Early spreading events of fibroblasts on microgrooved substrates.

We investigated the "contact guidance" phenomenon, shortly after cell attachment. For this purpose polystyrene substrates were produced, either smooth, or equipped with micogrooves (depth 0.5 micrometer, width 1-10 micrometer). On these substrates, fibroblasts were cultured for 15, 30, 45, 60, 120, or 240 min. Subsequently, they were studied with light microscopy, scanning electron microscopy, confocal laser scanning microscopy, and digital image analysis. Up to 1 h, cell attachment on the grooved substrates was impaired. Further, cells oriented to the direction of the microgrooves. This orientation was established fastest on the narrow grooves. After 30 min, cells showed abundant membrane extensions in all directions. Well-formed actin filaments were not present in the cell body at timepoints before 4 h. Furthermore, cells on smooth surfaces exhibited less filaments. The addition of cytochalasin-B only caused a delay of cell attachment and spreading. From these experiments, we conclude that a well-formed cellular actin cytoskeleton is no prerequisite for the occurrence of contact guidance. Actin microfilaments in the lamellipodia, and the interplay between the lamellipodium and extracellular matrix molecules seem to be the determining factor in the establishment of contact guidance.

The small GTPase Rab6B, a novel Rab6 subfamily member, is cell-type specifically expressed and localised to the Golgi apparatus.

Members of the Rab subfamily of small GTPases play an important role in the regulation of intracellular transport routes. Rab6A has been shown to be a regulator of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER). Here, we report on the identification of a Rab6 isoform, termed Rab6B. The corresponding full-length cDNA was isolated from a Caco-2 cell library. The deduced amino acid sequence showed 91% identity with the Rab6A protein and revealed that sequence divergence is dispersed over a large region of the COOH-terminal domain. Rab6B is encoded by an independent gene which is located on chromosome 3 region q21-q23. In contrast to Rab6A whose expression is ubiquitous, northern blot analysis, immunohistochemistry, and immunofluorescence demonstrated that Rab6B is expressed in a tissue and cell-type specific manner. Rab6B is predominantly expressed in brain and the neuroblastoma cell line SK-N-SH. In brain, Rab6B was found to be specifically expressed in microglia, pericytes and Purkinje cells. Endogenous Rab6B localises to the Golgi apparatus and to ERGIC-53-positive vesicles. Comparable studies between Rab6A and Rab6B revealed distinct biochemical and cellular properties. Rab6B displayed lower GTP-binding activities and in overexpression studies, the protein is distributed over Golgi and ER membranes, whereas Rab6A is more restricted to the Golgi apparatus. Since the GTP-bound form of Rab6B (Rab6B Q72L) does interact with all known Rab6A effectors, including Rabkinesin-6, the results suggest a cell-type specific role for Rab6B in retrograde membrane traffic at the level of the Golgi complex.

Expression of Rab small GTPases in epithelial Caco-2 cells: Rab21 is an apically located GTP-binding protein in polarised intestinal epithelial cells.

Rab proteins belong to a subfamily of small GTP-binding protein genes of the Ras superfamily and play an important role in intracellular vesicular targeting. The presence of members of this protein family was examined in Caco-2 cells by a PCR-based strategy. Twenty-five different partial cDNA sequences were isolated, including 18 Rab protein family members. Seven novel human sequences, representing Rab2B, Rab6A', Rab6B, Rab10, Rab19B, Rab21 and Rab22A, were identified. For one clone, encoding Rab21, full-length cDNA was isolated from a Caco-2 cDNA library. Northern blot analysis showed a ubiquitous expression pattern of Rab21. To study Rab21 protein expression in Caco-2 cells, polyclonal antibodies were raised against GST-Rab21 fusion protein and characterised. The antibodies recognised Rab21 as a protein of approximately 25 kDa. Interestingly, the protein shows a general ER-like staining in nonpolarised Caco-2 cells in contrast to an apically located vesicle-like staining in polarised Caco-2 cells. Furthermore, immunohistochemical staining on human jejunal tissue showed a predominant expression of Rab21 in the epithelial cell layer with high expression levels in the apical region, whereas stem cells in the crypts were negative. We therefore suggest an alternative role for Rab21 in the regulation of vesicular transport in polarised intestinal epithelial cells.

Contact guidance of rat fibroblasts on various implant materials.

Providing a substrate surface with micrometer-sized parallel grooves influences the behavior of cells growing on such substrates in vitro. Cells elongate in the direction of the groove and migrate guided by the grooves. It has been suggested that cellular alignment on microgrooves is predominantly dependent on groove dimensions and that surface chemical variation of the substrate material has little effect. Therefore we seeded primary rat dermal fibroblasts (RDF) on smooth and microgrooved (groove width 1-10 microm, depth 0.5 microm) polystyrene (PS), poly-L-lactic acid (PLA), silicone (SIL), and titanium (Ti) substrates. The production process was found to be more accurate for PS and PLA than for SIL and Ti substrates. A proliferation study, scanning electron microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed differences between RDF behavior on the materials. Our conclusions are (1) the accuracy of microtexture production by casting depends greatly on the material used; (2) even if no sharp discontinuities are present, microtextures still are potent tools for inducing contact guidance; and (3) besides surface texture, surface chemistry has a definitive influence on cell morphology.

Growth behavior of fibroblasts on microgrooved polystyrene.

We investigated the contact guidance phenomenon of rat dermal fibroblasts (RDF) on microgrooved polystyrene substrates. Grooves were 1 microm deep, and between 1 and 10 microm wide. Light microscopy and digital image analysis (DIA) showed that RDF were oriented on all microgrooved substrates. Scanning electron microscopy showed that RDF cultured on 1 or 2 microm wide grooves were positioned on top of the ridges. On the wider 5 and 10 microm grooves, the cells were able to descend into the grooves. In confocal laser scanning microscopy, focal adhesions were lying in the same direction as the actin filament where they attached to. DIA confirmed an orientational behavior of focal adhesions and actin filaments on microgrooves. There were no differences in the measured orientation between the different grooves. Besides, no obvious preference was found for focal adhesions to lie along edges of the surface ridges. Transmission electron microscopy showed that focal adhesions were able to bend along the edges of ridges. On the basis of our observations, we suggest that the breakdown and formation of fibrous cellular components, especially in the filopodium, is influenced by the microgrooves. The microgrooves create a pattern of mechanical stress, which influences cell spreading and cause the cell to be aligned with surface microgrooves.

Microgrooved subcutaneous implants in the goat.

We investigated the behavior of microgrooved implants in soft tissue using polystyrene implantable disks, either smooth or microgrooved (1-10 microm) on both sides. The implants were placed subcutaneously in a goat for 1, 4, or 12 weeks. Light and transmission electron microscopy showed that fibrous capsule formation around the implants was fairly uniform. After 1 week the implants were covered with a fibrous capsule about 80 microm thick. The collagen matrix was loose, and many inflammatory cells were present. After 4 weeks the matrix was more dense and contained many newly formed blood vessels. At the implant surface a layer of inflammatory cells about 10 microm thick had accumulated. Finally, after 12 weeks the matrix had densified. One cellular layer of inflammatory cells was present at the implant surface. We carried out histomorphometric measurements of capsule thickness, inflammatory layer thickness, and the number of blood vessels. Capsule thickness appeared not to decrease with time. Further, these measurements showed that there were no differences in tissue reaction between smooth and microgrooved implants. On the basis of our observations, we suggest that 1 microm deep and 1-10 microm wide microgrooves do not influence tissue response around polystyrene implants in soft tissue.

Long-term effects of clenbuterol on diaphragm morphology and contractile properties in emphysematous hamsters.

The aim of the present study was to investigate the effect of chronic long-term clenbuterol treatment (1 mg/kg subcutaneously twice a day for 12 wk) on diaphragm morphology and function in emphysematous (EH) and normal hamsters (NH). Clenbuterol increased body weight, diaphragm weight, and skeletal muscle weight in both EH and NH to a similar extent. In the diaphragm, clenbuterol significantly increased myosin heavy chain type I, IIa, and IIx muscle fiber cross-sectional areas by approximately 35-55% in both EH and NH. This response to clenbuterol treatment was not significantly different between EH and NH diaphragm. In EH, twitch force (Pt), maximal tetanic force, and force-frequency curve were significantly reduced compared with NH. In EH, clenbuterol increased Pt by approximately 10%, restoring Pt to NH level. A similar improvement was observed in the force-frequency characteristics. Clenbuterol did not alter contractile properties in NH. In conclusion, long-term clenbuterol treatment resulted in an increased size of all diaphragm muscle fiber types in both NH and EH. Clenbuterol completely abolished the reduced force generation induced by emphysema.

Analysis of a naturally occurring mutation in sucrase-isomaltase: glutamine 1098 is not essential for transport to the surface of COS-1 cells.

A glutamine for proline substitution at position 1098 was previously shown to result in accumulation of brush-border sucrase-isomaltase in the Golgi apparatus. The substitution is present in a highly homologous region of the protein, and results in a comparable accumulation when introduced into the same region in lysosomal alpha-glucosidase. To study the importance of the glutamine-1098, we analyzed the transport compatibility of two mutants in which glutamine-1098 is substituted by lysine or alanine. Both mutants were transported to the cell surface and processed comparable to wild type. We concluded that glutamine-1098 is not essential for transport to the cell surface.

Scanning electron microscopic, transmission electron microscopic, and confocal laser scanning microscopic observation of fibroblasts cultured on microgrooved surfaces of bulk titanium substrata.

During this study, microtechnology and plasma etching were used to produce gratings 1.0 (TiD01), 2.0 (TiD02), 5.0 (TiD05), and 10.0 microns wide (TiD10) into commercially pure titanium wafers. After incubation of rat dermal fibroblast (RDFs) on these surfaces for 3 days, the cells were observed with scanning electron (SEM), transmission electron (TEM), and confocal laser scanning microscopy (CLSM). Results showed that the RDFs as a whole and their stress fibers oriented strictly parallel to the surface pattern on the TiD01 and TiD02 surfaces. On the TiD05 and TiD10 surfaces, this orientation was not observed. In addition, TEM and CLSM demonstrated that the focal adhesion points (FAP) were located mainly on the surface pattern ridges. TEM revealed that FAP were wrapped occasionally around the edges of the ridges. Only the RDFs on both the TiD05 and TiD10 surfaces protruded into the grooves and possessed FAP on the walls of the grooves. Attachment to the groove floor was observed only on the TiD10 textures. Comparison of these results with earlier observations on microtextured silicone rubber substrata suggests that material-specific properties do not influence the orientational effect of the surface texture on the observed RDF cellular behavior. The proliferation rate of the RDFs, however, seems to be much higher on titanium than on silicone rubber substrata.

Interfacial phenomena: an in vitro study of the effect of calcium phosphate (Ca-P) ceramic on bone formation.

In previous studies we developed a RF magnetron sputter technique for the production of thin Ca-P coatings. With this technique coatings can be produced that vary in Ca/P ratio as well as in structural appearance. The aim of this investigation was to obtain more understanding of the biological behavior of these coatings by way of in vitro experiments. The effect of noncoated titanium (Ti) and three different Ca-P-sputtered surfaces on the proliferation and differentiation (morphology and matrix production) of osteoblast-like cells was studied. Proliferation was determined using counting procedures; morphology was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Fluorescent markers and energy-dispersive X-ray microanalysis (EDX) were used to obtain quantitative and compositional information about the resultant calcified extracellular matrix (ECM). Results demonstrated that proliferation of the osteoblast-like cells was significantly (p < 0.05) higher on noncoated than on Ca-P-coated samples. On the other hand, more mineralized ECM was formed on the coated surfaces. In addition, TEM confirmed that the cells on the coated substrates were surrounded by ECM with collagen fibers embedded in crystallized, needle-shaped structures. On the basis of these findings, we concluded that: (1) the investigated Ca-P sputter coatings possess the capacity to activate the differentiation and expression of osteogenic cells, and (2) bone formation proceeds faster on Ca-P surfaces than on Ti substrates. Further, this bone-inductive effect appeared to be dependent on the Ca-P ratio of the deposited coatings.

Routing and processing of lactase-phlorizin hydrolase in transfected Caco-2 cells.

Human lactase-phlorizin hydrolase (LPH) is a digestive enzyme that is expressed in the small intestinal brush-border membrane. After terminal glycosylation in the Golgi apparatus, the 230-kDa pro-LPH is cleaved into the 160-kDa brush-border LPHbeta and the 100-kDa profragment (LPHalpha). Since LPHbeta is not transport-competent when it is expressed separately from LPHalpha in COS-1 cells, it was suggested that LPHalpha functions as an intramolecular chaperone. What happens to LPHalpha after cleavage is still unclear. To analyze and localize LPHalpha in polarized epithelial cells, wild type and tagged LPH were stably expressed in Caco-2 cells. In tagged LPH, a vesicular stomatitis virus epitope tag was inserted into the LPHalpha region. Wild type and tagged proteins were processed at similar rates, and both cleaved LPHbeta forms were expressed at the apical cell surface. Pro-LPH was recognized by antibodies against LPH, a profragment epitope and the vesicular stomatitis virus tag. LPHalpha alone, however, could not be recovered by these antibodies. Our data suggest that LPHalpha is degraded immediately after cleavage.

Orientation of ECM protein deposition, fibroblast cytoskeleton, and attachment complex components on silicone microgrooved surfaces.

The microfilaments and vinculin-containing attachment complexes of rat dermal fibroblasts (RDF) incubated on microtextured surfaces were investigated with confocal laser scanning microscopy (CLSM) and digital image analysis (DIA). In addition, depositions of bovine and endogenous fibronectin and vitronectin were studied. Smooth and microtextured silicone substrata were produced that possessed parallel surface grooves with a groove and ridge width of 2.0, 5.0, and 10.0 microns. The groove depth was approximately 0.5 micron. CLSM and DIA make it possible to visualize and analyze intracellular and extracellular proteins and the underlying surface simultaneously. It was observed that the microfilaments and vinculin aggregates of the RDFs on the 2.0 microns grooved substrata were oriented along the surface grooves after 1, 3, 5, and 7 days of incubation while these proteins were significantly less oriented on the 5.0 and 10.0 microns grooved surfaces. Vinculin was located mainly on the surface ridges on all textured surfaces. In contrast, bovine and endogenous fibronectin and vitronectin were oriented along the surface grooves on all textured surfaces. These proteins did not seem to be hindered by the surface grooves since many groove-spanning filaments were found on all the microgrooved surfaces. In conclusion, it can be said that microtextured surfaces influence the orientation of intracellular and extracellular proteins. Although results corroborate three earlier published hypotheses, they do not justify a specific choice of any one of these hypotheses.

A mutation in a highly conserved region in brush-border sucrase-isomaltase and lysosomal alpha-glucosidase results in Golgi retention.

A point mutation in the cDNA of human intestinal sucrase-isomaltase has been recently identified in phenotype II of congenital sucrase-isomaltase deficiency. The mutation results in a substitution of glutamine by proline at position 1098 (Q1098P) in the sucrase subunit. Expression of this mutant sucrase-isomaltase cDNA in COS-1 cells results in an accumulation of sucrase-isomaltase in the ER, intermediate compartment and the cis-Golgi cisternae similar to the accumulation in phenotype II intestinal cells. An interesting feature of the Q1098P substitution is its location in a region of the sucrase subunit that shares striking similarities with the isomaltase subunit and other functionally related enzymes, such as human lysosomal acid alpha-glucosidase and Schwanniomyces occidentalis glucoamylase. We speculated that the Q-->P substitution in these highly conserved regions may result in a comparable accumulation. Here we examined this hypothesis using lysosomal alpha-glucosidase as a reporter gene. Mutagenesis of the glutamine residue at position 244 in the homologous region of alpha-glucosidase to proline results in a protein that is neither transported to the lysosomes nor secreted extracellularly but accumulates in the ER, intermediate compartment and cis-Golgi as a mannose-rich polypeptide similar to mutant sucrase-isomaltase in phenotype II. We propose that the Q1098P and Q244P mutations (in sucrase-isomaltase and alpha-glucosidase, respectively) generate structural alterations that are recognized by a control mechanism, operating beyond the ER in the intermediate compartment or cis-Golgi.

Quantitative analysis of fibroblast morphology on microgrooved surfaces with various groove and ridge dimensions.

Fibroblasts have been shown to respond to substratum surface roughness. The change in cell size, shape and orientation of rat dermal fibroblasts (RDF) was therefore studied using smooth and microtextured silicone rubber substrata. The microtextured substrata possessed parallel surface microgrooves that ranged in width from 1.0 to 10.0 microns, and were separated by ridges of 1.0 to 10.0 microns. The grooves were either 0.45 or 1.00 microns deep. Prior to incubation, the substrata were cleaned and given a radio frequency glow discharge treatment. After surface evaluation with scanning electron microscopy and confocal laser scanning microscopy, RDF were incubated on these substrata for 5 days. During this period of incubation, the RDF were photographed on days 1, 2, 3, 4, and 5, using phase contrast microscopy. Digital image analysis of these images revealed that on surfaces with a ridge width < or = 4.0 microns, cells were highly orientated (< 10 degrees) and elongated along the surface grooves. Protrusions contacting the ridges specifically could be seen. If the ridge width was larger than 4.0 microns, cellular orientation was random (approximately 45 degrees) and the shape of the RDF became more circular. Furthermore, results showed that the ridge width is the most important parameter, since varying the groove width and groove depth did not affect the RDF size, shape, nor the angle of cellular orientation.

Quantitative analysis of cell proliferation and orientation on substrata with uniform parallel surface micro-grooves.

In order to quantify the effect of the substrata surface topography on cellular behaviour, planar and micro-textured silicon substrata were produced and made suitable for cell culture by radio frequency glow discharge treatment. These substrata possessed parallel surface grooves with a groove and ridge width of 2.0 (SilD02), 5.0 (SilD05) and 10 microns (SilD10). Groove depth was approximately 0.5 micron. Rat dermal fibroblasts (RDFs) were cultured on these substrata and a tissue culture polystyrene control surface for 1, 2, 3, 5 and 7 days. After incubation the cell proliferation was quantified with a Coulter Counter, and RDF size, shape and orientation with digital image analysis. Cell counts proved that neither the presence of the surface grooves nor the dimension of these grooves had an effect on the cell proliferation. However, RDFs on SilD02, and to a lesser extent on SilD05 substrata, were elongated and aligned parallel to the surface grooves. Orientation of the RDFs on SilD10 substrata proved to be almost comparable to the SilD00 substrata. Finally, it was observed that the cells on the micro-textured substrata were capable of spanning the surface grooves.

Effects of different treatment regimens of methylprednisolone on rat diaphragm contractility, immunohistochemistry and biochemistry.

Systemic corticosteroid therapy may affect diaphragm structure and function. We postulated that functional, immunohistochemical and biochemical characteristics of rat diaphragm were less affected by alternate-day methylprednisolone (MP) administration, and more by repeated bursts of MP, in comparison to daily s.c MP. Sixty adult rats were randomized into four groups: saline s.c.; MP continuously (MP-C), 1 mg.kg-1 daily, MP alternate-day therapy (MP-A), 2 mg.kg-1 every other day; or MP in bursts (MP-B), MP 2 mg.kg-1 daily for 2 weeks, saline for 4 weeks, MP 2 mg.kg-1 daily for 2 weeks. The total treatment period was 8 weeks. Contractile properties of isolated diaphragm strips were measured. Antibodies reactive with type I, IIa, IIx and IIb myosin heavy chains were used for immunohistochemical analysis. Biochemical evaluation included markers of fast energy supply, glycogenolytic activity, beta-oxidation capacity and oxidative capacity. The force-frequency curve was depressed in all MP groups. Fibre type I, IIx and IIb cross-sectional area (CSA) decreased in all MP groups. Burst therapy decreased the contribution of type IIb fibres to total diaphragm muscle area. MP-A affected glycogenolytic activity less than MP-C. Burst MP therapy reduced creatine kinase (CK) activity and beta-oxidation capacity compared to MP-C. Oxidative capacity was increased in all MP groups. In conclusion, although the methylprednisolone treatment regimens affected diaphragm muscle morphology and bioenergetic enzyme activities in different ways, force generation decreased in all methylprednisolone treated groups to the same extent.

Congenital sucrase-isomaltase deficiency. Identification of a glutamine to proline substitution that leads to a transport block of sucrase-isomaltase in a pre-Golgi compartment.

Congenital sucrase-isomaltase deficiency is an example of a disease in which mutant phenotypes generate transport-incompetent molecules. Here, we analyze at the molecular level a phenotype of congenital sucrase-isomaltase deficiency in which sucrase-isomaltase (SI) is not transported to the brush border membrane but accumulates as a mannose-rich precursor in the endoplasmic reticulum (ER), ER-Golgi intermediate compartment, and the cis-Golgi, where it is finally degraded. A 6-kb clone containing the full-length cDNA encoding SI was isolated from the patient's intestinal tissue and from normal controls. Sequencing of the cDNA revealed a single mutation, A/C at nucleotide 3298 in the coding region of the sucrase subunit of the enzyme complex. The mutation leads to a substitution of the glutamine residue by a proline at amino acid 1098 (Q1098P). The Q1098P mutation lies in a region that is highly conserved between sucrase and isomaltase from different species and several other structurally and functionally related proteins. This is the first report that characterizes a point mutation in the SI gene that is responsible for the transport incompetence of SI and for its retention between the ER and the Golgi.

Ultrastructural immunogold labeling of lipid-laden enterocytes from patients with genetic malabsorption syndromes.

Intestinal biopsies from patients having genetic disorders of lipoprotein assembly and secretion, such as abetalipoproteinemia (ABL) or Anderson's disease (AD), contain large amounts of lipids which are accumulated in the enterocytes. Determination of the intracellular sites in which the lipids accumulate and to which apolipoproteins the lipids are bound would help to identify the defects in these diseases and further elucidate the mechanisms by which lipoprotein assembly and secretion occur normally. Ultrastructural immunogold labeling, however, is hampered by the poor preservation of the lipids accumulated in the enterocytes of these patients. We have used routine electron microscopy (fixation and ultra-thin sectioning) along with three methods for immunogold labeling of lipid-laden enterocytes: ultrathin cryosectioning, low temperature freeze substitution with embedding in Lowicryl K4M, and ultra-low temperature freeze substitution with embedding in Lowicryl HM20, to establish a protocol for investigating the intestinal tissue from these patients. Ultracryosectioning, while preserving the overall morphology of the lipid laden enterocytes, did not preserve the lipid content and the immunogold labeling of apolipoprotein B (ApoB) appeared dislocated. Freeze substitution and low temperature embedding in Lowicryl K4M, in contrast, appeared to better preserve the lipid and lipoprotein structures; however, the antigenicity of both apoAI and apoB appeared to be lost and no specific labeling could be obtained. Freeze substitution and embedding in Lowicryl HM20 best preserved the lipid and lipoprotein structures while maintaining apoprotein antigenicity. In conclusion, immunogold labeling of apolipoproteins on lipid structures in the lipid-laden enterocytes of patients with ABL and AD is best obtained by freeze substitution and embedding in Lowicryl HM20.

Biological evaluation of the effect of magnetron sputtered Ca/P coatings on osteoblast-like cells in vitro.

A rat bone marrow cell culture was used to evaluate the osteogenic potential of amorphous and crystalline thin calcium phosphate (Ca/P) coatings. The coatings were deposited on titanium discs using a radiofrequency magnetron sputter procedure. Amorphous and crystalline plasma spray Ca/P coated and noncoated titanium discs served as reference material. The cellular behavior was analyzed with quantitative (attachment and proliferation rates) and qualitative (scanning electron microscopy) techniques. No significant differences were found in cell attachment and proliferation rates between the various materials. Scanning electron microscopy showed extracellular matrix formation after 18 days of culture on amorphous plasma-sprayed and the two types of magnetron sputtered coatings. Furthermore, no severe degradation of the magnetron sputtered coatings was observed. They even appeared to induce apatite formation. On basis of the results, we conclude that magnetron sputtering appears to be a promising method to manufacture bioactive ceramic coatings.

Water channels encoded by mutant aquaporin-2 genes in nephrogenic diabetes insipidus are impaired in their cellular routing.

Congenital nephrogenic diabetes insipidus is a recessive hereditary disorder characterized by the inability of the kidney to concentrate urine in response to vasopressin. Recently, we reported mutations in the gene encoding the water channel of the collecting duct, aquaporin-2 (AQP-2) causing an autosomal recessive form of nephrogenic diabetes insipidus (NDI). Expression of these mutant AQP-2 proteins (Gly64Arg, Arg187Cys, Ser216Pro) in Xenopus oocytes revealed nonfunctional water channels. Here we report further studies into the inability of these missense AQP-2 proteins to facilitate water transport in Xenopus oocytes. cRNAs encoding the missense AQPs were translated with equal efficiency as cRNAs encoding wild-type AQP-2 and were equally stable. Arg187Cys AQP2 was more stable and Gly6-4Arg and Ser216Pro AQP2 were less stable when compared to wild-type AQP2 protein. On immunoblots, oocytes expressing missense AQP-2 showed, besides the wild-type 29 kDa band, an endoplasmic reticulum-retarded form of AQP-2 of approximately 32 kD. Immunoblots and immunocytochemistry demonstrated only intense labeling of the plasma membranes of oocytes expressing wild-type AQP-2. Therefore, we conclude that in Xenopus oocytes the inability of Gly64-Arg, Arg187Cys or Ser216Pro substituted AQP-2 proteins to facilitate water transport is caused by an impaired routing to the plasma membrane.