Changes In Cellular pH Research Articles

Phorbol esters and arginine vasopressin in vascular smooth muscle cell activation.

Vascular smooth muscle cell (VSMC) contraction is the result of several interacting mechanisms. In this study such interactions in rat aortic VSMC in culture were examined with a focus on the role of protein kinase C-mediated mechanisms. The change in shape of VSMC was used as a functional parameter representative of contraction. The protein kinase C agonist, phorbol myristate acetate (PMA), and arginine vasopressin (AVP) induced a dose-dependent, progressive change in VSMC shape. The effects of PMA differed from AVP in the delay time necessary to reach the plateau of the response and in the absence with PMA of a transient rise in cytosolic free Ca2+ [( Ca2+]i). The effect of PMA was potentiated by the Ca2+ ionophore, ionomycin, and involved a verapamil-inhibitable transmembrane Ca2+ transport system. Protein kinase C inhibition by either isoquinolin-sulfonyl-O-2-methylpiperazine or protein kinase C desensitization significantly reduced the cell shape change induced by either PMA or AVP. In the case of AVP, this inhibitory effect occurred without affecting the [Ca2+]i transient. Therefore, the [Ca2+]i transient appears to be independent of acute protein kinase C regulation, since it is apparently not affected by the absence of protein kinase C activity. Protein kinase C activation by PMA produced intracellular alkalinization that is blocked by the sodium transport antagonist, amiloride. Amiloride also blocked the cell shape change induced by PMA or AVP. The intracellular alkalinization, however, was not necessary for the cell shape change to occur. Specifically, with the use of VSMC preincubated with fetal calf serum, PMA did not induce cellular pH changes but still produced cell shape changes.

Hisactophilin, a histidine-rich actin-binding protein from Dictyostelium discoideum

The purification, cloning, and complete cDNA-derived sequence of a 17-kDa protein of Dictyostelium discoideum are described. This protein binds to F-actin in a pH-dependent and saturable manner. It induces actin polymerization in the absence of Mg2+ or K+, and is enriched in the submembranous region of the amoeboid cells as indicated by immunofluorescence labeling of cryosections. The mRNA as well as the protein are present throughout growth and all stages of development. The protein is detected in both soluble and particulate fractions of the cells. From a plasma membrane-enriched fraction, minor amounts of the protein are stepwise solubilized with 1.5 M KCl, 0.1 M NaOH, and Triton X-100, but most of the protein is only solubilized with 1% sodium dodecyl sulfate. As judged by the apparent molecular mass in sodium dodecyl sulfate-polyacrylamide gels, immunological cross-reactivity, and two-dimensional electrophoresis, the 17-kDa proteins from the soluble and particulate fraction resemble each other. The cDNA sequence does not reveal any signal peptide, trans-membrane region, or N-glycosylation site. Southern blots hybridized with a cDNA probe that spans the entire coding region show that the 17-kDa protein is encoded by a single gene. The most characteristic feature of the protein is its high content of 31 histidine residues out of 118 amino acids. We designate this protein as hisactophilin and suggest that this histidine-rich protein responds in its actin-binding activity to changes in cellular pH upon chemotactic signal reception.

Role of tyrosine kinase and membrane-spanning domains in signal transduction by the platelet-derived growth factor receptor.

Three types of mutations were introduced into the platelet-derived growth factor (PDGF) receptor to cause a loss of PDGF-stimulated tyrosine kinase activity: (i) a point mutation of the ATP-binding site, (ii) a deletion of the carboxyl-terminal region, and (iii) replacement of the membrane-spanning sequences by analogous transmembrane sequences of other receptors. Transfectants expressing mutated receptors bind, 125I-labeled PDGF with a high affinity but had no PDGF-sensitive tyrosine kinase activity, phosphatidylinositol turnover, increase in the intracellular calcium concentration, change in cellular pH, or stimulation of DNA synthesis. However, PDGF-induced receptor down regulation was normal in the mutant cells. These results indicate that the transmembrane sequence has a specific signal-transducing function other than merely serving as a membrane anchor and that the receptor kinase activity is necessary for most responses to PDGF but is not required for receptor down regulation.

Role of tyrosine kinase and membrane-spanning domains in signal transduction by the platelet-derived growth factor receptor.

Three types of mutations were introduced into the platelet-derived growth factor (PDGF) receptor to cause a loss of PDGF-stimulated tyrosine kinase activity: (i) a point mutation of the ATP-binding site, (ii) a deletion of the carboxyl-terminal region, and (iii) replacement of the membrane-spanning sequences by analogous transmembrane sequences of other receptors. Transfectants expressing mutated receptors bind, 125I-labeled PDGF with a high affinity but had no PDGF-sensitive tyrosine kinase activity, phosphatidylinositol turnover, increase in the intracellular calcium concentration, change in cellular pH, or stimulation of DNA synthesis. However, PDGF-induced receptor down regulation was normal in the mutant cells. These results indicate that the transmembrane sequence has a specific signal-transducing function other than merely serving as a membrane anchor and that the receptor kinase activity is necessary for most responses to PDGF but is not required for receptor down regulation.

The labile nature of the insulin signal(s) for the stimulation of DNA synthesis in mouse lens epithelial and 3T3 cells.

A kinetic study was carried out to assess the stability of the intracellular signal(s) generated by insulin in quiescent cells for the stimulation of DNA synthesis. Using murine lens epithelial cells and Swiss 3T3 cells in culture, it was found that insulin stimulated DNA synthesis after a lag of 14.5 h. If, however, 6 h after the addition of insulin to the cells, the insulin-containing media were totally removed, followed by the addition of fresh media (even if insulin was returned to the medium within approximately 10 min), a 14.5-h lag still remained after insulin readdition before DNA synthesis started. In another set of experiments, the insulin was removed after 6 h by diluting its concentration approximately 60,000-fold. In this case, if insulin was at the diluted concentration for approximately 60 min before being added back, a full 14.5 h was necessary for the start of DNA synthesis. The half-time for loss of signal was 2 +/- 1 min for total washout and 18.4 +/- 0.5 min for the dilution experiment. These results indicate that the intracellular signal(s) for DNA synthesis produced by the binding of insulin to its cellular receptor are extremely transitory in nature. The signal disappears at approximately the same rate that insulin dissociates from the receptor. Thus, insulin must be constantly binding to the membrane receptor in order to keep the key signal(s) at a high enough level for the cell to progress on to S phase. Early events, such as specific protein synthesis, changes in ion flux, changes in cellular metabolism, and changes in cellular pH, may be essential, but they are not sufficient to cause a cell to progress on to S phase. Addition of sodium vanadate to the cell is found to stabilize the messenger such that there is no loss of signal when insulin is removed. These data are consistent with the tyrosine-phosphorylated insulin receptor or a product of its action being the signal.

Methylamines and islet function: cationic aspects

Methylamine (2 to 10 mM) caused a dose-related inhibition of insulin release evoked in rat pancreatic islets by nutrient or non nutrient secretagogues. Trimethylamine exerted comparable effects upon insulin release. Methylamine (2 mM) inhibited insulin secretion but failed to affect 45Ca uptake and efflux in response to a rise in extracellular K+ concentration, suggesting that methylamine acts, to a certain extent at least, at a distal site in the secretory sequence. Methylamine, however, also exerted untoward ionic effects. First, methylamine (2 to 10 mM) apparently caused a dose-related increase in cellular pH. Second, methylamine (2mM) augmented 86Rb outflow from islets perifused either in the absence or presence of glucose or gliclazide, and inhibited Ca2+ inflow (as judged from the net uptake or efflux of 45Ca) in islets stimulated by D-glucose, L-leucine or 2-ketoisocaproate. This multiplicity of ionic and other effects may account for the fact that, in the presence of distinct secretagogues, the secretory process appeared more or less sensitive towards methylamine, depending on the relative importance of changes in cellular pH, K+ permeability and intracellular Ca2+ distribution as determinants of the secretory response.

Skeletal Muscle Energetics and Metabolism

When muscles contract, chemical energy is converted into mechanical en­ ergy and heat, physical quantities necessary for locomotion, circulation, digestion, respiration, reproduction, and thermal regulation. The task of the muscle energeticist is to learn how much chemical energy is used [and/or heat and work (enthalpy) produced], how chemical energy utilization varies and is regulated, what the mechanism of chemomechanical transduction is, and how substrates used during contraction are restored. To answer these questions, the extent of chemical change (and/or enthalpy production) must be measured. To this end, a variety of techniques have been developed, including measurement of: (a) the muscle's enthalpy production (myother­ mometry); (b) chemical change by quantitative analysis of frozen muscle extracts; (c) chemical change by 31p nuclear magnetic resonance studies of the whole living tissue; (d) oxygen consumption; (e) lactic acid production; (j) the intracellular concentration of NADH; and (g) cellular pH changes. No single technique alone is wholly satisfactory as the general tool for the study of energetics. In some cases a technique lacks the requisite time resolution and/or sensitivity. Other methods require assumptions about the

Mechanism of drug-induced vacuolization in tissue culture

Procaine, procaine amide, cocaine, pilocarpine, ephedrine, and atropine all produced marked cytoplasmic vacuolization in cells cultured in vitro. The accumulation of vacuoles did not seem to affect either the motility or mitosis of the cells. After the drug was removed by washing, vacuoles eventually disappeared completely. Ammonium chloride produced essentially the same phenomenon as the drugs. The process of vacuolization induced by the compounds tested was influenced by the pH of the external medium; in media adjusted to an acid pH where the undissociated base was in relatively low concentration, vacuolization was reduced. It was postulated that penetration of weak bases was the factor responsible for vacuole formation which in turn might be a means of counteracting cellular pH changes.