Characterization of Ryanodine-sensitive Ca2+ Release from Microsomal Vesicles of Rat Parotid Acinar Cells: Regulation by Cyclic ADP-ribose

We have measured ryanodine (caffeine)-sensitive ⁴⁵Ca²⁺ release from isolated microsomal vesicles of endoplasmic reticulum prepared from rat parotid acinar cells. After a steady state of ATP-dependent ⁴⁵Ca²⁺ uptake, the addition of caffeine (40 mm), ryanodine (10∼500 μm) or an NAD⁺ metabolite, cyclic ADP-ribose (cADPR, 4 μm) released about 10% of the ⁴⁵Ca²⁺ that had been taken up. The ⁴⁵Ca²⁺ release was not inhibited by heparin, an antagonist of IP₃ receptor. The effects of caffeine, ryanodine and cADPR on ⁴⁵Ca²⁺ release were also tested in the presence of thapsigargin (TG), an inhibitor of microsomal Ca²⁺-ATPase. When caffeine (10∼40 mm), ryanodine (10 μm) or cADPR (1∼10 μm) was added in the medium with 100 nm TG, a significant ⁴⁵Ca²⁺ release was seen, while higher concentrations of ryanodine (>100 μm) did not cause any ⁴⁵Ca²⁺ release in the presence of TG. The initial rate of caffeine (40 mm)-induced ⁴⁵Ca²⁺ release was increased by a pretreatment with 10 μm ryanodine, whereas the caffeine-induced ⁴⁵Ca²⁺ release was strongly inhibited by the presence of a higher concentration (500 μm) of ryanodine. cADPR-induced ⁴⁵Ca²⁺ release was also inhibited by 500 μm ryanodine. Caffeine (40 mm)- or cADPR (4 μm)-induced ⁴⁵Ca²⁺ release was abolished by a presence of ruthenium red (50∼100 μm). The presence of a low concentration (0.5 μm) of cADPR shifted the dose-response curve of caffeine-induced ⁴⁵Ca²⁺ release to the left. These results indicate the presence of a ryanodine sensitive Ca²⁺ release mechanism in the endoplasmic reticulum of rat parotid acinar cells that is distinct from the IP₃-sensitive Ca²⁺ channel and is activated by caffeine, cADPR and a low concentration (10 μm) of ryanodine, but is inhibited by higher concentrations (>100 μm) of ryanodine and ruthenium red. The properties of the ryanodine-sensitive mechanism are similar to that of the ryanodine receptor as described in muscle cells. Received: 11 June 1996/Revised: 14 November 1996     

Ca2+ Uptake Through Voltage-gated L-type Ca2+ Channels by Polarized Enterocytes from Atlantic Cod Gadus morhua

The presence and localization of voltage-gated Ca²⁺ channels of L-type were investigated in intestinal cells of the Atlantic cod. Enterocytes were loaded with the fluorescent Ca²⁺ probe, fure-2/AM and changes in intracellular Ca²⁺ concentrations ([Ca²⁺] _i ) were measured, in cell suspensions, in the presence of high potassium levels (100 mm), BAY K-8644 (5 μm), nifedipine (5 μm) or ω-conotoxin (1 μm). L-type Ca²⁺ channels were visualized on intestinal sections using the fluorescent dihydropyridine (-)-STBodipy. Depolarization of the plasma membrane produced a rapid (within 5 sec) and transient (at basal levels after 21 sec) increase in [Ca²⁺] _i . BAY K-8644 increased the [Ca²⁺] _i by 7.2%. Cells in a Ca²⁺-free buffer increased [Ca²⁺] _i after addition of 10 mm Ca²⁺, and this increase was abolished by nifedipine in both depolarizing and normal medium but not by ω-conotoxin. Single cell experiments using video microscopy revealed that enterocytes remained polarized several hours after preparation and that the Ca²⁺ entry and extrusion occurred at specific and different regions of the enterocyte outer membrane. Fluorescent staining of L-type Ca²⁺ channels in the intestinal mucosa showed the most intense staining at the brushborder membrane. These results demonstrate the presence of voltage gated L-type Ca²⁺ channels in enterocytes from the Atlantic cod. The channels are mainly located at the apical side of the cells, and there is a polarized uptake of Ca²⁺ into the enterocytes. This suggests that the L-type Ca²⁺ channels are involved in the transcellular Ca²⁺ entry into the enterocytes. Received: 21 August 1997/Revised: 15 April 1998     

Chloride-induced Ca2+ Release from the Sarcoplasmic Reticulum of Chemically Skinned Rabbit Psoas Fibers and Isolated Vesicles of Terminal Cisternae

There is increasing evidence that Ca²⁺ release from sarcoplasmic reticulum (SR) of mammalian skeletal muscle is regulated or modified by several factors including ionic composition of the myoplasm. We have studied the effect of Cl⁻ on the release of Ca²⁺ from the SR of rabbit skeletal muscle in both skinned psoas fibers and in isolated terminal cisternae vesicles. Ca²⁺ release from the SR in skinned fibers was inferred from increases in isometric tension and the amount of release was assessed by integrating the area under each tension transient. Ca²⁺ release from isolated SR was measured by rapid filtration of vesicles passively loaded with ⁴⁵Ca²⁺. Ca²⁺ release from SR was stimulated in both preparations by exposure to a solution containing 191 mm choline-Cl, following pre-equilibration in Ca²⁺-loading solution that had propionate as the major anion. Controls using saponin (50 μg/ml), indicated that the release of Ca²⁺ was due to direct action of Cl⁻ on the SR rather than via depolarization of T-tubules. Procaine (10 mm) totally blocked Cl⁻- and caffeine-elicited tension transients recorded using loading and release solutions having ([Na⁺] + [K⁺]) × [Cl⁻] product of 6487.69 mm ² and 12361.52 mm ², respectively, and blocked 60% of Ca²⁺ release in isolated SR vesicles. Surprisingly, procaine had only a minor effect on tension transients elicited by Cl⁻ and caffeine together. The data from both preparations suggests that Cl⁻ induces a relatively small amount of Ca²⁺ release from the SR by activating receptors other than RYR-1. In addition, Cl⁻ may increase the Ca²⁺ sensitivity of RYR-1, which would then allow the small initial release of Ca²⁺ to facilitate further release of Ca²⁺ from the SR by Ca²⁺-induced Ca²⁺ release. Received: 6 February 1996/Revised: 17 July 1996     

Muscarinic acetylcholine receptor compounds alter net Ca<Superscript>2+</Superscript> flux and contractility in an invertebrate smooth muscle

Responses of a holothurian smooth muscle to a range of muscarinic (M₁ to M₅) acetylcholine receptor (mAChR) agonists and antagonists were surveyed using calcium (Ca²⁺)-selective electrodes and a mechanical recording technique. Most of the mAChR agonists and antagonists tested increased both contractility and net Ca²⁺ efflux, with M₁-specific agents like oxotremorine M being the most potent in their action. To investigate the possible sources of Ca²⁺ used during mAChR activation, agents that disrupt intracellular Ca²⁺ ion sequestration [cyclopiazonic acid (CPA), caffeine, ryanodine], the phosphoinositide signaling pathway [lithium chloride (LiCl)], and L-type Ca²⁺ channels (diltiazem and verapamil) were used to challenge contractions induced by oxotremorine M. These contractions were blocked by treatment with CPA, caffeine, LiCl, and by channel blockers, diltiazem and verapamil, but were unaltered by ryanodine. Our data suggest that this smooth muscle had an M_1,3,5-like receptor that was associated with the phosphoinositide signaling pathway that relied on intracellular Ca²⁺ stores, but secondarily used extracellular Ca²⁺ via the opening of L-type channels.     

Silent Calcium Channels in Skeletal Muscle Fibers of the Crustacean Atya lanipes

The superficial (tonic) abdominal flexor muscles of Atya lanipes do not generate Ca²⁺ action potentials when depolarized and have no detectable inward Ca²⁺ current. These fibers, however, are strictly dependent on Ca²⁺ influx for contraction, suggesting that they depend on Ca²⁺-induced Ca²⁺ release for contractile activation. The nature of the communication between Ca²⁺ channels in the sarcolemmal/tubular membrane and Ca²⁺ release channels in the sarcoplasmic reticulum in this crustacean muscle was investigated. The effects of dihydropyridines on tension generation and the passive electrical response were examined in current-clamped fibers: Bay K 8644 enhanced tension about 100% but did not alter the passive electrical response; nifedipine inhibited tension by about 70%. Sr²⁺ and Ba²⁺ action potentials could be elicited in Ca²⁺-free solutions. The spikes generated by these divalent cations were abolished by nifedipine. As the Sr²⁺ or Ba²⁺ concentrations were increased, the amplitudes of the action potentials and their maximum rate of rise, V _max , increased and tended towards saturation. Three-microelectrode voltage-clamp experiments showed that even at high (138 mm) extracellular Ca²⁺ concentration the channels were silent, i.e., no inward Ca²⁺ current was detected. In Ca²⁺-free solutions, inward currents carried by 138 mm Sr²⁺ or Ba²⁺ were observed. The currents activated at voltages above −40 mV and peaked at about 0 mV. This voltage-activation profile and the sensitivity of the channels to dihydropyridines indicate that they resemble L-type Ca²⁺ channels. Peak inward current density values were low, ca.−33 μA/cm² for Sr²⁺ and −14 μA/cm² for Ba²⁺, suggesting that Ca²⁺ channels are present at a very low density. It is concluded that Ca²⁺-induced Ca²⁺ release in this crustacean muscle operates with an unusually high gain: Ca²⁺ influx through the silent Ca²⁺ channels is too low to generate a macroscopic inward current, but increases sufficiently the local concentration of Ca²⁺ in the immediate vicinity of the sarcoplasmic reticulum Ca²⁺ release channels to trigger the highly amplified release of Ca²⁺ required for tension generation. Received: 5 April 1999/Revised: 15 September 1999     

Effects of 2,3-Butanedione 2-Monoxime on Ca2+ Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle

Single channel and [³H]ryanodine binding measurements were performed to test for a direct functional interaction between 2,3-butanedione 2-monoxime (BDM) and the skeletal and cardiac muscle sarcoplasmic reticulum Ca²⁺ release channels (ryanodine receptors). Single channel measurements were carried out in symmetric 0.25 m KCl media using the planar lipid bilayer method. BDM (1–10 mm) activated suboptimally Ca²⁺-activated (0.5–1 μm free Ca²⁺) single, purified and native cardiac and skeletal release channels in a concentration-dependent manner by increasing the number of channel events without a change of single channel conductances. BDM activated the two channel isoforms when added to either side of the bilayer. At a maximally activating cytosolic Ca²⁺ concentration of 20 μm, BDM was without effect on the cardiac channel, whereas it inhibited skeletal channel activities with IC₅₀≈ 2.5 mm. In agreement with single channel measurements, high-affinity [³H]ryanodine binding to the two channel isoforms was increased in a concentration-dependent manner at ≤1 μm Ca²⁺. BDM was without a noticeable effect at low (≤0.01 μm) Ca²⁺ concentrations. At 20 μm Ca²⁺, BDM inhibited the skeletal but not cardiac channel. These results suggest that BDM regulates the Ca²⁺ release channels from the sarcoplasmic reticulum of skeletal and cardiac muscle in a concentration, Ca²⁺ and tissue-dependent manner. Received: 31 December 1998/Revised: 9 March 1999     

Two Types of Pharmacologically Distinct Ca2+ Currents with Voltage-dependent Similarities in Zona Fasciculata Cells Isolated from Bovine Adrenal Gland

Voltage-activated Ca²⁺ currents, in zona fasciculata cells isolated from calf adrenal gland, were characterized using perforated patch-clamp recording. In control solution (Ca²⁺: 2.5 mm) a transient inward current was followed, in 40% of the cells, by a sustained one. In 20 mm Ba²⁺, 61% of the cells displayed an inward current, which consisted of transient and sustained components. The other cells produced either a sustained or a transient inward current. These different patterns were dependent upon time in culture. Current-voltage relationships show that both the transient and sustained components activated, peaked and reversed at similar potentials: −40, 0 and +60 mV, respectively. The two components, fully inactivated at −10 mV, were separated by double-pulse protocols from different holding potentials where the transient component could be inactivated or reactivated. The decaying phase of the sustained component was fitted by a double exponential (time constants: 1.9 and 20 sec at +10 mV); that of the transient component was fitted by a single exponential (time constant: 19 msec at +10 mV). Steady-state activation and inactivation curves of the two components were superimposed. Their half activation and inactivation potentials were similar, about −15 and −34 mV, respectively. The sustained component was larger in Ba²⁺ than in Sr²⁺ and Ca²⁺. Ni²⁺ (20 μm) selectively blocked the transient component while Cd²⁺ (10 μm) selectively blocked the sustained one. (±)Bay K 8644 (0.5 μm) increased the sustained component and nitrendipine (0.5–1 μm) blocked it selectively. The sustained component was inhibited by calciseptine (1 μm). Both components were unaffected by ω-conotoxin GVIA and MVIIC (0.5 μm). These results show that two distinct populations of Ca²⁺ channels coexist in this cell type. Although the voltage dependence of their activation and inactivation are comparable, these two components of the inward current are similar to T- and L-type currents described in other cells. Received: 12 July 1999/Revised: 5 October 1999     

Mechanotransduction in Colonic Smooth Muscle Cells

We evaluated mechanisms which mediate alterations in intracellular biochemical events in response to transient mechanical stimulation of colonic smooth muscle cells. Cultured myocytes from the circular muscle layer of the rabbit distal colon responded to brief focal mechanical deformation of the plasma membrane with a transient increase in intracellular calcium concentration ([Ca²⁺] _i ) with peak of 422.7 ± 43.8 nm above an average resting [Ca²⁺] _i of 104.8 ± 10.9 nm (n= 57) followed by both rapid and prolonged recovery phases. The peak [Ca²⁺] _i increase was reduced by 50% in the absence of extracellular Ca²⁺, while the prolonged [Ca²⁺] _i recovery was either abolished or reduced to ≤15% of control values. In contrast, no significant effect of gadolinium chloride (100 μm) or lanthanum chloride (25 μm) on either peak transient or prolonged [Ca²⁺] _i recovery was observed. Pretreatment of cells with thapsigargin (1 μm) resulted in a 25% reduction of the mechanically induced peak [Ca²⁺] _i response, while the phospholipase C inhibitor U-73122 had no effect on the [Ca²⁺] _i transient peak. [Ca²⁺] _i transients were abolished when cells previously treated with thapsigargin were mechanically stimulated in Ca²⁺-free solution, or when Ca²⁺ stores were depleted by thapsigargin in Ca²⁺-free solution. Pretreatment with the microfilament disrupting drug cytochalasin D (10 μm) or microinjection of myocytes with an intracellular saline resulted in complete inhibition of the transient. The effect of cytochalasin D was reversible and did not prevent the [Ca²⁺] _i increases in response to thapsigargin. These results suggest a communication, which may be mediated by direct mechanical link via actin filaments, between the plasma membrane and an internal Ca²⁺ store. Received: 24 March 1997/Revised: 21 July 1997     

Caffeine inhibition of calcium accumulation by the sarcoplasmic reticulum in mammalian skinned fibers

Summary Oxalate-supported Ca accumulation by the sarcoplasmic reticulum (SR) of chemically skinned mammalian skeletal muscle fibers is activated by MgATP and Ca²⁺ and partially inhibited by caffeine. Inhibition by caffeine is greatest when Ca²⁺ exceeds 0.3 to 0.4 m, when free ATP exceeds 0.8 to 1mm, and when the inhibitor is present from the beginning of the loading period rather than when it is added after Ca oxalate has already begun to precipitate within the SR. Under the most favorable combination of these conditions, this effect of caffeine is maximal at 2.5 to 5mm and is half-maximal at approximately 0.5mm. For a given concentration of caffeine, inhibition decreases to one-half of its maximum value when free ATP is reduced to 0.2 to 0.3mm. Varying free Mg²⁺ (0.1 to 2mm) or MgATP (0.03 to 10mm) has no effect on inhibition. Average residual uptake rates in the presence of 5mm caffeine atpCa 6.4 range from 32 to 70% of the control rates in fibers from different animals. The extent of inhibition in whole-muscle homogenates is similar to that observed in skinned fibers, but further purification of SR membranes by differential centrifugation reduces their ability to respond to caffeine. In skinned fibers, caffeine does not alter the Ca²⁺ concentration dependence of Ca uptake (K _0.5, 0.5 to 0.8 m; Hilln, 1.5 to 2.1). Reductions in rate due to caffeine are accompanied by proportional reductions in maximum capacity of the fibers, and this configuration can be mimicked by treating fibers with the ionophore A23187. Caffeine induces a sustained release of Ca from fibers loaded with Ca oxalate. However, caffeine-induced Ca release is transient when fibers are loaded without oxalate. The effects of caffeine on rate and capacity of Ca uptake as well as the sustained and transient effects on uptake and release observed under different conditions can be accounted for by a single mode of action of caffeine: it increases Ca permeability in a limited population of SR membranes, and these membranes coexist with a population of caffeine-insensitive membranes within the same fiber.     

Inositol 1,4,5-Trisphosphate But Not Ryanodine-Receptor Agonists Induces Calcium Release from Rat Liver Golgi Apparatus Membrane Vesicles

We investigated the direct effect of inositol 1,4,5-trisphosphate (IP₃) and ryanodine receptor agonists on Ca²⁺ release from vesicles of a rat liver Golgi apparatus (GA) enriched fraction, which were actively loaded with ⁴⁵Ca²⁺. Results in GA were compared with those obtained in a rat liver endoplasmic reticulum (ER) enriched fraction. The addition of IP₃ at concentrations ranging from 100 nm to 100 μm, in the presence of thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPases, promoted a rapid decrease in the Ca²⁺ content of GA vesicles. The amount of Ca²⁺ released from the vesicles was a function of IP₃ concentration, reaching about 60% in both GA and ER fractions at 100 μm IP₃. Calcium release was inhibited by heparin, an antagonist of IP₃ receptors. Calcium exhibited a bell-shaped effect on IP₃-dependent Ca²⁺ released from GA vesicles: it activated Ca²⁺ release at concentrations up to 1 μm, and inhibited it at higher concentrations. In contrast to that found in the endoplasmic reticulum fraction, none of the ryanodine receptor agonists tested (cyclic ADP-ribose, caffeine and ryanodine) significantly induced Ca²⁺ release from GA fraction vesicles in the presence of thapsigargin. Our results indicate the presence of an IP₃-sensitive Ca²⁺ release mechanism in the Golgi apparatus membrane analogous to that of the ER. However, a Ca²⁺ release mechanism sensitive to ryanodine receptor agonists like that of ER is not evident in the GA membrane. Received: 13 March 2000/Revised: 13 July 2000     

K+ Secretion in Rat Distal Jejunum

The Ussing chamber technique was used to measure unidirectional Rb⁺ fluxes under short-circuit conditions across tissue sheets from proximal, central, and distal jejunum of rats. Whereas the proximal and central parts of the jejunum did not show any net transport of Rb⁺, there was a net secretion of around 0.2 μmol hr⁻¹ cm⁻² in the distal segment. This secretion could not be influenced significantly by mucosal application of K⁺ channel blockers such as Ba²⁺ (5 mm), tetraethylammonium (20 mm) or quinine (1 mm). Serosal ouabain (1 mm) blocked net secretion by increasing mucoserosal flux. Blockers of H⁺/K⁺ ATPases could not alter net fluxes of Rb⁺. Stimulation of Cl⁻ secretion by forskolin (10 μm) or of Na⁺ absorption by serine (10 mm) failed to influence the observed secretion of Rb⁺. Adrenaline (10 μm) also had no effect on Rb⁺ fluxes. Blocking Na⁺/H⁺ exchange by 5-(N-Ethyl-N-isopropyl)-amilorid (100 μm) blocked net secretion by increasing mucoserosal flux, as did the addition of Na⁺ acetate (30 mm) to the mucosal solution. We conclude that the distal jejunum of the rat secretes K⁺ under short-circuit conditions. This secretion does not seem to occur via K⁺ channels, but through a pH dependent mechanism. Received: 16 February 1999/Revised: 29 June 1999     

Involvement of Protein Tyrosine Kinase in the InsP3-Induced Activation of Ca2+-Dependent Cl− Currents in Cultured Cells of the Rat Retinal Pigment Epithelium

This combined study of patch-clamp and intracellular Ca²⁺ ([Ca²⁺] _i ) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca²⁺-dependent Cl⁻ channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca²⁺] _i and activation of Cl⁻ currents. In contrast, intracellular application of Ca²⁺ (10 μm) only induced transient activation of Cl⁻ currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca²⁺ and to the blocker of I _CRAC, La³⁺ (10 μm), despite the fact that both maneuvers led to a decline in [Ca²⁺] _i . The InsP3-induced rise in Cl⁻ conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca²⁺ stores or by removal of Ca²⁺ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca²⁺-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca²⁺/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl⁻-conductance in 50% of the cells investigated without affecting [Ca²⁺] _i . Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca²⁺] _i and Cl⁻ conductance. In summary, elevation of [Ca] _i by InsP3 leads to activation of Cl⁻ channels involving cytosolic Ca²⁺ stores and Ca²⁺ influx from extracellular space. Tyrosine kinases are essential for the Ca²⁺-independent maintenance of this conductance. Received: 15 October 1998/Revised: 3 March 1999     

Adenosine discriminates between the caffeine and adenine nucleotide sites on the sheep cardiac sarcoplasmic reticulum calcium-release channel

Calcium-release channels of sheep cardiac sarcoplasmic reticulum were incorporated into phosphatidylethanolamine bilayers and single channel currents were recorded under voltage-clamp conditions. The effect of adenosine on single channel conductance and gating was investigated, as were the interactions between adenosine and caffeine and adenosine and ,-methylene ATP.Addition of adenosine (0.5–5 mm) to the cytosolic but not the luminal side of the membrane increased the open probability of single calcium-activated calcium-release channels by increasing the frequency and duration of open events, yielding an EC₅₀ of 0.75 mm at 10 m activating Ca²⁺.Addition of 1 mm caffeine potentiated the effects of adenosine at 10 or 100 m-activating cytosolic calcium, but had no effect on the inability of adenosine to activate the channel at 80 pmcalcium, suggesting discrete sites of action on the calcium-release channel for adenosine and caffeine. In contrast, addition of 100 m ,-methylene-ATP decreased single channel open probability in the presence of adenosine, suggesting that these compounds act on the same site on the channel.Activation of single channel opening by adenosine, or by adenosine together with caffeine, had no effect on single channel conductance or the Ca²⁺/Tris⁺ permeability ratio. Channels activated by adenosine were characteristically modified by ryanodine and blocked by m ruthenium red or mm magnesium.These results show that adenosine activates the sheep cardiac sarcoplasmic reticulum Ca²⁺-release channel by increasing the frequency and duration of open events in a Ca²⁺-dependent manner. The receptor site on the channel for adenosine is distinct from that for caffeine but probably the same as that for adenine nucleotides.This work was supported by the British Heart Foundation.     

Regulation of Tight Junction Resistance in T84 Monolayers by Elevation in Intracellular Ca2+: A Protein Kinase C Effect

Elevation in intracellular Ca²⁺ acting via protein kinase C (PKC) is shown to regulate tight junction resistance in T₈₄ cells, a human colon cancer line and a model Cl⁻ secretory epithelial cell. The Ca²⁺ ionophore A23187, which was used to increase the intracellular Ca²⁺ concentration, caused a decrease in tight junction resistance in a concentration- and time-dependent manner. Dual Na⁺/mannitol serosal-to-mucosal flux analysis performed across the T₈₄ monolayers treated with 2 μm A23187 revealed that A23187 increased both fluxes and that in the presence of ionophore there was a linear relationship between the Na⁺ and mannitol fluxes with a slope of 56.4, indicating that the decrease in transepithelial resistance was due to a decrease in tight junction resistance. Whereas there was no effect of 0.1 μm A23187, 1 or 2 μm produced a 55% decrease in baseline resistance in 1 hr and 10 μm decreased resistance more than 80%. The A23187-induced decrease in tight junction resistance was partially reversible by washing 3 times with a Ringer's-HCO₃ solution containing 1% BSA. The A23187 effect on resistance was dependent on intracellular Ca²⁺; loading the T₈₄ cells with the intracellular Ca²⁺ chelator BAPTA significantly reduced the decrease in tight junction resistance caused by A23187. This intracellular Ca²⁺ effect was mediated by protein kinase C and not calmodulin. While the protein kinase C antagonist H-7 totally prevented the action of A23187 on tight junction resistance, the Ca²⁺/calmodulin inhibitor W13 did not have any effect. Sphingosine, another inhibitor of PKC, partially reduced the A23187-induced decline in tight junction resistance. The PKC agonist PMA mimicked the A23187 effect on resistance, although the effect was delayed up to 1 hr after exposure. In addition, however, PMA also caused an earlier increase in resistance, indicating it had an additional effect in addition to mimicking the effect of elevating Ca²⁺. The effects of a phospholipase inhibitor (mepacrine) and of inhibitors of arachidonic acid metabolism (indomethacin for the cyclooxygenase pathway, NDGA for the lipoxygenase pathway, and SKF 525A for the epoxygenase pathway) on the A23187 action were also examined. None of these agents altered the A23187-induced decrease in resistance. Monolayers exposed to 2 μm A23187 for 1 hr were stained with fluorescein conjugated phalloidin, revealing that neighboring cells did not part one from another and that A23187 did not have a detectable effect on distribution of F-actin in the perijunctional actomyosin ring. The results indicate that elevation in intracellular Ca²⁺ decreases tight junction resistance in the T₈₄ monolayer, acting through protein kinase C by a mechanism which does not involve visible changes in the perijunctional actomyosin ring. Received: 14 July 1995/Revised: 25 September 1995     

A9 Fibroblasts Transfected with the m3 Muscarinic Receptor Clone Express a Ca2+ Channel Activated by Carbachol,GTP and GDP

Muscarinic m3 receptor-mediated changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_l) occur by activation of Ca²⁺ release channels present in the endoplasmic reticulum membrane and Ca²⁺ entry pathways across the plasma membrane. In this report we demonstrate the coupling of m3 muscarinic receptors to the activation of a voltage-insensitive, cation-selective channel of low conductance (3.2 ± 0.6 pS; 25 mm Ca²⁺ as charge carrier) in a fibroblast cell line expressing m3 muscarinic receptor clone (A9m3 cells). Carbachol (CCh)-induced activation of the cation-selective channel occurred both in whole cell and excised membrane patches (CCh on the external side), suggesting that the underlying mechanism involves receptor-channel coupling independent of intracellular messengers. In excised inside-out membrane patches from nonstimulated A9m3 cells GTP (10 μm) and GDP (10 μm) activated cation-selective channels with conductances of approximately 4.3 and 3.3 pS, (25 mm Ca²⁺ as charge carrier) respectively. In contrast, ATP (10 μm), UTP (10 μm) or CTP (10 μm) failed to activate the channel. Taken together, these results suggest that carbachol and guanine nucleotides regulate the activation of a cation channel that conducts calcium. Received: 14 November 1996/Revised: 4 April 1997     

A Bicarbonate- and Weak Acid-permeable Chloride Conductance Controlled by Cytosolic Ca2+ and ATP in Rat Submandibular Acinar Cells

A Ca²⁺-activated Cl⁻ conductance in rat submandibular acinar cells was identified and characterized using whole-cell patch-clamp technique. When the cells were dialyzed with Cs-glutamate-rich pipette solutions containing 2 mm ATP and 1 μm free Ca²⁺ and bathed in N-methyl-d-glucamine chloride (NMDG-Cl) or Choline-Cl-rich solutions, they mainly exhibited slowly activating currents. Dialysis of the cells with pipette solutions containing 300 nm or less than 1 nm free Ca²⁺ strongly reduced the Cl⁻ currents, indicating the currents were Ca²⁺-dependent. Relaxation analysis of the ``on' currents of slowly activating currents suggested that the channels were voltage-dependent. The anion permeability sequence of the Cl⁻ channels was: NO⁻ ₃ (2.00) > I⁻ (1.85) ≥ Br⁻ (1.69) > Cl⁻ (1.00) > bicarbonate (0.77) ≥ acetate (0.70) > propionate (0.41) ≫ glutamate (0.09). When the ATP concentration in the pipette solutions was increased from 0 to 10 mm, the Ca²⁺-dependency of the Cl⁻ current amplitude shifted to lower free Ca²⁺ concentrations by about two orders of magnitude. Cells dialyzed with a pipette solution (pCa = 6) containing ATP-γS (2 mm) exhibited currents of similar magnitude to those observed with the solution containing ATP (2 mm). The addition of the calmodulin inhibitors trifluoperazine (100 μm) or calmidazolium (25 μm) to the bath solution and the inclusion of KN-62 (1 μm), a specific inhibitor of calmodulin kinase, or staurosporin (10 nm), an inhibitor of protein kinase C to the pipette solution had little, if any, effect on the Ca²⁺-activated Cl⁻ currents. This suggests that Ca²⁺/Calmodulin or calmodulin kinase II and protein kinase C are not involved in Ca²⁺-activated Cl⁻ currents. The outward Cl⁻ currents at +69 mV were inhibited by NPPB (100 μm), IAA-94 (100 μm), DIDS (0.03–1 mm), 9-AC (300 μm and 1 mm) and DPC (1 mm), whereas the inward currents at −101 mV were not. These results demonstrate the presence of a bicarbonate- and weak acid-permeable Cl⁻ conductance controlled by cytosolic Ca²⁺ and ATP levels in rat submandibular acinar cells. Received: 9 January 1996/Revised: 20 May 1996     

Activation of K+ channels by lanthanum contributes to the block of transmitter release in chick and rat sympathetic neurons

Summary We studied the effects of lanthanum (La³⁺) on the release of ³H-norepinephrine(³H-NE), intracellular Ca²⁺ concentration, and voltage clamped Ca²⁺ and K⁺ currents in cultured sympathetic neurons. La³⁺ (0.1 to 10 m) produced concentration-dependent inhibition of depolarization induced Ca²⁺ influx and ³H-NE release. La³⁺ was more potent and more efficacious in blocking ³H-NE release than the Ca²⁺-channel blockers cadmium and verapamil, which never blocked more than 70% of the release. At 3 m, La³⁺ produced a complete block of the electrically stimulated rise in intracellular free Ca²⁺ ([Ca²⁺] _i ) in the cell body and the growth cone. The stimulation-evoked release of ³H-NE was also completely blocked by 3 m La³⁺. However, 3 m La³⁺ produced only a partial block of voltage clamped Ca²⁺ current (I _Ca). Following La³⁺ (10 m) treatment ³H-NE release could be evoked by high K⁺ stimulation of neurons which were refractory to electrical stimulation. La³⁺ (1 m) increased the hyperpolarization activated, 4-aminopyridine (4-AP) sensitive, transient K⁺ current (I _A ) with little effect on the late outward current elicited from depolarized holding potentials. We conclude that the effective block of electrically stimulated ³H-NE release is a result of the unique ability of La³⁺ to activate a stabilizing, outward K⁺ current at the same concentration that it blocks inward Ca²⁺ current.     

Exocytosis Coupled to Mobilization of Intracellular Calcium by Muscarine and Caffeine in Rat Chromaffin Cells

Abstract: We used cultured rat chromaffin cells to test the hypothesis that Ca²⁺ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca²⁺ sources: (a) Ca²⁺ surrounding single cells was transiently displaced by applying agonist with or without Ca²⁺ from an ejection pipette. (b) Intracellular stores of Ca²⁺ were depleted by soaking cells in Ca²⁺-free plus 1 mM EGTA solution before transient exposure to agonist plus Ca²⁺. Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured by indo-1 fluorescence. KCl (35 mM) and nicotine (10 µM) caused an immediate increase in [Ca²⁺]_i and secretion in cells with or without internal Ca²⁺ stores, but only when applied with Ca²⁺ in the ejection pipette. Caffeine (10 mM) and muscarine (30 µM) evoked exocytosis whether or not Ca²⁺ was included in the pipette, but neither produced responses in cells depleted of internal Ca²⁺ stores. Pretreatment with ryanodine (0.1 µM) inhibited caffeine- but not muscarine-stimulated responses. Elevated [Ca²⁺]_i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 ± 0.38 s) or muscarine (2.2 ± 0.25 s). However, the duration of caffeine-evoked exocytosis (7.1 ± 0.8 s) was significantly shorter than that evoked by muscarine (33.1 ± 3.5 s). The duration of caffeine-evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An ～20-s refractory period was found between repeated caffeine-evoked exocytotic bursts even though [Ca²⁺]_i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca²⁺ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca²⁺. These results and conclusions are consistent with our original observations in the perfused adrenal gland.     

Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line

Summary We have investigated muscarinic receptor-operated Ca²⁺ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The carbachol (Cch) dose response of intracellular Ca²⁺ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K _0.510 or 30 m, depending on the method for [Ca²⁺] _i determination). However, similar data for Ca²⁺ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca²⁺ release and a second higher affinity site withK _0.52.5 m. In addition, the Ca²⁺ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K⁺ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca²⁺ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca²⁺] _i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca²⁺ channel blocker La³⁺ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca²⁺ in these cells by increasing Ca²⁺ entry. Organic Ca²⁺ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca²⁺ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K⁺ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP₃) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP₃ formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca²⁺ entry in HSG-PA cells. One is associated with IP₃ formation and intracellular Ca²⁺ release and is independent of membrane potential; the other is less dependent on IP₃ formation and intracellular Ca²⁺ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.     

Silver ions induce a rapid Ca2+ release from isolated intact bovine rod outer segments by a cooperative mechanism

Summary Micromolar concentrations of silver ion activate large Ca²⁺ fluxes across the plasma membrane of intact rod outer segments isolated from bovine retinas (intact ROS). The rate of Ag⁺-induced Ca²⁺ efflux from intact ROS depended on the Ag⁺ concentration in a sigmoidal manner suggesting a cooperative mechanism with a Hill coefficient between 2 and 3. At a concentration of 50 m Ag⁺ the rate of Ca²⁺ efflux was 7×10⁶ Ca²⁺/outer segment/sec; this represents a change in total intracellular Ca²⁺ by 0.7mm/outer segment/sec. Addition of the nonselective ionophore gramicidin in the absence of external alkali cations greatly reduced the Ag⁺-induced Ca²⁺ efflux from intact ROS, apparently by enabling internal alkali cations to leak out. Adding back alkali cations to the external medium restored Ag⁺-induced Ca²⁺ efflux when gramicidin was present. In the presence of gramicidin, Ag⁺-induced Ca²⁺ efflux from intact ROS was blocked by 50 m tetracaine orl-cis diltiazem, whereas without gramicidin both blockers were ineffective. Bothl-cis diltiazem and tetracaine are blockers of one kinetic component of cGMP-induced Ca²⁺ flux across ROS disk membranes. The ion selectivity of the Ag⁺-induced pathway proved to be broad with little discrimination between the alkali cations Li⁺, Na⁺, K⁺, and Cs⁺ or between Ca²⁺ and Mg²⁺. The properties of the Ag⁺-induced pathway(s) suggest that it may reflect the cGMP-dependent conductance opened in the absence of cGMP by silver ions.