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Regional differences in ciliary epithelial cell transport properties.

Experiments were performed to determine whether the transport properties of the ciliary epithelium vary over different regions. Rabbit iris-ciliary bodies were incubated under experimental or control conditions for 30 min before quick freezing, cryosectioning, dehydration and electron probe X-ray microanalysis. Cryosections were cut from three regions along the major axis of the iris-ciliary body, i.e., the anterior, middle and posterior (pars plicata) regions. In bicarbonate/CO2 solution, the epithelial cells of the anterior and middle regions contained more Cl and K than did those of the posterior region. These higher levels of Cl and K were reduced by the carbonic anhydrase inhibitor acetazolamide. Application of bumetanide, an inhibitor of the Na+-K+-2Cl- cotransporter, resulted in significant increases in Cl and K in the anterior and middle regions but not in the posterior region. In bicarbonate-free solution, the ratio for K/Na contents was higher in the posterior than in the two more anterior regions; Na, K and Cl contents of epithelial cells in the three regions were otherwise similar. Cell composition did not differ significantly between the crests and valleys of the posterior region. The divergent responses to perturbation of epithelial transport in the different regions provide the first demonstration of functional heterogeneity along the major axis of the iris-ciliary body. The response to inhibition of carbonic anhydrase raises the possibility that the anterior aspect of the ciliary epithelium may be the major site of aqueous humor secretion.

Timolol may inhibit aqueous humor secretion by cAMP-independent action on ciliary epithelial cells.

The beta-adrenergic antagonist timolol reduces ciliary epithelial secretion in glaucomatous patients. Whether inhibition is mediated by reducing cAMP is unknown. Elemental composition of rabbit ciliary epithelium was studied by electron probe X-ray microanalysis. Volume of cultured bovine pigmented ciliary epithelial (PE) cells was measured by electronic cell sizing; Ca(2+) activity and pH were monitored with fura 2 and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Timolol (10 microM) produced similar K and Cl losses from ciliary epithelia in HCO/CO(2) solution but had no effect in HCO/CO(2)-free solution or in HCO/CO(2) solution containing the carbonic anhydrase inhibitor acetazolamide. Inhibition of Na(+)/H(+) exchange by dimethylamiloride in HCO/CO(2) solution reduced Cl and K comparably to timolol. cAMP did not reverse timolol's effects. Timolol (100 nM, 10 microM) and levobunolol (10 microM) produced cAMP-independent inhibition of the regulatory volume increase (RVI) in PE cells and increased intracellular Ca(2+) and pH. Increasing Ca(2+) with ionomycin also blocked the RVI. The results document a previously unrecognized cAMP-independent transport effect of timolol. Inhibition of Cl(-)/HCO exchange may mediate timolol's inhibition of aqueous humor formation.

Formation of the aqueous humor.

1. Glaucoma is a worldwide disease affecting approximately 1-2% of the population aged over 35 years in industrial countries and is a major cause of blindness. 2. Glaucoma is usually associated with an increased intraocular pressure reflecting an imbalance between the rate of production of fluid (the aqueous humor) by the ciliary epithelial cells and its drainage from the eye. Therefore, it is important to understand how this secretion is produced. This requires a knowledge of ciliary epithelial cell composition, which has, in the past, proved difficult to obtain in mammalian preparations. 3. We have recently used the technique of electron-probe X-ray microanalysis to determine this composition under a variety of in vitro conditions. 4. Our results have led to a new model for this secretion that emphasizes the potential secretory role of the Na+/K+/2Cl- cotransporter.

Effects of eicosapentaenoic acid on the contraction of intact, and spontaneous contraction of chemically permeabilized mammalian ventricular myocytes.

The n-3 polyunsaturated fatty acids appear to protect the heart from ischaemia-induced arrhythmias. We have used single adult guinea-pig and rat ventricular myocytes to investigate the effects of the n-3 polyunsaturated fatty acid eicosapentaenoic acid on, (i) the l -type Ca2+current, (ii) twitch contraction, and (iii) the spontaneous mechanical activity induced in chemically skinned myocytes by an elevation of the superfusing [Ca2+]. Eicosapentaenoic acid reduced the size of the l -type Ca2+current in a dose-dependent manner in myocytes from both species. Inclusion of delipidated bovine serum albumin (BSA) to the Tyrode, which binds eicosapentaenoic acid, completely reversed the inhibition of the Ca2+current in both guinea-pig and rat cells. The effects of eicosapentaenoic acid on contraction were species dependent. In guinea-pig myocytes it produced a reduction in contraction size which was complex, being described by three phases. In rat cells there was an initial increase in the size of contractions, followed by a simple reduction in contraction strength. Delipidated BSA completely reversed these effects in rat cells but only partially restored twitch contraction in guinea-pig cells (60%). In saponin permeabilized cells, the frequency of the spontaneous activity evoked by elevation of [Ca2+] was reduced by micromolar concentrations of eicosapentaenoic acid in cells from both species. The reduction in the amplitude of contractions caused by eicosapentaenoic acid can be explained by an inhibition of the l -type Ca2+current, and by a reduction in Ca2+released from the sarcoplasmic reticulum (SR). The inhibition of the release of Ca2+from the SR reduces the frequency of [Ca2+] dependent spontaneous contractions in chemically skinned guinea-pig and rat ventricular myocytes.

The electrical and mechanical response of adult guinea pig and rat ventricular myocytes to omega3 polyunsaturated fatty acids.

Single adult guinea-pig and rat ventricular cardiac myocytes were used to study the effects of two members of the omega3 class of polyunsaturated fatty acids, docosahexaenoic acid and eicosapentaenoic acid, on the electrical and mechanical activity of cardiac muscle. Docosahexaenoic acid and eicosapentaenoic acid reduced the electrical excitability of both guinea-pig and rat cells in a dose-dependent manner. Both agents produced a dose-dependent negative inotropic response in guinea-pig cells but in the rat cells there was first a dose-dependent positive inotropic effect at low concentrations (< 10 microM) followed by a negative inotropic effect at higher concentrations (> 10 microM). Possible mechanisms by which these agents affect contraction were studied using conventional electrophysiological techniques. The polyunsaturated fatty acids reduced the action potential duration and the plateau potential of the guinea-pig cells in a simple, dose-dependent manner. In contrast, the effect on the rat action potential mirrored the inotropic effect. At low concentrations (< 10 microM) there was a concentration-dependent increase in action potential duration followed by a concentration-dependent decrease at higher concentrations (> 10 microM). Both polyunsaturated fatty acids decreased the fast Na+ current and the L-type Ca2+ current in a concentration-dependent but not use-dependent manner in cells from both species. In the rat cells these agents inhibited the transient outward current resulting in an increase in the duration of the rat action potential. The effects of polyunsaturated fatty acids on the Ca2+, Na+ and K+ currents underlie these changes in the action potentials in guinea-pig and rat heart cells. The effects on the L-type Ca2+ current and action potential duration can also explain both the simple negative inotropic effects of the agents on the guinea-pig cells and the more complex effects on the rat cells. These effects of polyunsaturated fatty acids on membrane currents may account for their anti-arrhythmic properties.

Effects of HCO3- on cell composition of rabbit ciliary epithelium: a new model for aqueous humor secretion.

PURPOSE: To determine whether the Na+-K+-2Cl- symport or the parallel Na+/H+ and Cl-/HCO3- antiports provide the dominant pathway for NaCl uptake into the ciliary epithelium. Both pathways are known to support NaCl entry from the stroma into the pigmented ciliary epithelial (PE) cells, after which Na+ and Cl- diffuse across the gap junctions into the nonpigmented ciliary epithelial (NPE) cells and are released into the aqueous humor. METHODS: Rabbit iris ciliary bodies were preincubated in HCO3-/CO2-containing or HCO3-/CO2-free solutions before quick freezing, cryosectioning, dehydration, and electron probe x-ray microanalysis. RESULTS: The NPE and the PE cells contained more K and Cl when incubated with bicarbonate. Inhibition of carbonic anhydrase with 0.5 mM acetazolamide had little effect in HCO3--free medium but prevented the increase in Cl in both cell types in HCO3-/CO2 solution. Inhibition of the Na+-K+-2Cl- symport with 10 to 500 microM bumetanide caused Cl loss from both cell types in HCO3--free solution, but bumetanide produced a paradoxical increase in Cl and Na in HCO3-/CO2 solution. Together, acetazolamide and bumetanide resulted in significant Cl loss in HCO3--free solution and prevented the gains of Cl and Na in HCO3-/CO2 solution. CONCLUSIONS: The present results indicate that the dominant entry pathway of NaCl from the stroma into the ciliary epithelial syncytium is through an acetazolamide-inhibitable Cl-/HCO3 and a parallel Na+/H+ antiport. The dominant release pathways into the aqueous humor appear to be a Na+-K+-2Cl-symport, which can be outwardly directed under physiological conditions, together with the Na+/K+-exchange pumps and Cl- channels.

Electron probe X-ray microanalysis of rabbit ciliary epithelium.

Rabbit iris-ciliary bodies were preincubated in control and experimental Ringer's solutions before quick freezing, cryosectioning, dehydration and electron probe X-ray microanalysis. After preincubation in a baseline bicarbonate-free Cl- Ringer's solution, the ciliary epithelial intracellular Na+, K+ and Cl- concentrations were estimated to be 15 +/- 3, 162 +/- 14 and 46 +/- 5 mmol kg-1 intracellular water, respectively. The water and elemental Na, K, Cl and P contents were similar in the non-pigmented (NPE) and pigmented (PE) ciliary epithelial cells. As expected, inhibition of the Na,K-exchange pump by preincubation with ouabain markedly increased the intracellular Na content, and markedly reduced the intracellular K content, verifying the validity of the experimental analysis. The Cl- channels of the NPE cells likely play a critical role in determining the rate of aqueous humor formation. Therefore, we have examined the effects of altering Cl- transport on the intracellular composition in this initial microprobe study of the ciliary epithelium. As expected, exposure to bicarbonate increased the intracellular Cl and water contents. Replacement of external Cl- by NO3- was twice as effective as replacement by gluconate in leaching Cl- out of the intracellular compartment. An unexpected finding was that NO3- replacement of internal Cl- substantially increased the intracellular Na and decreased the intracellular K content, possibly by stabilizing the Na,K-pump in the E1P form and inhibiting enzyme activity.

Effects of voltage clamping on epithelial cell composition in toad urinary bladder studied with x-ray microanalysis.

Toad urinary bladder epithelial cells were incubated in Na Ringer's with the serosal surface of the epithelium clamped at either +50 mV, 0 mV (short-circuited) or -50 mV with respect to the mucosal surface. Following incubation, portions of tissue were coated with an external albumin standard and rapidly frozen. Cryosections were freeze-dried and cell composition determined by x-ray microanalysis. Cell water and ion contents were unaffected when tissues were short-circuited rather than clamped close to their open-circuit potential difference (+50 mV). Incubation with vasopressin at +50 mV, and under short-circuit conditions, caused Na uptake without cell swelling or gain in Cl. Clamping at -50 mV resulted in uptake of water and ions, with considerable variation from cell to cell. These variations in cell composition were exacerbated by vasopressin. The greater the increase in water content, the greater the rise in cell Cl. However, there was no consistent pattern to the associated changes in cation contents. Most cells gained some Na. In some cells, this gain was accompanied by an increase in K. In others, the gain of Na was predominant and cell K content actually fell. At -50 mV with ouabain, many of the cells also gained water. As was found in our earlier study with ouabain under short circuit conditions (Bowler et al., 1991), there was considerable variation in the extent of the Na gain and K loss; some cells were largely depleted of K while in others the K content remained relatively normal. These results indicate differences between granular cells in the availabilities in the plasma membranes of ion pathways, either as a consequence of differences in the numbers of such pathways or in their control.

Cell Cl and transepithelial na transport in toad urinary bladder.

Relationships between short-circuit current (Isc), cell Cl and the mechanism(s) of Cl accumulation in toad bladder epithelial cells were investigated. In serosal Cl-free gluconate Ringer, 80% of the cell Cl (measured by x-ray microanalysis) was lost over 30-60 min with an associated decrease in cell water content. concomitantly, Isc fell to 20% of its initial value within 10 min but then recovered to 45% of its initial value despite continued Cl loss. With the reintroduction of Cl, cell Cl and Isc both recovered within 10 min. Serosal SITS (4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonate; 0.5 mM) plus bumetanide (0.1 mM), did not prevent the fall in Isc or the loss of cell Cl in gluconate medium, although they did inhibit subsequent recovery of Isc in this medium. They also prevented the recovery of Isc in Cl medium but not the reaccumulation of Cl by the cells. Although SITS and bumetanide did not prevent the loss or recovery of Cl, they modified the pattern of the ion changes. In their absence, changes in cellular Cl were twice that of the changes in measured cellular cations implicating basolateral Cl/HCO3 exchange in Cl movement. With SITS plus bumetanide present, changes of similar magnitude in Cl were associated with equivalent changes in cation, consistent with the inhibition of Cl/HCO3 exchange.

Problems in the understanding of cell volume regulation.

The constancy of cell volume under physiological conditions is generally thought to reflect a balance between solute influx and efflux and is therefore critically dependent on the properties of the plasma membrane. Despite a number of unanswered questions, a large amount of experimental data can be accommodated within this pump-leak framework and can by analysed using the simple assumptions of osmotic equality between cells and their surrounding fluid, and fluid electroneutrality. Experimentally, cell volume may be altered in vitro either by changing cell solute content under isosmotic conditions or by changing medium osmolality. Exposure to anisosmotic media may provoke a variety of cell responses that minimise the volume change. However, much of this experimental work has been performed under extreme conditions in vitro that would never be experienced by vertebrate cells in vivo; its relevance to pathophysiological situations is questionable. It is argued that regulation of cell volume should not be seen in isolation but as part of the process, cell homeostasis, by which cells attempt to minimise changes in composition when faced with perturbations in their environment. Given the variety of processes and the large numbers of membrane transporters, an understanding of how cells respond to such perturbations requires a combination of modelling and experimentation. A simple example of this approach is presented.

Effects of potassium-free media and ouabain on epithelial cell composition in toad urinary bladder studied with X-ray microanalysis.

The technique of X-ray microanalysis was used to study the composition of toad urinary bladder epithelial cells incubated in Na Ringer's and K-free medium, with and without ouabain. Following incubation under short-circuit conditions, portions of tissue were coated with an external albumin standard and plunge-frozen. Cryosections were freeze-dried and analyzed. In Na Ringer's, granular and basal cells, and also the basal portion of the goblet cells, had similar water and ion compositions. In contrast, mitochondria-rich cells contained less Cl and Na. On average, the granular cells and a subpopulation of the basal cells lost K and gained Na after ouabain and in K-free medium alone. However, there was considerable variation from cell to cell in the responses, indicating differences between cells in the availabilities of ion pathways, either as a consequence of differences in the numbers of such pathways or in their control. In contrast, the compositions of both the low Cl, mitochondria-rich cells and a sub-population of the basal cells were little affected by the different incubation conditions. This is consistent with a comparatively low Na permeability of these cells. The results also indicate that (i) much, if not all, of the K in the dominant cell type, the granular cells, is potentially exchangeable with serosal medium Na, and (ii) Na is accumulated from the serosal medium under K-free conditions. They also provide information about the role of the (Na-K)-ATPase in the maintenance of cellular K in K-free medium, being consistent with other evidence that removal of serosal medium K inhibits transepithelial Na transport by decreasing Na entry to the cells from the mucosal medium, rather than solely by inhibiting the basolateral membrane (Na-K)-ATPase.

Contributions of secondary active transport processes to membrane potentials.

Equations are developed to examine the effects of secondary active transport processes on the steady-state membrane potential of symmetrical cells. It is shown that, with suitable modifications, equations of the type developed by Goldman, Hodgkin and Katz may be derived to accommodate the contributions to the membrane potential of both electroneutral and electrogenic transporters. Where the membrane potential is a function of the dominant medium ions (Na, K, and Cl), other contributions can come only from an electrogenic Na pump and from neutral co- and counter-transporters if, and only if, these involve the dominant ions. Experimental approaches to measure the parameters necessary to solve the equations developed here are discussed.

Application of membrane potential equations to tight epithelia.

It is shown that equations developed to analyze the contributions of secondary active transport processes to symmetrical cells (Gordon, L.G.M., Macknight, A.D.C., 1991, J. Membrane Biol. 120:139-152) can be used, with minor modifications, to analyze the steady-state membrane potential in epithelia under the unique situation of short circuiting. Only under such conditions is there a single intracellular potential relative to both the mucosal and serosal media. The equations are investigated in relation to a model tight epithelium--the toad urinary bladder. It is shown that the properties of the membrane transport pathways are such that the intracellular potential under short-circuit conditions must be more negative than often reported. Given measurements of membrane potential and of voltage-divider ratio, it is possible to use the equations to estimate the absolute values of the membrane permeabilities and conductances under short-circuit conditions.

Ion channels in urinary bladder.

Urinary epithelia separate urine from interstitial fluid. In the mammal, this tight epithelium has a limited transport capacity but is capable of moving sodium from urine to blood through an aldosterone-sensitive cellular pathway. In lower vertebrates, absorption of ions and water from the urine can contribute significantly to fluid and electrolyte homeostasis. Transepithelial ion transport and maintenance of cellular composition are interdependent, requiring a balance between movements across the apical and basolateral plasma membranes through a variety of pathways including electrodiffusion through ion channels. A variety of such channels has been identified in urinary epithelia. Apical membranes contain amiloride-sensitive, highly selective sodium channels of low conductance (approximately 5-10 pS). There is evidence that in mammalian bladders trypsin-like enzymes in the urine continually degrade these channels, decrease in cation selectivity being followed by loss of the channels from the membrane. New channels stored in the cytoplasm appear to provide a source for replenishment of the membrane. Other channels of higher conductance and lower selectivity have also been described in both mammalian and amphibian bladders, but their physiological significance remains to be established. Basolateral membranes contain potassium channels. In the mammalian bladder, in which chloride appears to be distributed at electrochemical equilibrium, chloride conductance exceeds potassium conductance and patch clamp studies have revealed a chloride channel of conductance approximately 60 pS detectable immediately on patch excision and active at normal membrane potentials. In the amphibian bladder, a variety of findings indicates the presence of a basolateral membrane chloride conductance, but patch clamp data are not yet available.

Effects of impermeant medium ions on the composition of rabbit renal cortical slices.

When incubated in isosmotic oxygenated medium in which chloride was completely replaced by gluconate, rabbit renal cortical slices lost chloride with sodium, potassium and water before reaching a new steady-state composition after 15-30 min. When corrected for extracellular space, there was an electroneutral loss of alkali metal cations (Na + K) with chloride, accompanied by isosmotic loss of water from the cells. The losses of chloride and water were independent of medium pH over the range of 6.4-8.2, and were the same with potassium rather than sodium as the dominant medium cation. Incubation in isosmotic sodium chloride medium restored tissue composition of slices transferred from gluconate medium. This recovery was not dependent specifically upon medium chloride, for slice water content also recovered when nitrate rather than chloride was substituted for medium gluconate. With sodium completely replaced by n-methyl d-glucamine (nmdG+), cells in slices lost far more sodium and potassium than chloride before reaching a new steady-state composition after some 30 min. However, the loss of water was as predicted from the total losses of measured inorganic ions. With sodium and chloride completely replaced by nmdG+ and gluconate, there was a greater loss of water than found with unilateral substitutions. Again, the combined loss of diffusible inorganic cations exceeded the loss of chloride but the water loss was that expected for isosmotic loss accompanying the measured losses of ions. These results reveal that both gluconate and nmdG+ behave as impermeant ions in this tissue preparation. It is suggested that, in the absence of medium sodium, sodium-hydrogen exchange is inhibited. Retained hydrogen ions are buffered on charged cellular non-diffusible solutes and the associated hydroxyl (or bicarbonate) ions are lost from the cells accompanied by the inorganic univalent cations lost in excess of chloride in nmdG+ medium.

Single-channel recordings from the apical membrane of the toad urinary bladder epithelial cell.

The patch-clamp technique for the recording of single-channel currents was used to investigate the activity of ion channels in the intact epithelium of the toad urinary bladder. High resistance seals were obtained from the apical membrane of tightly stretched tissue. Single-channel recordings revealed the activity of a variety of ion channels that could be classified in 4 groups according to their mean ion conductances, ranging from 5 to 59 pS. In particular, we observed highly selective, amiloride-sensitive Na channels with a mean conductance of 4.8 pS, channels with a similar conductance that were not Na-selective and channels with mean conductance values of 17-58 pS that were mostly seen after stimulation of the tissue with vasopressin or cAMP. When inside-out patches from the apical membrane were exposed to 110 mM fluoride, large conductances (86-490 pS) appeared.

Principles of cell volume regulation.

Cell volume is determined by the content of osmotically active solute (cell osmoles) and the osmolarity of the extracellular fluid. Cell osmoles consist of non-diffusible and diffusible solutes. A large fraction of the diffusible cation content balances negative charges on the non-diffusible solutes. The content of diffusible solutes is determined by the electrochemical gradients driving them across the plasma membrane and the availability and activity of transport pathways in the membrane. The classical view that the sodium pump offsets passive leaks must be modified to accommodate the contributions of a number of secondary active transport processes, as well as to allow for changes in cell nondiffusible osmoles and in their net negative charge. The behaviour of cells in anisosmotic media is often different from that predicted for a perfect osmometer. In many cases this is a consequence of changes in cell osmole content. However, caution is required in extrapolating from in vitro responses of isolated cells to large, acutely induced changes in medium osmolality to the responses of tissues in vivo to more subtle changes in extracellular osmolality.