Perinatal PTX-sensitive G-protein expression and regulation of conductive 22Na+ transport in lung apical membrane vesicles.
Using apical membrane vesicles (AMV) prepared from mature foetal and early neonatal guinea pig lung we show that pertussis toxin (PTX)-sensitive G-protein regulation of conductive 22Na+ uptake undergoes rapid changes following birth. Thus, G-protein activation by intravesicular incorporation of 100 microM GTPgammaS into vesicles resuspended in NaCl, which in late gestation stimulated uptake, consistently induced inhibition of conductive Na+ uptake into AMV prepared from neonatal lung at 4 days of age (N4) (52+/-9%, n=8, P<0.05). This response was not significantly different in the presence of the relatively impermeant anion isethionate (Ise-) (69+/-9%, n=7, P<0.05). Changes in the regulation of uptake were already detectable on the day of birth (N0) in AMV resuspended in NaCl, with GTPgammaS inducing both stimulatory and inhibitory responses. These data indicate that the processes by which 22Na+ uptake into AMV is regulated by G-proteins undergoes a change at birth and by 4 days of age, G-protein regulation of uptake occurs predominantly via modulation of co-localised Na+ channels. Intravesicular incorporation of GDPbetaS or pre-treatment with PTX did not significantly alter conductive 22Na+ uptake in the presence of NaCl or NaIse suggesting that constitutively active G-proteins are not involved in this process. Pre-treatment of AMV with PTX prevented the inhibition of conductive 22Na+ uptake by GTPgammaS (105+/-16% n=7) indicating that a PTX-sensitive G-protein mediates the inhibition of channels in neonatal AMV. Western blotting demonstrated enrichment of Gialpha1, Gialpha2, Gialpha3 and Goalpha in the apical membrane preparations. We also show that there is a significant rise in the levels of Gialpha3 during the early neonatal period providing a potential candidate for the G-protein mediated changes in regulation of conductive 22Na+ uptake in neonatal AMV. (+info)
RNAs that bind and change the permeability of phospholipid membranes.
The RNA world hypothesis presumes that RNA will be competent for varied essential cellular functions. One such indispensable cell function is regulation of membrane permeability. Though this was not a known RNA activity, selection-amplification yielded RNAs that bound phosphatidylcholine:cholesterol liposomes. At least eight distinct, approximately 95-mer sequences bind well to the outside of the lipid bilayer, though randomized sequences had no such activity. No distinct sequence motif for lipid binding was found. However, truncation of one such RNA shows that a smaller, 44-nucleotide irregular RNA hairpin is an active membrane binding domain. Bound RNA increases the permeability of liposomes to (22)Na(+). In addition, using voltage clamp technique, four individual RNAs increase the ion permeability of the plasma membrane of cultured human cells. The existence of multiple sequences that bind membranes and provoke permeability changes suggests that these may be elementary RNA functions that could be selected in vivo. (+info)
Modelling cortical cataractogenesis XXIV: uptake by the lens of glutathione injected into the rat.
PURPOSE: Work of several groups including ours has shown that injection of glutathione may help to prevent the formation of cataract in the rat lens both in vitro and in vivo. These experiments were initiated to investigate the mechanism by which injected glutathione reaches the lens in vivo. The route is uncertain, but might involve either aqueous or vitreous humors, in contact with the lens anterior and posterior, respectively. Kannan's work has indicated that glutathione can be taken up ex vivo from the aqueous, by perfused isolated lens, but has not investigated; (1) whole animal glutathione injections, (2) the relative proportion of reduced and oxidized glutathione, and (3) the possibility that uptake can occur from the vitreous (in contrast to the aqueous humor) route. METHODS: 3H- or 35S-glutathione was injected into rats intraperitoneally and the radioactivity in serum and lens homogenates followed. RESULTS: The 3H-radioactivity reached a peak in the serum approximately 20-30 min after injection. Counts were also found in the lens, aqueous and vitreous humors. HPLC using a C18 Bondapak column (37 x 300 mm) indicated that the majority of the 3H-radioactivity in the lens was found in a component of a lower molecular weight than glutathione, but 8.1% of the counts occurred in the peak corresponding to reduced glutathione. Analysis of the unidentified radioactive component revealed a mobility the same as that of a dipeptide. Further analysis suggested this contained the amino acids cysteine and glycine bound in peptide linkage. These results suggest that glutathione may be degraded by the gamma-glutamyl cycle, and the action of transpeptidase produced cysteinylglycine. To confirm these results, similar experiments were undertaken using 35S-glutathione injection, to test whether a differently labelled form would be able to enter the lens. Homogenates prepared from the lens 20 min after 35S-glutathione injection were fractionated by HPLC. The glutathione peak contained 4.5% of the radioactivity in the lens extract. This amount was similar in quantity to the value for 3H-glutathione uptake by the lens. The average of the two values indicated that 6.3% of the total lens label was glutathione. The source of the labelled glutathione taken up by the lens was investigated by determining its concentration in the aqueous and vitreous humors and serum. The dipeptide appeared to be the major radiolabelled form occurring in the serum. This may explain its high level in the lens, as a result of uptake from other sources. Analysis using HPLC revealed that reduced glutathione (GSH) was the predominant chemical species of glutathione in the aqueous humor. In the vitreous humor, oxidized glutathione (GSSG) was the major species. The ratio of GSSG:GSH in the vitreous varied between 2:1 and 4:1. CONCLUSIONS: Over a 4 h period the lens could obtain 12.3% of its total GSH from the injected GSH, using the specific activity of the labelled glutathione to calculate the actual uptake of glutathione by the lens, suggesting a half-time of 16.25 h for replenishing GSH from external sources. The probable route of glutathione entry was by blood plasma and aqueous since the specific activity of the vitreous humor was too low for the vitreous to be a possible source of the lens GSH. (+info)
Kinetic and pharmacological properties of human brain Na(+)/H(+) exchanger isoform 5 stably expressed in Chinese hamster ovary cells.
The recently cloned Na(+)/H(+) exchanger isoform 5 (NHE5) is expressed predominantly in brain, yet little is known about its functional properties. To facilitate its characterization, a full-length cDNA encoding human NHE5 was stably transfected into NHE-deficient Chinese hamster ovary AP-1 cells. Pharmacological analyses revealed that H(+)(i)-activated (22)Na(+) influx mediated by NHE5 was inhibited by several classes of drugs (amiloride compounds, 3-methylsulfonyl-4-piperidinobenzoyl guanidine methanesulfonate, cimetidine, and harmaline) at half-maximal concentrations that were intermediate to those determined for the high affinity NHE1 and the low affinity NHE3 isoforms, but closer to the latter. Kinetic analyses showed that the extracellular Na(+) dependence of NHE5 activity followed a simple hyperbolic relationship with an apparent affinity constant (K(Na)) of 18.6 +/- 1.6 mM. By contrast to other NHE isoforms, NHE5 also exhibited a first-order dependence on the intracellular H(+) concentration, achieving half-maximal activation at pH 6.43 +/- 0.08. Extracellular monovalent cations, such as H(+) and Li(+), but not K(+), acted as effective competitive inhibitors of (22)Na(+) influx by NHE5. In addition, the transport activity of NHE5 was highly dependent on cellular ATP levels. Overall, these functional features distinguish NHE5 from other family members and closely resemble those of an amiloride-resistant NHE isoform identified in hippocampal neurons. (+info)
Betaxolol, a beta(1)-adrenoceptor antagonist, reduces Na(+) influx into cortical synaptosomes by direct interaction with Na(+) channels: comparison with other beta-adrenoceptor antagonists.
Betaxolol, a beta(1)-adrenoceptor antagonist used for the treatment of glaucoma, is known to be neuroprotective in paradigms of ischaemia/excitotoxicity. In this study, we examined whether betaxolol and other beta-adrenoceptor antagonists interact directly with neurotoxin binding to sites 1 and 2 of the voltage-sensitive sodium channel (Na(+) channel) in rat cerebrocortical synaptosomes. Betaxolol inhibited specific [(3)H]-batrachotoxinin-A 20-alpha-benzoate ([(3)H]-BTX-B) binding to neurotoxin site 2 in a concentration-dependent manner with an IC(50) value of 9.8 microM. Comparison of all the beta-adrenoceptor antagonists tested revealed a potency order of propranolol>betaxolol approximately levobetaxolol>levobunolol approximately carteolol>/=timolol>atenolol. None of the drugs caused a significant inhibition of [(3)H]-saxitoxin binding to neurotoxin receptor site 1, even at concentrations as high as 250 microM. Saturation experiments showed that betaxolol increased the K(D) of [(3)H]-BTX-B binding but had no effect on the B(max). The association kinetics of [(3)H]-BTX-B were unaffected by betaxolol, but the drug significantly accelerated the dissociation rate of the radioligand. These findings argue for a competitive, indirect, allosteric mode of inhibition of [(3)H]-BTX-B binding by betaxolol. Betaxolol inhibited veratridine-stimulated Na(+) influx in rat cortical synaptosomes with an IC(50) value of 28. 3 microM. Carteolol, levobunolol, timolol and atenolol were significantly less effective than betaxolol at reducing veratridine-evoked Na(+) influx. The ability of betaxolol to interact with neurotoxin site 2 of the Na(+) channel and inhibit Na(+) influx may have a role in its neuroprotective action in paradigms of excitotoxicity/ischaemia and in its therapeutic effect in glaucoma. (+info)
Critical evaluation of the one- versus the two-channel model for the operation of the ATP synthase's F(o) motor.
The mechanism of converting an electrochemical gradient of protons or Na(+) ions across the membrane into rotational torque by the F(o) motor of the ATP synthase has been described by a two-channel model or by a one-channel model. Experimental evidence obtained with the F(o) motor from the Propionigenium modestum ATP synthase is described which is in accordance with the one-channel model, but not with the two-channel model. This evidence includes the ATP-dependent occlusion of one (22)Na(+) per ATP synthase with a mutated Na(+)-impermeable a subunit or the Na(+)(in)/(22)Na(+)(out) exchange which is not affected by modifying part of the c subunit sites with dicyclohexylcarbodiimide. (+info)
Chronic effect of parathyroid hormone on NHE3 expression in rat renal proximal tubules.
BACKGROUND: The most abundant Na+/H+ exchanger in the apical membrane of proximal tubules is the type 3 isoform (NHE3), and its activity is acutely inhibited by parathyroid hormone (PTH). In the present study, we investigate whether changes in protein abundance as well as in mRNA levels play a significant role in the long-term modulation of NHE3 by PTH. METHODS: Three groups of animals were compared: (1) HP: animals submitted to hyperparathyroidism by subcutaneous implantation of PTH pellets, providing threefold basal levels of this hormone (2.1 U. h-1); (2) control: sham-operated rats in which placebo pellets were implanted; (3) PTX: animals submitted to hypoparathyroidism by thyroparathyroidectomy followed by subcutaneous implantation of thyroxin pellets, which provided basal levels of thyroid hormone. After eight days, we measured bicarbonate reabsorption in renal proximal tubules by in vivo microperfusion. NHE3 activity was also measured in brush border membrane (BBM) vesicles by proton dependent uptake of 22Na. NHE3 expression was evaluated by Northern blot, Western blot and immunohistochemistry. RESULTS: Bicarbonate reabsorption in renal proximal tubules was significantly decreased in HP rats. Na+/H+ exchange activity in isolated BBM vesicles was 6400 +/- 840, 9225 +/- 505, and 12205 +/- 690 cpm. mg-1. 15 s-1 in HP, sham, and PTX groups, respectively. BBM NHE3 protein abundance decreased 39.3 +/- 8.2% in HP rats and increased 54.6 +/- 7.8% in PTX rats. Immunohistochemistry showed that expression of NHE3 protein in apical BBM was decreased in HP rats and was increased in PTX rats. Northern blot analysis of total kidney RNA showed that the abundance of NHE3 mRNA was 20.3 +/- 1.3% decreased in HP rats and 27. 7 +/- 2.1% increased in PTX. CONCLUSIONS: Our results indicate that the chronic inhibitory effect of PTH on the renal proximal tubule NHE3 is associated with changes in the expression of NHE3 mRNA levels and protein abundance. (+info)
Portal hypertension induces sodium channel expression in colonocytes from the distal colon of the rat.
Cellular mechanisms for Na(+) retention in portal hypertension are undefined, but epithelial Na(+) channels (ENaC) may be involved. Under high-salt diet, ENaC are absent from distal colon of rat but can be induced by mineralocorticoids such as aldosterone. Presence of rat ENaC was determined by amiloride inhibition of (22)Na(+) uptake in surface colonocytes 7 and 14 days after partial portal vein ligation (PVL) or sham surgery. At both times, uptake inhibition was significantly increased in PVL rats. Presence of mRNA transcripts, determined by RT-PCR, demonstrated that channel alpha- and gamma-subunits were similarly expressed in both groups but that beta-subunit mRNA was increased in PVL rats. This confirms that there was induction of rat ENaC and indicates that beta-subunit has a regulatory role. Urinary Na(+) was decreased for 3 days after PVL but was not different at other times, and serum aldosterone levels were elevated at 7 days, at a time when urinary Na(+) output was similar to that of sham-operated rats. We conclude that PVL leads to induction of ENaC in rat distal colon. An increase in aldosterone levels may prevent natiuresis and is probably one of several control mechanisms involved in Na(+) retention in portal hypertension. (+info)