Regulation of cytosolic calcium by cAMP and cGMP in freshly isolated smooth muscle cells from bovine trachea

The regulation of the cytosolic calcium concentration was investigated in freshly isolated adult bovine tracheal smooth muscle cells using fura 2. These cells contain 1.1 and 1.8 pmol of cGMP kinase and cAMP kinase per mg protein, respectively. Carbachol, histamine, serotonin, isoproterenol, and salbutamol increased the cytosolic calcium in a dose-dependent manner from 79 nM to about 650 nM. Preincubation of these cells for 20 min with isoproterenol, forskolin, 8-Br-cAMP and 8-(4-Cl-phenyl)thio-cAMP did not lower carbachol-induced increases in cytosolic calcium concentration, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the atrionatriuretic factor, isobutylmethylxanthine, and 8-Br-cGMP lowered cytosolic calcium. The active fragment of cGMP kinase, but not the catalytic subunit of cAMP kinase lowered carbachol-induced calcium levels. Carbachol released calcium from intracellular stores and increased calcium influx from the extracellular space. The influx was inhibited by preincubation with the calcium channel blockers nitrendipine or gallopamil. Both carbachol-stimulated pathways were suppressed by 8-Br-cGMP. Isoproterenol increased only the influx of calcium from the outside by a channel which was blocked by calcium channel blockers or 8-Br-cGMP. Forskolin and 8-Br-cAMP lowered carbachol- and isoproterenol-stimulated increases in calcium when added shortly before or after the addition of the agonist. In addition, isoproterenol decreased carbachol-stimulated calcium levels when added 10 s after carbachol. The calcium stimulatory effect of isoproterenol was abolished by preincubation of the cells with pertussis toxin or cholera toxin. These results show (a) that the beta 2-adrenoceptor couples in isolated tracheal smooth muscle cells to a dihydropyridine- and pertussis toxin-sensitive calcium channel; (b) that the same channel is opened by carbachol; (c) that cGMP kinase is very effective in decreasing elevated cytosolic calcium concentrations, whereas cAMP-dependent protein kinase has a variable effect on stimulated cytosolic calcium levels.     

Regulation of intracellular calcium in epithelial cells

The role of [Ca2+]i as a second messenger in non-excitable cells has been appreciated for almost 3 decades. The advent of fluorescent Ca2+ indicators has allowed the monitoring of Ca2+ signalling in suspensions of these cells. Agonist mediated changes in [Ca2+]i usually show an initial Ca2+ transient followed by a maintained increase. The former has been shown to be due to Ca2+ release from one or more intracellular stores, the latter due to activation of receptor operated Ca2+ entry (ROCE). More recently it has been recognized that many cells show distinct maintained oscillatory behavior when examined by single cell optical methods. It is proposed here that these oscillations are the consequence of IP3 and Ca2+ stimulation of Ca2+ release and ligand activation of ROCE followed by Ca2+ inhibition of Ca2+ and ROCE as Ca2+ pumps are activated. These oscillations allow more exact regulation of a pump/leak controlled second messenger such as [Ca2+]i.     

Changes in cytosolic free calcium concentration in isolated rat parotid cells by cholinergic and beta-adrenergic agonists

The alteration in the concentration of cytosolic free calcium ([Ca2+]i) in isolated rat parotid cells caused by autonomic agents was directly measured using the Ca-sensitive fluorescent probe, quin2. [Ca2+]i of unstimulated cells was estimated to be 162.7 +/- 3.2 nM in normal medium. Carbachol (CCh) and isoproterenol (ISP) caused a rapid rise in [Ca2+]i in a dose-dependent manner. Maximum increases in [Ca2+]i induced by CCh and ISP were approximately 100% and 25% of resting level, respectively. In Ca-free medium, CCh produced a small, rapid rise in [Ca2+]i, followed by a slow decay and a return to resting level within 3-4 min, while all doses of ISP tested failed to change [Ca2+]i. These results suggest that CCh mobilizes Ca2+ from both extracellular and intracellular pools and then results in a rise in [Ca2+]i, whereas ISP may slightly mobilize only the extracellular Ca pool.     

Regulation of ion permeabilities of isolated rat liver cells by external calcium concentration and temperature.

Regulation of ion transport through the plasma membrane was studied on single cell suspensions of hepatocytes, obtained after perfusion of rat liver with collagenase/hyaluronidase solution. Steady-state intracellular K and Na contents were shown to be markedly dependent on external Ca concentration and temperature, the sum of both ion concentrations remaining nearly constant. In contrast, steady-state intracellular chloride content was found to be independent of external Ca concentration, but dependent on temperature. Using the constant field relations, the passive permeabilities PK and PCl for potassium and chloride, respectively, were derived from the experimental data. At temperatures at and above 37 degrees C, with increasing external Ca concentration, PK, exhibits a sharp decrease at about 10(-4)M. In contrast, PCl at 37 degrees C was found to be independent of Ca concentration within experimental error. Earth alkali ions other than Ca, show marked but different effects on PK if compared at equal concentrations. Preincubation of the cells with cholesterol leads to a broadening of the dependence of PK on external Ca concentration. The above results, as well as those on the dependence of PK on external Ca concentration obtained by other authors, could be quantitatively described by a theoretical model of the plasma membrane proposed earlier. This model postulates regulatory binding sites, which cooperatively undergo a cation exchange of divalent cations by K+ ions from the external medium if the cation composition of the latter is altered.     

Regulation of calcium efflux from isolated rat parotid cells

Calcium efflux from isolated rat parotid acinar cells was studied with 45Ca. Carbachol, phenylephrine, substance P, monobutyryl cyclic AMP and isoproterenol stimulated 45Ca efflux. It is suggested that carbachol, phenylephrine and substance P mobilize the same pool of cellular Ca. This suggestion is based on two observations. Firstly, combinations of any two of these three agonists at saturating concentrations result in no more 45Ca efflux than either agonist alone. Secondly, stimulation of 45Ca efflux by any one of the three agonists prevents further stimulation of 45Ca efflux by the same or one of the other two agonists. The pool of calcium mobilized by isoproterenol or monobutyryl cyclic AMP is different from the pool mobilized by carbachol. This conclusion is based on the observation that stimulation of 45Ca efflux by a saturating concentration of carbachol did not inhibit stimulation of 45Ca efflux by isoproterenol. Furthermore the effect of a saturating concentration of isoproterenol on 45Ca efflux is additive with that caused by a saturating concentration of carbachol. The effect of carbachol, phenylephrine and substance P on 45Ca2+ efflux did not require extracellular Ca2+.     

Potassium channel activity recorded from the apical membrane of freshly isolated epithelial cells in rat caudal epididymis.

K+ channels were recorded in excised, inside-out patches from the apical membrane of the freshly isolated tubule of the caudal portion of the rat epididymis. With asymmetric K+ concentrations in bath and pipette (140 mM K+in/6 mM K+out), the channels had a slope conductance of 54.2 pS at 0 mV. The relative permeability of K+ over Na+ was about 171 to 1. The channels were activated by intracellular Ca2+ and by membrane depolarization. These channels belong to a class defined as "intermediate-conductance Ca2+-activated K+ channel. " External tetraethylammonium ions (TEA+) caused a flickery block of the channel with reduction in single-channel current amplitude measured at a range of holding membrane potentials (-40 to 60 mV). Activity of the K+ channels was inhibited by intracellular ATP (KD =1.188 mM). The channel activity was detected only occasionally in patches from the apical membrane (about 1 in 17 patches containing active channels). The presence of the intermediate-conductance Ca2+-activated K+ channels indicates that they could provide a route for K+ secretion in a Ca2+-dependent process responsible for a high luminal K+ concentration found in the epididymal duct of the rat.     

Synchronous transporting activity in epithelial cells in relation to intracellular calcium concentration.

Cultured monolayers of human sweat-gland epithelia have been used to measure electrogenic sodium transport, as short-circuit current, and intracellular Ca2+ concentration ([Ca]i) from Fura-2 fluorescence. The sodium currents in response to the agonists lysylbradykinin, histamine and carbachol show oscillatory behaviour in the 1-2 per minute frequency range. The oscillations can be terminated either by using specific antagonists or with amiloride, which prevents sodium entry into the epithelium. Oscillatory behaviour is also seen when [Ca]i is measured and occurs in the same frequency range. Sodium transport in these cultured epithelia is thought to result from an increase in [Ca]i, which in turn activates calcium-sensitive potassium channels, so increasing the electrochemical gradient for sodium entry. The oscillatory behaviour implies that the epithelial cells behave in synchrony to increase [Ca]i, so inducing synchronous changes in sodium current. It is shown that the behaviour is not unique to sodium-absorbing epithelia, and the possible utility of synchronous behaviour in epithelial tissues is discussed.     

Regulation of the intracellular calcium concentration in MMQ pituitary cells by dopamine and protein kinase C.

The MMQ pituitary cell line, which expresses a membranal dopamine receptor, was used to examine the individual contributions of dopamine and protein kinase C (PKC) to control of the intracellular calcium concentration. The calcium concentrations, monitored with the fluorescent dye Indo-1, increased in response to elevated K+, BAY K8644, and maitotoxin, implicating the presence of voltage-dependent calcium channels. Dopamine had no detectable independent effect, but significantly inhibited the rise in intracellular calcium mediated by activation of voltage-dependent calcium channels; this dopaminergic action was prevented by haloperidol. Acute pharmacological activation of PKC for 60 s inhibited the stimulatory effects of these calcium channel activators, and this acute inhibitory action was abolished by prior depletion of PKC. In contrast, however, PKC depletion did not alter the calcium response to BAY K8644 or maitotoxin. Thus, MMQ cells appear to have voltage-dependent calcium channels which, at rest, are either at low density or in a closed state. The rise in intracellular calcium resulting from stimulation of the channels is under inhibitory control by an apparent D-2 dopamine receptor. When pharmacologically activated, phorbol diester-sensitive PKC limits the rise in the cellular calcium level associated with calcium uptake. In the absence of pharmacological activation, however, this enzyme system does not appear to play a role in the cellular calcium response to BAY K8644 or maitotoxin.     

Regulation of intracellular calcium concentration by nanosecond pulsed electric fields

Changes in [Ca²⁺]_i response of individual Jurkat cells to nanosecond pulsed electric fields (nsPEFs) of 60 ns and field strengths of 25, 50, and 100 kV/cm were investigated. The magnitude of the nsPEF-induced rise in [Ca²⁺]_i was dependent on the electric field strength. With 25 and 50 kV/cm, the [Ca²⁺]_i response was due to the release of Ca²⁺ from intracellular stores and occurred in less than 18 ms. With 100 kV/cm, the increase in [Ca²⁺]_i was due to both internal release and to influx across the plasma membrane. Spontaneous changes in [Ca²⁺]_i exhibited a more gradual increase over several seconds. The initial, pulse-induced [Ca²⁺]_i response initiates at the poles of the cell with respect to electrode placement and co-localizes with the endoplasmic reticulum. The results suggest that nsPEFs target both the plasma membrane and subcellular membranes and that one of the mechanisms for Ca²⁺ release may be due to nanopore formation in the endoplasmic reticulum.     

Synthesis of mucin glycoproteins by epithelial cells isolated from swine trachea by specific proteolysis

Summary Mucus-producing cells were isolated from swine trachea mucosa by a method that included enzymatic digestion of the epithelial surface with Dispase, a neutral protease fromBacillus polymyxa, and differential attachment of the washed cells to culture flasks coated with collagen. Epithelial cells were the major cell type isolated by these procedures. Ciliated cells that did not attach to the flasks were removed by decantation, and fibroblasts were destroyed by the bacterial protease. The isolated cells synthesized respiratory mucins and the rate of secretion was increased about threefold when tracheas were exposed to sulfur dioxide. The cultured cells incorporated both [³⁵S]O₄ and [I-¹⁴C]N-acetylglucosamine into secreted mucin glycoproteins. The secretion of glycoprotein increased for about 3 d until the cells became confluent, and then a constant rate was observed for a period of at least 7 d. This increase in the output of mucin glycoprotein during the initial 3 d of culture was accompanied by a corresponding increase in the number of mucus-producing cells in the flasks. The results obtained in these and subsequent studies suggest that the rate of formation of mucus-producing cells may be a rate limiting step in the regulation of mucin glycoprotein synthesis in tracheal epithelium. The chemical, physical, and immunological properties of the glycoprotein secreted by isolated tracheal epithelial cells were very similar to the mucin glycoprotein purified from washes of swine trachea epithelium. The purified mucin glycoproteins showed complete cross-reaction with antibodies to trachea mucin glycoprotein. They were eluted near the void volume during gel filtration on Sepharose CL-6B columns. The glycoprotein isolated from culture media under the standard assay conditions had nearly the same carbohydrate composition as samples purified from washes of trachea epithelium. Reduced oligosaccharides released by β-elimination with dilute alkaline borohydride showed similar elution profiles during chromatography on Bio Gel P-6 colums. Taken collectively, these results suggest that the isolated epithelial cells secreted mucin glycoproteins that were very similar to those synthesized by the intact trachea epithelium under standard incubation conditions. This investigation was supported by United States Public Health Service Grant HL 20868 from the National Heart, Lung and Blood Institute and AM 28187 from the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases.     

Synthesis of mucin glycoproteins by epithelial cells isolated from swine trachea by specific proteolysis

Mucus-producing cells were isolated from swine trachea mucosa by a method that included enzymatic digestion of the epithelial surface with Dispase, a neutral protease from Bacillus polymyxa, and differential attachment of the washed cells to culture flasks coated with collagen. Epithelial cells were the major cell type isolated by these procedures. Ciliated cells that did not attach to the flasks were removed by decantation , and fibroblasts were destroyed by the bacterial protease. The isolated cells synthesized respiratory mucins and the rate of secretion was increased about threefold when tracheas were exposed to sulfur dioxide. The cultured cells incorporated both [35S]O4 and [I-14C]N-acetylglucosamine into secreted mucin glycoproteins. The secretion of glycoprotein increased for about 3 d until the cells became confluent, and then a constant rate was observed for a period of at least 7 d. This increase in the output of mucin glycoprotein during the initial 3 d of culture was accompanied by a corresponding increase in the number of mucus-producing cells in the flasks. The results obtained in these and subsequent studies suggest that the rate of formation of mucus-producing cells may be a rate limiting step in the regulation of mucin glycoprotein synthesis in tracheal epithelium. The chemical, physical, and immunological properties of the glycoprotein secreted by isolated tracheal epithelial cells were very similar to the mucin glycoprotein purified from washes of swine trachea epithelium. The purified mucin glycoproteins showed complete cross-reaction with antibodies to trachea mucin glycoprotein. They were eluted near the void volume during gel filtration of Sepharose CL-6B columns. The glycoprotein isolated from culture media under the standard assay conditions had nearly the same carbohydrate composition as samples purified from washes of trachea epithelium. Reduced oligosaccharides released by beta-elimination with dilute alkaline borohydride showed similar elution profiles during chromatography on Bio Gel P-6 columns. Taken collectively, these results suggest that the isolated epithelial cells secreted mucin glycoproteins that were very similar to those synthesized by the intact trachea epithelium under standard incubation conditions.     

胍丁胺对大鼠心室肌细胞内游离钙浓度的影响

本研究旨在观察胍丁胺 (agmatine ,Agm)对分离大鼠心室肌细胞内游离钙浓度 ( [Ca2 +]i)的影响。用酶解方法分离大鼠心室肌细胞 ,用Fluo 3 AM负载 ,然后用激光共聚焦法测定单个心室肌细胞 [Ca2 +]i 的荧光强度 (fluorescenceintensity ,FI) ,结果以FI或相对荧光强度 (F/F0 % )表示。实验结果表明 ,在正常台氏液 (含钙 1 0mmol/L)和无钙台氏液中 ,单个大鼠心室肌细胞的荧光密度分别为 12 8 8± 13 8和 119 6± 13 6,两者无差异。Agm 0 1、1和 10mmol/L浓度依赖性地显著降低细胞的钙浓度 ;在正常台氏液中加入EGTA 3mmol/L ,Agm同样降低细胞的钙浓度。KCl 60mmol/L ,PE 3 0 μmol/L ,和Bay K 864 410 μmol/L均升高心室肌细胞的[Ca2 +]i。Agm同样降低高浓度KCl、Bay K 864 4和PE诱发的心室肌细胞 [Ca2 +]i 升高。当细胞外液钙浓度由 1mmol/L增加到 10mmol/L时 ,诱发心室肌细胞钙超载 ,同时部分心室肌细胞产生可传播的钙波 (Ca2 +wave) ,Agm 1mmol/L降低钙波的传播速度和持续时间 ,最终阻断钙波。以上结果提示 ,Agm对心室肌细胞的胞浆[Ca2 +]i具有抑制作用 ,此作用通过阻断电压依赖性钙通道而实现 ;并可能与抑制大鼠心室肌细胞内钙释放有关     

Effect of dephostatin on intracellular free calcium concentration and amylase secretion in isolated rat pancreatic acinar cells

This study investigates the effects of dephostatin, a new tyrosine phosphatase inhibitor, on intracellular free calcium concentration ([Ca²⁺]_i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. Dephostatin evoked a sustained elevation in [Ca²⁺]_i by mobilizing calcium from intracellular calcium stores in either the absence of extracellular calcium or the presence of lanthanium chloride (LaCl₃). Pretreatment of acinar cells with dephostatin prevented cholecystokinin-octapeptide (CCK-8)-induced signal of [Ca²⁺]_i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF^- ₄), whereas co-incubation with CCK-8 enhances the plateau phase of calcium response to CCK-8 without modifying the transient calcium spike. The effects of dephostatin on calcium mobilization were reversed by the presence of the sulfhydryl reducing agent, dithiothreitol. Stimulation of acinar cells with thapsigargin in the absence of extracellular Ca²⁺ resulted in a transient rise in [Ca²⁺]_i . Application of dephostatin in the continuous presence of thapsigargin caused a small but sustained elevation in [Ca²⁺]_i . These results suggest that dephostatin can mobilize Ca²⁺ from both a thapsigargin-sensitive and thapsigargin-insensitive intracellular stores in pancreatic acinar cells. In addition, dephostatin can stimulate the release of amylase from pancreatic acinar cells and moreover, reduce the secretory response to CCK-8. The results indicate that dephostatin can release calcium from intracellular calcium pools and consequently induces amylase secretion in pancreatic acinar cells. These effects are likely due to the oxidizing effects of this compound.     

Exogenous ATP-stimulated calcium uptake in isolated rat intestinal epithelial cells

ATP in the extracellular medium is known to stimulate Ca uptake into avian intestinal epithelial cells. We have now demonstrated a similar effect of ATP in mammalian intestinal epithelial cells and have further characterized this effect. Exogenous ATP increased 45Ca uptake 2-6 fold in isolated rat small intestinal epithelial cells, with a maximal effect at 1 mM and an ED50 of 290 microM. A strict structural requirement for nucleotide-stimulated 45Ca uptake was observed. ADP was much less effective than ATP and gamma-thio-ATP, and 5'-AMP, cyclic AMP, adenosine, non-adenine nucleotides, non-hydrolyzable ATP analogs and ATP analogs with ring substitutions at the 8 position were inactive. Prenylamine (100 microM) completely inhibited ATP-stimulated 45Ca uptake, while verapamil (100 microM) had only a small effect. In the intact intestine, ATP increased short-circuit current (Isc) when added to the mucosal side of the tissue. This effect was reduced by 10 microM and abolished by 100 microM prenylamine. The effect of ATP on Isc was markedly reduced in Cl-free solutions and in reduced-Ca solutions. Serosal and mucosal addition of the nonhydrolyzable ATP analog, beta, gamma-methylene-ATP, and serosal addition of ATP had little or no effect on Isc. The similarities between the effects of ATP in isolated cells and in the intact intestine suggest that the effect of ATP on Isc may be at least partially mediated through stimulation of Ca uptake into the epithelial cells.     

Fluorescent measurements of intracellular free calcium in isolated toad urinary bladder epithelial cells

Summary Sodium-calcium exchange has been suggested to play a pivotal role in the regulation of cytosolic free calcium (Ca _f ) by epithelial cells. Using isolated epithelial cells from the toad urinary bladder, Ca _f has been measured using the intracellular Casensitive fluorescent dyes Fura 2 and Quin. 2. Dye loading did not alter cell viability as assessed by measurements of ATP and ADP content or cell oxygen consumption. When basal Ca _f was examined over a wide range of cell dye content (from 0.04 to 180 nmol dye/mg protein) an inverse relationship was observed. At low dye content, Ca _f was 300–380 nM and, as dye content was increased, Ca _f progressively fell to 60 nM. Using low dye content cells, in which minimal alteration in Ca steady state would be expected, the role for plasma membrane Na–Ca exchange was examined using either medium sodium substitution or ouabain. While medium sodium substitution increased Ca _f , prolonged treatment with ouabain had no effect on Ca _f despite a clear increase in cell sodium content. The lack of effect of ouabain suggests that Na–Ca exchange-mediated Ca efflux plays a minimal role in the regulation of basal Ca _f . However, exchange-mediated Ca efflux may play a role in Ca _f regulation when cytosolic calcium is elevated.     

Properties of freshly isolated type II alveolar epithelial cells

The biochemical characteristics of type II alveolar epithelial cells dissociated from adult rabbit lung by instillation of low concentrations of an elastase trypsin mixture are reported. Cells studied immediately (within 4 h) after isolation were found to incorporate the radioactively labelled precursors [U-¹⁴C]glucose, [methyl-³H]choline and [³H]palmitate into cellular phosphatidylcholine at rates 2–10-fold higher than previously reported for cells not subject to short-term cell culture. Secretion of phosphatidylcholine was stimulated by beta-adrenergic agonists. Measurement of specific activities of enzymes of phospholipid biosynthesis in subcellular fractions of isolated lung cells showed a significant enrichment of acyl coenzyme A-lysophosphatidylcholine acyltransferase, an enzyme believed to be involved in pulmonary surfactant phosphatidylcholine remodeling, in the endoplasmic reticulum of type II cells. These observations support the utility of freshly isolated type II cells as a model system for the study of the functions of the alveolar epithelium.     

Plasma membrane specialization and intracellular polarity of freshly isolated rat hepatocytes

It was investigated whether rat hepatocytes maintain their plasma membrane specialization (sinusoidal, lateral and bile canalicular sites) and their intracellular polarity (peribiliary region, rich in lysosomes and poor in mitochondria) after isolation. The morphology of the hepatocytes and the cytochemical localization of marker enzymes for the bile canalicular membrane (alkaline phosphatase, adenosine triphosphatase and 5' nucleotidase), for the lysosomes (acid phosphatase) and for the mitochondria (beta-hydroxybutyrate dehydrogenase and succinate dehydrogenase) were studied in situ and directly after isolation using both light and electron microscopy. The morphology of the cells and the cytochemical activity of acid phosphatase, succinate dehydrogenase and beta-hydroxybutyrate dehydrogenase showed that in isolated cells, as in situ, the lysosomes were concentrated in bands, devoid of mitochondria. Unlike in situ the reaction product of alkaline phosphatase, adenosine triphosphatase and 5'nucleotidase was evenly distributed along the entire plasma membrane of the isolated cells. Morphologically, no tight or gap junctions or desmosomes could be detected in the isolated cells, while the plasma membrane appeared to be homogeneously covered with uniform microvilli. In conclusion it can be stated that during isolation the hepatocytes loose their distinct plasma membrane specialization, but maintain their peribiliary region rich in lysosomes and poor in mitochondria.