Selective Increase of Tetrameric (G4) Acetylcholinesterase Activity in Rat Hindlimb Skeletal Muscle Term Denervation

Acetylcholinesterase (AChE; EC 3.1.1.7) isoenzymes in gracilis muscles from adult Sprague-Dawley rats were studied 24-96 h after obturator nerve transection. Results show a selective denervation-induced increase in the globular G4 isoform, which is predominantly associated with the plasmalemma. This enzymatic increase was (a) transient (occurring between 24 and 60 h) and accompanied by declines in all other identifiable AChE isoforms; (b) observed after concurrent denervation and inactivation of the enzyme with diisopropylfluorophosphate, but not following treatment with cycloheximide; and (c) more prominent in the extracellular compartment of muscle endplate regions. Aside from this transient change, G4 activity did not fall below control levels, indicating that at least the short-term maintenance of G4 AChE (i.e., at both normal and temporarily elevated levels) does not critically depend on the presence of the motor nerve. In addition, this isoform's activity increases in response to perturbations of the neuromuscular system that are known to produce elevated levels of acetylcholine (ACh), such as short-term denervation and exercise-induced enhancement of motor activity. The present study is consistent with the hypothesis that individual AChE isoforms in gracilis muscle are subject to distinct modes of neural regulation and suggests a role for ACh in modulating the activity of G4 AChE at the motor endplate.     

Interactions of Muscarinic Receptors with the Heterotrimeric G Proteins Gq and G12: Transduction of Proliferative Signals

Abstract: The proliferative and transforming properties of m2 and m5 muscarinic acetylcholine receptors and a series of wild-type, chimeric, and mutant G proteins were measured alone or in combination in NIH 3T3 cells to determine which G proteins mediate these signals and to what extent these signals can be influenced by changing the stoichiometry of receptors and G proteins. Responses were measured using the focus-forming assay and a novel assay called R-SAT (R eceptor S election and A mplification T echnology) in which proliferative responses are monitored using a reporter gene. Individually, GTPase-deficient mutants (^?) of Gαq and Gα12, wild-type Gαq, and m5 were active in R-SAT. Gα12^? and m5 also induced focus formation. m2 was inactive in both assays. The ability of m5 to induce foci was significantly reduced by coexpression of Gαq^?. Synergistic effects of receptor/G protein combinations were not observed in focus-forming assays but were readily detected by R-SAT. Coexpression of Gαq with m5 induced constitutive activity in R-SAT and increased the potency of agonists at m5 by 90-fold. Gαq also evoked agonist-dependent responses from m2 but not constitutive activity. Agonist potency was increased 10-fold at m2 and decreased 15-fold at m5 when these receptors were coexpressed with Gαqi5, a chimeric G protein containing the five C-terminal residues of Gαi2, compared with coexpression with Gαq. Both Gαq and Gαqi5 had biphasic effects on the proliferative responses to m5 and m2, respectively, inhibiting responses at high agonist concentrations. Coexpression of Gα12 or Gα12i5 had no effect on the concentration-response relationships of m5, but both elicited weak responses from m2. We conclude that although Gα12 is a more potent oncogene, Gαq transduces m5-driven cellular responses. The demonstrations that proliferative responses can be elicited from a nonmitogenic receptor by altering the type and concentration of available G proteins and that constitutive responses can be induced by G proteins imply that both the magnitude and type of receptor-initiated signal can be regulated at the level of G proteins in vivo.     

Characterization of the Solubilized A1 Adenosine Receptor from Rat Brain Membranes

A1 adenosine receptors from rat brain membranes were solubilized with the zwitterionic detergent 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized receptors retained all the characteristics of membrane-bound A1 adenosine receptors. A high and a low agonist affinity state for the radiolabelled agonist (R)-N6-[3H]phenylisopropyladenosine([3H]PIA) with KD values of 0.3 and 12 nM, respectively, were detected. High-affinity agonist binding was regulated by guanine nucleotides. In addition agonist binding was still modulated by divalent cations. The solubilized A1 adenosine receptors could be labelled not only with the agonist [3H]PIA but also with the antagonist 1,3-diethyl-8-[3H]phenylxanthine. Guanine nucleotides did not affect antagonist binding as reported for membrane-bound receptors. These results suggest that the solubilized receptors are still coupled to the guanine nucleotide binding protein Ni and that all regulatory functions are retained on solubilization.     

Desensitisation of the Adenosine A1 Receptor by the A2A Receptor in the Rat Striatum

Abstract: The influence of the adenosine A_2A receptor on the A₁ receptor was examined in rat striatal nerve terminals, a model for other cells in which these receptors are coexpressed. Incubation of striatal synaptosomes with the A_2A receptor agonist 2- p -(2-carboxyethyl)phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS 21680) caused the appearance of a low-affinity binding site for the A₁ receptor agonist 2-chloro- N ⁶-cyclopentyladenosine (CCPA). This effect was blocked by the A_2A receptor antagonist ZM241385 and by the protein kinase C inhibitor chelerythrine, but not by the protein kinase A inhibitor N -(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004). The effect was not seen with striatal membranes or with hypotonically lysed synaptosomes. These results demonstrate a protein kinase C-mediated heterologous desensitisation of the A₁ receptor by the A_2A receptor.     

Pertussis Toxin-Insensitive Signaling of the ORL1 Receptor: Coupling to Gz and G16 Proteins

Abstract: Nociceptin/OFQ is the endogenous ligand for the G protein-coupled opioid receptor-like (ORL₁) receptor. To elucidate the cellular functions of the ORL₁ receptor, we examined its ability to interact with G_z and G₁₆, two pertussis toxin (PTX)-insensitive G proteins that are known molecular partners for the opioid receptors. In HEK 293 cells transiently expressing the ORL₁ and dopamine D₁ receptors, nociceptin/OFQ dose-dependently inhibited dopamine-stimulated cyclic AMP (cAMP) accumulation in a PTX-sensitive manner. However, PTX failed to block the nociceptin/OFQ-induced inhibition of dopamine-stimulated cAMP accumulation in HEK 293 cells co-expressing the α-subunit of G_z. This result indicates functional interaction between the ORL₁ receptor and G_z. A similar result was obtained with retinoic acid-differentiated SH-SY5Y cells, which endogenously express both the ORL₁ receptor and G_z. When the ORL₁ receptor was transiently co-expressed in COS-7 cells with the α-subunit of G₁₆, nociceptin/OFQ dose-dependently stimulated the formation of inositol phosphates. Nociceptin-induced stimulation of phospholipase C was absolutely dependent on the co-expression of α₁₆ and exhibited the appropriate ligand selectivity. In terms of its ability to interact with PTX-insensitive G proteins, the ORL₁ receptor behaves very much like the opioid receptors.     

[3H]SCH 58261, a Selective Adenosine A2A Receptor Antagonist, Is a Useful Ligand in Autoradiographic Studies

Abstract: We have characterized the new potent and selective nonxanthine adenosine A_2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A_2A receptors ([³H]CGS 21680) or A₁ receptors ( N ⁶-[³H]cyclohexyladenosine), it was found that SCH 58261 is close to 800-fold selective for rat brain A_2A versus A₁ receptors ( K _i values of 1.2 n M versus 0.8 µ M ). Moreover, receptor autoradiography showed that [³H]SCH 58261, in concentrations below 2 n M , binds only to the dopamine-rich regions of the rat brain, with a K _D value of 1.4 (0.8–1.8) n M . The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 n M , the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [³H]SCH 58261 with the following estimated K _i values (n M ): 2-hex-1-ynyl-5'- N -ethylcarboxamidoadenosine, 3.9 (1.8–8.4); CGS 21680, 130 (42–405); N ⁶-cyclohexyladenosine, 9,985 (3,169–31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 µ M ) or Mg²⁺ (10 m M ). The present results show that in its tritium-labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [³H]CGS 21680.     

Effect of Pertussis Toxin on Radioligand Binding to Rat Brain Adenosine A1 Receptors

In a previous study we showed that in vivo treatment with pertussis toxin could inhibit some, but not all, effects of adenosine in the rat hippocampus. In this study we investigated the effect of pertussis toxin on the binding of adenosine analogues to A1 receptors in rat brain. Intraventricular injection of pertussis toxin (10 micrograms into the lateral ventricle) did not affect A1 receptor binding in any brain region studied, as evaluated by autoradiography. In vitro treatment of brain sections (10 microns) with pertussis toxin for 5 h, under conditions when greater than 80% of the G proteins were ADP ribosylated, did not alter radioligand binding to adenosine A1 receptors. GTP (10 microM) virtually abolished the high-affinity agonist binding to the A1 receptor. On the other hand, in solubilized cortical membrane preparations, pertussis toxin pretreatment induced a complete shift of the A1 receptors to the low-affinity state. This suggests that the ability of pertussis toxin to affect G proteins coupled to A1 receptors in brain depends not only on the distribution of the toxin but also on the configuration of receptors and G proteins.     

Contribution of Noradrenergic Neurons to the Regulation of Dopaminergic (D1) Receptor Denervation Supersensitivity in Rat Prefrontal Cortex

3,4-Dihydroxyphenylethylamine (DA, dopamine) levels in the rat prefrontal cortex were selectively decreased by 52%, leaving noradrenaline (NA) levels unaffected, 4 weeks following restricted bilateral electrolytic lesions of the ventral mesencephalic tegmentum (VMT). These lesions also induced a significant increase in DA-sensitive, but not isoproterenol-sensitive, adenylate cyclase activity in tissue homogenates (+38%). We had shown previously that chemical (6-hydroxydopamine, 6-OHDA) lesions of the VMT destroy both ascending DA and NA fibers but do not alter the D1-receptor density in the prefrontal cortex. In this study, electrolytic lesions of the VMT were combined with bilateral injections of 6-OHDA made laterally in the pedunculus cerebellaris superior to assess the role of NA fibers in the development of D1-receptor supersensitivity. This combined treatment produces a large decrease of cortical NA levels (-95%), an increase in beta-adrenergic-sensitive adenylate cyclase activity (+110%), and a decrease in DA levels (-60%), but does not alter D1-receptor density in the prefrontal cortex. These results indicate that the development of D1-receptor supersensitivity in the prefrontal cortex following electrolytic lesion of the VMT depends on the presence of an intact NA innervation.     

A Procedure for Purification of the Ryanodine Receptor from Skeletal Muscle

In this paper, we describe a simple and reproducible method for purifying large quantities of ryanodine receptor from skeletal muscle membranes. The procedure involves the use of ion exchange chromatography and sucrose gradient centrifugation to purify the protein which has been identified as the calcium release protein of the sarcoplasmic reticulum (Imagawa, T., Smith, J., Coronado, R. and Campbell, K. (1987) J. Biol. Chem. 262:16,636–16,643). Addition of micromolar quantities of unlabeled ryanodine prior to solubilization and throughout the isolation procedure appears to stabilize the tetrameric structure of the ryanodine receptor. The purified receptor, consisting predominantly of a 400K polypeptide on SDS-PAGE, binds [³H]ryanodine with a binding affinity similar to that in membranes. Overall recovery of ryanodine binding activity was 21% of the initial activity with a 30-fold purification of the receptor.     

Cloning and Expression of a Human Serotonin 5-HT4 Receptor cDNA

Abstract: Using a combination of library screening and nested PCR based on a partial human serotonin 5-HT₄ receptor sequence, we have cloned the complete coding region for a human 5-HT₄ receptor. The sequence shows extensive similarity to the published porcine 5-HT_4A and rat 5-HT_4L receptor cDNA; however, in comparison with the latter, we find an open reading frame corresponding to only 388 amino acids instead of 406 amino acids. This difference is due to a frame shift caused by an additional cytosine found in the human sequence after position 1,154. Moreover, we also found the same additional cytosine in the rat 5-HT₄ sequence. We confirmed the occurrence of the sequence by examining this part of the sequence in genomic DNA of 10 human volunteers and in rat genomic DNA. Based on a part of the genomic 5-HT₄ receptor sequence that was identified in the cloning process, there seem to be at least two possible splice sites in the coding region of the gene. The human 5-HT₄ receptor, transiently expressed in COS-7 cells, showed radioligand binding properties similar to 5-HT₄ receptors in guinea pig striatal tissue. [³H]GR 113808 revealed K _D values of 0.15 ± 0.01 n M for the human receptor and 0.3 ± 0.1 n M in the guinea pig tissue. Binding constants were determined for four investigated 5-HT₄ antagonists and three agonists, and appropriate binding inhibition constants were found in each case. Stimulation of transfected COS-7 cells with 5-HT₄-specific agonists caused an increase in cyclic AMP levels.     

Lasting Increase in Serotonin 5-HT1A but Not 5-HT4 Receptor Subtypes in the Kindled Rat Dentate Gyrus: Dissociation from Local Presynaptic Effects

Abstract: We examined the effect of kindling on serotonergic neurotransmission in the hippocampus by measuring serotonin (5-HT) release and uptake in hippocampal synaptosomes and 5-HT_1A and 5-HT₄ receptor subtypes during and at different times after electrical kindling of the dentate gyrus. Using quantitative receptor autoradiography, we found that binding of 8-[³H]hydroxy-2-(di- n -propylamino)tetralin ([³H]8-OH-DPAT) to 5-HT_1A receptors was selectively increased by 20% on average ( p < 0.05) in the dentate gyrus of the stimulated and contralateral hippocampus 2 days after stage 2 (stereotypes and occasional retraction of a forelimb) and by 100% on average ( p < 0.05) 1 week after stage 5 (tonic-clonic seizures) compared with sham-stimulated rats. A 20% increase ( p < 0.05) was observed 1 month after the last generalized seizure. No changes were found after a single afterdischarge. 5-HT₄ receptors, which colocalize with 5-HT_1A receptors on hippocampal neurons, were not modified in kindled tissue. [³H]5-HT uptake and its release as well as the 5-HT_1B autoreceptor function did not differ from shams in hippocampal synaptosomes at stages 2 and 5. Systemic administration of 100 and 1,000 µg kg⁻¹ 8-OH-DPAT or 1,000 µg kg⁻¹ WAY-100,635, 30 min before each electrical stimulation, did not significantly alter kindling progression or the occurrence of stage 5 seizures in fully kindled rats. The changes in 5-HT_1A receptor density in the dentate gyrus are part of the plastic modifications occurring during kindling and may contribute to modulating tissue hyperexcitability.     

Pertussis Toxin Attenuates D2 Inhibition and Enhances D1 Stimulation of Adenylate Cyclase by Dopamine in Rat Striatum

The response of adenylate cyclase to GTP and to dopamine (DA) was investigated in synaptic plasma membranes isolated from rat striatum injected with pertussis toxin, which inactivates the inhibitory guanine nucleotide-binding regulatory protein (Ni) of adenylate cyclase. Pertussis toxin treatment reverted the inhibitory effects on the enzyme activity elicited by micromolar concentrations of GTP and reduced by 50% the DA inhibition of cyclase activity via D2 receptors. The toxin treatment enhanced the net stimulation of enzyme activity by DA in the presence of micromolar concentrations of GTP. However, the stimulatory effect of the selective D1 receptor agonist SKF 38393 was not significantly affected. The data indicate that Ni mediates D2 inhibition of striatal adenylate cyclase and participates in the modulation of D1 stimulation of the enzyme activity by DA.     

Evidence for D4 Receptor Regulation of Retinomotor Movement in Isolated Teleost Cone Inner-Outer Segments

Abstract: In the retinas of teleost fish, cone photoreceptors change shape in response to light and circadian signals. They elongate in the dark, contract in the light, and under conditions of constant darkness undergo appropriate movements at expected dusk and dawn. Dopamine induces cones to contract, thus mimicking the effect of light or expected dawn. To identify the receptor subtype responsible for mediating dopamine regulation of cone retinomotor movements, we have carried out pharmacological studies using isolated fragments of teleost cones consisting of cone inner segments-cone outer segments (CIS-COS). Isolated CIS-COS retain the ability to elongate in dark culture and contract when subsequently exposed to light or dopamine. We report that dark-induced elongation of CIS-COS was inhibited by dopamine and its agonists with an effectiveness ranking of dopamine = quinpirole > bromocriptine ⋙SKF-38393. After 60 min of elongation in dark culture, CIS-COS myoids contracted when subsequently cultured in the dark with dopamine or quinpirole. Quinpirole-induced inhibition of elongation and quinpirole-induced contraction were completely blocked by clozapine at 1 µ M or by sulpiride at 100 µ M . These effectiveness profiles for dopamine agonists and antagonists suggest that dopamine regulation of cone retinomotor movement is mediated by a D₄-like receptor.     

Surgical Sympathetic Denervation Increases α1Adrenoceptor-Mediated Accumulation of myo-Inositol Trisphosphate and Muscle Contraction in Rabbit Iris Dilator Smooth Muscle

Sympathetic denervation of the iris muscle produces increases in both the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) and in muscle contraction in response to norepinephrine (NE). To shed more light on the biochemical basis underlying this supersensitivity we investigated: the effects of NE on PIP2 breakdown, measured as myo-inositol trisphosphate (IP3) accumulation, and on muscle contraction in normal and denervated rabbit iris dilator; and the effects of denervation on selected biochemical properties of this muscle. The data obtained from these studies can be summarized as follows: The EC50 values (microM) for NE-induced IP3 accumulation in normal and denervated dilators were 14 and 3, respectively. This accumulation of IP3 was blocked by prazosin (1 microM). The EC50 values (microM) for NE-induced contraction for the normal and denervated muscles were 10 and 0.6, respectively. The NE-induced muscle contraction was blocked by prazosin (1 microM). The t1/2 values (s) for IP3 accumulation in normal and denervated muscles were 31 and 11, respectively, and for contraction the values were 19 and 9, respectively. Denervation increased significantly (15-18%) the basal labelling of phosphoinositides from myo-[3H]inositol, but not from 32P or [14C]arachidonic acid. Denervation had little effect on the activities of the enzymes involved in phosphoinositide metabolism. However, the activities of protein kinase C and Ca2+-ATPase increased in the denervated muscle. It is concluded that sympathetic denervation of the iris dilator renders the coupling between alpha1 receptors and PIP2 breakdown into IP3 and 1,2-diacylglycerol (DG) more efficient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase in Striatal [3H]Muscimol Binding Following Intrastriatal Injection of Kainic Acid: A Denervation Supersensitivity Phenomenon

The effect of intrastriatal microinjection of kainic acid (KA) on specific binding of [³H]muscimol to the particulate fractions obtained from corpus striatum (CS), globus pallidus (GP), substantia nigra (SN), and cerebral cortex (CC) was examined. Seven days after the unilateral intrastriatal microinjection of KA, the amount of specifically bound [³H]muscimol was significantly increased at the injected site, whereas no significant alteration of [³H]muscimol binding was found in GP, SN, or CC. Scatchard analysis of striatal binding revealed that microinjection of KA significantly increased the affinity (K_D) of GABA receptors on the injected (lesioned) side of the CS without affecting the total number of binding sites (B_max) therein. This significant increase in [³H]muscimol binding, however, was eliminated by pretreating particulate fractions from the CS with Triton X-100, a non-ionic detergent. No statistically significant difference in amounts of [³H]muscimol binding was detected when the preparations from the KA-treated and non-treated CS were preincubated with 0.05% Triton X-100, respectively. Scatchard analysis using CS preparations treated with 0.05% Triton X-100 revealed that the affinity of the GABA receptor was increased by treatment with Triton X-100, while the total number of binding sites (B_max) was unchanged by this treatment. These results suggest that neuronal degeneration produced by KA in vivo and pretreatment of particulate preparations with Triton X-100 in vitro may increase the amount of specifically bound [³H]muscimol to CS preparations by a similar molecular mechanism.     

NMDA Receptors with Different Sensitivities to Magnesium and Ifenprodil Control the Release of [14C]Acetylcholine and [3H]Spermidine from Rat Striatal Slices in Vitro

Abstract: KCI (20–100 mM) and W-methyl-D-aspartate (NMDA, 100–1,000 μM) produce concomitant concentration-dependent increases in the release of previously captured [¹⁴C]acetylcholine and [³H]spermidine from rat striatal slices in vitro. The effects of NMDA (300μM) on striatal [¹⁴C]acetylcholine and [³H]spermidine release were blocked with equal potencies by the competitive NMDA antagonist CGP 37849, the glycine site antagonist L-689,560, and the NMDA channel blocker dizocilpine. In contrast, although NMDA-evoked [¹⁴C]acetylcholine release was antagonized by ifenprodil (IC₅₀= 5.3 μM) and MgCl₂, (IC₅₀= 200 μM), neither compound antagonized the NMDA-evoked release of [³H]spermidine at concentrations up to 100 μM (ifenprodil) or 1 mM (MgCl₂). Distinct NMDA receptor subtypes with different sensitivities to magnesium and ifenprodil therefore exist in the rat striaturn.     

Alterations in the Production of 14CO2 and [14C]Acetylcholine from [U-14C]Glucose in Brain Subregions Following Transient Forebrain Ischemia in the Rat

Abstract: The production of ¹⁴CO₂ and [¹⁴C]acetylcholine from [U-¹⁴C]glucose was determined in vitro using tissue prisms prepared from the dorsolateral striatum (a region developing extensive neuronal loss following ischemia) and the paramedian neocortex (an ischemia-resistant region) following 30 min of forebrain ischemia and recirculation up to 24 h. Measurements were determined under basal conditions (5 mMK⁺) and following K⁺ depolarization (31 mM K⁺). The production of ¹⁴CO₂ by the dorsolateral striatum was significantly reduced following 30 min of ischemia for measurements in either 5 or 31 mM K⁺ but recovered toward preischemic control values during the first hour of recirculation. Further recirculation resulted in ¹⁴CO₂ production again being reduced relative to control values but with larger differences (20–27% reductions) detectable under depolarized conditions at recirculation times up to 6 h. Samples from the paramedian neocortex showed no significant changes from control values at all time points examined. [¹⁴C]Acetylcholine synthesis, a marker of cholinergic terminals that is sensitive to changes in glucose metabolism in these structures, was again significantly reduced only in the dorsolateral striatum. However, even in this tissue, only small (nonstatistically significant) differences were seen during the first 6 h of recirculation, a finding suggesting that changes in glucose oxidation during this period were not uniform within all tissue components. The results of this study provide evidence that in a region susceptible to ischemic damage there were specific changes during early recirculation in the metabolic response to depolarization. This apparent inability to respond appropriately to an increased need for energy production could contribute to the further deterioration of cell function in vivo and ultimately to the death of some cells.     

Regulation of L-Type Calcium Channels in GH4 Cells via A1 Adenosine Receptors

Abstract: Identification of A₁ adenosine receptors (A₁Rs) in a tumor cell line derived from rat pituitary (GH₄ cells) was performed by ligand binding and immunological experiments. Subsequently, the involvement of A₁Rs in the regulation of calcium conductance was studied in these cells. The agonist N ⁶-( R )-(2-phenylisopropyl)adenosine ( R -PIA) did not modify the intracellular calcium basal levels, whereas it inhibited the increase produced by 15 m M KCl depolarization. The antagonist 1,3-dipropyl-8-cyclopentylxanthine led to the opening of voltage-dependent cell surface calcium channels in the absence of exogenous KCl. The channels were of the L type because the effect was abolished by calciseptine and by verapamil. These results suggest that endogenous adenosine exerts a tonic inhibitory effect on calcium transport. This was confirmed by the high adenosine concentration found in cell supernatants (up to 1 µ M ) and by the calcium mobilization produced by exogenously added adenosine deaminase. In depolarizing conditions, the calcium peak in the presence of adenosine deaminase was reduced when cells were preincubated with R -PIA, thus suggesting that A₁R activation regulates the intensity of depolarization. These results demonstrate that adenosine is an important regulator of the physiological state of pituitary tumor cells by modulating, in an autocrine manner, the activity of L-type voltage-dependent calcium channels.     

Autoradiographic Visualization of A1 Adenosine Receptors in Rat Brain with [3H]8-Cyclopentyl-1,3-Dipropylxanthine

A1 adenosine receptors were labeled in rat brain sections with the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) and visualized at the light microscopic level using autoradiography. The specific binding of [3H]DPCPX to the sections showed the pharmacological characteristics of A1 adenosine receptors and was accompanied by very low levels of nonspecific binding. Whereas GTP had no significant effect on [3H]DPCPX binding to rat brain membranes, the addition of 100 microM GTP increased the apparent affinity of [3H]DPCPX to tissue sections fivefold (from 1.83 to 0.35 nM), enhancing it to the affinity measured in membranes. However, GTP altered neither the binding capacity nor the distribution of binding sites in tissue sections. It is suggested that a competitive antagonism with endogenous adenosine explains the lower affinity of [3H]DPCPX in the absence of GTP. The autoradiographic pattern of [3H]DPCPX binding was characteristic for A1 adenosine receptors. Distinct labeling of the different layers of the cerebellar cortex was shown by photomicrographs generated with the coverslip technique. In addition, several fiber tracts were found to be labeled. The high selectivity for A1 adenosine receptors and low nonspecific binding of [3H]DPCPX, the ability to produce high-resolution autoradiograms, together with the fact that the effects of endogenous adenosine can be eliminated by the addition of GTP make [3H]DPCPX a very useful tool in the autoradiographic study of A1 adenosine receptors.     

Conserved Extracellular Cysteine Pair in the M3 Muscarinic Acetylcholine Receptor Is Essential for Proper Receptor Cell Surface Localization but Not for G Protein Coupling

Most G protein-coupled receptors contain a conserved pair of extracellular cysteine residues that are predicted to form a disulfide bond linking the first and second extracellular loops. Previous studies have shown that this disulfide bond may be critical for ligand binding, receptor activation, and/or proper receptor folding. However, the potential importance of the two conserved cysteine residues for proper receptor cell surface localization has not been investigated systematically. To address this issue, we used the rat M3 muscarinic receptor as a model system. Most studies were carried out with a modified version of this receptor subtype (lacking potential N-glycosylation sites and the central portion of the third intracellular loop) that could be readily detected via western blot analysis. Cys-->Ala mutant receptors were generated, transiently expressed in COS-7 cells, and then examined for their subcellular distribution and functional properties. ELISA and immunofluorescence studies showed that the presence of both conserved cysteine residues (corresponding to C140 and C220 in the rat M3 muscarinic receptor sequence) is required for efficient expression of the M3 muscarinic receptor on the cell surface. On the other hand, these residues were found not to be essential for protein stability (determined via immunoblotting) and receptor-mediated G protein activation (studied in second messenger assays). These results shed new light on the functional role of the two extracellular cysteine residues present in most G protein-coupled receptors.