Cross talk between serine/threonine and tyrosine kinases regulates ADP-induced thromboxane generation in platelets

SummaryADP-induced thromboxane generation depends on Src family kinases (SFKs) and is enhanced with pan-protein kinase C (PKC) inhibitors, but it is not clear how these two events are linked. The aim of the current study is to investigate the role of Y311 phosphorylated PKCδ in regulating ADP-induced platelet activation. In the current study, we employed various inhibitors and murine platelets from mice deficient in specific molecules to evaluate the role of PKCδ in ADP-induced platelet responses. We show that, upon stimulation of platelets with 2MeSADP, Y311 on PKCδ is phosphorylated in a P2Y1/Gq and Lyn-dependent manner. By using PKCδ and Lyn knockout murine platelets, we also show that tyrosine phosphorylated PKCδ plays a functional role in mediating 2MeSADP-induced thromboxane generation. 2MeSADP-induced PKCδ Y311 phosphorylation and thromboxane generation were potentiated in human platelets pre-treated with either a pan-PKC inhibitor, GF109203X or a PKC α/β inhibitor and in PKC α or β knockout murine platelets compared to controls. Furthermore, we show that PKC α/β inhibition potentiates the activity of SFK, which further hyper-phosphorylates PKCδ and potentiates thromboxane generation. These results show for the first time that tyrosine phosphorylated PKCδ regulates ADP-induced thromboxane generation independent of its catalytic activity and that classical PKC isoforms α/β regulate the tyrosine phosphorylation on PKCδ and subsequent thromboxane generation through tyrosine kinase, Lyn, in platelets.

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Thrombin Mediated Tyrosine Phosphorylation of PKCδ in Human Platelets Occurs in a Calcium- and Src Family Tyrosine Kinase- Dependent Manner.

Blood ◽

10.1182/blood.v104.11.3541.3541 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3541-3541

Author(s):

Swaminathan Murugappan ◽

Haripriya Shankar ◽

Satya Kunapuli

Keyword(s):

Tyrosine Kinase ◽

Intracellular Calcium ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Kinase Inhibitors ◽

Human Platelets ◽

Dependent Manner ◽

Protein Signaling ◽

Glycoprotein Vi ◽

Pkc Isoforms

Abstract Protein kinase C (PKC)-δ is a novel PKC that has been shown to be tyrosine phosphorylated upon stimulation with agonists in platelets. Tyrosine phosphorylation of PKCδ has been shown to occur in a Fyn-dependent manner downstream of glycoprotein VI (GPVI) signaling in platelets. Although thrombin causes tyrosine phosphorylation of PKCδ in platelets, the mechanism of this event is not elucidated. In this study, we investigated whether G-protein signaling pathways utilize similar pathways as GPVI in tyrosine phosphorylation of PKCδ. Protease activated receptor (PAR) -1 selective peptide, SFLLRN and PAR - 4 selective peptide, AYPGKF caused a time- and concentration-dependent increase in tyrosine phosphorylation of PKCδ in human platelets. However, AYPGKF failed to cause tyrosine phosphorylation of PKCδ in Gq-deficient mouse platelets. Both U73122, a phospholipase C (PLC) inhibitor, and dimethyl-BAPTA, an intracellular calcium chelator, inhibited the tyrosine phosphorylation of PKCδ downstream of the PAR activation suggesting a role for Gq/PLC pathways and intracellular calcium in mediating this event. Inhibition of PKC isoforms using GF109203X potentiated the tyrosine phosphorylation of PKCδ. The Src family tyrosine kinase inhibitors, PP1 and PP2 inhibited the tyrosine phosphorylation of PKCδ suggesting a role for Src family tyrosine kinase members in this event. We also found that both Lyn and Src are physically associated with PKCδ in a constitutive manner in platelets. Finally we found that there was a time-dependent activation of Src following activation of platelets with thrombin. Thus, the precomplexed Src and Lyn tyrosine kinases get activated following PAR stimulation resulting in the tyrosine phosphorylation of PKCδ. All these data indicate that tyrosine phosphorylation of PKCδ downstream of thrombin occurs in a calcium- and Src-family kinase dependent manner in human platelets.

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1,25-Dihydroxyvitamin D3 and TPA activate phospholipase D in Caco-2 cells: role of PKC-α

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1999.276.4.g993 ◽

1999 ◽

Vol 276 (4) ◽

pp. G993-G1004 ◽

Cited By ~ 7

Author(s):

Sharad Khare ◽

Marc Bissonnette ◽

Beth Scaglione-Sewell ◽

Ramesh K. Wali ◽

Michael D. Sitrin ◽

...

Keyword(s):

Phospholipase D ◽

Dihydroxyvitamin D3 ◽

Dihydroxyvitamin D ◽

1,25 Dihydroxyvitamin D ◽

Kinase C ◽

Pkc Isoforms ◽

Gf 109203X ◽

Pkc Β ◽

Stimulation Of

1,25-Dihydroxyvitamin D3[1,25(OH)2D3] and 12- O-tetradecanoylphorbol 13-acetate (TPA) both activated phospholipase D (PLD) in Caco-2 cells. GF-109203x, an inhibitor of protein kinase C (PKC) isoforms, inhibited this activation by both of these agonists. 1,25(OH)2D3activated PKC-α, but not PKC-β1, -βII, -δ, or -ζ, whereas TPA activated PKC-α, -β1, and -δ. Chronic treatment with TPA (1 μM, 24 h) significantly reduced the expression of PKC-α, -βI, and -δ and markedly reduced the ability of 1,25(OH)2D3or TPA to acutely stimulate PLD. Removal of Ca2+ from the medium, as well as preincubation of cells with Gö-6976, an inhibitor of Ca2+-dependent PKC isoforms, significantly reduced the stimulation of PLD by 1,25(OH)2D3or TPA. Treatment with 12-deoxyphorbol-13-phenylacetate-20-acetate, which specifically activates PKC-βI and -βII, however, failed to stimulate PLD. In addition, the activation of PLD by 1,25(OH)2D3or TPA was markedly reduced or accentuated in stably transfected cells with inhibited or amplified PKC-α expression, respectively. Taken together, these observations indicate that PKC-α is intimately involved in the stimulation of PLD in Caco-2 cells by 1,25(OH)2D3or TPA.

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PROTEIN KINASE C CONTROLS CA2+ MOBILIZATION IN HUMAN PLATELETS

10.1055/s-0038-1644509 ◽

1987 ◽

Cited By ~ 1

Author(s):

W Siffert ◽

P Scheid ◽

JW N Akkerman

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Human Platelets ◽

Cytosolic Ph ◽

Fluorescent Indicators ◽

Kinase C ◽

Deutsche Forschungsgemeinschaft ◽

Ionophore Monensin ◽

Stimulation Of

Platelet stimulation has been shown to result in a rise of cytosolic pH (pHi) as a result of an activation of a Na+/H+ antiport. We have investigated the role of pH in Ca2+ mobilization in human platelets. pHi and free Ca2+, {Ca2+)i, were measured in platelets loaded with the fluorescent indicators BCECF and quin2, respectively. Stimulation of platelets by either thrombin or OAG, an activator of protein kinase C (Pk-C), increased pHi. Pretreatment of platelets with inhibitors of Pk-C, trifluoperazine (TFP) or sphingosine (SPH), blocked the stimulus-induced rise in pHi, suggesting a role of Pk-C in the activation of Na+/H+ exchange. Blocking Na+/H+ exchange by an amiloride analogue or by TFP similarly suppressed the thrombin-induced increase in {Ca2*}i. This effect could be prevented by increasing pHi with the Na+/H+ ionophore monensin or with NH4Cl. The thrombin-induced (0.05 U/ml) rise in {Ca2+}i was more than 3-fold enhanced when the pH was raised from 6.8 to 7.4.Our results demonstrate that pHi controls Ca2+ mobilization in human platelets and suggest that Pk-C contributes to this control by activating the Na+/H+ exchanger.Supported by the Deutsche Forschungsgemeinschaft. No Sche 46/5-2.

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Genetic and Pharmacological Analyses of Involvement of Src-family, Syk and Btk Tyrosine Kinases in Platelet Shape Change

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615689 ◽

2001 ◽

Vol 85 (02) ◽

pp. 331-340 ◽

Cited By ~ 20

Author(s):

Markus Bauer ◽

Petra Maschberger ◽

Lynn Quek ◽

Stephen Briddon ◽

Debabrata Dash ◽

...

Keyword(s):

Tyrosine Kinases ◽

Shape Change ◽

Thromboxane A2 ◽

Human Platelets ◽

Src Family Kinases ◽

G Protein Coupled Receptors ◽

Protein Tyrosine Phosphorylation ◽

G Protein Coupled ◽

Platelet Shape ◽

Stimulation Of

SummaryPlatelet shape change was found to be associated with an increase in protein tyrosine phosphorylation upon stimulation of thrombin-, ADPand thromboxane A2-G-protein coupled receptors in human platelets and thromboxane A2 receptors in mouse platelets. By using PP1 and PD173956, two structurally unrelated specific inhibitors of Src-family tyrosine kinases, and mouse platelets deficient in the Src-kinase Fyn or Lyn, we show that Src-family kinases cause the increase in protein tyrosine phosphorylation. We further detected that the non-Src tyrosine kinase Syk was activated during shape change in a manner dependent on Src-family kinaseactivation. The pharmacological experiments and the studies on Fyn-, Lyn- and Syk-deficient mouse platelets showed that neither Src-family kinases nor Syk are functionally involved in shape change. Also human platelets deficient of the tyrosine kinase Btk showed a normal shape change. Binding of PAC-1 that recognizes activated integrin αIIb β3 complexes on the platelet surface was enhanced during shape change and blocked by inhibition of Src-kinases. We conclude that the activation of Src-kinases and the subsequent Syk stimulation upon activation of G-protein coupled receptors are not involved in the cytoskeletal changes underlying shape change of human and mouse platelets, but that the stimulation of this evolutionary conserved pathway leads to integrin αIIb β3 exposure during shape change.

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Inhibitory role of Src family tyrosine kinases on Ca2+-dependent insulin release

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00103.2006 ◽

2007 ◽

Vol 292 (3) ◽

pp. E845-E852 ◽

Cited By ~ 12

Author(s):

Haiying Cheng ◽

Susanne G. Straub ◽

Geoffrey W. G. Sharp

Keyword(s):

Insulin Secretion ◽

Tyrosine Kinases ◽

Neurotransmitter Release ◽

Src Family Kinases ◽

Dependent Manner ◽

Inhibitory Role ◽

Intracellular Ca ◽

Src Family Tyrosine Kinases ◽

Sites Of Action

Both neurotransmitter release and insulin secretion occur via regulated exocytosis and share a variety of similar regulatory mechanisms. It has been suggested that Src family tyrosine kinases inhibit neurotransmitter release from neuronal cells (H. Ohnishi, S. Yamamori, K. Ono, K. Aoyagi, S. Kondo, and M. Takahashi. Proc Natl Acad Sci USA 98: 10930–10935, 2001). Thus the potential role of Src family kinases in the regulation of insulin secretion was investigated in this study. Two structurally different inhibitors of Src family kinases, SU-6656 and PP2, but not the inactive compound, PP3, enhanced Ca2+-induced insulin secretion in both rat pancreatic islets and INS-1 cells in a concentration-dependent and time-dependent manner. Furthermore, Src family kinase-mediated insulin secretion appears to be dependent on elevated intracellular Ca2+ and independent of glucose metabolism, the ATP-dependent K+ channel, adenylyl cyclase, classical PKC isoforms, extracellular signal-regulated kinase 1/2, and insulin synthesis. The sites of action for Src family kinases seem to be distal to the elevation of intracellular Ca2+ level. These results indicate that one or more Src family tyrosine kinases exert a tonic inhibitory role on Ca2+-dependent insulin secretion.

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Classical PKCs Regulate ADP-Induced Thromboxane Generation by Modulating Tyrosine Phosphorylation On Novel PKC Isoform Delta Through Shptp-1

Blood ◽

10.1182/blood.v120.21.1064.1064 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1064-1064

Author(s):

Dheeraj Bhavanasi ◽

Carol T Dangelmaier ◽

Jin Jianguo ◽

Soochong Kim ◽

Satya P. Kunapuli

Keyword(s):

Tyrosine Phosphorylation ◽

Conflicts Of Interest ◽

Tyrosine Phosphatase ◽

Src Family Kinases ◽

Dependent Manner ◽

Functional Responses ◽

Pkc Isoforms ◽

Adp Receptors ◽

Gf 109203X

Abstract Abstract 1064 Introduction: Adenosine Di-phosphate (ADP) is stored in dense granules of platelets and is released upon platelet activation acting as a feedback activator by binding to G-protein coupled P2Y1 and P2Y12 receptors. ADP stimulation causes platelets to change shape, aggregate, release dense and a-granule contents and synthesize thromboxane A2 that can further act as a feedback activator potentiating platelet responses by binding to thromboxane receptor (TP). Protein kinase C is a serine threonine specific kinase that regulates multiple platelet functional responses. Specific PKC isoforms regulating platelet responses downstream of ADP receptors are not completely known. Aim: The aim of the current study is to elucidate the role of PKC isoforms in regulating ADP-induced platelet functional responses in platelets. Methods: We sought to delineate the mechanism of ADP-induced platelet responses by performing platelet aggregation (aggregometry), ATP secretion (luciferin-luciferase reaction) and thromboxane generation (ELISA kit measuring TxB2) in human or murine platelets by pre-incubating the platelets with control (DMSO) or inhibitors wherever mentioned. We also evaluated the role of PKCd to ADP-induced platelet responses by using murine platelets lacking PKCd. Background and Results: Murugappan et al have shown that PKCd was not activated downstream of ADP receptors based on the inability of ADP to cause threonine 507 phosphorylation on PKCd in platelets. However, studies from other labs have shown that PKCd can be activated when it is phosphorylated on its tyrosine residues. In the current study we show that, upon stimulation with 2MeSADP, PKCd is phosphorylated on tyrosine residue 311 in a time-dependent manner in platelets (Fig A). Also, ADP-induced thromboxane generation (Fig B) and ADP-induced thromboxane-mediated dense granule secretion were significantly inhibited in PKCd knockout murine platelets compared to those of wild type platelets. Similarly, thromboxane generation downstream of ADP receptors in human platelets pre-incubated with a PKCd inhibitor is significantly inhibited compared to control indicating a role for PKCd in mediating ADP-induced responses in platelets. Bynagari et al have shown that ADP-induced thromboxane generation is potentiated in the presence of the pan-PKC inhibitor, GF 109203X and the isoform regulating this effect is PKCe. We observed that pre-incubation of PKCe knockout murine platelets with GF 109203X further potentiated ADP-induced thromboxane generation suggesting that there are other PKC isoforms negatively regulating ADP-induced thromboxane generation. We show that this potentiating effect of thromboxane generation with GF 109203X in WT or PKCe KO murine platelets correlate with an increase in the phosphorylation of Y311 on PKCd (Fig C) suggesting that ADP-induced thromboxane generation is regulated through PKCd Y311 phosphorylation. Tyrosine phosphorylation on PKCd is mediated by Src family kinases (SFKs) as the phosphorylation is abolished with PP2, a SFK inhibitor and is only partially inhibited in Fyn knockout murine platelets suggesting that other SFKs also mediate this tyrosine phosphorylation. Surprisingly, pre-incubation of platelets with LY-333531, a classical PKC isoform (a/b) inhibitor potentiated PKCd Y311 phosphorylation (Fig D) as well as thromboxane generation (Fig E) downstream of ADP receptors suggesting a role for classical PKCs. Also, platelets pre-incubated with LY-333531 showed a decrease in the phosphorylation of SHPTP-1 (Fig F), a tyrosine phosphatase, rendering it active. The active SHPTP-1 phosphatase may dephosphorylate and activate SFKs, which can now phosphorylate PKCd on Y311 in platelets. Conclusions: In the current study, we report for the first time that the novel PKC isoform d is tyrosine phosphorylated downstream of ADP receptors through which it mediates ADP-induced thromboxane generation. We also show a novel role for classical PKC isoforms a/b in regulating tyrosine phosphorylation on novel isoform, PKCd possibly through the tyrosine phosphatase SHPTP-1 and Src family kinases in platelets. Disclosures: No relevant conflicts of interest to declare.

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Mastoparan promotes exocytosis and increases intracellular cyclic AMP in human platelets. Evidence for the existence of a Ge-like mechanism of secretion

Biochemical Journal ◽

10.1042/bj2810465 ◽

1992 ◽

Vol 281 (2) ◽

pp. 465-472 ◽

Cited By ~ 29

Author(s):

C P D Wheeler-Jones ◽

T Saermark ◽

V V Kakkar ◽

K S Authi

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cyclic Amp ◽

G Protein ◽

Human Platelets ◽

Dependent Manner ◽

Kinase C ◽

Reversible Electroporation ◽

Dose Dependent ◽

Stimulation Of

Recent studies have shown that mastoparan, an amphiphilic peptide derived from wasp venom, accelerates guanine nucleotide exchange and GTPase activity of purified GTP-binding proteins. In the present study we have examined the functional consequences of exposure of intact human platelets to mastoparan. Mastoparan promoted rapid (less than or equal to 1 min) dose-dependent increases in 5-hydroxy[14C]tryptamine and beta-thromboglobulin release from dense-granule and alpha-granule populations respectively. The exocytotic response did not result from a lytic effect of mastoparan and occurred in the complete absence of platelet shape change and aggregation. Liberation of [3H]arachidonate and increases in cytosolic [Ca2+] (detected with fura 2) were not observed in platelets stimulated with mastoparan. Similarly, in platelets preloaded with [3H]inositol during reversible electroporation, mastoparan did not cause the accumulation of [3H]inositol phosphates. Mastoparan-induced secretion was unaffected by preincubation with either the protein kinase C inhibitor staurosporine (10 nM-10 microM) or prostacyclin (PGI2; 100 ng/ml) and was not accompanied by phosphorylation of the 45 kDa protein kinase C substrate or the 20 kDa protein normally associated with platelet activation. The G-protein inhibitor guanosine 5′-[beta-thio]diphosphate (GDP[S]; 1 mM) attenuated the secretion induced by mastoparan in both intact and saponin-permeabilized platelets. Encapsulation of GDP[S] during reversible permeabilization inhibited mastoparan-induced secretion, providing evidence for an intracellular action of GDP[S]. In all these studies thrombin (0.05-0.2 unit/ml) elicited characteristic responses, and thrombin-induced secretion was inhibited by staurosporine, PGI2 and GDP[S]. Mastoparan also increased intra-platelet cyclic AMP in a dose-dependent manner. Mastoparan and PGI2 increased 32P incorporation into a protein of approx. 24 kDa, whereas phosphorylation of a 50 kDa substrate was only seen in PGI2-stimulated platelets. These results indicate that mastoparan promotes secretion by a mechanism which does not involve stimulation of phospholipase C and suggest that the secretory event may result either from a direct fusogenic action of mastoparan and/or from stimulation of the putative exocytosis-linked G-protein, Ge.

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Secreted KIAA1199 promotes the progression of rheumatoid arthritis by mediating hyaluronic acid degradation in an ANXA1-dependent manner

Cell Death and Disease ◽

10.1038/s41419-021-03393-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Wei Zhang ◽

Guoyu Yin ◽

Heping Zhao ◽

Hanzhi Ling ◽

Zhen Xie ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Hyaluronic Acid ◽

Dependent Manner ◽

Collagen Induced Arthritis ◽

Intracellular Degradation ◽

Main Pathway ◽

First Time

AbstractIn inflamed joints, enhanced hyaluronic acid (HA) degradation is closely related to the pathogenesis of rheumatoid arthritis (RA). KIAA1199 has been identified as a hyaladherin that mediates the intracellular degradation of HA, but its extracellular function remains unclear. In this study, we found that the serum and synovial levels of secreted KIAA1199 (sKIAA1199) and low-molecular-weight HA (LMW-HA, MW < 100 kDa) in RA patients were significantly increased, and the positive correlation between them was shown for the first time. Of note, treatment with anti-KIAA1199 mAb effectively alleviated the severity of arthritis and reduced serum LMW-HA levels and cytokine secretion in collagen-induced arthritis (CIA) mice. In vitro, sKIAA1199 was shown to mediate exogenous HA degradation by attaching to the cell membrane of RA fibroblast-like synoviosytes (RA FLS). Furthermore, the HA-degrading activity of sKIAA1199 depended largely on its adhesion to the membrane, which was achieved by its G8 domain binding to ANXA1. In vivo, kiaa1199-KO mice exhibited greater resistance to collagen-induced arthritis. Interestingly, this resistance could be partially reversed by intra-articular injection of vectors encoding full-length KIAA1199 instead of G8-deleted KIAA119 mutant, which further confirmed the indispensable role of G8 domain in KIAA1199 involvement in RA pathological processes. Mechanically, the activation of NF-κB by interleukin-6 (IL-6) through PI3K/Akt signaling is suggested to be the main pathway to induce KIAA1199 expression in RA FLS. In conclusion, our study supported the contribution of sKIAA1199 to RA pathogenesis, providing a new therapeutic target for RA by blocking sKIAA1199-mediated HA degradation.

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The zona incerta modulates spontaneous spike-wave discharges in the rat

Journal of Neurophysiology ◽

10.1152/jn.00750.2011 ◽

2013 ◽

Vol 109 (10) ◽

pp. 2505-2516 ◽

Cited By ~ 8

Author(s):

Fu-Zen Shaw ◽

Yi-Fang Liao ◽

Ruei-Feng Chen ◽

Yu-Hsing Huang ◽

Rick C. S. Lin

Keyword(s):

Functional Role ◽

Significant Inhibition ◽

Zona Incerta ◽

Obvious Effect ◽

Spike Wave Discharges ◽

Long Evans ◽

Oscillation Frequencies ◽

Spike Wave ◽

Stimulation Of

The contribution of the zona incerta (ZI) of the thalamus on spike-wave discharges (SWDs) was investigated. Chronic recordings of bilateral cortices, bilateral vibrissa muscle, and unilateral ZI were performed in Long-Evans rats to examine the functional role of SWDs. Rhythmic ZI activity appeared at the beginning of SWD and was accompanied by higher-oscillation frequencies and larger spike magnitudes. Bilateral lidocaine injections into the mystacial pads led to a decreased oscillation frequency of SWDs, but the phenomenon of ZI-related spike magnitude enhancement was preserved. Moreover, 800-Hz ZI microstimulation terminates most of the SWDs and whisker twitching (WT; >80%). In contrast, 200-Hz ZI microstimulation selectively stops WTs but not SWDs. Stimulation of the thalamic ventroposteriomedial nucleus showed no obvious effect on terminating SWDs. A unilateral ZI lesion resulted in a significant reduction of 7- to 12-Hz power of both the ipsilateral cortical and contralateral vibrissae muscle activities during SWDs. Intraincertal microinfusion of muscimol showed a significant inhibition on SWDs. Our present data suggest that the ZI actively modulates the SWD magnitude and WT behavior.

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Cytoplasmic [Ca2+] and intracellular pH in lymphocytes. Role of membrane potential and volume-activated Na+/H+ exchange.

The Journal of General Physiology ◽

10.1085/jgp.89.2.185 ◽

1987 ◽

Vol 89 (2) ◽

pp. 185-213 ◽

Cited By ~ 70

Author(s):

S Grinstein ◽

S Cohen

Keyword(s):

Protein Kinase C ◽

Membrane Potential ◽

Protein Kinase ◽

Intracellular Ph ◽

Enzyme Treatment ◽

Membrane Hyperpolarization ◽

Kinase C ◽

Free Media ◽

Stimulation Of

The effect of elevating cytoplasmic Ca2+ [( Ca2+]i) on the intracellular pH (pHi) of thymic lymphocytes was investigated. In Na+-containing media, treatment of the cells with ionomycin, a divalent cation ionophore, induced a moderate cytoplasmic alkalinization. In the presence of amiloride or in Na+-free media, an acidification was observed. This acidification is at least partly due to H+ (equivalent) uptake in response to membrane hyperpolarization since: it was enhanced by pretreatment with conductive protonophores, it could be mimicked by valinomycin, and it was decreased by depolarization with K+ or gramicidin. In addition, activation of metabolic H+ production also contributes to the acidification. The alkalinization is due to Na+/H+ exchange inasmuch as it is Na+ dependent, amiloride sensitive, and accompanied by H+ efflux and net Na+ gain. A shift in the pHi dependence underlies the activation of the antiport. The effect of [Ca2+]i on Na+/H+ exchange was not associated with redistribution of protein kinase C and was also observed in cells previously depleted of this enzyme. Treatment with ionomycin induced significant cell shrinking. Prevention of shrinking largely eliminated the activation of the antiport. Moreover, a comparable shrinking produced by hypertonic media also activated the antiport. It is concluded that stimulation of Na+/H+ exchange by elevation of [Ca2+]i is due, at least in part, to cell shrinking and does not require stimulation of protein kinase C.

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