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     

Hypertonic cell shrinkage reduces the K+ conductance of rat colonic crypts

 It has previously been shown in studies of a renal epithelial cell line that nonselective cation (NSC) channels are activated by exposure to hypertonic solution. We have also found such channels in excised patches of colonic crypt cells. They require high Ca²⁺ activities on the cytosolic side and a low ATP concentration for their activation and have not been recorded from cell-attached patches of colonic crypts. We examine here whether this type of channel is activated by hypertonic cell shrinkage. Bath osmolality was increased by addition of 25, 50 or 100 mmol/l mannitol. Cell-attached and whole-cell patch recordings were obtained from rat base and mid-crypt cells. In whole-cell recordings we found that addition of 50 or 100 mmol/l mannitol depolarized these cells significantly from –78±2.0 to –66±3.8 mV (n=22) and from –78±1.3 to –56±2.6 mV (n=61), respectively, and reduced the whole-cell conductance from 20±8.0 to 14±6.6 nS (n=7) and from 20±3.0 to 9.8±1.6 nS (n=19), respectively. In cell-attached patches K⁺ channels with a single-channel conductance of ≈16 pS were found in most recordings. The activity of these channels (N×P _o, N=number, P _o=open channel probability) was reduced from 2.08±0.37 to 0.98±0.23 (n=15) by the addition of 50 mmol/l mannitol and from 1.75±0.26 to 0.77±0.20 (n=12) by 100 mmol/l mannitol. No NSC channel activity was apparent in any of these recordings. Previously we have shown that the 16-pS K⁺ channel is controlled by cytosolic Ca²⁺ ([Ca²⁺]_i). Therefore we measured [Ca²⁺]_i by the fura-2 method and found that hypertonic solution reduced [Ca²⁺]_i significantly (n=16). These data indicate that exposure of rat colonic crypts to hypertonic solutions does not activate NSC channels; [Ca²⁺]_i falls in hypertonic solution leading to a reduction in the value of K⁺ channel N×Po, a reduced whole-cell conductance and depolarization of mid-crypt cells. These processes probably assist volume regulation inasmuch as they reduce KCl losses from the cell. Received: 21 July 1997 / Received after revision: 24 November 1997 / Accepted: 15 December 1997     

Ion channels in isolated mouse jejunal crypts

 The patch-clamp technique was used to characterise the ion channels in cells located in the mid region of mouse jejunal crypts. Six different channels were seen. A large outwardly rectified K⁺ channel (BK) (conductance, g at 0 mV = 92 ± 6 pS), which was highly selective for K⁺ [P _K ⁺ (1) > P _Rb ⁺ (0.6) >> P _Cs ⁺ (0.09) ≈ P _Na ⁺ (0.07) > P _Li ⁺ (0.04)], had a low, voltage-independent open probability (P _o) in the on-cell (O/C) configuration and appeared in 66% of the patches. In inside-out (I/O) patches, this channel had a linear current/voltage (I/V) relationship (g = 132 ± 3 pS), P _o was voltage dependent and it was blocked by cytoplasmic Ba²⁺ (5 mmol/l). An intermediate K⁺ channel (IK) which was present in 49% of O/C patches, had a linear I/V (g = 38 ± 3 pS), ran-down in O/C patches, and was not seen in I/O patches. A number of smaller channels (SC) with conductances ranging from 5 to 20 pS were seen in 16% of O/C patches. Also present in the basolateral membrane were a Cl^– channel (ICOR) and a nonselective cation channel (NSCC). These channels were only seen in I/O patches. ICOR had an outwardly rectified conductance (g at 0 mV = 36 ± 2 pS), its P _o was independent of voltage and unaffected by variations in cytoplasmic Ca²⁺ (100 nmol/l to 1 mmol/l) or ATP (0–1 mmol/l). The NSCC had a linear conductance (20 ± 1 pS), its P _o increased with depolarisation and elevation of cytoplasmic [Ca²⁺] (≥ 10 μmol/l), but was reduced by cytoplasmic ATP. None of the basolateral channels described here were activated by cAMP-dependent secretagogues, although a Cl^– conductance was activated. This cAMP-dependent Cl^– conductance was distinct from the basolateral Cl^– channel and thus is most likely located in the apical membrane. Received: 25 June 1997 / Accepted: 14 October 1997     

Attenuation of stimulated Ca2+ influx in colonie epithelial (HT29) cells by cAMP

In HT₂₉ colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca²⁺ activity ([Ca²⁺]_i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P ₃-) mediated Ca²⁺ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca²⁺ influx. The present study was undertaken to examine the effect of increased adenosine 3′,5′-cyclic monophasphate (cAMP) (forskolin 10 μmol/l = FOR) on the Ca²⁺ transient in the presence of CCH (100 μmol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca²⁺ ([Ca²⁺]_i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K⁺ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca²⁺]_i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca²⁺]_i uniformly from a fluorescence ratio (345/380) of 2.9 ± 0.12 to 1.8 ± 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (V _m) of the patch-clamped cells significantly and reversibly from −54 ± 7.4 to −27 ± 5.9 mV (n = 6). In additional experiments V _m was depolarized by 15–54 mV by various increments in the bath K⁺ concentration. This led to corresponding reductions in [Ca²⁺]_i. Irrespective of the cause of depolarization (high K⁺ or FOR) there was a significant correlation between the change in V _m and change in [Ca²⁺]_i. These data indicate that the cAMP-mediated attenuation of Ca²⁺ influx is caused by the depolarization produced by this second messenger. Received: 12 March 1996/Accepted: 2 April 1996     

Evidence for a Na+/Ca2+ exchange mechanism in frog skin epithelium

 In the present study we investigated the possible existence of a Na⁺/Ca²⁺ exchange mechanism in the basolateral membrane of the frog skin epithelium and whether such a mechanism plays a role in the regulation of transepithelial Na⁺ transport. Cytosolic calcium ([Ca²⁺]_i) was measured with the probe fura-2 in a set-up in which pieces of tissue were mounted on the stage of an epifluorescence microscope. Na⁺ transport was measured as the amiloride-sensitive short-circuit current (I _sc) using a conventional voltage clamp. Basal [Ca²⁺]_i was 65±6 nM (n=15). Removal of Na⁺ from the mucosal solution had no effect on [Ca²⁺]_i. When Na⁺ was removed from the serosal solution, [Ca²⁺]_i increased biphasically to a peak of 220±38 nM (n=8, P=0.006). Readdition of Na⁺ to the serosal solution returned [Ca²⁺]_i to control level. The serosal Na⁺ gradient and changes in [Ca²⁺]_i were closely correlated; stepwise changes in serosal Na⁺ were followed by stepwise changes in [Ca²⁺]_i. These observations indicate the existence of a Na⁺/Ca²⁺ exchange mechanism in the basolateral membrane of the frog skin epithelium. The transepithelial Na⁺ transport decreased from 13.2±1.8 to 9.2±1.5 μA cm^–2 (n=8, P=0.049) when Na⁺ was omitted from the serosal solution. When this protocol was repeated in the absence of serosal Ca²⁺, Na⁺ transport decreased similarly from 16.7±1.7 to 11.6 ±1.8 μA cm^–2 (n=6, P=0.004). We conclude that it is unlikely that the observed decrease in I _sc after removal of serosal Na⁺ is due to an increase in [Ca²⁺]_i per se. Received: 10 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998     

The sarcolemmal mechanisms involved in the control of diastolic intracellular calcium in isolated rat cardiac trabeculae

 We performed experiments using the calcium indicator Indo-1 to determine the relative roles of the sarcolemmal mechanisms involved in the regulation of diastolic intracellular calcium concentration ([Ca²⁺]_i) in trabeculae from the rat heart. Ryanodine was used to eliminate sarcoplasmic reticulum (SR) function. In the functional absence of the SR, 76.8 ± 3.9% of the calcium was extruded by the Na-Ca exchange carrier in the [Ca²⁺]_i range of diastolic concentration ± 200–400 nM. This was assessed by measuring the recovery of [Ca²⁺]_i from small perturbations in the presence and absence of extracellular sodium. The steady-state relationship between [Ca²⁺]_o and [Ca²⁺]_i was linear over the range of 1–40 mM, a 20-fold increase of [Ca²⁺]_o produced a 1.97-fold ± 0.13-fold increase in [Ca²⁺]_i (n = 5). In the absence of extracellular sodium raising [Ca²⁺]_o had a variable effect. In some preparations there was little change of [Ca²⁺]_i while in others the response was almost as large as in control conditions. We conclude that the Na-Ca exchanger contributes ≈ 77% of sarcolemmal calcium extrusion following small perturbations in [Ca²⁺]_i and that this fraction does not diminish as the [Ca²⁺]_i declines. In addition we have shown a sodium-independent entry of calcium into quiescent cardiac muscle under resting conditions. Received: 16 May 1996 / Received after revision and accepted: 28 June 1996     

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

Secretory apical Cl– channels in A6 cells: possible control by cell Ca2+ and cAMP

Distal kidney cells (A6) from Xenopus laevis were cultured to confluency on porous supports. Tissues were mounted in Ussing-type chambers to measure short-circuit current (I _sc), transepithelial conductance and capacitance, and to analyse the fluctuation in I _sc. In the absence of apical NaCl, but with normal basolateral NaCl Ringer’s solution, extracellular addition of ATP, oxytocin, a membrane-permeant cAMP derivative, and forskolin produced a transient increase of the electrical parameters. Noise analysis revealed a spontaneous Lorentzian component. All responses depend strictly on the presence of basolateral Cl^– and are caused by the activation of an apical (CFTR type) Cl^– permeability. Repetitive treatment with ATP (or oxytocin) resulted in refractoriness. ATP and oxytocin acted antagonistically, whereas cAMP and ATP had additive effects. Incubation with the vesicular Ca²⁺ pump inhibitor thapsigargin or application of the Ca²⁺ channel blocker nifedipine elicited finite but variable Cl^– channel activity. After treatment with nifedipine or thapsigargin, the response to oxytocin was severely impaired. We speculate that not only cAMP but also cell Ca²⁺ plays a crucial role in the activation of CFTR in A6. Ca²⁺ may be multifunctional but the rise in capacitance (apical area) observed with all stimulants strongly suggests its involvement in, and contribution to, exocytosis in the process of the CFTR-mediated transcellular Cl^– movements. Received: 30 November 1998 / Received after revision: 23 February 1999 / Accepted: 12 March 1999     

Phosphorylation modulates L-type Ca channels in frog ventricular myocytes by changes in sensitivity to Mg2+ block

 Maitotoxin (MTX) may exert its toxic effect by activating ion conductances and has been shown to elicit a fertilization-like response in Xenopus laevis oocytes. In the present study we investigated the electrophysiological response of stage V–VI Xenopus oocytes to MTX using the two-microelectrode voltage-clamp technique. Membrane voltage (V _m) measurements demonstrated that MTX (50 pM to 1 nM) depolarized the oocytes from –49±7 to –14±1 mV. Subsequent replacement of bath Na⁺ by the impermeant cation NMDG (N-methyl-d-glucamine) shifted V _m from –14±1 to –53±5 mV (n=29). This indicates that MTX activates a cation conductance. Indeed, current measurements at a holding potential of –60 or –100 mV showed that within 10 s of MTX application an inward current component developed which was largely abolished by extracellular Na⁺ removal. After a 1-min application of 1 nM MTX the NMDG-sensitive current increased more than 100-fold from 0.14±0.03 μA to a peak value of 21±3 μA (n=11). The effect of MTX was concentration dependent with an EC₅₀ of about 250 pM but only slowly reversible. Ion substitution experiments indicated that the stimulated conductance was nonselective for monovalent cations with a slight preference for NH₄ ⁺ (2.1) > K⁺ (1.5) > Na⁺ (1.0) > Li⁺ (0.7). Regarding divalent cations, a complex biphasic response to extracellular Na⁺ replacement by Ca²⁺ was observed, which suggests that the stimulated channels may have a small Ca²⁺ permeability but that exposure to high extracellular Ca²⁺ enhances recovery from MTX stimulation. No significant conductance for Mn²⁺ was observed. Application of 1 mM benzamil, 1 mM amiloride, or 100 μM 1-(β-[3-(4-Methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365) reduced the MTX-stimulated inward current by 81%, 62%, or 65%, respectively. Gd³⁺ had an inhibitory effect of 29% and 38% at concentrations of 10 μM or 100 μM, respectively. Flufenamic acid, niflumic acid, (RS)-(3,4-dihydro-6,7-dimethoxyisoquinoline-1-γ1)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)-ethyl]-acetamide (LOE908), and 3′,5′-dichlorodiphenylamine-2-carboxylic acid (DCDPC), known blockers of other nonselective cation channels, had no significant effect. We conclude that MTX activates a nonselective cation conductance in Xenopus oocytes. The underlying channels may be involved in changes in V _m that occur during the early stages of fertilization. Received: 30 December 1997 / Received after revision and accepted: 17 March 1998     

Intracellular cAMP potentiates voltage-dependent activation of L-type Ca2+ channels in rat islet β-cells

 Intracellular cAMP-dependent modulation of L-type Ca²⁺ channel activation in cultured rat islet β-cells has been investigated using the patch-clamp whole-cell current recording mode. The L-type voltage-dependent Ca²⁺ current (I _Ca) showed a fast activation followed by a slow inactivation, and was sensitive to Ca²⁺ channel blockers, for example nifedipine. Application of a cAMP analogue, dibutyryl cyclic AMP (db-cAMP), increased the magnitude of the peak I _Ca in a concentration-dependent manner. Values of the half-activation potentials (V _1/2), taken from activation curves for I _Ca, were –16.7 ± 1.8 and –21.9 ± 3.4 mV (P < 0.05) before and after application of db-cAMP, respectively, with no change of the slope factor (k) or the reversal potential. Pretreatment with a specific protein kinase A antagonist, Rp-cAMP, prevented the potentiating effect of db-cAMP. These results indicate that in rat islet β-cells, phosphorylation of cAMP-dependent kinase potentiates the voltage-dependent activation of L-type Ca²⁺ channels. Received: 9 September 1997 / Received after revision: 19 November 1997 / Accepted: 21 November 1997     

Ca2+ store determines gating of store operated calcium entry in mammalian skeletal muscle

This work describes the gating of the store operated calcium entry (SOCE) in adult mammalian skeletal muscle. Flexor digitorum brevis fibers (FDB) were isolated from adult mice and exposed to conditions to deplete the sarcoplasmic reticulum (SR). A transient SR depletion caused either by repetitive depolarizations, chlorocresol (CMC) or, cyclopiazonic acid (CPA) induced a bell shaped calcium entry that raised the [Ca²⁺]_i to a maximum of 27.09 ± 4.35 nM from the resting value. The activation time to reach 10–90% of the maximum amplitude was 112 ± 10 s (n = 22). On the other hand, any mechanism that caused a permanent SR depletion (like thapsigargin, continuous CPA, or continuous CMC) triggered a calcium entry pathway that lasted 325 ± 23 s and raised the [Ca²⁺]_i to 129.50 ± 13.05 nM from the resting level (n = 28). Then, a prolonged depletion triggered an increase in [Ca²⁺]_i to higher values and for a longer time than when the SR is transiently depleted (p < 0.001). Our results, in skeletal muscle, showed that calcium store depletion was the signal for SOCE activation and how the SR got depleted was not relevant. Also, we found that SOCE deactivation was not caused by [Ca²⁺]_i but by the SR content. Our results suggest that the SR calcium content plays an important role in SOCE gating in mammalian skeletal muscle and a calcium sensor is located inside the SR.     

Slow spontaneous [Ca2+]i oscillations reflect nucleotide release from renal epithelia

Renal epithelia can be provoked mechanically to release nucleotides, which subsequently increases the intracellular Ca²⁺ concentration [Ca²⁺]_i through activation of purinergic (P2) receptors. Cultured cells often show spontaneous [Ca²⁺]_i oscillations, a feature suggested to involve nucleotide signalling. In this study, fluo-4 loaded Madin–Darby canine kidney (MDCK) cells are used as a model for quantification and characterisation of spontaneous [Ca²⁺]_i increases in renal epithelia. Spontaneous [Ca²⁺]_i increases occurred randomly as single cell events. During an observation period of 1 min, 10.9 ± 6.7% (n = 23) of the cells showed spontaneous [Ca²⁺]_i increases. Spontaneous adenosine triphosphate (ATP) release from MDCK cells was detected directly by luciferin/luciferase. Scavenging of ATP by apyrase or hexokinase markedly reduced the [Ca²⁺]_i oscillatory activity, whereas inhibition of ecto-ATPases (ARL67156) enhanced the [Ca²⁺]_i oscillatory activity. The association between spontaneous [Ca²⁺]_i increases and nucleotide signalling was further tested in 132–1N1 cells lacking P2 receptors. These cells hardly showed any spontaneous [Ca²⁺]_i increases. Transfection with either hP2Y₆ or hP2Y₂ receptors revealed a striking degree of oscillations. Similar spontaneous [Ca²⁺]_i increases were observed in freshly isolated, perfused mouse medullary thick ascending limb (mTAL). The oscillatory activity was reduced by basolateral apyrase and substantially lower in mTAL from P2Y₂ knock out mice (0.050 ± 0.020 events per second, n = 8) compared to the wild type (0.147 ± 0.018 events per second, n = 9). These findings indicate that renal epithelia spontaneously release nucleotides leading to P2-receptor-dependent [Ca²⁺]_i oscillations. Thus, tonic nucleotide release is likely to modify steady state renal function. C. S. Geyti and E. Odgaard contributed equally to the publication.     

ATP increases [Ca2+]i and ion secretion via a basolateral P2Y-receptor in rat distal colonic mucosa

 Under resting conditions the mammalian distal colon is a NaCl-absorptive epithelium. NaCl absorption occurs at surface cells in colonic crypts. Intracellular Ca²⁺ or cAMP are important second messengers that activate NaCl secretion, a function that is most pronounced in crypt bases. In the present study we examined the effect of extracellular ATP on isolated crypts of rat distal colon using the fura-2 technique. Intracellular Ca²⁺ ([Ca²⁺]_i) was measured spectrofluorimetrically either by photon counting or video imaging. ATP reversibly increased [Ca²⁺]_i in crypt base cells with an EC₅₀ of 4.5 μmol/l (n = 11). This [Ca²⁺]_i increase was composed of an initial peak, reflecting intracellular store release, and a secondary plateau phase reflecting transmembrane influx. Digital video imaging revealed that agonist-induced [Ca²⁺]_i elevations were most marked at the crypt base. In the middle part of the crypt ATP induced smaller increases of [Ca²⁺]_i (peak and plateau) as compared to basal cells and in surface cells this [Ca²⁺]_i transient was even further reduced. Attempts to identify the relevant P₂-receptor demonstrated the following rank order of potency: 2MeS-ATP > ADP ≥ ATP >> AMP > UTP > AMP-PCP > adenosine. In Ussing chamber experiments ATP (1 mmol/l) functioned as a secretagogue, increasing transepithelial voltage (V _te) and equivalent short-circuit current (I _sc): ΔI _sc = –36.4 ± 5.4 μA/cm², n = 17. Adenosine itself (1 mmol/l) induced an increase of I _sc of similar magnitude to that induced by ATP: ΔI _sc = –55.1 ± 8.4 μA/cm², n = 9. The effect of adenosine, but not that of ATP, was fully inhibited by the A₁/A₂-receptor antagonist 8-(p-sulphophenyl)theophylline, 0.5 mmol/l, n = 4. Together these data indicate that: (1) basolateral ATP induces [Ca²⁺]_i in isolated rat colonic crypts and acts as a secretagogue in the distal rat colon; (2) a basolateral P2Y-receptor is responsible for this ATP-induced NaCl secretion; (3) the ability of ATP to increase I _sc in Ussing chamber experiments is not mediated via adenosine; and (4) the agonist-induced [Ca²⁺]_i signals are mostly located in the crypt base, which is the secretory part of the colonic crypt. Received: 17 September 1996 / Received after revision: 20 January 1997 / Accepted: 28 January 1997     

Ionic selectivity of the mechano-electrical transduction channels in the hair cells of the frog sacculus

In the isolated saccular macula of Ratio, esculenta extracellular hair cell receptor currents evoked by mechanical stimulation of the otolithic membrane were recorded under transepithelial voltage clamp conditions. The ionic selectivity of the mechano-electrical transduction channels of the hair cells was determined by examining the effects of different concentrations of Ca²⁺ and K⁺ in the apical solution on the transepithelial voltage at which the extracellular receptor current was zero (V_rev). Changing the concentration of Ca²⁺ from 0.26 mM to 0.026 and to 2.6 mM at a constant K⁺ concentration caused changes in V_rev of – 15 + 7 mV (mean±SD; n= 9) and 20±6mV (n= 13), respectively. The relative ionic permeabilities of the transduction channels were estimated from a modified Goldman, Hodgkin and Katz equation, assuming that 80% of the transepithelial resistance is located in the apical membranes of the hair cells. The permeability of the transduction channels for Ca²⁺ was found to be two orders of magnitude larger than that for K⁺. The measured effects on V_rev of changing the concentration of K⁺ at constant ionic strength and at different constant Ca²⁺ concentrations were well predicted by the same equation. These results indicate that the transduction channels of the frog saccular hair cells are highly selective to Ca²⁺.     

Ca2+-transients induced by K+ channel openers in isolated coronary capillaries

 To study the role of endothelial ATP-sensitive K⁺ channels in the regulation of vascular tone we examined the intracellular calcium concentration ([Ca²⁺]_i) in coronary capillaries consisting only of endothelial cells. Coronary capillary fragments were isolated enzymatically from the guinea-pig heart and [Ca²⁺]_i was determined by microfluorometry of fura-2 loaded cells. Low concentrations of the K⁺ channel opener diazoxide, which caused pronounced glibenclamide-sensitive hyperpolarization in capillaries, induced a rapid, transient rise in [Ca²⁺]_i followed by a sustained elevation of [Ca²⁺]_i (19 of 40 experiments). [Ca²⁺]_i in the endothelial cells increased from 32 ± 7 nM at rest to 66 ± 11 nM at the peak (n = 19). One third of the [Ca²⁺]_i-transients showed irregular oscillations of [Ca²⁺]_i. No significant difference in the [Ca²⁺]_i-response induced by 100 nM or 1 μM diazoxide was found. Similar results were obtained with the K⁺ channel opener rilmakalim. Simultaneous measurements of the membrane potential and [Ca²⁺]_i with fluorometric methods indicated that the hyperpolarization but not the [Ca²⁺]_i-transient could be repeatedly induced in a single capillary by the K⁺ channel openers. Electrophysiological recordings of the membrane potential using the ”perforated patch” method (n = 4), showed that rilmakalim (1 μM) induced hyperpolarization of capillaries towards the K⁺ equilibrium potential, confirming our fluorometric measurements. In conclusion, for the first time, these data indicate that K⁺ channel openers induce [Ca²⁺]_i-transients in microvascular endothelial cells. This raises the possibility that these drugs not only act as synthetic vasoactive factors via hyperpolarizing smooth muscle cells but also via NO release of microvascular endothelial cells. Interestingly, only 100 nM diazoxide was sufficient for a maximal response, suggesting the expression of a new type of K_ATP-channel in coronary capillaries characterised by high sensitivity to diazoxide. Received: 22 August 1997 / Received after revision and accepted: 7 November 1997     

Blockade of HERG channels expressed in Xenopus oocytes by external H+

 We have investigated the effect of external H⁺ concentration ([H⁺]_o)on the human-ether-a-go-go-related gene (HERG) current (I _HERG), the molecular equivalent of the cardiac delayed rectifier potassium current (I _Kr), expressed in Xenopus oocytes, using the two-microelectrode voltage-clamp technique. When [H⁺]_o was increased, the amplitude of the I _HERG elicited by depolarization decreased, and the rate of current decay on repolarization was accelerated. The activation curve shifted to a more positive potential at lower external pH (pH_o) values (the potential required for half-maximum activation, V _1/2, was: –41.8 mV, –38.0 mV, –33.7 mV, –26.7 mV in pH_o 8.0, 7.0, 6.6, 6.2, respectively). The maximum conductance (g _max) was also affected by [H⁺]_o: a reduction of 7.9%, 14.6%, and 22.8% was effected by decreasing pH_o from 8.0 to 7.0, 6.6, and 6.2, respectively. We then tested whether this pH effect was affected by the external Ca²⁺ concentration, which is also known to block HERG channels. When the extracellular Ca²⁺ concentration was increased from 0.5 mM to 5 mM, the shift in V _1/2 caused by increasing [H⁺]_o was attenuated, suggesting that these two ions compete for the same binding site. On the other hand, the decrease in g _max caused by increasing [H⁺]_o was not significantly affected by changing external Ca²⁺ levels. The results indicate that HERG channels are inhibited by [H⁺]_o by two different mechanisms: voltage-dependent blockade (shift of V _1/2) and the decrease in g _max. With respect to the voltage-dependent blockade, the interaction between H⁺ and Ca²⁺ is competitive, whereas for the decreasing g _max, their interaction is non-competitive. Received: 12 January 1999 / Received after revision: 15 February 1999 / Accepted: 16 February 1999     

Identification of a voltage- and calcium-dependent non-selective cation channel in cultured adult and fetal human nasal epithelial cells

The apical membranes of cultured human nasal epithelial cells from adults and fetuses were investigated with the patch-clamp technique. Amiloride-insensitive, calcium- and voltage-dependent, non-selective cation channels were found in 4% of the cell-attached, and 18% of the inside-out and outside-out patches (n=412). Multiple functional channels were present in more than 90% of these patches, with a mean of 3.9 channels per patch (n=55). The current-voltage relationship can be described by the Goldman equations and the single channel conductance was 20.1±0.3 pS (n=29) in adult and 20.7±0.4 pS (n=44) in fetal cells in symmetrical 150mM NaCl solutions. The channels were highly selective for cations: P_Na/P_Cl was 30 in adult and 45 in fetal experiments. They were equally permeable for K⁺ and Na⁺, somewhat less for Cs⁺, and impermeable for choline⁺ and tetraethylammonium⁺. The open probability was voltage dependent: it increased approximately 2-fold with 30mV depolarization in the potential range from −60mV to +60mV. The channels were activated by Ca²⁺ concentrations of about 10⁻⁴M at the cytoplasmic side, but were insensitive to extracellular Ca²⁺ and amiloride (10⁻⁴M). The non-selective cation channels found in apical membranes of cultured fetal nasal epithelial cells were not different from the adult ones.     

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pH_i) and intracellular Ca²⁺ activity ([Ca²⁺]_i) of HT₂₉ cells was investigated microspectrofluorimetrically using pH- and Ca²⁺- sensitive fluorescent indicators, [i.e. 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (V _m) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pK _a value. Trimethylamine (20 mmol/l) increased pH_i by 0.78 ± 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pH_i and a slow and incomplete recovery: pH_i stayed 0.26 ± 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pH_i. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca²⁺]_i transient which reached a peak value within 10–25 s and then slowly declined to a [Ca²⁺]_i plateau. The initial Δ[Ca²⁺]_i induced by trimethylamine (20 mmol/l) was 160 ± 15 nmol/l (n = 17). The [Ca²⁺]_i peak was independent of the Ca²⁺ activity in the bath solution, but the [Ca²⁺]_i plateau was significantly lower under Ca²⁺-free conditions and could be immediately interrupted by application of CO₂ (10%; n = 6), a manoeuvre to acidify pH_i in HT₂₉ cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca²⁺ stores completely abolished this [Ca²⁺]_i transient. Tetramethylamine led to higher [Ca²⁺]_i changes than the other amines tested and only this transient could be completely blocked by atropine (10⁻⁶ mol/l). Trimethylamine (20 mmol/l) hyperpolarized V _m by 22.5 ± 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 ± 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pH_i changes, release Ca²⁺ from intracellular, inositol-1,4,5-trisphosphate-sensitive Ca²⁺ stores and increase Ca²⁺ influx into HT₂₉ cells. The latter may be related to both the store depletion and the hyperpolarization. Received: 11 September 1995/Received after revision and accepted: 18 December 1995     

Extracellular zinc stimulates a calcium-activated chloride conductance through mobilisation of intracellular calcium in renal inner medullary collecting duct cells

We have used the perforated patch clamp and fura-2 fluorescence techniques to study the effect of extracellular Zn²⁺ on whole-cell Ca²⁺-activated Cl⁻ currents (I _CLCA) in mouse inner medullary collecting duct cells (mIMCD-3). I _CLCA was spontaneously active in 74% of cells under basal conditions and displayed time and voltage-independent kinetics and an outwardly rectifying current/voltage relationship (I/V). Addition of zinc chloride (10–400 μM) to the bathing solution resulted in a dose-dependent increase in I _CLCA with little change in Cl⁻ selectivity or biophysical characteristics, whereas gadolinium chloride (30 μM) and lanthanum chloride (100 μM) had no significant effect on the whole-cell current. Using fura-2-loaded mIMCD-3 cells, extracellular Zn²⁺ (400 μM) stimulated an increase in intracellular Ca²⁺ to an elevated plateau. The Zn²⁺-stimulated [Ca²⁺]_i increase was inhibited by thapsigargin (200 nM), the IP₃ receptor antagonist 2-aminoethoxydiphenyl borate (10 μM) and removal of bath Ca²⁺. Pre-exposure to Zn²⁺ (400 μM) markedly attenuated the ATP (100 μM)-stimulated [Ca²⁺]_i increase. These data are consistent with the hypothesis that extracellular Zn²⁺ stimulates an increase in [Ca²⁺]_i by a release of calcium from thapsigargin/IP₃ sensitive stores. A possible physiological role for a divalent metal ion receptor, distinct from the extracellular Ca²⁺-sensing receptor, in IMCD cells is discussed.     

Activation of non-selective cation conductance by carbachol in freshly isolated bovine ciliary muscle cells

 In smooth muscle cells freshly isolated from the bovine ciliary body, effects of carbachol (CCh) on the membrane potential and current were examined by the whole-cell clamp method. The resting membrane potential of the muscle cells used was –60 ± 1 mV (n=111). Extracellular application of CCh (2 μM) depolarized the cells to –15 ± 5 mV (n=50) with an apparent increase in membrane conductance. Under voltage-clamp conditions, CCh (2 μM) evoked an inward current which exhibited inward-going rectification and reversed the polarity at about 0 mV. Removal of Na⁺ from the external solution caused a reduction of the amplitude of the current and a shift of the reversal potential to the negative direction. CCh was able to elicit an inward current even under a condition where Ca²⁺ was the only cation producing an inwardly directed electrochemical gradient. The current was not affected by verapamil or by tetrodotoxin. The CCh-induced current was inhibited by antimuscarinic agents with the affinity sequence: atropine ≈4–DAMP >> pirenzepine > AF-DX116, indicating that the response is mediated by a muscarinic cholinoceptor that belongs to the M₃-subtype. Unlike the non-selective cation channel current in intestinal smooth muscles, which is activated by elevation of the intracellular Ca²⁺ concentration ([Ca²⁺]_i), the current of the ciliary muscle was inactivated when the [Ca²⁺]_i was increased. The conductance, which admits Ca²⁺, may serve as a pathway for Ca²⁺ entry required for contraction. Received: 2 December 1996 / Received after revision: 7 January 1997 / Accepted: 8 January 1997