Effects of inhibitors and ion substitutions on oscillations of cell membrane potential in cells expressing the RAS oncogene

Previous studies revealed that in NIH fibroblasts expressing the ras oncogene but not in other NIH fibroblasts, bradykinin leads to sustained, calcium dependent oscillations of cell membrane potential by repetitive activation of calcium-sensitive K⁺ channels. The present study has been performed to test for ion and inhibitor sensitivity of these oscillations. Both, Lys-bradykinin (kallidin) and bradykinin, but not any shorter peptide tested, maintained the oscillations. The oscillations are abolished in the presence of the K⁺ channel blocker barium (10 nmol/l). The amplitude but not the frequency of the oscillations is dependent on the extracellular potassium concentration. The oscillations are not dependent on the presence of extracellular sodium, bicarbonate or chloride. The oscillations are abolished in the absence of extracellular calcium and their frequency is significantly decreased at reduced extracellular calcium (to 0.2 mmol/l). The oscillations are not inhibited by acute administration of ouabain (0.1 mmol/l), by dimethylamiloride (100 mol/l), furosemide (1 mmol/l) and hydrochlorothiazide (100 mol/l), by cobalt (100 mol/l), zinc (100 mol/l), gadolinium (100 mol/l), verapamil (10 mol/l) and diltiazem (10 mol/l), but are abolished in the presence of 100 mol/l lanthanum, 1 mmol/l cadmium, 10 mol/l nifedipine, 25 mol/l SK & F 96365 and 200 mol/l TMB-8. Stimulation of calcium entry by 10 mol/l ionomycin is frequently followed by oscillations of cell membrane potential even in the absence of bradykinin. In conclusion, in cells expressing the ras oncogene bradykinin leads to sustained activation of calcium channels at the cell membrane, which cause oscillations of the cell membrane potential by triggering intracellular calcium release.     

Roles of inositol trisphosphate and protein kinase C in the spontaneous outward current modulated by calcium release in rabbit portal vein

We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutylate (PDBu) and H-7 on Ca²⁺-dependent K⁺ currents in smooth muscle cells of the rabbit portal vein using the whole-cell patch-clamp technique, to explore the effects of PKC on the oscillatory outward current (I _oo). Neomycin (30 M), an inhibitor of phospholipase C, and intracellular applications of heparin (10 g/ml) and guanosine 5-O-(2-thiodiphosphate) (GDP[S]; 1 mM) partly but consistently inhibited the generation of I _oo, whereas a higher concentration of heparin (100 g/ml) transiently enhanced then suppressed the generation of I _oo. Inhibition of I _oo generation by heparin was more powerful at the holding potential of + 20 mV than at –20 mV. Inositol 1,4,5-trisphosphate (InsP ₃; 30 M) continuously generated I _oo at holding potentials more positive than –60 mV. Noradrenaline (10 M) and caffeine (3–20 mM) transiently augmented, then reduced the generation of I _oo. Heparin (10 g/ml) completely inhibited responses induced by InsP ₃ and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5-triphosphate (GTP; 200 M) or low concentrations of guanosine 5-O-(3-thiotriphosphate) (GTP[S]; 3 M) continuously augmented the generation of I _oo. High concentrations of GTP[S] (10 M) transiently augmented, then inhibited I _oo. Neither GTP[S] nor noradrenaline induced the transient augmentation or the subsequent inhibition of I _oo when applied in the presence of GDP[S] (1 mM), neomycin (30 M) or heparin (10 g/ml). PDBu (0.1 M) reduced the generation of I _oo but failed to produce an outward current following application of caffeine (3–5 mM). This action of PDBu was inhibited by pretreatment with H-7 (20 M). In the presence of H-7, GTP[S] continuously enhanced the generation of I _oo. The suppression of the generation of I _oo during application of noradrenaline (10 M) was reduced by pretreatment with H-7. Thus both InsP₃ and protein kinase C contribute to the generation of I _oo in smooth muscle cells of the rabbit portal vein and heparin is not a specific InsP ₃ antagonist on the InsP ₃-induced Ca²⁺-release channel (PIRC). InsP ₃ opens PIRC and protein kinase C may deplete the stored Ca²⁺ by either inhibiting the reuptake of Ca²⁺ or by enhancement of the releasing actions of InsP ₃.     

Mechanisms of antagonistic action of internal Ca2+ on serotonin-induced potentiation of Ca2+ currents in Helix neurones

The influence of internal Ca²⁺ ions has been investigated during intracellular perfusion of isolated neurones from pedal ganglia of Helix pomatia in which serotonin (5-HT) induces a cyclic-adenosine-monophosphate-(cAMP)-dependent enhancement of high-threshold Ca²⁺ current (I _Ca). Internal free Ca²⁺ ([Ca²⁺]_i) was varied between 0.01 and 10 M by addition of Ca²⁺-EGTA [ethylenebis(oxonitrilo)tetraacetate] buffer. Elevation of [Ca²⁺]_i depressed the 5-HT effect. The dose/ effect curve for the Ca²⁺ blockade had a biphasic character and could be described by the sum of two Langmuir's isotherms for tetramolecular binding with dissociation constants K _d1=0.063 M and K _d2=1 M. Addition of calmodulin (CM) antagonists (50 M trifluoperazine or 50 M chlorpromazine), phosphodiesterase (PDE) antagonists [100 M isobutylmethylxanthine (IBMX) or 5 mM theophylline] and protein phosphatase antagonists [2 M okadaic acid (OA)] in the perfusion solution caused anticalcium action and modified the Ca²⁺ binding isotherm. Using the effect of OA and IBMX, two components of the total Ca²⁺ inhibition were separated and evaluated. In the presence of one of these blockers tetramolecular curves with K _d1=0.04 M and K _d2=0.69 M were obtained describing the activation of the retained unblocked enzyme — PDE or calcineurin (CN) correspondingly. The sum of these isotherms gave a biphasic curve similar to that in control. Leupeptin (100 M), a blocker of Ca²⁺-dependent proteases did not influence the amplitude of 5-HT effect, indicating that channel proteolysis is not involved in the depression. Our findings show that the molecular mechanism of Ca²⁺-induced suppression of the cAMP-dependent upregulation of Ca²⁺ channels is due to involvement of two Ca²⁺-CM-dependent enzymes: PDE reducing the cAMP level, and CN causing channel dephosphorylation. No other processes are involved in the investigated phenomenon at a Ca²⁺ concentration of less than or equal to 10 M.     

Positive cooperativity in activation of the cardiac muscarinic K+ channel by intracellular GTP

Concentration-dependent effects of intracellular GTP on activation of the muscarinic K⁺ channel were examined in inside-out patches of cardiac atrial myocytes. The pipette solution contained 0.1 M ACh. GTP (0.01–30 M) and 0.5 mM MgCl₂ were applied to the inside side of the patch membrane. K⁺ channels were activated with GTP concentration above 0.1 M. Channel activation reached a maximal value with 1–3 M GTP. It decreased at GTP concentrations larger than 3 M, probably due to desensitization. The dependence of the open probability of the channel on intracellular GTP showed a sigmoidal relationship with a Hill coefficient of around 3. A positive cooperative effect of intracellular GTP on the K⁺ channel may play an important role in amplifying the signal from the membrane receptor to the K⁺ channel.     

Properties of two calcium transport systems of isolated rat ileal epithelial cells: effects of Ca2+ channel modulators and membrane potential examined with fluorescent dye,fura-2

Calcium transport systems of isolated ileal epithelial cells were investigated. The concentration of cytosolic free calcium ions, [Ca²⁺]_i, was monitored with a fluorescent Ca²⁺ dye, fura-2. The fluorescence intensity ratio (I ₃₄₀/I ₃₈₀) was used as an index of [Ca²⁺]_i. [Ca²⁺]_i of the cells suspended in the nominally Ca²⁺-free solution was estimated at 52±3 nM. Ca²⁺ uptake was followed for as long as 5 min in the presence of 100–1000 M added CaCl₂. Most of the experiments were performed at 200 M CaCl₂. The Ca²⁺ uptake was abolished by 0.8 mM Ni²⁺ and 50 M Mn²⁺ and partitally antagonized by 50 M verapamil and 50 M diltiazem but not affected by 20 M nifedipine. The Ca²⁺ entry was reduced by increasing concentrations of extracellular K⁺ in the presence of valinomycin, suggesting a voltage-dependent nature of the uptake. On the other hand, the Ca²⁺ transport doubled in the presence of Bay K8644 (8 M), a Ca²⁺ channel agonist. The Bay-K-8644-induced uptake was inhibited by either 10 M nifedipine, 10 M verapamil or 10 M diltiazem and was relatively independent of extracellular K⁺ concentration. These results suggest that there are at least two distinct Ca²⁺ transport systems in the rat ileal epithelial cells, one resistant to organic Ca²⁺ channel blockers but relatively sensitive to membrane potential (basal uptake) and another inducible by Bay K 8644 and sensitive to the channel blockers but relatively independent of membrane potential.     

DSLET and ACTH(4-10) increase mitotic activity of hepatocytes and suppress antibody production

The mitotic index of hepatocytes remained unchanged after 10 intraperitoneal injections of DSLET and ACTH_4-10 in doses of 0.5, 1.5, and 5 g/kg, but increased after injection of these substances in doses of 50 and 150 g/kg. DSLET in doses of 5, 50, and 150 g/kg decreased the number of antibody-producing cells in the spleen. ACTH_4-10 possessed immunosuppressive activity not only in these doses, but also in a dose of 1.5 g/kg. As differentiated from mitotic activity of hepatocytes, the degree of immunosuppression increased with increasing the dose of test peptides.     

Characterization of the Cl− conductance in the granular duct cells of mouse mandibular glands

We have previously shown that mouse mandibular granular ducts contain a hyperpolarization-activated Cl^– conductance. We now show that the instantaneous current/voltage (I/V) relation of this Cl^– conductance is inwardly rectifying with a slope conductance of 15.4±1.8 nS (n=4) at negative potentials and of 6.7±0.9 nS (n=4) at positive potentials. Thus, the inward rectification seen in the steady-state I/V relation is due, not only to voltage activation of the Cl^– conductance, but also to the intrinsic conductance properties of the channel. We show further that the ductal Cl^– conductance is not activated by including ATP (10 mmol/l) in the pipette solution. Finally, we show that the conductance is not blocked by the addition of any of the following compounds to the extracellular solution: anthracene-9-carboxylate (A9C, 1 mmol/l), diphenylamine-2-carboxylate (DPC, 1 mmol/l), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 mol/l), 4,4-diisothiocyanato-stilbene-2,2-disulphonate (DIDS, 100 mol/l), indanyloxyacetic acid (IAA-94, 100 mol/l), verapamil (100 mol/l), glibenclamide (100 mol/l) and Ba²⁺ (5 mmol/l). The properties of the ductal Cl^– conductance most nearly resemble those of the ClC-2 channel. Both channel types have instantaneous I/V relations that are slightly inwardly rectifying, are activated by hyperpolarization with a time-course in the order of hundreds of milliseconds, have a selectivity sequence of Br^–>Cl^–>I^–, and are insensitive to DIDS. The only identified difference between the two is that the ClC-2 channel is 50% blocked both by DPC and A9C (1 mmol/l), whereas the ductal Cl^– conductance is insensitive to these compounds.     

Actin-dependent activation of ion conductances in bronchial epithelial cells

Activation of Cl^– and K⁺ channels is necessary to drive ion secretion in epithelia. There is substantial evidence from previous reports that vesicular transport and exocytosis are involved in the regulation of ion channels. In the present study we examined the role of cytoskeletal elements and components of intracellular vesicle transport on ion channel activation in bronchial epithelial cells. To this end, cells were incubated with a number of different compounds which interact with either microtubules or actin microfilaments, or which interfere with vesicle transport in the Golgi apparatus. The effectiveness of these agents was verified by fluorescence staining of cellular microtubules and actin. The function was examined in ³⁶Cl^– efflux studies as well as in whole-cell (WC) patch-clamp and cell-attached studies. The cells were studied under control conditions and after exposure to (in mmol/l) ATP (0.1), forskolin (0.01), histamine (0.01) and hypotonic bath solution (HBS, NaCl 72.5). In untreated control cells, ATP primarily activated a K⁺ conductance whilst histamine and forskolin induced mainly a Cl^– conductance. HBS activated both K⁺ and Cl^– conductances. Incubation of the cells with brefeldin A (up to 100 mol/l) did not inhibit WC current activation and ³⁶Cl^– efflux. Nocodazole (up to 170 mol/l) reduced the ATP-induced WC current, and mevastatin (up to 100 mol/l) the cell-swelling-induced WC current. Neither had any effect on the WC current induced by forskolin and histamine. Also ³⁶Cl^– efflux induced by HBS, ATP, forskolin and histamine was unaltered by these compounds. Similarly, colchicine (10 mol/l) and taxol (6 mol/l) affected neither ³⁶Cl^– efflux nor WC current induced by ATP, forskolin, histamine or HBS. In contrast, depolymerisation of actin by cytochalasin D (10 mol/l) significantly attenuated ³⁶Cl^– effluxes and WC current activation by the above-mentioned agonists. Incubation with a C2 clostridial toxin (5 nmol/l) showed similar effects on WC currents. Moreover, when cytochalasin D (10 mol/l), C2 clostridial toxins (5 nmol/l), or phalloidin (10 mol/l) were added to the pipette filling solution current activation was markedly reduced. However, in excised inside-out membrane patches, cytochalasin D (10 mol/l), G-actin (10 mol/l) and phalloidin (10 mol/l) had no effect. These data suggest that actin participates in the activation of ion channels in 16HBE14o- epithelial cells and support the concept that exocytosis is a crucial step in the regulation of Cl^– and K⁺ channels in these cells.     

The effect of hyperosmotic challenge upon ion transport in cultured renal epithelial layers (MDCK)

Exposure of the basal-lateral surfaces of MDCK epithelia, mounted in Ussing chambers, to medium made hyperosmotic by the non-electrolyte mannitol, resulted in a marked inhibition of the adrenalinestimulated inward short-circuit current (Cl^– secretion). This inhibition was unaccompanied by a reversal of the adrenaline-stimulated increment in tissue conductance, indicating that the inhibition was due to modulation of ion transport at the basal-lateral membranes. Loop-diuretic-sensitive ⁸⁶Rb(K⁺) efflux mediated by the Na⁺-K⁺ — 2 Cl^– cotransporter at the basal-lateral membranes was markedly stimulated by hypertonic exposure. A diuretic-sensitive K⁺ (Cl^–) loss was observed in shrunken cells upon prolonged exposure (20 min), showing that the net direction of cotransport flux was outward. ⁸⁶Rb(K⁺) efflux stimulated by adrenaline (100 M), exogenous ATP (100 M) and A23187 (10 M) was attenuated in shrunken cells, suggesting that basal-lateral K⁺ conductance is reduced in hyperosmotic media. Cotransport stimulation by hyperosmotic medium was asymmetric, apical bathing hypertonicity being ineffective. These data are consistent with a low hydraulic permeability of the apical membranes.     

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K _o ⁺ (80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca _i ²⁺ of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca _i ²⁺ to a mean of 266±83 nM (n=15). Reduction of Na _p ⁺ (96% Li⁺ replaced) significantly increased Ca _i ²⁺ to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na _o ⁺ replacement (Li⁺) increased Ca _i ²⁺ at a significantly faster rate [3.6 nM min^–1 (control) cf. 19.8 nM min^–1 (ouabain)].     

Direct measurement of K movement by39K NMR in perfused rat mandibular salivary gland stimulated with acetylcholine

The intracellular K content (K_in) of the isolated perfused rat mandibular gland was measured by³⁹K NMR spectroscopy at 25°C, using an inversion recovery technique based on the fact that the spin-lattice (T₁) relaxation of K_in is much faster than that of the extracellular K. K_in decreased by 30–34% of the resting level and reached a plateau level during secretion evoked by a sustained infusion of 1 mol/l acetylcholine. Addition of 1 mmol/l ouabain decreased K_in by an additional 41% of the resting level. The K net flux to the blood and saliva was calculated from the K concentrations and flow rates of the effluent and the saliva. At an initial stage of secretion the gland lost K to the vascular side at a rate of 12.6±1.8 mol/g-min (mean ± SEM,n=7). During sustained secretion, the gland took K up from the vascular side at a rate of 3.3±0.7 mol/g-min (n=7), and the same amount of K was secreted into the saliva (4.7±1.1 at 5–10 min, 2.8±0.8 mol/g-min at 20–30 min), resulting in no net K movement from the gland. Addition of 1 mmol/l ouabain stopped salivary secretion and caused a transient K release to the vascular side at a maximum rate of 12.8±1.1 mol/g-min. Withdrawal of acetylcholine and ouabain induced K uptake from the vascular side (6.5±0.7 mol/g-min) and the amount of K released was completely restored when K_in recovered completely. The ratio (0.38) of the acetylcholine-induced K loss (30 mol/g) to the ouabain-induced total K loss (80 mol/g) was very similar to the ratio (0.41–0.45) measured by³⁹K NMR. The present observations lead to the conclusion that the changes in³⁹L-NMR-visible intracellular K directly relate to K movement across both the basolateral and the luminal membranes of salivary epithelia.     

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.     

Basolateral K+ efflux is largely independent of maxi-K+ channels in rat submandibular glands during secretion

The involvement of large-conductance, voltage- and Ca²⁺-activated K⁺ channels (maxi-K⁺ channels) in basolateral Ca²⁺-dependent K⁺-efflux pathways and fluid secretion by the rat submandibular gland was investigated. Basolateral K⁺ efflux was monitored by measuring the change in K⁺ concentration in the perfusate collected from the vein of the isolated, perfused rat submandibular gland every 30 s. Under conditions in which the Na⁺/K⁺-ATPase and Na⁺-K⁺-2Cl^– cotransporter were inhibited by ouabain (1 mmol/l) and bumeta-nide (50 mol/l) respectively, continuous stimulation with acetylcholine (ACh) (1 mol/l) caused a transient large net K⁺ efflux, followed by a smaller K⁺ efflux, which gradually returned to the basal level within 10 min. These two components of the K⁺ efflux appear to be dependent on an increase in cytosolic Ca²⁺ concentration. The initial transient K⁺ efflux was not affected by charybdotoxin (100 nmol/l) or tetraethylammonium (TEA) (5 mmol/l) but the smaller second component was strongly and reversibly inhibited by charybdotoxin (100 nmol/l) and TEA (0.1 and 5 mmol/l). The initial K⁺ efflux transient induced by ACh was inhibited by quinine (0.1–3 mmol/l), quinidine (1–3 mmol/l) and Ba²⁺ (5 mmol/l), but not by verapamil (0.1 mmol/l), lidocaine (1 mmol/l), 4-aminopyridine (1 mmol/l) or apamin (1 mol/l). Ca²⁺-dependent transient large K⁺ effluxes induced by substance P (0.01 mol/l) and A23187 (3 mol/l) were not inhibited by TEA (5 mmol/l or 10 mmol/l). A23187 (3 mol/l) evoked a biphasic fluid-secretory response, which was not inhibited by TEA (5 mmol/l). Patch-clamp studies confirmed that the whole-cell outward K⁺ current attributable to maxi-K⁺ channels obtained from rat submandibular endpiece cells was strongly inhibited by the addition of TEA (1–10 mmol/l) to the bath. It is concluded that maxi-K⁺ channels are not responsible for the major part of the Ca²⁺-dependent basolateral K⁺ efflux and fluid secretion by the rat submandibular gland.     

ATP-sensitive K+ channels regulated by intracellular Ca2+ and phosphorylation in normal (T84) and cystic fibrosis (CFPAC-1) epithelial cells

The elementary K⁺ conductance activated by the cAMP or the Ca²⁺ second messenger pathways was investigated in the model salt-secreting epithelium, the human T84 cell line. Under Cl^–-free conditions, an inwardly rectifying whole-cell K⁺ current was evoked by either forskolin 10 (mol/l) or acetylcholine 1 (mol/l) and blocked by extracellular charybdotoxin 10 (nmol/l). In the cell-attached mode, both secretory agonists induced the opening of a channel showing inward rectification with a unitary chord conductance of 36.8±2.5 pS (n=26) for inward currents. In inside-out patches, a 35-pS inwardly rectifying K⁺ channel that corresponded to the channel recorded in the cell-attached configuration was recorded in the presence of 0.3 mol/l free Ca²⁺ at the inner side of the membrane. This channel was blocked by Ba²⁺ (5 mol/l) and by charybdotoxin (50 nmol/l). Its open probability was enhanced by intracellular Ca²⁺ with and EC₅₀ of 0.25 mol/l and strongly reduced by intracellular MgATP with an IC₅₀ of 600 mol/l. In the continuous presence of ATP, the channel activity was consistently increased by 125 kU/l catalytic subunit of cAMP-dependent protein kinase. In the cystic fibrosis pancreatic duct cell line CFPAC-1, a K⁺ channel was also recorded, with similar characterictics and regulation as the 35-pS channel in T84 cells. We conclude that an ATP-sensitive K⁺ channel regulated by intracellular Ca²⁺ and phosphorylation supports the main K⁺ current activated by secretory agonists in normal cystic fibrosis cell lines. This conductance possibly represents the major pathway for K⁺ recycling at the basolateral membrane during transepithelial fluid secretion.     

Mechanism of modulation of single sodium channels from skeletal muscle by the β 1-subunit from rat brain

We studied the molecular mechanism of the rat skeletal muscle -subunit (_I) gating kinetics modulation by the brain ₁-subunit by heterologous expression of single sodium channels from _I and ₁ in Xenopus laevis oocytes. Coexpression of ₁ reduced mean open time at –10 mV to 21% when compared to channels expressed by _I alone. Channels formed by _I exerted multiple openings per depolarization, which occurred in bursts, in contrast to the channels formed by the _I/₁ complex that opened in average only once per depolarizing voltage pulse. Macroscopic current decay (mcd), as evidenced by reconstructed open probability vs. time , was greatly accelerated by ₁, closely resembling mcd of sodium currents from native skeletal muscle. Generally was larger for channels expressed from the pure _I subunit.From our single channel data we conclude that ₁ accelerates the inactivation process of the sodium channel complex.     

Voltage-sensitive chloride channels of large conductance in the membrane of pig aortic endothelial cells

Single, large-conductance chloride-selective channels were studied in the membrane of pig aortic endothelial cells. These channels were usually inactive in cell-attached recordings and activated spontaneously upon formation of inside-out patches or amphothericin Bperforated vesicles. Channel activity was voltage dependent, with a maximum open probability within the range of –20 mV to +20 mV. Addition of 1 mM Zn²⁺ to either the cytoplasmic or extracellular side blocked channel activity reversibly. Extracellular 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) blocked the channels; the concentration necessary for half-maximum blockade was 100 mol/l. The frequency of observing channels in cell-attached patches increased from less than 5% to 27% when cells were treated for several minutes with 1 mol/l bradykinin and to 80% in the presence of the calcium ionophore A23187 (1 mol/l). Both agents increase the cytoplasmic Ca²⁺ concentration, thereby stimulating nitric oxide (NO) synthesis and cGMP formation in endothelial cells. Sodium nitroprusside (100 mol/l), which spontaneously releases NO, did not increase Cl^– channel activity in intact cells. Polymyxin B (100 mol/l), an inhibitor of protein kinase C, clearly enhanced Cl^– channel activity in intact cells, resulting in the observation of Cl^– channels in 70% of cell-attached patches. Our results demonstrate the existence of a large-conductance (LC-type) Cl^– channel in vascular endothelium which is subject to a complex cellular regulation, possibly involving inhibition via phosphorylation by protein kinase C, and activation by a Ca²⁺-dependent process which is different from the NO/cGMP pathway.     

Blockade of dopamine storage,but not of dopamine synthesis,prevents activation of a tolbutamide-sensitive K+ channel in the guinea-pig substantia nigra

The substantia nigra has one of the highest levels of ATP-sensitive K⁺ channel in the brain. Since this channel is controlled by cell metabolism, the aim of this study was to see how closely it is associated with nigral dopamine systems, which are decreased in Parkinson's disease. In a sub-population of neurons within the rostral substantia nigra pars compacta of the guinea-pig, a brief period of hypoxia resulted in a tolbutamide (100–500 M) sensitive hyperpolarisation [input resistance (IR) decrease from 144.88±14.04 M pre-hypoxia to 105.91±13.25 M during hypoxia]. Maximal blockade of this decrease was seen in presence of 500 m tolbutamide [IR decrease only from 161.35±32.82 M to 155.02±34.29 M]. Reserpine (which depletes dopamine stores) but not -methyl-para-tyrosine (which decreases de novo synthesis of dopamine) caused a marked attenuation of this hyperpolarisation [IR decrease only from 163.32±44.42 M pre-hypoxia to 154.42±50.97 M during hypoxia]. This observation suggests that blockade of dopamine storage, but not of de novo synthesis, leads to a loss of responsiveness of certain mid-brain neurons to hypoxia, rendering them potentially more susceptible to subsequent degeneration. The possible link between nigral dopamine systems and ATP-sensitive K⁺ channels is discussed.     

α-Adrenergic activation of the muscarinic K+ channel is mediated by arachidonic acid metabolites

Phenylephrine (10 M), added in the bathing solution, stimulated the cardiac muscarinic K⁺ channel (I_K.ACh) in the cell-attached patch. The pipette solution contained 10 M atropine and 100 M theophylline to block the muscarinic acetylcholine and adenosine receptors, respectively. The channel activation induced by phenylephrine was blocked by prazosin, an ₁-antagonist, indicating that ₁-adrenergic receptor mediates the response. Phenylephrine-induced activation was prevented by nordihydroguaiaretic acid, a lipoxygenase inhibitor, and AA-861, a 5-lipoxygenase inhibitor, but was not affected by indomethacin, a cyclooxygenase inhibitor. These observations suggest that 5-lipoxygenase metabolites of arachidonic acid may be involved in the -adrenergic activation of I_K.ACh.On leave from Kyowa Hakko Kogyo Co., Pharmaceutical Research Laboratories; Nagaizumi-cho, Sunto-gun, Shizuoka 411, Japan     

Expression of surface membrane IgG on pokeweed mitogen-reactive anti-tetanus toxoid antibody-producing cells

Anti-tetanus toxoid antibody-producing cells, differentially expressing surface membrane IgM, were analyzed for the additional expression of surface membrane IgG. ⁺ and ^– cells were rosetted with anti--ox red blood cells and separated by density centrifugation into fractions enriched or depleted or ⁺ cells. These B-cell subsets were assayed for the production of IgM and IgG anti-tetanus toxoid antibody and total IgM and IgG. The results indicated that the majority of anti-tetanus toxoid antibody synthesis in the ^– fraction was by ⁺ cells. In the ⁺ fraction, however, both IgM and IgG anti-tetanus toxoid antibody production was detected in the ^+– and ⁺⁺ fraction. The inclusion of isotype-specific antisera during the first 2 days of culture further established that was expressed on the surface of the majority of the precursors for IgG anti-tetanus antibody productionin vitro. Studies performed to determine the culture requirements of ^– and ⁺ cells revealed that production of IgG anti-tetanus toxoid antibody by both cell subsets was dependent on T cells and pokeweed mitogen. However, some ^– cells could produce IgG in the presence of T cells alone.     

Effect of Na+,K+,2Cl- Cotransport Inhibitor Bumetanide on Electrical and Contractile Activity of Smooth Muscle Cells in Guinea Pig Ureter

The effect of Na⁺,K⁺,2Cl^- cotransport inhibitor bumetanide on action potentials and contractions of smooth muscle cells in the ureter of guinea pigs evoked by electrical stimulation was studied by the method of double sucrose bridge. Bumetanide (10-100 M) dose-dependently suppressed action potential and contractions of smooth muscle cells induced by 1-10 M histamine, 10 M mesatone, 5 mM tetraethylammonium, and 100 M sodium nitroprusside. Our findings suggest that test substances modulate Na⁺,K⁺,2Cl^- cotransport in smooth muscle cells.