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It has been reported that lidocaine protects neurons against ischemic damage by inhibiting the release of Ca 2+ from intracellular Ca 2+ stores.[1] In our previous experiment, we showed that procaine also has this effect.[2 ]Two mechanisms for the release of Ca 2+ from the endoplasmic reticulum into cytosol have previously been identified.[3,4] The first is ryanodine-receptor mediated Ca 2+ release, which is activated by an increase in the cytosolic concentration of Ca 2+ and the second is inositol 1,4,5-trisphosphate (IP.3)-receptor mediated Ca 2+ release, which is activated by IP.3. However, the mechanisms by which procaine and lidocaine inhibit the release of Ca 2+ from intracellular stores have not yet been elucidated. It has been also demonstrated that caffeine induces a marked increase in intracellular Ca 2+ concentration (Ca 2+ .i) through activation of ryanodine-receptor mediated Ca 2+ release.[5,6 In this study, we investigated the in vitro effects of procaine and lidocaine on caffeine-evoked Ca 2+ release from ryanodine-sensitive Ca 2+ stores in gerbil hippocampal slices, using microfluorometry.
METHODS
Slice preparation
The experiment was performed on hippocampal slices from 60-80 g male Mongolian gerbils. The preparation of the hippocampal slices was performed as described in Chen et al.[2] Gerbils were anesthetized with ether and then decapitated. The brains were rapidly removed and placed in ice-cold physiological medium (NaCl 124 mmol/L; KCl 5 mmol/L; CaCl.2 2 mmol/L; MgCl.2 2 mmol/L; NaH.2PO.4 1.25 mmol/L; NaHCO.3 26 mmol/L; glucose 10 mmol/L). Then, 300 μm thick hippocampal transverse slices were cut out with a vibrating slicer (DTK-1000, Dosaka, Kyoto, Japan). The slices were incubated in physiological medium equilibrated with a 95% O.2 and 5% CO.2 gas mixture for 1 h at 26℃, at which point they were preloaded with the fluorescent indicator rhod-2 acetoxymethy ester (rhod-2 AM) for 45 min. Following the preloading, the slices were further incubated in the physiological medium for at least 30 min.
Measurement of intracellular Ca2+ concentration
The Ca 2+ ］.i levels were measured using an inverted fluorescence microscope, a high-performance video camera and an image processing system. A low-magnification objective lens (×4) and a side illumination system were used to visualize the fluoresced image of the slice. Each slice was transferred to a flow-through chamber mounted on the fluorescence microscope equipped with a heat plate stage (IMT2; Olympus, Japan) and superfused at 3 ml/min with the appropriate medium at 36.5℃. The slice was excited with 550 nm light produced by an ultraviolet (UV) lamp (100 W; Osram, Munich, Germany), which was run through an interference filter (550 nm, band width <16 nm), and conducted to the slice through an optical fiber (5 mm diameter). The fluorescence signals (>580 nm) were captured on a silicon-intensified target (SIT) camera (C2400-8, Hamamatsu Photonics, Hamamatsu, Japan), and processed using an image processor (Argus-100; Hamamatus Photonics).Prior to the measurement of Ca 2+i, the slice was excited with 550 nm light, and the image (on a TV monitor) was examined to confirm that the dye was uniformly distributed throughout the slice.After placement of the slice in the chamber, the slice was perfused with physiological medium for 15 min, and Ca 2+ .i in the basal state was measured. The slice was then perfused with 50 mmol/L KCl containing medium (NaCl 79 mmol/L; KCl 50 mmol/L; MgCl2 2 mmol/L; NaH.2PO.4 1.25 mmol/L; NaHCO3 26 mmol/L; glucose 10 mmol/L) for 30 seconds, at which point, the medium was switched to physiological medium. After 5 min of incubation, the slice was superfused with 20 mmol/L caffeine containing physiological medium for 2 min. Then, the slice was superfused with physiological medium until the end of the measurement. The fluorescence intensity of the image was measured, and the numerical values (pixels) were divided by the value of the corresponding element that had been taken prior to the measurement. Thus, the ratio of Ca2+ .i was obtained every 15 seconds. The entire measurement of Ca 2+ .i lasted 15 min, and the medium was equilibrated with 95% O.2 and 5% CO.2 throughout.
The effects of procaine and lidocaine (100 μmol/L) on the caffeine-induced Ca[2+] release were evaluated by adding those agents to the medium after high K+ medium perfusion.
Drugs and chemicals
Procaine, lidocaine and caffeine were purchased from Sigma (St. Louis, MO., USA). Ether was obtained from Takada (Osaka, Japan). Other chemicals were all of reagent grade.
Data analysis
The data were evaluated with repeated two-way analysis of variance to detect differences among groups. When differences were found, Scheffé's test was used post hoc to compare each value to the corresponding value in the control group.
RESULTS
The changes in Ca 2+ .i are shown in Fig. 50 mmol/L KCl elicited an acute and sizable increase in Ca 2+ .i in the hippocampal CA1 region. The peak value of the ratio of Ca 2+ .i reached 152% of the basal value. In control slices, 20 mmol/L of caffeine induced an increase in Ca 2+ but the increase was gradual. The maximum increase observed was (135±12)% (mean±SD, n=10) of the basal level. When the slices were perfused with procaine -containing medium (100 μmol/L), the caffeine-induced increase in the Ca 2+ .was inhibited significantly with the maximum increase at (121±6)% of the basal value （n=10, P<0.05）. 100 μmol/L of lidocaine did not demonstrate a similar inhibitory action (132±9%=10) (P>0.05). (Fig .)
DISCUSSION
I n the present study, we observed that procaine inhibited caffeine-stimulated Ca2+ release from ryanodine-sensitive Ca2+ store in gerbil hippocampal neurons, but lidocaine did not.Evidence has shown that lidocaine protects hippocampal neurons from ischemia-induced damage by inhibiting the increase of Ca2+ .i.[1] In a previous experiment, we found that procaine also has a similar effect.[2 In cerebral ischemia, the increase in Ca 2+ .i plays an important role in the process of neuronal damage.[7] Two mechanisms contribute to the increase of Ca[2+］.i: one is the Ca2+ influx from the extracellular space, and the other is release from intracellular Ca 2+ stores.[8] In regards to the Ca2+ release, two mechanisms have been determined: Ca 2+ -induced Ca[2+] release from ryanodine-sensitive Ca 2+ stores, and IP.3-induced Ca 2+ release.[3,4]
The ryanodine-sensitive Ca 2+ stores were first described in striated muscles. Recently, however, they were also found in other tissues such as neurons, cardiac muscles, mouse eggs, pancreatic acinar cells, and bovine chromaffin cells.[9,10] It has been demonstrated that caffeine stimulates ryanodine-receptor mediated Ca 2+ release. The extent of caffeine-induced Ca 2+ release depends on the amount of Ca 2+ in stores, which in turn is dependent on basal Ca 2+ .i. The depolarization of the cellular membrane by KCl, which induces Ca 2+ influx and increases the basal Ca 2+ .i, can multiply the caffeine-induced Ca 2+ increase.[5,6,11] In this experiment, when the slice was superfused with 50 mmol/L KCl-containing medium, an acute and sharp increase in Ca 2+ .i was observed. Caffeine elicited an increase in Ca 2+ .i, which was inhibited by procaine, but not lidocaine. A similar phenomenon has also been observed in purified ryanodine receptor.[12]The differences between the effects of procaine and lidocaine may be due to the structural variations of these two local anesthetics.[8,13,14] A structure-activity study has suggested that procaine and lidocaine have different distances between the dipole in the carbonyl group and the nitrogen (cation). When local anesthetics interact with the ryanodine-receptor, this distance is essential to the effect of the anesthetic. The longer distance in procaine demonstrates inhibitory activity, but the smaller distance in lidocaine does not affect such an action.[14]
Our findings indicate that procaine and lidocaine inhibit Ca 2+ release from intracellular stores through two different mechanisms. Procaine inhibits ryanodine-receptor mediated Ca 2+ release, while lidocaine inhibits Ca 2+ release through another, unknown mechanism.
REFERENCES
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