Presence Of Phloretin Research Articles

The Disordering Effect of Plant Metabolites on Model Lipid Membranes of Various Thickness

This study was focused on the effect of plant metabolites (phloretin, capsaicin, digitonin, diosgenin and betulin) on the model lipid membranes. The methods of assessing the permeability of lipid bilayers based on measuring the leakage of a fluorescent marker (calcein) from liposomes and differential scanning microcalorimetry of vesicle suspension were used. It was found that the release rate of calcein from 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) liposomes with test compounds added to the suspension at a ratio with lipid 1 : 50 decreased in the order capsaicin > phloretin >> betulin ≈ diosgenin ≈ digitonin. In the case of cholesterol- and ergosterol-containing POPC liposomes, the activity decreased as diosgenin ≈ digitonin > betulin > capsaicin > phloretin. It was demonstrated that phloretin and capsaicin significantly reduce the melting temperature (Tm) and to increase the half-width of the main peak on the endotherm (T1/2) of dipalmitoylphosphocholine (DPPC), distearoylphosphocholine (DSPC) and diarachidoylphosphocholine (DAPC). These results show that the incorporation of these small molecules between the polar “heads” of phosphocholine. It was found that the increase in the length of saturated chains of membrane-forming lipids (from 16 to 20 hydrocarbon units), the absolute values of ΔTm and ΔT1/2 decreased in the presence of phloretin and increased with capsaicin. This may be the result of differences in the localization of phloretin and capsaicin in the membrane. Steroid saponins exhibited a weak effect on the thermotropic behavior of phosphocholines. The absolute values of ΔTm and ΔT1/2 decreased in the order DPPC, DSPC, and DAPC and increased in the order betulin, diosgenin, and digitonin. Steroid saponins are characterized by a more pronounced effect on the thermotropic behavior of the sterol–phospholipid mixture. The findings are consistent with the assumption of a high affinity of the tested saponins for sterol-containing membranes.

Effect of AQP Inhibition on Boar Sperm Cryotolerance Depends on the Intrinsic Freezability of the Ejaculate.

Aquaporins (AQPs) are transmembrane channels with permeability to water and small solutes that can be classified according to their structure and permeability into orthodox AQPs, aquaglyceroporins (GLPs), and superAQPs. In boar spermatozoa, AQPs are related to osmoregulation and play a critical role in maturation and motility activation. In addition, their levels differ between ejaculates with good and poor cryotolerance (GFE and PFE, respectively). The aim of this work was to elucidate whether the involvement of AQPs in the sperm response to cryopreservation relies on the intrinsic freezability of the ejaculate. With this purpose, two different molecules: phloretin (PHL) and 1,3-propanediol (PDO), were used to inhibit sperm AQPs in GFE and PFE. Boar sperm samples were treated with three different concentrations of each inhibitor prior to cryopreservation, and sperm quality and functionality parameters were evaluated in fresh samples and after 30 and 240 min of thawing. Ejaculates were classified as GFE or PFE, according to their post-thaw sperm viability and motility. While the presence of PHL caused a decrease in sperm quality and function compared to the control, samples treated with PDO exhibited better quality and function parameters than the control. In addition, the effects of both inhibitors were more apparent in GFE than in PFE. In conclusion, AQP inhibition has more notable consequences in GFE than in PFE, which can be related to the difference in relative levels of AQPs between these two groups of samples.

Phloretin modulates the rate of channel formation by polyenes

The influence of flavonoids and polyene antibiotics on the permeability of membranes has been investigated through measurements of calcein leakage from large unilamellar vesicles composed of DOPC:cholesterol (67:33mol%). Phloretin and biochanin A have been shown to induce calcein release from liposomes, but quercetin, daidzein, and catechin have not. Differential scanning calorimetry has indicated a decreasing of melting temperature of DPPC vesicles by 1.5–2°C in the presence of phloretin and biochanin A. Quercetin, catechin, and daidzein have had almost no effect on the main transition temperature. Phloretin, biochanin A, and quercetin have significantly broadened the main transition peak of DPPC. Phloretin have increased a leakage induced by polyene antibiotics, whereas catechin and daidzein have not. Quercetin has slightly affected it. The effects of tested flavonoids on the polyene-induced calcein leakage and channel forming activity have been similar. The obtained data agree with the previously supposed hypothesis regarding the enhancement of polyene activity by reducing elastic stress near the lipid mouth of the nystatin pore. The inhibition of polyene channel forming activity by biochanin A observed in planar DOPC:cholesterol bilayers may be related to the flavonoid competition with cholesterol in the polyene–sterol channel complexes.

Effect of phloretin on the binding of 1-anilino-8-naphtalene sulfonate (ANS) to 1,2-Dimyristoyl-sn-glycero-3-phosphocoline (DMPC) vesicles in the gel and liquid-crystalline state.

Phloretin is a known modifier of the internal dipole potential of lipid membranes. We studied the interaction of phloretin with model lipid membranes and how it influences the membrane dipole organization using ANS as fluorescent probe. The fluorescence increase observed when ANS binds to DMPC liposomes in gel phase (13 °C) was 2.5 times larger in the presence of phloretin. This effect was due to an increase in ANS affinity, which can be related to the known capability of phloretin in decreasing the dipole potential. Conversely, when the experiments were carried out at 33 °C (liquid crystalline phase), phloretin completely inhibited the increase in ANS fluorescence. In addition, phloretin only affected the electrical properties of the membrane in the gel phase, whereas it modifies structural ones in the liquid-crystalline state. We postulate that phloretin was bound only to the DMPC interface in the gel phase decreasing the surface negative charge density without modifying the structural properties of the ANS binding sites. In the liquid-crystalline phase instead, it increased the accessibility of water to the ANS binding sites decreasing the intrinsic affinity and the fluorescence quantum yield of ANS.

The alteration of lipid bilayer dynamics by phloretin and 6-ketocholestanol

Lipid bilayer properties are quantified with a variety of arbitrary selected parameters such as molecular packing and dynamics, electrostatic potentials or permeability. In the paper we determined the effect of phloretin and 6-ketocholestanol (dipole potential modifying agents) on the membrane hydration and efficiency of the trans-membrane water flow. The dynamics of water molecules within the lipid bilayer interface was evaluated using solvent relaxation method, whereas the osmotically induced trans-membrane water flux was estimated with the stopped-flow method using the liposome shrinkage kinetics. The presence of phloretin or 6-ketocholestanol resulted in a change of both, the interfacial hydration level and osmotically driven water fluxes. Specifically, the presence of 6-ketocholestanol reduced the amount and mobility of water in the membrane interface. It also slows the osmotically induced water flow. The interfacial hydration change caused by phloretin was much smaller and the effect on osmotically induced water flow was opposite to that of 6-ketocholestanol.

In vitro attenuation of acrolein-induced toxicity by phloretin, a phenolic compound from apple

In the current study, the protective effects of phloretin were investigated in acrolein-challenged amino acid, protein, and cell models. It was found that the formation of FDP-lysine (a typical acrolein–lysine adduct) was strongly inhibited in the presence of phloretin and the remaining electrophilic site in FDP-lysine was also blocked by phloretin. Moreover, direct trapping of acrolein by phloretin was found to be responsible for inhibiting the incorporation of carbonyl groups into BSA and oligomerisation in RNase A. Subsequently, the reduction of LDH release in human neuroblastoma SH-SY5Y cells under acrolein challenge suggested the cytoprotective effects of phloretin. Such protection might be mediated through inhibiting the increased cellular protein carbonyl level as revealed by Western blotting analysis. The present study highlighted an apple phenolic compound, phloretin as a promising candidate in prevention or treatment of acrolein-associated human diseases.

Amphotericin B Channel-Forming Activity Depends on Membrane Dipole Potential

Amphotericin B (AmB) is the most effective polyene antibiotic used in treatment of systemic fungal infections. The activity of AmB results from its interaction with sterol-containing membranes and consequent pore formation. As some sterols influence membrane dipole potential (Vd) one can assume that Vd may regulate the ability of AmB to form ion channels. The aim of the present work was to study the channel-forming activity of AmB as well as the properties of its single pores in planar lipid bilayers of different dipole potentials. AmB (0.1-0.2 µM) was added on both sides of a membrane prepared from diphytanoylphosphocholine (67 mol %) and cholesterol (33 mol %) in 2 M KCl (5 mM Hepes, pH 7.0) solutions. A reduction of Vd by addition of 20 µM phloretin to the bilayer bathing solutions is accompanied by ∼3-fold decrease of a single channel conductance and ∼20-fold increase of a steady-state AmB-induced membrane conductance. Increasing the membrane dipole potential by adding 5 µM RH421 produces ∼2-fold increase of the conductance of AmB channels and practically does not affect the steady-state bilayer conductance. The observed changes in the channel-forming activity of AmB in the presence of dipole modifying agents correspond to ∼60-fold increase and ∼2 fold decreases in steady-state open channel number in the presence of phloretin and RH421, respectively. The obtained results allow us to speculate that AmB molecule possess a certain dipole moment. Therefore, the decrease of membrane dipole potential produces a reduction of the energy barrier for interfacial accumulation of AmB dipoles. These results may be instructive in understanding of the membrane activity of amphotericin B. The study was supported in part by RFBR (# 09-04-00883), SS-3796.2010.4, the Program “Molecular and Cell Biology”, RAS, and the State contract # P1372 (MES, FTP“SSEPIR”).

Insulin down-regulates the Na +/K + ATPase in enterocytes but increases intestinal glucose absorption

The effect of insulin on [ 14C] 3-O-methyl- d-glucose (3OMG) absorption in the rat jejunum was studied using an in situ perfusion technique. Insulin increased apical glucose entry into the cells and decreased intestinal retention suggesting that serosal glucose transport was enhanced by the hormone. This enhanced uptake was ascribed to an increase in the expression of glucose transporters as confirmed by Western blot analysis and not to a higher sodium gradient, since insulin reduced the activity and protein expression of the Na +/K + ATPase. To separate the glycemic from the insulinemic effect on glucose transport, the effect of the hormone was investigated in vitro using cultured Caco-2 cells. The cells also showed an increase in [ 14C] 3OMG uptake and intracellular glucose levels when treated with insulin and a lower Na +/K + ATPase activity. Phloretin, an inhibitor of GLUT2 was used to determine if these transporters are targeted by the hormone. The results showed that the effect of insulin on glucose uptake and intracellular glucose was still enhanced in presence of phloretin. Considering the inhibitory effect of the hormone on the Na +/K + ATPase, it was concluded that insulin acts by increasing the number of glucose transporters, a hypothesis that was confirmed by Western blot analysis.

The effect of phloretin on sphingolipid-containing membranes modified by syringomycin E

The effects of dipole modifier phloretin on the activity of syringomycin (SRE) channels in the lipid bilayers containing different sphingolipids, N-stearoyl-phytosphingosine (PSP) and N-stearoyl-D-erythrosphingosine (DSP), have been compared. It is shown that the addition of phloretin up to a concentration of 10 μM into solutions bathing the bilayers containing 20 mol.% PSP causes a 170-fold increase in the SRE channel-forming activity. In the case of DSP-containing membranes, a more significant (5200-fold) increase of the equilibrium number of open SRE channels is observed. The enhancement of SRE activity is accompanied by about 2-fold increase of the gating charge of SRE channels in the membranes with PSP, while in the bilayers with DSP the gating charge increases about 4-fold. The revealed differences in the parameters of SRE channels in the membranes including phloretin and PSP or DSP are accounted for by different partition coefficients of the toxin and dipole modifier between the lipid and water phases. The data suggest heterogeneity of dipole potential of the PSP-containing membranes in the presence of phloretin. This heterogeneity is due to the possibility of formation in these membranes of rafts with the dipole potential not affected by modifier.

Characterization of Fatty Acid Transport across Human Brain Microvessel Endothelial Cells (HBMECs)

Endothelial cells lining the blood capillaries of the Blood Brain Barrier (BBB) are tightly packed thus regulating the transport of substances from the blood into the brain, including fatty acids. Fatty acids are essential for both the developing and adult mammalian brain. Since most fatty acids in the brain enter from the blood, we examined the mechanism of their transport across Human Brain Microvessel Endothelial Cells (HBMECs). HBMECs were plated onto transwell plate inserts and then incubated for up to 4 h with [1‐14C]oleate in the apical medium. Radioactivity in the basolateral medium was temporally examined. There was a near linear increase in [1‐14C]oleate incorporation into the basolateral media in the presence of albumin indicating a protein acceptor is required for oleate transport. The presence of phloretin, a non‐specific fatty acid uptake inhibitor, significantly decreased [1‐14C]oleate transport into the basolateral medium. In addition, siRNA knockdown of fatty acid transport protein‐1 (FATP‐1) or fatty acid translocase (FAT/CD36) significantly decreased [1‐14C]oleate incorporation into the basolateral media. In summary, transport of oleate across HBMECs is, in part, a transcellular process. Oleate transport across HBMECs is mediated by both diffusion and carrier mediated processes. (Supported by the Manitoba Health Research Council and the Canadian Institutes of Health Research)

Effects of Phloretin and Phloridzin on Ca2+ Handling, the Action Potential, and Ion Currents in Rat Ventricular Myocytes

The effects of the phytoestrogens phloretin and phloridzin on Ca(2+) handling, cell shortening, the action potential, and Ca(2+) and K(+) currents in freshly isolated cardiac myocytes from rat ventricle were examined. Phloretin increased the amplitude and area and decreased the rate of decline of electrically evoked Ca(2+) transients in the myocytes. These effects were accompanied by an increase in the Ca(2+) load of the sarcoplasmic reticulum, as determined by the area of caffeine-evoked Ca(2+) transients. An increase in the extent of shortening of the myocytes in response to electrically evoked action potentials was also observed in the presence of phloretin. To further examine possible mechanisms contributing to the observed changes in Ca(2+) handling and contractility, the effects of phloretin on the cardiac action potential and plasma membrane Ca(2+) and K(+) currents were examined. Phloretin markedly increased the action potential duration in the myocytes, and it inhibited the Ca(2+)-independent transient outward K(+) current (I(to)). The inwardly rectifying K(+) current, the sustained outward delayed rectifier K(+) current, and L-type Ca(2+) currents were not significantly different in the presence and absence of phloretin, nor was there any evidence that the Na(+)/Ca(2+) exchanger was affected. The effects of phloretin on Ca(2+) handling in the myocytes are consistent with its effects on I(to). Phloridzin did not significantly alter the amplitude or area of electrically evoked Ca(2+) transients in the myocytes, nor did it have detectable effects on the sarcoplasmic reticulum Ca(2+) load, cell shortening, or the action potential.

Effect of phloretin and phloridzin on properties of digestion and absorption in the rat small intestine

Experiments in vitro on everted sacs of rat small intestine have shown that phloretin (an inhibitor of basolatheral glucose GLUT2 transporter) added from mucosal side of the sacs decreases release of glucose from enterocytes into serosal fluid without changing glucose accumulation in tissue of the preparations. Addition of phloridzin (an inhibitor of Na+ and glucose co-transporter SGLT1) from mucosal side inhibited both glucose accumulation in the tissue and its release into serosal fluid. Unspecific effects of phloretin and phloridzin on activities of several digestive enzymes (in particular, alkaline phosphatase, amino peptidase, and glycyl-L-leucine dipeptidase) has been revealed in homogenates of the rat small intestine mucosa. In chronic experiments on rats, absorption of glycine from the isolated small intestinal loop was inhibited in the presence of phloretin in perfusate. The obtained results indicate that the experimental approach of inhibition of glucose absorption by phloretin used from mucosal side in vitro appears to give a significant overestimation of contribution of facilitated diffusion (with participation of the GLUT2 transporter inserted in the apical enterocyte membrane) to glucose transport across this membrane. Thus, the role of the GLUT2 transporter in the mechanism of glucose absorption in the small intestine under its physiological conditions does not seem to be as great as it is thought by the authors of the recently proposed hypothesis.

Stimulation of non-sodium-dependent water, electrolyte, and glucose transport in rat small intestine by gum arabic.

In experimental models of gastroenterological disease, the soluble fiber gum arabic (GA) acts as a proabsorptive adjuvant. This study investigated which specific transport pathway(s) are affected by GA. Rat jejunum was perfused under anesthesia with a standardized oral rehydration solution (ORS) containing D-glucose, with or without GA (2.5 g/liter). In some preparations either phloridizin, a competitive inhibitor of Na+-coupled D-glucose transport, or phloretin, an inhibitor of basolateral glucose transport, were added to the ORS, with or without GA. Diffusion and paracellular transport changes due to GA were evaluated with L-glucose and [14C]polyethlyene glycol 4000 (PEG). GA partially reversed water, Na+, and D-glucose absorption inhibition induced by phloridzin and normalized water and Na+ absorption in the presence of phloretin. GA also increased absorption of water, Na+, and PEG from an L-glucose ORS. The data suggest that GA does not act via Na+ dependent mechanism(s), but stimulates transcellular and/or transjunctional transport pathways; therefore GA may be useful to increase absorption of solutes transported by diffusion.

Interaction of Phloretin with Lipid Monolayers: Relationship between Structural Changes and Dipole Potential Change

Phloretin is known to adsorb to lipid surfaces and alters the dipole potential of lipid monolayers and bilayers. Its adsorption to biological and artificial membranes results in a change of the membrane permeability for a variety of charged and neutral compounds. In this respect phloretin represents a model substance to study the effect of dipole potentials on membrane permeability. In this investigation we studied the interaction of phloretin with monolayers formed of different lipids in the liquid-expanded and the condensed state. Phloretin integrated into the monolayers as a function of the aqueous concentration of its neutral form, indicated by an increase of the surface pressure in the presence of phloretin. Simultaneous recording of the surface potential of the monolayers allowed us to correlate the degree of phloretin integration and the phloretin-induced dipole potential change. Increasing the surface pressure decreased the phloretin-induced shift of the isotherms, but did not influence the phloretin-induced surface potential change. This means that phloretin adsorption to the lipid surface can occur without affecting the lipid packing. The surface potential effect of phloretin is accompanied by a change of the lipid dipole moment vector dependent on the lipid packing. This means that the relation between the surface potential change and the lipid packing cannot be described by a static model alone. Taking into account the deviations of the surface potential change versus molecular area isotherms of the experimental data to the theoretically predicted course, we propose a model that relates the area change to the dipole moment in a dynamic manner. By using this model the experimental data can be described much better than with a static model.

Inhibitory effect of phloridzin and phloretin on glucuronidation of p-nitrophenol, acetaminophen and 1-naphthol: kinetic demonstration of the influence of glucuronidation metabolism on intestinal absorption in rats

Intestinal glucuronidation and absorption of p-nitrophenol ( p-NP), acetaminophen (APAP) and 1-naphthol (α-NA) in the presence of phloridzin (inhibitor of Na +/glucose cotransporter) and phloretin (aglycone of phloridzin) were studied. Glucuronides of p-NP, APAP and α-NA appeared on both the serosal and mucosal sides. The amounts of glucuronides on the serosal side were decreased in the presence of phloridzin and phloretin. p-NP, APAP and α-NA appeared on the serosal side as well, and the amounts of p-NP, APAP and α-NA on the serosal side were increased by the presence of phloridzin and phloretin. Furthermore, the intestinal glucuronidation and absorption of α-NA at various concentrations were studied in the presence and absence of phloretin. Metabolic clearance was decreased in the presence of phloretin, and the absorption clearance was increased. The higher concentrations of α-NA caused higher absorption clearance. The lower the metabolic clearance, the higher the absorption clearance. The relationship between glucuronidation metabolism and absorption in intestine was kinetically analyzed by the metabolic inhibition model. Complete inhibition of glucuronidation improved the intestinal absorption of α-NA, and the absorption clearance increased to 7.17 μl/min/cm. The formation of phloretin and an unknown metabolite from phloridzin were observed. An unknown metabolite from phloretin was observed, and was suppressed by the presence of α-NA. This suggests that phloridzin was hydrolyzed to phloretin, which was metabolized to glucuronide, and thereby inhibited glucuronidation of p-NP, APAP and α-NA.

Intramembrane Molecular Dipoles Affect the Membrane Insertion and Folding of a Model Amphiphilic Peptide

The relationship between the dipole potential and the interaction of the mitochondrial amphipathic signal sequence known as p25 with model membranes has been studied using 1-(3-sulfonatopropyl)-4-[β[2-(di-n-octyl-amino)-6-naphthyl]vinyl] pyridinium betaine (di-8-ANEPPS) as a fluorescent probe. The dipole potential of phosphatidylcholine membranes was modified by incorporating into the bilayer the sterols phloretin and 6-ketocholestanol (KC), which decrease and increase the dipole potential, respectively. The results derived from the application of a dual-wavelength ratiometric fluorescence method for following the variation of the membrane dipole potential have shown that when p25 inserts into the lipidic bilayer, a decrease in the dipole potential takes place. The magnitude of this decrease depends on the initial value of the dipole potential, i.e., before interaction with the peptide. Thus, when KC was incorporated into the bilayer, the decrease caused by the membrane insertion of p25 was larger than that caused in PC membranes. Alternatively, in the presence of phloretin, the decrease in the potential caused by the peptide insertion was smaller. Complementary studies involving attenuated total reflectance-Fourier transform infrared spectroscopy of the peptide membrane interactions have shown that modification of the dipole potential affects the conformation of the peptide during the course of its interaction with the membrane. The presence of KC induces a higher amount of helicoidal structure. The presence of phloretin, however, does not appear to affect the secondary structure of the peptide. The differences observed in the dipole potential decreases caused by the presence of the peptide with the PC membranes and phloretin-PC membranes, therefore, must involve differences in the tertiary and, perhaps, quaternary conformations of p25.

The Adsorption of Phloretin to Lipid Monolayers and Bilayers Cannot Be Explained by Langmuir Adsorption Isotherms Alone

Phloretin and its analogs adsorb to the surfaces of lipid monolayers and bilayers and decrease the dipole potential. This reduces the conductance for anions and increases that for cations on artificial and biological membranes. The relationship between the change in the dipole potential and the aqueous concentration of phloretin has been explained previously by a Langmuir adsorption isotherm and a weak and therefore negligible contribution of the dipole-dipole interactions in the lipid surface. We demonstrate here that the Langmuir adsorption isotherm alone is not able to properly describe the effects of dipole molecule binding to lipid surfaces—we found significant deviations between experimental data and the fit with the Langmuir adsorption isotherm. We present here an alternative theoretical treatment that takes into account the strong interaction between membrane (monolayer) dipole field and the dipole moment of the adsorbed molecule. This treatment provides a much better fit of the experimental results derived from the measurements of surface potentials of lipid monolayers in the presence of phloretin. Similarly, the theory provides a much better fit of the phloretin-induced changes in the dipole potential of lipid bilayers, as assessed by the transport kinetics of the lipophilic ion dipicrylamine.

Uptake and metabolism of palmitate by isolated cardiac myocytes from adult rats: involvement of sarcolemmal proteins

The precise mechanism of uptake of long-chain fatty acids (FA) by cardiac myocytes is incompletely understood. We examined the involvement of sarcolemmal proteins in the initial uptake of FA by isolated rat cardiac myocytes, and the relation between initial uptake and metabolism. Cardiac myocytes were incubated in the presence of 90 microns [1-14C]palmitate complexed to 300 microns bovine serum albumin (BSA), presenting a physiologically relevant condition. During initial palmitate uptake (3 min), 56% of the intracellularly sequestered palmitate was esterified, and an additional 21% converted into oxidation intermediates. Varying the palmitate/BSA molar ratio revealed saturation kinetics with the apparent Km for cellular palmitate uptake (435 micro M) to be comparable to those for esterification (465 micro M) and oxidation (222 micro M). Varying the BSA concentration at a fixed palmitate/BSA molar ratio also showed saturation of uptake at increasing concentrations, with an apparent Km for BSA of 23 micro M. Changes in palmitate metabolism induced by changes in glucose utilization were accompanied by identical effects on palmitate uptake. Addition of lactate also inhibited both oxidation and uptake of palmitate, but had no effect on esterification. Virtually complete inhibition of palmitate oxidation by etomoxir inhibited palmitate uptake for 50%, while decreasing esterification by 33%. In the presence of phloretin and trypsin, palmitate uptake and metabolism were inhibited 76-88%, and in the presence of sulfo-N-succinimidyloleate by 53%. It is concluded that a) the bulk of sarcolemmal palmitate translocation occurs by membrane-associated FA-binding proteins, most likely assisted by albumin binding proteins without regulatory function, and b) palmitate uptake is most likely driven by its rapid intracellular metabolic conversion.

Evidence for a phloretin-sensitive glycerol transport mechanism in the perfused rat liver.

Glycerol, water, and urea transport were studied in the perfused rat liver. When hypertonic glycerol (100 mM) was infused into the perfused rat liver, rapid equilibration of glycerol between the extracellular and intracellular space occurred and only small effects of osmotic water fluxes on liver mass and extracellular ion concentrations were observed. However, when phloretin (0.2 mmol/l) was present, addition (or removal) of glycerol (100 mmol/l) induced within seconds a marked and transient decrease (or increase) of both liver mass and effluent Na+ and K+ concentrations, suggestive of transient osmotic water shifts out of and/or into the cells. These effects were observed with glycerol but not with infusion of 1,2-propanediol, 1,3-propanediol, or ethanediol (100 mmol/l). Calculation of permeability coefficients for hepatocytes in the perfused rat liver revealed that in the absence of a transmembrane glycerol concentration gradient the glycerol permeability was decreased from 0.146 +/- 0.005 (n = 14) to 0.055 +/- 0.016 microm/s (n = 4) in the presence of phloretin (200 microM). Water permeability was not affected by phloretin [0.146 +/- 0.005 under control conditions (n = 12) vs. 0.157 +/- 0.006 microm/s (n = 4) in the presence of phloretin]. Urea permeability was 0.131 +/- 0.008 microm/s (n = 5) and decreased to 0.061 +/- 0.003 microm/s in the presence ofphloretin. Glycerol and water permeability were inhibited in the presence of hypertonic glucose (200 mM) by 47 and 27%, respectively, but urea permeability was not affected by glucose.

The presence of water channel proteins in ram and human sperm membranes.

Ram and human spermatozoa have a high coefficient of osmotic water permeability (Pf) with a low activation energy (Ea), suggesting the presence of water channels within their plasma membranes. Sperm membranes were examined for the presence of two known water channel proteins, CHIP28 and glucose transporters belonging to the GLUT family of proteins. The water permeability of ram spermatozoa was not inhibited by mercuric chloride to which the CHIP28 channel is sensitive. The CHIP28 protein was not located in western blots of ram sperm membrane preparations that used an anti-CHIP28 antibody. The water permeability of ram and human spermatozoa was inhibited in the presence of phloretin, an inhibitor of glucose transport. Rabbit spermatozoa, which have a low Pf and a high Ea value, suggesting a non-porous membrane, were unaffected by phloretin. These results indicate that the erythrocyte and proximal tubule water channel, CHIP28, is not present in sperm membranes but that sperm membrane glucose transporters may have a secondary water channel function.