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1.
The Na+-independent uptake of l-alanine has been studied in trout red blood cells, isolated hepatocytes and peripheral blood lymphocytes. The present study shows the existence of two functionally different Na+-independent systems for short chain neutral amino acids in these cells. They are designated as asc systems because of their resemblance to systems described in other cell types. Besides their independence of sodium and a rough similarity in substrate preference, the most important property shared by the two carriers is a lack of trans-stimulation, allowing further differentiation from system L. One of them is an unusually stereospecific carrier present in red blood cells, the other is less restrictive and present in hepatocytes and peripheral blood lymphocytes. Extracellular acid pH increases the incorporation to red blood cells, while it slightly depresses the uptake in the other cells. From the data presented, it is not possible, at first, to classify these carriers as asc
1 or asc
2 systems. Moreover, the system present in red cells resembles that found in the nonerythroid cells, BSC-1, while there is no clear parallelism between the system found in hepatocytes/lymphocytes and any of those described previously.This work was supported in part by a grant from the DGICYT (PB91-0235) of the Spanish Government and by a grant from the CIRIT (AR91-21) of the Generalitat de Catalunya. M.A.G. is a recipient of a fellowship from the Generalitat de Catalunya. We would like to express our sincere thanks to Mrs. Rosa Marsol and Mr. Antonino Clemente (Medi Natural, Generalitat de Catalunya) for their help and logistical assistance and to Mr. Robin Rycroft for his editorial help. J.L. Albi, P. Canals and M.A. Gallardo contributed equally to this study. 相似文献
2.
Although both amiloride- and phloretin-sensitive Na+/Li+ exchange activities have been reported in mammalian red blood cells, it is still unclear whether or not the two are mediated
by the same pathway. Also, little is known about the relative contribution of these transport mechanisms to the entry of therapeutic
concentrations of Li+ (0.2–2 mm) into cells other than erythrocytes. Here, we describe characteristics of these transport systems in rat isolated hepatocytes
in suspension. Uptake of Li+ by hepatocytes, preloaded with Na+ and incubated in the presence of ouabain and bumetanide, comprised three components. (a) An amiloride-sensitive component, with apparent K
m
1.2 mm Li+, V
max
40 μmol · (kg dry wt · min)−1, showed increased activity at low intracellular pH. The relationship of this component to the concentration of intracellular
H+ was curvilinear suggesting a modifier role of [H+]
i
. This system persisted in Na+-depleted cells, although with apparent K
m
3.8 mm. (b) A phloretin-sensitive component, with K
m
1.2 mm, V
max
21 μmol · (kg · min)−1, was unaffected by pH but was inactive in Na+-depleted cells. Phloretin inhibited Li+ uptake and Na+ efflux in parallel. (c) A residual uptake increased linearly with the external Li+ concentration and represented an increasing proportion of the total uptake. The results strongly suggest that the amiloride-sensitive
and the phloretin-sensitive Li+ uptake in rat liver are mediated by two separate pathways which can be distinguished by their sensitivity to inhibitors and
intracellular [H+].
Received: 8 April 1999/Revised: 19 July 1999 相似文献
3.
The uptake of 3H-labeled choline by a suspension of isolated type II epithelial cells from rat lung has been studied in a Ringer medium.
Uptake was linear for 4 min at both 0.1 μm and 5.0 μm medium choline; at 5 μm, only 10% of the label was recovered in a lipid fraction. Further experiments were conducted at the low concentration (0.1
μm), permitting characterization of the properties of high-affinity systems. Three fractions of choline uptake were detected:
(i) a sodium-dependent system that was totally inhibited by hemicholinium-3 (HC-3); (ii) a sodium-independent uptake, when
Na+ was replaced by Li+, K+ or Mg2+, inhibited by HC-3; (iii) a residual portion persisting in the absence of Na+ and unaffected by HC-3. Choline uptake was sigmoidally related to the medium Na+ concentration. Kinetic properties of the uptake of 0.1 μm
3H-choline in the presence and absence of medium Na+ were examined in two ways. (a) Inhibition by increasing concentrations of unlabeled choline (0.5–100 μm) was consistent with the presence of two Michaelis-Menten-type systems in the presence of Na+; a Na+-dependent portion (a mean of 0.52 of the total) had a K
m
for choline of 1.5 μm while K
m
in the absence of Na+ (Li+ substituting) was 18.6 μm. (b) Inhibition by HC-3 (0.3–300 μm) gave Ki values of 1.7 μm and 5.0 μm HC-3 for the Na+-dependent and -independent fractions. The apparent K
m
of the Na+-dependent uptake is lower than that reported previously for lung-derived cells and is in the range of the K
m
values reported for high-affinity, Na+-dependent choline uptake by neuronal cells.
Received: 18 February 1997/Revised: 7 December 1997 相似文献
4.
J.I. Kourie 《The Journal of membrane biology》1999,167(1):73-83
The understanding of the role of cytoplasmic pH in modulating sarcoplasmic reticulum (SR) ion channels involved in Ca2+ regulation is important for the understanding of the function of normal and adversely affected muscles. The dependency of
the SR small chloride (SCl) channel from rabbit skeletal muscle on cytoplasmic pH (pH
cis
) and luminal pH (pH
trans
) was investigated using the lipid bilayer-vesicle fusion technique. Low pH
cis
6.75–4.28 modifies the operational mode of this multiconductance channel (conductance levels between 5 and 75 pS). At pH
cis
7.26–7.37 the channel mode is dominated by the conductance and kinetics of the main conductance state (65–75 pS) whereas
at low pH
cis
6.75–4.28 the channel mode is dominated by the conductance and kinetics of subconductance states (5–40 pS). Similarly, low
pH
trans
4.07, but not pH
trans
6.28, modified the activity of SCl channels. The effects of low pH
cis
are pronounced at 10−3 and 10−4
m [Ca2+]
cis
but are not apparent at 10−5
m [Ca2+]
cis
, where the subconductances of the channel are already prominent. Low pH
cis
-induced mode shift in the SCl channel activity is due to modification of the channel proteins that cause the uncoupling of
the subconductance states. The results in this study suggest that low pH
cis
can modify the functional properties of the skeletal SR ion channels and hence contribute, at least partly, to the malfunction
in the contraction-relaxation mechanism in skeletal muscle under low cytoplasmic pH levels.
Received: 20 May 1998/Revised: 24 September 1998 相似文献
5.
M. Torras-Llort R. Ferrer J.F. Soriano-García M. Moretó 《The Journal of membrane biology》1996,152(3):183-193
The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles
can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (K
mA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence
of Na+ (l-methionine inhibition constant KiA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (KmB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence
of Na+ (KiB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described
for systems b
0,+ and y+, respectively.
Received: 14 August 1995/Revised: 2 April 1996 相似文献
6.
L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral
membrane vesicles l-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective.
The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is
saturable with respect to external lactate with a K
m
of 39.2 ± 4.8 mm and a J
max of 8.9 ± 0.7 nmoles mg protein−1 sec−1. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains
a H+-lactate cotransporter, whereas in the apical membrane both H+-lactate and Na+-lactate cotransporters are present, even if they exhibit a low transport rate.
Received: 22 October 1996/Revised: 11 March 1997 相似文献
7.
O. Ortiz-Carranza M.E. Miller N.C. Adragna P.K. Lauf 《The Journal of membrane biology》1997,156(3):287-295
We examined the effects of pH, internal ionized Ca (Ca2+
i
), cellular ATP, external divalent cations and quinine on Cl-independent ouabain-resistant K+ efflux in volume-clamped sheep red blood cells (SRBCs) of normal high (HK) and low (LK) intracellular K+ phenotypes. In LK SRBCs the K+ efflux was higher at pH 9.0 (350%) than at pHs 7.4 and 6.5, and was inhibited by external divalent cations, quinine, and
cellular ATP depletion. The above findings suggest that the increased K+ efflux at alkaline pH is due to the opening of ion channels or specific transporters in the cell membrane. In addition, K+ efflux was activated (100%) when Ca2+
i
was increased (+A23187, +Ca2+
o
) into the μm range. However, in comparison to human red blood cells, the Ca2+
i
-induced increase in K+ efflux in LK SRBCs was fourfold smaller and insensitive to quinine and charybdotoxin. The Na+ efflux was also higher at pH 9.0 than at pH 7.4, and activated (about 40%) by increasing Ca2+
i
. In contrast, in HK SRBCs the K+ efflux at pH 9.0 was neither inhibited by quinine nor activated by Ca2+
i
. These studies suggest the presence in LK SRBCs, of at least two pathways for Cl−-independent K+ and Na+ transport, of which one is unmasked by alkalinization, and the other by a rise in Ca2+
i
.
Received: 23 May 1996/Revised: 6 December 1996 相似文献
8.
Zvyagilskaya R Parchomenko O Abramova N Allard P Panaretakis T Pattison-Granberg J Persson BL 《The Journal of membrane biology》2001,183(1):39-50
In this study we have used a newly isolated Yarrowia lipolytica yeast strain with a unique capacity to grow over a wide pH range (3.5–10.5), which makes it an excellent model system for
studying H+- and Na+-coupled phosphate transport systems. Even at extreme growth conditions (low concentrations of extracellular phosphate, alkaline
pH values) Y. lipolytica preserved tightly-coupled mitochondria with the fully competent respiratory chain containing three points of energy conservation.
This was demonstrated for the first time for cells grown at pH 9.5–10.0. In cells grown at pH 4.5, inorganic phosphate (Pi) was accumulated by two kinetically discrete H+/Pi-cotransport systems. The low-affinity system is most likely constitutively expressed and operates at high Pi concentrations. The high-affinity system, subjected to regulation by both extracellular Pi availability and intracellular polyphosphate stores, is mobilized during Pi-starvation. In cells grown at pH 9.5–10, Pi uptake is mediated by several kinetically discrete Na+-dependent systems that are specifically activated by Na+ ions and insensitive to the protonophore CCCP. One of these, a low-affinity transporter operative at high Pi concentrations is kinetically characterized here for the first time. The other two, high-affinity, high-capacity systems,
are derepressible and functional during Pi-starvation and appear to be controlled by extracellular Pi. They represent the first examples of high-capacity, Na+-driven Pi transport systems in an organism belonging to neither the animal nor bacterial kingdoms. The contribution of the H+- and Na+-coupled Pi transport systems in Y. lipolytica cells grown at different pH values was quantified. In cells grown at pH values of 4.5 and 6.0, the H+-coupled Pi transport systems are predominant. The contribution of the Na+/Pi cotransport systems to the total cellular Pi uptake activity is progressively increased with increasing pH, reaching its maximum at pH 9 and higher.
Received: 15 December 2000/Revised: 14 May 2001 相似文献
9.
The present study shows that in trout red blood cells the activity of some amino acid carriers, not directly involved in
cell volume regulation, is affected by external osmolality. Glycine uptake has been used as the experimental approach because
it was shown previously that it is effected by different carriers, namely the Na+-dependent ASC and Gly systems, as well as the Na+-independent asc and L systems.
An increase in the uptake through the Gly system and the two Na+-independent carriers was found, while the ASC system appeared to be downregulated. Those systems whose activities were increased
by hypo-osmolality did not share the mechanism by which this increase was obtained. Thus, the Gly system was sensitive to
intracellular ionic changes, while the Na+- independent systems were mechanically stimulated, as assessed by the iso-osmotic swelling caused by ammonium chloride.
On the other hand, a volume-sensitive transporter may be present in trout red blood cells, which is involved in the swelling-induced
glycine movement, as can be deduced from the effect of some inhibitors such as pyridoxal phosphate, DIDS (4,4′-diisothiocyanate-stilbene-2,2′-disulfonic
acid) and quinine.
Received: 12 February 1996/Revised: 9 September 1996 相似文献
10.
When expressed in Xenopus oocytes KAAT1 increases tenfold the transport of l-leucine. Substitution of NaCl with 100 mm LiCl, RbCl or KCl allows a reduced but significant activation of l-leucine uptakes. Chloride-dependence is not strict since other pseudohalide anions such as thyocyanate are accepted. KAAT1
is highly sensitive to pH. It can transport l-leucine at pH 5.5 and 8, but the maximum uptake has been observed at pH 10, near to the physiological pH value, when amino
and carboxylic groups are both deprotonated. The pH value mainly influences the V
max
in Na+ activation curves and l-leucine kinetics. The kinetic parameters are K
mNa
= 4.6 ± 2 mm, V
maxNa
= 14.8 ± 1.7 pmol/oocyte/5 min for pH 8.0 and K
mNa
= 2.8 ± 0.7 mm, V
maxNa
= 31.3 ± 1.9 pmol/oocyte/5 min for pH 10.0. The kinetic parameters of l-leucine uptake are: K
m
= 120.4 ± 24.2 μm, V
max
= 23.2 ± 1.4 pmol/oocyte/5 min at pH 8.0 and K
m
= 81.3 ± 24.2 μm, V
max
= 65.6 ± 3.9 pmol/oocyte/5 min at pH 10.0.
On the basis of inhibition experiments, the structural features required for KAAT1 substrates are: (i) a carboxylic group,
(ii) an unsubstituted α-amino group, (iii) the side chain is unnecessary, if present it should be uncharged regardless of
length and ramification.
Received: 27 April 1999/Revised: 10 January 2000 相似文献
11.
J.I. Kourie 《The Journal of membrane biology》1999,172(1):37-45
The lipid bilayer technique is used to examine the biophysical properties of anion and cation channels frequently formed
by platypus (Ornithorhynchus anatinus) venom (OaV). The OaV-formed anion channel in 250/50 mm KCl cis/trans has a maximum conductance of 857 ± 23 pS (n= 5) in 250/50 mm KCl cis/trans. The current-voltage relationship of this channel shows strong inward rectification. The channel activity undergoes time-dependent
inactivation that can be removed by depolarizing voltage steps more positive than the reversal potential for chloride, E
Cl
, (+40 mV).
The reversal potential of the OaV-formed slow current activity in 250/50 mm KCl cis/trans is close to the potassium equilibrium potential (E
K
) of −40 mV. The conductance values for the slow channel are 22.5 ± 2.6 pS and 41.38 ± 4.2 pS in 250/50 and 750/50 mm
cis/trans, respectively. The gating kinetics of the slow ion channels are voltage-dependent. The channel open probability (P
o
) is between 0.1 and 0.8 at potentials between 0 and +140 mV. The channel frequency (F
o
) increases with depolarizing voltages between 0 and +140 mV, whereas mean open time (T
o
) and mean closed time (T
c
) decrease. Ion substitution experiments of the cis solution show that the channel has conductance values of 21.47 ± 2.3 and 0.53 ± 0.1 pS in 250 mm KCl and choline Cl, respectively. The amplitude of the single channel current is dependent on [K+]
cis
and the current reversal potential (E
rev
) responds to increases in [K+]
cis
by shifting to more negative voltages. The increase in current amplitude as a function of increasing [K+]
cis
can be best described by a third order polynomial fit. At +140 mV, the values of the maximal single channel conductance (γ
max
) and the concentration for half maximal γ (K
s
) are 38.6 pS and 380 mm and decline to 15.76 pS and 250 mm at 0 mV, respectively. The ion selectivity of the channel to K+, Na+, Cs+ and choline+ was determined in ion substitution experiments. The permeability values for P
K+
:P
Na+
:P
Cs+
:P
choline+
were 1:1:0.63:0.089, respectively. On the other hand, the activity of the slow channel was eliminated (Fig. 7B). The slow channel was reversibly inhibited by [TEA+]
trans
and the half-maximal inhibitory concentration (K
i
) was ∼48 mm.
Received: 26 April 1999/Revised: 19 July 1999 相似文献
12.
S.E. Gasanov M.A. Alsarraj N.E. Gasanov E.D. Rael 《The Journal of membrane biology》1997,155(2):133-142
Membrane-active toxins from snake venom have been used previously to study protein-lipid interactions and to probe the physical
and biochemical states of biomembranes. To extend these studies, we have isolated from Naja naja kaowthia (cobra) venom a cytotoxin free of detectable phospholipase A2 (PLA2). The amino acid composition, pI (10.2), and net charge of the cytotoxin compares well with membrane-active toxins isolated
from venoms of other cobras. The cytotoxin, shown by a spin label method, associates with PLA2 in buffers at pH values between 7.0 and 5.0, but not at pH 4.0. It is suggested that cytotoxin and PLA2 (pI close to 4.8) associate electrostatically in the native venom. The effect of the cytotoxin on model phospholipid membranes
was studied by EPR of spin probes in oriented lipid multilayers and 1H-NMR of sonicated liposomes. The cytotoxin did not significantly affect the packing of lipids in pure phosphatidylcholine
(PC) membranes and in PC membranes containing 10 mol% phosphatidic acid (PA) or cardiolipin (CL). However, the cytotoxin induced
an increase in membrane permeability and formation of nonbilayer structures in PC membranes containing 40 mol% of PA or CL.
The purified cytotoxin was cytocidal to Jurkat cells, but had little effect on normal human lymphocytes. However, both Jurkat
cells and normal lymphocytes were killed equivalently when treated with 10−9
m PLA2 and 10−5
m cytotoxin in combination. From its effect on model membranes and Jurkat cells, it is suggested that purified cytotoxin preferentially
targets and disrupts membranes that are rich in acidic phospholipids on the extracellular side of the plasma membrane.
Received: 20 March 1996/Revised: 25 September 1996 相似文献
13.
P.J. White 《The Journal of membrane biology》1996,152(1):89-99
Nitrogen is available to the plant in the form of NH+
4 in the soil solution. Here it is shown that a voltage-independent K+ channel in the plasma membrane of rye (Secale cereale L.) roots is permeable to NH+
4. The channel was studied following its incorporation into planar 1-palmitoyl-2-oleoyl phosphatidyl ethanolamine bilayers.
The unitary conductance of the channel was greater when assayed in the presence of 100 mm NH4Cl than 100 mm KCl. However, the probability of finding the channel open (P
o
) was lower in the presence of 100 mm NH4Cl (P
o
= 0.63) than in 100 mm KCl (P
o
= 0.8), suggesting that P
o
can be regulated by the (permeant) ions present in solution. When assayed in equimolar concentrations of NH4Cl (cis) and KCl (trans), the zero-current (reversal) potential for the channel (E
rev) exhibited a complex concentration dependence. At low cation concentrations, the apparent permeability of NH+
4 relative to K+ (PNH4/PK) was greater than 1.0. However, as the cation concentration was increased, PNH4/PK initially decreased to a minimum of 0.95 at 3 mm before increasing again to a maximum of 1.89 at 300 mm. At cation concentrations above 300 mm, PNH4/PK decreased slightly. This implies that the pore of the channel can be occupied by more than one cation simultaneously. Ammonium
permeation through the pore was simulated using a model which is composed of three energy barriers and two energy wells (the
ion-binding sites). The model (3B2S) allowed for single-file permeation, double cation occupancy, ion-ion repulsion within
the pore and surface potential effects. Results indicated that energy peaks and energy wells were situated asymmetrically
within the electrical distance of the pore, that cations repel each other within the pore and that the vestibules to the pore
contain negligible surface charge. The energy profile obtained for NH+
4 is compared with ones obtained for K+ and Na+. This information allows the fluxes through the K+ channel of the three major monovalent cations present in the soil solution to be predicted.
Received: 16 October 1995/Revised 12 March 1996 相似文献
14.
d-Aspartate (d-Asp) uptake by suspensions of cerebral rat brain astrocytes (RBA) maintained in long-term culture was studied as a means
of characterizing function and regulation of Glutamate/Aspartate (Glu/Asp) transporter isoforms in the cells. d-Asp influx is Na+-dependent with K
m
= 5 μm and V
max= 0.7 nmoles · min−1· mg protein−1. Influx is sigmoidal as f[Na+] with Na+
K
m
∼ 12 μm and Hill coefficient of 1.9. The cells establish steady-state d-Asp gradients >3,000-fold. Phorbol ester (PMA) enhances uptake, and gradients near 6,000-fold are achieved due to a 2-fold
increase in V
max, with no change in K
m
. At initial [d-Asp] = 10 μm, RBA take up more than 90% of total d-Asp, and extracellular levels are reduced to levels below 1 μm. Ionophores that dissipate the ΔμNa+ inhibit gradient formation. Genistein (GEN, 100 μm), a PTK inhibitor, causes a 40% decrease in d-Asp. Inactive analogs of PMA (4α-PMA) and GEN (daidzein) have no detectable effect, although the stimulatory PMA response
still occurs when GEN is present. Further specificity of action is indicated by the fact that PMA has no effect on Na+-coupled ALA uptake, but GEN is stimulatory. d-Asp uptake is strongly inhibited by serine-O-sulfate (S-O-S), threohydroxy-aspartate (THA), l-Asp, and l-Glu, but not by d-Glu, kainic acid (KA), or dihydrokainate (DHK), an inhibition pattern characteristic of GLAST and EAAC1 transporter isoforms.
mRNA for both isoforms was detected by RT-PCR, and Western blotting with appropriate antibodies shows that both proteins are
expressed in these cells.
Received: 11 January 2001/Revised: 26 March 2001 相似文献
15.
The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using
continuous short circuit current (I
SC
) measurements in HCO3
−/CO2 buffered solution. Steady state I
SC
averaged 73.8 ± 3.2 μA/cm2 (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na+ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y+ and a basal amino acid transport system y+L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic
amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I
SC
averaging 36.6 ± 5.4 μA/cm2 (n= 19) and 32.0 ± 7.2 μA/cm2 (n= 8), respectively. The response was preserved in the absence of bath Cl− (n= 4), but was abolished either in the absence of apical Na+ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I
SC
response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization
of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na+ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract
passive cationic amino acid leak into the CCD in the presence of electrogenic Na+ transport, consistent with reports of stimulated expression of Na+ and cationic amino acid transporters by aldosterone.
Received: 11 September 2000/Revised: 6 December 2000 相似文献
16.
D.T. Thwaites D. Markovich H. Murer N.L. Simmons 《The Journal of membrane biology》1996,151(3):215-224
The nature of transepithelial and cellular transport of the dibasic amino acid lysine in human intestinal epithelial Caco-2
cells has been characterized. Intracellular accumulation of lysine across both the apical and basolateral membranes consists
of a Na+-independent, membrane potential-sensitive uptake. Na+-independent lysine uptake at the basolateral membrane exceeds that at the apical membrane. Lysine uptake consists of both
saturable and nonsaturable components. Na+-independent lysine uptake at both membranes is inhibited by lysine, arginine, alanine, histidine, methionine, leucine, cystine,
cysteine and homoserine. In contrast, proline and taurine are without inhibitory effects at both membranes. Fractional Na+-independent lysine efflux from preloaded epithelial layers is greater at the basolateral membrane and shows trans-stimulation
across both epithelial borders by lysine, arginine, alanine, histidine, methionine, and leucine but not proline and taurine.
Na+-independent lysine influx (10 μm) in the presence of 10 mm homoserine shows further concentration dependent inhibition by lysine. Taken together, these data are consistent with lysine
transport being mediated by systems bo,+, y+ and a component of very low affinity (nonsaturable) at both membranes. The relative contribution to lysine uptake at each
membrane surface (at 10 μm lysine), normalized to total apical uptake (100%), is apical bo,+ (47%), y+ (27%) and the nonsaturable component (26%), and basal bo,+ (446%), y+ (276%) and the nonsaturable component (20%). Northern analysis shows hybridization of Caco-2 poly(A)+RNA with a human rBAT cDNA probe.
Received: 3 July 1995/Revised: 6 February 1996 相似文献
17.
Studies were conducted to examine the influence of the H+-ATPase inhibitor bafilomycin A1 on cultured rabbit nonpigmented ciliary epithelial cells (NPE). Cytoplasmic pH and sodium concentrations were measured by
digital fluorescence microscopy using BCECF and SBFI respectively. In some experiments, cell sodium content was measured by
atomic absorption spectroscopy. Added alone, bafilomycin A1 (100 nm) failed to change cytoplasmic pH but it caused an increase of cytoplasmic sodium concentration which occurred within 10 min.
It is likely that the rise of cytoplasmic sodium concentration was responsible for the stimulation of active sodium-potassium
transport which occurred in bafilomycin A1-treated cells as judged by a 50% increase of ouabain sensitive potassium (86Rb) uptake. In bafilomycin A1-treated cells, but not in control cells, dimethylamiloride (DMA) inhibited ouabain-sensitive potassium (86Rb) uptake in a dose-dependent manner with an IC50 of ∼2 μm. DMA (10 μm) also prevented the increase of cytoplasmic sodium caused by bafilomycin A1. Added alone, DMA (10 μm) failed to change cytoplasmic sodium content but reduced cytoplasmic pH by ∼0.4 pH units. In cells that first received 10
μm DMA, the subsequent addition of bafilomycin A1 (100 nm) caused a further cytoplasmic pH reduction of ∼0.3 pH units. Taken together, the results suggest H+-ATPase might contribute to the regulation of basal cytoplasmic pH in cultured NPE. In the presence of bafilomycin A1, Na-H exchanger activity appears to be stimulated, so stabilizing cytoplasmic pH but resulting in an increase of cytoplasmic
sodium concentration and consequent stimulation of active sodium-potassium transport.
Received: 19 March 1999/Revised: 20 September 1999 相似文献
18.
A fluorescence method was adapted to investigate active ion transport in membrane preparations of the SR-Ca-ATPase. The styryl
dye RH421 previously used to investigate the Na,K-ATPase was replaced by an analogue, 2BITC, to obtain optimized fluorescence
changes upon substrate-induced partial reactions. Assuming changes of the local electric field to be the source of fluorescence
changes that are produced by uptake/release or by movement of ions inside the protein, 2BITC allowed the determination of
electrogenic partial reactions in the pump cycle. It was found that Ca2+ binding on the cytoplasmic and on the lumenal side of the pump is electrogenic while phosphorylation and conformational transition
showed only minor electrogenicity. Ca2+ equilibrium titration experiments at pH 7.2 in the two major conformations of the protein indicated cooperative binding of
two Ca2+ ions in state E1 with an apparent half-saturation concentration, K
M
of 600 nm. In state P-E2 two K
M
values, 5 μm and 2.2 mM, were determined and are in fair agreement with published data. From Ca2+ titrations in buffers with various pH and from pH titrations in P-E2, it could be demonstrated that H+ binding is electrogenic and that Ca2+ and H+ compete for the same binding site(s). Tharpsigargin-induced inhibition of the Ca-ATPase led to a state with a specific fluorescence
level comparable to that of state E1 with unoccupied ion sites, independent of the buffer composition.
Received: 21 September 1998/Revised: 18 December 1998 相似文献
19.
In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO3) has been shown to mediate a large fraction of the total K+ transport. In the present study, Cl−-dependent and Cl−-independent K+ fluxes via frog erythrocyte membranes were investigated as a function of external and internal K+ ([K+]
e
and [K+]
i
) concentration. The dependence of ouabain-resistant Cl−-dependent K+ (86Rb) influx on [K+]
e
over the range 0–20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K
m
) of 8.2 ± 1.3 mm and maximal velocity (V
max
) of 10.4 ± 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl−-dependent K+ influx increased both K
m
(12.8 ± 1.7 mm, P < 0.05) and V
max
(20.2 ± 2.9 mmol/l/hr, P < 0.001). Raising [K+]
e
above 20 mm in isotonic media significantly reduced the Cl−-dependent K+ influx due to a reciprocal decrease of the external Na+ ([Na+]
e
) concentration below 50 mm. Replacing [Na+]
e
by NMDG+ markedly decreased V
max
(3.2 ± 0.7 mmol/l/hr, P < 0.001) and increased K
m
(15.7 ± 2.1 mm, P < 0.03) of Cl−-dependent K+ influx. Moreover, NMDG+ Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb+ uptake and K+ loss from red cells. Cell swelling did not affect the Na+-dependent changes in Rb+ uptake and K+ loss. In a nominally K+(Rb+)-free medium, net K+ loss was reduced after lowering [Na+]
e
below 50 mm. These results indicate that over 50 mm [Na+]
e
is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K+, Cl−-dependent K+ loss in K+-free media was a linear function of [K+]
i
, with a rate constant of 0.11 ± 0.01 and 0.18 ± 0.008 hr−1 (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry
with respect to transported K+ ions. The residual, ouabain-resistant K+ fluxes in NO3 were only 5–10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were
not different from those for K+ effluxes in isotonic (∼0.014 hr−1) and hypotonic (∼0.022 hr−1) media, but cell swelling resulted in a significant increase in the rate constants.
Received: 19 November 1998/Revised: 23 August 1999 相似文献
20.
The presence and localization of voltage-gated Ca2+ channels of L-type were investigated in intestinal cells of the Atlantic cod. Enterocytes were loaded with the fluorescent
Ca2+ probe, fure-2/AM and changes in intracellular Ca2+ concentrations ([Ca2+]
i
) were measured, in cell suspensions, in the presence of high potassium levels (100 mm), BAY K-8644 (5 μm), nifedipine (5 μm) or ω-conotoxin (1 μm). L-type Ca2+ channels were visualized on intestinal sections using the fluorescent dihydropyridine (-)-STBodipy.
Depolarization of the plasma membrane produced a rapid (within 5 sec) and transient (at basal levels after 21 sec) increase
in [Ca2+]
i
. BAY K-8644 increased the [Ca2+]
i
by 7.2%. Cells in a Ca2+-free buffer increased [Ca2+]
i
after addition of 10 mm Ca2+, and this increase was abolished by nifedipine in both depolarizing and normal medium but not by ω-conotoxin. Single cell
experiments using video microscopy revealed that enterocytes remained polarized several hours after preparation and that the
Ca2+ entry and extrusion occurred at specific and different regions of the enterocyte outer membrane. Fluorescent staining of
L-type Ca2+ channels in the intestinal mucosa showed the most intense staining at the brushborder membrane.
These results demonstrate the presence of voltage gated L-type Ca2+ channels in enterocytes from the Atlantic cod. The channels are mainly located at the apical side of the cells, and there
is a polarized uptake of Ca2+ into the enterocytes. This suggests that the L-type Ca2+ channels are involved in the transcellular Ca2+ entry into the enterocytes.
Received: 21 August 1997/Revised: 15 April 1998 相似文献