Regulation of ionic currents by protein kinase A and intracellular calcium in outer hair cells isolated from the guinea-pig cochlea

 Two prominent potassium currents, termed I _K and I _K,n, and a cation current are found in outer hair cells (OHCs) of the guinea-pig cochlea. We report here whole-cell recordings which indicate that the currents are regulated by intracellular factors. 8-bromo-cAMP (500 μM), a membrane-permeable cAMP analogue, activated potassium currents in OHCs in both apical and basal turns of the cochlea. In OHCs from the cochlear apex, the drug effect was largest at potentials positive to –40 mV, indicating I _K as the target. In short cells from the cochlear base, both I _K and I _K,n were affected. The effects of 8-bromo-cAMP could be blocked by the presence of 1 μM H-89 (a protein kinase A inhibitor) in the patch pipette solution. Extracellular application of 10 nM okadaic acid, a protein phosphatase inhibitor, also activated both potassium currents. Currents were also modulated by intracellular calcium. I _K was activated in long cells by photorelease of calcium from the caged compound nitr5. Cation current activation required calcium release by photolysis of DM-nitrophen, a compound releasing more calcium. The results show that OHC potassium channels are regulated by background phosphorylation through protein kinase A and dephosphorylation by protein phosphatase. Cellular calcium also activates I _K and the cation channel, but with different sensitivities. Received: 1 September 1998 / Received after revision: 21 October 1998 / Accepted: 22 October 1998     

Activation of nonselective cation channels in the basolateral membrane of rat distal colon crypt cells by prostaglandin E2

Ion channels in the basolateral membrane of colonic crypts were investigated with the patch-clamp technique during stimulation of secretion. Intact crypts were isolated from rat distal colon and the cell potential was recorded by addition of nystatin to the pipette solution. The cell resting potential in the base of the crypt was –74±1 mV (n=90). Addition of 100 M carbachol to the bath resulted in a transient hyperpolarization by 9 mV, which was probably due to the opening of basolateral K⁺ channels. In contrast, application of prostaglandin E₂ (PGE₂, 1 nM–1 M) caused a dose-dependent depolarization in the base of the crypt. With 1 M PGE₂ cells depolarized from -74±1 to –27±2 mV (n=26). Cell potential recordings in the midcrypt showed only a slight and transient depolarization after application of PGE₂, whereas cells close to the surface of the crypt had no response. In the base of the crypt the PGE₂-induced depolarization could be completely inhibited by addition of 50 M flufenamic acid, a known blocker of nonselective cation channels. After substitution of all monovalent cations by N-methyl-D-glucamine in the bath, PGE₂ had no significant effect on the cell potential. Cell-attached experiments with no nystatin in the patch pipette revealed the activation of ion channels in the basolateral membrane after application of PGE₂. After excision of the membrane patch, these channels could be identified as nonselective cation channels. Experiments involving substitution of the bath solution showed that the channel is impermeable for Cl^– and scarcely permeable for Ca²⁺ ions. The permeability sequence for monovalent cations, as calculated from reversal potentials, is NH ₄ ⁺ >Na⁺=K⁺>Rb⁺=Li⁺TRIS⁺=NMDG⁺. Single channels are completely inhibited by flufenamic acid (50 M), mefenamic acid (200 M), as well as by 3, 5-dichlorodiphenylamine-2-carboxylate. In conclusion, PGE₂ activates nonselective cation channels in the basolateral membrane of cells in the base of colonic crypts. It is suggested that this mechanism initiates the secretion of K⁺ ions. Na⁺ influx through the nonselective cation channel will stimulate the Na⁺/K⁺ pump and active uptake of K⁺ at the basolateral side. K⁺ can leave the cell at the luminal side through K⁺-selective channels.     

The outer hair cell motor in membrane patches

 We have used patch-clamp techniques to record the charge movement associated with motility in patches of basolateral membrane from isolated outer hair cells. Charge movement has been measured from the voltage-dependent capacitance. Using 3 to 4 MΩ pipettes with tip diameters of 3 μm the measured maximum voltage-dependent capacitance was 56 ± 6 fF at –36 mV when the resting membrane potential was –20 mV. The calculated total charge movement was 5.6 ± 0.6 fC (n = 13) and the inferred density of univalent motor elements was 8400/μm². Negative pressure (applied via the pipette) increased membrane tension and shifted the capacitance peak to depolarised potentials. Under conditions of isotropic membrane stress there was no change in the peak measured capacitance in contrast to that measured in previous whole-cell recordings. Received: 19 December 1996 / Received after revision: 21 February 1997 / Accepted: 5 March 1997     

Protein kinase C mediates the desensitization of CCh-activated nonselective cationic current in guinea-pig gastric myocytes

 The possibility of the protein kinase C (PKC) pathway being a mechanism underlying the desensitization of carbachol- (CCh-)activated nonselective cationic current (I _CCh) was investigated in a study of guinea-pig gastric myocytes. Using the conventional whole-cell patch-clamp technique with symmetrical CsCl-rich solution in pipette and bath, I _CCh was induced by bath application of 50 μM CCh. With 0.5 mM EGTA [ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid] in the pipette solution (0.5 mM [EGTA]_i), I _CCh decayed spontaneously (desensitization of I _CCh) to around 20% within 10 min. Desensitization of I _CCh was significantly attenuated with 2 mM [EGTA]_i. At a concentration of 20 μM OAG (1-oleoyl-2-acetyl-sn-glycerol), a PKC activator, inhibited I _CCh at 0.5 mM [EGTA]_i but far less at 2 mM [EGTA]_i (18% and 81% of control, respectively). The same cationic current induced by intracellular guanosine-5′-O-(3-thiotriphosphate) (GTP[γ-S]) was not inhibited by OAG with 0.5 mM [EGTA]_i. The pretreatment of gastric myocytes with PKC inhibitors, either 1 μM chelerythrine or 1 μM peptide inhibitor, attenuated the desensitization of I _CCh. [Ca²⁺]_i was also measured by single cell microfluorometry using fura-2. Under CCh stimulation with 2 mM [EGTA]_i, [Ca²⁺]_i did not increase above 100 nM while it increased to around 260 nM with 0.5 mM [EGTA]_i. These results suggest that the desensitization of I _CCh is partly due to the Ca²⁺-dependent PKC pathway in guinea-pig gastric myocytes. Received: 27 August 1997 / Received after revision: 2 January 1998 / Accepted: 21 January 1998     

Inhibitory effect of phorbol 12, 13 dibutyrate on carbachol-activated nonselective cationic current in guinea-pig gastric myocytes

 The effect of protein kinase C (PKC) on carbachol (CCh)-activated nonselective cationic current (I _CCh) was investigated in guinea-pig gastric myocytes using a PKC activator, phorbol 12, 13 dibutyrate (PDBu). Pretreatment with 1 μ M PDBu suppressed I _CCh by 96.5 ± 2.9% (n = 14) in a reversible manner in nystatin-perforated mode. In the presence of 1 μM chelerythrine , a PKC inhibitor, inhibition of I _CChby PDBu was not seen. In whole-cell mode, the inhibition of I _CCh by PDBu was dependent on intracellular MgATP. In the presence of MgATP in the pipette, PDBu decreased I _CCh by 98.8 ± 1.2% (n = 5) as was observed in nystatin-perforated mode. However, PDBu had little effect on I _CCh in the absence of MgATP in the pipette; the extent of inhibition was 12.7 ± 4.3% (n = 8). PDBu also suppressed the generation of cationic current induced by intracellularly perfused GTP[γS]. In the PDBu-pretreated group (n = 9) and PDBu-untreated control group (n = 6), GTP[γS]-induced currents were 6.7 ± 2.4 pA and 236 ± 23 pA, respectively. These results suggest that PKC modulates I _CCh at postreceptor sites via protein phosphorylation. Received: 4 April 1997 / Received after revision: 27 June 1997 / Accepted: 3 June 1997     

Suppression of the carbachol-activated nonselective cationic current by antibody against alpha subunit of Go protein in guinea-pig gastric myocytes

 In this study, we investigated which subtype of GTP-binding protein (G protein) is related to muscarinic activation of nonselective cation (NSC) channels in gastric smooth muscle. Inward cationic current was activated by the application of 50 μM carbachol (I _CCh) at a holding potential of –60 mV with the same CsCl-rich solution in both pipette and bath. The same cationic current as I _CCh was slowly activated by the dialysis of guanosine 5′-O-(3-thiotriphosphate) (GTP[γ-S]) through the pipette. Since it is known that pertussis toxin pretreatment can block I _CCh, antibodies (Abs) against G_α,i (anti-G_α,i) or G_α,o (anti-G_α,o) were tested. Activation of I _CCh was blocked by the addition of anti-G_α,o. However, anti-G_α,i Abs had no significant effect on I _CCh.The expression of G_α,o in guinea-pig gastric smooth muscle was confirmed by Western immunoblot analysis. These results suggest that G_o-type protein may mediate signals from the muscarinic receptor to NSC channel in guinea-pig gastric myocytes. Received: 16 January 1998 / Received after revision: 16 April 1998 / Accepted: 20 April 1998     

Effects of myosin light chain kinase inhibitors on carbachol-activated nonselective cationic current in guinea-pig gastric myocytes

 The effects of myosin light chain kinase inhibitors on muscarinic stimulation-activated nonselective cationic current (I _CCh) in guinea-pig gastric antral myocytes were studied using the whole-cell patch-clamp technique. I _CCh was induced by carbachol (CCh, 50 μM) at a holding potential of –30 mV or –60 mV. ML-7, a chemical inhibitor of myosin light chain kinase (MLCK), inhibited I _CCh concentration dependently in a reversible manner (53 ± 8.6% at 1 μM, mean ± SE, n = 11). In addition, amplitudes of I _CCh were only 37 ± 2.7% of the daily control values following the addition of a peptide inhibitor of MLCK to the pipette solution. On the other hand, ML-7 had an inhibitory effect on voltage-operated Ca²⁺ channel current. The peak value of Ba²⁺ current at 0 mV was reduced to 35 ± 7.4% (n = 9) by 3 μM of ML-7. As I _CCh is known to have an intracellular Ca²⁺ dependence, we tried to exclude the possibility that ML-7 inhibited I _CCh indirectly via suppression of Ca²⁺ current and the similar inhibitory effects of ML-7 on I _CChwere confirmed under the following conditions: (1) clamp of membrane potential at –60 mV; (2) clamp of intracellular [Ca²⁺] to 1 μM by 10 mM BAPTA; (3) pre-inhibition of Ca²⁺ channel by verapamil. Different from the effects on I _CCh, ML-7 barely inhibited the same cationic current induced by guanosine 5’-O-(3-thiotriphosphate) (GTP[γS], 0.2 mM) in the pipette solution. These results suggest that a Ca²⁺/calmodulin-MLCK-dependent pathway can modulate the activation of I _CCh in guinea-pig gastric antral myocytes. Received: 21 January 1997 / Received after revision and accepted: 2 April 1997     

The nonselective cation channel in the basolateral membrane of rat exocrine pancreas

Nonselective Ca²⁺-sensitive cation channels in the basolateral membrane of isolated cells of the rat exocrine pancreas were investigated with the patch clamp technique. With 1.3 mmol/l Ca²⁺ on the cytosolic side, the mean openstate probabilityP _o of one channel was about 0.5. In insideout oriented cell-excised membrane patches the substances diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 3,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) were applied to the cytosolic side. These compounds inhibited the nonselective cation channels by increasing the mean channel closed time (slow block). 100 mol/l of NPPB or DPC decreasedP _o from 0.5 (control conditions) to 0.2 and 0.04, respectively, whereas 100 mol/l of DCDPC blocked the channel completely. All effects were reversible. 1 mmol/l quinine also reducedP _o, but in contrast to the abov mentioned substances, it induced fast flickering. Ba²⁺ (70 mmol/l) and tetraethylammonium (TEA⁺; 20 mmol/l) had no effects. We investigated also the stilbene disulfonates 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and 4,4-dinitro-2,2-stilbenedisulfonate (DNDS). 10 mol/l SITS applied to the cytosolic side increasedP _o from 0.5 to 0.7 and with 100 mol/l SITS the channels remained nearly permanently in its open state (P _o1). A similar activation of the channels was also observed with DIDS and DNDS. These effects were poorly reversible. The stilbene disulfonates acted by increasing the channel mean open time. When the channel was inactivated by decreasing bath Ca²⁺ concentration to 0.1 mol/l, addition of 100 mol/l of SITS had no effect. Similarly, reducing bath Ca²⁺ concentration from 1.3 mmol/l in presence of 100 mol/l SITS (channels are maximally activated) to 0.1 mol/l, inactivated the channels completely. These results demonstrate, that SITS can only activate the channels in the presence of Ca²⁺. SITS had no effects, when applied to the extracellular side in outside out patches. In summary, the substances DPC, NPPB and DCDPC inhibit nonselective cation channels, where DCDPC has the most potent and NPPB the smallest effect; whereas SITS, DIDS and DNDS activate the channel when applied from the cytosolic side in the presence of Ca²⁺ ions.     

Role of calmodulin in the activation of carbachol-activated cationic current in guinea-pig gastric antral myocytes

In mammalian gastrointestinal myocytes, it is known that muscarinic stimulation activates nonselective cation channels through a G-protein and a Ca²⁺dependent pathway. We recorded inward cationic currents following application of carbachol (I _CCh) to guinea-pig gastric myocytes, which were held at –20 mV using the whole-cell patch-clamp method. I _CCh was suppressed by nicardipine or removal of Ca²⁺ from the bath solution. The peak value of inward current induced by repetitive applications of carbachol (CCh) decreased progressively (run-down phenomenon). This run-down was significantly alleviated by the addition of calmodulin to the pipette solution (0.15 mg/ml) or by using the perforated-patch whole-cell voltage-clamp technique. Moreover, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide], a calmodulin antagonist, was a reversible inhibitor of I _CCh. However, W-7 had only a weak inhibitory effect on the same cationic current which was induced by guanosine 5-O-(3-thiotriphosphate) (GTP[S] 0.2 mM) in the pipette solution. This GTP[S]-induced cationic current was still markedly suppressed by the Ca²⁺-free bath solution. W-7 itself had a weak inhibitory effect on voltage-operated Ca²⁺ channels as well as the effects on I _CCh. These data suggest that multiple Ca²⁺-dependent pathways are involved in the activation of CCh-gated cation channels in guinea-pig antral myocytes and a Ca²⁺/calmodulin-dependent pathway would be one of them.     

庆大霉素中毒后豚鼠耳蜗毛细胞的再生

目的：通过庆大霉素中毒后不同时间豚鼠耳蜗毛细胞变化的观察，研究哺乳动物耳蜗毛细胞受损后能否再生。方法：将60只豚鼠随机分成庆大霉素组和生理盐水对照组。应用扫描电镜（SEM）技术并结合听脑干反应（ABR）测试，观察耳蜗毛细胞情况和ABR阀值变化。结果：庆大霉素组耳蜗毛细胞在30 d时ABR阈值有明显恢复，但未达到正常水平，同时耳蜗第三转有新生的静纤毛出现。结论：庆大霉素损伤后的毛细胞可以再生。