Reduced water permeability and altered ultrastructure in thin descending limb of Henle in aquaporin-1 null mice.
It has been controversial whether high water permeability in the thin descending limb of Henle (TDLH) is required for formation of a concentrated urine by the kidney. Freeze-fracture electron microscopy (FFEM) of rat TDLH has shown an exceptionally high density of intramembrane particles (IMPs), which were proposed to consist of tetramers of aquaporin-1 (AQP1) water channels. In this study, transepithelial osmotic water permeability (Pf) was measured in isolated perfused segments (0.5-1 mm) of TDLH in wild-type (+/+), AQP1 heterozygous (+/-), and AQP1 null (-/-) mice. Pf was measured at 37 degrees C using a 100 mM bath-to-lumen osmotic gradient of raffinose, and fluorescein isothiocyanate (FITC)-dextran as the luminal volume marker. Pf was (in cm/s): 0.26 +/- 0.02 ([+/+]; SE, n = 9 tubules), 0.21 +/- 0.01 ([+/-]; n = 12), and 0.031 +/- 0.007 ([-/-]; n = 6) (P < 0.02, [+/+] vs. [+/-]; P < 0.0001, [+/+] vs. [-/-]). FFEM of kidney medulla showed remarkably fewer IMPs in TDLH from (-/-) vs. (+/+) and (+/-) mice. IMP densities were (in microm-2, SD, 5-12 micrographs): 5,880 +/- 238 (+/+); 5,780 +/- 450 (+/-); and 877 +/- 420 (-/-). IMP size distribution analysis revealed mean IMP diameters of 8.4 nm ([+/+] and [+/-]) and 5.2 nm ([-/-]). These results demonstrate that AQP1 is the principal water channel in TDLH and support the view that osmotic equilibration along TDLH by water transport plays a key role in the renal countercurrent concentrating mechanism. The similar Pf and AQP1 expression in TDLH of (+/+) and (+/-) mice was an unexpected finding that probably accounts for the unimpaired urinary concentrating ability in (+/-) mice. (+info)
Involvement of tumor necrosis factor alpha and interleukin-1beta in enhancement of pentylenetetrazole-induced seizures caused by Shigella dysenteriae.
Neurologic manifestations, mainly convulsions, are the most frequent extraintestinal complications of shigellosis. We used an animal model to study the roles of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in Shigella-related seizures. Administration of Shigella dysenteriae 60R sonicate enhanced the sensitivity of mice to the proconvulsant pentylenetetrazole (PTZ) within 7 h. This was indicated by a significantly higher mean convulsion score and an increased number of mice responding with clonic-tonic seizures in the Shigella-pretreated group. Preinjection of mice with anti-murine TNF-alpha (anti-mTNF-alpha) or anti-murine IL-1beta (anti-mIL-1beta) 30 min prior to administration of Shigella sonicate abolished their enhanced response to PTZ at 7 h. Mean convulsion scores were reduced by anti-mTNF-alpha from 1.2 to 0.8 (P = 0.017) and by anti-mIL-1beta from 1.3 to 0.7 (P = 0.008). Preinjection of anti-mTNF-alpha also reduced the percentage of mice responding with clonic-tonic seizures, from 48 to 29% (P = 0.002), and preinjection of anti-mIL-1beta reduced it from 53 to 21% (P = 0. 012). Neutralization of TNF-alpha or IL-1beta did not protect the mice from death due to S. dysenteriae 60R. These findings indicate that TNF-alpha and IL-1beta play a role in the very early sensitization of the central nervous system to convulsive activity after S. dysenteriae administration. Similar mechanisms may trigger neurologic disturbances in other infectious diseases. (+info)
Transport of solutes through cartilage: permeability to large molecules.
A review of the transport of solutes through articular cartilage is given, with special reference to the effect of variations in matrix composition. Some physiological implications of our findings are discussed. Also, results of an experimental study of the permeability of articular cartilage to large globular proteins are presented. Because of the very low partition coefficients of large solutes between cartilage and an external solution new experimental techniques had to be devised, particularly for the study of diffusion. The partition coefficients of solutes were found to decrease very steeply with increase in size, up to serum albumin. There was, however, no further decrease for IGG. The diffusion coefficient of serum albumin in cartilage was relatively high (one quarter of the value in aqueous solution). These two facts taken together suggest that there may be a very small fraction of relatively large pores in cartilage through which the transport of large molecules is taking place. The permeability of cartilage to large molecules is extremely sensitive to variations in the glycosaminoglycan content: for a threefold increase in the latter there is a hundredfold decrease in the partition coefficient. For cartilage of fixed charge density around 0-19 m-equiv/g, there is no penetration at all of globular proteins of size equal to or larger than serum albumin. (+info)
Drug-protein binding and blood-brain barrier permeability.
The permeability surface area (PS) product, an index of permeability of the blood-brain barrier (BBB), was measured by using the in situ perfusion method. In the cerebral circulation, the fraction of drug that permeates into the brain through the BBB is not only the unbound fraction but also the fraction dissociated from the protein in the perfusate. The sum of these two fractions, the apparent exchangeable fraction, was estimated by fitting the parameters of the BBB permeability under the condition of varying BSA concentrations in the perfusate. The unbound fraction of drugs in a buffer containing 0.5 mM BSA was measured by using the ultrafiltration method in vitro, and the apparent exchangeable fraction was measured in vivo by using the intracarotid artery injection method. The apparent exchange fraction was 100% for S-8510, 96.5% for diazepam, 90.9% for caffeine, 38.3% for S-312-d, 33.1% for propranolol, and 6.68% for (+)-S-145 Na, and each of these was higher than the corresponding unbound fraction in vitro in all drugs. The apparent exchangeable fractions, for example, were 8 times higher for diazepam and 38 times for S-312-d than the unbound fractions in vitro. The apparent exchangeable fraction of drugs was also estimated from the parameters obtained with the perfusion method. Because drugs can be infused for an arbitrary length of time in the perfusion method, substances with low permeability can be measured. The apparent exchangeable fractions obtained with this method were almost the same as those obtained with the intracarotid artery injection method. (+info)
Effects of inhibitors and substitutes for chloride in lumen on p-aminohippurate transport by isolated perfused rabbit renal proximal tubules.
The transport step for p-aminohippurate (PAH) from cell to lumen across the luminal membrane of rabbit proximal tubules has not been adequately defined. To examine this process more closely, we determined the effects of possible transport inhibitors and substitutes for chloride on PAH secretion in isolated perfused S2 segments of rabbit proximal tubules. The addition of 4-acetamido-4'-isothiocyano-2,2' disulfonic stilbene (10(-4) M) to the perfusate irreversibly inhibited PAH secretion, whereas the addition of probenecid (10(-4) M) to the perfusate reversibly inhibited PAH secretion. PAH secretion was unaffected by thiocyanate replacement of chloride in the luminal perfusate, reversibly inhibited by 15 to 20% by methyl sulfate replacement, and irreversibly inhibited by isethionate replacement. Because the luminal membrane is at least as permeable to thiocyanate as to chloride, less permeable to methyl sulfate, and much less permeable to isethionate, these data suggest that the PAH transport step from cells to lumen does not require chloride in the lumen but does require a highly permeant anion. During inhibition of PAH transport from cells to lumen, PAH uptake across the basolateral membrane was also reduced, suggesting some type of feedback inhibition. The data are compatible with PAH transport across the luminal membrane by an anion exchanger, a potential-driven uniporter, both carriers, or a carrier that can function in both modes. (+info)
Selectivity and permeation in calcium release channel of cardiac muscle: alkali metal ions.
Current was measured from single open channels of the calcium release channel (CRC) of cardiac sarcoplasmic reticulum (over the range +/-180 mV) in pure and mixed solutions (e.g., biionic conditions) of the alkali metal ions Li+, K+, Na+, Rb+, Cs+, ranging in concentration from 25 mM to 2 M. The current-voltage (I-V) relations were analyzed by an extension of the Poisson-Nernst-Planck (PNP) formulation of electrodiffusion, which includes local chemical interaction described by an offset in chemical potential, which likely reflects the difference in dehydration/solvation/rehydration energies in the entry/exit steps of permeation. The theory fits all of the data with few adjustable parameters: the diffusion coefficient of each ion species, the average effective charge distribution on the wall of the pore, and an offset in chemical potential for lithium and sodium ions. In particular, the theory explains the discrepancy between "selectivities" defined by conductance sequence and "selectivities" determined by the permeability ratios (i.e., reversal potentials) in biionic conditions. The extended PNP formulation seems to offer a successful combined treatment of selectivity and permeation. Conductance selectivity in this channel arises mostly from friction: different species of ions have different diffusion coefficients in the channel. Permeability selectivity of an ion is determined by its electrochemical potential gradient and local chemical interaction with the channel. Neither selectivity (in CRC) seems to involve different electrostatic interaction of different ions with the channel protein, even though the ions have widely varying diameters. (+info)
Modulation of slow inactivation in human cardiac Kv1.5 channels by extra- and intracellular permeant cations.
1. The properties and regulation of slow inactivation by intracellular and extracellular cations in the human heart K+ channel hKv1.5 have been investigated. Extensive NH2- and COOH-terminal deletions outside the central core of transmembrane domains did not affect the degree of inactivation. 2. The voltage dependence of steady-state inactivation curves of hKv1.5 channels was unchanged in Rb+ and Cs+, compared with K+, but biexponential inactivation over 10 s was reduced from approximately 100 % of peak current in Na+ to approximately 65 % in K+, approximately 50 % in Rb+ and approximately 30 % in Cs+. This occurred as a result of a decrease in both fast and slow components of inactivation, with little change in inactivation time constants. 3. Changes in extracellular cation species and concentration (5-300 mM) had only small effects on the rates of inactivation and recovery from inactivation (tau recovery approximately 1 s). Mutation of residues at a putative regulatory site at R487 in the outer pore mouth did not affect slow inactivation or recovery from inactivation of hKv1.5, although sensitivity to extracellular TEA was conferred. 4. Symmetrical reduction of both intra- and extracellular cation concentrations accelerated and augmented both components of inactivation of K+ (Kd = 34.7 mM) and Cs+ (Kd = 20.5 mM) currents. These effects could be quantitatively accounted for by unilateral reduction of intracellular K+ (K+i) (Kd = 43.4 mM) or Cs+i with constant 135 mM external ion concentrations. 5. We conclude that inactivation and recovery from inactivation in hKv1.5 were not typically C-type in nature. However, the ion species dependence of inactivation was still closely coupled to ion permeation through the pore. Intracellular ion modulatory actions were more potent than extracellular actions, although still of relatively low affinity. These results suggest the presence of ion binding sites capable of regulating inactivation located on both intracellular and extracellular sides of the pore selectivity filter. (+info)
Genetic selection of mutations in the high affinity K+ transporter HKT1 that define functions of a loop site for reduced Na+ permeability and increased Na+ tolerance.
Potassium is an important macronutrient required for plant growth, whereas sodium (Na+) can be toxic at high concentrations. The wheat K+ uptake transporter HKT1 has been shown to function in yeast and oocytes as a high affinity K+-Na+ cotransporter, and as a low affinity Na+ transporter at high external Na+. A previous study showed that point mutations in HKT1, which confer enhancement of Na+ tolerance to yeast, can be isolated by genetic selection. Here we report on the isolation of mutations in new domains of HKT1 showing further large increases in Na+ tolerance. By selection in a Na+ ATPase deletion mutant of yeast that shows a high Na+ sensitivity, new HKT1 mutants at positions Gln-270 and Asn-365 were isolated. Several independent mutations were isolated at the Asn-365 site. N365S dramatically increased Na+ tolerance in yeast compared with all other HKT1 mutants. Cation uptake experiments in yeast and biophysical characterization in Xenopus oocytes showed that the mechanisms underlying the Na+ tolerance conferred by the N365S mutant were: reduced inhibition of high affinity Rb+ (K+) uptake at high Na+ concentrations, reduced low affinity Na+ uptake, and reduced Na+ to K+ content ratios in yeast. In addition, the N365S mutant could be clearly distinguished from less Na+-tolerant HKT1 mutants by a markedly decreased relative permeability for Na+ at high Na+ concentrations. The new mutations contribute to the identification of new functional domains and an amino acid in a loop domain that is involved in cation specificity of a plant high affinity K+ transporter and will be valuable for molecular analyses of Na+ transport mechanisms and stress in plants. (+info)