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H Garty

Publications and source records attributed to H Garty.

At least 37 records · Page 2Linked to original sources
PubMed1995

A corticosteroid-induced gene expressing an "IsK-like" K+ channel activity in Xenopus oocytes.

Screening a rat colon cDNA library for aldosterone-induced genes resulted in the molecular cloning of a cDNA whose corresponding mRNA is strongly induced in the colon by dexamethasone, aldosterone, and a low NaCl diet. A similar mRNA was detected in kidney papilla but not in brain, heart, or skeletal muscle. Xenopus laevis oocytes injected with cRNA synthesized from this clone, designated CHIF (channel-inducing factor), express a K(+)-specific channel activity. The biophysical, pharmacological, and regulatory characteristics of this channel are very similar to those reported before for IsK (minK). These include: slow (tau > 20 s) activation by membrane depolarization with a threshold potential above -50 mV, blockade by clofilium, inhibition by phorbol ester, and activation by 8-bromoadenosine 3',5'-cyclic monophosphate and high cytoplasmic Ca2+. The primary structure of this clone, however, shows no homology to IsK. Instead, CHIF exhibits > 50% similarity to two other short bitopic membrane proteins, phospholemman and the gamma subunit of Na+K(+)-ATPase. The data are consistent with the possibility that CHIF is a member of a family of transmembrane regulators capable of activating endogenous oocyte transport proteins.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Dexamethasone enhances expression of mitochondrial oxidative phosphorylation genes in rat distal colon.

Dexamethasone and aldosterone are major activators of Na+ reabsorption in tight epithelia. The genes whose expression mediates the steroid actions are mostly unknown. To identify such genes, we performed differential screening of a rat colon cDNA library with total 32P-labeled cDNA probes reverse transcribed from steroid-stimulated and steroid-depleted poly(A)+ RNA. Several cDNAs whose corresponding mRNA is enhanced two- to threefold after dexamethasone injection were identified. Partial sequencing indicated that four of them code for subunits of cytochrome-c oxidase and 16S mitochondrial mRNA. The dexamethasone-induced increase in mitochondrial RNA abundance could not be mimicked by a low-salt diet, found to increase plasma aldosterone from 1.0 +/- 0.1 to 12.8 +/- 1.4 nM. Induction of mitochondrial genes by adrenal steroids may serve to prevent limitation of transport by the ATP supply to the Na(+)-K+ pump under conditions of maximal stimulation of Na+ transport.

Animals↗
PubMed1994

Molecular properties of epithelial, amiloride-blockable Na+ channels.

The apical membrane of many tight epithelia contains Na+ channels that are primarily characterized by their high affinity to the diuretic blocker amiloride. These channels mediate the first step of active Na+ reabsorption essential for the maintenance of body salt and water homeostasis. They are regulated by mineralocorticoids, antidiuretic peptides, atrial natriuretic peptides, and other factors. The molecular events that mediate the hormonal actions are poorly understood. In addition, patch clamp studies have established that amiloride-sensitive channels in different epithelia may differ in their regulatory mechanisms and biophysical properties. Several groups have reported the biochemical purification and/or molecular cloning of putative channel components. Of particular importance is the recent cloning of three cDNAs, whose coexpression in Xenopus oocytes evokes a large amiloride-blockable Na+ specific conductance (Canesa et al. (1994) Nature (London), 367, 463-467. This review summarizes existing data on properties, regulatory mechanisms, and diversity of amiloride-blockable channels, describes the different putative channel components identified, and examines possible relationships among them.

Amiloride↗
PubMed1994

Effects of corticoid agonists and antagonists on apical Na+ permeability of toad urinary bladder.

Effects of RU-28362 (glucocorticoid agonist), RU-38486 (glucocorticoid antagonist), and RU-26752 (mineralocorticoid antagonist) on the apical Na+ permeability of toad bladder were measured and correlated with occupancies of cytosolic type I (mineralocorticoid) and type II (glucocorticoid) receptors. Effects of the above steroids were measured in whole bladders, plasma membrane vesicles, and RNA-injected Xenopus oocytes. RU-38486 was found to fully displace aldosterone from type II receptors without affecting type I occupancy. Under these conditions, RU-38486 inhibited approximately 35% of the effect of aldosterone measured in the whole tissue and isolated membranes. Unexpectedly, oocytes injected with RNA from tissue stimulated with aldosterone plus RU-38486 expressed channel activity that was much higher than the sum of activities induced by either steroid alone. RU-28362 and RU-26752 at concentrations sufficient to fully occupy both receptors had only partial agonistic and antagonistic effects, respectively. The results suggest that at least one-third of the natriferic action of aldosterone measured in the amphibian urinary bladder is mediated by the glucocorticoid receptor. However, some of the effects observed cannot be accounted for by a simple receptor occupancy-response scheme.

Adrenal Cortex Hormones↗
PubMed1993

An epithelial high-affinity amiloride-binding site, different from the Na+ channel.

Specific binding of the radioactive amiloride analogues [3H]phenamil and [3H]benzamil was studied in plasma membrane from chicken lower intestine. A single population of sites whose affinities and specificities towards pyrazinecarboxamides roughly resemble those of the epithelial Na+ channel, was identified. However, a matched comparison of pyrazinecarboxamide binding and Na+ transport inhibition revealed substantial differences between the high-affinity [3H]phenamil-binding site detected, and the site whose occupancy by phenamil blocks Na+ transport. First, 5-(N-ethyl-N-isopropyl)-amiloride was found to displace bound [3H]phenamil at concentrations that are at least 10-fold lower than those needed to block the channel. Second, the rates at which [3H]phenamil associates and dissociates from this site are lower than the rates at which Na+ channels are inhibited and reactivated, under similar conditions. A site with high affinity to both amiloride and 5-(N-ethyl-N-isopropyl)-amiloride was detected also in membranes from other epithelia. We conclude that tight epithelia contain a major high-affinity amiloride receptor other than the Na(+)-conducting channel, the Na+/H+ antiport or the Na+/Ca2+ exchanger. This site could be associated with a pool of nonconducting channels, another (but structurally related) channel, or a totally unrelated protein.

Aldosterone↗
PubMed1992

Expression of amiloride-sensitive Na+ channels of hen lower intestine in Xenopus oocytes: electrophysiological studies on the dependence of varying NaCl intake.

Epithelial Na+ channels were incorporated into the plasma membrane of Xenopus laevis oocytes after micro-injection of RNA from hen lower intestinal epithelium (colon and coprodeum). The animals were fed either a normal poultry food which contained NaCl (HS), or a similar food devoid of NaCl (LS). Oocytes were monitored for the expression of amiloride-sensitive sodium channels by measuring membrane potentials and currents. Oocytes injected with poly(A)+RNA prepared from HS animals or non-injected control oocytes showed no detectable sodium currents, whereas oocytes injected with LS-poly(A)+RNA had large amiloride-blockable sodium currents. These currents were almost completely saturated by sodium concentrations of 20 mM with a Km of about 2.6 mM sodium. Amiloride (10 microM) inhibits the expressed sodium channels entirely and examination of dose response relationships yielded a half-maximal inhibition concentration (Ki) of 120 nM amiloride. I-V difference curves in the presence or absence of sodium or amiloride (10 microM) indicate a potential dependence of the sodium transport which can be described by the Goldman equation. When Na+ is replaced by K+, no amiloride response was detected indicating a high selectivity for Na+ over K+. These results provide strong evidence that intestinal Na+ channels are regulated by dietary salt intake on the RNA level.

Amiloride↗
PubMed1992

Expression of the amiloride-blockable Na+ channel by RNA from control versus aldosterone-stimulated tissue.

The amiloride-blockable Na+ channel was expressed in Xenopus oocytes injected with total RNA isolated from the toad urinary bladder. This system was used to investigate mechanisms that mediate the natriferic action of aldosterone. Incubation of the epithelium with aldosterone for 3 h doubled its channel activity but did not increase the ability of isolated RNA to express functional channels in oocytes. A 20-h incubation with the hormone produced an additional increase of Na+ transport across the intact epithelium and also augmented the channel activity expressed in oocytes by nearly 10-fold. The data are in agreement with our model that aldosterone enhances the apical Na+ permeability of tight epithelia by a short term activation of pre-existing channels, followed by chronic induction of new channel protein. Blocking methyl transfer reactions, previously shown to inhibit the natriferic action of aldosterone in tight epithelia, did not alter the basal or aldosterone-induced response in oocytes.

Aldosterone↗
PubMed1992

NaCl-dependent expression of amiloride-blockable Na+ channel in Xenopus oocytes.

RNA was isolated from chicken lower intestine (both colon and coprodeum) and injected into Xenopus oocytes. 22Na+ fluxes measured after 1-4 days demonstrated the induction of an amiloride-blockable pathway. The Na+ transporter expressed by the exogenous RNA had a high affinity to amiloride (inhibitory constant less than 0.1 microM), but was insensitive to ethylisopropyl amiloride, i.e., it is likely to be the apical Na+ channel. Functional channels were readily expressed in oocytes injected with RNA derived from chickens fed a low-NaCl diet. On the other hand, no channel activity was detected in oocytes injected with RNA isolated from chickens fed a high-NaCl diet. Thus the previously reported regulation of transport by the dietary NaCl intake involves modulations in the level of mRNA that codes either for the Na+ channel or a posttranscriptional regulator of the channel.

Amiloride↗
PubMed1991

Sorbitol permease: an apical membrane transporter in cultured renal papillary epithelial cells.

The efflux of sorbitol from the rabbit papillary epithelial cell line PAP-HT25 occurs through a specific transport pathway, which we denote the "sorbitol permease." The permease was studied by measuring cell volume changes that accompanied osmotic swelling and by determination of the sorbitol efflux from plasma membrane vesicles. The cell volume studies showed that sorbitol efflux in response to hypotonicity occurred only across the apical membrane of the cells and that loss of sorbitol was the primary mechanism for regulatory volume decrease (RVD) by these cells. Quinidine, a permeant inhibitor of the sorbitol permease, was shown to prevent RVD when added to either apical or basolateral bathing solution. Cell volume experiments also showed that the permease was present only on the apical membrane of cells that had been grown in isotonic medium and did not accumulate sorbitol. The permease could be demonstrated in membrane vesicles obtained from cells exposed to a hypotonic environment before being homogenized. Quinidine blocked the sorbitol efflux from vesicles indicating that it either directly inhibited the permease or a membrane-associated activation step.

Animals↗
PubMed1990

Toad urinary bladder as a model for studying transepithelial sodium transport.

Sodium ion transport across tight epithelia has been investigated particularly extensively by studying two model systems: the urinary bladder of the toad and the frog skin. The greatest advantage presented by these models is the capability of monitoring net transepithelial Na+ flux simply, precisely, and instantaneously by measurement of the short circuit current (ISC). Many of the caveats involved in the measurement are discussed in detail. In order to fully characterize the forces driving Na+ movement across the series apical and basolateral membranes, it is necessary to measure intracellular potential and ionic composition. Such measurements are far more easily conducted with frog skin than with toad bladder, using the major biophysical techniques currently available. Regulation of transepithelial Na+ movement across tight epithelia is largely conducted at the apical membranes. This regulation can be clarified by study of the isolated Na+ channels in membrane vesicles. Such vesicles are far more easily prepared from toad urinary bladder than from frog skin. The strengths and potential misappropriations of this technique are considered in detail.

Animals↗
PubMed1989

Ion channel-mediated fluxes in membrane vesicles: selective amplification of isotope uptake by electrical diffusion potentials.

The procedure we have described provides a simple, convenient, and sensitive method to assay conductive ion fluxes in membrane vesicles. It is particularly useful for detecting channels in heterogeneous populations of vesicles. The principal advantages are similar to those of sensitive enzyme assays, namely, screening for existence of channels in different membrane fractions, assaying purified channel proteins, large-scale testing of pharmacological agents, antibodies, etc. and in studies of macroscopic regulatory features, including channel activity or density in different states and interaction with regulatory ligands. In the future one can expect further applications in detecting synthesis of channel proteins, gene expression, etc. The tracer assay does not provide much information on molecular characteristics such as single-channel conductance, voltage sensitivity, and ion specificity. It therefore serves other purposes to those of the modern biophysical methods such as patch-clamp, noise analysis, and study of channels incorporated into bilayers.

Amiloride↗
PubMed1989

Guanosine nucleotide-dependent activation of the amiloride-blockable Na+ channel.

Effects of guanosine nucleotides on the epithelial Na+ channel were studied in apical membrane vesicles derived from the toad bladder epithelium. Trapping 10 microM guanosine-5'-O-(thiotriphosphate) (GTP gamma S) in vesicles evoked two- to fourfold increase in the amiloride-sensitive (Na+ channel-mediated) 22Na+ uptake. The nucleotide had no significant effect on the amiloride-insensitive 22Na+ uptake or the valinomycin-mediated 86Rb+ uptake in the same membranes. The stimulatory action of GTP gamma S was mimicked by 5'-guanylylimidiodiphosphate (GppNHp) and could at least partly be reversed by guanosine-5'-O-(thiodiphosphate) (GDP beta S) (10-fold excess). GTP itself and adenosine-5'-O-(thiotriphosphate) (ATP gamma S) had no sustained effect on Na+ transport in vesicles. Thus it appears that the epithelial Na+ channel is directly or indirectly regulated by the occupancy of a guanosine-specific site, probably the alpha subunit of a G protein. The possibility that GTP gamma S acts indirectly by activating a membrane-bound, GTP-dependent enzyme the product of which modulates the channel conductance was assessed by measuring 22Na+ fluxes in membrane vesicles prepared to contain products of such enzymes. None of the reagents tested [adenosine 3',5' cyclic monophosphate (cAMP), guanosine 3',5' cyclic monophosphate (cGMP), inositol 1,4,5-trisphosphate (IP3), and diacylglycerol (DAG)] increased the tracer flux in vesicles or altered its response to GTP gamma S.

Amiloride↗
PubMed1988

Conductive sodium pathway with low affinity to amiloride in LLC-PK1 cells and other epithelia.

Electrical potential driven 22Na+ fluxes were measured in membrane vesicles prepared from a number of cultured and naturally occurring epithelia. In all preparations a rheogenic pathway blocked by 200 microM (but not by 1.5 microM) amiloride was noted. This transporter was characterized in membranes prepared from cultured LLC-PK1 cells. In this preparation more than 50% of the rheogenic 22Na+ uptake was blocked by amiloride (IC50 approximately 30 microM), phenamil (IC50 approximately 66 microM), or ethylisopropylamiloride (IC50 approximately 5 microM). This amiloride-sensitive flux was not seen if the vesicles were partially depolarized by external Na+ or K+. It could not be driven by a pH gradient, did not require the presence of Ca2+, sugars, or amino acids, and showed little dependence on temperature (25 versus 0 degrees C). The data suggest the existence of an epithelial amiloride-blockable Na+ transporter different from the previously characterized Na+ channel, Na+/H+ and Na+/Ca2+ exchangers, and the Na+-hexose co-transporter. In rat kidney cortex membranes prepared by Mn2+ precipitation, this transporter is primarily located in the brush-border fraction.

Amiloride↗
PubMed1988

Sodium-dependent inhibition of the epithelial sodium channel by an arginyl-specific reagent.

Effects of the arginyl- and lysyl-specific reagent phenylglyoxal (PGO) on the epithelial Na+ channel were evaluated by measuring the amiloride-blockable 22Na+ fluxes in membrane vesicles derived from the toad bladder epithelium. Incubating whole cells or isolated membranes with PGO readily and irreversibly blocked the channel-mediated tracer flux. Na+ ions present during the interaction of membranes with PGO could protect channels from inactivation by PGO. This effect required the presence of Na+ at the luminal side of the membrane and was characterized by an IC50 of 79 mM Na+. Amiloride, too, could desensitize channels to PGO, but its effect was significant only when whole cells were interacted with the protein-modifying reagent. The data are compatible with a model in which the conductive path of the channel contains a functional arginine, possibly forming a salt bridge with a carboxylic group, which is involved in Na+ translocation and amiloride binding. It was also shown that the augmentation of transport induced by incubating whole cells in Ca2+-free solution (Garty, H., and Asher, C. (1985) J. Biol. Chem. 260, 8330-8335) involves the activation or recruitment of channels that are not vulnerable to PGO prior to incubation.

Aldehydes↗
PubMed1988

G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na+ channel interaction.

Our previous experiments in membranes prepared from rat heart and brain led us to suggest that the binding of agonists to the muscarinic receptors and to the Na+ channels is a coupled event mediated by guanine nucleotide binding protein(s) [G-protein(s)]. These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of [3H]acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of [3H]batrachotoxin to Na+ channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced 22Na+ uptake in the presence and absence of tetrodotoxin, which blocks Na+ channels. Our findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na+ channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components--receptor, G-protein, and Na+ channel--is such that at resting potential the muscarinic receptor induces opening of Na+ channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues.

Acetylcholine↗
PubMed1988

Aldosterone increases the apical Na+ permeability of toad bladder by two different mechanisms.

The aldosterone-induced augmentation of Na+ transport in toad bladder was analyzed by comparing the hormonal actions on the transepithelial short-circuit current and on the amiloride-sensitive 22Na+ uptake in isolated membrane vesicles. Incubating bladders with 0.5 microM aldosterone for 3 hr evoked more than a 2-fold increase of the short-circuit current (because of the activation or insertion of apical amiloride-blockable channels) but had no effect on the amiloride-sensitive Na+ transport in apical vesicles derived from the treated tissue. A longer incubation (e.g., 6 hr) produced an additional augmentation of the short-circuit current, which was accompanied by about a 3-fold increase of the channel activity in isolated membranes. The stimulatory effect of aldosterone sustained in vesicles was inhibited by the antagonist spironolactone (present at 1000-fold excess) and the protein synthesis inhibitor cycloheximide (1 microM). In addition, triiodothyronine and butyrate, previously reported to partly inhibit the aldosterone-induced increase in short-circuit current, blocked the hormonal effect in vesicles. It is suggested that aldosterone elevates the apical Na+ permeability of target epithelia by two different mechanisms: a relatively fast effect (less than or equal to 3 hr), which is insensitive to triiodothyronine or butyrate and is not sustained by the isolated membrane, and a slower or later (greater than 3 hr) response blocked by these reagents, which is preserved by the isolated membrane. The data also indicate that these processes are mediated by different nuclear receptors.

Aldosterone↗
PubMed1988

Na+ uptake into colonic enterocyte membrane vesicles.

Na+ uptake was studied in colonic enterocyte membrane vesicles prepared from normal and dexamethasone-treated rats. Vesicles from rats treated with dexamethasone demonstrated a fivefold greater 22Na+ uptake compared with vesicles from normal rats. Most of the tracer uptake in membranes derived from treated rats occurred through a conductive, amiloride-blockable pathway located in vesicles with low native K+ permeability and high Cl- permeability. Kinetic analysis of the amiloride inhibition curve revealed the presence of two amiloride-blockable pathways, one with a high affinity (Ki = 9 +/- 1.8 nM), accounting for 85% of the uptake, and one with a low affinity (Ki = 2.2 +/- 0.71 microM), accounting for only 12% of the uptake. Only the low-affinity pathway was detected with vesicles from normal rats. The high sensitivity to amiloride, the dependence on dexamethasone pretreatment, and the relative permeabilities to K+ and Cl- indicate that most of the 22Na+ uptake in membranes derived from treated rats is through a Na+-specific channel located in apical membrane vesicles. Preincubation of the isolated cells from dexamethasone-treated rats at 37 degrees C in Ca2+-free solutions before homogenization and membrane vesicle purification caused a 5- to 10-fold increase in amiloride-blockable 22Na+ uptake compared with vesicles derived from cells maintained at 0 degrees C. The addition of Ca2+, but not of Mg2+, to the incubation solution markedly reduced this temperature-dependent enhancement in 22Na+ uptake. The uptake of 22Na+ into vesicles from normal rats was unaffected by preincubation at 37 degrees C or the addition of Ca+ to the incubation solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗