Skip to main content
SpringerLink
Log in

Microfluorimetric imaging study of the mechanism of activation of the Na+/H+ antiport by muscarinic agonist in rat mandibular acinar cells

  • Published:
Pflügers Archiv Aims and scope Submit manuscript

Abstract

The mechanism of regulation of intracellular pH (pHi) in dispersed acini from the rat mandibular salivary gland has been studied with a microfluorimetric imaging method and the pH probe 2′,7′-bis(2-carboxyethyl)-5(and −6)-carboxyfluorescein. The pHi in the TRIS/HEPES-buffered standard solution was 7.29±0.01. Addition of 1 μmol/l acetylcholine (ACh) or ionomycin caused a sustained increase in the pHi. These agents decreased pHi in the absence of external Na+ or in the presence of amiloride. The rate of pHi recovery from an acid load after NH +4 prepulse was a linear function of pHi and increased as pHi became more acidic. Addition of ACh shifted the relationship towards a more alkaline pHi range. The increase in pHi induced by ACh or ionomycin was not inhibited by the protein kinase C inhibitors staurosporine (10 nM) and 1-(5-isoquinolinesulfonyl)-1-methylpiperazine (50 μmol/l). Addition of 0.1–1 μmol/l phorbol 12-myristate 13-acetate (TPA) had little effect on pHi within 10 min; however, exposure to TPA for 120 min resulted in a significant rise in pHi. In Ca2+-free solution with 50 μmol/l 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, the ACh-induced rise in both pHi and cytosolic Ca2+ concentration was suppressed. ACh and ionomycin caused an increment of amiloride-sensitive acid output into the extracellular fluid, while 20 μmol/l 1-oleoyl-2-acetylglycerol had little effect on it. It was concluded that (a) stimulation with ACh activated the Na+/H+ antiport in the plasma membrane, (b) ACh also stimulated the intracellular acid production but acid extrusion by the Na+/H+ antiport prevented the cell from intracellular acidification, and (c) the major route of signal transduction for the ACh-induced activation of the Na+/H+ antiport was independent of protein kinase C but was dependent on the rise in cytosolic Ca2+ concentration. The implication of the cytosolic acidification and cell volume change in pHi regulation is discussed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Arkle S, Michalek R, Templeton D (1987) Effects of C kinase activators and inhibitors on amylase secretion by isolated rat salivary glands. Br J Pharmacol 91:492p

    Google Scholar 

  2. Aronson PS, Nee J, Suhm MA (1982) Modifier role of internal H+ in activating Na+-H+ exchanger in renal microvillus membrane vesicles. Nature 299:161–163

    Google Scholar 

  3. Berridge MJ (1984) Inositol trisphosphate and diacylglycerol as second messengers. Biochem J 220:345–360

    Google Scholar 

  4. Boron WF, DeWeer P (1976) Intracellular pH transients in squid giant axons caused by CO2, NH3 and metabolic inhibitors. J Gen Physiol 67:91–112

    Google Scholar 

  5. Boron WF, Russell JM (1983) Stoichiometry and ion dependencies of the intracellular-pH-regulatory mechanism in squid giant axons. J Gen Physiol 81:373–399

    Google Scholar 

  6. Busa WB (1986) Mechanisms and consequences of pH-mediated cell regulation. Annu Rev Physiol 48:389–402

    Google Scholar 

  7. Chiou CY, Malagodi MH (1975) Studies on the mechanism of action of a new Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride in smooth and skeletal muscles. Br J Pharmacol 53:279–285

    Google Scholar 

  8. Emmer E, Rood RP, Wesolek, JH, Cohen ME, Braithwaik RS, Sharp GWG, Murer H, Donowitz M (1989) Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+/H+ antiporter. J Membr Biol 108:207–215

    Google Scholar 

  9. Foskett JK, Melvin JE (1989) Activation of salivary secretion coupling of cell volume and [Ca2+]i in single cells. Science 30:1582–1585

    Google Scholar 

  10. Graber ML, DiLillo DC, Friedman BL, Pastoriza-Munoz E (1986) Characteristics of fluoroprobes for measuring intracellular pH. Anal Biochem 156:202–212

    Google Scholar 

  11. Green J, Yamaguchi DT, Kleeman CR, Muallem S (1988) Cytosolic pH regulation in osteoblasts. Interaction of Na+ and H+ with the extracellular and intracellular faces of the Na+/H+ exchanger. J Gen Physiol 92:239–261

    Google Scholar 

  12. Grinstein S, Rothstein A (1986) Mechanisms of regulation of the Na+/H+ exchanger. J Membr Biol 90:1–12

    Google Scholar 

  13. Grinstein S, Cohen S (1987) Cytoplasmic [Ca2+] and intracellular pH in lymphocytes. Role of membrane potential and volume-activated Na+/H+ exchange. J Gen Physiol 89:185–213

    Google Scholar 

  14. Grinstein S, Cohen S, Goetz JD, Gelfand EW (1985) Characterization of the activation of Na+/H+ exchange in lymphocytes by phorbol esters: change in cytoplasmic pH dependence of the antiport. Proc Natl Acad Sci USA 82:1429–1433

    Google Scholar 

  15. Grinstein S, Goetz-Smith DJ, Steward D, Beresford B, Mellors A (1986) Protein phosphorylation during activation of Na+/H+ exchange by phorbol esters and by osmotic shrinkage. Possible relation to cell pH and volume regulation. J Biol Chem 261:8009–8016

    Google Scholar 

  16. Grinstein S, Rotin D, Mason MJ (1989) Na+/H+ exchange and growth factor-induced cytosolic pH changes. Role in cellular proliferation. Biochim Biophys Acta 988:73–97

    Google Scholar 

  17. Helmle-Kolb C, Montrose MH, Stange G, Murer H (1990) Regulation of Na+/H+ exchange in opossum kidney cells by parathyroid hormone, cyclic AMP and phorbol esters. Pflügers Arch 415:461–470

    Google Scholar 

  18. Hendey B, Mamrack MD, Putmann RW (1989) Thrombin induces a calcium transient that mediates an activation of the Na+/H+ exchanger in human fibroblasts. J Biol Chem 264:19540–19547

    Google Scholar 

  19. Hesketh TR, Moore JP, Morris JDH, Taylor MV, Rogers J, Smith GA, Metcalfe JC (1985) A common sequence of calcium and pH signals in the mitogenic stimulation of eukaryotic cells. Nature 313:481–484

    Google Scholar 

  20. Kauffmann RF, Taylor RW, Pfeiffer DR (1980) Cation transport and specificity of ionomycin. J Biol Chem 255:2735–2739

    Google Scholar 

  21. Komabayashi T, Yakata A, Izawa T, Suda K, Rubin RP, Tsuboi M (1991) Protein-kinase C-dependent diacylglycerol formation is mediated by Ca2+/calmodulin in parotid cells. Eur J Pharmacol (in press)

  22. Kramhoft B, Lambert IH, Hoffmann EK (1988) Na+/H+ exchange in Ehrlich ascites tumor cells: activation by cytoplasmic acidification and by treatment with cupric sulphate. J Membr Biol 102:35–48

    Google Scholar 

  23. Lau KR, Elliot AC, Brown PD (1989) Acetylcholine causes a transient intracellular acidosis in isolated acini from rabbit mandibular salivary gland. Am J Physiol 256:C288-C295

    Google Scholar 

  24. Mahnensmith RL, Aronson PS (1985) The plasma membrane sodium-hydrogen exchanger and its role in physiological pathophysiological processes. Circ Res 56:773–788

    Google Scholar 

  25. Manganel M, Turner RJ (1989) Agonist-induced activation of Na+/H+ exchange in rat parotid acinar cells. J Membr Biol 111:191–198

    Google Scholar 

  26. Manganel M, Turner RJ (1990) Agonist-induced activation of Na+/H+ exchange in rat parotid acinar cells is dependent on cellular calcium but not on protein kinase C. J Biol Chem 265:4284–4289

    Google Scholar 

  27. Melvin JE, Moran A, Turner RJ (1988) The role of HCO −3 and Na+/H+ exchange in the response of rat parotid acinar cells to muscarinic stimulation. J Biol Chem 263:19564–19569

    Google Scholar 

  28. Mitsuhashi T, Ives HE (1988) Intracellular Ca2+ requirement for activation of the Na+/H+ exchanger in vascular smooth muscle cells. J Biol Chem 263:8790–8795

    Google Scholar 

  29. Moolenaar WH (1986) Effects of growth factors on intracellular pH regulation. Annu Rev Physiol 48:363–376

    Google Scholar 

  30. Moolenaar WH, Tertoolen LGJ, De Laat SW (1984) Phorbol ester and diacylglycerol mimic growth factors in raising cytoplasmic pH. Nature 312:371–374

    Google Scholar 

  31. Nishizuka Y (1984) The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature 308:693–698

    Google Scholar 

  32. Ober SS, Pardee AB (1987) Both protein kinase C and calcium mediate activation of the Na+/H+ antiport in Chinese hamster embryo fibroblasts. J Cell Physiol 132:311–317

    Google Scholar 

  33. Okada M (1990) Evaluation of the role of cytosolic calcium and protein kinase C in the activation of the Na+-H+ antiport induced by cholinergic stimulation in the rat submandibular acinar cells. PhD thesis, Tohoku University

  34. Owen NE, Villereal ML (1982) Evidence for a role of calmodulin in stimulation of Na+ influx in human fibroblasts. Proc Natl Acad Sci USA 79:3537–3541

    Google Scholar 

  35. Ozawa T, Saito Y, Nishiyama A (1988) Evidence for an anion exchanger in the mouse lacrimal gland acinar cell membrane. J Membr Biol 105:273–280

    Google Scholar 

  36. Parod RJ, Putney JW Jr (1980) Stimulus-permeability coupling in rat lacrimal gland. Am J Physiol 239:G106-G113

    Google Scholar 

  37. Pirani D, Evans LAR, Cook DI, Young JA (1987) Intracellular pH in the rat mandibular salivary gland: the role of Na-H and Cl-HCO3 antiports in secretion. Pflügers Arch 408:178–184

    Google Scholar 

  38. Putney JW, McKinney JS, Aub DL, Leslie BA (1984) Phorbol ester-induced protein secretion in rat parotid gland. Relationship to the roll of inositol lipid breakdown and protein kinase C activation in stimulus-secretion coupling. Mol Pharmacol 26:261–266

    Google Scholar 

  39. Reuss L, Petersen K (1985) Cyclic AMP inhibits Na+/H+ exchange at the apical membrane of Necturus gallbladder epithelium. J Gen Physiol 85:409–429

    Google Scholar 

  40. Roos A, Boron WF (1981) Intracellular pH. Physiol Rev 61:296–434

    Google Scholar 

  41. Saito Y, Ozawa T, Nishiyama A (1987) Acetylcholine-induced Na+ influx in the mouse lacrimal gland acinar cells. Demonstration of multiple Na+ transport mechanisms by intracellular Na+ activity measurements. J Membr Biol 98:135–144

    Google Scholar 

  42. Saito Y, Ozawa T, Suzuki S, Nishiyama A (1987) Regulation of intracellular pH of the mouse lacrimal gland acinar cells. J Physiol Soc Jpn 49:345

    Google Scholar 

  43. Saito Y, Ozawa T, Nishiyama A (1990) Effect of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells. Pflügers Arch 417:382–390

    Google Scholar 

  44. Sergent S, Sohn DH, Kim HD (1989) Volume activated Na/H exchange activity in fetal and adult pig cells: inhibition by cyclic AMP. J Membr Biol 109:209–220

    Google Scholar 

  45. Sklar LA, Oades ZG, Jesaitis AJ, Painter RG, Cockrane CG (1981) Fluoresceinated chemotactic peptides and high-affintiy antifluorescein antibody as a probe of the temporal characteristics of neutrophil stimulation. Proc Natl Acad Sci USA 78:7540–7544

    Google Scholar 

  46. Soltoff SP, McMillan MK, Cantley LC, Cragoe EJ Jr, Talamo BR (1989) Effects of muscarinic, alpha-adrenergic and substance P agonists and ionomycin on ion transport mechanisms in the rat parotid acinar cells. The dependence of ion transport on intracellular calcium. J Gen Physiol 93:285–319

    Google Scholar 

  47. Thomas JA, Buchsbaum RN, Zimniak A, Racker E (1979) Intracellular pH measurements in Ehrlich-ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18:2210–2218

    Google Scholar 

  48. Wall SM, Kraut JA, Muallem S (1988) Modulation of Na+-H+ exchange activity by intracellular Na+, H+ and Li+ in IMCD cells. Am J Physiol 255:F331-F339

    Google Scholar 

  49. Weinmann EJ, Shenolikar S (1986) Protein kinase C activates the renal apical membrane Na+/H+ exchanger. J Membr Biol 93:133–139

    Google Scholar 

  50. Young JA, Cook DI, Lennep EW van, Roberts M (1987) Secretion by the major salivary glands. In: Johnson R (ed) Physiology of the gastrointestinal tract, 2nd edn. Raven, New York, pp 773–815

    Google Scholar 

Download references

Author information

Author notes

  1. M. Okada

    Present address: Department of Pediatrics, Tohoku University School of Medicine, 1-1 Seiryo-cho, 980, Sendai, Japan

Authors and Affiliations

  1. Department of Physiology, Tohoku University School of Medicine, Seiryo-cho, 980, Sendai, Japan

    M. Okada, Y. Saito, E. Sawada & A. Nishiyama

Authors
  1. M. Okada
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Y. Saito
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. E. Sawada
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. Nishiyama
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Okada, M., Saito, Y., Sawada, E. et al. Microfluorimetric imaging study of the mechanism of activation of the Na+/H+ antiport by muscarinic agonist in rat mandibular acinar cells. Pflügers Arch. 419, 338–348 (1991). https://doi.org/10.1007/BF00371116

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00371116

Key words

Search

Navigation