Does stimulation of NaCl secretion in in vitro perfused rectal gland tubules of Squalus acanthias increase membrane capacitance?

Abstract

NaCl secretion in rectal gland tubules (RGT) of Squalus acanthias requires the activation of Cl– channels in the luminal membrane. The RGT and its mechanism of activation are an early evolutionary paradigm of exocrine secretion. The respective Cl– channels probably resemble the shark equivalent of the cystic fibrosis transmembrane conductance regulator (CFTR). Activation of these Cl– channels occurs via cAMP. It has been hypothesized that the activation of CFTR occurs via exocytosis or inhibited endocytosis. To examine this question directly by electrical measurements we have performed whole-cell patch-clamp analyses of in vitro perfused RGT. NaCl secretion was stimulated by a solution (Stim) containing forskolin (10 µmol/l), dibutyryl-cAMP (0.5 mmol/l) and adenosine (0.5 mmol/l). This led to the expected strong depolarization and an increase in membrane conductance (G m). The membrane capacitance (C m) was measured by a newly devised two-frequency synchronous detector method. It was increased by Stim significantly from 5.00±0.22 to 5.17±0.21 pF (n=50). The increase in C m correlated with the increase in G m with a slope of 51 fF/nS. Next the effect of furosemide (500 µmol/l) was examined in previously stimulated RGT. Furosemide was supposed to inhibit coupled Na+2Cl–K+ uptake and to reduce cell volume but not membrane trafficking of Cl– channels. Furosemide reduced G m slightly (due to the fall in cytosolic Cl– concentration) and C m to the same extent by which Stim had increased it. Both changes were statistically significant, and the slope of ΔC m/ΔG m was similar to that caused by Stim. Inhibitors of microtubules or actin (colchicine, phalloidin and cytochalasin D added at 10 µmol/l to the pipette solution and dialysed for >10 min) did not alter cell voltage, G m or C m, nor did these inhibitors abolish the stimulatory effect of cAMP. These data suggest that the small C m changes observed with Stim reflect a minor cell volume increase and an ”unfolding” of the plasma membrane. The present data do not support the exocytosis/endocytosis hypothesis of cAMP-mediated activation of Cl– channels in these cells.

Osmotically Induced Conductance and Capacitance Changes in in vitro Perfused Rectal Gland Tubules of Squalus acanthias

“…The slope indicates that an increase in C m by 141 fF is found for a 1 nS increase in G m . This value is approximately three times larger than the slope obtained in experiments where RGT are stimulated by secretagogues [13], reflecting the fact that the volume increase induced by the present osmolality changes is much larger than the volume increase induced by stimulation by cAMP.…”

“…apparent membrane surface. Moreover, the volume changes, as measured by 360 nm fluorescence, and the C m changes correlate with each other: stimulation by cAMP reduced 360 nm fluorescence (enhanced cell volume) by 4% and increased C m by a b 51 fF/nS [13]; hypotonic cell swelling reduced 360 nm fluorescence by 16% and increased C m by 141 fF/nS. These data are of specific importance because they highlight that C m changes cannot a priori be taken as evidence for membrane insertion or retrieval.…”

“…This interpretation is favored by the following findings: (i) the changes occurred instantaneously with the change in the bath osmolality; (ii) they were paralleled by a fall or increase in fura-2 fluorescence measured at the isosbestic wavelength of 360 nm, respectively ( fig. 6 and 7a), and (iii) furosemide caused opposite C m changes [13]. Furosemide inhibits the Na + 2Cl -K + cotransporter specifically [12] and hence reduces ion and water uptake.…”

Construction and function of a recombinant adenovirus encoding a human aquaporin 1-green fluorescent protein fusion product

“…28,29 There is a positive linear correlation between C m and cell volume. C m was measured for individual SMIE cells infected with 0.5 MOI of AdhAQP1GFP and compared with that of individual control cells.…”

“…This is comparable with values typically found in other epithelial cells. 29,30 When SMIE cells were exposed to a hypotonic solution (270 mOsm), swelling was observed; in addition, the C m increased slightly in control cells but markedly in infected cells (Fig 7). The difference in the extent to which C m was increased in infected cells (148 Ϯ 28%, n ϭ 4) compared with control cells (108 Ϯ 14%, n ϭ 4) was statistically significant (Figure 7, P Ͻ .05).…”