Specific strychnine binding sites on acrosome-associated membranes of golden hamster spermatozoa

This study demonstrates for the first time, that membrane vesicles originated from the hamster sperm head after the occurrence of the acrosome reaction, possess specific strychnine binding sites. [3H]Strychnine binding was saturable and reversible, being displaced by unlabeled strychnine (IC(50)=26.7+/-2.3 microM). Kinetic analysis revealed one binding site with K(d)=120nM and B(max)=142fmol/10(6) spermatozoa. Glycine receptor agonists beta-alanine and taurine inhibited strychnine binding by 20-30%. Surprisingly, glycine stimulated binding by about 40-50%. Results obtained in this study strongly suggest the presence of glycine receptors-with distinctive kinetic properties on the periacrosomal plasma membrane of hamster spermatozoa. Localization of this receptor fits well with its previously proposed role in acrosomal exocytosis during mammalian fertilization.     

A role for the human sperm glycine receptor/Cl(-) channel in the acrosome reaction initiated by recombinant ZP3.

Previously, we have demonstrated an essential role for the neuronal glycine receptor (GlyR) in the acrosome reaction (AR) of mouse and porcine sperm initiated by the egg zona pellucida (ZP). In the present study, we have demonstrated presence of the GlyR in human sperm by immunoprecipitation and Western blot analysis, investigated the potential of a recombinant human ZP3 (rhZP3) preparation as an alternative research tool to solubilized human ZP, and shown that the human sperm GlyR is essential to the human AR initiated by rhZP3. Additionally, we have been able to demonstrate that rhZP3 possesses biological activity, because it is able to rapidly stimulate the AR in capacitated human sperm and its action is blocked by the addition of pertussis toxin. Moreover, spectrofluorometric studies using fura-2-loaded human sperm have shown that rhZP3 triggers a peak-and-plateau rise in intracellular Ca(2+) levels similar to that seen with solubilized mammalian ZP. These results suggest that the actions of rhZP3 and solubilized ZP are elicited via the same signal transduction pathways. Furthermore, incubation of human sperm with an antibody directed against the alpha1 subunit of the human spinal cord GlyR or with 50 nM strychnine caused significant inhibition in the rhZP3-initated AR. Finally, studies using fura-2-loaded human sperm showed that 50 nM strychnine was also able to inhibit the Ca(2+) influx associated with addition of rhZP3. These results further support the view that rhZP3 and the ZP work through the same mechanisms, show that the GlyR is involved in rhZP3-initiated AR, and suggest that the GlyR may also play a role in the early signal transduction cascades associated with ZP-initiated AR in vivo.     

The zona pellucida-initiated acrosome reaction: defect due to mutations in the sperm glycine receptor/Cl(-) channel

The mammalian sperm acrosome reaction (AR) is essential to fertilization, and the egg zona pellucida (ZP) is generally believed to be an in vivo initiator of the fertilizing sperm AR. Previously a neuronal glycine receptor/Cl(-) channel (GlyR) was detected on the plasma membrane of mammalian sperm and earlier pharmacological studies suggested that this receptor/channel is important to the ZP-initiated AR. Here, sperm from mice with mutations in the neuronal GlyR alpha or beta subunits (spasmodic and spastic) were shown to be deficient in their ability to undergo the AR initiated in vitro by glycine or by solubilized ZP from mouse eggs. However, both spontaneous and calcium ionophore (A23187)-initiated AR were unaffected. The ZP-initiated AR in wild-type sperm was maximal after 2 h of capacitation, but capacitation of sperm from spasmodic mice for up to 3 h did not result in significant ZP-initiated AR. Similar results were observed when sperm from wild-type and spastic mice were compared. Testis from mice with the beta subunit mutation contained truncated beta subunit mRNAs. Moreover, a monoclonal antibody against GlyR completely blocked ZP initiation of AR in normal mouse sperm. Our results are consistent with an essential role for the sperm GlyR in the ZP-initiated AR.     

Expression of the human glycine receptor alpha 1 subunit in Xenopus oocytes: apparent affinities of agonists increase at high receptor density.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel, which mediates post-synaptic inhibition in spinal cord and other brain regions. Heterologous expression of the ligand binding alpha subunits of the GlyR generates functional agonist-gated chloride channels that mimic most of the pharmacological properties of the receptor in vivo. Here, nuclear injection into Xenopus oocytes of a human alpha 1 subunit cDNA, engineered for efficient expression, was used to create GlyR channels over a wide density range, resulting in whole-cell glycine currents of 10 nA to 25 microA. Notably, the pharmacology of these channels changed at high expression levels, with the appearance of a novel receptor subpopulation of 5- to 6-fold higher apparent agonist affinity at current values > 4 microA. The low-affinity receptors were readily blocked by nM concentrations of the competitive antagonist strychnine, whereas the high-affinity receptors were more resistant to antagonism by this alkaloid. Picrotoxinin, a chloride channel blocker, inhibited both GlyR populations with equal potency. Our data suggest that receptor interactions, occurring at high receptor density, modify the agonist response of the GlyR. This phenomenon may contribute to neurotransmitter efficacy at fast synapses.     

Sulfhydryl Groups Modulate the Allosteric Interaction Between Glycine Binding Sites at the Inhibitory Glycine Receptor

We have investigated the effect of chemical reagents that modify sulfhydryl groups on the ligand binding properties of the glycine receptor (GlyR). The Hill coefficient (nH) for the displacement of [3H]strychnine binding by glycine was increased from approximately 0.8 to values significantly above 1 (approximately 1.2-1.4) in membranes pretreated with the disulfide-reducing agent dithiothreitol or glutathione. However, the affinity of strychnine or glycine for the GlyR was not affected by these treatments. This indicates that several glycine binding sites interact cooperatively for displacing bound strychnine under such experimental circumstances. A similar increase in the nH for glycine has been observed when the temperature of the binding assay was increased to 37 degrees C. Combination of dithiothreitol pretreatment and increased binding temperature led to nH variations similar to those observed with either of these treatments alone, a finding suggesting that their mechanisms of action are not independent. Conversely, modification of rat spinal cord membranes or of purified and reconstituted GlyR preparations with the sulfhydryl-alkylating agent N-ethylmaleimide or fluorescein-maleimide decreased nH values to approximately 0.5, without affecting glycine or strychnine affinities. This effect may be caused by an increased heterogeneity of GlyR populations. It is interesting that occupancy of the receptor by glycine or beta-alanine (but not by antagonists) specifically protects from the effects of the different sulfhydryl reagents. Moreover, the presence of some of the Eccles' anions, i.e., anions that permeate through the channels associated with GlyRs and gamma-aminobutyric acidA receptors, seems to be required for the action of both dithiothreitol and N-ethylmaleimide.(ABSTRACT TRUNCATED AT 250 WORDS)     

The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element.

A loop structure, formed by the putative disulfide bridging of Cys198 and Cys209, is a principal element of the ligand binding site in the glycine receptor (GlyR). Disruption of the loop's tertiary structure by Ser mutations of these Cys residues either prevented receptor assembly on the cell surface, or created receptors unable to be activated by agonists or to bind the competitive antagonist, strychnine. Mutation of residues Lys200, Tyr202 and Thr204 within this loop reduced agonist binding and channel activation sensitivities by up to 55-, 520- and 190-fold, respectively, without altering maximal current sizes, and mutations of Lys200 and Tyr202 abolished strychnine binding to the receptor. Removal of the hydroxyl moiety from Tyr202 by mutation to Phe profoundly reduced agonist sensitivity, whilst removal of the benzene ring abolished strychnine binding, thus demonstrating that Tyr202 is crucial for both agonist and antagonist binding to the GlyR. Tyr202 also influences receptor assembly on the cell surface, with only large chain substitutions (Phe, Leu and Arg, but not Thr, Ser and Ala) forming functional receptors. Our data demonstrate the presence of a second ligand binding site in the GlyR, consistent with the three-loop model of ligand binding to the ligand-gated ion channel superfamily.     

Localization of the strychnine binding site on the 48-kilodalton subunit of the glycine receptor

Amino acid residues that participate in antagonist binding to the strychnine-sensitive glycine receptor (GlyR) have been identified by selectively modifying functional groups with chemical reagents. Moreover, a region directly involved with strychnine binding has been localized in the 48-kDa subunit of this receptor by covalent labeling and proteolytic mapping. Modification of tyrosyl or arginyl residues promotes a marked decrease of specific [3H]strychnine binding either to rat spinal cord plasma membranes or to the purified GlyR incorporated into phospholipid vesicles. Occupancy of the receptor by strychnine, but not by glycine, completely protects from the inhibition caused by chemical reagents. Furthermore, these tyrosine- or arginine-specific reagents decrease the number of binding sites (Bmax) for [3H]strychnine binding without affecting the affinity for the ligand (Kd). These observations strongly suggest that such residues are present at, or very close to, the antagonist binding site. In order to localize the strychnine binding domain within the GlyR, purified and reconstituted receptor preparations were photoaffinity labeled with [3H]strychnine. The radiolabeled 48-kDa subunit was then digested with specific chemical proteolytic reagents, and the peptides containing the covalently bound radioligand were identified by fluorography after gel electrophoresis. N-Chlorosuccinimide treatment of [3H]strychnine-labeled 48K polypeptide yielded a single labeled peptide of Mr approximately 7300, and cyanogen bromide gave a labeled peptide of Mr 6200.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional chloride channels by mammalian cell expression of rat glycine receptor subunit

Cultured human cells were transfected with cloned rat glycine receptor (GlyR) 48 kd subunit cDNA. In these cells glycine elicited large chloride currents (up to 1.5 nA), which were blocked by nanomolar concentrations of strychnine. However, no corresponding high-affinity binding of [3H]strychnine was detected in membrane preparations of the transfected cells. Analysis by monoclonal antibodies specific for the 48 kd subunit revealed high expression levels of this membrane protein. After solubilization, the 48 kd subunit behaved as a macromolecular complex when analyzed by sucrose density centrifugation. Approximately 50% of the solubilized complex bound specifically to a 2-aminostrychnine affinity column, indicating the existence of low-affinity antagonist binding sites on most of the expressed GlyR protein. Thus, the 48 kd strychnine binding subunit efficiently assembles into high molecular weight complexes, resembling the native spinal cord GlyR. However, formation of functional receptor channels of high affinity for strychnine occurs with low efficiency.     

Characterization of Strychnine-sensitive Glycine Receptor in the Intact Frog Retina: Modulation by Protein Kinases

We studied ³H-glycine and ³H-strychnine specific binding to glycine receptor (GlyR) in intact isolated frog retinas. To avoid glycine binding to glycine uptake sites, experiments were performed at low ligand concentrations in a sodium-free medium. The binding of both radiolabeled ligands was saturated. Scatchard analysis of bound glycine and strychnine revealed a K_D of 2.5 and 2.0 M, respectively. Specific binding of glycine was displaced by -alanine, sarcosine, and strychnine. Strychnine binding was displaced 50% by glycine, and sarcosine. Properties of the strychnine-binding site in the GlyR were modified by sarcosine. Binding of both radioligands was considerably reduced by compounds that inhibit or activate adenylate cyclase and increased cAMP levels. A phorbol ester activator of PKC remarkably decreased glycine and strychnine binding. These results suggest modulation of GlyR in response to endogenous activation of protein kinases A and C, as well as protein phosphorylation modulating GlyR function in retina.     

Studies of sperm from mutant mice suggesting that two neurotransmitter receptors are important to the zona pellucida-initiated acrosome reaction

Two sperm neurotransmitter receptor/channels, the glycine receptor (GlyR) and a nicotinic acetylcholine receptor containing an alpha7 subunit (alpha7nAChR) were previously shown to be important to the mouse acrosome reaction (AR) initiated by solubilized egg zona pellucida (ZP). Here, we investigated whether sperm from homozygous mutant mice with a single amino acid mutation in the alpha subunit of their GlyR and sperm from homozygous mutant mice with an engineered disruption of the gene for the nicotinic acetylcholine receptor alpha7 subunit could undergo the AR on ZP-intact eggs. Wild-type and mutant sperm were treated with 3-quinuclidinyl benzilate (QNB), known to be an inhibitor of the ZP-initiated AR (but shown in the present work not to inhibit the acetylcholine-initiated AR). The ZP-initiated AR on ZP-intact eggs should occur only in sperm not treated with QNB. The absence of such an increase in the untreated mutant sperm would demonstrate that such sperm were unable to respond to the intact ZP. The results demonstrated for the first time that GlyR mutant sperm do not undergo the AR on ZP-intact mouse eggs, and that their ability to fertilize is inhibited by 63% in vitro. Moreover, we found that GlyR mutant sperm exhibited normal capacitation and confirmed that they not undergo the AR initiated by solubilized mouse ZP. Our studies demonstrated for the first time that sperm from mutant alpha7nAChR mice exhibit normal capacitation, do not undergo the AR in response to acetylcholine, solubilized ZP or on ZP-intact eggs, and display a 25% reduction in fertilization in vitro. This is the first genetic evidence for the importance of the alpha7nAChR in the ZP-initiated AR. While defects in either the GlyR or the alpha7nAChR completely inhibit the ZP-initiated AR, fertilization by these mutant sperm can still occur in vitro, probably due to sperm that complete spontaneous AR on the ZP.     

The neurotoxicants strychnine and bicuculline protect renal proximal tubules from mitochondrial inhibitor-induced cell death.

Glycine-induced cytoprotection of renal proximal tubules exposed to chemical- or hypoxic/anoxic-induced cell death is shared by a few amino acid agonists of the neuronal strychnine-sensitive glycine receptor. The goal of this study was to determine if antagonists of the strychnine-sensitive glycine receptor attenuated the cytoprotective effects of glycine. Strychnine did not antagonize the cytoprotective effects of glycine in proximal tubules exposed to antimycin A. In contrast, strychnine was cytoprotective, was equipotent as glycine (EC50 = 0.4 mM), and the combination of strychnine and glycine was additive. Likewise, bicuculline and norharmane were cytoprotective but 20-50% less potent than glycine. These results suggest that glycine and strychnine act as a common site to produce proximal tubule cytoprotection, but this site does not share the same potency and agonist/antagonist properties as the neuronal strychnine-sensitive glycine receptor.     

Studies related to progesterone-induced hamster sperm acrosome reaction

Experiments were designed to characterize the effect of progesterone on the hamster sperm acrosome reaction (AR). Progesterone stimulated exocytosis of previously capacitated spermatozoa in a dose-dependent manner Progesterone-3-(O-carboxymethyl)oxime:BSA conjugate also induced AR when added to capacitated sperm suspensions. EGTA and La³⁺, added 10 min before progesterone, completely abolished the steroid-stimulatory effect. Benzamidine, a trypsin inhibitor, also inhibited AR when added to sperm cells 10 min before progesterone. This effect was avoided when spermatozoa were treated with the Ca2+ ionophore ionomycin. Conversely, the H+ ionophore PCCP, or the Na⁺/K⁺ ionophore nigericin, did not prevent the effect of the inhibitor. Results suggest that progesterone acts on the hamster sperm plasma membrane to stimulate exocytosis, which requires external Ca2+ and presumably Ca2+ influx. In addition, a sperm trypsin like protease may be part of the mechanism by which progesterone stimulates AR. Since the ionomycin-induced AR does not require this proteolytic activity, the possible involvement of such an enzyme in the progesterone-stimulated Ca2+ influx necessary for the occurrence of AR is discussed. © 1996 Wiley-Liss, Inc.     

Glycine Antagonists Structurally Related to 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol Inhibit Binding of [3H]Strychnine to Rat Brain Membranes

[3H]Strychnine binding to rat pons + medulla membranes was used as a measure of glycine receptors or glycine receptor-coupled chloride channels in vitro. A series of compounds structurally related to 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), which previously were shown to antagonize glycine responses in cat spinal cord, inhibited [3H]strychnine binding in micromolar concentrations. The most potent of these glycine antagonists, 5,6,7,8-tetrahydro-4H-isoxazolo[3,4-d]azepin-3-ol (iso-THAZ), was also the most potent inhibitor of [3H]strychnine binding, with a Ki of 1,400 nM. The Ki value for strychnine was 7.0 nM, whereas the Ki value for the mixed gamma-aminobutyric acid (GABA)/glycine antagonist 3 alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (RU 5135) was only 4.6 nM. Sodium chloride (1,000 mM) enhanced the affinity of strychnine, brucine, isostrychnine, and the nonselective GABA antagonist pitrazepin for [3H]strychnine binding sites, whereas the affinities of glycine, beta-alanine, and taurine were reduced. These sodium chloride shifts, however, were not predictive of antagonist or agonist properties, since the sodium chloride shift for the glycine antagonist iso-THAZ and of the other THIP-related antagonists were similar to those of the glycine-like agonists. The various sodium chloride shifts show that different groups of ligands bind to glycine receptor sites in different ways.     

Bull sperm acrosome reaction induced by gamma-aminobutyric acid (GABA) is mediated by GABAergic receptors type A

The effect of gamma-aminobutyric acid (GABA) on the bull sperm acrosome reaction was evaluated, and the interaction of progesterone, a physiologic inducer of the acrosome reaction, with the GABA receptor was explored. The acrosome reaction was stimulated by GABA in a dose-dependent manner. This effect was inhibited completely by bicuculline, a GABA A receptor antagonist, but GABA B and C receptor antagonists had no effect. Accordingly, muscimol, a GABA A receptor agonist, stimulated the acrosome reaction to the same extent as GABA, whereas baclofen (GABA B receptor agonist) and CACA (GABA C receptor agonist), had no effect. Preincubation with progesterone followed by the addition of GABA resulted in a significant increase in the percentage of acrosome reacted spermatozoa compared with progesterone or GABA alone. Taking into account that this increase was less than a simple addition of effects, it might be suggested that GABA and progesterone act through the same receptor and/or use the same mechanism of action. To test this hypothesis, the abilities of GABA and progesterone to induce acrosome reaction were tested in the presence of bicuculline, which suppressed both stimulatory effects. Given that the GABA A receptor is linked to the Cl(-) channel, we tested whether picrotoxin, a blocker of this channel, could modulate the effects of progesterone or GABA. Cl(-) channel blocker picrotoxin dramatically reduced the GABA and progesterone-initiated AR. In conclusion: GABA and progesterone stimulate the acrosome reaction in bull spermatozoa acting through a classical GABA A receptor. The mechanism of action requires the functional integrity of the Ca(2+) Cl(-) channel.     

Ivermectin, an unconventional agonist of the glycine receptor chloride channel

The effects of the antihelmintic, ivermectin, were investigated in recombinantly expressed human alpha(1) homomeric and alpha(1)beta heteromeric glycine receptors (GlyRs). At low (0.03 microm) concentrations ivermectin potentiated the response to sub-saturating glycine concentrations, and at higher (> or =0.03 microm) concentrations it irreversibly activated both alpha(1) homomeric and alpha(1)beta heteromeric GlyRs. Relative to glycine-gated currents, ivermectin-gated currents exhibited a dramatically reduced sensitivity to inhibition by strychnine, picrotoxin, and zinc. The insensitivity to strychnine could not be explained by ivermectin preventing the access of strychnine to its binding site. Furthermore, the elimination of a known glycine- and strychnine-binding site by site-directed mutagenesis had little effect on ivermectin sensitivity, demonstrating that the ivermectin- and glycine-binding sites were not identical. Ivermectin strongly and irreversibly activated a fast-desensitizing mutant GlyR after it had been completely desensitized by a saturating concentration of glycine. Finally, a mutation known to impair dramatically the glycine signal transduction mechanism had little effect on the apparent affinity or efficacy of ivermectin. Together, these findings indicate that ivermectin activates the GlyR by a novel mechanism.     

Zinc Potentiation of the Glycine Receptor Chloride Channel Is Mediated by Allosteric Pathways

Abstract: Molecular mechanisms of zinc potentiation were investigated in recombinant human α1 glycine receptors (GlyRs) by whole-cell patch-clamp recording and [³H]strychnine binding assays. In the wild-type (WT) GlyR, 1 µ M zinc enhanced the apparent binding affinity of the agonists glycine and taurine and reduced their concentrations required for half-maximal activation. Thus, in the WT GlyR, zinc potentiation apparently occurs by enhancing agonist binding. However, analysis of GlyRs incorporating mutations in the membrane-spanning domain M1–M2 and M2–M3 loops, which are both components of the agonist gating mechanism, indicates that most mutations uncoupled zinc potentiation from glycine-gated currents but preserved zinc potentiation of taurine-gated currents. One such mutation in the M2–M3 loop, L274A, abolished the ability of zinc to potentiate taurine binding but did not inhibit zinc potentiation of taurine-gated currents. In this same mutant where taurine acts as a partial agonist, zinc potentiated taurine-gated currents but did not potentiate taurine antagonism of glycine-gated currents, suggesting that zinc interacts selectively with the agonist transduction pathway. The intracellular M246A mutation, which is unlikely to bind zinc, also disrupted zinc potentiation of glycine currents. Thus, zinc potentiation of the GlyR is mediated via allosteric mechanisms that are independent of its effects on agonist binding.     

A single amino acid exchange alters the pharmacology of neonatal rat glycine receptor subunit

Agonist activation of the inhibitory glycine receptor (GlyR) in the adult vertebrate CNS is efficiently antagonized by the alkaloid strychnine. Here, we describe a novel rat GlyR alpha subunit cDNA (alpha 2*) that generates chloride channels of low strychnine sensitivity upon expression in Xenopus oocytes. Comparison with the highly homologous human alpha 2 polypeptide and site-directed mutagenesis identified a single amino acid exchange at position 167 that causes the altered pharmacology of alpha 2* receptors. Amplification by the polymerase chain reaction revealed a strong decrease in alpha 2* mRNA abundancy during postnatal spinal cord development. These data indicate that alpha 2* represents a ligand binding subunit of the previously identified neonatal GlyR isoform of low strychnine affinity.     

Pacemaker activity in hydra is modulated by glycine receptor ligands

In the mammalian central nervous system, the neurotransmitter, glycine, acts both on an inhibitory, strychnine-sensitive receptor (GlyR) and an excitatory, strychnine-insensitive site at the NMDA receptor. Here we present electrophysiological evidence that the strychnine-sensitive glycine agonists, glycine and taurine, and the antagonist, strychnine, affect the endodermal rhythmic potential (RP) system and that the ectodermal contraction burst (CB) pacemaker system is modulated by glycine and strychnine in hydra. The RP and CB pacemaker systems are responsible for the respective elongation and contraction of hydra's body column. Activity of the CB system, quantified by the rate of contraction bursts (CBs), the number of pulses per contraction burst (P/CB), and the duration of bursts, was decreased by glycine. Glycine, coadministered with the strychnine-insensitive glycine site blocker, indole-2-carboxylic acid (I2CA), decreased RPs but not CBs or P/CB. The effect was mimicked by taurine. Strychnine increased the duration of RP production, and decreased CB duration. The effect of glycine with I2CA was counteracted by strychnine. The results support the idea that a vertebrate-like GlyR may be involved in modulating activity of the endodermal RP system and suggest that a glycine site on an NMDA receptor may be involved in the CB system.     

Progesterone induces acrosome reaction in stallion spermatozoa via a protein tyrosine kinase dependent pathway

Progesterone (P(4)) is a physiological inducer of the acrosome reaction (AR) in stallion spermatozoa. However, the capacitation-dependent changes that enable progesterone binding, and the nature of the signaling cascade that is triggered by progesterone and results in induction of the AR, are poorly understood. The aim of the current study was, therefore, to investigate the protein kinase dependent signaling cascades involved in progesterone-mediated induction of the AR in stallion spermatozoa. In addition, we aimed to determine whether bicarbonate, an inducer of sperm capacitation, acted via the same pathway as P(4) or whether it otherwise synergized P(4)-mediated induction of the AR. We examined the effect on AR progression of specific inhibitors and stimulators of protein kinase A (PKA), protein kinase C (PKC), protein kinase G (PKG), and protein tyrosine kinase (PTK), in the presence or absence of 15 mM bicarbonate and/or 1 microg/ml progesterone. Progression of the AR was assessed using the Pisum sativum agglutinin conjugated to fluorescein iso thiocyanate (PSA-FITC) staining technique. Bicarbonate specifically activated a PKA-dependent signaling pathway, whereas the effect of P(4) was independent of PKA. Conversely, while P(4)-mediated AR induction was dependent on PTK, the effects of bicarbonate were PTK-independent. Finally, although the AR inducing effects of both P(4) and bicarbonate were sensitive to staurosporin, a potent blocker of PKC activity at moderate (50 nM) concentrations, the effect of P(4) was completely blocked at 50 nM staurosporin, whereas that of bicarbonate was only completely inhibited by much higher concentrations (2 microM) where staurosporin also inhibits PKA activity. In conclusion, P(4)-mediated activation of the AR is dependent on a pathway that includes both PTK and PKC. While the effects of bicarbonate on the AR are mediated via a separate PKA-dependent signaling pathway, P(4) and bicarbonate have synergistic effects on the AR.