Yoshito Matsumoto

Mastoparan increases membrane permeability in rat parotid cells independently of action on G-proteins

Mastoparan, a peptide toxin from wasp venom, stimulated the accumulation of inositol phosphates in rat parotid acinar cells. Addition of this peptide to fura-2-loaded cells resulted in a rapid increase in the fura-2 fluorescence ratio (340 nm/380 nm), suggesting that mastoparan stimulates an increase in cytosolic Ca2+ concentration. However, this change in the ratio appears to be due, in part, to fura-2 leakage from the cells, because addition of Mn2+, which quenches extracellular fura-2 fluorescence, reduced the increased fluorescence ratio. In addition to the fura-2 leakage, mastoparan caused considerable leakage of lactate dehydrogenase, a cytosolic marker enzyme. Furthermore, mastoparan decreased the number of trypan blue-excluding cells, indicating a decrease in cell viability. These results suggest that mastoparan enhances the membrane permeability by a mechanism independent of the activation of G-proteins.

Imaging of intracellular Ca2+ waves induced by muscarinic receptor stimulation in rat parotid acinar cells

Changes in cytosolic Ca2+ concentration ([Ca2+]i) following muscarinic receptor stimulation were studied with digital imaging microscopy in small clusters of Fura-2 loaded rat parotid acinar cells. In the absence of extracellular Ca2+, the increase in [Ca2+]i evoked by a high concentration (10 microM) of carbachol (CCh) was initiated in the apical pole of the acinar cells about 0.4 s after stimulation and then rapidly spread as a Ca2+ wave toward the basolateral region. The [Ca2+]i reached the maximum high level throughout the cells 1-2 s after stimulation. As Ca2+ was eliminated from the extracellular medium, the Ca2+ wave was a result of Ca2+ release from intracellular stores. The magnitude and velocity of the Ca2+ wave decreased with decreasing concentration of CCh, and the increase in [Ca2+]i induced by low CCh concentrations (< or = 0.5 microM) was always larger in the apical region of acinar cells than in the basal region. The Ca2+ wave was also observed in isolated single acinar cells, indicating that the maintenance of acinar structure is not essential for the development of the Ca2+ wave. Thapsigargin (ThG), an inhibitor of the endoplasmic reticulum Ca2+ pump, caused a slow and homogeneous increase in [Ca2+]i throughout the cells. Addition of ThG after CCh, or addition of CCh after ThG, did not stimulate further increases in [Ca2+]i, suggesting that the inositol-1,4,5-trisphosphate (InsP3) and ThG-sensitive Ca2+ stores overlap in parotid acinar cells. The present study supports the hypothesis that formation of InsP3 is essential to trigger the Ca2+ wave and that the development of the Ca2+ wave may be attributed to regional differences in InsP3 sensitivity of Ca2+ stores. The agonist-induced Ca2+ wave is probably a general phenomenon in exocrine acinar cells.

Evidence that isoproterenol-induced Ca2+-mobilization in rat parotid acinar cells is not mediated by activation of β-adrenoceptors

The effects of isoproterenol (ISO), a beta-adrenoceptor agonist, on cytosolic free Ca2+ ([Ca2+]i) in rat parotid acinar cells were examined using the fluorescent Ca2(+)-indicator fura-2. At concentrations up to 1 mM, ISO caused a rapid increase in [Ca2+]i in a dose-dependent manner, while addition of 1 microM ISO, which evokes the maximum amylase secretion, had only a slight effect on [Ca2+]i. There was no such increase in [Ca2+]i with the addition (2 mM) of 8-bromo-cyclic AMP, a permeant cyclic AMP analogue. The alpha-adrenoceptor antagonist phentolamine blocked the ISO-induced [Ca2+]i increase better than the beta-adrenoceptor antagonist, propranol, and the muscarinic receptor antagonist, atropine. The IC50 value (the concentration which reduces the ISO-induced increase in [Ca2+]i by 50%) of phentolamine was estimated to be 7.6 nM, for propranolol 13.2 microM and for atropine 3.5 microM. The difference in potency between the three antagonists was similar to the difference in blocking the [Ca2+]i increase induced by phenylephrine, an alpha-adrenoceptor agonist. These results suggest that the Ca2(+)-mobilization in response to high concentrations of ISO results from an activation of alpha-adrenoceptors rather than beta-adrenoceptors.

Activation of β-adrenoceptors does not cause any change in cytosolic Ca2+ distribution in rat parotid acinar cells

The effects of the beta-adrenoceptor agonist isoproterenol on the distribution of cytosolic Ca2+ concentrations were studied with digital imaging microscopy in fura-2-loaded rat parotid acinar cells. At concentrations < 10 microM, isoproterenol did not cause any measurable change in cytosolic Ca2+ concentration ([Ca2+]i). Monitoring of [Ca2+]i in selected areas of the acinar cells failed to show that stimulation with isoproterenol causes a localized rise in [Ca2+]i at the apical region close to the lumen. As the maximum response of amylase exocytosis is observed at 0.1 or 1 microM isoproterenol [Tanimura, A., Matsumoto, Y., Tojyo, Y., 1990. Evidence that isoproterenol-induced Ca2+-mobilization in rat parotid acinar cells is not mediated by activation of beta-adrenoceptors. Biochim. Biophys. Acta, 1055, pp. 273-277], the data obtained here indicate that the isoproterenol-induced amylase exocytosis is not accompanied by Ca2+ mobilization. The high concentration (100 microM) of isoproterenol caused a small but significant increase in [Ca2+]i, particularly in the apical region. This response was completely attenuated by the alpha-adrenoceptor antagonist phentolamine, but not by the beta-adrenoceptor antagonist propranolol, indicating that the isoproterenol-induced increase in [Ca2+]i resulted from an activation of alpha-adrenoceptors. Further, the effect of cyclic AMP on Ca2+ release from intracellular Ca2+ stores was studied in saponin-permeabilized acinar cells using the lipophilic Ca2+ indicator Calcium Green C18. Cyclic AMP had no effect on the Ca2+ release, while the same acinar cells responded strongly to inositol 1,4,5-trisphosphate. This result does not support the hypothesis that cyclic AMP directly stimulates Ca2+ mobilization in rat parotid acinar cells.

Effect of cytochalasin D on acinar cell structure and secretion in rat parotid salivary glands in vitro

Cytochalasin D, a microfilament disrupting agent, considerably inhibited isoproterenol-stimulated amylase release from enzymatically dispersed parotid acini. Histologically cytochalasin D caused a loss of microvilli lining acinar lumina and luminal enlargement. Nearly empty vacuoles appeared near the luminal and lateral surface, and the membrane bordering on the vacuoles was often continuous with the plasma membrane. Therefore, the vacuolization probably resulted from an elongation of the membrane lining the lumen. Fluorescence staining with rhodamine-phalloidin showed that cytochalasin D caused disruption of microfilaments. When stimulating the cytochalasin D-treated cells with isoproterenol, the number of secretory granules in the cytoplasm diminished markedly and secretory material was observed in the vacuoles, indicating that inhibition of amylase release by cytochalasin D is not due to blocking of exocytosis but to the retention of amylase discharged into vacuoles. These findings suggest that microfilaments are essential in maintaining the parotid acinar structure but do not play a direct part in the movement of secretory granules and their fusion with the luminal membrane.

Effects of extracellular ATP on cytosolic Ca2+ concentration and secretory responses in rat parotid acinar cells

At concentrations >50 microM, extracellular ATP dose-dependently increased cytosolic Ca2+ concentration ([Ca2+]i) via activation of P(2Z) purinoceptors in rat parotid acinar cells. This increase in [Ca2+]i is primarily due to entry of Ca2+ from the extracellular medium. Inositol trisphosphate formation and Ca2+ release from intracellular Ca2+ stores were slightly stimulated by ATP. These responses seem to result from the activation of a P2 purinoceptor subtype different from P(2Z) because the maximum responses were induced by much lower concentrations ( <0.25 mM) of ATP than those (> 1 mM) required to produce the maximum activation of Ca2+ entry. ATP did not stimulate amylase release, supporting the view that an increase in [Ca2+]i is not a sufficient stimulus. In addition, ATP evoked a marked K+ release in the presence and absence of extracellular Ca2+, while in carbachol-stimulated cells. only a transient release of K+ was observed in the absence of extracellular Ca2+. The ATP-induced K+ release may be mediated by a mechanism independent of the opening of the Ca(2+)-sensitive K+ channels.

Staurosporine enhances Ca2+ entry induced by depletion of intracellular Ca2+ stores in rat parotid acinar cells

The effect of staurosporine on the Ca2+ signalling induced by the muscarinic receptor agonist carbachol (CCh) was studied in Fura-2-loaded rat parotid acinar cells. At concentrations > 1 nM, staurosporine dose-dependently enhanced the sustained increase in cytosolic free Ca2+ concentration ([Ca2+]i), but did not affect the peak [Ca2+]i seen just after stimulation. The enhancement of the sustained increase in [Ca2+]i was not attenuated by the protein kinase C activator, 4 beta-phorbol 12-myristate 13-acetate, and not mimicked by another inhibitor of protein kinase C, K-252a, suggesting that the effect of staurosporine on the CCh-induced Ca2+ signalling may be due to a mechanism independent of the inhibitory action on protein kinase C. Staurosporine also enhanced the increases in [Ca2+]i induced by the microsomal Ca(2+)-ATPase inhibitor thapsigargin (TG) and the Ca2+ ionophore ionomycin (Iono). When the cells were stimulated by CCh, TG, or Iono in the absence of extracellular Ca2+, a transient increase in [Ca2+]i due to Ca2+ release from intracellular stores was observed. This increase in [Ca2+]i was unaffected by preincubation with staurosporine. However, when Ca2+ was added to the extracellular medium after [Ca2+]i had returned to the resting level, the increase in [Ca2+]i was significantly enhanced by staurosporine. In addition, staurosporine accelerated the Mn2+ influx following the addition of CCh, TG, or Iono. These results suggest that staurosporine modulates the Ca2+ entry system activated by depletion of intracellular Ca2+ stores in rat parotid acinar cells.

Digital imaging of intracellular Ca2+ signaling in rat parotid acinar cells

Utilizing digital imaging microscopy, the receptor-mediated changes in cytosolic Ca2+ concentration ([Ca2+]i) were studied in fura-2-loaded rat parotid acinar cells. The increase in [Ca2+]i induced by carbachol was initiated in the apical pole of the acinar cells and then spread as a Ca2+ wave toward the basolateral region. A similar polarization of Ca2+ signal was observed when the acinar cells were stimulated with substance P or phenylephrine. As the microsomal Ca2+-ATPase inhibitor thapsigargin did not produce a Ca2+ wave, activation of phosphoinositide hydrolysis is probably essential to trigger the Ca2+ wave. Stimulation with 1 microM isoproterenol, a concentration which causes the maximum release of amylase, had no effect on [Ca2+]i. Extracellular ATP (0.5 mM) induced a homogeneous increase in [Ca2+]i throughout the cells in the presence of extracellular Ca2+ but did not change [Ca2+]i in the absence of extracellular Ca2+, indicating that the ATP-induced rise in [Ca2+]i, is due to Ca2+ entry.

Modulatory effect of 4 β-phorbol 12-myristate 13-acetate (PMA) on carbachol-induced Ca2+ mobilization in rat parotid acinar cells

Treatment of rat parotid acinar cells with 4 beta-phorbol 12-myristate 13-acetate (PMA) significantly inhibited an increase in cytosolic free Ca2+ concentration ([Ca2+]i) induced by carbachol (CCh), a muscarinic agonist. The CCh-induced increase in [Ca2+]i was also inhibited by another active phorbol ester, 4 beta-phorbol 12,13-dibutyrate, but not by 4 alpha-phorbol 12,13-didecanoate, which does not activate protein kinase C. The treatment with PMA had no effect on increases in [Ca2+]i evoked by ionomycin and thapsigargin, which do not stimulate phosphoinositide hydrolysis. In contrast, an increase in [Ca2+]i induced by NaF, a direct activator of GTP-binding proteins, was delayed in the presence of PMA. The formation of inositol phosphates in response to CCh was suppressed significantly by PMA treatment. In radioligand binding assays, PMA did not directly interfere with the specific binding of [3H]quinuclidinyl benzilate ([3H]QNB), a muscarinic antagonist, to plasma membranes. Furthermore, the [3H]QNB binding to plasma membranes prepared from the PMA-pretreated cells was not different from that to the control membranes. These results indicate that PMA attenuated the CCh-induced increase in [Ca2+]i through inhibition of phosphoinositide hydrolysis. Activation of protein kinase C may play a role in negative-feedback control of the muscarinic pathway in rat parotid acinar cells.

Evidence that substance-P receptors do not exist in mouse parotid and submandibular acinar cells

Substance P (SP) had no effect on cytosolic Ca2+ concentration ([Ca2+]i) and inositol phosphate formation in mouse parotid and submandibular acinar cells, but induced a rapid increase in [Ca2+]i and production of inositol phosphates in rat acinar cells. In addition, SP did not stimulate amylase release from mouse parotid acini, but SP-induced amylase release was seen in rats. These results indicate that the mouse lacks SP receptors on parotid and submandibular acinar cells.