Effects of Microinjected Cations on the Early Event of Fertilization in the Medaka Egg

Effects of microinjected cations on the early events of fertilization were examined using eggs of Oryzias latipes . Microinjection of either Ca²⁺, Ba²⁺ or Sr²⁺ into the thin cortical cytoplasm induced breakdown of cortical alveoli (vesicles) (CABD) under Ca-Mg-free conditions, but microinjection of Mg²⁺, Mn²⁺ or Co²⁺ prevented CABD at the injected region when the eggs were inseminated in regular saline. Under Ca-Mg-free conditions, CABD could also be induced by microinjection of various solutions (NaCl, choline chloride, sucrose, pH buffer) without any divalent cations or ionophore A23187. Ca²⁺ microinjected into the cortical cytoplasm did not play a role in sperm penetration. Upon microinjection with either Ca²⁺, Mg²⁺ or K⁺, the resting membrane potential leakage was transiently observed. However, depolarization of the membrane followed by slow hyperpolarization was observed only upon microinjection of Ca²⁺. From these experiments, it was inferred that microinjected divalent cations such as Ca²⁺, Ba²⁺ or Sr²⁺ do not act directly upon the cortical alveolus membrane, but trigger the induction of CABD via depolarization of the membrne and increase in intracellular Ca²⁺.     

Fertilization Reaction without Changes in Intracellular Ca2+ in Medaka Eggs—An Experiment with Acetone-Treated Eggs

To investigate whether or not causal relationship exists between the increase in intracellular Ca²⁺ and other cortical reactions at fertilization in the medaka, Oryzias latipes , intracellular Ca²⁺ was determined from luminescence of aequorin previously microinjected into cortical cytoplasm in acetone-treated eggs, when they were inseminated or activated by microinjection of Ca²⁺. Neither an increase in cytoplasmic calcium nor exocytosis of cortical alveoli occurred in eggs treated with acetone, though other events of fertilization i.e. completion of meiosis, fusion of pronuclei, and accumulation of cortical cytoplasm with intact cortical alveoli in the animal pole region were observed in normal time sequence in these eggs. When denuded eggs were treated with acetone, contraction of the egg and slow resumption of meiosis (extrusion of polar body) were observed without insemination. When denuded eggs were inseminated immediately after acetone-treatment, the number of spermatozoa that penetrated into the egg was greater in the animal hemisphere than in the vegetal hemisphere. These results may indicate that acetone inactivates the egg plasma membrane or its adjacent cortical cytoplasm so that it cannot participate in a propagative increase in intracellular Ca²⁺ and exocytosis, while it also induces cytoplasmic activation leading to egg contraction, resumption of meiosis and formation of pronuclei. The present results suggest that sperm penetration, resumption of meiosis and ooplasmic segregation are regulated separately from the release of intracellular Ca²⁺ and exocytosis.     

The Wave Pattern of Free Calcium Release Upon Fertilization in Medaka and Sand Dollar Eggs

A transient rise in the concentration of Ca²⁺ in the cortex upon fertilization was demonstrated in medaka eggs injected with aequorin. Detection of the aequorin luminescence with an ultra-high sensitivity photonic microscope system revealed a wave of increased Ca²⁺ concentration starting at the site of sperm entry (animal pole) and being propagated along the cortex of the egg toward the antipode. The wave traversed the entire egg surface within 2–3 min. The peak value of the aequorin luminescence, and therefore the peak value of the Ca²⁺ transient, was generally higher at the site of sperm entry than in other regions. The peak values of the luminescence (and therefore of the Ca²⁺ concentration in the cortex) remained fairly constant during propagation of the wave. Microinjection of Ca²⁺ into the cortex also induced a Ca²⁺ wave. When the egg was stimulated by microinjection of Ca²⁺ at the equatorial region, the Ca²⁺ wave was propagated at a fairly constant speed over the egg surface, except at the region near the vegetal pole where the wave was retarded. Simultaneous recording of the Ca²⁺ wave and the wave of cortical change (breakdown of cortical alveoli) in eggs during fertilization revealed that the Ca²⁺ wave preceded the wave of cortical change.
A Ca²⁺ wave was also demonstrated in sand dollar eggs, although due to their smaller size the phenomenon was not as clear as in medaka eggs.     

MAGNESIUM REQUIREMENT IN FERTILIZATION PROCESS OF SEA URCHINS

The Mg²⁺ requirement in fertilization was investigated in sea urchins. It was found that when sea urchin eggs were inseminated in sea water free of Mg²⁺, little fertilization took place. Even when spermatozoa pre-treated with dissolved egg-jelly to induce the acrosome reaction, which needs Ca²⁺, were used, the fertilization rate remained quite low in the absence of Mg²⁺. In Strongylo-centrotus intermedius , the lowest concentration of Mg²⁺ required for 50% fertilization was 0.05 mM in the presence of 10 mM Ca²⁺, whereas that of calcium was 3 mM in the presence of 49 mM Mg²⁺. These critical concentrations increased when the concentration of the other ion decreased. Removal of Mg²⁺ or Ca²⁺ or both from the suspending medium had little adverse effect on sperm motility. The elevation of the fertilization membrane was also induced by butyric acid independent of the presence or absence of Mg²⁺ and/or Ca²⁺. These results indicate that Mg²⁺ are required at least in some process(es) between acrosome reaction and fertilization membrane elevation, such as sperm penetration or membrane fusion.     

Formation of the enveloping layer of the chum salmon egg in isotonic salt solutions

After stimulation in a hypotonic solution (9.4 mOsm kg⁻¹), inseminated eggs of the chum salmon Oncorhynchus keta initiate cleavages in isotonic salmon Ringer's solution (267.3 mOsm kg⁻¹) containing 3.2 mM Ca²⁺ ions. Blastomeres of these eggs, however, separate from each other and the enveloping layer is not observed at the blastula stage. An increase in external divalent cations rescues the separation; the concentration of CaCl₂ in the external medium should be 25 mM or more to induce close contact of blastomeres and the formation of an enveloping layer in isotonic salt solutions. The effectiveness of Ca²⁺ ions can be substituted by Mg²⁺, Sr²⁺ and Zn²⁺ ions; the same results are obtained in isotonic MgCl₂ and SrCl₂ solutions (100 mM) or in isotonic salmon Ringer's solution containing Zn ions (6.2 mM). The close contact of blastomeres and the formation of an enveloping layer are also observed in a low Ca²⁺ concentration (< 0.1 mM) in a hypotonic salt solution (9.4 mOsm kg⁻¹). The Ca²⁺ level in the external medium to induce the enveloping layer formation seems to be correlated with the salinity of the incubation medium. It is suggested that adhesion molecules on the surface of blastomeres in the chum salmon eggs are different in properties from those found in sea urchin and other fish species.     

Comparative Detection of Calcium Fluctuations in Single Female Sex Cells of Tobacco to Distinguish Calcium Signals Triggered by in vitro Fertilization

Double fertilization is a key process of sexual reproduction in higher plants. The role of calcium in the activation of female sex cells through fertilization has recently received a great deal of attention. The establishment of a Ca²⁺-imaging technique for living, single, female sex cells is a difficult but necessary prerequisite for evaluating the role of Ca²⁺ in the transduction of external stimuli, including the fusion with the sperm cell, to internal cellular processes. The present study describes the use of Fluo-3 for reporting the Ca²⁺ signal in isolated, single, female sex cells, egg cells and central cells, of tobacco plants. A suitable loading protocol was optimized by loading the cells at pH 5.6 with 2 μM Fluo-3 for 30 min at 30  °C. Under these conditions, several key factors related to in vitro fertilization were also investigated in order to test their possible effects on the [Ca²⁺]_cyt of the female sex cells. The results indicated that the bovine serum albumin-fusion system was superior to the polyethlene glycol-fusion system for detecting calcium fluctuations in female sex cells during fertilization. The central cell was fertilized with the sperm cell in bovine serum albumin; however, no evident calcium dynamic was detected, implying that a transient calcium rise might be a specific signal for egg cell fertilization.     

Activation of Sea Urchin Eggs by Halothane and Its Inhibition by Dantrolene

Eggs of the sea urchin, Hemicentrotus pulcherrimus , were stimulated by halothane, known to induce Ca²⁺ release from sarcosome, to cause fertilization membrane formation in normal and Ca²⁺ free artificial sea water. In the absence of external Ca²⁺, halothane-induced formation of fertilization membrane was inhibited by dantrolene, an inhibitor of Ca²⁺ release from sarcosome, but was not blocked by nifedipine, a Ca²⁺ antagonist specific to Ca²⁺ channels in plasma membrane. Ca²⁺ release from sedimentable fraction isolated from eggs was induced by halothane and was inhibited by dantrolene, but was not blocked by nifedipine. In normal artificial sea water, halothane-caused egg activation was not inhibited either by dantrolene or by nifedipine, but was blocked in the presence of both compounds. ⁴⁵Ca²⁺ influx was substantially stimulated by halothane in eggs exposed to ⁴⁵CaCl₂. Halothane-induced ⁴⁵Ca²⁺ influx into eggs was inhibited by nifedipine but was not blocked by dantrolene. When Ca²⁺ release from intracellular organellae is blocked, Ca²⁺ transport through Ca²⁺ channels in plasma membrane probably acts as a "fail-safe" system to induce an increase in cytosolic Ca²⁺ level, resulting in egg activation.     

Ca2+-induced change of dorsal-ventral polarity in frog (Rana temporaria) eggs

Abstract. The effect of local Ca²⁺ administration 10–20 min after fertilization and during artificial activation was examined in Rana temporaria eggs. Ca²⁺ was injected into the pigmented region near the boundary between pigmented and unpigmented domains. The locations of egg gray crescent (GC) and dorsal lip of blastopore (DLB), as predictors of the dorsal region in embryos, as well as the measurements of angles between GC middle and sperm entry site were observed. In more than 70% of the cases, microinjection of Ca^{2 +} into subcortical cytoplasm and egg pricking in high-Ca^{2 +} solutions induced GC and DLB formation near the injection site. The formation of Ca²⁺-induced GC occurred mostly as in control eggs. In addition premature displacement of the egg surface was observed near the prick site in high-Ca^{2 +} solutions. GC formation occurred by displacement of the pigmented surface in the same direction as earlier wound translocations. These results show that Ca²⁺ injection determines the direction of the surface movement.     

Probable participation of phospholipase A2 reaction in the process of fertilization-induced activation of sea urchin eggs

In sea urchin eggs activated by sperm, A23187 or melittin, BPB (4-bromophenacyl bromide, a phospholipase A₂ inhibitor) blocked fertilization envelope formation and transient CN⁻-insensitive respiration in a concentration-dependent manner. BPB had virtually no effect on the increase in [Ca²⁺]_i, (cytosolic Ca²⁺ level), the activity of phosphorylase a and the rate of protein synthesis, as well as acid production and augmentation of CN⁻-sensitive respiration. BPB also inhibited fertilization envelope formation and augmentation of CN⁻-insensitive respiration induced by melittin. Melittin, known to be an activator of phospholipase A₂, induced the envelope formation, acid production, augmentation of CN⁻-insensitive and sensitive respiration, but did not cause any increase in [Ca²⁺]_i, the phosphorylase a activity and the rate of protein synthesis. An activation of phospholipase A₂ induced by Ca²⁺ or melittin seems to result in cortical vesicle discharge and production of fatty acids, which are to be utilized in CN⁻-insensitive lipid peroxidase reactions. Activation of other examined cell functions in eggs activated by sperm or A23187, probably results from Ca²⁺-triggered sequential reactions other than Ca²⁺-caused activation of phospholipase A₂.     

Fertilization Membrane Formation in Sea Urchin Eggs Induced by Drugs Known to Cause Ca2+Release from Isolated Sarcoplasmic Reticulum

Ryanodine, miconazole, clotrimazole, doxorubicin, quercetin, halothane, caffeine and chloroform, which activate Ca²⁺-induced Ca²⁺release from Ca²⁺stores, induced Ca²⁺release from a particulate fraction isolated from sea urchin eggs, Ca²⁺influx into eggs and formation of a fertilization membrane in an appreciable number of eggs. Their minimum effective concentrations for inducing a fertilization membrane increased in the order of these drugs listed above, and this order was also the same as that of their minimum effective concentrations for inducing Ca²⁺release from the isolated particulate fraction. Their effect in inducing a fertilization membrane was blocked by ruthenium red and procaine, which inhibit Ca²⁺release from Ca²⁺stores. Thus these drugs probably induced sufficient Ca²⁺release to make the cytosolic Ca²⁺level high enough in many eggs for formation of a fertilization membrane. In the absence of external Ca²⁺, fewer eggs treated with these drugs formed a fertilization membrane and more eggs did so on further treatment with either A23187 or carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP). Thus, a high level of Ca²⁺is probably derived from Ca²⁺release through Ca²⁺releasing channels (by A23187), from mitochondria (by FCCP) and its transport from the external medium.     

Intracellular Microinjection of Anticalmodulin Drugs Does not Inhibit the Cortical Reaction Induced by Fertilization, Ionophore A 23187 or Injection of Calcium Buffers in Sea Urchin Eggs

The drugs, fluphenazine, chlorpromazine, dibucaine, propranolol, vinblastine and W7[N-(6-arninohexyl)-5 chloro-1-napthalene-sulfonamide], which have been shown to prevent formation of the ternary activated complex of Ca⁺⁺-calmodulin with several soluble or membrane proteins, inhibit the cortical reaction induced by fertilization, by ionophore A 23187 or by the microinjection of Ca⁺⁺ buffers when applied from outside to sea urchin eggs. In contrast, direct intracellular microinjection of these drugs, even at concentrations much exceeding their I₅₀ for external application, does not suppress elevation of the fertilization membrane, although it prevents cleavage after fertilization. The implication is that intracellular calmodulin is not the receptor of Ca⁺⁺ in the Ca⁺⁺-dependent exocytosis of cortical granules induced by fertilization, by ionophore, or by the micro-injection of calcium buffers.     

Involvement of Wheat Germ Agglutinin (WGA)-Binding Protein in the Induction of the Acrosome Reaction of the Sea Urchin, Strongylocentrotus intermedius. II. Antibody against WGA-Binding Protein Induces the Acrosome Reaction

We obtained a polyclonal antibody against the WGA-binding protein (WGAbp) of Strongylocentrotus intermedius sperm, which is a membrane glycoprotein of 260 kD under non-reducing condition. Anti-WGAbp antibody induced increases in both intracellular Ca²⁺ ([Ca²⁺]i) and intracellular pH (pHi), resulting in the onset of the AR. The increases in [Ca²⁺]i and pHi required extracellular Ca²⁺ and Na⁺, respectively, and were suppressed by the pretreatment with WGA, resulting in the inhibition of the AR. Anti-WGAbp antibody-induced AR was inhibited also by lowered extracellular pH. elevated K⁺, removal of Na⁺ from seawater and the treatment with verapamil, a Ca²⁺ channel inhibitor. These inhibitory conditions are identical with those of the egg jelly-induced AR. Monovalent Fab fragments from anti-WGAbp antibody also induced the AR at relatively high concentration. These results suggest that the WGAbp on the sperm plasma membrane is involved in the regulation of Ca²⁺ influx and Na⁺/H⁺ exchange associated with the AR of S. intermedius sperm. It is a strong candidate for the receptor of the AR-inducing substance in the egg jelly.     

Activation of Ca2+ Transport System of Sea Urchin Sperm by High External pH: 220 kD Membrane Glycoprotein is Involved in the Regulation of the Ca2+ Entry

When sperm of the sea urchin, Hemicentrotus pulcherrimus , were exposed to high pH (9.0) sea water, they showed large increases in intracellular Ca²⁺ ([Ca²⁺]i) and pH (pHi) and underwent the acrosome reaction (AR) without the aid of the egg jelly. Not only [Ca²⁺]i increase but also pHi rise did not occur under Ca²⁺-free conditions. Both the increases in [Ca²⁺]i and pHi and the AR by high pH were inhibited by a Ca²⁺ channel blockers, verapamil and nisoldipine, and by a lectin, wheat germ agglutinin (WGA) which interacts with a 220 kD membrane glycoprotein of sperm. These reagents inhibited also the AR by the egg jelly. The inhibitory effects of WGA were immediately canceled by the addition of N-acetyl-D-glucosamine, a sugar which is known to remove WGA from its binding site. These results suggest that 1) the same Ca²⁺ transport system is activated by high external pH and the egg jelly, 2) increase in [Ca²⁺]i is prerequisite for the stimulation of the H⁺-efflux system(s) and 3) the 220 kD WGA-binding membrane protein functions as a regulator protein of Ca²⁺ transport system.     

Fertilization envelope formation induced by melittin in sea urchin eggs does not depend on Ca2+ signalling

In sea urchin eggs, 10 μg/mL melittin was found to induce fertilization envelope formation without any increase in [Ca²⁺]_i (the intracellular free Ca²⁺ level). On the other hand, 10 μmol/L Br-A23187 and 100 μg/mL SDS induced fertilization envelope formation associated with [Ca²⁺]_i increase. If EGTA was injected into eggs to make an intracellular concentration of 2 mmol/L, [Ca²⁺]_i became quite low and was not altered by melittin, or by Br-A23187 and SDS. In eggs containing EGTA, fertilization envelope formation was induced by melittin even in Ca²⁺-free artificial sea water, but not by Br-A23187 or SDS. Thus [Ca²⁺]_i is essential for induction of a fertilization envelope in sea urchin eggs by Br-A23187 or SDS but not by melittin. Melittin probably activates some Ca²⁺-independent reaction downstream of Ca²⁺-dependent reactions in a sequential reaction system that finally results in fertilization envelope formation.     

Ca2+ release from intracellular stores by thapsigargin in sea urchin eggs: Relationship to larval development and relevance in egg activation

Thapsigargin (Tg), an inhibitor of microsomal Ca²⁺ ATPase, is used as a tool to study the changes in Ca²⁺ sequestration in sea urchin eggs and their relationship to embryonic development. Micromolar amounts of Tg inhibit ATP-dependent Ca²⁺ sequestration in a dose-dependent and non-reversible manner, depending on the bulk of biological material used. IC_5O values are 1 nmol/L and 1–10μmol/L, respectively, in the cortical Ca²⁺ stores (isolated cortices preparation) and in digitonin-permeabilized eggs, a preparation giving access to the deeper reticulum compartment. Micromolar Tg does not induce Ca²⁺ release from ⁴⁵Ca pre-loaded cortices but leads to a loss of 25% of the total Ca²⁺ content from the cortical area. Using microspectrofluorimetry of fura-2-loaded eggs, we found that 10 μmol/L Tg induced a moderate rise in cytosolic Ca²⁺ activity as compared with the fertilization-induced Ca²⁺ transient whether eggs were fertilized or not. Early events related to fertilization as, for example, elevation of the fertilization envelope, proton excretion and sustained increase of amino acid uptake, are triggered by 10μmol/L Tg but with a delayed onset relative to sperm-induced effects. The present findings indicate that although it triggers most fertilization-related events, Tg cannot be considered as a true mitotic agent in sea urchin eggs. When added after fertilization, Tg affects cleavage and the further embryonic development giving rise to abnormalities comparable to the animalized larvae obtained with other compounds responsible for the inhibition of reticular Ca²⁺ sequestration.     

DIVALENT CATION-ACTIVATED ECTO-NUCLEOSIDE TRIPHOSPHATASE ACTIVITY OF NERVOUS SYSTEM CELLS IN TISSUE CULTURE

Abstract— Intact neuroblastoma and glial cells in monolayer culture hydrolysed ATP added to their medium. Evidence is presented that ATP is cleaved outside of the permeability barrier of the plasma membrane and the product is liberated in the extracellular medium, i.e. the enzyme is an ecto-enzyme. Divalent cations such as Mg²⁺, Ca²⁺, Mn²⁺ and Co²⁺ activate the enzyme. In neuroblastoma cells, Ca²⁺ is the preferential cation for activation; Mg²⁺ in glial cells. Substrate specificity was very low when different nucleoside-5'-triphosphates were examined. Competition studies have revealed that all of the nucleoside triphosphates are hydrolysed by the same enzyme: divalent cation-activated ecto-nucleoside-5'-triphosphate phosphohydrolase.
Developmental pattern of the enzyme in several lines was established. The role of enzyme in the transport of divalent cations across the plasma membrane and/or in the physical properties of the membrane is suggested.     

Activation of Xenopus Eggs by an Extract of Cynops Sperm

An extract obtained from Cynops sperm induced the activation of both Cynops and Xenopus eggs with accompanying changes in the potential of the egg membrane that were quite similar to those caused by the Cynops sperm. The activation-inducing properties of the extract were abolished by treatment with proteinase K or by heating (60°C, 15 min) and were associated with a protease activity against peptidyl Arg-MCA substrates. The activation of Xenopus eggs by the extract was inhibited by those substrates, or by protease inhibitors, aprotinin or leupeptin. The protease activity was localized in the acrosomal region of Cynops sperm. The activation of Xenopus eggs by the extract was prevented when the exterior concentration of Ca²⁺ions, [Ca²⁺]₀, was reduced to 1.5 μM, but it was enhanced when [Ca²⁺]₀ was increased to 340 μM. The activation of Xenopus eggs by the extract was not affected by positive clamping when [Ca²⁺]₀ was 340 μM. These results suggest that the sperm extract contains a protease that causes an increase in the influx of Ca²⁺ions that results in voltage-insensitive activation of the egg.     

Biochemical Studies on the Acrosome Reaction of the Starfish, Asterias Amurensis I. Factors Participating in the Acrosome Reaction

In contrast with the case in sea urchin sperm, in starfish the acrosome reaction is not spontaneously induced by simply increasing the extracellular Ca²⁺ concentration or pH. At higher pHs, starfish sperm undergo morphological changes accompanied by exocytosis of the acrosomal vacuole, but they do not form acrosomal filaments. Nomarski-microscopic observation confirmed that spermatozoa undergo the acrosome reaction within the jelly coat. Acrosome reaction-inducing substance, a glycoprotein from the egg jelly, required a diffusible cofactor(s) present in the egg jelly for full activity. Several lines of evidence showed that this diffusible factor(s) is not merely Ca²⁺.     

The Acrosome Reaction Induced by Dimethylsulfoxide in Sea Urchin Sperm

The acrosome reaction in sea urchin sperm, as judged by disappearance of the acrosomal vesicles in Nomarski optics, was induced by dimethylsulfoxide (DMSO) at concentration above 0.1% in normal artificial sea water. The number of the acrosome-reacted spermatozoa increased in proportion to DMSO concentration. The DMSO-induced acrosome reaction, as well as the jelly water- or A23187-induced one, was inhibited by nifedipine and hardly occurred in Ca²⁺-free artificial sea water. However, the DMSO-induced acrosome reaction was found in a few number of spermatozoa in the presence of Ca²⁺at above 0.5 mM, though the jelly water- or A23187-induced acrosome reaction did not occur at external Ca²⁺levels lower than 1 mM. Dependency of the acrosome reaction by DMSO on external Ca²⁺is somewhat lower than that of the reaction by jelly water. In Ca²⁺-free artificial sea water, the acrosomal regions of DMSO-treated spermatozoa attached to their own tails. In some cases, spermatozoa thus treated with DMSO in Ca²⁺free artificial sea water caused formation of fertilization membrane in a few number of eggs kept in Ca²⁺-free artificial sea water. Even in the absence of extermal Ca²⁺, preliminary step of the acrosome reaction seems to be completed probably by DMSO-induced weak Ca²⁺-mobilization in spermatozoa.     

Inhibitory Effects of Diltiazem and Verapamil, Calcium Antagonists, on Fertilization-Induced Increase in the Phosphorylase Reaction in Echiuroid Eggs

The calcium antagonists diltiazem and verapamil at 100 μM caused considerable inhibition of the glycolysis system in recently fertilized eggs of the echiuroid, Urechis unicinctus . The levels of glycolytic intermediates in eggs were found to be higher 5 min after insemination than before fertilization while the levels of adenine nucleotides and inorganic phosphate were almost the same before and after fertilization. Addition of diltiazem or verapamil 30 sec after insemination did not inhibit fertilization, but resulted in maintenance of as low levels of glycolytic intermediates as in unfertilized eggs. The apparent mass action ratio in the phosphorylase step, calculated from the levles of glucose-1-phosphate and inorganic phosphate was normally higher in fertilized eggs than in unfertilized eggs, but was maintained at as low a level as in unfertilized eggs by adding these compounds 30 sec after insemination. Phosphorylase a activity also normally increased after insemination, but was maintained at a low level in fertilized eggs by adding these compounds. These compounds also inhibited the increased ⁴⁵Ca²⁺ uptake normally observed after fertilization. These results suggest that after fertilization, the Ca²⁺ level increases associated with fertilization-induced Ca²⁺ influx and that this stimulates Ca²⁺ dependent protein kinase to phosphorylate phosphorylase b , resulting in an increased rate of the phosphorylase reaction.