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The cultured renal epithelial cell line MDCK has been used in a study of cell volume regulation, emphasis being placed upon cell swelling in hypotonic media. MDCK cell volume was measured directly by electronic cell sizing using a Coulter counter in MDCK cell suspensions; this method gave comparable values for cell water when compared with those obtained using an intracellular space marker [14C]3-O-methyl glucose. MDCK cells behaved as perfect osmometers when suspended in hypertonic fluid (cell shrinkage). Cellular swelling in hypotonic media, in certain conditions, was found to be less than expected for an ideal osmometer. Non-ideal swelling was found to be the result of a substantial loss of intracellular K+ (Cl-) due to a specific increase in membrane K+ permeability. The membrane channel mediating the increased net K+ loss was separate, by pharmacological identity, from the Na+-K+ pump, the diuretic-sensitive co-transport system and a Gardos-type channel inhibited by quinine. A role for increased Ca2+ influx mediating the increased K+ permeability is suggested by results from hypotonic exposure in nominally Ca2+-free solutions.

The inhibition of ouabain-insensitive 86Rb+ (K+) flux in a cultured renal cell line (MDCK) by a series of loop diuretics, including bumetanide, (the 'cotransport' flux component) has been determined in order to define the concentration range over which [3H]bumetanide would be expected to bind to the membrane transporter involved. Half-maximal inhibition by bumetanide was observed at 0.26 +/- 0.12 (S.D.) microM. The time and concentration dependence of [3H]bumetanide uptake in intact MDCK cells has been determined in experimental conditions, which were as far as possible, identical to inhibition of K+ flux. Total cellular uptake of [3H]bumetanide (0-1 microM) may be separated into a linear component and a component displaying sigmoidal saturation kinetics with a concentration giving half-maximal uptake of 0.33 +/- 0.17 (S.D.) microM, maximal uptake of 1.17 +/- 0.47 pmol/10(6) cells, and a Hill coefficient of 1.59 +/- 0.28. There is also evidence for a second component of [3H]bumetanide uptake of lower affinity (less than 5 microM). Competition of [3H]bumetanide uptake by a series of loop diuretics at varying concentrations gives an order of potency identical to that observed for inhibition of the ouabain-insensitive 86Rb+ influx. The magnitude of the saturable component of [3H]bumetanide uptake is correlated with the magnitude of the diuretic-sensitive 86Rb+ influx in MDCK cells and in a variety of other cultured cells. The relationship between the diuretic-sensitive transport, the saturable component of [3H]bumetanide uptake and the cellular location of bumetanide uptake is discussed.

Vasoactive intestinal peptide (VIP) has been shown to stimulate adenylate cyclase activity in plasma membranes isolated from canine renal cortex, outer and inner medulla in vitro. Though related hormones such as glucagon also stimulate adenylate cyclase in these membrane preparations, it is likely that VIP interacts with specific VIP receptors since the VIP receptor antagonist, (4Cl-D-Phe6, Leu17)-VIP, is capable of reducing the response to VIP, but not that to glucagon. Also binding of 125I-VIP to cortical renal plasma membranes shows competition by unlabelled VIP, but not by glucagon. Strain 1 (and clone CL(8)1b cells derived from the established cultured dog kidney cell line, MDCK, have been shown also to respond selectively to VIP by an increase in adenylate cyclase activity and cyclic AMP accumulation in intact cells. A physiological correlate of VIP activation of adenylate cyclase has been sought by addition of VIP to reconstituted epithelial monolayers of strain 1 MDCK cells clamped in Ussing chambers. VIP addition to the basal-lateral cell aspects generates an inward short-circuit current that is sensitive to replacement of medium Cl- by NO3-, and to inhibition by the Cl- channel blocker, 3-nitro-2(3-phenylpropylamino)-benzoic acid, consistent with VIP stimulation of transepithelial Cl- secretion.

A method is described for the preparation of an enriched population of proximal tubules from the cortices of neonate (21-28 d old) rabbits. The method uses collagenase-hyaluronidase digestion, followed by gentle shear to yield a suspension of tubules and glomeruli. Tubular enrichment is achieved by discontinuous density gradient centrifugation in a Percoll gradient. Two fractions were obtained by this method. The denser fraction contained predominantly proximal tubules, was depleted of glomeruli and was enriched in the proximal tubule marker enzyme alkaline phosphatase. Qualitatively similar results to those obtained with neonate animals were obtained with adult tissue. Neonate tubules from the denser gradient fraction grew readily in tissue culture. When examined by electron microscopy the cells exhibited a marked polarity of ultrastructural organization and retained apical tight junctions. Despite this, there was an obvious loss of structure in comparison with in vivo proximal tubule cells. The use of primary culture techniques to study in vitro renal epithelial function is discussed.

Monolayers of canine kidney (MDCK) and human lower intestinal (T84 and HCT-8) cell lines generated significant transepithelial electrical resistance (700-5000 omega cm2). Electrical integrity was maintained upon acidification of the apical and/or basolateral surfaces to pH 3.0, and this was associated with increased transepithelial electrical resistance, and generation of a potential difference at pH less than 4.5. These results indicate that resistance to acid is a general phenomenon of epithelial layers, and that monolayers of epithelial cells, including those of human origin, are a homogeneous and simple model for studying epithelial barrier function in vitro.

Cultured monolayers of Madin-Derby canine kidney (MDCK) cells grown upon permeable filter supports display many features of in vivo epithelia. Measurements of transmonolayer resistance, open-circuit p.d., dilution and bi-ionic p.d.s have been made of MDCK monolayers mounted in Ussing-type chamber (0.75 cm window radius). Previously reported values of transmonolayer resistance of 100 omega cm2 (Cereijido, Robbins, Dolan, Rotunno & Sabatini, 1978; Misfeldt, Hamamoto & Pitelka, 1976) indicate a 'leaky' epithelium. The major finding of this work is that MDCK monolayers can also display values of transmonolayer resistance similar to 'tight' epithelia (mean value = 4116 omega cm2). The reason for such differences is the existence of separate strains of MDCK cells. High-resistance monolayers originate from cell stocks of sixty serial passages whilst low-resistance monolayers originate from cell stocks of 109 serial passages. Measurements of transmonolayer dilution, and bi-ionic p.d.s together with Na ion tracer fluxes and electron microscope observations of La3+ penetration through the apical tight junctions, substantiate the finding of high-resistance properties in MDCK monolayers and confirm the existence of two separate MDCK cell strains displaying entirely different physiological properties. Differences extend to cellular morphology and cellular volume in the two strains of MDCK cells.

A catecholamine-sensitive adenylate cyclase activity was observed in cell homogenates of cultured renal epithelial (MDCK) cells. 10 microM isoprenaline gave a 2.85-fold increase in adenylate cyclase activity above basal levels. A series of adrenoceptor agonists gave a relative potency series of isoprenaline greater than adrenaline greater than noradrenaline (K1(9) values of 1.9 X 10(-7), 1.6 X 10(-6) and 1.9 X 10(-5) M respectively), consistent with activation of a beta-adrenoceptor. Intracellular accumulation of cyclic AMP was also stimulated by 10 microM isoprenaline, peak values being observed after 2 min, followed by a decline to lower maintained levels. The phosphodiesterase inhibitor isobutylmethylzanthine (1 mM) augmented isoprenaline-stimulated cyclic AMP accumulation. In epithelial preparations of MDCK cells grown upon Millipore filters and mounted in Ussing chambers isoprenaline was only effective in elevating intracellular cyclic AMP contents when applied to the basal cell surfaces. Direct measurement of beta-adrenoceptor density and subtype was determined by (+/-)-3-[125I]iodocyanopindolol binding to MDCK cell homogenates. Binding consisted of a saturable component (Vmax = 14.9 fmol/mg cell protein) of high molar affinity (Kd = 10.8 pM) and a non-saturable component which showed a linear dependence on iodocyanopindolol concentration. In addition to the high-affinity binding site, dissociation kinetics revealed a low-affinity component (Kd = 450 pM) comprising 24% of saturable binding. Competition of (+/-)-3-[125I]iodocyanopindolol binding with beta-adrenoceptor agonists and antagonists was entirely consistent with the existence of a beta 2-adrenoceptor. Examination of various MDCK cultures and clones revealed the existence of MDCK cultures whose adenylate cyclase activity was unresponsive to catecholamine stimulation; this correlated with a reduced or undetectable level of (+/-)-3-[125I]iodocyanopindolol binding. The control of transepithelial chloride transport in MDCK epithelia by catecholamines is discussed.

Cultured renal MDCK cells possess a Na+ + K+ + Cl- co-transport system which is inhibited with high affinity by loop diuretics (K0.5 for bumetanide inhibition = 0.28 microM). By the use of 'mutant' cell lines deficient in co-transport flux the specific interaction of [3H]bumetanide with the co-transporter has been identified. [3H]Bumetanide uptake in parental MDCK-N cells in the range 0-1 microM comprises a non-saturable linear component, assessed by the inclusion of 100 microM-unlabelled bumetanide and a saturable component (K0.5 = 0.19 microM and Bmax = 0.55 pmol/10(6) cells). Though the magnitude of the linear non-specific component was little different between the parental MDCK-N cell line and two co-transport-deficient mutant cell lines (LKC1 and LKA3), the magnitude of the saturable component was markedly reduced in both co-transport-deficient mutants. In addition to the saturable component associated with flux inhibition a lower-affinity uptake displaceable by excess unlabelled bumetanide was evident in MDCK-N cells comprising 8 pmol/10(6) cells measured at 10 microM-[3H]bumetanide. This lower-affinity uptake was present in both co-transport-deficient mutant cell lines confirming its lack of association with inhibition of co-transport flux. In MDCK cells possessing the co-transporter, an estimate of the turnover number was made when co-transport flux and specific bumetanide uptake at 0.5 microM-[3H]bumetanide were measured in the same cell batch. At 37 degrees C this was 113 K+ ions site-1 s-1.

Loop diuretics (furosemide, bumetanide, piretanide) inhibit the adrenaline-stimulated short-circuit current due to transepithelial Cl- secretion in cultured renal epithelial layers (MDCK). The inhibition of Cl- secretion by loop diuretics is consistent with the presence of basal-lateral 'cotransport' since inhibition is observed only with the basal applications of loop diuretics, is of high potency (half-maximal bumetanide inhibition being observed at 0.8 microM, bumetanide being more potent than furosemide) and is without effect upon the adrenaline-stimulated increase in tissue conductance. Loop diuretics are also shown to inhibit a component of K+ efflux across the basal-lateral surfaces. Cellular uptake of [3H]bumetanide across both apical and basal surfaces of intact epithelial layers was measured in order to localize the cotransport system. A component of cellular [3H]bumetanide uptake sensitive to competition by 0.1 mM unlabelled loop diuretic is only observed from the basal-lateral cell surfaces. There is no evidence for transepithelial bumetanide secretion as is seen in renal cortex.

The 3H-labelled loop diuretic bumetanide has been used to investigate loop diuretic binding to purified plasma membranes from rabbit kidney cortex (and outer medulla). Bumetanide binding to partially purified cortical plasma membranes in the range 0-10 microM, in a buffer containing principally Na, K and Cl ions, consists of a linear non-saturable component as assessed by 100 microM unlabelled bumetanide, and a saturable component consisting of high- and low-affinity binding sites, half-maximal binding being observed at 1.3 and 220 microM, respectively. The high-affinity site was found to be present in a fraction enriched in basolateral membrane markers when plasma membranes were further purified on a continuous Percoll gradient, whilst bumetanide binding to fractions enriched in brush-border or mitochondrial membrane markers was of lower affinity. Several features of bumetanide binding to basolateral membrane marker-enriched fractions are consistent with binding to the Na+K+Cl 'co-transporter' inhibited by loop diuretics: half-maximal binding was observed at 1.8 microM, with a finite maximal binding capacity of 78 pmol/mg. The relative efficacy of several loop diuretics for displacement of [3H]bumetanide was bumetanide greater than piretanide greater than furosemide = ethacrynic acid. Binding of loop diuretic was found to be dependent upon the medium ionic composition, Na, K and Cl being required to give maximal binding. The ability of probenecid and 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS) to compete with [3H]bumetanide was tested since these compounds are known inhibitors of anion secretion in the proximal nephron. Both DIDS and probenecid were able to effectively compete with [3H]bumetanide binding. A test of the ability of these compounds to inhibit 'co-transport' flux was made in intact MDCK cells using the ouabain-insensitive 86Rb (K) influx. Probenecid, at the concentrations seen to displace [3H]bumetanide binding to renal plasma membranes, was an effective inhibitor of 'co-transport' whereas DIDS was not. The adequacy of present criteria as to the identification of the 'co-transporter' in renal membranes using [3H]bumetanide binding are discussed in the light of this evidence.