Biomedical subjects

M M Civan

Publications and source records attributed to M M Civan.

At least 55 records · Page 3Linked to original sources

PubMed1991

Voltage dependence of current through the Na,K-exchange pump of Rana oocytes.

We have studied current (IStr) through the Na,K pump in amphibian oocytes under conditions designed to minimize parallel undesired currents. Specifically, IStr was measured as the strophanthidin-sensitive current in the presence of Ba2+, Cd2+ and gluconate (in place of external Cl-). In addition, IStr was studied only after the difference currents from successive applications and washouts of strophanthidin (Str) were reproducible. The dose-response relationship to Str in four oocytes displayed a mean K0.5 of 0.4 microM, with 2-5 microM producing 84-93% pump block. From baseline data with 12 Na(+)-preloaded oocytes, voltage clamped in the range [-170, +50 mV] with and without 2-5 microM Str, the average IStr depended directly on Vm up to a plateau at 0 mV with interpolated zero current at -165 mV. In three oocytes, lowering the external [Na+] markedly decreased the voltage sensitivity of Ip, while producing only a small change in the maximal outward IStr. In contrast, decreasing the external [K+] from 25 to 2.5 mM reduced IStr at 0 mV without substantially affecting its voltage dependence. At K+ concentrations of less than 1 mM, both the absolute value of IStr at 0 mV and the slope conductance were reduced. In eight oocytes, the activation of the averaged IStr by [K+]0 over the voltage interval [-30, +30 mV] was well fit by the Hill equation, with K' = 1.7 +/- 0.4 mM and nH (the minimum number of K+ binding sites) = 1.7 +/- 0.4. The results unequivocally establish that the cardiotonic-sensitive current of Rana oocytes displays only a positive slope conductance for [K+]0 greater than 1 mM. There is therefore no need to postulate more than one voltage-sensitive step in the cycling of the Na, K pump under physiologic conditions. The effects of varying external Na+ and K+ are consistent with results obtained in other tissues and may reflect an ion-well effect.

Animals↗

PubMed1991

Ca(2+)-independent form of protein kinase C may regulate Na+ transport across frog skin.

Activators of protein kinase C (PKC) stimulate Na+ transport (JNa) across frog skin. We have examined the effect of Ca2+ on PKC stimulation of JNa. Both the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and the diacyl-glycerol sn-1,2-dioctanoylglycerol (DiC8) were used as PKC activators. Blocking Ca2+ entry into the cytosol (either from external or internal stores) reduced the subsequent natriferic effect of the PKC activators. This negative interaction did not simply reflect saturation of activation of the apical Na+ channels, since the stimulations produced by blocking Ca2+ entry and adding cyclic AMP were simply additive. The Ca2+ dependence of the natriferic effect could have reflected either a direct action of cytosolic Ca2+ on PKC or an indirect action on the final receptor site (the Na+ channel). To distinguish between these possibilities, the TPA- and phospholipid-dependent kinase activity of broken-cell preparations was assayed. The kinase activity was not stimulated by physiological levels of Ca2+, and in fact was inhibited at millimolar concentrations of Ca2+. We conclude that the effects of Ca2+ on the natriferic response to PKC activators are indirect. Reducing cytosolic uptake of Ca2+ may have stimulated Na+ transport by a chemical modification of the apical channels observed in other tight epithelia. The usual stimulation of Na+ transport produced by PKC activators in frog skin may reflect the operation of a nonconventional form of PKC. This enzyme is Ca2+ independent and seems related to the nPKC or PKC epsilon observed in other systems.

Amiloride↗

PubMed1991

Effects of adrenergic agents on transepithelial electrical measurements across the isolated iris-ciliary body.

Transmembrane electrical measurements were performed on the isolated rabbit iris-ciliary body to study direct effects of adrenergic drugs on the ciliary epithelium. Alpha-adrenergic agonists (epinephrine, norepinephrine, or phenylephrine) lowered the short-circuit current (SCC) in a dose-dependent fashion relative to which chamber side the drug was added: simultaneous addition to both chambers greater than blood side only greater than aqueous side only. Pretreatment (5 x 10(-5) M) with the non-selective beta-adrenergic antagonist timolol had no effect while the non-selective alpha-adrenergic antagonist, phentolamine, completely prevented the alpha agonist-induced decrease in SCC. The alpha-adrenergic response was mediated by the alpha 1 subtype since prazosin, but not yohimbine, blocked the induced reduction in SCC. The beta-adrenergic agonist isoproterenol caused a dose-dependent decrease in the SCC. The decrease was similar when the drug was added to only the blood side or to both sides of the chamber. Addition to only the aqueous chamber had no effect. Pretreatment with beta-adrenergic antagonists blocked the isoproterenol response: non-selective = selective beta 2 greater than selective beta 1. The isoproterenol-induced decrease in SCC was also blocked by non-selective alpha-adrenergic antagonists. The response was mediated by the alpha 1 subtype since prazosin, but not yohimbine, blocked the isoproterenol response. This suggests that isoproterenol interacted with the alpha 1-adrenergic sensitive pathway in the rabbit ciliary process.

Animals↗

PubMed1991

Halogenated inhalation anesthetic agents decrease transepithelial electrical measurements across the isolated iris-ciliary body.

Transmembrane electrical measurements were performed on the isolated rabbit iris-ciliary body (I-CB) to study the direct effects of halogenated inhalation anesthetic agents on the ciliary epithelium. Addition of either halothane, enflurane, or isoflurane to the control 95% O2:5% CO2 gas mixture resulted in a dose-dependent decrease in the short-circuit current (SCC) and potential difference (PD). This response was reversible after the anesthetic gas was discontinued. Pretreatment with either alpha-adrenergic or beta-adrenergic antagonists (phentolamine or timolol) had no effect on the halothane-induced decrease in SCC. Delivery of the analgestic gas N2O did not alter baseline electrical measurements across the isolated I-CB.

Analysis of Variance↗

PubMed1990

Toad urinary bladder as a model for studying transepithelial sodium transport.

Sodium ion transport across tight epithelia has been investigated particularly extensively by studying two model systems: the urinary bladder of the toad and the frog skin. The greatest advantage presented by these models is the capability of monitoring net transepithelial Na+ flux simply, precisely, and instantaneously by measurement of the short circuit current (ISC). Many of the caveats involved in the measurement are discussed in detail. In order to fully characterize the forces driving Na+ movement across the series apical and basolateral membranes, it is necessary to measure intracellular potential and ionic composition. Such measurements are far more easily conducted with frog skin than with toad bladder, using the major biophysical techniques currently available. Regulation of transepithelial Na+ movement across tight epithelia is largely conducted at the apical membranes. This regulation can be clarified by study of the isolated Na+ channels in membrane vesicles. Such vesicles are far more easily prepared from toad urinary bladder than from frog skin. The strengths and potential misappropriations of this technique are considered in detail.

Animals↗

PubMed1989

Volume regulation of cultured, transformed, non-pigmented epithelial cells from human ciliary body.

Electronic cell sizing has been used to measure the volume of cells suspended in isosmotic and in hyposmotic solutions of identical ionic composition. Without inhibitors, the cells displayed a regulatory volume decrease (RVD) following anisosmotic cell swelling with a time constant (tau) of 6.3 +/- 0.9 min (mean +/- S.E.). The RVD was markedly impaired by substituting gluconate for external Cl-, and tau was prolonged by: (i) reducing the chemical gradient favoring K+ loss (by elevating the external [K+] and blocking the Na. K-exchange pump), (ii) blocking the K+ channels with Ba2+, (iii) blocking Cl- channels and Cl-/HCO3 = exchange with DIDS, and (iv) removing external HCO3-. Withdrawing HCO3- may have altered the RVD either directly by inhibiting a Cl-/HCO3- antiport, or indirectly by affecting intracellular pH. The regulatory volume response of ODM Cl-2/SV40 cells is in several respects qualitatively similar to that of non-pigmented epithelial cells of the intact ciliary body. These common characteristics suggest that the cultured cells can serve as a useful model for studying solute and fluid transport across the human ciliary epithelium. The basis for the RVD is likely to be activation of separate K+ and Cl- channels, with or without the parallel operation of coupled K+/H+ and Cl-/HCO3- antiports.

Cell Line, Transformed↗

PubMed1989

Interactions of TPA and insulin on Na+ transport across frog skin.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) activates protein kinase C (PKC) and produces an early stimulation of Na+ transport across frog skin. The ionic basis for this stimulation was studied with combined transepithelial and intracellular electrical measurements. In an initial series of experiments, TPA approximately doubled the amiloride-sensitive short-circuit current (ISC), apical Na+ permeability (PapNa), and apical membrane conductance without affecting the basolateral membrane conductance. The apical effects led to a marked depolarization of the short-circuited skin and a small increase in intracellular Na+ concentration. TPAs increase of PapNa was sufficient to explain the stimulation of basolateral Na+ transport when both the voltage and substrate dependence of the pump were taken into account. After the early stimulation, TPA later depressed ISC. Added at this point (congruent to 1-2 h after TPA administration), insulin had no effect on ISC, whereas a partial response to vasopressin was still observed. Measured either early or late after TPA addition, the phorbol ester reduced insulin binding by congruent to 40%. Insofar as 60% of the specific binding is retained, the abolishment of insulin's natriferic response is unlikely to result from the TPA-induced reduction in hormonal binding. The data provide further support for the concept that activation of PKC produces an early stimulation of Na+ transport by increasing apical Na+ permeability, and that part of insulin's natriferic effect may be mediated by PKC activation.

Amiloride↗

PubMed1989

Coupling of volume and Na+ transport in frog skin epithelium.

Whole skins and isolated epithelia were bathed with isotonic media (congruent to 244 mOsm) containing sucrose or glucose. The serosal osmolality was intermittently reduced (congruent to 137 mOsm) by removing the nonelectrolyte. Transepithelial and intracellular electrophysiological parameters were monitored while serosal osmolality was changed. Serosal hypotonicity increased the short-circuit current (ISC) and the basolateral conductance, hyperpolarized the apical membrane (psi mc), and increased the intracellular Na+ concentration. The increases in apical conductance and apical Na+ permeability (measured from Goldman fits of the relationship between amiloride-sensitive current and psi mc) were not statistically significant. To verify that the osmotically induced changes in ISC were mediated primarily at the basolateral membrane, the basolateral membrane potential of the experimental area was clamped close to 0 mV by replacing the serosal Na+ with K+ in Cl--free media. The adjoining control area was exposed to serosal Na+. Serosal hypotonicity produced a sustained stimulation of ISC across the control, but not across the adjoining depolarized tissue area. The current results support the concept that hypotonic cell swelling increases Na+ transport across frog skin epithelium by increasing the basolateral K+ permeability, hyperpolarizing the apical membrane, and increasing the electrical driving force for apical Na+ entry.

Amiloride↗

PubMed1988

Apical Na+ permeability of frog skin during serosal Cl- replacement.

Gluconate substitution for serosal Cl- reduces the transepithelial short-circuit current (Isc) and depolarizes short-circuited frog skins. These effects could result either from inhibition of basolateral K+ conductance, or from two actions to inhibit both apical Na+ permeability (PapNa) and basolateral pump activity. We have addressed this question by studying whole-and split-thickness frog skins. Intracellular Na+ concentration (CcNa) and PapNa have been monitored by measuring the current-voltage relationship for apical Na+ entry. This analysis was conducted by applying trains of voltage pulses, with pulse durations of 16 to 32 msec. Estimates of PapNa and CcNa were not detectably dependent on pulse duration over the range 16 to 80 msec. Serosal Cl- replacement uniformly depolarized short-circuited tissues. The depolarization was associated with inhibition of Isc across each split skin, but only occasionally across the whole-thickness preparations. This difference may reflect the better ionic exchange between the bulk medium and the extracellular fluid in contact with the basolateral membranes, following removal of the underlying dermis in the split-skin preparations. PapNa was either unchanged or increased, and CcNa either unchanged or reduced after the anionic replacement. These data are incompatible with the concept that serosal Cl- replacement inhibits PapNa and Na,K-pump activity. Gluconate substitution likely reduces cell volume, triggering inhibition of the basolateral K+ channels, consistent with the data and conclusions of S.A. Lewis, A.G. Butt, M.J. Bowler, J.P. Leader and A.D.C. Macknight (J. Membrane Biol. 83:119-137, 1985) for toad bladder. The resulting depolarization reduces the electrical force favoring apical Na+ entry. The volume-conductance coupling serves to conserve volume by reducing K+ solute loss. Its molecular basis remains to be identified.

Animals↗

PubMed1988

Insulin and phorbol ester stimulate conductive Na+ transport through a common pathway.

Insulin stimulates Na+ transport across frog skin, toad urinary bladder, and the distal renal nephron. This stimulation reflects an increase in apical membrane Na+ permeability and a stimulation of the basolateral membrane Na,K-exchange pump. Considerable indirect evidence has suggested that the apical natriferic effect of insulin is mediated by activation of protein kinase C. However, no direct information has been available documenting that insulin and protein kinase C indeed share a common pathway in stimulating Na+ transport across frog skin. In the present work, we have studied the interaction of insulin and phorbol 12-myristate 13-acetate (PMA), a documented activator of protein kinase C. Preincubation of skins with 1,2-dioctanoylglycerol, another activator of protein kinase C, increases baseline Na+ transport and reduces the subsequent natriferic response to PMA. Preincubation with PMA markedly reduces the subsequent natriferic action of insulin. This effect does not appear to primarily reflect PMA-induced internalization of insulin receptors. The insulin receptors are localized on the basolateral surface of frog skin, but the application of PMA to this surface is much less effective than mucosal treatment in reducing the response to insulin. Preincubation with D-sphingosine, an inhibitor of protein kinase C, also reduces the natriferic action of insulin. The current results provide documentation that insulin and protein kinase C share a common pathway in stimulating Na+ transport across frog skin. The data are consistent with the concept that the natriferic effect of insulin on frog skin is, at least in part, mediated by activation of protein kinase C.

Animals↗

PubMed1988

Intracellular pH in frog skin: effects of Na+, volume, and cAMP.

Single skins were analyzed by 31P-nuclear magnetic resonance (NMR) spectroscopy during alternate perfusion with control and experimental solutions. Intracellular (pHc) and extracellular (pHo) pH were monitored by measuring the spectral frequencies of intracellular Pi and external methylphosphonate, respectively. Base-line pHc was 7.20 +/- 0.02 (SE) when pHo was 6.99 +/- 0.02. A 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-inhibitable, HCO3--dependent alkaline shift in pHc can be elicited by replacing external Cl- by gluconate or sulfate. We now report that this effect is observed even in sodium-free media. The substitution of gluconate for external Cl- has also been reported to shrink cell volume. This shrinkage can be minimized by replacing Cl- with gluconate during perfusion with hypotonic, rather than isotonic, media. Conducted in this manner, the anionic substitution produces a much smaller alkaline shift in pHc. Replacement of external NaCl with N-methyl-D-glucamine chloride acidified the cells reversibly by 0.22 +/- 0.02. In the presence of the Na-H antiport blocker 5-(N-methyl-N-isobutyl)amiloride (MIA), restoration of external Na+ did not increase pHc. Separate addition of MIA acidified the cells by 0.18 +/- 0.03. Adenosine 3',5'-cyclic monophosphate (cAMP) also alters pHc. Addition of 1 mM 8(4-chlorophenylthio)cAMP or 100 mU/ml vasopressin acidified the cells by 0.22 +/- 0.03 and by 0.14 +/- 0.04, respectively. The data suggest that frog skin regulates pHc by the parallel operation of Na-H and Na+-independent Cl-HCO3 antiports. Cell volume and cAMP may play regulating roles in this epithelium.

Algorithms↗

PubMed1987

Diacylglycerols stimulate short-circuit current across frog skin by increasing apical Na+ permeability.

The phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) stimulates baseline Na+ transport across frog skin epithelium and partially inhibits the natriferic response to vasopressin. The effects are produced largely or solely when TPA is added to the mucosal surface of the tissue. Although TPA activates protein kinase C, it has other effects, as well. Thus, the biochemical basis for the effects and the ionic events involved have been unclear. Furthermore, the physiologic implications have been obscure because of the sidedness of TPA's actions. We now report that two synthetic diacylglycerols (DAG) replicate the stimulatory and inhibitory effects of TPA on frog skin. DAG is the physiologic activator of PKC. In this tissue, it produces half-maximal stimulation at a concentration of less than or equal to 19 microM. In contrast to TPA, DAG is about equally effective from either tissue surface. In a series of eight experiments, DAG was found to depolarize the apical membrane. Diacylglycerol also increases the paracellular conductance of frog skins bathed with mucosal Cl- Ringer's solution. The latter effect can be minimized by replacing NO3- for Cl- in the mucosal solution. Under these conditions, combined intracellular and transepithelial measurements indicated that DAG increased both the apical Na+ permeability and intracellular Na+ concentration. These results are qualitatively similar to the effects of cyclic 3',5'-AMP on this tissue, suggesting that activation of PKC by DAG causes phosphorylation of the same or nearby gating sites phosphorylated by cAMP. We propose that apical Na+ entry is regulated in part by activation of PKC, and that insulin may be a physiologic trigger of this activation.

Animals↗

PubMed1987

Ba2+-inhibitable 86Rb+ fluxes across membranes of vesicles from toad urinary bladder.

86Rb+ fluxes have been measured in suspensions of vesicles prepared from the epithelium of toad urinary bladder. A readily measurable barium-sensitive, ouabain-insensitive component has been identified; the concentration of external Ba2+ required for half-maximal inhibition was 0.6 mM. The effects of externally added cations on 86Rb+ influx and efflux have established that this pathway is conductive, with a selectivity for K+, Rb+ and Cs+ over Na+ and Li+. The Rb+ uptake is inversely dependent on external pH, but not significantly affected by internal Ca2+ or external amiloride, quinine, quinidine or lidocaine. It is likely, albeit not yet certain, that the conductive Rb+ pathway is incorporated in basolateral vesicles oriented right-side-out. It is also not yet clear whether this pathway comprises the principle basolateral K+ channel in vivo, and that its properties have been unchanged during the preparative procedures. Subject to these caveats, the data suggest that the inhibition by quinidine of Na+ transport across toad bladder does not arise primarily from membrane depolarization produced by a direct blockage of the basolateral channels. It now seems more likely that the quinidine-induced elevation of intracellular Ca2+ activity directly blocks apical Na+ entry.

Animals↗

PubMed1987

Effects of TPA on short-circuit current across frog skin.

TPA (12-O-tetradecanoylphorbol-13-acetate) is an effective tumor promoter that affects a variety of ion transport processes. To examine the relationship between effects on transport and growth and differentiation, we have been studying the actions of TPA on frog skin, a particularly well-characterized epithelium. We have reported that high concentrations of TPA stimulate base-line short-circuit current (ISC) and inhibit the subsequent natriferic action of vasopressin. The current study of 89 preparations extends those findings. The Km of the stimulatory effect of TPA is approximately 3 nM; this high affinity indicates that the transport phenomenon does not simply reflect a nonspecific interaction of phorbol ester with the plasma membranes. TPA acts largely or entirely at the mucosal surface of both split and whole skins; thus the sidedness of the effect does not arise from adsorption onto the underlying connective tissue when TPA is applied to the serosal surface of whole skin. Amiloride, an inhibitor of apical Na+ entry, abolishes ISC across frog skins pretreated with TPA. The phorbol ester also increases ISC across split skins, preparations which do not produce net Cl-transport. Indomethacin (1 microM) blocks PGE1 release, but does not alter the response to TPA at a fivefold lower concentration than previously used. NDGA (nordihydroguaretic acid, 10 microM), an inhibitor of the lipoxygenase pathway, partially inhibited the responses of ISC to 8 nM TPA. The present results indicate that frog skin is highly responsive to TPA at concentrations known to activate protein kinase C in broken-cell preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

31P NMR analysis of intracellular pH of Swiss Mouse 3T3 cells: effects of extracellular Na+ and K+ and mitogenic stimulation.

Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional 31P and 19F probes of intracellular pH (pHc) were found to be impracticable. Cells were therefore superfused with 1 to 4 mM 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na+ with choline or K+ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pHc on external Na+ concentration (CoNa). PHc also depended on intracellular Na+ concentration (CcNa). Increasing ccNa by withdrawing external K+ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducing CoNa produced a larger acid shift in pHc than with external K+ present. Comparison of separate preparations indicated that pHc was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pHc of Swiss mouse 3T3 cells using 31P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event.

Animals↗

PubMed1986

Microelectrode study of intracellular pH in frog skin: dependence on serosal chloride.

Replacement of external chloride has been known to reduce Na+ transport across whole frog skin. However, the sidedness and mechanism of the phenomenon have been unclear. In the present study, transepithelial current (IT), transepithelial resistance (RT), and basolateral membrane potential measured both with reference micropipettes (psi sc) and pH-selective microelectrodes (EscH) were monitored in isolated epithelial sheets from frog skin; removal of the underlying dermis facilitates ionic exchange across the basolateral membranes. The intracellular hydronium ion activity (acH) was 58 +/- 4 nM (means +/- SE) when the extracellular hydronium activity was 25 +/- 1 nM under base-line conditions. This measurement is equivalent to an intracellular pH (pHc) of 7.24 +/- 0.03 at an extracellular pH of 7.60 +/- 0.01, in reasonable agreement with estimates obtained by 31P- and 19F-nuclear magnetic resonance (NMR) analyses of frog skin. Complete replacement of mucosal Cl- by gluconate had variable effects on tissue current and resistance from preparation to preparation. The same ionic substitution on the serosal side uniformly produced a prompt reversible decrease in IT, increase in RT, and a substantial membrane depolarization of the short-circuited skins. In most of the preparations, the depolarization was preceded by a small hyperpolarization of 0.5-3.5 mV. The replacement of serosal Cl- also produced a fall in intracellular hydronium ion activity of 33 +/- 10 nM. The present date are consistent with the concept that serosal replacement of Cl- alkalinizes the cells by either favoring HCO3- entry or blocking HCO3- exit through a Cl- HCO3 antiport at the basolateral membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Intracellular pH regulation in frog skin: a 31P-nuclear magnetic resonance study.

The 31P-nuclear magnetic resonance spectra of single frog skins were monitored during perfusion with reference and experimental solutions, permitting each tissue to serve as its own series control. Over an external pH (pHo) range of approximately 7.25-7.64 U, the intracellular pH (pHc) displayed both homeostasis and response. External alkalinization produced a transient intracellular regulation, but pHc returned to its base-line value even in the absence of a further change in pHo. Below this range, pHc was linearly and strongly dependent on pHo with a slope close to 1, when the external pH was varied by addition of nonvolatile base. Measurements were generally performed with tissues bathed in Ringer solutions containing 2.5 mM HCO3- and bubbled with 99% O2-1% CO2. Replacement of external Cl- by gluconate reversibly increased pHc by 0.34 +/- 0.05 U. Substitution of SO4(2-) for Cl- also alkalinized the cells; pHc was increased by 0.16 +/- 0.01 U in the presence of the standard concentration of external HCO3-, but an alkaline shift was only just detectable (0.05 +/- 0.02 U) in its nominal absence. The application of 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid blocked the sustained intracellular alkalinization characteristically produced by external Cl- replacement but did not alter the reduction in short-circuit current and increase in transepithelial resistance caused by gluconate substitution.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1985

Intracellular calcium activity in split frog skin epithelium: effect of cAMP.

Measurement of intracellular calcium activity (acCa) by ion-selective microelectrodes has previously been technically limited to relatively large cells (greater than or equal to 20 micron). We now report results obtained with this technique in the small epithelial cells (less than or equal to 10 micron) of split frog skin using microelectrodes having an outer tip diameter of less than 0.2 micron. The basolateral membrane potential was measured with Ca2+-selective microelectrodes (EscCa) and with reference micropipettes (psi sc) either sequentially or simultaneously in 15 successful experiments. Under baseline conditions, acCa was measured to be 215 +/- 39 nM (mean +/- SE), in close agreement with the mean values estimated from published data obtained with Necturus proximal tubule. Stimulation of Na+ transport across six skins with 1 mM serosal 8 p-chlorophenylthio-3,5' cyclic AMP (CPTcAMP) increased acCa by a factor of 2.6 +/- 0.6. The increase in acCa preceded the CPTcAMP-induced increase in Isc. The results of the present study indicate that electrometric determination of intracellular calcium activity is now feasible in a much wider range of cell systems than heretofore possible. CPT cAMP elevates intracellular Ca2+ activity; this phenomenon is an early event, preceding the natriferic effect of CPTcAMP.

Animals↗