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S A Weinman

Publications and source records attributed to S A Weinman.

At least 19 recordsLinked to original sources
PubMed2000

Biophysical properties of ClC-3 differentiate it from swelling-activated chloride channels in Chinese hamster ovary-K1 cells.

ClC-3 is a highly conserved voltage-gated chloride channel, which together with ClC-4 and ClC-5 belongs to one subfamily of the larger group of ClC chloride channels. Whereas ClC-5 is localized intracellularly, ClC-3 has been reported to be a swelling-activated plasma membrane channel. However, recent studies have shown that native ClC-3 in hepatocytes is primarily intracellular. Therefore, we reexamined the properties of ClC-3 in a mammalian cell expression system and compared them with the properties of endogenous swelling-activated channels. Chinese hamster ovary (CHO)-K1 cells were transiently transfected with rat ClC-3. The resulting chloride currents were Cl(-) > I(-) selective, showed extreme outward rectification, and lacked inactivation at positive voltages. In addition, they were insensitive to the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and were not inhibited by phorbol esters or activated by osmotic swelling. These properties are identical to those of ClC-5 but differ from those previously attributed to ClC-3. In contrast, nontransfected CHO-K1 cells displayed an endogenous swelling-activated chloride current, which was weakly outward rectifying, inactivated at positive voltages, sensitive to NPPB and DIDS, and inhibited by phorbol esters. These properties are identical to those previously attributed to ClC-3. Therefore, we conclude that when expressed in CHO-K1 cells, ClC-3 is an extremely outward rectifying channel with similar properties to ClC-5 and is neither activated by cell swelling nor identical to the endogenous swelling-activated channel. These data suggest that ClC-3 cannot be responsible for the swelling-activated chloride channel under all circumstances.

Animals↗
PubMed2000

Expression and canalicular localization of two isoforms of the ClC-3 chloride channel from rat hepatocytes.

The molecular identities of functional chloride channels in hepatocytes are largely unknown. We examined the ClC-3 chloride channel in rat hepatocytes and found that mRNA for two different isoforms is present. A short form is identical to the previously reported sequence for rat ClC-3, and a long form contains a 176-bp insertion immediately upstream of the translation initiation site. This predicts a 58-amino acid NH(2) terminal insertion. Both long and short form mRNA was expressed in diverse tissues of the rat. Transient transfection of the long form in CHO-K1 cells resulted in currents with an I(-) > B(-) > Cl(-) selectivity sequence, outward rectification, and inactivation at positive voltages. Short form currents had identical ionic selectivity but displayed a more extreme outward rectification and showed no voltage-dependent inactivation. Immunofluorescence and immunoblots localized native ClC-3 preferentially but not exclusively to the canalicular membrane. We have therefore identified a new isoform of rat ClC-3 and shown that expression of both isoforms produces functional channels. In hepatocytes, ClC-3 is located in association with the canalicular membrane.

Animals↗
PubMed1998

Bile acid uptake via the human apical sodium-bile acid cotransporter is electrogenic.

Intestinal absorption of bile acids depends on a sodium-bile acid cotransport protein in the apical membrane of the ileal epithelial cell. Transport is Na+-dependent, but the Na+-bile acid stoichiometry and electrogenicity of transport are not known. Studies in whole intestine, isolated cells, and ileal membrane vesicles have been unable to resolve this issue because transport currents are small and can be obscured by other ionic conductances and transport proteins present in these membranes. In this study, the human apical sodium-bile acid transporter was expressed in stably transfected Chinese hamster ovary cells that lack other bile acid transporters. The Na+-dependent transport of a fluorescent bile acid analog, chenodeoxycholyl-Nepsilon-nitrobenzoxadiazol-lysine, was monitored by fluorescence microscopy in single, voltage-clamped cells. Bile acid movement was bidirectional and voltage-dependent with negative intracellular voltage-stimulating influx. A 3-fold reduction in extracellular Na+ produced a negative 52 mV shift of the flux-voltage relationship, consistent with a 2:1 Na+:bile acid coupling stoichiometry. No Na+- or voltage-dependent uptake was observed in nontransfected Chinese hamster ovary cells. These results indicate that the cotransport of bile acids and Na+ by human apical sodium-bile acid transporter is electrogenic and bidirectional and is best explained by a 2:1 Na+:bile acid coupling stoichiometry. These results suggest that membrane potential may regulate bile acid transport rates under physiological and pathophysiological conditions.

Animals↗
PubMed1998

Expression and regulation of leukotriene-synthesis enzymes in rat liver cells.

The liver plays a major role in metabolism and elimination of leukotrienes (LT). It produces cysteinyl leukotrienes (cLT), and cLT have been implicated in hepatocellular toxicity in several models of lipopolysaccharide (LPS)-associated liver injury. However, the liver cell types responsible for cLT production are poorly defined, and the expression of the LT-synthesis enzymes, 5-lipoxygenase (5-LO) and LTC4 synthase (LTC4-S), in liver cells has never been demonstrated. The aim of the present study was to examine the ability of rat liver cells to produce cLT by determining whether hepatocytes, Kupffer cells, and sinusoidal endothelial cells express mRNA and enzyme activities of the LT-synthesis enzymes and whether expression is altered by LPS. 5-LO mRNA was expressed in whole liver, and expression was enhanced by LPS. Cell fractionation studies demonstrated that expression was present in Kupffer cells and sinusoidal endothelial cells, but not in hepatocytes. LTC4-S mRNA was detected in whole liver, hepatocytes, and sinusoidal endothelial cells, but not in Kupffer cells. Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) showed that LPS increased LTC4-S expression in hepatocytes by a factor of 3 (n = 3; P < .03). LTC4-S enzyme activity in the microsomal fraction of hepatocytes was also increased from 0.52 +/- 0.13 to 1.90 +/- 0.66 nmol . mg protein-1 . 5 min-1 (n = 6; P < .015) after LPS treatment. These results indicate that hepatocytes do not possess the ability for de novo synthesis of cLT from arachidonic acid, but they may actively participate in cLT production by conjugation of LTA4 with glutathione to produce LTC4. LPS enhances LTC4-S expression in hepatocytes. This intrinsic cLT production may contribute to hepatocellular injury during inflammation.

Animals↗
PubMed1997

Leukotriene D4 activates a chloride conductance in hepatocytes from lipopolysaccharide-treated rats.

Endotoxin (LPS) can cause hepatocellular injury under several circumstances, and leukotrienes have been implicated as a contributing factor. Since ion channel activation has been associated with cytotoxicity, the aim of this study was to determine the circumstances under which LPS and/or leukotrienes activate ionic conductances in hepatocytes. LPS treatment of rats increased Cl- conductance in hepatocytes from 232+/-42 to 1236+/-134 pS/pF. Voltage dependence and inhibitor specificity of this conductance were similar to that of a swelling-activated Cl- conductance, and internal dialysis with nucleoside analogues suggested control by an inhibitory G protein. The lipoxygenase inhibitor nordihydroguaiaretic acid, the specific leukotriene D4 (LTD4) receptor antagonist MK-571, and the 5-lipoxygenase activating protein inhibitor MK-886 all significantly inhibited the conductance. Intracellular dialysis with LTD4 (1.5 microM) elevated intracellular Ca2+ from 143+/-6.5 to 388+/-114 nM within 6 min and stimulated an outwardly rectifying conductance from 642+/-159 to 1669+/-224 pS/pF (n = 9, P < 0.001). In hepatocytes prepared from untreated rats, this concentration of intracellular LTD4 neither raised intracellular Ca2+ nor activated the conductance. The LTD4 response could be induced in normal hepatocytes by culture with either conditioned medium from LPS-treated macrophages or purified TNF-alpha. In conclusion, intracellular LTD4 activates a chloride conductance in hepatocytes isolated from rats treated with LPS or primed in vitro with TNF-alpha. Changes in the hepatocellular accumulation of leukotrienes therefore mediate channel activation and may contribute to liver injury during sepsis and other inflammatory conditions.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Photosensitization of experimental hepatocellular carcinoma with protoporphyrin synthesized from administered delta-aminolevulinic acid: studies with cultured cells and implanted tumors.

BACKGROUND/AIMS: Photodynamic therapy using porphyrins or related compounds and laser light is an investigational treatment for neoplasms. The aim of this study was to establish whether this might be applicable for hepatocellular carcinoma using protoporphyrin synthesized in the tissue from administered delta-aminolevulinic acid. METHODS: We measured porphyrin accumulation in normal rat hepatocytes and Morris hepatoma cells in culture, and in subcutaneously implanted hepatomas and other tissues of the rat after administration of delta-aminolevulinic acid, and assessed cell and tissue damage after application of laser light. RESULTS: Porphyrin accumulation after delta-aminolevulinic acid was added to the medium was greater and continued to increase for a longer period of time in hepatoma cells than in hepatocytes (1337+/-42 vs 513+/-31 fluorescence units/cell at 8 h, means+/-SE, p<0.001). After intraperitoneal injection of delta-aminolevulinic acid to rats with subcutaneously growing hepatomas, porphyrin content in tumor and liver was similar at 4 h but was higher in tumor at 6 h. Laser light caused necrosis of normal and malignant liver cells in culture and subcutaneous hepatomas in vivo. CONCLUSIONS: We conclude from these in vitro and in vivo studies that porphyrin accumulation after administration of delta-aminolevulinic acid in this hepatoma is substantial and time dependent, and delivery of laser light locally can cause tumor photosensitization and necrosis.

Aminolevulinic Acid↗
PubMed1996

cAMP- and swelling-activated chloride conductance in rat hepatocytes.

An outwardly rectifying Cl- conductance was identified in primary isolated rat hepatocytes, and the whole cell patch-clamp technique was used to characterize its properties and mechanisms of activation. With symmetrical Cl(-)-containing solutions on both sides and adenosine 3',5'-cyclic monophosphate (cAMP; 100 microM) in the pipette solution, a large outwardly rectifying conductance (1,014 +/- 153 pS/pF, n = 20) developed in all cells within 3 min. This cAMP-activated conductance was highly anion selective and slowly inactivated at voltages > 80 mV. It was completely inhibited by the anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (200 microM, n = 6) and partially inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (150 microM, n = 7). It displayed a halide selectivity of I- > Br- > Cl-. In the absence of cAMP, a functionally similar conductance was activated by cell swelling. Reduction of bath osmolality from 300 to 250 mosmol/kg increased membrane conductance from 64 +/- 16.4 to 487 +/- 23 pS/pF (n = 4). This swelling-activated conductance was also highly anion selective and had identical halide selectivity and blocker sensitivity as the cAMP-activated conductance. Although cell swelling was not necessary for cAMP activation, cell shrinkage with hyperosmotic bath (350 mosmol/kg), either before or after exposure to cAMP, inhibited the cAMP-activated conductance. By the determination of conductance as a function of bath osmolality in the presence and absence of cAMP, it was observed that cAMP shifted the osmotic set point for conductance activation without changing either the maximum or minimum conductance. In conclusion, both cAMP and cell swelling activate a large outwardly rectifying Cl- conductance in rat hepatocytes. Its ionic selectivity and sensitivity to channel blockers are identical to those seen for swelling-activated Cl- conductances in many cell types. The conductive properties are not those of cystic fibrosis transmembrane conductance regulator-mediated Cl- conductance. cAMP appears to activate this conductance by altering the volume set point of a swelling-activated channel.

Animals↗
PubMed1996

cAMP stimulates fluorescent bile acid uptake into hepatocytes by membrane hyperpolarization.

Elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) hyperpolarizes hepatocytes and increases the uptake rate of bile acids. The purpose of this study was to determine to what extent these two phenomena are linked. Fluorescent bile acid analogues (FBA) were used to probe bile acid transport into whole cell patch-clamped hepatocytes. Na(+)-dependent uptake of cholyl-nitrobenz-2-oxa-1,3-diazol-4-yl-lysine (C-NBD-L), an FBA with a net charge of -1, was shown to be electrogenic, whereas uptake of cholylglycylamidofluorescein (CGamF), an FBA with a net charge of -2, was neutral. Incubation of hepatocytes with 8-bromo-cAMP (8-BrcAMP; 100 microM) increased the uptake rate of the electrogenically transported FBA by 25% (P = 0.002), but had no effect on the uptake rate of the electroneutrally transported FBA. Microelectrode impalements revealed that 8-BrcAMP or forskolin hyperpolarized hepatocytes by 6-8 mV. To determine if hyperpolarization is responsible for the cAMP-induced increase in uptake rate, cAMP was directly introduced into hepatocytes during whole cell patch clamp under voltage-clamp conditions. As long as voltage clamp was maintained at -30 mV there was no stimulation of C-NBD-L uptake. However, when voltage clamp was terminated by either pipette removal or current clamp, cAMP increased the uptake rate by 25-34% (P < 0.002). In both of these protocols, cAMP had no effect on uptake of the electroneutrally transported FBA, CGamF. Finally, in voltage-clamped hepatocytes in the absence of cAMP, a 10-mV hyperpolarization increased the uptake rate of C-NBD-L by 23%. We therefore conclude that short-term cAMP-induced stimulation of fluorescent bile acid uptake in hepatocytes is a direct consequence of membrane hyperpolarization.

Animals↗
PubMed1995

Automobile air bag-mediated injury: a case presentation.

Because of the growing number of automobiles equipped with air bags, it is virtually certain that patients will seek treatment in the emergency department for air bag-mediated injury. The emergency nurse must be aware of air bag-mediated injury patterns and prepared for triage and management of any of the potential concurrent injuries caused by air-bag deployment.

Accidents, Traffic↗
PubMed1995

Transport characteristics of three fluorescent conjugated bile acid analogs in isolated rat hepatocytes and couplets.

The transport properties of three different synthetically prepared fluorescent conjugated bile acid analogs (FBA), all with the fluorophore on the side chain, were determined using isolated rat hepatocytes and hepatocyte couplets. The compounds studied were cholylglycylamidofluorescein (CGamF), cholyl(N epsilon-nitrobenzoxadiazolyl [NBD])-lysine (C-NBD-L), and chenodeoxycholyl-(N epsilon-NBD)-lysine (CDC-NBD-L). When hepatocytes were incubated at 37 degrees C with 0.3 mumol/L of FBA and 0.15 mol/L of Na+, cell fluorescence increased linearly with time at a rate (U/min) of 7.8 +/- 0.5 for CGamF, 7.2 +/- 0.3 for C-NBD-L, and 13.7 +/- 1.0 for CDC-NBD-L (mean, +/- SE; n = 40 to 90). Uptake was concentration dependent for concentrations less than 20 mumol/L and was saturable. The Michaelis constant (Km) value (mumol/L) for CGamF was 10.8, for C-NBD-L was 3.8, and for CDC-NBD-L was 3.0. In the absence of Na+, the uptake rate was decreased by 50% for CGamF and by 38% for C-NBD-L; but uptake of CDC-NBD-L was unchanged and thus Na+ independent. Cellular uptake of all three derivatives was specific to hepatocytes and was absent in several nonhepatocyte cell lines. For CGamF and C-NBD-L, both Na(+)-dependent and Na(+)-independent uptake was inhibited by 200-fold excess concentrations of cholyltaurine, dehydrocholyltaurine, and cholate, but for CDC-NBD-L, these nonfluorescent bile acids did not inhibit initial uptake. The intracellular fluorescence of CGamF was strongly pH dependent at an excitation wavelength of 495 nm, but pH independent at 440 nm excitation. In contrast, intracellular fluorescence of C-NBD-L and CDC-NBD-L was pH independent. All three FBA were secreted into the canalicular space of approximately 50% to 60% of couplets. Cellular adenosine triphosphate (ATP) depletion with either CN- or atractyloside inhibited secretion of all three FBA. The multispecific organic anion transporter (MOAT) inhibitor, chlorodinitrobenzene, blocked secretion of fluorescent MOAT substrates at a concentration of 1 mumol/L. At this concentration it did not affect secretion of the three FBA. At higher concentrations, chlorodinitrobenzene partially inhibited the canalicular secretion of CGamF but not the other two FBA. In conclusion, all three FBA are secreted by canalicular membrane bile acid transporters, but the sinusoidal uptake characteristics of CGamF and C-NBD-L are more similar than those of CDC-NBD-L to the transport properties of cholyltaurine. Therefore, C-NBD-L appears to be the best of the three for studies of conjugated trihydroxy-bile acid transport in hepatocytes.

Animals↗
PubMed1994

Maintenance of cellular acidification in cyanide-treated hepatocytes results from inhibition of Na+/H+ exchange.

Inhibition of respiration by metabolic inhibitors or hypoxia is accompanied by intracellular acidification. Although this acidification is known to promote cell survival during hypoxia, little is known about its mechanism. Given that the Na+/H+ exchanger is known to be a major component of pH regulation in normal hepatocytes, the aim of this study was to determine the effects of inhibition of mitochondrial respiration on intracellular pH (pHi) regulation and Na+/H+ exchange. Cyanide (CN-; 5 mM) plus fructose (20 mM) were used as a model of hypoxic acidosis. pHi was measured with quantitative fluorescence microscopy of cells loaded with the pH indicator, 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein. In control cells, pHi was 7.09 +/- 0.01 SE (n = 106). After 60 min in CN(-)-fructose, pHi fell to 6.74 +/- 0.01 (n = 129, P < 0.001). The pHi recovery rate (expressed as mmol H+.l-1.min-1) was determined under both conditions after acid loading by transient exposure and removal of 20 mM NH4Cl. Control and CN(-)-treated cells recovered at 3.59 +/- 0.25 (n = 42) and 0.69 +/- 0.09 (n = 38, P < 0.001), respectively. Amiloride treatment (1 mM) in the absence of CN- reduced pHi recovery similarly to that caused by CN- (0.34 +/- 0.07, n = 14). CN(-)-treated cells exposed to amiloride demonstrated no additional inhibition (efflux rate 0.65 +/- 0.11, n = 27), suggesting that the inhibition is directed at Na+/H+ exchange. Twenty minutes after CN- removal, CN(-)-treated cells regained their ability to recover from an acid load, thus demonstrating the reversibility of this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium Chloride↗
PubMed1994

Free concentrations of intracellular fluorescent anions determined by cytoplasmic dialysis of isolated hepatocytes.

Intracellular organic ions exist in free solution bound to cytoplasmic proteins, partitioned within intracellular membranes, and enclosed in intracellular vesicles and organelles. The aim of this study was to develop a method for measurement of the free cytosolic concentration of organic ions. This was accomplished by measuring initial rates of diffusion between patch-clamp pipettes and cell cytoplasm and determining the null-point concentration of this process. Carboxydimethylfluorescein (CF) was used as a model compound. It readily diffused between cytoplasm and pipette, and there was a linear relationship between concentration in the pipette and equilibrium cell fluorescence. When cells previously loaded with CF were patched, intracellular fluorescence rapidly changed in a positive or a negative direction, depending on the concentration of CF in the pipette. The null point, defined as the concentration at which cells neither gained nor lost fluorescence, described the same relationship between free concentration and total cell fluorescence as that determined by direct loading of the cells to equilibrium. In hepatocytes preloaded with a fluorescent bile acid derivative, cholylglycylamidofluorescein (CGamF), by exposure (0.05 microM) for 30 min, the null point occurred at a CGamF concentration in the pipette of 0.6 microM. This value is 12 times greater than that in the bath. In conclusion, a new method is described that can measure free cytosolic concentrations of fluorescent molecules. It should prove useful in determining the intracellular location and state of transported organic ions.

Animals↗