Transmembrane 36CI− Flux Measurements and Desensitization of the γ-Aminobutyric AcidA Receptor

Abstract: Some data on the concentration range of response and the concentration for half-response (EC₅₀) of γ-aminobutyric acid (GABA) for the GABA_A receptor are reviewed and compared. An analysis of the ³⁶CI⁻ flux assay demonstrates that both the EC₅₀ and the slope of a Hill plot depend on the ion influx or efflux assay time. The effects of depletion of the ³⁶CI⁻ concentration gradient during the assay and of receptor desensitization on the result for a range of assay times are considered. The EC₅₀ can be decreased by orders of magnitude by increasing the assay time. The EC₅₀ measured in a finite time is less than the half-response concentration for the response(s) of the receptor. The extent of this difference depends on the receptor concentration per internal volume. The maximal decrease of EC₅₀ depends on the rate of receptor desensitization. The computer simulations showed that a GABA_A receptor with a half-response concentration of 100 μ M GABA can give ³⁶CI⁻ flux measurements with an EC₅₀ value 100-fold lower.     

Comparative repellency of commercial formulations of deet, permethrin and citronellal against the mosquito Aedes aegypti, using a collagen membrane technique compared with human arm tests

A collagen membrane technique, based on the membrane blood-feeding system of Cosgrove et al . (1994) , was used to compare repellents against Aedes aegypti mosquitoes. Repellency was defined in terms of inhibition of probing (ED₅₀ and ED₉₀) after 5 min exposure. A direct comparison was made with repellency from probing after 5 min on five male volunteers.
Four repellent products were compared with technical DEET as the standard. The liquid formulations tested were: Autan^® (20% deet; Bayer); Repel Plus^® (20% deet plus 0.05% permethrin; Boots); permethrin (Zeneca) and citronellal (Sigma) .
Membrane and arm tests gave similar results. Deet formulations required less active compound than citronellal for the same degree of repellency. Pure deet and Autan gave similar results, dose for dose. Permethrin was highly repellent at very low doses, but Repel Plus did not enhance the immediate repellency of deet. A technique using the same membrane system was developed to evaluate persistence of Autan, which declined to 75% after 1 h against Ae. aegypti , and to about 50% after 2–4 h.     

(+)-Anatoxin-a Is a Potent Agonist at Neuronal Nicotinic Acetylcholine Receptors

Abstract: The effects of the nicotinic agonist (+)-anatoxin-a have been examined in four different preparations, representing at least two classes of neuronal nicotinic receptors. (+)-Anatoxin-a was most potent (EC₅₀= 48 n M ) in stimulating ⁸⁶Rb⁺ influx into M10 cells, which express the nicotinic receptor subtype comprising α4 and β2 subunits. A presynaptic nicotinic receptor mediating acetylcholine release from hippocampal synaptosomes was similarly sensitive to (+)-anatoxin-a (EC₅₀= 140 n M ). α-Bungarotoxin-sensitive neuronal nicotinic receptors, studied using patch-clamp recording techniques, required slightly higher concentrations of this alkaloid for activation: Nicotinic currents in hippocampal neurons were activated by (+)-anatoxin-a with an EC₅₀ of 3.9 γ M , whereas α7 homooligomers reconstituted in Xenopus oocytes yielded an EC₅₀ value of 0.58 γ M for (+)-anatoxin-a. In these diverse preparations, (+)-anatoxin-a was between three and 50 times more potent than (–)-nicotine and ˜20 times more potent than acetylcholine, making it the most efficacious nicotinic agonist thus far described.     

The role of inositol lipids in the activation of monocytes by interleukin-1 and bacterial endotoxin

Abstract The effect of interleukin-1 (IL-1) and bacterial endotoxin (lipopolysaccharide, LPS) on the activation of phosphoinositidase C (PIC) and on prostaglandin E₂ release was studied in monocytes (Mø). Both IL-1α and IL-1β increased the release of PGE₂ in a concentration-dependent manner, with EC₅₀s of 0.48 nM and 0.12 nM, respectively. Intact Mø were prelabelled with [³H]inositol and the formation of inositol phosphates (IPs) was estimated by ion exchange chromatography. PIC activity was estimated directly by measuring the conversion of [³H]phosphatidylinositol-4,5,-bisphosphate to aqueous soluble radioactivity by Mø homogenates. IL-1α (5.8 nM) increased the accumulation of IPs within 1–4 minutes and increases in IP₃ and IP₄ occured before the increase in IP₁₊₂ whereas LPS only increased the IPs level after at least 30 min. IL-1α increased PIC activity in Mø homogenates within 15 min with an EC₅₀ of 0.58 nM and IL-1β (0.1 nM) also increased activity. Neither IL-1α nor IL-1β affected the PIC activity of membrane or cytosolic fractions. LPS decreased activity in all fractions. These data indicate that IL-1, but not LPS, can directly lead to an increased activity of PIC which may be involved in eicosanoid formation in Mø.     

The compatibility of the fungicide azoxystrobin with Pochonia chlamydosporia, a biological control agent for potato cyst nematodes (Globodera spp.)

The biological control potential of Pochonia chlamydosporia against root-knot ( Meloidogyne spp.) and cyst-forming ( Heterodera and Globodera spp.) nematodes is widely appreciated. In spite of this, little has been undertaken to determine the compatibility of this fungus with modern fungicides. A series of experiments were undertaken to investigate the effect of azoxystrobin on P. chlamydosporia . Initial Petri dish experiments found a significant reduction in the growth of P. chlamydosporia . EC₅₀ values were calculated at 7 and 14 mg L⁻¹, respectively, for hyphal growth and chlamydospore germination. A microcosm experiment based on counts of the colony-forming units of the re-isolated fungus showed a reduction in EC₅₀ values for azoxystrobin in soil after 12, 20 and 40 days incubation (1.25, 1.00 and 3.00 mg kg⁻¹ soil, respectively). There was evidence the fungus recovered over time in response to azoxystrobin application. This was also demonstrated in a glasshouse experiment where EC₅₀ values of 1.32 and 4.4 mg kg⁻¹ soil were obtained for 35 and 49 days after planting (DAP), respectively.     

Proadrenomedullin N-Terminal 20 Peptide (PAMP), an Endogenous Anticholinergic Peptide: Its Exocytotic Secretion and Inhibition of Catecholamine Secretion in Adrenal Medulla

Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca²⁺-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC₅₀ = 50.1 µ M ) and catecholamines (EC₅₀ = 63.0 µ M ), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH₂ inhibited carbachol-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀ = 2.5 µ M ) in a noncompetitive manner and thereby reduced carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels (IC₅₀ = 1.0 µ M ) and catecholamine secretion (IC₅₀ = 1.6 µ M ). It did not alter high K⁺-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels or veratridine-induced ²²Na⁺ influx via voltage-dependent Na⁺ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca²⁺-dependent exocytosis in response to nicotinic receptor stimulation.     

5-Methoxytryptamine Inhibits Cyclic AMP Accumulation in Cultured Retinal Neurons Through Activation of a Pertussis Toxin-Sensitive Site Distinct from the 2-[125I]Iodomelatonin Binding Site

Abstract: Melatonin and 5-methoxytryptamine inhibited forskolin-stimulated cyclic AMP formation in cultured neural cells prepared from embryonic chick retina. Both methoxyindoles exhibited similar potency and efficacy, with EC₅₀ values of 0.8 n M for melatonin and 7.2 n M for 5-methoxytryptamine. Inhibition of cyclic AMP formation by 5-methoxytryptamine or melatonin was prevented by pretreatment with pertussis toxin. Pretreatment of cultures with 5-methoxytryptamine for 24 h reduced the subsequent inhibitory cyclic AMP response to 5-methoxytryptamine but not that to 2-iodomelatonin. Putative melatonin receptors on cultured retinal cells were labeled with 2-[¹²⁵I]iodomelatonin. Melatonin displaced specific 2-[¹²⁵I]iodomelatonin with a K _i value (0.8 n M ) similar to the EC₅₀ for inhibition of cyclic AMP formation. In contrast, 5-methoxytryptamine only inhibited 2-[¹²⁵I]iodomelatonin binding at very high concentrations ( K _i = 650 n M ). Pretreating cultured cells for 24 h with 2-iodomelatonin or melatonin, but not with 5-methoxytryptamine, reduced subsequent 2-[¹²⁵I]iodomelatonin binding. Thus, 5-methoxytryptamine appears to inhibit forskolin-stimulated cyclic AMP formation at a site distinct from the 2-iodomelatonin binding site.     

Histamine H1 and Endothelin ETB Receptors Mediate Phospholipase D Stimulation in Rat Brain Hippocampal Slices

Abstract: Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin-1, and trans -(1 S ,3 R )-aminocyclopentyl-1,3-dicarboxylic acid ( trans -ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [³²P]P_i-prelabeled rat brain cortical and hippocampal slices. The accumulation of [³²P]phosphatidylethanol was measured as an index of phospholipase D-catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin-1, and trans -ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC₅₀ 18 µ M ) was inhibited by the H₁ receptor antagonist mepyramine with a K _i constant of 0.7 n M and was resistant to H₂ and H₃ receptor antagonists (ranitidine and tioperamide, respectively). Endothelin-1-stimulated phospholipase D (EC₅₀ 44 n M ) was not blocked by BQ-123, a specific antagonist of the ET_A receptor. Endothelin-3 and the specific ET_B receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin-1, and EC₅₀ values of 16 and 3 n M , respectively. These results show that histamine and endothelin-1 stimulate phospholipase D in rat brain through H₁ and ET_B receptors, respectively.     

A “Septide-Sensitive” Receptor Is Not Involved in Tachykinin-Mediated Secretory and Inositol Phosphate Responses in Rat Parotid Gland: Are Several Transduction Pathways Involved After the Stimulation of the NK1 Receptor?

Abstract: In the rat parotid gland, the neuropeptide substance P (SP), as well as SP(4–11), and septide elicited inositol phosphate production (EC₅₀ values 0.44, 2, and 20 n M , respectively). No additivity of the maximal response to the three agonists was observed. SP, SP(4–11), and septide also stimulated protein secretion; for SP, two EC₅₀ were determined (0.5 and 160 n M ), whereas a single one could be determined for SP(4–11) and septide (EC₅₀ values 15 and 20 n M , respectively). The selective tachykinin NK₁ receptor antagonist RP67580 acted as a competitive inhibitor of both SP- and SP(4–11)-induced inositol phosphate production. Its effect on septide-induced inositol phosphate production was noncompetitive. RP67580 is apparently as potent at antagonizing septide, SP, or SP(4–11) (in all cases K _B = 3 n M ). These results show that in parotid gland, only NK₁ receptors are activated by SP, SP(4–11), and septide. We also showed that the protein secretion stimulated by SP was inhibited competitively by RP67580, whereas the effect of RP67580 was noncompetitive on protein secretion when SP(4–11) or septide was used. Our data indicate that in rat parotid gland, the existence of a specific "septide-sensitive" receptor can be ruled out and that only the NK₁ receptor is present and mediates cellular responses. Taken together, these results show that in this tissue the NK₁ receptor would present at least two different binding sites that could be coupled to different transduction pathways and that would regulate protein secretion.     

Regulation of Multiple Effectors by the Cloned δ-Opioid Receptor: Stimulation of Phospholipase C and Type II Adenylyl Cyclase

Abstract: The δ-opioid receptor is known to regulate multiple effectors in various tissues. When expressed in human embryonic kidney 293 cells, the cloned δ-opioid receptor inhibited cyclic AMP (cAMP) accumulation in response to the δ-selective agonist [ d -Pen², d -Pen⁵]enkephalin. The inhibitory response of [ d -Pen², d -Pen⁵]enkephalin was dependent on the expression of the δ-opioid receptor and exhibited an EC₅₀ of 1 n M . The receptor showed ligand selectivity and a pharmacological profile that is appropriate for the δ-opioid subtype. The inhibition was blocked by the opiate antagonist naloxone or by pretreatment of the cells with pertussis toxin. Cotransfection of the δ-opioid receptor with type II adenylyl cyclase and an activated mutant of α_s converted the δ-opioid signal from inhibition to stimulation of cAMP accumulation. It is interesting that when transfected into Ltk⁻ fibroblasts, the cloned δ-opioid receptor was able to stimulate the formation of inositol phosphates (EC₅₀ = 8 n M ). This response was sensitive to pertussis toxin. The opioid-mediated formation of inositol phosphates exhibited the same ligand selectivity as seen with the inhibition of cAMP accumulation. The ability of the δ-opioid receptor to couple to G proteins other than G_i was also examined. Cotransfection studies revealed that the δ-opioid receptor can utilize G_z to regulate cAMP accumulation and to stimulate the formation of inositol phosphates.     

A Charybdotoxin-Sensitive, Ca2+-Activated K+ Channel with Inward Rectifying Properties in Brain Microvascular Endothelial Cells: Properties and Activation by Endothelins

Abstract: A charybdotoxin-sensitive, Ca²⁺-activated K⁺ channel was identified in cultured rat brain capillary endothelial cells by using conventional single-channel recording techniques and ⁸⁶Rb⁺-influx and efflux experiments. Channel activity was dependent on the presence of Ca²⁺ on the cytosolic face of the membrane with a threshold concentration of 100 n M . It was inhibited by charybdotoxin (IC₅₀ 30 n M ) and quinine (IC₅₀ 0.1 m M ) but not by apamin. K(Ca) channels showed unusual inward rectifying properties under asymmetrical ionic conditions. They were activated by endothelin-1 (EC₅₀ 0.7 n M ) and endothelin-3 (EC₅₀ 7–10 n M ). The actions of endothelins were prevented by BQ-123 ( K _i = 8 n M ) in a competitive fashion, hence suggesting the involvement of an ET_A-receptor subtype. The channel activity was unaffected by cyclic AMP- or cyclic GMP-elevating agents. The possible role of the intermediate conductance, Ca²⁺-activated K⁺ channels for mediating K⁺ movements across the blood-brain barrier is discussed.     

γ-Aminobutyric AcidA Receptor Function Is Desensitised in Rat Cultured Cerebellar Granule Cells Following Chronic Flunitrazepam Treatment

Abstract: This study examined γ-aminobutyric acid_A (GABA_A) receptor function in cultured rat cerebellar granule cells by using microphysiometry following chronic flunitrazepam exposure, and correlated the findings with the α1 and β2/3 subunit protein expression and [³H]muscimol binding after the same treatment paradigm. Flunitrazepam treatment reduced ( p < 0.05) the maximal GABA-stimulated increase in extracellular acidification rate ( E _max) (16.5 ± 1.2% and 11.3 ± 1.0%, 2-day control and treated cells, respectively; 17.4 ± 1.0% and 9.9 ± 0.7%, 7-day control and treated cells, respectively; best-fit E _max± SEM, n = 7), without affecting the GABA concentration required to elicit 50% of maximal response (EC₅₀) (1.2 ± 1.7 and 2.3 ± 1.8 µ M , 2-day control and treated cells, respectively; 1.7 ± 1.5 and 1.5 ± 1.5 µ M , 7-day control and treated cells, respectively; best-fit EC₅₀± SEM, n = 7). Flunitrazepam exposure also abolished the flunitrazepam potentiation of the GABA response, caused a transient reduction of the GABA_A receptor α1 and β2/3 subunit proteins over the initial 2 days, but did not alter [³H]muscimol binding compared with vehicle-treated cells. The results suggest that changes in GABA_A receptor subunit protein expression, rather than loss of [³H]muscimol binding sites, underlie the chronic flunitrazepam-mediated desensitisation of GABA_A receptor function.     

Effects of citrate, heparin and cation supplementation on mortality and egg production of laboratory-reared horn flies

Abstract. The effects of the blood anticoagulants sodium citrate and sodium heparin on horn fly, Haematobia irritans L., egg production were tested. Sodium citrate was added to freshly collected bovine blood to give final concentrations of 5-100mM while sodium heparin was used in concentrations of 10–70 USP units/ml blood. Small cages containing five male and ten female newly emerged laboratory-reared horn flies were maintained for 8–10 days on these blood samples, and mortality and egg production recorded daily. Results showed that as blood citrate concentration was increased, egg production decreased logarithmically. At sodium citrate concentrations of 50 mM and above, severe impacts on egg production and adult horn fly survival occurred. Although no dose-related response of egg production to increasing heparin concentrations was noted, the 25 USP units heparin/ml blood treatments gave the largest egg production, yielding approximately 28% more eggs than any other treatment. Since citrate is a known chelator of divalent metal cations, the effects of supplemental cation additions to citrated blood were tested for their ability to reverse the egg production decrease seen at 50 mM sodium citrate. Blood samples containing 50mM sodium citrate were supplemented with CaCl₂, calcium lactate, CuCl₂, cupric acetate, FeCl₃, ferric citrate, MgCl₂, magnesium acetate, MnCl₂, ZnSO₄, EGTA or EGTA plus calcium lactate, each at 1 mM except EGTA which was used at 2.5 mM. The magnesium acetate supplement and the combination of calcium lactate plus EGTA resulted in a statistically significant increase in egg production ( P < 0.05).     

[3H]GLYCOGEN HYDROLYSIS IN BRAIN SLICES: RESPONSES TO NEUROTRANSMITTERS AND MODULATION OF NORADRENALINE RECEPTORS

Abstract— Different agents have been investigated for their effects on [C³H]glycogen synthesized in mouse cortical slices. Of these noradrenaline, serotonin and histamine induced clear concentration-dependent glycogenolysis.
[C³H]Glycogen hydrolysis induced by noradrenaline appears to be mediated by beta-adrenergic receptors because it is completely prevented by timolol, while phentolamine is ineffective. It seems to involve cyclic AMP because it is potentiated in the presence of isobutylmethylxanthine; in addition dibutyryl cyclic AMP (but not dibutyryl cyclic GMP) promotes glycogenolysis.
Lower concentrations of noradrenaline were necessary for [C³H]glycogen hydrolysis (EC₅₀= 0.5μM) than for stimulation of cyclic AMP accumulation (EC₅₀= 8μM).
After subchronic reserpine treatment the concentration-response curve to noradrenaline was significantly shifted to the left (EC₅₀= 0.09 ± 0.02 μM as compared with 0.49 ± 0.08 μM in saline-pretreated mice) without modifications of either the basal [C³H]glycogen level, maximal glycogenolytic effect, or the dibutyryl cAMP-induced glycogenolytic response.
In addition to noradrenaline, clear concentration-dependent [³H]glycogen hydrolysis was observed in the presence of histamine or serotonin. In contrast to the partial [³H]glycogen hydrolysis elicited by these biogenic amines, depolarization of the slices by 50 mM K⁺ provoked a nearly total [C³H)glycogen hydrolysis.     

NMDA-mediated modulation of dopamine release is modified in rat prefrontal cortex and nucleus accumbens after chronic nicotine treatment

In this study, we investigate the effects of chronic administration of (−)nicotine on the function of the NMDA-mediated modulation of [³H]dopamine (DA) release in rat prefrontal cortex (PFC) and nucleus accumbens (NAc). In the PFC synaptosomes NMDA in a concentration-dependent manner evoked [³H]DA release in rats chronically treated with vehicle (14 days) with an EC₅₀ of 13.1 ± 2.0 μM. The NMDA-evoked overflow of the [³H]DA in PFC nerve endings of rats treated with (−)nicotine was significantly lower (−43%) than in vehicle treated rats. The EC₅₀ was 9.0 ± 1.4 μM. Exposure of NAc synaptosomes of rats treated with vehicle to NMDA produced an increase in [³H]DA overflow with an EC₅₀ of 14.5 ± 5.5 μM. This effect was significantly enhanced in chronically treated animals. The EC₅₀ was 10.5 ± 0.5 μM. The K⁺-evoked release of [³H]DA was not modified by the (−)nicotine administration. Both the changes of the NMDA-evoked [³H]DA overflow in the NAc and PFC disappeared after 14 days withdrawal. The results show that chronic (−)nicotine differentially affects the NMDA-mediated [³H]DA release in the PFC and NAc of the rat.     

Application of bioluminescence-based microbial biosensors to the ecotoxicity assessment of organotins

The toxicity of two common organotin pollutants and their initial breakdown products (tributyltin, dibutyltin, triphenyltin and diphenyltin) were assessed using two different bioluminescent microbial biosensors: Microtox and lux -modified Pseudomonas fluorescens pUCD 607. The organotins were made up as standards, and tested both in double-deionized water and in extracted soil solution, the latter representing a realistic matrix for terrestrial contamination. Microtox was especially sensitive to the organotins, with 50% effective concentration (EC₅₀) values (15 min) for tributyltin as low as 21·9 μg l⁻¹ in pure water, and 0·118 μg l⁻¹ in soil extract. The sensitivity of Microtox was increased by an order of magnitude in soil extract. The Ps. fluorescens was less sensitive, with EC₅₀ values (30 min) of around 800 μg l⁻¹ in pure water. The toxicity to Ps. fluorescens was decreased by around an order of magnitude in soil extract. The two biosensors showed different response patterns, with Microtox being more sensitive to the triorganotins and Ps. fluorescens being more sensitive to the diorganotins.     

Surfactants as Chemical Shark Repellents: Past, Present, and Future

The development of Shark Chaser by the U.S. Navy during World War II was the first serious effort to develop a chemical shark repellent. In the decade following the war reports of Shark Chaser ineffectiveness led the Office of Naval Research to search for a more efficacious shark repellent. After years without success, ONR eventually canceled the use of Shark Chaser and abandoned the search for a chemical shark repellent. In the early 1970s, interest in chemical shark repellents was renewed by the discovery of pardaxin, a natural shark repellent secreted by the Red Sea Moses sole, Pardachirus marmoratus. The surfactant-like nature of pardaxin led investigators to test the potential of various surfactants as repellents. Subsequent studies indicated that the shark repellent efficacy of the effective alkyl sulfate surfactants was due to their hydrophobic nature. Here we report tests conducted on juvenile swell sharks, Cephaloscyllium ventriosum, to determine if the noxious quality of alkyl sulfates is affected by surfactant hydrophobicity [carbon chain length and ethylene oxide (EO) groups] and counterions. Our results indicate that the aversive response of sharks to alkyl sulfate surfactants increases with carbon chain length from octyl to dodecyl, decreases with the addition of EO groups and is not affected by counterions. This study confirms that dodecyl sulfate is the most effective surfactant shark repellent, but it does not meet the Navy's potency requirement for a nondirectional surrounding-cloud type repellent of 100 parts per billion (0.1ugml^–1). Thus, dodecyl sulfate is only practical as a directional repellent such as in a squirt application. Future research should test the action of alkyl sulfates on cell membranes, the potential of other biotoxic agents, and semiochemicals in the search for an effective chemical shark repellent.     

Coupling of Serotonin 5-HT1B Receptors to Activation of Mitogen-Activated Protein Kinase (ERK-2) and p70 S6 Kinase Signaling Systems

Abstract: Little is known about the coupling of serotonin 5-HT_1B receptors to cellular signals other than cyclic AMP. In the present studies, the activation by 5-HT_1B receptors of p70 S6 kinase and the mitogen-activated protein kinase (MAP kinase) ERK-2 was investigated. Studies were performed by using both nontransfected Chinese hamster ovary (CHO) cells, which express endogenous receptors at a very low density, and a stable transfected CHO cell line expressing 5-HT_1B receptors at 230 fmol/mg of membrane protein, a density similar to that expressed in cortex. In nontransfected cells, 5-HT was found to stimulate a greater than twofold increase in MAP kinase activity with an EC₅₀ of 20 n M . Reflecting increased density of receptors, 5-HT caused a greater than eightfold activation of ERK-2 in transfected cells with an EC₅₀ of 2 n M . 5-HT was found to also stimulate p70 S6 kinase in both nontransfected and transfected cells. The stimulation was sixfold in both types of cells, but the EC₅₀ for 5-HT was fourfold lower in transfected cells. The coupling of 5-HT_1B receptors to ERK-2 and to p70 S6 kinase was inhibited by pertussis toxin, inhibitors of phosphatidylinositol 3-kinase, and by the inhibitor of MAP kinase kinase PD098059. Activation of p70 S6 kinase, but not ERK-2, was also inhibited by rapamycin. These findings demonstrate that 5-HT_1B receptors couple to ERK-2 and p70 S6 kinase through overlapping, but nonidentical, pathways.     

Desensitization of Nicotine-Stimulated [3H]Dopamine Release from Mouse Striatal Synaptosomes

Potential desensitization of brain nicotinic receptors was studied using a [³H]dopamine release assay. Nicotine-stimulated [³H]dopamine release from mouse striatal synaptosomes was concentration-dependent with an EC₅₀ of 0.33 ± 0.13 μ M and a Hill coefficient of 1.44 ± 0.18. Desensitization by activating concentrations of nicotine had a similar EC₅₀ and a half-time of 35 s. Concentrations of nicotine that evoked little release also induced a concentration-dependent desensitization (EC₅₀=6.9 plusmn; 3.6 n M , t_1/2= 1.6-2.0 min, n _H=1.02 ± 0.01). Both types of desensitization produced a maximum 75% decrease in [³H]dopamine release. Recovery from desensitization after exposure to low or activating concentrations of nicotine was time-dependent with half-times of 6.1 min and 12.4 min, respectively. Constants determined for binding of [³H]nicotine to striatal membrane at 22°C included a K _Dof 3.7 ± 0.5 n M , B_max of 67.5 ± 2.2 fmol/mg, and Hill coefficient of 1.07 ± 0.06. Association of nicotine with membrane binding sites was biphasic with half-times of 9 s and 1.8 min. The fast rate process contributed 37% of the total reaction. Dissociation was a uniphasic process with a half-time of 1.6 min. Comparison of constants determined by the release and binding assays indicated that the [³H]-nicotine binding site could be the presynaptic receptor involved in [³H]dopamine release in mouse striatal synaptosomes.