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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.

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.

The sections in this article are: 1 Structure of Toad Urinary Bladder 2 Ion Movements Across Epithelium 2.1 Apical Plasma Membrane 2.2 What ore the occurrence and characteristics of individual channels? 2.3 Basolateral Plasma Membrane 2.4 Paracellular Pathway 2.5 Representations of Transepithelial Transport 2.6 Membrane Potentials 3 Water Movements Across Epithelium 4 Composition of Epithelial Cells 4.1 Sodium 4.2 Potassium 4.3 Chloride 4.4 Calcium 4.5 pH 5 Metabolism and Transepithelial Transport 6 Regulation and Control of Transepithelial Transport and of Cell Composition 7 Hormonal Control of Transepithelial Transport 8 Summary

Publisher Summary The mammalian ciliary epithelium is often studied as though it were a homogeneous preparation. However, both anatomical and histological studies show significant differences among different regions. The minor, flat pars plana is posterior to two well-defined anatomic regions, the posterior and anterior portions of the ciliary epithelium. The posterior region, the posterior pars plicata, displays long ciliary processes reaching regularly down to the iris. In contrast, in the rabbit anterior region, comprising the iridial portion of the primary ciliary processes, the folds are more tortuous. From the histological studies, it is known that proteins and biologically active peptides are expressed nonuniformly in different regions of the ciliary epithelium. The ciliary epithelium secretes the aqueous humor into the posterior chamber of the eye. Despite the physiologic and pharmacologic importance of this epithelium, its complex structure and heterogeneous cell composition have impeded progress in studying the integrated functioning of the intact tissue. Electron probe X-ray microanalysis has provided an unusual opportunity to study the different cell populations in different regions of the intact ciliary epithelium. This chapter summarizes advances in the understanding made possible by exploiting this technique.

The rate of aqueous humor formation sequentially across the pigmented (PE) and nonpigmented (NPE) ciliary epithelial cell layers may not be uniform over the epithelial surface. Because of the tissue's small size and complex geometry, this possibility cannot be readily tested by conventional techniques. Rabbit iris-ciliary bodies were divided, incubated, quick-frozen, cryosectioned, and freeze-dried for electron probe X-ray microanalysis of the elemental contents of the PE and NPE cells. We confirmed that preincubation with ouabain to block Na+,K+-ATPase increases Na+and decreases K+contents far more anteriorly than posteriorly. The anterior and posterior regions were the iridial portion of the primary ciliary processes and the pars plicata, respectively. Following interruption of gap junctions with heptanol, ouabain produced smaller changes in anterior PE cells, possibly reflecting higher Na+or K+permeability of anterior NPE cells. Inhibiting Na+entry selectively with amiloride, benzamil, or dimethylamiloride reduced anterior effects of ouabain by ∼50%. Regional dependence of net secretion was also assessed with hypotonic stress, which stimulates ciliary epithelial cell regulatory volume decrease (RVD) and net Cl−secretion. In contrast to ouabain's actions, the RVD was far more marked posteriorly than anteriorly. These results suggest that 1) enhanced Na+reabsorption anteriorly, likely through Na+channels and Na+/H+exchange, mediates the regional dependence of ouabain's actions; and 2) secretion may proceed primarily posteriorly, with secondary processing and reabsorption anteriorly. Stimulation of anterior reabsorption might provide a novel strategy for reducing net secretion.

The intraocular pressure (IOP) reflects a balance between inflow and outflow of aqueous humour. A major strategy in the medical treatment of glaucoma is to reduce inflow and thereby IOP. Understanding the mechanisms and regulation of inflow is thus of clear clinical relevance. Many mechanisms underlying inflow have been identified. The integration and regulation of these mechanisms is less clear. Aqueous humour is secreted across the ciliary epithelium by transferring solute, chiefly NaCl, from the stroma to the posterior chamber of the eye, with water passively following. The epithelium consists of two layers: the pigmented ciliary epithelial (PE) cells abutting the stroma, and the non-pigmented ciliary epithelial (NPE) cells facing the aqueous humour. Gap junctions link adjacent cells within and between these layers. Secretion proceeds in three steps: (1) uptake of NaCl from stroma to PE cells by electroneutral transporters, (2) passage of NaCl from PE to NPE cells through gap junctions, and (3) release of Na+ and Cl- through Na+,K+-activated ATPase and Cl- channels, respectively. Most of our understanding of inflow mechanisms has been obtained by studying in vitro preparations at subcellular, cellular and tissue levels. A particularly productive approach has been the electron probe X-ray microanalysis (EPMA) of the elemental composition of excised ciliary epithelium. This technique permits analysis of adjacent cells within different regions of the ciliary epithelium. EPMA of rabbit preparations has supported the idea that paired activity of Na+/H+ and Cl-/HCO3- antiports can be the dominant mechanism underlying the first step in secretion, stromal NaCl uptake by PE cells. EPMA also indicates that Cl- turnover is faster in the anterior than the posterior region of the epithelium. At the opposite epithelial surface, release of Na+ through Na+,K+-activated ATPase of NPE cells is also greater anteriorly than posteriorly. The accompanying release of Cl- through ion channels is enhanced by agonists of A3 adenosine receptors (ARs). The concepts that paired antiport activity is important in stromal NaCl uptake and that A3ARs modulate NaCl release into the aqueous humour were based on in vitro studies. The potential relevance of these conclusions to in vivo conditions has been tested by measurements of IOP in the living mouse. The results have confirmed the predictions that inhibitors of Na+/H+ antiports lower IOP, and that A3AR agonists and antagonists raise and lower IOP, respectively.

The diagnosis and therapy of childhood brain tumors, most of which are low grade, can be complicated because of their frequent adjacent location to crucial structures, which limits diagnostic biopsy. Also, although new prognostic biomarkers identified by molecular analysis or DNA microarray gene profiling are promising, they too depend on invasive biopsy. Here, we test the hypothesis that combining information from biologically important intracellular molecules (biomarkers), noninvasively obtained by proton magnetic resonance spectroscopic imaging, will increase the diagnostic accuracy in determining the clinical grade of pediatric brain tumors. We evaluate the proton magnetic resonance spectroscopic imaging exams for 66 children with brain tumors. The intracellular biomarkers for choline-containing compounds (Cho), N-acetylaspartate, total creatine, and lipids and/or lactate were measured at the highest Cho region and normalized to the surrounding healthy tissue total creatine. Neuropathological grading was done with WHO criteria. Normalized Cho and lipids and/or lactate were elevated in high-grade (n = 23) versus low-grade (n = 43) tumors, which multiple logistic regression confirmed are independent predictors of tumor grade (for Cho, odds ratio 24.8, P < 0.001; and for lipids and/or lactate, odds ratio 4.4, P < 0.001). A linear combination of normalized Cho and lipids and/or lactate that maximizes diagnostic accuracy was calculated by maximizing the area under the receiver operating characteristic curve. Proton magnetic resonance spectroscopic imaging, although not a proxy for histology, provides noninvasive, in vivo biomarkers for predicting clinical grades of pediatric brain tumors. Clin Cancer Res

MR assessment of pediatric brain tumors has expanded to include physiologic information related to cellular metabolites, hemodynamic and diffusion parameters. The purpose of this study was to investigate the relationship between MR and proton MR spectroscopic imaging in children with primary brain tumors. Twenty-one patients (mean age 9 years) with histologically verified brain tumors underwent conventional MR imaging, hemodynamic MR imaging (HMRI) and proton MR spectroscopic imaging (MRSI). Fourteen patients also had diffusion-weighted MR imaging (DWMRI). Metabolic indices including choline-containing compounds (Cho), total creatine (tCr) and lipids/lactate (L) were derived by proton MRSI, relative cerebral blood volume (rCBV) by HMRI, and apparent tissue water diffusion coefficients (ADC) by DWMRI. Variables were examined by linear regression and correlation as well as by ANOVA. Cho (suggestive of tumor cellularity and proliferative activity) correlated positively with rCBV, while the relationship between Cho and ADC (suggestive of cellular density) was inverse ( P

The 34th Congress of the International Union of Physiological Sciences (IUPS) will be held in Christchurch, New Zealand, August 26—31, 2001. These Congresses are held every four years and represent a unique opportunity for physiologists and workers in associated disciplines to meet together on a

Two twins with late infantile globoid cell leukodystrophy of Krabbe's disease were studied with conventional magnetic resonance imaging (MRI) and proton magnetic resonance spectroscopy. Brain MRI demonstrated brain atrophy with extensive bilateral symmetric abnormal T2 signal in the posterior periventricular white matter, parietal lobes, corona radiata, centrum semiovale, and splenium of the corpus callosum. Magnetic resonance imaging-guided proton magnetic resonance spectroscopy revealed prominent peaks from choline-containing compounds, total creatine, and inositols. The N-acetylaspartate peak was markedly reduced, and the choline-to-N-acetylaspartate ratio was abnormally high; in one of the twins, lactic acid was also detected. The constellation of magnetic resonance spectroscopy findings is indicative of extensive demyelination, gliosis, and loss of axons in the involved white matter; the latter two events occur in the later stages of globoid cell leukodystrophy. In conjunction with brain MRI, these magnetic resonance spectroscopy findings may alert clinicians to the possibility of leukodystrophy in children with progressive encephalopathy. J Child Neurol

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.

SUMMARY 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 intra-ocular 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.

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.

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.

Relationships between short-circuit current (I sc), 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, I sc 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 I sc 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 I sc or the loss of cell Cl in gluconate medium, although they did inhibit subsequent recovery of I sc in this medium. They also prevented the recovery of I sc 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.

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.

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.

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.