Ca2+ mobilization induced by ouabain in thymocytes involves intracellular and extracellular Ca2+ pools

Immune dysfunction has been reported in hypertensive rats, and circulating levels of ouabain are increased in some experimental models of hypertension. Ouabain is an inhibitor of the Na+/K+-ATPase capable of diverse effects on cells of the immune system, but its mode of action on these cells is still unknown. The levels of cytoplasmic calcium ions play an important role in cell signaling, and ouabain may induce an increase in intracellular calcium indirectly through the Na+/Ca2+ exchanger. The current work examined the possibility that this drug could be exerting its effects on thymocytes through calcium mobilization and an increase in the cytosolic calcium concentration. Intracellular calcium was evaluated by using Balb-c mouse thymocytes loaded with FURA-2. Both intracellular and extracellular calcium pools were mobilized by ouabain (3 to 1000 nmol). The influx of extracellular calcium depended on the Na+/Ca2+ exchanger and on voltage-dependent calcium channels, as it was inhibited by amiloride and benzamil, consistent with the inhibition of the Na+/K+ pump. In addition, the increase of calcium from intracellular stores was extremely rapid. Furthermore, an increase in cytosolic calcium levels was obtained with the combination of ouabain and thapsigargin, which was greater than that seen with either drug alone. Our data suggest that low concentrations of ouabain may be acting on thymocytes through a mechanism different from the traditional inhibition of the Na+/K+-ATPase, as the cytosolic calcium rise was partly dependent on the release from intracellular stores.     

Ca2+-independent and Ca2+/GTP-binding protein-controlled exocytosis in a plant cell

Exocytosis allows the release of secretory products and the delivery of new membrane material to the plasma membrane. So far, little is known about the underlying molecular mechanism and its control in plant cells. We have used the whole-cell patch-clamp technique to monitor changes in membrane capacitance to study exocytosis in barley aleurone protoplasts. To investigate the involvement of Ca2+ and GTP-binding proteins in exocytosis, protoplasts were dialyzed with very low (<2 nM) and high (1 microM) free Ca2+ and nonhydrolyzable guanine nucleotides guanosine 5'-gamma-thio]triphosphate (GTP[gammaS]) or guanosine 5'-[beta-thio]diphosphate (GDP[betaS]). With less than 2 nM cytoplasmic free Ca2+, the membrane capacitance increased significantly over 20 min. This increase was not altered by GTP[gammaS] or GDP[betaS]. In contrast, dialyzing protoplasts with 1 microM free Ca2+ resulted in a large increase in membrane capacitance that was slightly reduced by GTP[gammaS] and strongly inhibited by GDP[betaS]. We conclude that two exocytotic pathways exist in barley aleurone protoplasts: one that is Ca2+-independent and whose regulation is currently not known and another that is stimulated by Ca2+ and modulated by GTP-binding proteins. We suggest that Ca2+-independent exocytosis may be involved in cell expansion in developing protoplasts. Ca2+-stimulated exocytosis may play a role in gibberellic acid-stimulated alpha-amylase secretion in barley aleurone and, more generally, may be involved in membrane resealing in response to cell damage.     

Sex differences in the extracellular Ca2+-independent release of LH and FSH

The present study was undertaken to investigate the effects of sex and estrous cycle on the manifestation of the extracellular Ca2+-independent component of gonadotropin secretion. Quartered pituitaries from male, ovariectomized (OVX) females +/- estradiol (E2) implants, and mature females at each stage of the estrous cycle were perifused with Ca2+-free medium. Gonadotropin releasing hormone (GnRH)-stimulated luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion from male and OVX pituitaries was inhibited in Ca2+-free medium. In contrast, only a partial inhibition was obtained from OVX + E2 or regularly cycling female pituitaries. This extracellular Ca2+-independent component of gonadotropin secretion was lowest at estrus and increased progressively during the estrous cycle. Estradiol replacement in OVX animals resulted in a response similar to that obtained on proestrus. These results indicate that the extracellular Ca2+-independent component of LH and FSH release is only manifest from intact female and not male pituitaries, and is dependent on estradiol.     

Immunohistochemical expression of extracellular matrix proteins and adhesion molecules in pancreatic carcinoma.

BACKGROUND/AIMS: Carcinoma invasion and metastasis in general involve multiple steps including dynamic changes in the composition and structure of extracellular matrix proteins and cell surface receptors. In the present study, the usually highly invasive carcinoma of the pancreas was investigated regarding the expression of various extracellular matrix proteins and their corresponding integrin receptors, as well as E-cadherin. METHODOLOGY: Phenotypic expression of various markers was investigated immunohistochemically in frozen sections of 16 pancreatic carcinomas and normal pancreatic tissue. RESULTS: An irregular and discontinuous deposition of type IV collagen and laminin in the basement membrane was found in cancer tissue and a pronounced desmoplastic reaction with deposition of type I, type III, and type IV collagen in the tumor stroma. In contrast, the noninvolved pancreas showed an intact basement membrane and a sparse stroma. The collagen type IV and laminin receptors alpha 2, alpha 3, and beta 1 integrin subunits were expressed on pancreatic cancer cells but not the alpha 6 integrin subunit normally present on epithelial cells, suggesting anchorage independence of the carcinoma cells. An increased capacity for cancer cell motility was suggested by the abundant expression of the "antiadhesive" extracellular matrix proteins, tenascin and vitronectin close to the cancer cells, and the expression of cell surface receptors such as alpha v (vitronectin-binding). Expression of the alpha 4 integrin subunit was also increased on cancer cells. CONCLUSIONS: The distribution of extracellular matrix proteins and the cell surface immune phenotype differed in pancreatic carcinoma as compared to normal pancreatic tissue. The present findings substantiate the notion that disseminated growth of highly malignant carcinomas of the pancreas reflects an invasive interaction of the tumor cells with extracellular matrix proteins of a well-established stroma. Similar findings were observed regardless of tumor histology and patient survival time.     

Phosphorylation and dephosphorylation of dihydropyridine-sensitive voltage-dependent Ca2+ channel in skeletal muscle membranes by cAMP- and Ca2+-dependent processes.

The phosphorylation and dephosphorylation of the dihydropyridine-sensitive Ca2+ channel was studied in transverse-tubule membranes isolated from rabbit skeletal muscle. Exposure of these membranes to either the cAMP-dependent protein kinase or a Ca2+/calmodulin-dependent protein kinase resulted in a rapid phosphorylation of a protein with properties similar to the major component of the skeletal muscle Ca2+ channel. The molecular mass of the phosphoprotein was 140 or 160 kDa, depending on the electrophoretic conditions. The stoichiometry of the phosphorylation was calculated to be 0.4-1.0 mol of phosphate per mol of protein. Neither the rate nor the extent of phosphorylation was affected by dihydropyridines. Limited proteolytic digestion of the protein that had been phosphorylated by either or both protein kinases yielded a single phosphopeptide of approximately equal to 5.4 kDa. The Ca2+-dependent phosphatase calcineurin dephosphorylated the membrane-bound Ca2+ channel that had been previously phosphorylated by either protein kinase. The results suggest that the major component of the dihydropyridine-sensitive Ca2+ channel from skeletal muscle can be effectively phosphorylated and dephosphorylated in its native state by cAMP- and Ca2+-dependent processes.     

Altered glycosylation of integrin adhesion molecules in colorectal cancer cells and decreased adhesion to the extracellular matrix.

The integrin mediated interactions between tumour cells and the surrounding extracellular matrix are thought to play crucial parts in the complex process of invasion and metastasis. It has been previously shown that the expression of integrins is differently diminished in a chain-specific manner in human colorectal cancer. To further characterise the integrins still expressed in colorectal carcinomas, immunoblots with monoclonal antibodies against the beta 1 integrin subunit have been performed. In isolated cell membranes of colorectal cancers a second smaller beta 1 chain (105 kD non-reduced) was found as well as the mature beta 1 chain (116 kD non-reduced) present in normal mucosa of the colon. This smaller beta 1 chain comigrates with the diminished glycosylated precursor form of the beta 1 chain. The role of N-glycosylation for the function and expression of integrins in vitro was therefore investigated, with deoxymannojirimycin (DMJ) and deoxynojirimycin (DNJ) as specific inhibitors of N-glycan processing. Pretreatment of human colon adenocarcinoma derived HT-29 cells with DMJ resulted in an expression of the 105 kD beta 1 precursor chain and of smaller forms of the alpha 1, alpha 3, alpha 6, and alpha v integrin subunits in a time and dose dependent manner. HT-29 cells treated with DMJ adhered poorly to laminin (8% of untreated controls), collagen type IV (40%), and fibronectin (35%). Pretreatment of the cells with DNJ did not alter the molecular weight of the integrin chains expressed and reduced HT-29 adhesion to laminin and fibronectin only to 68% and 49% respectively. Adhesion to collagen type IV was increased to 124% by DNJ. These results show that N-glycan processing is essential for the function and expression of integrins in human colorectal cancer cells. An altered glycosylation of these adhesion receptors may contribute to a more invasive or metastatic phenotype in colorectal cancer.     

Glutamate receptors activate Ca2+ mobilization and Ca2+ influx into astrocytes.

We measured changes in the molar concentration of cytosolic Ca2+ ([Ca2+]i) in individual astrocytes in culture produced by the glutamate analog quisqualate (QA) and related substances by using fura-2 digital fluorescence microscopy. In cells cultured from the cortex, hippocampus, and cerebellum, the QA analog alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA; 10 microM) produced a slow increase in [Ca2+]i that was modest in amplitude (approximately 200 nM). These effects were completely abolished by 10 microM 6-nitro-7-cyano-quinoxaline-2,3-dione (CNQX). In cerebellar astrocytes, similar effects were produced by QA. However, in cortical and hippocampal astrocytes, the response to QA was much more complex. In these cells, QA produced an initial [Ca2+]i spike that was followed by a sustained influx of Ca2+ ("plateau"). In the absence of extracellular Ca2+, this plateau was abolished but the spike remained. CNQX did not block the spike and only slightly reduced the size of the plateau in some cells. Ni2+ (10 microM) but not nimodipine (10 microM) reduced the amplitude of the plateau. Pretreatment with 100 nM phorbol 12-myristate 13-acetate for 15 min abolished the spike but not the plateau portion of the QA response. Treatment with pertussis toxin at 250 ng/ml for 12-16 hr failed to alter the response. In some instances, the latency of the QA response differed considerably for individual cells in a group. It appeared that the response began in one cell and then spread to neighboring cells. Thus, QA appears to trigger a complex response in some astrocytes consisting of Ca2+ mobilization from intracellular stores and also Ca2+ influx resulting from the activation of AMPA-sensitive and -insensitive pathways.     

Increases in muscle Ca2+ mediate changes in acetylcholinesterase and acetylcholine receptors caused by muscle contraction.

The synthesis of acetylcholinesterase (AcChoE; acetylcholine acetylhydrolase, EC 3.1.1.7) and of acetylcholine receptors (AcChoR) by cultured rat muscle fibers is influenced strongly by the level of muscle contractile activity. If fibers are grown in the presence of tetrodotoxin (TTX) to block spontaneous contraction, the total amount of AcChoE decreases markedly, as does the percentage of AcChoE assembled as the collagen-tailed presumed synaptic form of the enzyme. Under these conditions, however, the number of AcChoR increases. We demonstrate here that each effect of TTX can be prevented by treating the muscle cells with the calcium ionophore A23187. Thus, cells treated with A23187 and TTX have 30- to 40-fold higher levels of collagen-tailed AcChoE and lower levels of AcChoR by a factor of 4-5 than do cells grown in TTX alone. These results suggest that an increase in muscle cytoplasmic Ca2+ mediates the known effects of muscle contraction on these cholinergic macromolecules.     

Reciprocal regulation of two G protein-coupled receptors sensing extracellular concentrations of Ca2+ and H+

G protein-coupled receptors (GPCRs) are cell surface receptors that detect a wide range of extracellular messengers and convey this information to the inside of cells. Extracellular calcium-sensing receptor (CaSR) and ovarian cancer gene receptor 1 (OGR1) are two GPCRs that sense extracellular Ca²⁺ and H⁺, respectively. These two ions are key components of the interstitial fluid, and their concentrations change in an activity-dependent manner. Importantly, the interstitial fluid forms part of the microenvironment that influences cell function in health and disease; however, the exact mechanisms through which changes in the microenvironment influence cell function remain largely unknown. We show that CaSR and OGR1 reciprocally inhibit signaling through each other in central neurons, and that this is lost in their transformed counterparts. Furthermore, strong intracellular acidification impairs CaSR function, but potentiates OGR1 function. Thus, CaSR and OGR1 activities can be regulated in a seesaw manner, whereby conditions promoting signaling through one receptor simultaneously inhibit signaling through the other receptor, potentiating the difference in their relative signaling activity. Our results provide insight into how small but consistent changes in the ionic microenvironment of cells can significantly alter the balance between two signaling pathways, which may contribute to disease progression.Cells are surrounded by interstitial fluid, the composition of which is influenced by neighboring cells and which constitutes a key part of the microenvironment in which cells have to operate and survive. Changes in this microenvironment influence cell physiology (1, 2) and may promote disease (3, 4). Extracellular Ca²⁺ ([Ca²⁺]_o) and H⁺ ([H⁺]_o) concentrations are important components of the microenvironment, and their extracellular concentration changes in an activity- and state-dependent manner (5, 6). [Ca²⁺]_o is required for membrane stability, serves as a reservoir to allow Ca²⁺ influx into cells, and contributes to the membrane potential. [H⁺]_o sets the local pH, thereby influencing protein function as well as contributing to the membrane potential. Furthermore, Ca²⁺ and H⁺ can cross the membrane via ion channels and transporters, meaning that both can serve as intracellular and extracellular messengers (7–10).Levels of [Ca²⁺]_o and [H⁺]_o are communicated to cells via cell surface receptors that change their activity in a manner dependent on [Ca²⁺]_o and [H⁺]_o. These receptors include G protein-coupled receptors (GPCRs), such as the extracellular Ca²⁺-sensing receptor (CaSR), ovarian cancer gene receptor 1 (OGR1), G protein-coupled receptor 4 (GPR4), and T-cell death-associated gene 8 (TDAG8), all of which sense [H⁺]_o, as well as a range of ion channels (8).Intriguingly, Ca²⁺ and H⁺ signaling can be intimately linked, and changes in extracellular pH (pH_o) and intracellular pH (pH_i) may affect intracellular Ca²⁺ ([Ca²⁺]_i) signaling directly and indirectly (8, 11, 12). This is exemplified by OGR1, which, like CaSR (13), can couple to G_q and hence trigger Ca²⁺ release from intracellular Ca²⁺ stores via activation of the phospholipase C pathway (14). Neither CaSR nor OGR1 desensitizes (13, 14); thus, they continually monitor [Ca²⁺]_o and [H⁺]_o levels, respectively, and faithfully report any changes in their extracellular concentration. Because of the vital importance of Ca²⁺ and H⁺ to cells, information about their extracellular presence is crucial for cells, and lack of or altered signaling through these receptors may contribute to disease pathways.We have previously found that OGR1 activation in DAOY cells, a human cerebellar granule cancer cell line, leads to complex [Ca²⁺]_i signals and activation of the ERK signaling pathway, thereby providing a mechanistic explanation of how the acidic environment may influence transformed cell function and/or survival (15). This action is lost on differentiation, suggesting a link between OGR1 activity and proliferative behavior of the transformed neurons (16). To better understand the role played by OGR1 in central neurons, we investigated OGR1 activation in primary cerebellar granule cells, the nontransformed equivalent of DAOY cells. We found that OGR1 and CaSR reciprocally inhibit [Ca²⁺]_i signaling through each other, and that intracellular acidosis, which accompanies extracellular acidification, promotes OGR1 but inhibits CaSR activity. Finally, CaSR-dependent inhibition of OGR1activity is absent in DAOY cells.     

Beat-to-beat Ca2+-dependent regulation of sinoatrial nodal pacemaker cell rate and rhythm

Whether intracellular Ca²⁺ regulates sinoatrial node cell (SANC) action potential (AP) firing rate on a beat-to-beat basis is controversial. To directly test the hypothesis of beat-to-beat intracellular Ca²⁺ regulation of the rate and rhythm of SANC we loaded single isolated SANC with a caged Ca²⁺ buffer, NP-EGTA, and simultaneously recorded membrane potential and intracellular Ca²⁺. Prior to introduction of the caged Ca²⁺ buffer, spontaneous local Ca²⁺ releases (LCRs) during diastolic depolarization were tightly coupled to rhythmic APs (r² = 0.9). The buffer markedly prolonged the decay time (T₅₀) and moderately reduced the amplitude of the AP-induced Ca²⁺ transient and partially depleted the SR load, suppressed spontaneous diastolic LCRs and uncoupled them from AP generation, and caused AP firing to become markedly slower and dysrhythmic. When Ca²⁺ was acutely released from the caged compound by flash photolysis, intracellular Ca²⁺ dynamics were acutely restored and rhythmic APs resumed immediately at a normal rate. After a few rhythmic cycles, however, these effects of the flash waned as interference with Ca²⁺ dynamics by the caged buffer was reestablished. Our results directly support the hypothesis that intracellular Ca²⁺ regulates normal SANC automaticity on a beat-to-beat basis.     

Phosphodiesterase and its Ca2+-dependent activating factor in bovine thyroid.

Properties of cyclic 3',5'-nucleotide phosphodiesterase in the 100,500 X g supernatant of the bovine thyroid were investigated. The enzyme activity was measured by a radioisotopic method using an anionic-exchange resin, and it was found that the activity was stimulated by Mg2+. Sephadex G-200 gel filtration separated the supernatant into an activating factor, which required the presence of Ca2+, and an enzyme form dependent on the factor. The molecular weights were estimated to be 25,000 and 130,000, respectively. There appeared to be another enzyme form of cAMP phosphodiesterase with different dependence on the activating factor as suggested by gel filtration, but this enzyme form could not be clearly separated. cGMP phosphodiesterase purified by gel filtration showed biphasic kinetic behavior in the absence of Ca2+ and the activating factor, whereas, in their presence, the Lineweaver-Burk plot gave a single Km. The activating mechanism of phosphodiesterase may play a role in the control of concentrations of intracellular cyclic 3',5'-nucleotides in the bovine thyroid.     

Cytokines modulate MIA PaCa 2 and CAPAN-1 adhesion to extracellular matrix proteins.

Variations in cancer cell adhesion to extracellular matrix (ECM) proteins might underlie an enhanced metastatic potential. ECM binding is mediated by cell-adhesion molecules, the membrane expression of which might be influenced by soluble mediators, such as cytokines. The aims of our study were to ascertain whether epidermal growth factor (EGF), transforming growth factor beta1 (TGF-beta1), interleukin 1alpha (IL-1alpha), or interleukin 1beta (IL-1beta) can modify MIA PaCa 2 (pancreatic cancer cell line) and CAPAN-1 (metastatic pancreatic cancer cell line) adhesion to fibronectin, laminin, or type I collagen, and whether these cytokines can shift the membrane expression of the hyaluronic acid receptor (CD44). EGF significantly enhanced MIA PaCa 2, but not CAPAN-1, adhesion to fibronectin, laminin, and type I collagen. TGF-beta1 reduced MIA PaCa 2 adhesion to type I collagen, but enhanced CAPAN-1 adhesion to fibronectin and laminin. IL-1alpha was found to enhance MIA PaCa 2 adhesion to fibronectin, while reducing adhesion to type I collagen, whereas IL-1beta reduced the adhesion to laminin. IL-1alpha enhanced CAPAN-1 adhesion to laminin in a dose-dependent manner; IL-1beta slightly increased the adhesion of these cells to laminin at low dosage, and to type I collagen at high dosage. Both IL-1alpha and IL-1beta reduced CD44 membrane expression of MIA PaCa 2, while TGF-beta1 increased the percentage of CD44-positive CAPAN-1 cells. We suggest that the effects on cell adhesion induced by different cytokines depend on the status of the target pancreatic cancer cell. EGF and, in part, IL-1alpha can favor nonmetastatic pancreatic cancer cell adhesion to ECM, possibly favoring tumor spread. Metastatic cells seem to lose the responsiveness to EGF, while becoming hyperresponsive to IL-1alpha. TGF-beta1 might exert an antidiffusive effect on primary, and a prodiffusive effect on metastatic pancreatic cancer cells. Only IL-1alpha, IL-1beta, and TGF-beta1 seem to influence CD44 membrane expression. All the results presented in this study were obtained in vitro, and in vivo studies are needed to verify whether the studied cytokines can favor or counteract pancreatic cancer spread.     

Cytosolic Ca2+-dependent neutral proteinases from rabbit liver: activation of the proenzymes by Ca2+ and substrate.

Two neutral Ca2+-dependent proteinases, differing in molecular size, have been isolated from rabbit liver. Both are recovered as inactive proenzymes that can be converted to the active forms by high (0.1-1.0 mM) concentrations of Ca2+ in the absence of substrate or, in the presence of a protein substrate, by low (1-5 microM) concentrations of Ca2+. The activated proteinases required only 1-5 microM Ca2+ for maximal activity. Substrates hydrolyzed were denatured globin, globin, casein, and to a lesser extent, several extracellular proteins; no digestion was observed with several intracellular cytosolic enzymes tested. Only those proteins that served as substrates were capable of promoting conversion of the proenzymes to the active low-Ca2+-requiring proteinases.     

梗阻性不稳定膀胱逼尿肌细胞内Ca2+及N型Ca2+通道的变化及意义

目的对Ca2 和N型Ca2 通道在兔不全梗阻性不稳定膀胱逼尿肌细胞中的变化进行研究。方法成年同龄雄性新西兰白兔30只,随机分两组,15只中膀胱出口不全梗阻8w尿流动力学证实为不稳定膀胱者为实验组,15只为对照组(成年雄性新西兰白兔仅仅手术游离兔膀胱颈而不做结扎梗阻为对照组)。采用急性酶法分离及传代培养的方法获得单个膀胱逼尿肌细胞,采用激光共聚焦显微镜观察模型膀胱逼尿肌细胞静态Ca2 浓度;运用共聚焦显微镜及免疫组织化学方法观察N型Ca2 通道在梗阻性不稳定膀胱逼尿肌细胞中的变化。结果静息状态下不全梗阻性不稳定膀胱组逼尿肌细胞内游离钙离子浓度明显增高(Ca2 超负荷);共聚焦显微镜及免疫组化均证实不稳定膀胱逼尿肌细胞膜之N钙离子通道数量明显增多。结论膀胱逼尿肌细胞Ca2 及其N型Ca2 通道病理性改变是出现不稳定膀胱的重要因素。     

Models of the extracellular domain of the nicotinic receptors and of agonist- and Ca2+-binding sites

We constructed a three-dimensional model of the amino-terminal extracellular domain of three major types of nicotinic acetylcholine receptor, (alpha7)5, (alpha4)2(beta2)3, and (alpha1)2beta1gammadelta, on the basis of the recent x-ray structure determination of the molluscan acetylcholine-binding protein. Comparative analysis of the three models reveals that the agonist-binding pocket is much more conserved than the overall structure. Differences exist, however, in the side chains of several residues. In particular, a phenylalanine residue, present in beta2 but not in alpha7, is proposed to contribute to the high affinity for agonists in receptors containing the beta2 subunit. The semiautomatic docking of agonists in the ligand-binding pocket of (alpha7)5 led to positions consistent with labeling and mutagenesis experiments. Accordingly, the quaternary ammonium head group of nicotine makes a pi-cation interaction with W148 (alpha7 numbering), whereas the pyridine ring is close to both the cysteine pair 189-190 and the complementary component of the binding site. The intrinsic affinities inferred from docking give a rank order epibatidine > nicotine > acetylcholine, in agreement with experimental values. Finally, our models offer a structural basis for potentiation by external Ca2+.     

Quantification in crude homogenates of rat myocardial Na+, K+-and Ca2+-ATPase by K+ and Ca2+-dependent pNPPase. Age-dependent changes

Assays for complete quantification of Na⁺, K⁺-and Ca²⁺-ATPase in crude homogenates of rat ventricular myocardium by determination of K⁺-and Ca²⁺-dependentp-nitrophenyl phosphatase (pNPPase) activities were evaluated and optimized. Using these assays the total K⁺-and Ca²⁺-dependentpNPPase activities in ventricular myocardium of 11–12 week-old rats were found to be 2.98±0.10 and 0.29±0.02 mol×min^–1×g^–1 wet wt. (mean±SEM) (n=5), respectively. Coefficient of variance of interindividual determinations was 7 and 12%, respectively. The total Na⁺, K⁺-and Ca²⁺-ATPase concentrations were estimated to 2 and 10 nmol×g^–1 wet wt., respectively. Evaluation of a putative developmental variation revealed a biphasic age-related change in the rat myocardial Ca²⁺-dependentpNPPase activity with an increase from birth to around the third week of life followed by a decrease. By contrast, the K⁺-dependentpNPPase activity of the rat myocardium showed a decrease from birth to adulthood. It was excluded that the changes were simple out-come of variations in water and protein content of myocardium. Expressed per heart, the K⁺-and Ca²⁺-dependentpNPPase activity gradually increased to a plateau. The present assay for Na⁺, K⁺-ATPase quantification has the advantage over [³H] ouabain binding of being applicable on the ouabain-resistant rat myocardium, and is more simple and rapid than measurements of K⁺-dependent 3-0-methylfluorescein phosphatase (3-0-MFPase) in crude tissue homogenates. Furthermore, with few modifications thepNPPase assay allows quantification of Ca²⁺-ATPase on crude myocardial homogenates. Age-dependent changes in K⁺-and Ca²⁺-dependentpNPPase activities are of developmental interest and indicate the importance of close age match in studies of quantitative aspects of Na⁺, K⁺-and Ca²⁺-ATPase in excitable tissues.Abbreviations Na⁺, K⁺-ATPase sodium, potassium-dependent ATPase - Ca²⁺-ATPase caldium-dependent ATPase - pNP p-nitrophenyl - pNPP p-nitrophenyl phosphate - 3-0-MFP 3-0 methylfluorescein phosphate - DOC sodium deoxycholate     

Effect of extracellular phosphate on Ca2+ and K+ fluxes in pancreatic islets

The idea that a lowering in cytosolic Ca2+ concentration may cause a decrease in K+ conductance in the pancreatic B-cell was tested by investigating the effect of a high extracellular phosphate concentration on 45Ca and 86Rb efflux from prelabelled rat pancreatic islets. Whether in the absence or presence of glucose, 20 mM phosphate tended to decrease 45Ca efflux. This effect was not suppressed in the absence of extracellular Ca2+, at least in glucose-deprived islets, suggesting that it may reflect a fall in cytosolic Ca2+ concentration. The administration of phosphate failed, however, to decrease 86Rb efflux from the islets. In the presence of extracellular Ca2+, 20 mM phosphate also failed to stimulate insulin release from islets perifused at low glucose concentration and inhibited insulin release stimulated by a high glucose concentration. These data indicate that the sequestration of Ca2+ in intracellular organelles and concomitant decrease in cytosolic Ca2+ concentration, as presumably provoked by a rise in extracellular phosphate concentration, is not sufficient to simulate the effect of glucose on K+ conductance.     

Incorporation of cellular and plasma fibronectins into smooth muscle cell extracellular matrix in vitro.

Fibronectins isolated from the conditioned medium produced by cultures of undifferentiated (monolayer) and differentiated (nodular) swine vascular smooth muscle cells are similar but not identical. In general, the nodular-cell fibronectin has a smaller molecular mass than monolayer-cell fibronectin and appears to lack the COOH-terminal interchain disulfide linkage. We studied the incorporation of cellular and plasma fibronectins into the cell layer. Smooth muscle cells bound 2.5 times more monolayer-cell fibronectin than nodular-cell fibronectin. Polypeptide fragments of human plasma fibronectin were used as a model system to investigate fibronectin incorporation into the cell layer. Only intact molecules were incorporated into the cell layer and subsequently organized into fibers. Polypeptide fragments of molecular mass 205 kDa and 185 kDa were not incorporated even though they retained the collagen-, cell-, and heparin-binding regions. Incorporation appears to require an activity associated with either the NH2-terminal or COOH-terminal domains. We propose that fibronectin activity is lost during differentiation of smooth muscle cells.     

Regulation of airway smooth muscle cell contractility by Ca2+ signaling and sensitivity

Airway smooth muscle cell contraction is regulated by changes in intracellular Ca2+ concentration ([Ca2+]i) and the responsiveness of the airway smooth muscle cell to this Ca2+. The mechanism controlling [Ca2+]i primarily involves agonist-induced release of Ca2+ from internal stores to generate Ca2+ oscillations. The extent of contraction correlates with the persistence and frequency of these Ca2+ oscillations. The maintenance of the Ca2+ oscillations requires Ca2+ influx, but membrane depolarization appears to have a minor role in initiating or sustaining contraction. Contraction also requires agonist-induced Ca2+ sensitization, which is mediated mainly by decreases in myosin light-chain phosphatase activity. Although it is not clear if airway hyperresponsiveness associated with asthma results from the specific modulation of these Ca2+-based regulatory mechanisms, bronchodilators relax airways by both attenuating the Ca2+ oscillations and by decreasing the Ca2+ sensitivity.