Increase In pHi Research Articles

Inhibitory mechanisms of low concentrations of oxidized low-density lipoprotein on platelet aggregation

The intracellular mechanisms underlying oxidized low-density lipoprotein (oxLDL)-signaling pathways in platelets are not yet completely understood. Therefore, the aim of this study was to further examine the effects of oxLDL in prevention of platelet aggregation. In this study, oxLDL concentration-dependently (40-120 microg/ml) inhibited platelet aggregation in human platelet-rich plasma stimulated by agonists. Moreover, oxLDL (40 and 80 microg/ml) markedly decreased the fluorescence intensity of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation of a protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (150 nM). This phosphorylation was markedly inhibited by oxLDL (40 and 80 microg/ml) in phosphorus-32-labeled platelets. In addition, oxLDL (40 and 80 microg/ml) markedly increased levels of cyclic AMP and cyclic AMP-induced vasodilator-stimulated phosphoprotein (VASP) Ser(157) phosphorylation. The thrombin-evoked increase in pHi was inhibited in the presence of oxLDL (40 and 80 microg/ml). These results indicate that the antiplatelet activity of oxLDL may involve the following pathways. (1) oxLDL may initially induce conformational changes in platelet membranes, leading to inhibition of the activation of protein kinase C, followed by inhibition of P47 protein phosphorylation, and intracellular Ca(2+) mobilization. (2) oxLDL also activated formation of cyclic AMP and cyclic AMP-induced VASP Ser(157) phosphorylation, resulting in inhibition of the Na(+)/H(+)exchanger; this leads to reduced intracellular Ca(2+) mobilization, and ultimately to inhibition of platelet aggregation. This study further provides new insights concerning the effects of low concentrations of oxLDL on platelet aggregation.

The Role of Wall Ca2+ in the Regulation of Wall Extensibility During the Acid-induced Extension of Soybean Hypocotyl Cell Walls

We examined the acid-facilitated yielding properties of cell walls of soybean hypocotyls and the effects of Ca(2+) upon the properties by stress-strain analyses using glycerinated hollow cylinders (GHCs) from the elongating regions of the hypocotyls. Stress-extension rate curves of native GHCs showed characteristic changes with pH, all indicating the existence of yield threshold tension (y) as well as wall extensibility (phi), i.e. a downward shift of y and an increase in phi with wall acidification. The acid-induced downward shift of y was inhibited by boiling of GHCs. In contrast, a considerable increase in phi with acidification remained even after boiling. This indicates that phi consists of two components, i.e. heat-sensitive and heat-resistant, both being pH sensitive. A Ca(2+) chelator (Quin 2) dramatically increased phi at a neutral pH. Subsequent addition of Ca(2+) or ruthenium red suppressed the chelator-induced increase in phi. These findings suggest that wall Ca(2+) plays an important role in the regulation of wall extensibility during the acid-induced wall extension by reacting with carboxyl groups of wall pectin.

Functional interaction of carbonic anhydrase and chloride/bicarbonate exchange in human platelets

Recently, our laboratory has reported the presence of one acidifying Cl−/HC exchange mechanism in human platelets. This paper demonstrates that this exchanger decreases its activity after inhibition of carbonic anhydrase. BCECF-loaded platelets, previously equilibrated in a bicarbonate/CO2 buffered solution, were resuspended in a Hepes-buffered, chloride-free (glucuronate) medium to produce a pHi increase. After addition of 50 mM NaCl, pHi fell rapidly reaching steady state in the succeeding 400 s. The recovery in chloride-containing solution was in contrast to the effect of a similar change in osmolarity by addition of 50 mM sodium glucuronate that produced a significantly slower variation of pHi. Alkali loads produced by 25 mM TMA were also counteracted by HC equivalent efflux via Cl−/HC exchange. The present study shows that the efflux of HC was slower when the platelets were previously incubated in 100 µM methazolamide. As a conclusion, the recovery of pHi from alkalosis by Na-independent Cl−/HC exchange is facilitated in platelets by the enzymatic activity of the carbonic anhydrase.

The polymerization of actin: Extent of polymerization under pressure, volume change of polymerization, and relaxation after temperature jumps

The protein actin can polymerize from monomeric globular G-actin to polymeric filamentary F-actin, under the regulation of thermodynamic variables such as temperature, pressure, and compositions of G-actin and salts. We present here new measurements of the extent of polymerization (phi) of actin under pressure (P), for rabbit skeletal muscle actin in H2O buffer in the presence of adenosine triposphate and calcium ions and at low (5-15 mM) KCl concentrations. We measured phi using pyrene-labeled actin, as a function of time (t) and temperature (T), for samples of fixed concentrations of initial G-actin and KCl and at fixed pressure. The phi(T,P) measurements at equilibrium have the same form as reported previously at 1 atm: low levels of polymerization at low temperatures, representing dimerization of the actin; an increase in phi at the polymerization temperature (Tp); a maximum in phi(T) above Tp) with a decrease in phi(T) beyond the maximum, indicating a depolymerization at higher T. From phi(T,P) at temperatures below Tp, we estimate the change in volume for the dimerization of actin, DeltaVdim, to be -307+/-10 ml/mol at 279 K. The change of Tp with pressure dTp/dP=(0.3015+/-0.0009) K/MPa=(30.15+/-0.09) mK/atm. The phi(T,P) data at higher T indicate the change in volume on propagation, DeltaVprop, to be +401+/-48 ml/mol at 301 K. The phi(t) measurements yield initial relaxation times rp(T) that reflect the behavior of phi(T) and support the presence of a depolymerization temperature. We also measured the density of polymerizing actin with a vibrating tube density meter, the results of which confirm that the data from this instrument are affected by viscosity changes and can be erroneous.

Acute Insult of Ammonia Leads to Calcium-dependent Glutamate Release from Cultured Astrocytes, an Effect of pH

Hyperammonemia is a key factor in the pathogenesis of hepatic encephalopathy (HE) as well as other metabolic encephalopathies, such as those associated with inherited disorders of urea cycle enzymes and in Reye's syndrome. Acute HE results in increased brain ammonia (up to 5 mM), astrocytic swelling, and altered glutamatergic function. In the present study, using fluorescence imaging techniques, acute exposure (10 min) of ammonia (NH4+/NH3) to cultured astrocytes resulted in a concentration-dependent, transient increase in [Ca2+]i. This calcium transient was due to release from intracellular calcium stores, since the response was thapsigargin-sensitive and was still observed in calcium-free buffer. Using an enzyme-linked fluorescence assay, glutamate release was measured indirectly via the production of NADH (a naturally fluorescent product when excited with UV light). NH4+/NH3 (5 mM) stimulated a calcium-dependent glutamate release from cultured astrocytes, which was inhibited after preincubation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester but unaffected after preincubation with glutamate transport inhibitors dihydrokainate and DL-threo-beta-benzyloxyaspartate. NH4+/NH3 (5 mM) also induced a transient intracellular alkaline shift. To investigate whether the effects of NH4+/NH3 were mediated by an increase in pH(i), we applied trimethylamine (TMA+/TMA) as another weak base. TMA+/TMA (5 mM) induced a similar transient increase in both pH(i) and [Ca2+]i (mobilization from intracellular calcium stores) and resulted in calcium-dependent release of glutamate. These results indicate that an acute exposure to ammonia, resulting in cytosolic alkalinization, leads to calcium-dependent glutamate release from astrocytes. A deregulation of glutamate release from astrocytes by ammonia could contribute to glutamate dysfunction consistently observed in acute HE.

SLC26A7 Is a Cl– Channel Regulated by Intracellular pH

Members of the SLC26 transporter family play an essential role in several epithelial functions, as revealed by diseases associated with mutations in members of the family. Several members were shown to function as Cl(-) and HCO(3)(-) transporters that likely play an important role in epithelial Cl(-) absorption and HCO(3)(-) secretion. However, the mechanism of most transporters is not well understood. SLC26A7 is a member of the SLC26 transporter family reported to be expressed in the basolateral membrane of the cortical collecting duct and parietal cells and functions as a coupled Cl(-)/HCO(3)(-) exchanger. In the present work we examined the transport properties of SLC26A7 to determine its transport characteristics and electrogenicity. We found that when expressed in Xenopus oocytes or HEK293 cells SLC26A7 functions as a pH(i)-regulated Cl(-) channel with minimal OH(-)/HCO(3)(-) permeability. Expression of SLC26A7 in oocytes or HEK293 cells generated a Cl(-) current with linear I/V and an instantaneous current that was voltage- and time-independent. Based on measurement of reversal potential the selectivity of SLC26A7 is NO(3)(-)>>Cl(-)=Br(-)=I(-)>SO(4)(2-)=Glu(-), although I(-) partially inhibited the current. Incubating the cells with HCO(3)(-) or butyrate acidified the cytosol and increased the selectivity of SLC26A7 for Cl(-). Measurement of membrane potential and pH(i) showed minimal OH(-) and HCO(3)(-) transport by SLC26A7 when the cells were incubated in Cl(-)-containing or Cl(-)-free media. The activity of SLC26A7 was inhibited by all inhibitors of anion transporters tested, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, diphenylamine-2-carboxylic acid, and glybenclamide. These findings reveal that SLC26A7 functions as a unique Cl(-) channel that is regulated by intracellular H(+).

Intracellular pH homeostasis in cultured human placental syncytiotrophoblast cells: recovery from acidification

Resting or basal intracellular pH (pHi) measured in cultured human syncytiotrophoblast cells was 7.26+/-0.04 (without HCO3-) or 7.24+/-0.03 (with HCO3-). Ion substitution and inhibitor experiments were performed to determine whether common H+-transporting species were operating to maintain basal pHi. Removal of extracellular Na+ or Cl- or addition of amiloride or dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H2DIDS) had no effect. Acidification with the K+/H+ exchanger nigericin reduced pHi to 6.25+/-0.15 (without HCO3-) or 6.53+/-0.10 (with HCO3-). In the presence of extracellular Na+, recovery to basal pHi was prompt and occurred at similar rates in the absence and presence of HCO3-. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pHi after acidification. Recovery was inhibited by removal of Na+ or addition of amiloride, whereas removal of Cl- and addition of H2DIDS were ineffective. Addition of the Na+/H+ exchanger monensin to cells that had returned to basal pHi elicited a further increase in pHi to 7.48+/-0.07. Analysis of recovery data showed that there was a progressive decrease in DeltapH per minute as pHi approached the basal level, despite the continued presence of a driving force for H+ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pHi is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na+/H+ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load.

Probing Macromolecular Adsorbed Layer Structure and History Dependence via the Interfacial Cavity Function

Adsorbed layers of proteins and other macromolecules often relax structurally more slowly than they form, rendering layer growth an out-of-equilibrium process. We show here how the interfacial cavity function, Phi (the average Boltzmann factor for a single probe molecule), may be determined, using kinetic data available from optical waveguide lightmode spectroscopy, and used as a continuous, in situ measure of history dependent adsorbed layer structure. The increase of Phi observed with residence time for fibronectin and lysozyme layers suggests post-adsorption clustering on a time scale longer than that predicted by a surface diffusion model.

Levetiracetam inhibits Na+-dependent Cl-/HCO3- exchange of adult hippocampal CA3 neurons from guinea-pigs.

The novel anticonvulsant levetiracetam (LEV) was tested for effects on bioelectric activity and intracellular pH (pHi) regulation of hippocampal CA3 neurons from adult guinea-pigs. In 4-aminopyridine-treated slices, LEV (10-100 microm) reduced the frequency of action potentials and epileptiform bursts of CA3 neurons by 30-55%, while the shape of these potentials remained largely unchanged. Suppressive effects were reversed by an increase of pHi with trimethylamine (TMA). Using BCECF-AM-loaded slices, we found that LEV (10-50 microm) reversibly lowered neuronal steady-state pHi by 0.19 +/- 0.07 pH units in the presence of extracellular CO2/HCO3- buffer. In the nominal absence of extracellular CO2/HCO3- or in Na+-free CO2/HCO3(-)-buffered solution, LEV had no effect on steady-state pHi. Recovery of pHi subsequent to ammonium prepulses remained unchanged in the absence of CO2/HCO3- buffer, but was significantly reduced by LEV in the presence of CO2/HCO3- buffer. These findings show that LEV inhibits HCO3(-)-dependent acid extrusion, but has no effect on Na+/H+ exchange. LEV did not affect Na+-independent Cl-/HCO3- exchange because intracellular alkalosis upon withdrawal of extracellular Cl- remained unchanged. These data show that LEV at clinically relevant concentrations inhibits Na+-dependent Cl-/HCO3- exchange, lowers neuronal pHi, and thereby may contribute to its anticonvulsive activity.

Revisiting the role of H+ in chemotactic signaling of sperm.

Chemotaxis of sperm is an important step toward fertilization. During chemotaxis, sperm change their swimming behavior in a gradient of the chemoattractant that is released by the eggs, and finally sperm accumulate near the eggs. A well established model to study chemotaxis is the sea urchin Arbacia punctulata. Resact, the chemoattractant of Arbacia, is a peptide that binds to a receptor guanylyl cyclase. The signaling pathway underlying chemotaxis is still poorly understood. Stimulation of sperm with resact induces a variety of cellular events, including a rise in intracellular pH (pHi) and an influx of Ca2+; the Ca2+ entry is essential for the chemotactic behavior. Previous studies proposed that the influx of Ca2+ is initiated by the rise in pHi. According to this proposal, a cGMP-induced hyperpolarization activates a voltage-dependent Na+/H+ exchanger that expels H+ from the cell. Because some aspects of the proposed signaling pathway are inconsistent with recent results (Kaupp, U.B., J. Solzin, J.E. Brown, A. Helbig, V. Hagen, M. Beyermann, E. Hildebrand, and I. Weyand. 2003. Nat. Cell Biol. 5:109–117), we reexamined the role of protons in chemotaxis of sperm using kinetic measurements of the changes in pHi and intracellular Ca2+ concentration. We show that for physiological concentrations of resact (<25 pM), the influx of Ca2+ precedes the rise in pHi. Moreover, buffering of pHi completely abolishes the resact-induced pHi signal, but leaves the Ca2+ signal and the chemotactic motor response unaffected. We conclude that an elevation of pHi is required neither to open Ca2+-permeable channels nor to control the chemotactic behavior. Intracellular release of cGMP from a caged compound does not cause an increase in pHi, indicating that the rise in pHi is induced by cellular events unrelated to cGMP itself, but probably triggered by the consumption and subsequent replenishment of GTP. These results show that the resact-induced rise in pHi is not an obligatory step in sperm chemotactic signaling. A rise in pHi is also not required for peptide-induced Ca2+ entry into sperm of the sea urchin Strongylocentrotus purpuratus. Speract, a peptide of S. purpuratus may act as a chemoattractant as well or may serve functions other than chemotaxis.

Mechanisms of antiplatelet and antithrombotic activity of midazolam in in vitro and in vivo studies

Midazolam is widely used as a sedative and anesthetic induction agent. The aim of this study was to systematically examine the inhibitory mechanisms of midazolam in platelet aggregation. In this study, midazolam concentration-dependently (15 and 30 μM) inhibited platelet aggregation in washed human platelets stimulated by thrombin (0.05 U/ml). Midazolam (15 and 30 μM) also inhibited phosphoinositide breakdown and intracellular Ca +2 mobilization in platelets stimulated by thrombin (0.05 U/ml). In addition, midazolam (15 and 30 μM) increased the formation of cyclic AMP but not cyclic GMP or nitric oxide. The thrombin-evoked increase in pHi was markedly inhibited in the presence of midazolam (15 and 30 μM). Rapid phosphorylation of a platelet protein of molecular weight (Mr.) 47,000 (P47), a marker of protein kinase C activation, was triggered by thrombin (0.05 U/ml). This phosphorylation was markedly inhibited by midazolam (15 and 30 μM). Midazolam (30 μM) did not significantly reduce the electron spin resonance signal intensity of hydroxyl radicals in activated platelets. In the vivo study, intravenous injection of midazolam (10 μg/g) significantly prolonged the latent period of inducing platelet plug formation in mesenteric venules. These results indicate that midazolam can significantly prevent thrombus formation in vivo. Its antiplatelet activity may be involved in the inhibition of the activation of phospholipase C and the Na +/H + exchanger and increased cyclic AMP formation. These lead to lower intracellular Ca +2 mobilization and phosphorylation of P47.

A concept for controlling singlet oxygen (1 Delta g) yields using nitroxide radicals: phthalocyaninatosilicon covalently linked to nitroxide radicals.

In this study, we have investigated the singlet oxygen ((1)Delta(g)) generation mechanism using phthalocyaninatosilicon (SiPc) covalently linked to nitroxide radicals (NRs), and we succeeded in increasing the singlet oxygen quantum yield (Phi(Delta)) by linking the NRs. This originates from both an increase in the triplet quantum yield and excited-state lifetimes long enough to utilize photochemical reactions. Because the electron exchange interactions with paramagnetic species were known to result only in very fast excited-state relaxation, leading to a decrease in photochemical reaction yields, this increase in Phi(Delta) is an unusual and precious example for increasing photochemical reaction yields by electron exchange interactions with paramagnetic species. In addition, our experiments and theoretical analyses show that the spin-selective energy transfer rate constant is not influenced by linking the NRs and can be evaluated by the product of spin-statistical factors and matrix elements between the initial and final states.

Involvement of the Antiplatelet Activity of Magnesium Sulfate in Suppression of Protein Kinase C and the Na+/H+ Exchanger

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca⁺² mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of M_r 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca⁺² mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na⁺/H⁺ exchanger, leading to reduced intracellular Ca⁺² mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.

Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na+/H+ exchanger.

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6-3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of M(r) 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca+2 mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na+/H+ exchanger, leading to reduced intracellular Ca+2 mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.

Na-H Exchange-dependent Increase in Intracellular pH Times G2/M Entry and Transition

It is well established that activation of the Na-H exchanger NHE1 and increases in intracellular pH (pHi) are early and universal responses to mitogens and have permissive effects in promoting cell proliferation. Despite this evidence, a specific role for NHE1 or pHi in cell cycle progression remains undetermined. We now show that NHE1 activity and pHi regulate the timing of G2/M entry and transition. Prior to G2/M entry there is a rapid and transient increase in NHE1 activity and pHi, but in fibroblasts expressing a mutant NHE1 that lacks ion translocation activity, this increase in pHi is attenuated, S phase is delayed, and G2/M transition is impaired. In the absence of ion translocation by NHE1, expression of cyclin B1 and the kinase activity of Cdc2 are decreased and Wee1 kinase expression increases. Increasing pHi in the absence of NHE1 activity, however, is sufficient to restore Cdc2 activity and cyclin B1 expression and to promote G2/M entry and transition. These data indicate that a transient increase in pHi induced by NHE1 promotes the timing of G2/M, and they suggest that increases in pHi at the completion of S phase may constitute a previously unrecognized checkpoint for progression to G2 and mitosis.

Characterization of Cl-HCO3 exchange in basolateral membrane of rat distal colon.

Sodium-independent Cl movement (i.e., Cl-anion exchange) has not previously been identified in the basolateral membranes of rat colonic epithelial cells. The present study demonstrates Cl-HCO3 exchange as the mechanism for 36Cl uptake in basolateral membrane vesicles (BLMV) prepared in the presence of a protease inhibitor cocktail from rat distal colon. Studies of 36Cl uptake performed with BLMV prepared with different types of protease inhibitors indicate that preventing the cleavage of the COOH-terminal end of AE2 protein by serine-type proteases was responsible for the demonstration of Cl-HCO3 exchange. In the absence of voltage clamping, both outward OH gradient (pHout/pHin: 7.5/5.5) and outward HCO3 gradient stimulated transient 36Cl uptake accumulation. However, voltage clamping with K-ionophore, valinomycin, almost completely (87%) inhibited the OH gradient-driven 36Cl uptake, whereas HCO3 gradient-driven 36Cl uptake was only partially inhibited (38%). Both electroneutral HCO3 and OH gradient-driven 36Cl uptake were 1) completely inhibited by DIDS, an anion exchange inhibitor, with a half-maximal inhibitory constant (Ki) of approximately 26.9 and 30.6 microM, respectively, 2) not inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid(NPPB), a Cl channel blocker, 3) saturated by increasing extravesicular Cl concentration with a Km for Cl of approximately 12.6 and 14.2 mM, respectively, and 4) present in both surface and crypt cells. Intracellular pH (pHi) was also determined with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetomethylester (BCECF-AM) in an isolated superfused crypt preparation. Removal of Cl resulted in a DIDS-inhibitable increase in pHi both in HCO3-buffered and in the nominally HCO3-free buffered solutions (0.28 +/- 0.02 and 0.11 +/- 0.02 pH units, respectively). We conclude that a carrier-mediated electroneutral Cl-HCO3 exchange is present in basolateral membranes and that, in the absence of HCO3, Cl-HCO3 exchange can function as a Cl-OH exchange and regulate pHi across basolateral membranes of rat distal colon.

Modulation of the Na +–H + antiport activity by adrenaline on erythrocytes from normal and obese individuals

The effect of adrenaline on normal and obese human Na +–H + antiport (NHE 1) erythrocyte activity has been studied. Adrenaline increased both intracellular pH (pHi) and Na + influx in erythrocyte suspensions. This effect of adrenaline was inhibited by amiloride or EIPA, indicating that adrenaline stimulated NHE 1. Phorbol myristicate ester (PMA), a protein kinase C (PKC) stimulator, increased the activity of NHE 1 whereas calphostin C, a PKC inhibitor, partially inhibited NHE 1 activation induced by adrenaline. The effect of adrenaline to NHE 1 was counteracted by prazocin and by propranolol as well indicating the involvement of both alpha and beta 2 adrenergic receptors. The effect of adrenaline on erythrocyte NHE 1 activity was significantly more profound in obese compared to normal subjects. These data indicate that adrenaline induces an increase of pHi and Na + uptake of human erythrocytes through stimulation of NHE 1 activity. The significantly more profound stimulation of NHE 1 activity by adrenaline in obese as compared to normal subjects is discussed.

Inhibitory mechanisms of kinetin, a plant growth-promoting hormone, in platelet aggregation

Kinetin has been shown to have anti-aging effects on several different systems including plants and human cells. The aim of this study was to examine the detailed inhibitory mechanisms of kinetin in platelet aggregation. In this study, kinetin concentration-dependently (50-150 μM) inhibited platelet aggregation in human platelets stimulated by agonists. Kinetin (70 and 150 μM) also concentration-dependently inhibited intracellular Ca2+ mobilization and phosphoinositide breakdown in platelets stimulated by collagen (1 μg/ml). Kinetin (70 and 150 μM) significantly inhibited thromboxane A2 formation stimulated by collagen (1 μg/ml) and arachidonic acid (60 μM) in human platelets. In addition, kinetin (70 and 150 μM) significantly increased the formation of cyclic AMP. Intracellular pH values were measured spectrofluorometrically using the fluorescent probe BCECF-AM in platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of kinetin (70 and 150 μM). Rapid phosphorylation of a platelet protein of molecular weight (Mr) 47 000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 μg/ml). This phosphorylation was inhibited by kinetin (70 and 150 μM). In conclusion, these results indicate that the anti-platelet activity of kinetin may be involved in the following pathways: kinetin's effects may initially be due to inhibition of the activation of phospholipase C and the Na+/H+ exchanger. This leads to lower intracellular Ca2+ mobilization, followed by inhibition of TxA2 formation and then increased cyclic AMP formation, followed by a further inhibition of the Na+/H+ exchanger, ultimately resulting in markedly decreased intracellular Ca2+ mobilization and phosphorylation of P47. These results suggest that kinetin has an effective anti-platelet effect and that it may be a potential therapeutic agent for arterial thrombosis.

Changes in intracellular pH in apricot buds during the winter season.

The prevention of the symplastic entry of metabolites into bud cells may be indicative of dormancy status. Intracellular measurement allows, at least in part, quantification of the sink strength for nutrients of different studied tissues. The aim of this research was to study the changes in intracellular pH (pHi) in vegetative and flower buds of apricot. The pHi measurement, based on DMO (5,5 dimethyloxazolidine 2,4 dione) radioactive probe accumulation, was carried out periodically from December to February on buds collected from mixed twigs of San Castrese cultivar. The vegetative and flower buds and underlying parenchymatic tissues were located on the same node but in different combinations. The flower buds presented significant pHi changes in relation to the end of endo-dormancy and the evolution of phenological stages. In particular, when flower buds were still in deep dormancy, it was possible to record an increase of pHi in the flower primordia tissues just before the first sign of growth reactivation.

Dimethylsphingosine increases cytosolic calcium and intracellular pH in human T lymphocytes

N,N-Dimethyl- d-erythro-sphingosine (DMS) is the N-methyl derivative of sphingosine; both are activators of sphingosine-dependent protein kinases. The aim of this work was to study the effect of DMS on cytosolic calcium and intracellular pH (pHi) in human T lymphocytes. The variations of calcium and pH were determined by fluorescence digital imaging using Fura-2-AM and BCECF-AM, respectively. DMS increased both pHi and Ca 2+-cytoslic in human T lymphocytes. These effects were dose-dependent. This drug induced a fast increase in pHi and a release of calcium from different intracellular calcium pools than thapsigargin. DMS also induced a Ca 2+-influx different from the store-operated calcium channels, since drug effect was not modified by 30 μM SKF 96365. The influx of calcium induced by DMS was completely blocked by preincubation in the presence of nickel, or lanthanum, while the increase in pHi was no affected. However, the presence of cadmium reduced but does not block Ca 2+-influx. The inhibition of G-protein by 100 ng/mL pertussis toxin, and the inhibition of tyrosine kinases by genistein significantly reduced the cytosolic calcium increase induced by DMS by an inhibition of both, release of calcium from intracellular pools and influx from extracellular medium. The inhibition of pools emptiness by these drugs was related with the inhibition that they induce in the DMS cytosolic alcalinization. In summary, DMS increases pHi and as consequence releases calcium from intracellular pools, and it increases calcium-influx through a channel different from store-operated channel (SOC). Both cytosolic calcium and pHi increase are modulated by G-proteins and tyrosine kinases.