Adenosine deaminase normalizes cyclic AMP responses of hypothyroid rat fat cells to forskolin, but not beta-adrenergic agonists

Lipolysis and cyclic AMP accumulation in response to beta-adrenergic agonists or forskolin are severely impaired in fat cells from the hypothyroid rat. Incubating hypothyroid rat fat cells with adenosine deaminase normalizes the cyclic AMP response to forskolin, but not to beta-adrenergic agonists. Increased sensitivity to adenosine action in the hypothyroid state appears to be the basis for the impaired cyclic AMP response to forskolin, but does not appear to be the underlying defect responsible for the impaired response to beta-adrenergic agonists.     

Insulin release independent of a rise in cytosolic free Ca2+ by forskolin and phorbol ester

The role of cytosolic free Ca2+ in insulin release was evaluated using isolated rat pancreatic islets permeabilized with digitonin and incubated in Ca-EGTA buffers to fix free Ca2+ concentration at arbitrary levels. Ca2+ induced insulin release in a concentration-dependent manner with the threshold being between 0.1 and 1 microM. The hormone release was increased by forskolin and 12-O-tetradecanoyl phorbol-13-acetate (TPA), a potent activator of adenylate cyclase and that of protein kinase C, respectively. The findings suggest that activation of both protein kinase A and protein kinase C modulate insulin release without a concomitant increase in cytosolic free Ca2+.     

The effect of calcium on somatostatin inhibition of insulin release and cyclic AMP production in mouse pancreatic islets

The effect of somatostatin on glucose-induced insulin secretion and cyclic AMP accumation in isolated islets from obese, hyperglycemic ob/ob mice was studied in a microperifusion system. The normal biphasic pattern of insulin release as well as the inhibitory pattern of insulin release produced by somatostatin (0.5–1 μg/ml) was matched by similar changes in the intracellular concentration of cyclic AMP. When islets were stimulated by glucose (3 mg/ml) plus 3-isobutyl-1-methylxanthine (0.1 mM), somatostatin (0.5 μg/ml) failed to inhibit insulin secretion or cyclic AMP formation in the second phase whereas in the first phase both parameters were significantly reduced by somatostatin (0.5 μg/ml). In batch-type incubations it was shown that addition of excess calcium (to 6 mM) reversed this inhibition. In the second phase calcium potentiated the (glucose + 3-isobutyl-1-methylxanthine)-stimulated insulin secretion without affecting the cyclic AMP production. This potentiation was inhibited by somatostatin (0.1 μg/ml). Somatostatin (1 μg/ml) inhibited adenylate cyclase activity in islet homogenates. No effect of somatostatin on islet glucose utilization could be demonstrated.The results indicate a dual action of somatostatin in the inhibition of insulin release, one involving the islet adenylate cyclase and one affecting the islet uptake of calcium.     

Mastoparan, a peptide toxin from wasp venom, stimulates glycogenolysis mediated by an increase of the cytosolic free Ca concentration but not by an increase of cAMP in rat hepatocytes

A wasp venom, mastoparan, rapidly increased the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and activated phosphorylase in rat hepatocytes in a concentration-dependent manner. Mastoparan could increase [Ca²⁺]_i even in the absence of extracellular Ca²⁺, but a larger increase was observed in the presence of extracellular Ca²⁺. Thus, mastoparan mobilized Ca²⁺ from intracellular and extracellular Ca²⁺ stores. It also activated inositol triphosphate (IP₃) accumulation, but did not stimulate cAMP production. From these results, we conclude that mastoparan activates rat hepatic glycogenolysis mediated by the accumulation of IP₃, which causes an increase of [Ca²⁺]_i but not that mediated by cAMP.     

12-O-Tetradecanoylphorbol 13-Acetate and Forskolin Modify Muscarinic Receptor-Linked Ca2+ Mobilization in SH-SY5Y Neuroblastoma Cells Through Different Mechanisms

The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which causes differentiation of SH-SY5Y neuroblastoma cells, reduces carbachol binding and carbachol-stimulated Ca2+ mobilization in these cells. The decrease in responsiveness to carbachol is due partially to a reduction in the amount of Ca2+ released by the cells and partially to a decrease in the sensitivity of the cells to carbachol. These effects probably can be attributed to a reduction in muscarinic receptor number and a decrease in receptor affinity, respectively. Forskolin, an alkaloid known to cause an increase in cellular cyclic AMP, enhances Ca2+ influx into the cells without affecting the cytosolic free Ca2+ concentration. The alkaloid causes an apparent restoration of the reduced Ca2+ release, caused by TPA, but does not affect the sensitivity of the cells to carbachol. Forskolin increases the decay of carbachol-induced increase in cytosolic Ca2+. The effects of TPA appear to be linked directly to receptor function, whereas those of forskolin are due to the effect of cyclic AMP on cellular Ca2+ metabolism.     

Control of glycogen synthase and phosphorylase by insulin in rat adipocytes which is unrelated to the concentration of cyclic AMP

Incubation of adipocytes in glucose-free medium with adrenocorticotrophic hormone, epinephrine, isoproterenol, or norepinephrine increased the concentration of cyclic AMP and the percentage of phosphorylase a activity, and decreased the percentage of glycogen synthase I activity. Glucose was essentially without effect on glycogen synthase or phosphorylase in either the presence or absence of epinephrine. Although glucose potentiated the action of insulin to activate glycogen synthase, the hexose did not enhance the effectiveness of insulin in the presence of epinephrine. Likewise, glucose did not increase the ability of insulin to oppose the activation of phosphorylase by epinephrine.The activation of glycogen synthase by insulin was not associated with a decrease in the concentration of cyclic AMP. Insulin partially blocked the rise in cyclic AMP due to isoproterenol, adrenocorticotrophic hormone, and norepinephrine. The maximum effects of isoproterenol on glycogen synthase and phosphorylase were observed when the concentration of cyclic AMP was increased twofold. However, insulin clearly opposed the changes in enzyme activity produced by isoproterenol (and also adrenocorticotrophic hormone, epinephrine and norepinephrine) even though concentrations of cyclic AMP were still increased three- to fourfold. Nicotinic acid opposed the increases in cyclic AMP due to adrenocorticotrophic hormone, isoproterenol and norepinephrine to the same extent as insulin; however, nicotinic acid was ineffective in opposing the activation of phosphorylase and inactivation of glycogen synthase produced by these agents. Thus, it is unlikely that the effects of insulin on glycogen synthase and phosphorylase result from an action of the hormone to decrease the concentration of cyclic AMP.     

Increased mitochondrial free Ca2+ during ischemia is suppressed,but not eliminated by,germline deletion of the mitochondrial Ca2+ uniporter

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Activation,but not inhibition,by glucose of Ca2+-dependent K+ permeability in the rat pancreatic B-cell

The effect of glucose on the Ca²⁺-activated K⁺ permeability in pancreatic islet cells was investigated by measuring the rate of ⁸⁶Rb efflux, ⁴⁵Ca efflux and insulin release from perifused rat pancreatic islets exposed to step-wise increased in glucose concentration. When the glucose concentration was raised from intermediate (8.3 or 11.1 mM) to higher values, a rapid and sustained increase in ⁸⁶Rb outflow, ⁴⁵Ca outflow and insulin release was observed. Likewise, in the presence of 8.3 or 16.7 mM glucose, tolbutamide increased ⁸⁶Rb and ⁴⁵Ca efflux, as well as insulin release. In the two series of experiments, a tight correlation was found between the magnitude of the changes in ⁸⁶Rb and ⁴⁵Ca outflow, respectively. It is concluded that, at variance with current ideas, glucose does not inhibit the response to cytosolic Ca²⁺ of the Ca²⁺-sensitive modality of K⁺ extrusion. On the contrary, as a result of its effect upon Ca²⁺ handling, glucose stimulates the Ca²⁺-activated K⁺ permeability.     

Cytoplasmic Ca2+ does not inhibit the cardiac muscle sarcoplasmic reticulum ryanodine receptor Ca2+ channel,although Ca2+-induced Ca2+ inactivation of Ca2+ release is observed in native vesicles

Single channel properties of cardiac and fast-twitch skeletal muscle sarcoplasmic reticulum (SR) release channels were compared in a planar bilayer by fusing SR membranes in a Cs⁺-conducting medium. We found that the pharmacology, Cs⁺ conductance and selectivity to monovalent and divalent cations of the two channels were similar. The cardiac SR channel exhibited multiple kinetic states. The open and closed lifetimes were not altered from a range of 10^–7 to 10^–3 M Ca²⁺, but the proportion of closed and open states shifted to shorter closings and openings, respectively.However, while the single channel activity of the skeletal SR channel was activated and inactivated by micromolar and millimolar Ca²⁺, respectively, the cardiac SR channel remained activated in the presence of high [Ca²⁺]. In correlation to these studies, [³H]ryanodine binding by the receptors of the two channel receptors was inhibited by high [Ca²⁺] in skeletal but not in cardiac membranes in the presence of adenine nucleotides. There is, however, a minor inhibition of [³H]ryanodine binding of cardiac SR at millimolar Ca²⁺ in the absence of adenine nucleotides.When Ca²⁺-induced Ca²⁺ release was examined from preloaded native SR vesicles, the release rates followed a normal biphasic curve, with Ca²⁺-induced inactivation at high [Ca²⁺] for both cardiac and skeletal SR. Our data suggest that the molecular basis of regulation of the SR Ca²⁺ release channel in cardiac and skeletal muscle is different, and that the cardiac SR channel isoform lacks a Ca²⁺-inactivated site.This work was supported by research grants from the National Institutes of Health HL13870 and AR38970, and the Texas Affiliate of the American Heart Association, 91A-188. M. Fill was the recipient of an NIH fellowship AR01834.     

Stimulation-Evoked Ca2+ Fluxes in Cultured Bovine Adrenal Chromaffin Cells Are Enhanced by Forskolin

Forskolin, 1 microM, increased acetylcholine (ACh)-stimulated 45Ca uptake by chromaffin cells. The stimulatory effects of forskolin decreased with increasing concentration of ACh. The attenuation of the effect of forskolin on 45Ca uptake as a function of ACh concentration correlated well with changes in the forskolin effect on ACh-evoked catecholamine (CA) release. Forskolin increased excess KCl- and veratrine-evoked CA release and 45Ca uptake. Forskolin by itself stimulated 45Ca efflux and enhanced ACh-, excess KCl-, and veratrine-stimulated 45Ca efflux. High doses of forskolin inhibited both ACh-evoked 45Ca uptake and CA release. The inhibitory action of forskolin was specific to receptor-mediated response because excess KCl- and veratrine-stimulated 45Ca uptake and CA release were not inhibited. Forskolin, 0.3-30 microM, dose-dependently increased caffeine-stimulated CA release and 45Ca efflux in the absence of Ca2+ in the medium, and the effects were mimicked by dibutyryl cyclic AMP. These results suggest that cyclic AMP increases stimulation-induced CA release by enhancing calcium uptake across the plasma membrane and/or altering calcium flux in an intracellular calcium store.     

Connexin-43 hemichannels mediate cyclic ADP-ribose generation and its Ca2+-mobilizing activity by NAD+/cyclic ADP-ribose transport

The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD(+). cADPR increases intracellular Ca(2+) through the intracellular ryanodine receptor/Ca(2+) release channel (RyR). It has been known that intracellular NAD(+) approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. However, it is unclear how cADPR extracellularly generated by ecto-CD38 approaches intracellular RyR although CD38 itself or nucleoside transporter has been proposed to import cADPR. Moreover, it has been unknown what physiological stimulation can trigger Cx43-mediated export of NAD(+). Here we demonstrate that Cx43 hemichannels, but not CD38, import cADPR to increase intracellular calcium through RyR. We also demonstrate that physiological stimulation such as Fcγ receptor (FcγR) ligation induces calcium mobilization through three sequential steps, Cx43-mediated NAD(+) export, CD38-mediated generation of cADPR and Cx43-mediated cADPR import in J774 cells. Protein kinase A (PKA) activation also induced calcium mobilization in the same way as FcγR stimulation. FcγR stimulation-induced calcium mobilization was blocked by PKA inhibition, indicating that PKA is a linker between FcγR stimulation and NAD(+)/cADPR transport. Cx43 knockdown blocked extracellular cADPR import and extracellular cADPR-induced calcium mobilization in J774 cells. Cx43 overexpression in Cx43-negative cells conferred extracellular cADPR-induced calcium mobilization by the mediation of cADPR import. Our data suggest that Cx43 has a dual function exporting NAD(+) and importing cADPR into the cell to activate intracellular calcium mobilization.     

Synaptotagmin-1 is a Ca2+ sensor for somatodendritic dopamine release

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Inhibition of glucose-stimulated insulin release by alpha 2-adrenoceptor activation is parallelled by both a repolarization and a reduction in cytoplasmic free Ca2+ concentration

Effects of the alpha 2-adrenergic agonist clonidine on insulin release, membrane potential, and cytoplasmic free Ca2+ concentration ([Ca2+]i) were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Addition of 2 microM clonidine promptly inhibited glucose-stimulated insulin release, an effect accompanied by a lowering in both membrane potential and [Ca2+]i. Within minutes, the effect on Ca2+ was partly reversed, [Ca2+]i attaining a new level, although still significantly lower than in the absence of agonist. This late increase in [Ca2+]i was inhibited by 50 microM D-600, a blocker of voltage-activated Ca2+ channels. The inhibitory effects of clonidine on membrane potential, [Ca2+]i, and insulin release were abolished by 5 microM of the alpha 2-adrenergic antagonist yohimbine. Depolarization with high K+ increased [Ca2+]i also in the presence of clonidine, conditions accompanied by only a minute release of insulin. Secretion was, however, partly restored by subsequent addition of 20 mM glucose. Addition of 5 mM Ca2+ transiently reversed the effects of clonidine on both membrane potential and [Ca2+]i. Although the clonidine-induced repolarization should be enough for closing the voltage-activated Ca2+ channels with a resulting decrease in [Ca2+]i, a direct interaction of the agonist with these channels cannot be excluded. The fact that it was possible to increase [Ca2+]i with only a minor effect on insulin release suggests that the inhibitory effect of clonidine not only is due to a reduction in [Ca2+]i, but also involves interference with some more distal step in the insulin secretory machinery.     

Tributyltin increases cytosolic free Ca2+ concentration in thymocytes by mobilizing intracellular Ca2+, activating a Ca2+ entry pathway, and inhibiting Ca2+ efflux.

The immunotoxic environmental pollutant tri-n-butyltin (TBT) kills thymocytes by apoptosis through a mechanism that requires an increase in intracellular Ca2+ concentration. The addition of TBT (EC50 = 2 microM) to fura-2-loaded rat thymocytes resulted in a rapid and sustained increase in the cytosolic free Ca2+ concentration ([Ca2+]i) to greater than 1 microM. In nominally Ca(2+)-free medium, TBT slightly but consistently increased thymocyte [Ca2+]i by about 0.11 microM. The subsequent restoration of CaCl2 to the medium resulted in a sustained overshoot in [Ca2+]i; similarly, the addition of MnCl2 produced a rapid decrease in the intracellular fura-2 fluorescence in thymocytes exposed to TBT. The rates of Ca2+ and Mn2+ entry stimulated by TBT were essentially identical to the rates stimulated by 2,5-di-(tert.-butyl)-1,4-benzohydroquinone (tBuBHQ), which has previously been shown to empty the agonist-sensitive endoplasmic reticular Ca2+ store and to stimulate subsequent Ca2+ influx by a capacitative mechanism. The addition of excess [ethylenebis(oxyethylenenitrilo)]tetraacetic acid to thymocytes produced a rapid return to basal [Ca2+]i after tBuBHQ treatment but a similar rapid return to basal [Ca2+]i was not observed after TBT treatment. In addition, TBT produced a marked inhibition of both Ca2+ efflux from the cells and the plasma membrane Ca(2+)-ATPase activity. Also, TBT treatment resulted in a rapid decrease in thymocyte ATP level. Taken together, our results show that TBT increases [Ca2+]i in thymocytes by the combination of intracellular Ca2+ mobilization, stimulation of Ca2+ entry, and inhibition of the Ca2+ efflux process. Furthermore, the ability of TBT to apparently mobilize the tBuBHQ-sensitive intracellular Ca2+ store followed by Ca2+ and Mn2+ entry suggests that the TBT-induced [Ca2+]i increase involves a capacitative type of Ca2+ entry.     

Effects of a time varying strong magnetic field on release of cytosolic free Ca2+ from intracellular stores in cultured bovine adrenal chromaffin cells

This study was made to explain the mechanisms for the effects of exposure to a time varying 1.51 T magnetic field on the intracellular Ca(2+) signaling pathway. The exposure inhibited an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine chromaffin cells induced by addition of bradykinin (BK) to a Ca(2+) free medium. The exposure did not change BK induced production of inositol 1,4,5-trisphosphate (IP(3)). [Ca(2+)](i) was markedly increased in IP(3) loaded cells, and this increase was inhibited by the magnetic field exposure. A similar increase in [Ca(2+)](i) by other drugs, which stimulated Ca(2+) release from intracellular Ca(2+) stores, was again inhibited by the same exposure. However, transmembrane Ca(2+) fluxes caused in the presence of thapsigargin were not inhibited by the magnetic field exposure in a Ca(2+) containing medium. Inhibition of the BK induced increase in [Ca(2+)](i) by the exposure for 30 min was mostly recovered 1 h after exposure ended. Our results reveal that the magnetic field exposure inhibits Ca(2+) release from intracellular Ca(2+) stores, but that BK bindings to BK receptors of the cell membrane and intracellular inositol IP(3) production are not influenced.     

Termination of cytosolic Ca2+ signals: Ca2+ reuptake into intracellular stores is regulated by the free Ca2+ concentration in the store lumen.

The mechanism by which agonist-evoked cytosolic Ca2+ signals are terminated has been investigated. We measured the Ca2+ concentration inside the endoplasmic reticulum store of pancreatic acinar cells and monitored the cytoplasmic Ca2+ concentration by whole-cell patch-clamp recording of the Ca2+-sensitive currents. When the cytosolic Ca2+ concentration was clamped at the resting level by a high concentration of a selective Ca2+ buffer, acetylcholine evoked the usual depletion of intracellular Ca2+ stores, but without increasing the Ca2+-sensitive currents. Removal of acetylcholine allowed thapsigargin-sensitive Ca2+ reuptake into the stores, and this process stopped when the stores had been loaded to the pre-stimulation level. The apparent rate of Ca2+ reuptake decreased steeply with an increase in the Ca2+ concentration in the store lumen and it is this negative feedback on the Ca2+ pump that controls the Ca2+ store content. In the absence of a cytoplasmic Ca2+ clamp, acetylcholine removal resulted in a rapid return of the elevated cytoplasmic Ca2+ concentration to the pre-stimulation resting level, which was attained long before the endoplasmic reticulum Ca2+ store had been completely refilled. We conclude that control of Ca2+ reuptake by the Ca2+ concentration inside the intracellular store allows precise Ca2+ signal termination without interfering with store refilling.     

A role for cyclic AMP in entrainment of the circadian oscillator in Xenopus retinal photoreceptors by dopamine but not by light

The circadian oscillator in Xenopus retinal photoreceptor layers can be reset in similar ways by light and agonists of D2-like dopamine receptors. Treatments that increase cyclic AMP levels act on this oscillator in an opposite fashion, mimicking darkness in the induction of phase shifts. Light and dopamine have each been reported to inhibit adenylate cyclase in photoreceptors. Together, these data suggest that the transduction pathways for entrainment by dopamine and/or light include suppression of cyclic AMP or a cyclic AMP-sensitive step. In these studies, we examined this hypothesis by measuring the effects of treatment with a cyclic AMP analogue on the phase shifts induced in photoreceptor melatonin rhythms by light or a D2 receptor agonist (quinpirole). When photoreceptor layers were treated simultaneously with 8-(4-chlorophenylthio)cyclic AMP (8-CPT-cAMP) and quinpirole at any of three different phases of the circadian cycle, the resulting phase shifts of the melatonin rhythm were always the same as those caused by 8-CPT-cAMP alone. This indicates that there is a cyclic AMP-sensitive step in the dopamine entrainment pathway. In contrast, light pulses did reset the oscillator in the presence of elevated cyclic AMP. This suggests a separate cyclic AMP-insensitive transduction pathway for entrainment by light. Quinpirole reduced basal levels of cyclic AMP in photoreceptors, but light did not. These data suggest that cyclic AMP plays a role in the entrainment pathway activated by dopamine but not in the entrainment pathway activated by light.     

The de novo phospholipid effect of insulin is associated with increases in diacylglycerol, but not inositol phosphates or cytosolic Ca2+.

We have previously reported that insulin increases the synthesis de novo of phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) in BC3H-1 myocytes and/or rat adipose tissue. Here we have further characterized these effects of insulin and examined whether there are concomitant changes in inositol phosphate generation and Ca2+ mobilization. We found that insulin provoked very rapid increases in PI content (20% within 15 s in myocytes) and, after a slight lag, PIP and PIP2 content in both BC3H-1 myocytes and rat fat pads (measured by increases in 32P or 3H content after prelabelling phospholipids to constant specific radioactivity by prior incubation with 32Pi or [3H]inositol). Insulin also increased 32Pi incorporation into these phospholipids when 32Pi was added either simultaneously with insulin or 1 h after insulin. Thus, the insulin-induced increase in phospholipid content appeared to be due to an increase in phospholipid synthesis, which was maintained for at least 2 h. Insulin increased DAG content in BC3H-1 myocytes and adipose tissue, but failed to increase the levels of inositol monophosphate (IP), inositol bisphosphate (IP2) or inositol trisphosphate (IP3). The failure to observe an increase in IP3 (a postulated 'second messenger' which mobilizes intracellular Ca2+) was paralleled by a failure to observe an insulin-induced increase in the cytosolic concentration of Ca2+ in BC3H-1 myocytes as measured by Quin 2 fluorescence. Like insulin, the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the transport of 2-deoxyglucose and aminoisobutyric acid in BC3H-1 myocytes. These effects of insulin and TPA appeared to be independent of extracellular Ca2+. We conclude that the phospholipid synthesis de novo effect of insulin is provoked very rapidly, and is attended by increases in DAG but not IP3 or Ca2+ mobilization. The insulin-induced increase in DAG does not appear to be a consequence of phospholipase C acting upon the expanded PI + PIP + PIP2 pool, but may be derived directly from PA. Our findings suggest the possibility that DAG (through protein kinase C activation) may function as an important intracellular 'messenger' for controlling metabolic processes during insulin action.     

Galanin inhibits glucose-stimulated insulin release by a mechanism involving hyperpolarization and lowering of cytoplasmic free Ca2+ concentration

The intrapancreatic neuropeptide galanin has been demonstrated to lower plasma insulin levels in vivo. The effects of this peptide on insulin secretion, cytoplasmic free Ca2+ concentration and membrane potential have now been studied in vitro. Glucose-stimulated insulin secretion was inhibited by galanin under these conditions, indicating a direct effect of the peptide on the beta-cells. The neuropeptide reversed both the increase in membrane potential and cytoplasmic free Ca2+ in response to glucose stimulation. At a non-stimulatory concentration of the sugar, galanin induced a slight hyperpolarization without any effect on cytoplasmic free Ca2+. Galanin did not affect K+-induced increase in cytoplasmic free Ca2+, excluding a direct inhibitory effect on the voltage-activated Ca2+ channels. The results indicate that galanin inhibition of glucose-stimulated insulin release involves hyperpolarization with a subsequent decrease in cytoplasmic free Ca2+.     

NaCl-induced changes in cytosolic free Ca2+ in Arabidopsis thaliana are heterogeneous and modified by external ionic composition

Increases in cytosolic free Ca²⁺ ([Ca²⁺]_cyt) are common to many stress-activated signalling pathways, including the response to saline environments. We have investigated the nature of NaCl-induced [Ca²⁺]_cyt signals in whole Arabidopsis thaliana seedlings using aequorin. We found that NaCl-induced increases in [Ca²⁺]_cyt are heterogeneous and mainly restricted to the root. Both the concentration of NaCl and the composition of the solution bathing the root have profound effects on the magnitude and dynamics of NaCl-induced increases in [Ca²⁺]_cyt. Alteration of external K⁺ concentration caused changes in the temporal and spatial pattern of [Ca²⁺]_cyt increase, providing evidence for Na⁺-induced Ca²⁺ influx across the plasma membrane. The effects of various pharmacological agents on NaCl-induced increases in [Ca²⁺]_cyt indicate that NaCl may induce influx of Ca²⁺ through both plasma membrane and intracellular Ca²⁺-permeable channels. Analysis of spatiotemporal [Ca²⁺]_cyt dynamics using photon-counting imaging revealed additional levels of complexity in the [Ca²⁺]_cyt signal that may reflect the oscillatory nature of NaCl-induced changes in single cells.