We show that a non-inhibitory monoclonal antibody (MAB) can be selected that provides substantial and sustained protection against proteolytic inactivation of L-asparaginase by trypsin. Of six non-inhibitory, high affinity, monoclonal antibodies to L-asparaginase, one afforded approximately 70% protection. Inactivation of L-asparaginase is associated with a single cleavage adjacent to lysine-29 that results in loss of an N-terminal fragment with a calculated MW of 2,647. The protective MAB prevented this trypsin cleavage. The products of gene fusions of "humanized" fragments of such antibodies and L-asparaginase could have increased clinical utility.

Immediately after osmotic swelling of Madin-Darby canine kidney (MDCK) cells, a transient (1-2 min) increase in Ca2+ influx and internal Ca2+ (Ca2+i) is observed. The normal Ca2+ influx appears to be mediated by the 3Na(+)-Ca2+ exchange system [Borle et al. Am. J. Physiol. 259 (Cell Physiol. 28): C19-C25, 1990], but the swelling-induced component is different in 1) Na+ dependence, 2) affinity for Ca2+, 3) inhibition by La3+, and 4) direction of net flux at low external Ca2+. Swelling appears to activate an uncoupled Ca2+ flow, perhaps through cation-nonspecific stretch-activated channels. The regulatory volume decrease (RVD) is dependent on the swelling-induced pulse of Ca2+ influx and associated rise in Ca2+i. Swelling also induces a biphasic change in membrane potential, a hyperpolarization followed by depolarization, reflecting sequential increases in K+ and Cl- permeabilities. The time dependence of the former corresponds closely with the transient peak in Ca2+i, but the latter does not. Ca2+i appears to have a direct activating effect on K+ channels but an indirect effect on Cl- channels, mediated via other Ca(2+)-triggered systems. The sequence of events following cell swelling appears to be transient increases in Ca2+ permeability, Ca2+ influx, Ca2+i, K+ permeability, followed by triggering of a mediating system that increases Cl- permeability. The net result is KCl, osmotic water loss, and volume adjustment.

In nature, increased stability of enzymes has often been found to be associated with noncovalent protein-protein interactions. Specific antibodies should be suitable for this purpose. To test this hypothesis, we used a number of model enzymes, complexed them with their specific antibodies, and exposed them and the free enzymes to low and high temperature, lyophilization, oxidation, and alcohol. The retained activity of the antibody-complexed enzymes was substantially, and in some cases dramatically, higher. In general mechanistic terms, stabilization may have been accomplished either by noncovalent antibody crosslinking of discontinuous oligopeptide chains on the surface of the enzyme, thereby increasing resistance to unfolding of the enzyme, or by physical shielding by the antibodies of vulnerable sites on the surface of the enzyme.

The mercurial, p-chloromercuribenzoylsulfonate (PCMBS), blocks volume recovery of dissociated, osmotically swollen, Madin-Darby canine kidney cells (MDCK) and, at higher concentrations, induces substantial swelling. In the absence of Na+ the rate of volume recovery is, in contrast, substantially increased. PCMBS does not inhibit the "normal" volume-regulating pathways, K+ and Cl- conductances. Rather, its blocking action is due to substantial activation of Na+ and K+ permeabilities, especially the former. Consequently, the normal reshrinking mechanism, loss of KCl, is counterbalanced by PCMBS-induced gains of NaCl. In isotonic cells, PCMBS, at higher concentrations, induces cell swelling, indicating that Cl- permeability is also increased, a conclusion confirmed by direct measurement of 36Cl- fluxes. HgCl2 produces similar effects except that it is more potent and more rapid in its action. Activation of conductive ion permeabilities to Na+, K+, and Cl- are associated with appropriate changes in membrane potential. A small bumetanide-sensitive swelling component (Na(+)-Cl- cotransport) is activated by HgCl2 but not by PCMBS. Another effect is elevation of cytoplasmic Ca2+, apparently by mobilization from internal stores. Some of the functional sites (Na+ and K+) appear to be located externally, rapidly accessible to both HgCl2 and PCMBS, whereas others (Cl- and Ca2+) appear to be internal, rapidly accessible to the permeant HgCl2 but slowly to relatively impermeant PCMBS. In conclusion, the disturbances of volume regulation are largely due to the increases in conductive ion fluxes.

Osmotic swelling of dissociated Madin-Darby canine kidney (MDCK) cells in NaCl medium is followed by shrinking (regulatory volume decrease, or RVD) or in KCl medium by secondary swelling. The cation ionophore gramicidin has little effect on volumes of isotonic cells but accelerates volume-activated changes in either medium. Immediately after hypotonic exposure, the membrane becomes transiently hyperpolarized followed by depolarization. The depolarization phase is diminished by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Swelling is also associated with an almost immediate increase in Ca2+ influx and elevation of cytoplasmic Ca2+ ([Ca2+]i) preceding RVD. In Ca2(+)-free medium, [Ca2+]i rapidly declines to a low level. Osmotic swelling, under these circumstances, is associated with a small transient increase in [Ca2+]i, but RVD or secondary swelling (in KCl) are minimal. Under these conditions, addition of gramicidin or the Ca2(+)-ionophore A23187 induces significant volume changes, although not as large as those found in the presence of Ca2+. Quinine inhibits RVD in the absence of gramicidin, but not in its presence; oligomycin C, DIDS, and trifluoperazine, on the other hand, inhibit in the presence of the ionophore. These findings suggest that in MDCK cells RVD involves activation of distinct conductive K+ and Cl- pathways which allow escape of KCl and osmotically obligated water and that activation of both pathways is associated with elevated [Ca2+]i derived largely from volume activation of a Ca2(+)-influx pathway.

The effectiveness of a methodoogy designed to protect the antigen binding capacity of monoclonal antibodies undergoing labelling with a number of reagents was examined. The antigen binding sites of monoclonal antibodies were protected by complexing them with their antigen.Chemical modification with 6mM of the water soluble Bolton-Hunter reagent of site protected monoclonal antibodies to glucoamylase resulted in antibodies that could tolerate a four-fold increase in reagent incorporation, without any loss of antigen binding capacity. Iodination of these antibodies (modified under site protected conditions) yielded over 70% increase in radioactivity incorporated in the active antibody fraction, compared with the incorporation into unprotected antibodies. Site protected labeling was found to be effective in retaining the antigen binding capacity of monoclonal antibodies modified with all reagents tested with the exception of chloramine-T.

In dissociated MDCK cells, activators of the cyclic AMP system cause depolarization detectable by changes in fluorescence of the membrane potential sensitive dye bisoxonol. Addition of forskolin (60 μM), vasopressin (2 μM), 8-bromo-cyclic AMP (0.5 mM) or l-epinephrine (10 μM) depolarized the cells substantially in low Cl− (5 mM) but had little effect in high Cl− (140 mM) solution. These results are consistent with cyclic AMP activation of Cl− channels. The Ca2+-ionophore ionomycin (1 μM) produced a rapid hyperpolarization in low and high Cl− solutions, consistent with K+ channel opening. Using a clonal subline, MDCK-14, the magnitude of the ionomycin hyperpolarization was roughly proportional to the concomitant rise in [Ca2+]i as measured with the intracellular Ca2+ probe indo-I. Both l-epinephrine and isoproterenol appeared to activate the Cl− channels. However only l-epinephrine produced a [Ca2+]i rise and a transient hyperpolarization (due to K+ channel opening), which preceeded the depolarization due to Cl− channel opening. The l-epinephrine-induced [Ca2+]i response of the heterogeneous MDCK cell population but not of the clonal subline MDCK-14 was inhibited by removal of extracellular Ca2+. In the latter only the slow secondary phase of the [Ca2+]i rise was affected by Ca2+ removal. It is concluded that l-epinephrine activates K+ and Cl− channels in a sequential manner in MDCK cells by Ca2+ and cAMP signals, presumably via α- and β-adrenergic receptors located on the same cell.

Enhanced sodium-proton exchange may play a role in the pathogenesis of hypertension. Na+-H+ exchange was measured indirectly in platelets as the rate of amiloride-sensitive and sodium-dependent volume gain of cells suspended in sodium-propionate; the cytoplasmic acidification induced by the permeant propionic acid activated the exchanger and the volume changes coupled to Na+ uptake were measured by cell sizing with a Coulter counter and 'Channelyzer'. The test was rapid, simple, and reproducible. 20 normotensives; 8 normotensives with a family history of hypertension; 15 patients with essential hypertension receiving medication; and 7 hypertensives who had not received any antihypertensive drugs were studied. The exchange rate constants of these groups were (mean [SE] in s-1 × 10-3) 13·1 (0·6); 15·5 (0·7); 18·4 (0·9); and 25·6 (2·8), respectively. The differences between hypertensives and normotensives were significant. Measurement of sodium-proton exchange in platelets may help to clarify the pathogenesis of hypertension.

Summary Acidification of the cytoplasm of human blood platelets leads to activation of Na+/H+ exchange. As a result, alkalinization occurs that is detectable by an intracellular fluorescent pH indicator. The activity of the exchanger can also be measured by the swelling of platelets suspended in Na-propionate medium using a Coulter Counter: the rapid entry of propionic acid leads to acidification and activation of Na+/H+ exchange with the parallel entry of Na+ and propionic acid leading to osmotic swelling. The Na+/H+ exchanger is sensitive to amiloride and to derivatives that are reported to be more specific inhibitors; it is specific for Na+ and Li+ with no measurable transfer of K+, Rb+ or Cs+; its Km for Na+ is 75 to 90 mM; it displays competitive behavior between Na+ and amiloride; its activity is decreased in cells loaded with Na+ by prolonged ouabain treatment; and it has a high temperature coefficient. These properties are in general similar to those of the exchanger in other cells. It is suggested that the Na+/H+ exchanger plays a role in platelet pH regulation.

Cetiedil, a drug that is reported to block K+-channels, substantially increases the conductive Cl− permeability of Chinese hamster ovary (CHO) cells. The permeability was monitored by volume changes in cells treated with gramicidin to increase the cation permeability. Under this circumstance, increases in Cl− conductances result in volume changes detectable by electronic sizing, with the direction determined by the gradients of the permeating ions. In NaCl or KCl media, swelling occurs, but in N-methylglucamine chloride, shrinking. The increases in Cl− conductance could also be measured as an increased 36Cl− flux or by changes in membrane potential (measured by fluorescence of a potential-sensitive dye) toward the Cl− equilibrium potential. The effect of cetiedil was concentration dependent, with maximal effect at 50 μM. The anion specificity for the conductance was NO3− > Cl− = Br− ⪢ SO4−2 or isethionate. A number of other drugs that influence transport activities had no effect on Cl− conductance. The cetiedil effect on Cl− conductance was observed in one other cell line, but was absent in several other cell types. The cetiedil-induced Cl− conductance in CHO cells appears to involve a different pathway than that induced by exposure to hypotonic medium.

Human platelets exposed to hypotonic media undergo an initial swelling followed by shrinking (regulatory volume decrease [RVD]). If the RVD is blocked, the degree of swelling is in accord with osmotic behavior. The cells could swell at least threefold without significant lysis. Two methods were used to follow the volume changes, electronic sizing and turbidometery. Changes in shape produced only limited contribution to the measurements. The RVD was very rapid, essentially complete in 2 to 8 minutes, with a rate proportional to the degree of initial cell swelling. RVD involved a loss of KCI via volume‐activated conductive permeability pathways for K + and anions, presumably Cl − . In media containing > 50 mM KCI, the shrinking was inhibited and with higher concentrations was reversed (secondary swelling), suggesting that it is driven by the net gradient of K + plus Cl − . The K + pathway was specific for Rb + and K + compared to Li + and Na + . The Cl − pathway accepted NO 3 − and SCN − but not citrate or SO 2‐ 4 . In isotonic medium, the permeability of platelets to Cl − appeared to be low compared to that of K + . After hypotonic swelling both permeabilities were increased, but the Cl − permeability exceeded that of K + . The Cl − conductive pathway remained open as long as the cells were swollen. RVD was incomplete unless amiloride, an inhibitor of Na + /H + exchange, was present or unless Na + was replaced by an impermeant cation. In addition, acidification of the cytoplasm occurred upon cell swelling. This reduction in pH i appeared to activate Na + / H + exchange, with a resultant uptake of Na + and reduction in the rate and amount of shrinking. Like other cells, platelets responded to hypertonic shrinking with activation of Na + /H + exchange, but regulatory volume increase was not detectable.

A preparation of band 4.5 protein of the red cell membrane, containing largely the sugar transporter, was labelled with the sulfhydryl reagent N-ethyl [14C]maleimide. In preparations denatured with sodium dodecyl sulfate (SDS), all five sulfhydryl groups present in the peptide, Mr 45 000 to 60 000, react with the alkylating agent within 20 min at 37 degrees C. If the peptide is reconstituted in lipid vesicles and cleaved with trypsin before extraction and denaturation with SDS, three sulfhydryl groups are found in a 30 kDa fragment and two in a 19 kDa fragment. In 'native' reconstituted protein only three groups react, even after two hours of exposure, two in the 30 kDa fragment and one in the 19 kDa fragment. Thus, one sulfhydryl group is cryptic, inaccessible to N-ethylmaleimide in each fragment. In intact cells, the single reactive group of the 19 kDa fragment can be protected against reaction with N-ethylmaleimide by the impermeant sulfhydryl reagent, p-chloromercuribenzene sulfonate (PCMBS). It is, therefore, considered to be exposed on the outer face of the membrane. The two reactive groups of the 30 kDa fragment are not protected by PCMBS and are, therefore, not considered to be exposed to the outside medium. Cytochalasin B, a competitive inhibitor of sugar transport affords temporary protection of the exofacial group of the 19 kDa against reaction with N-ethylmaleimide, and affords longer term protection of one of the reactive groups of the 30 kDa fragment. These findings allow conclusions about the topology of the sugar transport protein in the bilayer. Both proteolytic fragments must cross the bilayer. One of three reactive sulfhydryl groups is exofacial and two may be cytoplasmic. The two cryptic groups may be located within the bilayer.

The activity of the Na'/H' exchange system of rat thymic lymphocytes was determined by means of intracellular (pHi) and extracellular pH (pH.) measurements . In isotonic media, the antiport is virtually quiescent at physiological pHi (7.0-7.1), but is greatly activated by cytoplasmic acidifica- tion . At normal pH i, the antiport can also be activated by osmotic shrinking . Osmotic activation occurs after a delay of 20-30 s and is reversed several minutes after iso-osmolarity is restored . The mechanism of activation was analyzed by comparing the kinetic parameters of transport in resting (isotonic) and hyperosmotically stressed cells. The affinities of the external substrate site for Na' and H+ are not altered in shrunken cells . In contrast, the H; sensitivity of the antiport (which is largely dictated by an allosteric modifier site) was increased, which accounted for the activation . The concentration of free cytoplasmicCa" ((Ca2+ );) increased after osmotic shrinking. This increase was dependent on the presence of extracellularCa" and Na' and was blocked by inhibitors of Na'/H' exchange, which suggests that it is a consequence, rather than the cause, of the activation of the antiport . It is concluded that the shift in the pHi dependence of the modifier site of the Na'/H' antiport is the primary eventunderlying the regulatory volume increase that followsosmotic shrinkage.

A method is described which allows studying specific cation transport pathways of the cell membranes by converting ion fluxes into volume changes. Lipophilic weak electrolytes, such as propionate, rapidly penetrate the cell membranes in their undissociated acid from but not as negatively charged ions. When human red cells are incubated in isoosmotic K-propionate media an intracellular acidification occurs with a limited propionate uptake and volume increase (corresponding to the buffering capacity of the cytoplasm). If both protons and alkali cations are rendered permeable, a rapid salt influx and volume increase is observed. The latter can be quantitatively followed by electronic sizing methods. A detailed characterization of the system is provided through studies with ionophores, inhibitors of the red cell anion exchange system and drugs which activate or inhibit the Ca2+-induced K+ transport. It is demonstrated that in K-propionate media the permeability of the Ca2+-induced K+ pathway can be directly estimated. The method is suitable to observe population (all-or-none) responses in the activation of the K+ pathway under certain experimental conditions. The application of the method in the search for cation-proton exchanger systems is discussed and its use for demonstration purposes by producing selective lysis of red cells is described.

The Na+/H+ antiport is stimulated by 12-O-tetradecanoylphorbol-13, acetate (TPA) and other phorbol esters in rat thymic lymphocytes. Mediation by protein kinase C is suggested by three findings: (a) 1-oleoyl-2-acetylglycerol also activated the antiport; (b) trifluoperazine, an inhibitor of protein kinase C, blocked the stimulation of Na+/H+ exchange; and (c) activation of countertransport was accompanied by increased phosphorylation of specific membrane proteins. The Na+/H+ antiport is also activated by osmotic cell shrinking. The time course, extent, and reversibility of the osmotically induced and phorbol ester-induced responses are similar. Moreover, the responses are not additive and they are equally susceptible to inhibition by trifluoperazine, N-ethylmaleimide, and ATP depletion. The extensive analogies between the TPA and osmotically induced effects suggested a common underlying mechanism, possibly activation of a protein kinase. It is conceivable that osmotic shrinkage initiates the following sequence of events: stimulation of protein kinase(s) followed by activation of the Na+/H+ antiport, resulting in cytoplasmic alkalinization. The Na+ taken up through the antiport, together with the HCO3- and Cl- accumulated in the cells as a result of the cytoplasmic alkalinization, would be followed by osmotically obliged water. This series of events could underlie the phenomenon of regulatory volume increase.

Annals of the New York Academy of SciencesVolume 456, Issue 1 p. 207-219 Regulation of Na+/H+ Exchange in Lymphocytesa S. GRINSTEIN, S. GRINSTEIN Departments of Cell Biology and bImmunology Research Institute Hospital for Sick Children 555 University Avenue Toronto, Ontario, Canada M5G 1X8Search for more papers by this authorJ. D. GOETZ, J. D. GOETZ Departments of Cell Biology and bImmunology Research Institute Hospital for Sick Children 555 University Avenue Toronto, Ontario, Canada M5G 1X8Search for more papers by this authorS. COHEN, S. COHEN Departments of Cell Biology and bImmunology Research Institute Hospital for Sick Children 555 University Avenue Toronto, Ontario, Canada M5G 1X8Search for more papers by this authorA. ROTHSTEIN, A. ROTHSTEIN Departments of Cell Biology and bImmunology Research Institute Hospital for Sick Children 555 University Avenue Toronto, Ontario, Canada M5G 1X8Search for more papers by this authorE. W. GELFAND, E. W. GELFAND Department of Biochemistry University of Toronto Toronto, Ontario, CanadaSearch for more papers by this author S. GRINSTEIN, S. GRINSTEIN Departments of Cell Biology and bImmunology Research Institute Hospital for Sick Children 555 University Avenue Toronto, Ontario, Canada M5G 1X8Search for more papers by this authorJ. D. GOETZ, J. D. GOETZ Departments of Cell Biology and bImmunology Research Institute Hospital for Sick Children 555 University Avenue Toronto, Ontario, Canada M5G 1X8Search for more papers by this authorS. COHEN, S. COHEN Departments of Cell Biology and bImmunology Research Institute Hospital for Sick Children 555 University Avenue Toronto, Ontario, Canada M5G 1X8Search for more papers by this authorA. ROTHSTEIN, A. ROTHSTEIN Departments of Cell Biology and bImmunology Research Institute Hospital for Sick Children 555 University Avenue Toronto, Ontario, Canada M5G 1X8Search for more papers by this authorE. W. GELFAND, E. W. GELFAND Department of Biochemistry University of Toronto Toronto, Ontario, CanadaSearch for more papers by this author First published: November 1985 https://doi.org/10.1111/j.1749-6632.1985.tb14866.xCitations: 22 a The original work reviewed was supported by the National Cancer Institute (Canada) and the Medical Research Council (Canada). S. G. is a Medical Research Council Scientist. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 Murer, H., U. Hopfer & R. Kinne 1976. Biochem. J. 154: 597–604. 2 Aronson, P. S. 1983. Am. J. Physiol. 245: F647–659. 3 Krulwich, T. A. 1983. Biochim Biophys. Acta 583: 245–267. 4 Grinstein, S., C. A. Clarke & A. Rothstein 1983. J. Gen. Physiol. 82: 619–657. 5 Grinstein, S., S. Cohen & A. Rothstein 1984. J. Gen. Physiol. 83: 341–369. 6 Cala, P. M. 1983. Mol. Physiol. 4: 33–52. 7 Piwnica-Worms, D. & M. Lieberman 1983. Am. J. Physiol. 244: C422–C428. 8 Moolenaar, W. H., R. Y. Tsien, P. T. VanderSaag & S. W. DeLaat 1983. Nature 304: 645–648. 9 Owen, N. E. & M. L. Villereal 1982. Proc Natl. Acad. Sci. USA 79: 3537–3541. 10 Grinstein, S., J. D. Goetz, W. Furuya, A. Rothstein & E. W. Gelfand 1984. Am. J. Physiol. 247: C293–C298. 11 Grinstein, S., J. D. Goetz & A. Rothstein 1984a. J. Gen. Physiol. 84: 585–600. 12 Benos, D. J. & V. S. Sapirstein 1983. J. Cell. Physiol. 116: 213–220. 13 Aronson, P. S., J. Nee & M. A. Suhm 1982. Nature 299: 161–163. 14 Grinstein, S., J. D. Goetz & A. Rothstein 1984b. J. Gen. Physiol. 84: 565–584. 15 Rothenberg, P., L. Glaser, P. Schlessinger & D. Cassel 1983. J. Biol. Chem. 258: 12644–12653. 16 Burns, C. P. & E. Rozengurt 1984. J. Cell Biol. 98: 1082–1089. 17 L'Allemain, G., A. Franchi, E. Cragoe & J. Pouyssegur 1984. J. Biol. Chem. 259: 4313–4319. 18 Cassel, D., Y. Zhuang & L. Glaser 1984. Biochem Biophys. Res. Commun. 118: 675–681. 19 Nishizuka, Y. 1984. Nature 308: 693–698. 20 Grinstein, S., S. Cohen, J. D. Goetz, A. Rothstein & E. W. Gelfand 1984. Proc Natl. Acad. Sci. USA. 82: 1429–1433. 21 Grinstein, S., A. Rothstein, B. Sarkadi & E. W. Gelfand 1984. Am. J. Physiol. 246: C204–C215. 22 Dise, C. A., D. B. P. Goodman & H. Rasmussen 1980. J. Biol. Chem. 255: 5201–5207. 23 Grinstein, S., S. Cohen, J. D. Goetz & A. Rothstein 1984. Fed. Proc. 44: 2508–2512. 24 Shoyab, M. & G. J. Todaro 1980. Nature 288: 451–455. 25 Kikkawa, Y., W. Takai, Y. Tanaka, R. Miyake & Y. Nishizuka 1983. J. Biol. Chem. 258: 11442–11445. Citing Literature Volume456, Issue1Membrane Transport Driven by Ion GradientsNovember 1985Pages 207-219 ReferencesRelatedInformation