Changes in sodium pool and kinetics of sodium transport in frog skin produced by amiloride

1. Amiloride produces a decrease in size of the active sodium transport pool of isolated frog skin.2. Rate coefficients for sodium movement into and out of the transporting cells across the outside membrane are decreased by amiloride. The rate coefficient for sodium extrusion across the inside membrane is not significantly affected.3. In the presence and in the absence of amiloride, the relation of sodium transport to outside sodium concentration exhibits similar saturation kinetics but amiloride reduces sodium transport rate at every sodium concentration of the outside solution.4. Labelling of skin with (14)C-amiloride from the outside solution is significantly greater than labelling with (14)C-inulin.5. The results of these studies suggest that amiloride reacts with sites on the outside membrane of the transporting cells as a result of which the rate of sodium movement across this membrane is diminished.     

Effects of amiloride on active sodium transport by the isolated frog skin: Evidence concerning site of action

1. Amiloride reduces short-circuit current and potential difference across the isolated frog skin.2. Isotopically measured sodium influx and efflux are diminished.3. Total electrical conductance and partial sodium conductance are diminished, the reduction in total conductance being entirely accounted for by the reduction in partial sodium conductance.4. The effect of antidiuretic hormone (ADH), cyclic 3'5'-adenosine monophosphate (cyclic AMP) and theophylline can be antagonized by pretreatment with amiloride but the antagonism can be abolished by increasing the concentration of these compounds.5. Amiloride has no effect on oxygen consumption in concentrations which inhibit sodium transport. However, it prevents the stimulatory effect of ADH on oxygen consumption.6. The results are consistent with an action of amiloride at the passive outside membrane of the transporting cells of isolated frog skin.     

Permeation enhancer strategies in transdermal drug delivery

Abstract

Today, ～74% of drugs are taken orally and are not found to be as effective as desired. To improve such characteristics, transdermal drug delivery was brought to existence. This delivery system is capable of transporting the drug or macromolecules painlessly through skin into the blood circulation at fixed rate. Topical administration of therapeutic agents offers many advantages over conventional oral and invasive techniques of drug delivery. Several important advantages of transdermal drug delivery are prevention from hepatic first pass metabolism, enhancement of therapeutic efficiency and maintenance of steady plasma level of the drug. Human skin surface, as a site of drug application for both local and systemic effects, is the most eligible candidate available. New controlled transdermal drug delivery systems (TDDS) technologies (electrically-based, structure-based and velocity-based) have been developed and commercialized for the transdermal delivery of troublesome drugs. This review article covers most of the new active transport technologies involved in enhancing the transdermal permeation via effective drug delivery system.     

The effect of ADH and insulin on active sodium transport across frog skin in the presence of alternariol mycotoxin

1. The effect of vasopressin and insulin on active sodium transport across frog skin in the presence of internal alternariol mycotoxin was studied, using the short-circuit technique. 2. Vasopressin stimulates sodium transport across frog skin by decreasing the resistance to sodium entry into the epithelial cells, thus partially removing the inhibition on the short-circuit current due to the action of Alternariol mycotoxin. 3. Even insulin which is known to increase the short-circuit current by a different mechanism, determines a rapid reversal effect on the inhibition due to Alternariol. 4. These data confirm the different action of the two hormones on active sodium transport across frog skin, and furthermore are indicative of an inhibition of transepithelial sodium transport by Alternariol mycotoxin probably via the sodium pump.     

In vitro/in vivo functionality of Catapres-TTS

The in vitro and in vivo functionality of Catapres-TTS, a transdermal therapeutic system that delivers the alpha adrenergic receptor agonist clonidine, is discussed in terms of the drug transport kinetics and resultant plasma drug concentration profiles. The design of Catapres-TTS is presented as an optimization by which the best combination of system performance characteristics is obtained within the inherent limitations of the transdermal drug transport properties and the known pharmacokinetic and pharmacodynamic properties of the drug. Clonidine is a potent antihypertensive agent with a relatively low therapeutic index. For Catapres-TTS, the majority of control over the drug input rate resides within the system, rather than within the skin, which significantly reduces the variability in drug input rate and resulting plasma drug concentration both within and between patients. Moreover, the presence of a rate-control element in the system allows for patterning of the drug release rate. An initial bolus of drug is placed in the contact adhesive layer, where its transport into the skin is not inhibited by the rate control element in the system, for reduction in the time needed to achieve steady state drug input. The selection of the loading dose of drug is described as an optimization between the minimization of the lag time and the maintenance of constant plasma drug concentrations during the crossover period between system applications in chronic therapy.     

Effects of bumetanide on sodium transport of the isolated frog skin and on renal Na-K-ATPase.

The effects of bumetanide, a new potent diuretic, on net sodium transport of the isolated frog skin and on rat renal Na-K-ATPase were studied. A dose-related decrease in short-circuit current and potential difference with increased electrical resistance was observed when bumetanide was added to the corial side of the skin. Addition to the epithelial side resulted in enhanced net sodium transport with decreased electrical resistance. When applied to the corial side it abolished vasopressin- and aldosterone-stimulated transport. Present in the epithelial bath ouabain-inhibited transport was unaffected by this drug, while triamterene-induced inhibition of sodium transport was completely abolished. In vitro, no significant effects on Na-K-ATPase were noted. It is concluded that bumetanide shares properties of both furosemide and ethacrynic acid and excerts its effects on epithelial sodium transport by altering membrane permeability and possibly by inhibition of some step in the active transport mechanism for sodium.     

Effects of furosemide on sodium content and transport pool in frog skin (Rana esculenta)

Summary The effects of ouabain, vasopressin, and furosemide on intracellular concentrations of total sodium ([Na]) and potassium [K]), on exchangeable sodium ([Na^*]) and the sodium transport pool ([Na _p ^*]) were investigated in isolated short circuited skins of rana esculenta. Furosemide was added to the epithelial bathing solution, vasopressin and ouabain to the corial bathing solution. Results were compared with the amount of net sodium transport measured by short circuit current (scc).Ouabain reduces scc and increases [Na] and [Na^*]; [K] is decreased. The administration of vasopressin leads to a sharp increase of scc, combined with an enhancement of [Na] and [Na^*]; [K] shows no significant change. [Na _p ^*] is significantly increased, too, and approximately to the same amount as [Na^*]. Furosemide causes an increase of scc, whereas a significant change of [Na], [Na^*] and [K] could not be detected. On the other hand, [Na _p ^*] was enhanced significantly.The results support the hypothesis that furosemide like vasopressin is acting by increasing the entry of sodium into the transport compartment of the active cell layer. The result is an increased transfer of sodium across the skin.Preliminary reports of the results were presented at the Pharmacology Meeting Graz 1974.     

Pharmacology of 5-hydroxytryptamine receptors in frog skin epithelium.

1. 5-Hydroxytryptamine (5-HT) stimulates active sodium transport and decreases the passive mucosal to serosal chloride permeability across frog skin. The relative importance of the different regions of the 5-HT molecule in the mediation of these responses has been studied using a range of structurally related compounds. 2. Substitution in the ethyl amine side chain of 5-HT (5-hydroxytryptophan) results in decreased receptor affinity and intrinsic activity; removal of the side chain (5-hydroxyindole) abolishes activity. Methoxy substitution of the 5-OH moiety of 5-HT has no effect on intrinsic activity but reduces affinity; displacement of the hydroxyl group to position 6 diminishes intrinsic activity and affinity. 3. It is concluded that both of the 5-HT-induced physiological effects are mediated via a single receptor which is distinct from alpha- and beta-adrenoceptors.     

Iontophoretic permeation of sodium cromoglycate through synthetic membrane and excised hairless mouse skin.

The iontophoretic transport properties of sodium cromoglycate were characterized using a synthetic membrane and excised hairless mouse skin. The permeation rate of sodium cromoglycate through the synthetic membrane was found to be linearly dependent on the density of electrical current applied. Passive diffusion through the excised hairless mouse skin was not demonstrated for sodium cromoglycate; however, under iontophoresis, an appreciable permeation was exhibited by the drug through the animal skin, which was also found to be a function of the electrical current density.     

The effects of anions on sodium transport

1. Substitution of chloride by isethionate reduces the short circuit current (SCC) and increases the potential of isolated frog skin. In sodium isethionate Ringer antidiuretic hormone and choline chloride increase the SCC, whereas theophylline is ineffective.2. Frog skins treated on the outside with copper ions always show an increased potential when bathed in normal Ringer solution. The SCC may be moderately increased or decreased.3. Theophylline increases skin thickness and cell volume in non-short-circuited skins.4. The ways in which the theophylline-induced increase in chloride permeability affects sodium transport is discussed, together with the requirements for a permeant anion in both short- and open-circuited skins.     

Release of salicylic acid, diclofenac acid and diclofenac acid salts from isotropic and anisotropic nonionic surfactant systems across rat skin

Release of salicylic acid, diclofenac acid, diclofenac diethylamine and diclofenac sodium, from lyotropic structured systems, namely; neat and middle liquid crystalline phases, across mid-dorsal hairless rat skin into aqueous buffer were studied. Release results were compared with those from the isotropic systems. The donor systems composed of the surfactant polyoxyethylene (20) isohexadecyl ether, HCl buffer of pH 1 or distilled water and the specific drug. High performance liquid chromatography (HPLC) methods were used to monitor the transfer of the drugs across the skin barrier. Results indicated that the rate-determining step in the transport process was the release of the drug from the specified donor system. Further, apparent zero order release was demonstrated with all systems. Except for diclofenac sodium, drug fluxes decreased as the donor medium changed from isotropic to anisotropic. The decrease in fluxes was probably due to the added constrains on the movement of drug molecules. By changing the anisotropic donor medium from neat to middle phase, drug flux decreased in case of salicylic acid and diclofenac sodium. In the mean time, flux increased in case of the diethylamine salt and appeared nearly similar in case of diclofenac acid. Rates of drug transfer across the skin from the anisotropic donors seemed to be largely controlled by the entropy contribution to the transport process. The type and extent of drug-liquid crystal interactions probably influenced the latter.     

Lithium treatment strongly inhibits choline transport in human erythrocytes

1 The influx of [¹⁴C]-choline and the efflux of ²²Na in human erythrocytes were measured in vitro using blood from patients treated with lithium, patients not on lithium and healthy individuals.

2 The administration of lithium to patients significantly reduces the transport of choline; during the first 6 weeks of treatment the influx of choline is about half the normal rate, later it falls to around 10%.

3 This inhibition of choline transport is not dependent on the presence of lithium in the incubation medium.

4 The active and passive efflux of sodium are apparently not affected by lithium treatment.

     

Irreversible inhibition of epithelial sodium channels by ultraviolet irradiation.

1 The effects of u.v. irradiation at 254 nm and 350 nm on sodium transport across frog skin epithelium have been investigated. 2 Irradiation at 254 nm but not at 350 nm produces a dose-dependent, functionally selective blockade of sodium transport. The effect is apparently due to the irreversible closure of apical sodium channels. 3 The amiloride-sensitive conductance was directly related to sodium transport as measured by short circuit current (SCC) both in normal and irradiated tissues, although both conductance and current were reduced in irradiated tissues. 4 The sensitivity of epithelia to irradiation at 254 nm was defined from the rate constants for the decline in SCC during three 2 min periods of irradiation at 1850 microW cm-2. The rate constant for the initial 2 min irradiation was 0.093 +/- 0.008 min-1. 5 Lowering the sodium concentration to 5.5 mM from 110 mM increased the rate constant to 0.141 +/- 0.014 min-1, consistent with the view that more functional sodium channels exist at lowered sodium concentration. 6 Lowering the temperature to 7 degrees C from 23 degrees C reduced the rate constant to 0.032 +/- 0.007 min-1 suggesting that blockade of channels is not due to a direct interaction with photons. 7 Using a variety of experimental protocols we were unable to demonstrate that bromamiloride or iodoamiloride can act as photoligands for sodium channels in the epithelium of Rana temporaria. This is in contrast to earlier reports with other epithelia.     

Quantitative assessment of the contribution of sodium‐dependent taurocholate co‐transporting polypeptide (NTCP) to the hepatic uptake of rosuvastatin,pitavastatin and fluvastatin

Hepatic uptake transport is often the rate‐determining step in the systemic clearance of drugs. The ability to predict uptake clearance and to determine the contribution of individual transporters to overall hepatic uptake is therefore critical in assessing the potential pharmacokinetic and pharmacodynamic variability associated with drug–drug interactions and pharmacogenetics. The present study revisited the interaction of statin drugs, including pitavastatin, fluvastatin and rosuvastatin, with the sodium‐dependent taurocholate co‐transporting polypeptide (NTCP) using gene transfected cell models. In addition, the uptake clearance and the contribution of NTCP to the overall hepatic uptake were assessed using in vitro hepatocyte models. Then NTCP protein expression was measured by a targeted proteomics transporter quantification method to confirm the presence and stability of NTCP expression in suspended and cultured hepatocyte models. It was concluded that NTCP‐mediated uptake contributed significantly to active hepatic uptake in hepatocyte models for all three statins. However, the contribution of NTCP‐mediated uptake to the overall active hepatic uptake was compound‐dependent and varied from about 24% to 45%. Understanding the contribution of individual transporter proteins to the overall hepatic uptake and its functional variability when other active hepatic uptake pathways are interrupted could improve the current prediction practice used to assess the pharmacokinetic variability due to drug–drug interactions, pharmacogenetics and physiopathological conditions in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

A novel method for culturing sweat gland epithelia: comparison of normal and cystic fibrosis tissues.

A new method is described to produce epithelial sheets by direct explantation of human sweat glands onto matrigel-coated millipore filters. The method is applicable to whole glands, separated coils or ducts and to normal and CF tissues. Electrogenic transport studies show that epithelia develop sodium transporting capability, even when explants are derived from secretory coils.     

Effect of intracerebroventricular vanadate administration on salt and water intake and excretion in the rat

The effect of vanadate (VO-3), an "in vitro" inhibitor of Na,K-ATPase activity, on sodium and water intake and excretion of Na-depleted and water deprived rats, was investigated. Injection of sodium orthovanadate Na3V04, H20 14) 1 microliter, 1.0 mM solution, 51 ng/microliter free base vanadium (V) into the 3rd brain ventricle (3BV) inhibited by 34% the sodium intake induced by peritoneal dialysis (PD). Urinary water and sodium excretion increased and potassium excretion decreased. The same concentration of vanadate administered by continuous infusion into the 3BV (1 microliter/hr, 24 hr, 51 ng/microliter, 1.2 micrograms/24 hr) during 24 hours after PD, decreased sodium intake by 69%. The same rate of infusion through the jugular vein failed to inhibit sodium intake or to increase urinary water and sodium excretion. Injections into lateral hypothalamus were also ineffective. Vanadyl (VO+2), the reduced form of vanadate, did not affect sodium intake. Similar or larger doses of vanadate injected into the 3BV of water deprived rats, did not modify water intake significantly. The present results suggest that the Na-K, active transport system is involved in salt and water balance regulation at the central nervous system level.     

Fenoterol stimulation of sodium transport in the isolated toad skin: a beta adrenergic effect

1. This work examines the effects of fenoterol on the isolated skin of the Chilean toad Pleurodema thaul. 2. A dose-dependent increase in the potential difference and in the short-circuit current of the skin was found. Furthermore, sodium potential (ENa) and sodium conductance (GNA) also rose. 3. The increase in bioelectric parameters was reversibly blocked by propranolol, was not significantly affected by reserpinization and was not calcium-dependent. 4. The skin response was significantly reduced in the presence of low Na+ in the outer bathing solution. Although the response was also reduced in the presence of isethionate Ringer's in the inner bathing solution, this reduction was less than the decrease in the presence of low Na+. 5. Fenoterol significantly increased toad skin oxygen consumption and net Na+ movement across the skin due to an increase in Na+ flux from mucosa to serosa. 6. These results show that fenoterol enhances active transport across the isolated toad skin probably through beta adrenergic stimulating effects which activate the sodium driving force, sodium conductance and net Na+ transepithelial flux.     

Skin penetration behaviour of liposomes as a function of their composition

Deformable liposomes have been developed and evaluated as a novel topical and transdermal delivery system. Their mechanism of drug transport into and through the skin has been investigated but remains a much debated question. The present study concerns ex vivo diffusion experiments using pig ear skin in order to explain the penetration mechanism of classical and deformable liposomes. Classical and deformable vesicles containing betamethasone in the aqueous compartment through the use of cyclodextrin inclusion complexes were compared to vesicles encapsulating betamethasone in their lipid bilayer. Deformable liposomes contained sodium deoxycholate as the edge activator. Liposomes were characterised by their diameter, encapsulation efficiency, deformability, stability (in terms of change in diameter) and release of encapsulated drug. Exvivo diffusion studies using Franz diffusion cells were performed. Confocal microscopy was performed to visualise the penetration of fluorescently labelled liposomes into the skin. This study showed that liposomes do not stay intact when they penetrate the deepest layers of the skin. Betamethasone is released from the vesicles after which free drug molecules can diffuse through the stratum corneum and partition into the viable skin tissue.     

Transport mechanism of ursodeoxycholic acid in human placental BeWo cells

Ursodeoxycholic acid (UDCA) is a first‐line drug to treat intrahepatic cholestasis of pregnancy (ICP). However, its effects on the fetus are not clearly known. To better guide its clinical use, we aimed to study the mechanism underlying the placental transport of UDCA. The uptake and efflux of UDCA across placental apical membranes were studied using BeWo cells; effects of different exposure durations, UDCA concentrations, temperatures, and inhibitors of transporters were studied. A transwell assay was performed, and UDCA concentration in both fetal and maternal sides was measured using LC–MS/MS. Higher unidirectional transport of UDCA was observed in the basolateral‐to‐apical direction than in the apical‐to‐basolateral direction. Ko143 and verapamil, which are typical inhibitors of efflux transporters, significantly increased UDCA transport from different directions. UDCA uptake from the apical membrane of BeWo cells was time‐dependent, but sodium‐independent. It was inhibited by inhibitors of energy metabolism and of organic anion transporters, indicating an active transport mechanism. UDCA uptake from the apical membranes of BeWo cells could be mediated by organic anion‐transporting polypeptides, whereas its efflux could be mediated by breast cancer resistance protein and multidrug resistant protein 3. The results of the present study may provide a basis for UDCA use in pregnancy.     

Effects of some pyrazinecarboxamides on sodium transport in frog skin.

1 The inhibitory effect of amiloride (N-amidino-3,5-diamino-6-chloropyrazinecarboxamide) on sodium transport in isolated skin of frog has been compared with 17 of its analogues. The dissociation constant of amiloride for passive sodium channels was 181.9 +/- 8.9 nM, and the maximal percentage inhibition of sodium transport was 101.3 +/- 0.4% (means of 123 measurements) when measured at a sodium concentration of 111 mM. 2 The N-benzylamidino and N-o-chlorobenzylamidino compounds had affinities approximately 20 times larger than those for amiloride, and produced maximal inhibition of transport. 3 Substitution of chlorine in the 6-position by other halogens showed that the bromo-compound was equally active to amiloride, whereas the iodo derivative had an affinity equal to 15% of that for amiloride. 4 Substitution in the 5-amino group in 10 compounds reduced the affinities to less than 1% of that of amiloride, without affecting their ability to produce complete inhibition of transport. 5 N-Amidino-3,5-diaminopyrazinecarboxamide was unique in that it produced an unusual concentration-response relationship.