The resolution of dopamine and β1- and β2-adrenergic-sensitive adenylate cyclase activities in homogenates of cat cerebellum, hippocampus and cerebral cortex

The stimulation of adenylate cyclase by dopamine and various β-adrenergic agonists has been investigated in homogenates from 3 areas of cat brain: the cerebral cortex, cerebellum and hippocampus. The purpose of the study was to determine whether the β-adrenergic receptors coupled to adenylate cyclase could be classified as either β₁ and β₂ subtypes in the different regions studied.The stimulation of adenylate cyclase by the β-adrenergic agonist, (−)isoproterenol (5 × 10⁻⁶M), was completely blocked by the specific β-adrenergic antagonist, (−)alprenolol (10⁻⁵ M), but not by the dopaminergic antagonist, fluphenazine (10⁻⁵ M), whereas the stimulation of adenylate cyclase by (−)epinephrine (10⁻⁴ M) was blocked to varying extents by these two drugs in each of the 3 regions studied. The (−)epinephrine effect was always blocked in the combined presence of (−)alprenolol and fluphenazine. The adenylate cyclase stimulation by (−)epinephrine which is not blocked by (−)alprenolol was due to interaction of (−)epinephrine with a dopaminergic-sensitive adenylate cyclase which has been characterized in cerebral cortex, hippocampus and cerebellum.Regional differences in the affinity of β-adrenergic-sensitive adenylate cyclase for various agonists were investigated in the presence of fluphenazine (10⁻⁵ M). In the cerebellum the potency order was (±)protokylol> (±)hydroxybenzylisoproterenol> (±)isoproterenol> (−)epinephrine> (±)salbutamol> (−)norepinephrine, indicating the presence of a β₂-adrenergic receptor. In the cerebral cortex the potency order was (−)isoproterenol> (±)protokylol> (±)hydroxybenzylisoproterenol> (−)epinephrine= (−)norepinephrine((±)salbutamol being inactive). A similar pattern was found in the hippocampus indicating the presence of a β₁-adrenergic receptor in these two regions. (±)Salbutamol was a partial agonist in the cerebellum and a competitive antagonist in the cerebral cortex.The ratio of the antagonist potencies of (±)practolol and (±)butoxamine preferential β₁- and β₂-adrenergic antagonists respectively, to block the stimulation of adenylate cyclase was 25 in the cerebellum, compared to 0.5 in the cerebral cortex and 1.6 in the hippocampus. These results confirm the presence of a β₂ subtype of receptor coupled to adenylate cyclase in the former and β₁ subtypes in the latter two regions. The comparison between the affinities of a series of β-adrenergic agonists and antagonists for the β-adrenergic receptors coupled with an adenylate cyclase in cerebral cortex and cerebellum with their affinities for well characterized β₂-adrenergic receptors in lung and β₁-adrenergic receptor in heart substantiated this conclusion.     

Binding of [3H]-dihydroalprenolol and [3H]-acetobutolol to human blood platelets is not related to occupancy of beta-adrenoceptors

Binding of {³H}-dihydroalprenolol to human platelet lysates is inhibited by (±)-propranolol and (±)-butoxamine, but less effectively by (±) practolol. (−)-Isoprenaline causes no significant inhibition of binding where stimulation of adenylate cyclase can be shown. Binding of {³H}-acetobutolol is also inhibited by (±)-propranolol. “Specific” binding of {³H}-dihydroalprenolol and {³H}-acetobutolol defined by (±) propranolol shows a non-classical saturation curve. 50% maximal binding is observed in the range 15 – 25 mM. The extent of “specific” binding is 2-fold greater for {³H}-dihydroalprenolol. Similar and rapid rates of binding of {³H}-dihydroalprenolol are observed at 4°C and 20°C. No stereoselectivity is observed for inhibition of {³H}-dihydroalprenolol binding by (+) and (−)-propranolol. Binding of {³H}-dihydroalprenolol and {³H}-acetobutolol may relate to the lipophilic character of these radioligands and does not represent interaction with β-adrenoceptors.     

Effects of prostaglandin E2, forskolin and cholera toxin on cAMP production and in vitro LH-RH release from the rat hypothalamus

The present study attempts to elucidate the possible role of adenosine 3′,5′-monophosphate (cAMP) and prostaglandin E₂ (PGE₂) in the function of the neural luteinizing hormone-releasing hormone (LH-RH) apparatus. To this end, in vitro LH-RH release from superfused hypothalamic fragments and cAMP production by hypothalamic P₂ membrane fractions were measured. Immature female rats (day 28) were ovariectomized and implanted with Silastic capsules containing estradiol (235 μg/ml). Two days later, animals were sacrificed and the mediobasal hypothalamic preoptic area (hypothalamic units or fragments) were removed. To examine in vitro LH-RH release from superfused hypothalamic fragments, effluents were collected into tubes on ice at 10-min intervals and LH-RH concentration was determined by radioimmunoassay (RIA). Following a 50-min control period, a step-wise increment in several doses of PGE₂ (each dose for a 50-min interval) evoked a dose-related increase in LH-RH release. PGE₂ induced significant (P<0.01) increments in LH-RH release at doses of 5.68 × 10⁻⁷, 5.68 × 10⁻⁶, and 5.68 × 10⁻⁵ M, respectively. When adenylate cyclase activators, such as forskolin and cholera toxin were infused in a step-wise manner (each dose for a 50-min interval) following a 50-min control period, a dose-related increase in LH-RH release was also obtained; forskolin and cholera toxin significantly (P<0.01) stimulated LH-RH release at doses of 1 × 10⁻⁴ and 5.4 × 10⁻¹⁰ M, respectively. These two substances were ineffective in stimulating LH-RH release when hypothalamic fragments were superfused in calcium-free plus EGTA (10 mM) containing medium. An intermittent infusion of dibutyryl cAMP (dbcAMP: 1 × 10⁻⁷ M, 10-min on, 20-min off) resulted in rhythmic LH-RH release from median eminences superfused in vitro. In separate experiments, to examine adenylate cyclase activity, P₂ membrane fractions from the mediobasal hypothalamus were preincubated with appropriate test agents. Adenylate cyclase reaction was initiated by adding adenosine triphosphate. After a 15-min incubation, the reaction was terminated by boiling, the supernatant recovered and subjected to cAMP determination by RIA. The following results were obtained: (1) in vivo E priming of ovariectomized animals significantly increased basal adenylate cyclase activity of P₂ membrane preparations as compared to those from unprimed rats; (2) known adenylate cyclase activators, such as forskolin and cholera toxin clearly produced a dose-related increase in cAMP production; and (3) PGE₂ at the concentration of 5.68 × 10⁻⁶ M stimulated cAMP production. It appears that cAMP and PGE₂ may be involved in the activation of the LH-RH neural apparatus. It is tempting to postulate that PGE₂ may stimulate adenylate cyclase to increase intracellular cAMP levels which, in turn, trigger the release of LH-RH from the median eminence nerve terminals.     

Photoaffinity labeling of the β-adrenergic receptor in synaptic membranes of rat cerebral cortex and cerebellum

The β-adrenergic receptor polypeptides in synaptic membranes of rat cerebral cortex and cerebellum have been identified using the photoaffinity label[¹²⁵I]iodoazidobenzylpindolol. The major receptor polypeptides possess apparent molecular weights of 62, 000 and 49,000 (cerebral cortex) or 59,000 and 46,000 (cerebellum). Treatment of the membranes with endoglycosidase F caused the receptor polypeptides from both tissues to exhibit lower apparent molecular weight (51,000) on sodium dodecyl sulfate polyacrylamide gels, indicating that β-adrenergic receptors in mammalian brain are glycoproteins as are the receptors in peripheral tissues.     

Alpha1-adrenergic stimulation of ACTH secretion in vivo in the rat

The α-adrenergio agonist phenylephrine (100 μg, i.v.) causes a rapid 5- to 10-fold elevation of plasma ACTH levels 5 min after its administration in adult ovariectomized rats, the concentration of the hormone remaining elevated up to at least 2h. Epinephrine (10 μg) causes also a rapid but shorter-lived stimulation of ACTH secretion. While having no effect alone under basal conditions (conscious freely-moving animals), the highly specific α₁-adrenergic antagonist prazosin (0.25 μg) almost completely reverses the stimulatory effect of phenylephrine. Pretreatment with dexamethasone inhibits basal plasma ACTH levels by 70% and almost completely prevents the stimulatory effect of phenylephrine on this parameter. Plasma levels of α-MSH, on the other hand, are only stimulated 1-fold above control 5 min after the administration of phenylephrine and are insensitive to corticosteroid treatment. Based on the specificity of action of prazosin on postsynaptic α₁-adrenergic receptors and of dexamethasone on the anterior lobe of the pituitary gland, the present data indicate that phenylephrine is a potent stimulator of ACTH secretion by a direct action on an α₁-adrenergic receptor in corticotrophs of the adenohypophysis. They also support the suggestion that epinephrine and/or norepinephrine could be involved as physiological corticotropin-releasing factor(s).     

Effect of EDTA and PGE1 on beta-thromboglobulin liberation from platelets

The aim of this study was to evaluate the effect of PGE₁ and EDTA on liberation of β-thromboglobulin (βTG) from platelets in vitro. Liberation of BTG was followed in citrated blood at room temperature for 120 minutes after venesection. PGE₁ reduced βTG liberation, and maximal inhibition was attained by concentrations greater than 2 × 10⁻⁶M. EDTA induced the efflux of βTG. This EDTA-induced efflux was delayed but not prevented by PGE₁ and by citrate; it was not found at 0–4°C. Therefore the use of EDTA to prevent βTG liberation during sampling for in vitro or in vivo studies depends heavily on modifying factors such as PGE₁ and low temperature, and on the time taken to process samples. Its effectiveness must be in some doubt where the platelets may be sufficiently stimulated to overcome these modifying influences, or where handling of samples is less than optimal.     

The release of B beta 1-42 from fibrinogen and fibrin by plasmin

The balance between thrombin and plasmin action has been postulated to be an important determinant of thrombosis. Measurement of plasma concentrations of fibrinopeptide A (FPA), which reflect thrombin action on the NH₂-terminal end of the A chain, and of Bβ 1–42 (thrombin-increasable fibrinopeptide B immunoreactivity - TIFPB) which reflect plasmin action on the NH₂-terminal end of the Bβ chain have shown systematic changes in the relative concentrations of the two peptides in thrombotic states. This paper reports kinetic data for TIFPB release by plasmin using fibrinogen, fibrin I monomer, and fibrin I polymer as substrates. For fibrinogen and fibrin I monomer the data fit the Michaelis-Menten equation. Experiments were performed with human proteins in 0.15M Trisbuffered saline at pH 7.4 and at 37°C. With fibrinogen as substrate the K_m was calculated to be 0.87 μM and the V_max 3.75 × 10⁻⁵ M/min/unit of plasmin. With fibrin I monomer as the substrate the K_m was calculated to be 1.25 μM and the V_max 5.5 × 10⁻⁵ M/min/unit of plasmin. With fibrin I polymer as substrate the data did not fit the Michaelis-Menten equation but there appeared to be no dramatic differences in rates from those obtained with the other two substrates. The influence of factor XIIIa-induced cross-linking of fibrin was not examined. It is concluded from these findings that fibrinogen and non-cross-linked fibrin I are equally good substrates for plasmin cleavage of the NH₂-terminal end of the Bβ chain.     

Receptor expression in primary cultures of neurons or astrocytes

1. β-Adrenergic receptors are abundant on astrocytes which contain mainly the β₁ subtype. These cells also possess ₁ and ₂ receptors.

2. Neurons display fewer β-adrenergic receptor sites and those present are of the β₂ subtype.

3. Dopaminergic receptors are found on astrocytes and show a regional variation.

4. Serotonergic receptors have been demonstrated on astrocytes but are expressed to a larger extent in neurons.

5. Astrocytes display muscarinic receptors but have no receptor sites for the transmitter amino acids glutamate and GABA.

6. GABA receptors are present on cerebellar granule cells, are induced by the presence of GABA agonists in the medium and mediate an evoked release of glutamate.

7. Glutamate receptors are found on cultured cortical interneurons. Some, but not all, peptide hormones have receptors on astrocytes and/or neurons in primary cultures.     

Beta2-glycoprotein I, anti-beta2-glycoprotein I, and fibrinolysis

β₂-glycoprotein-I (β₂GPI) is a phospholipid-binding plasma protein that consists of five homologous domains. Domain V is distinguished from others by bearing a positively charged lysine cluster and hydrophobic extra C-terminal loop. β₂GPI has been known as a natural anticoagulant regulator. β₂GPI exerts anticoagulant activity by inhibition of phospholipid-dependent coagulation reactions such as prothrombinase, tenase, and factor XII activation. It also binds factor XI and inhibits its activation. On the other hand, β₂GPI inhibits anticoagulant activity of activated protein C. According to the data from knockout mice, β₂GPI may contribute to thrombin generation in vivo. Phospholipid-bound β₂GPI is one of the major target antigens for antiphospholipid antibodies present in patients with antiphospholipid syndrome (APS). Binding of pathogenic anti-β₂GPI antibodies increases the affinity of β₂GPI to the cell surface and disrupts the coagulation/fibrinolysis balance on the cell surface. These pathogenic antibodies activate endothelial cells via signal transduction events in the presence of β₂GPI. Impaired fibrinolysis has been reported in patients with APS. Using a newly developed chromogenic assay, we demonstrated lower activity of intrinsic fibrinolysis in euglobulin fractions from APS patients. Addition of monoclonal anti-β₂GPI antibodies with β₂GPI also decreased fibrinolytic activity in this assay system. β₂GPI is proteolytically cleaved by plasmin in domain V (nicked β₂GPI) and becomes unable to bind to phospholipids, reducing antigenicity against antiphospholipid antibodies. This cleavage occurs in patients with increased fibrinolysis turnover. Nicked β₂GPI binds to plasminogen and suppresses plasmin generation in the presence of fibrin, plasminogen, and tissue plasminogen activator (tPA). Thus, nicked β₂GPI plays a role in the extrinsic fibrinolysis via a negative feedback pathway loop.     

Stereoisomeric tetrahydro-β-carbolines differ in their interaction with rat brain benzodiazepine receptors

1. 1. A series of (+) and (−) tetrahydro-β-carboline-3-carboxamide stereoisomers related to the ethyl ester of β-carboline-3-carboxylate (β-CCE) have been tested for their ability to displace ³H-flunitrazepam from binding sites prepared from rat cerebral cortex (benzodiazepine receptors).

2. 2. The (+) stereoisomers of ethyl and propyl 1,2,3,4-tetrahydro-β-carboline-3-carboxamides were the most potent inhibitors of specific ³H-flunitrazepam binding and had significantly lower IC₅₀s than the corresponding (−) isomers.

3. 3. Although the IC₅₀ values for this series of tetrahydro-β-carboline-3-carboxamides were about 1000 fold higher than the IC₅₀ value for β-CCE, these compounds were as potent as the β-carboline alkaloids harmane and harmine.

4. 4. The title compounds may be useful stereoisomeric probes for the characterization of subtypes of specific benzodiazepine binding sites.

Beta-agkistrodotoxin inhibits large-conductance calcium-activated potassium channels in rat hippocampal CA1 pyramidal neurons

Effect of β-agkistrodotoxin (β-AgTx), a presynaptic neurotoxin purified from snake venom, on large-conductance calcium-activated potassium channels (BK_Ca) was studied in rat hippocampal CA1 pyramidal neurons using inside-out configuration of patch-clamp technique. The results showed that in equimolar K⁺ (150 mM) and 1 μM intracellular Ca²⁺ conditions, internal application of β-AgTx inhibited the activity of BK_Ca by reducing open probability (P_o) of the channels in a concentration-dependent manner. High concentration (74 nM) of β-AgTx completely eliminated opening of the channels. However, 37 nM β-AgTx (at −40 mV) decreased P_o from 0.49±0.07 to 0.03±0.03, switched two open time constants (0.51±0.32 and 8.77±1.63 ms) to be a single time constant of 0.46±0.40 ms. The results indicate that inhibition of BK_Ca by β-AgTx may account for the facilitatory phase of the toxin on acetylcholine release from nerve terminals.     

Effects of thiopental, halothane and isoflurane on the calcium-dependent and -independent release of GABA from striatal synaptosomes in the rat

The effects of the anesthetic agents thiopental, halothane and isoflurane on the release of GABA induced by depolarization and/or reversal of the GABA carrier were investigated in a synaptosomal preparation obtained from the rat striatum. Veratridine (1 μM) and KCl (9 mM) elicited a significant Ca²⁺-dependent release of [³H]GABA. The KCl-evoked release was not significantly modified in the presence of nipecotic acid (10⁻⁵ M), a selective blocker of the neuronal GABA carrier. The [³H]GABA release was significantly decreased by ω-conotoxin (10⁻⁷ M, a blocker of the N voltage-dependent Ca²⁺ channels, but was affected by neither nifedipine (10⁻⁴ M) nor ω-Aga-IVA (10⁻⁷ M) which block the L and Ca²⁺ channels, respectively. Thiopental application (10⁻⁵ to 10⁻³ M) was followed by a dose-related, significant, decrease in both the veratridine and KCl-induced releases, whether nipecotic acid was present or not. In contrast, halothane and isoflurane (1–3%) failed to alter [³H]GABA release. Altogether, these results suggest that reduction of the depolarization-evoked GABA release might contribute to thiopental anesthesia, but this seems unlikely for volatile anesthetics.     

β-Adrenergic receptor binding in brain of alcoholics

The binding of agonists and antagonists to β-adrenergic receptors in brain tissue obtained postmortem in nonalcoholic controls and matched intoxicated and sober alcoholics was measured to assess the state of the receptors and their coupling to adenylate cyclase. Binding of antagonist, iodocyanopindolol, to cerebral cortical and cerebellar membrane preparations was not different in alcoholics compared to that in controls, suggesting that the number of β-adrenergic receptors was not affected by chronic ethanol ingestion. Agonist binding data, however, indicated the loss of the high-affinity agonist binding state of the β-adrenergic receptor, representing the receptor-guanine nucleotide binding protein (G_s) complex. Such changes were observed in cerebral cortex but not in cerebellum of intoxicated alcoholics. These data suggest that cerebral cortical β-adrenergic receptors are uncoupled from adenylate cyclase in these subjects. In cerebral cortical and cerebellar membranes of sober alcoholics both the high- and low-affinity agonist binding sites were observed. These findings are similar to those seen in animal studies and suggest that the effect of chronic ethanol ingestion on β-adrenergic receptor-adenylate cyclase coupling is brain region specific and reversible with abstinence. Ethanol-induced changes in the coupling of receptors to adenylate cyclase may contribute to the physiological and behavioral manifestations of alcohol abuse.     

Peripheral catecholamine administration does not alter cerebral β-adrenergic receptor density

Chronic peripheral administration of isoproterenol, epinephrine or norepinephrine to rats caused a decrease in β-adrenergic receptor density in heart and lung, but not in cerebral cortex, caudate or cerebellum. The results show that alterations in peripheral catecholamine levels have no direct or indirect effects on brain β-adrenergic receptors.     

Beta2-glycoprotein I: antiphospholipid syndrome and T-cell reactivity

There is increasing evidence showing that recurrent thrombosis and intrauterine fetal loss in antiphospholipid syndrome (APS) are attributable to antiphospholipid (aPL) antibodies. We have recently identified autoreactive CD4⁺ T cells to β₂-glycoprotein I (β₂GPI) that promote production of pathogenic antiphospholipid antibodies. β₂GPI-specific CD4⁺ T cells preferentially recognize the antigenic peptide containing the major phospholipid (PL)-binding site in the context of DR53. T-cell helper activity that stimulates B cells to produce IgG anti-β₂GPI antibodies is mediated through IL-6 and CD40–CD154 interaction. β₂GPI-specific T cells respond to reduced β₂GPI and recombinant β₂GPI fragments produced in a bacterial expression system but not to native β₂GPI, indicating that the epitopes recognized by β₂GPI-specific T cells are ‘cryptic’ determinants, which are generated at a subthreshold level by the processing of native β₂GPI under normal circumstances. Although β₂GPI-specific T cells are detected in both APS patients and healthy individuals, these autoreactive T cells are activated in vivo in APS patients but not in healthy individuals. These findings indicate activation of β₂GPI-specific T cells and subsequent production of pathogenic anti-β₂GPI antibodies can be induced by the exposure of such T cells to cryptic peptides of β₂GPI efficiently presented by functional antigen-presenting cells (APC). Delineating the mechanisms that induce the efficient processing and presentation of cryptic determinants of β₂GPI as a consequence of antigen processing would clarify the etiology that initiates the autoantibody response in APS.     

Mechanisms of pain-induced local cerebral blood flow changes in the rat sensory cortex and thalamus

It is a well-known phenomenon that cerebral blood flow is coupled to neural activation induced by non-noxious somatosensory stimulation. However, basic questions related to pain-induced cerebral blood flow changes remain unanswered. In the present study, the sciatic nerve of anesthetized rats was subjected to electric stimulation with noxious and non-noxious parameters. Changes in local cerebral blood flow and neuronal activity were determined simultaneously in the sensory cortex and in the thalamus by laser-Doppler flowmetry and c-fos immunohistochemistry, respectively. The role of different vasoregulatory mechanisms and the pain-induced increase in mean arterial blood pressure (MABP) were examined with specific blocking agents and by means of rapid intra-arterial transfusion. Noxious stimulation resulted in significant enhancement of neuronal activity both in the thalamus and in the somatosensory cortex indicated by marked c-fos expression in these areas. Cortical and thalamic blood flow (cBF and tBF) increased by 47±4 and 44±3% during the stimulation while the MABP elevated by 35±2%. Similar changes in MABP induced by intra-arterial transfusion had no effect on tBF, while cBF increased only by 18±5%. Blockade of ATP sensitive potassium channels (K⁺_ATP) and sympathetic β-receptors significantly attenuated the pain-induced blood flow increases in both investigated areas, while inhibition of nitric oxide synthase was effective only in the thalamus. The blockade of the sympathetic -receptors, opiate receptors, and the cyclooxygenase enzyme had no effect on the pain-induced cerebral blood flow elevations. These findings demonstrate that during noxious stimulation, cerebral blood flow is adjusted to the increased neural activity by the interaction of vasoconstrictor autoregulatory and specific vasodilator mechanisms, involving the activation of sympathetic β-receptors, K⁺_ATP-channels and the release of nitric oxide.     

-Adrenergic receptor-mediated depolarization of rat neocortical astrocytes in primary culture

The membrane potentials of rat neocortical astrocytes growing in primary cultures (mean resting potential; −79 mV at [K⁺]_o = 4.5 nM) were depolarized by up to 30 mV by 10⁻⁵ M norepinephrine added to the medium, or up to 11 mV by norepinephrine or phenylephrine applied by ionophoresis. This depolarization could be inhibited by the -adrenergic receptor antagonist phentolamine (10⁻⁵ M) but not by the β-adrenergic antagonist propranolol (10⁻⁵ M). These results suggest that the norepinephrine-evoked depolarizations seen in these cells may be mediated through an -adrenergic receptor.     

A natural scavenger of peroxynitrites, rosmarinic acid, protects against impairment of memory induced by Abeta(25-35)

Peroxynitrite (ONOO⁻)-mediated damage is regarded to be responsible for the cognitive dysfunction induced by amyloid beta protein (Aβ) in Alzheimer's disease (AD). In the present study, we examined the protective effects of rosmarinic acid (RA), a natural scavenger of ONOO⁻, on the memory impairment in a mouse model induced by acute i.c.v. injection of Aβ_25–35. Mice daily received i.p. several doses of RA after the injection of Aβ_25–35. RA prevented the memory impairments induced by Aβ_25–35 in the Y maze test and novel object recognition task. RA, at the effective lowest dose (0.25 mg/kg), prevented Aβ_25–35-induced nitration of proteins, an indirect indicator of ONOO⁻ damage, in the hippocampus. At this dose, RA also prevented nitration of proteins and impairment of recognition memory induced by ONOO⁻-i.c.v.-injection. Co-injection of the non-memory-impairing dose of ONOO⁻ with Aβ_25–35 blocked the protective effects of RA (0.25 mg/kg). These results demonstrated that the memory protective effects of RA in the neurotoxicity of Aβ_25–35 is due to its scavenging of ONOO⁻, and that daily consumption of RA may protect against memory impairments observed in AD.     

Effect of substance P and protein kinase inhibitors on β-amyloid peptide-induced proliferation of cultured brain cells

The present study investigated the effect of substance P (SP) and protein kinase inhibitors (H7 and HA1004) on β-amyloid peptide-induced proliferation of neonatal rat brain cells in primary cultures. The β-amyloid peptide_1–28 (designated as βAP28), at nanomolar concentrations (10⁻⁹ M), significantly (P 0.05) increased the proliferation of brain cells (presumably non-neuronal) as measured by [³H]thymidine uptake into DNA (mitogenesis). The effect was dependent on time of cultured, concentration of βAP28, and presence of fetal calf serum. The supplementation of SP into cell cultures at time zero reversed the proliferative response of βAP28. Moreover, the βAP28-induced proliferation was inhibited by protein kinase inhibitor H7, but not by HA1004. Since H7 is a selective protein kinase C (PKC) inhibitor and SP action involves PKC, we conclude that βAP28 induces normal brain cell proliferation through PKC pathway of cell signaling.