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Biomedical subjects

W B Guggino

Publications and source records attributed to W B Guggino.

At least 127 records · Page 7Linked to original sources
PubMed1990

Cell volume regulation in the nephron.

Nearly every cell in the kidney can volume regulate in response to a hypertonic challenge. Some are able to respond immediately to hyperosmotic media by a RVI. Other cells require stimulation prior to exposure to hyperosmolarity to demonstrate RVI. An increase of intracellular osmolytes during RVI usually occurs by an increase of NaCl influx either via the activation of parallel Na(+)-H+ and Cl(-)-HCO3- exchangers, or Na(+)-K(+)-2Cl- cotransporters. Medullary and papillary cells use organic solutes as well to increase the intracellular concentration of osmolytes. In response to a hypotonic challenge, a RVD response has been demonstrated in the majority of the kidney cells. The efflux of solute during RVD is usually via K+ loss by activation of conductance pathways. Stretch-activated K+ channels and Ca2(+)-activated-K+ channels have been shown to be stimulated in cells exposed in hyposmotic solutions and could thus be involved in RVD. The accompanying anion loss is less well-defined but could be either Cl- or HCO3- in different segments of the nephron. In some cells, the reduction of intracellular solute content is via an efflux of organic osmolytes. Thus it appears that cells in all segments of the nephron volume regulate in response to osmotic stresses. This regulation may be an essential part of transepithelial transport since the cells have to survive transcellular fluxes of osmolytes. It may be particularly important in the kidney to control cell volume both in response to changes in osmolarity and transcellular fluxes of solute in order to maintain proper flow of fluid through the nephron.

Animals↗
PubMed1989

Techniques for isolation of brush-border and basolateral membrane vesicles from dog kidney cortex.

Two methods are reported for renal membrane preparation from the dog kidney cortex. One method is a simultaneous preparation of brush-border (BBMV) and basolateral (BLMV) membranes. Using readily available laboratory equipment, differential centrifugation produced a supernatant which was treated with Mg2+. The Mg2+ treatment produced a pellet (crude BLMV) which was added to Percoll and centrifuged to produce purified BLMV. The supernatant after Mg2+ treatment eventually yielded pure BBMV after additional Mg2+ precipitations. The second method used an acidic medium in conjunction with divalent-cation precipitation to prepare BBMV. Whichever method was used, BBMV and BLMV showed appropriate enzyme and transport activities.

Animals↗
PubMed1989

Cl- channels in CF: lack of activation by protein kinase C and cAMP-dependent protein kinase.

Secretory chloride channels can be activated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase in normal airway epithelial cells but not in cells from individuals with cystic fibrosis (CF). In excised, inside-out patches of apical membrane of normal human airway cells and airway cells from three patients with CF, the chloride channels exhibited a characteristic outwardly rectifying current-voltage relation and depolarization-induced activation. Channels from normal tissues were activated by both cAMP-dependent protein kinase and protein kinase C. However, chloride channels from CF patients could not be activated by either kinase. Thus, gating of normal epithelial chloride channels is regulated by both cAMP-dependent protein kinase and protein kinase C, and regulation by both kinases is defective in CF.

Adult↗
PubMed1989

Gene transfer by lipofection in rabbit and human secretory epithelial cells.

Lipofection, a recently-developed method for gene transfer, was tested in secretory epithelial cells. Lipofection facilitated both transient DNA transfection with plasmids containing the chloramphenicol acetyltransferase gene and stable transfection with a plasmid containing the neomycin resistance gene, which confers resistance to the antibiotic G418 (Geneticin). Gene transfer occurred efficiently in a rabbit kidney medullary thick ascending limb cell line and in primary cultures of rabbit tracheal epithelial cells. The method was also effective in Simian virus 40-transformed human airway cells isolated from a normal individual and from a patient with cystic fibrosis (CF). Cytotoxicity was minimal, particularly when the time of exposure to the lipofectin-DNA was limited to 3-5 h (less than 5% cell loss). Thus, the lipofection method is useful for gene transfer in a variety of secretory epithelial cells and should be ideal for studies of defective secretory epithelial cell function in CF.

Animals↗
PubMed1989

Ca2+-activated K+ channels from cultured renal medullary thick ascending limb cells: effects of pH.

Ca2+-activated K+ channels were studied in cultured medullary thick ascending limb cells (MTAL) using the patch-clamp technique. The purpose was to determine the effect of acidic pH on channel properties in excised patches of apical cell membrane. At pH 7.4, increasing Ca2+ on the intracellular side or applying positive voltages increases channel open probability. Reducing pH to 5.8 on the intracellular face of the channel decreases channel open probability at each voltage and Ca2+ concentration. Channel mean open times display two distributions and mean closed times display three distributions. Increasing Ca2+ or applying depolarizing voltages lengthens each of the mean open times and shortens each of the closed times. Lowering pH to 5.8 decreases the mean open times and increases mean closed times at each Ca2+ and voltage with the greatest effect on the mean closed times. In contrast, both single-channel conductance and channel kinetics are unaffected when pH is reduced to 5.8 on the extracellular face of the membrane. We conclude that protons interfere with Ca2+ binding to the gate of Ca2+-activated K+ channels reducing the probability of channel opening.

Animals↗
PubMed1989

Isomeric yohimbine alkaloids block calcium-activated K+ channels in medullary thick ascending limb cells of rabbit kidney.

The alpha2-adrenergic antagonist yohimbine (YOH) and the closely related isomers corynanthine (COR) and rauwolscine (RAU) caused brief interruptions in current characteristic of a fast blocker Ca2+-activated K+ channels in cultured medullary thick ascending limb (MTAL) cells. The apparent dissociation constants (Kapp) for COR, YOH, and RAU, respectively, at the intracellular face of the channel in the presence of 200 mM K+ are 45 +/- 1, 98 +/- 2, and 310 +/- 33 microM. The Kapp for COR on the extracellular side also in the presence of 200 mM K+ was much greater at 1.6 +/- 0.17 mM. Increasing K+ on the same side as the blocker relieves the blocking reaction. The Kapp for the alkaloids varies with K+ in a manner quantitatively consistent with K+ and the alkaloids competing for a common binding site. Finally, blocking by the charged form of these alkaloids is voltage dependent with changes in Kapp of 86 +/- 7 and 94 +/- 6 microM per e-fold change in voltage for blockers applied either from the inside or outside. The alkaloids block at an electrical distance similar to tetraethylammonium, suggesting that the site within the channel pore of these molecules may be similar.

Animals↗
PubMed1989

Steroid hormones: modulators of Na+ absorption and Cl- secretion in cultured tracheal epithelia.

Cultures of tracheal epithelial cells from adult nonpregnant female rabbits maintained a different balance of Na+ absorption and Cl- secretion from cells of pregnant animals and fetal cultures. Cells of nonpregnant rabbits had a large Na+ absorptive current (11 microA/cm2 or 53% of baseline short circuit current) and a small Cl- secretory current (2.4 microA/cm2 or 11%). In contrast, fetal cells had greater Cl- secretion (15.1 microA/cm2 or 61%) and much less Na+ absorption (1.6 microA/cm2 or less than 10%). Cells of pregnant animals had amounts of Cl- secretion (11.4 microA/cm2 or 41%) and Na+ absorption (6.7 microA/cm2 or 22%) that were in between that of fetal and adult nonpregnant animals. In addition to these striking differences in baseline ion transport among the three groups, epinephrine was found to stimulate Cl- secretion in cells from pregnant rabbits, both Na+ absorption and Cl- secretion in cells from nonpregnant animals, and neither in fetal cells. Steroid hormones of pregnancy, progesterone and estradiol, when added to cells cultured from nonpregnant rabbits, altered the magnitudes of the individual transport pathways to mimic cells cultured from pregnant animals. In addition to changing baseline transport, these steroids modified the epinephrine-stimulated currents to resemble those in the fetus and in pregnancy. We conclude that steroid hormones regulate the balance of Na+ absorption and Cl- secretion in tracheal epithelia in utero and in adulthood.

Animals↗
PubMed1989

Membrane stretch: a physiological stimulator of Ca2+-activated K+ channels in thick ascending limb.

The effects of membrane stretch on Ca2+-activated (maxi) K+ channels were examined in the apical membrane of cultured medullary thick ascending limb (MTAL) cells. Using cell-attached patchclamp technique, we found that negative pressure (-33 +/- 5 cmH2O) applied to the patch membrane increased fractional open probability (NPo) from 0.3 +/- 0.2 to 29.9 +/- 7.6% (n = 12) in the presence of 1.8 mM Ca2+ in the pipette. The activity returned to control on releasing the negative pressure. Reduction of extracellular osmolality from 293.2 +/- 1.6 to 219.8 +/- 1.1 mosmol/kg also activated K+ channels (NPo = 43.8 +/- 12.2%, n = 8) in cell-attached patches. Removal of Ca2+ from both pipette and bathing solution inhibited osmotic activation of K+ channels. K+ channels were shown to be Ca2+-activated K+ channels by their conductance (146 +/- 7 pS, n = 5) and Ca2+ dependence. Our data suggest that membrane stretch caused by swelling or possibly by tubular flow enhances Ca2+ entry across the apical cell membrane of MTAL cells activating maxi K+ channels.

Animals↗
PubMed1988

Cell volume regulation by the thin descending limb of Henle's loop.

Thin descending limb cells from Henle's loop (from the inner strip of the outer medulla of long loops) were studied with optical and video techniques to identify the mechanisms of ion transport and cell volume regulation. Increasing the K+ concentration in the basolateral solution from 5 to 90 mM caused the cells to swell. This K+-induced swelling was inhibited by exposure of the basolateral membrane to 9 mM Ba2+ and was abolished by removing Cl- from the perfusion solutions. Decreasing the perfusion osmolality caused an increase in cell volume followed by a return to the preexposure volume. The latter regulatory decrease in hypoosmolality was slowed by basolateral Ba2+ and the removal of HCO-3 from the solutions. Further slowing occurred when both HCO-3 and Cl- were removed. Exposure of cells to ouabain abolished volume regulation. These data suggest that the basolateral cell membrane of the thin descending limb has a Cl- -dependent K+ permeability, which is important in cell volume regulation. The cells also possess Cl- and HCO-3 transport pathways that participate in volume regulation. Finally, volume regulation is dependent upon the operation of the Na/K pump.

Animals↗
PubMed1988

Ion transport in cultured fetal and adult rabbit tracheal epithelia.

Transepithelial potential difference (Vte), short circuit current (Isc), and transepithelial resistance (Rte) were measured to assess Na+ and Cl- transport in cultured tracheal epithelial cells from fetal and adult rabbits. Cultured fetal and adult cells developed differentiated morphology as determined by electron microscopy. Base-line Vte, Isc, and Rte were equivalent in late gestation (28-30 days) and adult cultures. Amiloride (10(-4) M) (Na+ transport inhibitor) reduced Isc by 7.4 +/- 1.2 microA/cm2 (24%) in adult cultures but only by 1.8 +/- 0.7 microA/cm2 (7%) in fetal cultures (P less than 0.001). Furosemide (10(-4) M) (Cl- transport inhibitor) diminished Isc by 16.5 +/- 3.2 microA/cm2 (53%) in adult cultures and 18.8 +/- 2.8 microA/cm2 (74%) in fetal cultures (P less than 0.01). Epinephrine (10(-6) M) stimulated Isc by 24.2 +/- 5.8 microA/cm2 (72%) in adult cells but only by 7.0 +/- 1.1 microA/cm2 (32%) in fetal cells (P less than 0.05). After epinephrine exposure, furosemide-sensitive Isc increased to 41.5 +/- 7.7 microA/cm2 in adult cells but remained unchanged in fetal cells (P less than 0.001). Our results suggest that fetal cells absorb less Na+ and secrete more Cl- than adult cells and that the Cl- secretory response to epinephrine is greater in adult than in fetal cells.

Amiloride↗
PubMed1988

The amphibian diluting segment.

Diluting segments function to reabsorb NaCl and to reduce the osmolality of tubule fluid. These segments in amphibians are important in the conservation of NaCl. The diluting segment of mammals, the thick ascending limb, besides being an important site for the reabsorption of NaCl, supplies the energy that enables the kidney to excrete a concentrated urine. We focus in this review on the mechanisms involved in cellular and paracellular transport of Na+, K+, Cl-, and H+ in the amphibian diluting segment and how the transport of these ions is regulated. Also discussed is the action of loop diuretics and hormones on both transepithelial and cellular function. The large size of amphibian cells and their viability in various artificial conditions have allowed experiments to be performed that are not possible in the mammalian kidney, providing important information on the mechanisms of ion transport common to both mammalian thick ascending limb and amphibian diluting segment.

Aldosterone↗
PubMed1987

Volume regulation in the early proximal tubule of the Necturus kidney.

The ability of early proximal tubule cells of the Necturus kidney to regulate volume was evaluated using light microscopy, video analysis and conventional microelectrodes. Necturus proximal tubule cells regulate volume in both hyper- and hyposmotic solutions. Volume regulation in hyperosmotic fluids is HCO3- dependent and is associated with a decrease in the relative K+ conductance of the basolateral cell membrane and a decrease in the resistance ratio, Ra/Rbl. Volume regulation in hyposmotic solutions is also dependent upon the presence of HCO3- but is also inhibited by 2 mM Ba2+ in the basolateral solution. Hyposmotic regulation is accompanied by an increase in the relative K+ conductance of the basolateral cell membrane and an increase in Ra/Rbl. Neither hypo- nor hyposmotic regulation have any affect on the depolarization of the basolateral cell membrane potential induced by HCO3- removal. We conclude that volume regulation in the early proximal tubule of the kidney involves both HCO3(-)-dependent transport systems and the baso-lateral K+ conductance.

Animals↗
PubMed1987

Modification of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells by N-bromoacetamide.

Ca2+-activated K+ channels were studied in cultured medullary thick ascending limb (MTAL) cells using the patch-clamp technique in the inside-out configuration. The Ca2+ activation site was modified using N-bromoacetamide (NBA). 1 mM NBA in the bath solution, at 2.5 microM Ca2+ reduces the open probability, Po, of the channel to less than 0.01, without an effect on single-channel conductance. NBA-modified channels are still Ca2+-sensitive, requiring 25 mM Ca2+ to raise Po to 0.2. Both before and after NBA modification channel openings display at least two distributions, indicative of more than one open state. High Ca2+ (1 mM) protects the channels from modification. Also presented is a second class of Ca2+-activated K+ channels which are normally present in MTAL cells which open infrequently at 10 microM Ca2+ (Po = 0.01) but have a Po of 0.08 at 1 mM Ca2+. We can conclude (i) that NBA modifies the channel by shifting Ca2+-sensitivity to very high Ca2+, (ii) that NBA acts on a site involved in Ca2+ gating, and (iii) that a low affinity channel is present in the apical cell membrane with characteristics similar to those of normal channels modified with NBA.

Acetamides↗
PubMed1987

Ca2+-activated K+ channels in cultured medullary thick ascending limb cells.

The conductive properties of a clone of medullary thick ascending limb (MTAL) cells (GRB-MAL1) were assessed using conventional microelectrodes and the patch clamp technique. The apical cell membrane potential (Va) of MTAL cells was -46 +/- 3 mV. Addition of Ba2+ (1 mM) to the apical solution induced a 22 +/- 2 mV depolarization of Va, whereas furosemide hyperpolarized Va by -5 +/- 1 mV. In the cell-attached patch configuration, the most frequently occurring channel had a single channel conductance of 121 +/- 5 pS and carried outward current. In excised patches, current movement was down the electrochemical K+ gradient. Fluctuations were activated by depolarization of Va and by increasing Ca2+ concentration on the intracellular face. Micromolar amounts of Ba2+ on the intracellular face of the membrane inhibited channel activity. We conclude that cultures of MTAL cells GRB-MAL1 retain at least two of the properties of the mature phenotype, namely, an apical K+ conductance and a sensitivity to loop diuretics; the most frequently occurring channel in the apical cell membrane is a Ca2+-activated, maxi-K+ channel; and, finally Ca2+-activated K+ channels may play a role in generating the apical K+ conductance in cultured MTAL cells.

Animals↗
PubMed1987

Blocking agents of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells.

Ca2+-activated K+ channels with estimated single channel conductances of 127 +/- 2 pS were identified in the apical cell membrane of clone A3 of cultured medullary thick ascending limb (MTAL) cells. Both Ba2+ and the scorpion toxin, charybdotoxin (CTX), are slow blockers of the channels. An application of 0.1 microM Ba2+ to the intracellular face caused a 50% reduction in fractional open time (fv). Ba2+ block is both concentration and voltage dependent. Concentrations of CTX as low as 2 nM in the extracellular solution caused a significant reduction in fv. Tetraethylammonium (TEA) and quinine are fast blockers of Ca2+-activated K+ channels in MTAL cells. TEA, 400 microM, in the extracellular solution caused a voltage-dependent reduction in channel amplitude, whereas it takes 10 mM in the intracellular solution to reduce channel amplitude by 30%. Micromolar amounts of quinine applied to the intracellular face caused the channels to flicker rapidly between open and blocked states. These results suggest that K+ channels in MTAL cells are homologous to those found in muscle cells, and that these blocking agents may be used to probe the nature of K+ conductances in several nephron segments.

Animals↗
PubMed1986

Functional heterogeneity in the early distal tubule of the Amphiuma kidney: evidence for two modes of Cl- and K+ transport across the basolateral cell membrane.

Measurements of cell volume and basolateral cell membrane potential difference (Vbl) were made simultaneously and on the same cell to study chloride and potassium transport across the basolateral cell membrane of individual cells from the Amphiuma early distal tubule or diluting segment. Two distinct types of responses of Vbl are evident when either chloride is lowered or potassium is raised in the basolateral solution. In one cell type (high basolateral conductance, HBC), these maneuvers cause large depolarizations in Vbl, suggesting that the basolateral cell membrane has significant partial conductances to both chloride and to potassium. In contrast, Vbl of another group (low basolateral conductance, LBC) is much less sensitive to the same maneuvers, indicating that the partial conductance of the basolateral cell membrane is quite low. Consistent with the difference in partial conductance is the ratio of resistances, which is immeasurably high in HBC cells and averages 1.2 +/- 0.4 in LBC cells. Increasing basolateral potassium to 98 mM causes both types to swell, with HBC cells swelling approximately two times faster than LBC cells. In both cell types, potassium-induced cell swelling is inhibited by removing chloride from the perfusion solutions. In animals kept in a low-potassium environment, the number of HBC cells is approximately equal to the number of LBC cells. The proportion of LBC cells increases following potassium adaptation. This data suggests that there are at least two types of cells present in Amphiuma early distal tubule, one cell type in which both chloride and potassium move across the basolateral cell membrane primarily via a conductive pathway and another in which the movements are mostly electroneutral, probably involving a K-Cl cotransport system, and that this heterogeneity may change in response to the metabolic state of the animal.

Animals↗
PubMed1985

Relationship between cell volume and ion transport in the early distal tubule of the Amphiuma kidney.

The roles of apical and basolateral transport mechanisms in the regulation of cell volume and the hydraulic water permeabilities (Lp) of the individual cell membranes of the Amphiuma early distal tubule (diluting segment) were evaluated using video and optical techniques as well as conventional and Cl-sensitive microelectrodes. The Lp of the apical cell membrane calculated per square centimeter of tubule is less than 3% that of the basolateral cell membrane. Calculated per square centimeter of membrane, the Lp of the apical cell membrane is less than 40% that of the basolateral cell membrane. Thus, two factors are responsible for the asymmetry in the Lp of the early distal tubule: an intrinsic difference in the Lp per square centimeter of membrane area, and a difference in the surface areas of the apical and basolateral cell membranes. Early distal tubule cells do not regulate volume after a reduction in bath osmolality. This cell swelling occurs without a change in the intracellular Cl content or the basolateral cell membrane potential. In contrast, reducing the osmolality of the basolateral solution in the presence of luminal furosemide diminishes the magnitude of the increase in cell volume to a value below that predicted from the change in osmolality. This osmotic swelling is associated with a reduction in the intracellular Cl content. Hence, early distal tubule cells can lose solute in response to osmotic swelling, but only after the apical Na/K/Cl transporter is blocked. Inhibition of basolateral Na/K ATPase with ouabain results in severe cell swelling. This swelling in response to ouabain can be inhibited by the prior application of furosemide, which suggests that the swelling is due to the continued entry of solutes, primarily through the apical cotransport pathway.

Animals↗