AVP V1 receptor-mediated decrease in Clminus efflux and increase in dark cell number in choroid plexus epithelium

Conrad E. Johanson1, Jane E. Preston1, Adam Chodobski1, Edward G. Stopa2, Joanna Szmydynger-Chodobska1, and Paul N. McMillan2

1 Program in Neurosurgery, Department of Clinical Neurosciences and 2 Department of Pathology, Brown University/Rhode Island Hospital, Providence, Rhode Island 02903

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The cerebrospinal fluid (CSF)-generating choroid plexus (CP) has many V1 binding sites for arginine vasopressin (AVP). AVP decreases CSF formation rate and choroidal blood flow, but little is known about how AVP alters ion transport across the blood-CSF barrier. Adult rat lateral ventricle CP was loaded with 36Cl-, exposed to AVP for 20 min, and then placed in isotope-free artificial CSF to measure release of 36Cl-. Effect of AVP at 10-12 to 10-7 M on the Cl- efflux rate coefficient (in s-1) was quantified. Maximal inhibition (by 20%) of Cl- extrusion at 10-9 M AVP was prevented by the V1 receptor antagonist [beta -mercapto-beta ,beta -cyclopentamethyleneproprionyl1,O-Me-Tyr2,Arg8]vasopressin. AVP also increased by more than twofold the number of dark and possibly dehydrated but otherwise morphologically normal choroid epithelial cells in adult CP. The V1 receptor antagonist prevented this AVP-induced increment in dark cell frequency. In infant rats (1 wk) with incomplete CSF secretory ability, 10-9 M AVP altered neither Cl- efflux nor dark cell frequency. The ability of AVP to elicit functional and structural changes in adult, but not infant, CP epithelium is discussed in regard to ion transport, CSF secretion, intracranial pressure, and hydrocephalus.

cerebrospinal fluid homeostasis; chloride-36 efflux rate coefficient; hydrocephalus; V1 receptor antagonist; neuroendocrine regulation; arginine vasopressin

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

ARGININE VASOPRESSIN (AVP) V1 binding sites in choroid plexus (CP) (25, 35, 36) and blood-brain barrier have a role in regulating central nervous system (CNS) extracellular fluid balance (22, 32). Cerebrospinal fluid (CSF) is produced mainly by CP (5, 11, 37, 38), and its formation rate can be altered by changes in choroidal hemodynamics (7, 21) and via alterations in ion transport by CP epithelium (1, 15, 27, 31). AVP modulates choroidal blood flow and CSF formation (3, 5, 7, 8, 20, 22, 32), and yet there is scant information on how AVP affects CP transport of Na+, K+, and Cl-, all of which are integral to CSF formation (1, 5, 9, 15, 17, 27, 31, 38).

Anion transport by CP is tightly linked to CSF production (9, 12, 13, 15, 31, 34). Cl- is the main anion in CSF, and its transport by CP has been investigated in vivo and in vitro (9, 12, 15, 27, 31, 37, 38). Agents inhibiting CSF production (3-5, 12, 13, 34) usually suppress extrusion or release of Cl- from in vitro CP (9, 27, 31, 34). Because AVP curtails CSF production (3, 8, 20, 22), we hypothesized that this neuropeptide, by way of V1 receptor activation, would reduce the ability of in vitro CP to transport Cl- into surrounding fluid.

The ability of AVP to alter H2O and Cl- fluxes across renal epithelial membranes has inspired studies of peptide modulation of the kidneylike CP (6, 19, 28, 29). Exposure of isolated CP to AVP increases the number of dark epithelial cells (19). Dark cells also occur in the CP of hydrocephalic animals (30). Dohrmann (6) has concluded that CP in mouse, dog, and human normally contains ultrastructurally similar light and dark cells, the latter possibly representing varying states of cellular hydration. The physiological significance of dark cells in CP needs elucidation because 1) they exist normally in vivo (6) and 2) AVP, which alters CP-CSF functions, also increases the number of dark epithelial cells in vitro (19) and in vivo (28).

Therefore, the dual aims of this investigation were to analyze the abilities of AVP to alter Cl- transport and the number of dark epithelial cells in CP and to ascertain whether both effects could be prevented by blockade of V1 receptors. We found that 20-min exposure of CP to AVP induced functional and structural changes mediated by V1 receptors. The AVP-modified Cl- transport and dark cell frequency in CP are discussed in regard to CSF dynamics and homeostasis. Our AVP findings are compared with similar observations with ANG II (3, 4, 21, 22, 34) to add further evidence for a neuroendocrine model of CSF regulation.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals, anesthesia, and surgery. Approval for all technical and surgical aspects of this investigation was given by the Rhode Island Hospital Institutional Animal Welfare Committee. Sprague-Dawley adult rats, 6-8 wk old, were obtained from Charles River Laboratories (Wilmington, MA). Infant rats, 7-8 days postnatal, were obtained from pregnant animals shipped 1 wk before parturition. After induction of anesthesia with Metofane (Pitman-Moore) administered by nose cone, each animal was perfused transcardially with ice-cold isotonic mannitol so that AVP in residual blood of the CP would not contribute to effects. Subsequently, the lateral ventricles were opened and CP tissues were removed and immediately placed in 290 mosmol/kg artificial CSF (aCSF) of previously described composition (27).

Incubation of CP in aCSF. One of each pair of plexuses was used for control, and the other was used for in vitro treatment with AVP and/or the V1 receptor antagonist. A V1 antagonist was selected because several autoradiographic studies have indicated that AVP binding sites in adult CP are of the V1 and not the V2 subtype (25). CP was incubated in aCSF, either for investigation of Cl- efflux or for analysis of ultrastructure by the Core Research Facility at Rhode Island Hospital.

Before experimental incubation, each CP was stabilized for 5 min in a separate incubation tube containing 1 ml of isotope- and drug-free aCSF saturated with 95% O2-5% CO2 at 37°C (27). The preincubated CP was transferred to 0.5 ml of aCSF medium containing 2.5 µCi of 36Cl- to load the radioisotope into tissue for 20 min. BSA (0.1%) was used as carrier in all incubations of CP in aCSF.

36Cl- efflux experiments. After the 20-min 36Cl- loading period, AVP was added to the aCSF containing the "hot" tissue to yield an AVP concentration of 10-12 to 10-7 M. In other incubations, the V1 antagonist [beta -mercapto-beta , beta -cyclopentamethyleneproprionyl1,O-Me-Tyr2,Arg8]vasopressin [d(CH2)5Tyr(Me)AVP] was tested at 10-8 M with or without AVP. AVP and d(CH2)5Tyr(Me)AVP were obtained from Sigma (St. Louis, MO). After a 20-min exposure to AVP plus antagonist, CP were removed, rinsed quickly in isotope-free aCSF, and placed in an efflux bath (35-mm tissue culture dish) containing 2 ml of warmed, gassed, and magnetic bar-stirred aCSF. Six 200-µl samples were taken at 20-s intervals from the efflux bath into which 36Cl- was released from the 36Cl--loaded CP. At the time of the sixth sampling, the CP was removed, wiped on a glass slide, and weighed in a tared aluminum foil boat to within 0.01 mg on a Cahn 4600 electrobalance (Cerritos, CA) to ascertain tissue weight.

Radioactivity counting and calculations. Samples of CP and aCSF were prepared for liquid scintillation analysis and then assayed for 36Cl- on a Beckman LS 5800 beta-counter. Procedures for quantifying the rate of efflux of 36Cl- from choroidal tissues have been described at length (27) and so are concisely described here. Total tissue 36Cl- was taken as 100% labeling at time zero. 36Cl- remaining in CP at each time point of the efflux analysis was calculated by consecutively subtracting, from the total, the efflux measured every 20 s. The 36Cl- efflux rate coefficient (k; in s-1), determined by linear regression analysis, was ascertained from the slope of the logarithmic plot of 36Cl- remaining in tissue vs. time. AVP-induced decrease of 36Cl- release from isolated CP was reflected by reduced values for k.

Electron microscopy: tissue preparation and morphometric analyses. One goal was to extensively analyze the ultrastructure of dark epithelial cells. CP lends itself to both immersion and perfusion fixation because it has a single layer of cells surrounding microvessels. For CP of several mammalian species, Dohrmann (6) found dark and light epithelial cells fixed with glutaraldehyde and/or osmium by immersion or perfusion. Therefore, he concluded that the dark-light cell phenomenon reflected the in vivo situation and not an artifact of fixation (6).

Lateral ventricle CP for ultrastructural analysis was incubated in aCSF, with the protocols described above for experiments to determine k. After incubation, CP was fixed in modified Karnovsky's medium, postfixed in 1% OsO4 (contained in 0.1 M sodium cacodylate buffer, pH 7.4) and then embedded in Spurr's epoxy resin. Analysis of CP ultrastructure was done on a Philips 300 electron microscope.

Video images from electron microscopic negatives were acquired at a standardized magnification via a Perceptics Hyperscope (Perceptics, Knoxville, TN) frame grabber and analyzed with image-processing software, including Perceptics Biovision and NIH Image (National Institutes of Health, Bethesda, MD). By combining thresholding and manual filling procedures, two sets of binary images were created: 1) the total combined area occupied by the mitochondria and 2) the total area taken up by the cytoplasm, not including the nucleus.

For morphometric analyses, the mitochondrion was selected as the "reference organelle" because of its 1) relative abundance (there were ~100 mitochondria vs. a single nucleus per field), 2) traceability (for image outlining, the mitochondria could be demarcated and thus traced more accurately than, for example, sparsely distributed short profiles of endoplasmic reticulum), and 3) stability [because mitochondria were stable following AVP treatment (see Ultrastructure of dark cells), their collective volume was used as a reference for evaluating the changing volume of cytoplasm].

Evaluation of light microscopic images. Semi-thin sections were cut from rat CP, embedded in Spurr's low-viscosity embedding resin, and stained with methylene blue and azure II dyes. Photomicrographs were taken and enlarged to a final magnification of ×450. With care exercised to avoid overlapping fields, micrographs were acquired to include as many of the CP epithelial cells in each semi-thin section as possible. Subsequently, the total number of epithelial cells (typically 3,000-5,000) and the subset composed of dark cells only were manually determined for each tissue section. Cell counts of each of the above categories from all sections were summed, and the percentage of dark cells was determined.

Statistical analyses. The availability of two choroid tissues in each animal, i.e., from right and left lateral ventricles, permitted paired analyses, by Student's t-test. For analysis of AVP and V1 antagonist effects vs. corresponding controls, one-way ANOVA and the post hoc Bonferroni test were also employed. Data are means ± SE.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

36Cl- washout from CP. Representative 36Cl- washout curves for CP from adult and infant rats are presented in Fig. 1. The magnitude of the slopes provides k values (s-1) for 36Cl- release as a function of developmental stage as well as exposure to AVP. Linear regression analyses of the points constituting each washout curve generally yielded R2 values >0.98. It was previously established that the extracellular component of 36Cl- washout is >90% complete by 20 s (27); thus the slopes in Fig. 1, and the associated average values for k in Figs. 2-4, represent release of 36Cl- from the epithelial compartment.


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Fig. 1.   Washout curves for release of 36Cl- from adult (A) and infant (B; 1 wk) rat lateral ventricle choroid plexus (CP) incubated in artificial cerebrospinal fluid (aCSF) at 37°C. Each slope reflects rate of extrusion of 36Cl- from a single tissue exposed (open circle ), or not (bullet ), to arginine vasopressin (AVP) at 10-9 M for 20 min. See METHODS for determination of percentage of 36Cl- remaining in CP. Points were fitted to each curve by least-squares linear regression. Efflux rate coefficient for 36Cl- (k) was determined as absolute value of negative slope. AVP at 10-9 M significantly reduced slope of 36Cl- washout from adult CP but not from infant CP. R2, correlation coefficient.


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Fig. 2.   Effect of AVP concentration on rate of release of 36Cl- from adult rat lateral ventricle CP in vitro (means ± SE of k for 3-7 CP tissues). bullet , AVP; open circle , control. * P < 0.05 by paired Student's t-test for control vs. corresponding AVP concentration.


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Fig. 3.   Age difference in response to AVP. Cl- efflux from lateral CP of infant vs. adult rats. Shown are means ± SE of k for 5 (infant) or 8 (adult) CP tissues incubated in aCSF with or without 10-9 M AVP. * P < 0.05 by paired Student's t-test for control vs. AVP. Infant rats were 7-8 days postnatal.


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Fig. 4.   Effect of V1 receptor blockade, in presence and absence of 10-9 M AVP, on k for adult CP. A and B were run on right and left lateral ventricle tissues from same rat; similarly, C and D were carried out on paired lateral ventricle plexuses. V1 antagonist (V1 ANTAG) was d(CH2)5Tyr(Me)AVP at 2 × 10-8 M (see METHODS). Data are means ± SE; n = 4 CP for A and B and 6 CP for C and D. * P < 0.05 for C vs. D by paired Student's t-test and for C vs. A by 1-way ANOVA and Bonferroni test.

AVP concentration and 36Cl- release. The effect of AVP concentration on 36Cl- release from adult rat CP was analyzed over a range from 10-12 to 10-7 M (Fig. 2). A significant reduction in 36Cl- release, by 20%, was found at the AVP concentration of 10-9 M (P < 0.05), and higher peptide concentrations also brought about a significant decrease in Cl- efflux (Fig. 2).

In infant rat CP, the mean control value of the efflux k for 36Cl- was 0.0280 ± 0.0004 s-1 (Fig. 3). An AVP effect on 36Cl- release from infant rat CP was tested at 10-9 M, i.e., the concentration that elicits the maximal response in suppressing Cl- efflux from adult tissue (Fig. 2). However, unlike the case for adult tissue, 10-9 M AVP did not alter 36Cl- release from infant CP (k = 0.0283 ± 0.0015 s-1; n = 5; P > 0.05 vs. age-matched control).

V1 antagonism of AVP-altered Cl- efflux. The V1 receptor antagonist was tested for its ability to alter Cl- efflux either by itself or in combination with AVP (Fig. 4). V1 receptor blockade did not alter 36Cl- efflux from CP; thus, at the d(CH2)5Tyr(Me)AVP concentration of 2 × 10-8 M, the k value of 0.0322 ± 0.0005 s-1 was not different from its paired control value of 0.0321 ± 0.0010 s-1. However, d(CH2)5Tyr(Me)AVP abolished the AVP-induced attenuation of 36Cl- efflux from adult CP (P < 0.05; Fig. 4). The V1 receptor antagonist was not used in immature rat CP because AVP did not alter 36Cl- release from infant tissues.

AVP-induced increase in dark cell occurrence and its prevention by V1 receptor blockade. CP exposure to 10-9 M AVP for 20 min resulted in an increased number of dark epithelial cells (Figs. 5 and 6). Basically, the CP epithelial monolayer is composed of light and dark cells (Fig. 5). Normally, in controls, the light cells outnumber dark cells by ~15 to 1. Light cells are characterized by a generous apical microvillous membrane, a basolateral labyrinth (BL) that interdigitates with its counterpart in the adjacent cell, and an abundance of mitochondria, Golgi apparatus, and rough endoplasmic reticulum (RER). Dark cells in mature (Fig. 5, A and B) and young (Fig. 5, C and D) rat CP contain mitochondria, Golgi apparatus, and RER of normal appearance but differ from their light counterparts in having a dark (electron-dense) cytoplasm that is generally uniform throughout the cell. Dark cells are sometimes contiguous, but more often than not they are surrounded by light cells, in both infant and adult CP (Fig. 5).


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Fig. 5.   Low-power (×1,500) electron micrographs of rat lateral ventricle CP epithelia from 4 cohorts. A: adult tissue incubated for 20 min in aCSF (control incubation). Note normal appearance of majority of light epithelial cells (LC), which exhibit abundant apical microvilli (Mv), numerous mitochondria (M), ovoid nuclei (Nu), and light to moderate electron density of cytoplasm. Note also single dark cell (DC), which exhibits an electron-dense, elongated nucleus (Nu) surrounded by markedly electron-dense cytoplasm extending into densely packed filiform apical microvilli as well as basolateral labyrinth (BL). BL is composed of complex foldings and convolutions of basal surface of cell and interdigitates with corresponding structures of adjacent light cells. P, pial cells; Cp, capillary; V, ventricular lumen. Bar, 5 µm. B: adult CP tissue incubated for 20 min in aCSF containing 10-9 M AVP; 5 epithelial cells exhibit a normal or typical appearance regarding cytoplasmic organelles and overall electron density, whereas 3 cells show a striking increase in electron density characteristic of dark cell category. IS, interstitial space. Bar, 5 µm. C: infant rat CP (7 days postnatal) after a control incubation. Epithelial cells are similar to adult light cells in A except for glycogen (Gl) depots that are typically located in basal portion of cells. Dark cells are infrequently seen in infant CP. Bar, 5 µm. D: infant rat CP after a 20-min incubation in aCSF containing 10-9 M AVP. A dark cell is shown; however, unlike finding when adult cells were treated with 10-9 M AVP, there was no significant AVP-induced increase in DC in infant CP. Gl pooling was observed at apical as well as basolateral pole after exposure of CP to AVP. Bar, 5 µm.


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Fig. 6.   Dark cell frequency analysis of CP epithelium exposed in vitro for 20 min to 10-9 M AVP, with or without V1 receptor antagonist d(CH2)5Tyr(Me)AVP at 2 × 10-8 M. All tissues, from lateral ventricle of adult rats, were incubated for 20 min at 37°C. Control, aCSF without AVP or d(CH2)5Tyr(Me)AVP. Limits are means ± SE for 5 CP. Each bar represents counts of at least 2,000 choroid epithelial cells. Ordinate represents percentage of epithelial cells that were dark. * P < 0.05 for AVP vs. control and for AVP vs. AVP + V1 ANTAG, by 1-way ANOVA and Bonferroni test.

With methylene blue-stained semi-thin sections (not shown), we did a frequency analysis of the relative numbers of light and dark cells in AVP-exposed tissues, incubated with and without the V1 antagonist. In adult CP exposed to AVP, there was more than a doubling in the number of dark cells (Fig. 6), which as a percentage of the total number of epithelial cells increased from 7 (control) to 18% after 10-9 M AVP (P < 0.05). The V1 antagonist substantially reduced the AVP-mediated induction of dark cells (P < 0.05; Fig. 6). However, in CP from infants, 10-9 M AVP did not significantly increase the number of dark cells above the baseline control value of 9% (not depicted). Therefore, there was not a significant dark cell induction response in immature animals to be analyzed for V1 antagonism.

Ultrastructure of dark cells. The fine structure of dark epithelial cells was extensively analyzed by electron microscopy, in control (Fig. 7A) as well as AVP-exposed (Fig. 7B) CP tissues, and compared with that of their light cell counterparts. Control adult tissue shows that choroidal cells conventionally designated as "light" typically have electron-lucent cytoplasm, tight junctions, clavate apical microvilli, and abundant mitochondria, Golgi apparatus, and RER (Fig. 7A, right). In comparison, "dark" cells in controls display some interesting differences, that is, their cytoplasm exhibits a markedly increased electron density that extends into the microvilli (Fig. 7A, left). Dark cells in controls (Fig. 7A) are ultrastructurally similar to dark cells in AVP-treated CP (Fig. 7B). The enhanced electron density in the cytoplasm of dark cells extends not only into apical microvilli but also into the fingerlike projections of the BL; moreover, the darkened microvilli and BL "fingers" are thinner than those seen in their light cell counterparts (Fig. 7A). The marked contrast between dark and light cytoplasm is strikingly evident where there is intertwining of the BL processes between a dark cell and its adjacent light cell neighbor (Fig. 7A, bottom).


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Fig. 7.   Electron micrographs at intermediate magnification (×15,500) of light (typical) and dark cells in CP of control (A) and AVP-treated (B) adult cells. Typical choroid epithelial cells from mature animals are characterized by electron-lucent cytoplasm, clavate apical microvilli, occasional cilia (Ci), an abundance of mitochondria with cristae in orthodox configuration, rough endoplasmic reticulum (RER), and perinuclear Golgi apparatus (Go). Dark cells, on other hand, have a greater cytoplasmic electron density [which extends into thinner microvilli as well as BL; A], irregular electron-dense nuclei, and an overall shrunken cytoplasmic profile. However, cytoplasmic organelles exhibit normal morphology. Although in vitro treatment with 10-9 M AVP causes a doubling in number of dark cells (see Fig. 6), ultrastructural features of dark cells from AVP-treated specimens are virtually identical to those seen in controls. S, secretory granule. Bars, 1 µm.

Because of prominent effects of AVP on choroidal cells of adult animals, we thought it useful to do morphometric analysis of epithelial ultrastructure. Mitochondria were selected as the organelle for morphometric analysis (Table 1). To address the issue of mitochondrial stability, in regard to shape and size (volume) after AVP treatment, we measured the surface area and perimeter of individual mitochondria by image processing and compiled the data for thousands of mitochondria in hundreds of cells. The ratio of area to perimeter was relatively constant, at a value of ~5.0, for all four groups analyzed (Table 1). Moreover, shrinkage of dark cells compared with control light cells was manifested in smaller values for the ratio of cytoplasmic area to collective mitochondrial area (Table 1). Overall, then, even though dark cells were consistently smaller, presumably due to loss of H2O from the cytoplasmic compartment, the mitochondria remained stable in size, as evidenced from the surface area-to-perimeter determinations.

                              
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Table 1.   Morphometric analyses of light and dark epithelial cells in adult rat lateral ventricle choroid plexus

To further assess the viability of the dark choroid epithelial cells, we analyzed various cellular organelles at very high magnification (Fig. 8). Although dark cells consistently presented a shrunken and shriveled appearance (Fig. 7), their Golgi apparatus, RER, and other organelles exhibited normal morphology (Fig. 8).


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Fig. 8.   High-magnification (×30,000) micrograph of organelles in dark epithelial cells of adult rat lateral ventricle CP exposed to 10-9 M AVP. Choroidal tissue incubation conditions are described in METHODS. Ultrastructure of Golgi apparatus, RER, and mitochondria is virtually unaltered by 10-9 M AVP concentration that increases number of dark cells and reduces 36Cl- efflux. Quantitative morphometric data for mitochondria and cytoplasm are given in Table 1. Bar, 0.5 µm.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Overview of CP as a target organ for AVP. In this acute study, we demonstrated that nanomolar AVP reduces 36Cl- efflux from isolated CP and doubles the number of dark epithelial cells with morphologically normal and apparently functional organelles. Both effects were prevented by V1 receptor blockade, thereby implicating V1 receptors in modulating CP responses to AVP (3, 8, 20, 23, 25, 36). Our in vitro findings with adults that AVP inhibits Cl- flux across CP and increases the number of dark epithelial cells, together with previous in vivo observations that AVP alters choroidal hemodynamics (7) and CSF formation (3, 8), point to a role for CP and AVP in CNS fluid homeostasis.

Inhibitory effects of AVP on CP transport and CSF formation. AVP diminished 36Cl- efflux in isolated CP from adult rats. Cl- efflux from CP is integral to CSF formation (9, 12, 15, 17, 18, 27, 31, 38). Agents that inhibit CSF formation also reduce 36Cl- transport from the CP blood compartment into cisternal CSF (12). The same drugs, i.e., acetazolamide, disulfonic stilbenes, furosemide, and bumetanide, reduce Cl- extrusion from isolated CP to aCSF (9, 27, 31). The ability of AVP to reduce 36Cl- efflux from adult CP is in line with AVP inhibition of CSF formation in mature mammals (3, 8). Therefore, the AVP-mediated inhibition of CP 36Cl- efflux corroborates the conclusion from ventriculo-cisternal perfusion experiments (3) that AVP acts on choroidal tissue via V1 receptors (Figs. 2 and 4) to decrease CSF formation (3, 8, 20).

Is AVP-mediated induction of dark epithelial cells linked to diminished CSF production? Dark epithelial cells, normally in CP (6, 19), can be enhanced more than twofold in isolated adult CP exposed to exogenous AVP (Ref. 19; Fig. 6). Dark cells are also induced in CP of adult animals administered AVP intravenously or subjected to dehydration that causes plasma AVP to rise (29). Thus dark cell induction occurs in vitro and with pathophysiological states like hydrocephalus when fluid imbalance (29, 30) leads to compensatory increases in extracellular AVP (22, 32). CP from rodents with hydrocephalus displays more dark cells (30) and reduced transport of Cl- (18). Such findings are congruent with decreased CSF production in hydrocephalus.

Dark cells in CP likely modify fluid transfer across the blood-CSF barrier (8, 19, 29). The ability of AVP in adult CP to increase dark cell number and reduce 36Cl- efflux and its inability to do so in the presence of the V1 antagonist indicate an intimate association between ultrastructural and functional phenomena. Although these dual effects on the same epithelial cells await proof, we postulate that altered transport phenomena are linked to dark cell induction.

Ultrastructural characteristics of AVP-induced dark cells. Dark epithelial cells were similar in control and AVP-treated tissues. Electron-dense cytoplasm extended from the cell soma into apical microvilli and the BL interdigitating processes. Darkened cytoplasm was likely due to loss of H2O, as reflected by smaller ratios for dark cell cytoplasmic area to collective mitochondrial area. Some light cells had attenuated volume after AVP (Table 1). Perhaps certain light cells, as they are losing volume, do not quite reach the degree of diminution associated with intense cell darkening; an intriguing but not mutually exclusive hypothesis is that a subset of epithelial cells with different transporter or receptor properties respond to AVP in a manner that does not lead to great darkening of the cytoplasm.

Reduced CSF secretion has been attributed to slender filiform microvilli (29). Other organelles, i.e., mitochondria, Golgi complexes, and RER, were stable in respect to shape and electron lucency. Mitochondrial stability was manifest as a stark negative contrast to surrounding cytoplasm (Fig. 7A). Except for shrinkage, the dark epithelia did not display apoptotic phenomena like blebbing, fragmented nuclei, dilation of RER, or detachment of neighboring cells (Table 2). Moreover, dark cells did not show necrotic signs such as dilation and fragmentation of the RER and high-amplitude swelling of mitochondria. Rather, the integrity of Golgi apparatus and RER in dark cells was manifest by their robust appearance (6, 24). Mitochondrial viability, a sensitive indicator of cell metabolism, was always maintained. Overall then, the dehydrated cytoplasm notwithstanding, the electron micrographs depicted healthy-looking dark epithelial cells.

                              
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Table 2.   Comparative analysis of ultrastructure of choroid epithelial dark cells vs. general characteristics of other cells undergoing apoptosis or acute necrosis

Epithelial ion transport and cell volume. To meld our in vitro CP findings with in vivo CSF observations (3, 5, 8, 12), we propose that the inhibitory effect of AVP on Cl- efflux could result from slower uptake of Cl- and Na+ across the basolateral membrane via Cl-/HCO-3 and Na+/H+ exchange in CP (11). This would lead to reduced Cl- release and fluid turnover across the apical membrane into CSF. In the kidney, AVP, also by a V1 receptor-mediated process (10), inhibits Cl- uptake by cells of the medullary thick ascending limb. Therefore, we hypothesize that shrunken dark cells in CP result from an AVP-associated inhibition of Cl- uptake, which causes decreased cell volume. Due to cell shrinkage, the reduced 36Cl- efflux in AVP treatment could result from an attenuated volume-sensitive release of Cl- via apically located Cl- channels (9) or K+-Cl- cotransport (14, 39). Reduced flux of Cl- from epithelial cells to CSF is consistent with observed V1-dependent inhibition of CSF formation (3, 8, 20).

Inhibited basolateral uptake of ions is also supported by the ability of AVP to suppress 22Na+ transport from blood to CSF by 50% (5) and to reduce CSF formation rate (3, 8). Thus AVP has marked transport effects (29) on the in vivo as well as in vitro CP. Apparent loss of volume regulation in dark cells is intriguing, especially in regard to interrupted transfer of ions and H2O across membranes of CP epithelium into CSF (12, 19, 22, 31). Moreover, inhibition of bumetanide-sensitive ion uptake by apical Na+-K+-2Cl- cotransport (17, 26) in rat CP in vitro also causes a net loss of H2O from choroidal tissue (1) and from isolated CP epithelial cells (K. Strange, personal communication). Studies are needed to ascertain how effects of AVP on ion cotransport in CP (1, 15, 17, 26) alter CSF reabsorptive transport and dark cell frequency.

Infant vs. adult rat CP response to AVP and other agents. Heretofore, the role of AVP in CP and CSF functions has been analyzed mainly in adult mammals (2, 3, 5, 7, 8, 19, 25, 29). Infant rat is also a useful model because immature CP (13, 14, 24, 27, 37) has less functional capacity (27, 33) and responds differently to drugs and peptides, compared with adult CP (24, 27). This provides insight about postnatal development of homeostatic mechanisms (37). Ontogenetically, AVP neither altered 36Cl- efflux from, nor induced dark cells in, 1-wk rat CP. Lack of response to AVP by infant CP may be due to fewer V1 receptors, consistent with observed sparser systems for neurohumoral ligand/receptors in postnatal rat CP-CSF (22). Factors such as incompletely developed neuropeptide receptor signal transduction systems and a metabolism dissimilar to adult (see glycogen in Fig. 5) probably also alter the infant response to AVP.

Is AVP modulation of CP-CSF needed in early CNS development? Due to lower blood flow (33), Cl- transport (27), and choroid cell enzyme activities [e.g., Na+-K+-ATPase (Na+ pump) (24) and carbonic anhydrase/HCO-3 generation (13)], the 1-wk rat CP has less capacity to secrete CSF (16, 27, 37) than the adult counterpart. In early development when CSF flow is sluggish (37) and intracranial pressure (ICP) is relatively low (16), the brain, with its greater tissue compliance, is probably less dependent on sympathetic and vasopressinergic mechanisms (22) to regulate CSF flow. Adult brain, with its greater vascular perfusion and CSF turnover (3, 5, 7, 8, 12, 17, 33) and lower tissue elasticity, needs homeostatic mechanisms to regulate ICP and fluid volume.

A model for CP-CSF involvement in neuroendocrine regulation. Centrally released peptides regulate fluid balance in adult brain (21, 22). One source of AVP for CSF is CP synthesis (2). AVP and ANG II both reduce CP blood flow, Cl- transport, and CSF secretion rate (4, 7, 8, 21, 34). AVP also interacts with ANG II, i.e., there is a V1 receptor-mediated inhibitory action of ANG II on CSF formation (3). Fluid balance in adult CNS stems from regulated transport and permeability at the blood-brain and blood-CSF barriers (3, 4, 8, 15, 21, 27, 38). One mechanism to alleviate ICP elevation (e.g., as occurring in hydrocephalus or cerebral edema) would be a downward adjustment in CSF formation. Interestingly, the CSF level of AVP is elevated in hydrocephalus and cerebral ischemia (32). This suggests a role of AVP synthesis/secretion in CP (2) as part of a neuroendocrine feedback loop for altering CSF production.

Perspective. Information presented herein provides further evidence that AVP in the CNS has a role in modulating CP transport and CSF dynamics. Epithelial cells in CP are responsive to AVP, and the dark cells induced by this peptide display ultrastructural evidence, i.e., shrinkage, that presumably reflects altered H2O movement. Analysis is now needed to delineate the epithelial cells, dark vs. light, in regard to their V1 receptors and ion transporters.

    ACKNOWLEDGEMENTS

We thank C. Thompson, M. Bridges, and M. Lizotte for helpful secretarial assistance, S. Spangenberger, C. Ayala, and P. Monfils for microscopy aid, M. Dyas for skillful technical support in the experiments, and K. Strange for providing critique of the manuscript.

    FOOTNOTES

This study was expedited by research funds from Lifespan, Rhode Island Hospital and by National Institute of Neurological Disorders and Stroke Grant NS-27601 (to C. E. Johanson) and National Science Foundation Grant IBN-9809907 (to A. Chodobski).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: C. E. Johanson, Dept. of Clinical Neurosciences, Rhode Island Hospital, 593 Eddy St., Providence, RI 02903.

Received 31 July 1998; accepted in final form 28 September 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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