Effects of local anesthetics on guanyl nucleotide modulation of the catecholamine-sensitive adenylate cyclase system and on beta-adrenergic receptors

Tetracaine and other local anesthetics exert multiple actions on the catecholamine-sensitive adenylate cyclase system of frog erythrocyte membranes. Tetracaine (0.2--20 mM) reduces the responsiveness of adenylate cyclase to (a) guanyl-5'-yl-imidodiphosphate and (b) isoproterenol in the presence of GTP or guanyl-5'-yl-imidodiphosphate. Local anesthetics did not affect (a) basal enzyme activity, and (b) enzyme responsiveness to NaF. Tetracaine inhibited stimulation of adenylate cyclase by guanyl-5'-yl-imidodiphosphate over the whole range of nucleotide concentrations. By contrast, inhibition by tetracaine of isoproterenol activity in the presence of GTP was significant only if GTP concentrations exceeded 10(-7) M. Tetracaine also competitively inhibited binding of both the antagonist [3H]dihydroalprenolol and the agonist [3H]hydroxybenzylisoproterenol to beta-adrenergic receptors. However, it was twice as potent in inhibiting [3H]hydroxybenzylisoproterenol as [3H]dihydroalprenolol binding. The greater potency for inhibition of agonist binding was due to the ability of the anesthetics to promote dissociation of the high-affinity nucleotide sensitive state of the beta-adrenergic receptor induced by agonists. Other local anesthetics mimicked the effects of tetracaine on adenylatecyclase and in dissociating high-affinity agonist-receptor complexes. The other of potency for both processes was dibucaine greater than tetracaine greater than bupivacaine greater than lidocaine which agrees with their relative potencies as local anesthetics. By contrast, a different order of potency was observed for competitive inhibition of [3H]dihydroalprenolol binding: dibucaine greater than tetracaine greater than greater than lidocaine greater than bupivacaine.     

Solubilization of receptors for thyrotropin-releasing hormone from GH4C1 rat pituitary cells: demonstration of guanyl nucleotide sensitivity

TRH receptors have been solubilized from GH4C1 cells using the plant glycoside digitonin. Solubilized receptors retain the principal binding characteristics exhibited by the TRH receptor in intact pituitary cells and their membranes. The binding of the methylhistidyl derivative of TRH [( 3H]MeTRH) attained equilibrium within 2-3 h at 4 C, and it was reversible, dissociating with a t1/2 of 7 h. Analysis of [3H]MeTRH binding to soluble receptors at 4 C yielded a dissociation constant (Kd) of 3.8 nM and a total binding capacity (Bmax) of 3.9 pmol/mg protein. Peptides known to interact with non-TRH receptors on GH cells failed to interfere with the binding of [3H]MeTRH, indicating that the TRH binding was specific. Chlordiazepoxide, a competitive antagonist for TRH action in GH cells, inhibited TRH binding to soluble receptors with an IC50 of 11 microM. When [3H]MeTRH was bound to membranes and the membrane proteins were then solubilized, we found enhanced dissociation of the prebound [3H]MeTRH from its solubilized receptor by guanyl nucleotides. Maximal enhancement of [3H]MeTRH dissociation by 10 microM GTP gamma S occurred within about 45 min at 22 C. GTP gamma S, GTP, GDP beta S, and GDP were all effectors of [3H]MeTRH dissociation, exhibiting EC50s in the range of 14-450 nM. The rank order of potency of the tested nucleotides was GTP gamma S greater than GTP congruent to GDP beta S greater than GDP much greater than ATP gamma S greater than GMP. We conclude that TRH receptors have been solubilized from GH cells with digitonin and retain the binding characteristics of TRH receptors in intact pituitary cells. Furthermore, prebinding [3H]MeTRH to GH4C1 cell membranes results in the solubilization of a complex in which the TRH receptor is linked functionally to a GTP binding protein.     

Thyrotropin releasing hormone (TRH) binding sites in the adult human brain: localization and characterization

In the current study, we found evidence for the existence of binding sites for TRH in synaptic membrane preparations of several regions of the postmortem adult human brain. High levels of specific binding (fmol [3H]Me-TRH/mg protein/2 hr) were found in limbic structures: amygdala (7.1 +/- 0.6, Mean +/- SE), hippocampus (2.8 +/- 0.3), and temporal cortex (2.4 +/- 0.8). Intermediate levels of binding were found in the hypothalamus and nucleus accumbens whereas binding was low to undetectable in frontal and occipital cortex, cerebellum, pons, medulla and corpus striatum. Binding of the radioligand was linear over protein concentrations of 0.05-1.5 mg, and greater than 6 hr of incubation was required to achieve maximal binding. In the amygdala, binding was inhibited in the presence of TRH and Me-TRH but not in the presence of up to 1 microM concentrations of cyclo (His-Pro), TRH-OH, pGlu-His or peptides unrelated to TRH. Pretreatment of amygdala synaptic membranes with detergents, proteases or phospholipases disrupted [3H]Me-TRH binding; pretreatment with DNase or collagenase had no effect on binding. Saturation and association/dissociation analyses of the binding of [3H]Me-TRH to purified amygdala synaptic membranes revealed the presence of a high affinity (KD = 2.0 nM), low capacity (Bmax = 180 +/- 16 fmoles/mg protein) binding site. These results demonstrate that a highly specific membrane associated receptor for TRH is present in the adult human brain. The specific role that this receptor plays in brain function remains to be elucidated.     

Effects of local anesthetics of guanyl nucleotide modulation of the catecholamine-sensitive adenylate cyclase system and on β-adrenergic receptors

Tetracaine and other local anesthetics exert multiple actions on the catecholamine-sensitive adenylate cyclase system of frog erythrocyte membranes. Tetracaine (0.2–2.0 mM) reduces the responsiveness of adenylate cyclose to (a) guanyl-5′-yl-imidodiphosphate and (b) isoproterenol in the presence of GTP or guanyl-5′-yl-imidodiphosphate. Local anesthetics did not affect (a) basal enzyme activity, and (b) enzyme responsiveness to NaF. Tetracaine inhibited stimulation of adenylate cyclase by guanyl-5′-yl-imidodiphosphate over the whole range of nucleotide concentrations. By contrast, inhibition by tetracaine of isoproterenol activity in the presence of GTP was significant only if GTP concentrations exceeded 10^?7 M.Tetracaine also competitively inhibited binding of both the antagonist [³H]-dihydroalprenolol and the agonist [³H]hydroxybenzylisoproterenol to β-adrenergic receptors. However, it was twice as potent in inhibiting [³H]-hydroxybenzylisoproterenol as [³H]dihydroalprenolol binding. The greater potency for inhibition of agonist binding was due to the ability of the anesthetics to promote dissociation of the high-affinity nucleotide sensitive state of the β-adrenergic receptor induced by agonists.Other local anesthetics mimicked the effects of tetracaine on adenylate     

Verapamil: influence upon basal and stimulated rat growth hormone and prolactin release in vitro

Verapamil is an organic calcium antagonist which is believed to prevent the passage of calcium (Ca2+) across the cell membrane into the cell. In a rat pituitary perifusion-immunoprecipitation system, verapamil (50 microM) prevents the inhibitory effect of increased extracellular Ca2+ (5.4 mM) on basal and stimulated release of stored, prelabeled [3H]GH and [3H]PRL. [3H]GH release from pituitary explants perifused in standard medium (GIBCO Minimum Essential Medium: 1.8 mM Ca2+) is transiently increased by 50 microM verapamil while [3H]PRL release is suppressed. With continued exposure to 50 microM verapamil, [3H]GH release rates fall below (89.8 +/- 2.1% of base) preverapamil levels while [3H]PRL release rates simply remain suppressed (48.2 +/- 7.3% of base). With 250 microM verapamil, poststimulatory inhibition of [3H]GH release occurs more quickly, and after its withdrawal rebound release of both GH and PRL occur. Inhibition of [3H]GH release by 25 nM somatostatin (SRIF) and post-SRIF rebound [3H]GH release is not prevented by 50 microM verapamil. The early, rapid [3H]GH release phase of 1 mM dibutyryl cyclic AMP (dbcAMP) stimulation is potentiated by verapamil pretreatment, but only if the verapamil is continued during dbcAMP stimulation. Potassium (21 mM K+)-stimulated release of both 3H-labeled hormones is inhibited after similar pretreatment 50 microM verapamil. Conclusions: (a) verapamil antagonizes the inhibitory effects of increased extracellular Ca2+ on basal or dbcAMP-stimulated [3H]GH and [3H]PRL release; (b) in standard medium (1.8 mM Ca2+), 50 microM verapamil increases basal [3H]GH release suggesting either a direct effect or an antagonism of 1.8 mM extracellular Ca2+; (c) although verapamil-sensitive Ca2+ movement is not necessary for dbcAMP stimulation of [3H]GH release, verapamil potentiates dbcAMP-stimulated release; (d) because verapamil also inhibits K+-stimulated [3H]GH and [3H]PRL release, these observations support previous suggestions that K+- and dbcAMP-stimulated rapid hormone release occurs from different intracellular sites; and (e) because verapamil does not prevent any phase of SRIF action and since these two agents differentially alter K+- and cAMP-stimulated release, their mechanisms of action must partially differ.     

Direct evidence for the interaction between papaverine and alpha 2-adrenergic receptors in canine platelets

The effects of papaverine on specific [3H]-yohimbine binding to canine platelet alpha 2-adrenergic receptors and on the platelet aggregation were assessed and compared with those of verapamil. Both compounds concentration-dependently inhibited [3H]-yohimbine binding with KI values for respective compounds of 0.39 +/- 0.05 microM (n = 3) and 15 +/- 0.19 microM (n = 3). In the presence of either compound KD values in Scatchard analysis of the equilibrium ligand binding increased in concentration-dependent manner, whereas Bmax did not change, indicating competitive inhibition of the ligand binding by these compounds. (-)-Epinephrine (3 microM) potentiation of adenosine diphosphate (ADP, 0.1 microM) aggregation was inhibited by papaverine with IC50 of 11 +/- 3.6 microM (n = 4). In the same experiments verapamil inhibited the platelet aggregation with lower IC50 (3.1 +/- 0.87 microM, n = 4) in comparison with that for papaverine. These results suggest that papaverine, like verapamil, inhibits physiological response of canine platelets through alpha-adrenergic receptor stimulation by direct interaction with the receptors.     

Studies on the mechanism of action of local anesthetics on proton translocating ATPase from Mycobacterium phlei

We have measured the inhibitory potencies of local anesthetics (procaine, lidocaine, tetracaine and dibucaine) on ATP-mediated H+-translocation, Ca2+-transport and ATPase activity in membrane vesicles from Mycobacterium phlei. Procaine and lidocaine up to 1 mM concentration did not inhibit ATP-dependent H+-translocation, Ca2+-transport and ATPase activity. However, tetracaine and dibucaine at 0.2 mM concentration caused dissipation of the proton gradient, measured by the reversal of the quenching of fluorescence of quinacrine, and inhibition of active Ca2+-transport. Tetracaine (1 mM) inhibited membrane-bound ATPase activity without affecting solubilized F1-ATPase activity. Studies show that these local anesthetics do not prevent the inactivation of F0-F1 ATPase by dicyclohexylcarbodiimide (DCCD). Binding of [14C]DCCD to F0-proteolipid component remained unchanged in the presence of tetracaine indicating that DCCD and tetracaine do not share common binding sites on the F0-proteolipid sector. The inhibition of H+-translocation and membrane-bound ATPase activity by tetracaine was substantially additive in the presence of vanadate.     

Na(+)- and Cl(-)-dependent [3H]GABA binding to catfish brain particles

The uptake of [3H]GABA by homogenates of catfish brain was previously shown to be temperature-sensitive and sodium-dependent, and to display saturation kinetics. The present study is a continuation of this work and was undertaken to characterize the initial binding of [3H]GABA to its transport system. [3H]GABA binding to catfish brain particles at 4 degrees C displayed saturability and was totally dependent on both Na+ and Cl-, the optimum concentrations of which were 150 mM and 75 mM, respectively. The effects of a number of drugs on binding were established. Unlabelled GABA was the most potent inhibitor (IC50 = 3.2 microM). The structural analogues nipecotic acid and guvacine were also strongly inhibitory. Interestingly, verapamil, a Ca2+ channel blocker, also inhibited [3H]GABA binding (IC50 = 38 microM). Harmaline, known to compete for Na+ binding in other transport systems, did not appear to influence Na+ binding but was effective at displacing [3H]GABA. These results suggest that the interaction of GABA with its carrier is similar to that found in the mammalian nervous system and is further evidence that GABA is involved in neurotransmission in catfish brain.     

Cocaine and its derivatives blunt neutrophil functions without influencing phosphorylation of a 47-kilodalton component of the reduced nicotinamide-adenine dinucleotide phosphate oxidase

Cocaine and its derivatives blunted responses of neutrophils (cell/cell aggregation, up-regulation of the receptor for C3bi (CR3, CD11b/CD18), generation of superoxide anion (O2-) and degranulation to various stimuli. The order of potency of these agents was the same as that for local anesthesia: tetracaine greater than bupivacaine greater than cocaine greater than lidocaine. Neutrophil aggregation elicited by the chemoattractant FMLP (10(-7) M) was inhibited by cocaine (10 mM) to 13.6 +/- 6% of control (p less than 0.002); the IC50 was approximately 4 mM. Cocaine and the other local anesthetics not only inhibited the upregulation of CR3 and O2- generation, but also blocked degranulation of cytochalasin B-treated cells. Cocaine (10 mM) reduced beta-glucuronidase and lysozyme secretion to 4.3 +/- 0.7 and 13 +/- 2.2% controls, respectively; its IC50 was 4 mM. Local anesthetics added after ligand/receptor engagement (FMLP) interrupted aggregation and halted generation of O2-. Moreover, local anesthetics rapidly inhibited aggregation, O2- generation, and degranulation elicited by PMA (1 microgram/ml) or the Ca ionophore A23187 (10 microM): the effects of cocaine could therefore not be attributed to unique actions at the FMLP receptor. Peak levels of intracellular Ca2+ ([Ca]i) at 5 to 10 s, and levels of [Ca]i 120 s after FMLP in Fura 2-loaded cells were significantly lower in cells treated with lidocaine, findings that could be explained by enhanced 45Ca2+ efflux from neutrophils. In cells loaded with bis(carboxyethyl)carboxyfluorescine (pH indicator) local anesthetics failed to affect the initial FMLP-induced (0 to 15 s) drop of pHi but inhibited the later (120 s) realkalinization of the cytosol (lidocaine, bupivacaine). Most remarkably, autoradiographs of SDS gels prepared from stimulated, 32P-labeled neutrophils treated with local anesthetics showed no difference from resting cells, either with respect to patterns of phosphorylation and dephosphorylation or their kinetics. Labeling of a 47-kDa protein, a component of the reduced nicotinamide-adenine dinucleotide phosphate-oxidase system, was unchanged. The effects of local anesthetics, which blunt neutrophil responses without affecting protein phosphorylation, suggest that protein phosphorylation is an insufficient signal for neutrophil activation. Inasmuch as cocaine and its derivatives affect cell functions at sites distal to activation of protein kinase C, these agents should prove useful in uncoupling protein phosphorylation from functional responses.     

Effects of Acetylethylcholine Mustard on [3H]Quinuclidinyl Benzilate Binding and Acetylcholine Release in Rat Brain Synaptosomes

The effects of acetylethylcholine mustard and its aziridinium derivative (AMMA) on acetylcholine (ACh) release and [3H]quinuclidinyl benzilate (QNB) binding were studied in rat cortical synaptosomes. After incubation for 5 min at 37 degrees C, AMMA reduced [3H]QNB binding with an IC50 of 9 microM. Following incubation for 5 min with 50 microM AMMA and washing, there was a 62% reduction in the [3H]QNB binding capacity with no change in the KD value for the remaining receptors, a result indicating the irreversibility of the AMMA binding. AMMA and oxotremorine both reduced the basal and 30 mM K+-induced release of newly synthesized [3H]ACh in dose-dependent manners over a 2.5-min period. At identical 50 microM concentrations, AMMA produced a much longer inhibition of basal [3H]ACh release than oxotremorine did. The inhibition of basal and 30 mM K+-induced [3H]ACh release by AMMA (10-250 microM) was blocked by 2 microM atropine during a 2.5-min release incubation, but not during a 30-min release incubation. After synaptosomes were treated with 50 microM AMMA for 5 min and the unbound drug was washed out from the tissue, [3H]ACh release (basal and K+-induced) was reduced. AMMA (50 microM) reduced high-affinity choline uptake and ACh synthesis by greater than 90% in this tissue, but these effects did not account for the [3H]ACh release inhibition, because they were not atropine sensitive and hemicholinium-3 had no effect on [3H]ACh release under the conditions used in these studies, i.e., after extracellular [3H]choline was washed out. Taken together, these results suggest that AMMA may be an irreversible agonist at presynaptic muscarinic autoreceptors.     

Stereoselectivity for the (R)-Enantiomer of HA-966 (l-Hydroxy-3-Aminopyrrolidone-2) at the Glycine Site of the N-Methyl-d-Aspartate Receptor Complex

HA-966 (1-hydroxy-3-aminopyrrolidone-2) is an antagonist at the glycine allosteric site of the N-methyl-D-aspartate receptor ionophore complex. Unlike presently known glycine antagonists, HA-966 is chiral. We report stereoselectivity for the (R)-enantiomer at the glycine antagonist site. In [3H]glycine binding, the (R)-enantiomer has an IC50 of 4.1 +/- 0.6 microM. The racemic mixture has an IC50 of 11.2 +/- 0.5 microM, whereas (S)-HA-966 has an IC50 greater than 900 microM. In glycine-stimulated [3H]1-[1-(2- thienyl)cyclohexyl]piperidine binding, the (R)-enantiomer inhibits with an IC50 of 121 +/- 61 microM, whereas the racemic mixture has an IC50 of 216 +/- 113 microM and (S)-HA-966 is inactive. The inhibition by (R)-HA-966 can be prevented by the addition of glycine. (R)-HA-966 and racemic HA-966, but not (S)-HA-966, also prevent N-methyl-D-aspartate cytotoxicity in cortical cultures. The (R)-enantiomer and, less potently, the (S)-enantiomer inhibit N-methyl-D-aspartate-evoked [3H]norepinephrine release from rat hippocampal slices (IC50 values of about 0.3 mM and 1.6 mM, respectively), but only the inhibition by (R)-HA-966 is reversed by added glycine. In glutamate-evoked contractions of the guinea pig ileum, (R)-HA-966 causes a glycine-reversible inhibition (IC50 of about 150 microM), whereas (S)-HA-966 is much less potent (IC50 of greater than 1 mM). These results demonstrate stereoselectivity of the glycine antagonist site of the N-methyl-D-aspartate receptor complex in a variety of tissues and assays. The stereoselectivity also confirms the specificity of N-methyl-D-aspartate receptors in glutamate-evoked contractions of the guinea pig ileum, and supports their similarity to central N-methyl-D-aspartate receptors.     

Some interactions of L-DOPA and its related compounds with glutamate receptors

L-DOPA is probably a transmitter and/or modulator in the central nervous system (1). L-DOPA methyl ester (DOPA ME) is a competitive L-DOPA antagonist. However, it remains to be clarified whether there exist L-DOPAergic receptors. In Xenopus laevis oocytes injected with rat brain poly(A)+ RNA, L-DOPA induced small inward currents with ED50 of 2.2 mM at a holding potential of -70 mV. The currents were abolished by kynurenic acid or CNQX. Similar L-DOPA-currents were seen in oocytes co-injected with AMPA receptors, GluRs1,2,3 and 4. In brain membrane preparations, L-DOPA inhibited specific binding of [3H]-AMPA with IC50 of 260 microM. This inhibition was not modified by 200 microM ascorbic acid, an antioxidant. L-DOPA did not inhibit binding of [3H]-ligands of MK-801, kainate, DCKA and CGP39653. DOPA ME and L-DOPA cyclohexyl ester, a novel, potent and competitive antagonist (2), inhibited specific binding of [3H]-MK-801 with respective IC50 of 1 and 0.68 mM, but elicited no effect on that of the other [3H]-ligands. With low affinities, L-DOPA acts on AMPA receptors, while competitive antagonists act on NMDA ion channel domain. L-DOPAergic agonist and antagonist may not interact on ionotropic glutamate receptors. DOPA ME-sensitive L-DOPA recognition sites (1) seem to differ from glutamate receptors.     

Inhibition by local anesthetics and barbiturates of the active transport of H+ in the synaptic vesicle membranes of the brain

The effects of local anesthetics and barbiturates on the ATP-dependent H+ transport in synaptic vesicle membranes from rat brain were studied using a fluorescent probe, acridine orange. Local anesthetics depressed the active H+ transport with the following order of potencies: tetracaine trimecaine lidocaine procaine. Respective IC50 values were 0.07, 0.28, 0.46 and 0.60 mM. The local anesthetics also disrupted the endogenous pH gradient seen in the absence of ATP. Barbiturates inhibited the active H+ transport showing IC50 values in the range of 2-5 mM except for benzobarbital and barbital characterized by IC50 values of 0.5 and 20 mM, respectively. The order of potencies was benzobarbital hexobarbital amobarbital pentobarbital phenobarbital barbital. The endogenous pH gradient was not affected by the barbiturates. The results show that local anesthetics disrupt the H+ transport by acting as permeable weak bases (uncouplers) whereas barbiturates are likely to block and anion channel which maintains electroneutrality of the H+ transport in the membrane of synaptic vesicles.     

Involvement of Ni protein in the functional coupling of the atrial natriuretic factor (ANF) receptor to adenylate cyclase in rat lung plasma membranes

In the presence of 1 microM atrial natriuretic factor (ANF) and low (0.1 mM) Mg2+ concentrations, the initial rate of binding of [3H]guanosine 5'-[beta, gamma-imido)triphosphate [( 3H]p[NH]ppG) to rat lung plasma membranes was increased twofold to threefold. ANF-dependent stimulation of the initial rate of [3H]p[NH]ppG binding was reduced at high (5 mM) Mg2+ concentrations. Preincubation of membranes with p[NH]ppG (5 min at 37 degrees C) eliminated the ANF-dependent effect on [3H]p[NH]ppG binding whereas ANF-dependent [3H]p[NH]ppG binding was unaffected by similar pretreatment with guanosine 5'-[beta-thio]diphosphate (GDP[beta S]). An increase in ANF concentration from 10 pM to 1 microM caused a 40% decrease in forskolin-stimulated or isoproterenol-stimulated adenylate cyclase activities (IC50 5 nM) in rat lung plasma membranes. GTP (100 microM) was obligatory for the ANF-dependent inhibition of adenylate cyclase, which could be completely overcome by the presence of 100 microM GDP[beta S] or the addition of 10 mM Mn2+. Reduction of Na2+ concentration from 120 mM to 20 mM had the same effect. Pertussis toxin eliminated ANF-dependent inhibition of adenylate cyclase by catalyzing ADP-ribosylation of membrane-bound Ni protein (41-kDa alpha subunit of the inhibitory guanyl-nucleotide-binding protein of adenylate cyclase). The data support the notion that one of the ANF receptors in rat lung plasma membranes is negatively coupled to a hormone-sensitive adenylate cyclase complex via the GTP-binding Ni protein.     

Dantrolene and azumolene inhibit [3H]PN200-110 binding to porcine skeletal muscle dihydropyridine receptors.

We tested whether the hydantoin muscle relaxants dantrolene, azumolene, or aminodantrolene could alter the binding of [3H]PN200-110 to transverse tubule dihydropyridine receptors or the binding of [3H]ryanodine to junctional sarcoplasmic reticulum Ca2+ release channels. All three drugs inhibited [3H]PN200-110 binding with azumolene (IC50 approximately 20 microM) 3-5 times more potent than dantrolene or aminodantrolene. In contrast, 100 microM azumolene and dantrolene produced a small inhibition of [3H]ryanodine binding (less than 25%) while aminodantrolene was essentially inert. Hence there was a preferential interaction of hydantoins with dihydropyridine receptors instead of ryanodine receptors. Skeletal muscle dihydropyridine receptors may participate in the mechanism of action of dantrolene and azumolene.     

Local anesthetics: stimulation of incorporation of inositol into phosphoinositides in guinea pig cerebral cortical synaptoneurosomes

Various local anesthetics enhanced the incorporation of [3H]inositol into phosphoinositides in guinea pig cerebral cortical synaptoneurosomes. Dibucaine, QX-572 and dimethisoquin showed maximum stimulation at 100 microM, tetracaine and diphenhydramine at 300 microM, and QX-314 at 1 mM, while quinacrine, lidocaine and cocaine showed no or only slight stimulation. There was no correlation between local anesthetic activity, estimated by inhibition of the 22Na+ flux elicited by the sodium channel activator batrachotoxin, and the potency for stimulation of inositol incorporation. A quaternary, relatively weak, local anesthetic, QX-572, was the most potent agent in stimulation of inositol incorporation, while the next most potent agent was dibucaine, a tertiary, very potent, local anesthetic. Dibucaine did not affect the uptake of [3H]inositol by synaptoneurosomes. The incorporation of [3H]inositol into phosphoinositides was increased in calcium-free buffer. The presence of dibucaine resulted in further stimulation of [3H]inositol incorporation in calcium-free buffer. Although dibucaine and QX-572 markedly stimulated incorporation of [3H]inositol into phosphoinositides, only QX-572 significantly enhanced the incorporation of 32PO4(3-) into phosphoinositides. The results suggest that certain local anesthetics enhance a pathway involving an exchange reaction between inositol and the phosphoinositol ester bond of phosphatidylinositol, but do not markedly affect the de novo pathway of phosphoinositide synthesis.     

Effect of tetracaine and lidocaine on insulin release in isolated mouse pancreatic islets

The effect of tetracaine and lidocaine on insulin secretion and glucose oxidation by islets of ob/ob-mice was measured. Tetracaine, at a concentration of 1 microM to 0.1 mM, did not markedly influence the basal (3 mM glucose) insulin secretion, whereas 0.5-3.5 mM induced a marked increase. At 7 mM glucose, there was a dose-dependent increase with 0.1-2.5 mM tetracaine. Insulin release induced by 20 mM glucose was potentiated by 0.1 mM and 0.5 mM tetracaine, but this effect disappeared at 1 mM tetracaine. The stimulatory effect of 0.5-1 mM tetracaine on basal insulin release was blocked by the secretory inhibitors, adrenaline (1 microM), clonidine (1 microM) and by Ca2+-deficiency, but the stimulation by 3.5 mM tetracaine was not reduced by 1 microM clonidine or Ca2+ deficiency. Atropine (10 microM) did not affect the stimulation by 0.5 mM tetracaine at 3 mM glucose or by 0.25 mM tetracaine at 20 mM glucose. Tetracaine, at 0.1 mM, potentiated the secretory stimulation of 20 mM L-leucine, 20 mM D-mannose, or 1 microM glibenclamide. Mannoheptulose, 10 mM, abolished the combined effects of 0.1 mM tetracaine and 10 mM glucose. Lidocaine, 1-5 mM, stimulated basal insulin release, but 1 microM-1 mM of the drug did not affect glucose-induced (20 mM glucose) insulin release and 5 mM lidocaine inhibited glucose stimulation. The oxidation of 10 mM D-[U-14C]glucose was slightly enhanced by 0.1 and 1 mM tetracaine. The results indicate that tetracaine and lidocaine, at certain concentrations, can induce insulin release and that tetracaine potentiates secretion induced by other secretagogues. It is concluded that these effects may be associated with beta-cell functions related to the adrenergic receptors but probably not to cholinergic receptors.     

Rat brain TRH receptors: kinetics, pharmacology, distribution and ionic effects

Optimal conditions for measuring receptor binding for thyrotropin-releasing hormone (TRH) in the rat central nervous system (CNS) have been determined using 3H-labelled [3-Me-His2]TRH [( 3H]MeTRH). Binding assays conducted at 0 degree C for 5-6 h using sodium phosphate- and/or Hepes-buffered tissue resuspensions, with subsequent filtration through Whatman GF/B filters, yielded the best results. Association and dissociation of [3H]MeTRH binding to amygdala membranes were time and temperature dependent. Dissociation kinetics appeared biphasic. Progressive reduction in receptor affinity and capacity and increased radioligand breakdown were observed at elevated temperatures. Bacitracin (25-1000 microM) prevented peptide degradation but inhibited receptor binding (8-37%). Detailed competition experiments using MeTRH and other drugs yielded a pharmacological profile similar to that observed previously in other tissues indicating TRH receptor identification. Highest density of TRH receptors was observed in the retina and numerous limbic areas. Monovalent and divalent cations modulated [3H]MeTRH binding by reducing apparent receptor number.     

Properties of receptors for the Ca2+-channel blocker verapamil in transverse-tubule membranes of skeletal muscle. Stereospecificity, effect of Ca2+ and other inorganic cations, evidence for two categories of sites and effect of nucleoside triphosphates

The verapamil receptor associated with the voltage-dependent calcium channel of rabbit skeletal muscle transverse tubule membranes has the following properties. (i) This receptor is stereospecific and discriminates between the different stereoisomers of verapamil, gallopamil and diltiazem. (ii) Inorganic divalent cations inhibit the binding of [3H]verapamil to its receptor in an apparently non-competitive fashion. The rank order of potency is: Ca2+ = Mn2+ greater than Mg2+ greater than Sr2+ greater than Ba2+ much greater than Co2+ much greater than Ni2+. Ca2+ and Mn2+ have inhibition constants of 0.3 mM. Binding of [3H]verapamil is also sensitive to monovalent cations such as Cs+, K+, Li+ and Na+. The most active of these cations (Cs+ and K+) have inhibition constants in the range of 30 mM. (iii) Binding of [3H]verapamil is pH-dependent and reveals the presence on the verapamil receptor of an essential ionizable group with a pKa of 6.5. (iv) A low-affinity binding site for verapamil and for some other Ca2+ channel blockers is detected by studies of dissociation kinetics of the [3H]verapamil receptor in the presence of high concentrations of verapamil, gallopamil, bepridil and diltiazem. (v) GTP and nucleoside analogs change the properties of [3H]verapamil binding to verapamil binding sites. High-affinity binding sites seem to be transferred into low-affinity sites. Dissociation constants obtained from inhibition studies of [3H]verapamil binding are in the range of 0.1-0.3 mM for GTP, ATP and Gpp(NH)p.     

Homocysteic Acid as a Putative Excitatory Amino Acid Neurotransmitter: I. Postsynaptic Characteristics at N-Methyl-D-Aspartate-Type Receptors on Striatal Cholinergic Interneurons

The actions of the stereoisomers of homocysteic acid (HCA) were characterized at N-methyl-D-aspartate (NMDA)-type receptors which mediate excitatory amino acid-evoked [3H]acetylcholine ([3H]ACh) release from striatal cholinergic interneurons. Like NMDA, L-HCA and D-HCA evoked the release of [3H]ACh formed from [3H]choline in striatal slices. The concentration-response curve for L-HCA was virtually superimposable on that for NMDA, yielding an equal EC50 value (56.1 microM) and maximal response. However, D-HCA was weaker, with an EC50 value of 81.1 microM, and an apparently smaller maximal response. L-HCA-evoked [3H]ACh release was inhibited by the same categories of compounds which inhibit NMDA-evoked [3H]ACh release: the divalent ion Mg2+ (IC50 = 25.8 microM); competitive NMDA antagonists 2-amino-7-phosphonoheptanoate (IC50 = 51.2 microM) and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (IC50 = 20.1 microM); and the dissociative anesthetics tiletamine (IC50 = 0.59 microM) and MK-801 (IC50 = 0.087 microM). Like NMDA, L-HCA produced a tachyphylaxis in this system. Tachyphylaxis to NMDA resulted in a decrease response to L-HCA, and conversely, tachyphylaxis to L-HCA resulted in a decrease response to NMDA. The results suggest that L-HCA is an agonist at the NMDA-type receptor and may represent an endogenous ligand for this excitatory amino acid receptor.