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M M Civan

Publications and source records attributed to M M Civan.

At least 73 records · Page 4Linked to original sources
PubMed1985

Effects of internal and external pH on amiloride-blockable Na+ transport across toad urinary bladder vesicles.

We have examined the effect of internal and external pH on Na+ transport across toad bladder membrane vesicles. Vesicles prepared and assayed with a recently modified procedure (Garty & Asher, 1985) exhibit large, rheogenic, amiloride-sensitive fluxes. Of the total 22Na uptake measured 0.5-2.0 min after introducing tracer, 80 +/- 4% (mean +/- SE, n = 9) is blocked by the diuretic with a KI of 2 X 10(-8) M. Thus, this amiloride-sensitive flux is mediated by the apical sodium-selective channels. Varying the internal (cytosolic) pH over the physiologic range 7.0-8.0 had no effect on sodium transport; this result suggests that variation of intracellular pH in vivo has no direct apical effect on modulating sodium uptake. On the other hand, 22Na was directly and monotonically dependent on external pH. External acidification also reduced the amiloride-sensitive efflux across the walls of the vesicles. This inhibition of 22Na efflux was noted at external Na+ concentrations of both 0.2 microM and 53 mM. These results are different from those reported with whole toad bladder. A number of possible bases for these differences are considered and discussed. We suggest that the natriferic response induced by mucosal acidification of whole toad urinary bladder appears to operate indirectly through one or more factors, presumably cytosolic, present in whole cells and absent from the vesicles.

Amiloride↗
PubMed1985

31P-nuclear magnetic resonance analysis of perfused single frog skins.

Perfusion of single frog skins has produced stability of the 31P-nuclear magnetic resonance (NMR) spectra over periods as long as 8 h at room temperature. With this approach, relatively large phosphocreatine (PCr) signals were recorded for each of the 12 frog skins studied. The ratio of the concentration of PCr to ATP was estimated to be 0.76 +/- 0.07, a value an order of magnitude larger than that previously reported. Comparison of the ratio of the intracellular concentrations of inorganic phosphate (Pi) to ATP determined in the present NMR study with that previously estimated by chemical analysis suggests that little intracellular Pi is likely to be immobilized. Inclusion of methylphosphonate in the perfusing solutions permitted simultaneous determination of extracellular and intracellular pH from the NMR spectra alone. The methylphosphonate has been found to be nontoxic. At an extracellular pH of 7.72 +/- 0.04, the intracellular pH was 7.47 +/- 0.06.

Adenosine Triphosphate↗
PubMed1985

Effects of tumor promoters on sodium ion transport across frog skin.

Phorbol esters are tumor promoters and mitogens whose effects may be mediated by changes in ion transport across membranes. Clarification of the transport effects of these agents should be facilitated by using a well-characterized model epithelial system whose intracellular and transmural parameters are readily measurable. The current results constitute a preliminary study of the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibutyrate (PDBU), and phorbol on the short-circuit current (Isc) across frog skin. TPA produced two effects: a stimulation of Isc of variable magnitude and a far more constant inhibition of the natriferic action of vasopressin. These effects appear related to the action of TPA as a tumor promoter insofar as PDBU (an active ester) also inhibited the natriferic response to vasopressin, whereas phorbol (inactive as a tumor promoter) had no significant effect. TPA is largely active from the mucosal medium, inhibits the natriferic response to adenosine 3',5'-cyclic monophosphate (cAMP) as well as that to vasopressin, and does not stimulate Isc in the presence of 10(-4) M mucosal amiloride. Inhibition of prostaglandin E1 production by indomethacin had no effect on the actions of TPA. The results indicate that frog skin is a promising model for studying the transport effects of the phorbol esters. The data further suggest that TPA acts on frog skin by activating the physiological amiloride- and cAMP-sensitive channels gating apical Na+ entry from the mucosal medium into the epithelial cells.

Amiloride↗
PubMed1985

cAMP- and beta-adrenergic-stimulated chloride-dependent Ca2+ secretion in frog skin.

This study examined the possible existence and nature of Ca2+ transport in frog skin using 45Ca fluxes and short-circuiting technique. Following the addition to full-thickness frog skin (FTFS) of 8-[p-chlorophenylthio]cAMP (8-CPT-cAMP), forskolin, or 1-methyl-3-isobutylxanthine, the secretory Ca2+ flux increased severalfold, inducing net Ca2+ secretion. The absorptive flux was unchanged. Isoproterenol (10(-6)M) reproduced the effects of cAMP on Ca2+ secretion (-3.76 +/- 0.80 nmol X cm-2 X h-1 vs. +0.04 +/- 0.05 in control) while vasopressin and parathyroid hormone did not alter Ca2+ fluxes. Because FTFS contains subepidermal glands capable of Cl- secretion in response to beta-adrenergic stimulation, split-thickness frog skin (STFS) consisting of the gland-free Na-absorbing surface epithelium was used to localize the anatomic site of Ca2+ secretion. In STFS, addition of 8-CPT-cAMP or isoproterenol failed to induce Ca2+ secretion, suggesting that this transport in FTFS is localized in skin glands. Additional studies explored the relationship between Ca2+ and Cl- transport in FTFS. Furosemide prevented the stimulation of both Ca2+ and Cl- secretion. Removal of Cl- from the bathing medium abolished Ca2+ secretion. Thus, FTFS exhibits a beta-adrenergic, cAMP-stimulated net Ca2+ secretion that is Cl- dependent. As this effect is not observed in STFS, the pathway of Ca2+ secretion in frog skin is probably localized in the subepidermal glandular epithelium in association with Cl- secretion. Frog skin glands may represent a useful model for the study of Ca2+ transport in Cl--transporting epithelia.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Apical sodium entry in split frog skin: current-voltage relationship.

Apical Na+ entry into frog skin epithelium is widely presumed to be electrodiffusive in nature, as for other tight epithelia. However, in contrast to rabbit descending colon and Necturus urinary bladder, the constant field equation has been reported to fit the apical sodium current (INa)-membrane potential (psi mc) relationship over only a narrow range of apical membrane potentials or to be inapplicable altogether. We have re-examined this issue by impaling split frog skins across the basolateral membrane and examining the current-voltage relationships at extremely early endpoints in time after initiating pulses of constant transepithelial voltage. In this study, the rapid transient responses in psi mc were completed within 0.5 to 3.5 msec. Using endpoints to 1 to 25 msec, the Goldman equation provided excellent fits of the data over large ranges in apical potential of 300 to 420 mV, from approximately -200 to about +145 mV (cell relative to mucosa). Split skins were also studied when superfused with high serosal K+ in order to determine whether the INapsi mc relationship could be generated purely by transepithelial measurements. Under these conditions, the basolateral membrane potential was found to be -10 +/- 3 mV (cell relative to serosa, mean +/- SE), the basolateral fractional resistance was greater than zero, and the transepithelial current was markedly and reversibly reduced. For these reasons, use of high serosal K+ is considered inadvisable for determining the INa-psi mc relationship, at least in those tissues (such as frog skin) where more direct measurements are technically feasible. Analysis of the INa-psi mc relationships under baseline conditions provided estimates of intracellular Na+ concentration and of apical Na+ permeability of 9 to 14 mM and of approximately 3 X 10(-7) cm X sec-1, respectively, in reasonable agreement with estimates obtained by different techniques.

Amiloride↗
PubMed1984

Kinetics of CO2 exchange in human erythrocytes analyzed by 13C-NMR.

The 13C-nuclear magnetic resonance signals of CO2 and bicarbonate have been studied in suspensions of mature human erythrocytes, in lysed suspensions, and in supernatant solutions. This technique requires no chemical perturbation of the samples analyzed. When care is taken to avoid technical artifacts, lysis produces only a slight broadening of the 13CO2 spectral line. The results are consistent with the concept that CO2 exchange is primarily determined by the kinetics of intracellular carbonic anhydrase and not by the permeability of the erythrocyte membrane.

Carbon Dioxide↗
PubMed1984

Intracellular pH of perfused single frog skin: combined 19F- and 31P-NMR analysis.

Intracellular pH (pHc) has been determined in frog skin by applying two different methods of pH measurement, 19F and 31P nuclear magnetic resonance (NMR) analysis, to the same tissues. Results from both NMR approaches confirm an observation by Lin, Shporer, and Civan [Am. J. Physiol. 248 (Cell Physiol. 17): 1985] that acidification of the extracellular medium reverses the sign of the pH gradient present under baseline conditions. The fluorinated probe, alpha-(difluoromethyl)-alanine methyl ester, was introduced into the epithelial cells by preincubating skins for 4.7-10.4 h at room temperature in Ringer solutions containing 1 mM ester. The free amino acid was subsequently released by intracellular esterase activity, thus providing a high enough probe concentration for NMR analysis to be practicable. From measurements of short-circuit current and transepithelial resistance under base-line and experimental conditions and the appearance of phosphocreatine (PCr) in the 31P spectrum of preloaded tissues, the fluorinated probe appears to be nontoxic to frog skin. Measurement of the chemical shift of methylphosphonate relative to PCr permitted calculation of extracellular pH. Estimation of the intracellular pH was performed both by measurement of the chemical shift of inorganic phosphate (Pi) relative to PCr and by measurement of the central peak spacing of the 19F spectrum. From four direct comparisons of the two techniques in two experiments, the difference in the estimated pH was only 0.03 +/- 0.07 pH units, supporting the concept that 31P-NMR analysis is a valid method of measuring pH in this tissue.

Animals↗
PubMed1983

Microelectrode study of K+ accumulation by tight epithelia: II. Effect of inhibiting transepithelial Na+ transport on reaccumulation following depletion.

The effects of restoring serosal potassium to potassium-depleted toad urinary bladders have been re-examined using double-barrelled microelectrodes. The data confirm the existence of a time-lag phenomenon, a dissociation between potassium reaccumulation and restoration of short-circuit current. Returning serosal potassium stimulates an increase in intracellular potassium activity 21-26 min before any increase can be detected in short-circuit current. The reaccumulation of potassium has been further studied using split frog skin, a far more suitable preparation for electrophysiologic study than toad bladder. Under baseline short-circuited conditions, potassium is accumulated against an electrochemical gradient of 22 +/- 4 mV. Reaccumulation of potassium by potassium-depleted tissues can be blocked by inhibiting the Na,K-exchange pump with high concentrations of ouabain. On the other hand, blocking apical sodium entry by the addition of 10(-4) M amiloride to the outer bathing medium does not interfere with reaccumulation of potassium. The data support the concept that the time-lag phenomenon of toad bladder reflects stimulation of potassium reaccumulation by the sodium pump in exchange for the extrusion of excess cell sodium collected during the period of potassium depletion. This reaccumulation of potassium can proceed before the entry of significant added amounts of sodium across the apical plasma membrane.

Animals↗
PubMed1983

Microelectrode study of K+ accumulation by tight epithelia: I. Baseline values of split frog skin and toad urinary bladder.

Toad bladder and split frog skin were impaled with fine-tipped single- and double-barrelled K+-selective microelectrodes. In order to circumvent membrane damage induced by impaling toad bladder, a null point method was developed, involving elevations of mucosal potassium concentration. The results suggest that intracellular potassium activity of short-circuited toad bladder is approximately 82 mM, twice as large as earlier estimates. Far more stable and rigorously defined intracellular measurements were recorded from short-circuited split frog skins. The intracellular positions of the micropipette and microelectrode tips were verified by transient hyperpolarizations of the membrane potential with mucosal amiloride or by transient depolarizations with serosal barium or strophanthidin. Simultaneous impalement of distant cells with separate micropipettes demonstrated that both the baseline membrane potentials and the responses to depolarizing agents were similar, further documenting that frog skin is a functional syncytium. Measurements with double-barrelled microelectrodes and simultaneous single-barrelled microelectrodes and reference micropipettes suggest that the intracellular potassium activity is about 104 mM, lower than previously reported. Taken together with measurements of intracellular potassium concentration, this datum suggests that potassium is uniformly distributed within the epithelial cells.

Animals↗
PubMed1983

Observations of 23Na in frog skin by NMR.

The shift reagent dysprosium tripolyphosphate [Dy(PPPi)7-2] has been applied to the nuclear magnetic resonance (NMR) study of 23Na in frog skin. The anion complex produces slight increases in the transcellular short-circuit current and conductance of the tissue. However, the electrophysiological responsiveness of the tissue to vasopressin, amiloride, and transient removal of extracellular potassium appear unimpaired. A large signal of [31P]phosphocreatine was also observed, further documenting that relatively little damage was produced by the reagent. Three signals of 23Na were observed, reflecting the sodium pools in an external standard solution, the extracellular space, and a medium relatively inaccessible to Dy(PPPi)7-2. The size of the reagent-insensitive signal could be increased by ouabain, a selective inhibitor of sodium extrusion, and could be decreased by amiloride, a selective inhibitor of sodium entry. The results indicate that the anion complex-insensitive signal reflects, at least in part, intracellular sodium and suggest that 23Na may become a useful complementary tool for monitoring the intracellular sodium content of transporting epithelia.

Amiloride↗
PubMed1983

Bioenergetics of Na+ transport across frog skin: chemical and electrical measurements.

Enzymatically prepared split frog skins consisted purely of epithelial cells. Electrical parameters and the cell contents of ATP, ADP, phosphocreatine (PCr), creatine, inorganic phosphate, protein, and water were measured in skins maintained at room temperature. Studies were conducted under base-line conditions, 15 and 60 min after adding vasopressin, and 30 min after adding amiloride. Intracellular ionic activities and concentrations were obtained from previous results. The data demonstrated that 1) the base-line concentration ratio of PCr/ATP was 0.53 +/- 0.03; 2) the average molar free energy of hydrolysis of intracellular ATP was approximately 15.0 kcal X mol-1 under control conditions, changing by less than or equal to 3% with changes in transport; and 3) the free energy of extruding 3 mol of Na+ and accumulating 2 mol of K+ was approximately 9.8 kcal X mol-1 under base-line conditions; the difference between the molar free energies of ATP hydrolysis and of transport work remained large, despite large changes in transepithelial transport. The simplest conclusion is that the Na+ pump of frog skin operates far from equilibrium.

Amiloride↗
PubMed1983

NMR analysis of transporting epithelia.

Three specific examples have been discussed, illustrating how NMR spectroscopy has, is, and will be providing useful complementary information in the study of transepithelial sodium transport. In a brief introduction to principles of NMR, the concepts have first been presented of: nuclear magnetic energy levels, dipolar and quadrupolar interactions, bulk magnetization, Larmor frequency, chemical shift, relative integrated area, longitudinal and transverse relaxations, line width, correlation time, immobilization, compartmentalization, and nuclear exchange. In the three succeeding sections of the review, examples have been provided of how NMR can be used to quantify the degree of intracellular binding of Na+ and K+, relate measurements of the intracellular phosphates to studies of transepithelial Na+ transport, and monitor the content of intracellular Na+. Emphasis has been placed on the value of NMR spectroscopy as a non-invasive probe of nuclides of biological interest. However, note has also been made of the limited sensitivity of the technique and the complexity in analysis posed by cellular and subcellular compartmentalization. Some of these complexities are now being addressed by spin transfer NMR techniques.

Animals↗
PubMed1982

Transepithelial Na+ transport and the intracellular fluids: a computer study.

Computer simulations of tight epithelia under three experimental conditions have been carried out, using the rheogenic nonlinear model of Lew, Ferreira and Moura (Proc. Roy. Soc. London. B 206:53-83, 1979) based largely on the formulation of Koefoed-Johnsen and Ussing (Acta Physiol. Scand. 42: 298-308. 1958). First, analysis of the transition between the short-circuited and open-circuited states has indicated that (i) apical Cl- permeability is a critical parameter requiring experimental definition in order to analyze cell volume regulation, and (ii) contrary to certain experimental reports, intracellular Na+ concentration (ccNa) is expected to be a strong function of transepithelial clamping voltage. Second, analysis of the effects of lowering serosal K+ concentration (csK) indicates that the basic model cannot simulate several well-documented observations; these defects can be overcome, at least qualitatively, by modifying the model to take account of the negative feedback interaction likely to exist between the apical Na+ permeability and ccNa. Third, analysis of the strongly supports the concept that osmotically induced permeability changes in the apical intercellular junctions play a physiological role in conserving the body's stores of NaCl. The analyses also demonstrate that the importance of Na+ entry across the basolateral membrane is strongly dependent upon transepithelial potential, cmNa and csK; under certain conditions, net Na+ entry could be appreciably greater across the basolateral than across the apical membrane.

Animals↗
PubMed1982

31P nuclear magnetic resonance analysis of frog skin.

The intracellular phosphate composition of whole and split frog skins has been studied by 31P nuclear magnetic resonance (NMR) analysis. The spectra were similar to those previously recorded from isolated epithelial cells of toad bladder. However, qualitative differences were noted in comparison with spectra from whole toad bladder. The 31P spectra from whole frog skin reflect the intracellular compositions of the epithelial cells, whereas subepithelial elements contribute significantly to the total observed 31P signals from toad bladder. Analyzed at 4 degrees C, the average phosphocreatine (PCr) and ATP concentrations of frog skin are of similar magnitude. The ratio of [PCr] to [ATP + ADP] depends on time, tissue oxygen tension, temperature, and extracellular inorganic phosphate concentration. Both this ratio and the short-circuit current (measured in parallel experiments) fell during the course of aerating frog skins in Ringer solution at room temperature. The intracellular inorganic phosphate (Pi) signal was identified. After reduction of extracellular pH, the signal did not shift immediately but subsequently did undergo an acid shift.

Amiloride↗
PubMed1980

Electrophysiologic changes associated with potassium depletion of frog skin.

Skins from the frog Rana pipiens pipiens were studied under short-circuited conditions during the course of removing and replacing potassium in the inner bathing media in 14 experiments. The intracellular potential (Vsc), fractional resistance (FR), short-circuit current (Isc) and total tissue conductance (gr) were constantly monitored during impalements of the epithelial cells. The mean value (+/- SE) for Vsc was --79 (+/- 3) mV under baseline conditions. Removal of potassium from the inner bathing solution transiently stimulated the short-circuit current and hyperpolarized the basolateral membrane; with sufficiently long incubations, the basolateral membrane was eventually depolarized. Restoration of potassium to the inner solution within 43 min after initiating the perfusion with K+-free solution depolarized the basolateral membrane. However, restoration of potassium after at least 1 1/2 hr of incubation hyperpolarized the membrane. Ouabain consistently depolarized the basolateral membrane, even after extended periods of potassium depletion as long as 320 min. In the presence of ouabain, restoration of potassium depolarized the basolateral membrane. The data provide further evidence for the concept that the Na--K exchange pump of frog skin is rheogenic. Furthermore, the results suggest that the pump continues to be active even during prolonged periods of potassium depletion, reaccumulating potassium which has leaked out of the epithelial cells.

Animals↗
PubMed1980

Microprobe study of toad urinary bladder in absence of serosal K+.

The bulk of the intracellular potassium in mucosal epithelial cells from toad urinary bladder has been previously reported to exchange very slowly with the serosal medium, with a half-time of some 9 hr. This observation, based on chemical analyses of mucosal cell scrapings, has been reexamined with stimultaneous diffractive and energy dispersive electron probe X-ray microanalysis. Fifty-three intracellular sites in hydrated sections and 286 sites in dehydrated sections were studied in bladders from eight toads under baseline conditions and after removal of serosal K+ for 83-133 min, with or without 10(-2) M ouabain. The baseline data confirm and extend previous examinations of the intracellular ionic composition, and provide the most direct measure of intracellular water thus far available for this tissue. Removal of serosal K+ reduced the intracellular K+ content by 20%, increased intracellular Na+ content threefold, and slightly reduced the intracellular Cl- and water contents, qualitatively consistent with published chemical analyses. The intracellular Na+ content of mucosal origin, measured by radioactive tracers and chemical analyses of cell scrapings, has been reported to be unchanged under these conditions. Simultaneous addition of ouabain and removal of external K+ produced a dramatic fall in intracellular K+ of more than 80% in a third of the cells and reduced the mean intracellular K+ content by 60%; 20% of the cells appeared to retain K+ more effectively than the bulk of the epithelial cell population. We conclude that: (i) the low rate of net exchange of intracellular K+ with the serosal bulk solution primarily reflects recycling of K+ across the basolateral membranes, (ii) radioactive tracer and chemical measurements of the intracellular Na+ pool of mucosal origin substantially understimate the total intracellular Na+ content under certain experimental conditions, and (iii) the epithelial cells display a functional heterogeneity of response to the effects of adding ouabain and withdrawing external K+.

Animals↗
PubMed1980

Relationship of transient electrical properties to active sodium transport by toad urinary bladder.

Application of voltage pulses of 10 mV for periods of 9 sec across toad urinary bladder elicits a rapid deflection in transepithelial current. Frequently, the current decays back towards its baseline value during the course of the polarizing pulse. This transient phenomenon can be induced, or its magnitude increased, by raising the mucosal or serosal Na+ concentration. The transient can be abolished by sufficiently hyperpolarizing the tissue (rendering serosa positive to mucosa), by inhibiting transcellular Na+ transport with amiloride or ouabain, and by increasing the serosal K+ concentration. Vasopressin increases net Na+ movement across toad bladder but does not elicit these transients. It is proposed as a working hypothesis for further study that the transient behavior characterized in this study reflects: (1) the partition of Na+ between the apical plasma membrane and contiguous fluid layers, (2) the partition of K+ between the basolateral plasma membrane and adjacent submucosal fluid layer, and (3) the negative feedback interaction between intracellular Na+ activity and Na+ permeability of the apical plasma membrane of the transporting cells.

Amiloride↗