Probe Indo-1 Research Articles

Cytosolic Ca2+ of excitable pituitary cells at resting potentials is controlled by steady state Ca2+ currents sensitive to dihydropyridines.

Different elements of voltage-gated Ca2+ influx and their role for cytosolic-free Ca2+, [Ca2+]i, were studied in cells of the pituitary line GH3B6. Single cell monitoring of [Ca2+]i with the fluorescent probe indo-1, as well as fast on-line ratio [Ca2+]i imaging with fura-2, were combined with electrophysiological recordings using the perforated patch configuration of the patch clamp technique. [Ca2+]i signals are generated by Ca2+ influx both during action potentials and in between. Steady state Ca2+ influx at resting potentials (-60 to -40 mV) was voltage-gated and sensitive to the dihydropyridine antagonist PN 200-110. Ca2+ influx could be demonstrated in physiological conditions as inward Ca2+ currents of maximally 10 pA, which were triggered when stepping up the holding voltage beyond a narrow threshold around -50 mV. Inward currents were well correlated with [Ca2+]i elevations (R = -0.8; p < 0.001). It is concluded that dihydropyridine-sensitive, low threshold voltage-gated steady state Ca2+ currents, which may be tonically activated at resting potentials, provide for [Ca2+]i signaling in excitable endocrine cells in a mode that parallels the modulation of Ca2+ influx during action potentials.

Intracellular Calcium and pH Alterations Induced by Tri- n-butyltin Chloride in Isolated Rainbow Trout Hepatocytes: A Flow Cytometric Analysis

The effects of tri- n-butyltin chloride (TBT) on ionic homeostasis on isolated trout hepatocytes were investigated by flow cytometry (FCM), using the Ca 2+-sensitive and pH-sensitive fluorescent probes Indo-1 and SNARF-1, respectively. Cell viability was monitored concurrently. Treatment of hepatocytes with 1 and 5 μM TBT caused a rapid and sustained elevation of cytosolic free Ca 2+ concentration [Ca 2+] i and an important cytoplasmic acidification. These changes were dependent upon TBT concentration and were maintained over 60 min, the maximum exposure period investigated. At 0.5 μM TBT, there was a slight but not significant increase in [Ca 2+] i and a significant reduction in intracellular pH (pH i) only after 60 min of exposure. A rise in [Ca 2+] i and cytoplasmic acidification were observed before loss of viability was detectable. Experiments carried out in Ca 2+-free medium suggest that TBT mainly mobilizes Ca 2+ from intracellular stores in trout hepatocytes. The cytoplasmic acidification following TBT exposure seems to be caused by the combination of intracellular Ca 2+ mobilization and by direct action of TBT. The present results suggest that ionic homeostasis perturbations could be early events in the mechanism of cell injury by TBT.

Antigen-Specific Cell Conjugate Formation and Long-Lasting Calcium Responses in Recognition of Mls Cellular Superantigen by Cloned Murine T Lymphocytes

T lymphocyte recognition of cell-associated minor lymphocyte stimulation (Mls) superantigen was studied by simultaneous flow cytometric measurement of T cell free ionized intracellular calcium ([Ca +]i) with the fluorescent probe indo-1 and T cell binding to antigen-presenting cells stained with a long-chained, membrane-fixed cyanine dye. Cloned T cell-B lymphocyte antigen-presenting cell conjugate formation and increased T cell [Ca 2+]i were antigen specific and tightly linked in four Mls-reactive T cell clones. The T cell-antigen-presenting cell conjugates were extremely stable and, like T cell [Ca 2+]i elevation, were maintained for more than 2 hr. Three ligand-receptor pairs, (i) T cell receptor/antigen + class II, (ii) CD4/class II, and (iii) LFA-1/ICAM-1, were obligatory participants in T cell recognition of cell-bound superantigen since monoclonal antibodies to any of them blocked formation of cell conjugates. Despite previous reports, Mls recognition occurred by the conventional T cell recognition pathway with easily detected phosphoinositide hydrolysis. B cell activation greatly enhances their recognition by Mls-specific T cells.

Effects of two neuronal antidiuretic molecules, neuroparsin and 5-hydroxytryptamine, on cytosolic free calcium monitored with indo-1 in epithelial and muscular cells of the African locust rectum

Using the probe indo-1 in a microspectrofluorimetric study, it was demonstrated that the locust antidiuretic neurohormone, neuroparsin, enhanced cytosolic free Ca 2+ concentrations, measured as percentages of the ratio F405 F480 ( R), in epithelial cells of the African locust rectum. 5-hydroxytryptamine, whose antidiuretic effect was previously established, enhanced R in longitudinal muscular cells, and was able to increase R slightly in epithelial cells. The possibility of reciprocal Ca 2+ movements between muscular and epithelial cells is discussed. Both of the neuronal molecules, which act via distinct transduction pathways (phosphoinositide turnover for neuroparsin, and Ca 2+-dependent adenylate cyclase for 5-hydroxytryptamine), stimulated an increase in R by causing Ca 2+ entry through dihydropyridine-sensitive Ca 2+ channels. Cyclic nucleotides (cAMP and cGMP) lowered R in epithelial cells, the cGMP effect being interpreted as a feedback control on phosphoinositide turnover and resulting in the ability to re-establish cAMP production to levels incompatible with high PLC activity. In longitudinal muscular cells, the increase in R due to cAMP suggests the involvement of 5-hydroxytryptamine in stimulation of adenylate cyclase activity. Furthermore, results enabled the localization in epithelial cells of the transduction pathways mediating the actions of another antidiuretic factor extracted from the glandular lobes of the locust corpora cardiaca.

Antiulcer drugs and gastric prostaglandin E2: an in vitro study.

Prostaglandin (PG) has been reported to be an important protective and acid-suppressive factor in the gastric mucosa. Although the mechanisms of some antiulcer drugs are attributed to their stimulatory effects on endogenous prostaglandins, an understanding of these actions has not been established. In the present study we investigated the effects of antiulcer drugs on PGE2 using cultured gastric mucosal cells. Rabbit gastric mucosal cells were cultured after isolation with collagenase and ethylenediaminetetraacetic acid. PGE2 was measured by enzyme-linked immunoassay. Histamine H2-blockers (cimetidine, ranitidine, famotidine), omeprazole, and sucralfate did not modulate the media content of PGE2, whereas sofalcone dose- and time-dependently increased it. Sofalcone-induced increase of PGE2 was dose-dependently prevented by indomethacin. Sofalcone did not affect intracellular Ca2+ as assessed by the calcium-sensitive probe indo-1. Deprivation of Ca2+ in the media did not modulate the action of sofalcone. Sofalcone significantly suppressed 15-OH-PG dehydrogenase. These results suggest that among the various antiulcer drugs only sofalcone increases PGE2, which may be a factor in its therapeutic effect against peptic ulcer diseases.

Pyrazine Compounds and the Measurement of Cytosolic Ca2+

Several of the pyrazine derivatives are widely used for inhibiting sodium flux via Na+/Ca2+ exchange or Na+/H+ exchangers or through the epithelial cation channels. These processes can profoundly affect cytosolic Ca2+. We found that the widely used fluorescent probes fura-2 and indo-1 could not he used to measure the effect of pyrazine analogs on the cytosolic free calcium ([Ca2+]i) of YAC-1 lymphoma cells treated with the pore-forming protein cytolysin/perforin. We show that the excitation spectra of pyrazine derivatives that specifically inhibit Na+/Ca2+ exchange [5-(N-4-chlorobenzyl)-2′,4′-dimethylbenzamil], Na+/H+ exchange [5-(N-ethyl-N-isopropyl)-amiloride], and Na+ channels (phenamil) overlap with those of fura-2 and indo-1. In the presence of Ca2+, fluorescence readings for fura-2 plus drug are greater than those of fura-2 alone with the typically used 340- and 380-nm excitation light wavelengths; F380 readings were more affected than F340 readings. The effect was drug dose dependent. Hence, calculations that use F340 readings in the presence of pyrazine derivatives will result in overestimates of [Ca2+]i, while those that use the corresponding ratio readings, R340/380, will result in underestimates of [Ca2+]i. We found that the luminescent intracellular Ca2+ indicator aequorin could be used successfully with pyrazine derivatives, and that the ability of these compounds to enhance cytolysin/perforin-mediated increases in [Ca2+]i corresponded to their previously reported ability to inhibit Na+/Ca2+ exchange in pituitary cell plasma membrane vesicles. YAC-1 lymphoma cells are easy to culture and handle and may be a useful model for the studies of the Na+/Ca2+ exchanger in situ.

The induction of lipid peroxidation by E. coli lipopolysaccharide on rat hepatocytes as an important factor in the etiology of endotoxic liver damage

Oxygen-derived radicals have been suggested to produce tissue injury during endotoxic shock by initiating lipid peroxidation. In order to investigate the induction of lipid peroxidation by Escherichia coli 0111:B4 lipopolysaccharide (LPS) on hepatocytes, malondialdehide (MDA) and superoxide dismutase (SOD) activity have been evaluated in vivo and in vitro using two experimental models: rat liver after the establishment of endotoxic reversible shock, and cultured hepatocytes after treatment with LPS. Liver MDA levels were increase in vivo during the acute-phase of endotoxic shock, decreasing below control values in the recovery phase. An inverse pattern was obtained when SOD activitiy was measured, consistent with an active system of cellular protection. Similar results were obtained in vitro after treatment of cultured hepatocytes with LPS (50 μg/ml), thus indicating that a direct LPS cytotoxic effect on hepatocytes exits during the endotoxic process. The direct LPS interaction induced alterations in Ca 2+ permeability of hepatocyte plasma membrane as detected by flow cytometry using the fluorescent probe Indo-1.

Changes in cytosolic resting ionized calcium level and in calcium transients during in vitro development of normal and Duchenne muscular dystrophy cultured skeletal muscle measured by laser cytofluorimetry using Indo-1

Intracellular calcium activity was recorded during in vitro myogenesis of human normal and DMD muscle, using the calcium probe Indo-1 under laser illumination, at rest and during different kinds of stimulation (acetylcholine, high K +, caffeine). In myoblasts, the resting intracellular calcium level was significantly larger in DMD cells (89 ± 9 nM; n = 40 vs 37 ± 5 nM; n = 22) but there was no significant difference in myotubes, after fusion (44 ± 4 nM; n = 34 vs 36 ± 4 nM; n = 52). A similar evolution was observed in cells cultured from FSH biopsies. The amplitude of ACh- and high K +-induced calcium transients was significantly halved in DMD myotubes as compared to control ones and non-significantly decreased for caffeine responses. Some alterations in the kinetics of responses were observed in DMD muscle: the rising phases of ACh- and high K +-elicited transients and the decaying phase of the ACh-responses were significantly slowed down. It is concluded that: (i) in aneurally cultured human muscle, an increase in the basal level of internal calcium can occur at early stages of myogenesis before the expression of the dystrophin gene; and (ii) the changes in calcium transients induced by depolarization or direct stimulation of sarcoplasmic reticulum are not susceptible of inducing a calcium overload in DMD cells.

Intracellular calcium transients induced by different kinds of stimulus during myogenesis of rat skeletal muscle cells studied by laser cytofluorimetry with Indo-1

Resting intracellular calcium levels and intracellular calcium transients induced by three types of stimulus (acetylcholine, high potassium and caffeine) were recorded, during in vitro myogenesis, by means of a ratiometric fluorescence method using the calcium probe Indo-1 under laser illumination. Resting levels seemed to decrease with the age of cultured cells and the depolarization-induced transients, through 100 mM K + or Ach application, were progressively faster and larger as the muscle cells developed. An additive mechanism, likely due to calcium entry into the cell through nicotinic acetylcholine receptors, could explain the differences observed in Ach-induced responses as compared with the 100 mM K +-induced ones. In myoballs (the older cells) the calcium transients exhibited progressively a biphasic shape. From data obtained in different conditions (tetrodotoxin, nifedipine, strontium and free Ca EGTA) and those indicating the appearance of caffeine-releasable intracellular calcium stores only at 2–3 days stage, and from the previously reported developmental appearance of calcium currents and contraction, it was proposed that, in young myotubes, the calcium transients were more dependent on extracellular calcium than in older cells. These developmental data are discussed in the light of a known model of the in situ biogenesis of the structures involved in excitation-contraction coupling (ECC) like transverse tubules and triads.

Characterization of Ca 2+ signals generated by extracellular nucleotides in supporting cells of the organ of Corti

ATP has been demonstrated to act as a co-transmitter or neuromodulator in various physiological processes. There is recent evidence that ATP receptors, characterized as P 2 purinergic receptors, are expressed in the sensory hair cells of the auditory organ. The aim of the present study was to know whether other cell types of the organ of Corti, the supporting cells, were also sensitive to external ATP. In both types of supporting cells considered in this study, Deiters' cells (DCs) and Hensen's cells (HEs), extracellular ATP at sub-micromolar concentrations evoked a transient increase in [Ca 2+] i as monitored with fluorescence microscopy using the calcium probe indo-1. An apparent Kd of 0.5 and 0.9 μM was determined for DCs and HEs, respectively. Furthermore, we demonstrated that ATP stimulated Ca 2+ release from internal stores in DCs, but not in HEs. Dynamic calcium imaging by confocal laser scanning microscopy of ATP induced Ca 2+ mobilization demonstrated a calcium wave propagation in the cell body of DCs which originated in the phalangeal processes, suggesting a functional organization of Ca 2+ sequestering stores in DCs.

Localization and quantitation of calcium pools and calcium binding sites in cultured human keratinocytes

Calcium plays a crucial role in regulating the growth and differentiation of cultured keratinocytes. However, the mechanism(s) of this regulation is not clear. Prior studies have shown that intracellular free calcium (Cai) increases with keratinocyte differentiation. In this study, in order to evaluate the role of cytosolic free calcium and organelle-bound calcium in keratinocyte differentiation, we quantitated and localized calcium pools in keratinocytes, utilizing the fluorescence probe indo-1 and ion-capture cytochemistry, respectively. Cai of undifferentiated keratinocytes was 80-120 nM, whereas Cai of differentiated keratinocytes was 200-300 nM depending on the extent of differentiation. The Cai of individual cells in an undifferentiated colony was heterogeneous (60-160 nM) with larger cells displaying higher Cai. Heterogeneity also was observed in the intracellular calcium-containing precipitates in the different layers of stratifying keratinocyte cultures using the cytochemical technique. Calcium precipitates were abundant in the lower cell layers, progressively decreasing apically, with the uppermost layer devoid of precipitates. Calcium-containing precipitates appeared as fine-to-coarse electron-dense granules on the plasma membrane, within the cytosol, mitochondria, nucleus, and vacuolar organelles. Whereas ionomycin in the presence of extracellular calcium increased the amount of intracellular calcium precipitates, EGTA removed calcium precipitates from organelles. Unlike intact epidermis, keratinocytes displayed no extracellular calcium reservoirs. Putative calcium binding sites, visualized by trivalent lanthanum (La) binding, were abundant on cell membranes and desmosomes of basaloid cells, but decreased in the upper cell layers. These studies revealed differences in the distribution of free ionic calcium (as determined by the fluorescence technique) and organelle-bound calcium (as determined by the cytochemical technique). Striking differences were also observed in calcium localization between intact epidermis and cultured epidermal cells. The localization pattern of calcium in cultured keratinocytes may reflect the hyperproliferative state of these cells, as in psoriatic epidermis, and/or the absence of a normal permeability barrier in these submerged cultures.

The effects of extracellular calcium and epinephrine on cytosolic-free calcium in single rat adipocytes

Changes in cytosolic calcium concentration ([Ca2+]i) in response to extracellular calcium and epinephrine were monitored in individual rat adipocytes by both photon counting and digital imaging techniques utilizing the intracellular fluorescent calcium probes Fura-2 and Indo-1. Adipocytes containing Fura-2 were attached to coverslips and shown to be as hormonally responsive to insulin as adipocytes in suspension [3.5 +/- 0.8 (n = 5) vs. 4.2 +/- 0.6 (n = 8)-fold increase in glucose oxidation over basal in response to 0.7 nM insulin]. Basal [Ca2+]i in single rat adipocytes was found to be 128 +/- 6 nM (n = 100). The addition of either extracellular calcium or epinephrine elicited transient, concentration-dependent increases in [Ca2+]i. Although the characteristics of calcium- and epinephrine-induced calcium transients are generally similar, the peak [Ca2+]i increase over basal is higher in response to calcium vs. epinephrine [37 and 64% (1 and 27 microM epinephrine), vs. 132 and 236% (2 and 4 mM calcium)]. All the cells tested responded to calcium but only 67% responded to epinephrine. Both alpha- and beta-adrenergic agonists were able to increase [Ca2+]i. The epinephrine-induced [Ca2+]i transients appear to be dependent upon extra-cellular calcium. Neither cholera nor pertussis toxin treatments altered basal [Ca2+]i. However, after treatment of adipocytes with either pertussis or cholera toxin, epinephrine stimulated oscillations in [Ca2+]i. Digital imaging revealed that adipocytes demonstrate a high degree of intracellular spatial heterogeneity and intercellular variability in the magnitude of response to both calcium and epinephrine. These studies demonstrate the feasibility of using single rat adipocytes to monitor intracellular free calcium, using both photon counting and digital imaging.

The effects of extracellular calcium and epinephrine on cytosolic-free calcium in single rat adipocytes.

Changes in cytosolic calcium concentration ([Ca2+]i) in response to extracellular calcium and epinephrine were monitored in individual rat adipocytes by both photon counting and digital imaging techniques utilizing the intracellular fluorescent calcium probes Fura-2 and Indo-1. Adipocytes containing Fura-2 were attached to coverslips and shown to be as hormonally responsive to insulin as adipocytes in suspension [3.5 +/- 0.8 (n = 5) vs. 4.2 +/- 0.6 (n = 8)-fold increase in glucose oxidation over basal in response to 0.7 nM insulin]. Basal [Ca2+]i in single rat adipocytes was found to be 128 +/- 6 nM (n = 100). The addition of either extracellular calcium or epinephrine elicited transient, concentration-dependent increases in [Ca2+]i. Although the characteristics of calcium- and epinephrine-induced calcium transients are generally similar, the peak [Ca2+]i increase over basal is higher in response to calcium vs. epinephrine [37 and 64% (1 and 27 microM epinephrine), vs. 132 and 236% (2 and 4 mM calcium)]. All the cells tested responded to calcium but only 67% responded to epinephrine. Both alpha- and beta-adrenergic agonists were able to increase [Ca2+]i. The epinephrine-induced [Ca2+]i transients appear to be dependent upon extra-cellular calcium. Neither cholera nor pertussis toxin treatments altered basal [Ca2+]i. However, after treatment of adipocytes with either pertussis or cholera toxin, epinephrine stimulated oscillations in [Ca2+]i. Digital imaging revealed that adipocytes demonstrate a high degree of intracellular spatial heterogeneity and intercellular variability in the magnitude of response to both calcium and epinephrine. These studies demonstrate the feasibility of using single rat adipocytes to monitor intracellular free calcium, using both photon counting and digital imaging.

Lanthanum influx into cultured human keratinocytes: Effect on calcium flux and terminal differentiation

Trivalent cation lanthanum (La) binds to calcium binding sites of cells and either mimics the properties of calcium or inhibits the effects of calcium by displacing calcium from its binding sites. Extracellular calcium induces differentiation of human epidermal keratinocytes in culture, in part by increasing the intracellular calcium levels (Cai). Therefore, in this study we determined the effect of La on differentiation and intracellular calcium levels of keratinocytes. We observed that La inhibited the production of cornified envelopes, a marker for terminal differentiation of keratinocytes. La inhibited the calcium requiring envelope cross-linking enzyme, transglutaminase, in a direct manner, presumably, by displacing calcium from its binding site on the enzyme. La inhibited the influx and the efflux of 45Ca from keratinocytes. Paradoxically, extracellular La appeared to increase the Cai levels of keratinocytes as measured by the fluorescent probe indo-1. However, subsequent experiments revealed that indo-1 bound La with a higher affinity than Ca and emitted fluorescence in the same wavelength as the Ca bound form. Using this probe, we observed that La enters keratinocytes in a dose-dependent fashion and achieves concentrations exceeding 80 nM when the external La concentration is raised to 300 microM. This fully accounted for the apparent increase in Cai when La was added to the cells. Treatment of cells with ionomycin increased indo-1 fluorescence maximally in the presence of La indicating influx of La via this Ca specific ionophore. Our results indicate that La enters cells and inhibits calcium mediated keratinocyte differentiation both by blocking Ca influx and by blocking calcium regulated intracellular processes such as transglutaminase directed cornified envelope formation.

Nicardipine prevents calcium loading and "oxygen paradox" in anoxic single rat myocytes by a mechanism independent of calcium channel blockade.

The protective effect of nicardipine (1 and 4 microM) against reoxygenation injury was studied in an unstimulated rat single myocyte oxygen paradox model in comparison with control (no drug) or nifedipine (1 microM). Either concentration of nicardipine was strongly protective, approximately doubling the duration of ATP depletion (rigor) that cells could withstand without undergoing hypercontracture when reoxygenated. Nifedipine (1 microM), which matched the negative inotropic effect of nicardipine (4 microM) (as measured by extent of shortening when stimulated), had no protective effect against reoxygenation injury. Neither drug affected the time to rigor, which is a measure of the rate at which the resting cell consumes its endogenous glycogen stores during anaerobic metabolism. Intracellular calcium, measured with the fluorescent probe indo-1, which partitions into both cytosol and mitochondria, rose progressively throughout the rigor period. This rise in calcium was almost totally suppressed by nicardipine (1 microM) but was unaffected by nifedipine. We conclude that nicardipine possesses a direct protective effect on the myocardium not shared by all dihydropyridines. This effect is associated with the prevention of intracellular, and probably mitochondrial, calcium loading but is probably not due to blockade of the L-type calcium channel or reduction of metabolic rate.

Regulation of ATP-dependent surfactant secretion and activation of second-messenger systems in alveolar type II cells

Several different classes of agonists are known to stimulate exocytosis in type II cells. These agonists cause increases in second messengers, such as adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca2+, and/or stimulate protein kinase C. We studied generation of cAMP and phosphoinositide (PI) turnover in monolayer cultures of type II cells and measured [Ca2+]i in single cultured cells. ATP [10-4 M], which stimulates secretion of phosphatidylcholine (PC) and increases cellular cAMP, also stimulated PI turnover and increased [Ca2+]i. 12-O-tetradecanoylphorbol-13-acetate (TPA), which stimulates PC secretion and activates protein kinase C, did not increase [Ca2+]i. Pretreatment of type II cells with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the PC secretion induced by ATP and TPA and blocked the increase in PI turnover caused by ATP. ATP-dependent surfactant secretion and stimulation of PI turnover could also be inhibited by pretreatment of the cells with pertussis toxin. We used the fluorescent probe indo-1 to measure [Ca2+]i in single cultured type II cells. ATP produced rapid transient increases in [Ca2+]i, which could be prevented by pretreatment of the cells with either TPA or W-7. Our data suggest that pertussis toxin-sensitive G protein(s) are involved in ATP-dependent activation of PI turnover and in secretion of surfactant in type II cells. Activation of protein kinase C blocks the ATP-stimulated increase in [Ca2+]i. Finally, calmodulin may be involved in the regulation of ATP-dependent increase in [Ca2+]i, the activation of PI turnover, and the secretion of surfactant in type II cells. lung; exocytosis; cell calcium; G proteins; phosphoinositide turnover

Regulation of ATP-dependent surfactant secretion and activation of second-messenger systems in alveolar type II cells

Several different classes of agonists are known to stimulate exocytosis in type II cells. These agonists cause increases in second messengers, such as adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca2+, and/or stimulate protein kinase C. We studied generation of cAMP and phosphoinositide (PI) turnover in monolayer cultures of type II cells and measured [Ca2+]i in single cultured cells. ATP [10(-4) M], which stimulates secretion of phosphatidylcholine (PC) and increases cellular cAMP, also stimulated PI turnover and increased [Ca2+]i. 12-O-tetradecanoylphorbol-13-acetate (TPA), which stimulates PC secretion and activates protein kinase C, did not increase [Ca2+]i. Pretreatment of type II cells with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the PC secretion induced by ATP and TPA and blocked the increase in PI turnover caused by ATP. ATP-dependent surfactant secretion and stimulation of PI turnover could also be inhibited by pretreatment of the cells with pertussis toxin. We used the fluorescent probe indo-1 to measure [Ca2+]i in single cultured type II cells. ATP produced rapid transient increases in [Ca2+]i, which could be prevented by pretreatment of the cells with either TPA or W-7. Our data suggest that pertussis toxin-sensitive G protein(s) are involved in ATP-dependent activation of PI turnover and in secretion of surfactant in type II cells. Activation of protein kinase C blocks the ATP-stimulated increase in [Ca2+]i. Finally, calmodulin may be involved in the regulation of ATP-dependent increase in [Ca2+]i, the activation of PI turnover, and the secretion of surfactant in type II cells.

Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding.

In order to identify residues required for the binding of interleukin-8 (IL-8) to its receptor, mutants were constructed in which clusters of charged amino acids were systematically replaced with alanine along the entire IL-8 sequence. The mutants were tested for their ability to induce a receptor-mediated rise in cytosolic free Ca2+, a property of wild-type IL-8 which can readily be detected by flow cytometry using neutrophils loaded with the calcium probe Indo-1. Eleven of the 12 mutants caused neutrophil calcium mobilization at 5 nM; the exception being a triple alanine mutant at positions K3, E4, and R6, which was inactive at all concentrations tested (150 nM maximum). A second set of mutants was generated in which residues 1-15 were individually mutated to alanine. Mutants E4A, L5A, or R6A were all inactive in the Ca2+ assay at 5 nM and competed poorly with 125I-IL-8 for neutrophil receptor binding; I10A, E4A, L5A, and R6A had approximately 30-, 100-, 100-, and 1000-fold reduced affinity, as compared with control IL-8, respectively. The nuclear magnetic resonance structure of IL-8 indicates that, in solution, the side chains of E4, L5, R6, and I10 point away from the core of the protein and do not participate in any intramolecular hydrogen bonds or salt bridges (Clore, G. M., and Gronenborn, A. M. (1991) J. Mol. Biol. 217, 611-620).

Cytosolic free Ca2+ during operation of sodium‐calcium exchange in guinea‐pig heart cells.

1. Membrane current generated by the Na(+)-Ca2+ exchange mechanism was recorded in single guinea-pig ventricular myocytes using the whole-cell voltage-clamp technique and the intracellular free calcium concentration ([Ca2+]i) was monitored using the fluorescent probe Indo-1, applied intracellularly through a perfused patch pipette. The reversal potential of the exchanger (ENa, Ca) was measured from records of the 2 mM-Ni(2+)-sensitive current and used in an attempt to clamp [Ca2+]i at a level determined by the ionic compositions of the external and pipette solutions. 2. Measurements of ENa, Ca indicated that [Ca2+]i was close to that in the pipette solution when the holding potential was set at the ENa, Ca expected for a 3Na+:1Ca2+ exchanger. The measured value of ENa, Ca was more positive than the theoretical value when the membrane potential was held positive to ENa, Ca and the opposite was true when the holding potential was more negative than the expected ENa, Ca. 3. As Indo-1 diffused into the cell from the whole-cell clamp electrode, the intensities of the fluorescent signals measured at 405 and 480 nm increased with time, with no obvious saturation over a 10-45 min recording period. However, the ratio of these two signals reached a steady level within 5 min after rupture of the patch membrane, when the holding potential was set at the expected ENa, Ca of the exchanger. The intensity ratios measured using pipette solutions containing 600 and 803 nM [Ca2+] were almost equal to the ratios obtained extracellularly from internal solutions of identical compositions, but in experiments using pipette solutions having lower [Ca2+] the intensity ratios measured in myocytes were higher than those obtained extracellularly. 4. If the membrane was depolarized or hyperpolarized, the fluorescence ratio either increased or decreased, respectively. These changes in the fluorescence ratio were virtually blocked by the extracellular application of 2 mM-Ni2+. 5. When the concentration of bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the recording pipette was reduced from 30 to 1 mM, an increase in [Ca2+]i was observed during a depolarizing ramp pulse. The Ca2+ influx estimated by integrating the 2 mM-Ni(2+)-sensitive current during the pulse correlated with the increase in [Ca2+]i estimated from Indo-1 using the extracellular calibration curve, but the values of the influx determined directly from Indo-1 fluorescence were always larger than those calculated from the exchanger current.(ABSTRACT TRUNCATED AT 400 WORDS)

Physiological characterization of two functional states in subpopulations of prolactin cells from lactating rats.

1. Lactotroph cells from lactating female rat pituitary glands were dissociated, separated and enriched on a continuous gradient of bovine serum albumin at unit gravity. Two lactotroph subpopulations were observed in the light (F(3-5)) and the heavy (F(7-9)) fractions of the gradient. Both populations were maintained for at least 6 days in culture before experiments were performed. 2. Patch-clamp recordings, in the whole-cell mode, were performed on both lactotroph subpopulations in order to measure passive membrane properties and Ca2+ currents. Resting membrane potential as well as membrane capacitance values were found to be lower in light fraction cells. The two components of Ca2+ currents, called fast and slow deactivating (FD and SD) currents, were present with different proportions in each subpopulation; the ratio of current amplitudes, SD/FD, was 2.42 +/- 0.41 (n = 18) in light fraction cells and 1.17 +/- 0.27 (n = 17) in heavy fraction cells (P less than 0.02). 3. Reverse haemolytic plaque assay showed that in the light and heavy fractions, 68 and 47% of the lactotroph cells, respectively, were secreting. Population analysis of the plaque areas revealed a bimodal frequency distribution of plaque sizes consisting of small (1500 microns 2) and large plaques (3995 microns 2). A majority of light fraction cells produced large plaques whereas most of the heavy fraction cells produced small plaques. 4. Perifusion experiments performed on enriched prolactin cells showed that (1) basal prolactin (PRL) release was higher in light fraction than in heavy fraction cells, (2) the dopamine (10(-8)M)-induced inhibition of PRL release was greater in light fraction cells (86 +/- 15%) than in heavy fraction cells (41 +/- 21%), and (3) the thyrotrophin-releasing hormone (TRH, 10(-8)M)-induced increase of PRL release was 150 +/- 60% in light fraction versus 330 +/- 82% in heavy fraction cells. 5. Current-clamp recordings were performed using the intracellular technique. Lactotrophs were categorized according to their electrophysiological response following application of dopamine or TRH (both 10(-8)M). In the light fractions, the majority of the cells tested were hyperpolarized by dopamine (68%), whereas only 7% were depolarized by TRH application. In the heavy fractions, most of the cells (63%) responded to TRH application, while only 13% were dopamine sensitive. 6. Cytosolic free Ca2+ concentration ([Ca2+]i) measurements with the fluorescent probe Indo-1 revealed two lactotroph subtypes. Most cells in the light fractions (sixteen of twenty-two tested cells) exhibited an unstable level of [Ca2+]i with values fluctuating between 114.1 +/- 34.3 and 221 +/- 50 nM (mean +/- S.D.). Application of dopamine or of the D2 receptor agonist RU 24213 (10(-8)M) resulted in the disappearance of these fluctuations and in an accompanying decrease in basal [Ca2+]i level.(ABSTRACT TRUNCATED AT 400 WORDS)