Biomedical subjects

M M Civan

Publications and source records attributed to M M Civan.

At least 19 recordsLinked to original sources

PubMed1992

Regulatory volume decrease by cultured non-pigmented ciliary epithelial cells.

Cells (ODM C1-2/SV40) derived from human non-pigmented ciliary epithelial cells were studied by electronic cell sizing. The time course of the cell volume (vc) was monitored after suspending cells in paired experimental and control, isosmotic and hyposmotic solutions of identical ionic composition. Following anisosmotic cell swelling, the cells displayed the regulatory volume decrease (RVD) previously described. The RVD primarily reflects loss of cell KCl since: (1) the K(+)-channel blockers quinidine and Ba2+ both inhibit the RVD; and (2) replacement of external Cl- with gluconate or addition of the Cl- channel blocker NPPB also inhibits the RVD. Bicarbonate has previously been reported to speed the RVD. This action likely reflects pH dependence of the channels since: (1) increasing the external pH speeds the RVD, whether or not HCO3- is present; and (2) DIDS (a blocker of Cl- channels and of Cl-/HCO3- exchange) is an effective inhibitor of the RVD, even after blocking Cl-/HCO3- exchange by removing external HCO3-. The RVD could also be inhibited by reducing the availability of Ca2+, either by omitting Ca2+ from the external medium or by blocking mobilization of intracellular Ca2+ with TMB-8. Furthermore, the RVD was slowed and incomplete in the presence of the calcium/calmodulin blocker trifluoperazine. We conclude that anisosmotic swelling triggers a series of events, mediated at least in part by calcium/calmodulin, leading to the extrusion of KCl through parallel K+ and Cl- channels.

Bicarbonates↗

PubMed1992

Whole cell patch clamping of ciliary epithelial cells during anisosmotic swelling.

Anisosmotic cell swelling triggers a regulatory volume decrease (RVD) in cell lines derived from human nonpigmented ciliary epithelium. Measurements of cell volume have indicated that the RVD reflects activation of K+ and/or Cl- channels. We have begun to characterize the putative channels by whole cell patch clamping. The results obtained by altering the external K+ and Cl- concentrations and by adding 20-50 microM quinidine or 1 mM Ba2+ indicate that K+ conductances contribute substantially and Cl- conductances contribute very little to the total membrane conductance (GT) under baseline isotonic conditions. Reducing the external osmolality by 20-50% reversibly and reproducibly increased GT by an order of magnitude. Data obtained from ion substitutions and the channel blockers quinidine and 5-nitro-2-(3-phenylpropylamino)-benzoate indicate that most of the hypotonicity-induced conductance reflects stationary Cl(-)-channel activity. The contribution of new K(+)-channel activity was small at intracellular free Ca2+ concentrations of 10 or 200 nM. We conclude that the RVD triggered by bath hypotonicity primarily reflects increased Cl(-)-channel activity.

Chlorides↗

PubMed1992

Effect of bicarbonate on intracellular potential of rabbit ciliary epithelium.

Extracellular HCO3- hyperpolarizes the intracellular potential and makes the aqueous medium negative with respect to the stromal surface of the rabbit ciliary epithelial syncytium. The bases for these observations have been unclear. We have been studying the bicarbonate-induced hyperpolarization (BIH) with sustained intracellular recordings for periods as long as 1-2 hrs. The BIH was observed [6.0 +/- 0.4 mV (mean +/- SE, N = 22)] even when the external pH was clamped constant by appropriately changing the CO2 tension. External HCO3- was required since aeration with CO2 at low external pH did not replicate the BIH. DIDS [4,4'-diisothiocyano-2,2'-disulfonic acid] did not abolish the effect. The hyperpolarization is unlikely to reflect the pH dependence of K+ channels alone, since the effect was not reduced by either 2 mM Ba2+ alone or 2 mM Ba2+ together with 50-100 microM quinidine. The BIH depends directly or indirectly on external Na+, since the sign of the polarization response was reversed either by replacing Na+ with N-methyl-D-glucamine or by blocking the Na+,K(+)-exchange pump with 50-100 microM ouabain. Replacement of external Cl- with NO3- or application of the Cl(-)channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate] depolarized the membrane and reversed the sign of the BIH. The response of the ciliary epithelium to HCO3- is complex and may arise from several mechanisms. We suggest that one important element is an anion channel whose conductance is reduced by bicarbonate and whose reversal potential is indirectly dependent on the operations of the Na+,K(+)-pump and a Cl(-)-linked symport.

Animals↗

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

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

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↗

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↗

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

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↗