microA Cm-2 Research Articles

The thyroid cell monolayer in culture

When cultured on collagen coated nitrocellulose filters, thyroid epithelial cells form morphologically and functionally polarized monolayers. The bioelectric parameters of these monolayers were measured after mounting in Ussing chambers; transepithelial potential (Vab), short circuit current (Isc) and transepithelial resistance were respectively 12 +/- 1 mV (apical side negative), 3.8 +/- 0.2 microA cm-2 and 3250 +/- 214 omega cm2 (mean +/- SEM, n = 75). Eighty two percent of the short circuit current was related to sodium absorption as shown by inhibition by apical amiloride (Km = 0.2 microM) and by basal ouabain (K1/2 = 0.3 microM). Amphotericin B (5-25 micrograms/ml) added to the apical bath increased Isc suggesting an apical rate-limiting step. Step by step replacement of choline by Na+ in a Na+-free medium resulted in a progressive increase in Vab and Isc with half maximal effect at 20 +/- 1 mM Na+. Thyrotropin (TSH) increased Isc and Vab in a biphasic way with a transient maximum after 5 min and a plateau after 20 min (about four times the basal level at 100 microU/ml TSH). This increase in sodium transport was also inhibited by apical amiloride. Thus, in culture, the thyroid cell monolayer behaves as a tight sodium absorbing epithelium controlled by TSH, with a rate limiting apical sodium channel as the entry mechanism and a basolateral Na+, K+-ATPase as the electromotive force.

Voltage dependence of Na/K pump current in Xenopus oocytes.

Stage V and VI (Dumont, J.N., 1972, J. Morphol. 136:153-180) oocytes of Xenopus laevis were treated with collagenase to remove follicular cells and were placed in K-free solution for 2 to 4 days to elevate internal [Na]. Na/K pump activity was studied by restoring the eggs to normal 3 mM K Barth's solution and measuring membrane current-voltage (I-V) relationships before and after the addition of 10 microM dihydroouabain (DHO) using a two-microelectrode voltage clamp. Two pulse protocols were used to measure membrane I-V relationships, both allowing membrane currents to be determined twice at each of a series of membrane potentials: (i) a down-up-down sequence of 5 mV, 1-sec stair steps and (ii) a similar sequence of 1-sec voltage pulses but with consecutive pulses separated by 4-sec recovery periods at the holding potential (-40 mV). The resulting membrane I-V relationships determined both before and during exposure to DHO showed significant hysteresis between the first and second current measurements at each voltage. DHO difference curves also usually showed hysteresis indicating that DHO caused a change in a component of current that varied with time. Since, by definition, the steady-state Na/K pump I-V relationship must be free of hysteresis, the presence of hysteresis in DHO difference I-V curves can be used as a criterion for excluding such data from consideration as a valie measure of the Na/K pump I-V relationship. DHO difference I-V relationships that did not show hysteresis were sigmoid functions of membrane potential when measured in normal (90 mM) external Na solution. The Na/K pump current magnitude saturated near 0 mV at a value of 1.0-1.5 microA cm-2, without evidence of negative slope conductance for potentials up to +55 mV. The Na/K pump current magnitude in Na-free external solution was approximately voltage independent. Since these forward-going Na/K pump I-V relationships do not show a region of negative slope over the voltage range -110 to +55 mV, it is not necessary to postulate the existence of more than one voltage-dependent step in the reaction cycle of the forward-going Na/K pump.

Evidence for reduced Cl- and increased Na+ permeability in cystic fibrosis human primary cell cultures.

1. Employing a primary cell culture system and intracellular microelectrodes, we quantitated and compared the Na+ and Cl- pathways in apical membranes of normal and cystic fibrosis (CF) human airway epithelia. 2. Like the transepithelial difference (PD) in situ, the PD of CF epithelia in culture (-27 +/- 4 mV, mean +/- S.E.M.; n = 28) exceeded the PD of normal epithelia (-10 +/- 1 mV; n = 22). The raised PD principally reflected an increase in the rate of active transport (equivalent short circuit, Ieq) for CF epithelia (61 +/- 9 microA cm-2) as compared with normal epithelia (23 +/- 3 microA cm-2). No significant differences in transepithelial resistance were detected. 3. As indicated by ion replacement studies (gluconate for Cl-), the apical membrane of normal cells exhibits an apical membrane Cl- conductance (GCl) that can be activated by isoprenaline. CF cells do not exhibit an apical membrane GCl, nor can a GCl be activated by isoprenaline. 4. CF cells exhibited a larger amiloride-sensitive Ieq and amiloride-sensitive apical membrane conductance (GNa) than normal cells. Further, the amiloride-sensitive Ieq was increased by isoprenaline in CF but not normal airway epithelia. 5. Equivalent circuit analysis yielded evidence for a more positive electromotive force (EMF) across the apical membrane and a more negative EMF across the basolateral membrane of CF cells as compared with normal cells. Baseline resistances of the apical (Ra) and basolateral (Rb) membranes did not differ for normal and CF cells. 6. Estimates of the resistance of the paracellular path to ion flow (Rs) by equivalent circuit analysis or ion substitution detected no differences in Rs between CF and normal cells. 7. We conclude that abnormalities in both cellular Cl- permeability (reduced) and Na+ permeability (increased) are characteristic of the cultured CF respiratory epithelial cell. These data suggest that a defect in the regulation of apical membrane permeabilities is a central feature of this disease.

Human eccrine sweat gland epithelial cultures express ductal characteristics.

1. Isolated human eccrine sweat glands were cultured in vitro. Cells were harvested and plated onto permeable supports to form confluent cell sheets, area 0.2 cm2. These were used to study the electrogenic transepithelial transport of ions by measurement of short-circuit current (SCC). Epithelial sheets had a basal SCC of 5.89 +/- 0.62 microA cm-2 (n = 33) and a transepithelial resistance of 74.1 +/- 5.6 omega cm2 (n = 33). The transepithelial potential difference varied between -0.2 and -1.8 mV with a mean value of -0.71 +/- 0.09 mV (n = 33). 2. The basal current was abolished by addition of 10 microM-amiloride to the apical bathing solution. The concentration of amiloride which inhibited basal SCC by 50% (EC50) was 0.4 microM. Cultures prepared from the secretory coil of sweat glands, rather than from whole glands, were similarly sensitive to amiloride (EC50 = 0.8 microM). 3. Lysylbradykinin (LBK), carbachol, isoprenaline, prostaglandin E2 (PGE2) and A23187 all increased SCC in cultures from whole glands. LBK responses were obtained with basolateral and not with apical application. Furthermore LBK actions were not substantially altered by cyclo-oxygenase inhibition but showed marked desensitization upon repeated application. Sheet cultures prepared from sweat gland coils also showed SCC responses to both carbachol and LBK. Forskolin, an activator of adenylate cyclase, did not alter SCC in either type of preparation. 4. Replacement of chloride and of chloride and bicarbonate in the bathing solution did not cause attenuation of the responses to LBK or carbachol in whole-gland sheet cultures. Furthermore responses were unaffected by piretanide or acetazolamide. These results were taken to indicate that anion secretion was not the basis for the SCC responses. 5. Responses to LBK and carbachol were significantly reduced by amiloride (10 microM), this effect being reversible. No responses to LBK or carbachol were seen when N-methyl-D-glucamine (NMDG) was used to replace sodium, whereas reintroduction of sodium ions restored responsiveness to these agents. 6. The SCC responses to the muscarinic agonist carbachol and to LBK appear to be due to stimulation of amiloride-sensitive, electrogenic sodium absorption in whole-gland sheet cultures. Further it would appear that, in culture, the pleuripotential capacity of the cells is revealed since both whole-gland and secretory coil cultures exhibit some properties usually associated in vivo with duct cells. Many mammalian epithelia show electrogenic chloride secretion both in response to carbachol and LBK but also in response to activation of adenylate cyclase with forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of a protein phosphatase inhibitor, okadaic acid, on membrane currents of isolated guinea-pig cardiac myocytes.

The effects of a protein phosphatase inhibitor, okadaic acid (OA), were studied on membrane currents of isolated myocytes from guinea-pig cardiac ventricle. The whole-cell Ca2+ current (ICa) was recorded as peak inward current in response to test pulse to 0 mV. Extracellular application of OA (5-100 microM) produced an increase of ICa. The effect was markedly enhanced when the myocyte was pretreated with threshold concentrations of isoprenaline. ICa was increased from 11.3 +/- 0.8 microA cm-2 to 19.0 +/- 1.1 microA cm-2 (n = 4) by 5 microM-OA in the presence of 1 nM-isoprenaline. The delayed rectifier current was also slightly increased. Furthermore, the wash-out time of the beta-adrenergic increase of ICa was markedly prolonged by OA. The beta-adrenergic stimulation of cardiac Ca2+ current is thought to be mediated by cAMP-dependent phosphorylation. The present results strongly suggest that the effect of OA on ICa is related to inhibition of endogenous protein phosphatase activity which is responsible for the dephosphorylation process. By the isotope method, the inhibitory effect of OA on different types of phosphatase was compared. OA had a relatively high specificity to type 1-, and type 2A-phosphatases.

Inhibition by chloride channel blockers of anion secretion in cultured epididymal epithelium and intact epididymis of rats

1. Anion secretion by primary monolayer cultures of rat epididymal cells was studied by the short circuit current technique. 2. Monolayers had a transepithelial potential difference of 1.34 mV, apical side negative and a short circuit current of 2.45 microA cm-2. The transepithelial resistance was 504 omega cm2. 3. Addition of anthracene-9-carboxylate (9-AC) to the apical side caused a biphasic response, a decrease followed by an increase in the short circuit current (SCC) which then returned to the basal level. Addition of 9-AC to the basolateral side also caused a biphasic response but the increase in current was sustained. 4. Addition of diphenylamine-2-carboxylate (DPC) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) caused an inhibition of the SCC when added to the apical or basolateral side. 5. When the epithelium was stimulated with adrenaline (0.23 microM, basolaterally), the SCC rose to a peak value of 10.36 microA cm-2 and then stabilized at 3.82 microA cm-2 after 15 min. Addition of 9-AC to the apical side caused a triphasic response: a decrease, reversal to the original level followed by a slow inhibition which was sustained. The inhibition achieved at the steady state was concentration-dependent with an apparent IC50 value of 2.51 mM. Addition of 9-AC to the basolateral side produced a similar response but a time lag of 20 s was observed. 6. DPC and NPPB also caused the SCC to decrease when added to the apical side of the monolayers stimulated with adrenaline. The IC50 values 0.148 mM and 0.049 mM for DPC and NPPB, respectively. Basolateral application also caused an inhibition but higher concentrations were required to inhibit 50% of the adrenaline stimulated SCC. 7. Serosal to mucosal flux of chloride was studied in intact epididymis luminally perfused in vivo. 9-AC added to the luminal perfusion solution inhibited the C1 flux in a dose-dependent manner with an IC50 value similar to that observed in cultured epithelium in vitro. 8. It is concluded that anion secretion by the epididymal cells may conform to models proposed for other Cl secreting epithelia and may involve anion diffusion across the apical channels which are blocked by Cl channel blockers.

Transcellular sodium fluxes and pump activity in Necturus gall‐bladder epithelial cells.

1. Transepithelial Na transport in Necturus was determined by measuring the rate of isotonic fluid flow. The rate at 20 degrees C was equivalent to 175 pmol cm-2 s-1. 2. Ouabain was effective in Necturus, binding to the Na pump in gall-bladder cells with a mean rate constant of 5.4 X 10(3) M-1 s-1. Measurement of the diffusive time constant of the free space for [3H]ouabain shows that the pump must be fully inhibited within 20 s when ouabain is applied to the serosa at 10(-3) M. 3. The serosal Na efflux from loaded cells was inhibited 36% by ouabain equal to a flux of 73 pmol cm-2 s-1. The remaining flux could not be attributed to either exchange diffusion or electrodiffusion induced by ouabain. 4. The transepithelial potential was 0.3 mV serosa positive. The short-circuit current measured was 6.33 +/- 1.9 microA cm-2, equal to a positive univalent ion flux of 65.6 pmol cm-2 s-1 or 38% of the net Na transfer. The current was inhibited within 1-5 min by 5 X 10(-5) M-amiloride. 5. Fluid secretion was immediately inhibited 34% by ouabain, equivalent to an isotonic transport of Na of 59.7 pmol cm-2 s-1. Thereafter it continued for at least an hour, sometimes declining slowly. Amiloride had little effect (13%). 6. The Na pump rate was measured by titrating the cell content with tracer Na at different times after ouabain treatment. The initial slope was equal to a rate of 61.6 pmol cm-2 s-1 or 35% of the net flux at time zero. 7. The Na pump rate has also been measured by recording the rise in cell Na activity with ion-specific micro-electrodes, and correcting for swelling effects. The Na pump rate was very similar to that estimated from the rise in tracer Na content, equal to 59.3 pmol cm-2 s-1 or 31.4% of the transepithelial rate. Examination of the same experiment in the literature shows a closely similar value, about one-third of that expected from fluid secretion or net flux measurements. 8. A scheme is proposed to explain the results, which requires a flow of NaCl through a parallel pathway of small Na content involving exchange en route with the cytoplasmic Na.

Neuropeptide Y: a powerful modulator of epithelial ion transport.

Neuropeptide Y (NPY) is a major gut peptide localized in the intestinal mucosa of several mammalian species. Ileal mucosa from rabbit and guinea-pig was mounted in Ussing chambers in order to study the effect of NPY on short circuit current. Neuropeptide Y inhibited the short circuit current when applied to the serosal side of the tissue. The maximum change in short circuit current was -50 +/- 6 microA cm-2 in the rabbit ileum and -49 +/- 14 microA cm-2 in the guinea-pig ileum. The EC50 was 3 X 10(-8) M in both species. Pretreatment of rabbit ileum with the alpha 2-adrenoceptor antagonist, yohimbine (1 X 10(-6) M) for 10 min did not reduce the response of the tissue to neuropeptide Y (1 X 10(-7) M). When applied serosally to rabbit ileal mucosa, the related peptide YY caused a maximum change in short circuit current of -60 +/- 13 microA cm-2; the EC50 was 2 X 10(-9) M. Isotopic flux studies in rabbit ileum showed that 1 X 10(-7) M neuropeptide Y enhanced mucosal-to-serosal Na+ and Cl- fluxes and reduced serosal-to-mucosal Cl- flux. Replacement of chloride with gluconate on both sides of the tissue significantly reduced the change in short circuit current produced by neuropeptide Y (1 X 10(-7) M), as did a similar replacement of bicarbonate. It is concluded that neuropeptide Y and peptide YY are the most potent neurotransmitters or hormones so far described in their ability to attenuate electrogenic transport in the small intestine.

Ion‐selective micro‐electrode studies of the electrochemical potentials in trout urinary bladder.

Intracellular micro-electrode techniques were used to measure the electrical resistances of the cell membranes and the shunt pathway and intracellular ionic activities in trout urinary bladder when the tissue was incubated in Ringer solution and in the presence of the polyene antibiotic ionophore amphotericin B. In control conditions the transepithelial potential was zero and the intracellular potential was -56 mV. The intracellular ionic activities measured with single- and double-barrel ion-sensitive micro-electrodes for the first time in a fish bladder (aiNa = 16 mM, aiK = 87 mM, and aiCl = 21 mM) indicate an active accumulation of K and Cl ions and an active extrusion of Na ions by the cell. The maintenance of intracellular Cl activity above its equilibrium value depended on the presence of Na ions in the mucosal medium, but was independent of the presence of K ions. Flat cable analysis yielded values for transepithelial, apical, basolateral and shunt resistances of 197, 2790, 1986 and 205 omega cm-2 respectively. Equivalent circuit analysis using amphotericin B yielded similar values for shunt resistance. The paracellular pathway accounts for 96% of transepithelial current flow and this epithelium may be classified as 'leaky'. The cells are electrically coupled with a space constant of 354 micron. Amphotericin B when added to the mucosal solution induced an immediate serosa positive transepithelial potential of about 9 mV and a short-circuit current of 64 microA cm-2. The Vt was ouabain sensitive and dependent on mucosal Na concentration. The origin of the antibiotic induced transepithelial potential was an increase in the sum of the cell membrane electromotive forces. The apical membrane potential depolarized to -7 mV and its resistance fell to 433 omega cm-2. During the first 10 min of exposure aiNa increased to 80 mM and aiK decreased to 7 mM with only a small change in aiCl. The changes in cellular Na+ and K+ activities were in accordance with their passive redistribution down their electrochemical gradients.

Prostaglandin release mediates drug-induced stimulation of sodium transport in frog skin: the effects of quinacrine.

Quinacrine markedly increased the release of prostaglandin E2 (PGE2) into the basolateral solution of the bullfrog skin from a control value of 32.7 +/- 21.7 pg per 20 min period to a stimulated value of 8593.1 +/- 4112.3 pg per 20 min period. Quinacrine increased the amiloride-sensitive short circuit current from 20.7 +/- 2.1 microA cm-2 to 45.4 +/- 6.5 microA cm-2. The stimulatory effects of quinacrine on both short circuit current and prostaglandin release were blocked in skins pretreated with indomethacin (10(-6) M). Quinacrine did not block either the stimulation of the short circuit current or the increase in PGE2 release caused by the calcium ionophore, ionomycin. These results suggest: (a) the release of PGE2 and the stimulation of the short circuit current caused by quinacrine are linked since blocking PGE2 release inhibits the stimulation of the short circuit current; (b) given the complexity of its actions, quinacrine is a poor tool to examine whether the effects of a given agent are mediated through the activation of endogenous phospholipases. In addition our results taken together with other findings in the literature suggest that there is a diverse group of compounds that stimulate transepithelial sodium transport by releasing PGE2.

Chloride transport across the integumentary epithelium of Manduca sexta (Lepidoptera: Sphingidae).

Moulting fluid of Manduca sexta contains high concentrations of potassium and bicarbonate (100 mM) and low concentrations of chloride (5 mM). This fluid begins to disappear from the exuvial space approximately 9-10 h before the actual shedding of the integument. During this time, the integument can be isolated in an Ussing cell and electrical properties measured in vitro. In a normal 32 mM KHCO3 saline, potential difference (PD) is around 10 mV, exuvial side positive, and short-circuit current (SCC) is 15-20 microA cm-2. Substitution of chloride slightly reduces both PD and SCC, although resistance does not change significantly. Measurement of chloride transport in the absence of K+ indicates that 100% of the SCC can be accounted for by the net chloride flux (approximately 2 microA cm-2). The Km and Jmax for transepithelial chloride transport are 14 mM and 0.1 microEq cm-2 h-1. Bilateral potassium addition stimulates chloride transport, doubling net chloride flux as potassium concentration increases from 2 to 5 mM. Chloride net flux is not inhibited by the presence of furosemide (1 mM), nor in HCO3- -free saline by thiocyanate (1 or 10 mM) or acetazolamide (0.1 mM), but is inhibited by 100% N2. The pattern of chloride transport in M. sexta is similar to that previously reported for the rectum of locusts. As chloride is normally at low concentrations in the moulting fluid, it is suggested that this transport system acts to maintain low intracellular concentrations which may be necessary for enzymatic functions in the epidermal cells and has little importance in fluid transport.

Trifluoperazine reduces inward ionic currents and secretion by separate mechanisms in bovine chromaffin cells.

Using patch-clamp techniques, excitation and secretion in chromaffin cells were studied by measurement of unitary inward currents and of stimulus-evoked increments in membrane capacitance. The effect of the calmodulin inhibitor trifluoperazine (TFP) on Na, Ca and acetylcholine-induced (ACh) currents as well as on capacitance increments was investigated. TFP in concentrations up to 10 microM had no effect on Na channel currents. TFP was a potent anticholinergic agent. TFP in concentrations of 100 nM-1 microM decreased net ACh-induced currents by a slow block or allosteric modification of the channel. The effect was only partially reversible. Recovery from desensitization was retarded in direct relation to [TFP]. At the single channel level, TFP was found to slightly shorten open times in 0.5 and 20 microM-ACh. As reported previously, desensitization can be modelled by at least two desensitized states, as reflected by the bursting and clustering behaviour of single channels. TFP shortened clusters mainly by reducing the number of bursts per cluster. Whole-cell Ca currents (ICa) were reduced in 10 microM-TFP from an average of 29 microA cm-2-13 microA cm-2. Changes in capacitance of 1-200 fF were elicited in controls by maximal activation of the Ca current. We interpreted these steps to be the summed result of many exocytotic vesicular fusion events. Capacitance steps depended on ICa and were absent when extracellular Ca was removed. Application of 10 microM-TFP inhibited capacitance steps. The block of capacitance steps by TFP was shown to be independent of the reduction of ACh and Ca inward ionic currents. We conclude that the prevention of exocytosis by TFP is not completely described by its inhibition of electrical excitability but also results from intracellular actions.

Current generated by backward-running electrogenic Na pump in squid giant axons.

The sodium pump of animal cells is electrogenic, that is, it normally exports more sodium ions than it imports potassium ions. In the squid giant axon, the resulting net outward electric current has a density of a few microA cm-2, and contributes 1-2 mV to the resting membrane potential. The pump is driven by the free energy of hydrolysis of ATP, and in some instances it has been possible to run the pump backwards and synthesize ATP by lowering the [ATP]/[ADP] X [Pi] ratio and steepening the transmembrane Na+ and K+ gradients. Here we have examined the question of whether a backward-running sodium pump conserves its Na+/K+ greater than 1 stoichiometry. We demonstrate reversal of the sodium pump of squid giant axon, and find that backward pumping indeed produces a net inward electric current. This current is voltage-sensitive. Our observations have mechanistic implications for models of the sodium pump.

Intracellular ion activities in frog skin in relation to external sodium and effects of amiloride and/or ouabain.

Intracellular activities of sodium, potassium and chloride ions, aiNa, aiK, and aiCl were measured with ion-selective single-, double- and triple-barrelled micro-electrodes in skin and isolated epithelia of Rana temporaria bathed on both sides with normal or modified physiological saline. Apical and basolateral membrane potentials, psi ac and psi cs and resistance Ra and Rb respectively were also measured and from the latter the fractional resistance of the apical membrane, F(Ra) and voltage divider ratio, delta psi ac/delta psi cs were measured as criteria of satisfactory membrane penetration by the micro-electrodes. Under control conditions, aiNa was 12.3 +/- 0.8 mM, aiK was 70.3 +/- 22 mM and aiCl was 20.3 +/- 1.6 mM with psi ac averaging -38.0 +/- 3.2 mV. When 10(-4) M-amiloride was added to the apical bathing fluid aiNa fell within 10 min to 1.18 +/- 0.1 mM and aiCl to 5.2 +/- 0.9 mM, while aiK increased to 86.2 +/- 3.8 mM as measured from the basolateral border of isolated epithelia. The sodium transport pool of the skin was measured from the fall in aiNa in the presence of amiloride and could be expressed as 33 X 10(-9) mol cm-2 of epithelium. The mean rate of fall of aiNa under these conditions corresponded to an efflux rate at the basolateral border of 30.1 X 10(-9) mol cm-2 min-1 (48 microA cm-2) giving a half-time for turnover of the sodium transport pool of 33 s. Reduction of sodium concentration in the apical fluid from the normal 79 mM-Na to 10, 1 and 0.1 mM caused aiNa to fall in stages to 2 mM. Because psi ac increased in negativity to -101 mV in the process, this driving force for passive sodium accumulation, more than offset the increased sodium gradient opposing sodium influx across the apical border.

Volume flow, hydraulic conductivity and electrical properties across bovine tracheal epithelium in vitro: effect of histamine.

Volume flow (Jv), potential difference (delta psi), short-circuit current (io) and electrical resistance (R) were measured simultaneously across bovine tracheal epithelium in vitro. Under basal conditions, with no applied hydrostatic or osmotic pressure gradients (delta P = 0, delta phi = 0), no spontaneous Jv was observed. delta psi was 31 +/- 2 mV (lumen negative ), io 161 +/- 8 microA cm-2 and R 202 +/- 9 omega cm2, n = 50. When a delta pi was applied, by adding 20 - 80 mM sucrose into the medium bathing either the luminal or the serosal side of the tissue, a linear relationship was found between delta pi and Jv toward the lumen or toward the serosa. The apparent hydraulic conductivity (apparent Lp) was 4.6 - 4.9 10(-6) cms-1 atm-1. Histamine 10(-4) M did not induce any spontaneous Jv under basal conditions and had no effect on io nor on R. However, histamine caused a 100% increase in Jv elicited by sucrose gradients. It was concluded that histamine exerts a selective action on the hydraulic conductivity of bovine tracheal epithelium. Experiments using H1-receptors antagonists (diphenhydramine, dimetindene, chloropyramine) and H2-antagonists (cimetidine, metiamide) or a H2-agonist (impromidine) showed that the increase of Lp induced by histamine was mediated via H2-receptors.

Transepithelial transport in cell culture: D-glucose transport by a pig kidney cell line (LLC-PK1).

The pig kidney cell line LLC-PK1 cultured on a collagen coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by occluding junctions. A transepithelial electrical potential (PD) and short-circuit current (SCC) were dependent on the presence of Na and sugar in the apical bathing solution. In the presence of 5.5 mM D-glucose, a PD of 2.8 mV. apical surface negative a SCC of 13 microA cm-2 and transepithelial resistance of 211 ohm.cm2 were recorded. The SCC was promptly reduced by the addition of phlorizin to the apical bath but unaffected when placed in the basolateral bath. The effect on SCC of various sugars was compared by the concentrations required for half-maximal SCC: 0.13 mM beta-methyl-D-glucoside, 0.28 mM D-glucose, 0.65 mM alpha-methyl-D-glucoside, 0.77 mM 6-deoxy-D-glucose, 4.8 mM D-galactose, and 29 mM 3-O-methyl-glucose. When [Na] was reduced, the concentration of D-glucose required for half-maximal SCC increase. Isotopically labeled 3H and 14C D-glucose were used to simultaneously determine bidirectional fluxes; a resultant net apical-to-basolateral transport was present and abolished by phlorizin. The transported isotope cochromatographed with labeled D-glucose, indicating negligible metabolism of transported glucose. The pig kidney cell line, LLC-PK1, provides a cell culture model for the investigation of mechanisms of transepithelial glucose transport.

Photoelectric conversion by bacteriorhodopsin in charged synthetic membranes

Photoelectroactivity of oriented purple membrane layers attached to an ion exchange film has been investigated. The action spectrum of the photocurrent followed the absorption spectrum of bacteriorhodopsin. The intactness of structure and function of bacteriorhodopsin was demonstrated by studies of absorption and photocycle kinetics. The direction of the photocurrent suggests that the extracellular surface of purple membrane is more positive. Photocurrents as high as 20 microA cm-2 were obtained in some preparations. The dependence of steady-state photocurrents on intensity of illumination and temperature was also studied. The initial rate of build-up of photocurrent depends linearly on the intensity of illumination while the off rate does not exhibit any dependence on the intensity of illumination. With rise in temperature an increase in the steady state photocurrent has been observed. This dependence was found to be linear when increase of the photocurrent due to proton translocation alone was considered.

Occluding junctions in a cultured transporting epithelium: structural and functional heterogeneity.

MDCK cells (epithelioid of renal origin) form monolayers which are structurally and functionally similar to transporting epithelia. One of these similarities is the ability to form occluding junctions and act as permeability barriers. This article studies the junctions of MDCK monolayers formed on a permeable and transparent support (a disk of nylon cloth coated with collagen) by combining two different approaches: (i) Scanning of the electric field: the disk is mounted as a flat sheet between two Lucite chambers and pulses of 20--50 microA cm-2 are passed across. The apical surface of the monolayer is then scanned with a microelectrode to detect those points where the current is flowing. This shows that the occluding junctions of this preparation are not homogeneous, but contain long segments of high resistance, intercalated with sites of high conductance. (ii) Freeze fracture electron microscopy: the junctions are composed of regions of eight to ten strands intercalated with others where the strands are reduced to one or two ridges. The sites of high conductance may correspond to those segments where the number of junctional strands is reduced to 1 or 2. It is concluded that the occluding junctions of MDCK monolayers are functionally and morphologically heterogeneous, with "tight" regions intermixed with "leaky" ones.