Regulation of the Na+2Cl–K+ cotransporter in in vitro perfused rectal gland tubules of Squalus acanthias

 Previously it has been shown that the Na⁺2Cl^–K⁺ cotransporter accepts NH₄ ⁺ at its K⁺ binding site. This property can be used to estimate its transport rates by adding NH₄ ⁺ to the bath and measuring the initial furosemide-dependent rates of change in BCECF fluorescence. We have utilized this technique to determine the regulation of the furosemide-inhibitable Na⁺2Cl^–K⁺ cotransporter in in vitroperfused rectal gland tubules (RGT) of Squalus acanthias. Addition of NH₄ ⁺ to the bath (20 mmol/l) led to an initial alkalinization, corresponding to NH₃ uptake. This was followed by an acidification, corresponding to NH₄ ⁺ uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (Δfluorescence/time). This acidification could be completely inhibited by furosemide. In the absence of any secretagogue preincubation of RGT in a low Cl^– solution (6 mmol/l, low Cl^–) for 10 min enhanced the uptake rate significantly from 4.04±0.51 to 12.7±1.30 (n=5). The addition of urea (200 mmol/l) was without effect, but the addition of 300 mmol/l mannitol (+300 mannitol) enhanced the rate significantly from 7.24±1.33 to 14.7±4.6 (n=6). Stimulation of NaCl secretion by a solution maximizing the cytosolic cAMP concentration (Stim) led to a significant increase in NH₄ ⁺ uptake rate from 5.00±1.33 to 13.3±1.54 (n=6). Similar results were obtained in the additional presence of Ba²⁺ (1 mmol/l): the uptake rate was increased significantly from 4.23±0.34 to 15.1±1.86 (n=16). In the presence of Stim low Cl^– had no additional effect on the uptake rate: 15.1±3.1 versus 15.2±2.8 in high Cl^– (n=6). The uptake rate in Stim containing additional +300 mannitol (22.3±4.0, n=5) was not significantly different from that obtained with Stim or +300 mannitol alone. By whatever mechanism the NH₄ ⁺ uptake rate was increased furosemide (500 μmol/l) always reduced this rate to control values. Hence three manoeuvres enhanced furosemide-inhibitable uptake rates of the Na⁺2Cl^–K⁺ cotransporter probably independently: (1) lowering of cytosolic Cl^– concentration; (2) cell shrinkage; and (3) activation by cAMP. Received: 3 March 1998 / Received after revision: 22 April 1998 / Accepted: 27 April 1998     

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

 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. Received: 11 March 1998 / Received after revision 15 April 1998 / Accepted: 20 April 1998     

pH-regulatory mechanisms in in vitro perfused rectal gland tubules of Squalus acanthias

 Isolated in vitro perfused rectal gland tubules (RGT) were preincubated with the pH-sensitive dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and pH-regulatory mechanisms were studied. A reduction of bath Cl^– concentration from 269 to 6 mmol/l increased the fluorescence ratio 488/436 [corresponding to cytosolic pH (pH_i)] slightly but significantly (n=10). Depolarization by Ba²⁺ (1 mmol/l) or a bath solution containing 30 mmol/l K⁺ (n=4–6) increased the fluorescence ratio (pH_i). These data suggest that HCO₃ ^– uptake and/or H⁺ extrusion is dependent on Cl^– and/or voltage. A reduction of bath Na⁺ from 278 to 5 mmol/l reduced the ratio significantly (n=3). Addition of trimethylamine (Trima⁺, 20 mmol/l) alkalinized cytosolic pH (n=7). Similarly, addition of NH₄ ⁺ (20 mmol/l) led to an initial alkalinization and a strong acidification when NH₄ ⁺ was removed (n=59). The initial pH_i-recovery rates after NH₄ ⁺ removal were quantified and the responsible H⁺ extrusion and/or HCO₃ ^– import systems were examined. The recovery was almost completely abolished when the extracellular Na⁺ concentration was reduced to 5 mmol/l. In the presence of normal Na⁺, recovery was slower in the absence as compared to the presence of HCO₃ ^– (n=5). It was inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) (0.5 mmol/l, n=11) in the presence of HCO₃ ^– and in the absence of HCO₃ ^– by the Na⁺/H⁺-exchange blocker HOE694 (0.5 mmol/l, n=6). These data suggest that acid extrusion probably occurs by basolateral Na⁺-2HCO₃ ^–/Cl^– exchange in the presence of HCO₃ ^– and by basolateral Na⁺/H⁺ exchange in the absence of HCO₃ ^–. Luminal perfusion with a solution containing a low Cl^– concentration (6 mmol/l) increased the fluorescence ratio (pH_i) (n=5). The ratio (pH_i) was further increased and pH recovery further delayed by basolateral addition of Trima⁺ (20 mmol/l, n=3). These data suggest that the HCO₃ ^–/Cl^– exchanger is present in the luminal membrane. Luminal HCO₃ ^–/Cl^– exchange and basolateral Na⁺-2HCO₃ ^–/Cl^– exchange may work in tandem to secrete HCO₃ ^– and exchange it for luminal Cl^–. Received: 7 January 1998 / Received after revision and accepted: 5 March 1998     

Ca2+ regulated K+ and non-selective cation channels in the basolateral membrane of rat colonic crypt base cells

 We have previously shown that a new type of K⁺ channel, present in the basolateral membrane of the colonic crypt base (blm), is necessary for cAMP-activated Cl^- secretion. Under basal conditions, and when stimulated by carbachol (CCH) alone, this channel is absent. In the present patch clamp-study we examined the ion channels present in the blm under cell-attached and in cell-excised conditions. In cell-attached recordings with NaCl-type solution in the pipette we measured activity of a K⁺ channel of 16 ± 0.3 pS (n = 168). The activity of this channel was sharply increased by CCH (0.1 mmol/l, n = 26). Reduction of extracellular Ca²⁺ to 0.1 mmol/l (n = 34) led to a reversible reduction of activity of this small channel (SK_Ca). It was also inactivated by forskolin (5 μmol/l, n = 38), whilst the K⁺ channel noise caused by the very small K⁺ channel increased. Activity of non-selective cation channels (NS_cat) was rarely observed immediately prior to the loss of attached basolateral patches and routinely in excised patches. The NS_cat, with a mean conductance of 49 ± 1.0 pS (n = 96), was Ca²⁺ activated and required >10 μmol/l Ca²⁺ (cytosolic side = cs). It was reversibly inhibited by ATP (<1 mmol/l, n = 13) and by 3′,5-dichloro-diphenylamine-2-carboxylate (10–100 μmol/l, n = 5). SK_Ca was also Ca²⁺ dependent in excised inside-out basolateral patches. Its activity stayed almost unaltered down to 1 μmol/l (cs) and then fell sharply to almost zero at 0.1 μmol/l Ca²⁺ (cs, n = 12). SK_Ca was inhibited by Ba²⁺ (n = 31) and was charybdotoxin sensitive (1 nmol/l) in outside-out basolateral patches (n = 3). Measurements of the Ca²⁺ activity ([Ca²⁺]_i) in these cells using fura-2 indicated that forskolin and depolarization, induced by an increase in bath K⁺ concentration to 30 mmol/l, reduced [Ca²⁺]_i markedly (n = 8–10). Hyperpolarization had the opposite effect. The present data indicate that the blm of these cells contains a small-conductance Ca²⁺-sensitive K⁺ channel. This channel is activated promptly by very small increments in [Ca²⁺]_i and is inactivated by a fall in [Ca²⁺]_i induced by forskolin. Received: 15 April 1996 / Received after revision and accepted: 17 June 1996     

Mechanism of NaCl secretion in rectal gland tubules of spiny dogfish (Squalus acanthias)

Segments of rectal gland tubules (RGT) the spiny dogfish (Squalus acanthias) were perfused in vitro to study the cellular mechanism by which NaCl secretion is stimulated. Transepithelial PD (PD_te), transepithelial resistance (R_te), the PD across the basolateral membrane (PD_bl), the fractional resistance of the lumen membrane (FR₁), and the cellular activities for Cl^–, Na⁺, and K⁺ (a _x ^cell ) were measured. In series 1 the effects of stimulation (S) (dbcAMP 10^–4, adenosine 10^–4, and forskolin 10^–6 mol · l^–1) on these parameters were recorded and compared to nonstimulated state (NS). PD_te increased from –1.9±0.2 mV to –11.0±0.9 mV (n=51). PD_bI depolarized from –86±1 to –74±1.4 mV (n=52). R_te fell from 29±2.8 to 21±2 cm² (n=23), and FR₁ fell from 0.96±0.005 to 0.79±0.04 (n=9).a _K+ ^cell was constant (123±13 versus 128±17 mmol · 1^–1) (n=6), buta _Cl– ^cell -fell significantly from 48±4 to 41±3 mmol · l^–1 (n=7).a _Na+ ^cell increased from 11±2.1 to 29.5±6.6 mmol · l^–1 (n=4). In series 2 the conductivity properties were examined by rapid K⁺, and Cl^– concentration steps on the basolateral and luminal cell side respectively in NS and S states. In NS-segments reduction of bath K⁺ led to a hyperpolarization of PD_bI with a mean slope of 28±1.3 mV/decade (n=9) (as compared to 19 mV/decade for S-state). Reduction of lumen Cl^– led to very little depolarization of the lumen membrane PD in NS-state: 6.5±2.3 mV/decade (n=4) (as compared to 13 mV/decade for S-state). In series 3 the effects of furosemide (7 · 10^–5 mol l^–1, bath) were examined in NS and S tubules. In NS RGT segments furosemide had no effect on PD_bI or PD_te;a _Cl– ^cell fell slowly after furosemide with an initial rate of 0.33 mmol · l^–1 s^–1, as compared to 1.5 mmol · l^–1 · s^–1 for S-state. The increase ina _Cl– ^cell after removal of furosemide from NS to S-states was examined in the presence of furosemide. Despite the presence of furosemide stimulation was accompanied by a fall in R_te, FR₁, anda _Cl– ^cell . From these data we conclude that (a) stimulation by cyclic AMP increases the Cl^–-conductance of the apical cell membrane at least by a factor of 10, that (b) in the NS-state the Na⁺2Cl^–K⁺ carrier can be triggered to work at rates similar to the S state by loweringa _Cl– ^cell , and that (c) the increase in apical Cl^–-conductance is the primary event in cyclic AMP mediated stimulation of NaCl secretion.Supported by Deutsche Forschungsgemeinschaft Gr 480/8-1, and by NIH Grant AM 34208     

The basolateral Ca2+-dependent K+ channel in rat colonic crypt cells

 Previous studies have indicated that a 16-pS K⁺ channel (KC_ca) in the basolateral membrane is responsible for the acetylcholine-induced whole-cell K⁺ conductance in these cells. In the present study we have examined this channel in excised inside-out patches of the basolateral membrane. Over a wide voltage range this channel showed inward rectification. The Ca²⁺ sensitivity was very marked, with a Hill coefficient of three and with half-maximal activation at 330 nmol/l. After several minutes most channels showed a slow run-down. Channel activity could be refreshed by addition of ATP (1 mmol/l) to the bath solution. The non-metabolizable derivative 5’-adenylylimidodiphosphate (AMP-PNP) had no such effect. In contrast, it inhibited channel activity by some 50%. ATP and its derivatives had no effect on the Ca²⁺ sensitivity. Channels activated by ATP were subsequently studied in the presence of alkaline (10 kU/l) or acidic (1 kU/l) phosphatase. Both phosphatases reduced channel activity significantly. These data suggest that the 16-pS K⁺ channel is directly controlled by cytosolic Ca²⁺. This regulatory step is probably distal to an activation produced by protein-kinase-C-dependent phosphorylation. As is the case for several other K⁺ channels, high concentrations of non-metabolizable ATP analogues inhibit this channel. Received: 23 July 1997 / Accepted: 17 September 1997     

Intracellular Ca2+ distribution in migrating transformed renal epithelial cells

 Migration of transformed Madin-Darby canine kidney (MDCK-F) cells depends on the polarized activity of a Ca²⁺-sensitive K⁺ channel. We tested whether a gradient of intracellular Ca²⁺ concentration ([Ca²⁺]_i) underlies the horizontal polarization of K⁺ channel activity. [Ca²⁺]_i was measured with the fluorescent dye fura-2/AM. Spatial analysis of [Ca²⁺]_i indicated that a horizontal gradient exists, with [Ca²⁺]_i being higher in the cell body than in the lamellipodium. Resting and maximal levels during oscillations of [Ca²⁺]_i in the cell body were found to be 135 ± 34 and 405 ± 59 nmol/l, respectively, whereas they were 79 ± 18 and 307 ± 102 nmol/l in the lamellipodium. This gradient can partially explain the preferential activation of K⁺ channels in the plasma membrane of the cell body. We applied a local superfusion technique during migration experiments and measurements of [Ca²⁺]_i to test whether its maintenance is due to an uneven distribution of Ca²⁺ influx into migrating MDCK-F cells. Locally superfusing the cell body of migrating MDCK-F cells with La³⁺ alone or together with charybdotoxin, a specific blocker of Ca²⁺-sensitive K⁺ channels, slowed migration to 47 ± 10% and 9 ± 5% of control, respectively. Local blockade of Ca²⁺ influx into the cell body and the lamellipodium with La³⁺ was followed by a decrease of [Ca²⁺]_i at both cell poles. This points to Ca²⁺ influx occurring over the entire cell surface. This conclusion was confirmed by locally superfusing Mn²⁺ over the cell body and the lamellipodium. Fura-2 fluorescence was quenched in both areas, the decrease of fluorescence being two to three times faster in the cell body than in the lamellipodium. However, this difference is insufficient to account for the observed gradient of [Ca²⁺]_i. We hypothesize that the polarized distribution of intracellular Ca²⁺ stores contributes significantly to the generation of a gradient of [Ca²⁺]_i. Received: 22 July 1996 / Received after revision: 17 December 1996 / Accepted: 10 January 1997     

The cyclic ADP ribose antagonist 8-NH2-cADP-ribose blocks cholecystokinin-evoked cytosolic Ca2+ spiking in pancreatic acinar cells

 In order to investigate the possible involvement of cyclic ADP ribose as an intracellular messenger for hormone-evoked cytosolic Ca²⁺ signalling, we performed experiments on intracellularly perfused mouse pancreatic acinar cells. Both a stable inositol 1,4,5 trisphosphate analogue (IP₃) and cyclic ADP ribose (cADPR) evoked regular spikes of Ca²⁺ dependent ion current, reflecting cytosolic Ca²⁺ spiking. The effect of cADPR, but not IP₃, was abolished by the presence intracellularly of the cADPR antagonist 8-NH₂-cADPR. External application of cholecystokinin (CCK) in a physiological concentration (2.5–5 pM) evoked a mixture of short-lasting and longer-lasting spikes of Ca²⁺-dependent ion current. These effects were abolished by the presence intracellularly of 8-NH₂-cADPR (18 μM). Increasing the CCK concentration to 15 pM could overcome the inhibition by 18 μM of the antagonist. These experiments provide fresh evidence for the involvement of cADPR receptors in the hormone-evoked cytosolic Ca²⁺ signalling process in pancreatic acinar cells. Received: 2 December 1997 / Received after revision and accepted: 8 January 1998     

Involvement of Ca2+-induced Ca2+ release in the biphasic Ca2+ response evoked by readdition of Ca2+ to the medium after UTP-induced store depletion in A431 cells

 We have recently shown that the Ca²⁺ response in endothelial cells evoked by readdition of Ca²⁺ to the medium after store depletion caused by a submaximal concentration of agonist can involve Ca²⁺ release from Ca²⁺ stores sensitive to both inositol 1,4,5-trisphosphate and ryanodine. The present experiments were performed to determine whether this mechanism might also exist in other types of cell. For this purpose, we used the human carcinoma cell line A431, which has a varied resting [Ca²⁺]_i. We found that the amplitude of the Ca²⁺ response evoked by Ca²⁺ readdition did not correlate with the amplitude of the preceding UTP-evoked Ca²⁺ release, but did positively correlate with the initial [Ca²⁺]_i. An inspection of the two patterns of response seen in this study (the large biphasic and small plateau-shaped Ca²⁺ responses) revealed that there is an accelerating rise in [Ca²⁺]_i during the biphasic response. Application of ryanodine during the plateau-shaped Ca²⁺ response reversibly transformed it into the biphasic type. Unlike ryanodine, caffeine did not itself evoke Ca²⁺ release, but it caused a further [Ca²⁺]_i rise when [Ca²⁺]_i had already been elevated by thapsigargin. These data suggest that in A431 cells, as in endothelial cells, the readdition of Ca²⁺ after agonist-evoked store depletion can evoke Ca²⁺-induced Ca²⁺ release. This indicates that Ca²⁺ entry may be overestimated by this widely used protocol. Received: 28 July 1997 / Received after revision: 25 November 1997 / Accepted: 26 November 1997     

Mechanism of NaCl secretion in the rectal gland of spiny dogfish (Squalus acanthias)

Rectal gland tubules (RGT) of spiny dogfish were dissected and perfused in vitro. Transepithelial PD (PD_te), resistance (R_te), the PD across the basolateral membrane (PD_bl) and intracellular chloride and potassium activities (a _Cl– ^cell ,a _K+ ^cell ) were measured. In a first series, 67 RGT segments were perfused with symmetric shark Ringers solution. The bath perfusate contained in addition db-cAMP 10^–4, forskolin 10^–6, and adenosine 10^–4 mol · l^–1. PD_te was –11±1 (n=67) mV lumen negative, R_te 27±2 (n=47) cm². PD_bl –75±0.4 (n=260) mV.a _K+ ^cell anda _Cl– ^cell were 109±22 (n=4) and 38±4 (n=36) mmol · l^–1 respectively. These data indicate that Cl^– secretion across the RGT must be an uphill transport process, whereas secretion of Na⁺ could be driven by the lumen negative PD_te. Intracellular K⁺ is 14 mV above equilibrium with respect to the basolateral membrane PD and Cl^– is 23 mV above equilibrium across the apical membrane. In series 2, the conductivity properties of the apical and basolateral membrane as well as that of the paracellular pathway were examined in concentration step experiments. Decrease of the basolateral K⁺ concentration led to a rapid hyperpolarization of PD_bt with a mean slope of 19 mV per decade of K⁺ concentration change. Addition of 0.5 mmol · l^–1 Ba²⁺ to the bath solution lead to a marked depolarization and abolished the response to K⁺ concentration steps. In the lumen a Cl^– concentration downward step led to a depolarization of the lumen membrane; resulting in a mean slope of 18 mV per decade of Cl^– concentration change. When dilution potentials were generated across the epithelium, the polarity indicated that the paracellular pathway is cation selective. In series 3 the equivalent short circuit current (I_sc=PD_te/R_te) was determined as a function of symmetrical changes in Na⁺ concentration, with Cl^– held at 276 mmol · l^–1, and as a function of symmetrical changes in Cl^– concentration, with Na⁺ held at 278 mmol · l^–1 I_sc was a saturable function of Na⁺ concentration (Hill coefficient 0.9±0.1,K _1/2 4.4 mmol · l^–1,n=7) and also a saturable function of Cl^– concentration (Hill coefficient 2.0±0.1,K _1/2 75 mmol · l^–1,n=11). These data are compatible with the assumption that the carrier responsible for NaCl uptake has a 1 Na⁺ per 2 Cl^– stoichiometry. In series 4, the effect of a K⁺ concentration downward step on PD_bl anda _Cl– ^cell transients was followed with high time resolution in the presence and absence of basolateral furosemide (5 · 10^–5 to 10^–4 mol · l^–1) in an attempt to examine whether K⁺ reduction on the bath side inhibits Na⁺Cl^– uptake by the carrier system as does e.g. furosemide. The data indicate that removal of K⁺ from the bath side exerts an effect comparable to that of furosemide, i.e. it inhibits the carrier. We conclude that NaCl secretion in the RGT cell comprises at the least the following components: In the basolateral membrane, the (Na⁺+K⁺)-ATPase, probably the Na⁺ 2 Cl^–K⁺ carrier, and a K⁺ conductance. In the apical membrane a Cl^– conductance; and a Na⁺ conductive paracellular pathway.Supported by Deutsche Forschungsgemeinschaft DFG-Gr 480/8-1. Parts of this study have been presented at the 3rd International Symposium on Ion Selective Electrodes, Burg Rabenstein 1983, 16th Annual Meeting American Society of Nephrology, Washington DC 1983, 49th Tagung der Deutschen Physiologischen Gesellschaft, Dortmund 1984. A summary of the present study was published in Bulletin Mount Desert Island Biological Laboratory (Vol. 83)     

[Ca2+]i elevation evoked by Ca2+ readdition to the medium after agonist-induced Ca2+ release can involve both IP 3-, and ryanodine-sensitive Ca2+ release

 Sustained Ca²⁺ elevation (”Ca²⁺ response”), caused by subsequent readdition of Ca²⁺ to the medium after application of adenosine 5’-triphosphate (ATP, 15 μM) in a Ca²⁺-free medium, was studied using single bovine aortic endothelial (BAE) cells. In cells in which the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) was between about 50 and 110 nM, a massive Ca²⁺ response occurred and consisted of phasic and sustained components, whereas cells with a resting [Ca²⁺]_i of over 110 nM displayed small plateau-like Ca²⁺ responses. An increase of internal store depletion resulted in loss of the phasic component. When the store was partly depleted, the dependence of the Ca²⁺ response amplitude on resting [Ca²⁺]_i was biphasic over the range of 50 to 110 nM. The greatest degree of store depletion was associated with small monophasic Ca²⁺ responses, the amplitudes of which were almost constant and in the same range as resting [Ca²⁺]_i. Ni²⁺, known to partly block Ca²⁺ entry, caused no change in the half-decay time of [Ca²⁺]_i down to the level of the sustained phase [57 ± 4 s in control and 54 ± 3 s (n = 13) in the presence of 10 mM Ni²⁺] when added at the peak of the phasic component of the Ca²⁺ response. However, it lowered the sustained phase of the Ca²⁺ response by 42%. When applied at the start of the readdition of Ca²⁺, Ni²⁺ blocked the phasic component of the Ca²⁺ response, there being a threefold decrease in the initial rate of [Ca²⁺]_i rise. In cells with a resting [Ca²⁺]_i of 75–80 nM, pre-treatment with ryanodine (10 μM) did not affect the peak amplitude of the Ca²⁺ response, but it did increase the level of the sustained component. In some cells, ryanodine caused an oscillatory Ca²⁺ response. In conclusion, partial depletion of the inositol 1,4,5-trisphosphate-(IP ₃-) sensitive store by a submaximal concentration of agonist (in Ca²⁺-free medium) was followed, on readdition of Ca²⁺, by Ca²⁺ entry, which appeared to trigger IP ₃-sensitive Ca²⁺ release (IICR) which, in turn, initiated Ca²⁺-sensitive Ca²⁺ release (CICR), thus resulting in a massive elevation of [Ca²⁺]_i. Received: 3 July 1996 / Received after revision and accepted: 9 September 1996     

The membrane transporters regulating epithelial NaCl secretion

Ten years ago, the basic principles operating in one specific, albeit non-mammalian, exocrine gland, the rectal gland of Squalus acanthias, were described in detail. The concept emerging from these studies appeared applicable to almost any other exocrine gland, because it involved membrane transporters which are also present in mammalian epithelial cells. Meanwhile, it has become clear that the mechanisms of NaCl secretion are diverse: the mechanisms of NaCl uptake; the ion channels involved; and also the mechanisms of hormonal control. Nevertheless, several steps in NaCl secretion still appear to be uniform: (1) several signalling pathways converge and act cooperatively, (2) one primary regulatory step is the upregulation of the luminal Cl⁻ conductance, (3) secondarily active NaCl uptake mechanisms are upregulated, (4) increasing evidence links NaCl secretion to membrane trafficking and (5) the entire machinery seems to be primed to secure cellular homeostasis in terms of cytosolic ion concentrations. This brief review summarizes the mechanisms of control of NaCl secretion. The major issues addressed are the NaCl uptake mechanisms, the ion channels involved and the cellular mechanisms coordinating secretion. The major NaCl secreting cells discussed here will be the respiratory epithelial cells, the exocrine cells of pancreatic acini and the cells of colonic crypts.     

Effect of uncoupling NO/cGMP pathways on carbachol- and CCK-stimulated Ca2+ entry and amylase secretion from the rat pancreas

 Nitric oxide (NO) production reportedly regulates guanosine 3′,5′-cyclic monophosphate (cGMP) formation and Ca²⁺ influx in pancreatic acini. We have investigated the functional roles of the NO/cGMP messenger system in rat pancreatic acini. In dispersed acini, the levels of amylase secretion, cytosolic [Ca²⁺]([Ca²⁺]_i), NO synthase, and cGMP were measured. The NO synthase inhibitor N ^G-nitro-L-arginine methyl ester (L-NAME, 0.01–100 μM) had no effect on amylase secretion induced by various concentrations of carbachol, cholecystokinin octapeptide (CCK-8) or the high affinity CCK agonist, JMV-180. Similarly, L-NAME up to 100 μM did not affect the changes in Ca²⁺ spiking evoked by these secretagogues; nor was Ca²⁺ entry, refilling or oscillation altered by L-NAME. Sub- and supramaximal concentrations of these secretagogues did not change NO synthase activities compared with basal levels. While sodium nitroprusside (SNP), a NO donor, caused a 9.4-fold increase in cGMP levels compared with basal levels, carbachol, CCK-8 and JMV-180 had no effect. In addition, the guanylate cyclase inhibitor LY 83583 (10 nM to 10 μM) altered neither amylase secretion nor Ca²⁺ signaling induced by these secretagogues. These findings indicate that the stimulatory action of carbachol or CCK-8 is not mediated by NO or cGMP. To investigate whether cGMP stimulates pancreatic secretion we showed that both SNP and a cell-permeant cGMP analog at 0.1–1 mM stimulated amylase secretion and Ca²⁺ transients to a level equal to 10–15% and 13–24%, respectively, of those observed with maximal concentrations of secretagogues. The guanylate cyclase activator guanylin (1–10 μM), which increased cGMP levels 2.4-fold compared with basal levels, elicited a small amount of amylase secretion and a small Ca²⁺ transient. In conclusion, exogenous NO is capable of increasing endogenous cGMP, which results in a modest increase in the [Ca²⁺]_i transient and pancreatic amylase secretion. However, the NO/cGMP system does not appear to be involved significantly in the mediation of Ca²⁺ signaling and amylase secretion stimulated by carbachol and CCK-8. Received: 30 October 1996 / Received after revision and accepted: 13 January 1997     

Effect of acidosis on Ca2+-activated K+ channels in cultured porcine coronary artery smooth muscle cells

 Although acidosis induces vasodilation, the vascular responses mediated by large-conductance Ca²⁺-activated K⁺ (K_Ca) channels have not been investigated in coronary artery smooth muscle cells. We therefore investigated the response of porcine coronary arteries and smooth muscle cells to acidosis, as well as the role of K_Ca channels in the regulation of muscular tone. Acidosis (pH 7.3–6.8), produced by adding HCl to the extravascular solution, elicited concentration-dependent relaxation of precontracted, endothelium-denuded arterial rings. Glibenclamide (20 μM) significantly inhibited the vasodilatory response to acidosis (pH 7.3-6.8). Charybdotoxin (100 nM) was effective only at pH 6.9–6.8. When we exposed porcine coronary artery smooth muscle cells to a low-pH solution, K_Ca channel activity in cell-attached patches increased. However, pretreatment of these cells with 10 or 30 μM O, O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM), a Ca²⁺ chelator for which the cell membrane is permeable, abolished the H⁺-mediated activation of K_Ca channels in cell-attached patches. Under these circumstances H⁺ actually inhibited K_Ca channel activity. When inside-out patches were exposed to a [Ca²⁺] of 10^–6 M [adjusted with ethyleneglycolbis(β-aminoethylester)-N,N,N′,N′-tetraacetic acid (EGTA) at pH 7.3], K_Ca channels were activated by H⁺ concentration dependently. However, when these patches were exposed to a [Ca²⁺] of 10^–6 M adjusted with BAPTA at pH 7.3, H⁺ inhibited K_Ca channel activity. Extracellular acidosis had no significant direct effect on K_Ca channels, suggesting that extracellular H⁺ exerts its effects after transport into the cell, and that K_Ca channels are regulated by intracellular H⁺ and by cytosolic free Ca²⁺ modulated by acute acidosis. These results indicate that the modulation of K_Ca channel kinetics by acidosis plays an important role in the determination of membrane potential and, hence, coronary arterial tone. Received: 20 January 1998 / Received after revision: 9 April 1998 / Accepted: 22 April 1998     

Short pulses of acetylcholine stimulation induce cytosolic Ca2+ signals that are excluded from the nuclear region in pancreatic acinar cells

 We have investigated the spreading of cytosolic Ca²⁺ signals generated by acetylcholine stimulation (using either microionophoresis or pressure application) of isolated pancreatic acinar cells (or small cell clusters) using confocal microscopy of Ca²⁺-sensitive fluorescence (fura red). We have been particularly interested in the effects of short vigorous pulses of acetylcholine (ACh) stimulation since, in the pancreas, ACh secreted from nerve endings is quickly eliminated by the action of ACh esterase. We focused on three regions: the secretory pole (secretory granule area), the nucleus and the basal area outside the nucleus. The nuclei were visualized by using the specific nuclear stain Hoechst 33342. With ionophoretic application, a long-lasting stimulation with ACh (10 s and longer) induces large Ca²⁺ transients of similar amplitude in all the three selected regions of the cell. Short applications (about 3 s) of ACh result in a Ca²⁺ rise in the secretory pole, whereas no changes in cytoplasmic Ca²⁺ were detected in the basal, nonnuclear region or in the nucleus. We found that at the peak of such localised Ca²⁺ responses, evoked either by ACh ionophoresis or pressure application, significant Ca²⁺ concentration gradients (up to 400 nM/μm) can be established along the line connecting the secretory pole with the nucleus. In some experiments slightly longer applications (about 5 s) of ACh produce Ca²⁺ transients in both the secretory region and in the basal, nonnuclear regions of the cells, whereas the nuclear [Ca²⁺] remained largely unaffected. Estimation of the ACh concentration in the vicinity of the cell under investigation indicated that values of about 1 μM were attained in the pressure application experiments. These results show directly that the nucleus of pancreatic acinar cells can be effectively protected from relatively large Ca²⁺ transients generated in the secretory pole of pancreatic acinar cells by short pulses of near-maximal ACh concentrations. This indicates that calcium-dependent secretion (both fluid and digestive enzymes) can occur without changes of the intranuclear [Ca²⁺] and consequently without activation of numerous calcium dependent nuclear processes. Received: 6 May 1996 / Accepted: 5 July 1996     

Ruthenium red reduces the Ca2+ sensitivity of Ca2+ uptake into cardiac sarcoplasmic reticulum

 Ruthenium red inhibits mitochondrial Ca²⁺ uptake and is widely used as an inhibitor of ryanodine-sensitive Ca²⁺ channels that function to release Ca²⁺ from the sarcoplasmic reticulum (SR) of muscle cells. It also has effects on other Ca²⁺ channels and ion transporters. To study the effects of ruthenium red on Ca²⁺ transport into the SR of cardiac muscle cells, fluorescence measurements of Ca²⁺ uptake into cardiac SR vesicles were made. Ruthenium red significantly decreased the Ca²⁺ sensitivity of SR uptake in a dose-dependent manner at concentrations ranging from 5 μM to 20 μM. There were no significant effects of ruthenium red on the maximum velocity or the Hill coefficient of SR Ca²⁺ uptake. Received: 14 January 1998 / Received after revision: 12 March 1998 / Accepted: 16 March 1998     

Estimation of myofibrillar responsiveness to Ca2+ in isolated rat ventricular myocytes

 To estimate myofibrillar responsiveness to Ca²⁺, we used the relation between cell length and intracellular [Ca²⁺] ([Ca²⁺]_i) during tetanic contractions of isolated ventricular myocytes. Enzymatically isolated rat ventricular myocytes were loaded with fura-2 AM (4 μM for 10 min) and excited alternately at 340 nm and 380 nm. The ratio (R) of fura-2 fluorescence at these wavelengths [F(340)/F(380), an index of [Ca²⁺]_i] and cell length (L) were measured simultaneously. Following treatment with thapsigargin (0.2 μM), myocytes were stimulated at 10 Hz for 10 s to produce a tetanic contraction every min and an instantaneous plot of R vs L (R-L trajectory) was constructed. The R-L trajectory followed the same path during cell shortening and re-lengthening, suggesting that dynamic equilibrium between R and L was achieved during tetanus. Increasing the extracellular [Ca²⁺] from 1 to 8 mM extended the R-L trajectory without a substantial shift of the relation. The Ca²⁺-sensitizing thiadiazinone derivative, EMD57033 (1 μM), shifted the R-L trajectory to the left (sensitization of the myofibrils to Ca²⁺), whereas the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxantine (200 μM), shifted the R-L trajectory to the right (desensitization of the myofibrils to Ca²⁺), in agreement with previous results obtained using skinned preparations. We conclude that the R-L trajectory is useful for estimating the myofibrillar responsiveness to Ca²⁺ in isolated myocytes and may be beneficial for the evaluation of inotropic agents. Received: 13 March 1998 / Received after revision: 4 May 1998 / Accepted: 2 June 1998     

Vasopressin-stimulated Ca2+ reabsorption in rabbit cortical collecting system: effects on cAMP and cytosolic Ca2+

 The effect of arginine vasopressin (AVP) on transepithelial Ca²⁺ transport in primary cultures of rabbit cortical collecting system cells was examined. Addition of AVP to the basolateral side of the monolayer dose-dependently (EC₅₀ = 0.7 nM) increased active Ca²⁺ reabsorption from a basal value of 85 ± 2 nmol·h^–1·cm^–2 to a maximum value of 124 ± 3 nmol·h^–1·cm^–2. This was paralleled by a dose-dependent (EC₅₀ = 1.1 nM) increase in cellular adenosine 3′,5′-cyclic monophosphate (cAMP) content. Both effects of AVP were mimicked by the V₂ agonist deamino-Cys,D-Arg⁸-vasopressin (dDAVP) and forskolin. Addition of either AVP or dDAVP to the basolateral side evoked a sustained increase in cytosolic free Ca²⁺ concentration, which resulted from both Ca²⁺ entry and release from internal stores. Only the effect on Ca²⁺ entry was mimicked by forskolin, demonstrating that cAMP acts by activating a Ca²⁺ influx pathway. The present findings demonstrate that AVP stimulates transcellular Ca²⁺ transport in the cortical collecting system through activation of basolateral V₂ receptors coupled to adenylyl cyclase to increase the cellular cAMP content. Received: 4 July 1996 / Received after revision and accepted: 3 September 1996