Evidence of a parathyroid hormone-independent chronic effect of estrogen on renal phosphate handling and sodium-dependent phosphate cotransporter type IIa expression.

The effects of estrogen on phosphate metabolism are not well understood. To better define the chronic effects of estrogen on phosphate balance and on renal phosphate handling, the following groups were examined: A. young male and female rats, age- and weight-matched (age 8-10 weeks, 1 (st) study), and B. ovariectomized female rats (OVX), 22 weeks old, ovariectomized aged-matched rats receiving estrogen replacement (15 micromol x 3/week) for 14 weeks (OVX+E), control female rats (intact ovaries), and male rats, both age matched to OVX and OVX+E (2 (nd) Study). In younger females (1 (st) study), plasma phosphate was lower, whereas the urinary excretion of phosphate was higher than in males. In adult intact females and in OVX+E urinary excretion of phosphate was higher than in males and OVX (2 (nd) Study). In these rats, a significant correlation between plasma phosphate and estrogen level was found. Sodium-dependent phosphate cotransporter (NaPiIIa) mRNA expression and protein abundance were higher in the renal cortex of younger male rats than in age- and weight-matched females. In adult rats, NaPiIIa mRNA and protein abundance were higher in OVX than in OVX+E, and in mature males as compared with age-matched females. These differences were not related to the parathyroid hormone (PTH) levels. Chronic estrogen administration was also associated with increased plasma calcium level and urinary calcium excretion. These results suggest that chronic estrogen treatment is associated with an inhibitory, PTH-independent effect on the expression of NaPiIIa in the kidney, leading to sex-related differences in phosphate balance.

Dietary phosphate-dependent growth is not mediated by changes in plasma phosphate concentration.

The present study was undertaken in order to assess the role of dietary phosphate in growth. A diet deficient in phosphate led to a suppression of growth in juvenile rats. The phenomenon is similar to that described for diets deficient in other essential single components such as Mg, Zn or K. However, unlike the other constituents, dietary phosphate restriction affected the growth rate much more than it altered the serum phosphate concentration; addition of phosphate to the drinking water of rats fed a low-phosphate diet restored the growth rate without a concomitant change in serum phosphate concentration. The suppression of growth rate by the deletion of phosphate was associated with a delayed decrease in food intake. This finding implies that the variation in appetite was secondary to the change in growth. The increase in body weight following phosphate supplementation was associated with a concomitant increment in food intake. The phosphate-dependent growth was, however, evident also in rats that were pair-fed with those that were not supplied with phosphate. It is concluded that dietary phosphate-dependent growth is not mediated by changes in phosphate concentrations in the extracellular fluid. It is plausible that signals arising from receptors for phosphate in the digestive system constitute part of the growth control apparatus in rats.

Nasal potential difference measurements in patients with atypical cystic fibrosis.

The diagnosis of cystic fibrosis (CF) is based on characteristic clinical and laboratory findings. However, a subgroup of patients present with an atypical phenotype that comprises partial CF phenotype, borderline sweat tests and one or even no common cystic fibrosis transmembrane conductance regulator (CFTR) mutations. The aim of this study was to evaluate the role of nasal potential difference (PD) measurements in the diagnosis of CF patients with an atypical presentation and in a population of patients suspected to have CF. Nasal PD was measured in 162 patients from four different groups: patients with classical CF (n = 31), atypical phenotype (n = 11), controls (n = 50), and patients with questionable CF (n = 70). The parameter, or combination of nasal PD parameters was calculated in order to best discriminate all CF patients (including atypical CF) from the non-CF group. The patients with atypical CF disease had intermediate values of PD measurements between the CF and non-CF groups. The best discriminate model that assigned all atypical CF patients as CF used: e(response to chloride-free and isoproterenol/response to amiloride) with a cut-off >0.70 to predict a CF diagnosis. When this model was applied to the group of 70 patients with questionable CF, 24 patients had abnormal PD similar to the atypical CF group. These patients had higher levels of sweat chloride concentration and increased rate of CFTR mutations. Nasal potential difference is useful in diagnosis of patients with atypical cystic fibrosis. Taking into account both the sodium and chloride transport elements of the potential difference allows for better differentiation between atypical cystic fibrosis and noncystic fibrosis patients. This calculation may assist in the diagnostic work-up of patients whose diagnosis is questionable.

Sodium-dependent transport of phosphate in neuronal and related cells.

Sodium-dependent phosphate entry into neuronal cells was demonstrated in synaptic plasma membrane vesicles and synaptosomes prepared from rat brains, in PC12 cells and in primary culture of pituitary cells. The extent of the sodium-dependent phosphate transport in the synaptic plasma membrane preparation, at [Na]out = 110 mM and [P(i)]out = 0.1 mM, varied between 0.28 to 1.02 nmol phosphate/mg membrane protein/min. In pituitary cells the value was only about 0.05 nmol P(i)/mg protein/min. In PC12 cells the activity increased from 0.0085 to 0.26 nmol P(i)/mg protein/min in the transit from undifferentiated to differentiated cells. The dependence of phosphate on sodium concentrations fits a model in which two sodium ions are required to transfer the phosphate into the cells with a K[Na]0.5 of 43 mM. The K(m) for the phosphate transport in the synaptic plasma membrane preparations was between 0.1 and 0.45 mM. It is concluded that sodium-driven active transport of phosphate is a ubiquitous activity in various types of neuronal cells.

Phosphate load as the evolutionary driving force for the development of a high filtration rate based excretory organ: a hypothesis.

It is proposed that the nutritional load of phosphate is the primary factor that led to the development of an excretory organ that is based on a high filtration rate of the extracellular fluid. This hypothesis is based on the following arguments: 1) phosphate and potassium impose the highest average nutritional load rates defined as the ratio between the average ion content in foods that satisfies the energy demands of the body per unit time and the respective ionic plasma concentration. Due to the much higher intracellular than extracellular potassium concentration it is argued that a secretory based excretory organ would be more useful for disposal of potassium. 2) Magnesium and calcium nutritional load rates are lower than that of phosphate; in addition the disposal of magnesium should have been dealt with preferably by secretion due to its slightly higher intracellular concentration. The nutritional load rate of calcium is tempered by the inherently low absorption capacity of the intestinal mucosa. 3) In aglomerular teleost fishes the phosphate plasma concentration is significantly elevated vis a vis the mammals and other marine fishes, in comparison to only slight differences in calcium, magnesium and potassium levels. It is suggested that the seemingly peculiar and costly way in which sodium excretion is accomplished in the kidney represents the outcome of the solution to the phosphate nutritional load.

Modulation of rat olfactory bulb mitochondrial function by atrial natriuretic peptide.

Atrial Natriuretic Peptide (ANP) and receptors for ANP are widely distributed in many tissues and cell types in vertebrates. ANP has been shown to be internalized into the cytoplasm in several cell types and thus it raises the possibility that it may act on intracellular receptors. Displacement experiments of [125I]-ANP binding to rat olfactory bulb mitochondrial fraction demonstrated the presence of high affinity (Kd < 10(-9)M) binding sites (Bmax, 112 fmol/mg protein) in this preparation. The addition of ANP (10(-8) M) to this mitochondrial preparation resulted in a 25% increase in TPP+ accumulation, signifying a striking hyperpolarization of the mitochondrial membrane. In contrast ANP did not increase TPP+ uptake to liver mitochondrial preparations. This direct effect of ANP on Olfactory bulb mitochondrial membrane potential may underly the known effects of this hormone on steroidogenesis.

The pH dependence of the hemolytic potency of bile salts.

The membrane damaging potential of dilute solutions of bile salts was evaluated by monitoring continuously the hemolysis of a small sample of red blood cells (RBC) introduced into a defined media containing the bile salts at various pH values. The strength of the hemolytic bile salt was characterized by the rate of the induced hemolysis and by the time that elapsed between the introduction of the RBC sample into the bile salt containing solution and the onset of hemolysis. The potency of the unconjugated bile acids was extremely sensitive to pH, e.g. the rate of hemolysis caused by a 7.5 mM cholate was 1.5%, 20% and 64% per min when the pH of the solution was 7.65, 7.3 and 6.85, respectively. At low pH values the membrane damaging effects of deoxycholate was clearly discerned at micromolar concentration range. The hemolytic potency of glycodeoxycholate was also enhanced significantly by lowering the pH. The taurine-conjugated cholate and deoxycholate were only slightly sensitive to variations in pH. Taurocholate at concentrations that were not hemolytic greatly enhanced the injurious potency of deoxycholate. These results imply that in acidic solutions the presence of bile acids can cause damage to cell membranes. It is suggested that the acidic environment in the proximal duodenum and acidosis developed during hypoxia in the liver are two situations in which the bile salts may constitute a pathogenic factor.

Photoinduced electron transfer across lipid bilayers containing magnesium octaethylporphyrin.

Both photoinitiated (thermodynamically downhill) and photodriven (thermodynamically uphill) electron transfer reactions across lipid bilayers are sensitized by magnesium octaethyl porphyrin (MgOEP). It is shown that the reaction mechanism is via reduction of photoexcited MgOEP at the reducing (ascorbate) side of the bilayer and the charge carrier is likely the neutral protonated MgOEP anion. The MgOEP cation (or its neutral form) does not contribute to charge passage across the bilayer even though it is readily formed at the acceptor (ferricyanide or methyl viologen) side of the membrane. Photoelectric measurements on planar bilayers show that the time constant for reduction of excited MgOEP is about 10 microseconds with 10 mM ascorbate. The membrane transport of the mediator appears to be rate limiting when the reaction is photoinitiated and the interfacial reaction appears to be limiting when the reaction is photodriven. The quantum yield of the process is about 0.1 in the latter case and about 0.02 in the former. The former yield is increased to about 0.15 in the presence of a redox mediator, duroquinone. In these systems, the magnesium porphyrin is both sensitizer and trans membrane redox mediator.

Diffusion- and reaction rate-limited redox processes mediated by quinones through bilayer lipid membranes.

The mediation of redox reactions through bilayer lipid membranes was studied. With an appropriate choice of electron acceptors the redox process can be limited either by the chemical reaction rate between the mediator and the reactants or by the shuttle frequency of the mediator through the membrane. Both modes were demonstrated for redox reactions mediated by 2,6 dichlorobenzoquinone (DCBQ) and by alpha-tocopherol with ascorbate entrapped inside vesicles using ferricyanide (a mild oxidant) or hexachloroiridate (a strong oxidant) in the external solution. The redox processes were reaction rate-limited and diffusion-limited for ferricyanide and hexachloroiridate, respectively. The kinetics of the redox processes in the diffusion- and the reaction rate-limited modes allows the determination of the shuttle frequencies and of the interfacial reaction rates of the mediators, respectively. The shuttle frequencies of DCBQ and alpha-tocopherol were approximately 8 and 0.08 s-1, respectively, in L-alpha-dipalmitoyl phosphatidylcholine (DPPC) cholesterol vesicles at 25 degrees C. Interfacial reaction rates between the mediators and ferricyanide were about two- and tenfold lower compared with bulk reaction rates for DCBQ (water) and tocopherol (50% ethanol solution), respectively, i.e., tocopherol is relatively less accessible to aqueous oxidants at the membrane interface. Tocopherol and oxidized tocopherol are reversible hydrophobic redox couples that interact very rapidly with strong oxidants. In both modes of mediation DCBQ was more effective than alpha-tocopherol.

The effect of oxygen on the amplitude of photodriven electron transfer across the lipid bilayer-water interface.

The surprisingly small effect of oxygen on photoelectron transfer in pigmented lipid bilayers is traced to a short lifetime of the excited states. Decreasing the oxygen concentration by greater than 100-fold decreases the half saturating concentration of acceptor by only threefold and has no effect on the maximum photovoltage observed at acceptor saturation. This holds true for both magnesium octaethylporphyrin and chlorophyll with both ferricyanide and methyl viologen as acceptors. Since oxygen quenches excited states at near the encounter limit, the lifetime of reactive state must be short, less than 100 ns. About 100-fold higher concentrations of acceptor are required to quench the fluorescence (in liposomes) than to saturate the photoeffect. Thus the reactive state is most likely the triplet. The short life of the excited state is caused by concentration quenching, i.e., their reaction with ground state molecules. The increase of photovoltage with increasing pigment concentration shows that this quenching in a condensed form of the pigment produces ions that lead to the observed photovoltage by interfacial reaction of the anion with acceptor.

Comparison between bretylium and diphenylhydantoin interaction with mucosal sodium-channels.

The antifibrillatory drug bretylium and the antiepileptic drug diphenylhydantoin cause an increase in the potential different and in the short-circuit current (SCC) across frog skin when added to the outer surface. The effect of both drugs depends upon the presence of sodium ions in the outer medium and is blocked by the specific sodium channel blocker, amiloride. Quantitative analysis shows that amiloride binds to open as well as closed mucosal sodium channel with the same affinity. The effects of diphenylhydantoin and bretylium differ with respect to their dependence on external pH. The diphenylhydantoin or the bretylium stimulatory effects are additive to the effects of oxytocin. In most cases the diphenylhydantoin and bretylium effects are also additive. It is concluded that the external side of the mucosal Na+ channels contains sites which interact specifically with either bretylium or diphenylhydantoin and thus remove the sodium induced closure of the channels.

Genetic control of the organ specificity of lymphoproliferative disease virus (LPDV) of turkeys.

In a previous study based on the kinetics of virus replication and tumor formation (Gazit et al., 1982), it was shown that the organotropism of lymphoproliferative disease virus (LPDV) is confined to lymphoid tissues. The present paper demonstrates that this organ specificity is controlled at the level of infection and integration, that is, the lymphoid organs, which are the only organs sustaining virus replication, and also the only organs in whose cells integrated LPDV proviruses are detectable. At the same time, the efficiency of virus replication within the various target organs is regulated both at the level of infection and integration and at the level of viral gene transcription.

Bretylium opens mucosal amiloride-sensitive sodium channels.

Addition of the quanternary ammonium compound, bretylium, to the outer surface of a frog skin leads to an increase in the potential difference and in the short circuit current across the skin. Bretylium does not have any effect when applied to the inside face of the frog skin. The effect of bretylium is dependent upon the presence of sodium ions in the outer medium; it is depressed when sodium is replaced by choline or potassium but not when lithium substitutes for sodium. The bretylium effect is blocked by the specific sodium channel blocker, amiloride. It is proposed that bretylium opens mucosal, amiloride-sensitive sodium channels.

On the mechanism of osmotic phenomena.

When solute particles are distributed inhomogeneously in a solution due to the action of an external force, the pressure difference across a semipermeable membrane is determined by the concentration of the impermeant particles in the immediate vicinity of the membrane. In line with this conclusion, it is shown that in a Donnan system containing a porous membrane, the concentration of the impermeant polyelectrolyte at the membrane interface is equal to the difference between the osmolar concentrations of the solutions present on the two sides of the membrane. That is, the impermeant species are accumulated at the membrane interface in proportion to their density in the solution. The well-known effect of the concentration of the permeant ions on the equilibrium pressure difference in Donnan systems is related to the influence of the ionic strength on the extent of accumulation of the impermeant electrolyte at the membrane interface. Predictions derived from these theoretical considerations were verified experimentally.

The potential span of photoredox reactions of porphyrins and chlorophyll at the lipid bilayer-water interface.

Lipid bilayers containing chlorophyll (Chl) or magnesium octaethylporphyrin (MgOEP) and separating solutions containing varying amounts of differing acceptors are illuminated by a dye laser pulse (FWHM 0.3 microseconds) at 590 mm. Interfacial charge transfer is measured at the first current peak in a voltage clamp circuit. The constants describing the hyperbolic saturations of the charge transferred by differing acceptors are only weakly related to the redox potential of the acceptors. An assymetric molecule, anthraquinone-2-sulfonate, is over 20 times as effective in accepting the electron as is the symmetrical anthraquinone-2,6-disulfonate. In contrast to this variable effectiveness, the maximum amount of charge transferred as a function of acceptor redox potential is constant up to a cut-off value: -0.6 V (vs. standard hydrogen electrode) for MgOEP and -0.5 V for Chl. The reversible redox potential of MgOEP in the bilayer was determined by following both the decrease in photoactivity and the transmembrane potential as a function of aqueous redox potential. It is +0.77 V for MgOEP and approximately 0.7 V for Chl (limited by stability). Thus, a total of 1.4 V of reversible redox potential (free energy) is obtained from 1.8 eV (internal energy) of the triplet excited state of MgOEP.

Apparent inhibition of photoredox reactions of magnesium octaethylporphyrin at the lipid bilayer-water interface by neutral quinones.

Neutral quinones rapidly equilibrate across the lipid bilayer, hereby rendering the photoeffects seen in pigmented bilayers sensitive to the redox properties at both interfaces. The lack of photoeffect by quinones themselves and their apparent quenching reactions with aqueous acceptors is thus explained. An aqueous donor is needed on one side to break the symmetry and to allow vectorial electron transfer to be recorded. It is concluded that the neutral quinone accumulates on the polar side of the interface with respect to the hydrophobic pigment. The system may allow the study of kinetics of proton transfer accompanying the redox reactions of the quinones.

Dependence of photosensitivity of bileaflet lipid membranes upon the chlorophyll and carotenoid content.

Bileaflet lipid membranes were formed from solutions containing lecithin, chlorophyll and carotene in various concentrations. If all the above components were present at sufficient concentrations the membranes were photosensitive; i.e., a photocurrent was produced if a redox potential gradient was present across the membranes. The presence of chlorophyll and carotene were essential for the photosensitivity of the membranes. Photoresponse could be elicited by illuminating the membrane with light which did not excite carotene. On the other hand, elimination of the part of the light spectrum which excites chlorophyll led to the abolition of the photoresponse. The findings of this study are consistent with the assumption that the excited chlorophyll chromophores allow electron exchange at the membrane-water interface while the presence of carotene allows electron movement across the "bulk" lipid membrane.

Relation between the dielectric constant of hydrophobic cation exchange membrane and membrane permeability to counterions.

Filters made of cellulose acetate-nitrate when saturated with organic solvents and interposed between aqueous solutions form membranes which behave like cation exchangers. The diffusion coefficients of counterions in such membranes are strongly dependent upon the dielectric constant of the saturating solvent. The results obtained suggest that a linear relationship between the log of the cation's diffusion coefficient (or membrane conductance) and the reciprocal value of the dielectric constant of the saturating solvent exists. There is also a good correlation between the relative membrane permeability to organic cations and the solubility of the cations in the pure solvent phase. These studies indicate that there are two routes for cation movement through the membrane: (a) the bulk hydrophobic phase and (b) continuous narrow aqueous channels.