EP1 and EP4 receptors mediate exocytosis evoked by prostaglandin E(2) in guinea-pig antral mucous cells.

Effects of prostaglandin E(2) (PGE(2)) on exocytosis of mucin were studied in mucous cells isolated from guinea-pig antrum using video-microscopy. Stimulation with PGE(2) elicited a sustained increase in the frequency of exocytotic events in a dose-dependent manner, which was under regulation by both Ca(2+) and cAMP. Stimulation with a selective prostanoid EP4 receptor agonist (ONO-AEI-329, 10 microM), which activates cAMP signals, elicited a sustained increase in the frequency of exocytotic events (30 % of that evoked by 1 microM PGE(2)). Stimulation with an EP1 agonist (17-P-T-PGE(2), 1 microM), which activates Ca(2+) signals, increased the frequency of exocytotic events to a lesser extent (5 % of that evoked by 1 microM PGE(2)), while addition of an EP1 antagonist (ONO-8713, 10 microM) decreased the frequency of exocytotic events (approximately 40 % of that evoked by 1 microM PGE(2)). However, addition of the EP1 agonist potentiated the frequency of exocytotic events evoked by the EP4 agonist or forskolin (which elevates cAMP levels) and increased the sensitivity of the exocytotic events to forskolin. These results suggest that the Ca(2+) signal activated via the EP1 receptor potentiates the cAMP-regulated exocytotic events activated via the EP4 receptor during PGE(2) stimulation, by increasing the sensitivity of the exocytotic response to cAMP. In conclusion, exocytotic events in PGE(2)-stimulated antral mucous cells were regulated by interactions between EP1 and EP4 receptors. Experimental Physiology (2001) 86.4, 451-460.

[Na+]i, [K+]i and [Cl-]i regulation of exocytosis in guinea-pig antral mucous cells.

Effects of intracellular Na+, K+ and Cl- on Ca(2+)-regulated exocytosis activated by 10 microM acetylcholine (ACh) were studied in guinea-pig antral mucous cells which are permeabilized by nystatin treatment. Ca(2+)-regulated exocytotic events were modulated by [Na+]i, [K+]i and [Cl-]i via mediation of PTX-sensitive G proteins. Increases in [Na+]i and PTX inhibit G protein (G(Na)), which suppressed the exocytosis. Increases in [K+]i caused the exchange of G proteins (from G(Na) to G(K)) to increase, and GK evoked activation of the exocytosis and was inhibited by PTX. Increases in [Cl-]i and PTX inhibit G protein (G(Cl)), which stimulates exocytotic events. Based on these observations, the exocytosis in antral mucous cells were modulated by intracellular ions, concentration of which were increased or decreased by cell volume changes caused by Ach.

Visualization of the secretory process involved in Ca2+-activated fluid secretion from rat submandibular glands using the fluorescent dye, calcein.

The central feature of fluid and electrolyte secretion by salivary acinar cells is transepithelial Cl- movement as a driving force for the secretion. However, little is known about the membrane localization and regulation by agonists of various anion channels. To characterize the anion transport and fluid secretion, we visualized the secretory process induced by the cholinergic agonist, carbachol (CCh), using the anionic fluorescent dye, calcein, under a confocal laser scanning microscope. The fluorescence of calcein loaded into the isolated acini was spread diffusely throughout the cytoplasm and was less intense in the secretory vesicles which occupied the apical pole. Cytoplasmic calcein was released into intercellular canaliculi just after the addition of CCh, depending upon a rise in [Ca2+]i by Ca2+ release from intracellular stores. Thereafter, the formation of watery vacuoles connected with intercellular canaliculi was visualized in the calcein-loaded acini, depending upon external Ca2+. Both the calcein release and vacuole formation were inhibited by suppressing the Ca(2+)-activated K+ efflux. The calcein release was also affected by the external anion substitution, suggesting that calcein is released through an anion channel. In the isolated, perfused glands, CCh-induced fluid secretion was sustained in two phases, whereas the loaded calcein was initially and transiently released into the saliva. By revealing the [Ca2+]i dependence and sensitivities to channel blockers, our results suggest that the initial phase of CCh-induced fluid secretion was evoked in association with the release of the organic anion, calcein, and the late phase of fluid secretion, during which calcein is less permeable, was associated with the formation of watery vacuoles. Thus, the anion channels possessing the distinct property of anion permeation may be activated in the initial phase and late phase. These results indicate that the anionic fluorescent dye, calcein, is useful for visualizing the process of Ca(2+)-dependent fluid secretion, and for clarifying the relation between fluid secretion and anion transport.

Accumulation of cAMP evoked by acetylcholine stimulation in rat submandibular acinar cells: observation of exocytosis, fluid secretion and [Ca2+]i.

The effects of cAMP accumulation evoked by acetylcholine (ACh) stimulation were studied in rat submandibular acinar cells by observing the exocytotic events, swelling of intercellular canaliculi (IC) and intracellular Ca2+ concentration ([Ca2+]i), which were monitored using an optical microscope. ACh stimulation evoked transient increases followed by sustained increases in the frequency of exocytotic events and IC swelling, while isoproterenol (isoprenaline; IPR) stimulation evoked sustained increases in these parameters. BAPTA treatment reduced the frequency of exocytotic events evoked by 5 microM ACh in the absence of extracellular Ca2+, and further addition of Rp-cAMPS or H-89 (protein kinase A (PKA) inhibitors) eliminated the remaining ACh-evoked responses (50 %). Addition of PKA inhibitors in the presence of extracellular Ca2+ reduced the frequency of exocytotic events evoked by 500 microM ACh in non-BAPTA-loaded cells. However, IC swelling evoked by 5 microM ACh was not affected by addition of PKA inhibitors, and was eliminated in BAPTA-loaded cells perfused with Ca2+-free solution. These results indicate that the IC swelling is regulated by [Ca2+]i and the frequency of exocytotic events is regulated by both [Ca2+]i and [cAMP]i during ACh stimulation. Addition of H-89 inhibited the capacitative Ca2+ entry into ACh-stimulated acinar cells. Biochemical analysis revealed that ACh stimulation increased the cAMP content in perfused submandibular glands. These results indicate that ACh stimulates the accumulation of cAMP in submandibular acinar cells and that this accumulation of cAMP modulates Ca2+-regulated exocytosis.

Inhibition of Ca(2+) entry caused by depolarization in acetylcholine-stimulated antral mucous cells of guinea pig: G protein regulation of Ca(2+) permeable channels.

The effects of depolarizing conditions resulting from increasing extracellular K(+) concentration or nystatin treatment on intracellular Ca(2+) concentration ([Ca(2+)](i)) were studied in guinea pig antral mucous cells following acetylcholine (ACh) stimulation. ACh stimulation evoked a biphasic increase in [Ca(2+)](i), that is, an initial transient increase followed by a plateau. Depolarizing conditions reduced the [Ca(2+)](i) in the plateau phase during ACh stimulation. However, pertussis toxin (PTX, a G protein inhibitor) treatment caused [Ca(2+)](i) in the ACh-evoked plateau phase to increase under depolarizing conditions, while it had no effect on [Ca(2+)](i) under hyperpolarized conditions. Based on these observations, Ca(2+) permeable channels are regulated by a G protein which is activated by depolarized conditions and inhibited by hyperpolarized conditions and PTX; activation of the G protein (depolarization) causes Ca(2+) permeable channels to inhibit, and in turn, inhibition of the G protein (hyperpolarization) causes them to activate.

Isosmotic modulation of Ca2+-regulated exocytosis in guinea-pig antral mucous cells: role of cell volume.

1. Exocytotic events and changes of cell volume in mucous cells from guinea-pig antrum were examined by video-enhanced optical microscopy. 2. Acetylcholine (ACh) evoked exocytotic events following cell shrinkage, the frequency and extent of which depended on the ACh concentration. ACh actions were mimicked by ionomycin and thapsigargin, and inhibited by Ca2+-free solution and Ca2+ channel blockers (Ni2+, Cd2+ and nifedipine). Application of 100 microM W-7, a calmodulin inhibitor, also inhibited the ACh-induced exocytotic events. These results indicate that ACh actions are mediated by intracellular Ca2+ concentration ([Ca2+]i) in antral mucous cells. 3. The effects of ion channel blockers on exocytotic events and cell shrinkage evoked by ACh were examined. Inhibition of KCl release (quinine, Ba2+, NPPB or KCl solution) suppressed both the exocytotic events and cell shrinkage evoked by ACh. 4. Bumetanide (inhibition of NaCl entry) or Cl--free solution (increasing Cl- release and inhibition of NaCl entry) evoked exocytotic events following cell shrinkage in unstimulated antral mucous cells and caused further cell shrinkage and increases in the frequency of exocytotic events in ACh-stimulated cells. However, Cl--free solution did not evoke exocytotic events in unstimulated cells in the absence of extracellular Ca2+, although cell shrinkage occurred. 5. To examine the effects of cell volume on ACh-evoked exocytosis, the cell volume was altered by increasing the extracellular K+ concentration. The results showed that cell shrinkage increases the frequency of ACh-evoked exocytotic events and cell swelling decreases them. 6. Osmotic shrinkage or swelling caused the frequency of ACh-evoked exocytotic events to increase. This suggests that the effects of cell volume on ACh-evoked exocytosis under anisosmotic conditions may not be the same as those under isosmotic conditions. 7. In antral mucous cells, Ca2+-regulated exocytosis is modulated by cell shrinkage under isosmotic conditions.

Inhibition of salivary fluid secretion by occlusion of the intercellular space.

The effects of HCO3- on fluid secretory rate, cell volume and tissue structure were studied in perfused submandibular salivary glands under hyposmotic conditions (240 mosM). Fluid secretion was elicited by acetylcholine (ACh) in a hyposmotic HCO3(-)-free solution. Upon switching the perfusate from the HCO3(-)-free to the HCO3(-)-containing solution during ACh stimulation, the fluid secretory rate exhibited a small transient increase followed by a sharp decrease. ACh stimulation evoked rapid cell shrinkage in the absence of HCO3-, but upon switching from the HCO3(-)-free to the HCO3(-)-containing solution during ACh stimulation, the acinar cells exhibited increases in volume. In laser confocal microscopic examination of isolated acini, fluorescence of lucifer yellow was detected in the lateral intercellular spaces during ACh stimulation in the absence of HCO3-, but not in the presence of HCO3-. Electron microscopic examination revealed similar findings. We propose that the occlusion of the lateral intercellular space induced by cell swelling may result in an increase in the resistance to fluid flow in the space and consequently a decrease in the fluid secretion rate. Maintenance of an open lateral intercellular space may be an important requirement for fluid secretion. These observations suggest that a paracellular pathway may play a significant role in salivary fluid secretion.

Classification of the frog lingual gland cells and their exocytotic features.

We examined under light and electron microscopes the frog lingual gland before and after acetylcholine-stimulation. The lingual gland is composed of five kinds of secretory cells (type 1-5) and ciliated cells. Each secretory cell had numerous cell-type characteristic secretory granules, probably in which both serous and mucous secretory products were contained. The ciliated cells had lysosomes, cytoplasmic filaments and on their apical surface microvilli in addition to cilia. Two kinds of exocytotic features were demonstrated in the secretory cells. One of them, termed multigranular exocytosis, was whole-cell discharge of secretory products in which intracellular huge vacuoles continuous to the cell exterior were formed as the result of multiple granule-to-granule fusion as well as granule-to-plasma membrane fusion. The other type was local exocytotic release of a single or a few secretory granules near the plasma membrane. The multigranular exocytosis was restricted to type 4 and 5 secretory cells while single or a few granular exocytosis was seen in type 1, 2, 4 and 5 secretory cells. This may be because secretory granules are larger and more closely packed in type 4 and 5 secretory cells than in other types of secretory cells. Type 3 secretory cells contained abundant cytoplasmic filaments, and showed no exocytotic feature, suggesting transitional cells between secretory cells and ciliated cells.

Transient swelling of salivary acinus induced by acetylcholine stimulation: water secretion pathway in rat submandibular gland.

The volume changes of isolated acini and acinar cells from rat submandibular glands were measured from digitized images recorded upon stimulation of acetylcholine (ACh) or reduction of the perfusate osmolarity and water secretion pathway in salivary gland was studied. When acinus is exposed to a hyposmotic solution, water flows into the acinar cells and into the lumen via acinar epithelia. If the water enters the lumen chiefly via the cells, the swelling of the lumen would follow the same time course as the cell swelling or slower. The results show that reduction of the perfusate osmolarity evoked a transient increase followed by a gradual increase in the volume of unstimulated acinus, while it evoked only a gradual increase in the volumes of unstimulated acinar cells. Thus, the time course of the acinar swelling is faster than that of the acinar cell swelling. Reduction of the perfusate osmolarity also evoked a transient swelling in ACh stimulated acini. When acinus is stimulated by ACh, water also flows into the lumen via acinar epithelia according to the osmotic gradient which was generated by the active electrolyte transport of acinar cells. If the water enters the lumen chiefly from the cells, there would be no overall change in acinar volume. The results show that stimulation of ACh (5 microM) evoked a transient increase followed by a gradual decrease in the volume of the acinus, while it evoked only a decrease in the volume of acinar cells. Video-enhanced optical microscopy exhibited that ACh stimulation caused transient swelling of the luminal space, prior to causing the volume of acinar cells to decrease and the transient swelling of the lumen followed the same time course as that of acinus. Thus, the transient acinar swelling is explained by the transient swelling of luminar volume. These results suggest that water is probably drawn into the lumen from interstitial space directly in the salivary acinus.

Beta 2-agonist regulation of cell volume in fetal distal lung epithelium by cAMP-independent Ca2+ release from intracellular stores.

The effects of beta 2-adrenoceptor agonist (beta 2 agonist) and cAMP on cytosolic Ca2+ concentration ([Ca2+]c) and cell volume were studied in fetal distal lung epithelial cells. Both terbutaline (a specific beta 2 agonist, 10 microM) and dibutyryl cAMP (DBcAMP, 1 mM) increased [Ca2+]c in the presence of extracellular Ca2+. Even in the absence of extracellular Ca2+, the terbutaline-induced increase in [Ca2+]c was still observed, although the increase was transient. However, DBcAMP caused no significant change in [Ca2+]c. In the presence of 1 mM extracellular Ca2+, terbutaline and DBcAMP induced quinine (a blocker of K+ channel) sensitive cell shrinkage. However, in a Ca2(+)-free solution, terbutaline induced rapid cell shrinkage, followed by benzamil (a specific blocker of Na+ channel, an analogue of amiloride) sensitive transient cell swelling. In a Ca2(+)-free solution, DBcAMP induced benzamil-sensitive transient cell swelling without cell shrinkage. Taken together, our observations indicate that the beta 2 agonist induced an elevation of [Ca2+]c by increasing both a Ca2+ influx from the extracellular space and a Ca2+ release from intracellular Ca2+ stores, whereas DBcAMP only stimulated Ca2+ influx from the extracellular space. Furthermore, it is suggested that terbutaline and DBcAMP activated benzamil-sensitive channels independently of an increase in [Ca2+]c.

A method to estimate the transient interstitial osmolarity change upon application of osmotic stress in perfused rat submandibular gland.

Transient osmolarity changes in the interstitium of perfused rat submandibular glands were estimated by measurement of the interstitial Cl- concentration ([Cl-]ECF) with an Ag-AgCl electrode upon application of osmotic stress by the addition or subtraction of NaCl. A simple model was used to describe the interstitial osmolarity changes upon the application of osmotic stress. The measured [Cl-]ECF values and interstitial osmolarities were fit well by an exponential function derived from the model. The good fit of the theoretical curve indicates that this model describes adequately the changes in interstitial osmolarity after switching the perfusate osmolarity to a new value.

Beta-agonist-induced activation of Na+ absorption and KCl release in rat fetal distal lung epithelium: a study of cell volume regulation.

The effects of terbutaline (a selective beta 2-adrenoceptor agonist) on cell volume and ion transport in rat fetal distal lung epithelial (FDLE) cells were studied. In FDLE cells, benzamil (1 microM) induced cell shrinkage, while cell volume was increased by 1 mM quinine or 2 mM Ba2+ but was not affected by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 20 microM). Terbutaline (10 nM) induced transient cell swelling, which was suppressed by benzamil, while 10 microM terbutaline induced initial rapid cell shrinkage followed by delayed slow cell shrinkage, which was suppressed by 1 mM quinine or 20 microM NPPB but not by 2 mM Ba2+. Application of benzamil enhanced the cell shrinkage induced by 10 microM terbutaline. These observations suggest that: (1) benzamil-blockable Na(+)-permeable channels and quinine- and Ba(2+)-blockable K+ channels contribute to maintenance of cell volume of FDLE cells; (2) terbutaline at both 10 nM and 10 microM activates benzamil-blockable Na(+)-permeable channels; (3) quinine-blockable K+ channels are activated by 10 microM terbutaline; and (4) NPPB-blockable Cl- channels are responsible for Cl- movement in cell volume changes induced by terbutaline. The present study demonstrates that the cellular mechanisms of volume change induced by terbutaline are closely related to activation of Na+ absorption and KCl release.

Osmotic flow transients during acetylcholine stimulation in the perfused rat submandibular gland.

Osmotic stress was applied to the perfused rat submandibular gland during steady-state fluid secretion. Alterations of perfusate osmolarity, by addition or withdrawal of sucrose or NaCl, caused transient changes in secretory rate during continuous stimulation with 1 microM acetylcholine (ACh). Hyposmotic perfusates transiently increased, and hyperosmotic perfusates transiently reduced, the secretory rate. The transients were attributed to changes in osmotic flow resulting from changes in the instantaneous transepithelial osmotic gradient. The time course of the change in interstitial osmolarity was determined by using a Cl- electrode to record the changes in interstitial Cl- concentration following a step change in perfusate Cl- concentration. From the calculated changes in interstitial osmolarity and the resulting changes in secretory rate, the osmotic water permeability of the secretory pathway was estimated to be greater than 15.0 +/- 1.2 microliter (mosmol 1-1)-1 min-1 (g wet weight)-1 (9.8 x 10(-6) +/- 0.8 x 10(-6) l atm-1s-1g-1). The transepithelial gradient required to sustain steady state, ACh-evoked secretion would therefore be less than 16 mosmol l-1 NaCl, which is consistent with previous micropuncture data indicating that the luminal fluid is approximately isosmotic.

ADH action on whole-cell currents by cytosolic Ca2+-dependent pathways in aldosterone-treated A6 cells.

We studied the characteristics of the basal and antidiuretic hormone (arginine vasotocin, AVT)-activated whole cell currents of an aldosterone-treated distal nephron cell line (A6) at two different cytosolic Ca2+ concentrations ([Ca2+]c, 2 and 30 nm). A6 cells were cultured on a permeable support filter for 10 approximately 14 days in media with supplemental aldosterone (1 microM). At 30 nm [Ca2+]c, basal conductances mainly consisted of Cl- conductances, which were sensitive to 5-nitro-2-(3-phenyl-propylamino)-benzoate. Reduction of [Ca2+]c to 2 nm abolished the basal Cl- conductance. AVT evoked Cl- conductances at 2 as well as 30 nm [Ca2+]c. In addition to Cl- conductances, AVT induced benzamil-insensitive nonselective cation (NSC) conductances. This action on NSC conductances was observed at 30 nm [Ca2+]c but not at 2 nm [Ca2+]c. Thus, cytosolic Ca2+ regulates NSC and Cl- conductances in a distal nephron cell line (A6) in response to AVT. Keeping [Ca2+]c at an adequate level seems likely to be an important requirement for AVT regulation of ion conductances in aldosterone-treated A6 cells.

Transepithelial fluid shift generated by osmolarity gradients in unstimulated perfused rat submandibular glands.

The effects of osmotic gradients on transepithelial water movements were examined in unstimulated perfused submandibular glands of the rat. Osmotic gradients were applied transepithelially by adding sucrose to or removing it from the perfusate. An infusion of hypotonic perfusate shifted fluid from the interstitium to the lumen (luminal fluid shift) transiently, whereas an infusion of hypertonic perfusate shifted fluid from the lumen to the interstitium (interstitial fluid shift) transiently. The amount of fluid shifted from lumen to interstitium increased as the luminal fluid osmolarity was raised or as the perfusate osmolarity was reduced. Thus, fluid movements across the salivary epithelium were shown to be simply dependent on the osmolarity difference between lumen and interstitium. To estimate the effective pore radius of the epithelium, non-electrolyte solutions (urea, dimethylurea, diethylurea, mannitol, sucrose and maltotriose) were also used as luminal solutions. The results from non-electrolyte experiments showed that the effective pore radius of the passage for non-electrolytes was slightly larger than 0.38 nm. Solutes smaller than mannitol were less effective in opposing the interstitial fluid shift, and the value of effective pore radius in this report was similar to that of the secretory water pathway that has been measured in solvent drag studies (0.4 0.45 nm). These findings suggest that the passage for non-electrolytes may be water transport pathway in salivary epithelium.

Regulation of cell volume by beta2-adrenergic stimulation in rat fetal distal lung epithelial cells.

Cell-volume changes induced by terbutaline (a specific beta2-agonist) were studied morphometrically in rat fetal distal lung epithelium (FDLE) cells. Cell-volume changes qualitatively differed with the concentration of terbutaline. Terbutaline of 10(-10)-10(-8) M induced transient cell swelling. Terbutaline of 10(-7) M induced transient cell swelling followed by slow cell shrinkage. Terbutaline of 10(-6)-10(-5) M induced rapid cell shrinkage followed by slow cell shrinkage. Terbutaline of 10(-3) M induced transient cell shrinkage; then cell volume oscillated during stimulation. Benzamil of 10(-6) M suppressed the cell swelling induced by 10(-10)-10(-8) M terbutaline and quinine of 10(-3) M inhibited the cell shrinkage induced by 10(-6)-10(-5) M terbutaline. These results suggest that cell swelling would be induced by NaCl influx and the cell shrinkage is by KCl efflux. Dibutyryl cyclic AMP (DBcAMP) also induced similar cell-volume changes over a wide range of concentrations (10(-9)-10(-3) M): a low concentration induced transient cell swelling; a high concentration, rapid and slow cell shrinkage. Forskolin (10(-4) M), like terbutaline (10(-5) M), induced rapid cell shrinkage followed by slow cell shrinkage, and this decrease in the cell volume was enhanced by the presence of benzamil. On the other hand, cell shrinkage was induced by ionomycin (even low concentration; 3 x 10(-10) M ionomycin), and after that cell volume remained at a plateau level. Removal of extracellular Ca2+ abolished the cell swelling caused by terbutaline of 10(-10)-10(-8) M. With removal of extracellular Ca2+, the initial, rapid cell shrinkage induced by 10(-5) M terbutaline became transient, but we still detected slow cell shrinkage similar to that in the presence of extracellular Ca2+. Overall, at low concentrations (10(-10)-10(-8) M), terbutaline induced benzamil-sensitive cell swelling in FDLE cells, which was cAMP- and Ca2+-dependent; high concentrations (> or =10(-6)) induced quinine-sensitive rapid cell shrinkage, which was Ca2+-dependent; high concentrations (> or = 10(-7)) induced slow cell shrinkage, which was cAMP-dependent. These findings suggest that terbutaline regulates cell volume in FDLE cells by cytosolic cAMP and Ca2+ through activation of Na+ and K+ channels.

Bumetanide and bicarbonate increase short-circuit current in fetal lung epithelium.

1. We studied the effects of bumetanide (a Na+/K+/2Cl- cotransport inhibitor) and HCO3(-) on beta-adrenergic-agonist-stimulated short-circuit current (beta-Isc) in rat fetal distal lung epithelium (FDLE). 2. Bumetanide significantly increased beta-Isc in the absence of amiloride but decreased it in its presence. The amiloride- and bumetanide-insensitive beta-Isc was diminished by a removal of HCO3(-) in the bathing solution. 3. Our results suggest that bumetanide stimulates the amiloride-sensitive beta-Isc in FDLE and that the amiloride-insensitive beta-Isc is composed of two different pathways: bumetanide-sensitive and HCO3(-)-dependent Cl- secretion in FDLE.

Regulation of whole cell currents by cytosolic cAMP, Ca2+, and Cl- in rat fetal distal lung epithelium.

The whole cell patch-clamp technique was used to study ionic conductances in fetal distal lung epithelial (FDLE) cells. In unstimulated FDLE cells, K+ conductances were detected in lowered intracellular Cl- concentration ([Cl-]i, < or = 50 mM). The whole cell currents of FDLE cells were increased by elevation of intracellular Ca2+ concentration ([Ca2+]i) or intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration ([cAMP]i). The elevation of [Ca2+]i activated the K+ currents. The amiloride-blockable whole cell currents were activated by [cAMP]i of 1 mM with [Cl-]i of 20 mM and were more frequently detected in the pipette solution without ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) than with it (0.5 mM). When the [Cl-]i was fixed at 50 or 145 mM, however, the increase in these currents was not detected even with cAMP and without EGTA. The amiloride-blockable currents were detected in both the Na+ and K+ pipette solutions. Thus the increase in amiloride-blockable whole cell currents was due to the activation of nonselective cation channels. In FDLE cells treated with terbutaline, which is a beta 2-adrenergic receptor agonist, or forskolin, these currents were detected in the pipette solution containing 20 mM Cl- but were suppressed with time when the pipette solution contained 50 or 145 mM Cl-. It seems likely that maintenance of [Cl-]i at the lowered level is an important requirement for the FDLE cells to activate the amiloride-blockable whole cell currents. It is proposed that cellular mechanisms, such as cell shrinkage, exist to reduce the [Cl-]i in response to cAMP.

ADH-evoked [Cl-]i-dependent transient in whole cell current of distal nephron cell line A6.

The effect of antidiuretic hormone (ADH) on a distal nephron cell line (A6) was studied using the whole cell patch-clamp technique. A6 cells were cultured on a permeable support filter for 10-14 days in media containing 10% fetal bovine serum without supplemental aldosterone. In the unstimulated condition A6 cells had very small conductances of Na+,K+, and Cl-. Arginine vasotocin (AVT, 140 mU/ml, 280 nM) evoked a "transient" increase in whole cell currents as did dibutyryl-adenosine 3',5'-cyclic monophosphate (5 mM). These transients consisted of two components; one was the nonselective cation conductance, and the other was the Cl- conductance. Activation of these conductances was dependent on intracellular Cl- concentration ([Cl-]i). At low [Cl-]i (< or = 50 mM) both conductances were activated, whereas when [Cl-]i was 80 mM, only the Cl- conductance was activated. At high [Cl-]i (125 mM), both conductances were inhibited. It seems likely that the [Cl-]i maintained at a low level (< or = 50 mM) is an important requirement for A6 cells to respond to AVT.

Two types of exocytosis evoked by ACh in salivary acinar cells: a morphological observation in perfused submandibular gland of rats.

Exocytosis in the perfused rat submandibular gland was studied using electron microscopy. Stimulation with 1 microM acetylcholine (ACh) evoked exocytosis; i.e., the secretory granules fused with the luminal membrane and their contents were extruded into the luminal space, and watery vacuoles were observed in some acinar cells. Stimulation with 1 mM ACh also induced exocytosis, and led to a marked increase in the number of watery vacuoles. Some watery vacuoles were connected to the lumen and were thus suggested to be formed by the fusion of the granules. On the other hand, no watery vacuoles were observed in the acinar cells during 1 microM isoproterenol stimulation, while isoproterenol induced exocytosis and led to a marked decrease in the number of intracellular granules. Thus, the watery vacuolation is a characteristic response to the cholinergic stimulation, is dependent on the ACh dose in the submandibular acinar cells, and is regarded as a kind of exocytosis.