Release of endogenous glutamate from rat cerebellar synaptosomes: interactions with adenosine and ethanol

The effects of ethanol and adenosine receptor agonist R-PIA and antagonist theophylline on release of endogenous glutamate were tested in rat cerebellar synaptosomal preparation. Release was carried out for 5 to 60 sec after which time the released glutamate was separated from the synaptosomal membranes by rapid filtration. The amount of released glutamate in the filtrate was measured by an enzyme-linked fluorometric assay. Basal endogenous glutamate release was estimated as 3.7 +/- 0.3 nmol/mg protein/5 sec and was stimulated by high K+. Glutamate release consisted of an initial rapid phase for the first 10 sec that was followed by a relatively slower phase. Both Ca2+-dependent and Ca2+-independent glutamate release were observed which suggested the involvement of both neuronal and glial constituents of the synaptosomal preparation, respectively. Pharmacologically relevant concentrations of ethanol (25-100 mM) caused a trend toward a dose-dependent inhibition of glutamate release. R-PIA and theophylline inhibited and stimulated, respectively, basal release of glutamate and R-PIA-inhibited release was blocked by theophylline. Ethanol (25 mM) blocked the stimulatory effect of theophylline and the results of experiments following the inclusion of adenosine deaminase suggested the involvement of adenosine in this effect of ethanol. The results support our previous findings that suggest an involvement of cerebellar adenosine in the motor disturbing effects of acute ethanol and extend those findings by indicating that ethanol inhibits glutamate release from granule cells of the cerebellar cortex through an adenosine-sensitive mechanism.     

Dose-Dependent, K+-Stimulated Efflux of Endogenous Taurine from Primary Astrocyte Cultures Is Ca2+-Dependent

The K+-stimulated efflux of endogenous taurine from primary rat cerebellar astrocyte cultures prepared from 7-9-day-old rats was studied at 16-18 days in vitro using HPLC analysis. Taurine efflux was dose-dependent at K+ concentrations between 10 mM and 80 mM, with an EC50 of approximately 50 mM. Maximum stimulation of efflux above basal levels ranged from 56% at 10 mM K+ (204 pmol/min/mg protein) to 470% at 80 mM K+ (960 pmol/min/mg protein). Removal of Ca2+ from the buffer and the addition of either 1 mM EGTA or 10 mM Mg2+ abolished K+-stimulated efflux. Taurine efflux peaked and fell in parallel with the K+ concentration, but with an approximate lag of 3-5 min. The time course and amount of preloaded [3H]taurine released did not differ significantly from that seen for endogenous efflux. Basal taurine efflux varied inversely with the extracellular concentration of Ca2+ over the concentration range 0-5.0 mM. The observed Ca2+ dependence is consistent with a role for Ca2+ in the regulation of taurine release. Furthermore, taurine release from astrocytes in response to elevated K+ may reflect a neuromodulatory role for this amino acid in the CNS.     

Nature of Extrasynaptosomal Accumulation of Endogenous Adenosine Evoked by K+ and Veratridine

When rat brain synaptosomes were incubated for 10 min at 37 degrees C, basal accumulation of adenosine in the medium was 66 pmol/mg of protein. An elevated K+ level (24 mM) evoked an additional accumulation of 200 pmol/mg of protein, and 50 microM veratridine evoked 583 pmol of adenosine accumulation/mg of protein. K+- and veratridine-evoked accumulation of adenosine did not arise from microsomal or mitochondrial contaminants of the synaptosomal preparation, because purified microsomes and mitochondria did not exhibit evoked accumulation of adenosine in the medium. K+-evoked accumulation of extrasynaptosomal adenosine was Ca2+-dependent, whereas veratridine-evoked accumulation of adenosine was increased in Ca2+-free medium. In the presence of alpha,beta-methylene ADP and GMP, which inhibit ecto-5'-nucleotidase, conversion of added ATP and AMP to adenosine was inhibited by 90% in synaptosomal suspensions. However, inhibition of ecto-5'-nucleotidase only reduced basal extrasynaptosomal accumulation of adenosine by 74%, veratridine-evoked accumulation of adenosine by 46%, and K+-evoked accumulation by 33%. Most of the basal accumulation of extrasynaptosomal adenosine appears to be derived from released nucleotide, probably ATP, but about half of the veratridine-evoked accumulation of adenosine and most of the K+-evoked accumulation may arise from adenosine released in its own right, rather than from a released nucleotide.     

Distribution and subtype determination ofβ-receptors in the spinal cord of the adult rat

1. We determined the number of beta-receptors in the whole spinal cord of the adult rat and in the cervical, thoracal, and lumbal/sacral parts. 2. The undivided spinal cord contains 47 +/- 10 fmol/mg beta-receptors (KD = 2066 +/- 982 pmol/liter), and the cervical part of the spinal cord contains 53 +/- 8 fmol/mg protein (KD = 3224 +/- 1775 pmol/liter). The thoracal part shows 40 +/- 1 fmol/mg protein (KD = 3229 +/- 104 pmol/liter), and the lumbal/sacral spinal cord contains 48 +/- 8 fmol/mg protein (KD = 3610 +/- 1610 pmol/liter). 3. Competitive inhibition studies with l-practolol, dl-atenolol, and ICI 118,551 were performed and we calculated by a computer program in the whole spinal cord the following ratio of beta-receptor subtypes: 80 +/- 5% Beta 1-receptors and 20 +/- 5% beta 2-receptors. 4. The basal and (-)-isoproterenol- and NaF-stimulated activity of adenylate cyclase was highest in the cervical part of the spinal cord and equally distributed between the thoracal and the lumbal/sacral parts. 5. The whole synaptosomal protein of the cervical part of the spinal cord contained 132 +/- 20 fmol, the thoracal part 117 +/- 3 fmol, and the lumbal/sacral part 133 +/- 22 fmol.     

Insulin-stimulated glucose transport in rat adipose cells. Modulation of transporter intrinsic activity by isoproterenol and adenosine

The mechanism of modulation of insulin-stimulated glucose transport activity in isolated rat adipose cells by lipolytic and antilipolytic agents has been examined. We have measured glucose transport activity in intact cells with 3-O-methylglucose and in plasma membranes with D-glucose, and the concentration of glucose transporters in plasma membranes using a cytochalasin B binding assay. In intact cells, isoproterenol reduced insulin-stimulated transport activity by 60%. This effect was lost after cooling and washing the cells with homogenization buffer, and neither the concentration of glucose transporters nor transport activity in the plasma membranes differed from control. However, treatment of cells with KCN prior to homogenization preserved the isoproterenol effect through the fractionation procedure. Plasma membranes from these cells contained an unchanged number of transporters (31 +/- 7, mean +/- S.E., versus 31 +/- 4 pmol/mg of protein in controls) but transported glucose at a reduced rate (19 +/- 6 versus 48 +/- 9 pmol/mg of protein/s). Conversely, incubation of intact cells in the presence of adenosine stimulated plasma membrane glucose transport activity compared to that in the absence of adenosine (44 +/- 6 versus 36 +/- 6 pmol/mg of protein/s). Kinetic studies of isoproterenol-inhibited glucose transport in plasma membranes revealed a 60% decrease in Vmax (2900 +/- 350 versus 7200 +/- 1000 pmol/mg of protein/s) and a small increase in Km (15.1 +/- 1 versus 13.0 +/- 0.6 mM). These data indicate that modifications of glucose transport activity produced by lipolytic and antilipolytic agents in intact adipose cells can be fully retained in plasma membranes isolated under appropriate conditions. Furthermore, the effects of these agents occur through a modification of the glucose transporter intrinsic activity.     

Neurotensin potentiates the endogenous dopamine release from striatal synaptosomes of rat

The effect of neurotensin (NT) on the release of endogenous dopamine (DA) of rat striatal synaptosomes was studied. In the basic medium with Ca++ (5mM K+ and 1.2 mM Ca++), spontaneous release of DA was determined to be 12.03 +/- 1.12 pmol/mg protein, while in the Ca++-free basic medium containing EGTA (2.0 mM), the amount of DA released was still up to 11.2 +/- 1.06 pmol/mg protein. NT in 10(-4)-10(-6) M range tested potentiated both the spontaneous and K+-induced release of DA in Ca++-free medium. In addition, NT in 10(-4) M, but not in lower concentrations tested, potentiated the spontaneous, Ca++-dependent release of DA. It is suggested that the effect of NT on DA release is mediated by the specific NT receptors at the DA axonal terminals. The possibility, however, that NT has some influence on the carrier-mediated process of the membrane might not be ruled out.     

Determination of Endogenous Acetylcholine Release in Freely Moving Rats by Transstriatal Dialysis Coupled to a Radioenzymatic Assay: Effect of Drugs

The technique of intracerebral dialysis in combination with a sensitive and specific radioenzymatic method was used for recovery and quantification of endogenous extracellular acetylcholine from the striata of freely moving rats. A thin dialysis tube was inserted transversally through the caudate nuclei, and the tube was perfused with Ringer solution, pH 6.1, at a constant rate of 2 microliter min-1. The perfusates were collected at 10-min intervals. In the presence of 1 and 10 microM physostigmine, acetylcholine release was 4.5 +/- 0.02 and 7.3 +/- 0.3 pmol/10 min, respectively (not corrected for recovery). The latter concentration of the acetylcholinesterase inhibitor was used in all experiments. Under basal conditions, acetylcholine output was stable over at least 4 h. A depolarizing K+ concentration produced a sharp, reversible 87% increase in acetylcholine output. Both the basal and K+-stimulated release were Ca2+ dependent. The choline uptake inhibitor hemicholinium-3 (20 micrograms intracerebroventricularly) reduced striatal acetylcholine output to 35% of the basal value within 90 min. Scopolamine (0.34 mg/kg s.c.) provoked a sharp enhancement of acetylcholine release of approximately 63% over basal values, whereas oxotremorine (0.53 mg/kg i.p.) transiently reduced acetylcholine release by 54%. These results indicate the physiological and pharmacological suitability of transstriatal dialysis for monitoring endogenous acetylcholine release.     

Release of non-mast cell histamine from rat aorta

We previously reported that A23187 induces release of histamine from bovine intrapulmonary vein and provided pharmacological evidence against an involvement of mast cells as the source of histamine. This study was conducted to test more definitively the hypothesis that histamine is released from non-mast cell sources in blood vessels. The effects of A23187 on release of histamine were determined using rat aorta which does not contain mast cells. Aortic rings were mounted for recording of isometric tension, and following exposure to A23187 or vehicle, histamine in the bathing media was measured using enzyme immunoassay. A23187 (100 nmol/l - 10 micromol/l) induced concentration-related release of histamine from rings with endothelium. The accumulation of histamine in the bathing media induced by 10 microM A23187 reached plateau at 60 min (6.2 +/- 1.1 pmol/mg) and was markedly and significantly higher than vehicle control (0.4 +/- 0.1 pmol/mg, p < 0.05). Destruction of endothelium significantly inhibited A23187-induced histamine release (5.5 +/- 1.5 pmol/mg with endothelium, 1.1 +/- 0.3 pmol/mg without endothelium, p < 0.05). The results demonstrate that A23187 induces release of histamine from rat aorta which does not contain mast cells and that the release of histamine is largely dependent on the presence of endothelium.     

Cyclic nucleotide function in trachealis muscle of dogs with and without airway hyperresponsiveness

We examined basal adenosine 3',5'-cyclic monophosphate (cAMP) levels, isoproterenol (ISO)-stimulated cAMP responses, basal cAMP, and guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase (PDE) activities and protein-kinase (PK) activities in trachealis muscle from five Basenji-greyhound (BG) and four greyhound dogs to determine whether the inverse relationship between in vivo and in vitro airway responsiveness could be due to altered cyclic nucleotide metabolism. Basal cAMP levels were not significantly different (PNS) in muscle from BG (11.6 +/- 0.53 pmol/mg protein) and greyhound dogs (10.30 +/- 1.60 pmol/mg protein). The cAMP responses to stimulation with ISO were enhanced in BG compared with greyhound dogs. The low Michaelis constant (1) for Km-cAMP PDE activity (Km = 0.63 microM) was significantly less (P less than 0.005) in BG dogs (1.54 +/- 0.28 pmol.min-1.mg protein-1) than greyhounds (11.76 +/- 2.48). Endogenously active PK activity was significantly greater (P less than 0.005) in BG (54.74 +/- 5.39 pmol.min-1.mg protein-1) than in greyhound dogs (15.50 +/0 2.20). Increases in PK activity with 5 microM cAMP added were not significantly different between BG (14.79 +/- 6.00) and greyhound dogs (7.04 +/- 2.14). Approximately 90% of both endogenous PK activity and cAMP-activated PK activity in BG and greyhound dogs was inhibited by a cAMP-dependent PK inhibitor (PKI'). These data suggest that decreased cyclic nucleotide degradation due to decreased cyclic nucleotide PDE activity with increased PK could account for the in vitro hyporesponsiveness of airway smooth muscle in BG dogs as a protective adaptive mechanism.     

Impaired parathyroid hormone action on urinary phosphorus excretion in isolated perfused kidney of streptozotocin-induced diabetic rats.

To study the effects of diabetes on the renal actions of parathyroid hormone (PTH), we observed urinary excretion of cyclic adenosine monophosphate (cAMP) and phosphorus in isolated perfused rat kidney. Diabetic rats were kept for 7 days after an intraperitoneal injection of 70 mg/kg streptozotocin (STZ). STZ-induced diabetic rats were treated with a daily injection of 20 U/kg lente-type insulin for 7 days. Plasma albumin, calcium, phosphorus, and PTH levels were not different among normal control, diabetic and insulin-treated diabetic groups. In the control rat kidney, the addition of PTH increased urinary cAMP excretion from 8 +/- 3 to 190 +/- 49 pmol/5 min and urinary phosphorus excretion from 11.3 +/- 4.4 to 33.6 +/- 10.8 microg/5 min. In the STZ-diabetic rat kidney, basal urinary cAMP was impaired, and PTH altered neither urinary cAMP nor phosphorus excretion (from below 0.7 to below 0.7 pmol/5 min, and from 15.5 +/-4.5 to 13.6 +/- 8.1 microg/5 min, respectively). Insulin treatment completely recovered the PTH actions. These results show that insulinopenic diabetes induces PTH resistance in the kidney.     

Effect of bile salts on the plasma concentration of immunoreactive vasoactive intestinal polypeptide in man

The effects of bile salts on the release of immunoreactive vasoactive intestinal polypeptide (IR-VIP) were investigated in men using a specific radioimmunoassay. Plasma IR-VIP was determined after extraction by the acid-acetone method (recovery 75 +/- 5%). Oral administration of 400 mg sodium taurocholate caused a rise in plasma IR-VIP from 18.5 +/- 1.3 pmol/l to 31.1 +/- 2.1 pmol/l after 30 min and 39.0 +/- 1.7 pmol/l after 60 min and return to the initial value after 120 min. Oral administration of chenodeoxycholic acid (CDCA) also increased plasma IR-VIP from a basal level of 14.5 +/- 1.5 pmol/l to 36.3 +/- 1.2 pmol/l after 60 min. Oral administration of ursodeoxycholic acid (UDCA) increased plasma IR-VIP from 11.9 +/- 1.1 pmol/l to 25.6 +/- 1.8 pmol/l after 30 min. Perifusion of 1 mM taurocholate stimulated release of IR-VIP from human duodenal mucosa into the perifusate. These results suggest that bile salts may participate, at least in part, in the release of IR-VIP from the gut.     

Adenosine transport in bovine chromaffin cells in culture

Bovine adrenal chromaffin cells in culture have a high capacity and affinity for adenosine uptake with Vmax = 14 +/- 2.4 pmol/10(6) cells/min (133 pmol/mg of protein/min) and Km = 1 +/- 0.2 microM. Transport studies, at short time periods, in recently isolated chromaffin cells have Vmax = 15 pmol/10(6) cells/min and Km = 1.1 microM in ATP-depleted cells. Endogenous levels of the various purine nucleosides and bases were determined by high pressure liquid chromatography, with adenosine (3 +/- 1 nmol/10(6) cells), inosine (5.3 +/- 1.2 nmol/10(6) cells), and hypoxanthine (2.1 +/- 0.8 nmol/10(6) cells) being the purine metabolites found in the highest concentration. Taking into account the intracellular water, endogenous levels of 2.1, 3.8, and 1.5 mM, respectively, were obtained. Radioactively labeled adenosine inside the cell underwent enzymatic transformations, producing inosine, hypoxanthine, xanthine, and nucleotides, with their appearance and distribution being a function of the incubation time. When nicotine was used as a secretagogue, the adenosine transformed into the nucleotide pool was released, reaching 18 +/- 8% of the total adenosine found in the nucleotides. Dipyridamole, extensively used clinically, was a strong inhibitor for the adenosine uptake into these cells, with Ki = 5 +/- 0.5 nM and noncompetitive kinetically.     

Involvement of the 60 kDa phosphoprotein in the regulation of Ca2+ release from sarcoplasmic reticulum of normal and malignant hyperthermia susceptible pig muscles

Junctional sarcoplasmic reticulum (SR) vesicles isolated from back muscles of normal and malignant hyperthermia susceptible (MHS) pigs were phosphorylated by addition of MgATP in the presence of 5 mM Ca2+ and 1 microM calmodulin (CaM). The major site of phosphorylation was a 60 kDa protein both in normal and MHS SR. The maximal amount of phosphorylation in MHS SR (5 pmol P/mg SR) was significantly lower than that in the normal SR (12 pmol P/mg SR). The phosphorylated 60 kDa protein was spontaneously dephosphorylated both in normal and MHS SR. Ca2+ release from the passively loaded SR was induced by a Ca2+-jump, and monitored by stopped-flow fluorometry using chlorotetracycline. In the absence of preincubation with MgATP, no significant difference was found in any of the kinetic parameters of Ca2+ release between normal and MHS SR. Upon addition of 20 microM MgATP to the passively loaded SR to phosphorylate the 60 kDa protein, the initial rate of Ca2+ release in normal SR significantly decreased from 659 +/- 102 to 361 +/- 105 nmol Ca2+/mg SR per s, whereas in MHS SR the rate decreased from 749 +/- 124 to 652 +/- 179 nmol Ca2+/mg SR per s. Addition of 20 microM adenosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppA) did not significantly alter the initial rate of Ca2+ release both in normal and MHS SR. These results suggest that the previously reported higher Ca2+ release rate in MHS SR (Kim et al. (1984) Biochim. Biophys. Acta 775, 320-327) is at least partly due to the reduced extent of the Ca2+/CaM-dependent phosphorylation of the 60 kDa protein. Two-dimensional gel electrophoresis study showed that amount of a protein with Mr = 55,000 was significantly lower in MHS SR than in normal SR suggesting that the abnormally lower amount of 55 kDa protein would cause the lower amount of phosphorylation of the 60 kDa protein in MHS SR.     

A sensitive assay of GTP cyclohydrolase I activity in rat and human tissues using radioimmunoassay of neopterin

A highly sensitive and simple assay for the activity of GTP cyclohydrolase I (EC 3.5.4.16) was established using a newly developed radioimmunoassay. D-erythro-7,8-Dihydroneopterin triphosphate formed from GTP by GTP cyclohydrolase I was oxidized by iodine and dephosphorylated by alkaline phosphatase to D-erythro-neopterin, and quantified by a radioimmunoassay for D-erythro-neopterin. This method was highly sensitive and required only 0.2 mg of rat liver tissues for the measurement of the activity. It was reproducible and can be applied for the simultaneous assay of many samples. The activity of GTP cyclohydrolase I was measured in several rat tissues. For example, the enzyme activity in rat striatum (n = 5) was 13.7 +/- 1.5 pmol/mg protein per hour (mean +/- SE), and agreed well with those obtained by high-performance liquid chromatography with fluorescence detection. The activity in the autopsy human brains (caudate nucleus) was measured by this new method for the first time. The activity in the caudate nucleus from parkinsonian patients (n = 6) was 0.82 +/- 0.56 pmol/mg protein per hour which was significantly lower than the control value, 4.22 +/- 0.43 pmol/mg protein per hour (n = 10).     

L-[3H]Adenosine, a New Metabolically Stable Enantiomeric Probe for Adenosine Transport Systems in Rat Brain Synaptoneurosomes

The stereoenantimers D-[3H]adenosine and L-[3H]adenosine were used to study adenosine accumulation in rat cerebral cortical synaptoneurosomes. L-Adenosine very weakly inhibited rat brain adenosine deaminase (ADA) activity with a Ki value of 385 microM. It did not inhibit rat brain adenosine kinase (AK) activity, nor was it utilized as a substrate for either ADA or AK. The rate constants (fmol/mg of protein/s) for L-[3H]adenosine accumulation measured in assays where transport was stopped either with inhibitor-stop centrifugation or with rapid filtration methods were 82 +/- 14 and 75 +/- 10, respectively. Using the filtration method, the rates of L-[3H]adenosine accumulation were not significantly different from the value of 105 +/- 15 fmol/mg of protein/s measured for D-[3H]adenosine transport. Unlabeled D-adenosine and nitrobenzylthiolnosine, both at a concentration of 100 microM, reduced the levels and rates of L-[3H]adenosine accumulation by greater than 44%. These findings suggest that L-adenosine, a metabolically stable enantiomeric analog, and the naturally occurring D-adenosine are both taken up by rat brain synaptoneurosomes by similar processes, and as such L-adenosine may represent an important new probe with which adenosine uptake may be studied.     

Cholate-independent retinyl ester hydrolysis. Stimulation by Apo-cellular retinol-binding protein

Apo-cellular retinol-binding protein (apoCRBP) activated the hydrolysis of endogenous retinyl esters in rat liver microsomes by a cholate independent retinyl ester hydrolase. A Michaelis-Menten relationship was observed between the apoCRBP concentration and the rate of retinol formation, with half-maximum stimulation at 2.6 +/- 0.6 microM (mean +/- S.D., n = 5). Two other retinol-binding proteins, bovine serum albumin and beta-lactoglobulin, acceptors for the rapid and spontaneous hydration of retinol from membranes, had no effect up to 90 microM. These data suggest activation of the hydrolase by apoCRBP directly, rather than by facilitating removal of retinol from membranes. The hydrolase responding was the cholate-independent/cholate-inhibited retinyl ester hydrolase as shown by: 60% inhibition of the apoCRBP effect by 3 mM cholate; apoCRBP enhancement of retinyl ester hydrolysis in liver microsomes that had no detectable cholate-enhanced activity; inhibition of cholate-dependent, but not apoCRBP-stimulated retinyl ester hydrolysis by rabbit anti-rat cholesteryl esterase. Compared to the rate (mean +/- S.D. of [n] different preparations) supported by 5 microM apoCRBP in liver microsomes of 6.7 +/- 3.7 pmol/min/mg protein [10], microsomes from rat lung, kidney, and testes had endogenous retinyl ester hydrolysis rates of 1.8 +/- 0.3 [5], 0.5 +/- 0.2 [3], and 0.3 +/- 0.2 [5] pmol/min/mg protein, respectively. N-Ethylmaleimide and N-tosyl-L-phenylalanine chloromethyl ketone were potent inhibitors of apoCRBP-stimulated hydrolysis with IC50 values of 0.25 and 0.15 mM, respectively, but phenylmethylsulfonyl fluoride and diisopropyl-fluorophosphate were less effective with IC50 values of 1 mM, indicating the importance of imidazole and sulfhydryl groups to the activity. These data provide evidence of a physiological role for the cholate-independent hydrolase in retinoid metabolism and suggest that apoCRBP is a signal for retinyl ester mobilization.     

Biochemical Mapping of Peptidyl-Glycine α-Amidation Activity in the Rat CNS

Peptidyl-glycine alpha-amidation enzyme activity has been measured in 36 nuclei or areas in the rat CNS and pituitary using D-Tyr-Phe-Gly as the substrate. The distribution of this enzyme is highly uneven, with highest activity levels (greater than 30 pmol/mg of protein/h) in hypothalamic nuclei, substantia grisea centralis, and nucleus ruber; moderate activity levels (10-30 pmol/mg of protein/h) in globus pallidus, septum, midbrain, pons, medulla oblongata, and cervical spinal cord; and low activity levels (1-10 pmol/mg of protein/h) in other telencephalic and thalamic structures. Almost no alpha-amidation activity (less than 0.5 pmol/mg of protein/h) was detected in cerebellar cortex. The Km values in several brain regions are of the same order.     

Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [3H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with (R,S)-[3H]α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitro-quinoxaline-2,3-dione ([3H]-CNQX) in rat brain sections. [3H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of [3H]CNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a KD = 67 +/- 9.0 nM and Bmax = 3.56 +/- 0.34 pmol/mg protein. In displacement studies, quisqualate, L-glutamate, and kainate also appeared to bind to a single class of sites. However, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) displacement of [3H]CNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of [3H]AMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with KD1 = 9.0 +/- 3.5 nM, Bmax = 0.15 +/- 0.05 pmol/mg protein, KD2 = 278 +/- 50 nM, and Bmax = 1.54 +/- 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [3H]CNQX binding sites correlated to the binding of L-[3H]glutamate to quisqualate receptors and to sites labeled with [3H]AMPA. These results suggest that the non-NMDA glutamate receptor antagonist [3H]CNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [3H]AMPA.     

Characterization of Nucleotide Transport into Rat Brain Synaptic Vesicles

ATP transport to synaptic vesicles from rat brain has been studied using the fluorescent substrate analogue 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP). The increase in intravesicular concentration was time dependent for the first 30 min, epsilon-ATP being the most abundant nucleotide. The complexity of the saturation curve indicates the existence of kinetic and allosteric cooperativity in the nucleotide transport, which exhibits various affinity states with K0.5 values of 0.39 +/- 0.06 and 3.8 +/- 0.1 mM with epsilon-ATP as substrate. The Vmax values obtained were 13.5 +/- 1.4 pmol x min(-1) x mg of protein(-1) for the first curve and 28.3 +/- 1.6 pmol x min(-1) x mg of protein(-1) considering both components. This kinetic behavior can be explained on the basis of a mnemonic model. The nonhydrolyzable adenine nucleotide analogues adenosine 5'-O-3-(thiotriphosphate), adenosine 5'-O-2-(thiodiphosphate), and adenosine 5'-(beta,gamma-imino)triphosphate and the diadenosine polyphosphates P1,P3-di(adenosine)triphosphate, P1,P4-di(adenosine)tetraphosphate, and P1,P5-di(adenosine)pentaphosphate inhibited the nucleotide transport. The mitochondrial ATP/ADP exchange inhibitor atractyloside, N-ethylmaleimide, and polysulfonic aromatic compounds such as Evans blue and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid also inhibit epsilon-ATP vesicular transport.     

Multiple sources of 1,2-diacylglycerol in isolated rat pancreatic acini stimulated by cholecystokinin. Involvement of phosphatidylinositol bisphosphate and phosphatidylcholine hydrolysis

Changes in the cellular content of 1,2-diacylglycerol (DAG) in isolated rat pancreatic acini in response to agonist stimulation were studied using a sensitive mass assay. When acini were stimulated by 10 nM COOH-terminal cholecystokinin-octapeptide (CCK8), the increase in DAG was biphasic, consisting of an early peak at 5 s and a second, larger, gradual increase that was maximal by 15 min. The basal level of DAG in acini was 1.04 nmol/mg of protein, which was increased to 1.24 nmol/mg of protein at 5 s and 2.76 nmol/mg of protein at 30 min. In comparison, the increase in DAG stimulated by 30 pM CCK8, a submaximal concentration for amylase release, was monophasic, increasing without an early peak but sustained to 60 min. Other Ca2+-mobilizing secretagogues such as carbamylcholine and bombesin increased DAG in acini, whereas vasoactive intestinal peptide, which acts to increase cAMP, had no effect. Phorbol ester and Ca2+ ionophore also stimulated DAG production. Analysis of the mass level of inositol 1,4,5-trisphosphate (1,4,5-IP3) showed that the generation of 1,4,5-IP3 stimulated by 10 nM CCK8 peaked at 5 s, a finding consistent with the early peak of DAG. The basal level was 4.7 pmol/mg of protein, which was increased to 144.6 pmol/mg of protein at 5 s by 10 nM CCK8. The levels of 1,4,5-IP3 then returned toward basal in contrast to the gradual and sustained increase of DAG. The dose dependencies of 1,4,5-IP3 and DAG formation at 5 s with respect to CCK8 were almost identical. This suggests that phosphatidylinositol 4,5-bisphosphate hydrolysis is a major source of the early increase in DAG but not of the sustained increase in DAG. Therefore, a possible contribution of phosphatidylcholine hydrolysis to DAG formation was examined utilizing acini prelabeled with [3H]choline. CCK8 (1 nM) maximally increased [3H]choline metabolite release by 133% of control at 30 min. Separation of these metabolites by thin layer chromatography showed that the products of CCK8-stimulated release were almost entirely phosphorylcholine, indicating the activation of a phospholipase C specific for phosphatidylcholine. By comparison, 1 nM CCK8 stimulated [3H]ethanolamine metabolite release from [3H]ethanolamine-labeled acini by only 22% of control. These data suggest that CCK stimulates both phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine hydrolysis; the latter may contribute to the sustained generation of DAG and hence the maintained activation of protein kinase C.