共查询到20条相似文献,搜索用时 46 毫秒
1.
The Role of MIP in Lens Fiber Cell Membrane Transport 总被引:1,自引:1,他引:0
K. Varadaraj C. Kushmerick G.J. Baldo S. Bassnett A. Shiels R.T. Mathias 《The Journal of membrane biology》1999,170(3):191-203
MIP has been hypothesized to be a gap junction protein, a membrane ion channel, a membrane water channel and a facilitator
of glycerol transport and metabolism. These possible roles have been indirectly suggested by the localization of MIP in lens
gap junctional plaques and the properties of MIP when reconstituted into artificial membranes or exogenously expressed in
oocytes. We have examined lens fiber cells to see if these functions are present and whether they are affected by a mutation
of MIP found in Cat
Fr
mouse lens. Of these five hypothesized functions, only one, the role of water channel, appears to be true of fiber cells
in situ. Based on the rate of volume change of vesicles placed in a hypertonic solution, fiber cell membrane lipids have a low water
permeability (p
H2O
) on the order of 1 μm/sec whereas normal fiber cell membrane p
H2O
was 17 μm/sec frog, 32 μm/sec rabbit and 43 μm/sec mouse. Cat
Fr
mouse lens fiber cell p
H2O
was reduced by 13 μm/sec for heterozygous and 30 μm/sec for homozygous mutants when compared to wild type. Lastly, when expressed
in oocytes, the p
H2O
conferred by MIP is not sensitive to Hg2+ whereas that of CHIP28 (AQP1) is blocked by Hg2+. The fiber cell membrane p
H2O
was also not sensitive to Hg2+ whereas lens epithelial cell p
H2O
(136 μm/sec in rabbit) was blocked by Hg2+. With regard to the other hypothesized roles, fiber cell membrane or lipid vesicles had a glycerol permeability on the order
of 1 nm/sec, an order of magnitude less than that conferred by MIP when expressed in oocytes. Impedance studies were employed
to determine gap junctional coupling and fiber cell membrane conductance in wild-type and heterozygous Cat
Fr
mouse lenses. There was no detectable difference in either coupling or conductance between the wild-type and the mutant lenses.
Received: 17 February 1999/Revised: 16 April 1999 相似文献
2.
3.
Mouse myeloma cells were electropermeabilized by single square-wave electric pulses with amplitudes of up to ∼150 kV/cm and
durations of 10–100 nsec. The effects of the field intensity, pulse duration and medium conductivity on cell viability and
field-induced uptake of molecules were analyzed by quantitative flow cytometry using the membrane-impermeable fluorescent
dye propidium iodide as indicator molecule. Despite the extremely large field strengths, the majority of cells survived the
exposure to ultra-short field pulses. The electrically induced dye uptake increased markedly with decreasing conductivity
of the suspending medium. We assigned this phenomenon to the transient electrodeformation (stretching) force that assumes
its maximum value if cells are suspended in low-conductivity media, i.e., if the external conductivity σe is smaller than that of the cytosol σi. The stretching force vanishes when σe is equal to or larger than σi. Due to their capability of delivering extremely large electric fields, the pulse power systems used here appear to be a
promising tool for the electropermeabilization of very small cells and vesicles (including intracellular organelles, liposomes,
etc.).
Received: 15 May 2001/Revised: 20 July 2001 相似文献
4.
Fluid and electrolyte secretion from secretory epithelia is a highly regulated process. Chloride channel activity at the
apical membrane determines the rate and direction of salt and water secretion. Multiple classes of Cl− channels with distinct gating mechanisms are involved in moving ions and water. Secretory agonists that induce intracellular
increases in two second messenger systems, cAMP and [Ca2+]
i
, are generally associated with secretion. However, changes in cell volume and the membrane potential may also play a role
in regulating fluid and electrolyte secretion in some tissues. In this review we discuss the regulation of the different types
of Cl− channels found in secretory epithelia.
Received: 16 September 1997/Revised: 13 November 1997 相似文献
5.
In cystic fibrosis, the mutation of the CFTR protein causes reduced transepithelial Cl− secretion. As recently proposed, beside its role of Cl− channel, CFTR may regulate the activity of other channels such as a Ca2+-activated Cl− channel. Using a calcium imaging system, we show, in adenovirus-CFTR infected Chinese Hamster Ovary (CHO) cell monolayers,
that CFTR can act as a regulator of intracellular [Ca2+]
i
([Ca2+]
i
), involving purino-receptors. Apical exposure to ATP or UTP produced an increase in ([Ca2+]
i
in noninfected CHO cell monolayers (CHO-WT), in CHO monolayers infected with an adenovirus-CFTR (CHO-CFTR) or infected with
an adenovirus-LacZ (CHO-LacZ). The transient [Ca2+]
i
increase produced by ATP or UTP could be mimicked by activation of CFTR with forskolin (20 μm) in CHO-CFTR confluent monolayers. However, forskolin had no significant effect on [Ca2+]
i
in noninfected CHO-WT or in CHO-LacZ cells. Pretreatment with purino-receptor antagonists such as suramin (100 μm) or reactive blue-2. (100 μm), and with hexokinase (0.28 U/mg) inhibited the [Ca2+]
i
response to forskolin in CHO-CFTR infected cells. Taken together, our experiments provide evidence for purino-receptor activation
by ATP released from the cell and regulation of [Ca2+]
i
by CFTR in CHO epithelial cell membranes.
Received: 5 April 1999/Revised: 28 June 1999 相似文献
6.
Caffeine causes a [Ca2+]
i
increase in the cortex of Paramecium cells, followed by spillover with considerable attenuation, into central cell regions. From [Ca2+]rest
i
∼50 to 80 nm, [Ca2+]act
i
rises within ≤3 sec to 500 (trichocyst-free strain tl) or 220 nm (nondischarge strain nd9–28°C) in the cortex. Rapid confocal analysis of wildtype cells (7S) showed only a 2-fold cortical
increase within 2 sec, accompanied by trichocyst exocytosis and a central Ca2+ spread during the subsequent ≥2 sec. Chelation of Ca2+
o
considerably attenuated [Ca2+]
i
increase. Therefore, caffeine may primarily mobilize cortical Ca2+ pools, superimposed by Ca2+ influx and spillover (particularly in tl cells with empty trichocyst docking sites). In nd cells, caffeine caused trichocyst
contents to decondense internally (Ca2+-dependent stretching, normally occurring only after membrane fusion). With 7S cells this usually occurred only to a small
extent, but with increasing frequency as [Ca2+]
i
signals were reduced by [Ca2+]
o
chelation. In this case, quenched-flow and ultrathin section or freeze-fracture analysis revealed dispersal of membrane components
(without fusion) subsequent to internal contents decondensation, opposite to normal membrane fusion when a full [Ca2+]
i
signal was generated by caffeine stimulation (with Ca2+
i
and Ca2+
o
available). We conclude the following. (i) Caffeine can mobilize Ca2+ from cortical stores independent of the presence of Ca2+
o
. (ii) To yield adequate signals for normal exocytosis, Ca2+ release and Ca2+ influx both have to occur during caffeine stimulation. (iii) Insufficient [Ca2+]
i
increase entails caffeine-mediated access of Ca2+ to the secretory contents, thus causing their decondensation before membrane fusion can occur. (iv) Trichocyst decondensation
in turn gives a signal for an unusual dissociation of docking/fusion components at the cell membrane. These observations imply
different threshold [Ca2+]
i
-values for membrane fusion and contents discharge.
Received: 23 May 1997/Revised: 18 August 1997 相似文献
7.
Brush border membrane vesicles, BBMV, from eel intestinal cells or kidney proximal tubule cells were prepared in a low osmolarity
cellobiose buffer. The osmotic water permeability coefficient P
f
for eel vesicles was not affected by pCMBS and was measured at 1.6 × 10−3 cm sec−1 at 23°C, a value lower than 3.6 × 10−3 cm sec−1 exhibited by the kidney vesicles and similar to published values for lipid bilayers. An activation energy E
a
of 14.7 Kcal mol−1 for water transport was obtained for eel intestine, contrasting with 4.8 Kcal mol−1 determined for rabbit kidney proximal tubule vesicles using the same method of analysis. The high value of E
a
, as well as the low P
f
for the eel intestine is compatible with the absence of water channels in these membrane vesicles and is consistent with
the view that water permeates by dissolution and diffusion in the membrane. Further, the initial transient observed in the
osmotic response of kidney vesicles, which is presumed to reflect the inhibition of water channels by membrane stress, could
not be observed in the eel intestinal vesicles. The P
f
dependence on the tonicity of the osmotic shock, described for kidney vesicles and related to the dissipation of pressure
and stress at low tonicity shocks, was not seen with eel vesicles. These results indicate that the membranes from two volume
transporter epithelia have different mechanisms of water permeation. Presumably the functional water channels observed in
kidney vesicles are not present in eel intestine vesicles. The elastic modulus of the membrane was estimated by analysis of
swelling kinetics of eel vesicles following hypotonic shock. The value obtained, 0.79 × 10−3 N cm−1, compares favorably with the corresponding value, 0.87 × 10−3 N cm−1, estimated from measurements at osmotic equilibrium.
Received: 28 January 1999/Revised: 15 June 1999 相似文献
8.
Nucleotide-dependent gating of ΔF508-CFTR was evaluated in membrane patches excised from HEK 293 and mouse L-cells and compared
to observations on wt-CFTR channels recorded in the same expression systems. ΔF508-CFTR exhibited PKA activated, ATP-dependent channel gating.
When compared to wt-CFTR, the K
m
for ATP was increased by ninefold (260 μm
vs. 28 μm) and maximal open probability (P
o
) was reduced by 49% (0.21 ± 0.06 vs. 0.41 ± 0.02). Additionally, in the absence of PKA, ΔF508-CFTR inactivated over a 1 to 5 min period whereas wt-CFTR remained active. Time-dependent inactivation could be mimicked in wt-CFTR by the intermittent absence of ATP in the cytosolic solution. The effects of 3-isobutyl-1-methyl xanthine (IBMX), a
compound reported to stimulate ΔF508-CFTR, were evaluated on wt- and ΔF508-CFTR channels. At concentrations up to 5 mm, IBMX caused a concentration dependent reduction in the observed single channel amplitude (i) of wt-CFTR (maximal observed reduction 35 ± 3%). However, IBMX failed to significantly alter total patch current because of a concomitant
30% increase in P
o
. The effects of IBMX on ΔF508-CFTR were similar to effects on wt-CFTR in that i was reduced and P
o
was increased by similar magnitudes. Additionally, ΔF508-CFTR channel inactivation was dramatically slowed by IBMX. These
results suggest that IBMX interacts with the ATP-bound open state of CFTR to introduce a short-lived nonconducting state which
prolongs burst duration and reduces apparent single channel amplitude. A secondary effect observed in ΔF508-CFTR, which may
result from this interaction, is a prolongation of the activated state. In light of previously proposed linear kinetic models
of CFTR gating, these results suggest that IBMX traps CFTR in an ATP-bound state which may preclude inactivation of ΔF508-CFTR.
Received: 5 February 1999/Revised: 25 March 1999 相似文献
9.
M.-P. Blanchard N. Klauke S. Zitzmann H. Plattner 《The Journal of membrane biology》1999,169(3):155-165
We analyzed [Ca2+]
i
transients in Paramecium cells in response to veratridine for which we had previously established an agonist effect for trichocyst exocytosis (Erxleben
& Plattner, 1994. J. Cell Biol.
127:935–945; Plattner et al., 1994. J. Membrane Biol.
158:197–208). Wild-type cells (7S), nondischarge strain nd9–28°C and trichocyst-free strain ``trichless' (tl), respectively,
displayed similar, though somewhat diverging time course and plateau values of [Ca2+]
i
transients with moderate [Ca2+]
o
in the culture/assay fluid (50 μm or 1 mm). In 7S cells which are representative for a normal reaction, at [Ca2+]
o
= 30 nm (c.f. [Ca2+]
rest
i
=∼50 to 100 nm), veratridine produced only a small cortical [Ca2+]
i
transient. This increased in size and spatial distribution at [Ca2+]
o
= 50 μm of 1 mm. Interestingly with unusually high yet nontoxic [Ca2+]
o
= 10 mm, [Ca2+]
i
transients were much delayed and also reduced, as is trichocyst exocytosis. We interpret our results as follows. (i) With
[Ca2+]
o
= 30 nm, the restricted residual response observed is due to Ca2+ mobilization from subplasmalemmal stores. (ii) With moderate [Ca2+]
o
= 50 μm to 1 mm, the established membrane labilizing effect of veratridine may activate not only subplasmalemmal stores but also Ca2+
o influx from the medium via so far unidentified (anteriorly enriched) channels. Visibility of these phenomena is best in tl
cells, where free docking sites allow for rapid Ca2+ spread, and least in 7S cells, whose perfectly assembled docking sites may ``consume' a large part of the [Ca2+]
i
increase. (iii) With unusually high [Ca2+]
o
, mobilization of cortical stores and/or Ca2+
o
influx may be impeded by the known membrane stabilizing effect of Ca2+
o
counteracting the labilizing/channel activating effect of veratridine. (iv) We show these effects to be reversible, and,
hence, not to be toxic side-effects, as confirmed by retention of injected calcein.
(v) Finally, Mn2+ entry during veratridine stimulation, documented by Fura-2 fluorescence quenching, may indicate activation of unspecific
Me2+ channels by veratridine. Our data have some bearing on analysis of other cells, notably neurons, whose response to veratridine
is of particular and continous interest.
Received: 8 December 1998/Revised: 2 March 1999 相似文献
10.
An apical membrane ouabain-sensitive H-K exchange and a barium-sensitive basolateral membrane potassium channel are present
in colonic crypt cells and may play a role in both K absorption and intracellular pH (pHi) regulation. To examine the possible interrelationship between apical membrane H-K exchange and basolateral membrane K movement
in rat distal colon in the regulation of pHi, experiments were designed to assess whether changes in extracellular potassium can alter pHi. pHi in isolated rat crypts was determined using microspectrofluorimetric measurements of the pH-sensitive dye BCECF-AM (2′,7′-bis(carboxyethyl-5(6)-carboxy-fluorescein
acetoxy methylester). After loading with the dye, crypts were superfused with a Na-free solution which resulted in a rapid
and reversible fall in pHi (7.36 ± 0.02 to 6.98 ± 0.03). Following an increase in extracellular [K] to 20 mm, in the continued absence of Na, there was a further decrease in pHi (0.20 ± 0.02, P < 0.01). K-induced acidification was blocked both by 2 mm bath barium, a K channel blocker, and by 0.5 mm lumen ouabain. K-induced acidification was also observed when intracellular acidification was induced by a NH4Cl prepulse. These observations suggest that increased basolateral K movement increases intracellular [K] resulting in a decrease
in pHi that is mediated by a ouabain-sensitive apical membrane H,K-ATPase. Our results demonstrate an interrelationship between
basolateral K movement and apical H-K exchange in the regulation of pHi and apical K entry in rat distal colon.
Received: 31 March 1998/Revised: 8 September 1998 相似文献
11.
In cystic fibrosis airway epithelia, mutation of the CFTR protein causes a reduced response of Cl− secretion to secretagogues acting via cAMP. Using a Ca2+ imaging system, the hypothesis that CFTR activation may permit ATP release and regulate [Ca2+]
i
via a receptor-mediated mechanism, is tested in this study. Application of external nucleotides produced a significant increase
in [Ca2+]
i
in normal (16HBE14o− cell line and primary lung culture) and in cystic fibrosis (CFTE29o− cell line) human airway epithelia. The potency order of nucleotides on [Ca2+]
i
variation was UTP ≫ ATP > UDP > ADP > AMP > adenosine in both cell types. The nucleotide [Ca2+]
i
response could be mimicked by activation of CFTR with forskolin (20 μm) in a temperature-dependent manner. In 16HBE14o− cells, the forskolin-induced [Ca2+]
i
response increased with increasing temperature. In CFTE29o− cells, forskolin had no effect on [Ca2+]
i
at body temperature-forskolin-induced [Ca2+]
i
response in CF cells could only be observed at low experimental temperature (14°C) or when cells were cultured at 26°C instead
of 37°C. Pretreatment with CFTR channel blockers glibenclamide (100 μm) and DPC (100 μm), with hexokinase (0.5 U/mg), and with the purinoceptor antagonist suramin (100 μm), inhibited the forskolin [Ca2+]
i
response. Together, these results demonstrate that once activated, CFTR regulates [Ca2+]
i
by mediating nucleotide release and activating cell surface purinoceptors in normal and CF human airway epithelia.
Received: 3 April 2000/Revised: 30 June 2000 相似文献
12.
P.M. Dunn 《The Journal of membrane biology》1998,165(2):133-143
The actions of clotrimazole and cetiedil, two drugs known to inhibit the Gardos channel, have been studied on single intermediate
conductance calcium-activated potassium (IKCa) channels in inside out patches from human red blood cells, and compared with those of TEA and Ba2+ applied to the cytoplasmic face of the membrane. TEA produced a fast block which was observed as a reduction in the amplitude
of the single channel current. This effect was weakly voltage dependent with the fraction of the membrane potential sensed
by TEA at its binding site (δ) of 0.18 and a K
d
at 0 mV of 20.5 mm. Ba2+ was a very potent blocker of the channel, breaking the single channel activity up into bursts, interspersed with silent periods
lasting several seconds. The effect of Ba2+ was very voltage sensitive, δ= 0.44, and a K
d
at 0 mV of 0.15 μm. Clotrimazole applied to the inner face of the membrane at a concentration ≤1 μm produced a slow block resulting in bursts of channel activity separated by quiescent periods lasting many seconds. The effect
of clotrimazole was mimicked by a quaternary derivative UCL 1559, in keeping with an action at the cytoplasmic face of the
channel. A high concentration of cetiedil (100 μm) produced only a weak block of the channel. The kinetics of this action were very slow, with burst and inter-burst intervals
lasting several minutes. While inhibition of the Gardos channel by cetiedil is unlikely to involve an intracellular site of
action, if clotrimazole is able to penetrate the membrane, part of its effect may result from binding to an intracellular
site on the channel.
Received; 18 February 1998/Received: 5 June 1998 相似文献
13.
S. Bröer A. Schuster C.A. Wagner A. Bröer I. Forster J. Biber H. Murer A. Werner F. Lang A.E. Busch 《The Journal of membrane biology》1998,164(1):71-77
Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl− conductance (GCl), organic anion transport and Na+-dependent P
i
-uptake. In the present study we investigated the relation between the NaPi-1 induced GCl and P
i
-induced currents and transport. NaPi-1 expression induced P
i
-transport, which was not different at 1–20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1–2 days after cRNA
injection. In contrast, GCl was augmented at increased amounts of cRNA injection (1–20 ng/oocyte) and over a five day expression period. Subsequently
all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. P
i
-induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of P
i
(≥ 1 mm P
i
). The amplitudes of Ip correlated well with GCl. Ip was blocked by the Cl− channel blocker NPPB, partially Na+-dependent and completely abolished in Cl− free solution. In contrast, P
i
-transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na+ and weakly affected by Cl− substitution. Endogenous P
i
-uptake in water-injected oocytes amounted in all experiments to 30–50% of the Na+-dependent P
i
-transport observed in NaPi-1 expressing oocytes. The properties of the endogenous P
i
-uptake system (K
m
for P
i
> 1 mm; partial Na+- and Cl−-dependence; lack of NPPB block) were similar to the NaPi-1 induced P
i
-uptake, but no Ip could be recorded at P
i
-concentrations ≤3 mm. In summary, the present data suggest that Ip does not reflect charge transfer related to P
i
-uptake, but a P
i
-mediated modulation of GCl.
Received: 22 October 1997/Revised: 24 March 1998 相似文献
14.
Muscarinic receptor-linked G protein, G
i
, can directely activate the specific K+ channel (I
K(ACh)) in the atrium and in pacemaker tissues in the heart. Coupling of G
i
to the K+ channel in the ventricle has not been well defined. G protein regulation of K+ channels in isolated human ventricular myocytes was examined using the patch-clamp technique. Bath application of 1 μm acetylcholine (ACh) reversibly shortened the action potential duration to 74.4 ± 12.1% of control (at 90% repolarization,
mean ±sd, n= 8) and increased the whole-cell membrane current conductance without prior β-adrenergic stimulation in human ventricular
myocytes. The ACh effect was reversed by atropine (1 μm). In excised inside-out patch configurations, application of GTPγS (100 μm) to the bath solution (internal surface) caused activation of I
K(ACh) and/or the background inwardly-rectifying K+ channel (I
K1) in ventricular cell membranes. I
K(ACh) exhibited rapid gating behavior with a slope conductance of 44 ± 2 pS (n= 25) and a mean open lifetime of 1.8 ± 0.3 msec (n= 21). Single channel activity of GTPγS-activated I
K1 demonstrated long-lasting bursts with a slope conductance of 30 ± 2 pS (n= 16) and a mean open lifetime of 36.4 ± 4.1 msec (n= 12). Unlike I
K(ACh), G protein-activated I
K1 did not require GTP to maintain channel activity, suggesting that these two channels may be controlled by G proteins with
different underlying mechanisms. The concentration of GTP at half-maximal channel activation was 0.22 μm in I
K(ACh) and 1.2 μm in I
K1. Myocytes pretreated with pertussis toxin (PTX) prevented GTP from activating these channels, indicating that muscarinic
receptor-linked PTX-sensitive G protein, G
i
, is essential for activation of both channels. G protein-activated channel characteristics from patients with terminal heart
failure did not differ from those without heart failure or guinea pig. These results suggest that ACh can shorten the action
potential by activating I
K(ACh) and I
K1 via muscarinic receptor-linked G
i
proteins in human ventricular myocytes.
Received: 23 September 1996/Revised: 18 December 1996 相似文献
15.
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
16.
A voltage-activated Ca++ channel has been identified in the apical membranes of cultured rabbit proximal tubule cells using the patch-clamp technique.
With 105 mm CaCl2 solution in the pipette and 180 NaAsp in the bath, the channel had a conductance of 10.4 ± 1.0 pS (n= 8) in on-cell patches, and 9.8 ± 1.1 pS (n= 8) in inside-out patches. In both on-cell and inside-out patches, the channel is active by membrane depolarization. For
this channel, the permeation to Ba++ and Ca++ is highly selective over Na+ and K+ (PCa(Ba):PNa(K) >200:1). The sensitivity to dihydropyridines is similar to that for L-type channels where the channel was blocked by nifedipine
(10 μm), and activated by Bay K 8644 (5 μm). When activated by Bay K 8644, the channel showed subconductance levels. Treatment with forskolin (12.5 μm), phorbol ester (1 μm), or stretching (40 cm water) did not activate this channel. These results indicate that this Ca++ channel is mostly regulated by membrane voltage, and appears to be an epithelial class of L-type Ca++ channel. As such, it may participate in calcium reabsorption during periods of enhanced sodium reabsorption, or calcium signaling
in volume regulation, where membrane depolarization occurs for prolonged periods.
Received: 1 April 1996/Revised: 5 August 1996 相似文献
17.
I.I. Ismailov B.K. Berdiev A.L. Bradford M.S. Awayda C.M. Fuller D.J. Benos 《The Journal of membrane biology》1996,149(2):123-132
The hypothesis that amiloride-sensitive Na+ channel complexes immunopurified from bovine renal papillary collecting tubules contain, as their core conduction component,
an ENaC subunit, was tested by functional and immunological criteria. Disulfide bond reduction with dithiothreitol (DTT) of
renal Na+ channels incorporated into planar lipid bilayers caused a reduction of single channel conductance from 40 pS to 13 pS, and
uncoupled PKA regulation of this channel. The cation permeability sequence, as assessed from bi-ionic reversal potential measurements,
and apparent amiloride equilibrium dissociation constant (K
amil
i
) of the Na+ channels were unaltered by DTT treatment. Like ENaC, the DTT treated renal channel became mechanosensitive, and displayed
a substantial decrease in K
amil
i
following stretch (0.44 ± 0.12 μm versus 6.9 ± 1.0 μm). Moreover, stretch activation induced a loss in the channel's ability to discriminate between monovalent cations, and even
allowed Ca2+ to permeate. Polyclonal antibodies generated against a fusion protein of αbENaC recognized a 70 kDa polypeptide component
of the renal Na+ channel complex. These data suggest that ENaC is present in the immunopurified renal Na+ channel protein complex, and that PKA sensitivity is conferred by other associated proteins.
Received: 5 June 1995/Revised: 29 September 1995 相似文献
18.
The aim of this study was to clarify the mechanism of isotonic fluid transport in frog skin glands. Stationary ion secretion
by the glands was studied by measuring unidirectional fluxes of 24Na+, 42K+, and carrier-free 134Cs+ in paired frog skins bathed on both sides with Ringer's solution, and with 10−5
m noradrenaline on the inside and 10−4
m amiloride on the outside. At transepithelial thermodynamic equilibrium conditions, the 134Cs+ flux ratio, J
out
Cs/J
in
Cs, varied in seven pairs of preparations from 6 to 36. Since carrier-free 134Cs+ entering the cells is irreversibly trapped in the cellular compartment (Ussing & Lind, 1996), the transepithelial net flux
of 134Cs+ indicates that a paracellular flow of water is dragging 134Cs+ in the direction from the serosal- to outside solution. From the measured flux ratios it was calculated that the force driving
the secretory flux of Cs+ varied from 30 to 61 mV among preparations. In the same experiments unidirectional Na+ fluxes were measured as well, and it was found that also Na+ was subjected to secretion. The ratio of unidirectional Na+ fluxes, however, was significantly smaller than would be predicted if the two ions were both flowing along the paracellular
route dragged by the flow of water. This result indicates that Na+ and Cs+ do not take the same pathway through the glands. The flux ratio of unidirectional K+ fluxes indicated active secretion of K+. The time it takes for steady-state K+ fluxes to be established was significantly longer than that of the simultaneously measured Cs+ fluxes. These results allow the conclusion that — in addition to being transported between cells — K+ is submitted to active transport along a cellular pathway.Based on the recirculation theory, we propose a new model which
accounts for stationary Na+, K+, Cl− and water secretion under thermodynamic equilibrium conditions. The new features of the model, as compared to the classical
Silva-model for the shark-rectal gland, are: (i) the sodium pumps in the activated gland transport Na+ into the lateral intercellular space only. (ii) A barrier at the level of the basement membrane prevents the major fraction
of Na+ entering the lateral space from returning to the serosal bath. Thus, Na+ is secreted into the outside bath. It has to be assumed then that the Na+ permeability of the basement membrane barrier (P
BM
Na) is smaller than the Na+ permeability of the junctional membrane (P
JM
Na), i.e., P
JM
Na/P
BM
Na > 1. The secretory paracellular flow of water further requires that the Na+ reflection coefficients (σNa) of the two barriers are governed by the conditions, σBM
Na > 0, and σBM
Na > σJM
Na. (iii) Na+ channels are located in the apical membrane of the activated gland cells, so that a fraction of the Na+ outflux appearing downstream the lateral intercellular space is recirculated by the gland cells. Based on measured unidirectional
fluxes, a set of equations is developed from which we estimate the ion fluxes flowing through major pathways during stationary
secretion. It is shown that 80% of the sodium ions flowing downstream the lateral intercellular space is recycled by the gland
cells. Our calculations also indicate that under the conditions prevailing in the present experiments 1.8 ATP molecule would
be hydrolyzed for every Na+ secreted to the outside bath.
Received: 30 January 1996/Revised: 12 March 1996 相似文献
19.
Nigericin is an ionophore commonly used at the end of experiments to calibrate intracellularly trapped pH-sensitive dyes.
In the present study, we explore the possibility that residual nigericin from dye calibration in one experiment might interfere
with intracellular pH (pH
i
) changes in the next. Using the pH-sensitive fluorescent dye 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), we measured
pH
i
in cultured rat renal mesangial cells. Nigericin contamination caused: (i) an increase in acid loading during the pH
i
decrease elicited by removing extracellular Na+, (ii) an increase in acid extrusion during the pH
i
increase caused by elevating extracellular [K+], and (iii) an acid shift in the pH
i
dependence of the background intracellular acid loading unmasked by inhibiting Na-H exchange with ethylisopropylamiloride
(EIPA). However, contamination had no effect on the pH
i
dependence of Na-H exchange, computed by adding the pH
i
dependencies of total acid extrusion and background acid loading. Nigericin contamination can be conveniently minimized by
using a separate line to deliver nigericin to the cells, and by briefly washing the tubing with ethanol and water after each
experiment.
Received: 14 October 1998/Revised: 2 March 1999 相似文献
20.
J.I. Kourie 《The Journal of membrane biology》1998,164(1):47-58
The lipid bilayer technique was used to examine the effects of the ATP-sensitive K+ channel inhibitor (glibenclamide) and openers (diazoxide, minoxidil and cromakalim) and Cl− channel activators (GABA and diazepam) on two types of chloride channels in the sarcoplasmic reticulum (SR) from rabbit skeletal
muscle. Neither diazepam at 100 μm nor GABA at 150 μm had any significant effect on the conductance and kinetics of the 75 pS small chloride (SCl) channel.
Unlike the 150 pS channel, the SCl channel is sensitive to cytoplasmic glibenclamide with K
i
∼ 30 μm. Glibenclamide induced reversible decline in the values of current (maximal current amplitude, I
max and average mean current, I′) and kinetic parameters (frequency of opening F
o
, probability of the channel being open P
o
and mean open time, T
o
, of the SCl channel. Glibenclamide increased mean closed time, T
c
, and was a more potent blocker from the cytoplasmic side (cis) than from the luminal side (trans) of the channel.
Diazoxide increased I′, P
o
, and T
o
in the absence of ATP and Mg2+ but it had no effect on I
max and also failed to activate or remove the glibenclamide- and ATP-induced inhibition of the SCl channel. Minoxidil induced
a transient increase in I′ followed by an inhibition of I
max, whereas cromakalim reduced P
o
and I′ by increasing channel transitions to the closed state and reducing T
o
without affecting I
max. The presence of diazoxide, minoxidil or cromakalim on the cytoplasmic side of the channel did not prevent [ATP]
cis
or [glibenclamide]
cis
from blocking the channel.
The data suggest that the action(s) of these drugs are not due to their effects on the phosphorylation of the channel protein.
The glibenclamide- and cromakalim-induced effects on the SCl channel are mediated via a ``flicker' type block mechanism.
Modulation of the SCl channel by [diazoxide]
cis
and [glibenclamide]
cis
highlights the therapeutic potential of these drugs in regulating the Ca2+-counter current through this channel.
Received: 2 September 1997/Revised: 20 March 1998 相似文献