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PubMed2004

Effects of trans- and n-3 unsaturated fatty acids on cardiovascular risk markers in healthy males. An 8 weeks dietary intervention study.

BACKGROUND: Studies of long-term intake of industrially produced trans fatty acids (TFA) and n-3 polyunsaturated fatty acids (PUFA) suggest opposite effects on cardiovascular disease risk. Common mechanisms of action are probable. OBJECTIVE: To examine the effects on cardiovascular risk markers of dietary enrichment with TFA or n-3 PUFA. DESIGN: Randomized, double-blind, parallel intervention trial. SETTING: Department of Human Nutrition, The Royal Veterinary and Agricultural University. SUBJECTS: In all, 87 healthy males included, 79 completed. INTERVENTION: Subjects were randomly assigned to 8 weeks of a daily intake of 33 g of experimental fats from either partially hydrogenated soy oil containing 20 g of TFA, 12 g of fish oil with approximately 4 g of n-3 PUFA and 21 g of control fat, or 33 g of control fat. The experimental fats were incorporated into bakery products. Plasma lipids, blood pressure, heart rate variability (HRV), arterial dilatory capacity, compliance, and distensibility were recorded before and after intervention and at follow-up 12 weeks after the intervention. RESULTS: High-density lipoprotein cholesterol (HDL-C) decreased in the TFA group and triglycerides and mean arterial blood pressure decreased in the n-3 PUFA group compared to the control group. HRV, arterial dilatory capacity, compliance, and distensibility were unchanged. CONCLUSION: The results indicate that the association between coronary heart disease risk and intake of TFA and n-3 PUFA relates only modestly to changes in traditional risk markers. SPONSORSHIP: Danish Medical Research Council (Grant no. 22-01-0390), Center of Advanced Food Research (Copenhagen, Denmark) (Grant no. KVL-R-2001-107), the Danish Heart Association (Grant no. 99-2-3-45-22748), Novozymes (Bagsvaerd, Denmark), Aarhus Olie (Aarhus, Denmark), and from private sources. The experimental fats were provided by Pronova Biocare (Aalesund, Norway) and Aarhus Olie (Aarhus, Denmark).

Adult↗
PubMed1995

Milk fat depression, the glucogenic theory, and trans-C18:1 fatty acids.

Metabolic and endocrinological characteristics were compared for cows that differed in the extent of milk fat depression. Forty-one multiparous Holstein cows were fed control (40% concentrate and 60% forage) and high concentrate (80% concentrate and 20% forage) diets in a doubale-reversal design. Cows showing one or more percentage units of depression in milk fat were arbitrarily classified as responders (n = 26); those remaining were classified as nonresponders (n = 15). Compared with nonresponders, responders had greater increases in DMI, estimated NEL intake and balance, BW, milk yield, protein and lactose yields in milk, weight percentage of trans-C18:1 fatty acids in milk, and concentrations of triiodothyronine and thyroxine in serum when switched from the control diet to the 80% concentrate diet. Lack of an increase in concentrations of glucose and insulin in serum of cows with the greatest decline in percentage of milk fat casts doubt on the ability of the glucogenic theory to explain milk fat depression completely.

Animal Feed↗
PubMed2006

Effects of roasting on hazelnut lipids.

The effect of roasting on some nutritional characteristics of hazelnut lipidic fraction was investigated. Hazelnuts (Corylus avellana L.) were submitted to several different thermal treatments, comprising different temperatures (125-200 degrees C) and times of exposure (5, 15, and 30 min) and analyzed for their moisture and crude fat. Raw and roasted hazelnuts were also analyzed for their compositions in phytosterols and fatty acids (including trans isomers) by GC-FID, triacylglycerols by HPLC-ELSD, and tocopherols and tocotrienols by HPLC-DAD/fluorescence spectroscopy. Minor changes occurred in the fatty acid and triacylglycerol compositions. As temperatures and roasting periods increased, generally, a modest increase of oleic and saturated fatty acids and a decrease of linoleic acid, expressed as relative percentages, occurred. Similarly, an increase of triacylglycerols containing oleic acid moieties and a decrease of those containing linoleic acid moieties were found in the roasted samples. Roasting caused a modest decrease of the beneficial phytosterols (maximum 14.4%) and vitamin E homologues (maximum 10.0%) and a negligible increase of the trans fatty acids.

Corylus↗
PubMed1998

Desaturation and chain elongation of [1-14C]mono-trans isomers of linoleic and alpha-linolenic acids in perfused rat liver.

Trans polyunsaturated fatty acids are produced during heat treatment of oils, such as deodorization and frying. The detailed metabolic pathways of these trans isomers are not fully understood. In the present work, the desaturation and chain elongation of [1-14C]linoleic acid, 9cis,12trans -18:2, 9trans,12cis -18:2, alpha-linolenic acid, 9cis, 12cis,15trans -18:3 and 9trans,12cis, 15cis -18:3 were studied using a perfused rat liver model. After perfusion with both trans isomers of 18:2n-6, the 14C was equally distributed between phospholipids and triacylglycerols, compared to the 70:30 distribution (phospholipids:triacylglycerols) observed after infusing linoleic acid. The corresponding distribution of 14C after perfusion with both trans isomers of 18:3n-3 was comparable to what was obtained for alpha-linolenic acid. The products of conversion were analyzed by combination of different radio chromatographic methods. 9cis,12trans -18:2 was 16 times more converted into a C18:3n-6 fatty acid than linoleic acid into gamma-linolenic acid. Trans -18:2 isomers were more elongated into "dead-end products" when compared to the conversion of linoleic acid into 20:2n-6 (from 2- to 5-times more). 9cis,12cis,15trans -18:3 and 9trans,12cis, 15cis-18:3 were 2- and 10-times less converted to trans -20:5, respectively, than alpha-linolenic acid into eicosapentaenoic acid. Moreover, 9cis,12cis,15trans -18:3 and 9trans, 12cis, 15cis -18:3 were equally and 2.5-times more elongated into "dead-end products", respectively, than alpha-linolenic acid into 20:3n-3. The partitioning of the conversion between formation of desaturated and chain elongated products on the one hand and production of "dead-end products" on the other was also calculated. Compared to their cis analogs, 9trans,12cis -18:2 and 9trans,12cis, 15cis -18:3 were elongated into trans "dead-end products" rather than being converted to desaturated and chain elongated trans-metabolites. On the other hand 9cis,12cis,15trans -18:3 was more desaturated and chain elongated into 17trans 20:5 rather than elongated into 17trans -20:3.

Animals↗
PubMed2002

Long-term intake of trans (n-3) polyunsaturated fatty acids reduces the b-wave amplitude of electroretinograms in rats.

Weanling rats were fed three diets differing in their concentrations of the cis- and trans-isomers of alpha-linolenic acid [18:3(n-3)] for 12 mo to study the long-term effects of these fatty acids on the electroretinogram (ERG). The diets contained 18:3(n-3) in its natural form at 2.0 g/100 g total fatty acids (C group), partially isomerized 18:3(n-3) [1.3 g/100 g cis 18:3(n-3) + 0.7 g/100 g trans 18:3(n-3); cT group] and the control level of cis 18:3(n-3) with trans 18:3(n-3) [2.0 g/100 g cis 18:3(n-3) + 0.7 g/100 g trans 18:3(n-3); CT group]. The ERG and the levels of trans-isomers of the polyunsaturated fatty acids (PUFA) of retinal and hepatic phospholipids were determined after 3, 6, 9 and 12 mo of feeding the experimental diets. Dietary trans alpha-linolenic acid altered the fatty acid composition of retinal and hepatic phospholipids by significantly increasing the Delta19trans-isomer of docosahexaenoic acid. Moreover, dietary trans-isomers of alpha-linolenic acid significantly decreased the b-wave amplitude of the ERG by 9 mo of feeding. We conclude that long-term intake of small amounts of trans-isomers of alpha-linolenic acid could disturb visual function. However, further studies are required to determine the mechanisms responsible for this phenomenon.

Animals↗
PubMed2005

The short-term effect of fatty acids on glucagon secretion is influenced by their chain length, spatial configuration, and degree of unsaturation: studies in vitro.

The influence of fatty acids on beta cell function has been well established whereas little is known about the role of fatty acids on alpha cell function. The aim of our study was to investigate the short-term effects of chain length, spatial configuration, and degree of unsaturation of fatty acids on glucagon secretion from isolated mouse islets and alpha tumor cell 1 clone 6 cells (alpha TC1-6 cells). Glucagon release was measured with different saturated and unsaturated fatty acids as well as cis and trans isomers of fatty acids at low and high glucose. Palmitate (0.1-0.5 mmol/L) immediately stimulated glucagon release in a dose-dependent manner from both isolated islets and alpha TC 1-6 cells. The longer chain length of saturated fatty acids, the higher glucagon responses were obtained. The average fold increase in glucagon to saturated fatty acids (0.3 mmol/L) compared to control was octanoate 1.5, laurate 2.0, myristate 2.9, palmitate 5.4, and stearate 6.2, respectively. Saturated fatty acids were more effective than unsaturated fatty acids in stimulating glucagon secretion. At an equimolar concentration, trans-fatty acids were more potent than their cis isomers. Fatty acids immediately stimulate glucagon secretion from isolated mouse islets pancreatic alpha cells. The chain length, spatial configuration, and degree of unsaturation of fatty acids influence the glucagonotropic effect.

Animals↗
PubMed1985

Biochemical studies on cell fusion. II. Control of fusion response by lipid alteration.

The preceding communication (Roos, D.S. and P.W. Choppin, 1985, J. Cell Biol. 101:1578-1590) described the lipid composition of a series of mouse fibroblast cell lines which vary in susceptibility to the fusogenic effects of polyethylene glycol (PEG). Two alterations in lipid content were found to be directly correlated with resistance to PEG-induced cell fusion: increases in fatty acyl chain saturation, and the elevation of neutral glycerides, including an unusual ether-linked compound. In this study, we have probed the association between lipid composition and cell fusion through the use of fatty acid supplements to the cellular growth medium, and show that the fusibility of cells can be controlled by altering their acyl chain composition. The parental Clone 1D cells contain moderately unsaturated fatty acids with a ratio of saturates to polyunsaturates (S/P) approximately 1 and fuse virtually to completion following a standard PEG treatment. By contrast, the lipids of a highly fusion-resistant mutant cell line, F40, are highly saturated (S/P approximately 4). When the S/P ratio of Clone 1D cells was increased to approximate that normally found in F40 cells by growth in the presence of high concentrations of saturated fatty acids, they became highly resistant to PEG. Reduction of the S/P ratio of F40 cells by growth in cis-polyunsaturated fatty acids rendered them susceptible to fusion. Cell lines F8, F16, etc., which are normally intermediate between Clone 1D and F40 in both lipid composition and fusion response, can be altered in either direction (towards either increased or decreased susceptibility to fusion) by the addition of appropriate fatty acids to the growth medium. Although trans-unsaturated fatty acids have phase-transition temperatures roughly similar to saturated compounds, and might therefore be expected to affect membrane fluidity in a similar manner, trans-unsaturated fatty acids exerted the same effect as cis-unsaturates on the control of PEG-induced cell fusion. This observation suggests that the control of cell fusion by alteration of fatty acid content is not due to changes in membrane fluidity, and thus that the fatty acids are involved in some other way in the modulation of cell fusion.

Animals↗
PubMed1989

Fat content and fatty acid composition of infant formulas.

Fat content and fatty acid composition of 25 commercial infant formulas sold in the Federal Republic of Germany and of 3 home-made milk formulas were analysed, using gravimetry of extracted lipids and high-resolution capillary gas-chromatography. Results were compared with the composition of human milk. Fat contents of all commercial formulas were similar to human milk values and met current recommendations, but 2 home-made preparations were at the upper and lower limits of the recommended range. Milk formulas tended to contain higher percentages of saturated and lower ones of cis-monounsaturated and trans-isomeric fatty acids than human milk. Linoleic acid (C18:2n-6) content was similar to human milk in most products but deviated clearly from recommended values in 2 home-made mixtures. Alpha-linolenic acid (C18:3n-3) values were often low in formulas, resulting a high n-6/n-3-ratios. In contrast to human milk, all formulas contained only minor amounts of the physiologically important long-chain polyunsaturated fatty acids with 20 and 22 carbon atoms. Some seasonal variation in the content of palmitic (C16:0), oleic (C18:1n-9), linoleic and trans-fatty acids was found when five arbitrarily selected adapted formulas were analysed repeatedly over one year. The composition of a home-made formula made from fresh cow's milk was markedly different in winter and in summer, when percentages of saturated and trans-fatty acids were higher and of linoleic acid were lower. We conclude that the composition of most commercial formulas is better suited to meet the lipid requirements of young infants than the home-made preparations investigated.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary Fats↗
PubMed2006

[Quality of shortenings available on the home market].

The aim of this work was to examine the quality of shortenings available on Polish market, produced home or imported. The quality of twelve 100% vegetable fats and lard was estimated. Both chemical (fatty acids composition, especially trans isomers content, acid value, peroxide value, anisidine value, Totox, iodine value and oxidative stability--Rancimat test) and physical (melting point, solid fat content--at temperatures from 5 to 50 degrees C) properties were measured. The fats were subject to sensoric examination. The parameters defining the freshness of examined fats and their shelf life for all examined samples were good and proved the good quality. Induction time (150 degrees C) for examined fats varied from 1,79 to 4,29h. Examined fats differed significantly in saturated fatty acids content (from 14,0 do 60,2%) and trans isomers (from 0,1 to 56,6%). Fats produced from palm oil are also present and there are fats with smaller trans fat acids content. Examined shortenings contained very small content of essential fatty acids (from 0,5 to 10,4), and they showed very different melting points (from 19,6 to 42,1 degrees C) and solid phase contents. In general the examined fats were of good sensoric value. Summing up the received results, it should be underlined that large disparity in the content of trans isomers in analysed samples was observed and definitely TFA content should be lowered.

Chromatography, Gas↗
PubMed1992

Margarines and coronary artery disease.

In a previous paper we predicted that health effects of dietary fats in humans would require half a century or more to be understood, instead of the decade or so predicted during 1956 by an Editorial in The Lancet. It would seem that our prediction may have been optimistic since it has now been reported that trans unsaturated fatty acids present in high concentrations in margarines promote hypercholesterolemia in humans. Consequently, there has been a call for the reclassification of dietary fats upon the basis of their hypercholesterolemic properties. Using the latter criterion, therefore, many margarine brands would be classified as coronary artery disease risk foods. The primary adverse metabolic action of trans unsaturated fatty acids is the competitive inhibition of delta-6-desaturase, the hepatic enzyme responsible for the initial metabolic desaturation of the essential fatty acids cis linoleic and cis alpha-linolenic acid. In addition to margarines, many other common foods such as deep-fried foods, many convenience foods and bakery products contain relatively high levels of trans fatty acids. Therefore, since it has become virtually impossible to avoid a consistent, daily dietary intake of trans fatty acids, it would appear that a precautionary, preventative supplementation of the diet with supplements containing the direct metabolic products of delta-6-desaturation of the essential fatty acids, would be prudent. Such supplements are readily available.

Coronary Disease↗
PubMed2005

Dietary trans octadecenoic acids upregulate the liver gene encoding peroxisome proliferator-activated receptor-alpha in transition dairy cows.

Effects of feeding calcium salts of conjugated linoleic acid (CLA) or trans octadecenoic acids (trans 18:1) on lipid metabolism and hepatic contents of mRNA encoding carnitine palmitoyltransferase 1 (CPT1), microsomal triglyceride transfer protein (MTP) and peroxisome proliferator-activated receptor alpha (PPARalpha) were examined in 15 early post-partum Holstein cows. Dietary treatments were initiated at approximately 4 weeks prior to expected calving dates and continued for 7 weeks post partum. Treatments prepartum consisted of 1) a basal diet (Control), 2) basal diet+150 g/d of CLA mix (CLA), or 3) basal diet+150 g/d of trans 18:1 mix (TRANS). Intakes of calcium salts of CLA and trans 18:1 mixes were adjusted to 225 g/d during the 7-week postpartum treatment period. Blood samples were collected at weeks 1, 2 and 4 post partum and plasma was harvested immediately for subsequent hormone and metabolite assays. Concentrations of insulin, insulin-like growth factor-I (IGF-I), and leptin in blood did not vary among cows fed the three diets. Plasma nonesterified fatty acid (NEFA) concentrations decreased between weeks 1 and 4 of lactation and were lower in cows fed the diet supplemented with trans 18:1 than in those fed a control diet at week 2 post partum. Periparturient fat supplementation had no detectable effects on CPT1 mRNA content in the liver. Steady-state concentration of MTP mRNA in the liver was greater in the TRANS treatment group than in the control group at week 1 postpartum. Feeding trans 18:1 supplements to transition dairy cows upregulated hepatic PPARalpha mRNA content during the first month of lactation. Under the present experimental conditions, dietary CLA had minimal effects on plasma and hepatic lipid metabolite concentrations in early lactation Holstein cows. Results indicate that dietary trans fatty acids may affect liver lipid metabolism in post-partum dairy cows through alterations in PPARalpha gene expression.

Administration, Oral↗
PubMed1993

Trans-monoenoic and polyunsaturated fatty acids in phospholipids of a Vibrio species of bacterium in relation to growth conditions.

A Vibrio species of bacterium known to contain the polyunsaturated fatty acid 20:5n-3 was grown in both freshwater and seawater media at 5 and 20 degrees C and examined for adaptive changes in lipid composition. Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), together with a smaller proportion of nonesterified fatty acids (NEFA), comprised almost all the lipid under all growth conditions examined. Temperature had a more pronounced effect than the salinity of the medium on lipid composition. The proportion of PE in total lipid was always higher at 5 than at 20 degrees C. Conversely, the proportion of NEFA was lower at 5 than 20 degrees C whereas that of PG was not altered. The levels of saturated fatty acids in total lipid, PE and PG were all decreased by growth at 5 degrees C. No differences were observed with respect to growth temperature in the levels of cis 16:1n-7, the principal monoenoic fatty acid in both PE and PG. Trans 16:1n-7 was found to comprise 12.8-15.2% of fatty acids in PE and PG of bacteria grown at 5 degrees C but only 4.4-8.5% of phospholipid fatty acids in bacteria cultured at 20 degrees C. Regardless of medium composition, a reduction in growth temperature from 20 to 5 degrees C also caused the proportions of 20:5n-3 to increase from around 0.8 to 4.4% in PE and from around 4 to 20% in PG. The simultaneous occurrence of trans 16:1n-7 and 20:5n-3 is unique to this Vibrio species of bacterium.(ABSTRACT TRUNCATED AT 250 WORDS)

Fatty Acids↗
PubMed2004

Changes in milk fat in response to dietary supplementation with calcium salts of trans-18:1 or conjugated linoleic fatty acids in lactating dairy cows.

Milk fat was investigated in lactating dairy cows fed diets supplemented with Ca salts of trans fatty acids (Ca-tFA) or Ca salts of conjugated linoleic acids (Ca-CLA). Forty-five Holstein cows (115 days in milk) were fed a control diet (51% forage; dry matter basis) supplemented with 400 g of EnerG II (Ca salts of palm oil fatty acids) for 2 wk; subsequently, 5 groups of 9 cows each were assigned for 4 wk to the control diet or diets containing 100 g of Ca-CLA or 100, 200, or 400 g of Ca-tFA in a randomized block design. Treatments had no effect on dry matter intake, milk production, protein, lactose, or somatic cell count. Milk fat percentage was reduced from 3.39% in controls to 3.30, 3.04, and 2.98%, respectively, by the Ca-tFA diets and to 2.54% by the Ca-CLA diet. Milk fat yield (1.24 kg/d in controls) was decreased by 60, 130, and 190 g/d with increasing dose of Ca-tFA and by 290 g/d with the Ca-CLA supplement. Consistent with increased endogenous synthesis of cis-9-containing CLA from precursors provided by the Ca-tFA diets, total CLA were similar in milk of cows fed Ca-CLA or Ca-tFA. Compared with controls, the Ca-CLA diet increased trans-10, cis-12-18:2 yield in milk, without altering levels of trans-18:1 isomers. In contrast, yields of most trans-18:1 isomers were elevated in milk of cows fed Ca-tFA diets, whereas yields of trans-10, cis-12-18:2 remained similar to control values. We conclude that milk fat depression can occur without an increase in trans-10, cis-12-18:2 in milk and that other components, perhaps the trans-10-18:1 isomer, may be involved.

Animal Feed↗
PubMed1983

Modulation of membrane transport by free fatty acids: inhibition of synaptosomal sodium-dependent amino acid uptake.

High-affinity, Na+-dependent synaptosomal amino acid uptake systems are strongly stimulated by proteins which are known to bind free fatty acids. The rate of uptake as well as the overall level of accumulation is increased by such proteins as bovine serum albumin, hepatic fatty acid binding protein, beta-lactoglobulin, and fetuin. Such a stimulation is not observed with proteins which do not bind fatty acids. The transport activity of synaptosomal preparations can be directly correlated with the free fatty acid content of the preparation. Thus, incubation with albumin reduces the free fatty acid content of synaptosomal preparations, suggesting that the stimulatory effects of the proteins are related to their removal of inhibitory fatty acids formed by hydrolysis of membrane lipids during incubation. Inhibition of amino acid uptake is seen with most cis-unsaturated long chain fatty acids while saturated and trans-unsaturated fatty acids have relatively little or no effect. Under conditions in which the ionophore gramicidin D causes an increase of 22Na flux into synaptosomes, oleic acid (50 microM) has no effect on the influx. These data are consistent with the hypothesis proposed earlier by us [Rhoads, D. E., Peterson, N. A., & Raghupathy, E. (1982) Biochemistry 21, 4782] that Na+-dependent amino acid transport carrier proteins reside in a relatively fluid lipid domain in the synaptosomal membrane and that the effects of cis-unsaturated fatty acids are mediated by interactions with such domains.

Amino Acids↗
PubMed1990

Arachidonic acid inhibits glycine transport in cultured glial cells.

The effects of arachidonic acid on glycine uptake, exchange and efflux in C6 glioma cells were investigated. Arachidonic acid produced a dose-dependent inhibition of high-affinity glycine uptake. This effect was not due to a simple detergent-like action on membranes, as the inhibition of glycine transport was most pronounced with cis-unsaturated long-chain fatty acids, whereas saturated and trans-unsaturated fatty acids had relatively little or no effect. Endogenous unsaturated non-esterified fatty acids may exert a similar inhibitory effect on the transport of glycine. The mechanism for this inhibitory effect has been examined in a plasma membrane vesicle preparation derived from C6 cells, which avoids metabolic or compartmentation interferences. The results suggest that part of the selective inhibition of glycine transport by arachidonic acid could be due to the effects of the arachidonic acid on the lipid domain surrounding the carrier.

Arachidonic Acid↗
PubMed1981

Effects of free fatty acids on the organization of cytoskeletal elements in lymphocytes.

Treatment of mouse lymphocytes with cis-unsaturated free fatty acids produced alterations in the immunofluorescence patterns of the cytoskeleton and contractile proteins. Saturated free fatty acids and trans-unsaturated free fatty acids had no effect. In untreated cells, the microtubular pattern exhibited radiation from an organizing center, resembling the spokes of an umbrella. The addition of linoleic acid produced a polarized submembranous aggregate. Under control conditions, staining for actin revealed a diffuse pattern over the entire cell, but the addition of linoleic acid caused the formation of a single large patch, or polarized submembranous aggregate. The pattern for alpha-actinin normally revealed intense perinuclear staining on a diffuse background. Linoleic acid caused the loss of this pattern and the formation of a polarized submembranous aggregate. Linoleic acid treatment also caused the pattern for myosin to change from diffuse to uniform submembranous patching around the periphery of the cell. For all of these proteins, calcium (8 mM), but not magnesium, partially reversed the effects of linoleic acid. Sodium azide had little effect on the normal distribution of actin, tubulin, and alpha-actinin; however, myosin staining revealed prominent patch formation. Colchicine treatment caused diffuse staining, some polarized submembranous aggregate formation of tubulin, and some patching of myosin, but not as extensively as did treatment with linoleic acid. Actin and alpha-actinin were unaffected. These results, in view of the previously shown facts that pretreatment of cells with linoleic acid followed by anti-immunoglobulin inhibits capping of surface immunoglobulin (Klausner, et al., Proc. Natl. Acad. Sci. U.S.A. 77:437-441, 1980) and that free fatty acids partition into the surface membrane (Klausner et al., J. Biol. Chem. 255:1286-1295, 1980), suggest that the perturbation of the plasma membrane with unsaturated free fatty acids alters the interaction of surface receptors with the cytoskeleton, which in turn affects cytoplasmic distribution of the proteins.

Actins↗
PubMed1998

Modulation of K+ channels by arachidonic acid in T84 cells. II. Activation of a Ca(2+)-independent K+ channel.

We used single-channel recording techniques to identify and characterize a large-conductance, Ca(2+)-independent K+ channel in the colonic secretory cell line T84. In symmetric potassium gluconate, this channel had a linear current-voltage relationship with a single-channel conductance of 161 pS. Channel open probability (Po) was increased at depolarizing potentials. Partial substitution of bath K+ with Na+ indicated a permeability ratio of K+ to Na+ of 25:1. Channel Po was reduced by extracellular Ba2+. Event-duration analysis suggested a linear kinetic model for channel gating having a single open state and three closed states: C3<-->C2<-->C1<-->O. Arachidonic acid (AA) increased the Po of the channel, with an apparent stimulatory constant (Ks) of 1.39 microM. Neither channel open time (O) nor the fast closed time (C1) was affected by AA. In contrast, AA dramatically reduced mean closed time by decreasing both C3 and C2. The cis-unsaturated fatty acid linoleate increased Po also, whereas the saturated fatty acid myristate and the trans-unsaturated fatty acid elaidate did not affect Po. This channel is activated also by negative pressure applied to the pipette during inside-out recording. Thus we determined the effect of the stretch-activated channel blockers amiloride and Gd3+ on the K+ channel after activation by AA. Amiloride (2 mM) on the extracellular side reduced single-channel amplitude in a voltage-dependent manner, whereas Gd3+ (100 microM) had no effect on channel activity. Activation of this K+ channel may be important during stimulation of Cl- secretion by agonists that use AA as a second messenger (e.g., vasoactive intestinal polypeptide, adenosine) or during the volume regulatory response to cell swelling.

Amiloride↗