Cyclic AMP‐and beta‐agonist‐activated chloride conductance of a toad skin epithelium.

Abstract

SUMMARY1. The control by intracellular cyclic AMP and ,-adrenergic stimulation of chloride conductance was studied in toad skin epithelium mounted in a chamber on the stage of an upright microscope. Impalement of identified principal cells from the serosal side with single-barrelled conventional or double-barrelled Cl--sensitive microelectrodes was performed at x 500 magnification. For blocking the active sodium current 50 ftM-amiloride was present in the mucosal bath.2. When clamped at transepithelial potential difference V = 0 mV, the preparations generated clamping currents of 0 9 +1 tA/cm2 (mean + S.E.M.; number of observations n = 55). The intracellular potential of principal cells (Vb) was -96+2 mV with a fractional resistance of the basolateral membrane (fRb) of 0-016 + 0-003 (n = 54), and an intracellular Cl-activity of 40 + 2 mm (n = 24).3. At V = 0 mV, serosal application of a cyclic AMP analogue, dibutyryl cyclic AMP (500 /iM) or a ,-adrenergic agonist, isoprenaline (5 /tM) resulted in a sixfold increase in transepithelial Cl-conductance identified by standard 36Cl-tracer technique.4. The clamping current at V = 0 mV was unaffected by cyclic AMP (short-circuit current ISC = 0 ±+0-3 jtA/cm2, n = 16) indicating that subepidermal Cl--secreting glands are not functioning in our preparations obtained by collagenase treatment. 5. Cyclic AMP-or isoprenaline-induced chloride conductance (GC1) activation (V = 0 mV) was not reflected in membrane potential and intracellular Cl-activity in principal cells. Intracellular chloride activity was constant at ; 40 mm at membrane potentials between -90 and -100 mV. Therefore, it can be concluded that the principal cells are not contributing to activated Cl-currents.6. At V = -100 mV where the voltage-dependent chloride conductance of mitochondria-rich (MR) cells was already fully activated, GC1 was unaffected by cyclic AMP or isoprenaline. The major effect of these treatments was a rightward displacement of the MR cell-generated Gc1-V relationship along the V axis.7. Our results indicate that the fl-adrenergically controlled cyclic AMP-mediated chloride conductance is localized to the mitochondria-rich cells.MS 9518 642 N. J. WILLUMSEN, L. VESTERGAARD AND E. H. LARSEN

α 1 -Adrenoceptors antagonize activated chloride conductance of amphibian skin epithelium

“…No effect is observed with agonists of α 2 -receptors. These effects are fundamentally different from those that are observed following β-adrenoceptor stimulation, which increases the baseline level of G Cl through elevation of cellular cAMP, and may result both from stimulation of glandular Cl -secretion [23] and activation of baseline current in the gland-free epithelium [27]. The mechanism of the voltage-activated G Cl appears to be more complex than the sole gating of an anion channel.…”

“…A special property of the Cl -pathway is the development of a large transepithelial current, which is due to the activation of a conductive pathway in response to serosa-positive electrical potentials and is carried by the flow of Cl -from the mucosal to the serosal side [13]. Since G Cl can also be activated after inhibition of phosphodiesterase by theophylline [10], β-adrenergic agonists and membrane-permeable analogues of cAMP [27], it has been proposed that cAMP regulates the conductive Cl -pathway by virtue of a shift in the voltage sensitivity of the activation. However, a subsequent study has shown that cAMP alters the properties of the Cl -pathway far more fundamentally [9].…”

Transepithelial chloride conductance in amphibian skin: regulatory mechanisms and localization

“…The view of control by cAMP was originally based on the observation that activation of G Cl could be enhanced by theophylline, an inhibitor of PDE, in frog skin [35] or restored in the toad skin in which conductance had been adaptationally reduced [24]. This was corroborated by the finding that application of the β-adrenergic agonist, isoproterenol, or membrane-permeable analogues of cAMP appear to shift the conductance/voltage relationship of apical Cl -channels towards lower voltages [89]. Subsequent studies [26] indicate that this effect, which looks like a shift, actually reflects a fundamental change of the kinetics and selectivity patterns of the anion pathway in response to high concentrations of cAMP.…”

“…Whilst earlier studies suggested that cAMP is a decisive messenger at the apical Cl -channels of MRC [39,89], it now appears that the role of cAMP is considerably more complex. The view of control by cAMP was originally based on the observation that activation of G Cl could be enhanced by theophylline, an inhibitor of PDE, in frog skin [35] or restored in the toad skin in which conductance had been adaptationally reduced [24].…”

“…The voltage-activated G Cl can be enhanced by theophylline; this response has been associated with an increase in cellular cAMP due to the inhibition of phosphodiesterases (PDE) [28]. Experiments with a cellpermeating cAMP analogue (N-2-O-dibutyryl-adenosine 3′,5′-cyclic monophosphate, db-cAMP) and β-adrenergic agonists suggest that an increase in cellular cAMP shifts the conductance/voltage relationship to lower voltages [89]. G Cl can be activated similarly by transepithelial voltage perturbation or by exposure to solutions with elevated serosal K + concentration [38].…”