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Regulation of the kinetics of intracellular Ca(2+) signals with a novel, membrane-penetrable, inositol 1,4,5-trisphosphate (InsP(3)) receptor/Ca(2+) channel modulator, 2-amino-ethoxydiphenyl borate (2APB), has been investigated using patch-clamp, whole-cell recording to monitor Ca(2+)-activated Cl(-) currents in single isolated pancreatic acinar cells. 2APB itself fails to evoke a detectable current response but it dramatically changes the kinetics of agonist-induced Ca(2+) release from pulsatile spikes to long-lasting, huge Ca(2+) waves, suggesting that 2APB coordinates local Ca(2+) release to generate global Ca(2+) signals. The regulation by 2APB can be elicited by internal perfusion of InsP(3) in a concentration-dependent manner, indicating that this regulation is not mediated through membrane receptors or G protein signal transduction. The InsP(3) receptor blocker heparin, but not the ryanodine-sensitive receptor blockers ruthenium red or ryanodine, abolishes 2APB-mediated regulation of Ca(2+) release. This results also suggest that 2APB effects are mediated through InsP(3) receptors. 2APB substantially modifies single inward Cl(-) current pulse evoked by the photolytic release of caged InsP(3) but not by caged Ca(2+). These data indicate that 2APB-induced regulation is mediated neither by Ca(2+)-induced Ca(2+) release nor by affecting Cl(-) channel activity directly. We conclude that 2APB regulates the kinetics of intracellular Ca(2+) signals, represented as the change in the Ca(2+) oscillation patterns from brief pulsatile spikes to huge, long-lasting Ca(2+) waves. Moreover, this regulation seems to be mediated through InsP(3)-sensitive Ca(2+) pools. 2APB may act as a novel, useful pharmacological tool to study the genesis of intracellular Ca(2+) signals.

The effects of corticotropin-releasing factor (CRF) on the intracellular concentration of Ca2+ were studied in isolated single beta-cells of the rat islet. Immunohistochemical staining using CRF-receptor antibodies revealed the presence of both type 1 (CRF-R1) and type 2 (CRF-R2) receptors for CRF in the majority of islet cells. CRF (2 nmol/l) increased cytosolic Ca2+ concentration under 2.8 mmol/l glucose, dependent upon extracellular Ca2+. CRF caused depolarization of the cell membrane, which was followed by action potentials under 2.8 mmol/l glucose. The dose-response relationships of CRF-induced depolarization in the presence of 1 micromol/l nifedipine produced a bell-shaped curve, showing the peak response at 2 nmol/l. In the whole-cell patch-clamp recording, CRF enhanced Ca2+ currents through L-type Ca2+ channels in a dose-dependent manner similar to that for depolarization. In cells pretreated with Rp-deastereomer of adenosine cyclic 3',5'-phosphorothiolate (100 micromol/l), neither depolarization nor an increase in the Ca2+ current was caused by CRF at concentrations <2 nmol/l. In these cells, CRF at 20 nmol/l reduced the Ca2+ current. These results suggest that in single beta-cells of rat islets, CRF, through its own receptor, potentiates Ca2+ influx through the L-type Ca2+ channel by activation of the cAMP/protein kinase A signaling pathway. CRF at a high concentration also shows an inhibitory effect on the Ca2+ current through an unknown signaling pathway.

Modulation of the agonist-specific cytosolic Ca2+ oscillatory pattern by thimerosal has been investigated in single pancreatic acinar cells using patch-clamp perforated whole-cell recording to measure the calcium-dependent chloride current (I(C1)(Ca2+)). 1 microM thimerosal, which fails to evoke Ca2+ oscillation alone, clearly changed the pattern of Ca2+ oscillation from pulsatile spikes (evoked by low concentrations of activators) to sinusoidal or transient oscillations. The mimetic action of thimerosal was independent of extracellular Ca2+, was blocked by extracellular application of dithiothreitol or 10 mM caffeine, as well as by internal perfusion with heparin; but was unaffected by ruthenium red. We conclude that thimerosal modulates the agonist-specific cytosolic Ca2+ oscillatory patterns mediated by sensitizing the InsP3-induced Ca2+ release.

Acetylcholine (ACh) caused repetitive transient Cl- currents activated by intracellular Ca2+ in single rat submandibular grand acinar cells. As the concentration of ACh increased the amplitude and the frequency of the transient Cl- currents increased. These responses occurred also in the absence of extracellular Ca2+ but disappeared after several minutes. Repetitive transient Cl- currents were restored by readmission of Ca2+ to the extracellular solution. The higher the concentration of extracellular Ca2+ readmitted, the larger the amplitude of the transient Cl- currents. Ca2+ entry through a store-coupled pathway was detected by application of Ca2+ to the extracellular solution during a brief cessation of stimulation with ACh. In these experiments too, the higher the concentration of Ca2+, the larger the transient Cl- currents activated by Ca2+ released from the stores. The time course of decrease in total charge movements of repetitive transient responses to ACh with removal of extracellular Ca2+ depended on a decrease in charge movements of each transient event rather than a decrease in frequency of the repetitive events. The decrease of charge movements of each transient event was due to a decrease in its amplitude rather than its duration. The results suggest that in this cell type and amplitude-modulated mechanism is involved in repetitive Ca2+ release and that Ca2+ entry is essential to maintain the repetitive release of Ca2+. The results further suggest that the magnitude of Ca2+ entry determines the number of unitary stores filled with Ca2+ which can synchronously respond to ACh.

The store-mediated Ca2+ entry was detected in single and cluster of rat submandibular acinar cells by measuring the Ca2+ activated ionic membrane currents. In the cells where intracellular Ca2+ was partly depleted by stimulation with submaximal concentration of acetylcholine (ACh) under a Ca2(+)-free extracellular condition, an employment of external Ca2+ in the absence of ACh caused a sustained increase of the K+ current without affecting the Cl- current. A renewed ACh challenge without external Ca2+ caused repetitive spikes of both K+ and Cl- currents due to the Ca2+ release. SK & F 96365 inhibited the generation of the sustained K+ current and refilling of the Ca2+ store following the Ca2+ readmission. It is suggested that the Ca2+ enters the cell through the store-mediated pathway new the K+ channels and is taken up by the store. Thus, only Ca2+ released from the store can activate both the K+ and Cl- currents.

In order to examine whether Ca2+ entry is directly involved in controlling exocrine secretion, the Ca(2+)-activated Cl- currents were recorded in single and clusters of rat submandibular gland cells using the whole-cell patch-clamp method. Extracellularly applied acetylcholine (ACh, 10 nM) as well as intracellularly applied GTP gamma S and InsP3 caused repetitive transients of the Cl- currents activated by intracellular Ca2+. These responses occurred also in the absence of external Ca2+, but disappeared after several minutes. Readmission of Ca2+ to the extracellular solution restored the repetitive current transients, while introduction of Sr2+ failed to restore the current signals in spite of the presence of Sr2+ entry detected by microfluorimetry. On the other hand, direct application of Sr2+ to the cell inside caused activation of the Cl- currents although less effectively than Ca2+. When Ca2+ was introduced to the extracellular solution during an interruption of ACh stimulation after the ACh-induced depletion of intracellular Ca2+ store, the Cl- current was not elicited. However, a subsequent challenge with ACh at the same concentration in the absence of extracellular Ca2+ caused repetitive transient Cl- currents. The results suggest that in this cell type the stimulated Ca2+ entry does not by itself activate the Cl- currents but activates them indirectly by triggering Ca2+ release from the intracellular Ca2+ store which may take up Ca2+ soon after the Ca2+ entry.