Dual-wavelength Microfluorometry Research Articles

Glucagon amplifies tetrodotoxin-resistant Na+ oscillations in glucose-stimulated pancreatic beta-cells.

The cytoplasmic concentration of Na+ ([Na+]i) was measured in individual mouse beta-cells with dual-wavelength microfluorometry using the indicator SBFI. The addition of 10 nM glucagon to a medium containing 11 mM glucose and 1.3 mM Ca2+ resulted in a 24% increase of [Na+]i often associated with superimposed oscillations. When replacing Ca2+ with 5 mM Sr2+, the presence of glucagon resulted in an increase of the amplitude of the [Na+]i oscillations with decrease of their frequency. Similar effects as with glucagon were obtained with 1 mM 8-Br-cAMP or 5 microM of the Ca2+ channel agonist BAY K8644. The glucose-induced oscillations of [Na+]i were resistant to 3 microM tetrodotoxin and disappeared after addition of 100 nM clonidine, 10 microM methoxyverapamil or 400 microM diazoxide. Studies of cell aggregates revealed the existence of well synchronized [Na+]i oscillations similar to those in individual beta-cells. The results provide evidence for tetrodotoxin-resistant [Na+]i rhythmicity in glucose-stimulated pancreatic beta-cells subject to regulation with cAMP.

Insulin-secreting INS-1 cells generate a novel type of poorly synchronized [formula omitted] transients

Pancreatic β-cells have an intrinsic oscillatory Ca 2+ activity supposed to be synchronized among the islets by cytoplasmic Ca 2+ transients elicited by nonadrenergic, noncholinergic (NANC) neurons. To improve the understanding of this process, the cytoplasmic Ca 2+ concentration ([Ca 2+] i) was measured in two insulin-releasing cell lines using dual wavelength microfluorometry and the indicator fura-2. INS-1 cells but not RINm5F cells were found to generate transients of [Ca 2+] i in the presence of the Ca 2+ channel blocker methoxyverapamil. These transients differed from those occurring in native β-cells persisting in the presence of thapsigargin or during prolonged exposure to ATP. Moreover, the [Ca 2+] i transients were poorly synchronized whether or not the INS-1 cells had physical contact. If appearing in native β-cells, the type of [Ca 2+] i transients now observed may interfere with the coordination of the β-cell rhythmicity evoked by NANC neurons.

Pancreatic beta-cells from obese-hyperglycemic mice are characterized by excessive firing of cytoplasmic Ca2+ transients.

Pancreatic beta-cells from obese-hyperglycemic (ob/ob) mice are widely used for studying the mechanisms of insulin release, including its regulation by the cytoplasmic Ca2+ concentration ([Ca2+]i). In this study, we compared changes of [Ca2+]i in single beta-cells isolated from ob/ob mice with those from lean mice using dual-wavelength microfluorometry and the indicator fura-2. There were no differences in the frequency, amplitude, and half-width of the slow oscillations induced by glucose. Most beta-cells from the obese mice responded to 10 mM caffeine with transformation of the oscillations into sustained elevation of [Ca2+]i, a process counteracted by ryanodine. The beta-cells from the obese mice were characterized by ample generation of [Ca2+]i transients, which increased in number in the presence of glucagon. The transients became less frequent when leptin was added at a concentration as low as 1 nM. It is suggested that the excessive firing of [Ca2+]i transients in the ob/ob mice is owing to the absence of leptin and is mediated by activation of the phospholipase C signaling pathway.

Mobilization of Ca2+stores in individual pancreatic β -cells permeabilized or not with digitonin or α -toxin

The concentration of free Ca2+in the cytoplasm and organelles of individual mouse pancreatic β-cells was estimated with dual wavelength microfluorometry and the indicators Fura-2 and furaptra. Measuring the increase of cytoplasmic Ca2+resulting from intracellular mobilization of the ion in ob/ob mouse β-cells, most organelle calcium (92%) was found in acidic compartments released when combining the Ca2+ionophore Br-A23187 with a protonophore. Only 3–4% of organelle calcium was recovered from a pool sensitive to the Ca2+-ATPase inhibitor thapsigargin. Organelle Ca2+was also measured directly in furaptra-loaded β-cells after controlled plasma membrane permeabilization. The permeabilizing agent α-toxin was superior to digitonin in preserving the integrity of intracellular membranes, but digitonin provided more reproducible access to intracellular sites. After permeabilization, the thapsigargin-sensitive fraction of Ca2+detected by furaptra was as high as 90%, suggesting that the indicator essentially measures Ca2+in endoplasmic reticulum (ER). Both α-toxin- and digitonin-permeabilized cells exhibited ATP-dependent uptake of Ca2+into thapsigargin-sensitive stores with half-maximal and maximal filling at 6–11 μM and 1 mM ATP respectively. Most of the thapsigargin-sensitive Ca2+was mobilized by inositol 1,4,5-trisphosphate (IP3), whereas caffeine, ryanodine, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate lacked effects both in β-cells from ob/ob mice and normal NMRI mice. Mobilization of organelle Ca2+by 4-chloro-3-methylphenol was attributed to interference with the integrity of the ER rather than to activation of ryanodine receptors. The observations emphasize the importance of IP3for Ca2+mobilization in pancreatic β-cells, but question a role for ryanodine receptor agonists.

Effect of bepridil on intracellular calcium concentration and contraction in cultured rat ventricular myocytes.

We studied the effects of a new antiarrhythmic and antianginal agent, bepridil, on the intracellular calcium transient and contraction of cultured neonatal rat ventricular cells, and compared the effects with those caused by an authentic Ca2+ -entry blocker, D600 (methoxyverapamil). The Ca2+ transient was measured by using dual-wavelength microfluorometry of fura-2. The contraction was measured as a shortening of cell aggregates with the use of a video image-analyzing system. Both bepridil (1-30 microM) and D600 (1-30 microM) decreased the peak systolic amplitude of the Ca2+ transient in a concentration- and frequency-dependent manner. Bepridil, but not D600, significantly shortened the half-decay time of the Ca2+ transient and prolonged the time course of the contraction. D600 decreased the contraction in parallel with the decrease in the peak Ca2+ transient, whereas bepridil exerted no significant effect on the contraction. Bepridil (10 microM) induced a leftward shift (to lower amplitude of peak systolic Ca2+ transient) of the relation between the magnitude of contraction and the peak systolic Ca2+ transient, which was obtained by changing external Ca2+ concentration. In contrast, D600 (10 microM) did not affect the relation. The results suggest that the negative inotropic effect of bepridil (caused by its Ca2+ channel-blocking effect) is offset by its simultaneous increase in the sensitivity of contractile protein(s) to intracellular Ca2+, which may be a unique characteristic of this antiarrhythmic agent in a clinical setting.

Synchronization of Glucose-Induced Ca2+Transients in Pancreatic β-Cells by a Diffusible Factor

Glucose is known to induce transients of cytoplasmic Ca2+by mobilizing intracellular stores when pancreatic β-cells are exposed to glucagon. Dual wavelength microfluorometry with fura-2 was used to study such transients in individual β-cells isolated from ob/ob-mice. The Ca2+transients were often synchronized in β-cells situated up to 80 μm apart. The messenger might be nitric oxide, as indicated from a decreased number of synchronized transients in the presence of 500 μmol/l oxyhemoglobin or 10 mmol/l Nω-nitro-L-arginine methyl ester. The discovery that Ca2+transients are synchronized in the absence of cell contact indicates the involvement of a diffusible factor in coordinating the activity of the insulin-releasing β-cells.

In situ characterization of nonmitochondrial Ca2+ stores in individual pancreatic beta-cells.

Free Ca2+ was measured in intracellular stores of individual mouse pancreatic beta-cells using dual-wavelength microfluorometry and the low-affinity Ca2+ indicator furaptra. Controlled permeabilization of the plasma membrane with 4 micromol/l digitonin revealed that 22% of the furaptra was trapped in intracellular nonnuclear compartments. When 3 mmol/l ATP and 200 nmol/l Ca2+ were simultaneously present, this cation rapidly accumulated in the organelle pool, reaching an average concentration of 200-500 micromol/l. Whereas agents affecting the mitochondrial function (5 mmol/l succinate, 2 micromol/l ruthenium red, or 10 micromol/l antimycin A + 2 microg/ml oligomycin) had little effects, the Ca2+-ATPase inhibitor thapsigargin released 92% of the Ca2+ mobilizable with the ionophore Br-A23187. Digital imaging revealed regional differences in the organelle Ca2+. The regions with the highest Ca2+ concentration were particularly responsive to inositol 1,4,5-trisphosphate (IP3). IP3 mobilized Ca2+ in a dose-dependent way with half-maximal and maximal effects at about 1 and 5 micromol/l, respectively. High concentrations of IP3 released about half of the thapsigargin-sensitive Ca2+, but there were no responses to agents known to activate ryanodine receptors, such as 10 mmol/l caffeine, 0.1-1 micromol/l ryanodine, or 1-5 micromol/l cyclic ADP ribose. The results reinforce the concept that mobilization of intracellular Ca2+ in the pancreatic beta-cell is mediated by IP3 receptors rather than ryanodine receptors.

Unmasking of a periodic Na+ entry into glucose-stimulated pancreatic beta-cells after partial inhibition of the Na/K pump.

The cytoplasmic concentration of Na+ ([Na+]i) was measured in individual mouse beta-cells using dual wavelength microfluorometry and the indicator sodium-binding benzofuran isophtalate. Under conditions known to induce large amplitude oscillations in cytoplasmic Ca2+ (1.3 mM Ca2+; 11 mM glucose), [Na+]i remained low and stable at 10-14 mM. Partial suppression of the Na/K pump with 50 microM ouabain resulted in oscillations of [Na+]i in 65% of the cells (frequency, 0.13+/-O.O1 min(-l); amplitude, 4.4 +/-0.3 mM). The oscillations were unaffected by the presence of 3 microM tetrodotoxin, but disappeared when the medium was depleted of Ca2+ or supplemented with 10 microM methoxyverapamil. The analysis of the ouabain effect was facilitated by replacing extracellular Ca2+ with 5 mM Sr2+. In the Sr2+-containing medium, oscillations of [Na+]i were seen in more than 70% of the beta-cells exposed to 11 mM glucose. Ouabain (50 microM) modified the [Na+]i oscillations by increasing their amplitudes almost 3-fold and reducing the frequency from once every 3 min to once every 10 min. A relationship between oscillations of cytoplasmic Sr2+ and Na+ was apparent both from observations of similar frequencies and for the modifications obtained with ouabain. It is concluded that the glucose-induced oscillations of cytoplasmic Ca2+ result in a rhythmic entry of Na+, usually balanced by the Na/K pump. A resulting periodic consumption of ATP in the Na/K pump might have implications for the release of insulin by affecting ATP-dependent processes associated with the plasma membrane.

Calcitonin down-regulates immediate cell signals induced in human osteoclast-like cells by the bone sialoprotein-IIA fragment through a postintegrin receptor mechanism.

Calcitonin (CT) is a peptide hormone that interacts with the cAMP-and phospholipase C-associated CT receptor subtypes. We investigated whether CT modulates the interaction of human tumoral osteoclast-like (GCT23) cells with a protein of the bone matrix, bone sialoprotein-II (BSP-II). Single GCT23 cells loaded with the intracellular Ca2+ indicator fura-2 were treated with the maximal active dose (300 micrograms/ml) of the 18-mer Arg-Gly-Asp (RGD)-containing BSP-IIA fragment, and the cytosolic free Ca2+ concentration ([Ca2+]i) was measured by dual wavelength microfluorometry. BSP-IIA stimulated an elevation in [Ca2+]i, consisting mainly of a peak, followed by a rapid return toward baseline. Pretreatment with CT induced a modest elevation of [Ca2+]i. However, CT significantly inhibited the response to BSP-IIA in a dose-dependent manner. Maximal inhibition (90% vs. untreated) was observed in the micromolar range. The intracellular mechanisms leading to this effect were investigated by pretreatment of GCT23 cells with the cAMP permeant analog, (Bu2)cAMP, and the protein kinase-C-activating agent, 12-O-tetradecanoylphorbol 13-acetate. Similar to CT, both agents inhibited the response to 300 micrograms/ml BSP-IIA. The effect induced by CT was specific, because an increase in the extracellular Ca2+ concentration, which is also known to inhibit bone resorption, failed to modify the ability of BSP-IIA to alter [Ca2+]i in GCT23 cells. To investigate whether the CT-induced alteration of BSP-IIA-dependent cell signals was due to a modification in the synthesis of cell surface receptors (integrins) for the extracellular matrix macromolecules, 1-h CT-treated [35S]methionine metabolically labeled GCT23 cell lysates were immunoprecipitated with anti-alpha 3-, -alpha v-, -beta 1-, and -beta 3-integrin subunit antibodies. Autoradiography demonstrated that 10(-7)-10(-6) M CT did not alter new synthesis of the alpha v beta 3 and the alpha 3 beta 1 receptors. Similarly, CT did not affect surface expression of these receptors, assessed by enzyme-linked immunosorbent assay. Finally, no alteration of the adhesion rate and spreading of GCT23 cells onto BSP-IIA-coated substrates was observed. This indicates that CT-induced down-regulation of immediate cell signals prompted by BSP-IIA in GCT23 cells is a postintegrin receptor event.

Glutamate receptor agonists modulate [Ca2+]i in isolated rat melanotropes.

Although glutamate is the predominant excitatory amino acid in the vertebrate central nervous system (CNS) where it affects a variety of physiological processes and pathophysiological states, the role that glutamate receptors may play outside the CNS has not been clearly established. In the present study, the effects of N-methyl-D-aspartate (NMDA), alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) kainate, and metabotropic glutamate receptor agonists and antagonists were investigated on neuroendocrine melanotropes of the rat pars intermedia using single-cell dual-wavelength microfluorometry and the Ca(2+)-sensitive probe, fura-2, to monitor changes in [Ca2+]i. Glutamate induced a rapid, concentration-dependent rise in [Ca2+]i with an EC50 of 24 microM that was Mg(2+)-sensitive and dependent on the presence of extracellular Ca2+. NMDA increased [Ca2+]i in a glycine-dependent manner with an EC50 of 83 microM that was blocked by 1 microM MK-801 and 1 mM Mg2+. The non-NMDA receptor agonists kainate, AMPA, and quisqualate increased [Ca2+]i with an EC50 of 124, 5 and 8 microM, respectively. Responses to kainic acid were blocked by 10 microM CNQX and were shown to be sensitive to Mg2+ and dihydropyridine. AMPA stimulation was the most potent, and glutamate stimulation was the most efficacious at mediating increases in [Ca2+]i. The metabotropic receptor-specific agonist, trans-ACPD, failed to induce a change in [Ca2+]i. The glutamate-induced Ca2+ influx was about half of that elicited by a 50 mM K(+)-induced membrane depolarization and activation of voltage-sensitive Ca2+ channels. These results demonstrate the presence of glutamate receptors on rat melanotropes and suggest that glutamate receptors in the intermediate lobe of the pituitary may provide the excitatory counterbalance to the well-described secretoinhibiting input via dopamine and gamma-aminobutyric acid receptors.

Intracellular Mg2+ movement during muscarinic stimulation. Mg2+ uptake by the intracellular Ca2+ store in rat sublingual mucous acini.

The muscarinic agonist carbachol produces a sustained elevation in the cytosolic free Mg2+ concentration ([Mg2+]i) by mobilizing an intracellular Mg2+ pool in rat sublingual mucous acini (Zhang, G. H., and Melvin, J.E. (1992) J. Biol. Chem. 267, 20721-20727). In the present study, we investigated the relationship between the agonist-induced mobilization of the intracellular Ca2+ store and Mg2+ movement in acini loaded with the Ca(2+)-selective chelator bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) by monitoring [Mg2+]i using dual wavelength microfluorometry of the Mg(2+)-sensitive fluorescent indicator mag-fura-2. The resting [Mg2+]i in BAPTA-loaded acini was comparable to unloaded acini (0.36 +/- 0.01 mM (n = 29) versus 0.35 +/- 0.01 mM (n = 119)). In contrast to the > 40% sustained increase in [Mg2+]i in unloaded acini, carbachol stimulation of BAPTA-loaded acini induced a rapid transient [Mg2+]i decrease (approximately 30%), followed by a slower recovery to the prestimulated [Mg2+]i in 3-4 min. Furthermore, in Ca(2+)-free medium or when Ca2+ influx was blocked with La3+ or Ni2+, carbachol induced a sustained decrease in [Mg2+]i (approximately 40%). Reintroducing extracellular Ca2+ resulted in recovery of [Mg2+]i, even in the absence of extracellular Mg2+. 8-(Di-ethylamino)octyl 3,4,5-trimethoxybenzoate, an inhibitor of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ release pathway, completely blocked the carbachol-induced decrease in [Mg2+]i, whereas thapsigargin, a Ca(2+)-ATPase inhibitor that empties the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store, stimulated a decrease in [Mg2+]i. The thapsigargin-induced Mg2+ uptake was into the same intracellular pool as that activated by carbachol because stimulation with thapsigargin after carbachol did not induce a further decrease in [Mg2+]i. Taken together, these results strongly suggest that depletion of the inositol 1,4,5-trisphosphate-sensitive Ca2+ store activates Mg2+ uptake, most likely to maintain charge balance of the intracellular Ca2+ pool.

Glucagon-like peptide-1-(7-36)amide and a rise in cyclic adenosine 3',5'-monophosphate increase cytosolic free Ca2+ in rat pancreatic beta-cells by enhancing Ca2+ channel activity.

Glucagon-like peptide-1 (GLP-1), in the form of either GLP-1-(7-36)amide or GLP-1-(7-37), has been shown to potently stimulate insulin release in a glucose-dependent manner and is suggested to be a physiological incretin. To explore the mechanisms by which GLP-1-(7-36)amide stimulates insulin release, we investigated its action on the cytosolic free Ca2+ concentration ([Ca2+]i) in single rat pancreatic beta-cells by the dual wavelength microfluorometry with fura-2. In the presence of 8.3 mM glucose, GLP-1-(7-36)amide at a concentration as low as 3 x 10(-12) M produced a rapid transient increase in [Ca2+]i in some of the single beta-cells. GLP-1-(7-36)amide at 10(-11) M or more evoked the [Ca2+]i response in the majority of beta-cells. In the presence of 2.8 mM glucose, GLP-1-(7-36)amide was without effect. The [Ca2+]i response to GLP-1-(7-36)amide was completely and reversibly inhibited under Ca(2+)-free conditions and by 1 microM nitrendipine, a blocker of L-type Ca2+ channels. Elevation of cAMP in beta-cells by either 10 microM forskolin, an activator of adenylyl cyclase, or 5 mM (bu)2cAMP (db-cAMP) produced an increase in [Ca2+]i similar to that caused by GLP-1-(7-36)amide. The db-cAMP-induced increase in [Ca2+]i was also completely blocked by nitrendipine. In the continuous presence of GLP-1-(7-36)amide and after the transient [Ca2+]i increase it elicited, db-cAMP failed to evoke the [Ca2+]i response. It is concluded that GLP-1-(7-36)amide at physiological concentrations and a rise in cAMP increase [Ca2+]i in pancreatic beta-cells by enhancing the activity of L-type Ca2+ channels in the beta-cell plasma membrane. It is suggested that the cAMP-operative mechanism is involved in the GLP-1-(7-36)amide action to increase [Ca2+]i in beta-cells.

Altered responses to agonists after chronic in vivo atropine administration in rat parotid acini.

Salivary gland hypofunction, resulting from a variety of perturbations including prescribed medications, is associated with adverse effects on the health of the oral cavity. In the present study, we investigated the in vivo effects of chronic administration of atropine, a muscarinic antagonist, on the acute response of rat parotid acini to alpha-adrenergic and muscarinic stimulation. The regulation of intracellular pH (pHi) and cytosolic free Ca2+ ([Ca2+]i) were monitored using dual wavelength microfluorometry of the ion-sensitive fluorescent dyes, BCECF and fura-2, respectively. Chronic atropine treatment (40 mg/kg/d for 4 weeks) significantly increased the magnitude of the initial (< 30 s) agonist-induced rise in [Ca2+]i, but did not alter the sustained increase in [Ca2+]i (> 2 min). The generation of inositol trisphosphates and inositol tetrakisphosphates after 30 s of muscarinic stimulation was not significantly altered. The resting Cl- content as well as the stimulated Cl- loss, were reduced in parotid acini after chronic atropine administration. In addition, the muscarinic- and alpha-adrenergic-induced intracellular acidification was blunted, suggesting that reduced HCO3- efflux occurs in acini isolated from atropine-treated animals. Our results indicate (1) that chronic atropine treatment does not inhibit the receptor-coupled generation of inositol phosphates or the resulting rise in [Ca2+]i and (2) chronic treatment may prevent the production of saliva either by reducing the driving force for anion-dependent fluid secretion or by preventing the activation of the anion efflux pathway.

Na+ influx is mediated by Na(+)-K(+)-2Cl- cotransport and Na(+)-H+ exchange in sublingual mucous acini

The intracellular free Na+ concentration ([Na+]i) was studied using dual-wavelength microfluorometry of the fluorescent Na+ indicator sodium-binding benzofuran isophthalate (SBFI) to determine the mechanism(s) by which muscarinic stimulation increases the Na+ content in rat sublingual mucous acini. [Na+]i was 15.5 +/- 0.7 mM in acini superfused with a Na(+)-containing medium (135 mM Na+). Application of ouabain, a Na(+)-K(+)-adenosinetriphosphatase inhibitor, resulted in an increase in [Na+]i (approximately 75% in 10 min), whereas replacement of extracellular Na+ with N-methyl-D-glucamine induced a gradual decrease in [Na+]i (approximately 55% decrease in 5 min). The recovery of [Na+]i in Na(+)-depleted acini was K+ and Cl- dependent and was inhibited by bumetanide (Bum), a specific Na(+)-K(+)-2Cl- cotransport inhibitor, and by 5-(N-methyl-N-isobutyl)amiloride (MIBA), an amiloride derivative that specifically blocks Na(+)-H+ exchange. Stimulation with a muscarinic agonist (10 microM carbachol) resulted in a dramatic increase in the [Na+]i [approximately 180%, half time (t1/2) approximately 1 min] and a net increase in Na+ content, as measured with 22Na+ (approximately 110%, t1/2 approximately 1 min). Both the initial rate of the increase in [Na+]i and the magnitude of the net increase in Na+ content were dramatically blunted by Bum and MIBA. Increasing the intracellular free Ca2+ concentration ([Ca2+]i) with ionomycin, a Ca2+ ionophore, resulted in an increase in [Na+]i. Preventing the [Ca2+]i increase by chelating cytosolic Ca2+ with bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid completely abolished the agonist-induced evaluation in [Na+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretagogue-induced mobilization of an intracellular Mg2+ pool in rat sublingual mucous acini.

The regulation of the intracellular free Mg2+ concentration ([Mg2+]i) was monitored in rat sublingual mucous acini using dual wavelength microfluorometry of the Mg(2+)-sensitive dye mag-fura-2. Acini attached to coverslips and superfused continuously with a Mg(2+)-containing medium (0.8 mM) have a steady-state [Mg2+]i of 0.35 +/- 0.01 mM. Adjusting the extracellular Mg2+ concentration to 0 and 10 mM or removing extracellular Na+ did not alter the resting [Mg2+]i. Stimulation with the Ca(2+)-mobilizing, muscarinic agonist, carbachol, induced a sustained increase in [Mg2+]i (approximately 50%; t1/2 < 20 s; Kd approximately 1.5 microM), the magnitude and the duration of which were unchanged in Mg(2+)-depleted medium indicating that the rise in [Mg2+]i was generated by Mg2+ release from an intracellular Mg2+ pool. Forskolin, which increases the intracellular cAMP content, produced a small, transient increase in the [Mg2+]i (< 10%). Muscarinic stimulation in a Ca(2+)-free medium blunted the initial increase in [Mg2+]i by approximately 50%, whereas the sustained increase in [Mg2+]i was lost. When the muscarinic-induced increase in [Ca2+]i was blocked by 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate, an inhibitor of the agonist-sensitive intracellular Ca2+ release pathway, both the initial and the sustained phases of the increase in [Mg2+]i were virtually eliminated. Thapsigargin and 2,5-di-(terbutyl)-1,4-benzohydroquinone, which increase [Ca2+]i by inhibiting microsomal Ca(2+)-ATPase, caused a dramatic increase in [Mg2+]i. Stimulation in a Na(+)-free medium or in the presence of bumetanide, an inhibitor of Na+/K+/2Cl- cotransport, blunted the agonist-induced rise in [Mg2+]i (approximately 50%), whereas ouabain, a Na+,K(+)-ATPase inhibitor, had no significant effect. FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), a mitochondrial uncoupler, mobilized an intracellular Mg2+ pool as well. The carbachol-induced increase in [Mg2+]i was markedly inhibited by FCCP (approximately 80%), suggesting that the same pool(s) of Mg2+ were primarily involved. The above results provide strong evidence that Ca(2+)-mobilizing agonists increase cytoplasmic free [Mg2+] by releasing an intracellular pool of Mg2+ that is associated with a rise in the [Na+]i.

Regulation of cytoplasmic pH in rat sublingual mucous acini at rest and during muscarinic stimulation.

The regulation of intracellular pH (pHi) in rat sublingual mucous acini was monitored using dual-wavelength microfluorometry of the pH-sensitive dye BCECF (2',7'-biscarboxyethyl-5(6)-carboxyfluorescein). Acini attached to coverslips and continuously superfused with HCO3(-)-containing medium (25 mM NaHCO3/5% CO2; pH 7.4) have a steady-state pHi of 7.25 +/- 0.02. Acid loading of acinar cells using the NH4+/NH3 prepulse technique resulted in a Na(+)-dependent, MIBA-inhibitable (5-(N-methyl-N-isobutyl) amiloride, Ki approximately 0.42 microM) pHi recovery, the kinetics of which were not influenced by the absence of extracellular Cl-. The rate and magnitude of the pHi recovery were dependent on the extracellular Na+ concentration, indicating that Na+/H+ exchange plays a critical role in maintaining pHi above the pH predicted for electrochemical equilibrium. When the NH4+/NH3 concentration was varied, the rate of pHi recovery was enhanced as the extent of the intracellular acidification increased, demonstrating that the activity of the Na+/H+ exchanger is regulated by the concentration of intracellular protons. Switching BCECF-loaded acini to a Cl(-)-free medium did not significantly alter resting pHi, suggesting the absence of Cl-/HCO3- exchange activity. Muscarinic stimulation resulted in a rapid and sustained cytosolic acidification (t 1/2 < 30 sec; 0.16 +/- 0.02 pH unit), the magnitude of which was amplified greater than two-fold in the presence of MIBA (0.37 +/- 0.05 pH unit) or in the absence of extracellular Na+ (0.34 +/- 0.03 pH unit). The agonist-induced intracellular acidification was blunted in HCO3(-)-free media and was inhibited by DPC (diphenylamine-2-carboxylate), an anion channel blocker. In contrast, the acidification was not influenced by removal of extracellular Cl-. The Ca2+ ionophore, ionomycin, mimicked the effects of stimulation, whereas preloading acini with BAPTA (bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid) to chelate intracellular Ca2+ blocked the agonist-induced cytoplasmic acidification. The above results indicate that during muscarinic stimulation an intracellular acidification occurs which: (i) is partially buffered by increased Na+/H+ exchange activity; (ii) is most likely mediated by HCO3- efflux via an anion channel; and (iii) requires an increase in cytosolic free [Ca2+].

Characterization of oscillations in cytosolic free Ca2+ concentration and measurement of cytosolic Na+ concentration changes evoked by angiotensin II and vasopressin in individual rat aortic smooth muscle cells. Use of microfluorometry and digital imaging

Dual wavelength microfluorometry was used to characterize the changes in cytosolic free Ca2+ concentration [( Ca2+]i) in individual cultured rat aortic vascular smooth muscle cells (VSMC). Angiotensin II (ANG II) at 10(-8) M induced a transient rise in [Ca2+]i from 43 +/- 2 to 245 +/- 23 nM, lasting for approximately 60 s (n = 42). In half of the population, discrete oscillations in [Ca2+]i of smaller amplitude occurred after the initial [Ca2+]i peak, with a period of 58 +/- 8 s and a maximum height of 132 +/- 24 nM. A similar oscillatory pattern was observed with arginine vasopressin (AVP). The oscillations depended upon the presence of extracellular Ca2+. Cytosolic free Na+ concentration ([Na+]i) in VSMC was also measured using the fluorescent Na+ probe sodium-binding benzofuran isophthalate. ANG II induced a gradual and sustained elevation of [Na+]i, from 24.0 +/- 6.2 to 36 +/- 9.7 mM. In response to AVP, [Na+]i rose to 41.0 +/- 11.6 mM. Video imaging of individual VSMC, with on-line ratio calibration of [Ca2+]i, revealed an inhomogeneous distribution of Ca2+ within the cell. [Ca2+] in the nucleus was invariably lower than in the cytoplasm in resting cells. In the cytoplasm, there were small regions in which [Ca2+] was elevated, or "hot spots." In Ca(2+)-containing medium, the initial rise in [Ca2+]i triggered by ANG II and AVP appeared to emanate from the hot spots and to spread evenly throughout the cytoplasm. Between [Ca2+]i oscillations, Ca2+ retreated back to the original hot spots. This study demonstrates the cellular and subcellular heterogeneity of [Ca2+]i both in resting VSMC and during stimulation by ANG II and AVP and reports the direct measurement of [Na+]i in VSMC. The results suggest an action of Ca2+ in both the initial and sustained phases of the response in VSMC and a link between changes in [Ca2+]i and [Na+]i.

Effect of angiotensin II on cytosolic free calcium in neonatal rat cardiomyocytes

The effect of angiotensin II (ANG II) on cytosolic free Ca2+ concentration ([Ca2+]i) was studied in cultured neonatal rat ventricular myocytes. [Ca2+]i was estimated in groups of one to three cells by dual-wavelength microfluorometry or in cell populations using conventional fluorometry. ANG II (10(-8) M) produced an acute short-lived increase over the control basal diastolic [Ca2+]i and increased the frequency of the [Ca2+]i transients. The amplitude of the [Ca2+]i transients was decreased to 64.4% of basal values. The effect of ANG II on [Ca2+]i was blocked by the selective AT1 receptor subtype antagonist Du Pont 753 but not by the AT2 antagonist PD 123319. Removal of extracellular Ca2+ or blockade of voltage-gated Ca2+ channels in cells cultured for 5-7 days abolished the [Ca2+]i transients, but only partially diminished the effect of ANG II on [Ca2+]i. Thapsigargin, an inhibitor of sarcoplasmic reticulum Ca(2+)-Mg(2+)-ATPase, reduced or abolished the [Ca2+]i response to ANG II. Phorbol 12-myristate 13-acetate (PMA), 10(-6) and 10(-7) M, also decreased the amplitude of the Ca2+ transients similar to ANG II. Pretreatment with 10(-6) M PMA or 10(-6) M 1-oleoyl-2-acetyl-glycerol (OAG) inhibited the initial rise in [Ca2+]i and the Ca2+ transients. Thus ANG II produces an acute rise in [Ca2+]i which is derived predominantly from sarcoplasmic reticulum intracellular stores. This acute effect is followed by a significant reduction in the amplitude for the Ca2+ transient and may be mediated by activation of protein kinase C.

Disappearance of glucose-induced oscillations of cytoplasmic Ca 2+ in pancreatic β-cells exposed to streptozotocin or alloxan

Dual wavelength microfluorometry and the indicator fura-2 were employed for measuring cytoplasmic Ca 2+ (Ca 2+ i) in individual pancreatic β-cells isolated from ob/ob-mice. In most β-cells, a rise of external glucose from 3 to 20 mM resulted in large amplitude oscillations in Ca 2+ i, superimposed on a basal level of 60–90 nM. The diabetogenic agents streptozotocin and alloxan (1–4.4 mM) rapidly abolished the glucose-induced oscillations of Ca 2+ i. The presence of a high glucose concentration during the exposure to the drugs counteracted the action of alloxan but not that of streptozotocin. Perturbation of the cyclic variations of Ca 2+ i by streptozotocin did not interfere with a glucose-induced increase of the ion in mildly affected β-cells. The most advanced lesions obtained with the exposure to the diabetogenic agents were manifested as uncontrolled and sustained increases of Ca 2+. Although disrupting the intracellular Ca 2+ homeostasis by separate mechanisms, streptozotocin and alloxan may finally kill the β-cells by activating a common suicidal process due to an excessive rise of Ca 2+ i.

Glucose induces temperature-dependent oscillations of cytoplasmic Ca 2+ in single pancreatic β-cells related to their electrical activity

Glucose induces large amplitude oscillations of the cytoplasmic Ca 2+ concentration ([Ca 2+] i) in pancreatic β-cells. The effects of temperature on these oscillations were examined by monitoring [Ca 2+] i continuously in single β-cells from ob ob - mice using dual wavelength microfluorometry. The oscillations of [ca 2+] i disappeared when the temperature was increased above 42°C and were reversibly inhibited below 30°C. However, cooling did not prevent a glucose response in terms of the average rise of [Ca 2+] i. Since patch clamp studies of single β-cells have indicated a random occurrence of glucose-induced action potentials at room temperature, it was important to explore how the sugar affected the electrical activity at 37°C. Using the cell-attached configuration of the patch clamp technique for such analyses, the action potentials were found to occur in bursts with durations similar to the large amplitude oscillations of [Ca 2+] i.