Basolateral Membrane Vesicles Research Articles

Gender differences in expression of organic cation transporter OCT2 in rat kidney

The organic cation transporter (OCT) mediates translocation of various cationic molecules including drugs, toxins and endogenous substances. We examined gender differences in the expression of rat (r) OCT2 in the kidney. Slices and basolateral membrane vesicles of male rat kidney showed a higher transport activity for tetraethylammonium than those of female rat kidney. The expression levels of rOCT2 mRNA and protein in the kidney of males were much higher than those in females. There was no gender difference in mRNA expression of rOCT1 and rOCT3. These findings suggest that rOCT2 is responsible for the gender differences in renal basolateral membrane organic cation transport activity.

Polarized Expression of Different Monocarboxylate Transporters in Rat Medullary Thick Limbs of Henle

Extracellular lactic acid is a major fuel for the mammalian medullary thick ascending limb (MTAL), whereas under anoxic conditions, this nephron segment generates a large amount of lactic acid, which needs to be excreted. We therefore evaluated, at both the functional and molecular levels, the possible presence of monocarboxylate transporters in basolateral (BLMVs) and luminal (LMVs) membrane vesicles isolated from rat MTALs. Imposing an inward H(+) gradient induced the transient uphill accumulation of L-[(14)C]lactate in both types of vesicles. However, whereas the pH gradient-stimulated uptake of L-[(14)C]lactate in BLMVs was inhibited by anion transport blockers such as alpha-cyano-4-hydroxycinnamate, 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS), and furosemide, it was unaffected by these agents in LMVs, indicating the presence of a L-lactate/H(+) cotransporter in BLMVs, but not in LMVs. Under non-pH gradient conditions, however, the uptake of L-[(14)C]lactate in LMVs was transstimulated 100% by L-lactate, but by only 30% by D-lactate. Furthermore, this L-lactate self-exchange was markedly inhibited by alpha-cyano-4-hydroxycinnamate and DIDS and almost completely by 1 mM furosemide, findings consistent with the existence of a stereospecific carrier-mediated lactate transport system in LMVs. Using immunofluorescence confocal microscopy and immunoblotting, the monocarboxylate transporter (MCT)-2 isoform was shown to be specifically expressed on the basolateral domain of the rat MTAL, whereas the MCT1 isoform could not be detected in this nephron segment. This study thus demonstrates the presence of different monocarboxylate transporters in rat MTALs; the basolateral H(+)/L-lactate cotransporter (MCT2) and the luminal H(+)-independent organic anion exchanger are adapted to play distinct roles in the transport of monocarboxylates in MTALs.

Effects of sea water and stanniectomy on branchial Ca(2+) handling and drinking rate in eel (Anguilla anguilla L.).

We examined the effects of seawater adaptation and extirpation of the Stannius corpuscles on branchial Ca(2+) flows, gill plasma membrane Ca(2+) transporters and drinking rate of European eels, Anguilla anguilla. Transepithelial Ca(2+) inflow in the gills increased 2 weeks after transfer of the eels from fresh water to sea water and after stanniectomy. Neither of these treatments changed the membrane density or the affinity of the Ca(2+)-extrusion mechanisms (Ca(2+)-ATPase and Na(+)/Ca(2+)-exchanger) in the gill cells, as measured in basolateral plasma membrane vesicles. We conclude that the increase in the Ca(2+)-transporting capacity observed in the gills of fish exposed to the larger transepithelial Ca(2+) fluxes, resulting from exposure to sea water or stanniectomy, involves an increase in number and/or size of the Ca(2+)-transporting cells, but not in the membrane density of Ca(2+) transporters. Branchial Ca(2+) outflow was higher in stanniectomised than in sham-operated fish. Changes in electrochemical driving forces as well as plasma stanniocalcin or teleocalcin levels may be the basis for the observed differences. Stanniectomy enhanced drinking in stanniectomised eels. Drinking was not further affected by transfer to sea water. These observations suggest that the corpuscles of Stannius are involved in the water balance.

Age-Related Changes in Renal Function, Membrane Protein Metabolism, and Na,K-ATPase Activity and Abundance in Hypokalemic F344 × BNF1 Rats

Background: Potassium depletion is a common electrolyte abnormality in elderly humans, usually as a consequence of diuretic use or poor oral intake. Hypokalemia is associated with a number of changes in renal function and an increase in some renal membrane transporters; its growth-promoting effect in young animals is well known. With aging, the renal adaptation to a number of challenges is often diminished. We hypothesized that aging is related to decreases in renal function, renal membrane protein metabolism, as well as Na,K-ATPase protein abundance and activity in both control animals as well as in those with potassium depletion. Objective: We examined the effects of dietary-induced hypokalemia in true-aged nonobese rats (30 months old) on renal function, cortical brush border membrane (BBM) and basolateral membrane (BLM) protein metabolism, and Na,K-ATPase protein abundance and activity. We compared the results obtained to those seen in their 4-month-old counterparts similarly treated. Methods: Young (4-month-old) and senescent (30-month-old) male Fisher 344 × Brown-Norway F₁ rats (F344 × BNF₁) were fed either a normal or potassium-deficient diet for 7 days. At 24 h, the U-¹⁴C-leucine incorporation was measured for determination of protein metabolism in renal BBM and BLM. Cortical BLM vesicle and microdissected proximal convoluted tubule (PCT) Na,K-ATPase activities were determined along with Western blot analysis of the cortical BLM α₁ subunit of Na,K-ATPase. Metabolic and renal function parameters were also examined. Results: Hypokalemia caused hyperbicarbonatemia, hyperglycemia, and azotemia, but only in the senescent animals. The aged control rats had a higher basal level of urine volume, ammonium excretion, and fractional excretion of chloride. By contrast, aging in the F344 × BNF₁ rats was associated with a decrease in plasma aldosterone (by 35%) and phosphate (by 40%) levels as compared with their young controls. Hypokalemia resulted in a significant reduction of plasma aldosterone and a rise in muscle sodium concentration in both age groups; it significantly increased renal BBM and BLM protein concentrations in the young group, while these parameters remained unchanged in the senescent rats. The aged potassium-depleted animals showed a 14% decrease in BBM protein biosynthesis, but there were no changes in the young hypokalemic rats. Both potassium-depleted elderly and young rats had a significant reduction (by 33%) in BLM protein biosynthesis. Hypokalemia significantly increased the Na,K-ATPase activity in both cortical BLM vesicles and in microdissected PCT. The percentage increase in microdissected PCT segments (Na,K-ATPase activity) in elderly potassium-depleted animals was significantly less than that seen in hypokalemic young ones. Aging, per se, was associated with decreased basal microdissected PCT Na,K-ATPase activity in control animals. Hypokalemia had no effect on cortical BLM α₁ subunit Na,K-ATPase protein abundance in either age group. Conclusions: The present study provides the first evidence in nonobese aged rats as to the metabolic parameters, renal function, renal cortical membrane protein metabolism, and transporter Na,K-ATPase activity and abundance during potassium depletion. The aged nonobese F344 × BNF₁ rats responded differently from their young nonobese counterparts following potassium depletion. These differences may contribute substantially to the effects often encountered in elderly humans receiving diuretics or having a poor dietary potassium intake.

Volume regulatory responses of basolateral membrane vesicles from Necturus enterocytes: role of the cytoskeleton.

Previous studies from this laboratory have demonstrated that basolateral membrane vesicles isolated from Necturus maculosus small intestinal epithelial cells possess a K(+) channel that is inhibited by ATP. In the present studies, we demonstrate that these vesicles, which are essentially devoid of soluble cytoplasmic contaminants, exhibit volume regulatory responses that parallel those of intact epithelial cells. Thus, suspension of these vesicles in a solution that is hypotonic to the intravesicular solution increases channel activity whereas suspension in a solution that is hypertonic to the intravesicular solution decreases, and may abolish, channel activity. These volume regulatory responses appear to be mediated by the same K(ATP) channel and depend on an intact actin cytoskeletal network. The responses to both hypotonic and hypertonic challenge are abolished by cytochalasin D or by incubating the vesicles under conditions that are known to depolymerize actin. Phalloidin, which is known to stabilize actin filaments, partially prevents the action of cytochalasin D. Thus, the present results indicate that the K(ATP) channel activity of basolateral membrane vesicles from Necturus basolateral membranes respond to hypo- and hypertonic challenge monotonically around an isotonic "set point" and that these responses depend on an intact actin cytoskeleton.

ATP-Dependent Silver Transport across the Basolateral Membrane of Rainbow Trout Gills

Silver has been shown to be extremely toxic to freshwater teleosts, acting to inhibit Na+ uptake at the gills, due to the inactivation of branchial Na+/K+-ATPase activity. However, the gills are also a route by which silver may enter the fish. Therefore, this study focuses on the mechanism of transport of this nonessential metal across the basolateral membrane of the gill cell, using basolateral membrane vesicles (BLMV) prepared from the gills of freshwater rainbow trout. Uptake of silver by BLMV was via a carrier-mediated process, which was ATP-dependent, reached equilibium over time, and followed Michaelis–Menten kinetics, with maximal transport capacity (Vmax) of 14.3 ± 5.5 (SE) nmol mg membrane protein-1 min-1 and an affinity (Km) of 62.6 ± 43.7 μM, and was inhibited by 100 μM sodium orthovanadate (Na3VO4). The ionophore monensin (10 μM) released transported silver from the BLMV. Acylphosphate intermediates, of a 104 kDa size, were formed from the BLMV preparations in the presence of ATP plus Ag. These results demonstrate that there is a P-type ATPase present in the basolateral membrane of the gills of rainbow trout that can actively transport silver, a process which will remove this heavy metal from its site of toxic action, the gill.

Sulfate/bicarbonate antiport by lobster hepatopancreatic basolateral membrane vesicles

Sulfate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Sulfate transport can be coupled to proton or sodium symport or antiport processes involving a variety of anions. It had been previously observed in lobster hepatopancreas that sulfate could be secreted, however, the mechanisms for this potential secretory process had not been fully described. Therefore, the present study was done to delineate one of the potential processes for this transport. Purified basolateral membrane vesicles (BLMV) were prepared from lobster hepatopancreas by osmotic disruption and discontinuous sucrose gradient centrifugation. Transport of (35S) sulfate into BLMV was stimulated by an outwardly directed bicarbonate gradient compared with gluconate-loaded vesicles. An inside-positive membrane potential (valinomycin and K+) stimulated sulfate/bicarbonate exchange; whereas an inside-negative membrane potential was inhibitory. Sulfate/sulfate exchange was not affected by alterations of transmembrane potential difference. External protons stimulated sulfate/bicarbonate exchange although proton gradients had no effect on sulfate/bicarbonate exchange in the BLMV. The stilbenes, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), strongly inhibited sulfate/bicarbonate exchange. These results suggest that sulfate/bicarbonate exchange in hepatopancreatic BLMV occurred by an electrogenic carrier mechanism exhibiting a 1:1 flux ratio. The possible physiological role of this antiport process in sulfate transport across the crustacean hepatopancreas is discussed. J. Exp. Zool. 284:158–167, 1999. © 1999 Wiley-Liss, Inc.

Pathways for HCO-3 exit across the basolateral membrane in rat thick limbs.

We studied the pathways for HCO-3 transport in basolateral membrane vesicles (BLMV) purified from rat medullary thick ascending limbs (MTAL). An inward HCO-3 gradient in the presence of an inside-positive potential stimulated the rate of 22Na uptake minimally and did not induce a 22Na overshoot, arguing against the presence of electrogenic Na+-HCO-3 cotransport in these membranes. An inside-acid pH gradient stimulated to the same degree uptake of 86Rb+ (a K+ analog) with or without HCO-3. Conversely, applying an outward K+ gradient caused a modest intracellular pH (pHi) decrease of approximately 0.38 pH units/min, as monitored by quenching of carboxyfluorescein; its rate was unaffected by HCO-3, indicating the absence of appreciable K+-HCO-3 cotransport. On the other hand, imposing an inward Cl- gradient in the presence of HCO-3 caused a marked pHi decrease of approximately 1.68 pH units/min; its rate was inhibited by a stilbene derivative. Finally, we could not demonstrate the presence of a HCO-3/lactate exchanger in BLMV. In conclusion, the presence of significant Na+-, K+-, or lactate-linked HCO-3 transport could not be demonstrated. These and other data suggest that basolateral Cl-/HCO-3 exchange could be the major pathway for HCO-3 transport in the MTAL.

Molecular Mechanisms for the Hepatic Uptake of Magnetic Resonance Imaging Contrast Agents

The mechanisms were investigated for the hepatic transport of 4 different gadolinium complexes used as contrast agents for magnetic resonance imaging (MRI). In basolateral rat hepatocyte plasma membrane vesicles, Gd-DTPA uptake was indistinguishable from non-specific binding to vesicles; Gd-BOPTA and Gd-EOB-DTPA entered plasma membrane vesicles following a linear, concentration-dependent mechanism up to 1.5 mM of substrate. By contrast, Gd-B 20790 uptake followed a saturative kinetic with an apparent Kmof 92 ± 15 μM and a Vmaxof 143 ± 42 pmol/mg prot/15 sec, and it occurred into an osmotic-sensitive space. Sulfobromophthalein ant taurocholate, but not unconjugated bilirubin inhibited the uptake rate of Gd-B 20790 but not that of the other three compounds. Injection intoXenopus laevisoocytes of 5 ng of human OATP cRNA resulted, after 3 days, in a ≥2-fold stimulation (p < 0.001) of transport of Gd-B 20790 but not of Gd-BOPTA or Gd-EOB-DTPA. Collectively, these data indicate that the hepatic uptake of the MRI contrast agent Gd-B 20790 is a carrier-mediated mechanism operated by OATP while MRI compounds with other chemical structures enter the hepatocyte by other mechanisms.

ATP-dependent calcium uptake into basolateral vesicles from transporting epithelia of intermolt crayfish.

ATP-dependent Ca2+ uptake was determined into inside-out basolateral membrane vesicles (BLMV) from intermolt crayfish (Procambarus clarkii) Ca2+-transporting epithelia: gill, hepatopancreas (liver), and antennal gland (kidney). Extravesicular (EV) ATP (5 mM) increased 45Ca2+ uptake (free Ca2+ 5 microM) by fivefold but was abolished by pretreatment with either vanadate or the ionophore A-23187. Addition of A-23187 to Ca2+-loaded vesicles produced 70% efflux. The saturable carrier exhibited a Km for Ca2+ of 0.11-0.27 microM and maximal influx of 20-123 pmol. mg-1. min-1. The Km for ATP was 0.01-0.04 mM. The temperature coefficient ranged from 1.43 to 2.06. EGTA treatment of hepatopancreas and antennal gland vesicles decreased 45Ca2+ uptake by 50-90%; uptake was restorable by calmodulin. However, in gill, 45Ca2+ uptake was unaffected by EGTA treatment and calmodulin decreased uptake in both EGTA-treated and untreated vesicles. Addition of EV Na+ (5 mM) increased ATP-dependent Ca2+ uptake into hepatopancreas and antennal gland BLMV by 60%; in hepatopancreas BLMV, this increase was inhibitable by ouabain. However, ATP-dependent Ca2+ uptake in gill vesicles was Na+ independent. The relative role of each epithelium in whole animal Ca2+ homeostasis has been interpreted based on in vitro characteristics.

Effect of experimentally induced hypothyroidism on sulfate renal transport in rats.

Decreased serum sulfate concentrations are observed in hypothyroid patients. However, the mechanism involved in thyroid hormone-induced alterations of renal sulfate homeostasis is unknown. The objectives of this investigation were to determine the effect of 6-propyl-2-thiouracil (PTU)-induced hypothyroidism in rats on 1) the in vivo serum concentrations, renal clearance, and renal reabsorption of sulfate, 2) the in vitro renal transport in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, and 3) the cellular mechanism of the hypothyroid-induced alteration in sulfate renal transport. Serum sulfate concentrations, renal fractional reabsorption of sulfate, and creatinine clearance were decreased significantly in the hypothyroid group. The Vmax values for sodium-sulfate cotransport in BBM were significantly decreased in the kidney cortex from the hypothyroid animals (0.90 +/- 0.31 vs. 0.49 +/- 0.08 nmol. mg-1. 10 s-1, n = 5-6, P < 0.05) without changes in Km. There were no significant differences in Vmax and Km for sulfate/anion exchange transport in BLM. Sodium-dependent sulfate transporter (NaSi-1) mRNA and protein levels were significantly lower in the kidney cortex from hypothyroid rats. Hypothyroidism did not alter the membrane motional order (fluidity) in BBM and BLM, which indicates that the changes in the membrane fluidity do not represent the mechanism for the altered renal transport. These results demonstrate that PTU-induced hypothyroidism decreases sodium-sulfate cotransport by downregulation of the NaSi-1 gene.

Taurocholate induces preferential release of phosphatidylcholine from rat liver canalicular vesicles.

Biliary phospholipid secretion involves predominant segregation of canalicular phosphatidylcholine into bile. We tested the hypothesis that micellar concentrations of the major physiologic bile salt taurocholate can preferentially solubilize phosphatidylcholine from the canalicular rat liver plasma membrane. Subcellular fractions from rat liver and kidney were isolated with standardized procedures, incubated in vitro with taurocholate or 3-[(3-cholamidopropyl)dimethylammonio]-propane-1-sulphonate (CHAPS) and released phospholipids determined after centrifugation. After incubation of canalicular (cLPM) and basolateral (blLPM) rat liver plasma membrane vesicles with 6 and 8 mM taurocholate, the proportion of phosphatidylcholine released was about two-fold higher as compared with its relative contribution to the overall lipid composition of the membranes. Quantitatively, this taurocholate-induced preferential phosphatidylcholine release was about four-fold higher in cLPM (117 nmol) as compared with blLPM (28 nmol). Comparison of membranes from different organs showed that increased sphingomyelin content reduced taurocholate-induced phosphatidylcholine release. Furthermore, phosphatidylcholine release from cLPM did not fit an inverse exponential relationship between membrane sphingomyelin content and phosphatidylcholine release from different starting material, indicating that cLPM is especially prone to taurocholate-induced phosphatidylcholine release. In contrast, in rat liver microsomes and kidney brush border membranes, taurocholate released phospholipids in proportion of their membrane contents, indicating an unspecific membrane solubilizing effect only. Similarly, CHAPS had an unselective lipid solubilizing effects in cLPM and blLPM. These results support the concept that the very last step of canalicular phospholipid secretion is mediated in vivo by bile salt-induced vesiculation of phosphatidylcholine-enriched microdomains from the outer leaflet of cLPM.

Functional and molecular characterization of luminal and basolateral Cl-/HCO-3 exchangers of rat thick limbs.

Cl-/HCO-3 exchange was measured in luminal (LMV) and basolateral (BLMV) membrane vesicles purified from rat medullary thick ascending limb (MTAL). Cl-/HCO-3 exchange in BLMV and LMV was inhibited by DIDS, with respective IC50 values of 3.2 +/- 0.9 and 15.2 +/- 5.2 microM, whereas Cl- conductances were DIDS insensitive. At constant external pH, BLMV 36Cl-/HCO-3 and 36Cl-/Cl- exchanges exhibited a sigmoidal pattern of activation as internal pH (pHi) increased from 6.1 to 8.0, whereas LMV 36Cl-/Cl- exchange was unchanged between pHi 6.7 and 7.8. The 165-kDa AE2 polypeptide and approximately 115-kDa AE1-related polypeptide were present only in BLMV. In contrast, AE1-related polypeptides of approximately 90 and 95 kDa were present not only in BLMV but also (in variable abundance) in LMV. We conclude that rat MTAL BLMV and LMV express distinct anion exchange activities and distinct sets of AE polypeptides. AE2 (and perhaps AE1) in BLMV likely contribute to HCO-3 absorption. In contrast, LMV exchangers may contribute to NaCl absorption via parallel coupling with the luminal Na+/H+ antiporters and/or may provide negative feedback regulation of HCO-3 absorption.

Reconstitution of single potassium channels from bovine gall-bladder epithelium.

The study of molecular transport across gall-bladder epithelium may contribute to our understanding of the pathophysiology of gall-bladder disease. The aim of this study was to reconstitute and characterize single potassium ion channels in bovine gall-bladder epithelial mucosa - both apical and basolateral aspects. Standard subcellular fractionation techniques were used to form either apical or basolateral closed-membrane vesicles from the mucosal epithelium of fresh gall bladders from healthy young adult cattle. Vesicular ion channels were incorporated into voltage-clamped planar lipid bilayers under known ionic conditions and their conductances, reversal potentials, and voltages were characterized. Low-conductance voltage-insensitive apical membrane vesicle channels of at least four conductance levels were found (mean +/- SD): 12+/-4 pS, n = 10; 40+/-12 pS, n = 4; 273+/-31 pS, n = 3; and 151+/-24 pS, n = 5. Conductances of potassium ion channels in basolateral membrane vesicles were in the range 9 - 450 pS, and these channels included high-conductance calcium-activated potassium-ion channels 'K(Ca)' which were voltage- and calcium-dependent.

PH dependence of Cl/HCO 3 exchanger in the rat jejunal enterocyte

During bicarbonate absorption in rat jejunum, a Cl/HCO 3 exchanger mediates bicarbonate extrusion across the basolateral membrane of the enterocyte. Previous studies demonstrated that anion antiport exhibits a particular behaviour: its activity is positively affected by the presence of sodium, but the cation is not translocated by the carrier protein. In view of the particular features of the jejunal Cl/HCO 3 antiporter, first we performed a pharmacological characterisation of the transport protein using various Cl channels blockers. Then, since it is well known that anion exchangers play a substantial role in cell pH regulation, we investigated the possible involvement of jejunal basolateral Cl/HCO 3 antiporter in intracellular pH maintenance. The sensitivity of the exchanger to pH was investigated by measuring 36Cl uptake into basolateral membrane vesicles either varying simultaneously intra- and extravesicular pH, or presetting at 7.4 external pH and varying only the internal one. Experiments were performed both in the absence and in the presence of Na. In all the tested conditions, uptake peaked at pH of about 7.3–7.4 and then decreased, suggesting that the main function of Cl/HCO 3 exchanger is related to HCO 3 absorption rather than to intracellular pH control. Since pH-regulating mechanisms counteracting acidification are well known in the jejunal enterocyte, we investigated how it regulates pH after alkalinisation of the cytosol. We tested both basolateral and brush border membrane vesicles for the presence of a K/H exchanger, but we could not give evidence for its presence by means of 86Rb uptake experiments. In conclusion, the jejunal enterocyte seems to lack a mechanism counteracting cellular alkalinisation: the main purpose of pH homeostasis might be to hinder acidification of the cytosol due to influx of protons and production of acid by the metabolism.

Lack of renal secretion of carbenicillin in rats: poor affinity to the organic anion transporter at renal brush border membrane.

The renal secretion of carbenicillin (CBPC) was studied in rats. The results obtained in the in vivo study indicated very poor renal secretion of CBPC in rats, which was entirely different from those observed in humans and rabbits. In humans and rabbits, significant and stereoselective renal secretion of CBPC was observed in vivo. In order to verify the poor renal secretion of CBPC in rats, the transport characteristics of the organic anion transporters were studied in vitro using basolateral and brush border membrane vesicles. Transport of p-aminohippuric acid (PAH) into the basolateral membrane vesicles (BLMVs) was inhibited by CBPC, indicating that the organic anion transporter located at the BLM may have affinity to CBPC. In contrast, the transport of PAH into the brush border membrane vesicles (BBMVs) was not inhibited by CBPC, suggesting that the organic anion transporter located at the BBM may not have affinity to CBPC. Similar results were obtained for sulbenicillin (SBPC). Since CBPC and SBPC exist as di-anions at physiological pH, the organic anion transporter located at the rat renal BBM may not exhibit affinity to water-soluble di-anions, which in turn will result in poor renal secretion of these compounds.

Renal Transport Systems for Organic Anions and Cations in Cadmium-Exposed Rats

To evaluate the effect of cadmium intoxication on renal transport systems for organic anions and cations, transport ofp-aminohippurate (PAH) and tetraethylammonium (TEA) were studied in renal cortical plasma membrane vesicles isolated from cadmium-intoxicated rats. Cadmium intoxication was induced by daily injections of CdCl2(2 mg Cd/kg · day sc) for 2–3 weeks. Renal plasma membrane vesicles were prepared by Percoll gradient centrifugation and magnesium precipitation method. Vesicular uptake of substrate was determined by rapid filtration technique using Millipore filter. The cadmium treatment resulted in a marked attenuation of Na+-dependent, α-ketoglutarate (αKG)-driven PAH uptake in the basolateral membrane vesicle (BLMV), and this was due to a reduction inVmaxand notKm. The Na+-αKG symport activity of the BLMV was not affected by 2-week cadmium treatment, but it was significantly inhibited by 3-week cadmium treatment. On the other hand, the αKG-PAH antiport activity of the BLMV appeared to be markedly suppressed in 2-week as well as 3-week cadmium-treated animals. The cadmium treatment inhibited the proton gradient-dependent TEA transport in the brush-border membrane vesicle (BBMV), and this was associated with a reduction inVmaxwith no change inKm. These results indicate that cadmium exposures may impair the capacities for organic anion transport in the proximal tubular basolateral membrane and organic cation transport in the luminal membrane. The cadmium effect on organic anion transport is attributed mainly to an inhibition of dicarboxylate–organic anion antiport system.

Characteristics of a low affinity passive Ca2+ influx component in rat parotid gland basolateral plasma membrane vesicles.

We have previously reported the presence of two Ca2+ influx components with relatively high (KCa = 152 +/- 79 microM) and low (KCa = 2.4 +/- 0.9 mM) affinities for Ca2+ in internal Ca2+ pool-depleted rat parotid acinar cells [Chauthaiwale et al. (1996) Pfluegers Arch. 432: 105-111]. We have also reported the presence of a high affinity Ca2+ influx component with KCa = 279 +/- 43 microM in rat parotid gland basolateral plasma membrane vesicles (BLMV). [Lockwich, Kim & Ambudkar (1994) J. Membrane Biol. 141:289-296]. The present studies show that a low affinity Ca2+ influx component is also present in BLMV with KCa = 2.3 +/- 0.41 mM (Vmax = 16.36 +/- 4.11 nmoles of Ca2+/mg protein/min). Our data demonstrate that this low affinity component is similar to the low affinity Ca2+ influx component that is activated by internal Ca2+ store depletion in dispersed parotid gland acini by the following criteria: (i) similar KCa for calcium flux, (ii) similar IC50 for inhibition by Ni2+ and Zn2+; (iii) increase in KCa at high external K+, (iv) similar effects of external pH. The high affinity Ca2+ influx in cells is different from the low affinity Ca2+ influx component cells in its sensitivity to pH, KCl, Zn2+ and Ni2+. The low and high affinity Ca2+ influx components in BLMV can also be distinguished from each other based on the effects of Zn2+, Ni2+, KCl, and dicyclohexylcarbodiimide. In aggregate, these data demonstrate the presence of a low affinity passive Ca2+ influx pathway in BLMV which displays characteristics similar to the low affinity Ca2+ influx component detected in parotid acinar cells following internal Ca2+ store depletion.

Isolation, visualization, characterization, and osmotic reactivity of crayfish BLMV

Procedures were developed to isolate basolateral membrane vesicles (BLMV) from gill, hepatopancreas, and antennal gland of intermolt freshwater crayfish, Procambarus clarkii. Individual procedures involved a discontinuous sucrose gradient (gill), a 65% sucrose cushion (hepatopancreas), or differential centrifugation (antennal gland). BLMV were visualized, characterized (37 degrees C), and tested for osmotic reactivity with a view to using them for Ca2+ uptake studies. Mean diameters of BLMV were 159 nm (gill), 363 nm (hepatopancreas), and 226 nm (antennal gland). Enrichments of basolateral membranes and mitochondria in BLMV were, respectively, 18- and 1.7-fold for gill, 9- and 0.4-fold for hepatopancreas, and 10- and 1-fold for antennal gland. Apical contamination was negligible in BLMV. Percentages of resealing of vesicles as inside out, right side out, or leaky/sheets were 17:27:56% (gill), 14:26:60% (hepatopancreas), and 21:39:40% (antennal gland). Vesicles exhibited osmotic reactivity, as indicated by a linear relationship between vesicular 45Ca2+ uptake and osmolality. Nonspecific 45Ca2+ binding was 20% in gill, 39% in hepatopancreas, and 31% in antennal gland. Data were compared with published values for marine crustaceans.

Ontogeny of basolateral membrane sodium-hydrogen exchange (NHE) activity and mRNA expression of NHE-1 and NHE-4 in rat kidney and jejunum

Na +/H + exchange (NHE) activity varies with ontogenic state in rat intestinal basolateral membrane vesicles (BLMV). The current investigation sought to determine if these observations are due to differential expression of BLM NHE isoforms, NHE-1 and NHE-4. In rat kidney, BLMV sodium uptake levels were similar in 2, 3 and 6 week rats (13.28±0.68, 14.03±0.84, and 11.71±0.66 nmol Na +/mg protein/30 s, respectively), and lower in adults (5.53±0.24) ( n=4; p<0.001 between 2 week rats and adults, and between 3 week rats and adults; p<0.01 between 6 week rats and adults). In rat jejunum, BLMV uptake was highest in adults (13.07±0.86 nmol Na +/mg protein/30 s), and decreased in 6, 3, and 2 week rats (4.48±0.75, 2.94±0.68, and 1.59±0.58, respectively) ( n=4; p<0.001 between all groups and adults). Control immunoblot experiments with NHE-3 antiserum showed that BLMV preps were not contaminated with significant amounts of this brush-border membrane specific protein. Northern blots with isoform-specific probes showed highest renal NHE-1 hybridization intensities in 2 and 3 week rats (11.00±0.25 and 12.07±0.16 phosphorimage units, respectively), and lower intensities in 6 week and adult animals (4.30±0.95, and 4.40±1.40, respectively) ( n=4; p<0.01 between 2 week animals and 6 week and adult animals, and between 3 week animals and 6 week and adult animals). NHE-1 probes in the intestine showed no hybridization intensity differences between groups: 2 week-7.09±1.10, 3 week-5.39±0.56, 6 week-8.24±1.57, and adult-8.99±2.20 ( n=3). NHE-4 specific probes in the kidney showed hybridization intensity levels of 9.22±0.35 in 2 week animals, 12.12±1.26 in 3 week animals, 5.63±0.81 in 6 week animals, and 3.52±0.57 in adults ( n=4; p<0.05 between 2 week and adults; p<0.01 between 3 week and 6 week animals, and between 3 week and adults). No NHE-4 message was detected in rat jejunum by Northern blot analysis or by reverse transcriptase-PCR. These results suggest that ontogenic NHE activity at the jejunal BLM is not related to differential expression of NHE-1, while NHE activity at the renal BLM may in part be related to differential ontogenic expression of NHE-1 and NHE-4.