[Effect of high calcium intake on blood pressure and intracellular pH, Na(+)-H+ exchange activity in stroke-prone spontaneously hypertensive rats].

Effect of high calcium (3%) intake on blood pressure and intracellular pH, Na(+)-H+ exchange activity, cytosolic free calcium concentration in stroke-prone spontaneously hypertensive rats (SHRSP-Ca) is compared with sex-, ag-matched SHRSP and WKY rats without high calcium intake. The results showed that high calcium intake significantly suppressed increase in blood pressure and decreased cytosolic free calcium concentration after it was given to SHRSP-Ca for 6 weeks, while plasma calcium was increased. The intracellular pH tended to be in the normal range of WKY rats. Na(+)-H+ exchange activity was slightly decreased. Correlationship between blood pressure and intracellular pH, Na(+)-H+ exchange activity and cytosolic free calcium concentration were discussed.

Multiple forms of the G protein-related beta subunit in Daudi lymphoblastoid cells.

We have explored the forms of the G protein-related beta subunit which are present in Daudi lymphoblastoid cells. Northern blotting with labeled beta-1 and beta-2 probes indicates that two messages of 3.3 kb and 1.7 kb are present for both beta-1 and beta-2, implying that multiple forms of the beta subunit are present. Antibodies were raised against two peptides of the beta subunit (residues 1-23 and 127-145). Both antibodies detected subunits at 35 kDa and 31 kDa, of which the 35-kDa form predominates in the membrane fraction and the 31-kDa one in the cell cytosol. Crosslinking of the membrane fraction with the cleavable crosslinker (DTSSP) caused a simultaneous diminution in the 31-kDa form while increasing the amount of the 35-kDa form--a pattern which was reversed upon the reduction of these crosslinks with DTT. Studies of the soluble form indicate that this is truly a soluble protein since centrifugation at 200,000 x g for 2 h did not diminish the levels of the protein in the soluble fraction. Sedimentation analysis indicates that the soluble beta-homologue is found in fractions which overlap with those which contain the mu chain of immunoglobulin at a position clearly distinct from the expected positions of free mu or free beta. Our results suggest that at least two forms of a subunit which is closely related to, or identical with, the beta subunit of G proteins are present in Daudi cells.

Induction of FMLP-mediated calcium mobilization requires crosslinking of surface immunoglobulin in Daudi cells.

We have explored the requirements for the induction of the N-formyl-methionyl-leucyl-phenylalanine (FMLP) response in Daudi cells after anti-immunoglobulin treatment. Our results indicate that (a) induction of responsiveness to FMLP was observed in Daudi only after crosslinking of surface immunoglobulin by anti-immunoglobulin; (b) this induced responsiveness was not observed in Ramos or Wil-2 cells; (c) the F(ab')2 fragment was sufficient for the induction of the FMLP response, but the Fab fragment and the Fc fragment were ineffective; (d) of the many agents active in B lymphocyte regulation which were tested, none were as effective as anti-immunoglobulin in the induction of the FMLP response; and (e) three inhibitors of calcium mobilization (W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide), PMA (phorbol 12-myristate 13-acetate), and colchicine), acting on distinct mechanisms, inhibited both the calcium mobilization due to anti-immunoglobulin and the induction of responsiveness to FMLP. Our results suggest important determinants in the induction of a calcium-mobilizing FMLP response in cells of B lymphocyte lineage include (a) the cell type, (b) a selective requirement for activation via surface immunoglobulin, and (c) crosslinking of the surface immunoglobulin.

G-proteins and the role of second messengers in the regulation of the human neutrophil.

The ideas discussed above clearly point to increasingly complex and interactive transduction mechanisms for the regulation of the neutrophil. The central challenges to be met include the following: 1. Better assays are needed for the study of physiological parameters such as adherence, aggregation, shape change, and cytoskeletal rearrangements, assays which are not prohibitively complex and expensive while still allowing for more detailed physiological observations. 2. The neutrophil receptors need to be characterized in greater detail at the molecular level. Protein purification, sequencing, and cloning approaches are needed. Given the inherent shortcomings of working with the neutrophil system due to the presence of proteases and the problems of obtaining sufficient amounts of plasma membranes as source material for receptor purification, this is a difficult task. Advances in micropurification and sequencing may alleviate some of the difficulties here. 3. The size and complexity of the G-protein family continue to expand. However, as pointed out earlier, stimulus-responsive enzymes without G-protein-associated regulation, and G-proteins without clearly identified targets, remain. A better definition and description of the G-protein family will be required if cellular regulation is to be understood at the molecular level. In terms of second messengers and their role in cellular regulation, the main questions which remain to be answered concern identification of the precise pathways which are important to cellular regulation. In order to understand the complex cascades of arachidonate metabolism, phospholipid turnover, and calcium homeostasis, it is all the more important that the manner in which second messengers may regulate particular cell functions be better understood. An omission in this review is the role of kinases in cellular regulation. Activation of kinase C (through calcium and diacylglycerol) and kinase A (through cAMP) has been demonstrated. The substrates for these kinases have been described by various investigators. However, relating phosphorylation changes in a particular protein to the activity of the protein, and assignment of activity to particular physiological roles, has not been satisfactorily accomplished and remains a challenge for the future.