Gallbladder epithelial cell hydraulic water permeability and volume regulation

The hydraulic water permeability (Lp) of the cell membranes of Necturus gallbladder epithelial cells was estimated from the rate of change of cell volume after a change in the osmolality of the bathing solution. Cell volume was calculated from computer reconstruction of light microscopic images of epithelial cells obtained by the "optical slice" technique. The tissue was mounted in a miniature Ussing chamber designed to achieve optimal optical properties, rapid bath exchange, and negligible unstirred layer thickness. The control solution contained only 80% of the normal NaCl concentration, the remainder of the osmolality was made up by mannitol, a condition that did not significantly decrease the fluid absorption rate in gallbladder sac preparations. The osmotic gradient ranged from 11.5 to 41 mosmol and was achieved by the addition or removal of mannitol from the perfusion solutions. The Lp of the apical membrane of the cell was 1.0 X 10(-3) cm/s . osmol (Posm = 0.055 cm/s) and that of the basolateral membrane was 2.2 X 10(-3) cm/s . osmol (Posm = 0.12 cm/s). These values were sufficiently high so that normal fluid absorption by Necturus gallbladder could be accomplished by a 2.4-mosmol solute gradient across the apical membrane and a 1.1-mosmol gradient across the basolateral membrane. After the initial cell shrinkage or swelling resulting from the anisotonic mucosal or serosal medium, cell volume returned rapidly toward the control value despite the fact that one bathing solution remained anisotonic. This volume regulatory response was not influenced by serosal ouabain or reduction of bath NaCl concentration to 10 mM. Complete removal of mucosal perfusate NaCl abolished volume regulation after cell shrinkage. Estimates were also made of the reflection coefficient for NaCl and urea at the apical cell membrane and of the velocity of water flow across the cytoplasm.     

Comparative permeability of canine visceral and parietal pleura

To determine the permeability of canine pleural mesothelium, visceral and intercostal parietal pleura from mongrel dogs was carefully stripped from the underlying tissue and mounted as a planar sheet in a Ussing-type chamber. The hydraulic conductivity (Lp) was determined from the rate of volume flux in response to hydrostatic pressure gradients applied to either the mucosal or serosal surface of the pleural membrane. The diffusional permeability (Pd) of radiolabeled water, sucrose, inulin, and albumin was determined under equilibrium conditions from the unidirectional tracer flux. The Lp of the visceral pleura was 0.39 +/- 0.032 (SE) X 10(-4) ml.s-1.cmH2O-1.cm-2 and that Lp of parietal pleura was 1.93 +/- 0.93 X 10(-4) ml.s-1.cmH2O-1.cm-2 (P less than 0.001). The Pd of the visceral pleura ranged from 12.21 +/- 0.45 X 10(-4) cm/s for 3H2O to 0.34 +/- 0.03 X 10(-4) cm/s for [3H]albumin. The Pd of the parietal pleura for water and sucrose was similar to that of the visceral membrane, whereas its Pd for the larger inulin and albumin molecules was greater than that of visceral pleura (P less than 0.01). A spontaneous potential difference could not be detected across either membrane. The relatively higher parietal pleural Lp and Pd for larger solutes is probably due to the presence of stomata in this membrane. These results indicate that both the parietal and the visceral pleura are extremely permeable tissues which offer little resistance to water and solute flux.     

Analysis of the introduction and removal of glycerol in rabbit kidneys using a Krogh cylinder model

In 1982, Rubinsky and Cravalho described a Krogh cylinder model for the analysis of cryoprotectant transport in a perfused organ. By application of the Kedem-Katchalsky equations, changes in tissue volume caused by movements of water and solute were used to predict changes in capillary radius (Cryobiology 19, 70-82, 1982). We have now measured the changes in vascular resistance that are produced when sucrose or glycerol is introduced into the perfusate flowing through rabbit kidneys at 10 degrees C, and have analyzed these data by means of the Rubinsky-Cravalho semiempirical model. The sucrose data provided an estimate of hydraulic conductivity and the dimensions of the Krogh tissue units. Three rates of addition of glycerol, 10, 30, and 90 mM/min to a final concentration of 3 M, were studied. The vascular resistance fell to approximately 40% of its initial value (radius approximately 128% of initial value) with all three rates of addition, and then returned toward its normal value while the glycerol concentration was still increasing. This behavior could be explained either by a sudden change in solute permeability at that capillary radius, or by an inverse dependence of reflection coefficient upon solute concentration. Evidence is presented that favors the latter interpretation. The best fits for the apparent hydraulic conductivity and apparent solute permeability for glycerol are 1 X 10(-6) cm/sec atm and 6 X 10(-8) cm/sec, respectively, with the reflection coefficient falling from 1.0 when the glycerol concentration is zero to 0.1 when it is 3 M. The model is used to predict tissue concentrations of glycerol throughout each experiment.     

The influence of differing connective tissue substrates on the maintenance of adult stratified squamous epithelia

Summary The interaction between adult stratified squamous epithelium and its supporting connective tissue possibly involves both permissive and directive influences. To examine the effect of vitality and specificity of connective tissue on the maintenance of epithelial structure and histo-differentiation, specimens of skin and oral mucosa from various regions of adult mice were separated using either EDTA or trypsin. Prior to transplantation, the epithelium was recombined with either inverted homologous connective tissue or with connective tissue that had been killed either by heating or repeated freeze-thawing. Epithelial sheets were also transplanted onto the graft bed alone or in combination with striated muscle or tendon.Normal patterns of cytodifferentiation were maintained when the epithelium was recombined with inverted or frozen-thawed subepithelial connective tissue but there was a loss of spatial organization on the frozen-thawed connective tissue. In contrast, heat-killed or trypsin-treated frozen-thawed subepithelial connective tissue and non-dermal connective tissue failed to maintain a viable epithelium. These observations suggest that subepithelial connective tissues (dermis, lamina propria) but not deep connective tissues facilitate epithelial proliferation and histodifferentiation.Supported by NIH/NIDR RO1 DEO5190     

Permeability parameters of the toad isolated stratum corneum.

A technique for isolating the stratum corneum from the subjacent layers of the epithelium was developed which permits studying the stratum corneum as an isolated membrane mounted between half-chambers. The method basically consists of an osmotic shock induced by immersing a piece of skin in distilled water at 50 degrees C for 2 min. When the membrane is bathed on each surface by NaCl-Ringer's solution, its electrical resistance is 14.1 +/- 1.3 omega cm2 (n=10). This value is about 1/100 of the whole skin resistance in the presence of the same solution. The hydraulic filtration coefficient (Lp) measured by a hydrostatic pressure method, with identical solutions on each side of the membrane, is 8.8 X 10(-5) +/- 1.5 X 10(-5) cm sec-1 atm-1 (n=10) in distilled water and 9.2 X 10(-5) +/- 1.4 X 10(-5) cm sec-1 atm-1 (n=10) in NaCl-Ringer's solution. These values are not statistically different and are within the range of 1/80 to 1/120 of the whole skin Lp. The stratum corneum shows an amphoteric character when studied by KCl diffusion potentials at different pH'S. The membrane presents an isoelectric pH of 4.6 +/- 0.3 (n=10). Above the isoelectric pH the potassium transport number is higher than the chloride transport number; below it, the reverse situation is valid. Divalent cations (Ca++ or Cu++) reduce membrane ionic discrimination when the membrane is negatively charged and are ineffective when the membrane fixed charges are protonated at low pH.     

Effect of hypoxia on permeability of pulmonary endothelium of canine visceral pleura

To determine if hypoxia increases the permeability of the pulmonary capillaries of the visceral pleura, water and protein movement across visceral pleura of isolated blood-perfused lungs ventilated with 20% O2-5% CO2 or 0% O2-5% CO2 was analyzed in terms of a two-compartment model of fluid exchange. Lungs from mongrel dogs were enclosed in a water-impermeable membrane, thereby creating an artificial visceral pleural space (VPS); fluid flux was determined as the filtration or reabsorption of water and protein in the VPS. Hypoxic vasoconstriction was prevented by adding verapamil to the perfusate. Hydrostatic pressures were continuously monitored and samples of perfusate and pleural fluid were obtained for protein determinations. Pulmonary capillary pressure was varied between 5 and 20 Torr by changing venous pressure while the protein concentration gradient was varied from 0.5 to 6.6 g/dl by introducing different solutions of plasma mixed with saline into the VPS. The hydraulic conductivity (Lp) increased from 4.25 +/- 0.74 to 9.18 +/- 0.67 X 10(-7) ml X s-1 X mmHg-1 X cm-2 and the diffusional permeability (Pd) of protein increased from 1.29 +/- 0.28 to 4.06 +/- 0.44 X 10(-6) cm/s under hypoxic conditions (P less than 0.05). Inhibition of xanthine oxidase by the addition of allopurinol (10 mg/kg body wt) to the perfusate prevented the increase in Lp and Pd observed under hypoxic conditions. We conclude that free radicals generated via xanthine oxidase may be responsible for the increased permeability observed during severe hypoxia.     

Light and electron microscopic observations of the autonomic innervation of the mouse gallbladder mucosa. A histochemical, cytochemical, and secretory study.

The autonomic innervation of the mouse gallbladder mucosa was studied using histo- and cytochemical methods. In a light microscopic investigation the distribution of acetylcholinesterase (AChE) activity and formaldehyde-induced fluorescence was studied histochemically. Nerve fibres and small varicosities showed concentrations of AChE activity very close to the epithelium in the subepithelial connective tissue. No adrenergic nerves were observed in the mucosa. When using the electron microscope and employing the potassium permanganate fixation/staining technique only one sort of axonal enlargement was encountered, viz. the cholinergic type. These varicosities contained numerous agranular vesicles (500-600 A in diameter). No varicosities of the adrenergic (dense-cored vesicles) type were observed. Signs of increased secretory activity in the epithelium were observed in the first few minutes after cholinergic stimulation. After repeated in vivo stimulation, there was an almost total depletion of glycoprotein granules, best seen when using the cytochemical PA-CrA-silver technique. The findings suggest that the subepithelial connective tissue and the epithelium of the mouse gallbladder mucosa have a cholinergic innervation.     

Specific hydraulic conductivity of corneal stroma as seen by quick-freeze/deep-etch

Previous studies of the hydraulic conductivity of connective tissues have failed to show a correspondence between ultrastructure and specific hydraulic conductivity. We used the technique of quick-freeze/deep-etch to examine the ultrastructure of the corneal stroma and then utilized morphometric studies to compute the specific hydraulic conductivity of the corneal stroma. Our studies demonstrated ultrastructural elements of the extracellular matrix of the corneal stroma that are not seen using conventional electron microscopic techniques. Furthermore, we found that these structures may be responsible for generating the high flow resistance characteristic of connective tissues. From analysis of micrographs corrected for depth-of-field effects, we used Carmen-Kozeny theory to bound a morphometrically determined specific hydraulic conductivity of the corneal stroma between 0.46 x 10(-14) and 10.3 x 10(-14) cm2. These bounds encompass experimentally measured values in the literature of 0.5 x 10(-14) to 2 x 10(-14) cm2. The largest source of uncertainty was due to the depth-of-field estimates that ranged from 15 to 51 nm; a better estimate would substantially reduce the uncertainty of these morphometrically determined values.     

Electrical resistance of muscle capillary endothelium.

A recently developed technique for in vivo determination of the electrical resistance of vascular endothelium in microvessels was applied to the vessels in a thin frog muscle, m. cutaneus pectoris. The technique consists of injection of current via a glass micropipette into a capillary and measurement of the resulting intra- and extravascular potential profiles with another micropipette placed at various distances from the current source. The theory of Peskoff and Eisenberg (1974) was used to handle the problems arising from distributed extravascular resistances and was experimentally shown to describe the external field satisfactorily. With this extension of one-dimensional cable theory the specific electrical resistance of arterial microvessels was 33 omega cm2 and of venous capillaries 23 omega cm2. The "length constants" were 135 and 112 micrometers, respectively. If results from arterial and venous vessels are taken together, the ionic permeabilities at 20 degrees C were PNa = 3.9 X 10(-5) cm X s-1, PK = 5.7 X 10(-5) cm X s-1, PCl = 5.9 X 10(-5) cm X s-1 and PHCO3 = 3.4 X 10(-5) cm X s-1. These figures agree with figures for capillary permeability obtained in tracer experiments on whole muscle. The study bridges a gap between single capillary and whole organ techniques with the conclusion that the two different approaches lead to similar results in muscle capillaries.     

Improved electrical coupling in uterine smooth muscle is associated with increased numbers of gap junctions at parturition

We have studied some passive electrical properties of uterine smooth muscle to determine whether a change in electrical parameters accompanies gap junction formation at delivery. The length constant of the longitudinal myometrium increased from 2.6 +/- 0.8 mm (X +/- SD) before term to 3.7 +/- 1 mm in tissues from delivering animals. The basis of the change was a 33% decrease in internal resistance and a 46% increase in membrane resistance. Axial current flow in an electrical syncytium such as myometrium is impeded by the cytoplasm of individual cells plus the junctions between cells. Measurement of the longitudinal impedance indicated that the specific resistance of the myoplasmic component was constant at 319 +/- 113 omega . cm before term and 340 +/- 93 omega . cm at delivery. However, a decrease in junctional resistance was apparent from 323 +/- 161 omega . cm to 134 +/- 64 omega . cm at delivery. 1.5-2 d after delivery, the junctional resistance was increased, as was the myoplasmic resistance. Thin-section electron microscopy of some of the same muscle samples showed that gap junctions were present in significantly greater numbers in the delivering tissues. Therefore, our results support the hypothesis that gap junction formation at delivery is associated with improved electrical coupling of uterine smooth muscle.     

Osmotic water permeability of Necturus gallbladder epithelium

An electrophysiological technique that is sensitive to small changes in cell water content and has good temporal resolution was used to determine the hydraulic permeability (Lp) of Necturus gallbladder epithelium. The epithelial cells were loaded with the impermeant cation tetramethylammonium (TMA+) by transient exposure to the pore-forming ionophore nystatin in the presence of bathing solution TMA+. Upon removal of the nystatin a small amount of TMA+ is trapped within the cell. Changes in cell water content result in changes in intracellular TMA+ activity which are measured with intracellular ion-sensitive microelectrodes. We describe a method that allows us to determine the time course for the increase or decrease in the concentration of osmotic solute at the membrane surface, which allows for continuous monitoring of the difference in osmolality across the apical membrane. We also describe a new method for the determination of transepithelial hydraulic permeability (Ltp). Apical and basolateral membrane Lp's were assessed from the initial rates of change in cell water volume in response to anisosmotic mucosal or serosal bathing solutions, respectively. The corresponding values for apical and basolateral membrane Lp's were 0.66 x 10(-3) and 0.38 x 10(-3) cm/s.osmol/kg, respectively. This method underestimates the true Lp values because the nominal osmotic differences (delta II) cannot be imposed instantaneously, and because it is not possible to measure the true initial rate of volume change. A model was developed that allows for the simultaneous determination of both apical and basal membrane Lp's from a unilateral exposure to an anisosmotic bathing solution (mucosal). The estimates of apical and basal Lp with this method were 1.16 x 10(-3) and 0.84 x 10(-3) cm/s.osmol/kg, respectively. The values of Lp for the apical and basal cell membranes are sufficiently large that only a small (less than 3 mosmol/kg) transepithelial difference in osmolality is required to drive the observed rate of spontaneous fluid absorption by the gallbladder. Furthermore, comparison of membrane and transepithelial Lp's suggests that a large fraction of the transepithelial water flow is across the cells rather than across the tight junctions.     

Asymmetric [14C]albumin transport across bullfrog alveolar epithelium

Bullfrog lungs were prepared as planar sheets and bathed with Ringer solution in Ussing chambers. In the presence of a constant electrical gradient (20, 0, or -20 mV) across the tissue, 14C-labeled bovine serum albumin or inulin was instilled into the upstream reservoir and the rate of appearance of the tracer in the downstream reservoir was monitored. Two lungs from the same animal were used to determine any directional difference in tracer fluxes. An apparent permeability coefficient was estimated from a relationship between normalized downstream radioactivities and time. Results showed that the apparent permeability of albumin in the alveolar to pleural direction across the alveolar epithelial barrier is 2.3 X 10(-7) cm/s, significantly greater (P less than 0.0005) than that in the pleural to alveolar direction (5.3 X 10(-8) cm/s) when the tissue was short circuited. Permeability of inulin, on the other hand, did not show any directional dependence and averaged 3.1 X 10(-8) cm/s in both directions. There was no effect on radiotracer fluxes permeabilities of different electrical gradients across the tissue. Gel electrophoretograms and corresponding radiochromatograms suggest that the large and asymmetric isotope fluxes are not primarily due to digestion or degradation of labeled molecules. Inulin appears to traverse the alveolar epithelial barrier by simple diffusion through hydrated paracellular pathways. On the other hand, [14C]albumin crosses the alveolar epithelium more rapidly than would be expected by simple diffusion. These asymmetric and large tracer fluxes suggest that a specialized mechanism is present in alveolar epithelium that may be capable of helping to remove albumin from the alveolar space.     

Light and electron microscopic observations of the autonomic innervation of the mouse gallbladder mucosa

Summary The autonomic innervation of the mouse gallbladder mucosa was studied using histo-and cytochemical methods. In a light microscopic investigation the distribution of acetylcholinesterase (AChE) activity and formaldehyde-induced fluorescence was studied histochemically. Nerve fibres and small varicosities showed concentrations of AChE activity very close to the epithelium in the subepithelial connective tissue. No adrenergic nerves were observed in the mucosa.When using the electron microscope and employing the potassium permanganate fixation/staining technique only one sort of axonal enlargement was encountered, viz. the cholinergic type. These varicosities contained numerous agranular vesicles (500–600 Å in diameter). No varicosities of the adrenergic (dense-cored vesicles) type were observed.Signs of increased secretory activity in the epithelium were observed in the first few minutes after cholinergic stimulation. After repeated in vivo stimulation, there was an almost total depletion of glycoprotein granules, best seen when using the cytochemical PA-CrA-silver technique. The findings suggest that the subepithelial connective tissue and the epithelium of the mouse gallbladder mucosa have a cholinergic innervation.     

Solutes in the free space of growing stem tissues

The concentration of osmotically active solutes in the cell wall free space of young stem tissues was studied using a variety of extraction methods. When the intercellular air spaces of etiolated pea (Pisum sativum L.) internodes were perfused with distilled H₂O, the resulting solution contained a solute concentration of about 70 milliosmoles per kilogram. A second procedure involving vacuum infiltration of segments followed by centrifugation to collect the free space solution gave similar results. Apical stem segments yielded free space extracts about twice as concentrated as those from basal portions of the stem. After correcting for dilution of the free space solution by the infiltrated water, the osmotic pressure of the undiluted free space in pea stem tissue was estimated to be 2.9 bars for apical segments, 1.8 bars for basal regions. These values may be somewhat overestimated due to solute efflux from intracellular pools during the extraction procedure. Similar results were obtained for stem regions of etiolated soybean (Glycine max [L.] Merr.) and cucumber (Cucumis sativus L.) seedlings.

From measurements of the electrical conductivity and refractive index of free space extracts before and after ashing, it appears that 25% of the solutes are inorganic electrolytes and 75% are organic nonelectrolytes with an average size similar to that of glucose.

A significant osmotic pressure in the wall space offers an explanation for the frequent observation that nontranspiring plants have negative water potentials. Calculations of hydraulic resistance from water potential data must take into account solutes in the free space, else `apparent,' but unreal, changes in resistance may be calculated.

     

Effects of metamorphosis on water permeability of skin in the salamander, Ambystoma tigrinum

Developmentally associated changes in the pressure driven water permeability of the skin of the salamander Ambystoma tigrinum were measured at 20 degrees C in neotenic (gilled), transitional, and fully transformed adults. Mean values for the hydraulic conductivity of the skin (Lp, X 10(-5) cm.sec-1.ATM-1) were, respectively, 1.54, 0.54 and 0.13. This nearly 12-fold decrease in the H2O permeability coincides with the transition from aquatic to terrestrial life and may be related to the changing role of the skin in water conservation. The increase in hydraulic conductivity is opposite to the decrease in H2O diffusion rates reported by others. We suggest a theoretical basis for the apparently conflicting results.     

Determination of the permeability of human lymphocytes with a microscope diffusion chamber

A diffusion chamber similar to that proposed by J.J. McGrath (J. Microsc., in press) was constructed which allows microscopic observation of osmotically induced volume changes of individual cells in small (microliter) sample volumes. The cells are kept fixed in position in the upper compartment of the chamber by means of a highly permeable membrane and exposed to a step-like change in concentration generated in the lower compartment. An electrical conductivity probe in the upper compartment was used to monitor the temporal change of salt concentration as experienced by the cells. The rise from isotonic to hypertonic can be approximated by an exponential function. Its time constant of tau = 2.08 sec seems to be mainly determined by the change in flushing solution as tau = 1.48 sec was measured with no membrane installed. With human lymphocytes, no loss of cell volume was detected before 5 sec, i.e., when 95% of the final concentration was reached extracellularly. A step change can hence be assumed when modeling exosmosis for determining the lymphocyte membrane permeability. The equations for coupled transport of water and salt were solved numerically and fitted to the experimental data. The results were also compared to various other transport models described in the literature. Human lymphocytes are almost ideally semipermeable with a hydraulic reference permeability of Lp = 4.23 X 10(-4) cm/sec (3.13 X 10(-3) micron X atm-1 X sec-1) at T = 23 degrees C. The temperature and concentration dependence are described by an activation energy Ea = 14.3 kJ/mol and a concentration coefficient alpha 2 = 0.261 osmol/kg. An osmotically inactive volume fraction of 36.9% was determined from the final cell volumes reached asymptotically after shrinkage.     

The specific hydraulic conductivity of bovine serum albumin.

Previous studies of extracellular matrix hydraulic conductivity have characterized the flow resistance of glycosaminoglycans, proteoglycans and collagen. This work focuses on serum albumin, present in significant quantities in many connective tissues, but not previously considered for its role in determining connective tissue flow resistance. The specific hydraulic conductivity of bovine serum albumin solutions, as a function of concentration, was calculated from sedimentation and ultrafiltration data available in the literature. A rigid particle hydrodynamic model compared favorably with these results. Experimental measurements on an albumin ultrafiltration cell were in agreement with this model (within experimental error); furthermore, the experimental data confirmed the theoretical prediction that there is no (or negligible) pressure drop through the concentration polarization layer. Use of the hydrodynamic model for albumin specific hydraulic conductivity with literature values for the hindrance of albumin when passing through a glycosaminoglycan (GAG) matrix allows an estimate of the relative importance of the albumin on tissue hydraulic conductivity: in non-cartilaginous tissues with moderate GAG concentrations, tissue levels of albumin can generate flow resistance effects comparable to those of the GAGs, although well less than the flow resistance of these tissues.     

Simultaneous diffusion of inositol and mannitol in the rat brain

The diffusion of both inositol and mannitol has been determined simultaneously by the integral bolus method in rat brain. The permeability constant (Kin) of inositol averaged 0.27 +/- 0.02 ml X (100 g)-1 X min-1 or 4 X 10(-7) cm X s-1 at a cerebral capillary surface area of 100 cm2 x g-1. The permeability of mannitol was 0.08 +/- 0.01 ml X (100 g)-1. min-1 or 1 X 10(-7) cm X s-1. Neither glucose nor galactose affected the inositol permeability. Hypoglycemia increased somewhat the Km value for mannitol. The basal ganglia showed an increase Km for both substrates as compared with those obtained for cortex, temporal and parietal tissues.     

Determination of the membrane permeability coefficient and the reflection coefficient by the two-dimensional laminar flow model for intestinal perfusion experiments

We performed single perfusion experiments in the small intestine of rats in order to prove that the two-dimensional laminar flow model is suitable to determine the membrane permeability coefficient and the reflection coefficient. We used progesterone as an aqueous-diffusion-limited drug, urea as a membrane transport-limited drug and the tritiated water as an intermediate substance. The membrane permeability coefficient for progesterone was calculated to be 3.6 X 10(-4) cm/s. This value did not change when the thickness of the aqueous diffusion layer was altered by increasing the perfusion rate 10-fold. It was directly demonstrated that the two-dimensional laminar flow model was suitable to analyze the data of intestinal perfusion experiments. Membrane permeability coefficients for urea and tritiated water were determined to be 3.4 X 10(-5) cm/s and 8.9 X 10(-5) cm/s, respectively. In the presence of water absorption with the hypotonic perfusion solution, the reflection coefficient for urea was 0.84. This value is thought to be theoretically reasonable, suggesting the usefullness of the two-dimensional laminar flow model to obtain the reflection coefficient in the intestinal membrane.     

The influence of subepithelial connective tissues on epithelial proliferation in the adult mouse

Summary Subepithelial connective tissue is capable of modulating the pattern of histodifferentiation of stratified epithelia from adult animals, but it is not known whether the supporting connective tissue also influences epithelial proliferative activity. Epithelial and connective tissues of murine skin and oral mucosa, differing in their morphology and proliferative activity, were separated and heterotypically recombined prior to grafting to histocompatible hosts. After 3 or 8 weeks in situ, mitotic activity was determined following the administration of vinblastine sulfate. Although the mitotic activity in each of the epithelia could be modulated by some connective tissues, there was no distinct pattern of behavior. In combination with connective tissues from tongue or palate, the ear epidermis acquired a significantly increased mitotic activity. In contrast, when oral epithelia with high mitotic activity were recombined with dermal connective tissue, there was usually a significant reduction in proliferative activity. As there was no apparent association between mitotic activity and the induced changes in either organization or histodifferentiation, it is suggested that subepithelial connective tissue is capable of directly influencing the mitotic activity in the overlying epithelium.