Biomedical subjects

N L Simmons

Publications and source records attributed to N L Simmons.

At least 109 records · Page 6Linked to original sources

PubMed1990

Vasoactive intestinal peptide stimulation of human renal adenylate cyclase in vitro.

1. A direct action of vasoactive intestinal peptide (VIP) upon human kidney was sought by measurement of renal adenylate cyclase in tissue homogenates and plasma membranes isolated from tissue samples excised for therapeutic reasons. 2. VIP (1 microM) produced a mean stimulation of adenylate cyclase activity of 3.5-fold compared to basal values in cortical plasma membranes; comparative stimulations of 2.8-fold and 27.3-fold were obtained with 1 microM-glucagon and 1 microM-h(1-34) parathyroid hormone respectively. 3. Half-maximal stimulation of human renal cortical plasma membrane adenylate cyclase was observed with a mean value of 35 nM-VIP. 4. The stimulation of renal adenylate cyclase by VIP appeared to be specific because stimulation by glucagon was additive to that obtained with VIP, and the VIP receptor antagonist (4 Cl-D-Phe6, Leu17)-VIP inhibited the VIP-dependent stimulation of adenylate cyclase activity.

Adenylyl Cyclases↗

PubMed1989

Vasoactive intestinal peptide stimulation of renal adenylate cyclase and antagonism by (4Cl-D-Phe6Leu17)VIP.

The effect of vasoactive intestinal peptide (VIP) and related peptides [glucagon, secretin, PHI 1-27 (peptide with N-terminal histidine and C-terminal isoleucine)] on renal adenylate cyclase (AC) has been determined in several species. The largest stimulation (4.1 +/- 0.5-fold basal) of AC by 1 mumol.l-1 VIP was observed in feline cortical plasma membranes. In rabbit and guinea-pig, VIP increased AC activity 1.5 +/- 0.3- and 1.8 +/- 0.3-fold respectively but glucagon had no such action. Conversely in the rat glucagon stimulated AC some 3-fold over basal activity whereas VIP had little effect. In dog, cat and mouse both peptides were effective in increasing AC activity. For cat, half-maximal stimulation of cortical plasma membrane AC by VIP was seen at 27.0 +/- 9.0 nmol.l-1 (SE N = 9 animals). VIP also increased AC activity in both outer (red) and inner (white) medulla. In feline cortical membranes VIP and PTH (parathyroid hormone) when added in combination were fully additive. However for VIP and glucagon in combination there was no cumulative increase in AC activity, indeed the resultant activity was less than that attained by VIP alone. The VIP analogue (4Cl-D-Phe6Leu17)VIP at 10 mumol.l-1 produced a right shift in the VIP-dose response curve and increased the EC50 from 17.2 +/- 5.8 nmol.l-1 to 132.0 +/- 22.2 nmol..-1 VIP (SE N = 4). There was no reduction in the maximum response elicited by VIP consistent with a competitive type of antagonism by this analogue. PHI-stimulated AC was also reduced by (4Cl-D-Phe6Leu17)VIP resulting in a similar right shift in the dose response curve.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclase Inhibitors↗

PubMed1989

Vasoactive intestinal peptide control of renal adenylate cyclase: in vitro studies of canine renal membranes and cultured canine renal epithelial (MDCK) cells.

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.

Adenylyl Cyclases↗

PubMed1989

Resistance to acid of canine kidney (MDCK) and human colonic (T84) and ileo-caecal (HCT-8) adenocarcinoma epithelial cell monolayers in vitro.

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.

Adenocarcinoma↗

PubMed1988

Distribution of vasoactive intestinal peptide-sensitive adenylate cyclase activity along the rabbit nephron.

The effect of vasoactive intestinal peptide (VIP) upon adenylate cyclase (AC) activity has been determined in defined microdissected renal tubules isolated from collagenase-treated rabbit kidneys. In the presence of 10 microM GTP, 1 microM VIP gave marked stimulations of AC over basal values in the bright portion of the distal convoluted tubule (DCTb) (10.1-fold), and in the collecting tubule isolated from the inner stripe of the outer medulla (OMCTi, 7.8-fold). Less pronounced effects of VIP were found in the medullary collecting tubule isolated from the outer stripe (2.5-fold) and in the granular portion of the distal convoluted tubule (2.0-fold). VIP stimulation of AC activity in these segments amounted to 25 to 40% of the effect elicited by other agonists (arginine vasopressin, calcitonin or parathyroid hormone) in their respective target segments. A low response to VIP was observed in the cortical thick ascending limb (1.8-fold) which represented less than 5% of the calcitonin-stimulated AC activity. In the thin descending limb VIP produced a slight and variable stimulation of AC. VIP was without effect upon AC in the convoluted and straight portions of the proximal tubule, the medullary thick ascending limb and the cortical collecting tubule. Half-maximal stimulation of AC by VIP was observed at 26 +/- 10 nM (n = 3) in OMCTi and at 19 nM (n = 2) in DCTb. Related peptides glucagon, secretin and PHI gave lower stimulations of AC compared to VIP in OMCTi. Conversely for rat OMCTi, under identical conditions, glucagon was much more effective than VIP.

Adenylyl Cyclases↗

PubMed1988

[3H]bumetanide binding and inhibition of Na+ + K+ + Cl- co-transport: demonstration of specificity by the use of MDCK cells deficient in co-transport activity.

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.

Animals↗

PubMed1987

Attribution of [3H]bumetanide binding to the Na+K+Cl 'co-transporter' in rabbit renal cortical plasma membranes: a caveat.

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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1987

Vasoactive intestinal polypeptide regulation of rabbit renal adenylate cyclase activity in vitro.

1. The effect of vasoactive intestinal polypeptide (VIP) upon adenylate cyclase activity was determined in purified cortical basolateral membranes and in glomeruli and tubular elements obtained from rabbit kidney. 2. In purified basolateral membranes prepared from cortex, 1 microM-VIP consistently stimulated adenylate cyclase activity above basal levels (1.55 +/- 0.09-fold (mean +/- S.E. of mean), n = 10 animals). Half-maximal stimulation was observed at 17 +/- 11 nM-VIP (S.D., n = 9). 3. Related peptides, e.g. secretin, glucagon, gastric inhibitory peptide, human pancreatic growth hormone releasing factor, and peptide having N-terminal histidine and C-terminal isoleucine amide (PHI), were without effect or gave lower stimulations of adenylate cyclase activity when tested at 1 microM. 4. Significant VIP degradation was observed under the assay conditions used but this did not substantially alter the response or selectivity to VIP. 5. In separate preparations of isolated glomeruli and proximal tubules addition of 1 microM-VIP resulted in a 3.3 +/- 1.1-fold (S.D., n = 3) and 2.2 +/- 1.0-fold (S.D., n = 3) stimulation (respectively) of adenylate cyclase activity. 6. In isolated medullary tubule suspensions, isolated by collagenase-hyaluronidase digestion of outer (red) medulla, and in thick ascending-limb-enriched preparations prepared by Percoll density gradient fractionation, 1 microM-VIP significantly increased adenylate cyclase activity by 2.4 +/- 0.6-fold (S.D., n = 3) and 2.1 +/- 0.7-fold (S.D., n = 3) respectively. 7. A possible role for VIP in the regulation of renal function in the rabbit is discussed in relation to the occurrence of VIP stimulation of adenylate cyclase activity in several renal cellular elements.

Adenylyl Cyclases↗

PubMed1986

An investigation of [3H]bumetanide uptake in a cultured renal cell line (MDCK).

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.

Animals↗

PubMed1986

Loop-diuretic inhibition of adrenaline-stimulated Cl- secretion in a cultured epithelium of renal origin (MDCK).

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.

Animals↗

PubMed1985

Role of passive potassium fluxes in cell volume regulation in cultured HeLa cells.

Cultured HeLa cells behave as ideal osmometers when subjected to hyperosmolar media, and show no volume regulatory behavior. In hypoosmolar solutions, cell swelling is not as great as predicted, and this is due largely to a loss of intracellular KCl. In hyperosmolar solutions there is a stimulation of the ouabain-insensitive but loop diuretic-sensitive 86Rb+ (K+) pathway. Analysis of the K+, Na+ and Cl- dependency of this K+ flux pathway demonstrates that the increase is principally due to an increase in its maximal velocity (Vmax). The sensitivity of this pathway to diuretic inhibition is unchanged in hyperosmolar media. Diuretic-sensitive 86Rb+ (K+) efflux stimulated by hypertonicity shows no marked dependence on external K+. The K+ loss observed in hypoosmolar media is distinct from the K+ transport pathway stimulated by hyperosmolar media on the basis of its sensitivity to furosemide and anion dependence.

Cell Membrane Permeability↗

PubMed1984

An effect of piretanide upon the intracellular cation contents of cells subjected to partial chronic (Na-K) pump blockade by ouabain.

Cultured cells have been used to study the contribution made by the ouabain-insensitive but diuretic (piretanide)-sensitive K transport system (so-called cotransport) to the maintenance of intracellular Na+ and K+ contents in normal cells and in cells whose Na-pump sites have been subjected to chronic partial inhibition. In cells which have normally directed gradients of Na+ and K+, chronic incubation in piretanide (10(-4) M) for up to 24 hr has no significant effect on the internal ion contents of HeLa (human carcinoma), MDCK (dog kidney epithelium) or BC3H1 (mouse smooth muscle) cell lines. This observation is consistent with the notion that when the intracellular ion contents are in a normal steady state the net driving force acting upon the diuretic-sensitive K transport (Na + K + Cl cotransport system) is zero or very close to zero. When cells are subjected to chronic partial inhibition of the sodium pump as a consequence of growth in sublethal concentrations of ouabain (10(-9)-3 X 10(-7) M), the number of functional Na-pump sites decreases, the intracellular Na+ content increases and the intracellular K+ decreases in a dose dependent manner. Under these conditions, inclusion of piretanide (10(-4) M) causes a significant retardation of Na+ gain and K+ loss from the cells. This response is of high molar affinity (EC50 = 3-4 X 10(-6) M) and can be obtained with the other loop diuretics, furosemide and bumetanide. The data presented are consistent with the idea that in cells subjected to chronic partial inhibition of the Na-pump, there is a piretanide-sensitive exchange of intracellular K+ for extracellular Na+. Such an effect of the cotransport system would not be predicted on the basis of a tightly coupled electroneutral cotransport of Na+, K+ and Cl- with a stoichiometry of 1Na:1K:2Cl. The data are discussed in relation to the possible role of putative circulating endogenous inhibitors of the Na-pump.

Animals↗

PubMed1984

Biochemical and physiological adaptation to chronic propranolol treatment in the rat.

The biochemical and physiological aspects of isoprenaline sensitivity in normotensive rats were examined during and after abrupt withdrawal of chronic propranolol treatment. Serum propranolol concentrations in rats chronically treated for one month (0.125% propranolol in drinking water: 75-100 mg/kg/day) ranged from 7 to 23 ng/ml. At the height of the blockade, rats showed a decreased responsiveness in vivo to isoprenaline-induced increase in heart rate and fall in blood pressure; the ED50 values for isoprenaline being increased some 20- and 4-fold respectively. There was a 180% increase in beta-receptor number in sarcolemmal membranes isolated from ventricular muscle of these animals, together with increased basal (290%), fluoride- (100%), forskolin- (80%) and isoprenaline-stimulated (125%) adenylate cyclase activity. Twenty-four hours after propranolol withdrawal, serum propranolol concentrations were reduced by over 95%. At this time rats exhibited increased chronotropic and blood pressure responses to i.v. isoprenaline, indicated by the reduced ED50 values (2-fold and 12-fold respectively compared to controls). In addition, cardiac sarcolemmal beta-receptor number and adenylate cyclase activities were still significantly elevated above those of controls; 35% increase in beta-receptor number and increases of 96, 26, 13 and 37% in basal, fluoride-, forskolin- and isoprenaline-stimulated adenylate cyclase activities respectively. Forty-eight hours after drug withdrawal serum propranolol concentrations were only just detectable at 0.5 +/- 0.1 ng/ml. Although sarcolemmal beta-receptor numbers were still elevated (23%) isoprenaline-stimulated adenylate cyclase activity had returned to control values. However, both the fluoride- and forskolin-stimulated enzyme activities were decreased below control values by 12 and 23% respectively, suggestive of a reduction in the catalytic capacity of the adenylate cyclase complex. In parallel with the reduction in beta-receptor number and adenylate cyclase activity, the chronotropic response to i.v. isoprenaline had also returned to control values. In contrast, the blood pressure response to i.v. isoprenaline was still elevated in these animals indicated by the 5-fold reduction in the ED50 value compared with control animals.

Adaptation, Physiological↗

PubMed1984

The action of epinephrine on Madin-Darby canine kidney cells.

We have used cultured monolayers of Madin-Darby canine kidney (MDCK) cells, which form epithelial layers of high transepithelial resistance, grown on Millipore filters, for transport studies. In the absence of hormones net ion transport is of small magnitude and is consistent with a net absorptive flow (apical to basal) of Na+. Epinephrine, effective only from the basolateral cell surface, stimulates a net secretion (basal to apical) of Cl-. A substantial portion of net Cl- secretion is inhibited by loop diuretics such as furosemide applied to the basolateral cell aspects. The participation of a diuretic-sensitive cotransport system for Na+, K+, and Cl-, similar to that found in other cells, in transepithelial Cl- flux is postulated. The action of catecholamines on MDCK cell adenylate cyclase and on a Ca2+-activated K+ conductance is described.

Adenylyl Cyclases↗

PubMed1984

Epithelial cell volume regulation in hypotonic fluids: studies using a model tissue culture renal epithelial cell system.

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.

Animals↗

PubMed1984

Control of cultured epithelial (MDCK) cell transport function: identification of a beta-adrenoceptor coupled to adenylate cyclase.

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.

Adenylyl Cyclases↗

PubMed1983

Effect of ouabain upon diuretic-sensitive K+ transport in cultured cells. Evidence for separate modes of operation of the transporter.

(1) Unidirectional K+ (86Rb) influx and efflux were measured in subconfluent layers of MDCK renal epithelial cells and HeLa carcinoma cells. (2) In both MDCK and HeLa cells, the furosemide-inhibitable and chloride-dependent component of K+ influx/efflux was stimulated 2-fold by a 30 min incubation in 1 . 10(-3) M ouabain. (3) Measurements of net K+ loss and Na+ gain in ouabain-treated cells at 1 h failed to show any diuretic sensitive component, confirming the exchange character of the diuretic-sensitive fluxes. (4) Prolonged incubations for 2.5 h in ouabain revealed a furosemide- and anion-dependent K+ (Cl-) outward net flux uncoupled from net Na+ movement. Net K+ (Cl-) outward flux was half-maximally inhibited by 2 microM furosemide. (5) After 2.5 h ouabain treatment, the anion and cation dependence of the diuretic-sensitive K+ influx/efflux were essentially unchanged when compared to untreated controls.

Animals↗

PubMed1983

Transepithelial chemotaxis of rat peritoneal exudate cells.

The migration of peritoneal exudate (PE) cells into plain Millipore filters mounted in Boyden chambers occurs under random, chemokinetic and chemotactic conditions. Significant migration of such cells in vivo, however, involves both transendothelial and transepithelial penetration and occurs predominantly under pathological conditions where chemotactic agents are presumed to be present in gradient form. When Madin-Darby canine kidney (MDCK) epithelial cells are grown as a confluent monolayer on the Millipore filter of a Boyden chamber, transepithelial migration is seen only under chemotactic conditions thus modelling in vivo behaviour more effectively. The MDCK cell line exists as 2 variant strains which model different regions of the mammalian nephron. Strain I MDCK cells share features of the distal and collecting tubules and have relatively high junctional resistance (greater than 1k omega cm2). Strain II MDCK cells model the proximal segment of the nephron and have relatively low junctional resistance (c. 70 omega cm2). We have found that PE cells penetrate the less resistant strain II MDCK monolayer at a faster rate (as assessed by leading front migration) than they penetrate the tighter strain I monolayer. We have also utilized the electrophysiological features of MDCK monolayers to monitor transepithelial penetration. Our electrophysiological data indicate that rat PE cells penetrate MDCK monolayers of either strain by a transjunctional route with consequent reversible dissolution of the junctional complex. This extracellular path of PE cell migration was confirmed by ultrastructural observations. The extent of junctional dissolution and the delay in re-establishment of monolayer integrity (as assessed by electrophysiological means) are related to the concentration of PE cells added to the MDCK monolayer. Brief treatment (10 min) of the MDCK monolayer with the cation chelating agent EDTA also disrupts monolayer integrity, although its re-establishment is significantly faster than when disruption occurs by PE cell transmigration. Our results show a clear parallel between PE cell migration across an MDCK monolayer and changes in its electrophysiological parameters and thus suggest that transepithelial chemotaxis may be directly assessed by electrophysiological means. The use of Boyden chambers modified by the incorporation of epithelial monolayers may prove useful in in vitro studies of inflammation and could be adapted for studies of other pathological processes, such as metastasis, where considerable cell invasion is involved.

Animals↗