A synthetic peptide of the pseudosubstrate domain of protein kinase C blocks cytoplasmic alkalinization during activation of the sea urchin egg

Multiple second messenger pathways have been proposed for transduction of the sperm-egg fusion event during fertilization of sea urchin eggs. Cytoplasmic alkalinization due to increased Na(+)-H+ antiport has been causally linked to many of the metabolic events during fertilization. Two possible second messenger pathways coupling sperm-egg fusion and antiporter activity are activation of protein kinase C (PKC) and Ca2(+)-calmodulin kinase. A selective inhibitor of PKC is PKC(19-36), a synthetic peptide of the pseudosubstrate domain of the kinase. Injection of PKC(19-36) into unfertilized sea urchin eggs blocked cytoplasmic alkalinization during activation by phorbol 12-myristate 13-acetate, a PKC agonist. The rise in pH during fertilization was partially blocked by PKC(19-36), which suggested that multiple pathways regulate the antiporter during fertilization. The use of fluorescein chromophores to measure intracellular pH in sea urchin eggs is also discussed.     

Na+-H+ antiport during fertilization of the sea urchin egg is blocked by W-7 but is insensitive to K252a and H-7

Fertilization of the sea urchin egg initiates or accelerates a number of metabolic activities, which have been causally linked to a rise in cytoplasmic pH due to increased Na+-H+ antiport. Two possible regulatory pathways linking sperm-egg fusion to the activity of the antiporter are activation of protein kinase C (PKC) and Ca2+, calmodulin (CaM)-dependent kinase. This report presents the effects of protein kinase inhibitors on antiporter activation during fertilization and treatment with PKC agonists, dioctanoylglycerol or phorbol diester. Protein kinase inhibitors, K252a and H-7 blocked the action of PKC agonists, without inhibiting cytoplasmic alkalinization during fertilization. In contrast, W-7 blocked fertilization-induced rise in cytoplasmic pH, without altering the actions of PKC agonists. These results suggest that the Na+-H+ antiporter may be regulated by PKC or Ca2+, CaM-dependent kinase activities, but activation of the antiporter during fertilization is Ca2+, CaM-dependent, despite production of diacylglycerols by hydrolysis of phosphatidylinositols.     

Phorbol Myristate Acetate Induces the Phosphorylation of Plasma Membrane-Associated Proteins in Sea Urchin Eggs

Immediately after fertilization sea urchin eggs undergo an increase in cytoplasmic pH from 6.8 to 7.2. This pH change occurs by activation of a Na⁺/H⁺ antiporter, and is a necessary signal for later steps in metabolic activation of development. Activators of protein kinase C such as phorbol myristate acetate (PMA) and diacylglycerol produce a similar pH increase in eggs. Phosphorylation of the antiporter or a regulatory protein may be a step in activating Na⁺/H⁺ exchange. Here we show that treatment of sea urchin eggs ( S. purpuratus ) with PMA results in increased phosphorylation of over a dozen proteins. Of these, three proteins of Mr=240, 92 and 80 kD are located in the egg cortex; under-representation of these bands in isolated cortical granules suggests that they are plasma membrane-associated. Phosphorylation of the 92 kD band is concentration-dependent over a range of 10 to 1000 nM PMA and occurs over a time-course of 1 to 3 min. Phosphoamino acid analysis indicates that phosphorylation is on serine residues. Phosphorylation appeares to be mediated by protein kinase C since the inactive PMA analogue, 4α-phorbol 12, 13-didecanoate, does not induce phosphorylation nor does experimental alkalinization of the egg cytoplasm.     

Intracellular Ca2+ potentiates Na+/H+ exchange and cell differentiation induced by phorbol ester in U937 cells

The human cell line U937 differentiates to monocyte macrophage-like cells in response to tumour-promoting phorbol esters. This effect is attributed to activation of protein kinase C. We show here that U937 cell differentiation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) is associated with cytoplasmic alkalinization. Ethyl-isopropyl-amiloride (EIPA), a potent inhibitor of Na+/H+ exchange, blocked both cytoplasmic alkalinization and cell differentiation. Cell acidification by addition of 2-4 mM sodium propionate also blocked TPA-induced U937 cell differentiation. These results suggest that a sustained cell alkalinization mediated by activation of Na+/H+ exchange is essential for TPA-induced differentiation in U937 cells. The increase of cytoplasmic free calcium concentration ([Ca2+]i) by addition of the calcium ionophore ionomycin enhanced TPA-induced alkalinization by increasing the apparent affinity of the Na+/H+ antiporter for intracellular H+. Treatment with ionomycin also potentiated differentiation of U937 cells induced by TPA. This synergism suggests that [Ca2+]i either potentiates the activation of protein kinase C or triggers additional transducing mechanisms. The key events of this interaction occur during the first 30 min of treatment, even though cell differentiation manifests much later.     

Activation of amino acid uptake at fertilization in the sea urchin egg. Requirement for proton compartmentalization during cytosolic alkalosis

The comparative importance of the release of intracellular ionic calcium, Na+/H+ exchange and cytosolic alkalosis as activator signals was studied on the development of amino acid uptake at fertilization in sea urchin eggs. We show that, once stimulated, the rate of valine uptake is greatly dependent upon intracellular pH. Suppression of the Na+/H+ exchange at the time of activation, by applying ionophore (A23187) in sodium-free artificial sea water (ONaASW), inhibits the development of valine influx. This cannot be restored by a further (30 min later) alkalosis by transferring eggs into sea water. Suppressing the alkalosis in the presence of Na+/H+ exchange at fertilization by simultaneous addition of acid into sea water results in activation of the amino acid carrier which exhibits an increased rate of transport as soon as the eggs are replaced in sea water at pH 8.0. The absence of alkalosis in eggs activated in ONaASW can be counterbalanced either by adding NH4Cl 10 mM or by transfer into ASW at pH 9.0 at activation. Ammonia-treated eggs absorbed amino acid as controls, whereas eggs in sea water at pH 9.0 failed to develop a valine uptake system, suggesting that ammonia can completely replace the effect of Na+/H+ exchange. Furthermore, addition of NH4Cl immediately before fertilization conceals the Na+/H+ exchange but stimulates valine uptake as in controls. These data suggest that: the occurrence of the intracellular calcium increase alone is not sufficient for the develpment of the amino acid transport system; cell alkalinization at fertilization derives from the cytoplasmic membrane-located Na+/H+ exchange and an inward movement of protons into a cortical acidic compartment, which is discussed.     

Protein tyrosine kinase-dependent release of intracellular calcium in the sea urchin egg

The aminoguanide, methylglyoxal bis(guanylhydrazone) (MGBG), was shown to stimulate phosphorylation of RR-SRC, a synthetic protein tyrosine kinase (PTK) substrate, and different levels of tyrosyl phosphorylation of endogenous proteins in a sea urchin egg membrane-cortex preparation. Stimulating protein tyrosine kinase activity in the sea urchin egg stimulated intracellular Ca2+ release, because microinjection of 1-5 mM of MGBG into unfertilized eggs triggered a transient rise in intracellular Ca2+ activity ([Ca2+]i) after a brief latent period. Pretreating eggs with PTK-specific inhibitors, genistein or tyrphostin B42, significantly inhibited the MGBG-induced rise in [Ca2+]i. Methylglyoxal bis(guanylhydrazone) stimulation of PTK activities in the unfertilized sea urchin egg appeared to trigger Ca2+ release through phospholipase C (PLC)-dependent inositol 1,4,5-trisphosphate (InsP3) production. The MGBG-induced Ca2+ response could be suppressed in eggs preloaded with the InsP3 receptor antagonist, heparin, and was reduced in eggs pretreated with U73122, a PLC inhibitor. However, the response was unchanged in eggs treated with nicotinamide, an inhibitor of ADP-ribosyl cyclase, or nifedipine, an inhibitor of nicotinic acid adenine dinucleotide phosphate activity. These results suggest that MGBG may be useful as a chemical agonist of PTK in sea urchin eggs and allow direct testing of the PTK requirement for the transient rise in [Ca2+]i in sea urchin eggs during fertilization. Although genistein was observed to significantly delay the onset, the sperm-induced Ca2+ response in PTK inhibitor-loaded eggs otherwise appeared normal. Therefore, it was concluded that sea urchin eggs contain a PTK-dependent pathway that can mediate intracellular Ca2+ release, but PTK activity does not appear to be required for the fertilization response.     

Relation of cytoplasmic alkalinization to microvillar elongation and microfilament formation in the sea urchin egg

Experiments have been carried out to test the proposal that the pH increase at fertilization in sea urchin eggs promotes microvillar elongation. Results presented herein show that microvillar elongation and microfilament formation occurred when sea urchin eggs were incubated in sodium-free seawater containing the calcium ionophore A23187, a treatment which initiates activation, i.e., induces a transient increase in intracellular free calcium, but prevents subsequent cytoplasmic alkalinization. Within elongated microvilli and cortices of these eggs, microfilaments were arranged in a loose meshwork. However, if the pH of the egg cytoplasm was increased experimentally, microfilament bundles appeared within individual microvilli. These findings suggest that: (1) microvillar elongation and microfilament formation in the sea urchin egg at fertilization may occur when cytoplasmic alkalinization is inhibited, and (2) formation of the microvillus bundle of microfilaments at egg activation is pH sensitive. Additionally, if the cytoplasmic pH of unfertilized eggs was experimentally elevated by NH₄Cl, microvilli failed to elongate. These data indicate that elevation of intracellular pH by this method is not sufficient to induce microvillar elongation.     

Stimulation of leucine transport by a phorbol ester through activation of protein kinase C and Na+/H+ exchanger

A synthetic diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG), as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) has been found to elevate the cytoplasmic pH and increase leucine uptake dose-dependently, when added to quiescent cultures of Chang liver cell. Addition of either a protein kinase C inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), or an Na+/H+ antiporter inhibitor, ethylisopropylamiloride (EIPA), abolished completely or incompletely the TPA-stimulated leucine uptake and TPA-induced cytoplasmic alkalinization. Therefore the stimulation of leucine uptake by OAG and TPA is proposed to be elicited at least partly through activation of Na+/H+ antiporter. We suggest that activation of protein kinase C by the phorbol ester is responsible for the stimulation of Na+/H+ exchange system and also leucine uptake in the cell.     

A novel 15 kDa Ca2+-binding protein present in the eggs of the sea urchin, Hemicentrotus pulcherrimus

A novel Ca2+-binding protein, different from calmodulin, has been purified to homogeneity from the soluble cytoplasmic protein fraction of the egg of the sea urchin, Hemicentrotus pulcherrimus. This protein, designated as 15 kDa protein, shows a Ca2+-dependent mobility shift upon SDS-gel electrophoresis and has Ca2+-binding ability. This protein did not resemble the sea urchin egg calmodulin in either molecular mass or amino acid composition. The 15 kDa protein could not activate cyclic adenosine 3',5'-monophosphate-dependent phosphodiesterase from bovine brain and did not bind to fluphenazine-Sepharose 6B. Antibodies against the 15 kDa protein did not react with sea urchin egg calmodulin. These results suggest that the 15 kDa protein is a novel Ca2+-binding protein in the sea urchin egg.     

Phorbol ester treatment stimulates tyrosine phosphorylation of a sea urchin egg cortex protein

Fertilization of the sea urchin egg results in the phosphorylation, on tyrosine, of a high molecular weight protein localized in the egg cortex. In the present study, treatment of unfertilized eggs with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate stimulated tyrosine phosphorylation of the high molecular weight cortical protein to levels three- to fivefold higher than that occurring in response to fertilization. Experiments using agents that inhibit the egg Na+/H+ exchange system or mimic the fertilization-induced shift in cytoplasmic pHi, suggest a signal transduction pathway in which protein kinase C activates the egg Na+/H+ exchange system and the resultant cytoplasmic pHi shift promotes tyrosine phosphorylation of the high molecular weight cortical protein.     

Possible involvement of a Na+/H+ antiporter in the stimulation of hexose transport in Swiss 3T3 cells by a phorbol ester and growth factors

Phorbol-12,13-dibutyrate, epidermal growth factor, and insulin raised the intracellular pH ([pH]i), presumably through the activation of a Na+/H+ antiporter. Addition of amiloride or replacement of extra-cellular Na+ by choline which abolishes the cytoplasmic alkalinization prevented the stimulation of hexose transport by these agents. Furthermore, monensin, a Na+/H+ ionophore which increases the [pH]i, stimulated hexose transport. This stimulation was also prevented by the replacement of extra-cellular Na+ by choline. These observations suggest that stimulation of the Na+/H+ antiporter may have stimulated the increase in hexose transport.     

Changes in intracellular acidic compartments in sea urchin eggs after activation

Acridine orange (AO) was used as a vital probe for looking at acidic intracellular compartments in sea urchin eggs. This weak base is concentrated by acidic compartments, shifting its fluorescence from green to red due to the formation of dye aggregates. Fertilization or parthenogenetic activation with ionophore A23187 resulted in the appearance of orange fluorescent granules of sizes ranging from 1 to 2 microns at the cortical region of the egg. In one species of sea urchin (Lytechinus pictus), these granules migrate inward before cell division and associate with the forming mitotic apparatus. Treatments that discharge the transmembrane pH gradient (NH4Cl, nigericin, monensin, and acidic external pH) eliminate the orange fluorescence, indicating they are acidic compartments. Spectrofluorimetric measurements showed a decrease in monomer fluorescence accompanying egg activation which is reversible by similar treatments as seen with the fluorescence microscopic observations. Stratified eggs which were subsequently fertilized had acidic granules concentrated at the centripetal pole. This allowed the electron microscopic identification of the granules and showed they are present in the unfertilized egg, although not able to concentrate the AO. Activation of eggs in the absence of Na+ prevented the cytoplasmic alkalinization and also inhibited the appearance of acidic granules. The results indicate that the internal pH rises after egg activation triggers the acidification of these granules. Their possible functions, as in intracellular pH regulation, are discussed.     

Multifunctional Ca2+/calmodulin-dependent protein kinase is necessary for nuclear envelope breakdown

The role of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in nuclear envelope breakdown (NEB) was investigated in sea urchin eggs. The eggs contain a 56-kD polypeptide which appears to be a homologue of neuronal CaM kinase. For example, it undergoes Ca2+/calmodulin-dependent autophosphorylation that converts it to a Ca2(+)-independent species, a hallmark of multifunctional CaM kinase. It is homologous to the alpha subunit of rat brain CaM kinase. Autophosphorylation and substrate phosphorylation by the sea urchin egg kinase are inhibited in vitro by CaMK(273-302), a synthetic peptide corresponding to the autoinhibitory domain of the neuronal CaM kinase. This peptide inhibited NEB when microinjected into sea urchin eggs. Only one mAb to the neuronal enzyme immunoprecipitated the 56-kD polypeptide. Only this antibody blocked or significantly delayed NEB when microinjected into sea urchin eggs. These results suggest that sea urchin eggs contain multifunctional CaM kinase, and that this enzyme is involved in the control of NEB during mitotic division.     

Ha-ras activates the Na+/H+ antiporter by a protein kinase C-independent mechanism

In quiescent Ha-ras-transfected NIH 3T3 cells, addition of serum growth factors, bombesin or 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to a dimethylamiloride-sensitive intracellular alkalinization which can be inhibited by staurosporine, a potent inhibitor of protein kinase C. Expression of the transforming Ha-ras gene causes a growth factor-independent increase in cytoplasmic pH. This Ha-ras-induced alkalinization is sensitive to dimethylamiloride but is not affected by staurosporine concentrations which prevent the pH response after addition of growth factors or TPA. Protein kinase C depletion by long term exposure to TPA eliminates the pH response to bombesin and phorbol ester but does not effect the Ha-ras-induced intracellular alkalinization. It is concluded that expression of Ha-ras causes an activation of the Na+/H+ antiporter by an as yet unknown protein kinase C-independent mechanism.     

Two phases of inositol polyphosphate and diacylglycerol production at fertilisation

[3H]Inositol and [3H]arachidonic acid were used to label polyphosphoinositide phospholipids in sea urchin eggs. Both [3H]inositol polyphosphate (InsP3) and [3H]diacylglycerol (DAG) increase at fertilisation. An early increase in InsP3 occurs as the sperm-induced calcium transient crosses the egg and exocytosis occurs; a later increase in InsP3 as calcium declines and the protein kinase C-dependent Na/H antiporter causes the cytoplasmic pH in increase. These results support suggestions that a calcium-induced hydrolysis of phosphatidylinositol bisphosphate occurs at fertilisation, that the production of diacylglycerol may be essential for exocytosis and that diacylglycerol production at fertilisation stimulates the Na/H antiporter. The increase in [3H]inositol polyphosphate as calcium declines indicates that this second messenger may have some function later in the cell cycle.     

The Na+/H+ antiporter is not involved in potentiation of thrombin-induced responses by epinephrine

Stimulation of platelets with thrombin, ADP and epinephrine has recently been shown to activate a Na+/H+ antiporter, with a resulting alkalinization of the cytoplasm. Unlike thrombin, however, epinephrine is incapable of directly activating phospholipase C, but is well known to potentiate the effects of thrombin on this enzyme and other subsequent steps of platelet activation. Therefore, we have studied the involvement of the Na+/H+ antiporter in this aspect of epinephrine action to see whether alkalinization of platelet cytosol could be a requirement for agonists to stimulate inositol phospholipid hydrolysis and mobilize cellular Ca2+ stores. Alpha-thrombin induced the rapid formation of inositol trisphosphate with a parallel mobilization of intracellular Ca2+ stores. Epinephrine alone had no effect on either of these parameters. The response to thrombin desensitized over a period of minutes, and after this had occurred, epinephrine was able to activate phospholipase C and induce the release of intracellular Ca2+. This showed that epinephrine was able to recouple thrombin receptors to phospholipase C, and this appeared to be mediated by the same mechanism which is involved in potentiation by epinephrine of thrombin-stimulation of phospholipase C. These effects of epinephrine were not altered by inhibition of the Na+/H+ antiporter with ethylisopropylamiloride or by use of the Na+/H+ ionophore, monensin. We conclude that epinephrine potentiates thrombin-induced responses by a mechanism which is unrelated to its effects on the Na+/H+ antiporter, and this is not a requirement for thrombin-induced phospholipase C activation.     

Role of Na+/H+ exchange in thrombin-induced platelet-activating factor production by human endothelial cells

Thrombin-stimulated endothelial cells produce platelet-activating factor (PAF) in a dose-dependent manner: the activation of a Ca2+-dependent lyso-PAF acetyltransferase is the rate-limiting step in this process. The present study shows that acetyltransferase activation and consequent PAF production induced by thrombin in human endothelial cells are markedly inhibited in Na+-free media or after addition of the amiloride analog 5-(N-ethyl-N-isopropyl)amiloride, suggesting that a Na+/H+ antiport system is present in endothelial cells and plays a prominent role in thrombin-induced PAF synthesis. Accordingly, thrombin elicits a sustained alkalinization in 6-carboxyfluorescein-loaded endothelial cells, that is abolished in either Na+-free or 5-(N-ethyl-N-isopropyl)amiloride-containing medium. Extracellular Ca2+ influx induced by thrombin (as measured by quin2 and 45Ca methods) is completely blocked in the same experimental conditions, and monensin, a Na+/H+ ionophore mimicking the effects of the antiporter activation, evokes a dose-dependent PAF synthesis and a marked Ca2+ influx, which are abolished in Ca2+-free medium. An amiloride-inhibitable Na+/H+ exchanger is present in the membrane of human endothelial cells, its apparent Km for extracellular Na+ is 25 mM, and its activity is greatly enhanced when the cytoplasm is acidified. These results suggest that Na+/H+ exchange activation by thrombin and the resulting intracellular alkalinization play a direct role in the induction of Ca2+ influx and PAF synthesis in human endothelial cells.     

The Na+/Ca2+ antiporter in aortic smooth muscle cells. Characterization and demonstration of an activation by phorbol esters

The Na+/Ca2+ antiporter is present in aortic smooth muscle cells of the A7r5 cell line. Imposing an outward Na+ gradient to the cells promoted a 45Ca2+ uptake component which was sensitive to amiloride derivatives and insensitive to blockers of the voltage-dependent Ca2+ channel. The Ca2+ uptake system was dependent on intracellular Na+ concentration; it was inactive when Li+ replaced intracellular Na+ and it was electrogenic. Flow cytometric analysis of cells that had been loaded with the Ca2+ indicator indo-1 showed that all conditions that promoted Ca2+ influx led to corresponding increases in the free cytoplasmic Ca2+ concentration. Treatment of the A7r5 cells with phorbol myristate acetate, a known activator of protein kinase C (Ca2+/phospholipid-dependent enzyme), led to a two-fold activation of the system and to larger intracellular Ca2+ transients when cells were shifted to Na+-free solutions. Activation was observed at all intracellular Na+ concentrations. Changing the activity of the Na+/Ca2+ system did not affect the size and duration of intracellular Ca2+ transients elicited by the Ca2+ mobilizing hormone vasopressin. It is concluded that the Na+/Ca2+ antiporter in smooth muscle cells is a target for protein kinase C but that the system is not involved in the regulation of Ca2+ transients induced by vasopressin.     

T cell stimulation via CD2 molecules is regularly accompanied by an increase in cytoplasmic pH. Different effects of lectins and CD3 antibodies

The stimulation of different cell types with growth factors is often accompanied by a rapid intracellular alkalinization. By using mitogenic lectins, cluster of differentiation (CD)2 and CD3 mAb, as stimuli, we studied early changes of the intracellular pH in the activation process of resting human PBL. We found increases in free cytoplasmic Ca2+ levels and DNA synthesis but no intracellular alkalinization in the early activation phase upon stimulation with the mitogenic lectins, Con A, and PHA. Similarly stimulation with CD3 mAb led in most instances to no detectable pH shifts. Only in 7 out of 30 experiments was CD3 mAb-induced alkalinization observed. In contrast, stimulation with mitogenic combinations of anti-CD2 mAb led in all instances to rapid and clear-cut intracellular pH shifts very similar to those observed upon stimulation with PMA. In medium lacking sodium bicarbonate the intracellular alkalinization via the CD2 structure could be blocked by the amiloride analogue 5-(N-methyl-N-isobutyl)amiloride (MIA), which indicates that this increase in pH is mediated by the amiloride-sensitive Na+/H+ antiporter. Blockade of this antiporter had no negative effect, however, on T cell proliferation as measured by thymidine incorporation. In contrast, significantly enhanced proliferation rates were observed after stimulation with mitogenic combinations of anti-CD2 antibodies in the presence of MIA. No such effect of MIA could be observed in lectin induced T cell stimulation. These findings indicate that stimulation of the Na+/H+ antiporter via the CD2 structure is neither a prerequisite for T cell proliferation nor does it promote T cell growth. It rather seems to function in a regulatory role. In its absence, superinduction of proliferation can be achieved.     

Inositol phosphates turnover, cytosolic Ca++ and pH: putative signals for the control of cell growth

The signals that induce a cell to divide are usually external and in the form of a binding of growth factors. We focussed our attention in defining the sequence of events which occurs after the binding of the mitogens to their surface receptors. One of the early membrane events stimulated by growth factors is a Na+ flux coupled to a H+ efflux that is typically inhibited by amiloride. The importance of this event and of the consequent cytoplasmic alkalinization for the cell proliferation is discussed. Recent data indicate that mitogens increase intracellular Ca++ levels and activate protein kinase C by inducing the hydrolysis of membrane phosphoinositides. A role for Ca++ and protein kinase C in activating Na+/H+ A role for Ca++ and protein kinase C in activating Na+/H+ exchange system is discussed. Finally a model is presented that illustrates the first membrane events stimulated by the growth factors. The model reveals an intimate interconnections between phosphoinositide metabolism, cytosolic Ca++ rise, protein kinase C and cytoplasmic alkalinization.