Cell signalling through phospholipid breakdown

There is much evidence that G-proteins transduce the signal from receptors for Ca²⁺-mobilizing agonists to the phospholipase C that catalyzes the hydrolysis of phosphoinositides. However, the specific G-proteins involved have not been identified. We have recently purified a 42 kDa protein from liver that activates phosphoinositide phospholipase C and cross-reacts with antisera to a peptide common to G-protein -subunits. It is proposed that this protein is the a-subunit of the G-protein that regulates the phospholipase in this tissue.Ca²⁺-mobilizing agonists and certain growth factors also promote the hydrolysis of phosphatidylcholine through the activation of phospholipases C and D in many cell types. This yields a larger amount of diacylglycerol for a longer time than does the hydrolysis of inositol phospholipids. Consequently phosphatidylcholine breakdown is probably a major factor in long-term regulation of protein kinase C. The functions of phosphatidic acid produced by phospholipase D are speculative, but there is evidence that it is a major source of diacylglycerol in many cell types. The regulation of phosphatidylcholine phospholipases is multiple and involves direct activation by G-proteins, and regulation by Ca²⁺ protein kinase C and perhaps growth factor receptor tyrosine kinases.     

Ca2+ transport activities of inside-out vesicles prepared from density-separated erythrocytes from rat and human

The plasma membrane Ca²⁺ ATPase in erythrocytes is vital for the maintenance of intracellular Ca²⁺ levels. Since the cytoplasmic Ca²⁺ concentration is elevated in older erythrocytes, the properties of the Ca²⁺ transport ATPase were examined during cell aging using inside-out vesicles (IOVs) prepared from density-separated, young (less dense, Ey) and old (more dense, Eo) rat and human erythrocytes. The transport of Ca²⁺ and the coupled hydrolysis of ATP were measured using radiolabeled substrates. The calmodulin-independent Ca²⁺ transport activity (Ey, 38.8 vs. Eo, 23.3 nmols/min/mg IOV protein) and the Ca²⁺ dependent ATP phosphohydrolase activity (Ey, 53.5 vs. Eo, 48.8 nmols/min/mg protein) were greater in IOVs prepared from younger (less dense) rat erythrocytes. The calmodulin-independent Ca²⁺ transport activity in IOVs from human erythrocytes was 12.9 nmols/min/mg IOV protein for Ey and 10.7 nmols/min/mg IOV protein for Eo. Inside-out vesicles from older (more dense) cells exhibited a lower pumping efficiency as determined by the calculated stoichiometry, molecule of Ca²⁺ transported per molecule of ATP hydrolyzed (rat: Ey, 0.74 vs. Eo, 0.49; human: Ey, 1.22 vs. Eo, 0.77). The enzymatic activity of rat and human Ey IOVs was labile. The Ca²⁺ transport activity in Ey but not Eo IOVs rapidly declined during cold storage (4°C). The decrease in Ca²⁺ transport activity during aging may accentuate the age-related decline in several erythrocytic properties.Abbreviations IOV Inside-Out Vesicles - Ey Erythrocytes enriched with young (less dense) cells - Eo Erythrocytes enriched with old (more dense) cells - ACEase Acetylcholinesterase     

Parathyroid hormone-induced changes of the brush border topography and cytoskeleton in cultured renal proximal tubular cells

Summary In order to examine the possibility of parathyroid hormone-mediated ultrastructural rearrangements in target epithelium, isolated canine renal proximal tubular cells were grown on a collagen-coated semipermeable membrane in a defined medium. Scanning and transmission electron microscopy of these monolayers revealed abundant microvilli. Exposure of the proximal tubular cells to parathyroid hormone resulted in a biphasic changes involving: (1) dramatic shortening and rarefaction of microvilli within 1 min; and (2) recovery of microvillar topography after 5 min. A similar shortening of microvilli was observed following exposure to ionomycin, whereas incubation with cyclic AMP resulted in an elongation of microvilli. Parathyroid hormone stimulated cyclic AMP production and increased cytoplasmic free calcium concentration in cultured proximal tubular cells. Pretreatment of cells with a calmodulin inhibitor abolished the effect of parathyroid hormone on brush border topography. Shortening of microvilli was associated with a disappearance of microvillar core filaments. Staining of F-actin with fluoresceinphalloidin showed that parathyroid hormone resulted in fragmentation of stress fibers. It is concluded that parathyroid hormoneinduced cell activation involves cytoplasmic-free calcium, calmodulin, and the cytoskeleton.     

钙通道阻断剂异搏定对缺氧性肺动脉高压形成的影响

为考察Ca~( )在缺氧性肺动脉高压形成中的作用,我们观察了钙通道阻断剂异搏定对慢性连续性缺氧大鼠肺动脉压及左右心功能的影响。将动物置于模拟海拔5000m高原的低压舱内,腹腔注射异搏定,剂量为4mg/kg BW,每日两次。实验结果表明:异搏定可以减弱缺氧15天所引起的肺动脉压升高和右心功能加强的程度,对颈动脉压及左心功能无明显影响,提示Ca~( )的跨膜内流是构成缺氧性肺动脉高压形成的重要基础之一。我们还比较了异搏定对缺氧持续时间不同(15天、10天、5天)的大鼠肺循环的影响,并讨论了异搏定发生作用的机制。     

Cationic probes: Specificity of distribution of metal-binding sites in bovine sperm

Salts of transition elements that alter the rate of sperm cell movement act at or near calcium-binding sites. After living bull sperm cells had been preincubated in VO₄^3?, Ni²⁺, Zn²⁺, Mn²⁺, and also La³⁺, they were then fixed. Crisply defined organelles and the absence of particulate deposits in the morphological controls contrasted sharply with the treated specimens; the latter contained regions of increased electron density, the nature and distribution of which depended on the test substance, reflecting the differential affinities of the specific ions. La³⁺ formed fine dense areas, mainly at the exocytic surface of the plasma membrane. VO₄^3? marks the cell surface but also left particulate densities within the cell. Ni²⁺ caused a nearly uniformly dense deposit at the surface and on the satellite fibers and axonemal microtubules. Zn²⁺ formed less uniform but coarser deposits, while in Mn²⁺ the distribution was similar to that in Zn²⁺ but much denser in the axonemal matrix and on the satellite fibers. Verapamil restricted the size and number of the opacities, while procaine permitted a similar distribution of slightly larger size reaction product. The differences in size and distribution of the enhanced densities were consistent and replicable for the individual assay substances. Vanadate, which specifically inhibits Na, K-ATPase, bound to ouabain-sensitive enzyme loci, however, completely disrupting the axonemal complex. This suggests that an important role of dynein in flagellar motion may relate to intracellular transport of Ca²⁺.     

Altered growth kinetics precede calcium-induced differentiation in mouse epidermal cells

Summary Primary cultures of newborn mouse epidermal cells proliferate rapidly and with a high growth fraction for several months when grown in medium with low calcium (0.02 to 0.1 mM). Addition of calcium to levels generally used in culture medium (1.2 mM) was followed by rapid changes in the pattern of proliferation. By using a combination of technics (a stathmokinetic method, autoradiography, [³H]thymidine incorporation into DNA, DNA flow cytometry) it was found that cell flux was blocked for 5 to 6 h, followed by a short rise in the mitotic rate at 10 h, and a gradual fall in all growth parameters until about 32 h after the calcium switch. There was no accumulation of cells in any particular cell cycle phase. The results indicate that the calcium switch is followed by a strong reduction in cell flux from G₁ whereas the majority of the cells that had left G₁ at the time of the switch completed one cell division before cessation of all proliferative activity. Both before and after the switch the primary epidermal cultures consisted of one diploid and one tetraploid G₁ DNA stemline that seemed to react in the same way to calcium. This work reported in this paper was undertaken during the tenure of an American Cancer Society-Eleanor Roosevelt-International Cancer Fellowship awarded by the International Union Against Cancer (K. E.). The project was supported by funds partly provided by the International Cancer Research Data Bank Program of the National Cancer Institute, National Institutes of Health, Bethesda, MD, under contract N01-C0-65341 (International Cancer Research Technology Transfer) and partly by the International Union Against Cancer (O.P.F.C.).     

Cellular mechanism of HCO 3 − and Cl− transport in insect salt gland

Summary Active HCO ₃ ^t- secretion in the anterior rectal salt gland of the mosquito larva,Aedes dorsalis, is mediated by a 11 Cl^–/HCO ₃ ^– exchanger. The cellular mechanisms of HCO ₃ ^– and Cl^– transport are examined using ion- and voltage-sensitive microelectrodes in conjunction with a microperfused preparation which allowed rapid saline changes. Addition of DIDS or acetazolamide to, or removal of CO₂ and HCO ₃ ^– from, the serosal bath caused large (20 to 50 mV) hyperpolarizations of apical membrane potential (V _a) and had little effect on basolateral potential (V _bl). Changes in luminal Cl^– concentration alteredV _a in a repid, linear manner with a slope of 42.2 mV/decaloga _Cl ^l –. Intracellular Cl^– activity was 23.5mm and was approximately 10mm lower than that predicted for a passive distribution across the apical membrane. Changes in serosal Cl^– concentration had no effect onV _bl, indicating an electrically silent basolateral Cl^– exit step. Intracellular pH in anterior rectal cells was 7.67 and the calculated was 14.4mm. These results show that under control conditions HCO₃ enters the anterior rectal cell by an active mechanism against an electrochemical gradient of 77.1 mV and exits the cell at the apical membrane down a favorable electrochemical gradient of 27.6 mV. A tentative cellular model is proposed in which Cl enters the apical membrane of the anterior rectal cells by passive, electrodiffusive movement through a Cl^–-selective channel, and HCO ₃ ^– exits the cell by an active or passive electrogenic transport mechanism. The electrically silent nature of basolateral Cl^– exit and HCO₃ entry, and the effects of serosal addition of the Cl^–/HCO₃ exchange inhibitor, DIDS, on and transepithelial potential (V _ic) suggest strongly that the basolateral membrane is the site of a direct coupling between Cl^– and HCO ₃ ^– movements.     

Mechanism of inhibition of net ion transport across frog corneal epithelium by calcium channel antagonists

Summary In the isolated bullfrog cornea, three calcium channel antagonists had dose-dependent inhibitory effects on the Cl-originated short-circuit current (SCC). Their order of decreasing potency was bepridil, verapamil and diltiazem. One millimolar diltiazem inhibited the SCC by 98% and subsequent incubation with the calcium ionophore A23187 had no restorative effect. Increasing the bathing solution Ca concentration from 0.05 to 15mm, however, decreased diltiazem's inhibitory efficacy. This antagonist depolarized the intracellular potential differenceV _m from –54 to –18 mV (tear: reference) and the voltage divider ratioFR ₀ decreased from 0.58 to 0.30, suggesting an increase in basolateral membrane electrical resistance. Additional indication of a basolateral membrane effect by the drug was that preincubation with 10⁵ m amphotericin B in Cl-free Ringer's did not eliminate the inhibitory effect of the drug on the Na- and K-elicited SCC. In the absence of amphotericin B in Cl-free Ringer's (SCC=0), 1 ×10³ m diltiazem depolarized theV _m from –78 to –9 mV suggesting that the increase in basolateral membrane resistance was due to K channel blockade. Diltiazem (1×10³ m) significantly decreased cyclic AMP content; however, isoproterenol in the presence of the drug increased cyclic AMP fourfold without having any restorative effect on the inhibited SCC. Therefore, the inhibition of the Cl-originated SCC resulting from an increase in basolateral membrane K resistance is not caused by a decline in cyclic AMP content. In plasma membrane-enriched fractions prepared from broken cell preparations of bovine corneal epithelium, 1×10³ m diltiazem had no inhibitory effects on either Na,K-ATPase or Ca,Mg-ATPase activities. These latter effects further point to the selectivity of diltiazem as an inhibitor of K-channel activity, but do not preclude a Ca-channel blocker effect by the drug in the micromolar range.