Ca2+ dependency of Na+ transport by rabbit renal brush border membrane

Summary Intracellular Ca²⁺ has been suggested to play an important role in the regulation of epithelial Na⁺ transport. Previous studies showed that preincubation of toad urinary bladder, a tight epithelium, in Ca²⁺-free medium enhanced Na⁺ uptake by the subsequently isolated apical membrane vesicles, suggesting the downregulation of Na⁺ entry across the apical membrane by intracellular Ca²⁺. In the present study, we have examined the effect of Ca²⁺-free preincubation on apical membrane Na⁺ transport in a leaky epithelium, i.e., brush border membrane (BBM) of rabbit renal proximal tubule. In contrast to toad urinary bladder, it was found that BBM vesicles derived from proximal tubules incubated in 1mm Ca²⁺ medium exhibited higher Na⁺ uptake than those derived from proximal tubules incubated in Ca²⁺-free EGTA medium. Such effect of Ca²⁺ in the preincubation medium was temperature dependent and could not be replaced by another divalent cation, Ba²⁺ (1mm). Ca²⁺ in the preincubation medium did not affect Na⁺-dependent BBM glucose uptake, and its effect on BBM Na⁺ uptake was pH gradient dependent and amiloride (10^–3 m) sensitive, suggesting the involvement of Na⁺/H⁺ antiport system. Addition of verapamil (10^–4 m) to 1mm Ca²⁺ preincubation medium abolished while ionomycin (10^–6 m) potentiated the effect of Ca²⁺ to increase BBM Na⁺ uptake, suggesting that the effect of Ca²⁺ in the preincubation medium is likely to be mediated by Ca²⁺-dependent cellular pathways and not due to a direct effect of extracellular Ca²⁺ on BBM. Neither the proximal tubule content of cAMP nor the inhibitory effect of 8, bromo-cAMP (0.1mm) on BBM Na⁺ uptake was affected by the presence of Ca²⁺ in the preincubation medium, suggesting that Ca²⁺ in the preincubation medium did not increase BBM Na⁺ uptake by removing the inhibitory effect of cAMP. Addition of calmodulin inhibitor, trifluoperazine (10^–4 m) to 1mm Ca²⁺ preincubation medium did not prevent the increase in BBM Na⁺ uptake. The effect of Ca²⁺ was, however, abolished when protein kinase C in the proximal tubule was downregulated by prolonged (24 hr) incubation with phorbol 12-myristate 13-acetate (10^–6 m). In summary, these results show the Ca²⁺ dependency of Na⁺ transport by renal BBM, possibly through stimulation of Na⁺/H⁺ exchanger by protein kinase C.     

A patch-clamp study of the Ca2+ mobilization from internal stores in bovine aortic endothelial cells. I. Effects of caffeine on intracellular Ca2+ stores

Summary The effects of agents known to interfere with Ca²⁺ release processes of endoplasmic reticulum were investigated in bradykinin (BK)-stimulated bovine aortic endothelial cells (BAE cells), via the activation of Ca²⁺-activated potassium channels [K(Ca²⁺) channels]. In cell-attached patch experiments, the external application of caffeine (1 mm) caused a brief activation of K(Ca²⁺) channels in Ca²⁺-free and Ca²⁺-containing external solutions. The application of BK (10 nm) during cell stimulation by caffeine (1–20 mm) invariably led to a drastic channel activation which was maintained during a recording period longer than that observed in caffeine-free conditions. In addition, the cell exposure to caffeine (20 mm) during the BK stimulation enhanced systematically the channel activation process. Since a rapid inhibition of BK-evoked channel activity was also produced by removing caffeine from the bath medium, it is proposed that the sustained single-channel response recorded in the concomittant presence of both agents was due to their synergic action on internal stores and/or the external Ca²⁺ entry pathway resulting in an increased [Ca²⁺]_i. In addition, the local anesthetic, procaine, depressed the initial BK-induced K(Ca²⁺) channel activity and completely blocked the secondary phase of the channel activation process related to the external Ca²⁺ influx into stimulated cells. In contrast, this blocking effect of procaine was not observed on the initial caffeine-elicited channel activity and could not suppress the external Ca²⁺-dependent phase of this channel activation process. Our results confirm the existence of at least two pharmacologically distinct types of Ca²⁺-release from internal stores in BAE cells: an inositol 1,4,5-triphosphate (InsP₃)-dependent and a caffeine-induced Ca²⁺-release process.The authors would like to thank Dr. A. Diarra for his contribution to the fluorescence measurements and Diane Vallerand for preparing cell cultures. These data were presented in part at the 14th Scientific Meeting of the International Society of Hypertension (Madrid, Spain, June 14–18, 1992), and have been published in abstract form in the Journal of Hypertension (1992). Dominique Thuringer is a fellow of the Heart and Stroke Foundation of Canada. Rémy Sauvé is a senior fellow from the Fonds de la Recherche en Santé du Québec. This work was supported by a grant from the Medical Research Council of Canada.     

Roles of Ca2+ and Protein Tyrosine Kinase in Insulin Action on Cell Volume via Na+ and K+ Channels and Na+/K+/2Cl− Cotransporter in Fetal Rat Alveolar Type II Pneumocyte

The aim of the present study was to investigate the roles of Ca²⁺ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes. Insulin (100 nm) increased cell volume in the presence of extracellular Ca²⁺ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca²⁺ (<1 nm). This insulin action in a Ca²⁺-containing solution was completely blocked by co-application of bumetanide (50 μm, an inhibitor of Na⁺/K⁺/2Cl⁻ cotransporter) and amiloride (10 μm, an inhibitor of epithelial Na⁺ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action on cell volume in a Ca²⁺-free solution was completely blocked by quinine (1 mm, a blocker of Ca²⁺-activated K⁺ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive Na⁺ channel and a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter in the presence of 1 mm extracellular Ca²⁺, that the stimulatory action of insulin on an amiloride-sensitive Na⁺ channel and a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter requires Ca²⁺, and that in a Ca²⁺-free solution insulin activates a quinine-sensitive K⁺ channel but not in the presence of 1 mm Ca²⁺. The insulin action on cell volume in a Ca²⁺-free solution was almost completely blocked by treatment with BAPTA (10 μm) or thapsigargin (1 μM, an inhibitor of Ca²⁺-ATPase which depletes the intracellular Ca²⁺ pool). Further, lavendustin A (10 μm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca²⁺-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K⁺ channel is mediated through PTK activity in a cytosolic Ca²⁺-dependent manner. Lavendustin A, further, completely blocked the activity of the Na⁺/K⁺/2Cl⁻ cotransporter in a Ca²⁺-free solution, but only partially blocked the activity of the Na⁺/K⁺/2Cl⁻ cotransporter in the presence of 1 mm Ca²⁺. This observation suggests that the activity of the Na⁺/K⁺/2Cl⁻ cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca²⁺-independent pathway and the other is a PTK-independent, Ca²⁺-dependent pathway. Further, we observed that removal of extracellular Ca²⁺ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na⁺ channel and the bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter, and that removal of extracellular Ca²⁺ abolished the activity of the quinine-sensitive K⁺ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca²⁺ results from diverse effects on the cotransporter and Na⁺ and K⁺ channels. Received: 2 September 1998/Revised: 30 November 1998     

Glutamate receptor agonists evoked Ca2+-dependent and Ca2+-independent release of [3H]d-Aspartate from cultured chick retina cells

We studied the release of [³H]d-aspartate evoked by glutamate receptor agonists from monolayer cultures of chick retina cells, and found that activation of the glutamate receptors can evoke both Ca²⁺-dependent and Ca²⁺-independent release of [³H]d-aspartate. In Ca²⁺-free (no added Ca²⁺) Na⁺ medium, the agonists of the glutamate receptors induced the release of [³H]d-aspartate with the following rank order of potency: kainate>α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)∼N-methyl-d-aspartate (NMDA). In media containing 1 mM CaCl₂ the release of [³H]d-aspartate evoked by NMDA, kainate and AMPA was increased by about 112%, 20% and 39%, respectively, as compared to the release evoked by the same agonists in Ca²⁺-free medium. NMDA was the most potent agonist in stimulating the Ca²⁺-dependent release of [³H]d-aspartate, possibly by exocytosis, and AMPA was as potent as kainate. The Ca²⁺-dependent release of [³H]d-aspartate evoked by kainate was dependent on the influx of Ca²⁺ through the receptor associated channel, as well as through the N- (ω-Conotoxin GVIA-sensitive) and L- (nitrendipine-sensitive)type voltage-sensitive Ca²⁺ channels (VSCC). The exocytotic release of [³H]d-aspartate evoked by AMPA relied exclusively on Ca²⁺ entry through the L-type VSCC, whereas the effect of NMDA was partially mediated by the influx of Ca²⁺ through the receptor-associated channel, but not through L- or N-type VSCC. Thus, activation of these different glutamate receptors under physiological conditions is expected to cause the release of cytosolic and vesicular glutamate, and the routes of Ca²⁺ entry modulating vesicular release may be selectively recruited.     

Store-Dependent Entry of Ca<Superscript>2+</Superscript> into Sensory Neurons of the Rat

We studied store-dependent (activated by depletion of the endoplasmic reticulum, ER, store) entry of Ca²⁺ from the extracellular medium into neurons of the rat spinal ganglia (small- and medium-sized cells; diameter, 18 to 36 μm). Activation of ryanodine-sensitive receptors of the ER in the studied neurons superfused by Tyrode solutions containing Ca²⁺ or with no Ca²⁺ was provided by application of 10 mM caffeine. The decay phase of caffeine-induced calcium transients in a Ca²⁺-containing solution was significantly longer than that in a Ca²⁺-free solution. This fact allows us to suppose that such a phenomenon is determined by Ca²⁺ entry into the neuron from the extracellular medium activated by caffeine-induced depletion of the ER store. Substitution of Ca²⁺-free extracellular solution by Ca²⁺-containing Tyrode solution, after depletion of the ER stores induced by applications of 100 nM ryanodine, 200 μM ATP, or 1 μM thapsigargin, resulted in increases in the concentration of intracellular Ca²⁺. These observations allow us to postulate that store-dependent Ca²⁺ entry into the studied neurons is activated after depletion not only of the inositol trisphosphate-sensitive ER store but also of the ryanodine-sensitive store. This entry also occurs after blocking of ATPases of the ER by thapsigargin. The kinetic characteristics of the rising phase of store-dependent Ca²⁺ entry induced by depletion of the ER stores under the influence of various agents are dissimilar; this can be related to different mechanisms of activation of such signals and/or to a compartmental organization of the ER. Neirofiziologiya/Neurophysiology, Vol. 37, No. 3, pp. 277–283, May–June, 2005.     

Cytoplasmic Ca+ signals evoked by activation of cholecystokinin receptors: Ca2+-Dependent current recording in internally perfused pancreatic acinar cells

Summary The effects on the cytosolic Ca²⁺ concentration of activating cholecystokinin receptors on single mouse pancreatic acinar cells have been investigated using patch-clamp whole-cell recording of Ca²⁺-dependent Cl^– current. We used the nonsulphated octapeptide of cholecystokinin (CCK8-NS) since the effects of even high concentrations were rapidly reversible which was not the case for the sulphated octapeptide. A submaximal concentration of CCK8-NS (10nm) evoked a current response consisting of short-lasting (a few seconds) spikes, and some of these spikes were seen to trigger larger and longer (about half a minute) current pulses. At a higher concentration (100nm) CCK8-NS evoked smooth and sustained responses. The effect of CCK8-NS was almost abolished when the internal perfusion solution contained a high concentration of the Ca²⁺ chelator EGTA (5mm). The responses evoked by CCK8-NS were independent of the presence of Ca²⁺ in the external solution at least for the first 5 min of stimulation. Internal perfusion with GTP--S markedly potentiated the effect of CCK8-NS or at a higher concentration itself induced responses very similar to those normally evoked by CCK8-NS. Caffeine added to the external solution at a low concentration (0.2–1mm) enhanced weak CCK8-NS responses, whereas high caffeine concentrations always inhibited the CCK8-NS-evoked responses. These inhibitory caffeine effects were quickly reversible. Forskolin evoked a similar inhibitory effect. Intracellular heparin (200 g/ml) infusion markedly inhibited the response to CCK8-NS stimulation. We conclude that the primary effect of activating CCK receptors is to induced inositoltrisphosphate (IP₃) production. IP₃ evokes a small and steady Ca²⁺ release, and this in turn evokes pulsatile release of a larger magnitude from a caffeine-sensitive Ca²⁺ pool. The action of CCK is thus very similar to that previously established for muscarinic receptor activation in the same cells. Nevertheless, the pattern of the cytosolic Ca²⁺ fluctuations are different, and the basic process of Ca²⁺-induced Ca²⁺ release and Ca²⁺ signal spreading must therefore be modulated by a messenger yet unknown.     

Extracellular Ca2+ and the effect of antidiuretic hormone on the water permeability of the toad urinary bladder: An example of flow-induced alteration of flow

Summary The extracellular Ca²⁺ requirement for antidiuretic hormone (ADH) stimulation of water permeability in the toad urinary bladder has been critically examined. The polarity of the tissue was maintained with 1mm Ca²⁺ in the mucosal bathing medium and a serosal bath nominally free of Ca²⁺. Under these condition, ADH-induced osmotic water flow was inhibited by more than 60% while enhancement of the diffusional permeability to water was unaffected. Structural studies revealed that low serosal Ca²⁺ led to parallel alterations in epithelial architecture that amounted to a significant distorition of the osmotic water pathway. Prevention of these alterations, or restoration of normal cell-cell contact showed that the reduction of serosal Ca²⁺ did not restrict hormonal action,per se, but that it resulted in a weakening of cell-cell junctions such that intercellular space distension during water flow occurred to a point where the geometric conditions for maintenance of osmotic flow were compromised. We conclude that extracellular Ca²⁺ is not a requirement for the molecular aspects of ADH action but that, in its absence, a direct measurement of ADH-induced osmotic flow proves to be an inaccurate index of the hormone-generated changes in epithelial transport characteristics. Under certain conditions the ADH-effect on the tissue's hydraulic permeability is probably best assessed by measurement of the diffusional permability to water; although accuracy in this determination is difficult, it is not as strongly dependent on tissue geometry.     

Demarcation of Ca2+ transport processes in guinea pig stomach smooth muscle

Summary Microsomal fractions were isolated from gastric antrum and fundus smooth muscle of guinea pigs. Ca²⁺ uptake into and Ca²⁺ release from the membrane vesicles were studied by a rapid filtration method, and Ca²⁺ transport properties of the different regions of the stomach were compared. ATP-dependent Ca²⁺ uptake was similar in microsomes isolated from both regions. This uptake was increased by oxalate and was not affected by NaN₃. Oxalate affected Ca²⁺ permeability of both antrum and fundus microsome vesicles similarly. Fundus microsome vesicles preincubated in 100mm NaCl and then diluted to 1/20 concentration with Na⁺-free medium had significantly higher ATP-independent Ca²⁺ uptake than vesicles preincubated in 100mm KCl and treated the same way. This was not true for antrum vesicles. Monensin abolished Na⁺-dependent Ca²⁺ uptake, and NaCl enhanced Ca²⁺ efflux from fundus microsome vesicles. The halflife values of Ca²⁺ loss from fundus vesicles in the presence of NaCl were significantly smaller than those in the presence of KCl. The release of Ca²⁺ from the vesicles within the first 3 min was accelerated by NaCl to three times that by KCl. However, NaCl had ro effect on Ca²⁺ release from antrum microsome vesicles.Results suggest two distinct mechanisms of stomach membrane Ca²⁺ transport: (1) ATP-dependent Ca²⁺ uptake and (2) Na⁺–Ca²⁺ exchange; the latter in the fundus only.     

Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells

We have examined the effect of the Ca²⁺ (Mg²⁺)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent ⁴⁵Ca²⁺ uptake into IP₃-sensitive Ca²⁺ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40–50% of total Ca²⁺ uptake was inhibited by TG up to 10 nm (apparent K_i4.2 nm, Ca²⁺ pool I). An additional increase of inhibition up to 85–90% of total Ca²⁺ uptake could be achieved at 15 to 20 nm of TG (apparent K_i12.1 nm, Ca²⁺ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent K_i10 m). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca²⁺ uptake. About 30–40% of total Ca²⁺ uptake was inhibited by 100 m of vanadate (apparent K_i18 m, Ca²⁺ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent K_i300 m) or by TG up to 10 nm (Ca²⁺ pool I). The amount of IP₃-induced Ca²⁺ release was constant at 25% over a wide range of Ca²⁺ filling. About 10–20% remained unreleasable by IP₃. Reduction of IP₃ releasable Ca²⁺ in the presence of inhibitors showed similar dose-response curves as Ca²⁺ uptake (apparent K_i 3.0 nm for IP₃-induced Ca²⁺ release as compared to 4.2 nm for Ca²⁺ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca²⁺ pump fills the IP₃-sensitive Ca²⁺ pool I. At TG concentrations >10 nm which blocked Ca²⁺ pool II the apparent K_i values were 11.3 and 12.1 nm, respectively. For inhibition by vanadate up to 100 m the apparent K_i values were 18 m for Ca²⁺ uptake and 7 m for Ca²⁺ release (Ca²⁺ pool II). At vanadate concentrations up to 1 mm the apparent K_i values were 300 and 200 m, respectively (Ca²⁺ pool I). Both Ca²⁺ pools I and II also showed different sensitivities to IP₃. Dose-response curves for IP₃ in the absence of inhibitors (control) showed an apparent K_m value for IP₃ at 0.6 m. In the presence of TG (inhibition of Ca²⁺ pool I) the curve was shifted to the left with an apparent K_m for IP₃ at 0.08 m. In the presence of vanadate (inhibition of Ca²⁺ pool II), the apparent K_m for IP₃ was 2.1 m. These data allow the conclusion that there are at least three different Ca²⁺ uptake mechanisms present in pancreatic acinar cells: TG- and IP₃ insensitive but highly vanadate-sensitive Ca²⁺ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca²⁺ pools with different TG-, vanadate- and IP₃-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 246. The authors wish to thank Dr. KlausDieter Preuß for valuable discussions and Mrs. Gabriele Mörschbächer for excellent secretarial help.     

Ruthenium red selectively depletes inositol 1,4,5-trisphosphate-sensitive calcium stores in permeabilized rabbit pancreatic acinar cells

Rabbit pancreatic acinar cells, permeabilized by saponin treatment, rapidly accumulated 3.5 nmol of Ca²⁺/mg protein in an energy-dependent pool when incubated at an ambient free Ca²⁺ concentration of 100 nm. Maximal loading of the internal stores was reached at 10 min and remained unchanged thereafter. Complete inhibition of the Ca²⁺ pump with thapsigargin revealed that this plateau was the result of a steady-state between slow Ca²⁺ efflux and ATP-driven Ca²⁺ uptake. Sixty percent of the pool could be released by Ins(1,4,5)P₃, whereas GTP released another twenty percent. The striking finding of this study is that the energy-dependent store could also be released by ruthenium red. Uptake experiments in the presence of ruthenium red revealed that the dye, at concentrations below 100 m, selectively reduced the size of the Ins(1,4,5)P₃-releasable pool. Ruthenium red had no effect on the half-maximal stimulatory concentration of Ins(1,4,5)P₃. At concentrations beyond 100 m, the dye also affected the GTP-releasable pool. Comparison with thapsigargin revealed that ruthenium red released Ca²⁺ from stores loaded to steady-state at a rate markedly faster than can be explained by inhibition of the ATPase alone. From the data presented, we concluded that ruthenium red selectively releases Ca²⁺ from the Ins(1,4,5)P₃-sensitive store by activating a Ca²⁺ release channel, whereas Ca²⁺ release from the GTP-sensitive store is predominantly caused by inhibition of the Ca²⁺ pump. The postulated ruthenium red-sensitive Ca²⁺ release channel might be similar to the ryanodine-receptor in muscle.The research of Dr. P.H.G.M. Willems has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences.     

H+/Ca2+ exchange in rabbit renal cortical endosomes

Summary We have examined the effect of second messengers on ATP-driven H⁺ transport in an H⁺ ATPase-bearing endosomal fraction isolated from rabbit renal cortex. cAMP (0.1mm) had no effect on H⁺ transport. Acridine orange fluorescence in the presence of 0.5mm Ca²⁺ (+1mm EGTA) was 19±6% of control. Inhibition of ATP-driven H⁺ transport by Ca²⁺ was concentration dependent; 0.25 and 0.5mm Ca²⁺ (+1mm EGTA) inhibited acridine orange fluorescence by 50 and 80%, respectively. Ca²⁺ also produced a concentration-dependent increase in the rate of pH-gradient dissipation. Ca²⁺ did not affect ATP hydrolysis. ATP-dependent Br^– uptake was virtually unchanged in the presence of 0.5mm Ca²⁺ (+1mm EGTA). These vesicles were also shown to transport Ca²⁺ in an ATP-dependent mode. Inositol 1, 4, 5-trisphosphate had no effect on ATP-dependent Ca²⁺ uptake. These results are consistent with the co-existence of an H⁺ ATPase and an H⁺/Ca²⁺ exchanger on these endosomes, the latter transport system using the H⁺ gradient to energize Ca²⁺ uptake. Attempts to demonstrate an H⁺/Ca²⁺ antiporter in the absence of ATP have been unsuccessful. Yet, when a pH gradient was established by preincubation with ATP and residual ATP was subsequently removed by hexokinase + glucose, stimulation of Ca²⁺ uptake could be demonstrated. A Ca²⁺-dependent increase in H⁺ permeability and an ATP-dependent Ca²⁺ uptake might have important implications for the regulation of vacuolar H⁺ ATPase activity as well as the homeostasis of cytosolic Ca²⁺ concentration.     

Temperature-dependent modification of divalent cation flux in the rat parotid gland basolateral membrane

Divalent cation (Mn²⁺, Ca²⁺) entry into rat parotid acinar cells is stimulated by the release of Ca²⁺ from the internal agonist-sensitive Ca²⁺ pool via a mechanism which is not yet defined. This study examines the effect of temperature on Mn²⁺ influx into internal Ca²⁺ pool-depleted acini (depl-acini, as a result of carbachol stimulation of acini in a Ca²⁺-free medium for 10 min) and passive ⁴⁵Ca²⁺ influx in basolateral membrane vesicles (BLMV). Mn²⁺ entry into deplacini was decreased when the incubation temperature was lowered from 37 to 4°C. At 4°C, Mn²⁺ entry appeared to be inactivated since it was not increased by raising extracellular [Mn²⁺] from 50 m up to 1 mm. The Arrhenius plot of depletion-activated Mn²⁺ entry between 37 and 8°C was nonlinear, with a change in the slope at about 21°C. The activation energy (Ea) increased from 10 kcal/mol (Q₁₀=1.7) at 21–37°C to 25 kcal/mol (Q₁₀=3.0) at 21-8°C. Under the same conditions, Mn²⁺ entry into basal (unstimulated) cells and ionomycin (5 m) permeabilized depl-acini exhibit a linear decrease, with E _a of 7.8 kcal/mol (Q₁₀=1.5) and 6.2 kcal/mol (Q₁₀ < 1.5), respectively. These data suggest that depletion-activated Mn²⁺ entry into parotid acini is regulated by a mechanism which is strongly temperature dependent and distinct from Mn²⁺ entry into unstimulated acini.As in intact acini, Ca²⁺ influx into BLMV was decreased (by 40%) when the temperature of the reaction medium was lowered from 37 to 4°C. Kinetic analysis of the initial rates of Ca²⁺ influx in BLMV at 37°C demonstrated the presence of two Ca²⁺ influx components: a saturable component, with K _Ca =279 ± 43 m, V_max = 3.38 ± 0.4 nmol Ca²⁺/mg protein/min, and an apparently unsaturable component. At 4°C, there was no significant change in the affinity of the saturable component, but V_max decreased by 61% to 1.3 ± 0.4 nmol Ca²⁺/mg protein/min. There was no detectable change in the unsaturable component. When BLMV were treated with DCCD (5 mm) or trypsin (1100, enzyme to membrane) for 30 min at 37°C there was a 40% decrease in Ca²⁺ influx. When BLMV were treated with DCCD or trypsin at 4°C and subsequently assayed for Ca²⁺ uptake at 37°C there was no significant loss of Ca²⁺ influx. These data suggest that the temperature sensitive high affinity Ca²⁺ flux component in BLMV is mediated by a protein which undergoes a modification at low temperatures, resulting in decreased Ca²⁺ transport.We thank Dr. Bruce Baum, Dr. Yukiharu Hiramatsu, Dr. Ofer Eidelman, and our other colleagues for their support during this work.     

Participation of Ca2+ in cessation of cytoplasmic streaming induced by membrane excitation inCharaceae internodal cells

Summary The mechanism of the cessation of cytoplasmic streaming upon membrane excitation inCharaceae internodal cells was investigated.Cell fragments containing only cytoplasm were prepared by collecting the endoplasm at one cell end by centrifugation. In such cell fragments lacking the tonoplast, an action potential induced streaming cessation, indicating that an action potential at the plasmalemma alone is enough to stop the streaming.The active rotation of chloroplasts passively flowing together with the endoplasm also stopped simultaneously with the streaming cessation upon excitation. The time lag or interval between the rotation cessation and the electrical stimulation for inducing the action potential increased with the distance of the chloroplasts from the cortex. The time lag was about 1 second/15 m, suggesting that an agent causing the rotation cessation is diffused throughout the endoplasm.Using internodes whose tonoplast was removed by replacing the cell sap with EGTA-containing solution (tonoplast-free cells,Tazawa et al. 1976), we investigated the streaming rate with respect to the internal Ca²⁺ concentration. The rate was roughly identical to that of normal cells at a Ca²⁺ concentration of less than 10^–7 M. It decreased with an increase in the internal Ca²⁺ concentration and was zero at 1 mM Ca²⁺.The above results, together with the two facts that Ca²⁺ reversibly inhibits chloroplast rotation (Hayama andTazawa, unpublished) and the streaming in tonoplast-free cells does not stop upon excitation (Tazawa et al. 1976), lead us to conclude that a transient increase in the Ca²⁺ concentration in the cytoplasm directly stops the cytoplasmic streaming. Both Ca influxes across the resting and active membranes were roughly proportional to the external Ca²⁺ concentration, which did not affect the rate of streaming recovery. Based on these results, several possibilities for the increase in Ca²⁺ concentration in the cytoplasm causing streaming cessation were discussed.     

Gonadotropin releasing hormone stimulation of pituitary cell 3′–5′ cyclic AMP in a carp(Cyprinus carpio) is dependent on extracellular calcium

Pituitaries were collected from a common carp,yprinss carpi, belonging to vitellogenic phase and cells were disaggregated by using 0.3% collagenase and 0.05% tsypsin. Enzymatically dispersed cells were incubatedin vitro in Ca²⁺-free medium to observe the effect ofCanna punctatus GnRH (cGnRH) and Ca²⁺ on pituitary cell cAMP accumulation. Addition of cGnRH (20 Big) to pituitary cell incubation (6 × 10⁴ cells/well) containing 4 mM theophylline, a phosphodiesterase inhibitor, caused two-fold increase of cAMP accumulation in comparison to control, Addition of Ca²⁺ (2 mM) to cGnRH further augmented cAMP accumulation, i.e., four-fold as compared to control. Increasing concentrations of cGnRH in the presence of Ca²⁺ resulted in a dose-dependent increase in cAMP accumulation. To examine the specificity of Ca²⁺ augmentory effect on cGnRH-stimulated pituitary cell cAMP accumulation, a specific Ca²⁺-channel blocker, verapamil was used, At 3 μM dose verapamil completely waived Ca²⁺-augmentation of cGnRH stimulatory effect on cAMP. Interestingly, verapamil also significantly inhibited cGnRH stimulation of cAMP in the Ca²⁺-free medium. Extent of Ca²⁺ plus cGnRH stimulatory effect on cAMP was further increased by the addition of 25 pmol of calmodulin, a Ca²⁺-carrier protein, Addition of verapamil to this system strongly inhibited Ca²⁺ and ealmodulin augnientory effect on cGnRH. Reduced level of cAMP in the pituitary cell due to verapamil was even lower than that of cGnRH plus ealmodulin incubation. Data indicates a contamination of Ca²⁺ in an apparently Ca²⁺-free medium, Results suggest that in lower vertebrate, i.e., fish, GnRH stimulation of pituitary cell cAMP is dependent on extracellulnr Ca²⁺ and incubation of pituitary cell in Ca²⁺-free medium is truly not free of Ca²⁺.     

Participation of the microtubular-microfilamentous system on intracellular Ca transport and acid secretion in dispersed parietal cells

Biphasic responses of amino[¹⁴C]pyrine accumulation and oxygen consumption were registered by gastrin stimulation in dispersed parietal cells from guinea pig gastric mucosa, and this was mimicked with the calcium ionophore A23187. The characteristics of these phases (first phase and second phase) were distinguished by the differences in the requirements of extracellular Ca²⁺. The first phase evoked by gastrin or ionophore A23187 was independent of extracellular Ca²⁺, whereas the second phase was not. In the first phase, fluorescence of a cytosolic Ca²⁺ indicator (quin2-AM) increased with the stimulation of ionophore A23187 and carbamylcholine chloride in the presence of extracellular Ca²⁺. In addition, an increase in cytosolic Ca²⁺ induced by ionophore A23187, but not by carbamylcholine chloride was also observed in the absence of extracellular Ca²⁺, suggesting that Ca²⁺ pool(s) in parietal cells might be present in the intracellular organelle. Cytochalasin B and colchicine, but not oligomycin, could eliminate this cytosolic Ca²⁺ increase induced by A23187 in a Ca²⁺-free medium. On the other hand, in a Ca²⁺-free medium, addition of ATP after pretreatment with digitonin could diminish the cytosolic Ca²⁺ increase brought about by A23187. This was also observed with oligomycin-treated cells, but not with cytochalasin B-treated cells. Similarly, subcellular fractionation of a parietal cell which had been pretreated with cytochalasin B or colchicine in an intact cell system reduced the rate of ATP-dependent Ca²⁺ uptake. These observations indicate that intracellular Ca²⁺ transport in dispersed parietal cells may be regulated by the microtubular-microfilamentous system. In conclusion, this study demonstrates the possibility of the existence of intracellular Ca²⁺ transport mediated by gastrin or ionophore A23187 and regulated by the microtubular-microfilamentous system in parietal cells.     

Characterization of the 1,25-dihydroxycholecalciferol-stimulated calcium influx pathway in CaCo-2 cells

The present studies were conducted to investigate the mechanisms underlying the 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃)-induced increase in intracellular Ca²⁺ ([Ca²⁺] _i ) in individual CaCo-2 cells. In the presence of 2mm Ca²⁺, 1,25(OH)₂D₃-induced a rapid transient rise in [Ca²⁺] _i in Fura-2-loaded cells in a concentration-dependent manner, which decreased, but did not return to baseline levels. In Ca²⁺-free buffer, this hormone still induced a transient rise in [Ca²⁺] _i , although of lower magnitude, but [Ca²⁺] _i then subsequently fell to baseline. In addition, 1,25(OH)₂D₃ also rapidly induced⁴⁵Ca uptake by these cells, indicating that the sustained rise in [Ca²⁺] _i was due to Ca²⁺ entry. In Mn²⁺-containing solutions, 1,25(OH)₂D₃ increased the rate of Mn²⁺ influx which was temporally preceded by an increase in [Ca²⁺] _i . The sustained rise in [Ca²⁺] _i was inhibited in the presence of external La³⁺ (0.5mm). 1,25(OH)₂D₃ did not increase Ba²⁺ entry into the cells. Moreover, neither high external K⁺ (75mm), nor the addition of Bay K 8644 (1 μm), an L-type, voltage-dependent Ca²⁺ channel agonist, alone or in combination, were found to increase [Ca²⁺] _i , 1,25(OH)₂D₃ did, however, increase intracellular Na⁺ in the absence, but not in the presence of 2mm Ca²⁺, as assessed by the sodium-sensitive dye, sodium-binding benzofuran isophthalate. These data, therefore, indicate that CaCo-2 cells do not express L-type, voltage-dependent Ca²⁺ channels. 1,25(OH)₂D₃ does appear to activate a La³⁺-inhibitable, cation influx pathway in CaCo-2 cells.     

K+-conductance and electrogenic Na+/K+ transport of cultured bovine pigmented ciliary epithelium

Summary Using intracellular microelectrode technique, we investigated the changes in membrane voltage (V) of cultured bovine pigmented ciliary epithelial cells induced by different extracellular solutions. (1)V in 213 cells under steady-state conditions averaged –46.1±0.6 mV (sem). (2) Increasing extracellular K⁺ concentration ([K⁺] _o ) depolarizedV. Addition of Ba²⁺ could diminish this response. (3) Depolarization on doubling [K⁺] _o was increased at higher [K⁺] _o (or low voltage). (4) Removing extracellular Ca²⁺ decreasedV and reduced theV amplitude on increasing [K⁺] _o . (5)V was pH sensitive. Extra-and intracellular acidification depolarizedV; alkalinization induced a hyperpolarization.V responses to high [K⁺] _o were reduced at acidic extracellular pH. (6) Removing K _o ⁺ depolarized, K _o ⁺ readdition after K⁺ depletion transiently hyperpolarizedV. These responses were insensitive to Ba²⁺ but were abolished in the presence of ouabain or in Na⁺-free medium. (7) Na⁺ readdition after Na⁺ depletion transiently hyperpolarizedV. This reaction was markedly reduced in the presence of ouabain or in K⁺-free solution but unchanged by Ba²⁺. It is concluded that in cultured bovine pigmented ciliary epithelial cells K⁺ conductance depends on Ca²⁺, pH and [K⁺] _o (or voltage). An electrogenic Na⁺/K⁺-transport is present, which is stimulated during recovery from K⁺ or Na⁺ depletion. This transport is inhibited by ouabain and in K⁺-or Na⁺-free medium.     

Monovalent amidiniums block calcium channels in chick sensory neurons

The effect of amidiniums on high-threshold Ca²⁺ channel currents (I _Ca) was studied in chick dorsal root ganglion neurons. Guanidinium reduced I _Ca in a dose-dependent fashion. The block was relieved by increasing the concentration of the permeant ions, Ba²⁺ or Ca²⁺, suggesting a competition for a common binding site within the channel. Formamidinium and methyl-guanidinium suppressed I _Ca with similar potencies, whereas l-arginine had no effect. A neutral amidine, urea, increased I _Ca. In Ca²⁺-free solutions guanidinium and Na⁺ permeated through the Ca²⁺ channel equally well. Structure-activity relationship obtained for blocking efficacies of different amidiniums are used to discuss possible configurations of the selectivity filter in the Ca²⁺ channel.The author wishes to thank Ms. S. Engers for the cell isolation and making of the electrodes.     

Cytosolic calcium,oxygen consumption and the intracellular pH of stimulated neutrophils

The cytoplasmic pH undergoes a biphasic change when neutrophils are activated. The role of Ca²⁺ in initiating these changes was investigated. No correlation was found between the increased cytosolic [Ca²⁺] and the stimulation of the Na⁺/H⁺ antiport. Similarly, the cytoplasmic acidification elicited by activation in Na⁺-free media was found to be unrelated to [Ca²⁺]. Reversal of Na⁺/H⁺ exchange was also ruled out as the source of the acidification. Data using a variety of soluble activators indicate that metabolic acid generation is largely responsible for the observed drop in cytoplasmic pH.     

Mechanosensitive Calcium Entry and Mobilization in Renal A6 Cells

Using spectrofluorescence imaging of fura-2 loaded renal A6 cells, we have investigated the generation of the cytosolic Ca²⁺ signal in response to osmotic shock and localized membrane stretch. Upon hypotonic exposure, the cells began to swell prior to a transient increase in [Ca²⁺] _i and the cells remained swollen after [Ca²⁺] _i had returned towards basal levels. Exposure to 2/3rd strength Ringer produced a cell volume increase within 3 min, followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also produced a transient increase in [Ca²⁺] after a delay of 22 sec. Both the RVD and [Ca²⁺] _i response to hypotonicity were inhibited in a Ca²⁺-free bathing solution and by gadolinium (10 μm), an inhibitor of stretch-activated channels. Stretching the membrane by application of subatmospheric pressure (-2 kPa) inside a cell-attached patch-pipette induced a similar global increase in [Ca²⁺] _i as occurred after hypotonic shock. A stretch-sensitive [Ca²⁺] _i increase was also observed in a Ca²⁺-free bathing solution, provided the patch-pipette contained Ca²⁺. The mechanosensitive [Ca²⁺] _i response was by gadolinium (10 μm) or Ca²⁺-free pipette solutions, even when Ca²⁺ (2 mm) was present in the bath. Long-term (>10 min) pretreatment of the cells with thapsigargin inhibited the [Ca²⁺] _i response to hypotonicity. These results provide evidence that cell swelling or mechanical stimulation can activate a powerful amplification system linked to intracellular Ca²⁺ release mechanisms. Received: 3 August 1998/Revised: 19 November 1998