Sensitivity to dihydropyridines, ω-conotoxin and noradrenaline reveals multiple high-voltage-activated Ca2+ channels in rat insulinoma and human pancreatic β-cells

High-voltage-activated (HVA) Ba²⁺ currents of rat insulinoma (RINm5F) and human pancreatic -cells were tested for their sensitivity to dihydropyridines (DHPs), -conotoxin (-CgTx) and noradrenaline. In RINm5F cells, block of HVA currents by nimodipine, nitrendipine and nifedipine was voltage- and dose-dependent (apparent K _D<37 nM) and largely incomplete even at saturating doses of DHPs (mean 53%, at 10 M and 0 mV). Analysis of slow tail currents in Bay K 8644-treated cells indicated the existence of Bay K 8644-insensitive channels that turned on at slightly more positive voltages and deactivated more quickly than Bay K 8644-modified channels. DHP Ca²⁺ agonists and antagonists in human -cells had similar features to RINm5F cells except that DHP block was more pronounced (76%, at 10 M and 0 mV) and Bay K 8644 action was more effective, suggesting a higher density of L-type Ca²⁺ channels in these cells. In RINm5F cells, but not in human -cells, DHP-resistant currents were sensitive to -CgTx. The toxin depressed 10–20% of the DHP-resistant currents sparing a residual current (25–35%) with similar voltage-dependent characteristics and Ca²⁺/Ba²⁺ permeability. Noradrenaline (10 M) exhibited different actions on the various HVA current components: (1) it prolonged the activation kinetics of -CgTx-sensitive currents, (2) it depressed by about 20% the size of DHP-sensitive currents, and (3) it had little or no effects on the residual DHP- and -CgTx-resistant current although intracellularly applied guanosine 5-O-(3-thiotriphosphate) (GTP--S) prolonged its activation time course. The first action was clearly voltage-dependent and most evident in RINm5F cells that displayed neuronal-like processes. The second was observed more frequently, was voltage-independent and fully blocked by saturating doses of nifedipine (10 M). Both actions were prevented by intracellular perfusion with guanosine 5-O-(2-thiodiphosphate) (GDP--S). Our data suggest that beside a majority of L-type channels, RINm5F and human pancreatic -cells may express a variable fraction of DHP-insensitive channels that may be involved in the control of insulin secretion during -cell activity.     

Dual effect of nitric oxide on ATP-sensitive K+ channels in rat pancreatic β cells

We have previously shown that NO has stimulatory and inhibitory effects on insulin secretion at low and high concentrations, respectively. The present study investigated effects of NO on K_ATP channels of rat β cells by patch clamp analysis to elucidate the mechanism for the dual effect. NOC7 at 0.5 μM suppressed K_ATP channels activated by diazoxide in the cell-attached and perforated whole-cell modes but failed to suppress them in the inside-out mode. The inhibitory effect in the cell-attached mode was abolished by the soluble guanylate cyclase inhibitor ODQ and by the protein kinase G inhibitor KT5823. Moreover, 0.5 μM NOC7 failed to suppress the channel activity in the presence of the mitochondrial uncoupler FCCP. In contrast, 10 μM NOC7 activated K_ATP channels in the cell-attached and perforated whole-cell modes, although it had no effect on the channels in the inside-out mode. The K_ATP currents evoked by 10 μM NOC7 in the cell-attached mode were not inhibited by ODQ. The dual effect of NOC7 at 0.5 and 10 μM was observed in the same patch. Taken together, these results suggest that low-concentration NO exerts an inhibitory effect on K_ATP channels of β cells, which is induced through the cGMP/protein kinase G pathway, whereas high-concentration NO activates K_ATP channels through the mechanism independent of cGMP.     

cAMP-independent decrease of ATP-sensitive K+ channel activity by GLP-1 in rat pancreatic β-cells

Using the patch-clamp method, we studied the mechanism of depolarization of rat pancreatic beta-cells induced by glucagon-like peptide 1 (7-36) amide (GLP-1). GLP-1 caused depolarization in a concentration-dependent manner (0.2-100 nM). Exendin (9-39) amide, a GLP-1 receptor antagonist, prevented the GLP-1-induced depolarization. GLP-1 reduced tolbutamide-sensitive membrane currents evoked by voltage ramps from -90 to -50 mV, recorded in the perforated whole-cell configuration, suggesting that GLP-1 decreased the activity of the ATP-sensitive K+ channel (KATP). This GLP-1 effect was prevented by exendin (9-39) amide. In cells treated with Rp-cAMPS, an inhibitor of the cAMP-dependent protein kinase (PKA), GLP-1 still caused depolarization and reduced the whole-cell membrane current through KATP. Examined in the cell-attached configuration, 20 nM GLP-1, applied out of the patch, had little effect on KATP activity. In the inside-out configuration, the open time probability and the single-channel conductance of KATP in the absence of ATP inside the membrane were unaffected by the presence of 20 nM GLP-1 in the pipette. In both conditions, application of ATP to the inside of the membrane reduced KATP activity. The half-maximal concentrations (ki) of ATP were 11.6 microM without and 5.6 microM with 20 nM GLP-1 in the pipette (P<0.05). The values of the Hill coefficient (h) were 1.03 without and 1.01 with GLP-1. We conclude that GLP-1 reduces KATP activity by elevating the sensitivity of KATP to ATP, resulting in depolarization of pancreatic beta-cells. This GLP-1 action is independent of the cAMP signalling pathway.     

Phosphorylation-regulated low-conductance Cl− channels in a human pancreatic duct cell line

A low-conductance Cl^– channel has been identified in the apical membrane of the human pancreatic duct cell Capan-1 using patch-clamp techniques. Cell-attached channels were activated by the vasoactive intestinal polypeptide (VIP, 0.1 mol/l), dibutyryl-adenosine 3,5-cyclic monophosphate (db-cAMP, 1 mmol/l), 8-bromo adenosine 3,5-cyclic monophosphate (8-BrcAMP, 1 mmol/l), 3-isobutyl-1-methyl-xanthine (IBMX, 100 mol/l) and forskolin (10 mol/l). No channel activity was observed in non-stimulated control cells. In both cell-attached and excised inside-out patches, the channel had a linear current/voltage relationship and a unitary conductance of 9 pS at 23°C and 12 pS at 37°C. Its opening probability was not voltage dependent although pronounced flickering was induced at negative potentials. Anionic substitution led to the selectivity sequence Cl^–>I^–>HCO₃ ^–>gluconate. In insideout excised patches, the channel activity declined spontaneously within a few minutes. Reactivation of silent excised channels was achieved by adding protein kinase A (PKA, in the presence of ATP, cAMP and Mg²⁺). Conversely, active channels were silenced in the presence of alkaline phosphatase. The PKA-activated Cl^– channel was 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS, 100 mol/l) and 4-acetamido-4-isothiocyanatostilbene-2, 2-disulphonic acid (SITS, 100 mol/l) insensitive, but was blocked by diphenylamine-2-carboxylic acid (DPC, 100 mol/l). These results demonstrate that the apical low-conductance Cl^– channel in Capan-1 is regulated on-cell by VIP receptors via cAMP and off-cell by PKA and phosphatases. They provide evidence that this channel is closely related to the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel.     

Modification of K-ATP channels in pancreatic β-cells by trypsin

The inside-out configuration of the patchclamp method was used to study the effects of trypsin on the activity of ATP-sensitive potassium (K-ATP) channels from isolated mouse pancreatic -cells. Trypsin (20 g/ml) irreversibly enhanced channel activity around twofold by reducing the interburst intervals without altering the burst kinetics. No effect on the single channel conductance or the inward rectification produced by internal Mg²⁺ was observed: however, the protease did reduce the inhibitory effect of Mg²⁺ on channel activity. Trypsin both prevented rundown of K-ATP channel activity and reactivated the channels after complete rundown. These effects of trypsin were absent in the presence of trypsin inhibitor. The protease also reduced the inhibitory effect of ATP on channel activity, increasing the dissociation constant from 7 to 49 M. Trypsin removed the activating effect of ADP (0.1 mmol/l) on channel activity and reduced the inhibitory effect of tolbutamide (0.5 mmol/l). Carboxypeptidase A did not activate K-ATP channels in excised patches, although it was able to slightly reactivate channels after complete rundown, whereas chymotrypsin increased K-ATP channel activity but it did not produce reactivation. The effects of papain were similar to those of trypsin.     

A Gi1–2-protein is required for α2A-adrenoceptor-induced stimulation of voltage-dependent Ca2+ channels in rat portal vein myocytes

In rat portal vein myocytes, _2A-adrenoceptors activate voltage-dependent Ca²⁺ channels via a transduction pathway requiring protein kinase C activation mediated by a pertussis-toxin-sensitive G-protein. As revealed by the use of antibodies directed against the different -subunits expressed in portal vein myocytes, we show that the clonidine-induced stimulation of voltage-dependent Ca²⁺ channels is mainly mediated by a G_i1–2-protein.     

Coupling of exocytosis to depolarization in rat pancreatic islet β-cells: effects of Ca2+, Sr2+ and Ba2+-containing extracellular solutions

Summary Using rat -cells we present evidence that Sr²⁺ and Ba²⁺, like Ca²⁺, support depolarization-induced increases in membrane capacitance which reflect insulin granule exocytosis. Even with identical total charge entry, Sr²⁺ and Ba²⁺ are 3–5 and 20-fold less effective than Ca²⁺ in supporting release. While exocytosis supported by Sr²⁺ is graded with cation entry and complete within 250ms of depolarization, exocytosis supported by Ba²⁺ begins abrupty after a threshold of charge entry and continues for many seconds. Ba²⁺-supported release continues in the presence of greatly enhanced cytosolic Ca²⁺ buffering, arguing against release of Ca²⁺ from stores as its principal action. These results suggest that Sr²⁺ and Ba²⁺ support exocytosis largely by binding to Ca²⁺-dependent release-activating sites, though with less affinity than Ca²⁺.     

Bursting electrical activity in pancreatic β-cells: evidence that the channel underlying the burst is sensitive to Ca2+ influx through L-type Ca2+ channels

In glucose-stimulated pancreatic -cells, the membrane potential alternates between a hyperpolarized silent phase and a depolarized phase with Ca²⁺ action potentials. The molecular and ionic mechanisms underlying these bursts of electrical activity remain unknown. We have observed that 10.2–12.8 mM Ca²⁺, 1 M Bay K 8644 and 2 mM tetraethylammonium (TEA) trigger bursts of electrical activity and oscillations of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the presence of 100 M tolbutamide. The [Ca²⁺]_i was monitored from single islets of Langerhans using fura-2 microfluorescence techniques. Both the high-Ca²⁺ and Bay-K-8644 evoked [Ca²⁺]_i oscillations overshot the [Ca²⁺]_i recorded in tolbutamide. Nifedipine (10–20 M) caused an immediate membrane hyperpolarization, which was followed by a slow depolarization to a level close to the burst active phase potential. The latter depolarization was accompanied by suppression of spiking activity. Exposure to high Ca²⁺ in the presence of nifedipine caused a steady depolarization of approximately 8 mV. Ionomycin (10 M) caused membrane hyperpolarization in the presence of 7.7 mM Ca²⁺, which was not abolished by nifedipine. Charybdotoxin (CTX, 40–80 nM), TEA (2 mM) and quinine (200 M) did not suppress the high-Ca²⁺-evoked bursts. It is concluded that: (1) the channel underlying the burst is sensitive to [Ca²⁺]_i rises mediated by Ca²⁺ influx through L-type Ca²⁺ channels, (2) both the ATP-dependent K⁺ channel and the CTX and TEA-sensitive Ca²⁺-dependent K⁺ channel are highly unlikely to provide the pacemaker current underlying the burst. We propose that the burst is mediated by a distinct Ca²⁺-dependent K⁺ channel and/or by [Ca²⁺]_idependent slow processes of inactivation of Ca²⁺ currents.     

The Ba2+ block of the ATP-sensitive K+ current of mouse pancreaticβ-cells

We studied the block of whole-cell ATP-sensitive K⁺ (K_ATP) currents in mouse pancreatic-cells produced by external Ba²⁺. Ba²⁺ produced a time- and voltage-dependent block of K_ATP currents, both the rate and extent of the block increasing with hyperpolarization. With 5.6 mM [K⁺]_o, the relationship between the steady-state KATP current and [Ba²⁺]_o, was fit by the Hill equation with aK _d of 12.5 ± 2.8 M at –123 mV and of 0.18 ± 0.02 mM at –62 mV The Hill coefficient (n) was close to 1 at all potentials indicating that binding of a single Ba²⁺ ion is sufficient to block the channel. When [K⁺]_o was raised to 28 mM the K_d was little changed (12.4 ± 4.1 gM at –123 mV 0.27 ± 0.05 mM at –62 mV) and n was unaffected, suggesting that K⁺ does not interact with the Ba²⁺ binding site. The kinetics of Ba²⁺ block were slow, 10 M Ba²⁺ blocking the K_ATP current with a time constant of 20 ms at –123 mV in 28 mM [K⁺]_o. The blocking rate constant was calculated as 1.7 mM^–1 ms^–1 and the unblocking rate as 0.02 ms^–1, at –123 mV The data are discussed in terms of a model in which Ba²⁺ binds to a site at the external mouth of the channel to inhibit the K_ATP channel.     

Inhibition of voltage-dependent Ca2+ channels via α2-adrenergic and opioid receptors in cultured bovine adrenal chromaffin cells

Adrenal chromaffin cells secrete catecholamindes and opioids. The effects of these agents on whole-cell Ca²⁺ channel currents were studied, using bovine adrenal chromaffin cells kept in short term culture. Ca²⁺ channel currents recorded during voltageclamp pulses from a holding potential of –80 mV to 0 mV were reversibly reduced by 10 M epinephrine (in the presence of 1 M propranolol) or 5 M of the synthetic opioid, d-Ala²-d-Leu⁵-enkephalin (DADLE) by approximately 35% and 25%, respectively. The inhibitory action of epinephrine was mimicked by clonidine, reduced by yohimbine but not affected by prazosin. The DADLE-induced reduction of the Ca²⁺ channel current was antagonized by naloxone. The dihydropyridine (+)PN 200-110 (5 M) reduced the Ca²⁺ channel current by approximately 40%; the Ca²⁺ channel current inhibited by (+)PN 200-110 was not further reduced by epinephrine. Intracellular infusion of guanosine-5-O-(2-thiodiphosphate) and pretreatment of cells with pertussis toxin abolished the inhibitory effect of both epinephrine and DADLE. In membranes of adrenal chromaffin cells, four pertussis-toxin-sensitive G-proteins were identified, including G_i1, G_i2, G_o1 and another G_o subtype, possibly G_o2. The data show that activation of ₂-adrenergic and opioid receptors causes an inhibition of dihydropyridine-sensitive Ca²⁺ channels in adrenal chromaffm cells. These inhibitory modulations are mediated by pertussis-toxin-sensitive G-proteins and may represent a mechanism for a negative feedback signal by agents released from the adrenal medulla.     

Dysregulation of calcium channels decreases parasecretion in pancreatic β-cells in rats born small for gestational age

Objective: To investigate the role of intrauterine malnourishment in the development and function of pancreatic islet β-cells. Methods: Whole-cell patch clamping was used to record voltage-gated calcium channel (VGCC)-mediated currents. Insulin secretion was detected by measuring capacitance using a sequence of sine wave stimuli. VGCC currents and insulin secretion were measured in the small for gestational age (SGA) group treated with human recombinant growth hormone (hGH). Results: The membrane capacitance in the SGA group (6.4?±?0.9?fF/Pf) was significantly reduced. Calcium current density and peak current density in the SGA group were also markedly decreased, whereas other measurements of calcium channels were unaltered. Treatment with hGH significantly rescued the membrane capacitance, whereas calcium channels were not affected. Conclusion: Our data suggest that decreased β-cell secretion is caused by a decreased expression of calcium channels and reduced calcium currents. hGH restores β-cell secretion in SGA animals, possibly independently of VGCC.     

Long-term modulation of Na+ and K+ channels by TGF-��1 in neonatal rat cardiac myocytes

Previous work shows that transforming growth factor-β1 (TGF-β1) promotes several heart alterations, including atrial fibrillation (AF). In this work, we hypothesized that these effects might be associated with a potential modulation of Na(+) and K(+) channels. Atrial myocytes were cultured 1-2?days under either control conditions, or the presence of TGF-β1. Subsequently, Na(+) (I(Na)) and K(+) (I(K)) currents were investigated under whole-cell patch-clamp conditions. Three K(+) currents were isolated: inward rectifier (I(Kin)), outward transitory (I(to)), and outward sustained (I(Ksus)). Interestingly, TGF-β1 decreased (50%) the densities of I(Kin) and I(Ksus) but not of I(to). In addition, the growth factor reduced by 80% the amount of I(Na) available at -80?mV. This effect was due to a significant reduction (30%) in the maximum I(Na) recruited at very negative potentials or I(max), as well as to an increased fraction of inactivated Na(+) channels. The latter effect was, in turn, associated to a -7?mV shift in V(1/2) of inactivation. TGF-β1 also reduced by 60% the maximum amount of intramembrane charge movement of Na(+) channels or Q(max), but did not affect the corresponding voltage dependence of activation. This suggests that TGF-β1 promotes loss of Na(+) channels from the plasma membrane. Moreover, TGF-β1 also reduced (50%) the expression of the principal subunit of Na(+) channels, as indicated by western blot analysis. Thus, TGF-β1 inhibits the expression of Na(+) channels, as well as the activity of K(+) channels that give rise to I(Ksus) and I(Kin). These results may contribute to explaining the previously observed proarrhythmic effects of TGF-β1.     

Evidence for the involvement of K+ channels and K+-Cl– cotransport in the regulatory volume decrease of newborn rat cardiomyocytes

In order to delineate ion transport mechanisms involved in volume homeostasis of freshly isolated newborn rat ventricular myocytes, we investigated the effects of ion substitutions and pharmacological maneuvers upon (1) isotonic volume, (2) hypotonically induced initial swelling, and (3) the subsequent regulatory volume decrease (RVD), as determined by electronic cell sizing. Cardiomyocytes exposed to hypotonic medium (176 mosmol/l) swelled by 51+/-1% of isotonic volume, and they underwent a partial regulatory volume decrease (RVD), reaching a maximum regulation after 30 min (51+/-1% of initial swelling), with a half-time (t1/2) of 6+/-1 min (n=60). RVD was associated with significant cardiomyocyte K+ loss (12+/-4% at 5 min and 15+/-2% of isotonic control after 30 min: n=6, P<0.001), 71% of which was Cl- dependent (P<0.05). Within the 30-min experimental time frame, ouabain, a Na+/K+ pump inhibitor, had no significant effect on RVD (despite an inhibitory trend), cell swelling or on isotonic volume (n=6). Bumetanide (50 microM), a Na+-K+-Cl- co-transport blocker, induced a significant reduction of isotonic cell volume (3+/-2%, n=6. P<0.05), potentiated initial swelling by 16+/-1% (n=8, P<0.02), and it partially inhibited RVD (24+/-11% at 30 min, n=6), whereas Na+ omission had no significant effect on isotonic cell volume, cell swelling or RVD. The effects of bumetanide on initial swelling and RVD were prevented by gadolinium ion (10 microM), a stretch-activated cation channel blocker (n=5). Quinidine (500 microM), a non-selective Ca(2+)-activated potassium channel blocker with no side-effects on K(+)-Cl(-) cotransport, did not modify initial cell swelling, but inhibited RVD (50+/-3% at 5 min, n=9, P<0.01; 22+/-3% at 30 min), an effect which was cancelled by external Ca2+ chelation with EGTA (n=5), and reproduced by tetraethylammonium (TEA, 20 mM), another K+ channel blocker. 4,4'-Diisothiocyanatostilbene 2,2'-disulfonic acid (DIDS, 100 microM), a non-selective swelling-activated Cl- channel blocker with marginal side-effects on K(+)-Cl(-)cotransport, did not modify initial swelling, but inhibited RVD to the same extent as quinidine (42+/-3% at 5 min, and 23+/-3% at 30 min, n=15, P<0.05), whereas hypotonic Cl(-)-free solution had no effect on isotonic volume, but potentiated initial swelling by 16+/-2% (P<0.05) and fully inhibited RVD (n=5, P<0.001). R(+)-[(2-n-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inde n-5yl)-oxy] acetic acid) (DIOA, 80 microM), a K(+)-Cl- cotransport blocker (with inhibitory potency toward Ca(2+)-activated K+ channels), inhibited 87+/-5% of the RVD process at 5 min (P<0.001) and 56+/-16% at 30 min (P<0.001), whereas it had a small effect on isotonic volume (+4%, P<0.01) and initial cell swelling (+2%, N.S.; n=9). In contrast to quinidine, DIOA was able to inhibit Ca(2+)-omission-resistant RVD (full inhibition at 5 min, and 56+/-9% at 30 min; P<0.01, n=5). In conclusion, our results suggest that at least three distinct ion transport mechanisms are involved in the RVD in newborn rat cardiomyocytes: (1) K+ and Cl-channels, (2) K(+)-Cl- cotransport, and (3) Na(+)-K(+)-Cl- co-transport.     

Adrenal α2-adrenergic receptors in the aging normotensive and spontaneously hypertensive rat

This study investigates α₂-adrenergic receptor (α₂AR) mediated feedback inhibition of catecholamine release from the adrenal medulla of adult (52 weeks) and old (98 weeks) spontaneously hypertensive rats (SHR) and normotensive controls Wistar Kyoto (WKY) rats. Adrenal epinephrine content as well as the spontaneous and the nicotinic-evoked release of epinephrine were similar between adult SHR and WKY rats. Aging produced a significant reduction in epinephrine synthesis in WKY rats. In contrast, in SHR aging produced a significant increase in epinephrine release without significant changes in epinephrine synthesis. The α₂AR agonist medetomidine abolished (80–90% inhibition) the nicotinic-evoked release of epinephrine in adult SHR and WKY rats. With aging, this effect was unaltered in WKY rats but was significantly decreased in SHR (30% inhibition). Adrenal α_2AAR mRNA levels were significantly reduced in old SHR compared with age matched WKY rats. In conclusion, in aging the α₂AR mediated feedback inhibition of epinephrine release from the adrenal medulla is preserved in WKY rats but compromised in SHR, resulting in increased epinephrine release.     

HCO 3 − -dependent volume regulation in α-cells of the rat endocrine pancreas

Ion transport activity in pancreatic α-cells was assessed by studying cell volume regulation in response to anisotonic solutions. Cell volume was measured by a video imaging method, and cells were superfused with either 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid-buffered or HCO₃⁻-buffered solutions. α-Cells did not exhibit a regulatory volume increase (RVI) in response to cell shrinkage caused by hypertonic solutions. A RVI was observed, however, in cells that had first undergone a regulatory volume decrease (RVD), but only in HCO₃⁻-buffered solutions. RVI was also observed in response to a HCO₃⁻-buffered hypertonic solution in which the glucose concentration was increased from 4 to 20 mM. The post-RVD RVI and the glucose-induced RVI were both inhibited by 10 μM 5-(N-methyl-N-isobutyl) amiloride or 100 μM 2,2′-(1,2-ethenediyl) bis (5-isothio-cyanatobenzenesulfonic acid), but not by 10 μM benzamil nor 10 μM bumetanide. These data suggest that Na⁺–H⁺ exchangers and Cl⁻–HCO₃⁻ exchangers contribute to volume regulation in α-cells.     

Direct effects of tolbutamide on mitochondrial function, intracellular Ca2+ and exocytosis in pancreatic β-cells

 Using the whole-cell voltage-clamp method to measure ATP-sensitive K⁺(K_ATP) currents, changes in cell capacitance to measure secretion and microfluorimetry to monitor intracellular Ca²⁺ and mitochondrial function, we have investigated the direct effect of sulphonylureas on exocytosis in pancreatic β-cells. Tolbutamide (100 μM) and 100 nM 4-β-12-phorbolmyristate-13-acetate (PMA), which activates the protein kinase C (PKC) isoforms found in β-cells, potentiated exocytosis in a non-additive manner. These effects were blocked by down-regulation of PKC. Our data support the idea that tolbutamide can potentiate secretion from β-cells via a PKC-dependent pathway. Because PKC and sulphonylureas can modulate the activity of K_ATP channels, we explored whether the above effects are caused by inhibition of this channel. PMA increased whole-cell K_ATP currents but did not affect their sensitivity to tolbutamide. Down-regulation of PKC affected neither the magnitude nor the tolbutamide sensitivity of the K_ATP current. Both tolbutamide and the mitochondrial uncoupler FCCP (1 μM) mobilized intracellular Ca²⁺ and prolonged Ca²⁺ transients elicited by cholinergic mobilization of intracellular Ca²⁺ stores. Tolbutamide (0.1–0.5 mM), like FCCP, depolarized the mitochondrial membrane potential and activated K_ATP currents. We suggest that sulphonylureas can directly potentiate exocytosis by impairing mitochondrial function and Ca²⁺ handling, which ultimately leads to activation of Ca²⁺-dependent enzymes such as PKC. Received: 1 September 1998 / Received after revision: 9 November 1998 / Accepted: 10 November 1998     

Constitutive nitric oxide synthases in rat pancreatic islets: direct imaging of glucose-induced nitric oxide production in β-cells

The production of nitric oxide (NO) by constitutive nitric oxide synthase (NOS) was investigated in isolated rat pancreatic islets and dispersed -cells. Double-immunocytochemical analyses with a confocal microscope demonstrated the presence of NOS1 in -, -, -, and PP-cells and that of NOS3 in -, -, and PP-cells, but not -cells, in the isolated rat islets. Image analyses with the NO-reactive fluorescence dye DAF-2 clearly showed that an elevation in glucose concentrations from 0 to 11.1 mM increased intracellular NO in most cells of the isolated islets. The glucose-induced elevation of intracellular NO in the islet cells was abolished in the presence of the Ca²⁺ channel blocker nicardipine and after treatment with the NOS inhibitor N ^G-nitro-l-arginine. Similarly, the ATP-sensitive K⁺ channel blocker tolbutamide, which elevates intracellular Ca²⁺ concentrations, increased DAF-2 fluorescence in most cells of the isolated islets. In isolated -cells, 11.1 mM glucose increased DAF-2 fluorescence, which was suppressed by N ^G-nitro-l-arginine and by reducing the glucose concentration to 0 mM. DAF-2 fluorescence in -cells was also increased by 50 mM K⁺, which was suppressed by N ^G-nitro-l-arginine. These results suggest that NOS1 and NOS3 are present in rat pancreatic -cells, and that glucose produces NO by Ca²⁺-dependent activation of the constitutive NOS isoforms.     

Regulation of the Na+2Cl–K+ cotransporter in isolated rat colon crypts

The Na(+2)Cl(-)K+ cotransporter accepts NH4+ at its K+-binding site. Therefore, the rate of cytosolic acidification after NH4+ addition to the bath (20 mmol/l) measured by BCECF fluorescence can be used to quantify the rate of this cotransporter. In isolated colon crypts of rat distal colon (RCC) addition of NH4+ 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 (delta fluorescence ratio units/1000 s). In pilot experiments it was shown that the pH signal caused by the Na(+)2Cl(-)K+ co-transporter could be amplified if the experiments were carried out in the presence of bath Ba2+ to inhibit NH4+ uptake via K+ channels. Therefore all subsequent experiments were performed in the presence of 1 mmol/l Ba2+. In the absence of any secretagogue, preincubation of RCC in a low-Cl- solution (4 mmol/l) for 10 min enhanced the uptake rate significantly from 1.70+/-0.11 to 2.54+/-0.27 U/1000 s (n=20). The addition of 100 mmol/l mannitol (hypertonic solution) enhanced the rate significantly from 1.93+/-0.17 to 2.84+/-0.43 U/1000 s (n=5). Stimulation of NaCl secretion by a solution containing 100 micromol/l carbachol (CCH) led to a small but significant increase in NH4+ uptake rate from 2.06+/-0.34 to 2.40+/-0.30 U/1000 s (n= 11). The increase in uptake rate observed with stimulation of the cAMP pathway by isobutylmethylxanthine (IBMX) and forskolin (100 micromol/l and 5 micromol/l, respectively) was from 2.39+/-0.24 to 3.06+/-0.36 U/1000 s (n=24). Whatever the mechanism used to increase the NH4+ uptake rate, azosemide (500 micromol/l) always reduced this rate to control values. Hence three manoeuvres enhanced loop-diuretic-inhibitable uptake rates of the Na(+)2Cl(-)K+ cotransporter: (1) lowering of cytosolic Cl- concentration; (2) cell shrinkage; (3) activation of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a transient fall in cell volume.     

Trypsin and α-chymotrypsin treatment abolishes glibenclamide sensitivity of KATP channels in rat ventricular myocytes

Cytoplasmic trypsin-treatment of voltagesensitive potassium channels has been shown to cleave domains of the channel responsible for inactivation of the channel. Trypsin has also been reported to remove slow, irreversible inactivation, or run-down in ATP-sensitive potassium (K_ATP) channels. Cytoplasmic treatment of rat ventricular K_ATP channels with either crude, or pure trypsin (1–2 mg/ml) failed to prevent a slow run-down of channel activity. However, trypsin (porcine pancreatic type IX, or type II (Sigma Chem. Co.), or chymotrypsin (Sigma Chem. Co.) rapidly and irreversibly removed, or substantially decreased glibenclamide and tolbutamide-sensitivity of the channels without removing sensitivity to ATP. We conclude that glibenclamide must bind to either a separate protein, or to a separate domain on the channel in order to effect channel inhibition, and this domain is functionally disconnected from the channel by trypsin-, or -chymotrypsin treatment.     

K+ and Cl− currents in freshly isolated rat osteoclasts

Membrane electrical properties of freshly isolated rat osteoclasts were studied using patch-clamp recording methods. Characterization of the passive membrane properties indicated that the osteoclast cell membrane behaved as an isopotential surface. The specific membrane capacitance was 1.2±0.3 F/cm² (mean ±SD), with no difference between cells plated on glass and those adhering to a permeable collagen substrate. The current/voltage (I/V) relationship of all cells showed inward rectification and I/V curves shifted 51 mV positive per tenfold increase of [K⁺]_out, indicating an inwardly rectifying K⁺ conductance. The voltage dependence of the K⁺ chord conductance (g _K) also shifted positive along the voltage axis, and the maximum conductance increased, with elevation of [K⁺]_out. g _K for cells bathed in 4.7 mM [K⁺]_out increased e-fold per 12mV hyperpolarization, and half-maximal activation was at –89 mV. Approximately 18% (50 pS/pF) of the maximum g _K was active at –70 mV. Inward single-channel currents were recorded in cell-attached patches at hyperpolarizing potentials. With symmetrical K⁺, channel conductance was 25±3 pS and reversal was close to the K⁺ equilibrium potential, consistent with this K⁺ channel underlying the whole-cell K⁺ currents. With both conventional whole-cell and perforated-patch recording, no voltage-activated Ca²⁺ current was detected. In approximately 30% of osteoclasts studied, an outwardly rectifying current was observed, which was reversibly blocked by 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene2,2-disulphonic acid (SITS). This DIDS- and SITS-sensitive current reversed direction at the chloride equilibrium potential. We conclude that an inwardly rectifying K⁺ current is present in all rat osteoclasts and that some osteoclasts also exhibit an outwardly rectifying Cl^– current. Both these membrane conductances may play an important physiological role by dissipating the potential that arises from the electrogenic transport of H⁺ across the ruffled membrane of the osteoclast.