Kenneth R. Spring

Bending the MDCK cell primary cilium increases intracellular calcium.

Abstract. We tested the hypothesis that the primary cilium of renal epithelia is mechanically sensitive and serves as a flow sensor in MDCK cells using differential interference contrast and fluorescence microscopy. Bending the cilium, either by suction with a micropipette or by increasing the flow rate of perfusate, causes intracellular calcium to substantially increase as indicated by the fluorescent indicator, Fluo-4. This calcium signal is initiated by Ca2+-influx through mechanically sensitive channels that probably reside in the cilium or its base. The influx is followed by calcium release from IP3-sensitive stores. The calcium signal then spreads as a wave from the perturbed cell to its neighbors by diffusion of a second messenger through gap junctions. This spreading of the calcium wave points to flow sensing as a coordinated event within the tissue, rather than an isolated phenomenon in a single cell. Measurement of the membrane potential difference by microelectrode during perfusate flow reveals a profound hyperpolarization during the period of elevated intracellular calcium. We conclude that the primary cilium in MDCK cells is mechanically sensitive and responds to flow by greatly increasing intracellular calcium.

Removal of the MDCK Cell Primary Cilium Abolishes Flow Sensing

The hypothesis that cell primary cilium is solely responsible for the flow-induced Ca2+ response in MDCK cells was tested by removal of the cilia from mature, responsive cells. Incubation of the cells with 4 mM chloral hydrate for 68 hours resulted in the complete loss of the primary cilia and in disorganization of microtubules, as visualized by immunofluorescence. When intracellular Ca2+ concentration was measured with Fluo-4, the elevation that normally accompanies an increase in fluid flow was abolished after 20 hours exposure to chloral hydrate. At this time, the primary cilia still remained attached to the cells but had become twisted and flexible. Twentyfour hours after return of the deciliated cells to normal medium, intracellular microtubule organization appeared normal, but primary cilia had not yet been expressed. The cells failed to increase intracellular Ca2+ in response to fluid flow until after they had been in normal medium for 120 hours, at which time the primary cilia were 3–4 mm long. Chloral hydrate did not impair the Ca2+ mobilization machinery, as the Ca2+ response to mechanical contact and the spread to neighboring cells was unaffected by the drug. We conclude that the primary cilium is the only sensor for the flow-induced Ca2+ response in MDCK cells and estimate that a single mechanically sensitive channel in the cilium could provide the requisite Ca2+ influx.

Bending the primary cilium opens Ca2+-sensitive intermediate-conductance K+ channels in MDCK cells

Increasing tubular fluid flow rate has previously been shown to induce K+ secretion in mammalian cortical collecting duct. The mechanism responsible was examined in the present study using MDCK cells as a model. The change in membrane potential difference (EM) of MDCK cells was measured with a fluorescent voltage-sensitive dye, DiBAC4(3), when the cells primary cilium was continuously bent with a micropipette or by the flow of perfusate. Bending the cilium produced a hyperpolarization of the membrane that lagged behind the increase in intracellular Ca2+ concentration by an average of 36 seconds. Gd3+ , an inhibitor of the flow-induced Ca2+ increase, prevented the hyperpolarization. Blocking K+ channels with Ba2+ reduced the flow-induced hyperpolarization, implying that it resulted from activation of Ca2+-sensitive K+ channels. Further studies demonstrated that the hyperpolarization was diminished by the blocker of Ca2+-activated K+ channels, charybdotoxin, whereas iberiotoxin or apamin had no effect, results consistent with the activation of intermediate-conductance Ca2+-sensitive K+ channels. RT-PCR analysis and sequencing confirmed the presence of intermediate-conductance K+ channels in MDCK cells. We conclude that the increase in intracellular Ca2+ associated with bending of the primary cilium is the cause of the hyperpolarization and increased K+ conductance in MDCK cells.

Epithelial cell volume modulation and regulation

SummaryEpithelial cell volume is a sensitive indicator of the balance between solute entry into the cell and solute exit. Solute accumulation in the cell leads to cell swelling because the water permeability of the cell membranes is high. Similarly, solute depletion leads to cell shrinkage. The rate of volume change under a variety of experimental conditions may be utilized to study the rate and direction of solute transport by an epithelial cell. The pathways of water movement across an epithelium may also be deduced from the changes in cellular volume. A technique for the measurement of the volume of living epithelial cells is described, and a number of experiments are discussed in which cell volume determination provided significant new information about the dynamic behavior of epithelia. The mechanism of volume regulation of epithelial cells exposed to anisotonic bathing solution is discussed and shown to involve the transient stimulation of normally dormant ion exchangers in the cell membrane.

Chloride reabsorption by renal proximal tubules of Necturus.

SummaryMovement of Cl from the lumen ofNecturus proximal tubule into the cells is mediated and dependent on the presence of luminal Na. Intracellular Cl activity was monitored with ion selective microelectrodes. In Cl Ringers perfused kidneys, cell Cl activity was 24.5±1.1mm, 2 to 3 times higher than that predicted for passive distribution. When luminal NaCl was partially replaced by mannitol (capillaries perfused with Cl Ringers) cell Cl decreased showing a sigmoidal dependence on luminal NaCl. Peritubular membrane potential was unaltered. Sulfate Ringers perfusion of the kidneys washed out all cell Cl but did not alter peritubular membrane potential. Chloride did not enter the cell when the tubule lumen was perfused with 100mm KCl, LiCl, or tetramethylammonium Cl. Luminal perfusion of NaCl caused cell Cl to rise rapidly to the same value as the controls in the Cl Ringers experiments. Perfusion of the tubule lumen with mixtures of NaCl and Na2SO4, while the capillaries contained sulfate Ringers yielded a sigmoidal dependence of cell Cl on luminal NaCl activity. Chloride movement from the lumen into the proximal tubule cells required approximately equal concentrations of Na and Cl. Current clamp experiments indicated that intracellular chloride activity was insensitive to alterations in liminal membrane potential, suggesting that chloride entry was electrically neutral. The transcellular chloride flux was calculated to constitute about one half of the normal chloride reabsorption rate. We conclude that the cell Cl activity is primarily determined by the NaCl concentration in the tubule lumen and that Cl entry across the luminal membrane is mediated.

Chloride transport activation by plasma osmolarity during rapid adaptation to high salinity of Fundulus heteroclitus

Transition from low salt water to sea water of the euryhaline fish, Fundulus heteroclitus, involves a rapid signal that induces salt secretion by the gill chloride cells. An increase of 65 mOsm in plasma osmolarity was found during the transition. The isolated, chloridecell-rich opercular epithelium of sea-water-adapted Fundulus exposed to 50 mOsm mannitol on the basolateral side showed a 100% increase in chloride secretion, which was inhibited by bumetanide 10−4m and 10−4m DPC (N-Phenylanthranilic acid). No effect of these drugs was found on apical side exposure. A Na+/H+ exchanger, demonstrated by NH4Cl exposure, was inhibited by amiloride and its analogues and stimulated by IBMX, phorbol esters, and epithelial growth factor (EGF). Inhibition of the Na+/H+ exchanger blocks the chloride secretion increase due to basolateral hypertonicity. A Cl−/HCO3−exchanger was also found in the chloride cells, inhibited by 10−4m DIDS but not involved in the hyperosmotic response. Ca2+ concentration in the medium was critical for the stimulation of Cl− secretion to occur. Chloride cell volume shrinks in response to hypertonicity of the basolateral side in sea-water-adapted operculi; no effect was found on the apical side. Freshwater-adapted fish chloride cells show increased water permeability of the apical side. It is concluded that the rapid signal for adaptation to higher salinities is an increased tonicity of the plasma that induces chloride cell shrinkage, increased chloride secretion with activation of the Na+K+2Cl− cotransporter, the Na+/H+ exchanger and opening of Cl− channels.

Volume regulation by Necturus gallbladder: basolateral KCl exit

SummarySwelling of the epithelial cells ofNecturus gallbladder caused by an 18% reduction in the osmolality of the mucosal bath is followed by rapid volume readjustment. This volume regulatory decrease requires Cl and is sensitive to the K and Cl gradients across the basolateral cell membrane. Volume regulatory decrease is not inhibited by amiloride, SITS, ouabain or bicarbonate removal. The process is blocked by bumetanide in the serosal bath. Measurement of the intracellular activities of K and Cl and the rate of volume regulation under five different experimental conditions showed that KCl exited from the cell across the basolateral membrane with a stoichiometry of 3 K to 2 Cl. This KCl exit process appears to be transiently activated following the reduction in osmolality of the mucosal perfusate.

Cell membrane water permeability of rabbit cortical collecting duct.

SummaryThe water permeability (Posm) of the cell membranes of isolated perfused rabbit cortical collecting ducts was measured by quantitative light microscopy. Water permeability of the basolateral membrane, corrected for surface area, was 66 μm·sec−1 for principal cells and 62.3 μm·sec−1 for intercalated cells. Apical membranePosm values corrected for surface area, were 19.2 and 25 μm·sec−1 for principal and intercalated cells, respectively, in the absence of antidiuretic hormone (ADH). Principal and intercalated cells both responded to ADH by increasingPosm of their apical membranes to 92.2 and 86.2 μ·sec−1 respectively. The ratio of the total basolateral cell membrane osmotic water permeability to that of the apical cell membrane was ∼27∶1 in the absence of ADH and ∼7∶1 in the presence of the hormone for both cell types. This asymmetry in water permeability is most likely due to the fact that basolateral membrane surface area is at least 7 to 8 times greater than that of the apical membrane. Both cell types exhibited volume regulatory decrease when exposed to dilute serosal bathing solutions. Upon exposure to a hyperosmotic serosal bath (390 mosm), pricipal cells did not volume regulate while two physiologically distinct groups of intercalated cells were observed. One group of intercalated cells failed to volume regulate; the second group showed almost complete volume regulatory increase behavior.

Ion transport by mitochondria-rich cells in toad skin

SummaryThe optical sectioning video imaging technique was used for measurements of the volume of mitochondria-rich (m.r.) cells of the isolated epithelium of toad skin. Under short-circuit conditions, cell volume decreased by about 14% in response to bilateral exposure to Cl-free (gluconate substitution) solutions, apical exposure to ouabain resulted in a large increase in volume, which could be prevented either by the simultaneous application of amiloride in the apical solution or by the exposure of the epithelium to bilateral Cl-free solutions. Unilateral exposure to a Cl-free solution did not prevent ouabain-induced cell swelling. It is concluded that m.r. cells have an amiloride-blockable Na conductance in the apical membrane, a ouabain-sensitive Na pump in the basolateral membrane, and a passive Cl permeability in both membranes. From the initial rate of ouabain-induced cell volume increase the active Na current carried by a single m.r. cell was estimated to be 9.9±1.3 pA. Voltage clamping of the preparation in the physiological range of potentials (0 to −100 mV, serosa grounded) resulted in a cell volume increase with a time course similar to that of the stimulation of the voltage-dependent activation were prevented by exposure of the tissue to a Cl-free apical solution. The steady-state volume of the m.r. cells increased with the clamping voltage, and at −100 mV the volume was about 1.15 times that under short-circuit conditions. The rate of volume increase during current passage was significantly decreased by lowering the serosal K concentration (Ki) to 0.5mm, but was independent of whether Ki was 2.4, 5, or 10mm. This indicates that the K conductance of the serosal membrane becomes rate limiting for the uptake of KCl when Ki is significantly lower than its physiological value. It is concluded that the voltage-activated Cl currents flow through the m.r. cells and that swelling is caused by an uptake of Cl ions from the apical bath and K ions from the serosal bath. Bilateral exposure of the tissue to hypo- or hypertonic bathing solutions changed cell volume without detectable changes in the Cl conductance. The volume response to external osmotic perturbations followed that of an osmometer with an osmotically inactive volume of 21%. Using this value and the change in cell volume in response to bilateral Cl-free solutions, we calculated an intracellular steady-state Cl concentration of 19.8±1.7mm (n=6) of the short-circuited cell.

Chloride movement across the basolateral membrane of proximal tubule cells

SummaryElectrophysiologic and tracer experiments have shown that Cl− entersNecturus proximal tubule cells from the tubule lumen by a process coupled to the flow of Na+, and that Cl− entry is electrically silent. The mechanism of Cl− exit from the cell across the basolateral membrane has not been directly studied. To evaluate the importance of the movement of Cl− ions across the basolateral membrane, the relative conductance of Cl− to K+ was determined by a new method. Single-barrel ion-selective microelectrodes were used to measure intracellular Cl− and K+ as a function of basolateral membrane PD as it varied normally from tubule to tubule. Basolateral membrane Cl− conductance was about 10% of K+ conductance by this method. A second approach was to voltage clamp the basolateral PD to 20 mV above and below the spontaneous PD, while sensing intracellular Cl− activity with the second barrel of a double-barrel microelectrode. An axial wire electrode in the tubule lumen was used to pass current across the tubular wall and thereby vary the basolateral membrane PD. Cell Cl− activity was virtually unaffected by the PD changes. We conclude that Cl− leavesNecturus proximal tubule cells by a neutral mechanism, possibly coupled to the efflux of Na+ or K+.