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T E Andreoli

Publications and source records attributed to T E Andreoli.

At least 55 records · Page 3Linked to original sources
PubMed1992

Cl- channels in basolateral renal medullary membranes, VI. Cl- conductance expression in Xenopus oocytes.

Cl- channels from basolateral membranes of the mammalian thick ascending limb of Henle differ significantly, in certain of their functional characteristics, from Cl- channels in apical membranes of secretory epithelia such as trachea or small intestine and from certain Cl- channels in the central nervous system. Yet there is no sequence information available about basolateral thick ascending limb Cl- channels. As an initial step in the isolation of a Cl- channel from the thick ascending limb of Henle's loop, we attempted a functional expression of a Cl- conductance in oocytes from Xenopus laevis. Oocytes injected with mRNA isolated from the outer medulla of rabbit kidney had a membrane conductance, measured using a two-electrode voltage clamp, which was sixfold greater than in water-injected oocytes (9.05 +/- 1.56 vs. 1.43 +/- 0.15 microS, respectively). Ion substitution experiments showed the conductance in the RNA-injected oocytes to be Cl- selective. In addition, the Cl(-)-channel blocker diphenylamine-2-carboxylate (1 mM) almost completely inhibited (79 +/- 6%) the increased conductance seen in the RNA-injected oocytes. Fractionation of the mRNA by sucrose gradient centrifugation revealed that peak Cl- channel activity was expressed using an mRNA fraction of 1.8-3.2 kb in size. These results demonstrate that a membrane Cl- conductance can be expressed in X. laevis oocytes injected with size-selected fractions of mRNA from rabbit outer renal medulla.

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PubMed1991

Cl- channels in basolateral renal medullary membrane vesicles: IV. Analogous channel activation by Cl- or cAMP-dependent protein kinase.

We examined the interactions of cAMP-dependent protein kinase and varying aqueous Cl- concentrations in modulating the activity of Cl- channels obtained by fusing basolaterally enriched renal outer medullary vesicles into planar lipid bilayers. Under the present experimental conditions, the cis and trans solutions face the extracellular and intracellular aspects of these Cl- channels, respectively. Raising the trans Cl- concentration from 2 to 50 mM increased the channel open-time probability, raised the unit channel conductance, and affected the voltage-independent determinant (delta G) of channel activity but not the gating charge (Winters, C.J., Reeves, W.B., Andreoli, T.E. 1990. J. Membrane Biol. 118:269-278). With 2 mM trans KCl, trans addition of the catalytic subunit of PKA (C-PKA) plus ATP increased channel open-time probability and altered the voltage-independent determinant of channel activity without affecting either unit channel conductance or gating charge. The effect was ATP specific, did not occur with (C-PKA plus ATP) addition to cis solutions, and was abolished by denaturing C-PKA. Finally, (C-PKA plus ATP) activation of channel activity was not detected with relatively high (50 mM) trans Cl- concentrations. These data indicate that (C-PKA plus ATP) might modulate Cl- channel activity by phosphorylation at or near the Cl(-)-sensitive site on the intracellular face of these channels.

Adenosine Triphosphate↗
PubMed1990

Cl- channels in basolateral renal medullary membranes: III. Determinants of single-channel activity.

We evaluated the effects of varying aqueous Cl- concentrations, and of the arginyl- and lysyl-specific reagent phenylglyoxal (PGO), on the properties of Cl- channels fused from basolaterally enriched renal medullary vesicles into planar lipid bilayers. The major channel properties studied were the anion selectivity sequence, anionic requirements for channel activity, and the effects of varying Cl- concentrations and/or PGO on the relation between holding voltage (VH, mV) and open-time probability (Po). Reducing cis Cl- concentrations, in the range 50-320 mM, produced a linear reduction in fractional open time (Po) with a half-maximal reduction in Po at cis Cl- approximately 170 mM. Channel activity was sustained by equimolar replacement of cis Cl- with F-, but not with impermeant isethionate. For trans solutions, the relation between Cl- concentration and Po was negatively cooperative, with 50% reduction in po at 10 mM Cl-. Reducing cis Cl- had no effect on the gating charge (Z) for channel opening, but altered significantly the voltage-independent energy (delta G) for channel opening. Phenylglyoxal (PGO) reduced Z and altered delta G for Cl- channel activity when added to cis, but not trans solutions. Furthermore, in the presence of cis PGO, reducing the cis Cl- concentration had no effect on Z but altered delta G. Thus we propose that cis PGO and cis Cl- concentrations affect separate sites determining channel activity at the extracellular faces of these Cl- channels.

Animals↗
PubMed1990

Cl- transport in basolateral renal medullary vesicles: I. Cl- transport in intact vesicles.

This paper provides the results of studies which characterized conductive 36Cl- flux in basolaterally enriched membrane vesicles prepared from rabbit renal outer medulla. Conductive 36Cl- uptake was studied under two different experimental conditions. In the first, 36Cl- flux was driven by an inside positive voltage created with oppositely directed Cl- and gluconate gradients. In the second, an inwardly direct K+ gradient was used to drive 36Cl- uptake. By these two methods, voltage-sensitive 36Cl- uptake was shown to comprise about 45 and 65%, respectively, of the initial rates of total 36Cl- flux. Separate paired studies demonstrated that the conductive 36Cl- uptake was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate (DPC) with an IC50 for DPC of 154 microM. The voltage-dependent 36Cl- uptake had an activation energy of 6.4 kcal/mole. This 36Cl- conductance had an anion selectivity sequence of I- greater than Cl- greater than or equal to NO3- much greater than gluconate.

Animals↗
PubMed1990

Cl- transport in basolateral renal medullary vesicles: II. Cl- channels in planar lipid bilayers.

The present studies examined some of the properties of Cl- channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having a PCl/PK ratio of 10 and a unit conductance of 93 pS in symmetric 320 mM KCl. In asymmetric KCl solutions, the I-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl- conductance of isolated medullary thick ascending limb segments. The Cl- channels incorporated into the planar bilayers were asymmetrical: the trans surface was sensitive to changes in ionized Ca2+ concentrations and insensitive to reducing KCl concentrations to 10 mM, while the cis side was insensitive to changes in ionized Ca2+ concentrations, but was inactivated by reducing KCl concentrations to 50 mM.

Animals↗
PubMed1989

Activation of K+ channels in renal medullary vesicles by cAMP-dependent protein kinase.

ADH, acting through cAMP, increases the potassium conductance of apical membranes of mouse medullary thick ascending limbs of Henle. The present studies tested whether exposure of renal medullary apical membranes in vitro to the catalytic subunit of cAMP-dependent protein kinase resulted in an increase in potassium conductance. Apical membrane vesicles prepared from rabbit outer renal medulla demonstrated bumetanide- and chloride-sensitive 22Na+ uptake and barium-sensitive, voltage-dependent 86Rb+ influx. When vesicles were loaded with purified catalytic subunit of cAMP-dependent protein kinase (150 mU/ml), 1 mM ATP, and 50 mM KCl, the barium-sensitive 86Rb+ influx increased from 361 +/- 138 to 528 +/- 120 pM/mg prot.30 sec (P less than 0.01). This increase was inhibited completely when heat-stable protein kinase inhibitor (1 microgram/ml) was also present in the vesicle solutions. The stimulation of 86Rb+ uptake by protein kinase required ATP rather than ADP. It also required opening of the vesicles by hypotonic shock, presumably to allow the kinase free access to the cytoplasmic face of the membranes. We conclude that cAMP-dependent protein kinase-mediated phosphorylation of apical membranes from the renal medulla increases the potassium conductance of these membranes. This mechanism may account for the ADH-mediated increase in potassium conductance in the mouse mTALH.

Adenosine Triphosphate↗
PubMed1988

Diluting power of thick limbs of Henle. I. Peritubular hypertonicity blocks basolateral Cl- channels.

This paper provides the results of experiments intended to assess the mechanism responsible for the suppression of net salt absorption and the attendant spontaneous voltage (Ve, mV) that occurs when isolated thick ascending limbs of Henle (TAL) are exposed to a hypertonic environment. In isolated mouse medullary (MTAL) and cortical (CTAL) segments, as well as in rabbit MTAL segments, increases in peritubular osmolality with urea produced a graded suppression of Ve. This effect was evaluated in further detail in isolated mouse MTAL segments, where 600 mM peritubular urea produced a reversible reduction in Ve and a reversible reduction in the transcellular electrical conductance (Gc; mS.cm-2). There was no detectable effect on the paracellular conductance (Gs; mS.cm-2). Simultaneously, 600 mM peritubular urea also produced hyperpolarization of the voltage across basolateral membranes (mV). Moreover, 600 mM peritubular urea produced virtually the same magnitude reduction in Gc either in the absence or presence of 10(-4) M luminal furosemide. Thus we conclude that peritubular urea hypertonicity directly suppresses the Cl- conductance of basolateral membranes (mS.cm-2).

Animals↗
PubMed1988

Diluting power of thick limbs of Henle. II. Bumetanide-sensitive 22Na+ influx in medullary vesicles.

We evaluated the effects of osmotic gradients on 22Na+ influx in vesicles prepared from rat outer renal medulla. 22Na+ influx driven in a coflow mode by an inwardly directed 100 mM KCl gradient was measured at 20 and 60 s; 1 mM bumetanide inhibited approximately 30% of 22Na+ influx. The bumetanide-sensitive 22Na+ influx was reduced by approximately 65% when either K+ or Cl- was omitted from the aqueous phases. We found that an osmotic gradient for vesicle shrinkage, that is, 600 mM urea in the extravesicular medium, enhanced the bumetanide-sensitive 22Na+ influx twofold. Conversely, an osmotic gradient for vesicle swelling, that is, with vesicles but not extravesicular media loaded with 600 mM urea, produced a 50% suppression of bumetanide-sensitive 22Na+ influx. Moreover, 600 mM extravesicular urea, an osmotic gradient for vesicle shrinkage, also reduced uptake of the nonspecific marker [14C]mannitol. These effects of osmotic gradients were not due to alterations in ionic driving forces, since bumetanide-sensitive 22Na+ influx driven in a counterflow mode by loading the vesicles with 100 mM NaCl also was activated or suppressed by osmotic gradients for vesicle shrinkage or swelling, respectively. We conclude that osmotic gradients, and/or vesicle volume changes, modulate bumetanide-sensitive Na+:K+:2Cl- activity.

Alkaline Phosphatase↗
PubMed1988

Diluting power of thick limbs of Henle. III. Modulation of in vitro diluting power.

This paper considers the quantitative interplay of various factors in modulating diluting power of in vitro medullary and cortical thick ascending limbs of Henle (MTAL and CTAL, respectively) segments from mouse and rabbit. Experimentally, the measured diluting power of the in vitro rabbit CTAL is greater than that of the rabbit MTAL, although the inherent rate of net Cl- absorption at high perfusion rates is considerably greater in the rabbit MTAL than in the rabbit CTAL. Similar results apply when comparing the rabbit CTAL to the mouse MTAL exposed to antidiuretic hormone (ADH). Our calculations show that, in the rabbit CTAL, the measured static head luminal salt concentration can be accounted for quantitatively by the measured rate of net salt absorption at a high perfusion rate together with the passive permeability coefficients for Na+ and Cl-. Moreover, with perfusion rates of 10% of single-nephron glomerular filtration rate, the transport properties of the CTAL predict that, at the end of the CTAL, the static head luminal Cl- concentration occurs if the initial perfusate contains either 50 or 150 mM Cl-. Thus one can argue that, in vivo the CTAL may be the cardinal determinant of the TAL contribution to diluting power and to external salt balance. The relatively blunted diluting power of in vitro MTAL segments can be accounted for quantitatively by assuming that luminal dilution, and the attendant osmotic gradient from lumen to cell, suppresses the inherent rate of transcellular Cl- transport. Our calculations also show that prostaglandin E2 and peritubular osmolality increases blunt tubular diluting power. Thus in vivo, the MTAL segment may be the cardinal determinant of TAL contribution to concentrating power and to intrarenal salt balance.

Animals↗