURPOSE: We tested the hypothesis that the increases in the porosity of the skin during iontophoresis would not significantly increase the transport of peptides due to the small size of electrically induced pores. To investigate this mechanistically, we used human epidermal membrane under constant voltage conditions, applying the Nernst-Planck equation to the transport of a small ionic solute, tetraethylammonium bromide (TEAB), and a model peptide, luteinizing hormone releasing hormone. METHODS: Steady-state flux of the drugs was determined under passive conditions and also during iontophoresis using constant DC voltages applied across side-by-side diffusion cells. Electrical conductance measurements were used to monitor the porosity changes that occur during electrical field application. RESULTS: Porosity increases observed in the membrane substantially increased the permeability enhancement of the small ionic solute TEAB. The permeability enhancement was well described by Nernst-Planck model predictions after porosity changes in the membrane were taken into account. Enhancement of luteinizing hormone releasing hormone under identical conditions was much less than TEAB. The porosity increases induced by iontophoresis had little or no effect on the permeability enhancement of the larger molecule. CONCLUSIONS: These findings closely parallel those reports that have found electrically induced pores to be significantly smaller than preexisting pores in the human epidermal membrane. The data obtained also support the view that iontophoresis-induced pores, alone, may provide limited benefit for macromolecule transport across the skin.

Alternating current (AC) iontophoretic transport across human epidermal membrane: effects of AC frequency and amplitude.

PURPOSE: As a continuing effort to understand the mechanisms of alternating current (AC) transdermal iontophoresis and the iontophoretic transport pathways in the stratum corneum (SC), the objectives of the present study were to determine the interplay of AC frequency, AC voltage, and iontophoretic transport of ionic and neutral permeants across human epidermal membrane (HEM) and use AC as a means to characterize the transport pathways. MATERIALS AND METHODS: Constant AC voltage iontophoresis experiments were conducted with HEM in 0.10 M tetraethyl ammonium pivalate (TEAP). AC frequencies ranging from 0.0001 to 25 Hz and AC applied voltages of 0.5 and 2.5 V were investigated. Tetraethyl ammonium (TEA) and arabinose (ARA) were the ionic and neutral model permeants, respectively. In data analysis, the logarithm of the permeability coefficients of HEM for the model permeants was plotted against the logarithm of the HEM electrical resistance for each AC condition. RESULTS: As expected, linear correlations between the logarithms of permeability coefficients and the logarithms of resistances of HEM were observed, and the permeability data were first normalized and then compared at the same HEM electrical resistance using these correlations. Transport enhancement of the ionic permeant was significantly larger than that of the neutral permeant during AC iontophoresis. The fluxes of the ionic permeant during AC iontophoresis of 2.5 V in the frequency range from 5 to 1,000 Hz were relatively constant and were approximately 4 times over those of passive transport. When the AC frequency decreased from 5 to 0.001 Hz at 2.5 V, flux enhancement increased to around 50 times over passive transport. CONCLUSION: While the AC frequency for achieving the full effect of iontophoretic enhancement at low AC frequency was lower than anticipated, the frequency for approaching passive diffusion transport at high frequency was higher than expected from the HEM morphology. These observations are consistent with a transport model of multiple barriers in series and the previous hypothesis that the iontophoresis pathways across HEM under AC behave like a series of reservoirs interconnected by short pore pathways.

Role of different types of potassium channels in the antidepressant-like effect of agmatine in the mouse forced swimming test.

The administration of agmatine elicits an antidepressant-like effect in the mouse forced swimming test by a mechanism dependent on the inhibition of the NMDA receptors and the L-arginine-nitric oxide (NO) pathway. Since it has been reported that the NO can activate different types of potassium (K(+)) channels in several tissues, the present study investigates the possibility of synergistic interactions between different types of K(+) channel inhibitors and agmatine in the forced swimming test. Treatment of mice by i.c.v. route with subeffective doses of tetraethylammonium (a non specific inhibitor of K(+) channels, 25 pg/site), glibenclamide (an ATP-sensitive K(+) channels inhibitor, 0.5 pg/site), charybdotoxin (a large- and intermediate-conductance calcium-activated K(+) channel inhibitor, 25 pg/site) or apamin (a small-conductance calcium-activated K(+) channel inhibitor, 10 pg/site), augmented the effect of agmatine (0.001 mg/kg, i.p.) in the forced swimming test. Furthermore, the administration of agmatine and the K(+) channel inhibitors, alone or in combination, did not affect locomotion in the open-field test. Moreover, the reduction in the immobility time elicited by an active dose of agmatine (10 mg/kg, i.p.) in the forced swimming test was prevented by the pre-treatment of mice with the K(+) channel openers cromakalim (10 microg/site, i.c.v.) and minoxidil (10 microg/site, i.c.v.), without affecting locomotion. Together these data raise the possibility that the antidepressant-like effect of agmatine in the forced swimming test is related to its modulatory effects on neuronal excitability, via inhibition of K(+) channels.

K+ channel modulation of slow wave activity in the guinea-pig prostate.

BACKGROUND AND PURPOSE: The aim of this study was to investigate the role of different K(+) channel populations and the inhibitory effect of various exogenously applied K(+) channel openers in the regulation of slow wave activity in the guinea-pig prostate. EXPERIMENTAL APPROACH: Recordings of membrane potential were made using intracellular microelectrodes. KEY RESULTS: Tetraethylammonium (TEA 300 micro M and 1 mM), iberiotoxin (150 nM) and 4-aminopyridine (4-AP 1 mM) increased the frequency of slow wave discharge. Apamin (1-200 nM) and glibenclamide (1 micro M) had no effect on slow wave activity. Lemakalim (1 micro M) and PCO-400 (1 micro M) abolished the slow waves, as did sodium nitroprusside (SNP 10 micro M) and calcitonin gene-related peptide (CGRP 100 nM). The inhibitory effect of these agents was independent of a significant change in membrane potential. In the presence of 4-AP (1 mM), TEA (1 mM) or glibenclamide (1 micro M) the inhibitory actions of SNP (10 micro M) were attenuated. The inhibitory actions of CGRP (100 nM) were also reversed by glibenclamide (1 micro M). In contrast, isoprenaline (1 micro M) did not alter the frequency of slow wave discharge. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that BK(Ca) and 4-AP-sensitive K(+) channels regulate the frequency of prostatic slow wave discharge. SNP and CGRP abolish slow waves in a hyperpolarisation-independent manner, partially via opening of K(ATP) channels. BK(Ca) and 4-AP-sensitive K(+) channels also play an important role in the SNP-induced inhibition of slow wave activity. The lack of membrane hyperpolarisation associated with the SNP- and CGRP-induced inhibition implies that the channels involved in this action are not predominantly located on the smooth muscle cells.

Enhanced transscleral lontophoretic transport with ion-exchange membrane.

PURPOSE: Transscleral iontophoresis has been recently re-examined for drug delivery to the back of the eye. In conventional iontophoresis, due to the relatively high electromobility of the endogenous competing ions (counterions) relative to that of the drug ion in the tissue barrier, the efficiency of iontophoretic drug delivery is generally low. The objective of the present study was to examine ion-exchange membrane-enhanced transscleral iontophoretic transport in which the ion-exchange membrane in series with the sclera can hinder the transport of the competing counterions and selectively allow the transport of the permeant across the sclera. METHODS: The physical properties of the Ionac ion-exchange membrane and excised rabbit sclera were determined in equilibrium uptake experiments and in passive and iontophoretic transport experiments with salicylate, tetraethylammonium, urea, and mannitol. Transscleral experiments with the ion-exchange membrane were conducted with salicylate and excised rabbit sclera in vitro. The contribution of electroosmosis to electrotransport during transscleral iontophoresis was assessed with urea and mannitol. RESULTS: The ion-exchange membrane is highly positively charged and has a small effective pore size. The sclera is relatively porous with a large effective pore size and low pore tortuosity. The sclera is also net negatively charged but this does not significantly affect the transport of small ions. A three-fold steady-state transscleral flux enhancement of salicylate was observed in ion-exchange membrane-enhanced iontophoresis over conventional transscleral iontophoresis without the membrane. Such enhancement was relatively independent of the applied electric current density and the thickness of the studied ion-exchange membrane assembly. Although the ion-exchange membrane altered transscleral electroosmosis, the contribution of electroosmosis to electrotransport was not significant. CONCLUSIONS: The present study has demonstrated the potential of ion-exchange membranes for enhancing iontophoretic transport and drug delivery.

[Inhibitory effects of tetraethylammonium on proliferation and voltage-gated potassium channels in human cervical carcinoma cell line SiHa].

BACKGROUND & OBJECTIVE: Various potassium channels are known to be involved in proliferation of many malignant cell lines. This study was to explore the role of voltage-gated potassium channels in proliferation of human cervical carcinoma cells through observing the effects of tetraethylammonium (TEA) on proliferation and outward potassium currents in human cervical carcinoma cell line SiHa. METHODS: SiHa cells were treated with TEA. The effect of TEA on proliferation of SiHa cells was assessed by MTT assay. Cell apoptosis and cell cycle were detected by flow cytometry with Hoechst 33258 staining. The outward potassium currents were recorded by patch clamp technique. RESULTS: TEA inhibited the proliferation of SiHa cells in dose-and time-dependent manners, and induced cell apoptosis. The cell cycle was arrested at G0/G1 phase after treatment with TEA. Exposure of SiHa cells to 10 mmol/L TEA reduced the peak outward potassium currents significantly from (260+/-12) pA to (58+/-6) pA (P<0.01). CONCLUSIONS: Voltage-gated potassium channels play an important role in regulating proliferation of cervical carcinoma SiHa cells. Blocking voltage-gated potassium channels could inhibit proliferation of SiHa cells.

Evaluation of constant current alternating current iontophoresis for transdermal drug delivery.

Previous studies in our laboratory have demonstrated that alternating current (AC) iontophoresis can significantly decrease skin electric resistance and enhance the transport of charged permeants across skin. Flux variability of neutral permeants during AC iontophoresis was also found to be less than that of conventional direct current (DC) iontophoresis. The objectives of the present study were to evaluate flux enhancement of constant current AC transdermal iontophoresis and compare the AC flux with that of constant current DC iontophoresis. Iontophoresis studies of AC amplitude of 1, 2, and 5 mA were conducted in side-by-side diffusion cells with donor solution of 0.015, 0.15, and 1.0 M tetraethylammonium (TEA) chloride and receiver solution of phosphate buffered saline (PBS) using human epidermal membrane (HEM). Conventional constant current DC iontophoresis of 0.2 mA was also performed under similar conditions. TEA and mannitol were the model permeants. The following are the major findings in the present study. The flux of TEA increased proportionally with the AC current for all three TEA chloride concentrations and at the AC frequency used in the present study. When the permeant and its counter ion were the only ionic species in the donor chamber, the fluxes during DC iontophoresis were weakly dependent of its donor concentration. The fluxes of TEA during constant current AC iontophoresis were moderately related to the donor concentration with the highest TEA flux observed under the 1.0 M TEA chloride condition although the relationship between flux and donor concentration was not linear. A trend of decreasing electroosmotic transport with increasing donor TEA chloride concentration was observed with significant sample-to-sample variability during DC iontophoresis. Mannitol permeability was also observed to decrease with increasing TEA chloride concentration in the donor under the AC conditions, but data variability under AC was significantly smaller than that under DC. The results in the present study indicate that constant current AC iontophoresis under conditions tolerable to human (2 and 5 mA) can provide predictable fluxes that were lower than but of comparable magnitude as those of conventional constant current DC iontophoresis (0.2 mA).

Effects of dietary or injected organic cations on larval Drosophila melanogaster: mortality and elimination of tetraethylammonium from the hemolymph.

This study of larval Drosophila melanogaster examined the effects of injecting the prototypical organic cation tetraethylammonium (TEA) into the hemocoel or adding TEA and/or other organic cations to the diet. Mortality, hemolymph TEA levels, and Malpighian tubule TEA secretion rates were measured. The LD50 for dietary TEA was 158.4 mM and mortality increased if competitive inhibitors of organic cation transporters were also included in the diet. Mortality increased from 24% on TEA (100 mM) alone to 83 and 67% when the diet contained both TEA and quinidine (10 mM) or cimetidine (100 mM), respectively. TEA-selective microelectrode measurements indicated that hemolymph TEA concentration was approximately 3% of that in the diet for larvae maintained on TEA-enriched diet for 24 h. Malpighian tubules isolated from larvae exposed to dietary TEA excreted more TEA than did tubules from controls fed a TEA-free diet. However, the rate of decline of hemolymph TEA concentration following ingestion or injection of TEA into the hemocoel was greater than that explicable by rates of active transport by the gut and Malpighian tubules (MTs). We propose that TEA concentrations in the hemolymph are reduced not only by active transport across the MTs and gut, but also by diffusion into the gut. The latter pathway is particularly important when larvae previously maintained upon TEA-enriched diet are transferred to a TEA-free diet. The ingestion of TEA-free food not only clears the gut lumen, but also creates a TEA-free compartment into which TEA may passively diffuse from the hemolymph.

Delayed-rectifier (KV2.1) regulation of pancreatic beta-cell calcium responses to glucose: inhibitor specificity and modeling.

The delayed-rectifier (voltage-activated) K(+) conductance (K(V)) in pancreatic islet beta-cells has been proposed to regulate plasma membrane repolarization during responses to glucose, thereby determining bursting and Ca(2+) oscillations. Here, we verified the expression of K(V)2.1 channel protein in mouse and human islets of Langerhans. We then probed the function of K(V)2.1 channels in islet glucose responses by comparing the effect of hanatoxin (HaTx), a specific blocker of K(V)2.1 channels, with a nonspecific K(+) channel blocker, tetraethylammonium (TEA). Application of HaTx (1 microM) blocked delayed-rectifier currents in mouse beta-cells, resulting in a 40-mV rightward shift in threshold of activation of the voltage-dependent outward current. In the presence of HaTx, there was negligible voltage-activated outward current below 0 mV, suggesting that K(V)2.1 channels form the predominant part of this current in the physiologically relevant range. We then employed HaTx to study the role of K(V)2.1 in the beta-cell Ca(2+) responses to elevated glucose in comparison with TEA. Only HaTx was able to induce slow intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations in cells stimulated with 20 mM glucose, whereas TEA induced an immediate rise in [Ca(2+)](i) followed by rapid oscillations. In human islets, HaTx acted in a similar fashion. The data were analyzed using a detailed mathematical model of ionic flux and Ca(2+) regulation in beta-cells. The results can be explained by a specific HaTx effect on the K(V) current, whereas TEA affects multiple K(+) conductances. The results underscore the importance of K(V)2.1 channel in repolarization of the pancreatic beta-cell plasma membrane and its role in regulating insulin secretion.

Heterogeneity of neuronal and smooth muscle receptors involved in the VIP- and PACAP-induced relaxations of the pig intravesical ureter.

1. The mechanisms and receptors involved in the vasoactive intestinal peptide (VIP)- and pituitary adenylate cyclase-activating polypeptide (PACAP)-induced relaxations of the pig intravesical ureter were investigated. 2. VIP, PACAP 38 and PACAP 27 concentration-dependently relaxed U46619-contracted ureteral strips with a similar potency. [Ala(11,22,28)]-VIP, a VPAC(1) agonist, showed inconsistent relaxations. 3. The neuronal voltage-gated Ca(2+) channel inhibitor, omega-conotoxin GVIA (omega-CgTX, 1 microm), reduced the VIP relaxations. Urothelium removal or blockade of capsaicin-sensitive primary afferents, nitric oxide (NO) synthase and guanylate cyclase with capsaicin (10 microm), N(G)-nitro-l-arginine (l-NOARG, 100 microm) and 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 microm), respectively, did not change the VIP relaxations. However, the PACAP 38 relaxations were reduced by omega-CgTX, capsaicin, l-NOARG and ODQ. 4. The VIP and VIP/PACAP receptor antagonists, [Lys(1), Pro(2,5), Arg(3,4), Tyr(6)]-VIP (1 microm) and PACAP (6-38) (0.4 microm), inhibited VIP and VIP and PACAP 38, respectively, relaxations. 5. The nonselective and large-conductance Ca(2)-activated K(+) channel blockers, tetraethylammonium (3 mm) and charybdotoxin (0.1 microm), respectively, and neuropeptide Y (0.1 microm) did not modify the VIP relaxations. The small-conductance Ca(2)-activated K(+) channel blocker apamin (1 microm) did not change the PACAP 27 relaxations. 6. The cAMP-dependent protein kinase A (PKA) blocker, 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate (Rp-8-CPT-cAMPS, 100 microm), reduced VIP relaxations. The phosphodiesterase 4 inhibitor rolipram and the adenylate cyclase activator forskolin relaxed ureteral preparations. The rolipram relaxations were reduced by Rp-8-CPT-cAMPS. Forskolin (30 nm) evoked a potentiation of VIP relaxations. 7. These results suggest that VIP and PACAP relax the pig ureter through smooth muscle receptors, probably of the VPAC(2) subtype, linked to a cAMP-PKA pathway. Neuronal VPAC receptors localized at motor nerves and PAC(1) receptors placed at sensory nerves and coupled to NO release, seem also to be involved in the VIP and PACAP 38 relaxations.